WO2022042537A1 - APPLICATION OF COMBINATION OF FUSION PROTEIN OF TGF-β RECEPTOR AND MULTI-TARGETED TYROSINE KINASE INHIBITOR IN PREPARING ANTI-TUMOR DRUG - Google Patents

Abstract

Disclosed is an application of a combination of a fusion protein of TGF-β receptor and multi-targeted tyrosine kinase inhibitor in preparing an anti-tumor drug.

Description

Use of fusion protein of TGF-β receptor in combination with multi-target tyrosine kinase inhibitor in the preparation of antitumor drugs

This application requires the Chinese patent application (application number 202010854875.3) filed on August 24, 2020, the Chinese patent application (application number 202110476314.9) filed on April 29, 2021, and the Chinese patent application (application number 202110476314.9) filed on June 16, 2021 No. 202110664907.8) priority. This application cites the full text of the above Chinese patent application.

technical field

The present disclosure belongs to the field of pharmacy, in particular to the use of a fusion protein containing a TGF-β receptor in combination with a multi-target tyrosine kinase inhibitor in the preparation of a medicament for treating tumors.

Background technique

Programmed death 1 (PD-1) is a member of the CD28 superfamily. PD-1 is expressed in activated T cells, B cells and myeloid cells, and it has two ligands, programmed death ligand 1 (PD-L1) and PD-L2. PD-L1 interacts with the receptor PD-1 on T cells and plays an important role in the negative regulation of immune responses. The expression of PD-L1 protein can be detected in many human tumor tissues. The microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells. The expressed PD-L1 is beneficial to the occurrence and growth of tumors and induces anti-tumor effects. Apoptosis of T cells. PD-1/PD-L1 pathway inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, and restore the activity of T cells, thereby enhancing the immune response. Therefore, PD-1/PD- L1-targeted immune regulation has important implications for tumor suppression.

Transforming growth factor-β (TGF-β) belongs to the TGF-β superfamily that regulates cell growth and differentiation. TGF-beta signals through a heterotetrameric receptor complex consisting of two type I and two type II transmembrane serine/threonine kinase receptors.

Studies have found that blocking the TGF-β signaling pathway can reduce tumor metastasis. Using truncated Smad2/3 dominant-negative mutants to inhibit the TGF-β signaling pathway in breast tumor cell lines, it was found that the metastatic ability of tumor cells was inhibited. Microsatellite instability studies in colon cancer found that inactive mutations in TGF-βRII resulted in reduced metastasis and increased postoperative survival in subjects in need. However, in general, the inhibitor that inhibits the TGF-β signaling pathway alone has a weak effect in clinical treatment, which may be related to the abnormally high expression of TGF-β in tumor cells, while the inhibitor of the TGF-β signaling pathway alone is very effective. It is difficult to focus on tumor, resulting in low efficacy or bioavailability of signaling pathway inhibitors.

Therefore, inhibiting the PD-1/PD-L1 pathway on the basis of targeting and neutralizing TGF-β in the tumor microenvironment can restore the activity of T cells, enhance the immune response, and more effectively improve the effect of inhibiting the occurrence and development of tumors. The PD-L1/TGF-β dual antibody M-7824 developed by Merck has entered clinical phase III. In addition to being used as a single drug for the treatment of tumors, this product has also been developed in combination with eribulin mesylate for the treatment of metastasis triple-negative breast cancer (NCT03579472); combined with gemcitabine for the treatment of previously treated advanced pancreatic adenocarcinoma (NCT03451773); combined with topotecan or temozolomide for the treatment of recurrent small cell lung cancer (NCT0-3554473) and other indications and, M7824 in NSCLC (NCT03631706) has been terminated early, and a Phase II/III clinical trial of M7824 in combination with gemcitabine and cisplatin in biliary tract cancer (NCT04066491) is currently recruiting.

WO2018205985A discloses a novel PD-L1/TGF-β fusion protein. CN101007815A provides multi-target tyrosine kinase inhibitors, including compounds of formula (I), and discloses that the compounds have VEGF and EGFR inhibitory activities. CN106535896A discloses that compounds of formula (I) are useful for the treatment of fibrotic diseases, such as pulmonary fibrosis, liver cirrhosis, scleroderma or renal fibrosis. The contents of the aforementioned patents are incorporated in this disclosure in their entirety.

