CN114652851A - Antibody conjugate of anti-TROP2 protein - Google Patents

Antibody conjugate of anti-TROP2 protein Download PDF

Info

Publication number
CN114652851A
CN114652851A CN202111155804.5A CN202111155804A CN114652851A CN 114652851 A CN114652851 A CN 114652851A CN 202111155804 A CN202111155804 A CN 202111155804A CN 114652851 A CN114652851 A CN 114652851A
Authority
CN
China
Prior art keywords
cancer
trop2
ser
gly
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202111155804.5A
Other languages
Chinese (zh)
Other versions
CN114652851B (en
Inventor
姚兵
高晓
淡墨
袁灿
胡喜新
孙召朋
惠希武
刘伯宁
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jushi Biopharmaceutical Co ltd
Original Assignee
Jushi Biopharmaceutical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jushi Biopharmaceutical Co ltd filed Critical Jushi Biopharmaceutical Co ltd
Priority to CN202111155804.5A priority Critical patent/CN114652851B/en
Publication of CN114652851A publication Critical patent/CN114652851A/en
Application granted granted Critical
Publication of CN114652851B publication Critical patent/CN114652851B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Landscapes

  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Hematology (AREA)
  • Oncology (AREA)

Abstract

Providing an antibody conjugate drug targeting TROP2 shown in formula (I), and pharmaceutically acceptable salts, hydrates, solvates or isotopically labeled analogues thereof: ab- (L-D)n(I) And uses and methods of making thereof, wherein Ab, L, D, and n are as defined in the specification.

