WO2019072220A1 - Use of pd-1 antibody combined with epigenetic regulator in preparation of drug for treating tumors - Google Patents

Abstract

Provided is a use of a PD-1 antibody combined with an epigenetic regulator in the preparation of a drug for treating tumors. In particular, provided is a use of the PD-1 antibody combined with the epigenetic regulator in the preparation of a drug for treating tumors and/or enhancing T-cell activity.

Description

Use of PD-1 antibody and epigenetic regulator in combination for preparing medicine for treating tumor Technical field

Use of an anti-PD-1 antibody in combination with an epigenetic modulator in the manufacture of a medicament for treating tumors and/or enhancing T-cell activity.

Background technique

PD-1 (programmed death receptor 1) antibody can specifically recognize and bind to PD-1 on the surface of lymphocytes, block PD-1/PD-L1 signaling pathway, and then activate T cell immune killing effect on tumors. The immune system removes tumor cells from the body. Epigenetic changes are closely related to cancer development and drug resistance. As the study progressed, it was found that there was resistance after repeated administration of PD-1 antibody to patients. DNA methylation inhibitor decitabine

It has been approved for hematologic treatment of malignant tumors and its clinical efficacy in solid tumors. Various animal models and cell line studies have shown that decitabine induces genes that control apoptosis, cell cycle arrest, and tumor surface antigens. Expression; induction of major histocompatibility complex (MHC) and costimulatory molecules by DNA demethylation effects, which resulted in decitabine increasing the response rate of anti-tumor immunotherapy and inhibiting tumor growth. Studies have found that methylation modifications and silencing of specific genes may play a key role in the maturation and typing of T-cells. It has been reported that 5-azacitidine can be increased by PD-1/PD-L1 (programmed death Body 1) is the sensitivity of the target immunological checkpoint treatment, so epigenetic treatment combined with immunotherapy can improve the effectiveness of clinical treatment of cancer (Cancer letters 354.1 (2014): 12-20). Riccadonna et al. showed that demethylating agents have improved immunotherapy for the recognition and subsequent destruction of various glioma cells, and their cellular and animal studies have shown that decitabine combined with multiple immunotherapies for malignant glioma models Has a good anti-tumor effect ([J]. PloS one, 2016, 11 (8): e0162105); Tamas A et al. found that decitabine combined with immunological checkpoint inhibitor (Anti-PD-1H), especially In the KBC pancreatic cancer model mice, the tumor inhibition efficiency was significantly increased. After the administration of decitabine, the expression of PD-1H was significantly increased. The anti-PD-1H treatment of decitabine was significantly prolonged. Mouse survival ([J]. Gastroenterology, 2017, 152(5): S42-S43); WO2015035112 discloses an epigenetic modulator combined with an immunomodulator for treating a tumor, wherein the epigenetic regulator is selected from the group consisting of For citrate, the immunomodulator may be selected from the group consisting of PD-1/PD-L1 antibodies.

At present, clinical research has gradually carried out the treatment of decitabine combined with anti-PD-1 antibody, and 18% of the clinical studies of PD-1 antibody Nivolumab combined with azacitidine in the treatment of relapsed and refractory AML (acute myeloid lymphocytic leukemia) The patient's efficacy was evaluated as CR ([J]. Blood 2016 128:763); Chunmeng Wang reported a phase I clinical study of PD-1 antibody combined with decitabine in the treatment of advanced and untreated malignancies. One patient in the patient was evaluated as CR ([J]. Journal of Clinical Oncology 35 (2017): e14555-e14555). Phase II clinical trial of PD-L1 inhibitor Durvalumab in combination with oral azacitidine in the treatment of myelodysplastic syndrome previously insensitive to decitabine or azacitidine (NCT02281084); PD-1 inhibitor Pembrolizumab Combined with decitabine in the treatment of relapsed and refractory AML is also underway (NCT02996474), but the clinical results reported so far are still not satisfactory, and the extension of the overall survival of refractory tumors is not satisfactory, but The results of clinical studies have not yet been known, how to screen the best PD-1/PD-L1 inhibitors and DNA demethylation inhibitors to obtain the best anti-tumor effect and at the same time take into account the safety is still a clinical problem to be solved.

The anti-PD-1 antibody provided by the present invention, WO2015085847 discloses the sequence and preparation method of the antibody, and the PD-1 antibody is currently in the clinical phase I of China, and the safety is good, and the reported clinical research results have shown that it has certain Antitumor effect ([J]. Journal of Clinical Oncology 35 (2017): e15572-e15572).

Summary of the invention

The invention provides the use of an immunotherapeutic agent in combination with an epigenetic modulator in the manufacture of a medicament for treating a tumor and/or enhancing T-cells, the immunotherapeutic agent being selected from the group consisting of PD-1 antibodies.

A PD-1 antibody is known, and preferably the light chain variable region of the PD-1 antibody comprises LCDR1, LCDR2 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. LCDR3.