Pancreatic cancer and biliary tract malignant tumors have a high degree of malignancy and rapid progression, but their onset is insidious and their early symptoms are not typical. The treatment of pancreatic cancer and biliary tract malignancies is a systematic project, which often requires a multidisciplinary consultation model (MDT), which mainly includes surgical treatment, radiotherapy, chemotherapy, interventional therapy and supportive care, in which surgical resection is pancreatic cancer and biliary tract. Subjects in need of malignancy have the only chance for cure and long-term survival. For advanced pancreatic cancer that has failed first-line treatment, the available treatment options are limited. According to the physical function status and tumor progression of the subjects in need, gemcitabine or fluorouracil-based single-agent chemotherapy or combination chemotherapy and second-line chemotherapy can be selected. ORR < 10%. For advanced biliary tract malignancies that have failed first-line treatment, the preferred chemotherapy regimen is oxaliplatin combined with 5-FU, and immunotherapy regimens include pembrolizumab combined with lenvatinib, pembrolizumab ( Only tumors with high microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) and nivolumab have been recommended by the Chinese Society of Clinical Oncology (CSCO) and the National Comprehensive Cancer Network (NCCN) guidelines. However, no phase III study data has been published yet, and the efficacy of immunotherapy needs further exploration.

Therefore, to find more effective new treatment approaches or drug combinations to provide new evidence-based medical evidence for the clinical treatment of pancreatic cancer and biliary tract tumors, so as to guide the clinical diagnosis and treatment of subjects in need. important clinical significance.

SUMMARY OF THE INVENTION

The present disclosure relates to the use of a fusion protein containing a TGF-β receptor in combination with a multi-target tyrosine kinase inhibitor in the preparation of a medicament for treating tumors or cancer.

The present disclosure provides a method of treating, alleviating or preventing a tumor or cancer, comprising administering to a subject in need thereof a therapeutically, alleviating or prophylactically effective amount of a multi-targeted tyrosine kinase inhibitor and a therapeutically, alleviating or prophylactically effective amount The fusion protein containing TGF-β receptor.

In some embodiments, there is provided a method of treating a tumor or cancer, comprising administering to a subject in need thereof a therapeutically, ameliorating or prophylactically effective amount of the above-described multi-targeted tyrosine kinase inhibitor, said multi-targeted tyrosine kinase inhibitor The agent is used in combination with the fusion protein containing the TGF-β receptor.

In some embodiments, there is provided a method of treating a tumor or cancer comprising administering to a subject in need thereof a therapeutically, ameliorating or prophylactically effective amount of a TGF-beta receptor-containing fusion protein, the TGF-beta receptor-containing fusion protein In combination with the multi-targeted tyrosine kinase inhibitor.

In certain embodiments, the multi-target tyrosine kinase inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

In certain embodiments, the multi-target tyrosine kinase inhibitor is famitinib or a pharmaceutically acceptable salt thereof.

In certain embodiments, the fusion protein of TGF-β receptor comprises PD-L1 antibody or antigen-binding fragment thereof and TGF-β receptor, wherein the part of TGF-β receptor is the extracellular domain of TGF-βRII N-terminal truncated form.

In certain embodiments, the TGF-β receptor fusion protein is shown in general formula (II):

Ab-L-TGF-βRII ECD (II)

wherein TGF-βRII ECD is a truncated form of the extracellular domain of TGF-βRII;

Ab is a PD-L1 antibody or an antigen-binding fragment thereof;

L is the linking sequence.

In certain embodiments, the linker sequence is (G4S)xG, wherein x is 3-6, eg, ₄ or 5.

In certain embodiments, the PD-L1 antibody or antigen-binding fragment thereof comprises:

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and HCDR1 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively of LCDR1, LCDR2 and LCDR3,

Wherein, each CDR sequence mentioned above is as follows:

HCDR1: SYWMH SEQ ID NO: 1

HCDR2:RIGPNSGFTSYNEKFKN SEQ ID NO:2

HCDR3: GGSSYDYFDY SEQ ID NO:3

LCDR1: RASESVSIHGTHLMH SEQ ID NO:4

LCDR2: AASNLES SEQ ID NO:5

LCDR3: QQSFEDPLT SEQ ID NO:6

In certain embodiments, the PD-L1 antibody or antigen-binding fragment thereof is referred to as a chimeric antibody or a functional fragment thereof, a humanized antibody or a functional fragment thereof, or a human antibody or a functional fragment thereof.

In certain embodiments, the sequences of the humanized PD-L1 antibody heavy and light chains are as follows:

PD-L1 antibody heavy chain variable region:

PD-L1 antibody light chain variable region:

Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic in the sequence is the FR sequence; the underline is the CDR sequence, and the double underlined site is the site obtained after affinity maturation screening.

