CN114652851B - Anti-TROP 2 protein antibody conjugate - Google Patents
Anti-TROP 2 protein antibody conjugate Download PDFInfo
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Abstract
提供式(I)所示的靶向TROP2的抗体偶联药物，及其药用盐、水合物、溶剂合物或同位素标记的类似物：Ab‑(L‑D)n(I)，及其用途和制备方法，其中Ab、L、D和n如说明书中所定义。Provided are an antibody-drug conjugate targeting TROP2 as shown in formula (I), and a pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled analog thereof: Ab-(L-D) n (I), and uses and preparation methods thereof, wherein Ab, L, D and n are as defined in the specification.
Description
Background
TROP2, which is known as a human trophoblast surface antigen (Trophoblast cell surface antigen 2) belongs to the TACSTD (Tumor-Associated Calcium Signal Transducer) family, is a cell surface glycoprotein encoded and expressed by the TACSTD gene, and is also known as Tumor associated calcium signal transducer 2 (TACSTD 2), epidermal glycoprotein 1 (EGP-1), gastrointestinal Tumor associated antigen (GA 733-1), and membrane fraction chromosome 1 surface marker 1 (M1S 1). It was originally identified as an antigen present on human gastrointestinal tumors, and another family member is GA733-2, also known as the epithelial cell adhesion molecule (EpCAM), trop-1 or KSA. The TROP2 gene is unique in that it contains no introns, and studies of both genes indicate that TROP2 is the result of EpCAM gene relocation. TROP2 and EpCAM have about 49% amino acid homology and about 67% sequence similarity. The human and mouse Trop-2 have 87% sequence similarity, and the human Trop2 protein consists of a signal peptide of 26aa, an extracellular domain of 248aa, a transmembrane region of 23aa and a cytoplasmic region of 26 aa.
It has been found that TROP2 is more highly expressed in various human epithelial cancers, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, ovarian cancer, and the like, as compared to normal tissues. While the biological role of TROP2 is still under investigation, various studies have shown that in various human cancers, TROP2 overexpression is associated with increased tumor growth, tumor invasiveness, metastasis and poor prognosis. 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 proliferation and stem cell self-renewal through β -catenin signaling.
In patients with invasive ductal carcinoma breast cancer, the increase in TROP2 mRNA expression is a strong predictor of poor survival and lymph node metastasis, while the Kaplan-Meier survival curve also shows that patients with breast cancer with high TROP2 expression have significantly shorter survival. In endometrial cancer tissue, there is a positive correlation between TROP2 expression and pathological grading, i.e. the extent of TROP2 expression is correlated with the malignancy of the disease. The survival rate of TROP2 protein high-expressive person is obviously reduced, so that TROP2 expression can be used as an independent prognosis judgment index for patients with shortened survival time. High expression of TROP2 protein is associated with high aggressiveness of ovarian cancer. In summary, there is a close relationship between increased expression of TROP2 and malignancy of the female reproductive system and is associated with the malignancy of the disease. The presence of TROP2 over-expression in pancreatic adenocarcinoma is clearly associated with lymph node metastasis, tumor grade and poor patient survival, suggesting that TROP2 may serve as a unique pancreatic cancer prognostic biomarker. In colon cancer, the expression of TROP2 protein in cancer tissues is significantly higher than that in normal tissues. TROP2 high expression increases the chance of liver metastasis and the patient's mortality, and the depth of metastasis is also deeper, with more lymph node metastasis and poor prognosis. Expression of TROP2 is necessary for obtaining tumor formation and invasion ability of colon cancer cells, and after the RNA interference means is used for inhibiting TROP2, both tumor formation and invasion ability of the cancer cells are inhibited, so that proliferation ability of colon cancer cells which externally express TROP2 is enhanced. In gastric cancer, TROP2 also appears to be highly expressed. Treatment of gastric adenocarcinoma cell lines with apoptosis activator 2 directed against TROP2 antigen significantly promotes apoptosis. TROP2 is overexpressed in tumor tissue of patients with oral cancer. TROP2 is also expressed in squamous cell carcinoma tissue of the throat and is associated with the extent of tissue differentiation and lymph node metastasis. Expression of TROP2 can be used as an independent prognostic factor for lymph node metastasis, extent of differentiation, tumor size and T-staging. TROP2 is closely related to malignancy, proliferation and angiogenesis of brain gliomas, and may help in targeted treatment of brain gliomas.