Description

Antibody conjugate of anti-TROP2 protein
Background
TROP2 is called human Trophoblast cell surface antigen (2), belongs to TACTD (Tumor-Associated Calcium Signal Transducer) family, is a cell surface glycoprotein encoded and expressed by TACTD 2 gene, and is also named as Tumor-Associated Calcium ion Signal Transducer 2 (TACTD 2), epidermal glycoprotein 1(EGP-1), gastrointestinal Tumor-Associated antigen (733 GA-1), and membrane component chromosome 1 surface marker 1(M1S 1). It was originally identified as an antigen present on human gastrointestinal tumors, and another family member was GA733-2, also known as epithelial cell adhesion molecule (EpCAM), Trop-1, or KSA. The TROP2 gene is unique in that it does not contain introns, and studies of both genes have shown that TROP2 is the result of the EpCAM gene relocation. TROP2 and EpCAM have about 49% amino acid homology and about 67% sequence similarity. The Trop-2 of human origin and mouse has 87% sequence similarity, and the human Trop2 protein consists of a 26aa signal peptide, 248aa extracellular domain, 23aa transmembrane region and 26aa cytoplasmic region.
It was found that TROP2 is more highly expressed in various human epithelial cancers, including breast, lung, stomach, colorectal, pancreatic, prostate, cervical, head and neck, and ovarian cancers, etc., than in normal tissues. While the biological role of TROP2 is still under investigation, various studies have shown that TROP2 overexpression is associated with increased tumor growth, tumor invasiveness, metastasis and poor prognosis in various human cancers. Studies have also shown that TROP2 is involved in tumor pathogenesis, at least in part, by activating the ERK1/2MAPK pathway, which is important in cancer cell proliferation, migration, invasion, and survival. TROP2 proteolytic regulation may drive epithelial cell proliferation and stem cell self-renewal through β -catenin signaling.
In patients with invasive ductal carcinoma breast cancer, increased expression of TROP2 mRNA is a strong predictor of poor survival and lymph node metastasis, while the Kaplan-Meier survival curve also shows that breast cancer patients with high TROP2 expression have significantly shorter survival. In endometrial cancer tissues, expression of TROP2 is positively correlated with pathological grade, i.e., the degree of expression of TROP2 is correlated with the degree of malignancy of the disease. The survival rate of the TROP2 protein high-expression patient is obviously reduced, so that the TROP2 expression can be used as an independent prognosis judgment index for the patients with shortened survival period. High expression of TROP2 protein is associated with high aggressiveness of ovarian cancer. In conclusion, there is a close relationship between elevated expression of TROP2 and malignancy of the female reproductive system and is associated with the malignancy of the disease. The over-expression of TROP2 exists in pancreatic adenocarcinoma, and is obviously related to lymph node metastasis, tumor grade and poor survival rate of patients, and the TROP2 can be used as a unique pancreatic cancer prognosis judgment biomarker. In colon cancer, expression of TROP2 protein was significantly higher in cancer tissues than in normal tissues. High expression of TROP2 increases the probability of liver metastasis and patient mortality, and the depth of metastasis is also deeper, with more lymph node metastasis and a poor prognosis. Expression of TROP2 is essential for obtaining the capacity of forming and invading colon cancer cell tumor, and after TROP2 is inhibited by RNA interference, the capacity of forming and invading cancer cell tumor is inhibited, so that the proliferation capacity of colon cancer cells which externally express TROP2 is enhanced. In gastric cancer, TROP2 was also highly expressed. The apoptosis of gastric adenocarcinoma cell lines can be obviously promoted by using apoptosis activator 2 aiming at TROP2 antigen. TROP2 was overexpressed in tumor tissues of oral cancer patients. Expression of TROP2 was also elevated in squamous cell carcinoma tissue of the throat and correlated with the degree of tissue differentiation and lymph node metastasis. Expression of TROP2 served as an independent prognostic factor for lymph node metastasis, degree of differentiation, tumor size and T grade. TROP2 is closely linked to the malignancy, proliferation and angiogenesis of brain gliomas and may contribute to the targeted treatment of brain gliomas.
Currently, 7 biological drugs targeting TROP2 are in clinical stage at home and abroad (see table 1 for detailed information), the most rapidly developing ADC molecule targeting TROP2, Trodelvy, 2021 year 04, day 08, the american Food and Drug Administration (FDA) awarding full approval to Trodelvy for treating adult patients with unresectable locally advanced or metastatic Triple Negative Breast Cancer (TNBC) who have previously received at least 2 therapies, at least 1 of which treats metastatic disease, developed by immunology.
Trodelvy is a novel and original antibody conjugate drug (ADC) targeting TROP2, and is prepared by conjugating a humanized IgG1 antibody targeting TROP2 antigen and a metabolic active product SN-38 of a chemotherapeutic drug irinotecan, a topoisomerase I inhibitor, and the drug-antibody ratio (DAR) is as high as 7.6: 1. TROP2 is a cell surface protein frequently expressed in many epithelial tumors, expressed in more than 90% of TNBC. Trodelvy targets TROP2 to bind and deliver the anti-cancer agent SN-38 to kill cancer cells. At the same time, the core of its proprietary ADC platform is the use of a novel linker that does not require enzymes to release the payload and can deliver active drugs within tumor cells and in the tumor microenvironment, thus creating a bystander effect.
In 4 months 2020, Trodelvy received US FDA accelerated approval with the indication of mTNBC. The accelerated approval was based on the total remission rate (ORR 33.3%) and duration of remission (median DOR 7.7 months) for the one-armed multicenter phase II study. Full approval at 08.04.2021 was supported by the results of the phase 3 clinical study ASCENT (NCT02574455) trial. The results show that the patient's risk of disease progression or death is reduced by 57%, both statistically and clinically significant, extending median progression-free survival (PFS) from 1.7 to 4.8 months of chemotherapy (HR: 0.43; 95% Cl: 0.35-0.54; P <0.0001), Trodelvy also extends median Overall Survival (OS) from 6.9 to 11.8 months of chemotherapy (HR:0.51, 95% Cl: 0.41-0.62; P <0.0001), reducing the risk of death by 49%.
Among the three studies of IMMU-132-01, IMMU-132-05(ASCENT) and IMMU-132-06, 795 patients received TRODELVY treatment, with the most common adverse effects (> 25%) being neutropenia (61%), nausea (66%), diarrhea (65%), fatigue (62%), alopecia (45%), anemia (42%), vomiting (39%), constipation (37%), decreased appetite (34%), rash (32%) and abdominal pain (28%). In IMMU-132-05, 63% of patients discontinued treatment with TRODELVY due to adverse effects, the most common (5%) effects that caused discontinuation of treatment were neutropenia (47%), diarrhea (5%), respiratory infections (5%) and leukopenia (5%). 22% of patients decreased the TRODELVY dose due to adverse effects, the most common (> 4%) adverse effects that led to dose reduction were neutropenia (11%) and diarrhea (5%). Therefore, adverse reactions of Trodelvy are relatively serious, and based on the current research situation of a TROP2 target spot, patients still have unmet treatment requirements for related antibody drug conjugates, and high-efficiency and high-safety targeted drugs are urgently needed.