In certain embodiments, the heavy chain amino acid sequence of the PD-L1 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 9 or at least 85%, 86% with the sequence shown in SEQ ID NO: 9, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, the PD- The light chain amino acid sequence of the L1 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 10 or has at least 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO: 10 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, where

PD-L1 antibody heavy chain sequence: IgG4(AA)(S228P)

PD-L1 antibody light chain sequence:

Non-limiting example sequences of the TGF-βRII extracellular domain and truncated forms thereof in the present disclosure are as follows:

TGF-βRII extracellular domain sequence: ECD(1-136)

TGF-βRII extracellular domain sequence with 19 amino acid truncation or deletion at the N-terminus: ECD(20-136)

TGF-βRII extracellular domain sequence with a 21 amino acid truncation or deletion at the N-terminus: ECD(22-136)

TGF-βRII extracellular domain sequence with a 14 amino acid truncation or deletion at the N-terminus: ECD(15-136)

Using homologous recombination technology, the C-terminal amino acid of the heavy chain of PD-L1 antibody was connected to the extracellular region of TGF-βRII of different lengths through (G ₄ S) _x G, and together with the light chain, it was routinely expressed through the 293 expression system, and the results were as shown in the table below. Fusion protein shown in 1:

Table 1. PD-L1 antibody/TGF-βRII extracellular domain fusion protein

Note: Ab is the PD-L1 antibody described in this disclosure. In the sequence description, ECD(n-136) is the full-length or truncated form of the extracellular region of TGF-βRII, and n is the truncated amino acid of the extracellular region of TGF-βRII. starting number of digits.

The PD-L1/TGF-β fusion protein in WO2018205985A is incorporated herein in its entirety.

In some specific embodiments, the heavy chain amino acid sequence of the PD-L1 antibody or its antigen-binding fragment in the fusion protein of TGF-β receptor is shown in SEQ ID NO:9, and the light chain amino acid sequence is shown in SEQ ID NO:10. As shown, the linking sequence L is (G ₄ S) ₄ G, and the extracellular region of TGF-βRII is shown in SEQ ID NO: 12.

In some embodiments, the PD-L1 antibody or antigen-binding fragment thereof is selected from the group consisting of avelumab, atezolizumab, durvalumab, CS-1001, M-7824, KL-A167, CX-072, BGB-A333, GNS-1480, CA-170, BMS-936559, preferably avelumab, atezolizumab, durvalumab.

In some embodiments, the administered dose of the multi-targeted tyrosine kinase inhibitor is selected from 1-1600 mg, for example: 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg , 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg , 190mg, 195mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280mg, 290mg, 300mg, 310mg, 320mg, 330mg, 340mg, 350mg, 360mg, 370mg, 380mg, 390mg, 400mg, 410mg, 420mg , 430mg, 440mg, 450mg, 460mg, 470mg, 480mg, 490mg, 500mg, 510mg, 520mg, 530mg, 540mg, 550mg, 560mg, 570mg, 580mg, 590mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg , 800mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg, 1000mg, 1025mg, 1050mg, 1075mg, 1100mg, 1125mg, 1150mg, 1175mg, 1200mg, 1225mg, 1250mg, 1275mg, 1300mg, 1375mg, 1300mg , 1425mg, 1450mg, 1475mg, 1500mg, 1525mg, 1550mg, 1575mg, 1600mg, the frequency of administration is twice a day, once a day, once every two days, once every three days, once every four days, once every five days, Once every six days, once a week, once every two weeks, once every three weeks or once every four weeks, preferably twice a day or once a day.

In some embodiments, the dosage of the multi-targeted tyrosine kinase inhibitor is selected from 1-800 mg, and the dosing frequency is twice a day, once a day, once every two days, once every three days, Once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks or once every four weeks, preferably twice a day or once a day.

In some embodiments, the multi-targeted tyrosine kinase inhibitor is administered at a dose selected from 5 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg , 23mg, 24mg, 25mg, 26mg, 27mg, 28mg, 29mg, 30mg, 35mg, 40mg, 45mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg , 750mg, 800mg, the dosing frequency is twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks Once, every three weeks or every four weeks, preferably twice a day or once a day.

In some embodiments, the administration dose of the fusion protein containing TGF-β receptor is selected from 0.1-500 mg/kg, and the administration frequency is once a week, once every two weeks, once every three weeks or once every four weeks . Specifically, the administration dose of the fusion protein containing TGF-β receptor can be selected from 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg , 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg /kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg, 32mg/kg, 33mg/kg , 34mg/kg, 35mg/kg, 36mg/kg, 37mg/kg, 38mg/kg, 39mg/kg, 40mg/kg, 41mg/kg, 42mg/kg, 43mg/kg, 44mg/kg, 45mg/kg, 46mg /kg, 47mg/kg, 48mg/kg, 49mg/kg, 50mg/kg, 51mg/kg, 52mg/kg, 53mg/kg, 54mg/kg, 55mg/kg, 56mg/kg, 57mg/kg, 58mg/kg , 59mg/kg, 60mg/kg, 61mg/kg, 62mg/kg, 63mg/kg, 64mg/kg, 65mg/kg, 66mg/kg, 67mg/kg, 68mg/kg, 69mg/kg, 70mg/kg, 71mg /kg, 72mg/kg, 73mg/kg, 74mg/kg, 75mg/kg, 76mg/kg, 77mg/kg, 78mg/kg, 79mg/kg, 80mg/kg, 81mg/kg, 82mg/kg, 83mg/kg , 84mg/kg, 85mg/kg, 86mg/kg, 87mg/kg, 88mg/kg, 89mg/kg, 90mg/kg, 91mg/kg, 92mg/kg, 93mg/kg, 94mg/kg, 95mg/kg, 96mg /kg, 97mg/kg, 98mg/kg, 99mg/kg, 100mg/kg, 105mg/kg, 110mg/kg, 115mg/kg, 120mg/kg, 125mg/kg, 130mg/kg, 135mg/kg, 140mg/kg , 145mg/kg, 150mg/kg, 155mg/kg, 160mg/kg, 165mg/kg, 170mg/kg, 175mg/kg, 180mg/kg, 185mg/kg, 190mg/kg, 195mg/kg, 200mg/kg, 2 05mg/kg, 210mg/kg, 215mg/kg, 220mg/kg, 225mg/kg, 230mg/kg, 235mg/kg, 240mg/kg, 245mg/kg, 250mg/kg, 260mg/kg, 270mg/kg, 280mg/kg kg, 290mg/kg, 300mg/kg, 310mg/kg, 320mg/kg, 330mg/kg, 340mg/kg, 350mg/kg, 360mg/kg, 370mg/kg, 380mg/kg, 390mg/kg, 400mg/kg, 410mg/kg, 420mg/kg, 430mg/kg, 440mg/kg, 450mg/kg, 460mg/kg, 470mg/kg, 480mg/kg, 490mg/kg, 500mg/kg.

In the above embodiment, the multi-targeted tyrosine kinase inhibitor is selected from c-Kit, VEGFR2, PDGFR inhibitor. In some embodiments, the multi-targeted tyrosine kinase inhibitor is a VEGFR2 inhibitor, such as sunitinib, a compound of formula (I) or a pharmaceutically acceptable salt thereof, preferably a compound of formula (I) or a pharmaceutically acceptable salt thereof Salt,

In some embodiments, the administration dose of the fusion protein containing TGF-β receptor (eg fusion protein 9) is selected from 0.1-200 mg/kg, and the administration frequency is once a week, once every two weeks, every three Once a week or every four weeks.

In some embodiments, the administration dose of the fusion protein containing TGF-β receptor (eg fusion protein 9) is selected from 0.1-100 mg/kg, and the administration frequency is once a week, once every two weeks, every three Once a week or every four weeks.

In some embodiments, the TGF-beta receptor-containing fusion protein (eg, fusion protein 9) is administered at a dose selected from 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg /kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg , 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg /kg, 32mg/kg, 33mg/kg, 34mg/kg, 35mg/kg, 36mg/kg, 37mg/kg, 38mg/kg, 39mg/kg, 40mg/kg, 41mg/kg, 42mg/kg, 43mg/kg , 44mg/kg, 45mg/kg, 46mg/kg, 47mg/kg, 48mg/kg, 49mg/kg, 50mg/kg, 51mg/kg, 52mg/kg, 53mg/kg, 54mg/kg, 55mg/kg, 56mg /kg, 57mg/kg, 58mg/kg, 59mg/kg, 60mg/kg, dosing frequency is once every two weeks, once every three weeks or once every four weeks.

In some embodiments, the TGF-beta receptor-containing fusion protein (eg, fusion protein 9) is administered at a dose selected from 1 mg/kg, 3 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg /kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 55mg/kg, 60mg/kg, dosing frequency is once every two weeks, once every three weeks or once every four weeks.

In some embodiments, the TGF-beta receptor-containing fusion protein (eg, fusion protein 9) is administered at a dose selected from 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg /kg, dosing frequency is once every two weeks, once every three weeks or once every four weeks.

In some embodiments, the TGF-beta receptor-containing fusion protein (eg, fusion protein 9) is administered at a dose of 30 mg/kg at a frequency of once every three weeks. A multi-targeted tyrosine kinase inhibitor (eg, formula (I)) is 20 mg once a day, administered continuously, every 3 weeks (21 days) as a cycle.

In some embodiments, when administered to a subject in need or a fusion protein containing a TGF-beta receptor (eg, fusion protein 9) once a week, once every two weeks, once every three weeks, or once every four weeks , once a week, once every two weeks, once every three weeks or once every four weeks as a dosing cycle, respectively, administered on the first day of each cycle.

In some embodiments, the dose of the fusion protein containing TGF-β receptor (eg fusion protein 9) is 1-4000mg, specifically 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg , 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1450mg, 1500mg, 1550mg, 160mg , 1750mg, 1800mg, 1850mg, 1900mg, 1950mg, 2000mg, 2050mg, 2100mg, 2150mg, 2200mg, 2250mg, 2300mg, 2350mg, 2400mg, 2450mg, 2500mg, 2550mg, 2600mg, 2650mg, 2700mg, 2750mg, 2800mg, 2850mg, 2900mg, 2950mg , 3000mg, 3050mg, 3100mg, 3150mg, 3200mg, 3250mg, 3300mg, 3350mg, 3400mg, 3450mg, 3500mg, 3550mg, 3600mg, 3650mg, 3700mg, 3750mg, 3800mg, 3850mg, 3900mg, 3950mg, 4000mg once a week , every two weeks, every three weeks or every four weeks.

In some embodiments, the dose of the TGF-beta receptor-containing fusion protein (eg, fusion protein 9) is 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, 2400 mg, 2700 mg, 3000 mg, 3300 mg, 3600 mg, Dosing frequency is every two weeks, every three weeks, or every four weeks.

In some embodiments, the dose of the TGF-beta receptor-containing fusion protein (eg, fusion protein 9) is 600 mg, 1200 mg, 1800 mg, 2400 mg, 3000 mg, and the dosing frequency is once every two weeks or once every three weeks.

In an optional embodiment, the TGF-β receptor-containing fusion protein (eg, fusion protein 9) is administered by injection, such as subcutaneous or intravenous injection, and the TGF-β receptor-containing fusion protein needs to be injected before injection. The protein is formulated into an injectable form, and the injectable form of the fusion protein containing the TGF-β receptor can be an injection or a freeze-dried powder, which comprises the fusion protein containing the TGF-β receptor, a buffer, and a stabilizer, Optionally also contain surfactants. The buffer can be selected from one or more of acetate, citrate, succinate, and phosphate. Stabilizers may be selected from sugars or amino acids, preferably disaccharides such as sucrose, lactose, trehalose, maltose. The surfactant is selected from polyoxyethylene hydrogenated castor oil, glycerol fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like.

The present disclosure provides a method of treating a tumor or cancer, comprising administering to a subject in need thereof an effective amount of the above-described multi-targeted tyrosine kinase inhibitor and an effective amount of the above-described TGF-beta receptor-containing fusion protein (eg, a fusion protein 9).

In some embodiments, a method of treating a tumor or cancer is provided, comprising administering to a subject in need an effective amount of the above-mentioned multi-targeted tyrosine kinase inhibitor (eg, famitinib or a compound of formula (I) or its pharmaceutically acceptable salt), the multi-targeted tyrosine kinase inhibitor is used in combination with the above-mentioned fusion protein (eg, fusion protein 9) containing the TGF-β receptor.

In some embodiments, there is provided a method of treating a tumor or cancer, comprising administering to a subject in need thereof an effective amount of the above-described TGF-beta receptor-containing fusion protein (eg, fusion protein 9), the TGF-beta receptor-containing fusion protein The fusion protein is used in combination with the above-mentioned multi-target tyrosine kinase inhibitor (eg, famitinib or the compound represented by formula (I) or a pharmaceutically acceptable salt thereof).

In an optional embodiment, in the aforementioned method or use for treating tumor or cancer, the subject in need is a human.

The tumors or cancers described in the present disclosure are selected from tumors or cancers of the following sites: colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, myeloma, lymphoma, leukemia, thyroid, endometrium, uterus, Bladder, neuroendocrine, head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, meke cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

In some specific embodiments, the tumor or cancer is pancreatic cancer, biliary tract tumor.

In some specific embodiments, the biliary tract tumor is a biliary tract malignancy selected from the group consisting of intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and gallbladder cancer.

In some specific embodiments, the pancreatic cancer is advanced or metastatic pancreatic cancer and the biliary tract tumor (eg, intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, and gallbladder cancer) is an advanced or metastatic biliary tract tumor.

In some specific embodiments, the biliary tract tumor is selected from biliary tract malignant tumors with mutations in RAS (including KRAS, NRAS, HRAS), BRAF, and PI3KCA genes.

In some specific embodiments, the pancreatic cancer or biliary tract tumor is progressive or metastatic or treatment failure following surgery or first-line therapy.

In some specific embodiments, the biliary tract tumor is one or more of uracil, doxorubicin, mitomycin, siggio or capecitabine combination therapy continues to progress or metastasize or Treatment failed.

detailed description

the term

In this disclosure, "combining" a mode of administration refers to the administration of at least one dose of a multitargeted tyrosine kinase inhibitor and at least one dose of a TGF-beta receptor-containing fusion over a period of time proteins, both of which exhibit pharmacological effects. Said time period can be within one dosing cycle, optionally within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 24 hours, within 2 hours. The multi-targeted tyrosine kinase inhibitor and the fusion protein containing the TGF-beta receptor can be administered simultaneously or sequentially. This term includes treatment in which the multi-targeted tyrosine kinase inhibitor and the fusion protein containing the TGF-beta receptor are administered by the same route of administration or by different routes of administration. The modes of administration of the combinations described in this disclosure are selected from simultaneous administration, separate formulation and co-administration, or separate formulation and sequential administration.

The "antibody" referred to in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

In the present disclosure, the antibody light chain of the present disclosure may further comprise a light chain constant region comprising human or murine κ, λ chains or variants thereof.

In the present disclosure, the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions. The sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present disclosure conform to the known Kabat numbering conventions (LCDR1-3, HCDE2-3), or the numbering conventions of Kabat and Chothia (HCDR1).

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

"Antigen-binding fragments" of the present disclosure include: single-chain antibodies (ie, full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (eg VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope binding fragments of any of the above (See, eg, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods of producing and preparing these antibody fragments are well known in the art (see, eg, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv form was first disclosed in WO2009/040562 and its disulfide stabilized form, Fab-dsFv, was first disclosed in WO2010/035012. Antigen-binding fragments of the present disclosure also include Fab and Fab&apos; fragments described in WO2005/003169, WO2005/003170 and WO2005/003171. Multivalent antibodies may comprise multispecific, eg bispecific, or may be monospecific (see eg WO92/22583 and WO05/113605), an example of the latter being the Tri-Fab (or Tri-Fab) described in WO 92/22583 TFM).

The term "binding to PD-L1" means capable of interacting with PD-L1 or an epitope thereof, which may be of human origin.

The term "antigen-binding site" refers to a discrete three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the present disclosure.

The term "murine antibody" in the present disclosure is a monoclonal antibody to human PD-L1 prepared according to the knowledge and skill in the art. In preparation, test subjects are injected with PD-L1 antigen, and hybridomas expressing antibodies with desired sequence or functional properties are isolated.

The term "chimeric antibody" is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody. To establish a chimeric antibody, first establish a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and then clone the variable region of the mouse. The gene is linked with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. In a preferred embodiment of the present disclosure, the antibody light chain of the PCSK-9 chimeric antibody further comprises a light chain constant region of a human κ, λ chain or a variant thereof. The antibody heavy chain of the PCSK-9 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising the heavy chain constant region of human IgG2 or IgG4, or without ADCC (antibody-dependent) after amino acid mutation. cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxicity of IgG4.

The term "humanized antibody", also known as CDR-grafted antibody, refers to the grafting of mouse CDR sequences into a human antibody variable region framework, i.e. a different type of human germline Antibody produced in antibody framework sequences. The strong antibody variable antibody response induced by chimeric antibodies can be overcome because they carry a large number of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th ed. In order to avoid the decrease of the activity while the immunogenicity is decreased, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain the activity. The humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation of the CDRs by phage display.

"Identity" as used in this disclosure refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. Sequence identity in the present disclosure may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%. Sequence comparison and determination of percent identity between two sequences can be performed with the default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.

The term "TGF-beta receptor II" or "TGFbetaRII" or "transforming growth factor beta receptor II" refers to binding ligands (including but not limited to TGFbeta1, TGFbeta2 and TGFbeta3) and thereby triggering intracellular signal transduction pathway's cell surface receptors.

The term "PD-L1" refers to programmed death ligand 1, also known as CD274 and B7H1. PD-L1 is a 290 amino acid protein with extracellular IgV-like and IgC-like domains (amino acids 19-239 of full-length PD-L1), a transmembrane domain, and an intracellular domain of approximately 30 amino acids. PD-L1 is constitutively expressed on many cells such as antigen-presenting cells (eg, dendritic cells, macrophages, and B cells), as well as on hematopoietic and non-hematopoietic cells (eg, vascular endothelial cells, pancreatic islets, and sites of immune forgiveness) . PD-L1 is also expressed on a variety of tumors and virus-infected cells and is a component of an immunosuppressive milieu (Ribas 2012, NEJM 366:2517-2519). PD-L1 binds to one of two T-cell co-inhibitors, PD-1 and B7-1.

The fusion protein described in the present disclosure is a protein product of co-expression of two genes obtained by DNA recombination. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, eg, Cold Spring Harbor's Technical Guide to Antibody Assays, Chapters 5-8 and 15. For example, mice can be immunized with human PD-L1 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibody or antigen-binding fragment of the present invention uses genetic engineering to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software, from the website of ImMunoGeneTics (IMGT) at http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001 ISBN012441351 get.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or disorder of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject in need or veterinary medicine may vary depending on factors such as the condition to be treated, the general health of the subject in need, the method, route and dosage of administration and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

ORR is the objective response rate. PFS is progression-free survival. OS is overall survival. DCR is the disease control rate.

Complete remission (CR): All target lesions disappeared, and the short diameter of all pathological lymph nodes (including target and non-target nodules) must be reduced to <10mm.

Partial remission (PR): At least 30% reduction in the sum of target lesion diameters from baseline.

Disease progression (PD): At least a 20% relative increase in diameter and relative increase in diameter and relative increase of at least 20% (or baseline value if the baseline measurement value is the smallest), with reference to the minimum value of the sum of all measured target lesion diameters throughout the experimental study; otherwise In addition, the absolute value of the sum of the diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered as disease progression).

Stable disease (SD): The degree of reduction of target lesions does not reach the PR level, and the degree of increase does not reach the level of PD. Between the two, the minimum sum of diameters can be used as a reference in the study.

The following examples are used to further describe the present invention, but these examples do not limit the scope of the present disclosure.

Example Evaluation of the efficacy of test drugs in humans

20-30 patients with advanced pancreatic cancer and biliary tract tumors were recruited to preliminarily evaluate the efficacy of TGF-β receptor fusion protein (fusion protein 9) combined with multi-target tyrosine kinase inhibitors in advanced pancreatic cancer and biliary tract tumors. .

1. Test antibodies and compounds

TGF-β receptor fusion protein: use fusion protein 9, 50mg/mL;

Multi-target tyrosine kinase inhibitor: compound represented by formula (I).

2. Criteria for entering subjects

1) Age 18-75 years old, gender is not limited;

2) Histologically or cytologically diagnosed pancreatic cancer or biliary tract tumor (TNM stage: IV); ≥1 line of treatment failure; according to the solid tumor response evaluation criteria (RECIST1.1), imaging diagnosis has at least one measurable lesion.

3. Method of administration

TGF-β receptor fusion protein (fusion protein 9): intravenously administered at a dose of 30 mg/kg, administered once every 3 weeks, every 3 weeks (21 days) as a cycle, and administered on the first day of each cycle. It is recommended to administer the drug through an infusion pump, and the intravenous infusion should be completed within 30-60 minutes. Intravenous bolus injection and rapid bolus injection are not allowed. After the infusion is completed, flush the infusion tube with a sufficient amount of normal saline or 5% glucose solution.

Multi-targeted tyrosine kinase inhibitor represented by formula (I): 20 mg (initial dose), taken orally before or after meals, recommended daily at a fixed time and within 0.5 hours after meals, once a day; continuous Take the medicine every 3 weeks (21 days) as a cycle.

Study drug was used until protocol-specified treatment termination criteria were met or subjects were withdrawn from the study.

After the subject has progressed according to the definition of Response Evaluation Criteria for Solid Tumors Version 1.1 (RECIST 1.1), if the investigator evaluates the subject's clinical benefit and can tolerate the study treatment and the subject in need is voluntary Under these conditions, the treatment with the TGF-β receptor fusion protein and/or the multi-targeted tyrosine kinase inhibitor represented by formula (I) can still be continued.

The primary endpoint was objective response rate (ORR) based on RECIST 1.1 criteria, and secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and safety. The safety indicators include vital signs, laboratory indicators, adverse events (AEs), serious adverse events (SAEs), drug-related AEs and SAEs.

A total of 18 patients with pancreatic cancer and 6 patients with biliary tract cancer were enrolled. Of the 12 needy subjects evaluable in the pancreatic cancer cohort, 1 CR, 4 SD, 7 PD, ORR 8.3%, DCR 41.6%; biliary tract cohort evaluable 3, including 1 PR (sustained). 3.3+ months, tumor shrinkage 46.48%), 1 case of SD, ORR 33.3%, DCR 66.7%. The incidence of treatment-related adverse events (TRAEs) was 66.67%. The most common TRAEs were hypertension (38.10%), proteinuria (38.10%) and elevated alanine aminotransferase (33.33%). The most common grade 3 TRAEs were blood pressure. Bilirubin increased (9.52%). No grade 4/5 AEs occurred.

The results showed that fusion protein 9 combined with famitinib had good efficacy and safety in subjects with pancreatic cancer and biliary tract cancer who had failed previous standard treatments. Since the enrolled cases are stage IV subjects in need who have failed at least first-line treatment, CR and PR cases have appeared in a small number of enrolled cases, indicating that the combination administration has a good prospect, and this study is expected to The combination used can extend PFS, OS.

Claims (19)

1. Use of a fusion protein containing a TGF-β receptor in combination with a multi-target tyrosine kinase inhibitor in the preparation of a medicament for the treatment of tumors or cancers, wherein the tumors or cancers are preferably pancreatic cancer and biliary tract tumors; the biliary tract The tumor is preferably selected from intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma and gallbladder carcinoma.
2. The use according to claim 1, wherein the pancreatic cancer and biliary tract tumor are advanced or metastatic.
3. The use according to claim 1 or 2, wherein the fusion protein of the TGF-β receptor comprises a PD-L1 antibody or an antigen-binding fragment thereof and a TGF-β receptor, wherein the TGF-β receptor part It is an N-terminal truncated form of the extracellular domain of TGF-βRII.
4. The use according to claim 3, wherein the TGF-β receptor fusion protein is shown in general formula (II):

   Ab-L-TGF-βRII ECD (II)

   wherein TGF-βRII ECD is a truncated form of the extracellular domain of TGF-βRII;

   Ab is a PD-L1 antibody or an antigen-binding fragment thereof;

   L is the linking sequence.
5. The use according to claim 4, wherein the linking sequence is (G ₄ S)×G, wherein x is 3-6, preferably 4.
6. The use according to claim 5, wherein the extracellular region of TGF-βRII comprises the sequences shown in SEQ ID NOs: 11, 12, 13 and 14; preferably, it comprises the sequences shown in SEQ ID NO: 12 sequence.
7. The use according to claim 4, wherein the PD-L1 antibody or antigen-binding fragment thereof comprises:

   HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and HCDR1 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively of LCDR1, LCDR2 and LCDR3.
8. The use according to any one of claims 4-7, wherein the PD-L1 antibody or an antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof.
9. The use according to claim 8, wherein the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:7, and a heavy chain variable region as shown in SEQ ID NO:8 light chain variable region.
10. The use according to any one of claims 4-9, wherein the heavy chain amino acid sequence of the PD-L1 antibody or its antigen-binding fragment is as shown in SEQ ID NO:9 or as shown in SEQ ID NO:9 The amino acid sequence shown is at least 95% identical, and the light chain amino acid sequence is shown in SEQ ID NO: 10 or has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 10.
11. The use according to claim 4, wherein the PD-L1 antibody or its antigen-binding fragment is selected from the group consisting of avelumab, atezolizumab, durvalumab, CS-1001, M-7824, KL-A167, CX-072, BGB - A333, GNS-1480, CA-170, BMS-936559, preferably avelumab, atezolizumab, durvalumab.
12. The use according to any one of claims 4-10, wherein in the fusion protein of TGF-β receptor, the heavy chain amino acid sequence of PD-L1 antibody or its antigen-binding fragment is as SEQ ID NO:9 As shown, the amino acid sequence of the light chain is shown in SEQ ID NO: 10, the linking sequence L is (G ₄ S) ₄ G, and the amino acid sequence of the extracellular region of TGF-βRII is shown in SEQ ID NO: 12.
13. The use according to claim 1 or 2, wherein the multi-target tyrosine kinase inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

    
14. The use according to any one of claims 1-13, wherein the administration dose of the fusion protein containing TGF-β receptor is selected from 0.1-500 mg/kg, and the administration frequency is once a week, every Every two weeks, every three weeks or every four weeks.
15. The use according to claim 14, wherein the administration dose of the fusion protein containing TGF-β receptor is selected from 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg at a frequency of once every two weeks, once every three weeks or once every four weeks;

    Preferably, the TGF-beta receptor fusion protein is administered at a dose of 30 mg/kg once every three weeks.
16. The use according to any one of claims 1-13, wherein the administration dose of the fusion protein containing TGF-β receptor is selected from 1-4000 mg, and the administration frequency is once a week, every two weeks Once, once every three weeks or once every four weeks, the preferred dosage is selected from 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, 2400 mg, 2700 mg, 3000 mg, 3300 mg, 3600 mg.
17. The use according to any one of claims 1-16, wherein the administration dose of the multi-target tyrosine kinase inhibitor is selected from 1-1600 mg, and the administration frequency is twice a day, one day Once, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks or once every four weeks, preferably once a day or twice a day;

    Preferably, the dosage of the multi-target tyrosine kinase inhibitor is 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, once a day;

    More preferably, the multi-targeted tyrosine kinase inhibitor is administered at a dose of 20 mg once a day.
18. The use of claim 17, wherein the multi-targeted tyrosine kinase inhibitor is administered for three consecutive weeks.
19. A medicine package, which contains the fusion protein of TGF-β receptor and the multi-target tyrosine kinase inhibitor in the use according to any one of claims 1-18.