Currently, 7 biological agents targeting TROP2 are in clinical stages (see table 1 for detailed information) at home and abroad, and the fastest development progress is that one ADC molecule targeting TROP2 developed by Immunomedics-Trodelvy, month 08 of 2021, U.S. Food and Drug Administration (FDA) grant Trodelvy (sacituzumab govitecan-hziy) is fully approved for the treatment of unresectable locally advanced or metastatic Triple Negative Breast Cancer (TNBC) adult patients who have previously received at least 2 therapies, at least 1 of which treat metastatic disease.
Trodelvy is a novel, first-initiated antibody-coupled drug (ADC) targeting TROP2, formed by coupling a humanized IgG1 antibody targeting TROP2 antigen with the metabolically active product SN-38 of the chemotherapeutic drug irinotecan, a topoisomerase I inhibitor, with a drug-to-antibody ratio (DAR) 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 target bind to TROP2 and deliver anticancer 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, which can deliver active drugs within tumor cells and in the tumor microenvironment, creating bystander effects (bystander effect).
Month 4 of 2020, trodelvy was approved by the FDA in the united states with the indication mTNBC. The accelerated approval was based on the total remission rate (orr=33.3%) and duration of remission (median dor=7.7 months) of the single arm multicentric phase II study. Full approval at week 08, 04, 2021 was supported by the results of phase 3 clinical study ASCENT (NCT 02574455) trials. The results showed that the patients had a 57% decrease in disease progression or mortality risk, with statistical and clinical significance, extending median Progression Free Survival (PFS) from 1.7 months to 4.8 months of chemotherapy (HR: 0.43;95% Cl:0.35-0.54; p < 0.0001), trodelvy also extended median total survival (OS) from 6.9 months to 11.8 months of chemotherapy (HR: 0.51,95% Cl:0.41-0.62; P < 0.0001), and 49% decrease in mortality risk.
795 Patients in three studies, IMMU-132-01, IMMU-132-05 (ASCENT) and IMMU-132-06, received TRODELVY treatment, the most common adverse effects (. Gtoreq.25%) were neutropenia (61%), nausea (66%), diarrhea (65%), fatigue (62%), hair loss (45%), anemia (42%), vomiting (39%), constipation (37%), loss of appetite (34%), rash (32%) and abdominal pain (28%). In IMMU-132-05, 63% of patients experience treatment interruption of TRODELVY due to adverse reactions, the most common (5% or more) adverse reactions that lead to treatment interruption being neutropenia (47%), diarrhea (5%), respiratory tract infection (5%) and leukopenia (5%). The dose of TRODELVY was reduced by adverse effects in 22% of patients, most commonly (> 4%) resulting in dose-reduced adverse effects of neutrophil reduction (11%) and diarrhea (5%). Therefore, trodelvy has serious adverse reaction, and based on the current research situation of TROP2 target, patients still have unmet treatment requirements on related antibody drug conjugates, and high-efficiency and high-safety targeted drugs are urgently needed.
Table 1: biological medicine for targeting TROP2 at home and abroad
Currently, the Trodelvy structure on the market is that anti-TROP2 antibody hRS7 is coupled to topoisomerase I inhibitor SN-38 via a CL2A linker, and the DAR value is about 7.6. An important feature of the ADC is that more than 90% of SN-38 is released within 3 days after administration, free SN-38 is rapidly cleared, reducing off-target toxicity, but this instability can potentially increase the risk of systemic toxicity, such as neutropenia and diarrhea, both of which are included by the "black box warning" in the Trodelvy specification.