Table 1: biological medicine targeting TROP2 at home and abroad
Figure BDA0003287333030000031
Currently, the structure of Trodelvy on the market is that anti-TROP2 antibody hRS7 is coupled with topoisomerase I inhibitor SN-38 through CL2A linker, and DAR value is about 7.6. An important feature of the ADC is that more than 90% of the SN-38 is released within 3 days after administration, free SN-38 is cleared rapidly, off-target toxicity is reduced, but this instability potentially increases the risk of systemic toxicity, such as neutropenia and diarrhea, both side effects are included in the "black box warning" in the specification of Trodelvy.
The first three co-pending company, Datopotamab derxtecan (DS-1062a), an ADC targeting TROP2 antibody conjugate DXd. The in vitro cell proliferation inhibitory activity of DS-1062a on TROP2 positive tumor cells (CFPAC-1, BxPC-3 (pancreatic cancer cells)) was reported to be represented by IC50 as 706ng/mL and 74.6ng/mL (Okajima, D., et al, "Clinical efficacy students of DS-1062a, a novel TROP2-targeting antibody-drug conjugate with a novel DNA topoisomerometer I inhibitor DXd." Journal of Clinical Oncology 36.15_ Suppl (2018): e 06-e24206. ").
ROPION-Pan Tumor01 is a phase 1 clinical trial for TNBC with a preliminary Objective Remission Rate (ORR) of 43% by blind independent central review in 21 evaluable patients receiving Datopotamab dermatan treatment (6mg/kg, n-19; 8mg/kg, n-2). By 8 days 1 month 2021, 5 patients confirmed complete or partial remission (CR/PR), and 4 others confirmed CR/PR with a Disease Control Rate (DCR) of 95%. No patients withdrew treatment due to Adverse Events (AEs); however, 6 patients (25%) experienced dose reduction by AEs, the most common causes being stomatitis (13%) and mucosal inflammation (8%). 33% of patients develop grade 3 or higher emergency treatment adverse events (TEAEs), including stomatitis (13%), fatigue (4%) and anemia (4%), with no grade 3 or higher diarrhea or neutropenia TEAEs. The most common TEAEs in 25% of patients are stomatitis, nausea, fatigue, vomiting and alopecia.
RN927C is an ADC with TOPR 2-targeted antibody site-directed conjugation Aur0101 (an auristatin microtubule inhibitor) developed by fevered, DAR value of 2, which has currently terminated clinical development due to toxicity problems. In the phase 1 clinical study, 31 patients with metastatic solid tumors received increasing doses (0.15-4.8mg/kg) of RN927C, and 11 patients had stable disease and had no complete or partial remission. The most common Treatment Emergent Adverse Events (TEAEs) were fatigue (42%), constipation (36%), nausea (32%), chills (29%), infusion related reactions (26%), neutropenia (26%), rash (26%), weight loss (26%), arthralgia (23%), appetite loss (23%), diarrhea (20%), dyspnea (20%), mucosal inflammation (20%) and itching (20%). In the 3.6mg/kg dose group, 33% of patients experienced dose-limiting toxicity, grade 4 neutropenia and grade 3 mucosal inflammation. At the 4.2mg/kg dose, 1/1 (100%) patients presented with dose-limiting toxicity, grade 3 maculopapular papules. At the 4.8mg/kg dose, 4 of 8 patients (50%) developed dose-limiting toxicities, including grade 4 febrile neutropenia, grade 4 toxic epidermal necrolysis, grade 4 dehydration and grade 3 rash.
Trodelvy has potential off-target toxicity, and DS-1062a has about two orders of magnitude lower affinity than Trodelvy in clinical stage, and affects antitumor activity. In order to solve the technical problems, the invention uses naked anti SY02 with the affinity equivalent to that of hRS7 combined with human monkey TROP2 to couple cytotoxic drugs through a linker of interchain cysteine and maleimide joint, so as to obtain antibody coupling drugs. The compound shows excellent activity in vivo and in vitro antitumor effect and cynomolgus monkey PK experiments.
Disclosure of Invention
The invention provides an antibody coupling drug targeting TROP2 shown in formula (I), and a pharmaceutically acceptable salt, hydrate, solvate or isotope labeled analogue thereof:
Ab-(L-D)n(I)
wherein
D is a cytotoxic drug selected from the group consisting of D1 and D2:
Figure BDA0003287333030000051
l is a linking unit selected from:
Figure BDA0003287333030000052
Figure BDA0003287333030000061
wherein n is an integer from 1 to 10; m is an integer from 1 to 10;
in L, the-S-terminus is linked to an antibody and the-C (O) -terminus is linked to a cytotoxic drug D;
ab is an antibody or antigen-binding fragment thereof targeting Trop2, which comprises heavy chain complementarity determining region 1(HCDR1), heavy chain complementarity determining region 2(HCDR2), heavy chain complementarity determining region 3(HCDR3), light chain complementarity determining region 1(LCDR1), light chain complementarity determining region 2(LCDR2), and light chain complementarity determining region 3(LCDR3), wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, the amino acid sequence of LCDR3 is shown in SEQ ID NO:6, and the numbering is determined according to the IMGT rules.
In a preferred embodiment of the invention, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 2 to 8, further preferably 4.
In a preferred embodiment of the invention, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, further preferred 8.
In a preferred embodiment of the invention, D is selected from D2.
In a preferred embodiment of the invention, L is selected from
Figure BDA0003287333030000062
Figure BDA0003287333030000071
D is selected from D2.
In a preferred embodiment of the invention, L is selected from
Figure BDA0003287333030000072
D is selected from D2.
In a preferred embodiment of the invention, L is selected from
Figure BDA0003287333030000073
D is selected from D1.
In a preferred embodiment of the invention, the antibody-conjugated drug targeting TROP2 has the following structure:
Figure BDA0003287333030000074
wherein n is selected from an integer of 1 to 10, n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, further preferably 2 to 8, more preferably 4, and m is selected from an integer of 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, further preferably 8.
In a preferred embodiment of the invention, the antibody-conjugated drug targeting TROP2 has the following structure:
Figure BDA0003287333030000081
wherein n is an integer of 1 to 10, and n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, more preferably 2 to 8, and still more preferably 4.
In a preferred embodiment of the invention, Ab is an antibody or antigen-binding fragment thereof targeting Trop2, comprising a heavy chain variable region (HV) as set forth in SEQ ID NO:7 and a light chain variable region (LV) as set forth in SEQ ID NO: 8.
