CN110234342A - Combination treatment - Google Patents

Abstract

The present invention provides the combination of II type protein arginine transmethylase (II type PRMT) inhibitor and immunomodulator, wherein the immunomodulator is anti-OX40 antibody or its antigen-binding fragment.The present invention also provides the methods of the treating cancer in the people of needs, the method includes administering to the human the combination of II type PRMT inhibitor and immunomodulator, wherein the immunomodulator is anti-OX40 antibody or its antigen-binding fragment, and it is following at least one: pharmaceutically acceptable carrier and pharmaceutically acceptable diluent, to treat the cancer of people.The present invention further provides pharmaceutical compositions, it includes the II type PRMT inhibitor of therapeutically effective amount and the second pharmaceutical compositions, second pharmaceutical composition includes the immunomodulator of therapeutically effective amount, wherein the immunomodulator is anti-OX40 antibody or its antigen-binding fragment.

Description

Combination treatment

Technical field

The present invention relates to the method for the treatment of mammalian cancer and it is related to can be used for the combination of this treatment.In particular, this Invention is related to II type protein arginine transmethylase (II type PRMT) inhibitor and immunomodulator, such as anti-OX40 antibody Combination.

Background technique

Effectively treatment hyperproliferative disease, including cancer, are the persistent goals of oncology.In general, cancer is by controlling The imbalance of the normal processes of cell division processed, differentiation and apoptotic cell death causes, and it is characterized in that malignant cell increasing It grows, the potentiality with the transfer of indeterminate growth, differentially expanding and whole body.The imbalance of normal processes includes the different of signal transduction pathway The normal and response to the factor different from the factor found in normal cell.

Arginine methylation is the important posttranslational modification to the protein for participating in various kinds of cell process, such as gene tune Control, RNA processing, DNA damage response and signal transduction.Containing methylating, arginic protein is present in nucleus and cytoplasm group In point, this shows that the enzyme that catalysis methyl is transferred on arginine exists in these subcellular lacunas and (summarizes in Yang, Y.& Bedford,M.T.Protein arginine methyltransferases and cancer.Nat Rev Cancer13, 37-50,doi:10.1038/nrc3409(2013)；Lee,Y.H.&Stallcup,M.R.Minireview:protein arginine methylation of nonhistone proteins in transcriptional regulation.Mol Endocrinol23,425-433,doi:10.1210/me.2008-0380(2009)).In mammalian cells, it methylates Arginine exists with three kinds of principal modes: ω-N^GMonomethyl-arginine (MMA), ω-N^G,N^GAsymmetric dimethylarginine (ADMA) or ω-N^G,N’^GSymmetrical diethylarginine (SDMA).Every kind of methylation state can influence in different ways Protein-protein interaction assigns different functional consequences (Yang, Y.& it is therefore possible to the bioactivity for substrate Bedford,M.T.Protein arginine methyltransferases and cancer.Nat Rev Cancer13, 37-50,doi:10.1038/nrc3409(2013))。

Arginine methylation mainly passes through protein arginine in the case where being rich in glycine, arginine (GAR) motif The activity of transmethylase (PRMT) family occurs, and the protein arginine transmethylase is by methyl from S- adenosine-L- first Methyllanthionine (SAM) is transferred to substrate arginine side chain, generates S- adenosyl-homocysteine (SAH) and methylation arginine.The egg White matter family by 10 member compositions, wherein 9 have been demonstrated with enzymatic activity (Bedford, M.T.&Clarke, S.G.Protein arginine methylation in mammals:who,what,and why.Mol Cell33,1-13, doi:10.1016/j.molcel.2008.12.013(2009)).According to the product of enzymatic reaction, PRMT family is divided into four kinds of Asias Type (I-IV type).IV type enzyme methylate internal guanidine radicals nitrogen and only in yeast description (Fisk, J.C.&Read, L.K.Protein arginine methylation in parasitic protozoa.Eukaryot Cell10,1013- 1022,doi:10.1128/EC.05103-11(2011))；I-III type enzyme generates monomethyl-essence by single methylation event Propylhomoserin (MMA, Rme1).MMA intermediate is considered as relatively low-abundance intermediate, however, the main type III activity of PRMT7 Selection substrate can keep monomethylation, and I type and II type enzyme are catalyzed respectively from MMA to asymmetric dimethylarginine The progress of (ADMA, Rme2a) or symmetrical diethylarginine (SDMA, Rme2s).II type PRMT includes PRMT5 and PRMT9, However, PRMT5 is responsible for forming the Major Enzymes of symmetric dimethyl.I type enzyme include PRMT1, PRMT3, PRMT4, PRMT6 and PRMT8.PRMT1, PRMT3, PRMT4 and PRMT6 are generally expressed, and PRMT8 is limited primarily to brain and (summarizes in Bedford, M.T.& Clarke,S.G.Protein arginine methylation in mammals:who,what,and why.Mol Cell33,1-13,doi:10.1016/j.molcel.2008.12.013(2009))。

PRMT5 works in the compound of several types in cytoplasm and nucleus, and the combination spouse of PRMT5 Body is necessary to identifying and selecting property of substrate.Transmethylase complex (methylosome) albumen 50 (MEP50) is The known co-factor of PRMT5 is (Ho MC et al. necessary to PRMT5 combination and the activity to histone and other substrates Structure of the arginine methyltransferase PRMT5-MEP50reveals a mechanism for substrate specificity.PLoS One.2013；8(2)).

PRMT5 symmetrically methylates the arginine in multiple proteins, preferably rich in arginine and glycine residue (Karkhanis V et al. Versatility of PRMT5-induced methylation in growth in region control and development.Trends Biochem Sci.2011Dec；36(12):633-41).PRMT5 methylation (the Karkhanis V et al. such as arginine in various cell proteins, including splicing factor, histone, transcription factor, kinases Versatility of PRMT5-induced methylation in growth control and development.Trends Biochem Sci.2011Dec；36(12):633-41).The methyl of multiple components of spliceosome Change is the critical event in spliceosome assembling, and is caused by striking low or gene knockout decrease PRMT5 activity to cell montage Destruction (BezziM et al. Regulation of constitutive and alternative splicing by PRMT5reveals a role for Mdm4pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev.2013Sep 1；27(17):1903-16).PRMT5 also methylates histone arginine residues (H3R8, H2AR3 and H4R3), these histidine tags are related to the Transcriptional Silencing of tumor suppressor gene, such as RB and ST7 (Wang L,Pal S,Sif S.Protein arginine methyltransferase 5suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells.Mol Cell Biol.2008Oct；28(20):6262-77).In addition, the symmetric dimethylization of H2AR3 and embryo are dry In cell the silencing of differentiation gene it is related (Tee WW, Pardo M, Theunissen TW, Yu L, Choudhary JS, Hajkova P,Surani MA.Prmt5is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency.Genes Dev.2010Dec 15；24(24): 2772-7).The methylation that PRMT5 also passes through EGFR and PI3K work in cell signalling (Hsu JM, Chen CT, Chou CK,Kuo HP,Li LY,Lin CY,Lee HJ,Wang YN,Liu M,Liao HW,Shi B,Lai CC,Bedford MT,Tsai CH,Hung MC.Crosstalk between Arg 1175methylation and Tyr 1173phosphorylation negatively modulates EGFR-mediated ERK activation.Nat Cell Biol.2011Feb；13(2):174-81；Wei TY,Juan CC,Hisa JY,Su LJ,Lee YC,Chou HY, Chen JM,Wu YC,Chiu SC,Hsu CP,Liu KL,Yu CT.Protein arginine methyltransferase 5is a potential oncoprotein that upregulates G1cyclins/cyclin-dependent kinases and the phosphoinositide 3-kinase/AKT signaling cascade.Cancer Sci.2012Sep；103(9):1640-50).

More and more evidences show that PRMT5 participates in tumour.PRMT5 albumen is overexpressed in many cancer types, Including lymthoma, glioma, breast cancer and lung cancer, only PRMT5 overexpression is just enough to convert normal fibroblast (Pal S,Baiocchi RA,Byrd JC,Grever MR,Jacob ST,Sif S.Low levels of miR-92b/ 96induce PRMT5translation and H3R8/H4R3methylation in mantle cell lymphoma.EMBO J.2007Aug 8；26(15):3558-69；Ibrahim R et al. Expression of PRMT5in lung adenocarcinoma and its significance in epithelial-mesenchymal transition.HumPathol.2014Jul；45(7):1397-405；Powers MA et al. Protein arginine methyltransferase 5accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4.Cancer Res.2011Aug 15；71(16):5579- 87；Yan F et al. Genetic validation of the protein arginine methyltransferase PRMT5as a candidate therapeutic target in glioblastoma.Cancer Res.2014Mar15； 74(6):1752-65).The reduction of PRMT5 struck low pass and often result in cell growth and survival in cancerous cell line.In breast cancer, The overall bad survival of high PRMT5 expression and high PDCD4 (apoptosis 4) horizontal prediction together (Powers MA, et al. Cancer Res.On August 15th, 2011；71 (16): 5579-87).PRMT5 methylation PDCD4 changes tumour correlation function. Coexpression of the PRMT5 and PDCD4 in the model in situ of breast cancer promotes tumour growth.The height of PRMT5 in glioma Expression is related to high tumor grade and overall poor survival rate, and PRMT5 strikes in the low model of spongioblastoma in situ Survival benefit (Yan F et al. Genetic validation of the protein arginine is provided methyltransferase PRMT5as a candidate therapeutic target in glioblastoma.Cancer Res.2014Mar 15；74(6):1752-65).Increased PRMT5 expression and activity facilitate Several tumor suppressor genes in silencing glioma cell line.

The strongest mechanism connection described between PRMT5 and cancer at present is lymphoma mantle cell (MCL).PRMT5 warp It is often overexpressed in MCL, and is highly expressed in core compartment, wherein it increases histone methylated horizontal and silencing tumour suppression The subset of gene processed.Nearest research disclose miRNA raised in MCL PRMT5 expression in effect.It is expected that more than 50 kinds MiRNA is annealed to the 3' non-translational region of PRMT5mRNA.It is reported that the PRMT5 in miR-92b and miR-96 level and MCL is horizontal It is negatively correlated, and the downward of these miRNA leads to the up-regulation of PRMT5 protein level in MCL cell.Cyclin D1 is The oncogene of transposition, related to PRMT5 in most MCL patients, and increases PRMT5 by cdk4 dependent mechanism and live Property (Aggarwal P et al. Nuclear cyclin D1/CDK4kinase regulates CUL4expression and triggers neoplastic growth via activation of the PRMT5methyltransferase.Cancer Cell.2010Oct 19；18(4):329-40).PRMT5 mediates negative regulator The inhibition of the key gene of DNA replication dna, so that cyclin D1 dependent tumors be allowed to grow.PRMT5 strikes low inhibit The conversion of cyclin D1 dependent cell, leads to death of neoplastic cells.These data highlight weight of the PRMT5 in MCL It acts on, and shows that PRMT5 inhibits to can be used as the therapeutic strategy in MCL.

In other tumor types, it has been assumed that PRMT5 is in differentiation, cell death, cell cycle progress, cell growth and increases It works in growing.Although the main mechanism that PRMT5 and tumour are connected it is unclear that, it is emerging statistics indicate that PRMT5 helps to adjust gene expression (histone methylated, transcription factor combines or promoter combination), montage change and signal Transduction.The PRMT5 methylation of transcription factor E2F 1 reduces it and inhibits cell growth and promote ability (the Zheng S of Apoptosis Et al. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1.Mol Cell.2013Oct 10；52(1):37-51).PRMT5 also methylates p53 (Jansson M Et al. Arginine methylation regulates the p53response.Nat Cell Biol.2008Dec；10 (12): 1431-9) to respond DNA damage and reduce the ability of p53 induction of cell cycle arrest, while it is thin to increase p53 dependence Born of the same parents' apoptosis.These statistics indicate that, PRMT5 inhibit can by induce p53 dependent cell apoptosis keep cell quick to DNA damage agent Sense.

In addition to directly methylating other than p53, PRMT5 also passes through montage related mechanism up-regulation p53 approach.Mouse neural progenitor cells In PRMT5 knock out and lead to the change of cell montage, the isotype including MDM4 gene converts (Bezzi M et al. Regulation of constitutive and alternative splicing by PRMT5reveals a role for Mdm4pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev.2013Sep 1；27(17):1903-16).Bezzi et al. has found that PRMT5 knocks out cell and has the long MDM4 reduced of the same race The expression (leading to functional p53 ubiquitin ligase) and the expression of the short isotype of increased MDM4 of type (lead to inactive connection Enzyme).These variations of MDM4 montage lead to the inactivation of MDM4, increase the stability of p53 albumen, and then activation p53 approach and Cell death.It is struck in PRMT5 and also observes MDM4 alternative splicing in low cancerous cell line.These are statistics indicate that PRMT5 inhibits Multiple nodes of p53 approach can be activated.

Other than the adjusting of growth of cancer cells and survival, PRMT5 further relates to epithelial-mesenchymal conversion (EMT).PRMT5 In conjunction with transcription factor SNAIL, and the crucial co-suppression factor as CAM 120/80 expression；Striking for PRMT5 low leads to E- calcium Up-regulation (Hou Z et al. The LIM protein AJUBArecruits protein arginine of mucin levels methyltransferase 5to mediate SNAIL-dependent transcriptional repression.Mol Cell Biol.2008May；28(10):3198-207).

Immunotherapy is the method for another treatment hyperproliferative disease.Enhance antitumor T cell function and induction T is thin Born of the same parents' proliferation is the strong and new method for the treatment of of cancer.There are three types of immune-oncology antibody (for example, immunological regulation currently on the market Agent).Anti- CTLA-4 (YERVOY/ her monoclonal antibody) is considered the enhancing immune response when T cell causes, and anti-PD-1 antibody (OPDIVO/ receive military monoclonal antibody and KEYTRUDA/ pyridine aldoxime methyliodide (PAM) monoclonal antibody) is considered playing a role in local tumor microenvironment, by subtracting The inhibition checkpoint in tumor specific T cells gently caused and activated.

Although there are many latest developments in terms for the treatment of of cancer, there is still a need for carry out more on the individual influenced with cancer Effective and/or enhancing treatment.

Brief description

The arginine methylation of four kinds of protein of Fig. 1: PRMT catalysis.

Fig. 2: known PRMT5 substrate.PRMT5 symmetrically methylates the arginine in multiple proteins, is preferably being rich in (Karkhanis V et al. Versatility of PRMT5-induced in the region of arginine and glycine residue methylation in growth control and development.Trends Biochem Sci.2011Dec；36 (12):633-41).These most substrates are identified by the ability of they and PRMT5 interaction.

Molecule between Fig. 3: PRMT5/MEP50 complex activity and cyclin D1 oncogene driving approach closes System.MEP50, a kind of PRMT5 are total to regulatory factor, are cdk4 substrates, and MEP50 phosphorylation increases PRMT5/MEP50 activity.Increase PRMT5 activity mediate relevant to the growth of cyclin D1 dependent tumors critical event, including CUL4 (Cullin 4) inhibit, CDT1 is overexpressed and DNA is replicated again (from Aggarwal P et al. Nuclear cyclin D1/CDK4kinase regulates CUL4expression and triggers neoplastic growth via activation of the PRMT5methyltransferase.Cancer Cell.2010Oct 19；18(4):329-40).

Fig. 4: for the compound IC of PRMT5/MEP50₅₀Value.(K in equilibrium conditions_{M is apparent}Concentration of substrate) using radiation Property measurement monitoring PRMT5/MEP50 (4nM) activity, measurement with compound C, compound F, compound B or compound E processing Afterwards³The transfer of H peptide from SAM to H4.IC is determined by the way that data are fitted to 3 parameter dosage-response equation₅₀Value.

Fig. 5: the crystal structure of compound PRMT5/MEP50, resolution ratio are with compound C and SinefunginIllustration Show that the compound is incorporated in peptide binding pocket and carries out key interactions with PRMT5 skeleton.

Fig. 6: chadogram is highlighted the transmethylase tested in selective group.For PRMT5 (10^{- 8}M), compound C show than any other tested enzyme (>10^-5M) higher effect.PRMT9 is in merely for relationship purpose It shows in family tree, is not assessed in the group.Figure comes from Richon VM. et al..

Fig. 7: the gIC of growths in 6 days/death measurement compound C in one group of cancerous cell line₅₀Value.DLBCL- diffuses Property large B cell lymphoid tumor, GBM- spongioblastoma, MCL- lymphoma mantle cell, MM- Huppert's disease

Fig. 8: growths in 6 days/death measurement compound C gIC in one group of cancerous cell line₁₀₀(black square) and Y_min- T0 (item) value (top concentration used in the measurement is 30 μM).DLBCL diffusivity large B cell lymphoid tumor, GBM- plastic Cell plastid tumor, MCL- lymphoma mantle cell, MM Huppert's disease

Fig. 9: the gIC of the compound B in the cancerous cell line (n=240) from 10 days 2D growth measurements₅₀Value.ALL- is acute Lymphoblastic leukemia, AML- acute myelogenous leukemia, CML- chronic myelogenous leukemia, DLBCL- diffuse the big B- of type Cell lymphoma, HL- Hodgkin lymphoma, HN- incidence cancer, MM- Huppert's disease, NHL- non-Hodgkin lymphoma, NSCLC- non-small cell lung cancer, PEL- lymphoma primary effusion, SCLC- Small Cell Lung Cancer, TCL-T- cell lymphoma.

Figure 10: the compound E of the 8-13 days colony formation assays carried out derived from patient and in cell lines Tumor model Opposite IC₅₀Value.

Figure 11: the compound C inhibition to SDMA.(A) the 3rd day representative SDMA dose-response curve (is normalized to Total SDMA of GAPDH) (above) and the 1st day and the 3rd day Z138 cell IC₅₀It is worth (following figure).(B) in one group of MCL cell line SDMA IC₅₀It is worth (the 4th day).

Figure 12: with the changes in gene expression in the lymphoma cell line of PRMT5 inhibitor processing.A. compound B (0.1 He 0.5 μM) differential expression (DE) gene quantifies in lymphoma cell line afterwards for processing (the 2nd day and the 4th day).B. across lymphoma cell The overlapping of the DE gene of system.

Figure 13: pass through the compound C gene expression EC50 value in the group of 11 genes of RNA sequencing identification.CDKN1A's Representative dosage-response curve (the 2nd day and the 4th day, left figure) and genome EC50 summary sheet (right figure, the 4th day).

Figure 14: compound B weakens the montage of introne subset in lymphoma cell line.A. the mechanism of cell montage is adjusted (from Bezzi M. et al.).B. the analysis that introne is expressed in the lymphoma cell line handled with 0.1 or 0.5 μM of compound B.

The isotype conversion of gene subset in Figure 15: compound B induction lymphoma cell line.A. compound B (0.1 He is used 0.5 μM) isotype conversion quantifies in 2 and 4 days 4 kinds of lymphoma cell lines of processing.B.4 isotype in lymphoma cell line is planted The overlapping of conversion.C. list of genes (the weight of 4 kinds of cell line of alternative splicing is undergone in all 4 kinds of lymphoma cell lines It is folded).

Figure 16: with compound C handle MCL cell line in MDM4 alternative splicing and p53 activate.A. with 10 Hes 200nM compound C or 5 μM of Nutlin-3 handles the MDM4 isotype in 2 days and 3 days 4 kinds of lymphoma mantle cell cell line groups Expression analysis (MDM4-FL- long；MDM4-S- is short).B. 3 days MCL are handled with 10 and 200nM compound C or 5 μM of Nutlin-3 The Western analysis of p53 and p21 expression in cell line.

The dose-dependant of MDM4RNA (A) montage and SDMA/p53/p21 level in Figure 17: compound C induction Z138 cell Property variation (B).

The activity of Figure 18: PRMT5 inhibitor and Yi Lu for Buddhist nun as single medicament and combination in MCL cell line.A.6 day Compound C and Yi Lu replace the gIC of Buddhist nun in growth/death CTG measurement₅₀Value.B. compound B and Yi Lu is thin in REC1 for the combination of Buddhist nun Representative growth curve (the 6th day, 1:1 ratio) in born of the same parents.C. with the chemical combination of specified ratio in growth in 6 days/death CTG measurement Object B: the combinatorial index (CI) of Buddhist nun is replaced according to Shandong.

Compound C effect and PD in Figure 19: Z138 heteroplastic transplantation model.Compound C is in Z138 heteroplastic transplantation model 21 days efficacy studies.B. quantify SDMA from the tumour of harvest (3 hours after last time administration) at the end of efficacy study Western data.

Compound C effect and PD in Figure 20: Maver-1 heteroplastic transplantation model.A. in Maver-1 heteroplastic transplantation model 21 days efficacy studies of compound C.B. at the end of efficacy study (last time be administered after 3 hours) from the tumour of harvest Quantify SDMA western data.

Figure 21: the compound B in one group of breast cancer cell line from growth 2D measurement in 7 days grows IC₅₀It is worth (TNBC- tri- Negative breast cancer, HER2-Her2 is positive, HR- hormone receptor positive).

Figure 22: PRMT5 candidate compounds C and PRMT5 tool molecule compound B are used, in mammary gland and MCL cell line Growth in 10-12 days/death measurement Ymin-T0 value.

Figure 23: the propidium iodide of breast cancer cell line in different time periods is handled with 30,200 and 1000nM compound C Facs analysis (the 2nd day, the 7th day and the 10th day, biology n=2, error bars represent standard deviation).

Figure 24: the time course that SDMA inhibits after 1 μM of compound B processing in one group of breast cancer cell line.Cell is used DMSO or 1 μM of compound B processing specified period, and cell is analyzed with SDMA and actin antibodies by Western blotting Lysate.The last one swimming lane of each trace is the 1/2 of DMSO control.

Compound C effect (left side) and PK/PD (right side) in Figure 25: MDA-MB-468 heteroplastic transplantation model.

Figure 26: PRMT5 candidate, compound C and PRMT5 tool molecule compound B (Y are used_min- T0) in GBM cell line In growths in 14 days/death CTG measurement.

Figure 27: compound B (1 μM) reduction SDMA is horizontal (B), induces the alternative splicing (A) of MDM4, and activate GBM and P53 (B) in lymphoma cell line.

Figure 28: it is combined with immunotherapy.Single medicament and combined average viability in A20 tumor model.

Figure 29: it is combined with immunotherapy.Single medicine and combined average viability in CT26 tumor model.

Figure 30: 106-222, humanization 106-222 (Hu106) and human receptor X61012 (GenBank accession number) VH sequence Amino acid sequence comparison.

Figure 31: 106-222, humanization 106-222 (Hu106) and human receptor AJ388641 (GenBank accession number) VL sequence The comparison of the amino acid sequence of column.

The nucleotide sequence of Figure 32: Hu106VH gene and the amino acid sequence of derivation, flank be SpeI and The site HindIII.

The nucleotide sequence of Figure 33: Hu106-222VL gene and the amino acid sequence of derivation, flank be NheI and The site EcoRI.

Figure 34: 119-122, humanization 119-122 (Hu119) and human receptor Z14189 (GenBank accession number) VH sequence Amino acid sequence comparison.

Figure 35: 119-122, humanization 119-122 (Hu119) and human receptor M29469 (GenBank accession number) VL sequence Amino acid sequence comparison.

The nucleotide sequence of Figure 36: Hu119VH gene and the amino acid sequence of derivation, flank be SpeI and The site HindIII.

The nucleotide sequence of Figure 37: Hu119VL gene and the amino acid sequence of derivation, flank are NheI and EcoRI Site.

The nucleotide sequence of Figure 38: mouse 119-43-1VH cDNA and the amino acid sequence of derivation.

The nucleotide sequence of Figure 39: mouse 119-43-1VL cDNA and the amino acid sequence of derivation.

Figure 40: the nucleotide sequence of the 119-43-1VH gene of design and the amino acid sequence of derivation, flank are The site SpeI and HindIII.

Figure 41: the nucleotide sequence of the 119-43-1VL gene of design and the amino acid sequence of derivation, flank are The site NheI and EcoRI.

Summary of the invention

In one embodiment, the present invention provides II type protein arginine transmethylase (II type PRMT) inhibitor With the combination of immunomodulator, wherein the immunomodulator be anti-OX40 antibody or its antigen-binding fragment.

In one embodiment, the method for the treating cancer in the people of needs is provided, the method includes administering to the human The combination of II type protein arginine transmethylase (II type PRMT) inhibitor and immunomodulator, and following at least one Kind: pharmaceutically acceptable carrier and pharmaceutically acceptable diluent, so that the cancer of people is treated, wherein the immunological regulation Agent is anti-OX40 antibody or its antigen-binding fragment.

In one embodiment, the present invention provides pharmaceutical composition, and it includes the II type protein essences of therapeutically effective amount Second pharmaceutical composition of propylhomoserin transmethylase (II type PRMT) inhibitor and the immunomodulator comprising therapeutically effective amount, Described in immunomodulator be anti-OX40 antibody or its antigen-binding fragment.

In one embodiment, the method for the treating cancer in the people of needs is provided, the method includes administering to the human Therapeutically effective amount includes I type protein arginine transmethylase (II type PRMT) inhibitor and the medicine comprising immunomodulator The pharmaceutical composition of compositions, wherein the immunomodulator is anti-OX40 antibody or its antigen-binding fragment, to treat The cancer of the people.

In one embodiment, the present invention provides II type protein arginine transmethylase (II type PRMT) inhibitor The purposes of combination in medicine preparation with immunomodulator, wherein the immunomodulator is anti-OX40 antibody or its antigen knot Close segment.

In one embodiment, the present invention provides II type protein arginine transmethylase (II type PRMT) inhibitor The purposes for the treatment of cancer is used for the combination of immunomodulator, wherein the immunomodulator is anti-OX40 antibody or its antigen knot Close segment.

Detailed description of the invention

Definition

As used herein, " II type protein arginine methyltransferase inhibitors " or " II type PRMT inhibitor " refer to suppression The reagent of Protein Arginine Methyltransferase 5 (PRMT5) processed and/or protein arginine transmethylase 9 (PRMT9).? In some embodiments, the II type PRMT inhibitor is small molecule compound.In some embodiments, the II type PRMT Inhibitor selective depression Protein Arginine Methyltransferase 5 (PRMT5) and/or protein arginine transmethylase 9 (PRMT9).In some embodiments, the II type PRMT inhibitor is the inhibitor of PRMT5.In some embodiments, The II type PRMT inhibitor is the selective depressant of PRMT5.

In view of them in the effect in a variety of bioprocess that adjusts, arginine methyltransferase is the attractive of adjusting Target.It has been found that compound as described herein and its pharmaceutically acceptable salt and composition are shifted as arginine methyl The inhibitor of enzyme is effective.

The definition of specific functional group and the technical terms of chemistry is described in further detail below.Chemical element is according to Handbook of Chemistry and Physics, the CAS version element periodic table of page is defined in 75 editions, and specific functional group on the whole Also the justice depending on wherein described.In addition, in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999；Smith and March, March ' s Advanced Organic Chemistry, 5th edition, John Wiley&Sons, Inc., New York, 2001；Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989；And Carruthers, Some Modern Methods of Organic Synthesis, the 3rd edition, Cambridge University Press, Cambridge, 1987 In describe the rule and specific functional group and reactivity of organic chemistry.

Compound as described herein be may include one or more asymmetric centers, and therefore can be deposited in the form of different isomer Such as enantiomter and/or diastereoisomer.For example, compound as described herein can be single enantiomter, non-right The form of isomers or geometric isomer is reflected, or can be the form of stereoisomer mixture, including racemic mixture and richness Mixture containing one or more stereoisomers.It can be by methods known to those skilled in the art by isomers from mixture Middle separation, formation and crystallization including chiral high performance liquid chromatography (HPLC) and chiral salt；Or asymmetric syntheses can be passed through Prepare preferred isomers.For example, see: Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981)；Wilen et al., Tetrahedron 33:2725 (1977)；Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962)；And Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (E.L.Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, IN 1972).The invention also includes the compound herein as single isomers, It is substantially free of other isomers, or includes the compound as different isomer mixture.

It should be understood that the compound of the present invention can be portrayed as different tautomers.It is also understood that when compound has When tautomeric form, all tautomeric forms are included within the scope of the invention, and any compound described herein Name be not excluded for any tautomeric form.

Unless otherwise stated, structure described herein is also implied that including only former in one or more isotope enrichments Different compound in terms of the presence of son.For example, the compound with structure of the invention, the difference is that hydrogen is substituted with deuterium or tritium, With¹⁸F substitution¹⁹F, or with being rich in¹³C- or¹⁴The carbon of C- substitutes carbon, is within.These compounds can be used as example Such as the analysis tool or probe in bioassay.

Term " aliphatic group ", as described herein, including saturation and unsaturated, non-aromatic, straight chain are (that is, unbranched ), branch, acyclic and cyclic (that is, carbocyclic ring) hydrocarbon.In some embodiments, aliphatic group is optionally substituted with one Or multiple functional groups.It will be understood by those skilled in the art that " aliphatic group " is intended to include alkyl, alkenyl, alkynyl, naphthenic base herein And cyclo-alkenyl moieties.

When listing a series of values, it is intended to comprising each value and subrange within the scope of this.Such as " C_1-6Alkyl " expected packet It includes, C₁；C₂、C₃、C₄、C₅、C₆、C_1-6、C_1-5、C_1-4、C_1-3、C_1-2、C_2-6、C_2-5、C_2-4、C_2-3、C_3-6、C_3-5、C_3-4、C_4-6、C_4-5With C_5-6Alkyl.

" base (Radical) " refers to the tie point in special groups.Base includes the biradical in special groups.

" alkyl " refers to linear chain or branched chain saturated hydrocarbyl the group (" C with 1 to 20 carbon atom_1–20Alkyl ").One In a little embodiments, alkyl has 1 to 10 carbon atom (" C_1-10Alkyl ").In some embodiments, alkyl has 1 to 9 A carbon atom (" C_1-9Alkyl ").In some embodiments, alkyl has 1 to 8 carbon atom (" C_1-8Alkyl ").In some realities It applies in scheme, alkyl has 1 to 7 carbon atom (" C_1-7Alkyl ").In some embodiments, alkyl has 1 to 6 carbon original Son (" C_1-6Alkyl ").In some embodiments, alkyl has 1 to 5 carbon atom (" C_1-5Alkyl ").In some embodiments In, alkyl has 1 to 4 carbon atom (" C_1-4Alkyl ").In some embodiments, alkyl has 1 to 3 carbon atom (" C_1-3 Alkyl ").In some embodiments, alkyl has 1 to 2 carbon atom (" C_1-2Alkyl ").In some embodiments, alkyl With 1 carbon atom (" C₁Alkyl ").In some embodiments, alkyl has 2 to 6 carbon atom (" C_2-6Alkyl ").C_1-6 The example of alkyl group includes methyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), normal-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄), isobutyl group (C₄), n-pentyl (C₅), 3- amyl (C₅), amyl (C₅), neopentyl (C₅), 3- methyl -2- Butyl (C₅), tertiary pentyl (C₅) and n-hexyl (C₆).Other examples of alkyl include n-heptyl (C₇), n-octyl (C₈) etc..At certain In a little embodiments, each case of alkyl, which independently is, optionally to be replaced, for example, unsubstituted (" unsubstituted alkyl ") or Substitution has one or more substituent groups (" substituted alkyl ").In certain embodiments, alkyl is unsubstituted C_1-10Alkyl (such as-CH₃).In certain embodiments, alkyl is the C replaced_1-10Alkyl.

In some embodiments, alkyl substitution has one or more halogens." whole haloalkyl " is that wherein all hydrogen are former Son is independently by halogen, for example, the substituted alkyl defined herein that fluorine, bromine, chlorine or iodo replace.In some embodiments, Moieties have 1 to 8 carbon atom (" C_1-8Whole haloalkyl ").In some embodiments, moieties have 1 to 6 Carbon atom (" C_1-6Whole haloalkyl ").In some embodiments, moieties have 1 to 4 carbon atom (" C_1-4Perhalogeno Alkyl ").In some embodiments, moieties have 1 to 3 carbon atom (" C_1-3Whole haloalkyl ").In some implementations In scheme, moieties have 1 to 2 carbon atom (" C_1-2Whole haloalkyl ").In some embodiments, all hydrogen all by Fluorine atom replaces.In some embodiments, all hydrogen atoms are all replaced by chloro.The example of perhaloalkyl groups include- CF₃、-CF₂CF₃、-CF₂CF₂CF₃、-CCl₃、-CFCl₂、-CF₂Cl etc..

" alkenyl " refers to 2 to 20 carbon atoms and one or more carbon-to-carbon double bonds (for example, 1,2,3 or 4 double Key), and optionally one or more three keys (for example, 1,2,3 or 4 three key) linear chain or branched chain hydrocarbyl group (" C_2-20Alkene Base ").In certain embodiments, alkenyl does not include three keys.In some embodiments, alkenyl have 2 to 10 carbon atoms (" C_2-10Alkenyl ").In some embodiments, alkenyl has 2 to 9 carbon atom (" C_2-9Alkenyl ").In some embodiments, Alkenyl has 2 to 8 carbon atom (" C_2-8Alkenyl ").In some embodiments, alkenyl has 2 to 7 carbon atom (" C_2-7Alkene Base ") in some embodiments, alkenyl has 2 to 6 carbon atom (" C_2-6Alkenyl ").In some embodiments, alkenyl has There are 2 to 5 carbon atom (" C_2-5Alkenyl ").In some embodiments, alkenyl has 2 to 4 carbon atom (" C_2-4Alkenyl ").? In some embodiments, alkenyl has 2 to 3 carbon atom (" C_2-3Alkenyl ").In some embodiments, alkenyl has 2 carbon Atom (" C₂Alkenyl ").One or more carbon-to-carbon double bonds can be internal (such as in 2- cyclobutenyl) or end (such as in 1- fourth In alkenyl).C_2-4The example of alkenyl group includes vinyl (C₂), 1- acrylic (C₃), 2- acrylic (C₃), 1- cyclobutenyl (C₄), 2- cyclobutenyl (C₄), butadienyl (C₄) etc..C_2-6The example of alkenyl group includes above-mentioned C_2-4Alkenyl and pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), etc..Other examples of alkenyl include heptenyl (C₇), octenyl (C₈), sarohornene Base (C₈), etc..In certain embodiments, each case of alkenyl, which independently is, optionally replaces, for example, it is unsubstituted (" not Substituted alkenyl ") or replace and have one or more substituent groups (" substituted alkenyl ").In certain embodiments, alkenyl is not Substituted C_2-10Alkenyl.In certain embodiments, alkenyl is the C replaced_2-10Alkenyl.