Datopotamab deruxtecan (DS-1062 a), developed by the first Santa Classification, inc., is an ADC targeting TROP2 antibody conjugate DXd. The literature reports that the in vitro cell proliferation inhibition activity of DS-1062a on TROP 2-positive tumor cells (CFPAC-1, bxPC-3 (pancreatic cancer cells)) is expressed as IC50, respectively 706ng/mL and 74.6ng/mL(Okajima,D.,et al."Preclinical efficacy studies of DS-1062a,a novel TROP2-targeting antibody-drug conjugate with a novel DNA topoisomerase I inhibitor DXd."Journal of Clinical Oncology 36.15_suppl(2018):e24206-e24206.).
ROPION-PanTumor01 is a phase 1 clinical trial for TNBC with a preliminary objective remission (ORR) of 43% in 21 evaluable patients receiving Datopotamab deruxtecan treatment (6 mg/kg, n=19; 8mg/kg, n=2) by blind independent central screening. By day 8 of 1 in 2021, 5 patients confirmed complete or partial remission (CR/PR) and another 4 CR/PR patients were confirmed with Disease Control (DCR) up to 95%. No patient stopped treatment due to Adverse Events (AEs); however, 6 patients (25%) developed dose reduction due to AEs, most commonly due to stomatitis (13%) and mucosal inflammation (8%). Grade 3 or higher emergency treatment adverse events (TEAEs), including stomatitis (13%), fatigue (4%) and anemia (4%), were experienced by 33% of patients, and no grade 3 or higher diarrhea or neutropenia of TEAEs were seen. The most common TEAEs in 25% of patients are stomatitis, nausea, fatigue, vomiting and hair loss.
RN927C is an ADC with a target TOPR2 antibody site-directed conjugated Aur0101 (an auristatin microtubule inhibitor) developed with a DAR value of 2, and clinical development has been terminated at present due to toxicity problems. In phase 1 clinical studies, 31 patients with metastatic solid tumors received increasing doses (0.15-4.8 mg/kg) of RN927C treatment, 11 patients were stable and did not have complete or partial remission. The most common emergent adverse events of Treatment (TEAEs) were fatigue (42%), constipation (36%), nausea (32%), chills (29%), infusion-related reactions (26%), neutropenia (26%), rash (26%), weight loss (26%), joint pain (23%), loss of appetite (23%), diarrhea (20%), dyspnea (20%), mucosal inflammation (20%), and itching (20%). Dose limiting toxicity, grade 4 neutropenia and grade 3 mucositis, occurred in 33% of patients in the 3.6mg/kg dose group. At a dose of 4.2mg/kg, 1/1 (100%) of the patients developed dose-limiting toxicity, grade 3 maculopapules. At a dose of 4.8mg/kg, 4 out of 8 patients (50%) developed dose-limiting toxicities including grade 4 febrile neutropenia, grade 4 toxic epidermonecroporolysis, grade 4 dehydration and grade 3 rash.
Trodelvy, which is marketed as a potential off-target toxin, has an affinity of DS-1062a at the clinical stage of about two orders of magnitude lower than Trodelvy, affecting antitumor activity. In order to solve the technical problems, the invention utilizes the naked anti-SY 02 with the affinity equivalent to that of hRS7 combined with human monkey TROP2 to couple the cytotoxic drug through the linker of the interchain cysteine and the maleimide linker to obtain the antibody coupling drug. The anti-tumor drug has excellent activity in vivo and in vitro and in the experiments of cynomolgus monkey PK.