In some preferred embodiments of the invention, Ab is an antigen binding fragment targeting Trop2, including but not limited to Fab, Fab ', F (Ab') 2, Fv fragments scFv, and diabodies.
In some preferred embodiments of the invention, Ab is coupled to linker unit L via an interchain cysteine.
In a preferred embodiment of the invention, Ab is coupled to linker unit L via an interchain cysteine, which can achieve DAR1-10, preferably 2-8, further preferably 4.
In a preferred embodiment of the invention, Ab is a chimeric, humanized or fully human antibody.
In a preferred embodiment of the invention, Ab specifically binds to the extracellular region of human TROP 2. In a preferred embodiment of the invention, the monoclonal antibodies can be made in HEK293 cells using methods known in the art. The invention provides a pharmaceutical composition, which comprises an antibody-conjugated drug targeting TROP2 shown in a formula (I). The pharmaceutical composition further comprises pharmaceutically acceptable auxiliary materials and carriers.
The invention provides an antibody coupling drug for targeting TROP2 shown in formula (I), and application of a pharmaceutically acceptable salt, a hydrate, a solvate or an isotope labeled analogue thereof in preparing a drug for treating proliferative diseases. Preferably, the proliferative disease is a disease associated with aberrant expression of TROP2, including a cancer, preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma (e.g., hodgkin's lymphoma, non-hodgkin's lymphoma or recurrent anaplastic large cell lymphoma).
The present invention provides a method for treating or preventing a proliferative disease, the method comprising administering to a patient in need thereof a therapeutically effective dose of an antibody conjugate drug targeting TROP2 represented by formula (I), and pharmaceutically acceptable salts, hydrates, solvates or isotopically labeled analogs thereof, or pharmaceutical compositions comprising the same; the proliferative disease is preferably a disease associated with abnormal expression of TROP2, including a cancer, preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., hodgkin's lymphoma, non-hodgkin's lymphoma, or recurrent anaplastic large cell lymphoma).
The invention provides a preparation method of the TROP 2-targeted antibody conjugate drug and a medicinal salt, a hydrate, a solvate or an isotope-labeled analogue thereof, which comprises the following steps: the TROP2 antibody obtained after animal immunization and screening is humanized, a humanized sequence is constructed to a eukaryotic cell protein expression plasmid vector, plasmid transient transfection HEK293 cells are extracted to produce antibody protein, and the obtained antibody is catalyzed by an enzyme method or coupled chemically to obtain an antibody coupling drug.
The TROP 2-targeted antibody conjugated drug has lower off-target toxicity compared with Trodelvy and higher anti-tumor activity compared with DS-1062 a. The TROP 2-targeted antibody conjugate drug shows excellent activity in vivo and in vitro antitumor drug effects and cynomolgus monkey PK experiments.
Drawings
FIG. 1 shows SY02-ADC structure (a.SY02-SN-38, b.SY02-DXd, c.SY02-MMAE).
FIG. 2 shows the results of ELISA detection of human monkey crossover.
FIG. 3 shows the result of measurement of SY02-MMAE modification rate.
FIG. 4 shows the result of detection of SY02-SN-38DAR value.
FIG. 5 shows the detection results of SY02-DXd DAR values.
FIG. 6 shows the results of the CFPAC-1 in vitro killing experiment.
FIG. 7 shows the results of CFPAC-1 in vivo antitumor experiments.
FIG. 8 shows the results of in vivo anti-tumor experiments with MDA-MB-468.
Fig. 9 is a plasma concentration-time curve of SY02-ADC total anti-drug.
Figure 10 is a plasma concentration-time curve of SY02-ADC free toxin.
Detailed Description
SY02 humanized antibody heavy chain amino acid (HC) and light chain amino acid (LC) sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively.
Example 1 preparation of humanized anti-TROP2 antibody SY02
The heavy and light chain DNA sequences of SY02 are synthesized by the gene of Weitusheng engineering bioengineering (Shanghai) corporation (abbreviated as "worker") and constructed into eukaryotic expression vector pcDNA3.1 to obtain recombinant plasmid expression vector pcDNA3.1_ TROP 2.
1) Design and synthesis of heavy chain:
the artificially synthesized heavy chain was named TROP 2-HC. The 5 ' end is introduced with HindIII endonuclease site, the 3 ' end is introduced with EcoRI endonuclease site, and simultaneously, the 5 ' end is introduced with Kozak sequence behind the HindIII endonuclease site, and a signal peptide sequence (19 amino acids) MELGLCWVFLVAILEGVQC. The expression cassette for the heavy chain was designed to:
HindIII-Kozak sequence-Signal peptide-TROP 2-HC-stop codon-EcoRI
Design and synthesis of light chain:
the artificially synthesized light chain was named TROP 2-LC. During synthesis, HindIII endonuclease site was introduced at the 5 ' end of the light chain, EcoRI endonuclease site was introduced at the 3 ' end, while Kozak sequence was introduced after HindIII endonuclease site at the 5 ' end, and signal peptide sequence (amino acid position 22): MDMRVPAQLLGLLLLWFPGSRC. The expression cassette for the light chain was designed to:
HindIII-Kozak sequence-Signal peptide-TROP 2-LC-stop codon-EcoRI
2) Construction of recombinant plasmid
Carrying out HindIII/EcoRI double digestion and ligation transformation on the PCR amplification product TROP2-HC and the plasmid vector pcDNA3.1, and carrying out the transformation by Amp+Screening positive clones by the resistance marker to obtain a recombinant heavy chain expression vector with correct construction; performing HindIII/EcoRI double digestion and ligation transformation on the PCR amplification product TROP2-LC and the plasmid vector pcDNA3.1, and performing amplification transformation by Amp+Screening positive clones by the resistance marker to obtain a recombinant light chain expression vector with correct construction.
3) Expression of antibodies
The humanized antibody was expressed in this experiment by transient transformation of HEK293 cells. HEK293 cells were placed in 5% CO2Shaking and culturing in a constant temperature shaking table at constant temperature of 37 ℃ and 120 rpm. Cells were cultured to a density of 2.0X 106Antibody heavy and light chains were added at a ratio of 0.5mg Hc, 0.5mg Lc per liter of cells per mL. Firstly, uniformly mixing a transfection buffer solution (KPM) and sterile plasmids, then preparing a transfection reagent by the KPM and a TA-293 transfection reagent, slowly adding the transfection reagent into the KPM with the plasmids, slightly and uniformly mixing to prepare a plasmid-vector compound, standing for 10min, and then adding the plasmid-vector compound into cells; after 24h, cell protein expression enhancer and transient infectious nutritional additive were added, and cells were harvested on day 6 after transfection and purified.
Example 3 affinity and species Cross-detection of humanized antibodies