" alkynyl " refers to 2 to 20 carbon atoms and one or more carbon-carbon triple bonds (for example, 1,2,3 or 4 three Key), and optionally one or more double bonds (for example, 1,2,3 or 4 double bond) linear chain or branched chain hydrocarbyl group (" C_2-20Alkynes Base ").In certain embodiments, alkynyl does not include double bond.In some embodiments, alkynyl have 2 to 10 carbon atoms (" C_2-10Alkynyl ").In some embodiments, alkynyl has 2 to 9 carbon atom (" C_2-9Alkynyl ").In some embodiments, Alkynyl has 2 to 8 carbon atom (" C_2-8Alkynyl ").In some embodiments, alkynyl has 2 to 7 carbon atom (" C_2-7Alkynes Base ").In some embodiments, alkynyl has 2 to 6 carbon atom (" C_2-6Alkynyl ").In some embodiments, alkynyl has There are 2 to 5 carbon atom (" C_2-5Alkynyl ").In some embodiments, alkynyl has 2 to 4 carbon atom (" C_2-4Alkynyl ").? In some embodiments, alkynyl has 2 to 3 carbon atom (" C_2-3Alkynyl ").In some embodiments, alkynyl has 2 carbon Atom (" C₂Alkynyl ").One or more triple carbon-carbon bonds can be internal (such as in 2- butynyl) or end (such as in 1- fourth In alkynyl).C_2-4The example of alkynyl group includes, but are not limited to acetenyl (C₂), 1- propinyl (C₃), 2-propynyl (C₃)、1- Butynyl (C₄), 2- butynyl (C₄), etc..C_2-6The example of alkenyl group includes above-mentioned C_2-4Alkynyl and pentynyl (C₅), hexin Base (C₆), etc..Other examples of alkynyl include heptynyl (C₇), octynyl (C₈), etc..In certain embodiments, alkynyl is every Kind of situation, which independently is, optionally to be replaced, for example, unsubstituted (" unsubstituted alkynyl ") or substitution have one or more substitutions Base (" substituted alkynyl ").In certain embodiments, alkynyl is unsubstituted C_2-10Alkynyl.In certain embodiments, alkynes Base is the C replaced_2-10Alkynyl.

" condensed " or " ortho-condensed " is used interchangeably herein, and refers to two common atoms and one Two rings of key, for example,

" bridge joint " refers to following loop system, and it includes (1) bridgehead atom or atomic group, the bridgehead atom or atomic group connect Connect two or more non adjacent positions of same ring；Or (2) bridgehead atom or atomic group, the bridgehead atom or atomic group connect Two or more positions of the different rings of loop system are connect, and therefore do not form the ring of ortho-condensed, for example,

" spiral shell " or " spiro-condensed " refers to the one group of atom (connecting together with position) for the same atoms for being connected to carbocyclic ring or heterocyclic ring system, To form ring, for example,

Also contemplate the loop coil fusion at bridgehead atom.

" carbocylic radical " or " carbocyclic ring " refers to has 3 to 14 ring carbon atom (" C in aromatic cyclic hydrocarbon ring system_3-14Carbocylic radical ") With 0 heteroatomic non aromatic cyclic hydrocarbyl group.In certain embodiments, carbocylic radical refers to and has in aromatic cyclic hydrocarbon ring system 3 to 10 ring carbon atom (C_3-10Carbocylic radical ") and 0 heteroatomic non aromatic cyclic hydrocarbyl group.In some embodiments, Carbocylic radical has 3 to 8 ring carbon atom (" C_3-8Carbocylic radical ").In some embodiments, carbocylic radical has 3 to 6 ring carbon originals Son (" C_3-6Carbocylic radical ").In some embodiments, carbocylic radical has 3 to 6 ring carbon atom (" C_3-6Carbocylic radical ").Some In embodiment, carbocylic radical has 5 to 10 ring carbon atom (" C_5-10Carbocylic radical ").Exemplary C_3-6Carbocylic radical includes, but unlimited In cyclopropyl (C₃), cyclopropanyl (C₃), cyclobutyl (C₄), cyclobutane base (C₄), cyclopenta (C₅), cyclopentenyl (C₅), hexamethylene Base (C₆), cyclohexenyl group (C₆), cyclohexadienyl (C₆), etc..Exemplary C_3-8Carbocylic radical includes, but are not limited to above-mentioned C_3-6Carbocyclic ring Base and suberyl (C₇), cycloheptenyl (C₇), cycloheptadiene base (C₇), cycloheptatriene base (C₇), cyclooctyl (C₈), cyclo-octene base (C₈), bicyclic [2.2.1] heptyl (C₇), bicyclic [2.2.2] octyl (C₈), etc..Exemplary C₃-₁₀Carbocylic radical includes, but are not limited to Above-mentioned C₃_₈Carbocylic radical and cyclononyl (C₉), cyclonoene base (C₉), cyclodecyl (C₁₀), cyclodecene base (C₁₀), octahydro-lH- indenyl (C₉), decahydro naphthalene (C₁₀), spiral shell [4.5] decyl (C₁₀), etc..As described in example before this, in some embodiments, carbocylic radical base Group is monocycle (" monocyclic carbocyclyl residues ") or the system condensed for condensed, bridge joint or loop coil (such as second cycle line system (" bicyclic carbocycle Base ")), and can be saturation or can be that part is unsaturated." carbocylic radical " also includes loop system, wherein carbon as described above Ring group ring and one or more aryl or heteroaryl groups are condensed, and wherein tie point is located on carbocyclic ring basic ring, and in this case, The number of carbon continues the carbon number being designated as in carbocyclic ring system system.In certain embodiments, each case of carbocylic radical is only On the spot optionally replace, for example, unsubstituted (" unsubstituted carbocylic radical ") or substitution there are one or more substituent groups (" to take Generation carbocylic radical ").In certain embodiments, the carbocylic radical is unsubstituted C_3-10Carbocylic radical.In certain embodiments In, the carbocylic radical is the C replaced_3-10Carbocylic radical.

In some embodiments, " carbocylic radical " is the monocycle with 3 to 14 ring carbon atoms, saturated carbon ring group (" C_3-14 Naphthenic base ").In some embodiments, " carbocylic radical " be monocycle with 3 to 10 ring carbon atoms, saturated carbon ring group (" C_3-10Naphthenic base ").In some embodiments, naphthenic base has 3 to 8 ring carbon atom (" C_3-8Naphthenic base ").In some realities It applies in scheme, naphthenic base has 3 to 6 ring carbon atom (" C_3-6Naphthenic base ").In some embodiments, naphthenic base have 5 to 6 ring carbon atom (" C_5-6Naphthenic base ").In some embodiments, naphthenic base has 5 to 10 ring carbon atom (" C_5-10Cycloalkanes Base ").C_5-6The example of group of naphthene base includes cyclopenta (C₅) and cyclohexyl (C₅)。C_3-6The example of group of naphthene base includes above-mentioned C_5-6Naphthenic base and cyclopropyl (C₃) and cyclobutyl (C₄)。C_3-8The example of group of naphthene base includes above-mentioned C_3-6Naphthenic base and ring Heptyl (C₇) and cyclooctyl (C₈).In certain embodiments, each case of naphthenic base independently is unsubstituted (" unsubstituted Naphthenic base ") or replace have one or more substituent groups (" substituted naphthenic base ").In certain embodiments, naphthenic base is Unsubstituted C_3-10Naphthenic base.In certain embodiments, naphthenic base is the C replaced_3-10Naphthenic base.

" heterocycle " or " heterocycle " refers to the non-aromatic ring body of 3- to 14- member with ring carbon atom and 1 to 4 ring hetero atom The group of system, wherein each hetero atom is independently selected from nitrogen, oxygen and sulphur (" 3-14 circle heterocyclic ring base ").In certain embodiments, miscellaneous The group for referring to the 3-10 member aromatic cyclic hydrocarbon ring system with ring carbon atom and 1-4 ring hetero atom of ring group or heterocycle, wherein each miscellaneous Atom is independently selected from nitrogen, oxygen and sulphur (" 3-10 circle heterocyclic ring base ").In the heterocycle comprising one or more nitrogen-atoms, connection Point can be carbon or nitrogen-atoms, as long as chemical valence allows.Heterocycle can be monocycle (" monocyclic heterocycles base ") or condensed, bridge joint or loop coil Condensed ring system such as bicyclic system (" bicyclic heterocyclic radical "), and can be saturation or can be that part is unsaturated.Heterocycle two Ring loop system can include one or more hetero atoms in one or two ring." heterocycle " further includes loop system, wherein as above Defined heterocyclic ring, condensed with one or more carbocylic radical groups, wherein tie point is located on carbocylic radical or heterocyclic ring On, or including loop system, wherein heterocyclic ring as defined above and one or more aryl or heteroaryl groups are condensed, Middle tie point is located on heterocyclic ring, and in this case, and the number of ring members continues to be designated as in heterocyclic ring system Ring members number.In certain embodiments, each case of heterocycle, which independently is, optionally replaces, for example, unsubstituted (" unsubstituted heterocycle ") or replaces and have one or more substituent groups (" substituted heterocycle ").In certain embodiments, Heterocycle is unsubstituted 3-10 circle heterocyclic ring base.In certain embodiments, heterocycle is the 3-10 circle heterocyclic ring base replaced.

In some embodiments, heterocycle is the non-aromatic ring body of 5-10 member with ring carbon atom and 1-4 ring hetero atom System, wherein each hetero atom is independently selected from nitrogen, oxygen and sulphur (" 5-10 circle heterocyclic ring base ").In some embodiments, heterocycle is 5-8 member aromatic cyclic hydrocarbon ring system with ring carbon atom and 1-4 ring hetero atom, wherein each hetero atom is independently selected from nitrogen, oxygen and sulphur (" 5-8 circle heterocyclic ring base ").In some embodiments, heterocycle is that the 5-6 member with ring carbon atom and 1-4 ring hetero atom is non- Aromatic ring system, wherein each hetero atom is independently selected from nitrogen, oxygen and sulphur (" 5-6 circle heterocyclic ring base ").In some embodiments, described 5-6 circle heterocyclic ring base has the 1-3 ring hetero atoms for being independently selected from nitrogen, oxygen and sulphur.In some embodiments, the 5-6 member is miscellaneous Ring group has the 1-2 ring hetero atoms for being independently selected from nitrogen, oxygen and sulphur.In some embodiments, the 5-6 circle heterocyclic ring base has One ring hetero atom selected from nitrogen, oxygen and sulphur.

It include but is not limited to illustratively aziridine base, oxa- ring comprising a heteroatomic 3 circle heterocyclic ring base group Propyl, thiirane base.It include but is not limited to illustratively azetidin comprising a heteroatomic 4 circle heterocyclic ring base group Alkyl, oxetanyl and Thietane base.Include comprising a heteroatomic illustrative 5 circle heterocyclic ring base group but not It is limited to tetrahydrofuran base, dihydrofuryl, tetrahydro-thienyl, dihydrothiophene, pyrrolidinyl, pyrrolin base and pyrrole radicals- 2,5-diketone.It include but is not limited to illustratively dioxolane base, oxa- comprising two heteroatomic 5 circle heterocyclic ring base groups Tiacyclopentane base (oxasulfuranyl), dithiolane base (disulfuranyl) and oxazolidine -2- ketone.It is exemplary Comprising three heteroatomic 5 circle heterocyclic ring base groups include but is not limited to triazoline base, oxadiazoline base and Thiadiazoline base.Show Example property comprising a heteroatomic 6 circle heterocyclic ring base group include but is not limited to piperidyl, THP trtrahydropyranyl, dihydropyridine base and Thia cyclohexyl.It include but is not limited to illustratively piperazinyl, morpholinyl, two comprising two heteroatomic 6 circle heterocyclic ring base groups Thia cyclohexyl, dioxane base.It include but is not limited to illustratively three comprising three heteroatomic 6 circle heterocyclic ring base groups Piperidine base (triazinanyl).It include but is not limited to illustratively nitrogen comprising a heteroatomic 7 circle heterocyclic ring base group Trioxepane base, oxepane alkyl and thia cycloheptyl alkyl.It illustratively include a heteroatomic 8 circle heterocyclic ring Ji Jituanbao Include but be not limited to Azacyclooctane base, oxocane base and thia cyclooctane base.Illustratively and C₆Condensed 5 yuan of aryl rings Heterocyclyl groups (herein also referred to as 5,6- bicyclic heterocycles) include but is not limited to indolinyl, iso-dihydro-indole-group, dihydrobenzene And furyl, dihydrobenzo thienyl, benzoxazoles quinoline ketone group (benzoxazolinonyl) etc..It is illustratively thick with aryl rings The 6 circle heterocyclic ring base groups (herein also referred to as 6,6- bicyclic heterocycles) closed include but is not limited to tetrahydric quinoline group, tetrahydro isoquinolyl Deng.

" aryl " refer to monocycle or polycyclic (such as two rings or tricyclic) 4n+2 aromatic rings system (as have 6,10 or 14 In annular array share pi-electron) and in the aromatic rings system have 6-14 ring carbon atom and 0 heteroatomic group (“C_6–14Aryl ").In some embodiments, there are six ring carbon atom (" C for aryl group tool₆Aryl "；Such as phenyl).Some In embodiment, aryl group has ten ring carbon atom (" C₁₀Aryl "；Such as naphthalene such as 1-naphthalene and 2-naphthalenes).In some realities It applies in scheme, aryl group has 14 ring carbon atom (" C₁₄Aryl "；Such as anthryl)." aryl " further includes loop system, wherein Aryl rings, as defined above, condensed with one or more carbocylic radicals or heterocyclyl groups, wherein linking group or tie point are located at In aryl rings, and in this case, the number of carbon atom continues the carbon atom number being designated as in aryl loop system.Certain In embodiment, each case of aryl, which independently is, optionally to be replaced, for example, unsubstituted (" unsubstituted aryl ") or taking In generation, there is one or more substituent groups (" substituted aryl ").In certain embodiments, aryl is unsubstituted C_6-14Aryl.? In certain embodiments, aryl is the C replaced_6-14Aryl.

" heteroaryl " refer to 5-14 unit monocycle or polycyclic (such as two rings or tricyclic) 4n+2 aromatic rings system (as have 6 or 10 in annular array share pi-electrons) and in the aromatic rings system with ring carbon atom and 1-4 ring hetero atom base Group, wherein each hetero atom is independently selected from nitrogen, oxygen and sulphur (" 5-14 unit's heteroaryl ").In certain embodiments, heteroaryl is 5-10 unit monocycle or bicyclic 4n+2 aromatic ring system of the finger with ring carbon atom and the 1-4 ring hetero atom in aromatic ring system Group, wherein each hetero atom is independently selected from nitrogen, oxygen and sulphur (" 5-10 unit's heteroaryl ").Containing one or more nitrogen-atoms In heteroaryl groups, as long as valence allows, tie point can be carbon or nitrogen-atoms.Heteroaryl second cycle line system can be at one or two Include one or more hetero atoms in ring." heteroaryl " includes such loop system, wherein heteroaryl ring as defined above with One or more carbocylic radicals or heterocyclyl groups are condensed, and wherein tie point is located on heteroaryl ring, and in this case, ring members Number continue the number for being designated as heteroaryl ring-member ring members." heteroaryl " further includes such loop system, wherein Heteroaryl ring as defined above and one or more aryl groups are condensed, and wherein tie point is located on aryl or heteroaryl ring, And in this case, the number of ring members continues the number for being designated as condensing (aryl/hetaryl) loop system ring members.Two A ring in heteroaryl group does not contain hetero atom (such as indyl, quinolyl, carbazyl etc.), and tie point can be located at two rings One of on, such as on heteroatomic ring (such as 2-indyls) or be located at do not contain heteroatomic ring (such as 5-indyls) On.

In some embodiments, heteroaryl is with ring carbon atom and the miscellaneous original of 1-4 ring being arranged in aromatic ring system The 5-14 member aromatic ring system of son, wherein each hetero atom is independently selected from nitrogen, oxygen and sulphur (" 5-14 unit's heteroaryl ").In some implementations In scheme, heteroaryl is the 5-10 member aromatic ring system with ring carbon atom and 1-4 ring hetero atom being arranged in aromatic ring system, Wherein each hetero atom is independently selected from nitrogen, oxygen and sulphur (" 5-10 unit's heteroaryl ").In some embodiments, heteroaryl be with The 5-8 member aromatic ring system of ring carbon atom and 1-4 ring hetero atom being arranged in aromatic ring system, wherein each hetero atom independently selects From nitrogen, oxygen and sulphur (" 5-8 unit's heteroaryl ").In some embodiments, heteroaryl is with ring carbon atom and to be arranged in aromatic ring The 5-6 member aromatic ring system of 1-4 ring hetero atom in system, wherein each hetero atom is independently selected from nitrogen, oxygen and sulphur, (" 5-6 member is miscellaneous Aryl ").In some embodiments, the 5-6 unit's heteroaryl has the 1-3 ring hetero atoms for being independently selected from nitrogen, oxygen and sulphur. In some embodiments, the 5-6 unit's heteroaryl has the 1-2 ring hetero atoms for being independently selected from nitrogen, oxygen and sulphur.In some realities It applies in scheme, the 5-6 unit's heteroaryl has 1 ring hetero atom selected from nitrogen, oxygen and sulphur.In certain embodiments, heteroaryl The each case of base, which independently is, optionally to be replaced, for example, unsubstituted (" unsubstituted heteroaryl ") or substitution have one or Multiple substituent groups (" heteroaryl replaced ").In certain embodiments, heteroaryl is unsubstituted 5-14 unit's heteroaryl.At certain In a little embodiments, heteroaryl is the 5-14 unit's heteroaryl replaced.

Include, but are not limited to pyrrole radicals, furyl and thienyl comprising a heteroatomic exemplary 5- unit's heteroaryl. Include, but are not limited to imidazole radicals, pyrazolyl, oxazolyl, isoxazolyl, thiophene comprising 2 heteroatomic exemplary 5- unit's heteroaryls Oxazolyl and isothiazolyl.Comprising 3 heteroatomic exemplary 5- unit's heteroaryls include, but are not limited to triazolyl, oxadiazoles base and Thiadiazolyl group.Include, but are not limited to tetrazole radical comprising 4 heteroatomic exemplary 5- unit's heteroaryls.It is heteroatomic comprising 1 Exemplary 6- unit's heteroaryl includes, but are not limited to pyridyl group.Include comprising 2 heteroatomic exemplary 6- unit's heteroaryls, but not It is limited to, pyridazinyl, pyrimidine radicals and pyrazinyl.It is respectively included comprising 3 or 4 heteroatomic exemplary 6- unit's heteroaryls, but unlimited In triazine radical and tetrazine base.Include, but are not limited to azepine cycloheptatriene comprising 1 heteroatomic exemplary 7- unit's heteroaryl Base, oxepin base and thia cycloheptatriene base.Exemplary 6,6- bicyclic heteroaryl includes, but are not limited to benzodiazine Base, pteridyl, quinolyl, isoquinolyl, cinnoline base, quinoxalinyl, phthalazinyl and quinazolyl.The exemplary bicyclic heteroaryl of 5,6- It is any that base includes, but are not limited to following formula:

In either one or two of monocycle or bicyclic heteroaryl group, tie point can be any carbon or nitrogen-atoms, as long as chemical valence Allow.

" part is unsaturated " refers to the group comprising at least one double or triple bonds.Term " part is unsaturated " is intended to cover Ring with multiple unsaturated sites, but do not include aromatic group as herein defined (for example, aryl or heteroaryl group). Equally, " saturation " refers to the group not comprising double or triple bonds, i.e., entirely includes singly-bound.

In some embodiments, aliphatic group as defined herein, alkyl, alkenyl, alkynyl, carbocylic radical, heterocycle, virtue Base and heteroaryl, optionally to replace (for example, " substitution " or " unsubstituted " aliphatic group, " substitution " or " unsubstituted " alkane Base, " substitution " or " unsubstituted " alkenyl, " substitution " or " unsubstituted " alkynyl, " substitution " or " unsubstituted " carbocylic radical " take Generation " or " unsubstituted " heterocycle, " substitution " or " unsubstituted " aryl or " substitution " or " unsubstituted " heteroaryl).In general, Term " substituted " refers to present on group (such as carbon or nitrogen-atoms) at least regardless of whether the front has term " optionally " One hydrogen is replaced by admissible substituent group, and if it replaces the substituent group of generation stable compound, the compound is, for example, not The spontaneous compound converted, the conversion are, for example, rearrangement, cyclisation, elimination or other reactions.Unless otherwise stated, it " takes Generation " group in the substitutive positions of one or more of the group has substituent group, and when more in any given structure When a position is substituted, the substituent group on each position is identical or different.Term " substituted " is believed to comprise organic All admissible substituent groups of compound replace, any substituent group as described herein including resulting in stable compound. The present invention is in view of any and all combination is to obtain stable compound.For the purpose of this paper, such as the miscellaneous original of nitrogen Son can have hydrogen substituent group and/or as described herein any suitable substituent group, meet heteroatomic valence and cause Form steady component.

Exemplary carbon replacing group includes, but are not limited to halogen ,-CN ,-NO₂、-N₃、-SO₂H、-S0₃H、-OH、- OR^aa、-ON(R^bb)₂、-N(R^bb)₂、-N(R^bb)₃ ⁺X、-N(OR^cc)R^bb、-SH、-SR^aa、-SSR^CC,-C (=O) R^aa、-CO₂H、- CHO、-C(OR^cc)₂、-CO₂R^aa,-OC (=O) R^aa、-OCO₂R^aa,-C (=O) N (R^bb)₂,-OC (=O) N (R^bb)₂、-NR^bbC (= O)R^aa、-NR^bbCO₂R^aa、-NR^bbC (=O) N (R^bb)₂,-C (=NR^bb)R^aa,-C (=NR^bb)OR^aa,-OC (=NR^bb)R^aa、-OC (=NR^bb)OR^aa,-C (=NR^bb)N(R^bb)₂,-OC (=NR^bb)N(R^bb)₂、-NR^bbC (=NR^bb)N(R^bb)₂,-C (=O) NR^bbSO₂R^aa、-NR^bbSO₂R^aa、-SO₂N(R^bb)₂、-SO₂R^aa、-SO₂OR^aa、-OSO₂R^aa,-S (=O) R^aa,-OS (=O) R^aa、- Si(R^aa)₃、-OSi(R^aa)₃- C (=S) N (R^bb)₂,-C (=O) SR^aa,-C (=S) SR^aa,-SC (=S) SR^aa,-SC (=O) SR^aa,-OC (=O) SR^aa,-SC (=O) OR^aa,-SC (=O) R^aa,-P (=O)₂R^aa,-OP (=O)₂R^aa,-P (=O) (R^aa)₂、- OP (=O) (R^aa)₂,-OP (=O) (OR^cc)₂,-P (=O)₂N(R^bb)₂,-OP (=O)₂N(R^bb)₂,-P (=O) (NR^bb)₂、-OP (=O) (NR^bb)₂、-NR^bbP (=O) (OR^cc)₂、-NR^bbP (=O) (NR^bb)₂、-P(R^CC)₂、-P(R^CC)₃、-OP(R^cc)₂、-OP (R^cc)₃、-B(R^aa)₂、-B(OR^cc)₂、-BR^aa(OR^cc)、C_1-10Alkyl, C_1-10Whole haloalkyl, C_2-10Alkenyl, C_2-10Alkynyl, C_3-10Carbocylic radical, 3-14 circle heterocyclic ring base, C_6-14Aryl and 5-14 unit's heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocylic radical, Heterocycle, aryl and heteroaryl, which independently replace, 0,1,2,3,4 or 5 R^ddGroup；

Or two on carbon atom together with position hydrogen by group=O ,=S ,=NN (R^bb)₂,=NNR^bbC (=O) R^aa,=NNR^bbC (=O) OR^aa,=NNR^bbS (=O)₂R^aa,=NR^bbOr=NOR^ccSubstitution；R^aaEach case be independently selected from C_1-10Alkyl, C_1-10Whole haloalkyl, C_2-10Alkenyl, C_2-10Alkynyl, C_3-10Carbocylic radical, 3-14 circle heterocyclic ring base, C_6-14Aryl and 5-14 member heteroaryl Base or two R^aaGroup connect to form 3-14 circle heterocyclic ring base or 5-14 unit's heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, Carbocylic radical, heterocycle, aryl and heteroaryl, which independently replace, 0,1,2,3,4 or 5 R^ddGroup；

R^bbEach case be independently selected from hydrogen ,-OH ,-OR^aa、-N(R^CC)₂,-CN ,-C (=O) R^aa,-C (=O) N (R^cc)₂、-CO₂R^aa、-SO₂R^aa,-C (=NR^cc)OR^aa,-C (=NR^CC)N(R^CC)₂、-SO₂N(R^cc)₂、-SO₂R^cc、-SO₂OR^cc、- SOR^aa,-C (=S) N (R^CC)₂,-C (=O) SR^cc,-C (=S) SR^CC,-P (=O)₂R^aa,-P (=O) (R^aa)₂,-P (=O)₂N (R^cc)₂,-P (=O) (NR^cc)₂、C_1-10Alkyl, C_1-10Whole haloalkyl, C_2-10Alkenyl, C_2-10Alkynyl, C_3-10Carbocylic radical, 3-14 member Heterocycle, C_6-14Aryl and 5-14 unit's heteroaryl or two R^bbGroup is connected to form 3-14 circle heterocyclic ring base or 5-14 member heteroaryl Basic ring, wherein each alkyl, alkenyl, alkynyl, carbocylic radical, heterocycle, aryl and heteroaryl independently replace have 0,1,2,3,4 or 5 R^ddGroup；

R^ccEach case be independently selected from hydrogen, C_1-10Alkyl, C_1-10Whole haloalkyl, C_2-10Alkenyl, C_2-10Alkynyl, C_3-10 Carbocylic radical, 3-14 circle heterocyclic ring base, C_6-14Aryl and 5-14 unit's heteroaryl or two R^ccGroup is connected to form 3-14 circle heterocyclic ring base Or 5-14 unit's heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocylic radical, heterocycle, aryl and heteroaryl independently replace There is 0,1,2,3,4 or 5 R^ddGroup；

R^ddEach case be independently selected from halogen ,-CN ,-NO₂、-N₃、-SO₂H、-SO₃H、-OH、-OR^ee、-ON(R^ff)₂、-N (R^ff)₂、-N(R^ff)₃ ⁺X、-N(OR^ee)R^ff、-SH、-SR^ee、-SSR^ee,-C (=O) R^ee、-CO₂H、-CO₂R^ee,-OC (=O) R^ee、- OCO₂R^ee,-C (=O) N (R^ff)₂,-OC (=O) N (R^ff)₂、-NR^ffC (=O) R^ee、-NR^ffCO₂R^ee、-NR^ffC (=O) N (R^ff)₂,-C (=NR^ff)OR^ee,-OC (=NR^ff)R^ee,-OC (=NR^ff)OR^ee,-C (=NR^ff)N(R^ff)₂,-OC (=NR^ff)N (R^ff)₂、-NR^ffC (=NR^ff)N(R^ff)₂,-NR^ffSO₂R^ee、-SO₂N(R^ff)₂、-SO₂R^ee、-SO₂OR^ee、-OSO₂R^ee,-S (=O) R^ee、-Si(R^ee)₃、-OSi(R^ee)₃,-C (=S) N (R^ff)₂,-C (=O) SR^ee,-C (=S) SR^ee,-SC (=S) SR^ee,-P (= O)₂R^ee,-P (=O) (R^ee)₂,-OP (=O) (R^ee)₂,-OP (=O) (OR^ee)₂、C_1-6Alkyl, C_1-6Whole haloalkyl, C_2-6Alkene Base, C_2-6Alkynyl, C_3-10Carbocylic radical, 3-10 circle heterocyclic ring base, C_6-10Aryl, 5-10 unit's heteroaryl, wherein each alkyl, alkenyl, alkynes Base, carbocylic radical, heterocycle, aryl and heteroaryl, which independently replace, 0,1,2,3,4 or 5 R^ggGroup or two are together with position R^ddIt takes Dai Jike connection is to form=O or=S；

R^eeEach case be independently selected from C_1-6Alkyl, C_1-6Whole haloalkyl, C_2-6Alkenyl, C_2-6Alkynyl, C_3-10Carbocylic radical, C_6-10Aryl, 3-10 circle heterocyclic ring base and 3-10 unit's heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocylic radical, heterocycle, aryl Independently replacing with heteroaryl has 0,1,2,3,4 or 5 R^ggGroup；

R^ffEach case be independently selected from hydrogen, C_1-6Alkyl, C_1-6Whole haloalkyl, C_2-6Alkenyl, C_2-6Alkynyl, C_3-10Carbocyclic ring Base, 3-10 circle heterocyclic ring base, C_1-6Aryl and 5-10 unit's heteroaryl or two R^ffGroup is connected to form 3-14 circle heterocyclic ring base or 5- 14 unit's heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocylic radical, heterocycle, aryl and heteroaryl independently replace have 0, 1,2,3,4 or 5 R^ggGroup；And

R^ggEach case independently be, halogen ,-CN ,-NO₂、-N₃、-SO₂H、-SO₃H、-OH、-O_1-6Alkyl ,-ON (C_1-6Alkyl)₂、-N(C_1-6Alkyl)₂、-N(C_1-6Alkyl)₃ ⁺X^-、-NH(C_1-6Alkyl)₂ ⁺X^-、-NH₂(C_1-6Alkyl)⁺X^-、-NH₃ ⁺ X、-N(OC_1-6Alkyl) (C_1-6Alkyl) ,-N (OH) (C_1-6Alkyl) ,-NH (OH) ,-SH ,-S_1-6Alkyl ,-SS (C_1-6Alkyl) ,-C (=O) (C_1-6Alkyl) ,-CO₂H、-CO₂(C_1-6Alkyl) ,-OC (=O) (C_1-6Alkyl) ,-OCO₂(C_1-6Alkyl) ,-C (=O) NH₂,-C (=O) N (C_1-6Alkyl)₂,-OC (=O) NH (C_1-6Alkyl) ,-NHC (=O) (C_1-6Alkyl) ,-N (C_1-6Alkyl) C (= O)(C_1-6Alkyl) ,-NHCO₂(C_1-6Alkyl) ,-NHC (=O) N (C_1-6Alkyl)₂,-NHC (=O) NH (C_1-6Alkyl) ,-NHC (= O)NH₂,-C (=NH) O (C_1-6Alkyl) ,-OC (=NH) (C_1-6Alkyl) ,-OC (=NH) OC_1-6Alkyl ,-C (=NH) N (C_1-6Alkane Base)₂,-C (=NH) NH (C_1-6Alkyl) ,-C (=NH) NH₂,-OC (=NH) N (C_1-6Alkyl)₂、-OC(NH)NH(C_1-6Alkyl) ,- OC(NH)NH₂、-NHC(NH)N(C_1-6Alkyl)₂,-NHC (=NH) NH₂、-NHSO₂(C_1-6Alkyl) ,-SO₂N(C_1-6Alkyl)₂、- SO₂NH(C_1-6Alkyl) ,-SO₂NH₂,-SO₂C_1-6Alkyl ,-SO₂OC_1-6Alkyl ,-OSO₂C_1-6Alkyl ,-SOC_1-6Alkyl ,-Si (C_1-6 Alkyl)₃、-OSi(C_1-6Alkyl)₃- C (=S) N (C_1-6Alkyl)₂, C (=S) NH (C_1-6Alkyl), C (=S) NH₂,-C (=O) S (C_1-6Alkyl) ,-C (=S) SC_1-6Alkyl ,-SC (=S) SC_1-6Alkyl ,-P (=O)₂(C_1-6Alkyl) ,-P (=O) (C_1-6Alkyl)₂、- OP (=O) (C_1-6Alkyl)₂,-OP (=O) (OC_1-6Alkyl)₂、C_1-6Alkyl, C_1-6Whole haloalkyl, C_2-6Alkenyl, C_2-6Alkynyl, C_3-10Carbocylic radical, C_6-10Aryl, 3-10 circle heterocyclic ring base, 5-10 unit's heteroaryl；Or two together with position R^ggSubstituent group can be connected to be formed =O or=S；Wherein X is counter ion counterionsl gegenions.

" counter ion counterionsl gegenions " or " anionic counter-ion " are mutually to be associated with cationic quaternary ammonium to keep the band of electroneutral negative The group of charge.Illustrative counter ion counterionsl gegenions include halide ion (such as F^–、Cl^–、Br^–、I^–)、NO₃ ^–、ClO₄ ^–、OH^–、H₂PO₄ ^–、 HSO₄ ^–、SO₄ ^–2, sulfonate ion (such as methanesulfonate, trifluoromethanesulfonic acid root, p-methyl benzenesulfonic acid root, benzene sulfonic acid root, 10-camphor sulphurs Acid group, naphthalene-2-sulfonate radical ,-5-sulfonate radical of naphthalene-1-sulfonic acid, ethane-1-- 2-sulfonate radical of sulfonic acid etc.) and carboxylic acid ion (such as vinegar Acid group, acetate, propionate, benzoate anion, glycerol acid group, lactate, tartrate anion, glycolic acid root etc.).

" halogenated " or " halogen " refer to fluorine (fluoro ,-F), chlorine (chloro ,-CI), bromine (bromo ,-Br) or iodine (iodo ,- I)。

As long as chemical valence allows, nitrogen-atoms can be substituted or unsubstituted, and former including primary nitrogen, secondary nitrogen, tertiary carbon and quaternary nitrogen Son.Illustrative nitrogen-atoms substituent group includes but is not limited to hydrogen ,-OH ,-OR^aa、-N(R^CC)₂,-CN ,-C (=O) R^aa,-C (= O)N(R^cc)₂、-CO₂R^aa、-SO₂R^aa,-C (=NR^bb)R^aa,-C (=NR^cc)OR^aa,-C (=NR^CC)N(R^CC)₂、-SO₂N(R^cc)₂、- SO₂R^cc、-SO₂OR^cc、-SOR^aa,-C (=S) N (R^CC)₂,-C (=O) SR^cc,-C (=S) SR^CC,-P (=O)₂R^aa,-P (=O) (R^aa)₂,-P (=O)₂N(R^cc)₂,-P (=O) (NR^cc)₂、C_1-10Alkyl, C_1-10Whole haloalkyl, C_2-10Alkenyl, C_2-10Alkynyl, C_3-10Carbocylic radical, 3-14 circle heterocyclic ring base, C_6-14Aryl and 5-14 unit's heteroaryl, or it is connected to two R of nitrogen-atoms^ccGroup connection To form 3-14 circle heterocyclic ring base or 5-14 unit's heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocylic radical, heterocycle, aryl Independently replacing with heteroaryl has 0,1,2,3,4 or 5 R^ddGroup, and wherein R^aa、R^bb、R^ccAnd R^ddAs defined above.

In certain embodiments, substituent group present on nitrogen-atoms is nitrogen-protecting group (also referred to as amino protecting group).Nitrogen Protecting group includes, but are not limited to ,-OH ,-OR^aa、-N(R^CC)₂,-C (=O) R^aa,-C (=O) N (R^cc)₂、-CO₂R^aa、-SO₂R^aa、- C (=NR^cc)R^aa,-C (=NR^cc)OR^aa,-C (=NR^CC)N(R^CC)₂、-SO₂N(R^cc)₂、-SO₂R^cc、-SO₂OR^cc、-SOR^aa、-C (=S) N (R^CC)₂,-C (=O) SR^cc,-C (=S) SR^CC、C_1-10Alkyl for example, aralkyl, heteroarylalkyl), C_2-10Alkenyl, C_2-10Alkynyl, C_3-10Carbocylic radical, 3-14 circle heterocyclic ring base, C_6-14Aryl and 5-14 unit's heteroaryl group, wherein each alkyl, alkenyl, Alkynyl, carbocylic radical, heterocycle, aralkyl, aryl and heteroaryl, which independently replace, 0,1,2,3,4 or 5 R group, and wherein R^aa、R^bb、R^ccAnd R^ddAs defined herein.Nitrogen-protecting group is well-known in the art and including Protecting Groups In Organic Synthesis, T.W.Greene and P.G.M.Wuts, the 3rd edition, John Wiley&Sons, institute in 1999 Those of state, it is hereby incorporated by reference.

Amide nitrogen-protecting group (such as-C (=O) R^aa) include, but are not limited to formamide, acetamide, chloroacetamide, trichlorine Acetamide, trifluoroacetamide, phenyl-acetamides, 3- Phenylpropionamide, picolinamide, 3- pyridinyl carboxamide, N- benzoyl Phenylalanyl derivative, benzamide, to phenylbenzamaide, ortho-nitrophenyl yl acetamide, adjacent nitro phenoxyacetyl Amine, acetyl acetamide, (N'- disulfide group benzyl oxygroup acyl amino) acetamide, 3- p- hydroxy phenyl) propionamide, 3- be (o- Nitrobenzophenone) propionamide, 2- methyl -2- (ortho-nitrophenyl oxygroup) propionamide, 2- methyl -2- (o- phenylazo phenoxy group) third Amide, 4- chlorobutamide, 3- methyl-3-nitro butyramide, o- nitrocinnamyl amide, N- acetyl methionine, ortho-nitrophenyl Formamide and o- (benzoyl oxygroup methyl) benzamide.

Carbamate nitrogen-protecting group (such as-C (=O) OR^aa) include, but are not limited to methyl carbamate, amino first Acetoacetic ester, 9-fluorenyl methyl ester of carbamic acid (Fmoc), carbamic acid 9-(2-sulfo group) fluorenyl methyl ester, carbamic acid 9-(2,7- bis- Bromine) fluorenyl methyl ester, bis--tert-butyl of carbamic acid 2,7--[9-(10,10- dioxy-10,10,10,10-tetrahydro thioxanthene base)] methyl esters (DBD-Tmoc), 4-methoxyphenacyl of carbamic acid (Phenoc), carbamic acid 2,2,2- trichloro ethyl ester (Troc), ammonia Base formic acid 2- trimethylsilylethyl (Teoc), 2-phenyl chlorocarbonate of carbamic acid (hZ), carbamic acid 1-(1-adamantane Base)-1-Methylethyl (Adpoc), carbamic acid 1,1- dimethyl-2-halogenated ethyl ester, carbamic acid 1,1- dimethyl-2,2- two Bromine ethyl ester (DB-t-BOC), carbamic acid 1,1- dimethyl-2,2,2- trichloro ethyl ester (TCBOC), 1-methyl of carbamic acid-1- (4-xenyl) ethyl ester (Bpoc), carbamic acid 1-(bis--tert-butyl-phenyl of 3,5-)-1-Methylethyl (t-Bumeoc), amino Formic acid 2-(2 '-and 4 '-pyridyl groups) ethyl ester (Pyoc), carbamic acid 2-(N, N- dicyclohexyl formamido) ethyl ester, amino first Tert-butyl acrylate (BOC), 1-adamantane esters of carbamic acid (Adoc), carbamic acid vinyl acetate (Voc), allyl carbamate (Alloc), 1-isopropyl of carbamic acid allyl ester (Ipaoc), carbamic acid cinnamic ester (Coc), 4-nitrocinnamyl of carbamic acid Ester (Noc), 8-quinoline of carbamic acid base ester, carbamic acid N-hydroxy piperidine base ester, alkyl dithiocarbamate, amino first Acid benzyl ester (Cbz), the p- methoxy benzyl ester of carbamic acid (Moz), the p- p-Nitrobenzyl of carbamic acid, the p- bromobenzyl ester of carbamic acid, The p- benzyl chloride ester of carbamic acid, carbamic acid 2,4- dichloro benzyl ester, 4-methyl sulfinyl of carbamic acid benzyl ester (Msz), amino first Sour 9-anthrylmethyls, carbamic acid diphenyl methyl esters, 2-methyl thio of carbamic acid ethyl ester, carbamic acid 2-methyl sulphonyl second Ester, carbamic acid 2-(ptoluene-sulfonyl) ethyl ester, carbamic acid [2-(1,3- dithia cyclohexyl)] methyl esters (Dmoc), ammonia Base 4-methyl thio of formic acid phenyl ester (Mtpc), carbamic acid 2,4- dimethyl thio phenyl ester (Bmpc), 2-phosphorus of carbamic acidBase Ethyl ester (Peoc), carbamic acid 2- triphenyl phosphorusBase isopropyl ester (Ppoc) ,-2-cyanaoethyl methacrylate of carbamic acid 1,1- dimethyl, The m- chloro- p- acyloxy benzyl ester of carbamic acid, p- (dihydroxy boryl) benzyl ester of carbamic acid, 5-benzisoxa of carbamic acidAzoles Base methyl esters ,-6-color onylmethyl (Tcroc) of carbamic acid 2-(trifluoromethyl), the m- nitro phenyl ester of carbamic acid, carbamic acid 3,5- dimethoxy benzyl ester, the o- p-Nitrobenzyl of carbamic acid ,-6-p-Nitrobenzyl of carbamic acid 3,4- dimethoxy, carbamic acid Phenyl (o-nitrophenyl) methyl esters, tert.-amyl carbamate, thiocarbamic acid S-benzyl ester, the p- cyano benzyl ester of carbamic acid, Carbamic acid ring butyl ester, carbamic acid cyclohexyl ester, carbamic acid ring pentyl ester, carbamic acid cyclopropylmethyl ester, the carbamic acid p- last of the ten Heavenly stems Oxygroup benzyl ester, carbamic acid 2,2- dimethoxy acyl group vinyl acetate, o- (N,N-dimethylformamide base) benzyl ester of carbamic acid, Carbamic acid 1,1- dimethyl-3-(N,N-dimethylformamide base) propyl ester, carbamic acid 1,1- dimethyl propynyl ester, amino first Sour two (2-pyridyl group) methyl esters, 2-furyl of carbamic acid methyl esters, 2-iodo-ethyl ester of carbamic acid, carbamic acid isobornyl thiocyanoacetate, ammonia Base iso-butyl formate, carbamic acid nicotimine ester, carbamic acid it is p- (to '-methoxyphenyl azo) benzyl ester, carbamic acid 1- Methyl ring butyl ester, 1-methyl cyclohexyl of carbamic acid ,-1-cyclopropylmethyl ester of 1-methyl of carbamic acid, 1-methyl of carbamic acid-1- (3,5- Dimethoxyphenyl) ethyl ester, 1-methyl of carbamic acid-1-(p- phenylazo phenyl) ethyl ester, 1-methyl of carbamic acid- 1-phenyl chlorocarbonate, 1-methyl of carbamic acid-1-(4-pyridyl group) ethyl ester, phenyl carbamate, p- (phenyl) benzyl of carbamic acid Ester, carbamic acid tri--tert-butyl of 2,4,6- phenyl ester, carbamic acid 4-(trimethyl ammonium) benzyl ester and carbamic acid 2,4,6- trimethyl Benzyl ester.

Sulfonamide nitrogen-protecting group (such as-S (=O)₂R^aa) include, but are not limited to para toluene sulfonamide (Ts), benzene sulfonyl Amine, 2,3,6,-trimethyl -4- methoxybenzenesulphoismide (Mtr), 2,4,6- triimethoxvbenzenesulfonamide (Mtb), 2,6- diformazan Base -4- methoxybenzenesulphoismide (Pme), 2,3,5,6- tetramethyl -4- methoxybenzenesulphoismide (Mte), 4- methoxybenzene sulphonyl Amine (Mbs), 2,4,6- trimethylbenzene sulfonamide (Mts), 2,6- dimethoxy-4 '-methyl benzenesulfonamide (iMds), 2,2,5,7, 8- pentamethyl chroman -6- sulfonamide (Pmc), Methanesulfomide (Ms), β-trimethyl silyl ethane sulphonamide (SES), 9- anthracene Sulfonamide, 4- (4', 8'- dimethoxy naphthyl methyl) benzsulfamide (DNMBS), benzyl sulfonamide, trimethyl fluoride sulfonyl amine and Phenacyl sulfonamide.

Other nitrogen-protecting groups include, but are not limited to phenothiazinyl-(10)-acyl derivative, the p- Tosylamino of N '- Acyl derivative, N '-phenyl amino Thioacyl derivative, N-benzoylphenylalanyl radical derivative, N-acetyl group egg Threonine derivative, 4,5- diphenyl-3-Oxazoline-2-ketone, N-phthalimide, N- dithiosuccinimide (N- Dithiasuccinimide, Dts), N-2,3- diphenylmaleimide, N-2,5- dimethyl pyrrole, N-1, Isosorbide-5-Nitrae, 4-tetramethyls Base dimethyl silanyl aza-cyclopentane adduct (STABASE), the 5-1,3- dimethyl-1,3,5- Trianacyclohexanes-replaced - 4-pyridone of 3,5- dinitro of-2-ketone of 1,3- dibenzyl-1,3,5- Trianacyclohexane, 1-substitution that 2-ketone, 5-replace, N-methyl amine, N-allyl amine, N-[2-(trimethyl silyl) ethyoxyl] methyl amine (SEM), N-3-acetyloxypropyl Amine, N-(1-- 3-yl of isopropyl-4-nitro-3-pyrrolin of-2-oxo) amine, quaternary ammonium salt, N-benzyl amine, the (4-methoxybenzenes of N- bis- Base) methyl amine, N-5- dibenzocycloheptyl amine, N- trityl group amine (Tr), N-[(4-methoxyphenyl) diphenyl methyl] Amine (MMTr), N-9-phenylfluorenyl amine (PhF), the chloro- 9-fluorenyl benzylidene amino of N-2,7- two, N-ferrocenyl methylamino (Fcm), N-2-picolyl amino N '-oxide, N-1,1- dimethyl thio benzylidene amino, N-benzal amine, the p- methoxy of N- Base benzal amine, N- diphenylmethyleneamines, N-[(2-pyridyl group)Base] benzylidene amino, N-(N ', N '-dimethyl amino Asia Methyl) amine, N, the p- nitrobenzal amine of N '-isopropylidene diamines, N-, N-salicylidene amine, N-5-chlorine salicylidene amine, N- (5-chloro- 2-hydroxy phenyl) phenylmethylene amine, N-cyclohexylidene amine, N-(- 1-cyclohexenyl group of 5,5--3-oxo of dimethyl) Amine, N-borane derivative, N- diphenyl-borinic acids (borinic acid) derivative, N-[phenyl (five acyl group chromium-or tungsten) acyl group] Amine, N-copper chelate, N-chelates of zinc, N-nitra-amine, N-nitroso-amines, amine n-oxide, diphenylphosphine amide (Dpp), two Methyl thio-phosphoryl amine (Mpt), diphenyl thio-phosphamide (Ppt), phosphoramidic acid dialkyl ester, phosphoramidic acid dibenzyl base ester, Phosphoramidic acid diphenyl, phenylsulfinyl amine, o- nitrobenzene sulfenamide (Nps), 2,4- dinitrobenzene sulfenamide, pentachloro- Phenylsulfinyl amine, 2-- 4-methoxybenzene of nitro sulfenamides, trityl group sulfenamide and 3-nitropyridine sulfenamides (Npys)。

In some embodiments, the substituent group being present on oxygen atom is oxygen protecting group (also referred to as hydroxyl protection base). Oxygen protecting group includes, but are not limited to ,-R^aa、-N(R^bb)₂,-C (=O) SR^aa,-C (=O) R^aa、-C O ₂R^aa,-C (=O) N (R^bb)₂,-C (=NR^bb)R^aa,-C (=NR^bb)OR^aa,-C (=NR^bb)N(R^bb)₂,-S (=O) R^aa、-SO₂R^aa、-Si(R^aa)₃、-P (R^CC)₂、-P(R^CC)₃,-P (=O)₂R^aa,-P (=O) (R^aa)₂,-P (=O) (OR^cc)₂,-P (=O)₂N(R^bb)₂With-P (=O) (NR^bb)₂, wherein R^aa、R^bbAnd R^ccAs defined herein.Oxygen protecting group is as known in the art and is included in Protecting Groups in Organic Synthesis, T.W.Greene and P.G.M.Wuts, the 3rd edition, John Wiley&Sons, 1999 Those of middle detailed description, the document is incorporated by reference herein.

Illustrative oxygen protecting group includes, but are not limited to methyl, methoxy (MOM), methylthiomethyl (MTM), uncle Butyl sulfidomethyl, (phenyldimethylsilyl) methoxy (SMOM), benzyloxymethyl (BOM), p- methoxyl group benzyloxy Ylmethyl (PMBM), (4-methoxyphenoxy) methyl (p-AOM), guaiacol methyl (GUM), t-butoxymethyl, 4-penta Enyloxymethyl (POM), silanyloxymethyl, 2-methoxvethoxvmethvls (MEM), 2,2,2- tri-chloroethoxy Ji Jia Base, bis- (2-chloroethoxy) methyl, 2-(trimethyl silyl) ethoxyl methyls (SEMOR), THP trtrahydropyranyl (THP), 3- Bromine THP trtrahydropyranyl, tetrahydro thiapyran base, 1-methoxycyclohexyl, 4-methoxyl group THP trtrahydropyranyls (MTHP), 4-methoxyl group tetrahydros Thiapyran base, 4-methoxyl group tetrahydro thiapyran base S, S- dioxide, 1-[(2-chloro- 4-methyl) phenyl]-4-methoxy piperide-4- bases (CTMP), 1,4- bis-Alkane -2- base, tetrahydrofuran base, tetrahydro thiapyran base (tetrahydrothiofuranyl), 2,3,3a, 4,5,6,7,7a-octahydro-7,8,8- trimethyl-2-base of-4,7-endo-methylene group benzofuran (2,3,3a, 4,5,6,7,7a- - 2-yl of-4,7-methanobenzofuran of octahydro-7,8,8-trimethyl), 1-ethoxyethyl group, 1-(2-chloroethenes Oxygroup) ethyl, 1-- 1-methoxy ethyl of methyl, 1-- 1-Benzyloxyethyl of methyl, 1-- 2-fluoro ethyl of-1-benzyloxy of methyl, 2, 2,2- trichloroethyl, 2- trimethylsilyethyl, 2-(phenylselanyl) ethyls (2-(phenylselenyl) ethyl), uncle Butyl, allyl, p- chlorphenyl, p- methoxyphenyl, dinitrophenyl group, benzyl (Bn), p- methoxy-benzyl, 3, 4- dimethoxy-benzyl, o- nitrobenzyl, p- nitrobenzyl, p- halogeno-benzyl, 2,6- dichloro benzyl, p- cyanobenzyls, P- phenylbenzyl, 2-picolyls, 4-picolyls, 3-- 2-picolyl of methyl N-oxygen bridge (oxido), diphenyl methyl, P, p '-dinitro benzhydryl, 5- dibenzocycloheptyl, trityl group, α-naphthyldiphenylmethyl base, p- methoxyphenyl Diphenyl methyl, two (p- methoxyphenyl) phenyl methyls, three (p- methoxyphenyl) methyl, 4-(4 '-Bromophenac rLls Phenyl) diphenyl methyl, 4,4 ', 4 "-three (4,5- dichloro-benzenes imidodicarbonic diamide base phenyl) methyl, 4,4 ', 4 "-three (acetyl Propiono (levulinoyl) phenyl) methyl, 4,4 ', 4 "-three (benzoyloxyphenyl) methyl, 3-(imidazoles-1- bases) Bis- (4 ', 4 "-Dimethoxyphenyl) methyl, 1,1-bis- (4-methoxyphenyl)-1 '-pyrenylmethies, 9-anthryls, 9-(9-phenyl)Ton base, 9-(9-- 10-oxo of phenyl) anthryls, 1,3-benzodisulfuran-2- bases, benzisothia oxazolyl S, S- titanium dioxide Object, trimethyl silyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethyl isopropyl Base silicyl (IPDMS), diethyl isopropyl silyl (DEIPS), dimethylhexanyl (ethyl, thexyl) monosilane Base, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzyl silicyl, three- P-xylene base silicyl, triphenyl-silyl, diphenylmethylsilyl (DPMS), tert-butyl methoxyphenyl Silicyl (TBMPS), formic acid esters, benzoyl formate, acetic acid esters, chloracetate, dichloroacetic acid ester, trichloroacetic esters, Trifluoro-acetate, methoxyacetic acid ester, triphenylmethoxy acetic acid esters, phenoxyacetic acid ester, p- tomatotone ester, 3- Phenylpropionic acid ester, 4-oxopentanoic acid esters (levulinate), (levulinic acyl group two is thio for 4,4-(ethylene is thio) valerates Acetal), pivalate, adamantate (adamantoate), crotonates, 4-methoxyl group crotonates, benzoic ether, P- phenylbenzoate, 2,4,6- trimethylbenzoic acid ester (Acid esters (mesitoate)), t-butyl carbonate (BOC), carbonic acid Alkyl methacrylate ester, 9-fluorenyl methyl ester of carbonic acid (Fmoc), alkyl carbonate ethyl ester, alkyl carbonate 2,2,2- trichloro ethyl ester (Troc), carbon Sour 2-(trimethyl silyl) ethyl esters (TMSEC), carbonic acid 2-(phenyl sulfonyl) ethyl ester (Psec), carbonic acid 2-(triphenyl phosphorusBase) ethyl ester (Peoc), alkyl carbonate isobutyl ester, alkyl carbonate vinyl acetate, alkyl carbonate allyl ester, the p- nitro of alkyl carbonate Phenylester, alkyl carbonate benzyl ester, the p- methoxy benzyl ester of alkyl carbonate, alkyl carbonate 3,4- dimethoxy benzyl ester, alkyl carbonate The p- p-Nitrobenzyl of o- p-Nitrobenzyl, alkyl carbonate, thiocarbonic acid alkyl S-benzyl ester, carbonic acid-1-naphthalene of 4-ethyoxyl ester, two Thiocarbonic acid methyl esters, 2-iodo-benzoic acid esters, 4-azido butyrates, 4-- 4-methylpent acid esters of nitro, o- (two bromomethyls) benzene Formic acid esters, 2-formylbenzene sulfonates, 2-(methyl thio methoxyl group) ethyls, 4-(methyl thio methoxyl group) butyrates, 2-(first Base thiomethoxy ylmethyl) benzoic ether, the chloro- 4-methylphenoxyacetate of 2,6- bis-, 2,6- bis- chloro- 4-(1,1,3,3-four Methyl butyl) phenoxyacetic acid ester, 2,4-bis- (1,1- dimethyl propyl) phenoxyacetic acid esters, chlorodiphenyl yl acetate, isobutyl Acid esters, monosuccinic acid ester, (E)-2-- 2-butenoate of methyl, o- (methoxyl group acyl group) benzoic ether, α-naphthoate, nitric acid Ester, N, N, N ', N '-tetramethyl phosphorodiamidate Arrcostab, N-phenylcarbamic acid Arrcostab, borate, dimethyl disulfide phosphino-, Dinitrophenyl group sulfenic acids Arrcostab, sulfuric ester, methane sulfonate (methanesulfonates), benzyl sulfonic acid ester and tosylate (Ts)。

In certain embodiments, the substituent group being present on sulphur atom is sulfur protecting group (also referred to as thiol protective group). Sulfur protecting group includes, but are not limited to ,-R^aa、-N(R^bb)₂,-C (=O) SR^aa,-C (=O) R^aa、-CO ₂R^aa,-C (=O) N (R^bb)₂,-C (=NR^bb)R^aa,-C (=NR^bb)OR^aa,-C (=NR^bb)N(R^bb)₂,-S (=O) R^aa、-SO₂R^aa、-Si(R^aa)₃-P (R^CC)₂、-P(R^CC)₃,-P (=O)₂R^aa,-P (=O) (R^aa)₂,-P (=O) (OR^cc)₂,-P (=O)₂N(R^bb)₂With-P (=O) (NR^bb)₂, wherein R^aa、R^bbAnd R^ccAs defined herein.Sulfur protecting group is as known in the art and is included in Protecting Groups in OrganicSynthesis, T.W.Greene and P.G.M.Wuts, the 3rd edition, John Wiley&Sons, those of is described in detail in 1999, and the document is incorporated by reference herein.

As used herein, " leaving group " or " LG " is the term understood in this field, is referred in heterolytic fission bond cleavage solution Shi Yuyi To the molecule fragment being electrically separated, wherein molecule fragment is anion or neutral molecule.See, e.g., Smith, March Advanced Organic Chemistry the 6th edition (501-502).The example of suitable leaving group includes but is not limited to halogen (such as chlorine, bromine or iodine), alkoxy-carbonyl oxy, aryloxycarbonyl oxygroup, alkane sulfonyl oxygroup, arenesulfonyl oxygen Base, alkyl-carbonyloxy base (such as acetoxyl group), aryl carbonyl epoxide, aryloxy, methoxyl group, N, O- dimethyl hydroxyl ammonia Base, pixyl, haloformate ,-NO₂, trialkyl ammonium and aryl salt.In some embodiments, leaving group is sulfonic acid Ester.In some embodiments, sulphonic acid ester includes formula-OSO₂R^LG1, wherein R^LG1Selected from the alkyl optionally replaced, optionally replace Alkenyl, the miscellaneous alkyl optionally replaced, the aryl optionally replaced, the heteroaryl optionally replaced, the aralkyl optionally replaced and optionally Substituted heteroaryl alkyl.In some embodiments, R^LGlFor substituted or unsubstituted C₁-C₆Alkyl.In some embodiments In, R^LGlFor methyl.In some embodiments, R^LGlFor substituted or unsubstituted aryl.In some embodiments, R^LGlFor Substituted or unsubstituted phenyl.In some embodiments, R^LGlAre as follows:

In some cases, leaving group be tosylate (tosylate, Ts), methane sulfonate (methanesulfonates, Ms), brosyl (brosylate, Bs) or trifluoromethayl sulfonic acid ester (triflate, Tf).In some feelings Under condition, leaving group is brosylate (brosyl).In some cases, leaving group is nitrobenzene-sulfonic acid base (2- nitrobenzenesulfonyl).In some embodiments, the leaving group is the group containing sulphonic acid ester.In some embodiments In, the leaving group is tosylate group.Leaving group can also be phosphine oxide (for example, shape in Mitsunobu reaction At) or internal leaving group such as epoxides or cyclic sulfates.

" pharmaceutically acceptable salt " refers to that those are suitable for and people and other animals within a reasonable range of medical judgment Tissue contact without excessive toxicity, stimulation, allergic reaction etc., and the salt to match with reasonable interests/Hazard ratio.Pharmaceutically Acceptable salt is well known in the art.For example, Berge et al. is in J.Pharmaceutical Sciences (1977) 66:1- Pharmaceutically acceptable salt is described in detail in 19.The pharmaceutically acceptable salt of the compounds of this invention includes being originated from suitable nothing Those of machine and organic bronsted lowry acids and bases bronsted lowry.The example of pharmaceutically acceptable non-toxic acid addition salts is and inorganic acid (such as hydrochloric acid, hydrogen bromine Acid, phosphoric acid, sulfuric acid and perchloric acid) formed or with organic acid (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, amber Acid or malonic acid) or by using method used in this field (such as ion exchange) formed amino salt.Its other medicine On acceptable salt include adipate, alginate, ascorbate, aspartate, benzene sulfonate, benzoate, Disulfate, borate, butyrate, camphor hydrochlorate, camsilate, citrate, cyclopentane propionate, digluconate, Lauryl sulfate, esilate, formates, fumarate, gluceptate, glycerophosphate, gluconate, half sulphur Hydrochlorate, enanthate, caproate, hydriodide, 2-hydroxyls-esilate, lactobionate, lactate, laruate, lauryl Sulfate, malate, maleate, malonate, mesylate, 2-naphthalene sulfonates, nicotinate, nitrate, oleate, grass Hydrochlorate, palmitate, embonate, pectate (pectinate), persulfate, 3-phenylpropionic acid salt, phosphate, hardship Sour salt, pivalate, propionate, stearate, succinate, sulfate, tartrate, rhodanate, p-methyl benzenesulfonic acid Salt, undecylate, valerate etc..Salt from suitable alkali includes alkali metal, alkaline-earth metal, ammonium and N⁺(C_1–4Alkyl)₄Salt.Generation The alkaline or alkaline-earth salts of table include sodium salt, lithium salts, sylvite, calcium salt, magnesium salts etc..In due course, other are pharmaceutically acceptable Salt include quaternary ammonium salt.

The present invention provides II type PRMT inhibitor.In one embodiment, the II type PRMT inhibitor is formula (III) Compound:

Or its pharmaceutically acceptable salt,

Wherein

Indicate singly-bound or double bond；

R¹For hydrogen, R^zOr-C (O) R^z, wherein R^zFor the C optionally replaced_1-6Alkyl；

L is-N (R) C (O)-,-C (O) N (R)-,-N (R) C (O) N (R)-,-N (R) C (O) O- or-OC (O) N (R)-；

The C that each R independently is hydrogen or optionally replaces_1-6Aliphatic group；

Ar is the heteroatomic monocycle or Bicyclic aryl rings that nitrogen, oxygen and sulphur are independently selected from 0-4, wherein Ar substitution have 0, 1,2,3,4 or 5 R^yGroup, as long as chemical valence allows；

Each R^yIndependently selected from halogen ,-CN ,-NO₂, optionally replace aliphatic group, optionally replace carbocylic radical, optionally Substituted aryl, the heterocycle optionally replaced, the heteroaryl ,-OR optionally replaced^A、-N(R^B)₂、-SR^A,-C (=O) R^A、-C(O) OR^A、-C(O)SR^A、-C(O)N(R^B)₂、-C(O)N(R^B)N(R^B)₂、-OC(O)R^A、-OC(O)N(R^B)₂、-NR^BC(O)R^A、-NR^BC (O)N(R^B)₂、-NR^BC(O)N(R^B)N(R^B)₂、-NR^BC(O)OR^A、-SC(O)R^A,-C (=NR^B)R^A,-C (=NNR^B)R^A,-C (= NOR^A)R^A,-C (=NR^B)N(R^B)₂,-NRBC (=NR^B)R^B,-C (=S) R^A,-C (=S) N (R^B)₂、-NR^BC (=S) R^A、-S(O) R^A、-OS(O)₂R^A、-SO₂R^A、-NR^BSO₂R^AOr-SO₂N(R^B)₂；

Each R^AIndependently selected from hydrogen, optionally the aliphatic group that replaces, the carbocylic radical optionally replaced, the heterocycle optionally replaced Base, the aryl optionally replaced and the heteroaryl optionally replaced；

Each R^BIndependently selected from hydrogen, optionally the aliphatic group that replaces, the carbocylic radical optionally replaced, the heterocycle optionally replaced Base, the aryl optionally replaced and the heteroaryl optionally replaced or two R^BGroup is formed together with atom between them appoints Choose the heterocycle in generation；

R⁵、R⁶、R⁷And R⁸It independently is hydrogen, halogen or the aliphatic group optionally replaced；

Each R^XIndependently selected from halogen ,-CN, the optionally aliphatic group ,-OR' and the-N (R ") that replace₂；

R' is hydrogen or the aliphatic group optionally replaced；

Each R " independently be hydrogen or the aliphatic group optionally replaced or two R " shape together with atom between them At heterocycle；And

N is 0,1,2,3,4,5,6,7,8,9 or 10, as long as chemical valence allows.

On the one hand, L is-C (O) N (R)-.On the one hand, R¹For hydrogen.On the one hand, 0 n.

In one embodiment, the II type PRMT inhibitor is the compound of formula (IV):

Or its pharmaceutically acceptable salt.On the one hand, at least one R^yFor-NHR^B.On the one hand, R^BOptionally to replace Naphthenic base.

In one embodiment, the II type PRMT inhibitor is the compound of formula (VII):

Or its pharmaceutically acceptable salt.On the one hand, L is-C (O) N (R)-.On the one hand, R¹For hydrogen.On the one hand, N is 0.

In one embodiment, the II type PRMT inhibitor is the compound of formula (VIII):

Or its pharmaceutically acceptable salt.On the one hand, L is-C (O) N (R)-.On the one hand, R¹For hydrogen.On the one hand, N is 0.

In one embodiment, the II type PRMT inhibitor is the compound of formula (IX):

Or its pharmaceutically acceptable salt.On the one hand, R¹For hydrogen.On the one hand, 0 n.

In one embodiment, the II type PRMT inhibitor is compound B:

Or its pharmaceutically acceptable salt.

In one embodiment, the II type PRMT inhibitor is the compound of formula (X):

Or its pharmaceutically acceptable salt.On the one hand, R^yFor-NHR^B.On the one hand, R^BFor the heterocycle optionally replaced.

In certain embodiments, the II type PRMT inhibitor is the compound of formula (XI):

Or its pharmaceutically acceptable salt, wherein X is-C (R^XC)₂,-O- ,-S- or-NR^XN, wherein R^XCEvery kind of feelings Condition independently is hydrogen, optionally replaces alkyl, the carbocylic radical optionally replaced, the heterocycle optionally replaced, the aryl optionally replaced, Or the heteroaryl optionally replaced；R^XNIndependently be hydrogen, the alkyl that optionally replaces, the carbocylic radical optionally replaced, optionally replace it is miscellaneous Ring group, the aryl optionally replaced, the heteroaryl optionally replaced ,-C (=O) R^XAOr nitrogen-protecting group；R^XAFor the alkane optionally replaced Base, the carbocylic radical optionally replaced, the heterocycle optionally replaced, the aryl optionally replaced or the heteroaryl optionally replaced.

In one embodiment, the II type PRMT inhibitor is compound C:

Or its pharmaceutically acceptable salt.The method of compound C and prepare compound C are disclosed in PCT/US2013/ 077235, at least in page 141 (compounds 208) and [00464] Duan Zhi of page 291 [00469] sections of page 294.

In another embodiment, the II type PRMT inhibitor is compound E:

Or its pharmaceutically acceptable salt.

In another embodiment, the II type PRMT inhibitor is compound F:

Or its pharmaceutically acceptable salt.

II type PRMT inhibitor is further disclosed in PCT/US2013/077235 and PCT/US2015/043679, is drawn Enter reference.Exemplary II type PRMT inhibitor be disclosed in the table 1A of PCT/US2013/077235, table 1B, table 1C, table 1D, table 1E, Table 1F and table 1G, the method for preparing II type PRMT inhibitor are at least described in page 239 of PCT/US2013/077235 [00359] Duan Zhi [00485] sections of page 301.Other non-limiting examples of II type PRMT inhibitor or PRMT5 inhibitor are public Open patent application WO2011/079236, WO2014/100695, WO2014/100716, WO2014/ in following discloses 100730, WO2014/100764 and WO2014/100734 and U.S. Provisional Application No. 62/017,097 and 62/017,055.This The general formula and particular compound of a little patent applications are hereby incorporated by reference and can be used for treating cancer as described herein.Some In embodiment, the II type PRMT inhibitor is nucleic acid (for example, siRNA).Such as Mol is described in the siRNAs of PRMT5 Cancer Res.2009Apr；7 (4): 557-69 and Biochem J.2012Sep 1；446(2):235-41.

" antigen-binding proteins (ABP) " refers to the protein in conjunction with antigen, including is worked in a manner of similar with antibody Antibody or engineered molecule.It is such for select antibody formation include three chain antibodies, four chain antibodies, miniantibody and miniantibody.Also Including alternative bracket, wherein one or more CDR of any molecule according to the present invention can be arranged in it is suitable nonimmune On immunoglobulin protein bracket or skeleton, such as affine body, SpA bracket, ldl receptor A class formation domain, avimer are (referring to example Such as, U.S. Patent Application Publication No. 2005/0053973,2005/0089932,2005/0164301) or EGF structural domain.ABP is also Antigen-binding fragment including such antibody or other molecules.In addition, ABP may include the area VH of the invention, when with it is appropriate Light chain matches clock synchronization, is configured to full length antibody, (Fab ') 2 segment, Fab segment, bispecific or double paratopes (biparatopic) molecule or its equivalent (such as scFV, double-chain antibody, three chain antibodies or four chain antibodies, Tandabs, etc.). ABP may include antibody, be IgG1, IgG2, IgG3 or IgG4；Or IgM；IgA, IgE or IgD or its modification variant.It can phase Select the constant domain of heavy chain of antibody with answering.Light-chain constant domains can be κ or λ constant domain.ABP is also possible to WO86/01533 Described in type chimeric antibody, it includes antigen binding domains and non-immunoglobulin area.Term " ABP ", " antigen binding egg It is white " and " binding protein " be used interchangeably herein.