Disclosure of Invention
The invention provides a TROP 2-targeted antibody-coupled drug of formula (I), and pharmaceutically acceptable salts, hydrates, solvates or isotopically labeled analogues thereof:
Ab-(L-D)n(I)
Wherein the method comprises the steps of
D is a cytotoxic drug selected from the following formulas D1 and D2:
l is a linking unit selected from:
wherein n is selected from integers from 1 to 10; m is selected from integers from 1 to 10;
in L, -S-terminal is linked to antibody, -C (O) -terminal is linked to cytotoxic drug D;
ab is an antibody or antigen binding fragment thereof targeting Trop2, comprising heavy chain complementarity determining region 1 (HCDR 1), heavy chain complementarity determining region 2 (HCDR 2), heavy chain complementarity determining region 3 (HCDR 3), light chain complementarity determining region 1 (LCDR 1), light chain complementarity determining region 2 (LCDR 2) and light chain complementarity determining region 3 (LCDR 3), wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO:1, the amino acid sequence of HCDR2 is shown as SEQ ID NO:2, the amino acid sequence of HCDR3 is shown as SEQ ID NO:3, the amino acid sequence of LCDR1 is shown as SEQ ID NO:4, the amino acid sequence of LCDR2 is shown as SEQ ID NO:5, the amino acid sequence of LCDR3 is shown as SEQ ID NO:6, and the numbering is determined according to the IMGT rule.
In a preferred embodiment of the invention n is selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, preferably 2-8, 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 preferably 8.
In a preferred embodiment of the invention, D is selected from D2.
In a preferred embodiment of the invention, L is selected from
D is selected from D2.
In a preferred embodiment of the invention, L is selected fromD is selected from D2.
In a preferred embodiment of the invention, L is selected fromD is selected from D1.
In a preferred embodiment of the invention, the TROP 2-targeting antibody-conjugated drug has the following structure:
Wherein n is selected from integers of 1-10, n is preferably 1, 2,3, 4, 5, 6, 7, 8, 9, 10, more preferably 2-8, more preferably 4, m is selected from integers of 1-10, preferably 1, 2,3, 4, 5, 6, 7, 8, 9, 10, more preferably 8.
In a preferred embodiment of the invention, the TROP 2-targeting antibody-conjugated drug has the following structure:
Wherein n is an integer selected from 1-10, preferably 1, 2,3, 4, 5, 6,7, 8, 9, 10, more preferably 2-8, even more preferably 4.
In a preferred embodiment of the invention Ab is a Trop 2-targeting antibody or antigen binding fragment thereof comprising a heavy chain variable region (HV) as shown in SEQ ID NO:7 and a light chain variable region (LV) as shown in SEQ ID NO: 8.
In some preferred embodiments of the invention, ab is an antigen binding fragment that targets Trop2, including but not limited to Fab, fab ', F (Ab') 2, fv fragment scFv, and diabodies.
In some preferred embodiments of the invention, ab is coupled to linking unit L through an interchain cysteine.
In a preferred embodiment of the invention, ab is coupled to the linking unit L via an interchain cysteine, which can achieve DAR1-10, preferably 2-8, more 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 comprising a TROP 2-targeting antibody-conjugated drug of formula (I). The pharmaceutical composition further comprises pharmaceutically acceptable auxiliary materials and carriers.
The invention provides an antibody coupled drug for targeting TROP2 shown in a formula (I), and application of pharmaceutically acceptable salts, hydrates, solvates or isotopically-labeled analogues thereof in preparing drugs for treating proliferative diseases. The proliferative disease is preferably a disease associated with abnormal expression of TROP2, including cancers, 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 lymphoblastic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, chronic myelogenous 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 the treatment or prophylaxis of a proliferative disease comprising administering to a patient in need thereof a therapeutically effective dose of a TROP 2-targeting antibody conjugate drug of formula (I), and pharmaceutically acceptable salts, hydrates, solvates or isotopically labeled analogues thereof, or a pharmaceutical composition comprising the same; the proliferative disease is preferably a disease associated with abnormal expression of TROP2, including cancers, 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 lymphoblastic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, chronic myelogenous 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 an antibody coupling drug targeting TROP2 and a pharmaceutically acceptable salt, hydrate, solvate or isotopically labeled analogue thereof, which comprises the following steps: animal immunization and screening to obtain TROP2 antibody, humanizing the murine antibody, constructing humanized sequence into eukaryotic cell protein expression plasmid vector, extracting plasmid to transiently transfect HEK293 cell to produce antibody protein, and enzyme-catalyzed or chemically coupled the obtained antibody to obtain antibody coupled medicine.