The affinity of SY02 and hRS7 with human and cynomolgus monkey TROP2 protein is carried out by ELISA in the experiment. After incubation of TROP2 proteins (Human TROP2, Cynomolgus TROP2) of different species with Antibody samples of different concentrations, the samples were analyzed for affinity to TROP2 protein of different species by ELISA by incubating with a Secondary Antibody (Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody) that binds IgG and detecting the signal values at different concentrations. The experimental results are shown in fig. 2 or table 2, and show that SY02 has an affinity for human and cynomolgus monkey TROP2 protein equivalent to hRS 7.
Table 2: TROP2 antibody species crossover
Figure BDA0003287333030000111
Example 4 anti-TROP2 ADC preparation
1. SY02-MMAE enzymatic coupling (linker-toxin coupled to heavy chain Q295 (numbering according to EU numbering rules) by transglutaminase mTGase):
the Nanjing Lining biopharmaceutical Co., Ltd is entrusted to synthesize linker small molecule compounds (structure shown in figure 1c), and certain volumes of LND1002 (dissolved in 1g:5mL DMSO), 10 × reaction buffer, SY02 antibody, mTGase (preferably Streptomyces mobaraensis), and 20% H2And adding O into an EP tube in sequence, sealing, uniformly mixing, and then placing at 30 ℃ for reaction for no more than 72 hours. When the coupling rate is more than or equal to 95 percent, the reaction is finished and the purification is carried out immediately. The reaction conditions were as follows:
table 3: SY02-MMAE coupling reaction system
Figure BDA0003287333030000121
The mTGase sequence is shown in SEQ ID NO: 11:
DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP
2. SY02-SN-38 chemical coupling (coupling of linker-toxin to interchain disulfide bond of antibody using maleimide linker):
the antibody was replaced with PBS6.0/EDTA (10mM PB, 137mM NaCl, 5mM EDTA, pH 6.0) buffer, the concentration was adjusted to 10mg/mL, and 4.8 molar equivalents of TCEP reducing agent, 1/20 volumes of 1M K, was added based on the amount of the antibody substance2HPO4Water bath at 37 ℃ for 2 h; then adding 9.6 molar equivalents of CL2A-SN38 (synthesized by Shanghai Haoyuan medicine GmbH, structure shown in figure 1a), and reacting at room temperature for 30 min; adding 15 mol of the solutionThe equivalent of N-ethylmaleimide blocks the unreacted thiol, and the reaction is carried out at room temperature for 30 min.
3. SY02-DXd chemical coupling (coupling of linker-toxin to the interchain disulfide bond of the antibody using maleimide linker):
the antibody was replaced with PBS6.0/EDTA buffer, the concentration was adjusted to 10mg/mL, and 2.4 molar equivalents of TCEP reducing agent, 1/20 volumes of 1M K based on the amount of the antibody material2HPO4Water bath at 37 ℃ for 1 h; then, 5 molar equivalents of deuxtecan (synthesized by Haoyuan medicine, Inc., Shanghai, with the structure shown in FIG. 1b) was added and reacted at room temperature for 30 min; the reaction was stopped by adding 10 molar equivalents of L-acetylcysteine.
Example 5 SY02-ADC physicochemical Properties analysis
1. SY02-MMAE modification rate detection
The experimental steps are as follows:
1) sample reduction: the SY02-MMAE 72h reaction solution was treated with 50mM ammonium acetate, 20mM DTT, 55mM Tris-HCl buffer and incubated at 30 ℃. + -. 2 ℃ for 30 min.
2) Loading: a30. mu.g sample was taken and a C4 column (5 μm particle size; 4.6 mm. times.250 mm; HICHROM) was selected.
3) And (3) elution: mobile phase a solution was 0.1% TFA aqueous solution, mobile phase B solution was 0.1% acetonitrile solution, mobile phase a solution was adjusted at 0, 5, 8, 15, 20, 22, 25, 30 min: the solution B was eluted at a ratio of 90:10, 70:30, 65:35, 60:40, 55:45, 50:50, 10:90, 90:10, with a flow rate of 0.8mL/min, a column temperature of 60 ℃ and a detection wavelength of 280 nm.
The experimental results are shown in FIG. 3, which shows that SY02 reacted for 72h, the modification rate is 97.82%.
2. DAR value detection of SY02-SN-38 and SY02-DXd
The experimental steps are as follows:
according to the high performance liquid chromatography 0512 of the four parts of the 2020 edition of Chinese pharmacopoeia, the product adopts reversed phase chromatography (RP-HPLC) to determine the DAR value of the drug-antibody coupling ratio.
An experimental instrument: high performance liquid chromatograph Agilent 1260
A chromatographic column:
Figure BDA0003287333030000131
5um,50*2.1mm
mobile phase: a: 0.1% (v/v) aqueous TFA
B: 0.1% (v/v) TFA acetonitrile solution
The detection method comprises the following steps:
Figure BDA0003287333030000132
the gradient elution procedure was as follows:
time (min) Mobile phase a liquid (%) Mobile phase B liquid (%)
0 73 27
3 73 27
8 65 35
25 57 43
And analyzing the experimental result by adopting an area normalization method:
the calculation results are as follows:
DAR＝(DAR0％×0+DAR1％×1+DAR2％×2+DAR3％×3)×2
the experimental results are shown in fig. 4 and 5.
Table 4: SY02-ADC DAR value
Figure BDA0003287333030000141
Example 6 SY02-ADC inhibition of human pancreatic duct adenocarcinoma cell CFPAC-1 growth in vitro
Collecting target cells, resuspending the target cells into single cell suspension, and identifying cell viability and cell count by a trypan blue staining method; adjusting the cell density to 1 × 105cells/mL; add 100 μ L per well to a 96 well black flat bottom cell culture plate; adding 20 mu L of diluted test sample to a 96-well black flat-bottom cell culture plate inoculated with cells; incubating in cell culture box (37 deg.C, 5% CO2) for 66 + -3 hr; adding solution (0.03%) of sodium celosite into the solution, wherein each well is 20 μ L; acting at 37 deg.C for 3-4h, reading fluorescence value with ELISA reader at 550nm/610nm, and mapping with Prism or similar mapping software to obtain semi-inhibitory concentration IC of reference standard and sample50。
The experimental results are shown in Table 5, SY02-DXd and SY02-SN-38 can inhibit the growth of CFPAC-1 cells in vitro, and the IC50 value of SY02-DXd is obviously lower than that of SY02-SN-38, while SY02-MMAE hardly has the inhibition effect.
Table 5: in vitro inhibition of SY02-ADC on CFPAC-1
Figure BDA0003287333030000142
Example 7 in vivo efficacy of SY02-ADC
1. Drug effect experiment for inhibiting human pancreatic ductal carcinoma in vivo
In the experiment, female BALB/c NU/NU mice with proper age are selected to inoculate CFPAC-1 human pancreatic ductal carcinoma cells until the tumor volume is about 100-3When the temperature of the water is higher than the set temperature,42 animals with good tumor growth were selected and divided into 6 groups based on tumor volume balance. A: solvent control 7 mice were given 0.9% sodium chloride injection (0.9% INJ NS, solvent control); b: SY 02-7 MMAE group, 0.3mg/kg (once weekly administration); c: SY 02-7 MMAE group, 1.0mg/kg (once weekly administration); d: SY 02-7 MMAE group, 3.0mg/kg (once weekly administration) E: SY02-SN-38 group 7, 10mg/kg (once weekly administration); SY02-DXd group 7, 6mg/kg (single administration). After administration, the mice were weighed, data were recorded, and tumor growth was dynamically observed by measuring the tumor size at different times after administration. On day 22 after administration, the mice were asphyxiated with carbon dioxide, and then the tumors were removed and weighed.