The heavy chain variable region of the PD-1 antibody comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

Among them, the CDR sequences described above are shown in the following table:

name	sequence	Numbering
HCDR1	SYMMS	SEQID NO: 1
HCDR2	TISGGGANTYYPDSVKG	SEQID NO: 2
HCDR3	QLYYFDY	SEQID NO: 3
LCDR1	LASQTIGTWLT	SEQID NO: 4
LCDR2	TATSLAD	SEQID NO: 5

LCDR3

QQVYSIPWT

SEQID NO: 6

Preferably, the PD-1 antibody is a humanized antibody.

Preferably, the humanized antibody light chain variable region sequence is the sequence set forth in SEQ ID NO: 10 or a variant thereof; said variant preferably has a 0-10 amino acid change in the light chain variable region; Amino acid changes of A43S are preferred. The humanized antibody heavy chain variable region sequence is the sequence set forth in SEQ ID NO: 9 or a variant thereof; the variant preferably has an amino acid change of 0-10 in the heavy chain variable region; more preferably Amino acid changes in G44R.

The variable region sequences of the heavy and light chains of the aforementioned humanized antibodies are as follows:

Heavy chain variable region

Light chain variable region

Preferably, the humanized antibody light chain sequence is the sequence set forth in SEQ ID NO: 8 or a variant thereof; said variant preferably has a 0-10 amino acid change in the light chain variable region; more preferably A43S Amino acid changes. The humanized antibody heavy chain sequence is the sequence set forth in SEQ ID NO: 7 or a variant thereof; the variant preferably has an amino acid change of 0-10 in the heavy chain variable region; more preferably an amino acid of G44R Variety.

Particularly preferably, the humanized antibody light chain sequence is the sequence set forth in SEQ ID NO: 8, and the heavy chain sequence is the sequence set forth in SEQ ID NO: 7.

The sequences of the aforementioned humanized antibody heavy and light chains are as follows:

Heavy chain

Light chain

In a preferred embodiment of the present invention, the epigenetic regulator is selected from the group consisting of a demethylating agent, a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HDAC) inhibitor, or In many cases, wherein the DNMT inhibitor is selected from the group consisting of guadecitabine, RX-3117, Lenalidomide and azacitidine compound, EPI-01, decitabine and E-7727 compound, RRx-001, temozolomide, CM-272, KM -101, KRX-0402, TdCyd, UVI-5008, azacitidine prodrug, decitabine prodrug, aza-T-dCyd, XB-05, PMX-700, CP-4200; said HDAC inhibitor Selected from mocetinostat, entinostat, purinostat, largazole, largazole analog, ACY-738, resminostat, VRx-3996, givinostat, MPT-0E028, tucidinostat, TMB-ADC, romidepsin, panobinostat, 4SC-202, abexinostat, CUDC-907, Vorinostat, pracinostat, HLY-0019, DWP-0016, remetinostat, sodium phenylbutyrate, valproic acid, CKD-509, CKD-504, CKD-506, CKD-581, ricolinostat, CG-200745, AR-42, SP -2-59, citalinostat, vorinostat, AP-001, quisinostat, SP-1-161, RCY-1497, belinostat, trichostatin A TMP-195, RND-001, GSK-3117391, ACY-1083, tefinostat, tinostamustine, SF-2558HA, CXD-101, JW-1521, CG-1255, LB-205, LB-201, OCID-4681, QTX- 153, APH-0812, CX-1026, OBP-801, CS-3158, RG-2833, TL-112, HG-3001, KDAC-001, thailandepsin B, QTX-125, RGFP-966, SP-1003, BEBT -908, BRD-3308, BEBT-201, BEBT-906, ACY-257, NMB-T-BMX-OS01, NBM-HD-1, zebularine.

In another preferred embodiment of the present invention, the epigenetic regulator is selected from the group consisting of azacitidine, decitabine, Fludarabine, Guadecitabine, Zebularine, NPEOC-DAC, CP-4200, Vorinostat, Romidepsin, Panobinostat, CI -994,5,6-dihydro-5-azacytidine, 5-fluoro-2'-deoxycytidine, RX-3117, epigallocatechin gallate (EGCG), genistein, curcumin Oligonucleotides, preferably from decitabine, azacitidine, Guadecitabine, Romidepsin, Fludarabine.

In a preferred embodiment of the present invention, in a preferred embodiment of the present invention, the tumor is selected from the group consisting of a malignant tumor and a benign tumor; and the malignant tumor is selected from the group consisting of a malignant epithelial tumor, a sarcoma, a myeloma, a leukemia, a lymphoma, Melanoma, head and neck tumor, brain tumor, mixed tumor, childhood malignant tumor; the malignant epithelial tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, stomach cancer, gastroesophageal adenocarcinoma, esophageal cancer , small bowel cancer, cardia cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer, kidney cancer, bladder cancer, anal cancer, gallbladder cancer, cholangiocarcinoma, teratoma, heart Tumor; the head and neck tumor is selected from the group consisting of nasopharyngeal carcinoma, laryngeal cancer, thyroid cancer, tongue cancer, oral cancer; the sarcoma is selected from the group consisting of Askin tumor, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannomas , osteosarcoma, soft tissue sarcoma; the myeloma is selected from the group consisting of isolated myeloma, multi-mode myeloma, diffuse myeloma, leukemia-type myeloma, extramedullary myeloma; Selected from acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, hairy cell leukemia, T cell lymphocytic leukemia, large granular lymphocytic leukemia, adult T cell leukemia; From non-Hodgkin's lymphoma, Hodgkin's lymphoma; the brain tumor is selected from the group consisting of a neuroepithelial tumor, a cranial nerve and a spinal nerve tumor, a meningeal tumor; the child's malignant tumor is selected from a nephroblastoma, a nerve Maternal tumor, retinoblastoma, childhood germ cell tumor.

In another preferred embodiment of the present invention, the lung cancer is selected from the group consisting of the lung cancer selected from the group consisting of non-small cell lung cancer and small cell lung cancer; and the breast cancer is selected from the group consisting of the hormone receptor (HR) positive. Breast cancer, human epidermal growth factor receptor-2 (HER2)-positive breast cancer, triple-negative breast cancer; the renal cancer is selected from the group consisting of transparent renal cell carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, and collecting duct The cancer of the neuroepithelial tissue is selected from the group consisting of: preferably astrocytoma, anaplastic astrocytoma, glioblastoma; the liver cancer is selected from the group consisting of primary liver cancer, secondary liver cancer, and primary liver cancer. It is selected from the group consisting of hepatocellular carcinoma, cholangiocarcinoma, and mixed liver cancer; and the colorectal cancer is selected from the group consisting of colon cancer and rectal cancer.

In another preferred embodiment of the present invention, the tumor is selected from the group consisting of a solid tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostate cancer, pancreatic cancer, lung cancer, esophageal cancer, liver cancer, cholangiocarcinoma, breast. Cancer, colorectal cancer, gastric cancer, kidney cancer, acute myeloid lymphocytic leukemia, myelodysplastic syndrome, glioma.

In a preferred embodiment of the invention, the tumor is mediated and/or expresses PD-L1 by PD-1.

In a preferred embodiment of the present invention, the tumor is selected from the group consisting of a middle-stage tumor, a relapsed and refractory tumor, a chemotherapy-treated drug failure and/or a recurrent tumor, a radiotherapy failure and/or a recurrent tumor, and a targeted drug treatment. Failure and/or recurrence of the tumor, failure of immunotherapy, and/or recurrence of the tumor.

In a preferred embodiment of the invention, the tumor is either resistant or resistant to immunotherapeutics or immunotherapy, preferably, the immunotherapeutic agent is PD-1 and/or PD-L1 or CTLA- 4 (cytotoxic T lymphocyte-associated protein 4) as a target; the immunotherapy is selected from the group consisting of immunological checkpoint block (ICB) therapy, chimeric antigen receptor T cell immunotherapy (CAR-T therapy), autologous cellular immunity Therapy (CIK therapy).

In a preferred embodiment of the present invention, preferably, the immunotherapeutic agent is selected from the group consisting of a PD-1 antibody, a PD-L1 antibody, and a CTLA-4 antibody, and the PD-1 antibody includes, but is not limited to, Pidilizumab, MEDI-0680. , AMP-224, PF-06801591, TSR-042, JS-001, GLS-010, PDR-001, Genolimzumab, Camrelizumab, BGB-A317, IBI-308, REGN-2810, Pembrolizumab, Nivolumab; said PD-L1 Antibodies include, but are not limited to, MSB-0011359-C, CA-170, LY-3300054, BMS-936559, Durvalumab, Avelumab, Atezolizumab; the CTLA-4 antibodies include, but are not limited to, ipilimumab, AK-104, JHL-1155, ATOR -1015, AGEN-1884, PRS-010, tremelimumab, IBI-310, MK-1308, BMS-986218, SN-CA21, FPT-155, KN-044, CG-0161, ONC-392, AGEN-2041, PBI -5D3H5.

In a preferred embodiment of the invention, there is provided the use of a PD-1 antibody in combination with an epigenetic modulator for the preparation of a medicament for enhancing T-cell activity, preferably a peripheral T-cell.

In a preferred embodiment of the present invention, the PD-1 antibody dose is selected from the group consisting of 1-10 mg/kg, preferably 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, 9 mg/kg, 10 mg/kg, more preferably 3 mg/kg, 4 mg/kg, 5 mg/kg.

In a preferred embodiment of the present invention, the PD-1 antibody dose is selected from the group consisting of 50-600 mg, preferably from 50 mg, 60 mg, 70 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 375 mg, 400 mg. 425 mg, 450 mg, 475 mg, 500 mg, 600 mg, more preferably from 100 mg, 200 mg, 400 mg.

In a preferred embodiment of the present invention, the epigenetic modulator dose is selected from the group consisting of 5-100 mg/m ² , preferably from 5 mg/m ² , 6 mg/m ² , 7 mg/m ² , 8 mg/m ² , 9 mg/ m ² , 10 mg/m ² , 12 mg/m ² , 15 mg/m ² , 20 mg/m ² , 25 mg/m ² , 30 mg/m ² , 35 mg/m ² , 40 mg/m ² , 50 mg/m ² , 60 mg/ m ² , 75 mg/m ² ; more preferably 7 mg/m ² , 10 mg/m ² , 12 mg/m ² , 15 mg/m ² , 20 mg/m ² , 25 mg/m ² , 30 mg/m ² , 40 mg/m ² , 50 mg/m ² , 75 mg/m ² .

In a preferred embodiment of the present invention, the epigenetic modulator dose is selected from the group consisting of 5 to 500 mg, preferably from 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg. 275 mg, 300 mg, 400 mg, 500 mg, more preferably 10 mg, 20 mg, 100 mg, 200 mg, 300 mg.

In a preferred embodiment of the present invention, the weight ratio of the PD-1 antibody to the epigenetic regulator is selected from 0.01 to 100:1, preferably from 5:1, 4:1, 3:1, 5:2. , 2:1, 7:4, 3:2, 4:3, 5:4, 1:1, 3:4, 2:3, 3:5, 1:2, 2:5, 1:3, 3 :10, 1:4, 40:1, 25:1, 20:1, 15:1, 25:2, 12:1, 10:1, 8:1, 9:2, 25:4, 6:1 20:3, 15:4; more preferably from 5:1, 4:1, 3:1, 5:2, 2:1, 1:1, 2:3, 4:3, 40:1, 20: 1.

In a preferred embodiment of the present invention, the combination further comprises a third component selected from the group consisting of an alkylating agent, a platinum complexing agent, a metabolic antagonist, a plant alkaloid, a hormone anticancer agent, and a protease. One or more of a body inhibitor, an aromatase inhibitor, an immunomodulator, an EGFR inhibitor, an ALK inhibitor, a PARP inhibitor VEGF antibody, a VEGFR inhibitor, and an mTOR inhibitor.

Preferably, the chemotherapeutic agent is selected from the group consisting of an alkylating agent, a platinum complexing agent, a metabolic antagonist, a plant alkaloid (such as a vinblastine, a harringtonine), a hormone anticancer agent, a proteasome inhibitor, and a fragrance. One or more of an enzyme inhibitor, an immunomodulator; in another preferred embodiment, the chemotherapeutic agent includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan, busulfan, and nevi Mustidine, ramustine, dacarbazine, temozolomide, hydrochloric acid mustard, dibromomannitol, cisplatin, carboplatin, oxaliplatin, nedaplatin, methotrexate, 5-fluorouracil, tega Fluoride, gemcitabine, capecitabine, fulvestrant, pemetrexed, anthracyclines, mitomycin, bleomycin, actinomycin, vinblastine, camptothecin, paclitaxel , vincristine, vinblastine, vindesine, etoposide, docetaxel, paclitaxel, albumin-bound paclitaxel, paclitaxel liposome, irinotecan, vinorelbine, mitoxantrone, vinca fluoride Ning, topotecan, leuprolide, goserelin, dutasteride, fulvestrant, ground Dexamethasone, tamoxifen, bortezomib, lenalidomide, etc., exemestane, letrozole, anastrozole.

Preferably, the targeted drug is selected from the group consisting of an EGFR inhibitor, an ALK inhibitor, a PARP inhibitor, a VEGF antibody and a VEGFR inhibitor, an mTOR inhibitor, one or more of the treatments. These targeted drugs are well known in the art, for example, the EGFR inhibitor may be selected from the group consisting of gefitinib, erlotinib, ectinib, and afatinib, cetuximab, trastuzumab. One or more; ALK inhibitors may be selected from the group consisting of crizotinib, ceritinib, axitinib, Brigatinib; VEGF antibodies are selected from bevacizumab, Brolucizumab, Vanucizumab, Navicixizumab, Ranibizumab, Conbercept One or more; the VEGFR inhibitor is selected from one or more of sunitinib, apatinib, and faritinib;

In a preferred embodiment of the present invention, the third component is selected from the group consisting of gemcitabine, cisplatin, carboplatin, paclitaxel albumin, paclitaxel liposome, paclitaxel, docetaxel, cyclophosphamide, doxorubicin, One or more of epirubicin, vincristine, tegafur, tegafur, 5-fluorouracil, and Tetrahydrouridine.

In the above preferred embodiment of the present invention, the third component may be based on a patient's body surface area, body weight, or KPS functional status scoring standard or ECOG physical status scoring standard (Zubrod-ECOG-WHO) and various tumor diagnosis and treatment guidelines. The dosages and dosing regimens recommended for different types of tumor chemotherapy regimens are selected. The albumin-paclitaxel dose of 50-500mg / m ^2, preferably 125mg / m ^2; gemcitabine, gemcitabine dose of 500-2000mg / m ^2, preferably 1000mg / m ^2; cisplatin dose of 25-200mg / m ² , preferably 75 mg/m ² .

In the present invention, the treatment period may be 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 3-4 weeks, 4 weeks, preferably 3 weeks or 3-4 weeks or 4 weeks.

In the present invention, the treatment cycle includes, but is not limited to, a chemotherapy cycle or a radiation therapy cycle or other related targeted drug therapy cycle or immunotherapy cycle.

In the present invention, a PD-1 antibody is used in combination with an epigenetic modulator for a tumor, and the order of administration of the two is administered by an epigenetic modulator before administration of the PD-1 antibody, or both. Or an epigenetic modulator is administered after administration of the PD-1 antibody; preferably, the epigenetic modulator is administered prior to administration of the PD-1 antibody.

In the present invention, the epigenetic agent and the PD-1 antibody can be used in combination for treating tumors in the same or different treatment cycles, and the epigenetic agent is administered in combination with the PD-1 antibody during the treatment of the tumor. Simultaneously or before or after, the tumor may be treated in combination according to different tumor preferred chemotherapy regimens or radiotherapy treatment regimens or targeted small molecule drug treatment regimens or immunotherapy regimens, including but not limited to cellular immunotherapy (eg CAR- T therapy, tumor vaccine, CIK therapy, etc.); in addition, the combination of an epigenetic agent with a PD-1 antibody may also be administered alone in combination with other treatment regimens.

In the present invention, the PD-1 antibody and the epigenetic regulator can be combined with different pathological types and progression stages of tumors according to various tumor diagnosis norms or guidelines, the tumors. Medical treatment norms or guidelines include, but are not limited to, NCCN (National Comprehensive Cancer Network publishes guidelines for clinical practice of various malignancies) or the guidelines for the diagnosis and treatment of malignant tumors issued by the Chinese Ministry of Health.

In the present invention, the epigenetic regulator can be used in 1-2 treatment cycles prior to conventional tumor treatment protocols including, but not limited to, chemotherapy, radiotherapy, small molecule targeted therapy, surgical resection, endoscopic treatment. Continuous administration in any one of the 1-7 day period, preferably in a period of 1-5 days, during which the PD-1 antibody is administered concurrently with the epigenetic modulator or Preferably, the PD-1 antibody can be administered on the first day, the second day, the third day, the fourth day after the end of the administration of the decitabine, before the epigenetic modulator or after the epigenetic modulator. Dosing on days, 5th, 6th, and 7th, preferably on days 3, 4, and 5; preferably, PD-1 antibodies can be administered before the start of decitabine administration. Dosing on the 1st day, the first 2 days, the first 3 days, the first 4 days, the first 5 days, the first 6 days, the first 7 days, preferably the first 3 days, the first 4 days, the first 5 days; the epigenetic regulation The agent is selected from the group consisting of decitabine and azacitidine; the dose of decitabine is preferably 10 mg or 7 mg/m ² .

The present invention relates to "combination" as a mode of administration, which means administering at least one dose of PD-1 antibody and at least one dose of an epigenetic modulator over a certain period of time, both of which exhibit pharmacology. effect. The time period may be within one administration period, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours, within 12 hours. The PD-1 antibody and epigenetic modulator can be administered simultaneously or sequentially. Such a term includes a treatment in which a PD-1 antibody and an epigenetic modulator are administered by the same route of administration or by different routes of administration. The combined modes of administration of the present invention are selected from the group consisting of simultaneous administration, independent formulation and co-administration or independently formulated and administered sequentially. In the present invention, the invention further relates to the use of the medicament, wherein the frequency of administration of the PD-1 antibody is once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks, one time Once a month, the frequency of administration of epigenetic regulators is once a day, twice a day, three times a day, once a week, once every two weeks, once every three weeks, once a month.

In a preferred embodiment, decitabine is administered at a dose of 10 mg/day for 5 days per day for a period of 21 days, and a PD-1 antibody of 4 mg/kg is administered once per week; wherein the PD-1 antibody is also A fixed dose of 200 mg or 400 mg per patient (weight greater than 80 kg); 8 dosing cycles can be administered according to this method of administration.

In a most preferred embodiment, 21 days is a dosing cycle, 10th day / 5th day of the first dosing cycle is given 10 mg / day of decitabine; on day 8 is administered PD-1 antibody 4 mg / kg; The PD-1 antibody can also be administered in a fixed dose of 200 mg or 400 mg per patient (weight greater than 80 kg). In a preferred embodiment of the invention, the PD-1 antibody is administered by injection, for example subcutaneously or intravenously, and the PD-1 antibody is formulated in an injectable form prior to injection. A particularly preferred injectable form of the PD-1 antibody is an injection or lyophilized powder comprising a PD-1 antibody, a buffer, a stabilizer, and optionally a surfactant. The buffering agent may be selected from one or more of the group consisting of acetate, citrate, succinate, and phosphate. The stabilizer may be selected from sugars or amino acids, preferably disaccharides such as sucrose, lactose, trehalose, maltose. The surfactant is selected from the group consisting of polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, preferably the polyoxyethylene sorbitan fatty acid ester is polysorbate 20, 40, 60 or 80. Most preferred is polysorbate 20. Injectable forms of the most preferred PD-1 antibodies comprise PD-1 antibody, acetate buffer, trehalose and polysorbate 20.

The combined administration routes of the present invention are selected from the group consisting of oral administration, parenteral administration, and transdermal administration, and include, but are not limited to, intravenous injection, subcutaneous injection, and intramuscular injection.

The present invention provides the above immunotherapeutic agent in combination with the above epigenetic modulator as a therapeutic preparation for treating tumors and/or enhancing T-cell activity.

In the present invention, there is provided a method of treating tumors and/or enhancing T-cell activity comprising administering to a patient an immunotherapeutic agent as described above and the above epigenetic modulator.

The present invention also provides a pharmaceutical kit, or a pharmaceutical pack comprising the aforementioned epigenetic modulator and PD-1 antibody.

The invention also provides a pharmaceutical composition comprising the aforementioned effective amount of a PD-1 antibody and an epigenetic modulator, and one or more pharmaceutically acceptable excipients, diluents or carriers.

DRAWINGS

Figure 1. Comparison of drug resistance between PD-1 antibody and decitabine and PD-1 antibody

Figure 2. Tumor regression after 4 weeks of PD-1 antibody combined with decitabine

Figure 3. PD-1 antibody combined with decitabine in the treatment of tumor burden reduction in malignant solid tumors

Figure 4. Tumor load of malignant solid tumors treated with PD-1 antibody combined with decitabine as a function of treatment time

Detailed ways

Detailed description of the invention

First, the term

In order to more easily understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "immunotherapy" refers to the use of the immune system to treat diseases, and in the present invention mainly refers to stimulating and enhancing the body's anti-tumor immune response by increasing the immunogenicity of tumor cells and sensitivity to effector cell killing, and applying The immune cells and effector molecules are infused into the host to cooperate with the body's immune system to kill tumors and inhibit tumor growth.

The invention is further described in the following examples, but these examples are not intended to limit the scope of the invention.

Example 1: Decitabine combined with PD-1 antibody in the treatment of relapsed and refractory Hodgkin's lymphoma

1. Test antibodies and compounds

The PD-1 antibody was prepared according to the method disclosed in WO2015085847, corresponding to the code number H005-1, and the sequences of the heavy and light chains thereof are SEQ ID NO: 7 and SEQ ID NO: 8 in the present invention. Batch number: P1512, 200mg / bottle, with 20mg / ml spare.

Commercially available diazepam lyophilized powder injection, 50mg/bottle, reconstituted with sterile water for injection, formulated into a solution of about 5mg/mL, and then formulated with physiological saline, 0.5% glucose solution or lactic acid green solution according to clinical requirements. A 0.1-1 mg/mL solution was used.

2. Enrolled subjects

(1) Pathological diagnosis of Hodgkin's lymphoma;

(2) Age 12-80 years old;

(3) The ECOG score is less than 2 points;

(4) Life expectancy is at least 3 months;

(5) Lymphoma subjects define at least one 1 cm measurable lesion by lymphoma response criteria;

(6) must receive at least two chemotherapy and have experienced a washout period of at least 4 weeks; subjects with autologous hematopoietic stem cell transplantation meet the conditions for more than 3 months;

(7) Must have sufficient bone marrow, liver, kidney, lung and heart function.

3, grouping

As of September 27, 2017, a total of 19 subjects were enrolled, 10 of whom were resistant to PD-1 antibody, grouped with PD-1 antibody-resistant group; 9 patients without PD-1 Antibody treatment was divided into groups without PD-1 antibody treatment; the final effective evaluation subjects were: 9 patients with PD-1 antibody treatment and 9 patients without PD-1 antibody treatment. Of the subjects in the PD-1 antibody-resistant group, 8 had previously received 3-8 Nivolumab treatments, and 1 had previously received both Nivolumab and Pembrolizumab; no PD-1 Three subjects in the antibody-treated group had previously received CAR-T treatment; of the 18 patients who were ultimately evaluated for efficacy, 2 had previously received both Nivolumab and CAR-T.

4, the method of administration

The enrolled subjects were given 10 mg/d of decitabine 10 mg/d on the 1st to 5th day of the first administration cycle (21 days); the PD-1 antibody was administered 200 mg on the 8th day (if the body weight exceeded 80 kg, 400 mg) was administered, and 8 administration cycles were cycled according to this administration method.

5, test results

Clinical studies showed (see Table 1) that no serious adverse reactions occurred during the clinical study. The most common adverse reactions were cherry hemangioma, fever, nausea, and rash, all of which were 1-2 grade adverse reactions. The total remission rate of recurrent refractory Hodgkin's lymphoma treated with ceram+PD-1 antibody was as high as 78%, and there was still an objective response rate of 56% in the PD-1 antibody-resistant group. -1/PD-L1 provides a new therapeutic approach for target-resistant patients.

Table 1. Efficacy of PD-1 antibody combined with decitabine in the treatment of relapsed and refractory tumors

Remarks: The drug-resistant group treated with PD-1 antibody was treated with PD-1 antibody for ≥ four weeks without objective response (CR+PR); disease recurrence after treatment with PD-1 antibody

At the end of the experiment, 71 patients were enrolled, one of whom was treated with PD-1 antibody alone. The specific results are as follows:

Example 2: Decitabine combined with PD-1 antibody in the treatment of relapsed and refractory malignant tumors

1. Test antibodies and compounds

See example 1

2. Enrolled subjects

(1) pathological diagnosis of malignant tumors;

(2) Age 12-80 years old;

(3) The ECOG score is less than 2 points;

(4) Life expectancy is at least 3 months;

(5) defining at least one 1 cm measurable lesion by lymphoma response criteria;

(6) must fail through first-line chemotherapy and have experienced a washout period of at least 4 weeks;

(7) Must have sufficient bone marrow, liver, kidney, lung and heart function.

3, grouping

As of October 30, 2016, a total of 11 subjects were enrolled, including 6 diffuse large B-cell lymphomas, 1 B-cell lymphoma, 1 Hodgkin's lymphoma, and 4 solid tumors ( 2 gastric cancers, 1 esophageal cancer); the final evaluable target was 9 people. Two of the diffuse large B-cell lymphoma subjects were treated with CAR-T, and one gastric cancer subject had previously received CIK.

4, the method of administration

The subjects were given 10 mg/d of decitabine 10 mg/d on the 1st to 5th day of the first administration period (21 days), and the PD-1 antibody was administered 4 mg/kg on the 8th day. The drug method is cycled for 8 dosing cycles.

5, test results

Clinical studies have shown (see Table 2) that the total remission rate of decitabine + PD-1 antibody in the treatment of relapsed and refractory malignancies was 7/9 (78%), of which 4 subjects received PD-1 antibody The evaluation of the efficacy of complete or partial remission with PD-1 antibody combined with decitabine after drug treatment progress has undoubtedly provided a new choice for the survival of such patients.

Table 2 Evaluation of the efficacy of PD-1 antibody combined with decitabine in the treatment of relapsed and refractory malignant tumors

Example 3: Decitabine combined with PD-1 antibody in the treatment of relapsed and refractory malignant tumors

1. Test antibodies and compounds

See example 1

2. Enrolled subjects

(1) pathological diagnosis of malignant tumors;

(2) Age 12-80 years old;

(3) The ECOG score is less than 2 points;

(4) Life expectancy is at least 3 months;

(5) defining at least one 1 cm measurable lesion by lymphoma response criteria;

(6) must fail through first-line chemotherapy and have experienced a washout period of at least 4 weeks;

(7) Must have sufficient bone marrow, liver, kidney, lung and heart function.

3, grouping

As of September 26, 2017, a total of 24 subjects were enrolled, including 18 Hodgkin's lymphoma, 2 B-cell non-Hodgkin's lymphoma, and 4 solid tumors (2 colorectal cancer, 1 kidney cancer, 1 breast cancer); the final evaluable target was 23 people. Eight of the 18 Hodgkin's lymphoma subjects had previously received 3-13 Nivolumab or Pembrolizumab treatments, 5 had previously received CAR-T treatment, and 2 had B-cell non-Hodgkin's lymphoma subjects. He had previously received 13 treatments for Nivolumab, and one had previously received CAR-T treatment; 2 of the 4 solid tumor subjects had previously received 2 or 10 Nivolumab treatments, and 1 had previously received 4 CIK treatments.

4, the method of administration

The subjects were given 10 mg/d of decitabine 10 mg/d on the 1st to 5th day of the first administration period (21 days), and the PD-1 antibody was administered 4 mg/kg on the 8th day. The drug method is cycled for 8 dosing cycles.

5, test results

Clinical studies have shown (see Figure 3, Figure 4), according to the immune-related response criteria (irRC standard) decitabine + PD-1 antibody in the treatment of recurrent refractory malignant tumors The rate was 10/23 (44%), of which 5 subjects had complete remission (CR, tumor burden decreased by more than 100% compared to baseline), and 5 patients had partial remission (PR, compared to baseline levels) Tumor burden decreased by more than 50%), 10 subjects had stable disease (SD, tumor load decreased by less than 25% compared with baseline, but increased by no more than 25%), and 2 subjects had disease progression (PD, with baseline) Levels increased by more than 25% compared to tumor burden). According to the irRC criteria, even if the patient's efficacy is evaluated as SD, it is actually considered to be also a clinically beneficial population. Therefore, the control rate (DCR) of relapsed and refractory malignant tumors treated with decitabine + PD-1 antibody is as high as 21/23 (91 %). By the embodiment of the present invention, the treatment of relapsed and refractory malignant tumor with decitabine + PD-1 antibody is undoubtedly prolonging the patient who is tolerated or relapsed by PD-1 antibody treatment and tolerated or relapsed by CAR-T treatment or CIK treatment. Survival provides an important treatment path.

Claims (17)

1. Use of an immunotherapeutic agent and an epigenetic modulator in combination for the preparation of a medicament for treating tumors and/or enhancing T-cell activity, characterized in that the immunotherapeutic agent is selected from the group consisting of PD-1 antibodies, the PD-1 antibody The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the heavy chain variable region of the PD-1 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
2. The use according to claim 1, wherein the PD-1 antibody is a humanized antibody.
3. The use according to claim 2, wherein the light chain variable region sequence of the anti-PD-1 humanized antibody is the sequence shown in SEQ ID NO: 10 or a variant thereof, preferably in the variant The light chain variable region has an amino acid change of 0-10, more preferably an amino acid change of A43S; the heavy chain variable region sequence is the sequence set forth in SEQ ID NO: 9 or a variant thereof, preferably the heavy one The chain variable region has an amino acid change of 0-10, more preferably an amino acid change of G44R.
4. The use according to any one of claims 1 to 3, wherein the humanized antibody light chain sequence is the sequence shown in SEQ ID NO: 8, and the heavy chain sequence is as SEQ ID NO: 7. The sequence shown.
5. The use according to any one of claims 1 to 4, wherein the epigenetic regulator is selected from the group consisting of a demethylating agent, a DNA methyltransferase (DNMT) inhibitor, and a histone deacetylase. One or more of (HDAC) inhibitors.
6. The use according to any one of claims 1 to 5, wherein the epigenetic regulator is selected from the group consisting of azacitidine, decitabine, Fludarabine, Guadecitabine, Zebularine, NPEOC-DAC, CP-4200 , Vorinostat, Romidepsin, Panobinostat, CI-994, 5,6-dihydro-5-azacytidine, 5-fluoro-2'-deoxycytidine, RX-3117, epigallocatechin gallate (EGCG) ), genistein, curcumin, oligonucleotides, preferably from decitabine, azacitidine, Guadecitabine, Romidepsin, Fludarabine.
7. The use according to any one of claims 1 to 6, wherein the tumor is selected from the group consisting of a malignant tumor, a benign tumor; the malignant tumor is selected from the group consisting of a malignant epithelial tumor, a sarcoma, a myeloma, a leukemia, a lymphoma, and a melanin. Tumor, head and neck tumors, brain tumors, mixed tumors, childhood malignant tumors.
8. The use according to any one of claims 1 to 7, wherein the tumor is selected from the group consisting of a solid tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostate cancer, pancreatic cancer, lung cancer, esophageal cancer, Liver cancer, cholangiocarcinoma, breast cancer, colorectal cancer, gastric cancer, kidney cancer, acute myeloid lymphocytic leukemia, myelodysplastic syndrome, glioma.
9. Use according to any of claims 1-8, characterized in that the tumor is mediated and/or expresses PD-L1 by PD-1.
10. The use according to any one of claims 1 to 9, wherein the tumor is selected from the group consisting of a mid-stage tumor, a relapsed refractory tumor, a chemotherapy-resistant drug failure and/or a recurrence tumor, a radiotherapy failure, and/or Recurrent tumors, targeted drug therapy failures and/or recurrence of tumors, failure of immunotherapy, and/or recurrence of tumors.
11. The use according to any one of claims 1 to 10, wherein the tumor exhibits resistance or resistance to an immunotherapeutic agent or immunotherapy.
12. The use according to any one of claims 1 to 11, wherein the tumor exhibits resistance or resistance to an immunotherapeutic agent targeting PD-1 or PD-L1 or CTLA-4.
13. The use according to any one of claims 1 to 12, characterized in that the PD-1 antibody dose is selected from the group consisting of 1-10 mg/kg, preferably 3 mg/kg, 4 mg/kg, 5 mg/kg.
14. The use according to any one of claims 1 to 13, characterized in that the PD-1 antibody dose is selected from 50-600 mg, preferably 200 mg, 400 mg each time, once every three weeks.
15. The use according to any one of claims 1 to 14, wherein the epigenetic modulator is administered at a dose of 10 mg/day, wherein each day is administered for 5 days.
16. A pharmaceutical packaging comprising the epigenetic regulator according to any one of claims 1 to 15 and a PD-1 antibody.
17. A pharmaceutical composition comprising an effective amount of a PD-1 antibody and an epigenetic modulator according to any one of claims 1-15, and one or more pharmaceutically acceptable excipients , diluent or carrier.

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