Protein programmed death receptor 1 (PD-1) is the inhibition member of CD28 receptor family, further include CD28, CTLA-4, ICOS and BTLA.PD-1 expressed in the B cell, T cell and bone marrow cell of activation (Agata et al., ibid； Okazaki et al. (2002) Curr.Opin.Immunol14:391779-82；Bennett et al. (2003) J Immunol 170:711-8).The initial member CD28 and ICOS of the family is the function by enhancing T cell proliferation after addition monoclonal antibody Energy property acts on and (Hutloff et al. (1999) Nature 397:263-266 of discovery；Hansen et al. (1980) Immunogenics 10:247-260).PD-1 (Ishida et al. is found by the differential expression in screening apoptotic cell (1992)EMBO J 11:3887-95).Other members of the family, CTLA-4 and BTLA, by screening cytotoxic T respectively Differential expression in lymphocyte and TH1 cell is found.CD28, ICOS and CTLA-4 have unpaired cysteine residual Base allows homodimerization.On the contrary, implying that PD-1 exists as monomer, lack unpaired half in other CD28 family members Cystine residue feature.PD-1 antibody and method for treating disease are described in U.S. Patent number: US 7,595,048；US 8,168,179；US 8,728,474；US 7,722,868；US 8,008,449；US 7,488,802；US 7,521,051；US 8,088,905；US 8,168,757；US 8,354,509；With US publication US20110171220；US20110171215； And US20110271358.The combination of CTLA-4 and PD-1 antibody is described in U.S. Patent number 9,084,776.

As used herein, " PD-1 antagonist ", which refers to, blocks the PD-L1 expressed on cancer cell and immunocyte (T cell, B Cell or NKT cell) on any chemical compound for combining of the PD-1 that expresses or biomolecule, and preferably also block cancer cell The combination of the PD-1 of PD-L2 and the immunocyte expression of upper expression.The substitution title or synonym of PD-1 and its ligand include: pair In PDCD1, PD1, CD279 and SLEB2 of PD-1；For PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H of PD-L1； With PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2.People PD-1 amino acid sequence can be in NCBI locus Number (Locus No.): it is found in NP_005009.Human PD-L 1 and PD-L2 amino acid sequence can be respectively in NCBI gene seat numbers: It is found in NP_054862 and NP_079515.

The PD-1 antagonist that can be used for any aspect of the present invention includes monoclonal antibody (mAb) or its antigen-binding fragment, It specifically binds PD-1 or PD-L1, and preferably specifically binds people PD-1 or human PD-L 1.MAb can be human antibody, people Source antibody or chimeric antibody, and may include human constant region.In some embodiments, human constant region be selected from IgG1, IgG2, IgG3 and IgG4 constant region, and in preferred embodiments, human constant region is IgG1 or IgG4 constant region.One In a little embodiments, antigen-binding fragment is selected from Fab, Fab'-SH, F (ab') 2, scFv and Fv segment.

In conjunction with the example of people PD-1 and the mAb of various aspects for use in the present invention and embodiment, to be described in the U.S. special Benefit number 8,552,154；U.S. Patent number 8,354,509；U.S. Patent number 8,168,757；U.S. Patent number 8,008,449；Beauty State's patent No. 7,521,051；U.S. Patent number 7,488,802；WO2004072286；WO2004056875；With WO2004004771。

Other PD-1 antagonists that can be used for any aspect of the present invention and embodiment include specifically binding exempting from for PD-1 Epidemic disease adhesin, and preferably specifically bind people PD-1, such as the PD-1 bound fraction containing extracellular or PD-L1 or PD-L2 Fusion protein, merged with the constant region of the immunoglobulin molecules such as area Fc.In WO2010027827 and WO2011066342 In describe specific binding PD-1 immunoadhesin molecule example.In treatment method of the invention, drug and on the way Specific fusion protein as PD-1 antagonist includes AMP-224 (also referred to as B7-DCIg), is PD-L2-FC fusion protein And in conjunction with people PD-1.

Military monoclonal antibody of receiving is a kind of anti-PD-1 antibody of Humanized monoclonal, withIt is commercially available.Military monoclonal antibody of receiving is suitable for Treat some can not cut off or metastatic melanoma.Military monoclonal antibody of receiving combines by its ligand PD-L1 and PD-L2 and blocks PD-1 The activation of (Ig superfamily transmembrane protein) leads to the activation of T cell and exempts from for tumour cell or the cell-mediated of pathogen Epidemic disease response.The PD-1 of activation is by inhibiting P13k/Akt pathway activation come negative regulator T cell activation and effector function.Receive Wu Dankang Other titles include: BMS-936558, MDX-1106 and ONO-4538.Receive military monoclonal antibody amino acid sequence and use and make Preparation Method is disclosed in United States Patent (USP) US 8,008,449.

Pyridine aldoxime methyliodide (PAM) monoclonal antibody is the anti-PD-1 antibody of Humanized monoclonal, withIt is commercially available.Pyridine aldoxime methyliodide (PAM) monoclonal antibody is suitable for Treat some can not cut off or metastatic melanoma.The amino acid sequence and application method of pyridine aldoxime methyliodide (PAM) monoclonal antibody are disclosed in U.S. Patent number 8,168,757。

PD-L1 is B7 family member, is expressed on many cell types, the T cell including APC and activation (Yamazaki et al. (2002) J.Immunol.169:5538).PD-L1 is in conjunction with PD-1 and B7-1.It is thin by PD-L1 combination T The B7-1 of cellular expression and by B7-1 combination T cell express PD-L1 result in T cell inhibit (Butte et al. (2007) Immunity 27:111).There is also evidence that PD-L1 can also provide thorn altogether to T cell as other B7 family members Energizing signal (Subudhi et al. (2004) J.Clin.Invest.113:694；Tamura et al. (2001) Blood 97:1809). PD-L1 (human PD-L 1 cDNA base sequence group as shown in EMBL/GenBank accession number AF233516 of ligand as PD-1 At mouse PD-L1cDNA base sequence shown in NM.sub.--021893 forms) it is such as living in so-called antigen presenting cell Expressed in the monocyte and dendritic cells of change (Journal of Experimental Medicine (2000), volume 19, the 7 phases, the 1027-1034 pages).These presented by cells interacting molecules, to T lymphocyte induction panimmunity induction letter Number, and PD-L1 is one of these molecules, is induced by PD-1 and inhibits signal.Have revealed that PD-L1 ligand stimulation presses down The activation of the T lymphocyte of expression PD-1 has been made (cell Proliferation and the various cell factors of induction generate).PD-L1 expression not only exists It is confirmed in immunocompetent cell, and is confirmed in certain tumor cell line (from the thin of monocytic leukemia Born of the same parents system, carrys out the cell line of mast cell, the cell line from liver cancer, the cell line from neuroblast, and comes from mammary gland The cell line of cancer) (Nature Immunology (2001), volume 2, the 3rd phase, the 261-267 pages).

Anti- PD-L1 antibody and preparation method thereof is known in the art.This antibody for PD-L1 can be polyclonal Or monoclonal, and/or recombination and/or humanization.PD-L1 antibody is as the immunomodulator for being used for treating cancer Just in exploitation.

Exemplary PD-L1 antibody is disclosed in U.S. Patent number 9,212,224；U.S. Patent number 8,779,108；United States Patent (USP) No 8,552,154；U.S. Patent number 8,383,796；U.S. Patent number 8,217,149；U.S. Patent Publication No. 20110280877；WO2013079174；And WO2013019906.The other examples of PD-L1 (also referred to as CD274 or B7-H1) Antibody and application method are disclosed in U.S. Patent number 8,168,179；U.S. Patent number 7,943,743；U.S. Patent number 7,595, 048；WO2014055897；WO2013019906；And WO2010077634.It can be used as treatment method of the invention, drug and use The specific anti-human PD-L1 monoclonal antibody of PD-1 antagonist on the way include MPDL3280A, BMS-936559, MEDI4736, MSB0010718C。

Aunar pearl monoclonal antibody is the anti-PD-L1 antibody of full-length human monoclonal, with TECENTRIQ^TMIt is commercially available.Aunar pearl monoclonal antibody is suitable For treating some Locally Advanceds or metastatic bladder transitional cell carcinoma.Aunar pearl MAbs blocking PD-L1 and PD-1's and CD80 is mutual Effect.

CD134, also referred to as OX40 are the members of the TNFR- superfamily of receptor, different from CD28, in the inmature T of tranquillization Not constitutive expression on cell.OX40 is secondary costimulatory molecules, is expressed within 24 to 72 hours upon activation；Its ligand OX40L It does not express on tranquillization antigen presenting cell, but is expressed after its activation.The expression of OX40 depends on the complete work of T cell Change；There is no CD28, the expression of OX40 is delayed by and level is reduced to a quarter.OX40/OX40- ligand (OX40 receptor)/ It (OX40L) is a pair of to T cell proliferation, survival, cell factor generation and the vital costimulatory molecules of memory cell generation. Initial in vitro is it is demonstrated experimentally that by OX40 in CD4⁺Signal transduction in T cell leads to TH2, but does not cause TH1 to develop.These As a result the support of In vivo study has been obtained, these are research shows that the mistake for blocking OX40/OX40L interaction that TH2 is prevented to mediate The induction and maintenance of quick property immune response.However, blocking OX40/OX40L interaction that can improve or prevent the disease that TH1 is mediated Disease.In addition, the application of solubility OX40L or OX40L gene transfer show that strongly enhancing the antitumor of mouse exempts from into tumour Epidemic disease power.Nearest research is it is also shown that OX40/OX40L may play a role in the immune response for promoting cd8 t cell to mediate.Such as Discussed in this article, OX40 signal transduction blocks CD4⁺CD25⁺The inhibition function of naturally occurring regulatory T cells, and OX40/OX40L plays a crucial role in the immune the global regulation relative to tolerance in periphery.OX-40 antibody, OX-40 fusion Albumen and its application method are disclosed in U.S. Patent number: US 7,504,101；US7,758,852；US 7,858,765；US 7, 550,140；US 7,960,515；With US 9,006,399 and International Publication: WO 2003082919；WO 2003068819；WO 2006063067；WO2007084559；WO 2008051424；WO2012027328；And WO2013028231.

Herein, antigen-binding proteins of the invention (ABP) or anti-OX40 antigen-binding proteins are the albumen in conjunction with OX40, And in some embodiments, carry out one or more of: being transduceed by OX40 adjustment signal, adjust the function of OX40, Exciting OX40 signal transduction stimulates OX40 function or costimulation OX40 signal transduction.The embodiment 1 of United States Patent (USP) 9,006,399 It discloses OX40 and combines test.Those skilled in the art will readily recognize that establishing various other well known surveys of these functions Determine method.

In one embodiment, the OX40 antigen-binding proteins are WO2012/027328 (PCT/US2011/ 048752), albumen disclosed in international filing date on August 23rd, 2011.In another embodiment, the antigen-binding proteins Comprising WO2012/027328 (PCT/US2011/048752), antibody disclosed in international filing date on August 23rd, 2011 CDR, or the CDR with disclosed CDR sequence with 90% identity.The antigen-binding proteins include in another embodiment WO2012/027328 (PCT/US2011/048752), VH, VL of antibody disclosed in international filing date on August 23rd, 2011 or Both, or VH or VL with disclosed VH or VL sequence with 90% identity.

In another embodiment, the OX40 antigen-binding proteins are disclosed in WO2013/028231 (PCT/US2012/ 024570), international filing date on 2 9th, 2012.In another embodiment, the antigen-binding proteins include WO2013/ 028231 (PCT/US2012/024570), the CDR of antibody disclosed in international filing date on 2 9th, 2012, or with it is disclosed CDR sequence has the CDR of 90% identity.In another embodiment, the antigen-binding proteins include WO2013/028231 (PCT/US2012/024570), VH, VL of antibody disclosed in international filing date on 2 9th, 2012 or both, or with public affairs VH the or VL sequence opened has the VH or VL of 90% identity.

In another embodiment, anti-OX40ABP of the invention or antibody include CDR or VH shown in this paper Figure 30 to 41 Or VL sequence or there is one of the sequence of 90% identity or a variety of with it.

In one embodiment, anti-OX40ABP of the invention or antibody are any or combinations thereof comprising following CDR:

In some embodiments, anti-OX40ABP of the invention or antibody include to have at least 90% with SEQ ID NO:5 The heavy chain variable region of sequence identity.Suitably, OX40 binding protein of the invention may include having about with SEQ ID NO:5 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or The heavy chain variable region of 100% sequence identity.

Humanized heavy chain (V_H) variable region:

In one embodiment of the invention, the OX40ABP or antibody have the amino as shown in SEQ IDNO:11 The light chain variable region of acid sequence include CDRL1 (SEQ ID NO:7), CDRL2 (SEQ ID NO:8) and CDRL3 (SEQ ID NO: 9).In some embodiments, OX40 binding protein of the invention includes light chain variable region described in SEQ ID NO:11.One In a embodiment, OX40 binding protein of the invention includes the heavy chain variable region and SEQ ID NO:11 of SEQ ID NO:5 Light chain variable region.

Humanization light chain (V_L) variable region

In some embodiments, the OX40 binding protein of the invention includes and the ammonia as shown in SEQ IDNO:11 Base acid sequence has the light chain variable region of at least 90% sequence identity.Suitably, OX40 binding protein of the invention may include With SEQ ID NO:11 have about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, the light chain variable region of 97%, 98%, 99% or 100% sequence identity.

In another embodiment, anti-OX40ABP of the invention or antibody are any or combinations thereof comprising following CDR:

In some embodiments, anti-OX40ABP of the invention or antibody include to have and SEQ IDNO:17 at least 90% The heavy chain variable region of sequence identity.Suitably, OX40 binding protein of the invention may include having about with SEQ ID NO:17 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or The heavy chain variable region of 100% sequence identity.

Humanized heavy chain (V_H) variable region:

The OX40ABP described in one embodiment of the invention or antibody are with amino acid described in SEQ ID NO:23 The light chain variable region of sequence include CDRL1 (SEQ ID NO:19), CDRL2 (SEQID NO:20) and CDRL3 (SEQ ID NO: 21).In some embodiments, OX40 binding protein of the invention includes light chain variable region described in SEQ ID NO:23.? In one embodiment, OX40 binding protein of the invention includes the heavy chain variable region and SEQ ID NO:23 of SEQ ID NO:17 Light chain variable region.

Humanization light chain (V_L) variable region

In some embodiments, the OX40 binding protein of the invention includes and amino described in SEQ ID NO:23 Acid sequence has the light chain variable region of at least 90% sequence identity.Suitably, OX40 binding protein of the invention may include with SEQ ID NO:23 have about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, the light chain variable region of 97%, 98%, 99% or 100% sequence identity.

CDR or minimum combining unit can be modified by least one amino acid substitution, deletion or addition, wherein variant Antigen-binding proteins substantially retain the biological property of unmodified protein, such as include SEQ ID NO:5 and SEQ ID NO: 11 antibody or antibody comprising SEQ ID NO:17 and SEQ ID NO:23.

It should be appreciated that each of CDR H1, H2, H3, L1, L2, L3 can be modified individually in any combination or permutation Or modification is combined with any other CDR.In one embodiment, by substitution, delete or be added to more 3 amino acid (examples Such as 1 or 2 amino acid, such as 1 amino acid) modify CDR.In general, modification is to replace, especially conservative substitution, such as such as Shown in the following table 1.

Table 1

Side chain	Member
		Hydrophobic	Met, Ala, Val, Leu, Ile
Neutral hydrophilic	Cys, Ser, Thr
		It is acid	Asp, Glu
Alkalinity	Asn, Gln, His, Lys, Arg
		Influence the residue of chain orientation	Gly, Pro
Aromatics	Trp, Tyr, Phe

In one embodiment, ABP of the invention or antibody include the CDR of 106-222 antibody, for example, such as the present invention Shown in Figure 30-31, for example, CDRH1, CDRH2 with the amino acid sequence of SEQ ID NO 1,2 and 3 as disclosed in Figure 30 And CDRH3, and for example it is respectively provided with CDRL1, CDRL2 and CDRL3 of the sequence of SEQ IDNO 7,8 and 9.In an embodiment party In case, ABP of the invention or antibody include WO2012/027328 (PCT/US2011/048752), and international filing date 2011 8 The CDR of 106-222, Hu106 or Hu106-222 antibody disclosed in the moon 23.In another embodiment, of the invention anti- OX40ABP or antibody include the area VH and VL of 106-222 antibody shown in Figure 30-31 of the present invention, for example, having SEQ ID NO: The VH of 4 amino acid sequence and the as shown in figure 31 VL of the amino acid sequence with SEQ ID NO:10.In another embodiment In, ABP of the invention or antibody include the VH with the amino acid sequence of SEQ ID NO:5 in Figure 30 of the present invention, and have this The VL of the amino acid sequence of SEQ ID NO:11 in invention figure 31.In another embodiment, anti-OX40ABP of the invention or anti- Body includes WO2012/027328 (PCT/US2011/048752), Hu106-222 disclosed in international filing date on August 23rd, 2011 The area VH and VL of antibody or 106-222 antibody or Hu106 antibody.In another embodiment, anti-OX40ABP of the invention or anti- Body is 106-222, Hu106-222 or Hu106, for example, such as WO2012/027328 (PCT/US2011/048752), international application Disclosed on August 23rd, 2011 day.In another embodiment, ABP of the invention or antibody include and the sequence in the paragraph CDR or VH or VL or antibody sequence with 90% identity.

In another embodiment, anti-OX40ABP of the invention or antibody include the CDR of 119-122 antibody, for example, such as Shown in Figure 34-35 of the present invention, for example, be respectively provided with the amino acid sequence of SEQ ID NO 13,14 and 15 CDRH1, CDRH2 and CDRH3.In another embodiment, anti-OX40ABP of the invention or antibody include WO2012/027328 (PCT/US2011/ 048752), the CDR of 119-122 disclosed in international filing date on August 23rd, 2011 or Hu119 or Hu119-222 antibody.Another In one embodiment, anti-OX40ABP of the invention or antibody include the amino with the SEQ ID NO:16 in Figure 34 of the present invention The VH of acid sequence, and the VL of the amino acid sequence with the SEQ ID NO:22 in Figure 35 of the present invention.In another embodiment, Anti- OX40ABP of the invention or antibody include the VH of the amino acid sequence with SEQ ID NO:17 and with SEQ ID NO:23 Amino acid sequence VL.In another embodiment, anti-OX40ABP of the invention or antibody include WO2012/027328 (PCT/US2011/048752), 119-122 or Hu119 or Hu119-222 disclosed in international filing date on August 23rd, 2011 is anti- The area VH and VL of body.In another embodiment, ABP of the invention or antibody are anti-for 119-222 or Hu119 or Hu119-222 Body, for example, such as WO2012/027328 (PCT/US2011/048752), disclosed in international filing date on August 23rd, 2011.Another In one embodiment, ABP of the invention or antibody include the CDR or VH or VL for having 90% identity with the sequence in the paragraph Or antibody sequence.

In another embodiment, anti-OX40ABP of the invention or antibody include the CDR of 119-43-1 antibody, for example, such as Shown in Figure 38-39 of the present invention.In another embodiment, anti-OX40ABP of the invention or antibody include WO2013/028231 (PCT/US2012/024570), the CDR of 119-43-1 antibody disclosed in international filing date on 2 9th, 2012.In another implementation In scheme, anti-OX40ABP of the invention or antibody include one of area VH of 119-43-1 antibody shown in Figure 38-41 and the area VL it One.In another embodiment, anti-OX40ABP of the invention or antibody include WO2013/028231 (PCT/US2012/ 024570), the area VH and VL of 119-43-1 antibody disclosed in international filing date on 2 9th, 2012.In another embodiment, ABP or antibody of the invention is 119-43-1 or 119-43-1 chimera (chimeric) disclosed in Figure 38-41 of the present invention.Another In one embodiment, ABP of the invention or antibody such as WO2013/028231 (PCT/US2012/024570), international filing date On 2 9th, 2012 open.In other embodiments, either one or two of ABP or antibody described in the paragraph are humanizations.? In other embodiments, either one or two of ABP or antibody described in the paragraph are designed to prepare humanized antibody.In another implementation In scheme, ABP of the invention or antibody include the CDR or VH for having 90% identity with the sequence in the paragraph or VL or antibody Sequence.

In another embodiment, any mouse or chimeric sequences of any anti-OX40ABP of the present invention or antibody are modified To prepare humanized antibody.

In one embodiment, anti-OX40ABP of the invention or antibody include: (a) including the amino of SEQ ID NO:1 The heavy chain variable region CDR1 of acid sequence；(b) the heavy chain variable region CDR2 of the amino acid sequence comprising SEQ ID NO:2；(c) include The heavy chain variable region CDR3 of the amino acid sequence of SEQ ID NO.3；(d) light chain of the amino acid sequence comprising SEQ ID NO.7 Variable region CDR1；(e) the light chain variable region CDR2 of the amino acid sequence comprising SEQ ID NO.8；It (f) include SEQ ID The light chain variable region CDR3 of the amino acid sequence of NO.9.

In another embodiment, anti-OX40ABP of the invention or antibody include: (a) including the ammonia of SEQ ID NO:13 The heavy chain variable region CDR1 of base acid sequence；(b) the heavy chain variable region CDR2 of the amino acid sequence comprising SEQ ID NO:14；(c) The heavy chain variable region CDR3 of amino acid sequence comprising SEQ ID NO.15；(d) comprising the amino acid sequence of SEQ ID NO.19 Light chain variable region CDR1；(e) the light chain variable region CDR2 of the amino acid sequence comprising SEQ ID NO.20；It (f) include SEQ The light chain variable region CDR3 of the amino acid sequence of ID NO.21.

In another embodiment, anti-OX40ABP of the invention or antibody include: the ammonia comprising SEQ ID NO:1 or 13 The heavy chain variable region CDR1 of base acid sequence；The heavy chain variable region CDR2 of amino acid sequence comprising SEQ ID NO:2 or 14；With/ Or the heavy chain variable region CDR3 of the amino acid sequence comprising SEQ ID NO:3 or 15, or with its have 90% identity heavy chain Variable region CDR.

In another embodiment, anti-OX40ABP of the invention or antibody include: the ammonia comprising SEQ ID NO:7 or 19 The light chain variable region CDR1 of base acid sequence；The light chain variable region CDR2 of amino acid sequence comprising SEQ ID NO:8 or 20 and/or The light chain variable region CDR3 of amino acid sequence comprising SEQ ID NO:9 or 21, or have the heavy chain of 90% identity can with it Become area.

In another embodiment, anti-OX40ABP of the invention or antibody include: comprising SEQ ID NO:10,11,22 or The light chain variable region (" VL ") of 23 amino acid sequence, or with SEQ ID NO:10,11,22 or 23 amino acid sequence has extremely The amino acid sequence of few 90% identity.In another embodiment, anti-OX40ABP of the invention or antibody contain SEQ The amino acid sequence of ID NO:4,5,16 and 17, or have at least 90% with the amino acid sequence of SEQ ID NO:4,5,16 and 17 The heavy chain variable region (" VH ") of the amino acid sequence of identity.In another embodiment, anti-OX40ABP or antibody of the invention The variable light chain sequence of variable heavy chain sequence comprising SEQ ID NO:5 and SEQ ID NO:11, or it is same with 90% with it The sequence of property.In another embodiment, anti-OX40ABP of the invention or antibody include the variable heavy chain sequence of SEQ ID NO:17 Column and SEQ ID NO:23 variable light chain sequence or with its with 90% identity sequence.

In another embodiment, anti-OX40ABP of the invention or antibody include the nucleic acid by SEQ ID NO:12 or 24 Sequence has lightening for at least nucleic acid sequence encoding of 90% identity with the nucleotide sequence of SEQ ID NO:12 or 24 Chain.In another embodiment, anti-OX40ABP of the invention or antibody include SEQ ID NO:6 or 18 nucleic acid sequence or with The nucleotide sequence of SEQ ID NO:6 or 18 has the variable heavy chain of at least nucleic acid sequence encoding of 90% identity.

Monoclonal antibody is also provided herein.In one embodiment, the monoclonal antibody includes to contain SEQ ID The amino acid sequence of NO:10 or 22 or the amino with the amino acid sequence of SEQ ID NO:10 or 22 at least 90% identity The variable light of acid sequence.Also provide monoclonal antibody, it includes the amino acid sequence containing SEQ ID NO:4 or 16 or with The amino acid sequence of SEQ ID NO:4 or 16 has the variable heavy chain of at least amino acid sequence of 90% identity.

CTLA-4 is T cell surface molecular, initially by the differential screening of mouse cytolytic T cell cDNA library come It identifies (Brunet et al., Nature 328:267-270 (1987)).CTLA-4 be also immunoglobulin (Ig) superfamily at Member；CTLA-4 includes single extracellular Ig structural domain.CTLA-4 transcription is found in the T cell group with cytotoxic activity Object, show CTLA-4 may work in cytolytic reaction (Brunet et al., it is above；Brunet et al., Immunol.Rev.103-(21-36(1988)).Researcher report CTLA-4 (Dariavach et al., Eur.J.Immunol.18:1901-1905 (1988)) mankind's counterpart gene clone and navigate to same chromosomal region (2q33-34) is used as CD28 (Lafage-Pochitaloff et al., Immunogenetics 31:198-201 (1990)).It should People CTLA-4DNA discloses the significant homology of sequence compared with the sequence of coding CD28 albumen, in nearly film and cytosolic domain In have maximum homology (Brunet etc., 1988, ibid；Dariavach etc., 1988, ibid).Yervoy (she Monoclonal antibody) it is the complete people CTLA-4 antibody sold by Bristol Myers Squibb.The protein structure of her monoclonal antibody and make Method is described in U.S. Patent number 6,984,720 and 7,605,238.

Include, but are not limited to anti-CTLA 4 antibody, people's anti-CTLA 4 for the suitable anti-CTLA 4 antibody of method of the invention Antibody, mouse anti-CTLA 4 antibody, mammal anti-CTLA 4 antibody, humanization anti-CTLA 4 antibody, monoclonal anti-CTLA 4 antibody, It is Anti-TNF-α CTLA4 antibody, embedding and anti-CTLA 4 antibody, her monoclonal antibody, Sibutramine Hydrochloride mesh monoclonal antibody, anti-CD28 antibody, anti- CTLA4adnectins, anti-CTLA 4 domain antibodies, single-stranded anti-CTLA 4 segment, heavy chain anti-CTLA 4 segment, light chain anti-CTLA 4 Segment, the CTLA4 inhibitor of exciting costimulation approach, antibody, PCT Publication WO disclosed in PCT Publication WO2001/014424 Antibody disclosed in application number US 2005/0201994 disclosed in antibody, U.S. disclosed in 2004/035607 and authorization Europe are specially Antibody disclosed in benefit EP1212422B1.Other CTLA-4 antibody are described in U.S.Pat.Nos.5,811,097,5,855, 887,6,051,227 and 6,984,720；PCT Publication WO 01/14424 and WO 00/37504；With U.S. publication number US 2002/0039581 and US 2002/086014.The other anti-CTLA-4 antibody that can be used for the method for the present invention include, for example, below Those disclosed: WO 98/42752；U.S.Pat.Nos.6,682,736 and 6,207,156；Hurwitz et al., Proc.Natl.Acad.Sci.USA, 95 (17): 10067-10071 (1998)；Camacho et al., J.Clin.Oncology, 22 (145): Abstract No.2505 (2004) (antibody CP-675206)；Mokyr et al., cancer Res., 58:5301-5304 (1998) and U.S.Pat.Nos.5,977,318,6,682,736,7,109,003 and 7,132,281.

As used herein, " immunomodulator " or " immunomodulatory agents " refers to that the monoclonal including influencing immune system resists Any substance of body.In some embodiments, immunomodulator or immunomodulatory agents raise immune system.Immunomodulator It can be used as antitumor agent for treating cancer.For example, immunomodulator includes but is not limited to that (Opdivo/ receives force to anti-PD-1 antibody Monoclonal antibody and Keytruda/ pyridine aldoxime methyliodide (PAM) monoclonal antibody), anti-CTLA-4 antibody such as her monoclonal antibody (YERVOY) and anti-OX40 antibody.

As used in the present invention, term " agonist " refers to antigen-binding proteins, including but not limited to antibody, with it is common Signal transduction receptor causes one of following or a variety of when contacting: (1) stimulating or activate ICOS receptor；(2) enhance, increase or Promotion, induction or the activity, function or the presence that extend receptor；And/or (3) enhancing, increase, promotion or the expression of inducing receptor. Agonist activity can be measured in vitro by various analyses known in the art, such as, but not limited to measurement cell signal turns It leads, cell Proliferation, activated immune cell label, cell factor generate.It can also be by measurement surrogate end point (such as, but not limited to Measurement T cell proliferation or cell factor generate) various analyses measure agonist activity in vivo.

As used herein, term " antagonist " refers to antigen-binding proteins, including but not limited to antibody, in signal together Receptor causes one or more of when contacting: (1) weakening, blocks or inactivate receptor by its native ligand and/or or block The activation of receptor, (2) reduction, activity, function or the presence for reducing or shortening receptor and/or (3) reduction reduce, elimination receptor Expression.Antagonist activities can be measured in vitro by various measurements known in the art, such as, but not limited to measurement cell What signal transduction, cell Proliferation, activated immune cell label, cell factor generated increases or decreases.Antagonist activities can be with Various measurements by measuring surrogate end point measure in vivo, such as, but not limited to the survey of T cell proliferation or cell factor generation Amount.

As used in the present invention, term " cross competition combination " refer to by with any reagent competitive binding of the invention to target spot Reagent such as antibody.It can be tested by various methods known in the art for the combination competition between two kinds of antibody, including Flow cytometry, Meso Scale Discovery and ELISA.In conjunction with can be directly measured, it is meant that can be by two kinds Or more binding protein contacted with common signal transduction receptor, and one of or every kind of albumen combination can be measured. Alternatively, can in conjunction with or native ligand test the combination of interested molecule, and carry out quantitative comparison each other.

Term " antibody " refer to for broadest in the present invention with immunoglobulin like domain (such as IgG, IgM, IgA, IgD or IgE) molecule, and including monoclonal, recombination, polyclonal, chimeric, people, humanization, polyspecific Antibody includes bispecific antibody and Heteroconjugate antibodies；Single variable domains (such as V_H、V_HH、V_L, domain antibodies (dAb^TM)), antigen binding antibody fragment, Fab, F (ab') 2, Fv, disulfide bond connection Fv, scFv, disulfide bond connection ScFv, double antibody, TANDABS^TMDeng and any of the above-described kind of revision (summary of optional " antibody " form is referring to example Such as, Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No.9,1126-1136).

Selectable antibody formation includes that skeleton may be selected, and wherein one or more CDR of antigen-binding proteins can be by It is arranged in suitable non-immunoglobulin skeleton or essential part, such as affinity body, SpA skeleton, ldl receptor type A type structure Domain, avimer (see, e.g., U.S. Patent Application Publication No. 2005/0053973,2005/0089932,2005/0164301) Or EGF structural domain.

Term " structural domain " refers to the protein structure of folding, retains it independently of the three-level of the rest part of protein Structure.Structural domain is generally responsible for the discrete functional character of protein, and can be added in many cases, remove or Other protein are transferred to, the function of the rest part without losing protein and/or structural domain.

Term " single variable domain " refers to the folded polypeptide structural domain comprising antibody variable domains characteristic sequence.Therefore, it wraps Include such as V_H、V_HHAnd V_LComplete antibody variable domain and modified antibody variable domains (for example, wherein one or more rings are Through by antibody variable domains non-characteristic sequence substitution), be truncated or comprising the end N- or C- extend antibody variable domains, with And at least retain the fold segments of the combination activity of overall length structural domain and the variable domain of specificity.Single variable domain can be tied independently Close the antigen or epitope of different variable region or variable domain." domain antibodies " or " dAb^TM" may be considered that and " single variable domain " phase Together.Single variable domain can be the single variable domain of people, but also include the single variable domain from other species, as rodent cuts with scissors mouth Shark and Camelidae V_HH dAb^TM.Camelidae V_HHIt is derived from including camel, yamma, alpaca, the species of dromedary camel and guanaco Immunoglobulin single variable domain polypeptide, generate the natural heavy chain antibody for lacking light chain.Such V_HHStructural domain can root It is humanized according to the available standard technique in this field, and such structural domain is considered as " single variable domain ".Such as the present invention It is used, V_HIncluding Camelidae V_HHDomain.

Antigen-binding fragment can be provided by arranging one or more CDR on non-antibody protein skeleton.Such as this hair Bright used, " protein backbone " includes but is not limited to immunoglobulin (Ig) skeleton, such as can be four chains or two chain antibodies IgG skeleton, or can only the area Fc comprising antibody or its may include one or more constant regions from antibody, the wherein perseverance Determining area can be people or primate source, or can be artificial chimeric's body of people and primate constant region.

Protein backbone can be Ig skeleton, such as IgG or IgA skeleton.IgG skeleton may include some or complete of antibody Portion's structural domain is (that is, CH1, CH2, CH3, V_H、V_L).Antigen-binding proteins may include selected from IgG1, IgG2, IgG3, IgG4 or The IgG skeleton of IgG4PE.For example, skeleton can be IgG1.Skeleton can by antibody Fc district's groups at or comprising antibody the area Fc, Either part of it.

Affinity is a molecule (such as antigen-binding proteins of the invention) and another molecule (such as its target antigen) In the bond strength of single binding site.Balance method (such as enzyme linked immunosorbent assay (ELISA) (ELISA) or radioactivity can be passed through Immunoassays (RIA)) or dynamics (such as BIACORE^TMAnalysis) determine that the combination of antigen-binding proteins and its target is affine Power.For example, Biacore described in embodiment 5^TMMethod can be used for measuring binding affinity.

Affinity (avidity) is the summation for the intensity that two molecules are bonded to each other in multiple sites, for example, it is contemplated that arriving phase The valence mumber of interaction.

" separation " refers to the molecule, such as antigen-binding proteins or nucleic acid, removes from the nature environment for finding it. For example, molecule can be from therewith normally present in being purified in the substance in nature.For example, in sample molecule matter Amount can be the 95% of gross mass.

Term " expression vector " as used in the present invention, refers to the nucleic acid of separation, can be used for introducing interested nucleic acid In cell (such as eukaryocyte or prokaryotic cell) or Cell free expression system, wherein interested nucleic acid sequence is expressed as peptide Chain, such as protein.Such expression vector can be clay, plasmid, virus sequence, swivel base for example comprising interested nucleic acid Son and linear nucleic acid.Once expression vector is imported in cell or Cell free expression system (such as reticulocyte lysate), It is generated by the protein of interested nucleic acid encode by transcription/translating mechanism.Expression vector in the scope of the invention can be true Core or prokaryotic expression provide necessary element, and the carrier including viral promotors driving, such as the load of CMV promoter driving Body (such as pcDNA3.1, pCEP4 and its derivative), rhabdovirus expression vector, Drosophila expression vector；And by mammal Gene promoter, the expression vector driven such as people's Ig gene promoter.Other examples include prokaryotic expression carrier, such as T7 starting The carrier of the carrier (such as pET41) of son driving, the carrier of Lac operon driving and the driving of arabinose gene promoter.This Field is skilled artisan will realize that many other suitable expression vectors and expression system.

Term " recombinant host cell " as used in the present invention, refers to the cell comprising interested nucleic acid sequence, the core Acid sequence is separated before being introduced into cell.For example, interested nucleic acid sequence can be expression vector, and cell can be with It is protokaryon or eukaryon.Exemplary eukaryotic cell is mammalian cell, such as, but not limited to COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2,653, SP2/0, NS0,293, HeLa, myeloma cell, lymphoma cell or derivatives thereof. Most preferably, eukaryocyte is HEK293, NS0, SP2/0 or Chinese hamster ovary celI.Escherichia coli are exemplary prokaryotic cell.According to Recombination T cell of the invention can be generated by transfection, cell fusion, immortalization or other methods well known in the art.Transfection Enter the interested nucleic acid sequence of cell, such as expression vector, can be extrachromosomal or by stable integration to cell dyeing In body.

" chimeric antibody " refers to a kind of engineered antibody, contains the naturally occurring variable region derived from donor antibody (light chain and heavy chain) associates with the light chain and heavy chain constant region for being derived from receptor antibody.

" humanized antibody " refers to the engineered antibody type with the CDR derived from non-human donor immunoglobulin, should Other immunoglobulin-derived parts of molecule are derived from one or more human immunoglobulin(HIg)s.Furthermore it is possible to change frame branch Residue is supportted to keep binding affinity (see, for example, Queen et al., Proc.Natl Acad Sci USA, 86:10029- 10032 (1989), Hodgson et al., Bio/Technology, 9:421 (1991)).According to donor antibody nucleotide and The homology of amino acid sequence, can be by selecting suitable human receptor antibody, such as KABAT in routine data library^TMDatabase, Los Alamos database and Swiss Protein database.It is characterized in that homologous (based on amino with the framework region of donor antibody Acid) human antibody may be adapted to provide heavy chain constant region and/or weight chain variable framework region for insertion into donor CDR.It can be similar Mode select the suitable receptor antibody for being capable of providing constant region of light chain or variable framework region.It should be noted that receptor antibody Heavy chain and light chain are not needed from identical receptor antibody.Prior art describes several sides for producing this humanized antibody Method-is for example, see EP-A-0239400 and EP-A-054951.

Term " fully human antibodies " include with from human germline immunoglobulin's sequence variable region and constant region (if In the presence of) antibody.Human sequence's antibody of the invention may include not residual by the amino acid of human germline immunoglobulin's sequential coding Base (for example, the mutation introduced by external random or site-specific mutagenesis, or dashed forward by what internal somatic mutation introduced Become).Fully human antibodies include only by the amino acid sequence of the polynucleotide encoding of final human origin or identical as these sequences Amino acid sequence.As described herein, the antibody in the mouse genome generated in transgenic mice is inserted into (by encoding human The DNA encoding of immunoglobulin) it is fully human antibodies, because they are by being finally the DNA encoding of human origin.In such case Under, the DNA of encoding human immunoglobulin can be reset in mouse (with encoding antibody), and it can also happen that body cell is prominent Become.Antibody by undergoing the DNA encoding of the human origin of this variation in mouse is signified fully human antibodies of the invention.Make Allowed to select fully human antibodies for human antigen with such transgenic mice.As understood in the art, it can be used Display technique of bacteriophage prepares fully human antibodies, wherein people's DNA library is inserted into bacteriophage to generate includes ethnic group system DNA sequence dna Antibody.

Term " donor antibody " refers to the amino acid sequence tribute of its variable region, CDR or other function fragments or the like Dedicate the antibody of the first immunoglobulin partner to.Therefore, donor provides the immunoglobulin-encoding region of change, and leads to institute The antibody changed is expressed, the antibody which changes has the antigentic specificity and neutralization activity feature of donor antibody.

Term " receptor antibody " refers to the antibody heterologous with donor antibody, contributes and compiles to the first immunoglobulin partner Whole (or any part) amino acid sequence of its heavy chain of code and/or light chain framework region and/or its heavy chain and/or constant region of light chain Column.Human antibody can be receptor antibody.

Term used herein " V_H" and " V_L" respectively refer to the heavy chain variable region and light chain variable region of antigen-binding proteins.

" CDR " is defined as the complementary determining region amino acid sequence of antigen-binding proteins.These are heavy chain immunoglobulins With the hypervariable region of light chain.There are three heavy chain CDR and three light chain CDR (or CDR region) in the variable part of immunoglobulin. Therefore, " CDR " refers to all three heavy chain CDR, all three light chain CDR, whole heavy chain CDR and light chain as used in the present invention CDR or at least two CDR.

In the present specification, the amino acid residue in variable domain sequence and full length antibody sequence is according to Kabat numbering convention Number.Similarly, term used in embodiment " CDR ", " CDRL1 ", " CDRL2 ", " CDRL3 ", " CDRH1 ", " CDRH2 ", " CDRH3 " follows Kabat numbering convention.Related more information refers to Kabat et al., Sequences of Proteins Of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, National Institutes of Health(1991)。

It will be apparent to one skilled in the art that in variable domain sequence and full length antibody sequence, there are amino acid The alternative numbering convention of residue.Also there is the substitution numbering convention of CDR sequence, such as in Chothia et al. (1989) Nature Those of listed in 342:877-883.The structure and protein folding of antibody may imply that other residues are considered as CDR sequence A part, and understood by those skilled in the art.

Other numbering conventions of CDR sequence obtained by those skilled in the art include " AbM " (University of ) and " contact (contact) " (University College London) method Bath.Kabat, Chothia, AbM can be used Determine minimum overlay region to provide " minimum combining unit " at least two in contact method.Minimum combining unit can be The subdivision of CDR.

In one embodiment, the present invention provides pharmaceutical composition, and it includes the II type protein essences of therapeutically effective amount Propylhomoserin transmethylase (II type PRMT) inhibitor and the second pharmaceutical composition, second pharmaceutical composition include therapeutically effective amount Immunomodulator, wherein the immunomodulator is selected from: anti-CTLA 4 antibody or its antigen-binding fragment, anti-PD-1 antibody or Its antigen-binding fragment, anti-PDL1 antibody or its antigen-binding fragment and anti-OX40 antibody or its antigen-binding fragment.In a side Face, II type PRMT inhibitor are that Protein Arginine Methyltransferase 5 (PRMT5) inhibitor or protein arginine methyl shift Enzyme 9 (PRMT9) inhibitor.On the one hand, immunomodulator is anti-PD-1 antibody or its antigen-binding fragment.On the one hand, resist PD-1 antibody is pyridine aldoxime methyliodide (PAM) monoclonal antibody or the Wu Dankang that receives.On the other hand, immunomodulator is anti-OX40 antibody or its antigen binding fragment Section.On the other hand, immunomodulator is anti-OX40 antibody or its antigen-binding fragment, and it includes following one or more: such as CDRH1 shown in SEQ ID NO:1；The CDRH2 as shown in SEQ ID NO:2；The CDRH3 as shown in SEQ ID NO:3；Such as CDRL1 shown in SEQ ID NO:7；The CDRL2 as shown in the SEQ ID NO:8 and/or CDRL3 as shown in SEQ ID NO:9 Or the direct equivalent of each CDR, wherein direct equivalent has in the CDR is no more than two amino acid substitutions.Another Aspect, immunomodulator be anti-OX40 antibody or its antigen-binding fragment, it includes with the amino acid as shown in SEQ ID NO:5 Sequence has at least variable heavy chain sequence of 90% sequence identity and has with the amino acid sequence as shown in SEQ ID NO:11 There is the variable light chain sequence of at least 90% sequence identity.On the one hand, II type PRMT inhibitor be formula III, IV, VII, The compound of VIII, IX, X or XI.On the other hand, II type PRMT inhibitor is compound B.On the one hand, II type PRMT presses down Preparation is compound C.In one embodiment, II type protein arginine transmethylase (II type PRMT) inhibition is provided The combination of agent and immunomodulator, wherein the II type PRMT inhibitor is compound C and the immunomodulator is agonist Anti- OX40 antibody or its antigen-binding fragment.In one embodiment, II type protein arginine transmethylase is provided The combination of (II type PRMT) inhibitor and immunomodulator, wherein the II type PRMT inhibitor is compound C and described immune Regulator is anti-OX40 antibody or its antigen-binding fragment, and it includes following one or more: as shown in SEQ ID NO:1 CDRH1；The CDRH2 as shown in SEQ ID NO:2；The CDRH3 as shown in SEQ ID NO:3；As shown in SEQ ID NO:7 CDRL1；The CDRL2's as shown in the SEQ ID NO:8 and/or CDRL3 as shown in SEQ ID NO:9 or each CDR is directly equivalent Object, wherein direct equivalent has in the CDR is no more than two amino acid substitutions.In one embodiment, it provides The combination of II type protein arginine transmethylase (II type PRMT) inhibitor and immunomodulator, wherein the II type PRMT Inhibitor is compound C and the immunomodulator is anti-OX40 antibody or its antigen-binding fragment, it includes with SEQ ID NO:5 has the light of at least 90% identity at least heavy chain variable region of 90% sequence identity and with SEQ ID NO:11 Chain variable region.

In another embodiment, the present invention provides pharmaceutical composition, and it includes the II type protein essences of therapeutically effective amount Propylhomoserin transmethylase (II type PRMT) inhibitor and the second pharmaceutical composition, second pharmaceutical composition include therapeutically effective amount Immunomodulator selected from the following: anti-CTLA 4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen binding fragment Section, anti-PDL1 antibody or its antigen-binding fragment and anti-OX40 antibody or its antigen-binding fragment.On the one hand, II type PRMT Inhibitor is Protein Arginine Methyltransferase 5 (PRMT5) inhibitor or protein arginine transmethylase 9 (PRMT9) Inhibitor.On the one hand, immunomodulator is anti-PD-1 antibody or its antigen-binding fragment.On the one hand, anti-PD-1 antibody is Pyridine aldoxime methyliodide (PAM) monoclonal antibody or the Wu Dankang that receives.On the other hand, immunomodulator is anti-OX40 antibody or its antigen-binding fragment.In another party Face, immunomodulator are anti-OX40 antibody or its antigen-binding fragment, and it includes following one or more: such as SEQ ID NO:1 Shown in CDRH1；The CDRH2 as shown in SEQ ID NO:2；The CDRH3 as shown in SEQ ID NO:3；Such as SEQ ID NO:7 institute The CDRL1 shown；The CDRL2's as shown in the SEQ ID NO:8 and/or CDRL3 as shown in SEQ ID NO:9 or each CDR is direct Equivalent, wherein direct equivalent has in the CDR is no more than two amino acid substitutions.On the other hand, immunological regulation Agent is anti-OX40 antibody or its antigen-binding fragment, and it includes have at least with the amino acid sequence as shown in SEQ ID NO:5 The variable heavy chain sequence of 90% sequence identity and with the amino acid sequence as shown in SEQ ID NO:11 have at least 90% sequence The variable light chain sequence of column identity.On the one hand, II type PRMT inhibitor is formula III, IV, VII, VIII, IX, X or XI Compound.On the other hand, II type PRMT inhibitor is compound B.On the one hand, II type PRMT inhibitor is compound C.? In one embodiment, the present invention provides pharmaceutical composition, and it includes the II type protein arginine methyl of therapeutically effective amount to turn Enzyme (II type PRMT) inhibitor and the second pharmaceutical composition are moved, which includes the immunological regulation of therapeutically effective amount Agent, wherein the II type PRMT inhibitor is compound C and the immunomodulator is the anti-OX40 antibody of agonist or its antigen Binding fragment.In one embodiment, pharmaceutical composition is provided, it includes the II type protein arginine first of therapeutically effective amount Based transferase (II type PRMT) inhibitor and the second pharmaceutical composition, second pharmaceutical composition include the immune of therapeutically effective amount Regulator, wherein the II type PRMT inhibitor is compound C and the immunomodulator is anti-OX40 antibody or its antigen knot Segment is closed, it includes following one or more: the CDRH1 as shown in SEQ ID NO:1；As shown in SEQ ID NO:2 CDRH2；The CDRH3 as shown in SEQ ID NO:3；The CDRL1 as shown in SEQ ID NO:7；As shown in SEQ ID NO:8 The direct equivalent of CDRL2 and/or the CDRL3 as shown in SEQ ID NO:9 or each CDR, wherein direct equivalent is in the CDR In have be no more than two amino acid substitutions.In another embodiment, pharmaceutical composition is provided, it includes therapeutically effective amounts II type protein arginine transmethylase (II type PRMT) inhibitor and the second pharmaceutical composition, the second pharmaceutical composition packet Immunomodulator containing therapeutically effective amount, wherein the II type PRMT inhibitor is compound C and the immunomodulator is anti- OX40 antibody or its antigen-binding fragment, it includes the weight chain variables with SEQ ID NO:5 at least 90% sequence identity Area and the light chain variable region with SEQ ID NO:11 at least 90% identity.

In one embodiment, the method for the treating cancer in the people of needs is provided, the method includes applying to people With the combination of II type protein arginine transmethylase (II type PRMT) inhibitor and immunomodulator, and following at least one Kind: pharmaceutically acceptable carrier and pharmaceutically acceptable diluent, so that the cancer of people is treated, wherein the immunological regulation Agent is selected from: anti-CTLA 4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PDL1 antibody or its Antigen-binding fragment and anti-OX40 antibody or its antigen-binding fragment.On the one hand, II type PRMT inhibitor is protein essence ammonia (PRMT5) inhibitor of acid methyltransferase 5 or protein arginine transmethylase 9 (PRMT9) inhibitor.On the one hand, exempt from Epidemic disease regulator is anti-PD-1 antibody or its antigen-binding fragment.On the one hand, anti-PD-1 antibody is pyridine aldoxime methyliodide (PAM) monoclonal antibody or the Wu Dankang that receives. On the other hand, immunomodulator is anti-OX40 antibody or its antigen-binding fragment.On the other hand, immunomodulator is anti- OX40 antibody or its antigen-binding fragment, it includes following one or more: the CDRH1 as shown in SEQ ID NO:1；Such as SEQ CDRH2 shown in ID NO:2；The CDRH3 as shown in SEQ ID NO:3；The CDRL1 as shown in SEQ ID NO:7；Such as SEQ ID The direct equivalent of CDRL2 shown in NO:8 and/or the CDRL3 as shown in SEQ ID NO:9 or each CDR, wherein directly equivalent Object has in the CDR is no more than two amino acid substitutions.On the other hand, immunomodulator is anti-OX40 antibody or it is anti- Former binding fragment, it includes have the variable of at least 90% sequence identity with the amino acid sequence as shown in SEQ ID NO:5 Sequence of heavy chain and the variable light sequence with the amino acid sequence as shown in SEQ ID NO:11 at least 90% sequence identity Column.On the one hand, II type PRMT inhibitor is the compound of formula III, IV, VII, VIII, IX, X or XI.On the other hand, II Type PRMT inhibitor is compound B.On the one hand, II type PRMT inhibitor is compound C.On the one hand, II type PRMT inhibits Agent and immunomodulator deliver medicine to patient with approach selected from the following: simultaneously, in succession, in any order, whole body, oral, vein In interior and tumor.On the other hand, II type PRMT inhibitor is administered orally.In one embodiment, it provides in the people of needs The method for the treatment of cancer, the method includes administering to the human compound C and the anti-OX40 antibody of agonist or its antigen-binding fragment Combination.In another embodiment, the method for the treating cancer in the people of needs is provided, the method includes administering to the human The combination of object C and anti-OX40 antibody or its antigen-binding fragment are closed, the anti-OX40 antibody or its antigen-binding fragment include following It is one or more: the CDRH1 as shown in SEQ ID NO:1；The CDRH2 as shown in SEQ ID NO:2；Such as SEQ ID NO:3 institute The CDRH3 shown；The CDRL1 as shown in SEQ ID NO:7；The CDRL2 as shown in SEQ ID NO:8 and/or such as SEQ ID NO:9 Shown in CDRL3 or each CDR direct equivalent, wherein direct equivalent in the CDR have be no more than two amino acid Replace.In another embodiment, the method for the treating cancer in the people of needs is provided, the method includes administering to the human chemical combination The combination of object C and anti-OX40 antibody or its antigen-binding fragment, the anti-OX40 antibody or its antigen-binding fragment include and SEQ ID NO:5 has at least 90% identity at least heavy chain variable region of 90% sequence identity and with SEQ ID NO:11 Light chain variable region.

In another embodiment, the method for the treating cancer in the people of needs is provided, the method includes applying to people With the pharmaceutical composition and packet comprising II type protein arginine transmethylase (II type PRMT) inhibitor of therapeutically effective amount Pharmaceutical composition containing immunomodulator selected from the following: anti-CTLA 4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its Antigen-binding fragment, anti-PDL1 antibody or its antigen-binding fragment and anti-OX40 antibody or its antigen-binding fragment, to treat The cancer of people.On the one hand, II type PRMT inhibitor is Protein Arginine Methyltransferase 5 (PRMT5) inhibitor or albumen Matter arginine methyltransferase 9 (PRMT9) inhibitor.On the one hand, immunomodulator is anti-PD-1 antibody or its antigen binding Segment.On the one hand, anti-PD-1 antibody is pyridine aldoxime methyliodide (PAM) monoclonal antibody or the Wu Dankang that receives.On the other hand, immunomodulator is anti-for anti-OX40 Body or its antigen-binding fragment.On the other hand, immunomodulator be anti-OX40 antibody or its antigen-binding fragment, it includes with It is next or multiple: the CDRH1 as shown in SEQ ID NO:1；The CDRH2 as shown in SEQ ID NO:2；Such as SEQ ID NO:3 Shown in CDRH3；The CDRL1 as shown in SEQ ID NO:7；The CDRL2 as shown in SEQ ID NO:8 and/or such as SEQ ID The direct equivalent of CDRL3 shown in NO:9 or each CDR, wherein direct equivalent has in the CDR is no more than two ammonia Base acid replaces.On the other hand, immunomodulator be anti-OX40 antibody or its antigen-binding fragment, it includes with such as SEQ ID Amino acid sequence shown in NO:5 have at least the variable heavy chain sequence of 90% sequence identity and with such as SEQ ID NO:11 institute The amino acid sequence shown has the variable light chain sequence of at least 90% sequence identity.On the one hand, II type PRMT inhibitor is The compound of formula III, IV, VII, VIII, IX, X or XI.On the other hand, II type PRMT inhibitor is compound B.In a side Face, II type PRMT inhibitor are compound C.On the one hand, II type PRMT inhibitor and immunomodulator are with way selected from the following Diameter delivers medicine to patient: simultaneously, in succession, in any order, in whole body, oral, intravenous and tumor.On the one hand, II type PRMT presses down Preparation oral administration.In one embodiment, provide the method for the treating cancer in the people of needs, the method includes to People applies the pharmaceutical composition comprising compound C of therapeutically effective amount and includes the anti-OX40 antibody of agonist or its antigen binding fragment The pharmaceutical composition of section.In another embodiment, the method for the treating cancer in the people of needs is provided, the method includes Administer to the human the pharmaceutical composition comprising compound C of therapeutically effective amount and comprising anti-OX40 antibody or its antigen-binding fragment Pharmaceutical composition, the anti-OX40 antibody or its antigen-binding fragment include following one or more: as shown in SEQ ID NO:1 CDRH1；The CDRH2 as shown in SEQ ID NO:2；The CDRH3 as shown in SEQ ID NO:3；As shown in SEQ ID NO:7 CDRL1；The CDRL2's as shown in the SEQ ID NO:8 and/or CDRL3 as shown in SEQ ID NO:9 or each CDR is directly equivalent Object, wherein direct equivalent has in the CDR is no more than two amino acid substitutions.In another embodiment, it provides The method for the treatment of cancer in the people of needs, the method includes administering to the human the drug comprising compound C of therapeutically effective amount Composition and the second pharmaceutical composition comprising anti-OX40 antibody or its antigen-binding fragment, the anti-OX40 antibody or its antigen Binding fragment includes to have at least heavy chain variable region of 90% sequence identity with SEQ ID NO:5 and have with SEQ ID NO:11 There is the light chain variable region of at least 90% identity.

In another embodiment, the present invention provides II type protein arginine transmethylase (II type PRMT) inhibitor With the combination purposes in medicine preparation of immunomodulator, wherein the immunomodulator is selected from: anti-CTLA 4 antibody or it is anti- Former binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PDL1 antibody or its antigen-binding fragment and anti-OX40 antibody Or its antigen-binding fragment.In another embodiment, the present invention provides II type protein arginine transmethylase (II type PRMT) combination of inhibitor and immunomodulator is used for the purposes for the treatment of cancer, wherein the immunomodulator is selected from anti-CTLA 4 Antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PDL1 antibody or its antigen-binding fragment and Anti- OX40 antibody or its antigen-binding fragment.On the one hand, II type PRMT inhibitor is Protein Arginine Methyltransferase 5 (PRMT5) inhibitor or protein arginine transmethylase 9 (PRMT9) inhibitor.On the one hand, II type PRMT inhibitor is Protein Arginine Methyltransferase 5 (PRMT5) inhibitor or protein arginine transmethylase 9 (PRMT9) inhibitor.? On the one hand, immunomodulator is anti-PD-1 antibody or its antigen-binding fragment.On the one hand, anti-PD-1 antibody be pyridine aldoxime methyliodide (PAM) monoclonal antibody or Receive Wu Dankang.On the other hand, immunomodulator is anti-OX40 antibody or its antigen-binding fragment.On the other hand, it is immunized and adjusts Saving agent is anti-OX40 antibody or its antigen-binding fragment, and it includes following one or more: as shown in SEQ ID NO:1 CDRH1；The CDRH2 as shown in SEQ ID NO:2；The CDRH3 as shown in SEQ ID NO:3；As shown in SEQ ID NO:7 CDRL1；The CDRL2's as shown in the SEQ ID NO:8 and/or CDRL3 as shown in SEQ ID NO:9 or each CDR is directly equivalent Object, wherein direct equivalent has in the CDR is no more than two amino acid substitutions.On the other hand, immunomodulator is Anti- OX40 antibody or its antigen-binding fragment, it includes have at least 90% with the amino acid sequence as shown in SEQ ID NO:5 The variable heavy chain sequence of sequence identity and with the amino acid sequence as shown in SEQ ID NO:11 have at least 90% sequence it is same The variable light chain sequence of one property.On the one hand, II type PRMT inhibitor is the chemical combination of formula III, IV, VII, VIII, IX, X or XI Object.On the other hand, II type PRMT inhibitor is compound B.On the one hand, II type PRMT inhibitor is compound C.In a side Face, II type PRMT inhibitor and immunomodulator deliver medicine to patient with approach selected from the following: simultaneously, in succession, in any order, In whole body, oral, intravenous and tumor.On the one hand, II type PRMT inhibitor is administered orally.In one embodiment, it provides The use of the combination of II type protein arginine transmethylase (II type PRMT) inhibitor and immunomodulator in medicine preparation On the way, wherein the II type PRMT inhibitor is compound C and the immunomodulator is the anti-OX40 antibody of agonist or its antigen Binding fragment.In one embodiment, II type protein arginine transmethylase (II type PRMT) inhibitor is provided and is exempted from The purposes of the combination of epidemic disease regulator in medicine preparation, wherein the II type PRMT inhibitor is compound C and the immune tune Saving agent is anti-OX40 antibody or its antigen-binding fragment, and it includes following one or more: as shown in SEQ ID NO:1 CDRH1；The CDRH2 as shown in SEQ ID NO:2；The CDRH3 as shown in SEQ ID NO:3；As shown in SEQ ID NO:7 CDRL1；The CDRL2's as shown in the SEQ ID NO:8 and/or CDRL3 as shown in SEQ ID NO:9 or each CDR is directly equivalent Object, wherein direct equivalent has in the CDR is no more than two amino acid substitutions.In one embodiment, II is provided The purposes of the combination of type protein arginine transmethylase (II type PRMT) inhibitor and immunomodulator in medicine preparation, Wherein the II type PRMT inhibitor is compound C and the immunomodulator is anti-OX40 antibody or its antigen-binding fragment, It includes have at least with SEQ ID NO:5 at least heavy chain variable region of 90% sequence identity and with SEQ ID NO:11 The light chain variable region of 90% identity.

In another embodiment, the present invention provides II type protein arginine transmethylase (II type PRMT) inhibitor With the combination of immunomodulator, it is used for treating cancer, wherein the immunomodulator is selected from anti-CTLA 4 antibody or its antigen knot Close segment, anti-PD-1 antibody or its antigen-binding fragment, anti-PDL1 antibody or its antigen-binding fragment and anti-OX40 antibody or its Antigen-binding fragment.On the one hand, II type PRMT inhibitor be Protein Arginine Methyltransferase 5 (PRMT5) inhibitor or Protein arginine transmethylase 9 (PRMT9) inhibitor.On the one hand, immunomodulator is anti-PD-1 antibody or its antigen Binding fragment.On the one hand, anti-PD-1 antibody is pyridine aldoxime methyliodide (PAM) monoclonal antibody or the Wu Dankang that receives.On the other hand, immunomodulator is anti- OX40 antibody or its antigen-binding fragment.On the other hand, immunomodulator be anti-OX40 antibody or its antigen-binding fragment, Include following one or more: the CDRH1 as shown in SEQ ID NO:1；The CDRH2 as shown in SEQ ID NO:2；Such as SEQ ID CDRH3 shown in NO:3；The CDRL1 as shown in SEQ ID NO:7；The CDRL2 as shown in SEQ ID NO:8 and/or such as SEQ The direct equivalent of CDRL3 shown in ID NO:9 or each CDR, wherein direct equivalent has in the CDR is no more than two Amino acid substitution.On the other hand, immunomodulator be anti-OX40 antibody or its antigen-binding fragment, it includes with such as SEQ ID Amino acid sequence shown in NO:5 have at least the variable heavy chain sequence of 90% sequence identity and with such as SEQ ID NO:11 institute The amino acid sequence shown has the variable light chain sequence of at least 90% sequence identity.On the one hand, II type PRMT inhibitor is The compound of formula III, IV, VII, VIII, IX, X or XI.On the other hand, II type PRMT inhibitor is compound B.In a side Face, II type PRMT inhibitor are compound C.On the one hand, II type PRMT inhibitor and immunomodulator are with way selected from the following Diameter delivers medicine to patient: simultaneously, in succession, in any order, in whole body, oral, intravenous and tumor.On the one hand, II type PRMT presses down Preparation oral administration.In one embodiment, II type protein arginine transmethylase (II type PRMT) inhibitor is provided With the combination of immunomodulator, it is used for treating cancer, wherein the II type PRMT inhibitor is compound C and the immune tune Saving agent is the anti-OX40 antibody of agonist or its antigen-binding fragment.In one embodiment, II type protein arginine is provided The combination of transmethylase (II type PRMT) inhibitor and immunomodulator, is used for treating cancer, wherein the II type PRMT Inhibitor is compound C and the immunomodulator is anti-OX40 antibody or its antigen-binding fragment, it includes with next or It is multiple: the CDRH1 as shown in SEQ ID NO:1；The CDRH2 as shown in SEQ ID NO:2；As shown in SEQ ID NO:3 CDRH3；The CDRL1 as shown in SEQ ID NO:7；The CDRL2 as shown in SEQ ID NO:8 and/or as shown in SEQ ID NO:9 CDRL3 or each CDR direct equivalent, wherein direct equivalent in the CDR have be no more than two amino acid substitutions. In one embodiment, II type protein arginine transmethylase (II type PRMT) inhibitor and immunomodulator are provided Combination, is used for treating cancer, wherein the II type PRMT inhibitor is compound C and the immunomodulator is anti-OX40 anti- Body or its antigen-binding fragment, it includes with SEQ ID NO:5 have at least the heavy chain variable region of 90% sequence identity and with SEQ ID NO:11 has the light chain variable region of at least 90% identity.

In one embodiment, the production comprising II type PRMT inhibitor and immunomodulator selected from the following is provided Product: anti-CTLA 4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PDL1 antibody or its antigen Binding fragment and anti-OX40 antibody or its antigen-binding fragment are used in medication simultaneously, separately or phase as combination preparation After use.On the one hand, II type PRMT inhibitor is Protein Arginine Methyltransferase 5 (PRMT5) inhibitor or protein Arginine methyltransferase 9 (PRMT9) inhibitor.On the one hand, immunomodulator is anti-PD-1 antibody or its antigen binding fragment Section.On the one hand, anti-PD-1 antibody is pyridine aldoxime methyliodide (PAM) monoclonal antibody or the Wu Dankang that receives.On the other hand, immunomodulator is anti-OX40 antibody Or its antigen-binding fragment.On the other hand, immunomodulator is anti-OX40 antibody or its antigen-binding fragment, and it includes following It is one or more: the CDRH1 as shown in SEQ ID NO:1；The CDRH2 as shown in SEQ ID NO:2；Such as SEQ ID NO:3 institute The CDRH3 shown；The CDRL1 as shown in SEQ ID NO:7；The CDRL2 as shown in SEQ ID NO:8 and/or such as SEQ ID NO:9 Shown in CDRL3 or each CDR direct equivalent, wherein direct equivalent in the CDR have be no more than two amino acid Replace.On the other hand, immunomodulator be anti-OX40 antibody or its antigen-binding fragment, it includes with such as SEQ ID NO:5 Shown in amino acid sequence have at least the variable heavy chain sequence of 90% sequence identity and with as shown in SEQ ID NO:11 Amino acid sequence has the variable light chain sequence of at least 90% sequence identity.On the one hand, II type PRMT inhibitor is formula The compound of III, IV, VII, VIII, IX, X or XI.On the other hand, II type PRMT inhibitor is compound B.On the one hand, II type PRMT inhibitor is compound C.On the one hand, II type PRMT inhibitor and immunomodulator are given with approach selected from the following Medicine is in patient: simultaneously, in succession, in any order, in whole body, oral, intravenous and tumor.On the one hand, II type PRMT inhibitor Oral administration.In one embodiment, it provides comprising compound C and the anti-OX40 antibody of agonist or its antigen-binding fragment Product, be used in medication simultaneously, separately or sequentially with.In one embodiment, it provides comprising compound C and resists The product of OX40 antibody or its antigen-binding fragment is used in medication simultaneously, separately or sequentially with wherein described anti- OX40 antibody or its antigen-binding fragment include following one or more: the CDRH1 as shown in SEQ ID NO:1；Such as SEQ ID CDRH2 shown in NO:2；The CDRH3 as shown in SEQ ID NO:3；The CDRL1 as shown in SEQ ID NO:7；Such as SEQ ID The direct equivalent of CDRL2 shown in NO:8 and/or the CDRL3 as shown in SEQ ID NO:9 or each CDR, wherein directly equivalent Object has in the CDR is no more than two amino acid substitutions.In one embodiment, it provides comprising compound C and resists The product of OX40 antibody or its antigen-binding fragment is used in medication simultaneously, separately or sequentially with wherein described anti- OX40 antibody or its antigen-binding fragment include with SEQ ID NO:5 have at least heavy chain variable region of 90% sequence identity and There is the light chain variable region of at least 90% identity with SEQ ID NO:11.

In one embodiment, the product comprising II type PRMT inhibitor and immunomodulator selected from the following is provided: Anti-CTLA 4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PDL1 antibody or its antigen binding Segment and anti-OX40 antibody or its antigen-binding fragment, as combination preparation for making simultaneously, separately or in succession in medication With.On the one hand, II type PRMT inhibitor is Protein Arginine Methyltransferase 5 (PRMT5) inhibitor or protein essence ammonia Acid methyltransferase 9 (PRMT9) inhibitor.On the one hand, immunomodulator is anti-PD-1 antibody or its antigen-binding fragment.? On the one hand, anti-PD-1 antibody is pyridine aldoxime methyliodide (PAM) monoclonal antibody or the Wu Dankang that receives.On the other hand, immunomodulator is anti-OX40 antibody or it is anti- Former binding fragment.On the other hand, immunomodulator is anti-OX40 antibody or its antigen-binding fragment, it includes with next or It is multiple: the CDRH1 as shown in SEQ ID NO:1；The CDRH2 as shown in SEQ ID NO:2；As shown in SEQ ID NO:3 CDRH3；The CDRL1 as shown in SEQ ID NO:7；The CDRL2 as shown in SEQ ID NO:8 and/or as shown in SEQ ID NO:9 CDRL3 or each CDR direct equivalent, wherein direct equivalent in the CDR have be no more than two amino acid substitutions. On the other hand, immunomodulator be anti-OX40 antibody or its antigen-binding fragment, it includes with as shown in SEQ ID NO:5 Amino acid sequence have at least the variable heavy chain sequence of 90% sequence identity and with the amino acid as shown in SEQ ID NO:11 Sequence has the variable light chain sequence of at least 90% sequence identity.On the one hand, II type PRMT inhibitor be formula III, IV, The compound of VII, VIII, IX, X or XI.On the other hand, II type PRMT inhibitor is compound B.On the one hand, II type PRMT inhibitor is compound C.On the one hand, II type PRMT inhibitor and immunomodulator are delivered medicine to approach selected from the following Patient: simultaneously, in succession, in any order, in whole body, oral, intravenous and tumor.On the one hand, II type PRMT inhibitor is oral Administration.In one embodiment, the production comprising compound C and the anti-OX40 antibody of agonist or its antigen-binding fragment is provided Product, be used in medication simultaneously, separately or sequentially with.In one embodiment, it provides comprising compound C and anti-OX40 The product of antibody or its antigen-binding fragment is used in medication simultaneously, separately or sequentially with wherein the anti-OX40 is anti- Body or its antigen-binding fragment include following one or more: the CDRH1 as shown in SEQ ID NO:1；Such as SEQ ID NO:2 institute The CDRH2 shown；The CDRH3 as shown in SEQ ID NO:3；The CDRL1 as shown in SEQ ID NO:7；As shown in SEQ ID NO:8 CDRL2 and/or the CDRL3 as shown in SEQ ID NO:9 or each CDR direct equivalent, wherein direct equivalent is described Have in CDR and is no more than two amino acid substitutions.In one embodiment, provide comprising compound C and anti-OX40 antibody or The product of its antigen-binding fragment, be used in medication simultaneously, separately or sequentially with, wherein the anti-OX40 antibody or its Antigen-binding fragment include with SEQ ID NO:5 have at least the heavy chain variable region of 90% sequence identity and with SEQ ID NO: 11 light chain variable regions at least 90% identity.

In one embodiment, the product comprising II type PRMT inhibitor and immunomodulator selected from the following is provided: Anti-CTLA 4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PDL1 antibody or its antigen binding Segment and anti-OX40 antibody or its antigen-binding fragment are used for as combination preparation simultaneously, separately or sequentially with treatment The cancer of people experimenter.On the one hand, II type PRMT inhibitor is Protein Arginine Methyltransferase 5 (PRMT5) inhibitor Or protein arginine transmethylase 9 (PRMT9) inhibitor.On the one hand, immunomodulator is anti-PD-1 antibody or it is anti- Former binding fragment.On the one hand, anti-PD-1 antibody is pyridine aldoxime methyliodide (PAM) monoclonal antibody or the Wu Dankang that receives.On the other hand, immunomodulator is anti- OX40 antibody or its antigen-binding fragment.On the other hand, immunomodulator be anti-OX40 antibody or its antigen-binding fragment, Include following one or more: the CDRH1 as shown in SEQ ID NO:1；The CDRH2 as shown in SEQ ID NO:2；Such as SEQ ID CDRH3 shown in NO:3；The CDRL1 as shown in SEQ ID NO:7；The CDRL2 as shown in SEQ ID NO:8 and/or such as SEQ The direct equivalent of CDRL3 shown in ID NO:9 or each CDR, wherein direct equivalent has in the CDR is no more than two Amino acid substitution.On the other hand, immunomodulator be anti-OX40 antibody or its antigen-binding fragment, it includes with such as SEQ ID Amino acid sequence shown in NO:5 have at least the variable heavy chain sequence of 90% sequence identity and with such as SEQ ID NO:11 institute The amino acid sequence shown has the variable light chain sequence of at least 90% sequence identity.On the one hand, II type PRMT inhibitor is The compound of formula III, IV, VII, VIII, IX, X or XI.On the other hand, II type PRMT inhibitor is compound B.In a side Face, II type PRMT inhibitor are compound C.On the one hand, II type PRMT inhibitor and immunomodulator are with way selected from the following Diameter delivers medicine to patient: simultaneously, in succession, in any order, in whole body, oral, intravenous and tumor.On the one hand, II type PRMT presses down Preparation oral administration.In one embodiment, it provides comprising compound C and the anti-OX40 antibody of agonist or its antigen binding fragment The product of section, is used for simultaneously, separately or sequentially with the cancer to treat people experimenter.In one embodiment, it provides Product comprising compound C and anti-OX40 antibody or its antigen-binding fragment is used for simultaneously, separately or sequentially with treatment The cancer of people experimenter, wherein the anti-OX40 antibody or its antigen-binding fragment include following one or more: such as SEQ ID CDRH1 shown in NO:1；The CDRH2 as shown in SEQ ID NO:2；The CDRH3 as shown in SEQ ID NO:3；Such as SEQ ID CDRL1 shown in NO:7；The CDRL2 as shown in the SEQ ID NO:8 and/or CDRL3 as shown in SEQ ID NO:9 or each CDR Direct equivalent, wherein direct equivalent in the CDR have be no more than two amino acid substitutions.In an embodiment In, the product comprising compound C and anti-OX40 antibody or its antigen-binding fragment is provided, is used to make simultaneously, separately or in succession To treat the cancer of people experimenter, wherein the anti-OX40 antibody or its antigen-binding fragment include to have with SEQ ID NO:5 There is at least heavy chain variable region of 90% sequence identity and there is the light chain variable of at least 90% identity with SEQ ID NO:11 Area.

In one embodiment, the product comprising II type PRMT inhibitor and immunomodulator selected from the following is provided: Anti-CTLA 4 antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, anti-PDL1 antibody or its antigen binding Segment and anti-OX40 antibody or its antigen-binding fragment are used for as combination preparation simultaneously, separately or sequentially with treatment The cancer of people experimenter, wherein the cancer is colon cancer or lymthoma.On the one hand, II type PRMT inhibitor is protein essence (PRMT5) inhibitor of propylhomoserin transmethylase 5 or protein arginine transmethylase 9 (PRMT9) inhibitor.On the one hand, Immunomodulator is anti-PD-1 antibody or its antigen-binding fragment.On the one hand, anti-PD-1 antibody is pyridine aldoxime methyliodide (PAM) monoclonal antibody or the Wu Dan that receives It is anti-.On the other hand, immunomodulator is anti-OX40 antibody or its antigen-binding fragment.On the other hand, immunomodulator is Anti- OX40 antibody or its antigen-binding fragment, it includes following one or more: the CDRH1 as shown in SEQ ID NO:1；Such as CDRH2 shown in SEQ ID NO:2；The CDRH3 as shown in SEQ ID NO:3；The CDRL1 as shown in SEQ ID NO:7；Such as The direct equivalent of CDRL2 shown in SEQ ID NO:8 and/or the CDRL3 as shown in SEQ ID NO:9 or each CDR, wherein directly Connecing equivalent has in the CDR no more than two amino acid substitutions.On the other hand, immunomodulator is anti-OX40 antibody Or its antigen-binding fragment, it includes have at least 90% sequence identity with the amino acid sequence as shown in SEQ ID NO:5 Variable heavy chain sequence and there is the variable of at least 90% sequence identity with the amino acid sequence as shown in SEQ ID NO:11 Sequence of light chain.On the one hand, II type PRMT inhibitor is the compound of formula III, IV, VII, VIII, IX, X or XI.In another party Face, II type PRMT inhibitor are compound B.On the one hand, II type PRMT inhibitor is compound C.On the one hand, II type PRMT Inhibitor and immunomodulator deliver medicine to patient with approach selected from the following: simultaneously, in succession, in any order, whole body, it is oral, In intravenous and tumor.On the one hand, II type PRMT inhibitor is administered orally.In one embodiment, it provides comprising compound C With the product of the anti-OX40 antibody of agonist or its antigen-binding fragment, be used for simultaneously, separately or sequentially with treat people by The cancer of examination person, wherein the cancer is colon cancer or lymthoma.In one embodiment, it provides comprising compound C and resists The product of OX40 antibody or its antigen-binding fragment, be used for simultaneously, separately or sequentially with the cancer to treat people experimenter, Wherein the cancer is colon cancer or lymthoma, and wherein the anti-OX40 antibody or its antigen-binding fragment include with next It is or multiple: the CDRH1 as shown in SEQ ID NO:1；The CDRH2 as shown in SEQ ID NO:2；As shown in SEQ ID NO:3 CDRH3；The CDRL1 as shown in SEQ ID NO:7；The CDRL2 as shown in SEQ ID NO:8 and/or as shown in SEQ ID NO:9 CDRL3 or each CDR direct equivalent, wherein direct equivalent in the CDR have be no more than two amino acid substitutions. In one embodiment, the product comprising compound C and anti-OX40 antibody or its antigen-binding fragment is provided, is used for same When, separate or sequentially with the cancer to treat people experimenter, wherein the cancer is colon cancer or lymthoma, and wherein described Anti- OX40 antibody or its antigen-binding fragment include the heavy chain variable region for having at least 90% sequence identity with SEQ ID NO:5 With the light chain variable region with SEQ ID NO:11 at least 90% identity.

In any one one side of this paper embodiment, the cancer is solid tumor or hematologic cancers.It on the one hand, is black Plain tumor, breast cancer, lymthoma or bladder cancer.

On the one hand, cancer is selected from head and neck cancer, breast cancer, lung cancer, colon cancer, oophoroma, prostate cancer, neuroglia Tumor, spongioblastoma, astrocytoma, glioblastoma multiforme, Bannayan-Zonana syndrome, cowden's disease, Lhermitte-Duclos disease, inflammatory breast cancer, Weir nurse this tumour, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, at Medulloblastoma, kidney, liver cancer, melanoma, cancer of pancreas, sarcoma, osteosarcoma, the giant cell tumor of bone, thyroid cancer, Cheng Lin It is bar cellularity T cell leukaemia, chronic granulocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute Lymphoblastic leukemia, acute myelogenous leukemia, AML, chronic neutrophilic leukemia, acute lymphoblast Property T cell leukaemia, plasmacytoma, immunoblast mast cell leukemia, jacket cell leukaemia, Huppert's disease macronucleus Chronic myeloid leukemia, Huppert's disease, acute megakaryoblastic leukemia, promyelocytic leukemia, erythroleukemia, pernicious leaching It is bar tumor, Hodgkin lymphoma, non-Hodgkin lymphoma, lymphoblast property t cell lymphoma, Burkitt lymphoma, follicularis Lymthoma, neuroblastoma, bladder cancer, bladder transitional cell carcinoma, carcinoma of vulva, cervix cancer, carcinoma of endometrium, kidney, celiothelioma, The cancer of the esophagus, salivary-gland carcinoma, hepatocellular carcinoma, gastric cancer, carcinoma of testis, cheek cancer, carcinoma of mouth, GIST (gastrointestinal stromal tumor) and carcinoma of testis.

On the one hand, method of the invention further comprises that at least one tumour medicine is administered to the people.

The people has solid tumor on the one hand.On the one hand, it is thin to be selected from incidence cancer, gastric cancer, melanoma, kidney for tumour Born of the same parents' cancer (RCC), the cancer of the esophagus, non-small cell lung cancer, prostate cancer, colorectal cancer, oophoroma and cancer of pancreas.Institute on the other hand People is stated with liquid tumors such as diffusivity large B cell lymphoid tumor (DLBCL), Huppert's disease, the white blood of chronic lymphocytic Sick (CLL), follicular lymphoma, acute myelogenous leukemia and chronic granulocytic leukemia.

The invention further relates to treat or mitigate cancer selected from the following seriousness method: brain (glioma), at Spongiocytoma, Bannayan-Zonana syndrome, cowden's disease, Lhermitte-Duclos disease, breast cancer, inflammatory breast Cancer, Weir nurse this tumour, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, colon, H&N, kidney, Lung, liver, melanoma, ovary, pancreas, prostate, sarcoma, osteosarcoma, the giant cell tumor of bone, thyroid gland, lymphoblast property It is T- chronic myeloid leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute at lymph Cell leukemia, acute myelogenous leukemia, chronic neutrophilic leukemia, Acute Lymphoblastic T- cell are white Blood disease, plasmacytoma, immunoblast mast cell leukemia, jacket cell leukaemia, Huppert's disease, the white blood of megacaryocyte Disease, Huppert's disease, acute megakaryoblastic leukemia, promyelocytic leukemia, erythroleukemia, malignant lymphoma, suddenly Odd gold lymthoma, non-Hodgkin lymphoma, lymphoblast property t cell lymphoma, Burkitt lymphoma, follicular lymphoma, Neuroblastoma, bladder cancer, bladder transitional cell carcinoma, lung cancer, carcinoma of vulva, cervix cancer, carcinoma of endometrium, kidney, celiothelioma, food Pipe cancer, salivary-gland carcinoma, hepatocellular carcinoma, gastric cancer, nasopharyngeal carcinoma, cheek cancer, carcinoma of mouth, GIST (gastrointestinal stromal tumor) and carcinoma of testis.

Term used herein " treatment " and its grammatical variants refer to therapeutic treatment.When mentioning particular condition, Treatment is it is meant that (1) improves or one or more biological manifestations of prevention illness or the illness；(2) interference (a) causes or makes At one or more points in the biological cascade of the illness or (b) one or more biological manifestations of illness；(3) alleviate and disease Disease or its treatment-related one or more symptoms, effect or side effect；Or (4) delay the development of illness or one kind of the illness Or various biological performance.To can also guess prophylactic treatment.Technical staff will recognize that " prevention " is not absolute art Language.In medicine, " prevention " is understood to mean the preventive administration of drug, to significantly reduce illness or its biological manifestation Possibility or seriousness, or postpone the illness or the generation of its biological manifestation.Such as when think subject be in development be When the high risk of cancer, prophylactic treatment is appropriate (such as when subject has extremely strong cancer family's medical history or when tested When person is exposed to carcinogen).

As used in the present invention, term " cancer ", " neoplasm " and " tumour " can be used interchangeably with singular or plural form, be Refer to experience vicious transformation so that it becomes there is pathologic cell to host organisms.It can be by mature technology, especially Histological examination easily distinguishes Primary cancerous and non-cancerous cells.The definition of cancer cell as used in the present invention Not only include Primary cancerous, further includes any cell derived from forerunner's cancer cell.This includes the cancer cell of transfer, and In vitro culture and cell line derived from cancer cell.When referring to the cancer types for being usually expressed as solid tumor, " clinic can be examined The tumour of survey " is the tumour that can be detected based on tumor mass；For example, total by the scanning of such as computer tomography (CT), magnetic The program of (MRI), X-ray, ultrasound or physical inspection palpation is imaged in vibration, and/or since the sample obtained from patient can be detected In one or more cancer-specific antigens expression.Tumour may be hematopoietic (or hematology or blood or blood relevant) Cancer, such as the cancer derived from haemocyte or immunocyte, are referred to alternatively as " liquid tumors ".Clinic based on neoplastic hematologic disorder The specific example of illness includes: leukaemia, and such as chronic granulocytic leukemia, acute myelocytic leukemia, chronic lymphatic is thin Born of the same parents' property leukaemia and acute lymphatic leukemia；Plasma-cell malignancy, as Huppert's disease, MGUS and Walden this Special Lun Shi macroglobulinemia；Lymthoma, such as non-Hodgkin lymphoma, Hodgkin lymphoma；Etc..

Cancer can be the mother cell that wherein there is abnormal quantity or undesirable cell Proliferation or be diagnosed as hematopoiesis system Any cancer of system cancer (including lymphocytic and myeloide malignant tumour).Myeloide malignant tumour includes but is not limited to: acute Myeloide (or myeloid or myeloide or myeloblastic) leukaemia (undifferentiated or differentiation), acute progranulocyte (or promyelocytic leukemic cell or promyelocyte or preceding pith mother cells) leukaemia, Acute Meyloid monocarpotic cellularity (or marrow list Core mother cell) leukaemia, acute monocytic (or monoblastic) leukaemia, rubricyte leukocythemia and megacaryocyte Property (or megakaryoblast) leukaemia.These leukaemia can be collectively referred to as acute myeloid (or myeloid or myeloide ) leukaemia (AML).Hematological malignancy further includes myeloproliferative disease (MPD) comprising but be not limited to: Chronic Myeloid Property (or myeloid) leukaemia (CML), chronic myelomonocytic leukemia (CMML), primary thrombocytosis (or piastrenemia) and polycythemia vera (PCV).Hematological malignancy further include: osteomyelodysplasia (or bone Marrow hyperplasia exception syndrome or MDS), it is referred to alternatively as refractory anemia (RA), the refractory anemia with excessive mother cell (RAEB) and the refractory anemia (RAEBT) with the mother cell in excessive conversion；And with or without reason it is unknown The myelofibrosis (MFS) of property myeloid metaplasia.

Hematopoietic system cancer further includes lymphoid malignant disease, and it is outer to will affect lymph node, spleen, marrow, peripheral blood and/or knot Site.Lymph cancer includes B cell malignant diseases, including but not limited to B cell non-Hodgkin lymphoma (B-NHL).B-NHL can be Painless (or low), moderate (or invasion) or height (high invasion).Painless B cell lymphoma includes: follicularis lymph Tumor (FL)；Small lymphocytic lymphoma (SLL)；Marginal zone lymphoma (MZL), including tubercle MZL, the outer MZL of knot, spleen MZL and Spleen MZL with villiform lymphocyte；Lymphoma lymphoplasmacytic (LPL)；And mucosa-associated lymphoid tissue (MALT Or knot outer edge area) lymthoma.Moderate B-NHL includes: the jacket cell lymthoma (MCL) for being related to or not being related to leukaemia, more Unrestrained property large celllymphoma (DLBCL), follicularis maxicell (or 3 grades or 3B grades) lymthoma and Primary Mediastinal lymthoma (PML).Height B-NHL include Burkitt lymphoma (BL), Hugh Burkitt sample lymthoma, small non-cleaved cell lymphoma (SNCCL) and Lymphoblastic lymphoma.Other B-NHL include immunoblastic lymphoma (or immune cell tumor), primary effusion Lymphoproliferative conditions (PTLD) or lymthoma after the lymthoma and transplanting of lymthoma, HIV related (or AIDS is related).B cell Malignant diseases further include but are not limited to: chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Walden Si Telunshi macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukaemia, acute leaching Bar property (or lymphatic or lymphoblast property) leukaemia and Ka Siermanshi disease.NHL may also include that T cell Fei Huoqi Golden lymthoma (T-NHL) comprising but it is not limited to the T cell non-Hodgkin lymphoma of not otherwise enumerated (NOS), periphery T cell lymph Tumor (PTCL), primary cutaneous type (ALCL), Angioimmunoblast lymph sample sick (AILD), nasal cavity type kill naturally Hurt (NK) cell/t cell lymphoma, gamma/delta lymthoma, skin-type t cell lymphoma, mycosis fungoides and Sezary syndrome (Sezary syndrome)。

Hematopoietic system cancer further includes Hodgkin lymphoma (or disease) comprising hodgkin lymphoma classical type, tubercle are hard Change type Hodgkin lymphoma, hodgkin lymphoma mixed cellularity type, lymphocyte leading type (LP) Hodgkin lymphoma, tubercle LP Hodgkin lymphoma and alymphocytosis type Hodgkin lymphoma.Hematopoietic system cancer further includes plasma cell disorder or cancer, Such as Huppert's disease (MM) comprising smoulder type MM, meaning does not determine the monoclonal gamma globulin of (unknown or unknown) Sick (MGUS), plasmacytoma (bone, marrow outside), lymphoma lymphoplasmacytic (LPL), Walden Si Telunshi macroglobulinemia Disease, plasma cell leukemia and primary amyloidosis (AL).Hematopoietic system cancer may also include its of other hematopoietic cells Its cancer, including polymorphonuclear leukocyte (or neutrophil leucocyte), basophilic granulocyte, eosinophil, Dendritic Cells, blood Platelet, red blood cell and natural killer cells.Tissue including hematopoietic cell is referred to as " hematopoietic cell tissue " in the present invention, Including marrow；Peripheral blood；Thymus gland；And peripheral lymphoid tissue, such as spleen, lymph node, lymphoid tissue relevant to mucous membrane (such as With gut associated lymphoid tissue), tonsillotome, peyer's patch and appendix, and lymphoid tissue relevant to other mucous membranes, example Such as bronchus liner.

As used herein, term " compound A²" refer to immunomodulator selected from the following: anti-PD-1 antibody or its antigen Binding fragment, anti-PDL1 antibody or its antigen-binding fragment, anti-CTLA 4 antibody or its antigen-binding fragment or anti-OX40 antibody Or its antigen-binding fragment.In some embodiments, compound A²For anti-PD-1 antibody.Suitably, compound A²It can be selected from receiving Military monoclonal antibody and pyridine aldoxime methyliodide (PAM) monoclonal antibody.In some embodiments, compound A²For the agonist for OX40 or its antigen-binding portion thereof Antibody, the OX40 or its antigen-binding portion thereof include to contain and amino acid sequence at least 90% etc. as shown in SEQ ID NO:5 The V of same amino acid sequence_HStructural domain；With at least 90% amino being equal of the amino acid sequence as shown in SEQ ID NO:11 The V of acid sequence_LStructural domain.In other embodiments, compound A²It is anti-for the agonist of OX40 or its antigen-binding portion thereof Body, including anti-OX40 antibody or its antigen-binding fragment comprising following one or more: as shown in SEQ ID NO:1 CDRH1；The CDRH2 as shown in SEQ ID NO:2；The CDRH3 as shown in SEQ ID NO:3；As shown in SEQ ID NO:7 CDRL1；The CDRL2's as shown in the SEQ ID NO:8 and/or CDRL3 as shown in SEQID NO:9 or each CDR is directly equivalent Object, wherein direct equivalent has in the CDR is no more than two amino acid substitutions.

As used herein, term " compound B²" refer to II type PRMT inhibitor.In some embodiments, compound B² For formula III, the compound of IV, VII, VIII, IX, X or XI.Suitably, compound B²For compound C.

Suitably, combination of the invention " during defined " is administered.

As used herein term " defined period (specified period) " and its grammatical variants, indicate administration Compound A²With compound B²One of and give drug compound A²With compound B²In another kind between time interval. Unless otherwise indicated, it is specified that during may include being administered simultaneously.Unless otherwise indicated, it is specified that during refer in one day to Drug compound A²With compound B²。

Suitably, if for the administration compound without being administered simultaneously, they can be at that within " defined period " This is separated by administration-in about 24 hours, and in this case, the defined period would be about 24 hours；Suitably, they can be with Being separated by administration-in about 12 hours, in this case, the defined period be would be about 12 hours；Suitably, they are equal Administration-can be separated by about 11 hours in this case, it is described as defined in during would be about 11 hours；Suitably, it Can be separated by about 10 hours administration-in this case, it is described as defined in during would be about 10 hours；Suitably Ground, they can be separated by about 9 hours administration-in this case, it is described as defined in during would be about 9 hours；It is suitable Locality, they can be separated by about 8 hours administration-in this case, it is described as defined in during would be about 8 hours； Suitably, they can be separated by about 7 hours administration-in this case, it is described as defined in during to would be about 7 small When；Suitably, they can be separated by about 6 hours administration-in this case, it is described as defined in during would be about 6 Hour；Suitably, they can be separated by about 5 hours administration-in this case, it is described as defined in during would be about 5 hours；Suitably, they can be separated by about 4 hours administration-in this case, it is described as defined in during will be About 4 hours；Suitably, they can be separated by about 3 hours administration-in this case, it is described as defined in during will It is about 3 hours；Suitably, they can in this case, the defined period will being separated by administration-in about 2 hours It is about 2 hours；Suitably, they can be separated by about 1 hour administration-in this case, it is described as defined in during It would be about 1 hour.As used in the present invention, compound A²With compound B²Administration be separated by and be considered as less than about 45 minutes It is administered simultaneously.

Suitably, when combination of the invention is administered with one section " defined period ", one section of each compound co-administered " is held The continuous time ".

Term " duration " and its grammatical variants as used in the present invention, indicate that two kinds of compounds of the invention are continuous The number of days specified number is administered.Unless otherwise indicated, continuous number of days is necessarily to start or treat terminal knot in treatment starting point Beam, it only needs certain time point over the course for the treatment of continuous number of days occur.

About " defined period " be administered: suitably, two kinds of compounds be administered during defined at least one day-at this In situation, the duration is at least one day；Suitably, over the course for the treatment of, two kinds of compounds are given during defined Medicine at least for three days on end-in this case, the duration is at least 3 days；Suitably, over the course for the treatment of, two kinds of compounds Be administered during defined it is 5 days at least continuous-in this case, the duration is at least 5 days；Suitably, it is treating In the process, two kinds of compounds be administered during defined it is 7 days at least continuous-in this case, the duration is at least 7 It；Suitably, over the course for the treatment of, two kinds of compounds be administered during defined it is 14 days at least continuous-in this case, institute Stating the duration is at least 14 days；Suitably, over the course for the treatment of, two kinds of compounds are administered at least continuous during defined 30 days-in this case, the duration is at least 30 days.

Suitably, if the compound is not after " (during a specifiled period) during defined " Administration, then they are exactly order of administration.Term " order of administration " and its grammatical variants as used in the present invention, indicate daily Be administered once compound A²With compound B²One of, continuous two days or more, then it is administered once a day compound A²With Compound B²In another kind, continuous two days or more.Similarly, the invention also includes in order of administration compound A²And change Close object B²One of and give drug compound A²With compound B²In another kind between the used off-drug period.Such as the present invention Used, the off-drug period is in order of administration compound A²With compound B²One of after and give drug compound A²And change Close object B²In it is another before do not give drug compound A²Also drug compound B is not given²Interval number of days.Off-drug period is selected from following One section of number of days: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days and 14 days.

About order of administration (sequential administration): suitably, giving drug compound A²With compound B² One of continuous 1 to 30 day, followed by the optional off-drug period, then give drug compound A²With compound B²In it is another even It is 1 to 30 day continuous.Suitably, drug compound A is given²With compound B²One of continuous 1 to 21 day, followed by the optional off-drug period, Then drug compound A is given²With compound B²In another continuous 1 to 21 day.Suitably, drug compound A is given²With compound B² One of continuous 1 to 14 day, followed by 1 to 14 day off-drug period, then give drug compound A²With compound B²In another kind Continuous 1 to 14 day.Suitably, drug compound A is given²With compound B²One of continuous 1 to 7 day, followed by 1 to 10 day stop The medicine phase then gives drug compound A²With compound B²In another continuous 1 to 7 day.

Suitably, compound B in this sequence²It is administered first, followed by the optional off-drug period, then gives drug compound A²。 Suitably, drug compound B is given²Continuous 3 to 21 days, followed by the optional off-drug period, then give drug compound A²Continuous 3 to 21 days. Suitably, drug compound B is given²Continuous 3 to 21 days, followed by 1 to 14 day off-drug period, then give drug compound A²Continuous 3 to 21 It.Suitably, drug compound B is given²Continuous 3 to 21 days, followed by 3 to 14 days off-drug periods, then give drug compound A²Continuous 3 To 21 days.Suitably, drug compound B is given²Continuous 21 days, followed by the optional off-drug period, then give drug compound A²Continuous 14 It.Suitably, drug compound B is given²Continuous 14 days, followed by 1 to 14 day off-drug period, then give drug compound A²Continuous 14 days. Suitably, drug compound B is given²Continuous 7 days, followed by 3 to 10 days off-drug periods, then give drug compound A²Continuous 7 days.Suitably Drug compound B is given on ground²For three days on end, followed by 3 to 14 days off-drug periods, drug compound A is then given²Continuous 7 days.Suitably, it gives Drug compound B²For three days on end, followed by 3 to 10 days off-drug periods, drug compound A is then given²For three days on end.

It should be appreciated that can be repeat administration (dosing) or can after " defined period " administration and " sequence " administration With then alternate dosage regimen, the off-drug period can be before repeat administration or alternate dosage regimen.

Method of the invention can also be used together with the treatment method of other treatment cancer.

Compound A²With compound B²It can be administered by any suitable approach.Suitable approach includes orally, through straight Intestines, intranasal, part (including buccal (buccal) and sublingual), in tumor, Via vagina and parenteral (including subcutaneous, intramuscular, vein Interior, intradermal, intrathecal and Epidural cavity).It is understood that optimization approach can with the subject of such as combination situation and controlled The cancer for the treatment of and change.It is also understood that each drug applied can be administered by identical or different approach, compound A²With compound B²It can be configured to pharmaceutical composition/preparation together.

In one embodiment, one of combination of the invention or Multiple components intravenous administration.Implement at one In scheme, one of combination of the invention or Multiple components oral administration.In another embodiment, in combination of the invention One or more ingredients be administered in tumor.In another embodiment, one of combination of the invention or Multiple components are complete Body administration, one of such as intravenous and present invention combination or various other components are administered in tumor.In the embodiment Any one in, such as in this section, each ingredient of the invention is administered as one or more pharmaceutical compositions.

Typically, in the present invention, any antitumor agent active to the susceptible neoplasm treated can be in cancer It is co-administered in disease treatment.The example of the drug can be in Cancer Principles and Practice of Oncology by V.T.Devita, T.S.Lawrence and S.A.Rosenberg (editor), the 10th edition (December 5 in 2014 Day), it finds in Lippincott Williams&Wilkins Publishers.Those of ordinary skill in the art will be based on The special characteristic of drug and related cancer distinguish which pharmaceutical agent combinations can be used.It is for use in the present invention typical anti-swollen Tumor agent includes but is not limited to: anti-micro-pipe or antimitotic agent such as diterpene and vinca alkaloids；Platinum coordination complex；Alkylation Agent such as mustargen, oxynitride phosphor ring class (oxazaphosphorine), alkyl sulfonic ester, nitroso ureas and triazenes；Antibiotic is such as put Line rhzomorph, anthracycline and bleomycin；Topoisomerase I inhibitor such as camptothecine；Topoisomerase II inhibitors such as table Podophyllum emodi var chinense Toxin；Antimetabolite such as purine and pyrimidine analogue and anti-folic acid compound；Hormone and hormone analogs；Signal transduction pathway Inhibitor；Nonreceptor tyrosine kinase angiogenesis inhibitors；Immunotherapeutic agent；Promote apoptosis agent；Cell cycle signals transduction suppression Preparation；Proteasome inhibitor；Heat shock protein inhibitors；Cancer metabolic poison；It is repaired with gene therapy for cancer agent such as gene The T cell of decorations.

For being antitumor agent with the example of the method for the present invention or combinatorial association or the other active components of co-administration.It is anti- The example of tumour agent includes but is not limited to chemotherapeutant；Immunoregulation agent (immuno-modulatory agent)；It is immune to adjust It saves agent (immune-modulator)；With immunostimulation adjuvant.

Embodiment

Following embodiment shows multiple non-limiting aspects of the invention.

Embodiment 1

Background

PRMT5 is symmetrical protein arginine transmethylase

Protein arginine transmethylase (PRMT) is the subset of enzyme, containing rich in glycine and arginine residues Region (GAR motif) protein in methylate arginine.Being divided PRMT based on the product of enzymatic reaction is four kinds of hypotype (I- IV type) (Fig. 1, Fisk JC et al., A type III protein arginine methyltransferase from the protozoan parasite Trypanosoma brucei.J Biol Chem.2009Apr 24；284(17):11590- 600).I-III type enzyme generates ω-N- monomethyl-arginine (MMA).Maximum hypotype, I type (PRMT 1,3,4,6 and 8), makes MMA becomes asymmetric dimethylarginine (ADMA), and II type generates symmetrical diethylarginine (SDMA).Although PRMT9/ FBXO11 also can produce SDMA, but PRMT5 is responsible for the Major Enzymes of symmetric dimethyl.PRMT5 is in cytoplasm and nucleus In several types compound in work, and the binding partners of PRMT5 are necessary to identifying and selecting property of substrate. Transmethylase complex protein 50 (MEP50) is the known co-factor of PRMT5, is that PRMT5 is combined and to histone and its (Ho MC et al. Structure of the arginine methyltransferase necessary to the activity of his substrate PRMT5-MEP50reveals a mechanism for substrate specificity.PLoS One.2013；8(2)).

PRMT5 substrate

PRMT5 methylates arginine, including splicing factor, histone, transcription factor, kinases in various cell proteins etc. (Fig. 2) (Karkhanis V, et al. Trends Biochem Sci.2011Dec；36(12):633-41).Spliceosome it is a variety of The methylation of component is the critical event in spliceosome assembling, and is caused by striking low or gene knockout decrease PRMT5 activity Destruction (Bezzi M et al. Regulation of constitutive and alternative splicing of cell montage by PRMT5reveals a role for Mdm4pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev.2013Sep 1；27(17):1903-16).PRMT5 also methylation group egg White arginine residues (H3R8, H2AR3 and H4R3), these histidine tags are related to the Transcriptional Silencing of tumor suppressor gene, such as RB and ST7 (Wang L et al. Protein arginine methyltransferase 5suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells.Mol Cell Biol.2008Oct；28(20):6262-77；Pal S et al. Low levels ofmiR-92b/ 96induce PRMT5translation and H3R8/H4R3methylation in mantle cell lymphoma.EMBO J.2007Aug 8；26(15):3558-69).In addition, the symmetric dimethylization of H2AR3 and embryo are dry thin Related (Tee WW et al. the Prmt5is essential for early mouse of the silencing of differentiation gene in born of the same parents development and acts in the cytoplasm to maintain ES cell pluripotency.Genes Dev.2010Dec 15；24(24):2772-7).PRMT5 also passes through EGFR and the methylation of PI3K rises in cellular signal transduction Act on (Hsu JM et al. Crosstalk between Arg 1175methylation and Tyr 1173phosphorylation negatively modulates EGFR-mediated ERK activation.Nat Cell Biol.2011Feb；13(2):174-81；Wei TY,Juan CC,Hisa JY,Su LJ,Lee YC,Chou HY, Chen JM,Wu YC,Chiu SC,Hsu CP,Liu KL,Yu CT.Protein arginine methyltransferase 5is a potential oncoprotein that upregulates G1cyclins/cyclin-dependent kinases and the phosphoinositide 3-kinase/AKT signaling cascade.Cancer Sci.2012Sep；103(9):1640-50).Effect of the PRMT5 in the methylation of protein for participating in the relevant approach of cancer As described below.

PRMT5 knocks out model

Completely losing for PRMT5 is embryonic death.PRMT5 plays key effect in embryonic development, passes through PRMT5 Depleted mice it is dead between 3.5 to 6.5 days embryonic period, embryonic phases and prove (Tee WW, et al. Prmt5is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency.Genes Dev.2010Dec 15；24(24):2772-7).Earlier studies have shown that PRMT5 is in HSC (hematopoiesis Stem cell) and NPC (neural progenitor cell) development in play an important role.Human cord blood CD 34⁺In cell PRMT5 strike it is low cause it is red Cell differentiation increases (Liu F et al. JAK2V617F-mediatedphosphorylation of PRMT5downregulates its methyltransferase activity and promotes myeloproliferation.Cancer Cell.2011Feb 15；19(2):283-94).In NPC, PRMT5 adjusts Neural Differentiation, intracellular growth and survival (Bezzi M Et al. Regulation of constitutive and alternative splicing by PRMT5reveals a role for Mdm4pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev.2013Sep 1；27(17):1903-16).

PRMT5 in cancer

More and more evidences show that PRMT5 participates in tumour.PRMT5 albumen is overexpressed in many cancer types, Including lymthoma, glioma, breast cancer and lung cancer, only PRMT5 overexpression is just enough to convert normal fibroblast (Pal S et al. Low levels of miR-92b/96induce PRMT5translation and H3R8/H4R3methylation in mantle cell lymphoma.EMBO J.2007Aug 8；26(15):3558-69；Ibrahim R et al. Expression of PRMT5in lung adenocarcinoma and its significance in epithelial- mesenchymal transition.Hum Pathol.2014Jul；45(7):1397-405；Powers MA et al. Protein arginine methyltransferase 5accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4.Cancer Res.2011Aug15；71(16): 5579-87；Yan F et al. Genetic validation of the protein arginine methyltransferase PRMT5as a candidate therapeutic target in glioblastoma.Cancer Res.2014Mar 15； 74(6):1752-65).The reduction of PRMT5 struck low pass and often result in cell growth and survival in cancerous cell line.In breast cancer, High PRMT5 expression and high PDCD4 (programmed cell death factors 4) horizontal overall bad survival (Powers MA etc. of prediction together People Protein arginine methyltransferase 5accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4.Cancer Res.2011Aug15；71(16):5579-87).PRMT5 methylation PDCD4 changes tumour correlation function.PRMT5 and PDCD4 Coexpression in the model in situ of breast cancer promotes tumour growth.The high expression and high tumour of PRMT5 in glioma Grade is related to overall poor survival rate, and PRMT5 strikes in the low model of spongioblastoma in situ and provides survival benefit (Yan F et al. Genetic validation of the protein arginine methyltransferase PRMT5as a candidate therapeutic target in glioblastoma.Cancer Res.2014Mar 15； 74(6):1752-65).Increased PRMT5 expression and activity facilitate the suppression of several tumours in silencing glioma cell line Gene processed.

The strongest mechanism connection described between PRMT5 and cancer at present is lymphoma mantle cell (MCL).PRMT5 warp It is often overexpressed in MCL, and the high expression in core compartment, wherein height expression increases histone methylated horizontal and silencing The subset of tumor suppressor gene.Nearest research disclose miRNA raised in MCL PRMT5 expression in effect.It is expected that being more than 50 kinds of miRNA are annealed to the 3' non-translational region of PRMT5mRNA.It is reported that the PRMT5 in miR-92b and miR-96 level and MCL It is horizontal negatively correlated, and the downward of these miRNA leads to the up-regulation of PRMT5 protein level in MCL cell.Cyclin D1 is the oncogene of most MCL patient's transpositions, related to PRMT5, and increases PRMT5 activity by cdk4 dependent mechanism (Fig. 3, Aggarwal P et al. Cancer Cell.2010Oct 19；18(4):329-40).PRMT5 mediates negative regulator DNA multiple The inhibition of the key gene of system, so that cyclin D1 dependent tumors be allowed to grow.PRMT5 strikes the low inhibition cell cycle The conversion of 1 dependent cell of protein D, leads to death of neoplastic cells.These data highlight important function of the PRMT5 in MCL, and Show that PRMT5 inhibits to can be used as the therapeutic strategy in MCL.

In other tumor types, it has been assumed that PRMT5 is in differentiation, cell death, cell cycle progress, cell growth and increases It works in growing.Although the main mechanism that PRMT5 and tumour are connected it is unclear that, it is emerging statistics indicate that PRMT5 helps to adjust gene expression (histone methylated, transcription factor combines or promoter combination), montage change and signal Transduction.The PRMT5 methylation of transcription factor E2F 1 reduces it and inhibits cell growth and promote ability (the Zheng S of Apoptosis Et al. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1.Mol Cell.2013Oct 10；52(1):37-51).PRMT5 also methylates p53 (Jansson M Et al. Arginine methylation regulates the p53response.Nat Cell Biol.2008Dec；10 (12): 1431-9) to respond DNA damage and reduce the ability of p53 induction of cell cycle arrest, while it is thin to increase p53 dependence Born of the same parents' apoptosis.These statistics indicate that, PRMT5 inhibit can by induce p53 dependent cell apoptosis keep cell quick to DNA damage agent Sense.

In addition to directly methylating other than p53, PRMT5 also passes through montage related mechanism up-regulation p53 approach.Mouse neural progenitor cells In PRMT5 knock out and lead to the change of cell montage, the isotype including MDM4 gene converts (Bezzi M et al. Regulation of constitutive and alternative splicing by PRMT5reveals a role for Mdm4pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev.2013Sep 1；27(17):1903-16).Bezzi et al. has found that PRMT5 knocks out cell and has the long MDM4 reduced of the same race The expression (generating functionality p53 ubiquitin ligase) and the expression of the short isotype of increased MDM4 of type (generate inactive connection Enzyme).These variations of MDM4 montage lead to the inactivation of MDM4, increase the stability of p53 albumen, and then activation p53 approach and Cell death.It is struck in PRMT5 and also observes MDM4 alternative splicing in low cancerous cell line.These are statistics indicate that PRMT5 inhibits Multiple nodes of p53 approach can be activated.

Other than the adjusting of growth of cancer cells and survival, PRMT5 further relates to epithelial-mesenchymal conversion (EMT).PRMT5 In conjunction with transcription factor SNAIL, and the crucial co-suppression factor as CAM 120/80 expression；Striking for PRMT5 low leads to E- calcium Up-regulation (Hou Z et al. The LIM protein AJUBA recruits protein arginine of mucin levels methyltransferase 5to mediate SNAIL-dependent transcriptional repression.Mol Cell Biol.2008May；28(10):3198-207).

These data highlight effect of the PRMT5 as the key regulator of kinds cancer relational approach, and show PRMT5 inhibitor can have extensive activity in blood and solid cancer.PRMT5 inhibitor as MCL and breast cancer and The therapeutic strategy of the cancer of the brain has very strong theoretical basis.These data, which are further highlighted, to be pressed down in cellular environment appropriate using PRMT5 The mechanism principle of preparation:

Cyclin D1 in MCL is inhibited to rely on sexual function；

Activation and adjusting p53 pathway activities；

Adjust the cell growth for relying on E2F1 and apoptosis function；

It disinthibites the expression of (de-repress) CAM 120/80；

Compound C be (MW=452.55) of the intermediate molecular weight of PRMT5/MEP50 compound it is effective, it is selectivity, Emulative, the reversible inhibitor of peptide has good overall physical property and oral administration biaavailability.Compound C influences several Kind cancer relational approach, finally generates effective anticancer activity in vitro and in vivo in model, for treatment MCL, breast cancer and brain Cancer provides new therapy mechanism.

Biochemistry

Analysis of compounds C is in many external biological chemical assays to characterize effect, the invertibity, choosing that it inhibits PRMT5 Selecting property and mechanism.

Using the inhibition effect of radioactivity determination method assessment compound C, the radioactivity determination method measurement is from SAM to a group egg Peptide derived from white H4³H transfer, wherein peptide derived from the histone H 4 is identified by histone peptide library selection.Use 120 points Any time dependence that the long reaction time of clock captures effect increases.It was found that compound C is effective suppression of PRMT5/MEP50 Preparation, IC₅₀For 8.7 ± 5nM (n=3).The effect represents molecule and really imitates close to the limit of combining closely (2nM) measured The upper limit (Fig. 4) of power.The inhibition effect of the approximate analog of compound C is similar, including compound F, compound B and compound E (key difference is on the left of molecule) is used as tool compound in some biological studies.

In order to assess the ability that compound C inhibits the PRMT5 dependence of the cell substrate in addition to histone H 4 to methylate, One group of PRMT5 substrate is assembled for assessing, (participates in montage and Transcriptional Silencing including SmD3, Lsm4, hnRNPH1 and FUBP1 The major part of these substrates passes through cell Methylscan described in hereafter biological part retinal diseases^TMResearch is found).Compound C has Effect ground inhibits the methylation of all these substrates of PRMT5/MEP50 catalysis, although extremely low K_{M is apparent}Eliminate the standard to effect Really measurement.

It is also multiple for PRMT5/MEP50 under the conditions of similar with people PRMT5 measurement in order to explain safety research Close the rat of object and the effect of dog ortholog thing assessment compound C.Compound C effect changes < 3 times relative to all species (table 2).

Table 2. uses radioactivity determination method (K in equilibrium conditions_{M is apparent}Concentration of substrate) monitoring PRMT5/MEP50 activity, It is measured with after compound C processing³Transfer of the H from SAM to protein substrate.By the way that data are fitted to 3 parameter dosage-response Equation determines IC₅₀Value.

Compound

In order to determine suppression mechanism and inhibitor binding pattern, by compound C and PRMT5/MEP50 compound and Xi Naifen Only a kind of (natural products SAM analog) cocrystallization (Resolution ratio) (Fig. 5).The inhibitor is incorporated in usually by peptide substrate In the crack occupied and close to the Sinefungin for occupying SAM bags.The aryl rings of tetrahydroisoquinoline and the amino shape of Sinefungin At π-aryl accumulation interaction.Hydrogen bond is formed between the hydroxyl and Leu437 skeleton and Glu244 of compound C.In pyrimidine ring Amide and Phe580 skeleton NH group between also form interaction of hydrogen bond.End piperidineacetamide is located at solvent exposure table On face, without apparent critical contact.Generally speaking, a kind of suppression mechanism of the structural support, the mechanism and SAM are uncompetitive (uncompetitive) and and substrate competition.

In order to determine whether compound C is the reversible inhibitor of PRMT5/MEP50 and in order to further explore inhibition machine System measures the combination of compound C and various PRMT5/MEP50 compounds using affinity selection mass spectrography (ASMS).It can be with Detect positive combination following: the binary complex containing PRMT5/MEP50 Yu SAM, Sinefungin or SAH arrives PRMT5/ MEP50:H4 peptide: the dead end ternary complex of SAH or Sinefungin.Since ASMS can not detect the compound of Irreversible binding C, therefore these results are consistent with Reversible binding mechanism.When competing with 10 times of excessive H4 peptides, the combination of compound C exists PRMT5/MEP50:H4 peptide: it is reduced in Sinefungin compound.With PRMT5/MEP50:H4 peptide complexes or individually use PRMT5/ MEP50 can't detect the combination of compound C, demonstrate the need for occupying SAM binding pocket for compound C combination.These results with Following suppression mechanism most matches, i.e., with SAM be uncompetitive and with H4 peptide compete.

The selectivity of compound C is assessed in one group of enzyme, the enzyme includes I type and II type PRMT and the transfer of lysine methyl Enzyme (KMT).Do not include PRMT9/FBXO11 due to lacking functional enzyme measurement, is that another kind II type PRMT lacks with unique The PRMT of THW ring.Compound C does not inhibit any 19 kinds of enzymes in transmethylase selectivity group, IC₅₀> 40 μM of value, causes Selectivity > 4000 times (Fig. 6) of PRMT5/MEP50.For the PRMT5 tool chemical combination used in biological part retinal diseases of the invention Object (compound B, compound F and compound E), also observes it for PRMT5/MEP50 relative to other transmethylases Selectivity.

In short, compound C is the effective of PRMT5/MEP50 compound, selectivity, reversible inhibitor, IC₅₀For 8.7 ± 5nM.The crystal structure and ASMS combined data and SAM uncompetitive, protein bottom of PRMT5/MEP50 and compound C compound Object competitiveness mechanism is consistent.

Biology

Abstract

PRMT5 is overexpressed in many human cancers, and is related to kinds cancer relational approach.Use PRMT5 inhibitor Therapeutic strategy as MCL and breast cancer and the cancer of the brain has very strong theoretical basis.In order to understand that PRMT5 inhibitor antiproliferative is living The range of property, using 2D and 3D growth measurement method in various in vitro and in vivo tumor models analysis of compounds C.

The characteristic of the gene and approach that are influenced by PRMT5 inhibition is for understanding indication priority, predictive biological marker The mechanism of the PRMT5 inhibitor of the design of discovery and the reasonable combination research of object is vital.Several bodies are carried out Outer Mechanism Study is to assess the biology to the PRMT5 response inhibited.Assess the arginine methylation level of many PRMT5 substrates To monitor the compound C activity in cell and xenograft tumours for PRMT5.Perhaps polyclonal RNA sequencing is carried out to comment Estimate compound C is influenced to gene expression, montage and by other molecular mechanisms and approach of PRMT5 Active Regulation.With P53 pathway activities are monitored in the cell line of PRMT5 inhibitor processing.

Finally, testing compound C activity, in MCL and several heteroplastic transplantation models of breast cancer to assess PRMT5 inhibition Effect in preclinical cancer model and the potential source biomolecule marker for assessing molecular mechanism and response.

Line sensitive

Inhibit the antiproliferative activity in various tumor types to assess PRMT5, uses the cancer cell system organized extensively With the tumor model from patient in 2D and 3D external test analysis of compounds C.Firstly, in 2D growth in 6 days/death measurement In in one group of cancerous cell line assess compound C (Fig. 7).Select cell line to represent tumor type, in the tumor type Report PRMT5 Active Regulation critical path and/or cell growth and survival (such as lymthoma and MCL, glioma, mammary gland Cancer and lung cancer cell line).In general, the most cells system of test shows the gIC lower than 1 μM₅₀Value, and most sensitive leaching Bar oncocyte system (lymphoma mantle cell and Diffuse large B cell lymphoma cell line) has the gIC of several nM ranges₅₀Value.

In growth in 6 days/death measurement, compound C of the concentration higher than 100nM is in Diffuse large B cell lymthoma (DLBCL), inducing cytotoxic reacts in lymphoma mantle cell (MCL), spongioblastoma, breast cancer and bladder cancer cell lines (Fig. 8, negative Y_min- T0 value).In general, MCL and DLBLC cell line shows strongest cell-cytotoxic reaction.Most of mammary gland Cancerous cell line has low Y_min- T0 value shows that PRMT5 inhibits to cause complete growth inhibition in breast cancer model, and remaining cell System shows part cell and inhibits reaction (positive Y_min- T0 value).

In the large-scale cancerous cell line screening (240 kinds of cell lines, 10 days 2D growth measurements) carried out with PRMT5 tool molecule (biochemistry of compound C and compound B in Fig. 9, Fig. 4/cell are living for the antiproliferative activity that further test PRMT5 inhibits Property compares).In short, most cells system shows the gIC lower than 1 μM₅₀Value.Intermediate value gIC₅₀The tumor type of < 100nM is anxious Property myelomatosis (AML), chronic granulocytic leukemia (CML), Hodgkin lymphoma (HL), Huppert's disease (MM), breast cancer, glioma, kidney, melanoma and oophoroma.These statistics indicate that PRMT5 inhibitor to various blood Cancer (heme) and solid tumor types show extensive antiproliferative activity.

In soft agar 3D colony formation assay, used in one group of tumor model and cell line (n=73) from patient PRMT5 tool compound observes the antigrowth (Figure 10) of similar wide scope.It is observed in 37% model opposite Grow IC₅₀Value is lower than 1 μM, including non-small cell lung cancer (NSCLC), breast cancer, melanoma, colon cancer and glioma.Tool There is minimum intermediate value IC₅₀The tumor type of value is maxicell lung cancer, breast cancer, kidney and glioma.

In short, these are statistics indicate that PRMT5 inhibitor has effective anti-increasing in various entities and hematologic cancers model Grow activity.According to the activity observed in the studies above, document assumes and clinical development potentiality, and following indication is selected to carry out volume Outer research:

MCL and DLBCL (PRMT5 is inhibited to generate effective antiproliferative and cytotoxic response)

Breast cancer (low gIC in cell line₅₀Value and complete growth inhibition, and the collection in the model group from patient It falls to form IC in measurement₅₀It is worth low)

Spongioblastoma (the low IC in colony formation assay₅₀Value).

Lymthoma biology

As described above, compound C is in the subset of jacket cell and Diffuse large B cell lymphoma cell line induction of effective Cytotoxic response (Fig. 7-8).Since PRMT5 is often overexpressed and in MCL approach (such as cyclin in MCL White D1 and p53) in play an important role, therefore compound C activity and mechanism are had evaluated in the research of several cell mechanisms.It is thin in set The effect of compound C is assessed in two kinds of heteroplastic transplantation models of born of the same parents' lymthoma.

Cell mechanism data (lymthoma)

SDMA inhibits

PRMT5 is responsible for the symmetrical arginine di-methylation of most cells.In order to better understand by PRMT5 inhibit with The biological mechanism that anticancer phenotype connects uses the cell protein subgroup of identification symmetric dimethyl in arginine residues SDMA Identification of the antibodies substrate.By being precipitated and mass spectral analysis (Methylscan with SDMA antibody mediated immunity^TM), it is split in Z138 cell Object (cell from control and the processing of PRMT5 inhibitor) middle identification is solved by the protein of SDMA antibody test.Containing SDMA Protein in, the overwhelming majority be participate in cell montage and RNA processing (SmB, Lsm4, hnRNPH1 and other), transcription (FUBP1) and translation the factor, highlight effect of the PRMT5 as the important regulator of cell RNA stable state.

Then SDMA antibody western and ELISA measurement is used to inhibit with the methylation for measuring compound C dependence. Firstly, handling Z138MCL cell (compound C gIC with the compound C of progressive concentration₅₀ 2.7nM、gIC₉₅82nM and gIC₁₀₀ 880nM, the cytotoxic response in growth in 6 days/death measurement, Fig. 7-8) with the 1st day and the 3rd day after determining processing SDMA The cell IC of inhibition₅₀(Figure 11).SDMA ELISA shows the time dependence variation of SDMA level, the 3rd day IC₅₀Value is 4.79nM, the 1st day and the 3rd day EC₅₀Respectively 7.3 and 2.35 (Figure 11 schemes A).It is being higher than 19nM (EC on day 3₉₀) it is dense The complete inhibition of SDMA is observed under degree.In gIC₉₅(82nM) and gIC₁₀₀It is observed between (880nM) complete in Z138 cell Growth inhibition (in growth in 6 days/death measurement), concentration is higher than the EC that SDMA inhibits₉₀.These statistics indicate that, in order in Z138 Complete growth inhibition and cytotoxicity are triggered in cell, are needed PRMT5 activity suppression > 90%.

It is thin in one group of MCL in order to assess whether the horizontal inhibition of SDMA is the prediction for growing response to the cell of compound C SDMA IC is measured in born of the same parents system₅₀Value.In one group of 5 MCL cell line, SDMAIC₅₀Value in the range of 0.3 to 14nM (Figure 11, Scheme B) (Mino and Jeko-1, Fig. 7-8 of sensitive Z138, Granta-519, Maver-1 and moderate resistance), show SDMA Not instead of respond flag, the active label of PRMT5 can be used for monitoring the PRMT5 in sensitive and resistant models and inhibit.

The gene expression profile of lymphoma cell line

PRMT5 will participate in the histone and protein methylation of RNA processing, it is therefore contemplated that PRMT5 inhibits to cell mRNA Stable state has profound influence.In order to further decode the cell for the cell response for being adjusted by PRMT5 and being facilitated PRMT5 inhibitor Mechanism assesses global changes in gene expression in the lymphoma model for inhibiting sensitive to PRMT5.Inhibit to be illustrated in PRMT5 The changes in gene expression occurred in lymphoma cell line after agent processing, passes through 4 sensibility lymphoma cells of RNA sequencing analysis It is (2 MCL cell line-Z138 and Granta-519 and 2 DLBCL cell line-DOHH2 and RL).

Firstly, assessing gene in the lymphoma cell line for handling 2 days and 4 days with the PRMT5 tool molecule of progressive concentration Expression variation (Figure 12).Effect to rna expression is the time and dose-dependent, and usually in 4 lymphoma cell lines 48 kinds of genes of middle adjusting.These are statistics indicate that PRMT5 inhibits to cause the expression variation of hundreds of genes, and the son of these variations Collection is common for all 4 kinds of sensibility lymphoma cell lines tested.Assessing what these genes inhibited in PRMT5 Correlation in cell response mechanism.

SDMA and changes in gene expression

In order to confirm the changes in gene expression of RNA-seq experiment discovery, the qPCR analysis (tool of gene subset expression has been carried out There is the gene of strong variations and participate in the gene of p53 approach).It is handled Z138 cell 2 days and 4 days with the compound C of ascending-dose, It separates RNA and is analyzed by qPCR.Figure 13 shows representative dosage-response curve in left figure, and summarizes in right figure Gene expression EC₅₀It is worth (the 4th day).In general, all 11 genes of test show time and dose dependent expression variation, EC₅₀Value is in the range of 22 to 332nM, intermediate value gene expression EC₅₀For 212nM.Importantly, median gene expression EC₅₀Value pair Ying Yu leads to the compound C concentration (measuring by SDMA antibody ELISA, Figure 11) for the maximum suppression that cell methylates in Z138, It demonstrates the need for almost inhibiting PRMT5 activity, to establish the variation of gene expression program.These data highlight PRMT5 suppression The relationship that processing procedure degree and gene expression and growth phenotype change, both of them need access to complete inhibition PRMT5 activity.

PRMT5 inhibits and montage

Due to PRMT5 methylation spliceosome subunit and PRMT5 inhibiting effect reduces many protein for participating in montage Arginine methylation, therefore have studied influence of the PRMT5 inhibition to cell montage.In above-mentioned lymthoma RNA-seq data set Assess the variation of RNA montage.

There are the molecular mechanism of several adjustable cell montages (Figure 14 scheme A), and wherein introne retains (B) and usually leads The change of gene expression is caused, and the use of exon skipping or substitution splice site causes isotype to convert (A, C-E).PRMT5 Tool compound processing causes the dosage that introne retains in all lymphoma cell lines tested and time dependence to increase (Figure 14 schemes B).It is interesting that splicing factor map analysis shows the subset of splicing factor binding site in all four cell lines It is enriched with, including hnRNPH1 (directly being methylated by PRMT5), hnRNPF, SRSF1 and SRSF5, shows in the introne of middle reservation Influence of the PRMT5 to cell montage can be dependent on the methylation of the various ingredients (Sm and hnRNP albumen) of spliceosome mechanism. PRMT5 inhibits also to induce the isotype conversion (alternative splicing) (Figure 15 schemes A) in lymphoma cell line and 34 kinds of genes are in institute Have and show consistent alternative splicing variation in the cell line of test (Figure 15 schemes B and C).

In general it was observed that the montage of hundreds of genes changes, show influence of the PRMT5 to montage be not it is global, But there is specificity for the RNA of limited quantity.This species specific explanation is, PRMT5 directly adjust specific montage because The RNA of son is combined, such as hnRNPH1 etc..The effect that alternative splicing changes in PRMT5 inhibitor mechanism of action is being studied, A specific example is discussed below.

The activation of MDM4 montage and p53 approach

It is reported that PRMT5 knock out or strike it is low cause MDM4 isotype convert, cause MDM4 ubiquitin ligase activity inactivate It (is described in the background section) to p53.PRMT5 inhibits to cause to test the 4 kinds of lymthomas tested in (GSEA) in RNA-seq The activation of p53 approach in cell line.In order to understand whether p53 activation is related to the conversion of MDM4 isotype, MDM4 selection is analyzed Property montage.Observe that MDM4 isotype is converted in all 4 lymphoma cell lines.Next, by RT-PCR at one group 4 (Figure 16 schemes A, and Z138, JVM-2 and MAVER-1MCL cell line is to compound C for the variation of confirmation MDM4 montage in MCL cell line Sensitivity, and REC-1 is the MCL cell line of most resistance).In Z138 and JVM-2 cell (two kinds of p53 wild types), compound C Induce the conversion of MDM4 isotype.In MAVER-1 and REC-1 cell (two kinds of p53 mutant), the basal expression of MDM4 elongated It is low/undetectable, it is thus impossible to detect that MDM4 isotype is converted.Then, in JVM-2 and Z138 cell p53 and p21 (or CDKN1A, p53 target gene) protein expression increase (Figure 16 schemes B).Importantly, in Z138 cell, 200nM compound C and 5 The processing of μM MDM2 inhibitor (Nutlin-3) expresses p53 and p21 to increase to similar level.These are statistics indicate that PRMT5 inhibits The MDM4 montage in the cell line of expression high level MDM4 long isotype is adjusted, and induces the way p53 in p53 wild-type cell system Diameter activity.Assessing effect of the p53 approach in biology of the p53 wild type MCL cell to the PRMT5 response inhibited.

In addition, assessing in the Z138 cell with the compound C processing of progressive concentration, MDM4 montage, SDMA inhibits and p53 The dose response of the variation of expression is inhibited, the relationship of MDM4 montage and p53 activation with assessing PRMT5 (Figure 17 schemes A and B).? Under the concentration of compound C higher than 8nM, SDMA level can't detect by western blot.Meanwhile in the chemical combination for being higher than 8nM Under the concentration of object C, MDM4 montage and the variation of p53/p21 protein expression are apparent.These results indicate that in gene montage and Subsequent pathway activities change before (MDM4/p53/p21), need substantially to inhibit PRMT5 activity (without Western Detectable SDMA is horizontal).

These are statistics indicate that PRMT5 inhibits to activate wild type p53 by adjusting MDM4 montage.This mechanism can be used for it The cancer types that middle p53 is infrequently mutated, such as blood and pediatric malignancies.In lymphoma model, PRMT5 inhibition causes Significantly (GSEA analysis) and relatively quick p53 pathway activation, this can help in the cell line handled with PRMT5 inhibitor In growth/death phenotype for observing.Striking low/rescue experiment will be used to further assess MDM4/p53 approach in PRMT5 inhibition Effect in the cell response of agent induction.

The expression of MDM4 isotype and p53 mutation are the potential predictive biomarkers that PRMT5 inhibits response in MCL.? In MCL cell line group, only two kinds of wild type p53 system Z138 and JVM-2 are most sensitive cell line (minimum gIC₅₀Value, And only there are two types of the MCL cell lines for showing cytotoxicity in growth/Study on mortality at 6 days).In two kinds of cell lines, chemical combination Object C processing leads to the conversion of MDM4 isotype and p53 pathway activation.The MCL cell line of limited quantity and foundation primary MCL model Extremely low success rate makes us that can not further assess p53 predictive biomarkers hypothesis.Although p53 approach can be wild in p53 Type cell to playing an important role in the biology of the response of PRMT5 inhibitor, but our data highlight other strongly can be with The importance of the approach of antitumor effect is driven, because PRMT5 inhibition causes antiproliferative to be made in the case where lacking functionality p53 With (such as Maver-1 cell line).

Lymphoma mantle cell: compound C and Yi Lu replace the comparison and combined activity of Buddhist nun.

Bruton's tyrosine kinase (BTK) inhibitor is approved for MCL recently according to Shandong for Buddhist nun, in recurrence/intractable In environment with it is unprecedented close to 70% overall response rate (Wang ML, et al. N Engl J Med.2013Aug 8； 369(6):507-16).However, the Most patients received according to Shandong for Buddhist nun's treatment are not up to complete incidence graph, middle position Progression free survival Phase is about 14 months.To understand whether compound C can be used for assessing in growth in 6 days/death measurement according to Shandong for Buddhist nun's resistance MCL Compound C and Yi Lu are for Buddhist nun's sensibility (Figure 18 schemes A).With low compound C gIC₅₀Cell line (Z-138, the Maver-1 of value And JVM-2) to resistant for Buddhist nun according to Shandong, and in only having to compound C according to Shandong for Buddhist nun's sensitive cell line (Mino, Jeko-1) Spend sensibility (Figure 18 schemes A).This is not statistics indicate that the activity profile according to Shandong for Buddhist nun and compound C is overlapped, and replaces Buddhist nun's resistance according to Shandong MCL model inhibits sensitive to PRMT5.In addition, PRMT5 inhibitor and Yi Lu replace the combination of Buddhist nun in the MCL cells of most of tests The antiproliferative activity (combinatorial index (CI) < 1) (Figure 18 schemes B and C) that collaboration is shown in system, shows the combination of two kinds of compounds Increased treatment benefit can be provided.These are statistics indicate that PRMT5 inhibitor can be used for replacing the MCL patient population of Buddhist nun's resistance according to Shandong Body, and PRMT5 inhibitor can explored in intractable and sensibility environment according to Shandong for Buddhist nun and replacing the combination of Buddhist nun according to Shandong.

Effect in lymphoma mantle cell model

In order to test whether the effect of observing in growth in vitro/death measurement in lymphoma cell line model is suitable for Vivo environment carries out compound C function in the heteroplastic transplantation model of lymphoma mantle cell (sensitive Z138 and Maver-1 cell line) Effect research.Firstly, test compound C is treated to tumour growth in 21 days efficacy studies in Z-138MCL heteroplastic transplantation model Therapeutic effect.Compared with support samples, the tumour in all compound C dosage groups shows the significance difference of weight and volume It is different, from the minimum 40%TGI to up to > the 90% of highest 100mg/kg BID dosage group of lowest dose level group (25mg/kg BID) (in all efficacy studies, not observing weight loss in all groups, Figure 19 schemes A).Using SDMA western to swollen Tumor carry out PD analysis shows that, the methyl of all dosage groups label is reduced more than 70%, and > 98% is up in maximum dose level group and (is schemed 19, scheme B).

Next, the effect of assessing compound C in Maver-1MCL heteroplastic transplantation model (Figure 20).It was measured at the 18th day All compound C dosage groups in tumour significant difference is shown in volume compared with support samples, range is from minimum dose The minimum 50%TGI of amount group is up to > 90% into maximum dose level group.PD is carried out analysis shows that all dosage to tumour using SDMA The methyl label of group reduces 80-95%.

These are statistics indicate that compound C processing causes significant tumour raw in the heteroplastic transplantation model of lymphoma mantle cell It is long to inhibit (close to 100%TGI).SDMA signal (> 90%) is almost inhibited for maximum TGI (> 90%), is indicated that Remarkable efficacy is obtained in tumour, is needed PRMT5 activity suppression > 90%.

Lymthoma biology is summarized

It is in MCL that presently described strongest mechanism, which contacts, between PRMT5 and cancer.The frequent mistake in MCL of PRMT5 Expression, and the high expression in core compartment, wherein it increases histone methylated horizontal and silencing tumor suppressor gene subset. Importantly, cyclin D1 (oncogene of transposition in most MCL patients) is related to PRMT5 and passes through cdk4 Dependent mechanism increases PRMT5 activity.PRMT5 has mediated the inhibition of the key gene to negative regulator DNA replication dna, to allow thin The growth of born of the same parents' cyclin D1 dependent tumors.PRMT5 strikes the low cell transformation for inhibiting cyclin D1 dependence, causes Death of neoplastic cells.These data highlight important function of the PRMT5 in MCL, and show that PRMT5 inhibition can be used as in MCL Therapeutic strategy.

Compound C inhibits growth and inducing death in MCL cell line, and MCL cell line is to test so far most One of sensitive cell line (in growth in 6 days/death measurement).In the MCL cell line of the test at one group, the gIC of 3 cell line₅₀< 10nM, 2 cell lines show gIC₅₀≤ 100nM, the gIC of 1 cell line₅₀>1μM.Currently research compound C is to PRMT5 With the influence of the downstream targets of cyclin D1, to assess, whether it facilitates antibiosis length and rush Apoptosis reacts.

SDMA antibody Methylscan^TMFor assessing the PRMT5 substrate in MCL cell line.The overwhelming majority contains SDMA Protein be participate in cell montage and RNA processing (SmB, Lsm4, hnRNPH1 and other), transcription (FUBP1) and translate because Son highlights effect of the PRMT5 as the important regulator of cell RNA stable state.SDMA antibody is further used for assessing one group of MCL PRMT5 in cell line inhibits, wherein SDMA IC₅₀Value is similar in sensitive and resistant models, and showing SDMA not is the mark of reaction Note, but the label that PRMT5 inhibits.

Montage variation in compound C processing induction RNA subgroup, is especially observed in MCL and DLBCL cell line The conversion of MDM4 isotype shows that PRMT5 inhibits to activate p53 approach by adjusting MDM4 montage.Strike it is low/rescue experiment will be used for into One step assesses effect of the MDM4/p53 approach in the cell response that PRMT5 inhibitor induces.

The expression of MDM4 isotype and p53 mutation are the potential predictive biological markers for inhibiting reaction in MCL to PRMT5 Object.In MCL cell line group, two kinds of wild type p53 cell lines Z138 and JVM-2 are most sensitive cell line (minimum gIC₅₀ Value and only there are two the MCL cell lines that cytotoxicity is shown in growth in 6 days/death measures).

In recent years, the method for revolutionizing MCL treatment according to Shandong for the Clinical Exploration of Buddhist nun.Vitro data shows PRMT5 Inhibitor can be used for replacing the MCL PATIENT POPULATION of Buddhist nun's resistance according to Shandong, and can according to Shandong for Buddhist nun is intractable and sensibility environment in It explores PRMT5 inhibitor and replaces the combination of Buddhist nun according to Shandong.

It is significant that In vivo study shows that compound C treatment results in the heteroplastic transplantation model of lymphoma mantle cell Tumor growth inhibition (close to 100%TGI).In order to obtain maximum effect (TGI > 90%) in tumour, need almost to press down PRMT5 processed is active (> 90%).

Mammary gland cancer biology

Cell line selection data prove that breast cancer cell line inhibits sensitive to PRMT5, and survey in 2D growth in 6 days/death Growth inhibition (low Y almost is shown in fixed_min- T0, Fig. 7-9).In addition, in (PDX) tumor model group from patient Colony formation assay statistics indicate that, tumor of breast is one of tumour most sensitive in the group (based on compound E rel.IC₅₀ Value, Figure 10).Therefore, breast cancer cell line is assessed in several growth/death and Mechanism Study, is inhibited with assessing PRMT5 in cream Effect and treatment potentiality in gland cancer.

In order to understand the PRMT5 inhibitor activity across different tumor of breast hypotypes, using PRMT5 tool compound at 7 days One group of breast cancer cell line (Figure 21) is analyzed in growth measurement.PRMT5 inhibits in the various Asias for the breast cancer cell line tested With low IC in type₅₀Value weakens cell growth.Compared with HER2 or hormone receptor (HR) positive cell line, TNBC (three negative breasts Cancer) value IC in cell line₅₀It is worth minimum.

In growths in 6 days/death measurement, most of breast cancer cell lines show cell inhibitory effect.In order to assess Whether being exposed to compound C for a long time will affect the cell inhibiting and cytotoxic nature of response, in long term growth/death measurement Middle assessment PRMT5 inhibitor (Figure 22).In SKBR3, MDA-MB-468 and MCF-7 cell, with compound C (and tool point Sub- compound B) processing after being exposed to compound for a long time (7-10 days) lead to cell-cytotoxic reaction.In ZR-75-1 cell, PRMT5 inhibitor inhibits response in (the 3-12 days) all time points initiation cell, and Z-138 (MCL, including as control) is thin Born of the same parents show the net cell death of serious totality at all time points (the 3-10 days) of measurement.These are statistics indicate that PRMT5 presses down Make the net cell death (cytotoxic response) caused after long-time exposure (> 5 days) in a part of breast cancer cell line.

It is whether related to the variation of cell cycle distribution in order to test the influence that compound C grows cell, use iodate The influence (Figure 23) of third ingot FACS (fluorescence-activated cell sorting) analysis assessment compound C cell cycle.In general, FACS As a result consistent with Long-term Proliferation data, show in 3 in 4 breast cancer cell lines, long-term compound C processing was at 7-10 days Inducing cell death (< 2N increase) after processing.In MCF-7 cell (p53 wild type), compound C processing causes thin on day 2 For born of the same parents in the accumulation of G1 phase (2N) and the loss (>2N and<4N) of the S phase cell of cell cycle, subsequent cell death is such as the 10th day thin Born of the same parents are proved in sub- G1 phase (< 2N) accumulation.In ZR-75-1 cell (p53 wild type), compound C cell cycle is distributed shadow Sound is smaller.Wherein the 7th day and G1 (2N) reduction in the 10th day, and in > 4N cellular portions increase.MDA-MB-468 and SKBR-3 are thin The response that born of the same parents system handles compound C is similar, reduces at G1 (2N) phase (the 7th day or the 10th day), G2/M (4N) and > 4N DNA contain Amount increases, this is consistent with the accumulation of cell in subG1 (< 2N), shows cell death.These are statistics indicate that PRMT5 inhibits to influence The distribution of cell in cell cycle, and phenotypic results depend on cellular environment.

In order to assess whether PRMT5 activity is comparably inhibited in sensitive and resistant breast cancer cell line, press down in PRMT5 Measurement SDMA is horizontal (Figure 24) in preparation treated cell.In general, the time that SDMA is reduced is for the thin of all tests Born of the same parents system (sensitive and resistance) is similar.The maximum suppression of SDMA is observed on day 3.SDMA in MDA-MB-468 cell IC₅₀For 5.4nM, similar to the SDMA IC in Z138 cell₅₀.These are statistics indicate that SDMA is the mark of PRMT5 catalytic activity Object, and cannot predict the antiproliferative reaction that PRMT5 inhibits.SDMA IC₅₀It is worth just further in one group of breast cancer cell line Assessment.

Effect in internal breast cancer model

Next, assessing the effect that PRMT5 inhibits in the In vivo model of breast cancer.Firstly, with 100mg/kg (QD and BID) and the compound C of 200mg/kg (QD) handles MDA-MB-468, triple negative breast cancer heteroplastic transplantation model (Figure 25).? Maximum Tumor growth inhibition (TGI=83%) is observed in 100mg/kg BID processing group, wherein SDMA inhibits greater than 90%, and In the animal of 100mg/kg QD processing, compound C processing is invalid and SDMA inhibiting rate is lower than 80%.It should be statistics indicate that SDMA Level needs almost to be suppressed (> 90%), to see significant TGI in Breast Cancer Xenograft Model in vivo.

Breast cancer is summarized

In breast cancer, high PRMT5 expression and high PDCD4 (programmed cell death factors 4) horizontal forecast are overall not Good survival rate.

Breast cancer cell line and the derivative model of patient with breast cancer are tested in 2D growth/death and colony formation assay One of most sensitive model.

Compound C processing leads to complete growth inhibition in growth in 6 days/death measurement, and in 4 cells of test 3 in system medium-term and long-term to be exposed to PRMT5 inhibitor inducing cell death.

In 7 days proliferation tests, inhibiting effect ratio Her2 and hormone receptor positive cell of the TNBC cell line to PRMT5 It is more sensitive.

The SDMA level of the breast cancer cell line of the sensitivity and resistance that are handled with PRMT5 inhibitor reduces, and shows SDMA The not instead of marker of response, the active marker of PRMT5.

In MDA-MB-468 heteroplastic transplantation model, compound C treatment causes to swell in 100mg/kg BID treatment group Tumor growth inhibition (TGI=83%), wherein SDMA inhibits to be greater than 90%, and in the animal of 100mg/kg QD treatment, compound C treatment is invalid and SDMA inhibits less than 80%.Should statistics indicate that, in order to be observed in Breast Cancer Xenograft Model in vivo Significant TGI needs almost to inhibit SDMA horizontal (> 90%).

In general, these are statistics indicate that PRMT5 inhibits the potential treatment plan for being used as breast cancer, especially TNBC hypotype Slightly.

Spongioblastoma (GBM) biology

PRMT5 albumen is often overexpressed in spongioblastoma tumour, and high PRMT5 level and GBM patient etc. Grade (IV grade) and poor survival rate it is strongly related (Yan F, et al. Cancer Res.On March 15th, 2014；74 (6): 1752- 65).PRMT5 strikes the low growth for reducing GBM cell line and survival and significantly improves depositing in Gli36 heteroplastic transplantation model in situ It is living (Yan F, et al. CancerRes.2014 March 15；74(6):1752-65).PRMT5 also passes through adjusting neural progenitor cell Growth and differentiation play an important role in normal mouse brain growth (Bezzi M, et al., Genes Dev.2013 September 1 day； 27 (17): 1903-16).

Spongioblastoma cell line model is one of tumor type most sensitive in soft-fractrue rock mass measurement (figure 10).In 2D, growth in 6 days/death CTG measurement, GBM cell line has the gIC within the scope of 40-22000nM₅₀Value, wherein responding Mainly cell inhibits, except SF539 cell line (Fig. 7 and 8).In order to understand that PRMT5 inhibits to being exposed to PRMT5 for more time The influence of cell growth and survival after inhibitor, the test compound C activity (figure in 2D, growth in 14 days/death CTG measurement 26).In general, cell inhibition/cytotoxic response property does not change after being exposed to compound for a long time, and rings It should inhibit and undergo unique cell line of apoptosis to be SF539 in PRMT5.

Next, by measurement SDMA, p53 and p21 protein level and MDM4 montage, what is handled with PRMT5 inhibitor The influence (Figure 27) to cell methylation and p53 approach is assessed in GBM cell.PRMT5 inhibition causes in all test cell systems The reduction (Figure 27 schemes B) of SDMA signal, the sensibility that PRMT5 is inhibited without considering them.It is detected in all cell lines Alternative MDM4 montage, in addition to SF539, be p53 mutant and with long MDM4 isotype low basal expression (Figure 27, Scheme A).P53 level increases in all cell lines, and only with wild type p53 cell line (Z138 (MCL), U87-MG and A172 (GBM)) in observe the induction of p21 albumen.These are statistics indicate that PRMT5 inhibitor can pass through the active inactivation of MDM4 And the p53 approach in GBM model is activated, similar to the effect observed in lymphoma model.Importantly, GBM cell line Sensibility is unrelated with p53 mutation status, shows that other mechanism facilitate the growth inhibition phenotype that PRMT5 inhibits induction.Interesting It is that PRMT5 inhibits to cause the cell in wild type p53 GBM cell line to inhibit response.P53 inhibits PRMT5 in GBM cell line Response in effect will further be tested in following research.Inhibit in addition, exploring PRMT5 to thin in GBM model The influence in born of the same parents' period and Neural Differentiation.

Spongioblastoma is summarized

PRMT5 albumen is often overexpressed in spongioblastoma tumour, and high PRMT5 level is higher with GBM patient's Rank (IV grades) and poor survival rate are closely related.

Spongioblastoma cell line model is one of tumor type most sensitive in soft-fractrue rock mass measurement.

In 2D, 6 days and growth in 14 days/death CTG measurement, GBM is mainly that cell inhibits to the PRMT5 response inhibited (having 3 in 4 cell lines, 1 cell line has cytotoxic response).

PRMT5 inhibits the reduction for leading to SDMA signal in all test cell systems, without considering that they inhibit PRMT5 Sensibility.

Other sensibility tumor types

Model discrimination derived from cell line and patient is statistics indicate that PRMT5 inhibitor weakens carefully in extensive tumor type Intracellular growth and survival (Fig. 7-10).

Whole biology is summarized

Compound C inhibits the symmetrical arginine di-methylation on various kinds of cell albumen, including spliceosome ingredient, group egg White, transcription factor and kinases.Therefore, PRMT5 inhibitor influences RNA stable state, including transcription, montage and mRNA by number of mechanisms The variation of translation.

PRMT5 inhibition causes gene expression and montage to change, and eventually leads to the induction of p53.Compound C is in p53 ubiquitin Isotype conversion is induced in ligase MDM4, stablizes p53 albumen, and in jacket cell and diffusivity large B cell lymphoid tumor and cream P53 expression of target gene signal transduction is induced in gland cancer and glioma cancerous cell line (tumor type uniquely tested so far).

Compound C inhibits the proliferation in extensive entity and blood tumor cell system, and induce a part of jacket cell and The cell death of Diffuse large B cell lymthoma, breast cancer, bladder cancer and glioma cell line.In jacket cell and diffuse Most effective growth inhibition is observed in type large B cell lymphoid tumor cell line.It is moved in lymphoma mantle cell and the xenogenesis of breast cancer The effect that compound C is tested in model is planted, wherein it significantly inhibits tumour growth.These data are to use compound C as set Cell lymphoma, Diffuse large B cell lymthoma, breast cancer and the cancer of the brain therapeutic strategy provide strong theoretical foundation.

Embodiment 2

Combination

It is small to measure CT-26 (colon cancer) tumor model for using compound C and anti-OX40 as single agents and combined therapy The survival advantage of mouse and A20 (lymthoma) tumor model mouse.Mouse oral drug administration carrier or 126.1mg/kg compound C, often It is primary, for 3 weeks.To the anti-OX40 (clone OX86) or corresponding carrier for applying 5mg/kg in mouse peritoneum, twice a week, Continue 21 days.Cloning OX86 is rat anti-mouse OX40 receptor antibody.

Figure 28 is shown with respective carrier (the 1st group and the 3rd group), compound C (the 6th group), anti-OX40 (the 2nd group) and compound Average viability in the A20 tumor model of (the 11st group) of the combination treatment of C and anti-OX40.

Figure 29 is shown with respective carrier (the 1st group and the 3rd group), compound C (the 6th group), anti-OX40 (the 2nd group) and compound The average viability of the CT-26 tumor model of (the 11st group) of the combination treatment of C and anti-OX40.

Lead to moderate survival advantage with the combined therapy A20 xenograft tumours of anti-OX-40 antibody and compound C, dashes forward Potential cooperative interaction between two kinds of medicaments is gone out.

Sequence table

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gtcaaggttt cctgcaaggc ttctggttat accttcacag actattcaat gcactgggtg 180

cgacaggctc caggacaagg tttaaagtgg atgggctgga taaacactga gactggtgag 240

ccaacatatg cagatgactt caagggacgg tttgtcttct ctttggacac ctctgtcagc 300

actgcctatt tgcagatcag cagcctcaaa gctgaggaca cggctgtgta ttactgtgct 360

aatccctact atgattacgt ctcttactat gctatggact actggggtca gggaaccacg 420

gtcaccgtct cctcaggtaa gaatggcctc tcaagctt 458

<210> 7

<211> 11

<212> PRT

<213> Mus sp.

<400> 7

Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 8

<211> 7

<212> PRT

<213> Mus sp.

<400> 8

Ser Ala Ser Tyr Leu Tyr Thr

1 5

<210> 9

<211> 9

<212> PRT

<213> Mus sp.

<400> 9

Gln Gln His Tyr Ser Thr Pro Arg Thr

1 5

<210> 10

<211> 107

<212> PRT

<213> Mus sp.

<400> 10

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Arg

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 11

<211> 107

<212> PRT

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthesis polypeptide

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 416

<212> DNA

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthetic polyribonucleotides

<400> 12

gctagcacca ccatggagtc acagattcag gtctttgtat tcgtgtttct ctggttgtct 60

ggtgttgacg gagacattca gatgacccag tctccatcct ccctgtccgc atcagtggga 120

gacagggtca ccatcacctg caaggccagt caggatgtga gtactgctgt agcctggtat 180

caacagaaac caggaaaagc ccctaaacta ctgatttact cggcatccta cctctacact 240

ggagtccctt cacgcttcag tggcagtgga tctgggacgg atttcacttt caccatcagc 300

agtctgcagc ctgaagacat tgcaacatat tactgtcagc aacattatag tactcctcgg 360

acgttcggtc agggcaccaa gctggaaatc aaacgtaagt agaatccaaa gaattc 416

<210> 13

<211> 5

<212> PRT

<213> Mus sp.

<400> 13

Ser His Asp Met Ser

1 5

<210> 14

<211> 17

<212> PRT

<213> Mus sp.

<400> 14

Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met Glu

1 5 10 15

Arg

<210> 15

<211> 11

<212> PRT

<213> Mus sp.

<400> 15

His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr

1 5 10

<210> 16

<211> 120

<212> PRT

<213> Mus sp.

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Lys Leu Ser Cys Glu Ser Asn Glu Tyr Glu Phe Pro Ser His

20 25 30

Asp Met Ser Trp Val Arg Lys Thr Pro Glu Lys Arg Leu Glu Leu Val

35 40 45

Ala Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met

50 55 60

Glu Arg Arg Phe Ile Ile Ser Arg Asp Asn Thr Lys Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 17

<211> 120

<212> PRT

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthesis polypeptide

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Glu Phe Pro Ser His

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val

35 40 45

Ala Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met

50 55 60

Glu Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 18

<211> 451

<212> DNA

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthetic polyribonucleotides

<400> 18

actagtacca ccatggactt cgggctcagc ttggttttcc ttgtccttat tttaaaaagt 60

gtacagtgtg aggtgcagct ggtggagtct gggggaggct tagtgcagcc tggagggtcc 120

ctgagactct cctgtgcagc ctctgaatac gagttccctt cccatgacat gtcttgggtc 180

cgccaggctc cggggaaggg gctggagttg gtcgcagcca ttaatagtga tggtggtagc 240

acctactatc cagacaccat ggagagacga ttcaccatct ccagagacaa tgccaagaac 300

tcactgtacc tgcaaatgaa cagtctgagg gccgaggaca cagccgtgta ttactgtgca 360

agacactatg atgattacta cgcctggttt gcttactggg gccaagggac tatggtcact 420

gtctcttcag gtgagtccta acttcaagct t 451

<210> 19

<211> 15

<212> PRT

<213> Mus sp.

<400> 19

Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His

1 5 10 15

<210> 20

<211> 7

<212> PRT

<213> Mus sp.

<400> 20

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 21

<211> 9

<212> PRT

<213> Mus sp.

<400> 21

Gln His Ser Arg Glu Leu Pro Leu Thr

1 5

<210> 22

<211> 111

<212> PRT

<213> Mus sp.

<400> 22

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 23

<211> 111

<212> PRT

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthesis polypeptide

<400> 23

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 24

<211> 428

<212> DNA

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthetic polyribonucleotides

<400> 24

gctagcacca ccatggagac agacacactc ctgttatggg tactgctgct ctgggttcca 60

ggttccactg gtgaaattgt gctgacacag tctcctgcta ccttatcttt gtctccaggg 120

gaaagggcca ccctctcatg cagggccagc aaaagtgtca gtacatctgg ctatagttat 180

atgcactggt accaacagaa accaggacag gctcccagac tcctcatcta tcttgcatcc 240

aacctagaat ctggggtccc tgccaggttc agtggcagtg ggtctgggac agacttcacc 300

ctcaccatca gcagcctaga gcctgaggat tttgcagttt attactgtca gcacagtagg 360

gagcttccgc tcacgttcgg cggagggacc aaggtcgaga tcaaacgtaa gtacactttt 420

ctgaattc 428

<210> 25

<211> 5

<212> PRT

<213> Mus sp.

<400> 25

Asp Ala Trp Met Asp

1 5

<210> 26

<211> 19

<212> PRT

<213> Mus sp.

<400> 26

Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu Ser

1 5 10 15

Val Asn Gly

<210> 27

<211> 8

<212> PRT

<213> Mus sp.

<400> 27

Gly Glu Val Phe Tyr Phe Asp Tyr

1 5

<210> 28

<211> 414

<212> DNA

<213> Mus sp.

<400> 28

atgtacttgg gactgaacta tgtattcata gtttttctct taaatggtgt ccagagtgaa 60

gtgaagcttg aggagtctgg aggaggcttg gtgcaacctg gaggatccat gaaactctct 120

tgtgctgcct ctggattcac ttttagtgac gcctggatgg actgggtccg ccagtctcca 180

gagaaggggc ttgagtgggt tgctgaaatt agaagcaaag ctaataatca tgcaacatac 240

tatgctgagt ctgtgaatgg gaggttcacc atctcaagag atgattccaa aagtagtgtc 300

tacctgcaaa tgaacagctt aagagctgaa gacactggca tttattactg tacgtggggg 360

gaagtgttct actttgacta ctggggccaa ggcaccactc tcacagtctc ctca 414

<210> 29

<211> 138

<212> PRT

<213> Mus sp.

<400> 29

Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly

1 5 10 15

Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr

65 70 75 80

Tyr Ala Glu Ser Val Asn Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Gly Ile Tyr Tyr Cys Thr Trp Gly Glu Val Phe Tyr Phe Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210> 30

<211> 448

<212> DNA

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthetic polyribonucleotides

<400> 30

actagtacca ccatgtactt gggactgaac tatgtattca tagtttttct cttaaatggt 60

gtccagagtg aagtgaagct ggaggagtct ggaggaggct tggtgcaacc tggaggatcc 120

atgaaactct cttgtgctgc ctctggattc acttttagtg acgcctggat ggactgggtc 180

cgccagtctc cagagaaggg gcttgagtgg gttgctgaaa ttagaagcaa agctaataat 240

catgcaacat actatgctga gtctgtgaat gggaggttca ccatctcaag agatgattcc 300

aaaagtagtg tctacctgca aatgaacagc ttaagagctg aagacactgg catttattac 360

tgtacgtggg gggaagtgtt ctactttgac tactggggcc aaggcaccac tctcacagtc 420

tcctcaggtg agtccttaaa acaagctt 448

<210> 31

<211> 138

<212> PRT

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthesis polypeptide

<400> 31

Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly

1 5 10 15

Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr

65 70 75 80

Tyr Ala Glu Ser Val Asn Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Gly Ile Tyr Tyr Cys Thr Trp Gly Glu Val Phe Tyr Phe Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210> 32

<211> 11

<212> PRT

<213> Mus sp.

<400> 32

Lys Ser Ser Gln Asp Ile Asn Lys Tyr Ile Ala

1 5 10

<210> 33

<211> 7

<212> PRT

<213> Mus sp.

<400> 33

Tyr Thr Ser Thr Leu Gln Pro

1 5

<210> 34

<211> 8

<212> PRT

<213> Mus sp.

<400> 34

Leu Gln Tyr Asp Asn Leu Leu Thr

1 5

<210> 35

<211> 378

<212> DNA

<213> Mus sp.

<400> 35

atgagaccgt ctattcagtt cctggggctc ttgttgttct ggcttcatgg tgctcagtgt 60

gacatccaga tgacacagtc tccatcctca ctgtctgcat ctctgggagg caaagtcacc 120

atcacttgca agtcaagcca agacattaac aagtatatag cttggtacca acacaagcct 180

ggaaaaggtc ctaggctgct catacattac acatctacat tacagccagg catcccatca 240

aggttcagtg gaagtgggtc tgggagagat tattccttca gcatcagcaa cctggagcct 300

gaagatattg caacttatta ttgtctacag tatgataatc ttctcacgtt cggtgctggg 360

accaagctgg agctgaaa 378

<210> 36

<211> 126

<212> PRT

<213> Mus sp.

<400> 36

Met Arg Pro Ser Ile Gln Phe Leu Gly Leu Leu Leu Phe Trp Leu His

1 5 10 15

Gly Ala Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ser Ser Gln Asp

35 40 45

Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro

50 55 60

Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser

85 90 95

Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp

100 105 110

Asn Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

115 120 125

<210> 37

<211> 413

<212> DNA

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthetic polyribonucleotides

<400> 37

gctagcacca ccatgagacc gtctattcag ttcctggggc tcttgttgtt ctggcttcat 60

ggtgctcagt gtgacatcca gatgacacag tctccatcct cactgtctgc atctctggga 120

ggcaaagtca ccatcacttg caagtcaagc caagacatta acaagtatat agcttggtac 180

caacacaagc ctggaaaagg tcctaggctg ctcatacatt acacatctac attacagcca 240

ggcatcccat caaggttcag tggaagtggg tctgggagag attattcctt cagcatcagc 300

aacctggagc ctgaagatat tgcaacttat tattgtctac agtatgataa tcttctcacg 360

ttcggtgctg ggaccaagct ggagctgaaa cgtaagtaca cttttctgaa ttc 413

<210> 38

<211> 126

<212> PRT

<213>artificial sequence

<220>

<223>explanation of artificial sequence: synthesis polypeptide

<400> 38

Met Arg Pro Ser Ile Gln Phe Leu Gly Leu Leu Leu Phe Trp Leu His

1 5 10 15

Gly Ala Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ser Ser Gln Asp

35 40 45

Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro

50 55 60

Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser

85 90 95

Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp

100 105 110

Asn Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

115 120 125

Claims (27)

1. The combination of 1.II type protein arginine transmethylase (II type PRMT) and immunomodulator, wherein the immunological regulation Agent is anti-OX40 antibody or its antigen-binding fragment.
2. 2. the combination of claim 1, wherein the II type PRMT inhibitor is Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor or protein arginine transmethylase 9 (PRMT9) inhibitor.
3. 3. the combination of claims 1 or 2, wherein the II type PRMT inhibitor is the compound of formula (III):

   Or its pharmaceutically acceptable salt,

   Wherein

   Indicate singly-bound or double bond；

   R¹For hydrogen, R^zOr-C (O) R^z, wherein R^zFor the C optionally replaced_1-6Alkyl；

   L is-N (R) C (O)-,-C (O) N (R)-,-N (R) C (O) N (R)-,-N (R) C (O) O- or-OC (O) N (R)-；

   The C that each R independently is hydrogen or optionally replaces_1-6Aliphatic group；

   Ar is the heteroatomic monocycle or Bicyclic aryl rings that nitrogen, oxygen and sulphur are independently selected from 0-4, wherein Ar substitution have 0,1,2, 3,4 or 5 R^yGroup, as long as chemical valence allows；

   Each R^yIndependently selected from halogen ,-CN ,-NO₂, optionally replace aliphatic group, optionally replace carbocylic radical, optionally replace Aryl, the heterocycle optionally replaced, the heteroaryl ,-OR that optionally replace^A、-N(R^B)₂、-SR^A,-C (=O) R^A、-C(O)OR^A、- C(O)SR^A、-C(O)N(R^B)₂、-C(O)N(R^B)N(R^B)₂、-OC(O)R^A、-OC(O)N(R^B)₂、-NR^BC(O)R^A、-NR^BC(O)N (R^B)₂、-NR^BC(O)N(R^B)N(R^B)₂、-NR^BC(O)OR^A、-SC(O)R^A,-C (=NR^B)R^A,-C (=NNR^B)R^A,-C (=NOR^A) R^A,-C (=NR^B)N(R^B)₂,-NRBC (=NR^B)R^B,-C (=S) R^A,-C (=S) N (R^B)₂、-NR^BC (=S) R^A、-S(O)R^A、- OS(O)₂R^A、-SO₂R^A、-NR^BSO₂R^AOr-SO₂N(R^B)₂；

   Each R^AIndependently selected from hydrogen, optionally the aliphatic group that replaces, the heterocycle optionally replaced, is appointed at the carbocylic radical optionally replaced The heteroaryl choosing the aryl in generation and optionally replacing；

   Each R^BIndependently selected from hydrogen, optionally the aliphatic group that replaces, the heterocycle optionally replaced, is appointed at the carbocylic radical optionally replaced The aryl for choosing generation and the heteroaryl optionally replaced or two R^BGroup is formed together with atom between them and optionally takes The heterocycle in generation；

   R⁵、R⁶、R⁷And R⁸It independently is hydrogen, halogen or the aliphatic group optionally replaced；

   Each R^XIndependently selected from halogen ,-CN, the optionally aliphatic group ,-OR' and the-N (R ") that replace₂；

   R' is hydrogen or the aliphatic group optionally replaced；

   Each R " independently be hydrogen or the aliphatic group optionally replaced or two R " is formed together with atom between them Heterocycle；And

   N is 0,1,2,3,4,5,6,7,8,9 or 10, as long as chemical valence allows.
4. 4. the combination of any one of claim 1-3, wherein the II type PRMT inhibitor is the compound of formula (X):

   Or its pharmaceutically acceptable salt.
5. 5. the combination of any one of claim 1-4, wherein the II type PRMT inhibitor is compound C:

   Or its pharmaceutically acceptable salt.
6. 6. the combination of any one of claim 1-5, wherein the immunomodulator is OX40 agonist.
7. 7. the combination of claim 6, wherein the immunomodulator is anti-OX40 antibody or its antigen-binding fragment, it includes with It is next or multiple: CDRH1 shown in SEQ ID NO:1；CDRH2 shown in SEQ ID NO:2；Shown in SEQ ID NO:3 CDRH3；CDRL1 shown in SEQ ID NO:7；Shown in CDRL2 and/or SEQ ID NO:9 shown in SEQ ID NO:8 The direct equivalent of CDRL3 or each CDR, wherein direct equivalent has in the CDR is no more than two amino acid substitutions.
8. 8. the combination of any one of claim 6-7, wherein the immunomodulator is anti-OX40 antibody or its antigen-binding fragment, It includes with amino acid sequence shown in SEQ ID NO:5 have at least the variable heavy chain sequence of 90% sequence identity and with Amino acid sequence shown in SEQ ID NO:11 has the variable light chain sequence of at least 90% sequence identity.
9. The combination of 9.II type protein arginine transmethylase (II type PRMT) and immunomodulator, wherein the immunological regulation Agent is anti-OX40 antibody or its antigen-binding fragment, wherein the II type PRMT inhibitor is compound C:

   Or its pharmaceutically acceptable salt, and wherein the immunomodulator be anti-OX40 antibody or its antigen-binding fragment, Include following one or more: CDRH1 shown in SEQ ID NO:1；CDRH2 shown in SEQ ID NO:2；SEQ ID NO:3 Shown in CDRH3；CDRL1 shown in SEQ ID NO:7；Shown in CDRL2 and/or SEQ ID NO:9 shown in SEQ ID NO:8 CDRL3 or each CDR direct equivalent, wherein direct equivalent in the CDR have be no more than two amino acid substitutions.
10. The combination of 10.II type protein arginine transmethylase (II type PRMT) and immunomodulator, wherein the immune tune Saving agent is anti-OX40 antibody or its antigen-binding fragment, wherein the II type PRMT inhibitor is compound C:

    Or its pharmaceutically acceptable salt, and wherein the immunomodulator be anti-OX40 antibody or its antigen-binding fragment, Comprising there is at least variable heavy chain sequence of 90% sequence identity and and SEQ with amino acid sequence shown in SEQ ID NO:5 Amino acid sequence shown in ID NO:11 has the variable light chain sequence of at least 90% sequence identity.
11. 11. the method for the treatment of cancer in the people of needs, this method includes administering to the human the combination of any one of claim 1-10, And it is following at least one: pharmaceutically acceptable carrier and pharmaceutically acceptable diluent, to treat the cancer of the people Disease.
12. 12. pharmaceutical composition, it includes the inhibition of the II type protein arginine transmethylase (II type PRMT) of therapeutically effective amount Agent and the second pharmaceutical composition, which includes the immunomodulator of therapeutically effective amount, wherein the immune tune Saving agent is anti-OX40 antibody or its antigen-binding fragment.
13. 13. pharmaceutical composition, it includes the inhibition of the II type protein arginine transmethylase (II type PRMT) of therapeutically effective amount Agent and the second pharmaceutical composition, which includes the immunomodulator of therapeutically effective amount, wherein the immune tune Saving agent is anti-OX40 antibody or its antigen-binding fragment, wherein the immunomodulator is anti-OX40 antibody or its antigen binding fragment Section, wherein the II type PRMT inhibitor is compound C:

    Or its pharmaceutically acceptable salt, and wherein the immunomodulator be anti-OX40 antibody or its antigen-binding fragment, Include following one or more: CDRH1 shown in SEQ ID NO:1；CDRH2 shown in SEQ ID NO:2；SEQ ID NO:3 Shown in CDRH3；CDRL1 shown in SEQ ID NO:7；Shown in CDRL2 and/or SEQ ID NO:9 shown in SEQ ID NO:8 CDRL3 or each CDR direct equivalent, wherein direct equivalent in the CDR have be no more than two amino acid substitutions.
14. 14. pharmaceutical composition, it includes the inhibition of the II type protein arginine transmethylase (II type PRMT) of therapeutically effective amount Agent and the second pharmaceutical composition, which includes the immunomodulator of therapeutically effective amount, wherein the immune tune Saving agent is anti-OX40 antibody or its antigen-binding fragment, wherein the immunomodulator is anti-OX40 antibody or its antigen binding fragment Section, wherein the II type PRMT inhibitor is compound C:

    Or its pharmaceutically acceptable salt, and wherein the immunomodulator be anti-OX40 antibody or its antigen-binding fragment, Comprising there is at least variable heavy chain sequence of 90% sequence identity and and SEQ with amino acid sequence shown in SEQ ID NO:5 Amino acid sequence shown in ID NO:11 has the variable light chain sequence of at least 90% sequence identity.
15. 15. pharmaceutical composition described in claim 12, wherein the II type PRMT inhibitor turns for protein arginine methyl Move (PRMT5) inhibitor of enzyme 5 or protein arginine transmethylase 9 (PRMT9) inhibitor.
16. 16. pharmaceutical composition described in claim 12 or 15, wherein the II type PRMT inhibitor is the chemical combination of formula (III) Object:

    Or its pharmaceutically acceptable salt,

    Wherein

    Indicate singly-bound or double bond；

    R¹For hydrogen, R^zOr-C (O) R^z, wherein R^zFor the C optionally replaced_1-6Alkyl；

    L is-N (R) C (O)-,-C (O) N (R)-,-N (R) C (O) N (R)-,-N (R) C (O) O- or-OC (O) N (R)-；

    The C that each R independently is hydrogen or optionally replaces_1-6Aliphatic group；

    Ar is the heteroatomic monocycle or Bicyclic aryl rings that nitrogen, oxygen and sulphur are independently selected from 0-4, wherein Ar substitution have 0,1,2, 3,4 or 5 R^yGroup, as long as chemical valence allows；

    Each R^yIndependently selected from halogen ,-CN ,-NO₂, optionally replace aliphatic group, optionally replace carbocylic radical, optionally replace Aryl, the heterocycle optionally replaced, the heteroaryl ,-OR that optionally replace^A、-N(R^B)₂、-SR^A,-C (=O) R^A、-C(O)OR^A、- C(O)SR^A、-C(O)N(R^B)₂、-C(O)N(R^B)N(R^B)₂、-OC(O)R^A、-OC(O)N(R^B)₂、-NR^BC(O)R^A、-NR^BC(O)N (R^B)₂、-NR^BC(O)N(R^B)N(R^B)₂、-NR^BC(O)OR^A、-SC(O)R^A,-C (=NR^B)R^A,-C (=NNR^B)R^A,-C (=NOR^A) R^A,-C (=NR^B)N(R^B)₂、-NR^BC (=NR^B)R^B,-C (=S) R^A,-C (=S) N (R^B)₂、-NR^BC (=S) R^A、-S(O)R^A、- OS(O)₂R^A、-SO₂R^A、-NR^BSO₂R^AOr-SO₂N(R^B)₂；

    Each R^AIndependently selected from hydrogen, optionally the aliphatic group that replaces, the heterocycle optionally replaced, is appointed at the carbocylic radical optionally replaced The heteroaryl choosing the aryl in generation and optionally replacing；

    Each R^BIndependently selected from hydrogen, optionally the aliphatic group that replaces, the heterocycle optionally replaced, is appointed at the carbocylic radical optionally replaced The aryl for choosing generation and the heteroaryl optionally replaced or two R^BGroup is formed together optional substitution with atom between them Heterocycle；

    R⁵、R⁶、R⁷And R⁸It independently is hydrogen, halogen or the aliphatic group optionally replaced；

    Each R^XIndependently selected from halogen ,-CN, the optionally aliphatic group ,-OR' and the-N (R ") that replace₂；

    R' is hydrogen or the aliphatic group optionally replaced；

    Each R " independently be hydrogen or the aliphatic group optionally replaced or two R " is formed together miscellaneous with atom between them Ring；And

    N is 0,1,2,3,4,5,6,7,8,9 or 10, as long as chemical valence allows.
17. 17. pharmaceutical composition described in claim 12,15 or 16, wherein the II type PRMT inhibitor is the chemical combination of formula (X) Object:

    Or its pharmaceutically acceptable salt.
18. 18. claim 12 and the described in any item pharmaceutical compositions of 15-17, wherein the II type PRMT inhibitor is compound C:

    Or its pharmaceutically acceptable salt.
19. 19. claim 12 and the described in any item pharmaceutical compositions of 15-18, wherein the immunomodulator is OX40 excitement Agent.
20. 20. pharmaceutical composition described in claim 19, wherein the immunomodulator is anti-OX40 antibody or its antigen binding Segment, it includes following one or more: CDRH1 shown in SEQ ID NO:1；CDRH2 shown in SEQ ID NO:2；SEQ CDRH3 shown in ID NO:3；CDRL1 shown in SEQ ID NO:7；CDRL2 and/or SEQ ID shown in SEQ ID NO:8 The direct equivalent of CDRL3 shown in NO:9 or each CDR, wherein direct equivalent has in the CDR is no more than two ammonia Base acid replaces.
21. 21. the described in any item pharmaceutical compositions of claim 19-20, wherein the immunomodulator be anti-OX40 antibody or its Antigen-binding fragment, it includes have the variable of at least 90% sequence identity with amino acid sequence shown in SEQ ID NO:5 Sequence of heavy chain and the variable light chain sequence with amino acid sequence shown in SEQ ID NO:11 at least 90% sequence identity.
22. 22. the method for the treatment of cancer in the people of needs, this method includes the claim 12-21 for administering to the human therapeutically effective amount Described in any item pharmaceutical compositions, to treat the cancer of the people.
23. 23. method described in claim 22, wherein the II type PRMT inhibitor and immunomodulator are with way selected from the following Diameter delivers medicine to patient: simultaneously, in succession, in any order, in whole body, oral, intravenous and tumor.
24. 24. method described in claim 22 or 23, wherein the II type PRMT inhibitor is administered orally.
25. 25. the described in any item methods of claim 22-24, wherein the cancer is melanoma, lymthoma or colon cancer.
26. 26. the purposes of the combination of any one of claim 1-10 in medicine preparation.
27. 27. purposes of the combination of any one of claim 1-10 in treating cancer.