The experimental result is shown in figure 7, under the experimental condition, the tumor inhibition rates of SY02-DXd and SY02-SN-38 are respectively 98.2% and 87.3%, which are consistent with the in vitro experimental result, and SY02-MMAE can hardly inhibit the growth of tumor. Although SY02-SN-38 is administered at a dose and frequency higher than those of SY02-DXd, the tumor-inhibiting effect is inferior to that of SY 02-DXd.
2. Experiment of drug effect of inhibiting human breast cancer in vivo
In the experiment, female BALB/c NU/NU mice with suitable age are selected to be inoculated with MDA-MB-468 human breast cancer cells until the tumor volume is about 100-3In this case, 42 animals with good tumor growth were selected and divided into 5 groups based on the tumor volume balance. A: solvent control group 7, given 0.9% sodium chloride injection (0.9% INJ NS, solvent control); b: SY02-SN-38 group 7, 3.0mg/kg (once biweekly administration); c: SY02-SN-38 group 7, 6.0mg/kg (once biweekly dosing); d: SY02-DXd group 7, 3.0mg/kg (single dose) E: SY02-DXd group 7, 10.0mg/kg (single administration). After administration, the mice were weighed, data were recorded, and tumor growth was dynamically observed by measuring the tumor size at different times after administration. On day 28 after dosing, the mice were asphyxiated with carbon dioxide and the tumors were removed and weighed.
As shown in FIG. 8, under the present experimental conditions, a single administration of 10mg/kg SY02-DXd can completely regress the tumor, the tumor inhibition rate of 3mg/kg SY02-DXd is 63.6% while the tumor inhibition rate of 10mg/kg SY02-SN-38 administered once every two weeks is only 56.2%. As can be seen, the in vivo anti-tumor effect of SY02-DXd is far better than that of SY 02-SN-38.
Example 8 SY02-ADC Security evaluation study
8 cynomolgus monkeys of the appropriate age were selected for this experiment, and the toxic reaction was observed by intravenous injection of SY 02-ADC. The test substances are SY02-MMAE, SY02-SN-38 and SY02-DXd, and the dosage is designed as shown in the following table, and is administered in a single dose. And observing the weight change, and detecting hematology and blood biochemical indexes. Blood was collected at 0, 2, 4, 26, 50, 74 and 146 hours post-dose, and total antibody and free toxin were detected. According to experimental data, SY02-MMAE has more serious toxicity than SY02-DXd and SY02-SN-38, death of monkeys occurs under different doses, and the most common skin-related side effects are scabbing, swelling and ulceration, and the toxicity is probably target-mediated.
Multiple hematological indexes of SY02-SN-38 are abnormal, while SY02-DXd has no obvious abnormality.
Table 6: toxicity test design scheme
Figure BDA0003287333030000161
The experimental results are shown below:
table 7: results of toxicity test
Figure BDA0003287333030000162
Note: WBC, leukocyte; # NEUT, neutrophil; # RETIC is reticulocyte; # EOS eosinophils; HGB, hemoglobin; HCT is the specific volume of red blood cells; # MONO is a monocyte; ALT alanine aminotransferase; AST, aspartate aminotransferase; CK is creatine kinase.
The results of the above experiments are combined to show that: the antibody drug conjugate SY02-DXd has the overall effect remarkably superior to SY02-SN-38 and SY02-MMAE in-vitro tumor cell inhibition experiments and in-vivo tumor inhibition drug effect experiments (pancreatic duct cancer and breast cancer); in particular, preliminary tests in the evaluation of safety show that: the antibody drug conjugate SY02-DXd obtained in the application has better safety and smaller side effect, and has no obvious skin toxicity or target organ toxicity.
SEQUENCE LISTING
<110> Megaku biopharmaceutical Co., Ltd
<120> an antibody conjugate against TROP2 protein
<130> N2021092802
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> HCDR1
<400> 1
Gly Tyr Thr Phe Thr Asp Tyr Ser
1 5
<210> 2
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> HCDR2
<400> 2
Ile Ser Thr Tyr Tyr Gly Asp Ala
1 5
<210> 3
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> HCDR3
<400> 3
Ala Arg Tyr Gly Asp Gly Tyr Asn Gln Phe Asp Tyr
1 5 10
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> LCDR1
<400> 4
Glu Ser Val Asp Ser Tyr Gly Asn Asn Phe
1 5 10
<210> 5
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> LCDR2
<400> 5
Arg Ala Ser
1
<210> 6
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> LCDR3
<400> 6
Gln Gln Ser Tyr Glu Asp Pro Tyr Thr
1 5
<210> 7
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> HV
<400> 7
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Val Ile Ser Thr Tyr Tyr Gly Asp Ala Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Gly Asp Gly Tyr Asn Gln Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 8
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> LV
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Val Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Asn Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 9
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> HC
<400> 9
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Val Ile Ser Thr Tyr Tyr Gly Asp Ala Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Gly Asp Gly Tyr Asn Gln Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 10
<211> 218
<212> PRT
<213> Artificial Sequence
<220>
<223> LV
<400> 10
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Val Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Asn Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 11
<211> 331
<212> PRT
<213> Artificial Sequence
<220>
<223> mTGase
<400> 11
Asp Ser Asp Glu Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met
1 5 10 15
Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Ile Val Asn
20 25 30
Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg
35 40 45
Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys
50 55 60
Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu
65 70 75 80
Ala Phe Ala Phe Phe Asp Glu Asp Lys Tyr Lys Asn Glu Leu Lys Asn
85 90 95
Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val
100 105 110
Ala Lys Asp Ser Phe Asp Glu Ala Lys Gly Phe Gln Arg Ala Arg Asp
115 120 125
Val Ala Ser Val Met Asn Lys Ala Leu Glu Asn Ala His Asp Glu Gly
130 135 140
Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala
145 150 155 160
Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn
165 170 175
Thr Pro Ser Phe Lys Asp Arg Asn Gly Gly Asn His Asp Pro Ser Lys
180 185 190
Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg
195 200 205
Ser Gly Ser Ser Asp Lys Arg Lys Tyr Gly Asp Pro Glu Ala Phe Arg
210 215 220
Pro Asp Arg Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile
225 230 235 240
Pro Arg Ser Pro Thr Ser Pro Gly Glu Ser Phe Val Asn Phe Asp Tyr
245 250 255
Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp
260 265 270
Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met
275 280 285
His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Asp Gly Tyr Ser Asp
290 295 300
Phe Asp Arg Gly Ala Tyr Val Val Thr Phe Val Pro Lys Ser Trp Asn
305 310 315 320
Thr Ala Pro Asp Lys Val Thr Gln Gly Trp Pro
325 330