Compared with Trodelvy, the TROP 2-targeted antibody coupling drug has lower off-target toxicity and higher anti-tumor activity compared with DS-1062 a. The TROP 2-targeted antibody coupled drug has excellent activity in both in-vivo and in-vitro anti-tumor drug effect and cynomolgus monkey PK experiments.
Drawings
FIG. 1 is a schematic diagram of SY02-ADC (a.SY02-SN-38, b.SY02-DXD, c.SY02-MMAE).
FIG. 2 shows the results of ELISA detection of human-monkey crossing.
FIG. 3 shows the results of SY02-MMAE modification ratio detection.
FIG. 4 shows the detection result of SY02-SN-38DAR value.
FIG. 5 shows the results of SY02-DXd DAR value detection.
FIG. 6 shows CFPAC-1 in vitro killing test results.
FIG. 7 shows CFPAC-1 in vivo anti-tumor test results.
FIG. 8 shows the results of an in vivo anti-tumor assay for MDA-MB-468.
FIG. 9 is a graph of SY02-ADC total antibody blood concentration versus time.
FIG. 10 is a graph of blood concentration versus time for SY02-ADC free toxin.
Detailed Description
The heavy chain amino acid (HC) and light chain amino acid (LC) sequences of the SY02 humanized antibody are shown in SEQ ID NO 9 and SEQ ID NO 10 respectively.
EXAMPLE 1 preparation of humanized anti-TROP 2 antibody SY02
The heavy and light chain DNA sequence of SY02 is synthesized by the gene of the division of the biological engineering (Shanghai) of principal engineering (abbreviated as "engineering") and is constructed into eukaryotic expression vector pcDNA3.1 to obtain recombinant plasmid expression vector pcDNA3.1_TROP2.
1) Design and Synthesis of heavy chain:
The artificially synthesized heavy chain is named TROP2-HC. The 5' end was introduced with a HindIII endonuclease site, the 3' end was introduced with an EcoRI endonuclease site, and a Kozak sequence, and a signal peptide sequence (amino acid 19) MELGLCWVFLVAILEGVQC were introduced after the 5' end HindIII endonuclease site. The expression cassette of the heavy chain is designed as follows:
HindIII-Kozak sequence-Signal peptide-TROP 2-HC-stop codon-EcoRI
Light chain design and synthesis:
The artificially synthesized light chain is named TROP2-LC. In the synthesis process, a HindIII endonuclease site is introduced at the 5' end of the light chain, an EcoRI endonuclease site is introduced at the 3' end, and a Kozak sequence and a signal peptide sequence (22 amino acids) are introduced behind the HindIII endonuclease site at the 5' end: MDMRVPAQLLGLLLLWFPLGSRC. The expression cassette of the light chain is designed as follows:
HindIII-Kozak sequence-Signal peptide-TROP 2-LC-stop codon-EcoRI
2) Construction of recombinant plasmids
Carrying out HindIII/EcoRI double digestion and connection transformation on PCR amplification products TROP2-HC and a plasmid vector pcDNA3.1, and screening positive clones through an Amp + resistance marker to obtain a recombinant heavy chain expression vector with correct construction; the PCR amplified product TROP2-LC and the plasmid vector pcDNA3.1 are subjected to HindIII/EcoRI double digestion and ligation transformation, and positive clones are screened through an Amp + resistance marker, so that a recombinant light chain expression vector with correct construction is obtained.
3) Expression of antibodies
The experiment adopts a mode of transiently transforming HEK293 cells to express the humanized antibody. HEK293 cells were placed in a 5% CO 2 constant temperature shaker and incubated at 37℃with shaking at 120 rpm. Cells were cultured to a density of 2.0X10 6/mL and antibody heavy and light chains were added at a rate of 0.5mg Hc and 0.5mg Lc per liter of cells. Firstly, uniformly mixing a transfection buffer solution (KPM) with sterile plasmids, then preparing a transfection reagent by using the KPM and a TA-293 transfection reagent, slowly adding the transfection reagent into the KPM with plasmids, slightly uniformly mixing to prepare a plasmid-carrier compound, standing for 10min, and adding the plasmid-carrier compound into cells; after 24h, cell protein expression enhancers and transient infection nutrition additives were added, and cells were harvested and purified on day 6 after transfection.
Example 3 affinity and species Cross detection of humanized antibodies
The experiment uses ELISA to carry out the affinity of SY02, hRS7 and human and cynomolgus monkey TROP2 protein. After incubation of different species of TROP2 proteins (Human TROP2, cynomolgus TROP 2) with antibody samples of different concentrations, the samples were analyzed for affinity to different species of TROP2 proteins by ELISA detection of signal values at different concentrations by incubation with secondary antibodies (Goat anti-Human IgG (h+l) Cross-Adsorbed Secondary Antibody) that bind IgG. 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 comparable to hRS 7.
Table 2: TROP2 antibody species crossover
Example 4 anti-TROP 2 ADC preparation
1. SY02-MMAE enzymatic conjugation (linker-toxin conjugation to heavy chain Q295 (numbering according to the EU numbering convention) via the glutamine transaminase mTGase):
The Nanjing-bining biopharmaceutical Co., ltd is entrusted to synthesize a linker small molecular compound (structure shown in figure 1 c), and a certain volume of LND1002 (dissolved in 1g:5mL DMSO), 10 Xreaction buffer, SY02 antibody, mTGase (preferably Streptomyces mobaraensis (Streptoverticillium mobaraense)), and 20% H 2 O are added into an EP tube in sequence, and after sealing and mixing, the mixture is placed at 30 ℃ for a reaction time of not more than 72 hours. When the coupling rate is more than or equal to 95%, the reaction is finished, and the purification is immediately carried out. The reaction conditions were as follows:
table 3: SY02-MMAE coupling reaction system
The mTGase sequence is shown as SEQ ID NO. 11:
DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP
2. SY02-SN-38 chemical coupling (coupling linker-toxin to inter-antibody chain disulfide bond using maleimide linker):
The antibody was replaced to PBS6.0/EDTA (10mM PB,137mM NaCl,5mM EDTA,pH =6.0) buffer, the concentration was adjusted to 10mg/mL, 4.8 molar equivalents of TCEP reductant were added in the amount of antibody material, 1/20 volume of 1/M K 2HPO4, 37℃water bath for 2h; then adding 9.6 molar equivalents of CL2A-SN38 (consigned to be synthesized by Shanghai Haifeng medical Co., ltd., structure is shown in FIG. 1 a), and reacting for 30min at room temperature; 15 molar equivalents of N-ethylmaleimide are added to block unreacted mercapto groups, and the reaction is carried out at room temperature for 30min.
3. SY02-DXd chemical coupling (coupling linker-toxin to inter-antibody chain disulfide bond using maleimide linker):
The antibody is replaced into PBS6.0/EDTA buffer solution, the concentration is adjusted to 10mg/mL, 2.4 molar equivalents of TCEP reducer is added according to the amount of the substance of the antibody, 1/20 volume of 1M K 2HPO4 is added, and water bath is carried out for 1h at 37 ℃; then adding deruxtecan molar equivalents (consigned to be synthesized by Shanghai Haoyuan medicine Co., ltd., structure is shown in figure 1 b), and reacting for 30min at room temperature; the reaction was terminated by adding 10 molar equivalents of L-acetylcysteine.
EXAMPLE 5 SY02-ADC physicochemical Property analysis
1. SY02-MMAE modification rate detection
The experimental steps are as follows:
1) Sample reduction: SY02-MMAE 72h reaction was incubated with 50mM ammonium acetate, 20mM DTT,55mM Tris-HCl buffer at 30.+ -. 2 ℃ for 30min.
2) Loading: a30. Mu.g sample was loaded and a C4 column (5. Mu. M PARTICLE size;4.6 mm. Times.250 mm; HICHROM) was selected.
3) Eluting: mobile phase a was 0.1% tfa in water, mobile phase B was 0.1% acetonitrile, and mobile phase a was adjusted for 0, 5,8, 15, 20, 22, 25, 30 min: the ratio of the solution B is 90:10, 70:30, 65:35, 60:40, 55:45, 50:50, 10:90 and 90:10, the flow rate is controlled to be 0.8mL/min, the column temperature is 60 ℃, and the detection wavelength is 280nm.
The experimental results are shown in FIG. 3, and the modification rate of SY02 reaction 72h is 97.82%.
2. DAR value detection of SY02-SN-38 and SY02-DXd
The experimental steps are as follows:
referring to the high performance liquid chromatography of four 0512 parts of Chinese pharmacopoeia 2020 edition, the product adopts reverse phase chromatography (RP-HPLC) to measure the DAR value of the drug antibody coupling ratio.
Experimental instrument: high performance liquid chromatograph Agilent 1260
Chromatographic column:5um,50*2.1mm
mobile phase: a:0.1% (v/v) TFA aqueous solution
B:0.1% (v/v) TFA acetonitrile solution
The detection method comprises the following steps:
The gradient elution procedure was as follows:
| time (min) | Mobile phase liquid a (%) | Mobile phase B liquid (%) |
| 0 | 73 | 27 |
| 3 | 73 | 27 |
| 8 | 65 | 35 |
| 25 | 57 | 43 |
And (3) analyzing an 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
EXAMPLE 6 inhibition of SY02-ADC on in vitro growth of human pancreatic ductal adenocarcinoma cells CFPAC-1
Collecting target cells to be resuspended into single cell suspension, and identifying cell viability and cell count by trypan blue staining; the cell density was adjusted to 1X 10 5 cells/mL; 100 μl per well was added to a 96-well black flat bottom cell culture plate; adding 20 mu L of diluted test sample to the 96-well black flat bottom cell culture plate inoculated with cells; incubating in a cell incubator (37deg.C, 5% CO 2) for 66+ -3 hr; add sodium resazurin solution (0.03%), 20 μl per well; and (3) performing the action for 3-4 hours at 37 ℃, reading fluorescence values at 550nm/610nm by using an enzyme labeling instrument, and performing drawing by using Prism or similar drawing software to fit the semi-inhibitory concentration IC 50 of the reference standard and the sample.
As shown in Table 5, SY02-DXD and SY02-SN-38 can inhibit CFPAC-1 cells in vitro, and the IC50 value of SY02-DXd is obviously lower than that of SY02-SN-38, but SY02-MMAE has almost no inhibiting effect.
Table 5: in vitro inhibition of CFPAC-1 by SY02-ADC
EXAMPLE 7 in vivo efficacy of SY02-ADC
1. In vivo experiment for inhibiting human pancreatic duct cancer
In the experiment, a suitable-age female BALB/c NU/NU mouse is selected to be inoculated with CFPAC-1 human pancreatic duct cancer cells, 42 animals with good tumor growth are selected when the tumor volume is about 100-150mm 3, and the animals are divided into 6 groups according to the tumor volume balance. A: solvent control 7, 0.9% sodium chloride injection (0.9% inj NS, solvent control); b: SY02-MMAE group 7, 0.3mg/kg (once a week); c: SY02-MMAE group 7, 1.0mg/kg (once a week); d: SY02-MMAE group 7, 3.0mg/kg (once a week) E: SY02-SN-38 group 7, 10mg/kg (once a week); f SY02-DXd groups 7, 6mg/kg (single administration). The body weight of the mice is weighed after the administration, the data are recorded, and the growth condition of the tumor is dynamically observed by measuring tumor diameters at different times after the administration. Tumor weights were weighed by tumor stripping after carbon dioxide asphyxiation of mice on day 22 post-dosing.
As shown in FIG. 7, under the experimental conditions, the tumor inhibition rates of SY02-DXD and SY02-SN-38 are respectively 98.2% and 87.3%, and the in vitro experimental results are consistent, and SY02-MMAE hardly inhibits the growth of tumors. Although SY02-SN-38 is administered at a higher dose and frequency than SY02-DXd, the tumor inhibiting effect is inferior to SY02-DXd.
2. In vivo experiment of human breast cancer inhibition efficacy
In the experiment, female BALB/c NU/NU mice with proper ages are selected to inoculate MDA-MB-468 human breast cancer cells, 42 animals with good tumor growth are selected when the tumor volume is about 100-150mm 3, and the animals are divided into 5 groups according to the tumor volume balance. A: solvent control 7, 0.9% sodium chloride injection (0.9% inj NS, solvent control); b: SY02-SN-38 group 7, 3.0mg/kg (once every two weeks); c: SY02-SN-38 group 7, 6.0mg/kg (once every two weeks); d: SY02-DXd groups 7, 3.0mg/kg (single administration) E: SY02-DXd groups 7, 10.0mg/kg (single administration). The body weight of the mice is weighed after the administration, the data are recorded, and the growth condition of the tumor is dynamically observed by measuring tumor diameters at different times after the administration. Tumor weights were weighed by tumor stripping after carbon dioxide asphyxiation of mice on day 28 post-dosing.
As shown in FIG. 8, under the experimental conditions, the tumor can be completely resolved by single administration of 10mg/kg SY02-DXd, the tumor inhibition rate by single administration of 3mg/kg SY02-DXd is 63.6%, and the tumor inhibition rate by single administration of 10mg/kg SY02-SN-38 every two weeks is only 56.2%. It can be seen that the in vivo antitumor effect of SY02-DXd is far better than that of SY02-SN-38.
EXAMPLE 8 SY02-ADC safety evaluation study
In the experiment, 8 cynomolgus monkeys of proper age are selected, and the toxic reaction is observed through intravenous injection of SY 02-ADC. The test samples are SY02-MMAE, SY02-SN-38 and SY02-DXd, and the dosage design is shown in the following table, and the single administration is carried out. And observing the change of the body weight, and detecting hematology and blood biochemical indexes. Blood was collected at 0,2,4, 26, 50, 74 and 146 hours post-administration and tested for total antibodies and free toxins. The experimental data show that SY02-MMAE is significantly more toxic than SY02-DXD and SY02-SN-38, and that death of the monkeys occurs at different doses, and the most common skin-related side effects are crusting, swelling and ulceration, possibly as target-mediated toxicity.
SY02-SN-38 showed abnormalities in several hematological indices, whereas SY02-DXd showed no significant abnormalities.
Table 6: toxicity experiment design scheme
The experimental results are shown below:
Table 7: toxicity test results
Note that: WBC, white blood cells; neut, neutrophils; # RETIC reticulocytes; eosinophils, # EOS; HGB is hemoglobin; HCT: hematocrit; monocytes, # MONO; ALT is alanine aminotransferase; AST, aspartic acid aminotransferase; CK creatine kinase.
From the above experimental results, it can be seen that: the antibody drug conjugate SY02-DXd has overall effect obviously superior to SY02-SN-38 and SY02-MMAE in-vitro tumor cell inhibition experiments and in-vivo tumor inhibition efficacy experiments (pancreatic duct cancer and breast cancer); in particular, preliminary tests for safety evaluation show that: the antibody drug conjugate SY02-DXd obtained by the application has better safety and smaller side effect, and obvious skin toxicity and target organ toxicity are not seen.
SEQUENCE LISTING
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| 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 |
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| 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 |
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