Claims (10)

1. An antibody coupling drug targeting TROP2 shown in formula (I), and pharmaceutically acceptable salts, hydrates, solvates or isotopic labeled analogues thereof,
Ab-(L-D)n (I)
wherein
D is a cytotoxic drug selected from the group consisting of D1 and D2:
Figure FDA0003287333020000011
l is a linking unit selected from:
Figure FDA0003287333020000012
wherein n is an integer from 1 to 10; m is an integer from 1 to 10;
in L, the-S-terminus is linked to an antibody and the-C (O) -terminus is linked to a cytotoxic drug D;
ab is an antibody or antigen-binding fragment thereof targeting Trop2, which comprises heavy chain complementarity determining region 1(HCDR1), heavy chain complementarity determining region 2(HCDR2), heavy chain complementarity determining region 3(HCDR3), light chain complementarity determining region 1(LCDR1), light chain complementarity determining region 2(LCDR2), and light chain complementarity determining region 3(LCDR3), wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, the amino acid sequence of LCDR3 is shown in SEQ ID NO:6, and the numbering is determined according to the IMGT rules.
2. The TROP2-targeting antibody conjugate drug of claim 1, wherein D is selected from D2.
3. The TROP2-targeting antibody-conjugated drug of claim 1, wherein L is selected from the group consisting of
Figure FDA0003287333020000021
D is selected from D2; or
L is selected from:
Figure FDA0003287333020000022
d is selected from D1, wherein m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 8.
4. The TROP2-targeting antibody conjugate drug according to claim 1, having the structure:
Figure FDA0003287333020000031
5. the TROP2-targeting antibody conjugate drug according to any one of claims 1 to 4, wherein Ab is a TROP2-targeting antibody or antigen-binding fragment thereof comprising a heavy chain variable region as set forth in SEQ ID No. 7 and a light chain variable region as set forth in SEQ ID No. 8.
6. The TROP2-targeting antibody conjugate drug of claim 1, wherein the antigen-binding fragment comprises but is not limited to Fab, Fab ', F (ab') 2, Fv fragment scFv, and diabodies.
7. A pharmaceutical composition comprising the antibody conjugate drug of claims 1-6, and pharmaceutically acceptable salts, hydrates, solvates, or isotopically labeled analogs thereof.
8. Use of the antibody conjugate of claims 1-6, and pharmaceutically acceptable salts, hydrates, solvates, or isotopically labeled analogs thereof, or the pharmaceutical composition of claim 7, in the manufacture of a medicament for the treatment of a proliferative disease.
9. The use of claim 8, wherein the proliferative disease is preferably a disease associated with abnormal expression of TROP2, including cancer, preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemic bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma.
10. The use according to claim 9, wherein the lung cancer is preferably selected from the group consisting of small cell lung cancer and non-small cell lung cancer; the leukemia is preferably selected from acute lymphocytic leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia; the lymphoma is preferably selected from hodgkin's lymphoma, non-hodgkin's lymphoma or recurrent anaplastic large cell lymphoma.
CN202111155804.5A 2021-09-29 2021-09-29 Anti-TROP 2 protein antibody conjugate Active CN114652851B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111155804.5A CN114652851B (en) 2021-09-29 2021-09-29 Anti-TROP 2 protein antibody conjugate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111155804.5A CN114652851B (en) 2021-09-29 2021-09-29 Anti-TROP 2 protein antibody conjugate

Publications (2)

Publication Number Publication Date
CN114652851A true CN114652851A (en) 2022-06-24
CN114652851B CN114652851B (en) 2024-06-07

Family

ID=82026317

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111155804.5A Active CN114652851B (en) 2021-09-29 2021-09-29 Anti-TROP 2 protein antibody conjugate

Country Status (1)

Country Link
CN (1) CN114652851B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110903395A (en) * 2018-09-14 2020-03-24 四川科伦博泰生物医药股份有限公司 Antibody, conjugate, preparation method and application thereof
CN112390885A (en) * 2019-08-12 2021-02-23 凯惠科技发展(上海)有限公司 TROP2 antibody, preparation method thereof, conjugate thereof and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110903395A (en) * 2018-09-14 2020-03-24 四川科伦博泰生物医药股份有限公司 Antibody, conjugate, preparation method and application thereof
CN112390885A (en) * 2019-08-12 2021-02-23 凯惠科技发展(上海)有限公司 TROP2 antibody, preparation method thereof, conjugate thereof and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
A. SPIRA等: "Datopotamab Deruxtecan (Dato-DXd; DS-1062), a TROP2 ADC, in Patients With Advanced NSCLC: Updated Results of TROPION-PanTumor01 Phase 1 Study", 《JOURNAL OF THORACIC ONCOLOGY》, vol. 16, no. 35, pages 106 - 107 *

Also Published As

Publication number Publication date
CN114652851B (en) 2024-06-07

Similar Documents

Publication Publication Date Title
CA3076712C (en) Antibody-drug conjugates and uses thereof
US20210290775A1 (en) Combination of antibody-drug conjugate and tubulin inhibitor
WO2021151984A1 (en) Treatment of cancer
CN102573908A (en) Anti-gcc antibody molecules and related compositions and methods
Li et al. Development of a novel EGFR-targeting antibody-drug conjugate for pancreatic cancer therapy
SG194875A1 (en) Protein-active agent conjugates and method for preparing the same
JP2008515889A (en) A therapeutic agent with reduced toxicity
TWI822822B (en) Use of antibody-drug conjugate
WO2016108587A1 (en) Repebody derivative-drug conjugate, preparation method and use thereof
US10792370B2 (en) Antibody-drug conjugate
JP7440509B2 (en) CDCP1-targeted therapy
KR20200138759A (en) Humanized anti-prostate specific membrane antigen (PSMA) antibody drug conjugate
TW202327662A (en) Anti-her2 antibody drug conjugate and composition and use thereof
KR20230026983A (en) Drug Conjugates Containing Alpha-Enolase Antibodies and Their Uses
CN114652851B (en) Anti-TROP 2 protein antibody conjugate
KR20070003806A (en) Targeting of erb antigens
CA3206798A1 (en) Use of antibody-drug conjugate targeting her2 in treatment of specific breast cancer
CN115916822A (en) Methods of using anti-CD 79b immunoconjugates
IL292634A (en) Humanized anti-ca ix antibodies and methods of their use
WO2024116094A1 (en) Combination of antibody-drug conjugates and dnmt inhibitors
CN115884794A (en) Combination of an anti-HER 2 antibody drug conjugate with a HER dimerization inhibitor
EA046932B1 (en) TREATMENT OF METASTATIC BRAIN TUMOR BY ADMINISTRATION OF ANTIBODY-DRUG CONJUGATE
Li et al. a potent and selective cell permeable inhibitor Menu

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant