KR20190090823A - Combination therapy - Google Patents

Abstract

The present invention provides a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor with an immunomodulator, wherein the immunomodulator is an anti-OX40 antibody or antigen binding fragment thereof. The invention also relates to treating a cancer in a human in need thereof by administering a combination of a type II PRMT inhibitor and an immunomodulator with at least one of a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent. Including a method of treating cancer in a human being in need thereof, wherein the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. The invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a Type II PRMT inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulatory agent, wherein the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof.

Description

Combination therapy

The present invention relates to methods of treating cancer in a mammal and combinations useful for such treatment. In particular, the present invention relates to the combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor with an immunomodulator, such as an anti-OX40 antibody.

Effective treatment of hyperproliferative disorders, including cancer, is a continuing goal in the field of oncology. In general, cancer results from the deregulation of normal processes that control cell division, differentiation and apoptosis cell death, and is characterized by the proliferation of malignant cells with the potential for unlimited growth, local expansion and systemic metastasis. Deregulation of normal processes includes abnormalities in signal transduction pathways and responses to factors that are different from those found in normal cells.

Arginine methylation is an important post-translational modification to proteins involved in a wide range of cellular processes, such as gene regulation, RNA processing, DNA damage response, and signal transduction. Proteins containing methylated arginine are present in both nuclear and cytosol fractions, suggesting that enzymes that catalyze the transfer of methyl groups onto arginine are also present throughout these subcellular compartments (Yang) , Y. & Bedford, MT Protein arginine methyltransferases and cancer.Nat Rev Cancer 13, 37-50, doi: 10.1038 / nrc3409 (2013); Lee, YH & Stallcup, MR Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation. Mol Endocrinol 23, 425-433, doi: 10.1210 / me.2008-0380 (2009). In mammalian cells, methylated arginine exists in three main forms: ω-N ^G -monomethyl-arginine (MMA), ω-N ^G , N ^G -asymmetric dimethyl arginine (ADMA), or ω-N ^G , N ' ^G -symmetric dimethyl arginine (SDMA). Each methylation state can affect protein-protein interactions in different ways and thus has the potential to impart distinct functional consequences on the biological activity of the substrate (Yang, Y. & Bedford, MT Protein arginine methyltransferases and cancer.Nat Rev Cancer 13, 37-50, doi: 10.1038 / nrc3409 (2013)).

Arginine methylation mostly transfers methyl groups from S-adenosyl-L-methionine (SAM) to the substrate arginine side chains with respect to glycine-, arginine-rich (GAR) motifs, to S-adenosyl-homocysteine (SAH) and methylated Arginine occurs through the activity of a family of proteins arginine methyltransferase (PRMT). This protein family consists of 10 members, 9 of which have been shown to have enzymatic activity (Bedford, MT & Clarke, SG Protein arginine methylation in mammals: who, what, and why.Mol Cell 33 , 1-13, doi: 10.1016 / j.molcel. 2008.12.013 (2009)). The PRMT family is categorized into four subtypes (type I-IV) depending on the product of the enzymatic reaction. Type IV enzymes methylate internal guanidino nitrogen and are only described in yeast (Fisk, JC & Read, LK Protein arginine methylation in parasitic protozoa.Eukaryot Cell 10, 1013-1022, doi: 10.1128 / EC.05103-11 (2011)); Type I-III enzymes produce monomethyl-arginine (MMA, Rme1) via a single methylation event. MMA intermediates are considered to be relatively low abundance intermediates, but select substrates of the major type III activity of PRMT7 can remain monomethylated, while type I and II enzymes are each asymmetric dimethyl-arginine (ADMA, Rme2a) from MMA. Or catalyzes progress to symmetric dimethyl arginine (SDMA, Rme2s). Type II PRMTs include PRMT5 and PRMT9, although PRMT5 is the primary enzyme responsible for the formation of symmetric dimethylation. Type I enzymes include PRMT1, PRMT3, PRMT4, PRMT6, and PRMT8. PRMT1, PRMT3, PRMT4 and PRMT6 are ubiquitously expressed while PRMT8 is mostly restricted to the brain (Bedford, MT & Clarke, SG Protein arginine methylation in mammals: who, what, and why.Mol Cell 33, 1-13 , doi: 10.1016 / j.molcel. 2008.12.013 (2009)).

PRMT5 functions as several types of complexes in the cytoplasm and nucleus and requires a binding partner of PRMT5 for substrate recognition and selectivity. Methylosomal protein 50 (MEP50) is a known cofactor of PRMT5 that is required for PRMT5 binding and activity on histones and other substrates (Ho MC, et al. Structure of the arginine methyltransferase PRMT5-MEP50 reveals a mechanism for substrate specificity PLoS One. 2013; 8 (2)).

PRMT5 symmetrically methylates arginine in multiple proteins, preferentially in regions rich in arginine and glycine residues (Karkhanis V, et al. Versatility of PRMT5-induced methylation in growth control and development. Trends Biochem Sci. 2011 Dec; 36 (12): 633-41). PRMT5 methylates arginine in various cellular proteins, including splicing factors, histones, transcription factors, kinases, etc. (Karkhanis V, et al. Versatility of PRMT5-induced methylation in growth control and development. Trends Biochem Sci. 2011 Dec 36 (12): 633-41). Methylation of multiple components of spliceosomes is a major event in spliceosome assembly, and attenuation of PRMT5 activity through knockdown or gene knockout leads to disruption of cell splicing (Bezzi M, et al. Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev. 2013 Sep 1; 27 (17): 1903-16). PRMT5 also methylates histone arginine residues (H3R8, H2AR3 and H4R3), and these histone marks are associated with transcriptional silencing of tumor suppressor genes such as RB and ST7 (Wang L, Pal S, Sif S. Protein arginine methyltransferase 5 suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells.Mol Cell Biol. 2008 Oct; 28 (20): 6262-77). Additionally, symmetric dimethylation of H2AR3 has been implicated in the silencing of differentiation genes in embryonic stem cells (Tee WW, Pardo M, Theunissen TW, Yu L, Choudhary JS, Hajkova P, Surani MA. Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency.Genes Dev. 2010 Dec 15; 24 (24): 2772-7). PRMT5 also plays a role in cellular signaling through methylation of EGFR and PI3K (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 1175 methylation and Tyr 1173 phosphorylation negatively modulates EGFR-mediated ERK activation.Nat Cell Biol. 2011 Feb; 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 5 is a potential oncoprotein that upregulates G1 cyclins / cyclin-dependent kinases and the phosphoinositide 3-kinase / AKT signaling cascade.Cancer Sci. 2012 Sep; 103 (9): 1640-50).

Increasing evidence suggests that PRMT5 is involved in tumorigenesis. PRMT5 protein is overexpressed in many cancer types, including lymphoma, glioma, breast and lung cancer, and PRMT5 overexpression alone is sufficient to transform normal fibroblasts (Pal S, Baiocchi RA, Byrd JC, Grever MR) Jacob ST, Sif S. Low levels of miR-92b / 96 induce PRMT5 translation and H3R8 / H4R3 methylation in mantle cell lymphoma.EMBO J. 2007 Aug 8; 26 (15): 3558-69; Ibrahim R, et al. Expression of PRMT5 in lung adenocarcinoma and its significance in epithelial-mesenchymal transition.Hum Pathol. 2014 Jul; 45 (7): 1397-405; Powers MA, et al. Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4. Cancer Res. 2011 Aug 15; 71 (16): 5579-87; Yan F, et al. Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma.Cancer Res. 2014 Mar 15; 74 (6): 1752-65). Knockdown of PRMT5 often leads to a decrease in cell growth and survival in cancer cell lines. In breast cancer, high PRMT5 expression, combined with high PDCD4 (programmed cell death 4) levels, predicts overall poor survival (Powers MA, et al. Cancer Res. 2011 Aug 15; 71 (16): 5579-87). . PRMT5 methylates PDCD4, altering tumor-related function. Co-expression of PRMT5 and PDCD4 in the in situ model of breast cancer promotes tumor growth. High expression of PRMT5 in glioma is associated with high tumor grade and overall poor survival, while PRMT5 knockdown provides survival benefit in an allograft model (Yan F, et al. Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma.Cancer Res. 2014 Mar 15; 74 (6): 1752-65). Increased PRMT5 expression and activity contribute to the silencing of several tumor suppressor genes in glioma cell lines.

The strongest mechanism link currently described between PRMT5 and cancer is in mantle cell lymphoma (MCL). PRMT5 is frequently overexpressed in MCL and highly expressed in nuclear compartments, where it increases the level of histone methylation and silences a subset of tumor suppressor genes. Recent studies have revealed the role of miRNAs in the upregulation of PRMT5 expression in MCL. More than 50 miRNAs are expected to anneal to the 3 'untranslated region of PRMT5 mRNA. It has been reported that miR-92b and miR-96 levels are inversely correlated with PRMT5 levels in MCL and downregulation of these miRNAs in MCL cells results in upregulated PRMT5 protein levels. Cyclin D1, a translocated oncogene in the majority of MCL patients, associates with PRMT5 and increases PRMT5 activity through a cdk4-dependent mechanism (Aggarwal P, et al. Nuclear cyclin D1 / CDK4 kinase regulates CUL4 expression and triggers neoplastic growth via activation of the PRMT5 methyltransferase.Cancer Cell. 2010 Oct 19; 18 (4): 329-40). PRMT5 mediates the inhibition of key genes that negatively regulate DNA replication, enabling cyclin D1-dependent neoplasia growth. PRMT5 knockdown inhibits cyclin D1-dependent cell transformation resulting in death of tumor cells. These data highlight the important role of PRMT5 in MCL and suggest that PRMT5 inhibition can be used as a treatment strategy in MCL.

In other tumor types, PRMT5 has been assumed to play a role in differentiation, cell death, cell cycle progression, cell growth and proliferation. Although the main mechanisms that link PRMT5 to tumorigenesis are unknown, the neonatal data suggest that PRMT5 contributes to the regulation of gene expression (histone methylation, transcription factor binding or promoter binding), alteration of splicing, and signal transduction. . PRMT5 methylation of the transcription factor E2F1 decreases its ability to inhibit cell growth and promote apoptosis (Zheng S, et al. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1. Mol Cell. 2013 Oct 10; 52 ( 1): 37-51). PRMT5 also methylates p53 in response to DNA damage (Jansson M, et al. Arginine methylation regulates the p53 response. Nat Cell Biol. 2008 Dec; 10 (12): 1431-9), increasing p53-dependent apoptosis. Reduces the ability of p53 to induce cell cycle arrest. These data suggest that PRMT5 inhibition can sensitize cells to DNA damaging agents through induction of p53-dependent apoptosis.

In addition to directly methylating p53, PRMT5 upregulates the p53 pathway through a splicing-related mechanism. PRMT5 knockouts in mouse neural progenitor cells cause alterations in cell splicing, including isotype conversion of the MDM4 gene (Bezzi M, et al. Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev. 2013 Sep 1; 27 (17): 1903-16). PRMT5 knockout cells in Bezzi et al. Have reduced expression of the long MDM4 isotype (which produces functional p53 ubiquitin ligase) and increased expression of the short MDM4 isotype (which produces inactive ligase). Was found. These changes in MDM4 splicing lead to inactivation of MDM4, increased stability of the p53 protein, and subsequent activation of the p53 pathway and cell death. MDM4 alternative splicing was also observed in the PRMT5 knockdown cancer cell line. These data suggest that PRMT5 inhibition can activate multiple nodes of the p53 pathway.

In addition to regulating cancer cell growth and survival, PRMT5 is also involved in epithelial-mesenchymal transition (EMT). PRMT5 binds to the transcription factor SNAIL and acts as an important co-repressor of E-cadherin expression; Knockdown of PRMT5 causes upregulation of E-cadherin levels (Hou Z, et al. The LIM protein AJUBA recruits protein arginine methyltransferase 5 to mediate SNAIL-dependent transcriptional repression. Mol Cell Biol. 2008 May; 28 (10) : 3198-207).

Immunotherapy is another approach to treat hyperproliferative disorders. Enhancing anti-tumor T cell function and inducing T cell proliferation are powerful new approaches for cancer treatment. Three immuno-oncology antibodies (eg immunomodulators) are currently commercially available. Anti-CTLA-4 (YERVOY / ipilimumab) is thought to boost the immune response at the moment of T cell priming and anti-PD-1 antibodies (OPDIVO / nivolumab and Kitruda) (KEYTRUDA) / Pembrolizumab) are thought to act in the local tumor microenvironment by mitigating inhibition checkpoints in tumor-specific T cells that have already been primed and activated.

Although much progress has recently been made in the treatment of cancer, there remains a need for more effective and / or enhanced treatment of individuals suffering from the effects of cancer.

, > 10^-5 M)에 대해서보다 PRMT5 (

, 10^-8 M)에 대해 훨씬 더 큰 효력을 나타냈다. PRMT9는 단지 계통수 내에서의 관계 목적을 위해 제시되고, 패널에서 평가되지 않았다. 도면은 문헌 [Richon VM. et al.]으로부터 채용되었다.
도 7: 암 세포주 패널에서의 6-일 성장/사멸 검정으로부터의 화합물 C gIC₅₀ 값. DLBCL-미만성 대 B-세포 림프종, GBM-교모세포종, MCL-외투 세포 림프종, MM-다발성 골수종
도 8: 암 세포주 패널에서의 6-일 성장/사멸 검정으로부터의 화합물 C gIC₁₀₀ (흑색 사각형) 및 Y_분-T0 (막대) 값 (본 검정에서 사용된 최고 농도는 30 μM이었음). DLBCL-미만성 대 B-세포 림프종, GBM-교모세포종, MCL-외투 세포 림프종, MM-다발성 골수종
도 9: 10일 2D 성장 검정으로부터의 암 세포주 (n=240)에서의 화합물 B gIC₅₀ 값. ALL-급성 림프모구성 백혈병, AML-급성 골수성 백혈병, CML-만성 골수성 백혈병, DLBCL-미만성 대 B-세포 림프종, HL-호지킨 림프종, HN-두경부암, MM-다발성 골수종, NHL-비-호지킨 림프종, NSCLC-비소세포 폐암, PEL-원발성 삼출 림프종, SCLC-소세포 폐암, TCL-T-세포 림프종.
도 10: 환자-유래 및 세포주 종양 모델에서 수행된 8-13일 콜로니 형성 검정으로부터의 화합물 E 상대 IC₅₀ 값.
도 11: SDMA의 화합물 C 억제. (A) 제3일에서의 대표적인 SDMA 용량-반응 곡선 (GAPDH에 대해 정규화된 총 SDMA) (상단) 및 제1일 및 제3일에서의 Z138 세포로부터의 IC₅₀ 값 (하단). (B) MCL 세포주 패널에서의 SDMA IC₅₀ 값 (제4일).
도 12: PRMT5 억제제로 처리된 림프종 세포주에서의 유전자 발현 변화. A. 화합물 B (0.1 및 0.5 μM) 처리 (제2일 및 제4일) 후 림프종 세포주에서의 차등 발현된 (DE) 유전자의 정량화. B. 림프종 세포주에 걸친 DE 유전자의 중첩.
도 13: RNA-서열분석에 의해 확인된 11종 유전자 패널에서의 화합물 C 유전자 발현 EC₅₀ 값. CDKN1A에 대한 대표적인 용량-반응 곡선 (제2일 및 제4일, 좌측 패널) 및 유전자 패널 EC₅₀ 요약 표 (우측 패널, 제4일).
도 14: 화합물 B는 림프종 세포주에서 인트론의 하위세트의 스플라이싱을 감쇠시킨다. A. 세포 스플라이싱의 조절 메카니즘 (문헌 [Bezzi M. et al.]으로부터 채용됨). B. 0.1 또는 0.5 μM 화합물 B로 처리된 림프종 세포주에서의 인트론 발현의 분석.
도 15: 화합물 B는 림프종 세포주에서 유전자의 하위세트에 대한 이소형 전환을 유도한다. A. 2 및 4일 동안 화합물 B (0.1 및 0.5 μM)로 처리된 4종의 림프종 세포주에서의 이소형 전환의 정량화. B. 4종의 림프종 세포주에서의 이소형 전환의 중첩. C. 모든 4종의 림프종 세포주 (4종의 세포주의 중첩)에서 대안적 스플라이싱을 겪은 유전자의 목록.
도 16: 화합물 C로 처리된 MCL 세포주에서의 MDM4 대안적 스플라이싱 및 p53 활성화. A. 2 및 3일 동안 10 및 200 nM 화합물 C 또는 5 μM Nutlin-3으로 처리된 4종의 외투 세포 림프종 세포주 패널에서의 MDM4 이소형 발현 분석 (MDM4-FL-긴; MDM4-S-짧은). B. 3일 동안 10 및 200 nM 화합물 C 또는 5 μM Nutlin-3으로 처리된 MCL 세포주에서의 p53 및 p21 발현의 웨스턴 분석.
도 17: 화합물 C는 MDM4 RNA 스플라이싱 (A) 및 Z138 세포에서의 SDMA/p53/p21 수준 (B)에서의 용량-의존성 변화를 유도한다.
도 18: MCL 세포주에서 단일 작용제로서 및 조합된 PRMT5 억제제 및 이브루티닙의 활성. A. 6-일 성장/사멸 CTG 검정에서의 화합물 C 및 이브루티닙에 대한 gIC₅₀ 값. B. REC1 세포에서의 화합물 B와 이브루티닙의 조합물에 대한 대표적인 성장 곡선 (제6일, 1:1 비). C. 6-일 성장/사멸 CTG 검정에서 나타낸 비의 화합물 B:이브루티닙에 대한 조합 지수 (CI).
도 19: Z138 이종이식 모델에서의 화합물 C 효능 및 PD. A. Z138 이종이식 모델에서의 화합물 C 21-일 효능 연구. B. 효능 연구의 종료시 (마지막 투여 3시간 후)에 수거된 종양으로부터 정량화된 SDMA 웨스턴 데이터.
도 20: Maver-1 이종이식 모델에서의 화합물 C 효능 및 PD. A. Maver-1 이종이식 모델에서의 화합물 C 21-일 효능 연구. B. 효능 연구의 종료시 (마지막 투여 3시간 후)에 수거된 종양으로부터 정량화된 SDMA 웨스턴 데이터.
도 21: 7-일 성장 2D 검정으로부터의 유방암 세포주 패널 (TNBC-삼중 음성 유방암, HER2-Her2 양성, HR-호르몬 수용체 양성)에서의 화합물 B 성장 IC₅₀ 값.
도 22: PRMT5 후보인 화합물 C 및 PRMT5 도구 분자인 화합물 B를 사용하는, 유방 및 MCL 세포주에서의 10-12일 성장/사멸 검정으로부터의 Y_분-T0 값.
도 23: 다양한 기간 동안 30, 200 및 1000 nM 화합물 C로 처리된 유방암 세포주의 아이오딘화프로피듐 FACS 분석 (제2일, 제7일 및 제10일, 생물학적 n=2, 오차 막대는 표준 편차를 나타냄).
도 24: 유방암 세포주 패널에서의 1 μM 화합물 B 처리 후 SDMA 억제의 시간 과정. 세포를 나타낸 기간 동안 DMSO 또는 1 μM 화합물 B로 처리하고, 세포 용해물을 SDMA 및 액틴 항체를 사용한 웨스턴 블롯에 의해 분석하였다. 각각의 블롯 상의 마지막 레인은 DMSO 대조군의 절반이다.
도 25: MDA-MB-468 이종이식 모델에서의 화합물 C 효능 (좌측) 및 PK/PD (우측).
도 26: PRMT5 후보인 화합물 C 및 PRMT5 도구 분자인 화합물 B를 사용하는, GBM 세포주에서의 14일 성장/사멸 CTG 검정 (Y_분 - T0).
도 27: 화합물 B (1 μM)는 GBM 및 림프종 세포주에서 SDMA 수준을 감소시키고 (B), MDM4의 대안적 스플라이싱을 유도하고 (A), p53을 활성화시킨다 (B).
도 28: 면역요법과의 조합. A20 종양 모델에서 단일 작용제 및 조합물에 대한 평균 생존.
도 29: 면역요법과의 조합. CT26 종양 모델에서 단일 작용제 및 조합물에 대한 평균 생존.
도 30: 106-222, 인간화 106-222 (Hu106) 및 인간 수용자 X61012 (진뱅크 수탁 번호) VH 서열의 아미노산 서열의 정렬.
도 31: 106-222, 인간화 106-222 (Hu106) 및 인간 수용자 AJ388641 (진뱅크 수탁 번호) VL 서열의 아미노산 서열의 정렬.
도 32: 추정 아미노산 서열을 갖는, SpeI 및 HindIII 부위가 플랭킹된 Hu106 VH 유전자의 뉴클레오티드 서열.
도 33: 추정 아미노산 서열을 갖는, NheI 및 EcoRI 부위가 플랭킹된 Hu106-222 VL 유전자의 뉴클레오티드 서열.
도 34: 119-122, 인간화 119-122 (Hu119) 및 인간 수용자 Z14189 (진뱅크 수탁 번호) VH 서열의 아미노산 서열의 정렬.
도 35: 119-122, 인간화 119-122 (Hu119) 및 인간 수용자 M29469 (진뱅크 수탁 번호) VL 서열의 아미노산 서열의 정렬.
도 36: 추정 아미노산 서열을 갖는, SpeI 및 HindIII 부위가 플랭킹된 Hu119 VH 유전자의 뉴클레오티드 서열.
도 37: 추정 아미노산 서열을 갖는, NheI 및 EcoRI 부위가 플랭킹된 Hu119 VL 유전자의 뉴클레오티드 서열.
도 38: 추정 아미노산 서열을 갖는 마우스 119-43-1 VH cDNA의 뉴클레오티드 서열.
도 39: 마우스 119-43-1 VL cDNA의 뉴클레오티드 서열 및 추정 아미노산 서열.
도 40: 추정 아미노산 서열을 갖는, Spel 및 Hindlll 부위가 플랭킹된 설계된 119-43-1 VH 유전자의 뉴클레오티드 서열.
도 41: 추정 아미노산 서열을 갖는, Nhel 및 EcoRI 부위가 플랭킹된 설계된 119-43-1 VL 유전자의 뉴클레오티드 서열.1: Four types of protein arginine methylation catalyzed by PRMT.
2: Known PRMT5 substrate. PRMT5 symmetrically methylates arginine in many proteins, primarily in regions rich in arginine and glycine residues (Karkhanis V, et al. Versatility of PRMT5-induced methylation in growth control and development. Trends Biochem Sci. 2011 Dec 36 (12): 633-41). The majority of these substrates were identified through their ability to interact with PRMT5.
3: Molecular relationship between PRMT5 / MEP50 complex activity and Cyclin D1 oncogene drive pathway. The PRMT5 co-regulatory factor MEP50 is a cdk4 substrate and MEP50 phosphorylation increases PRMT5 / MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplasia growth, including CUL4 (Cullin 4) inhibition, CDT1 overexpression and DNA replication (Aggarwal P, et al. Nuclear cyclin D1 / CDK4). kinase regulates CUL4 expression and triggers neoplastic growth via activation of the PRMT5 methyltransferase.Harm Cell. 2010 Oct 19; 18 (4): 329-40).
Figure 4: Compound IC ₅₀ values for PRMT5 / MEP50. PRMT5 / MEP50 using a radiometric assay under equilibrium conditions (substrate concentration equal to K _{m apparent} ) measuring the transfer of ³ H from SAM to H4 peptide after treatment with Compound C, Compound F, Compound B or Compound E (4 nM) activity was monitored. IC ₅₀ values were determined by fitting the data to a 3-parameter dose-response equation.
Fig. 5: Crystal structure analyzed at 2.8 Hz for PRMT5 / MEP50 in complex with Compound C and cinefungin. Insets show that the compound binds to the peptide binding pocket and creates a major interaction with the PRMT5 backbone.
Figure 6: Phylogenetic tree highlighting methyltransferases tested in selectivity panel. Compound C may be any other test enzyme (

PRMT5 (for> 10 ^-5 M)

, 10 ^-8 M). PRMT9 is only presented for relationship purposes within the tree and has not been evaluated in the panel. The drawings are described in Richon VM. et al.].
7: Compound C gIC ₅₀ values from 6-day growth / kill assay in panel of cancer cell lines. DLBCL-Understanding vs B-Cell Lymphoma, GBM-Glioblastoma, MCL- Mantle Cell Lymphoma, MM-Multimyeloma
Figure 8: Compound C gIC ₁₀₀ (black squares) and Y _min -T0 (rods) values from 6-day growth / kill assays in cancer cell line panels (the highest concentration used in this assay was 30 μΜ). DLBCL-Understanding vs B-Cell Lymphoma, GBM-Glioblastoma, MCL- Mantle Cell Lymphoma, MM-Multimyeloma
9: Compound B gIC ₅₀ value in cancer cell line (n = 240) from 10 day 2D growth assay. ALL-acute lymphoblastic leukemia, AML-acute myeloid leukemia, CML-chronic myeloid leukemia, DLBCL-less to large B-cell lymphoma, HL-Hodgkin's lymphoma, HN-head and neck cancer, MM-multimyeloma, NHL-non-ho Keukin's lymphoma, NSCLC-non-small cell lung cancer, PEL-primary exudative lymphoma, SCLC-small cell lung cancer, TCL-T-cell lymphoma.
10: Compound E relative IC ₅₀ values from 8-13 day colony formation assay performed in patient-derived and cell line tumor models.
11: Compound C inhibition of SDMA. (A) Representative SDMA dose-response curves at day 3 (total SDMA normalized to GAPDH) (top) and IC ₅₀ values (bottom) from Z138 cells at days 1 and 3. (B) SDMA IC ₅₀ values in panel of MCL cells (day 4).
12: Gene expression changes in lymphoma cell lines treated with PRMT5 inhibitors. A. Quantification of differentially expressed (DE) genes in lymphoma cell lines after Compound B (0.1 and 0.5 μM) treatment (Days 2 and 4). B. Overlap of DE genes across lymphoma cell lines.
13: Compound C gene expression EC ₅₀ values in a panel of 11 genes identified by RNA-sequencing. Representative dose-response curves for CDKN1A (day 2 and 4, left panel) and Gene Panel EC ₅₀ Summary Table (right panel, day 4).
14: Compound B attenuates splicing of a subset of introns in lymphoma cell lines. A. Regulatory mechanism of cell splicing (adopted from Bezzi M. et al.). B. Analysis of intron expression in lymphoma cell lines treated with 0.1 or 0.5 μM Compound B.
Figure 15: Compound B induces isotype conversion for a subset of genes in lymphoma cell lines. A. Quantification of isotype conversion in four lymphoma cell lines treated with Compound B (0.1 and 0.5 μM) for 2 and 4 days. B. Overlap of isotype conversion in four lymphoma cell lines. C. List of genes that underwent alternative splicing in all four lymphoma cell lines (overlapping four cell lines).
16: MDM4 alternative splicing and p53 activation in MCL cell lines treated with Compound C. A. MDM4 Isotype Expression Analysis (MDM4-FL-Long; MDM4-S-Short) in a Panel of 4 Mantle Cell Lymphoma Cell Lines Treated with 10 and 200 nM Compound C or 5 μM Nutlin-3 for 2 and 3 Days . B. Western analysis of p53 and p21 expression in MCL cell lines treated with 10 and 200 nM Compound C or 5 μM Nutlin-3 for 3 days.
17: Compound C induces dose-dependent changes in SDM / p53 / p21 levels (B) in MDM4 RNA splicing (A) and Z138 cells.
18: Activity of PRMT5 inhibitors and ibrutinib as single agent and combined in MCL cell line. A. gIC ₅₀ values for Compound C and ibrutinib in 6-day growth / kill CTG assays. B. Representative Growth Curves for Combination of Compound B and Ibrutinib in REC1 Cells (Day 6, 1: 1 Ratio). C. Combination Index (CI) for Compound B: Ibrutinib at the ratio shown in the 6-day growth / kill CTG assay.
19: Compound C efficacy and PD in Z138 xenograft model. A. Study of Compound C 21-day efficacy in Z138 xenograft model. B. SDMA Western data quantified from tumors collected at end of efficacy study (3 hours after last dose).
Figure 20: Compound C efficacy and PD in Maver-1 xenograft model. A. Study of Compound C 21-day efficacy in Maver-1 xenograft model. B. SDMA Western data quantified from tumors collected at end of efficacy study (3 hours after last dose).
21: Compound B growth IC ₅₀ values in a panel of breast cancer cell lines (TNBC-triple negative breast cancer, HER2-Her2 positive, HR-hormone receptor positive) from 7-day growth 2D assay.
Figure 22: Y _min -T0 value from 10-12 days growth / kill assay in breast and MCL cell lines using Compound C as a PRMT5 candidate and Compound B as a PRMT5 tool molecule.
Figure 23: Propidium iodide FACS analysis of breast cancer cell lines treated with 30, 200 and 1000 nM Compound C for various time periods (day 2, 7 and 10, biological n = 2, error bars are standard deviation ).
24: Time course of SDMA inhibition after 1 μM Compound B treatment in breast cancer cell line panel. Cells were treated with DMSO or 1 μM Compound B for the indicated periods and cell lysates were analyzed by Western blot using SDMA and actin antibodies. The last lane on each blot is half of the DMSO control.
Figure 25: Compound C efficacy (left) and PK / PD (right) in MDA-MB-468 xenograft model.
FIG. 26: 14-day growth / kill CTG assay in GBM cell line using Compound C as PRMT5 candidate and Compound B as PRMT5 tool molecule (Y _min -T0).
27: Compound B (1 μM) reduces SDMA levels in GBM and lymphoma cell lines (B), induces alternative splicing of MDM4 (A), activates p53 (B).
28: Combination with immunotherapy. Average survival for single agent and combination in A20 tumor model.
29: Combination with immunotherapy. Average survival for single agent and combination in CT26 tumor model.
Figure 30: Alignment of amino acid sequences of 106-222, humanized 106-222 (Hu106) and human recipient X61012 (Genbank Accession No.) VH sequences.
Figure 31: Alignment of amino acid sequences of 106-222, humanized 106-222 (Hu106) and human recipient AJ388641 (Genbank Accession No.) VL sequences.
32: Nucleotide sequence of Hu106 VH gene flanked with SpeI and HindIII sites, with putative amino acid sequence.
33: Nucleotide sequence of Hu106-222 VL gene flanking the NheI and EcoRI sites with putative amino acid sequence.
34: Alignment of amino acid sequences of 119-122, humanized 119-122 (Hu119) and human recipient Z14189 (Genbank Accession No.) VH sequences.
Figure 35: Alignment of amino acid sequences of 119-122, humanized 119-122 (Hu119) and human recipient M29469 (Genbank Accession No.) VL sequences.
36: Nucleotide sequence of Hu119 VH gene flanked with SpeI and HindIII sites, with putative amino acid sequence.
37: Nucleotide sequence of Hu119 VL gene flanking the NheI and EcoRI sites with putative amino acid sequence.
38: Nucleotide sequence of mouse 119-43-1 VH cDNA with putative amino acid sequence.
39: Nucleotide sequence and putative amino acid sequence of mouse 119-43-1 VL cDNA.
Figure 40: Nucleotide sequence of designed 119-43-1 VH gene flanking the Spel and Hindlll sites with putative amino acid sequence.
Figure 41: Nucleotide sequence of designed 119-43-1 VL gene flanking the Nhel and EcoRI sites with putative amino acid sequence.

Summary of the Invention

In one embodiment the invention provides a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor with an immunomodulator, wherein the immunomodulator is an anti-OX40 antibody or antigen binding fragment thereof.

In one embodiment, a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulatory agent is administered to a human being in need thereof with at least one of a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent. Provided are methods of treating cancer in a human in need thereof, comprising administering together to treat the cancer in the human, wherein the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof.

In one embodiment, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a type II protein arginine methyltransferase (type II PRMT) inhibitor, and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulatory agent. Is an anti-OX40 antibody or antigen binding fragment thereof.

In one embodiment, a human in need thereof is administered a therapeutically effective amount of a pharmaceutical composition comprising a type II protein arginine methyltransferase (type II PRMT) inhibitor and a pharmaceutical composition comprising an immunomodulatory agent to treat the cancer in the human There is provided a method of treating cancer in a human being in need thereof, the method comprising treating the cancer, wherein the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof.

In one embodiment, the invention provides the use of a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator for the manufacture of a medicament, wherein the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof to be.

In one embodiment, the invention provides the use of a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulatory agent for the treatment of cancer, wherein the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof to be.

DETAILED DESCRIPTION OF THE INVENTION

Justice

As used herein, "type II protein arginine methyltransferase inhibitor" or "type II PRMT inhibitor" means an agent that inhibits protein arginine methyltransferase 5 (PRMT5) and / or protein arginine methyltransferase 9 (PRMT9). . In some embodiments, the type II PRMT inhibitor is a small molecule compound. In some embodiments, the type II PRMT inhibitor selectively inhibits protein arginine methyltransferase 5 (PRMT5) and / or protein arginine methyltransferase 9 (PRMT9). In some embodiments, the type II PRMT inhibitor is an inhibitor of PRMT5. In some embodiments, the type II PRMT inhibitor is a selective inhibitor of PRMT5.

Arginine methyltransferase is an attractive target for coordination given its role in the regulation of various biological processes. It has now been found that the compounds described herein and pharmaceutically acceptable salts and compositions thereof are effective as inhibitors of arginine methyltransferases.

Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 ^th Ed., Inside cover, and specific functional groups are generally defined as described above. In addition, the general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 th 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, 3rd Edition, Cambridge University Press, Cambridge, 1987.

The compounds described herein may include one or more asymmetric centers and may therefore exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers or geometric isomers, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. . Isomers may be isolated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; Or preferred isomers may be prepared by asymmetric synthesis. See, eg, Jacques et ah, Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et ah, 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 present disclosure additionally encompasses the compounds described herein as individual isomers substantially free of other isomers, and alternatively as mixtures of various isomers.

It is to be understood that the compounds of the present invention may be depicted as different tautomers. It is also to be understood that where a compound has a tautomeric form, all tautomeric forms are intended to be included within the scope of the present invention and the names of any of the compounds described herein do not exclude any tautomeric form.

Unless stated otherwise, the structures shown herein are also intended to include different compounds in the presence of only one or more isotopically enriched atoms. For example, compounds having the structure of the present invention, except for replacement of hydrogen by deuterium or tritium, replacement of ¹⁹ F by ¹⁸ F, or replacement of carbon by ¹³ C- or ¹⁴ C-enriched carbon, are disclosed herein. It is within the category of the content. Such compounds are useful, for example, as analytical tools or probes in biological assays.

As used herein, the term “aliphatic” includes both saturated and unsaturated, nonaromatic, straight chain (ie, unbranched), branched, acyclic, and cyclic (ie, carbocyclic) hydrocarbons. In some embodiments, aliphatic groups are optionally substituted with one or more functional groups. As will be appreciated by those skilled in the art, "aliphatic" is intended to include alkyl, alkenyl, alkynyl, cycloalkyl and cycloalkenyl moieties herein.

When a range of values is listed, it is intended to encompass each value and subrange within that range. For example, "C _1-6 alkyl" means C _1; C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C _1-6 , C _1-5 , C _1-4 , C _1-3 , C _1-2 , C _2-6 , C _2-5 , It is intended to encompass C _2-4 , C _2-3 , C _3-6 , C _3-5 , C _3-4 , C _4-6 , C _4-5 and C _5-6 alkyl.

"Radial" refers to the point of attachment on a particular substrate. Radicals include divalent radicals of certain groups.

"Alkyl" refers to a radical of a straight or branched saturated hydrocarbon group having 1 to 20 carbon atoms ("C _1-20 alkyl"). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C _1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C _1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C _1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C _1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C _1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C _1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C _1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C _1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C _1-2 alkyl”). In some embodiments, an alkyl group has one carbon atom (“C ₁ alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C _2-6 alkyl”). Examples of C _1-6 alkyl groups are methyl (C ₁ ), ethyl (C ₂ ), n-propyl (C ₃ ), isopropyl (C ₃ ), n-butyl (C ₄ ), tert-butyl (C ₄ ), sec-butyl (C ₄ ), iso-butyl (C ₄ ), n-pentyl (C ₅ ), 3-pentanyl (C ₅ ), amyl (C ₅ ), neopentyl (C ₅ ), 3-methyl -2-butanyl (C ₅ ), tertiary amyl (C ₅ ), and n-hexyl (C ₆ ). Further examples of alkyl groups include n-heptyl (C ₇ ), n-octyl (C ₈ ), and the like. In certain embodiments, each instance of an alkyl group is independently optionally substituted, eg, unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"). In certain embodiments, the alkyl group is unsubstituted C _1-10 alkyl (eg, —CH ₃ ). In certain embodiments, the alkyl group is substituted C _1-10 alkyl.

In some embodiments, an alkyl group is substituted with one or more halogens. "Perhaloalkyl" is a substituted alkyl group as defined herein in which all hydrogen atoms are independently replaced by halogen, eg, fluoro, bromo, chloro or iodo. In some embodiments, an alkyl moiety has 1 to 8 carbon atoms (“C _1-8 perhaloalkyl”). In some embodiments, an alkyl moiety has 1 to 6 carbon atoms (“C _1-6 perhaloalkyl”). In some embodiments, an alkyl moiety has 1 to 4 carbon atoms (“C _1-4 perhaloalkyl”). In some embodiments, an alkyl moiety has 1 to 3 carbon atoms (“C _1-3 perhaloalkyl”). In some embodiments, an alkyl moiety has 1 to 2 carbon atoms (“C _1-2 perhaloalkyl”). In some embodiments, all hydrogen atoms are replaced with fluoro. In some embodiments, all hydrogen atoms are replaced with chloro. Examples of perhaloalkyl groups include -CF ₃ , -CF ₂ CF ₃ , -CF ₂ CF ₂ CF ₃ , -CCl ₃ , -CFCl ₂ , -CF ₂ Cl, and the like.

"Alkenyl" refers to 2 to 20 carbon atoms and one or more carbon-carbon double bonds (eg, 1, 2, 3 or 4 double bonds) and optionally one or more triple bonds (eg, 1, Refers to a radical of a straight or branched hydrocarbon group having 2, 3 or 4 triple bonds (“C _2-20 alkenyl”). In certain embodiments, alkenyl does not include triple bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C _2-10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C _2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C _2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C _2-7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C _2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C _2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C _2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C _2-3 alkenyl”). In some embodiments, an alkenyl group has two carbon atoms (“C ₂ alkenyl”). One or more carbon-carbon double bonds may be internal (eg, in 2-butenyl) or terminal (eg, in 1-butenyl). Examples of C _2-4 alkenyl groups are ethenyl (C ₂ ), 1-propenyl (C ₃ ), 2-propenyl (C ₃ ), 1-butenyl (C ₄ ), 2-butenyl (C ₄ ), butadienyl (C ₄ ), and the like. Examples of C _2-6 alkenyl groups include pentenyl (C ₅ ), pentadienyl (C ₅ ), hexenyl (C ₆ ), and the like, as well as the C _2-4 alkenyl groups mentioned above. Further examples of alkenyl include heptenyl (C ₇ ), octenyl (C ₈ ), octatrienyl (C ₈ ), and the like. In certain embodiments, each instance of an alkenyl group is independently optionally substituted, that is, unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents ("substituted alkenyl"). In certain embodiments, the alkenyl group is unsubstituted C _2-10 alkenyl. In certain embodiments, the alkenyl group is substituted C _2-10 alkenyl.

“Alkynyl” means 2 to 20 carbon atoms and one or more carbon-carbon triple bonds (eg 1, 2, 3 or 4 triple bonds) and optionally one or more double bonds (eg 1, Refers to a radical of a straight or branched hydrocarbon group having 2, 3 or 4 double bonds (“C _2-20 alkynyl”). In certain embodiments, alkynyl does not include a double bond. In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C _2-10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C _2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C _2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C _2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C _2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C _2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C _2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C _2-3 alkynyl”). In some embodiments, an alkynyl group has two carbon atoms (“C ₂ alkynyl”). One or more carbon carbon triple bonds may be internal (eg, in 2-butynyl) or terminal (eg, in 1-butynyl). Examples of C _2-4 alkynyl groups include, but are not limited to, ethynyl (C ₂ ), 1-propynyl (C ₃ ), 2-propynyl (C ₃ ), 1-butynyl (C ₄ ), 2 -Butynyl (C ₄ ) and the like. Examples of C _2-6 alkenyl groups include the aforementioned C _2-4 alkynyl groups as well as pentynyl (C ₅ ), hexynyl (C ₆ ), and the like. Further examples of alkynyl include heptynyl (C ₇ ), octinyl (C ₈ ), and the like. In certain embodiments, each occurrence of an alkynyl group is independently optionally substituted, that is, unsubstituted ("unsubstituted alkynyl") or substituted with one or more substituents ("substituted alkynyl"). In certain embodiments, the alkynyl group is unsubstituted C _2-10 alkynyl. In certain embodiments, the alkynyl group is substituted C _2-10 alkynyl.

"Fusion" or "ortho-fusion" is used interchangeably herein and refers to two rings that commonly have two atoms and one bond, for example, below.

"Crosslinked" means (1) a bridgehead atom or group of atoms connecting two or more non-adjacent positions of the same ring; Or (2) a ring system containing a bridgehead atom or group of atoms connecting two or more positions of different rings of the ring system and thereby not forming an ortho-fused ring, for example:

"Spiro" or "spiro-fusion" refers to an atomic group that is linked to (identically attached to) the same atom of a carbocyclic or heterocyclic ring system, thereby forming a ring, for example:

Spiro-fusion at bridgehead atoms is also contemplated.

"Carbocyclyl" or "carbocyclic" refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 14 ring carbon atoms and 0 heteroatoms in the non-aromatic ring system ("C _3-14 Carbocyclyl "). In certain embodiments, carbocyclyl group refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and 0 heteroatoms in the non-aromatic ring system (“C _3-10 carbocyclyl "). In some embodiments, the carbocyclyl group has 3 to 8 ring carbon atoms (“C _3-8 carbocyclyl”). In some embodiments, the carbocyclyl group has 3 to 6 ring carbon atoms (“C _3-6 carbocyclyl”). In some embodiments, the carbocyclyl group has 3 to 6 ring carbon atoms (“C _3-6 carbocyclyl”). In some embodiments, the carbocyclyl group has 5 to 10 ring carbon atoms (“C _5-10 carbocyclyl”). Exemplary C _3-6 carbocyclyl groups include, but are not limited to, cyclopropyl (C ₃ ), cyclopropenyl (C ₃ ), cyclobutyl (C ₄ ), cyclobutenyl (C ₄ ), cyclopentyl (C ₅ ), Cyclopentenyl (C ₅ ), cyclohexyl (C ₆ ), cyclohexenyl (C ₆ ), cyclohexadienyl (C ₆ ), and the like. Exemplary C _3-8 carbocyclyl groups include, but are not limited to, the aforementioned C _3-6 carbocyclyl groups, as well as cycloheptyl (C ₇ ), cycloheptenyl (C ₇ ), cycloheptadienyl (C ₇ ), Cycloheptatrienyl (C ₇ ), cyclooctyl (C ₈ ), cyclooctenyl (C ₈ ), bicyclo [2.2.1] heptanyl (C ₇ ), bicyclo [2.2.2] octanyl (C ₈ ) and the like. Exemplary C _3-10 carbocyclyl groups include, but are not limited to, the aforementioned C _3-8 carbocyclyl groups, as well as cyclononyl (C ₉ ), cyclononenyl (C ₉ ), cyclodecyl (C ₁₀ ), Cyclodecenyl (C ₁₀ ), octahydro-1H-indenyl (C ₉ ), decahydronaphthalenyl (C ₁₀ ), spiro [4.5] decanyl (C ₁₀ ), and the like. As the above examples illustrate, in certain embodiments, the carbocyclyl group is monocyclic ("monocyclic carbocyclyl"), or a fused, bridged or spiro-fused ring system, such as a bicyclic system ("bicyclic"). Carbocyclyl ") and may be saturated or partially unsaturated. "Carbocyclyl" also includes a ring system in which a carbocyclyl ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on a carbocyclyl ring, in which case the number of carbons Subsequently specifies the number of carbons in the carbocyclic ring system. In certain embodiments, each carbocyclyl group is independently optionally substituted, that is, unsubstituted ("unsubstituted carbocyclyl") or substituted with one or more substituents ("substituted carbocyclyl"). In certain embodiments, the carbocyclyl group is unsubstituted C _3-10 carbocyclyl. In certain embodiments, the carbocyclyl group is substituted C _3-10 carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having 3 to 14 ring carbon atoms (“C _3-14 cycloalkyl”). In some embodiments, “carbocyclyl” is a monocyclic saturated carbocyclyl group having 3 to 10 ring carbon atoms (“C _3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C _3-8 cycloalkyl”). In some embodiments, cycloalkyl groups have 3 to 6 ring carbon atoms (“C _3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C _5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C _5-10 cycloalkyl”). Examples of C _5-6 cycloalkyl groups include cyclopentyl (C ₅ ) and cyclohexyl (C ₆ ). Examples of C _3-6 cycloalkyl groups include cyclopropyl (C ₃ ) and cyclobutyl (C ₄ ) as well as the C _5-6 cycloalkyl groups mentioned above. Examples of C _3-8 cycloalkyl groups include cycloheptyl (C ₇ ) and cyclooctyl (C ₈ ) as well as the C _3-6 cycloalkyl groups mentioned above. In certain embodiments, each instance of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, the cycloalkyl group is unsubstituted C _3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C _3-10 cycloalkyl.

"Heterocyclyl" or "heterocyclic" refers to a 3- to 14-membered non-aromatic having a ring carbon atom and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur Refers to a radical of the ring system (“3-14 membered heterocyclyl”). In certain embodiments, “heterocyclyl” or “heterocyclic” is 3-10 having a ring carbon atom and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur It refers to a radical of the original non-aromatic ring system (“3-10 membered heterocyclyl”). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valency permits. Heterocyclyl groups may be monocyclic ("monocyclic heterocyclyl") or fused, bridged or spiro-fused ring systems such as bicyclic systems ("bicyclic heterocyclyl"), may be saturated or It may be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also refers to a ring system in which a heterocyclyl ring as defined above is fused with one or more carbocyclyl groups, wherein the point of attachment is on a carbocyclyl ring or on a heterocyclyl ring; Or a ring system in which a heterocyclyl ring as defined above is fused with at least one aryl or heteroaryl group, wherein the point of attachment is on a heterocyclyl ring, in which case the number of ring members continues to be heterocycle Specifies the number of ring members in the reel ring system. In certain embodiments, each instance of heterocyclyl is independently optionally substituted, that is, unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.

In some embodiments, the heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ( "5-10 membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ( "5-8 membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ( "5-6 membered heterocyclyl"). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, aziridinyl, oxiranyl and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl and thiethananyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and Pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl and oxazolidin-2-ones. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, ditianyl and dioxanyl. Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C ₆ aryl ring (also referred to herein as 5,6-bicyclic heterocyclic rings) include, but are not limited to, indolinyl, isoindolinyl, dihydro Benzofuranyl, dihydrobenzothienyl, benzoxazolinonyl and the like. Exemplary 6-membered heterocyclyl groups fused to aryl rings (also referred to herein as 6,6-bicyclic heterocyclic rings) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl And the like.

“Aryl” is a monocyclic or polycyclic (eg bicyclic or tricyclic) 4n + 2 aromatic ring system having 6-14 ring carbon atoms and 0 heteroatoms provided in an aromatic ring system (eg , Having 6, 10 or 14 π electrons shared in a cyclic array) (“C _6-14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C ₆ aryl”; eg, phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C ₁₀ aryl”; eg, naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C ₁₄ aryl”; eg, anthracyl). “Aryl” also includes ring systems in which an aryl ring as defined above is fused with one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment or radical is on an aryl ring, in which case the number of carbon atoms Continually specifies the number of carbon atoms in the aryl ring system. In certain embodiments, each instance of an aryl group is independently optionally substituted, that is, unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, the aryl group is unsubstituted C _6-14 aryl. In certain embodiments, the aryl group is substituted C _6-14 aryl.

"Heteroaryl" is a 5-14 membered monocyclic or polycyclic having a ring carbon atom and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (Eg, bicyclic or tricyclic) refers to a radical of a 4n + 2 aromatic ring system (eg having 6 or 10 π electrons shared in a cyclic array) (“5-14 membered heteroaryl "). In certain embodiments, heteroaryl is a 5-10 membered monocyclic having a ring carbon atom and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur Or a radical of a bicyclic 4n + 2 aromatic ring system (“5-10 membered heteroaryl”). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" includes a ring system in which a heteroaryl ring as defined above is fused with one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on a heteroaryl ring, in which case the number of ring members Subsequently, the number of ring members in the heteroaryl ring system is specified. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused with one or more aryl groups, wherein the point of attachment is on an aryl or heteroaryl ring, in which case the number of ring members is fused ( Aryl / heteroaryl) designates the number of ring members in the ring system. In bicyclic heteroaryl groups (eg, indolyl, quinolinyl, carbazolyl, etc.) in which one ring does not contain heteroatoms, the point of attachment bears either ring, for example heteroatoms It may be on a ring (eg 2-indolyl) or on a ring containing no heteroatoms (eg 5-indolyl).

In some embodiments, the heteroaryl group is a 5-14 membered aromatic ring system having 1-4 ring heteroatoms and ring carbon atoms provided within the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided within the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system having 1-4 ring heteroatoms and ring carbon atoms provided within the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having 1-4 ring heteroatoms and ring carbon atoms provided within the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl"). In some embodiments, 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has one ring heteroatom selected from nitrogen, oxygen, and sulfur. In certain embodiments, each instance of a heteroaryl group is independently optionally substituted, eg, unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl"). In certain embodiments, the heteroaryl group is unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadizolyl and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, but are not limited to triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azefinyl, oxepinyl and thiepinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, putridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl and quinazolinyl do. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, any of the following formulas:

In any monocyclic or bicyclic heteroaryl group, the point of attachment may be a carbon or nitrogen atom, as valency permits.

"Partially unsaturated" refers to a group comprising at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple unsaturated sites, but is not intended to include aromatic groups (eg, aryl or heteroaryl groups) as defined herein. Likewise, "saturated" refers to groups that do not contain double or triple bonds, ie, all contain a single bond.

In some embodiments, aliphatic, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl groups, as defined herein, are optionally substituted (eg, “substituted” or “unsubstituted” Substituted, aliphatic, "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" Carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl or "substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted" refers to at least one hydrogen present on a group (eg, carbon or nitrogen atom), whether preceded by the term "optionally" or not, for example a substitution Substituent is substituted with a substituent which produces a compound that is stable at time, e.g., a compound that does not undergo spontaneous conversion, such as by rearrangement, cyclization, removal or other reactions. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of this group, and when more than one position in any given structure is substituted, the substituents are the same or different at each position . The term “substituted” is contemplated to include the substitution of all organic substituents with all acceptable substituents, including any substituents described herein resulting in the formation of stable compounds. The present disclosure contemplates any and all such combinations in order to reach stable compounds. For the purposes of the present disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents as described herein that meet the valence of the heteroatoms and result in the formation of stable moieties.

Exemplary carbon atom substituents are halogen, -CN, -NO ₂ , -N ₃ , -SO ₂ H, -SO ₃ H, -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 , -CO ₂ H, -CHO,- C (OR ^cc ) ₂ , -CO ₂ R ^aa , -OC (= O) R ^aa , -OCO ₂ R ^aa , -C (= O) N (R ^bb ) ₂ , -OC (= O) N (R ^bb ) ₂ , -NR ^bb C (= O) R ^aa , -NR ^bb CO ₂ R ^aa , -NR ^bb C (= 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 ^bb C (= NR ^bb ) N (R ^bb ) ₂ , -C (= O) NR ^bb SO ₂ R ^aa , -NR ^bb SO ₂ R ^aa , -SO ₂ N (R ^bb ) ₂ , -SO ₂ R ^aa , -SO ₂ OR ^aa , -OSO ₂ R ^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) ₂ R ^aa , -OP (= O) ₂ R ^aa , -P (= O) (R ^aa ) ₂ , -OP (= O) (R ^aa ) ₂ , -OP (= O) (OR ^cc ) ₂ , -P (= O) ₂ N (R ^bb ) ₂ , -OP (= O) ₂ N (R ^bb ) ₂ , -P (= O) (NR ^bb ) ₂ , -OP (= O) (NR ^bb ) ₂ , -NR ^bb P (= O) (O R ^cc ) ₂ , -NR ^bb P (= 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 _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 Alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl, including but not limited to the respective alkyl, alkenyl, alkynyl , Carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups;

Or two covalent hydrogens on a carbon atom are the groups = O, = S, = NN (R ^bb ) ₂ , = NNR ^bb C (= O) R ^aa , = NNR ^bb C (= O) OR ^aa , = NNR ^bb Is replaced by S (= 0) ₂ R ^aa , = NR ^bb , or = NOR ^cc ; Each occurrence of R ^aa is independently C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocycle Aryl, C _6-14 aryl and 5-14 membered heteroaryl, or two R ^aa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, Alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups;

R ^bb in each case is independently hydrogen, —OH, —OR ^aa , —N (R ^CC ) ₂ , —CN, —C (═O) R ^aa , —C ( ^═O ) N (R ^cc ) ₂ , -CO ₂ R ^aa , -SO ₂ R ^aa , -C (= NR ^cc ) OR ^aa , -C (= NR ^CC ) N (R ^CC ) ₂ , -SO ₂ N (R ^cc ) ₂ , -SO ₂ R ^cc , -SO ₂ OR ^cc , -SOR ^aa , -C (= S) N (R ^CC ) ₂ , -C (= O) SR ^cc , -C (= S) SR ^CC , -P (= O) ₂ R ^aa , -P (= O) (R ^aa ) ₂ , -P (= O) ₂ N (R ^cc ) ₂ , -P (= O) (NR ^cc ) ₂ , C _1-10 alkyl, C _1- Or selected from ₁₀ perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl Or two R ^bb groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and hetero Aryl is independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups;

Each occurrence of R ^cc is independently hydrogen, C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 member Heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl, or two R ^cc groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups;

R ^dd in each case is independently halogen, -CN, -NO ₂ , -N ₃ , -SO ₂ H, -SO ₃ H, -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 , -CO ₂ H, -CO ₂ R ^ee , -OC (= O) R ^ee , -OCO ₂ R ^ee , -C (= O) N (R ^ff ) ₂ , -OC (= O) N (R ^ff ) ₂ , -NR ^ff C ( = O) R ^ee , -NR ^ff CO ₂ R ^ee , -NR ^ff C (= 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 ^ff C (= NR ^ff ) N (R ^ff ) ₂ , -NR ^ff SO ₂ R ^ee , -SO ₂ N (R ^ff ) ₂ , -SO ₂ R ^ee , -SO ₂ OR ^ee , -OSO ₂ R ^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) ₂ R ^ee , -P (= O) (R ^ee ) ₂ , -OP (= O) (R ^ee ) ₂ , -OP (= O) (OR ^ee ) ₂ , C _1-6 alkyl, C _1-6 perhaloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-10 carbocyclyl, 3-10 membered heterocyclyl, C _6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbosi Reel, heterocyclyl, aryl and heteroaryl are independently 0, 1, 2, 3, 4 or 5 R substituted ^gg group, or two such position R ^dd substituents are connected to form = O or = S Can;

Each occurrence of R ^ee is independently C _1-6 alkyl, C _1-6 perhaloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-10 carbocyclyl, C _6-10 aryl, 3-10 membered heterocyclyl and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently 0, 1, 2, Substituted with 3, 4 or 5 R ^gg groups;

Each occurrence of R ^ff is independently hydrogen, C _1-6 alkyl, C _1-6 perhaloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-10 carbocyclyl, 3-10 member Heterocyclyl, C _1-6 aryl and 5-10 membered heteroaryl, or two R ^ff groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^gg groups;

Each occurrence of R ^gg is independently halogen, —CN, —NO ₂ , —N ₃ , —SO ₂ H, —SO ₃ H, —OH, —O _1-6 alkyl, —ON (C _1-6 alkyl) _2, -N (C _1-6 alkyl) _2, -N (C _{1-6 ^{alkyl) 3 + X -, -NH (}} C 1-6 _{^{alkyl) 2 + X -, -NH 2}} (C 1- _{6 ^{alkyl) + X -, -NH 3 +}} X, -N (OC 1-6 alkyl) (C _1-6 alkyl), -N (OH) (C 1-6 alkyl), -NH (OH), - SH, -S _1-6 alkyl, -SS (C _1-6 alkyl), -C (= 0) (C _1-6 alkyl), -CO ₂ H, -CO ₂ (C _1-6 alkyl),- OC (═O) (C _1-6 alkyl), —OCO ₂ (C _1-6 alkyl), —C (═O) NH ₂ , —C (═O) N (C _1-6 alkyl) ₂ ,- OC (= O) NH (C _1-6 alkyl), -NHC (= O) (C _1-6 alkyl), -N (C _1-6 alkyl) C (= O) (C _1-6 alkyl), -NHCO ₂ (C _1-6 alkyl), -NHC (= O) N (C _1-6 alkyl) ₂ , -NHC (= O) NH (C _1-6 alkyl), -NHC (= O) NH ₂ , -C (= NH) O (C _1-6 alkyl), -OC (= NH) (C _1-6 alkyl), -OC (= NH) OC _1-6 alkyl, -C (= NH) N ( C _1-6 alkyl) ₂ , -C (= NH) NH (C _1-6 alkyl), -C (= NH) NH ₂ , -OC (= NH) N (C _1-6 alkyl) ₂ , -OC (NH) NH (C _1-6 alkyl), -OC (NH) NH ₂ , -NHC (NH) N (C _1-6 alkyl) ₂ , -NHC (= NH) NH ₂ , -NHSO ₂ (C _{1 -6} alkyl), -SO ₂ N (C _1-6 alkyl) ₂ , -SO ₂ NH (C _1-6 alkyl), -SO ₂ NH ₂ , -SO ₂ C _1-6 alkyl, -SO ₂ OC _1-6 alkyl, -OSO ₂ C _1-6 alkyl, -SOC _1-6 alkyl, -Si (C _1-6 alkyl) ₃ , -OSi ( C _1-6 alkyl) ₃ -C (= S) N (C _1-6 alkyl) ₂ , C (= S) NH (C _1-6 alkyl), C (= S) NH ₂ , -C (= O ) S (C _1-6 alkyl), -C (= S) SC _1-6 alkyl, -SC (= S) SC _1-6 alkyl, -P (= O) ₂ (C _1-6 alkyl),- P (= O) (C _1-6 alkyl) ₂ , -OP (= O) (C _1-6 alkyl) ₂ , -OP (= O) (OC _1-6 alkyl) ₂ , C _1-6 alkyl, C _1-6 perhaloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-10 carbocyclyl, C _6-10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl Or; Or two identical-position R ^gg substituents may be joined to form = O or = S; Where X is the counterion.

"Counterions" or "anionic counterions" are negatively charged groups associated with cationic quaternary amino groups to maintain electron neutrality. Exemplary counterions include halide ions (eg, F ⁻ , Cl ⁻ , Br ⁻ , I ⁻ ), NO ₃ ⁻ , ClO ₄ ⁻ , OH ⁻ , H ₂ PO ₄ ⁻ , HSO ₄ ⁻ , sulfonate ions (eg methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1 Sulfonic acid-5-sulfonate, ethane-1-sulfonic acid-2-sulfonate, and the like, and carboxylate ions (eg, acetate, ethanoate, propanoate, benzoate, glycerate, lactate, Tartrate, glycolate, and the like).

"Halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

Nitrogen atoms may be substituted or unsubstituted as valency permits and include primary, secondary, tertiary or quaternary nitrogen atoms. Exemplary nitrogen atom substituents include hydrogen, —OH, —OR ^aa , —N (R ^CC ) ₂ , —CN, —C (═O) R ^aa , —C ( ^═O ) N (R ^cc ) ₂ , -CO ₂ R ^aa , -SO ₂ R ^aa , -C (= NR ^bb ) R ^aa , -C (= NR ^cc ) OR ^aa , -C (= NR ^CC ) N (R ^CC ) ₂ , -SO ₂ N (R ^cc ) ₂ , -SO ₂ R ^cc , -SO ₂ OR ^cc , -SOR ^aa , -C (= S) N (R ^CC ) ₂ , -C (= O) SR ^cc , -C (= S) SR ^CC , -P (= O) ₂ R ^aa , -P (= O) (R ^aa ) ₂ , -P (= O) ₂ N (R ^cc ) ₂ , -P (= O) (NR ^cc ) ₂ , C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5 Two R ^cc groups attached to a nitrogen atom, including but not limited to -14 membered heteroaryl, are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl , Alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 R ^dd groups, wherein R ^aa , R ^bb , R ^cc and R ^dd is defined in the As it described.

In certain embodiments, the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups are -OH, -OR ^aa , -N (R ^CC ) ₂ , -C (= O) R ^aa , -C (= O) N (R ^cc ) ₂ , -CO ₂ R ^aa , -SO ₂ R ^aa , -C (= NR ^cc ) R ^aa , -C (= NR ^cc ) OR ^aa , -C (= NR ^CC ) N (R ^CC ) ₂ , -SO ₂ N (R ^cc ) ₂ , -SO ₂ R ^cc , -SO ₂ OR ^cc , -SOR ^aa , -C (= S) N (R ^CC ) ₂ , -C (= O) SR ^cc , -C (= S) SR ^CC , C _1-10 alkyl (example For example, aralkyl, heteroaralkyl), C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 Including, but not limited to, membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl and heteroaryl is independently 0, 1, 2, 3, 4 Or substituted with 5 R groups, wherein R ^aa , R ^bb , R ^cc and R ^dd are as defined herein. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 rd edition, John Wiley & Sons, 1999. .

Amide nitrogen protecting groups (e.g., -C (= O) R ^aa ) are formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, p Cholineamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N ' -Dithiobenzyloxyacylamino) acetamide, 3- (p-hydroxyphenyl) propanamide, 3- (o-nitrophenyl) propanamide, 2-methyl-2- (o-nitrophenoxy) propanamide, 2-methyl-2- (o-phenylazophenoxy) propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamid, N-acetylmethionine, o-nitrobenzamide And o- (benzoyloxymethyl) benzamide.

Carbamate nitrogen protecting groups (eg, -C (= 0) OR ^aa ) are methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9- (2-sulfo) Fluorenylmethyl carbamate, 9- (2,7-dibromo) fluoroenylmethyl carbamate, 2,7-di-t-butyl- [9- (10,10-dioxo-10 , 10,10,10-tetrahydrothioxanthyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate ( Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamantyl) -1-methylethyl carbamate (Adpoc), 1,1 -Dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloro Ethyl carbamate (TCBOC), 1-methyl-1- (4-biphenylyl) ethyl carbamate (Bpoc), 1- (3,5-di-t-butylphenyl) -1-methylethyl carba Mate (t-Bumeoc), 2- (2'- and 4'-pyridyl) ethyl carbamate (Pyoc), 2- (N, N-dicyclohexylcarboxamido) ethyl carbamate, t-butyl carbamate (BOC), 1 -Adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamil carbamate (Coc), 4- Nitrocinnamil carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carba Bamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2- (p-toluenesulfonyl) ethyl Carbamate, [2- (1,3-ditianyl)] methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyl Oxybenzyl carbamate, p- (dihydroxyboryl) benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2- (trifluoromethyl) -6-chromemonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methyl Carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclophene Carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o- (N, N-dimethylcarboxamido) benzyl carbamate, 1,1-dimethyl-3- (N, N-dimethylcarboxamido) propyl carbamate, 1,1-dimethylpropynyl carbamate, di (2-pyridyl) methyl carbamate, 2-fura Nylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p- (p'-methoxyphenylazo) benzyl carbamate , 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1- (3,5-dimethoxyphenyl) ethyl carba Mate, 1-methyl-1- (p-phenylazophenyl) ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1- (4-pyridyl) ethyl carbamate, Nyl carbamate, p- (phenylazo) benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4- (trimethylammonium) benzyl carbamate, and 2,4,6- Trimethylbenzyl carbamate, including but not limited to.

Sulfonamide nitrogen protecting groups (e.g., -S (= O) ₂ R ^aa ) include p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6, -trimethyl-4-methoxybenzenesulfonamide ( Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5, 6-tetramethyl-4-meth Methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7, 8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4- ( 4 ', 8'-dimethoxynaphthylmethyl) benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include phenothiazinyl- (10) -acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5- Dimethylpyrrole, N-1,1,4,4-tetramethyldisylylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one , 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N- Allylamine, N- [2- (trimethylsilyl) ethoxy] methylamine (SEM), N-3-acetoxypropylamine, N- (1-isopropyl-4-nitro-2-oxo-3-proline- 3-yl) amine, quaternary ammonium salt, N-benzylamine, N-di (4-methoxyphenyl) methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N -[(4-methoxyphenyl) diphenylmethyl] a (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl) methyl] Methyleneamine, N- (N ', N'-dimethylaminomethylene) amine, N, N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicyli Denamine, N- (5-chloro-2-hydroxyphenyl) phenylmethyleneamine, N-cyclohexylideneamine, N- (5,5-dimethyl-3-oxo-1-cyclohexenyl) amine, N Borane derivatives, N-diphenylborinic acid derivatives, N- [phenyl (pentaacromium- or tungsten) acyl] amines, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosoamines, amines N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt) , Dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfen Amides, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting groups are -R ^aa , -N (R ^bb ) ₂ , -C (= O) SR ^aa , -C (= O) R ^aa , -CO ₂ R ^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 , -SO ₂ R ^aa , -Si (R ^aa ) ₃ , -P (R ^CC ) ₂ , -P (R ^CC ) ₃ , -P (= O) ₂ R ^aa , -P (= O) (R ^aa ) ₂ , -P ( = O) (OR ^cc ) ₂ , -P (= O) ₂ N (R ^bb ) ₂ , and -P (= O) (NR ^bb ) ₂ , including but not limited to R ^aa , R ^bb And R ^cc are as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 edition, John Wiley & Sons, 1999.

Exemplary oxygen protecting groups are methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxy Benzyloxymethyl (PMBM), (4-methoxyphenoxy) methyl (p-AOM), guoacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis (2-chloroethoxy) methyl, 2- (trimethylsilyl) ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- Methoxytetrahydrothiopyranyl S, S-dioxide, 1-[(2-chloro-4-methyl) phenyl] -4-methoxypiperidin-4-yl (CTMP), 1,4-dioxane- 2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a, 4,5,6,7,7a-octahydro-7,8, 8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1- Benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl) ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- Halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolinyl N-oxido, diphenylmethyl, p, p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di (p-methoxyphenyl) phenylmethyl, tri (p -Methoxyphenyl) methyl, 4- (4'-bromophenacyloxyphenyl) diphenylmethyl, 4,4 ', 4 "-tris (4,5-dichlorophthalimidophenyl) methyl, 4,4', 4 "-tris (levulinoyloxyphenyl) methyl, 4,4 ', 4 "-Tris (benzoyloxyphenyl) methyl, 3- (imidazol-1-yl) bis (4 ', 4" -dimethoxyphenyl) methyl, 1,1-bis (4-methoxyphenyl) -1'- Pyrenylmethyl, 9-anthryl, 9- (9-phenyl) xanthenyl, 9- (9-phenyl-10-oxo) anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S, S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltecsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanate (levulinate), 4,4- (ethylenedithio) pentanoate (levulinoyldithioacetal), pivalate, adamantatoate, cro Tonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), t-butyl carbonate (BOC), alkyl methyl carbonate , 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2- (trimethylsilyl) ethyl carbonate (TMSEC) , 2- (phenylsulfonyl) ethyl carbonate (Psec), 2- (triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate , Alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxy Benzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate , 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl) benzoate, 2-formylbenzenesulfonate, 2- (methylthiometh Methoxy) ethyl, 4- (methylthiomethoxy) butyrate, 2- (methylthiomethoxymethyl) benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4- (1, 1,3,3-tetramethylbutyl) phenoxyacetate, 2,4-bis (1,1-dimethylpropyl) phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E) -2- Methyl-2-butenoate, o- (methoxyacyl) benzoate, a-naphthoate, nitrate, alkyl N, N, N ', N'-tetramethylphosphorodiamidate, alkyl N-phenyl Carr Including but not limited to barmate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts) Do not.

In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups are -R ^aa , -N (R ^bb ) ₂ , -C (= O) SR ^aa , -C (= O) R ^aa , -CO ₂ R ^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 , -SO ₂ R ^aa , -Si (R ^aa ) ₃ -P (R ^CC ) ₂ , -P (R ^CC ) ₃ , -P (= O) ₂ R ^aa , -P (= O) (R ^aa ) ₂ , -P (= O) (OR ^cc ) ₂ , -P (= O) ₂ N (R ^bb ) ₂ , and -P (= O) (NR ^bb ) ₂ , including but not limited to R ^aa , R ^bb and R ^cc is as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 ^rd edition, John Wiley & Sons, 1999. .

As used herein, “leaving group” or “LG” is a term understood in the art to refer to a molecular fragment that is an anionic or neutral molecule that falls with a pair of electrons upon heterogeneous bond cleavage. See, eg, Smith, March Advanced Organic Chemistry 6th ed. (501-502). Examples of suitable leaving groups include halides (such as chloride, bromide, or iodide), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arerensulfonyloxy, alkyl-carbonyloxy (eg, Acetoxy), arylcarbonyloxy, aryloxy, methoxy, N, O-dimethylhydroxylamino, pixyl, haloformate, -NO ₂ , trialkylammonium, and aryliodonium salts Does not. In some embodiments, the leaving group is a sulfonic acid ester. In some embodiments, the sulfonic acid ester comprises the formula -OSO ₂ R ^LG1 , wherein R ^LG1 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted heteroaryl, Optionally substituted arylalkyl, and optionally substituted heteroarylalkyl. In some embodiments, R ^LGl is substituted or unsubstituted C ₁ -C ₆ alkyl. In some embodiments, R ^LGl is methyl. In some embodiments, R ^LGl is substituted or unsubstituted aryl. In some embodiments, R ^LGl is substituted or unsubstituted phenyl. In some embodiments, R ^LGl is:

In some cases, the leaving group is toluenesulfonate (tosylate, Ts), methanesulfonate (mesylate, Ms), p-bromobenzenesulfonyl (brosylate, Bs) or trifluoromethanesulfonate (triplate , Tf). In some cases, the leaving group is brosylate (p-bromobenzenesulfonyl). In some cases, the leaving group is nosylate (2-nitrobenzenesulfonyl). In some embodiments, the leaving group is a sulfonate-containing group. In some embodiments, the leaving group is a tosylate group. The leaving group may also be a phosphineoxide (eg, formed during the Mitsunobu reaction) or an internal leaving group such as epoxide or cyclic sulfate.

"Pharmaceutically acceptable salts" are those salts that are suitable for use in contact with human and other animal tissues without reasonable toxicity, irritation, allergic reactions, etc. and within reasonable benefit / risk ratios within the scope of sound medical judgment. Refers to. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. J. Pharmaceutical Sciences (1977) 66: 1-19, describes pharmaceutically acceptable salts in detail. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Salts of amino groups formed by using or formed by other methods used in the art, such as using ion exchange. Other pharmaceutically acceptable salts are adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropio Nate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- Hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, maleate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate , Oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pival Sites, propionitrile include carbonate, stearate, succinate, sulfate, tartrate, such as thiocyanate, p- toluenesulfonate, undecanoate, valerate salts. Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In addition, where appropriate, pharmaceutically acceptable salts include quaternary salts.

The present invention provides a type II PRMT inhibitor. In one embodiment, the Type II PRMT inhibitor is a compound of Formula (III) or a pharmaceutically acceptable salt thereof:

here

는 단일 또는 이중 결합을 나타내고;

Represents a single or double bond;

R ¹ is hydrogen, R ^z , or —C (O) R ^z , wherein R ^z is optionally substituted C _1-6 alkyl;

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)-;

Each R is independently hydrogen or optionally substituted C _1-6 aliphatic;

Ar is a monocyclic or bicyclic aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein Ar is 0, 1, 2, 3, 4, or 5, as valency permits Are substituted with R ^y groups;

Each R ^y is independently halo, —CN, —NO ₂ , optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —OR ^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 ^B C (O) R ^A , -NR ^B C (O) N (R ^B ) ₂ , -NR ^B C (O) N (R ^B ) N (R ^B ) ₂ , -NR ^B C (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 ^B C (= NR ^B ) R ^B , -C (= S) R ^A , -C (= S) N (R ^B ) ₂ , -NR ^B C (= S) R ^A , -S (O) R ^A , -OS (O) ₂ R ^A , -SO ₂ R ^A , -NR ^B SO ₂ R ^A , or -SO ₂ N (R ^B ) ₂ ;

Each R ^A is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;

Each R ^B is independently selected from hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two R ^{The B} group together with its intervening atoms form an optionally substituted heterocyclic ring;

R ⁵ , R ⁶ , R ⁷ and R ⁸ are independently hydrogen, halo, or optionally substituted aliphatic;

Each R ^X is independently selected from the group consisting of halo, —CN, optionally substituted aliphatic, —OR ′, and —N (R ″) ₂ ;

R ^' is hydrogen or optionally substituted aliphatic;

Each R ″ is independently hydrogen or an optionally substituted aliphatic, or two R ″ together with their intervening atoms form a heterocyclic ring;

n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, as valency permits.

In one aspect, L is -C (O) N (R)-. In one aspect, R ¹ is hydrogen. In one aspect, n is zero.

In one embodiment, the type II PRMT inhibitor is a compound of Formula (IV) or a pharmaceutically acceptable salt thereof:

In one aspect, at least one R ^y is -NHR ^B. In one aspect, R ^B is optionally substituted cycloalkyl.

In one embodiment, the Type II PRMT inhibitor is a compound of Formula (VII) or a pharmaceutically acceptable salt thereof:

In one aspect, L is -C (O) N (R)-. In one aspect, R ¹ is hydrogen. In one aspect, n is zero.

In one embodiment, the type II PRMT inhibitor is a compound of Formula (VIII) or a pharmaceutically acceptable salt thereof:

In one aspect, L is -C (O) N (R)-. In one aspect, R ¹ is hydrogen. In one aspect, n is zero.

In one embodiment, the type II PRMT inhibitor is a compound of Formula (IX) or a pharmaceutically acceptable salt thereof:

In one aspect, R ¹ is hydrogen. In one aspect, n is zero.

In one embodiment, the type II PRMT inhibitor is Compound B or a pharmaceutically acceptable salt thereof:

In one embodiment, the type II PRMT inhibitor is a compound of Formula (X) or a pharmaceutically acceptable salt thereof:

In one aspect, R ^y is -NHR ^B. In one aspect, R ^B is optionally substituted heterocyclyl.

In certain embodiments, the Type II PRMT inhibitor is a compound of Formula (XI) or a pharmaceutically acceptable salt thereof:

Wherein X is -C (R ^XC ) _2- , -O-, -S-, or -NR ^XN -wherein each occurrence of R ^XC is independently hydrogen, optionally substituted alkyl, optionally substituted carbocyclyl, Optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; R ^XN is independently hydrogen, optionally substituted alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -C (= 0) R ^XA , or a nitrogen protecting group ; R ^XA is optionally substituted alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

In one embodiment, the type II PRMT inhibitor is Compound C or a pharmaceutically acceptable salt thereof:

Compound C and methods of preparing Compound C are disclosed in at least page 141 (compound 208) and page 291, paragraphs 294, 294, paragraphs [00469] in PCT / US2013 / 077235.

In another embodiment, the type II PRMT inhibitor is Compound E or a pharmaceutically acceptable salt thereof:

In another embodiment, the type II PRMT inhibitor is Compound F or a pharmaceutically acceptable salt thereof:

Type II PRMT inhibitors are further disclosed in PCT / US2013 / 077235 and PCT / US2015 / 043679, which are incorporated herein by reference. Exemplary Type II PRMT inhibitors are disclosed in Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, Table 1F, and Table 1G of PCT / US2013 / 077235, and methods of making Type II PRMT inhibitors are described in PCT / US2013 / 077235. At least page 239, paragraphs [00359] to page 301, paragraphs [00485]. Other non-limiting examples of type II PRMT inhibitors or PRMT5 inhibitors include the following published patent applications WO2011 / 079236, WO2014 / 100695, WO2014 / 100716, WO2014 / 100730, WO2014 / 100764 and WO2014 / 100734 and US Provisional Application Nos. 62 / 017,097 and 62 / 017,055. General and specific compounds described in these patent applications are incorporated herein by reference and can be used to treat cancer as described herein. In some embodiments, the type II PRMT inhibitor is a nucleic acid (eg, siRNA). SiRNAs for PRMT5 are described, for example, in Mol Cancer Res. 2009 Apr; 7 (4): 557-69, and Biochem J. 2012 Sep 1; 446 (2): 235-41.

“Antigen binding protein (ABP)” means a protein that binds to an antigen, including an antibody or engineered molecule that functions in an antibody-like manner. Such alternative antibody formats include triabodies, tetrabodies, miniantibodies and minibodies, wherein one or more CDRs of any molecule according to the disclosure can be arranged in a suitable non-immunoglobulin protein scaffold or framework. Alternative scaffolds such as affibodies, SpA scaffolds, LDL receptor class A domains, avimers (see, eg, numbers 2005/0053973, 2005/0089932, 2005/0164301) or EGF domains are also included. ABPs also include antigen binding fragments of such antibodies or other molecules. In addition, when paired with a suitable light chain, ABPs may be full-length antibodies, (Fab ′) 2 fragments, Fab fragments, bispecific or biparatopic molecules or equivalents thereof (eg scFV, non-tri- or tetra-body). , Tandabs, etc.) may comprise the V _H region of the invention. ABP is IgG1, IgG2, IgG3, or IgG4; Or IgM; Antibodies or modified variants thereof that are IgA, IgE, or IgD. The constant domain of the antibody heavy chain can be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. ABPs may also be chimeric antibodies of the type described in WO86 / 01533 comprising an antigen binding region and a non-immunoglobulin region. The terms "ABP", "antigen binding protein" and "binding protein" are used interchangeably herein.

Protein programmed kill 1 (PD-1) is also an inhibitory member of the CD28 family of receptors, including CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed in activated B cells, T cells and bone marrow cells (Agata et al., Supra; Okazaki et al. (2002) Curr. Opin. Immunol 14: 391779-82; Bennett et al. (2003) J Immunol 170: 711-8). CD28 and ICOS, early members of the family, were found by functional effects on the enhancement of T cell proliferation after addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397: 263-266; Hansen et al. ( 1980) Immunogenics 10: 247-260). PD-1 was found through screening for differential expression in apoptosis cells (Ishida et al. (1992) EMBO J 11: 3887-95). Other members of the family, CTLA-4 and BTLA, were found through screening for differential expression in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS and CTLA-4 all have non-pairing cysteine residues that allow homodimerization. In contrast, PD-1 lacks the non-pairing cysteine residues characteristic of other CD28 family members, suggesting that it exists as a monomer. PD-1 antibodies and methods for use in the treatment of diseases are described in US Pat. No. 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; And US Publication No. US20110171220; US20110171215; And US20110271358. Combinations of CTLA-4 and PD-1 antibodies are described in US Pat. No. 9,084,776.

As used herein, "PD-1 antagonist" blocks PD-L1 expressed on cancer cells from binding to PD-1 expressed on immune cells (T cells, B cells or NKT cells), and preferably also, By any chemical compound or biological molecule that blocks the binding of PD-L2 expressed on cancer cells to immune cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; And PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. Human PD-1 amino acid sequences can be found in NCBI Locus No. NP — 005009. Human PD-L1 and PD-L2 amino acid sequences can be found in NCBI locus numbers NP_054862 and NP_079515, respectively.

PD-1 antagonists useful in any of the aspects of the invention, monoclonal antibodies that specifically bind to PD-1 or PD-L1 and preferably specifically to human PD-1 or human PD-L1 (mAb), or antigen binding fragments thereof. Such mAbs may be human antibodies, humanized antibodies or chimeric antibodies, and may comprise human constant regions. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 constant regions, and in preferred embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab'-SH, F (ab ') ₂ , scFv and Fv fragments.

Examples of mAbs that bind to human PD-1 useful in various aspects and embodiments of the present invention are described in US Pat. No. 8,552,154; US Patent No. 8,354,509; US Patent No. 8,168,757; US Patent No. 8,008,449; US Patent No. 7,521,051; US Patent No. 7,488,802; WO2004072286; WO2004056875; And WO2004004771.

Other PD-1 antagonists useful in any of the aspects and embodiments of the present invention are immunoadhesin, eg, constant, that bind specifically to PD-1 and preferably bind to human PD-1. Fusion proteins containing an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a region such as an Fc region of an immunoglobulin molecule. Examples of immunoadhesin molecules that specifically bind PD-1 are described in WO2010027827 and WO2011066342. Specific fusion proteins useful as PD-1 antagonists in the therapeutic methods, medicaments and uses of the present invention include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein and binds to human PD-1. .

Nivolumab is a humanized monoclonal anti-PD-1 antibody commercially available as Opdivo®. Nivolumab is recommended for the treatment of some unresectable or metastatic melanoma. Nivolumab binds to the Ig superfamily transmembrane protein PD-1 and blocks its activation by its ligands PD-L1 and PD-L2, thereby activating T-cells and cell-mediated immunity to tumor cells or pathogens. Cause a reaction. Activated PD-1 negatively regulates T-cell activation and effector function through inhibition of P13k / Akt pathway activation. Other names for nivolumab include BMS-936558, MDX-1106 and ONO-4538. Amino acid sequences for nivolumab, and methods of use and preparation are disclosed in US Pat. No. US 8,008,449.

Pembrolizumab is a humanized monoclonal anti-PD-1 antibody commercially available as Kitruda®. Pembrolizumab is recommended for the treatment of some nonresectable or metastatic melanoma. The amino acid sequence of pembrolizumab and methods of use are disclosed in US Pat. No. 8,168,757.

PD-L1 is a B7 family member expressed in many cell types, including APC and activated T cells (Yamazaki et al. (2002) J. Immunol. 169: 5538). PD-L1 binds to both PD-1 and B7-1. Binding of T-cell-expressed B7-1 by PD-L1 and binding of T-cell-expressed PD-L1 by B7-1 both induce T cell inhibition (Butte et al. (2007) Immunity 27: 111). As with other B7 family members, there is also evidence that PD-L1 can also provide co-stimulatory signals to T cells (Subudhi et al. (2004) J. Clin. Invest. 113: 694; Tamura et al. (2001) Blood 97: 1809). PD-L1, a ligand of PD-1 (human PD-L1 cDNA consists of the nucleotide sequence shown by EMBL / Ginbank Accession No. AF233516, and mouse PD-L1 cDNA is the nucleotide sequence shown by NM.sub .-- 021893 Consisting of so-called antigen-presenting cells such as activated monocytes and dendritic cells (Journal of Experimental Medicine (2000), vol. 19, issue 7, p 1027-1034). These cells provide interaction molecules that induce various immune-induced signals to T lymphocytes, and PD-L1 is one of these molecules that induces suppression signals by PD-1. It has been found that PD-L1 ligand stimulation inhibits the activation of PD-1 expressing T lymphocytes (induction of cell proliferation and various cytokine production). PD-L1 expression is derived from not only immunocompetent cells but also certain types of tumor cell lines (cell lines derived from mononuclear leukemia, cell lines derived from mast cells, cell lines derived from liver carcinomas, cell lines derived from neuroblastoma cells, and breast carcinomas). Cell lines) (Nature Immunology (2001), vol. 2, issue 3, p. 261-267).

Anti-PD-L1 antibodies and methods for their preparation are known in the art. Antibodies to such PD-L1 may be polyclonal or monoclonal, and / or recombinant and / or humanized antibodies. PD-L1 antibodies are under development as immunomodulators for the treatment of cancer.

Exemplary PD-L1 antibodies are described in US Pat. No. 9,212,224; US Patent No. 8,779,108; US Patent No. 8,552,154; US Patent No. 8,383,796; US Patent No. 8,217,149; US Patent Publication 20110280877; WO2013079®; And WO2013019906. Additional exemplary antibodies and methods of use for PD-L1 (also referred to as CD274 or B7-H1) are described in US Pat. No. 8,168,179; US Patent No. 7,943,743; US Patent No. 7,595,048; WO2014055897; WO2013019906 and WO2010077634. Specific anti-human PD-L1 monoclonal antibodies useful as PD-1 antagonists in the therapeutic methods, medicaments and uses of the present invention include MPDL3280A, BMS-936559, MEDI4736, MSB0010718C.

Atezolizumab is a fully humanized monoclonal anti-PD-L1 antibody commercially available as TECENTRIQ ™. Atezolizumab is recommended for the treatment of some locally advanced or metastatic urinary tract carcinoma. Atezolizumab blocks the interaction of PD-L1 with PD-1 and CD80.

CD134, also known as OX40, is a member of the TNFR-superfamily of receptors that, unlike CD28, are not constitutively expressed on resting naïve T cells. OX40 is a secondary costimulatory molecule expressed 24 to 72 hours after activation; Its ligand, OX40L, is also not expressed on resting antigen presenting cells but after its activation. Expression of OX40 is dependent on full activation of T cells; In the absence of CD28, expression of OX40 is delayed and four times lower. OX40 / OX40-ligand (OX40 receptor) / (OX40L) is a pair of costimulatory molecules critical for T cell proliferation, survival, cytokine production and memory cell production. Initial in vitro experiments demonstrated that signaling through OX40 on CD4 ⁺ T cells leads to TH2 rather than TH1 development. These results were supported by in vivo studies showing that blocking OX40 / OX40L interaction prevents the induction and maintenance of TH2-mediated allergic immune response. However, blocking the OX40 / OX40L interaction improves or prevents TH1-mediated disease. In addition, it has been suggested that administration of soluble OX40L or gene delivery of OX40L to tumors strongly enhances anti-tumor immunity in mice. Recent studies also suggest that OX40 / OX40L may play a role in promoting the CD8 T cell-mediated immune response. As discussed herein, OX40 signaling blocks the inhibitory function of CD4 ⁺ CD25 ⁺ naturally occurring regulatory T cells, and the OX40 / OX40L pair plays a critical role in the overall regulation of peripheral immune versus resistance. OX-40 antibodies, OX-40 fusion proteins and methods of their use are described in US Pat. No. US 7,504,101; US 7,758,852; US 7,858,765; US 7,550,140; US 7,960,515; And US 9,006,399 and International Publications: WO 2003082919; WO 2003068819; WO 2006063067; WO 2007084559; WO 2008051424; WO2012027328; And WO2013028231.

An antigen binding protein (ABP) or anti-OX40 antigen binding protein of the invention herein is one that binds OX40, and in some embodiments, performs one or more of the following: modulating signaling through OX40, or Modulates function, agonizes OX40 signaling, stimulates OX40 function, or co-stimulates OX40 signaling. Example 1 of US Pat. No. 9,006,399 discloses an OX40 binding assay. Those skilled in the art will readily recognize various other well known assays for establishing this function.

In one embodiment, the OX40 antigen binding protein comprises one disclosed in WO2012 / 027328 (PCT / US2011 / 048752), dated August 23, 2011. In another embodiment, the antigen binding protein comprises a CDR of an antibody disclosed in WO2012 / 027328 (PCT / US2011 / 048752) of August 23, 2011 International Application Date, or a CDR having 90% identity with the disclosed CDR sequences. In further embodiments, the antigen binding protein is a V _H , V _L or both, or a disclosed V _H or V _L sequence of an antibody disclosed in WO2012 / 027328 (PCT / US2011 / 048752), filed Aug. 23, 2011 And V _H or V _L having 90% identity with.

In another embodiment, the OX40 antigen binding protein is disclosed in WO2013 / 028231 (PCT / US2012 / 024570) on Feb. 9, 2012, international application date. In another embodiment, the antigen binding protein comprises a CDR of an antibody disclosed in WO2013 / 028231 (PCT / US2012 / 024570) on February 9, 2012, International Application Date, or a CDR having 90% identity with the disclosed CDR sequences. In a further embodiment, the antigen binding protein is a V _H , V _L or both, or a disclosed V _H or V _L sequence of an antibody disclosed in WO2013 / 028231 (PCT / US2012 / 024570) on Feb. 9, 2012, international application date. And V _H or V _L having 90% identity with.

In another embodiment, an anti-OX40 ABP or antibody of the invention comprises one or more of the CDR or VH or VL sequences set forth in FIGS. 30-41 herein, or sequences having 90% identity to them.

In one embodiment, an anti-OX40 ABP or antibody of the invention comprises any one or a combination of the following CDRs:

In some embodiments, an anti-OX40 ABP or antibody of the invention comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 5. Suitably, the OX40 binding protein of the invention is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 relative to SEQ ID NO: 5. Heavy chain variable regions with%, 96%, 97%, 98%, 99%, or 100% sequence identity.

Humanized heavy chain (V _H ) variable region:

In one embodiment of the invention the OX40 ABP or antibody comprises CDRL1 (SEQ ID NO: 7), CDRL2 (SEQ ID NO: 8), and CDRL3 (SEQ ID NO: 7) in a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 Number: 9). In some embodiments, the OX40 binding protein of the invention comprises the light chain variable region set forth in SEQ ID NO: 11. In one embodiment, the OX40 binding protein of the invention comprises a heavy chain variable region of SEQ ID NO: 5 and a light chain variable region of SEQ ID NO: 11.

Humanized Light Chain (V _L ) Variable Regions

In some embodiments, the OX40 binding protein of the invention comprises a light chain variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. Suitably, the OX40 binding protein of the invention is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 relative to SEQ ID NO: 11. Light chain variable regions with%, 96%, 97%, 98%, 99%, or 100% sequence identity.

In another embodiment, an anti-OX40 ABP or antibody of the invention comprises any one or a combination of the following CDRs:

In some embodiments, an anti-OX40 ABP or antibody of the invention comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 17. Suitably, the OX40 binding protein of the invention is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 relative to SEQ ID NO: 17. Heavy chain variable regions with%, 96%, 97%, 98%, 99%, or 100% sequence identity.

Humanized heavy chain (V _H ) variable region:

In one embodiment of the invention the OX40 ABP or antibody comprises CDRL1 (SEQ ID NO: 19), CDRL2 (SEQ ID NO: 20), and CDRL3 (SEQ ID NO: 20) in a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23 Number: 21). In some embodiments, the OX40 binding protein of the invention comprises the light chain variable region set forth in SEQ ID NO: 23. In one embodiment, the OX40 binding protein of the invention comprises a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 23.

Humanized Light Chain (V _L ) Variable Regions

In some embodiments, the OX40 binding protein of the invention comprises a light chain variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23. Suitably, the OX40 binding protein of the invention is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 relative to SEQ ID NO: 23. Light chain variable regions with%, 96%, 97%, 98%, 99%, or 100% sequence identity.

The CDR or minimum binding unit can be modified by at least one amino acid substitution, deletion or addition, wherein the variant antigen binding protein is an unmodified protein such as an antibody comprising SEQ ID NO: 5 and SEQ ID NO: 11 or Substantially retains the biological characteristics of the antibody comprising SEQ ID NO: 17 and SEQ ID NO: 23.

It will be appreciated that each CDR H1, H2, H3, L1, L2, L3, alone or in combination with any other CDR, may be modified in any permutation or combination. In one embodiment, the CDRs are modified by substitution, deletion or addition of up to three amino acids, for example one or two amino acids, for example one amino acid. Typically, the modification is a substitution, in particular a conservative substitution, for example as shown in Table 1 below.

Table 1

In one embodiment, an ABP or antibody of the invention is, for example, as set forth in CDRs of 106-222 antibodies of FIGS. 30-31 herein, eg, as set forth in SEQ ID NOs: 1, 2 and 3 as disclosed in FIG. CDRH1, CDRH2 and CDRH3 having the same amino acid sequence, and CDRL1, CDRL2 and CDRL3 having the sequences as set forth in, for example, SEQ ID NOs: 7, 8 and 9, respectively. In one embodiment, the ABP or antibody of the invention comprises the CDRs of the 106-222, Hu106 or Hu106-222 antibody as disclosed in WO2012 / 027328 (PCT / US2011 / 048752) on August 23, 2011, international application date. . In a further embodiment, the anti-OX40 ABP or antibody of the invention has the VH and VL regions of the 106-222 antibody as shown in FIGS. 30-31, eg, amino acid sequences as set forth in SEQ ID NO: 4. VH and VL in FIG. 31 having an amino acid sequence as set forth in SEQ ID NO: 10. In another embodiment, an ABP or antibody of the invention has a VH having an amino acid sequence as set forth in SEQ ID NO: 5 of FIG. 30 herein, and an amino acid sequence as set forth in SEQ ID NO: 11 of FIG. 31 herein. Contains VL. In further embodiments, an anti-OX40 ABP or antibody of the invention is a Hu106-222 antibody or 106-222 antibody or Hu106 antibody as disclosed in WO2012 / 027328 (PCT / US2011 / 048752), filed Aug. 23, 2011 VH and VL regions. In further embodiments, an anti-OX40 ABP or an antibody of the invention is disclosed, for example, in 106-222, Hu106-222, or as disclosed in WO2012 / 027328 (PCT / US2011 / 048752), filed August 23, 2011, International Application Date. Hu106. In further embodiments, an ABP or antibody of the invention comprises a CDR or VH or VL or antibody sequence having 90% identity to the sequence in this paragraph.

In another embodiment, an anti-OX40 ABP or antibody of the invention is, for example, the CDRs of the 119-122 antibody of FIGS. 34-35, eg, as shown in SEQ ID NOs: 13, 14 and 15, respectively. CDRH1, CDRH2 and CDRH3 having amino acid sequences. In another embodiment, an anti-OX40 ABP or antibody of the invention is a 119-122 or Hu119 or Hu119-222 antibody as disclosed in WO2012 / 027328 (PCT / US2011 / 048752), filed August 23, 2011 CDRs. In a further embodiment, an anti-OX40 ABP or antibody of the invention is a VH having an amino acid sequence as set forth in SEQ ID NO: 16 of FIG. 34 herein and as set forth in SEQ ID NO: 22 as shown in FIG. 35 herein. VL having an amino acid sequence. In another embodiment, an anti-OX40 ABP or antibody of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO: 17 and a VL having an amino acid sequence as set forth in SEQ ID NO: 23. In a further embodiment, the anti-OX40 ABP or antibody of the invention is a VH of a 119-122 or Hu119 or Hu119-222 antibody as disclosed in WO2012 / 027328 (PCT / US2011 / 048752), filed Aug. 23, 2011 And a VL region. In further embodiments, the ABP or antibody of the invention is a 119-222 or Hu119 or Hu119-222 antibody, eg, as disclosed in WO2012 / 027328 (PCT / US2011 / 048752), filed August 23, 2011 . In further embodiments, an ABP or antibody of the invention comprises a CDR or VH or VL or antibody sequence having 90% identity to the sequence in this paragraph.

In another embodiment, an anti-OX40 ABP or antibody of the invention comprises a CDR of a 119-43-1 antibody, eg, as shown in FIGS. 38-39 herein. In another embodiment, the anti-OX40 ABP or antibody of the invention comprises the CDRs of the 119-43-1 antibody as disclosed in WO2013 / 028231 (PCT / US2012 / 024570) on February 9, 2012, International Application Date. . In further embodiments, an anti-OX40 ABP or antibody of the invention comprises one of the VH regions and one of the VL regions of the 119-43-1 antibody as shown in FIGS. 38-41. In a further embodiment, the anti-OX40 ABP or antibody of the invention is directed to the VH and VL regions of the 119-43-1 antibody as disclosed in WO2013 / 028231 (PCT / US2012 / 024570) on February 9, 2012, International Application Date. Include. In further embodiments, the ABPs or antibodies of the invention are 119-43-1 or 119-43-1 chimeras as disclosed in FIGS. 38-41 herein. In further embodiments, the ABPs or antibodies of the present invention are as disclosed in WO2013 / 028231 (PCT / US2012 / 024570), dated February 9, 2012, international application date. In further embodiments, any one of the ABPs or antibodies described in this paragraph is humanized. In further embodiments, any one of the ABPs or antibodies described in this paragraph is engineered to produce a humanized antibody. In further embodiments, an ABP or antibody of the invention comprises a CDR or VH or VL or antibody sequence having 90% identity to the sequence in this paragraph.

In another embodiment, any mouse or chimeric sequence of any anti-OX40 ABP or antibody of the invention is engineered to produce a humanized antibody.

In one embodiment, an anti-OX40 ABP or antibody of the invention comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 7; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 8; And (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

In another embodiment, an anti-OX40 ABP or antibody of the invention comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 14; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 15; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 19; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 20; And (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 21.

In another embodiment, an anti-OX40 ABP or antibody of the invention comprises: a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 13; A heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 14; And / or heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 15, or a heavy chain variable region CDR having 90% identity to it.

In another embodiment, an anti-OX40 ABP or antibody of the invention comprises: a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 7 or 19; A light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 20 and / or a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 9 or 21, or a heavy chain variable region having 90 percent identity to it.

In further embodiments, an anti-OX40 ABP or antibody of the invention comprises: the amino acid sequence of SEQ ID NO: 10, 11, 22 or 23 or to the amino acid sequence of SEQ ID NO: 10, 11, 22 or 23 A light chain variable region (“VL”) comprising an amino acid sequence having at least 90 percent identity to. In another embodiment, an anti-OX40 ABP or antibody of the invention is at least 90 percent relative to amino acid sequences of SEQ ID NOs: 4, 5, 16, and 17 or amino acid sequences of SEQ ID NOs: 4, 5, 16, and 17 Heavy chain variable region (“VH”) comprising an amino acid sequence having identity. In another embodiment, an anti-OX40 ABP or antibody of the invention comprises a variable heavy chain sequence of SEQ ID NO: 5 and a variable light chain sequence of SEQ ID NO: 11, or a sequence having 90 percent identity to it. In another embodiment, an anti-OX40 ABP or antibody of the invention comprises a variable heavy chain sequence of SEQ ID NO: 17 and a variable light chain sequence of SEQ ID NO: 23, or a sequence having 90 percent identity to it.

In another embodiment, an anti-OX40 ABP or antibody of the invention is selected by a nucleic acid sequence having at least 90 percent identity to a nucleic acid sequence of SEQ ID NO: 12 or 24, or a nucleotide sequence of SEQ ID NO: 12 or 24 The variable light chain is encoded. In another embodiment, an anti-OX40 ABP or antibody of the invention is selected by a nucleic acid sequence having at least 90 percent identity to a nucleic acid sequence of SEQ ID NO: 6 or 18, or a nucleotide sequence of SEQ ID NO: 6 or 18. The variable heavy chain to be encoded.

Monoclonal antibodies are also provided herein. In one embodiment, the monoclonal antibody comprises a variable light chain comprising an amino acid sequence of SEQ ID NO: 10 or 22, or an amino acid sequence having at least 90 percent identity to an amino acid sequence of SEQ ID NO: 10 or 22. . Further provided is a monoclonal antibody comprising a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 4 or 16, or an amino acid sequence having at least 90 percent identity to an amino acid sequence of SEQ ID NO: 4 or 16 do.

CTLA-4 is a T cell surface molecule originally identified by differential screening of murine lysis T cell cDNA libraries (Brunet et al., Nature 328: 267-270 (1987)). CTLA-4 is also a member of the immunoglobulin (Ig) superfamily; CTLA-4 comprises a single extracellular Ig domain. CTLA-4 transcripts have been found in T cell populations with cytotoxic activity, suggesting that CTLA-4 can function in cytolytic responses (Brunet et al., Supra; Brunet et al., Immunol Rev. 103- (21-36 (1988)). The researchers studied CTLA-4 for the same chromosomal region (2q33-34) (Lafage-Pochitaloff et al., Immunogenetics, 31: 198-201 (1990)). Cloning and mapping of genes to human counterparts of (Dariavach et al., Eur. J. Immunol., 18: 1901-1905 (1988)) reported DNA encoding the human CTLA-4 DNA and CD28 protein. Sequence comparisons between the two showed significant homology to the sequences and had the greatest degree of homology in the membrane and cytoplasmic regions (Brunet et al. (1988), supra; Dariavach et al. (1988), supra). Boi (Ipilimumab) is a fully human CTLA-4 antibody marketed by Bristol Myers Scoop Protein Structure and Method of Use of Ipilimumab It is described in U.S. Patent No. 7,605,238 and 6.98472 million.

Anti-CTLA4 antibodies suitable for use in the methods of the invention include anti-CTLA4 antibodies, human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, monoclonal anti-CTLA4 Antibody, polyclonal anti-CTLA4 antibody, chimeric anti-CTLA4 antibody, ipilimumab, tremelimumab, anti-CD28 antibody, anti-CTLA4 adnectin, anti-CTLA4 domain antibody, single chain anti-CTLA4 fragment, heavy chain anti -CTLA4 fragments, light chain anti-CTLA4 fragments, inhibitors of CTLA4 that agonize co-stimulatory pathways, antibodies disclosed in PCT Publication No. WO 2001/014424, antibodies disclosed in PCT Publication No. WO 2004/035607, US Publication No. US 2005 Antibodies disclosed in / 0201994, and antibodies disclosed in approved European Patent No. EP1212422B1. Additional CTLA-4 antibodies are disclosed in US Pat. Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; PCT Publication Nos. WO 01/14424 and WO 00/37504; And US Publication Nos. US 2002/0039581 and US 2002/086014. Other anti-CTLA-4 antibodies that can be used in the methods of the invention include, for example, WO 98/42752; US Patent 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 number 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58: 5301-5304 (1998) and US Pat. Nos. 5,977,318, 6,682,736, 7,109,003 and 7,132,281.

As used herein, “immuno-modulator” or “immuno-modulatory agent” refers to any substance, including monoclonal antibodies that affect the immune system. In some embodiments, an immunomodulator or immunomodulatory agent upregulates the immune system. Immunomodulators can be used as antineoplastic agents for the treatment of cancer. For example, immunomodulatory agents include anti-PD-1 antibodies (opdibo / nivolumab and kitruda / pembrolizumab), anti-CTLA-4 antibodies such as ipilimumab (yerbois) and anti-OX40 antibodies However, it is not limited thereto.

As used herein, the term “agonist” refers to (1) stimulation or activation of a receptor upon contact with a co-signaling receptor, (2) enhancement, increase or promotion, induction or duration of activity, function or presence of the receptor and / or Or (3) an antigen binding protein, including, but not limited to, an antibody that causes one or more of the enhancement, increase, promotion or induction of expression of a receptor. Agonist activity can be measured in vitro by various assays known in the art such as, but not limited to, measurement of cell signaling, cell proliferation, immune cell activation markers, cytokine production. Agonist activity can also be measured in vivo by a variety of assays that measure substitution endpoints such as, but not limited to, measurement of T cell proliferation or cytokine production. As used herein, the term “antagonist” refers to (1) attenuation, blocking or inactivation of a receptor and / or blocking its activation by a natural ligand of a receptor upon contact with a co-signaling receptor; Antigen binding, including, but not limited to, antibodies that cause one or more of (2) a decrease, reduction or attenuation of the activity, function or presence of a receptor, and / or (3) a decrease, reduction, or abolition of receptor expression. Refers to a protein. Antagonist activity can be measured in vitro by measuring various assays known in the art such as, but not limited to, cell signaling, cell proliferation, immune cell activation markers, increase or decrease in cytokine production. Antagonist activity can also be measured in vivo by a variety of assays that measure substitution endpoints such as, but not limited to, measurement of T cell proliferation or cytokine production.

As used herein, the term “cross compete for binding” refers to any agent such as an antibody that will compete for binding to a target with any of the agents of the invention. Competition for binding between two antibodies can be tested by various methods known in the art, including flow cytometry, meso scale discovery, and ELISA. Binding can be measured directly, which means that two or more binding proteins can be contacted with a co-signaling receptor and the binding can be measured for one or each. Alternatively, the binding of the molecule of interest can be tested for binding or natural ligand and compared quantitatively to each other.

The term “antibody” is used herein in the broadest sense to refer to a molecule having an immunoglobulin-like domain (eg, IgG, IgM, IgA, IgD or IgE), monoclonal, recombinant, polyclonal, Multispecific antibodies, including chimeric, human, humanized, bispecific antibodies, and heteroconjugate antibodies; Single variable domain (eg, V _H , V _HH , V _L , domain antibody (dAb ™)), antigen binding antibody fragment, Fab, F (ab ') ₂ , Fv, disulfide linked Fv, single chain Fv, Disulfide-linked scFv, diabodies, TANDABS ™, and the like, as well as modified versions of any of the above. Vol 23, number 9, 1126-1136).

Alternative antibody formats include alternative scaffolds such as affibodies, SpA scaffolds, LDL receptor class A domains, in which one or more CDRs of an antigen binding protein can be arranged on a suitable non-immunoglobulin protein scaffold or backbone. Avimers (see, eg, US Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or EGF domains are also included.

The term "domain" refers to a folded protein structure that maintains its tertiary structure independently of the rest of the protein. In general, domains are responsible for the individual functional properties of proteins and in many cases can be added, removed or transferred to other proteins without loss of function of the protein and / or the rest of the domain.

The term “single variable domain” refers to a folded polypeptide domain that includes the sequence characteristics of the antibody variable domain. Thus, this means that the complete antibody variable domains, such as V _H , V _HH and V _L , and modified antibody variable domains, eg, where one or more loops have been replaced with sequences not characteristic of the antibody variable domain, or truncated or N- Or a folded fragment of an antibody variable domain comprising a C-terminal extension, as well as a variable domain that retains the binding activity and specificity of at least the full length domain. A single variable domain can bind an antigen or epitope independently of different variable regions or domains. A "domain antibody" or "dAb (™)" can be considered identical to a "single variable domain". The single variable domain may be a human single variable domain, but also includes a single variable domain from other species such as rodents sharks and camels V _HH dAb ™. Camel V _HH is an immunoglobulin single variable domain polypeptide derived from species including camel, llama, alpaca, dromedary and guanaco, which naturally produces heavy chain antibodies that lack light chains. Such V _HH domains can be humanized according to standard techniques available in the art, which domains are considered to be "single variable domains". As used herein, V _H comprises the camel V _HH domain.

Antigen binding fragments may be provided by the arrangement of one or more CDRs on a non-antibody protein scaffold. As used herein, a “protein scaffold” may be a four-chain or two-chain antibody, or may include only the Fc region of an antibody, or may include one or more constant regions from an antibody and the constant region is human or Immunoglobulin (Ig) scaffolds, such as IgG scaffolds, which may be of primate origin or may be artificial chimeras of human and primate constant regions.

The protein scaffold may be an Ig scaffold such as an IgG or IgA scaffold. IgG scaffolds may comprise some or all of the domains of an antibody (ie, CH1, CH2, CH3, V _H , V _L ). The antigen binding protein may comprise an IgG scaffold selected from IgG1, IgG2, IgG3, IgG4 or IgG4PE. For example, the scaffold may be IgG1. The scaffold may consist of, or include, an Fc region of an antibody.

Affinity is the strength of binding of one molecule, eg, an antigen binding protein of the invention, to a single binding site to another, eg, its target antigen. The binding affinity of an antigen binding protein for its target can be determined by an equilibrium method (eg, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or by kinetics (eg, BIACORE ™ assay). Can be determined by. For example, the Biacore ™ method described in Example 5 can be used to measure binding affinity.

The binding force is the total sum of the strengths of the two molecules in each other at multiple sites to each other, for example, the binding value of the interaction is considered.

"Isolated" is intended to remove molecules, such as antigen binding proteins or nucleic acids, from the environment in which they can be found in nature. For example, a molecule can be purified from substances that normally exist with it in nature. For example, the mass of the molecules in the sample can be 95% of the total mass.

As used herein, the term “expression vector” refers to an isolated nucleic acid that can be used to introduce a nucleic acid of interest into a cell, such as a eukaryotic cell or a prokaryotic cell, or into a cell-free expression system in which the nucleic acid sequence of interest is expressed as a peptide chain such as a protein. . Such expression vectors can be, for example, cosmids, plasmids, viral sequences, transposons, and linear nucleic acids comprising the nucleic acid of interest. When the expression vector is introduced into a cell or cell-free expression system (eg reticulocyte lysate), the protein encoded by the nucleic acid of interest is produced by a transcription / translation apparatus. Expression vectors within the scope of the present disclosure can provide the necessary elements for eukaryotic or prokaryotic expression and can include viral promoter driven vectors such as CMV promoter driven vectors such as pcDNA3.1, pCEP4, and derivatives thereof, baculovirus ( Baculovirus) expression vectors, Drosophila expression vectors, and expression vectors driven by mammalian gene promoters, such as the human Ig gene promoter. Other examples include prokaryotic expression vectors such as T7 promoter drive vectors such as pET41, lactose promoter drive vectors and arabinose gene promoter drive vectors. Those skilled in the art will recognize many other suitable expression vectors and expression systems.

As used herein, the term “recombinant host cell” means a cell comprising the nucleic acid sequence of interest isolated prior to its introduction into the cell. For example, the nucleic acid sequence of interest may be present in an expression vector and the cell may be prokaryotic or eukaryotic. Exemplary eukaryotic cells are mammalian cells such as but not limited to COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2, 653, SP2 / 0, NS0, 293, HeLa, myeloma, lymphoma cells or Any derivative thereof. Most preferably, the eukaryotic cells are HEK293, NS0, SP2 / 0, or CHO cells. this. E. coli is an exemplary prokaryotic cell. Recombinant cells according to the present disclosure can be generated by transfection, cell fusion, immortalization, or other procedures well known in the art. The nucleic acid sequence of interest, such as an expression vector, that is transfected into a cell may be present outside the chromosome or stably integrated into the cell's chromosome.

A “chimeric antibody” refers to an engineered antibody type that contains naturally occurring variable regions (light and heavy chains) derived from donor antibodies, associated with light and heavy chain constant regions derived from recipient antibodies.

A "humanized antibody" has its CDR derived from a non-human donor immunoglobulin and the type of engineered antibody wherein the remaining immunoglobulin-derived portion of the molecule is derived from one or more human immunoglobulin (s). Refers to. Framework support residues can also be altered to preserve binding affinity (see, eg, Queen et al. Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson, et al., Bio / Technology, 9: 421 (1991)). Suitable human acceptor antibodies are characterized by homology to the nucleotide and amino acid sequences of the donor antibody, such as in conventional databases such as the KABAT® database, the Los Alamos database, and the Swiss Protein database. It may be selected from. Human antibodies characterized by homology to the framework regions of the donor antibody (based on amino acids) may be suitable to provide heavy chain constant regions and / or heavy chain variable framework regions for insertion of donor CDRs. Suitable acceptor antibodies capable of donating light chain constant or variable framework regions can be selected in a similar manner. It should be noted that the recipient antibody heavy and light chains need not originate from the same recipient antibody. The prior art describes several ways of producing such humanized antibodies-see for example EP-A-0239400 and EP-A-054951.

The term “complete human antibody” includes antibodies having variable and constant regions (if any) derived from human germline immunoglobulin sequences. Human sequence antibodies of the invention comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutations). can do. Fully human antibodies include amino acid sequences encoded only by polynucleotides that are ultimately of human origin or of the same amino acid sequence as such sequences. As intended herein, antibodies encoded by human immunoglobulin-encoding DNA inserted into the mouse genome produced in transgenic mice are fully human antibodies because they are ultimately encoded by DNA of human origin. In this situation, human immunoglobulin-encoding DNA can be rearranged (to encode an antibody) in mice and somatic mutations can also occur. Antibodies encoded by the original human DNA that have undergone these changes in mice are fully human antibodies as intended herein. The use of such transgenic mice makes it possible to select fully human antibodies against human antigens. As will be appreciated in the art, fully human antibodies can be prepared using phage display technology, wherein a human DNA library is inserted into phage for the production of antibodies comprising human germline DNA sequences.

The term “donor antibody” refers to an antibody that provides a first immunoglobulin partner with an amino acid sequence of its variable region, CDR, or other functional fragment or analog thereof. Thus, the donor provides an altered immunoglobulin coding region and results in expression of the altered antibody having the antigen specificity and neutralizing activity characteristics of the donor antibody.

The term “receptor antibody” provides all (or any portion) of an amino acid sequence encoding for the first immunoglobulin partner its heavy and / or light chain framework region and / or its heavy and / or light chain constant region. Reference is made to antibodies that are heterologous to the donor antibody. Human antibodies can be recipient antibodies.

The terms "V _H " and "V _L " are used herein to refer to the heavy and light chain variable regions of an antigen binding protein, respectively.

"CDR" is defined as the complementarity determining region amino acid sequence of an antigen binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. Three heavy and three light chain CDRs (or CDR regions) are present in the variable portion of immunoglobulins. Thus, as used herein, “CDR” refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

Throughout this specification, amino acid residues in variable domain sequences and full length antibody sequences are numbered according to the Kabat numbering convention. Similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", and "CDRH3" used in the embodiments follow the Kabat numbering convention. For further information, see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1991).

It will be apparent to those skilled in the art that alternative numbering conventions exist for variable domain sequences and amino acid residues in full length antibody sequences. See also CDR sequences, for example Chothia et al. (1989) Nature 342: 877-883, there are alternative numbering provisions for that. The structure and protein folding of an antibody can mean that other residues are considered part of the CDR sequence and will be understood as such by those of skill in the art.

Other numbering conventions for CDR sequences available to those skilled in the art include the method "AbM" (University of Bath) and "Contact" (University College London). In order to provide a "minimum binding unit", at least two of the Kabat, Chothia, AbM and contact methods can be used to determine the minimum overlapping region. The minimum binding unit may be a sub-part of the CDR.

In one embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a type II protein arginine methyltransferase (type II PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulatory agent, wherein the immunomodulatory agent is Anti-CTLA4 antibody or antigen binding fragment thereof, anti-PD-1 antibody or antigen binding fragment thereof, anti-PDL1 antibody or antigen binding fragment thereof and anti-OX40 antibody or antigen binding fragment thereof. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect the anti-PD-1 antibody is pembrolizumab and / or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one embodiment, a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator is provided, wherein the type II PRMT inhibitor is Compound C and the immunomodulator is an agonist anti-OX40 antibody or antigen binding thereof It is a fragment. In one embodiment, a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator is provided, wherein the type II PRMT inhibitor is Compound C and the immunomodulatory agent comprises one or more of the following: OX40 antibody or antigen binding fragment thereof: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In one embodiment, a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator is provided, wherein the type II PRMT inhibitor is Compound C and the immunomodulator is at least 90% relative to SEQ ID NO: 5. An anti-OX40 antibody or antigen binding fragment thereof comprising a heavy chain variable region having sequence identity and a light chain variable region having at least 90% identity to SEQ ID NO: 11.

In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a type II protein arginine methyltransferase (type II PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulator selected from: Anti-CTLA4 antibody or antigen binding fragment thereof, anti-PD-1 antibody or antigen binding fragment thereof, anti-PDL1 antibody or antigen binding fragment thereof and anti-OX40 antibody or antigen binding fragment thereof. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a type II protein arginine methyltransferase (type II PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulatory agent. The inhibitor is Compound C and the immunomodulator is an agonist anti-OX40 antibody or antigen binding fragment thereof. In one embodiment is provided a pharmaceutical composition comprising a therapeutically effective amount of a type II protein arginine methyltransferase (type II PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulatory agent, wherein the type II PRMT inhibitor is a compound And an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another embodiment, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a type II protein arginine methyltransferase (type II PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulatory agent, wherein the type II PRMT inhibitor is An anti-OX40 antibody or antigen thereof comprising Compound C and an immunomodulatory agent comprising a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 5 and a light chain variable region having at least 90% identity to SEQ ID NO: 11 Is a binding fragment.

In one embodiment, a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulatory agent is administered to a human being in need thereof with at least one of a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent. A method of treating cancer in a human is provided comprising administering together to treat cancer in the human, wherein the immunomodulatory agent is selected from: an anti-CTLA4 antibody or antigen binding fragment thereof, an anti-PD-1 antibody Or an antigen binding fragment thereof, an anti-PDL1 antibody or antigen binding fragment thereof and an anti-OX40 antibody or antigen binding fragment thereof. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one aspect, the type II PRMT inhibitor and immunomodulatory agent are administered to the patient simultaneously, sequentially, in any order, systemically, orally, intravenously and intratumorally. In another aspect, the type II PRMT inhibitor is administered orally. In one embodiment, a method of treating cancer in a human is provided comprising administering a combination of Compound C and an agonist anti-OX40 antibody or antigen binding fragment thereof to a human in need thereof. In another embodiment, treating cancer in a human comprising administering to a human being in need thereof a combination of Compound C and an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: Methods are provided: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as set forth in SEQ ID NO: 8 and / or CDRL3 as set forth in SEQ ID NO: 9, or a direct equivalent of each CDR wherein the direct equivalent in said CDR has up to two amino acid substitutions. In another embodiment, a human in need of treatment of a cancer has a heavy chain variable region having at least 90% sequence identity to Compound C and at least 90% identity to SEQ ID NO: 11 with Compound C. A method of treating cancer in a human is provided comprising administering an anti-OX40 antibody comprising a light chain variable region or a combination of antigen binding fragments thereof.

In a further embodiment, a pharmaceutical composition comprising a type II protein arginine methyltransferase (type II PRMT) inhibitor to a human in need thereof, and an anti-CTLA4 antibody or antigen-binding fragment thereof, anti-PD-1 antibody Or treating a cancer in the human by administering a therapeutically effective amount of a pharmaceutical composition comprising an immunomodulatory agent selected from an antigen-binding fragment thereof, an anti-PDL1 antibody or antigen-binding fragment thereof, and an anti-OX40 antibody or antigen-binding fragment thereof. To provide a method of treating cancer in humans. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one aspect, the type II PRMT inhibitor and immunomodulatory agent are administered to the patient simultaneously, sequentially, in any order, systemically, orally, intravenously and intratumorally. In one aspect, the type II PRMT inhibitor is administered orally. In one embodiment, in a human, comprising administering to the human being in need thereof a therapeutically effective amount of a pharmaceutical composition comprising Compound C and a pharmaceutical composition comprising an agonist anti-OX40 antibody or antigen binding fragment thereof A method of treating cancer is provided. In another embodiment, a therapeutically effective amount of a pharmaceutical composition comprising Compound C, and a pharmaceutical composition comprising an anti-OX40 antibody or antigen-binding fragment thereof, comprising one or more of the following: A method of treating cancer in a human is provided comprising administering: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as set forth in SEQ ID NO: 8 and / or a direct equivalent of CDRL3 or each CDR as set forth in SEQ ID NO: 9, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another embodiment, a pharmaceutical composition comprising Compound C and a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 5 and at least 90 for SEQ ID NO: 11 to a human being in need thereof. Provided is a method of treating cancer in a human comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-OX40 antibody or antigen binding fragment thereof comprising a light chain variable region having% identity.

In another embodiment, the invention provides the use of a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator for the manufacture of a medicament, wherein the immunomodulatory agent is an anti-CTLA4 antibody or antigen binding thereof Fragments, anti-PD-1 antibodies or antigen binding fragments thereof, anti-PDL1 antibodies or antigen binding fragments thereof and anti-OX40 antibodies or antigen binding fragments thereof. In another embodiment, the present invention provides the use of a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulatory agent for the treatment of cancer, wherein the immunomodulatory agent is an anti-CTLA4 antibody or antigen binding thereof. Fragments, anti-PD-1 antibodies or antigen binding fragments thereof, anti-PDL1 antibodies or antigen binding fragments thereof and anti-OX40 antibodies or antigen binding fragments thereof. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one aspect, the type II PRMT inhibitor and immunomodulatory agent are administered to the patient simultaneously, sequentially, in any order, systemically, orally, intravenously and intratumorally. In one aspect, the type II PRMT inhibitor is administered orally. In one embodiment, there is provided the use of a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator for the manufacture of a medicament, wherein the type II PRMT inhibitor is Compound C and the immunomodulator is an agonist anti- OX40 antibody or antigen binding fragment thereof. In one embodiment, there is provided the use of a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator for the manufacture of a medicament, wherein the type II PRMT inhibitor is Compound C and the immunomodulatory agent is one of An anti-OX40 antibody or antigen-binding fragment thereof comprising: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as set forth in SEQ ID NO: 8 and / or CDRL3 as set forth in SEQ ID NO: 9 or a direct equivalent of each CDR wherein the direct equivalent has no more than two amino acid substitutions. In one embodiment, there is provided the use of a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator for the manufacture of a medicament, wherein the type II PRMT inhibitor is Compound C and the immunomodulator is SEQ ID NO: An anti-OX40 antibody or antigen binding fragment thereof comprising a heavy chain variable region having at least 90% sequence identity to 5 and a light chain variable region having at least 90% identity to SEQ ID NO: 11.

In another embodiment, the invention provides a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor with an immunomodulatory agent for use in the treatment of cancer, wherein the immunomodulatory agent is an anti-CTLA4 antibody or antigen thereof Binding fragment, anti-PD-1 antibody or antigen binding fragment thereof, anti-PDL1 antibody or antigen binding fragment thereof and anti-OX40 antibody or antigen binding fragment thereof. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one aspect, the type II PRMT inhibitor and immunomodulatory agent are administered to the patient simultaneously, sequentially, in any order, systemically, orally, intravenously and intratumorally. In one aspect, the type II PRMT inhibitor is administered orally. In one embodiment, a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator is provided for use in the treatment of cancer, wherein the type II PRMT inhibitor is Compound C and the immunomodulatory agent is -OX40 antibody or antigen binding fragment thereof. In one embodiment, a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator is provided for use in the treatment of cancer, wherein the type II PRMT inhibitor is Compound C and the immunomodulator is one of the following: An anti-OX40 antibody or antigen-binding fragment thereof comprising at least one species: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as set forth in SEQ ID NO: 8 and / or a direct equivalent of CDRL3 or each CDR as set forth in SEQ ID NO: 9, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In one embodiment, a combination of a type II protein arginine methyltransferase (type II PRMT) inhibitor and an immunomodulator is provided for use in the treatment of cancer, wherein the type II PRMT inhibitor is Compound C and the immunomodulator is SEQ ID NO: : An anti-OX40 antibody or antigen binding fragment thereof comprising a heavy chain variable region having at least 90% sequence identity to 5 and a light chain variable region having at least 90% identity to SEQ ID NO: 11.

In one embodiment, a type II PRMT inhibitor, and an anti-CTLA4 antibody or antigen binding fragment thereof, an anti-PD-1 antibody or antigen binding fragment thereof, anti-PDL1 antibody as a combination formulation for simultaneous, separate or sequential use in medicine Or an immunomodulator selected from an antigen binding fragment thereof and an anti-OX40 antibody or antigen binding fragment thereof. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one aspect, the type II PRMT inhibitor and immunomodulatory agent are administered to the patient simultaneously, sequentially, in any order, systemically, orally, intravenously and intratumorally. In one aspect, the type II PRMT inhibitor is administered orally. In one embodiment, a product is provided that contains Compound C and an agonist anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in medicine. In one embodiment there is provided a product containing Compound C and an anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in medicine, wherein the anti-OX40 antibody or antigen binding fragment thereof is one of Including: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as set forth in SEQ ID NO: 8 and / or CDRL3 as set forth in SEQ ID NO: 9 or a direct equivalent of each CDR wherein the direct equivalent has no more than two amino acid substitutions. In one embodiment, a product is provided containing a compound C and an anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in a medicament, wherein the anti-OX40 antibody or antigen binding fragment thereof is SEQ ID NO: A heavy chain variable region having at least 90% sequence identity to 5 and a light chain variable region having at least 90% identity to SEQ ID NO: 11.

In one embodiment, a product is provided comprising a Type II PRMT inhibitor, and an immunomodulator selected from the following as a combination formulation for simultaneous, separate or sequential use in medicine: an anti-CTLA4 antibody or antigen binding fragment thereof, anti-PD -1 antibody or antigen binding fragment thereof, anti-PDL1 antibody or antigen binding fragment thereof and anti-OX40 antibody or antigen binding fragment thereof. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one aspect, the type II PRMT inhibitor and immunomodulatory agent are administered to the patient simultaneously, sequentially, in any order, systemically, orally, intravenously and intratumorally. In one aspect, the type II PRMT inhibitor is administered orally. In one embodiment, a product is provided that contains Compound C and an agonist anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in medicine. In one embodiment there is provided a product containing Compound C and an anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in medicine, wherein the anti-OX40 antibody or antigen binding fragment thereof is one of Including: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as set forth in SEQ ID NO: 8 and / or a direct equivalent of CDRL3 or each CDR as set forth in SEQ ID NO: 9, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In one embodiment, a product is provided containing a compound C and an anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in a medicament, wherein the anti-OX40 antibody or antigen binding fragment thereof is SEQ ID NO: A heavy chain variable region having at least 90% sequence identity to 5 and a light chain variable region having at least 90% identity to SEQ ID NO: 11.

In one embodiment, a product is provided comprising a Type II PRMT inhibitor and an immunomodulator selected from the following as a combination formulation for simultaneous, separate or sequential use in the treatment of cancer in a human subject: an anti-CTLA4 antibody or antigen binding thereof Fragments, anti-PD-1 antibodies or antigen binding fragments thereof, anti-PDL1 antibodies or antigen binding fragments thereof and anti-OX40 antibodies or antigen binding fragments thereof. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one aspect, the type II PRMT inhibitor and immunomodulatory agent are administered to the patient simultaneously, sequentially, in any order, systemically, orally, intravenously and intratumorally. In one aspect, the type II PRMT inhibitor is administered orally. In one embodiment, a product containing Compound C and an agonist anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in the treatment of cancer in a human subject is provided. In one embodiment, a product containing Compound C and an anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in the treatment of cancer in a human subject is provided wherein the anti-OX40 antibody or antigen thereof Binding fragments comprise one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as set forth in SEQ ID NO: 8 and / or a direct equivalent of CDRL3 or each CDR as set forth in SEQ ID NO: 9, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In one embodiment, a product containing Compound C and an anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in the treatment of cancer in a human subject is provided wherein the anti-OX40 antibody or antigen thereof The binding fragment comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 5 and a light chain variable region having at least 90% identity to SEQ ID NO: 11.

In one embodiment, there is provided a product containing a Type II PRMT inhibitor and an immunomodulator selected from the following as a combination formulation for simultaneous, separate or sequential use in the treatment of cancer in a human subject, wherein the cancer is colon cancer or lymphoma : Anti-CTLA4 antibody or antigen binding fragment thereof, anti-PD-1 antibody or antigen binding fragment thereof, anti-PDL1 antibody or antigen binding fragment thereof and anti-OX40 antibody or antigen binding fragment thereof. In one aspect, the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. In one aspect, the immunomodulatory agent is an anti-PD-1 antibody or antigen binding fragment thereof. In one aspect, the anti-PD-1 antibody is pembrolizumab or nivolumab. In another aspect, the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof. In another aspect, an immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In another aspect, an immunomodulatory agent has a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 An anti-OX40 antibody or antigen binding fragment thereof comprising a sequence. In one aspect, the type II PRMT inhibitor is a compound of Formula III, IV, VII, VIII, IX, X or XI. In another aspect, the type II PRMT inhibitor is compound B. In one aspect, the type II PRMT inhibitor is compound C. In one aspect, the type II PRMT inhibitor and immunomodulatory agent are administered to the patient simultaneously, sequentially, in any order, systemically, orally, intravenously and intratumorally. In one aspect, the type II PRMT inhibitor is administered orally. In one embodiment, there is provided a product containing Compound C and an agonist anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in the treatment of cancer in a human subject, wherein the cancer is colon cancer or lymphoma to be. In one embodiment, there is provided a product containing Compound C and an anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in the treatment of cancer in a human subject, wherein the cancer is colon cancer or lymphoma, The anti-OX40 antibody or antigen binding fragment thereof comprises one or more of the following: CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR. In one embodiment is provided a product containing Compound C and an anti-OX40 antibody or antigen binding fragment thereof for simultaneous, separate or sequential use in the treatment of cancer in a human subject, wherein the cancer is colon cancer or lymphoma, and The -OX40 antibody or antigen binding fragment thereof comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 5 and a light chain variable region having at least 90% identity to SEQ ID NO: 11.

In one aspect of any of the embodiments herein, the cancer is a solid tumor or hematologic cancer. In one aspect, it is melanoma, breast cancer, lymphoma or bladder cancer.

In one aspect, the cancer may include head and neck cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, prostate cancer, glioma, glioblastoma, astrocytoma, glioblastoma multiforme, banyan-jona syndrome, Koden's disease, lermit-ducros disease, Inflammatory breast cancer, Wilms' tumor, Ewing's sarcoma, Rhabdomyosarcoma, Glioblastoma, Medulloblastoma, Kidney cancer, Liver cancer, Melanoma, Pancreatic cancer, Sarcoma, Osteosarcoma, Giant cell tumor of bone, Thyroid cancer, Lymphoblastic T cell leukemia, Chronic myeloma Leukemia, Chronic Lymphocytic Leukemia, Hairy-Cell Leukemia, Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, AML, Chronic Neutrophil Leukemia, Acute Lymphoblastic T Cell Leukemia, Plasmacoma, Immunoblastic Leukemia, Coat Cell Leukemia, multiple myeloma megaloblastic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, red leukemia, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, rim Maternal T-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urinary tract cancer, vulvar cancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular carcinoma, gastric cancer, nasopharyngeal cancer, isthmus cancer , Oral cancer, GIST (gastrointestinal stromal tumor) and testicular cancer.

In one aspect, the methods of the present invention further comprise administering at least one neoplastic agent to said human.

In one aspect, the human has a solid tumor. In one aspect, the tumor is selected from head and neck cancer, gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, non-small cell lung carcinoma, prostate cancer, colorectal cancer, ovarian cancer and pancreatic cancer. In another aspect humans have liquid tumors such as diffuse large B cell lymphoma (DLBCL), multiple myeloma, chronic lymphoblastic leukemia (CLL), follicular lymphoma, acute myeloid leukemia and chronic myeloid leukemia.

The present disclosure also relates to a method of treating or reducing the severity of a cancer selected from: brain cancer (gliomas), glioblastoma, banyanyan-jonana syndrome, Koden's disease, lermit-ducros disease, breast cancer , Inflammatory breast cancer, Wilms' tumor, Ewing's sarcoma, rhabdomyosarcoma, epidermoma, medulloblastoma, colon cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, osteosarcoma, bone giant Cell tumors, thyroid cancer, lymphocytic T-cell leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy-cell leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic neutrophil leukemia, acute lymphoblastic T-cell Leukemia, plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia, multiple myeloma megaloblastic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, Red Leukemia, Malignant Lymphoma, Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma, Lymphoblastic T Cell Lymphoma, Burkitt's Lymphoma, Follicle Lymphoma, Neuroblastoma, Bladder Cancer, Urinary Epithelial Cancer, Lung Cancer, Vulvar Cancer, Cervical Cancer, Endometrial Cancer, Renal cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharyngeal cancer, isthmus cancer, oral cancer, GIST (gastrointestinal stromal tumor) and testicular cancer.

As used herein, the term “treating” and its grammatical variations refers to a therapeutic regimen. With respect to a particular condition, treating means (1) ameliorates or prevents one or more of the biological signs of the condition, (2) (a) in the biological cascade causing or causing the condition. Interfere with one or more of the biological signs of one or more points or conditions, (3) alleviate one or more of the symptoms, effects, or side effects associated with the condition or treatment thereof, or (4) of the biological signs of the condition or condition Slow down one or more progress. Prophylactic therapy thereby is also contemplated. Those skilled in the art will appreciate that "prevention" is not an absolute term. In medicine, “prevention” is understood to refer to the prophylactic administration of a drug to substantially reduce the likelihood or severity of a condition or its biological manifestations, or to delay the onset of such a condition or its biological manifestations. For example, if the subject is considered to be at high risk of developing cancer, such as if the subject has a strong family history of cancer or if the subject has been exposed to carcinogens, prophylactic therapy is appropriate.

As used herein, the terms “cancer”, “neoplastic” and “tumor” are used interchangeably and refer to a cell that has undergone malignant transformation, in the singular or plural form, making the cell pathological to the host organism. Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, in particular histological examination. As used herein, the definition of cancer cells includes primary cancer cells, as well as any cells derived from cancer cell progenitors. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to the type of cancer that typically appears as a solid tumor, a "clinically detectable" tumor is based on a tumor mass, for example, computed tomography (CT) scans, magnetic resonance imaging (MRI), X-rays, Tumors that are detectable by procedures such as ultrasound or palpation upon physical examination and / or detectable due to the expression of one or more cancer-specific antigens in a sample obtainable from the patient. Tumors may be hematopoietic (or hematological or hematological or blood-related) cancers, for example cancers derived from blood cells or immune cells, which may be referred to as "liquid tumors". Specific examples of clinical conditions based on hematological tumors include leukemias such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; Plasma cell malignancies such as multiple myeloma, MGUS and Waldenstrom's macrobulinemia; Lymphomas such as non-Hodgkin's lymphoma, Hodgkin's lymphoma and the like.

The cancer may be any cancer that has an abnormal number of parental cells or unwanted cell proliferation or is diagnosed as a hematologic cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include acute myeloid (or myeloid or myeloid or myeloid) leukemia (undifferentiated or differentiated), acute promyelocytic (or promyelocytic or promyeloid or promyelocytic) leukemia, acute myeloid nucleus (or Osteoblastic) leukemia, acute mononuclear (or mononuclear) leukemia, red leukemia and megakaryocytic (or megakaryoblast) leukemia. These leukemias together may be referred to as acute myeloid (or myeloid or myeloid) leukemia (AML). Myeloid malignancies include, but are not limited to, chronic myeloid (or myeloid) leukemia (CML), chronic osteomyeloid leukemia (CMML), essential thrombocytopenia (or thrombocytopenia), and true polycythemia (PCV). Sex disorders (MPD) as well. Myeloid malignancies include myelodysplasia (or myelodysplastic syndromes or MDS), which may be referred to as refractory anemia (RA), hyperblast-associated refractory anemia (RAEB), and hyperblast-associated refractory anemia (RAEBT) during transformation; As well as unexplained myeloid metaplasia or unaccompanied myeloid fibrosis (MFS).

Hematopoietic cancers also include lymphoid malignancies, which can be affected by lymph nodes, spleen, bone marrow, peripheral blood and / or extranodal sites. Lymphoid cancers include B-cell malignancies, including but not limited to B-cell non-Hodgkin's lymphoma (B-NHL). B-NHL can be painless (or low grade), medium (or aggressive) or high grade (very aggressive). Painless B-cell lymphomas are follicular lymphomas (FL); Small lymphocytic lymphoma (SLL); MZL including nodular marginal lymphoma (MZL), extranodular MZL, spleen MZL, and spleen MZL with chorionic lymphocytes; Lymphoplasmacytic lymphoma (LPL); And mucosal-associated-lymphoid tissue (MALT or extra nodule margin) lymphoma. Medium grade B-NHL is associated with leukemia involvement or unaccompanied mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). Include. High grade B-NHL includes Burkitt's lymphoma (BL), Burkit-like lymphoma, undivided small cell lymphoma (SNCCL) and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytomas), primary exudative lymphoma, HIV-associated (or AIDS-related) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies include chronic lymphocytic leukemia (CLL), prelymphocytic leukemia (PLL), Waldenstrom's macrobulinemia (WM), hairy cell leukemia (HCL), giant granulocytes (LGL) leukemia, acute lymphoid (Or lymphocytic or lymphoblastic) leukemia, and Castleman's disease also include, but are not limited to. NHL is not specified otherwise (NOS) T-cell non-Hodgkin's lymphoma, peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmune lymphoid lymphoma (AILD), nasal natural T-cell non-Hodgkin's lymphoma (T-NHL) also includes, but is not limited to, killer (NK) cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T cell lymphoma, mycelial sarcoma, and Tridental syndrome. It may include.

Hematopoietic cancer is also typical of Hodgkin's lymphoma, including nodular sclerotic Hodgkin's lymphoma, mixed cytophilic Hodgkin's lymphoma, lymphocyte predominant (LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma and lymphocyte depleted Hodgkin's lymphoma. (Or disease). Hematopoietic cancers include plasma cell disease or cancer, such as MM, including asymptomatic multiple myeloma (MM), monoclonal gamma globulinopathy (MGUS) of unsigned (or unexplained or unclear), plasmacytoma (bone, extramedullary), Lymphoid cytoplasmic lymphoma (LPL), Waldenstrom's macrobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancer may also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes, and natural killer cells. Tissues comprising hematopoietic cells, referred to herein as "hematopoietic cell tissue," include bone marrow; Peripheral blood; Thymus; And peripheral lymphoid tissues such as the spleen, lymph nodes, lymphoid tissues associated with the mucous membranes (such as intestinal-associated lymphoid tissue), tonsils, fire plate and appendix, and lymphoid tissues associated with other mucous membranes, such as the bronchial lining do.

As used herein, the term “Compound A ² ” refers to an anti-PD-1 antibody or antigen binding fragment thereof, an anti-PDL1 antibody or antigen binding fragment thereof, an anti-CTLA4 antibody or antigen binding fragment thereof or an anti-OX40 antibody or antigen thereof. By immunomodulator selected from binding fragments. In some embodiments, Compound A ² is an anti-PD-1 antibody. Suitably Compound A ² may be selected from nivolumab and pembrolizumab. In some embodiments, Compound A ² comprises a V _H domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5; And an agonist antibody directed against OX40, or an antigen binding portion thereof, comprising a V _L domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11. In another embodiment, Compound A ² is an agonist antibody or antigen binding portion thereof directed against OX40, including an anti-OX40 antibody or antigen binding fragment thereof comprising one or more of the following: SEQ ID NO: CDRH1 as shown in 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR, wherein the direct equivalent is one having up to two amino acid substitutions in said CDR.

As used herein, the term “Compound B ² ” refers to a type II PRMT inhibitor. In some embodiments, Compound B ² is a compound of Formula III, IV, VII, VIII, IX, X or XI. Suitably compound B ² is compound C.

Suitably, the combinations of the present invention are administered within the "specified period".

As used herein, the term "specified time period" and its grammatical variants is meant the time between compound A and compound B ^{2 2} and one of the compound A ² B ^2, compounds of the administration of the others. Unless defined otherwise, specified periods may include simultaneous administration. Unless defined otherwise, the specified period refers to the administration of Compound A ² and Compound B ² during the day.

Suitably, if the compounds are administered within the "specified period" and not concurrently, they are both administered within about 24 hours with respect to each other-in this case, the specified period will be about 24 hours; Suitably they will both be administered within about 12 hours relative to each other—in this case, the specified period will be about 12 hours; Suitably they will both be administered within about 11 hours relative to each other—in this case, the specified period will be about 11 hours; Suitably they will both be administered within about 10 hours relative to each other—in this case, the specified period will be about 10 hours; Suitably they will both be administered within about 9 hours relative to each other—in this case, the specified period will be about 9 hours; Suitably they will both be administered within about 8 hours with respect to each other—in this case, the specified period will be about 8 hours; Suitably they will both be administered to each other within about 7 hours—in this case, the specified period will be about 7 hours; Suitably they will both be administered to each other within about 6 hours—in this case, the specified period will be about 6 hours; Suitably they will both be administered within about 5 hours of each other—in this case, the specified period will be about 5 hours; Suitably they will both be administered to each other within about 4 hours—in this case, the specified period will be about 4 hours; Suitably they will both be administered within about 3 hours relative to each other—in this case, the specified period will be about 3 hours; Suitably they will both be administered within about 2 hours relative to each other—in this case, the specified period will be about 2 hours; Suitably they will both be administered within about 1 hour with respect to each other—in this case, the specified period will be about 1 hour. As used herein, administration of Compound A ² and Compound B ² at intervals less than about 45 minutes is considered simultaneous administration.

Suitably, if a combination of the present invention is administered for a "specified period", the compound will be co-administered for a "durable period".

As used herein, the term “duration” and grammatical variations thereof means that both compounds of the invention are administered for the consecutive days indicated. Unless defined otherwise, consecutive days do not begin or end with the end of treatment, only the consecutive days are required to occur at some point during the course of treatment.

Regarding “specified period” administration:

Suitably, both compounds will be administered within the specified period for at least 1 day—in this case, the duration will be at least 1 day; Suitably, during the course of treatment, both compounds will be administered within a specified period for at least 3 consecutive days—in this case, the duration will be at least 3 days; Suitably during the course of treatment, both compounds will be administered within a specified period for at least 5 consecutive days—in this case, the duration will be at least 5 days; Suitably, during the course of treatment, both compounds will be administered within a specified period for at least 7 consecutive days—in this case, the duration will be at least 7 days; Suitably, during the course of treatment, both compounds will be administered within a specified period for at least 14 consecutive days—in this case, the duration will be at least 14 days; Suitably during the course of treatment, both compounds will be administered within a specified period for at least 30 consecutive days—in this case, the duration will be at least 30 days.

Suitably, if the compounds are not administered for a "specified period", they are administered sequentially. As used herein, the term “sequential administration” and its grammatical modifications refer to that one of Compound A ² and Compound B ² is administered once daily for at least two consecutive days, and the other of Compound A ² and Compound B ² is subsequently continuous It is meant to be administered once a day for two or more days. In addition, to use an Withdrawal between compound A and compound B ^{2 2} and one of the compound A ² B ^2, compounds of the other of sequential administration is contemplated herein. The Withdrawal used herein group would be a period of compound A ² and Compound B after a sequentially administration of the ^two, and the compound A ² and compound B ² compounds before other of the administration of A ² and compound B the number of days that are not administered anything ² which of . Suitably the holiday period is selected from one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, and fourteen days. It will be a term.

For sequential administration:

Suitably, one of Compound A ² and Compound B ² is administered for 1 to 30 days in a row, followed by an optional drug holiday, followed by another of Compound A ² and Compound B ² for 1 to 30 days in a row. . Suitably, one of Compound A ² and Compound B ² is administered for 1 to 21 days in a row, followed by an optional drug holiday, followed by another of Compound A ² and Compound B ² for 1 to 21 days in a row. . Suitably, one of Compound A ² and Compound B ² is administered for 1 to 14 consecutive days, followed by 1 to 14 days of drug holiday, followed by another of Compound A ² and Compound B ² for 1 to 14 consecutive days Is administered. Suitably, one of Compound A ² and Compound B ² is administered for 1 to 7 consecutive days, followed by 1-10 days of drug holiday, followed by another of Compound A ² and Compound B ² for 1-7 consecutive days Is administered.

Suitably, Compound B ² will be administered first in sequence, followed by an optional drug holiday, and then Compound A ² will be administered. Suitably, compound B ² is administered for 3 to 21 consecutive days, followed by an optional drug holiday, followed by compound A ² for 3 to 21 consecutive days. Suitably, compound B ² is administered for 3 to 21 days consecutively, followed by a drug holiday of 1 to 14 days, followed by compound A ² for 3 to 21 days continuous. Suitably, compound B ² is administered for 3 to 21 days consecutively, followed by a 3 to 14 day washout period, followed by compound A ² for 3 to 21 days continuous. Suitably, compound B ² is administered for 21 consecutive days, followed by an optional drug holiday, followed by compound A ² for 14 consecutive days. Suitably, Compound B ² is administered for 14 consecutive days, followed by a 1 to 14 day washout period, followed by Compound A ² for 14 consecutive days. Suitably, compound B ² is administered for 7 consecutive days, followed by a 3-10 day drug holiday, followed by compound A ² for 7 consecutive days. Suitably, Compound B ² is administered for 3 consecutive days, followed by a 3-14 day washout period, followed by Compound A ² for 7 consecutive days. Suitably, compound B ² is administered for 3 consecutive days, followed by a 3-10 day drug holiday, followed by compound A ² for 3 consecutive days.

It is understood that "specified period" administration and "sequential" administration may be followed by repeated administration or may be followed by a cross-dose protocol, followed by a drug holiday before the repeated or cross-dose protocol.

The methods of the present invention can also be used in conjunction with other therapeutic methods of cancer treatment.

Compound A ² and Compound B ² may be administered by any suitable route. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), intratumoral, vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural). It will be appreciated that the preferred route may vary depending, for example, on the condition of the recipient of the combination and the cancer to be treated. It will also be appreciated that each agent administered can be administered by the same or different routes and that Compound A ² and Compound B ² can be combined together in a pharmaceutical composition / formulation.

In one embodiment, one or more components of the combination of the present invention are administered intravenously. In one embodiment, at least one component of the combination of the present invention is administered orally. In another embodiment, at least one component of the combination of the present invention is administered intratumorally. In another embodiment, at least one component of the combination of the invention is administered systemically, for example intravenously, and at least one other component of the combination of the invention is administered intratumorally. For example, in any of the embodiments in this paragraph, the components of the invention are administered as one or more pharmaceutical compositions.

Typically, any anti-neoplastic agent that has activity against susceptible tumors to be treated can be co-administered in the treatment of cancer in the present invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by VT Devita, TS Lawrence, and SA Rosenberg (editors), 10 ^th edition (December 5, 2014), Lippincott Williams & Wilkins Publishers. One skilled in the art will be able to identify which combination of agents would be useful based on the particular characteristics of the drug and the cancer involved. Typical anti-neoplastic agents useful in the present invention include anti-microtubules or antimitotic agents such as diterpenoids and vinca alkaloids; Platinum coordination complex; Alkylating agents such as nitrogen mustards, oxazaphosphorins, alkylsulfonates, nitrosoureas, and triazenes; Antibiotics such as actinomycin, anthracycline, and bleomycin; Topoisomerase I inhibitors such as camptothecins; Topoisomerase II inhibitors such as epipodophyllotoxins; Anti metabolites such as purine and pyrimidine analogs and anti-folate compounds; Hormone and hormone analogs; Signaling pathway inhibitors; Non-receptor tyrosine kinase angiogenesis inhibitors; Immunotherapy agents; Apoptosis promoters; Cell cycle signaling inhibitors; Proteasome inhibitors; Heat shock protein inhibitors; Inhibitors of cancer metabolism; And cancer gene therapy agents such as genetically modified T cells.

An example of a further active ingredient or ingredients for use or co-administration in combination with the present method or combination is an anti-neoplastic agent. Examples of anti-neoplastic agents include chemotherapeutic agents; Immune-modulating agents; Immune-modulators; And immunostimulatory adjuvants.

Example

The following examples illustrate various non-limiting aspects of the present invention.

Example 1

Background technology

PRMT5 is a symmetric protein arginine methyltransferase.

Protein arginine methyltransferase (PRMT) is a subset of enzymes that methylate arginine in proteins containing regions rich in glycine and arginine residues (GAR motifs). PRMTs are categorized into four subtypes (type I-IV) based on the product of an enzymatic reaction (FIG. 1, Fisk JC, et al. A type III protein arginine methyltransferase from the protozoan parasite Trypanosoma brucei. J. Biol Chem. 2009 Apr 24; 284 (17): 11590-600)]. Type I-III enzymes produce ω-N-monomethyl-arginine (MMA). The largest subtype, Type I (PRMT1, 3, 4, 6 and 8), progresses MMA to asymmetric dimethyl arginine (ADMA), while type II produces symmetric dimethyl arginine (SDMA). While PRMT9 / FBXO11 can also produce SDMA, PRMT5 is the major enzyme responsible for symmetric dimethylation. PRMT5 functions as several types of complexes in the cytoplasm and nucleus and requires a binding partner of PRMT5 for substrate recognition and selectivity. Methylosomal protein 50 (MEP50) is a known cofactor of PRMT5 that is required for PRMT5 binding and activity on histones and other substrates (Ho MC, et al. Structure of the arginine methyltransferase PRMT5-MEP50 reveals a mechanism for substrate specificity PLoS One. 2013; 8 (2)).

PRMT5 substrate

PRMT5 methylates arginine in various cellular proteins including splicing factors, histones, transcription factors, kinases and the like (FIG. 2) (Karkhanis V, et al. Trends Biochem Sci. 2011 Dec; 36 (12): 633- 41). Methylation of multiple components of spliceosomes is a major event in spliceosome assembly, and attenuation of PRMT5 activity through knockdown or gene knockout leads to disruption of cell splicing (Bezzi M, et al. Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev. 2013 Sep 1; 27 (17): 1903-16). PRMT5 also methylates histone arginine residues (H3R8, H2AR3 and H4R3), and these histone marks are associated with transcriptional silencing of tumor suppressor genes such as RB and ST7 (Wang L, et al. Protein arginine methyltransferase 5 suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells.Mol Cell Biol. 2008 Oct; 28 (20): 6262-77; Pal S, et al. Low levels of miR-92b / 96 induce PRMT5 translation and H3R8 / H4R3 methylation in mantle cell lymphoma.EMBO J. 2007 Aug 8; 26 (15): 3558-69). Additionally, symmetric dimethylation of H2AR3 has been implicated in the silencing of differentiation genes in embryonic stem cells (Tee WW, et al. Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency.Genes Dev. 2010 Dec 15 24 (24): 2772-7). PRMT5 also plays a role in cell signaling through methylation of EGFR and PI3K (Hsu JM, et al. Crosstalk between Arg 1175 methylation and Tyr 1173 phosphorylation negatively modulates EGFR-mediated ERK activation. Nat Cell Biol. 2011 Feb; 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 5 is a potential oncoprotein that upregulates G1 cyclins / cyclin-dependent kinases and the phosphoinositide 3-kinase / AKT signaling cascade.Cancer Sci. 2012 Sep; 103 (9): 1640-50.). The role of PRMT5 in methylation of proteins involved in cancer-related pathways is described below.

PRMT5 knockout model

Complete loss of PRMT5 is embryonic lethality. PRMT5 plays an important role in embryonic development, evidenced by the fact that PRMT5-null mice die between 3.5 and 6.5 days of embryos (Tee WW, et al. Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency.Gennes Dev. 2010 Dec 15; 24 (24): 2772-7). Early studies suggest that PRMT5 plays an important role in HSC (hematopoietic stem cell) and NPC (neural progenitor cell) development. Knockdown of PRMT5 in human umbilical cord blood CD34 ⁺ cells induces increased red blood cell differentiation (Liu F, et al. JAK2V617F-mediatedphosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation. Cancer Cell. 2011 Feb 15; 19 (2): 283-94). In NPC, PRMT5 regulates neuronal differentiation, cell growth and survival (Bezzi M, et al. Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev. 2013 Sep 1; 27 (17): 1903-16).

PRMT5 in Cancer

Increasing evidence suggests that PRMT5 is involved in tumorigenesis. PRMT5 protein is overexpressed in many cancer types, including lymphoma, glioma, breast and lung cancer, and PRMT5 overexpression alone is sufficient to transform normal fibroblasts (Pal S, et al. Low levels of miR- 92b / 96 induce PRMT5 translation and H3R8 / H4R3 methylation in mantle cell lymphoma.EMBO J. 2007 Aug 8; 26 (15): 3558-69 .; Ibrahim R, et al. Expression of PRMT5 in lung adenocarcinoma and its significance in epithelial -mesenchymal transition.Hum Pathol. 2014 Jul; 45 (7): 1397-405; Powers MA, et al. Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4.Cancer Res. 2011 Aug 15 71 (16): 5579-87; Yan F, et al. Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma. Cancer Res. 2014 Mar 15; 74 (6): 1752-65). Knockdown of PRMT5 often leads to a decrease in cell growth and survival in cancer cell lines. In breast cancer, high PRMT5 expression, combined with high PDCD4 (programmed cell death 4) levels, predicts overall poor survival (Powers MA, et al. Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4. Cancer Res. 2011 Aug 15; 71 (16): 5579-87). PRMT5 methylates PDCD4, altering tumor-related function. Co-expression of PRMT5 and PDCD4 in the in situ model of breast cancer promotes tumor growth. High expression of PRMT5 in glioma is associated with high tumor grade and overall poor survival, while PRMT5 knockdown provides survival benefit in an allograft model (Yan F, et al. Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma.Cancer Res. 2014 Mar 15; 74 (6): 1752-65). Increased PRMT5 expression and activity contribute to the silencing of several tumor suppressor genes in glioma cell lines.

The strongest mechanism link currently described between PRMT5 and cancer is in mantle cell lymphoma (MCL). PRMT5 is frequently overexpressed in MCL and highly expressed in nuclear compartments, where it increases the level of histone methylation and silences a subset of tumor suppressor genes. Recent studies have revealed the role of miRNAs in the upregulation of PRMT5 expression in MCL. More than 50 miRNAs are expected to anneal to the 3 'untranslated region of PRMT5 mRNA. It has been reported that miR-92b and miR-96 levels are inversely correlated with PRMT5 levels in MCL and downregulation of these miRNAs in MCL cells results in upregulated PRMT5 protein levels. Cyclin D1, an oncogene translocated in the majority of MCL patients, associates with PRMT5 and increases PRMT5 activity through a cdk4-dependent mechanism (FIG. 3, Aggarwal P, et al. Cancer Cell. 2010 Oct 19; 18 ( 4): 329-40]). PRMT5 mediates the inhibition of key genes that negatively regulate DNA replication, enabling cyclin D1-dependent neoplasia growth. PRMT5 knockdown inhibits cyclin D1-dependent cell transformation resulting in death of tumor cells. These data highlight the important role of PRMT5 in MCL and suggest that PRMT5 inhibition can be used as a treatment strategy in MCL.

In other tumor types, PRMT5 has been assumed to play a role in differentiation, cell death, cell cycle progression, cell growth and proliferation. Although the main mechanisms that link PRMT5 to tumorigenesis are unknown, the neonatal data suggest that PRMT5 contributes to the regulation of gene expression (histone methylation, transcription factor binding or promoter binding), alteration of splicing, and signal transduction. . PRMT5 methylation of the transcription factor E2F1 decreases its ability to inhibit cell growth and promote apoptosis (Zheng S, et al. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1. Mol Cell. 2013 Oct 10; 52 ( 1): 37-51). PRMT5 also methylates p53 in response to DNA damage (Jansson M, et al. Arginine methylation regulates the p53 response. Nat Cell Biol. 2008 Dec; 10 (12): 1431-9), increasing p53-dependent apoptosis. Reduces the ability of p53 to induce cell cycle arrest. These data suggest that PRMT5 inhibition can sensitize cells to DNA damaging agents through induction of p53-dependent apoptosis.

In addition to directly methylating p53, PRMT5 upregulates the p53 pathway through a splicing-related mechanism. PRMT5 knockouts in mouse neural progenitor cells cause alterations in cell splicing, including isotype conversion of the MDM4 gene (Bezzi M, et al. Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.Genes Dev. 2013 Sep 1; 27 (17): 1903-16). PRMT5 knockout cells in Bezzi et al. Have reduced expression of the long MDM4 isotype (which produces functional p53 ubiquitin ligase) and increased expression of the short MDM4 isotype (which produces inactive ligase). Was found. These changes in MDM4 splicing lead to inactivation of MDM4, increased stability of the p53 protein, and subsequent activation of the p53 pathway and cell death. MDM4 alternative splicing was also observed in the PRMT5 knockdown cancer cell line. These data suggest that PRMT5 inhibition can activate multiple nodes of the p53 pathway.

In addition to regulating cancer cell growth and survival, PRMT5 is also involved in epithelial-mesenchymal transition (EMT). PRMT5 binds to the transcription factor SNAIL and acts as an important co-repressor of E-cadherin expression; Knockdown of PRMT5 causes upregulation of E-cadherin levels (Hou Z, et al. The LIM protein AJUBA recruits protein arginine methyltransferase 5 to mediate SNAIL-dependent transcriptional repression. Mol Cell Biol. 2008 May; 28 (10) : 3198-207).

These data highlight the role of PRMT5 as an important regulator of multiple cancer-related pathways and suggest that PRMT5 inhibitors can have a wide range of activity in heme and solid cancers. There is strong evidence for PRMT5 inhibitors as a therapeutic strategy in MCL as well as in breast and brain cancers. These data also highlight the mechanistic basis for the use of PRMT5 inhibitors for:

MCL에서 시클린 D1-의존성 기능을 억제함;

Inhibits cyclin D1-dependent function in MCL;

p53 경로 활성을 활성화 및 조정함;

activating and modulating p53 pathway activity;

E2F1-의존성 세포 성장 및 아폽토시스 기능을 조정함;

Modulating E2F1-dependent cell growth and apoptosis function;

E-카드헤린 발현을 탈억제함;

De-inhibiting E-cadherin expression;

Compound C is a potent, selective, peptide competitive, reversible PRMT5 / MEP50 complex of medium molecular weight (MW = 452.55) with good overall physical properties and oral bioavailability. Compound C affects several cancer related pathways, ultimately inducing potent anticancer activity in both in vitro and in vivo models, providing new therapeutic mechanisms for the treatment of MCL, breast cancer and brain cancer.

biochemistry

Compound C was profiled in a number of in vitro biochemical assays to characterize the potency, reversibility, selectivity, and mechanism of PRMT5 inhibition.

Inhibitory potency of Compound C was assessed using a radioactive assay measuring ³ H transfer from SAM to histone H4 derived peptides identified from histone peptide library screens. The long reaction time of 120 minutes was used to capture any time-dependent increase in potency. Compound C was found to be a potent inhibitor of PRMT5 / MEP50 with IC ₅₀ 8.7 ± 5 nM (n = 3). This potency is close to the dense-binding limit (2 nM) of the assay and thus represents an upper limit on the actual potency of the molecule (FIG. 4). The inhibitory potency was similar for the close analogues of Compound C, including Compound F, Compound B, and Compound E (with major differences on the left side of the molecule) used as tool compounds in some biological studies.

To assess Compound C's ability to inhibit PRMT5 dependent methylation of cell substrates other than histone H4, panels of PRMT5 substrates, including SmD3, Lsm4, hnRNPH1 and FUBP1, were assembled for evaluation (concomitant with splicing and transcriptional silencing) The majority of these substrates were found through the Cell MethylScan ™ study described in the Biology section below). Compound C effectively inhibited PRMT5 / MEP50 catalyzed methylation of both of these substrates, although the extremely low K _{m apparent} excluded the precise determination of potency.

To enable the interpretation of safety studies, the effect of Compound C was also evaluated on rat and dog orthologs of the PRMT5 / MEP50 complex under conditions similar to the human PRMT5 assay. Compound C potency varied <3 times for all species (Table 2).

Table 2. PRMT5 / MEP50 activity was monitored using a radioactivity assay under equilibrium conditions (substrate concentration in K _{m apparent} ), which measures the transfer of ³ H from SAM to protein substrate after treatment with Compound C. IC ₅₀ values were determined by fitting the data to a 3-parameter dose-response equation.

To determine the mechanism of the inhibitory and inhibitory binding mode, Compound C was co-crystallized with cinefungin, a PRMT5 / MEP50 complex and a natural product SAM analog (2.8 kPa resolution) (FIG. 5). Inhibitors usually bind in close proximity to the cinefungin occupying the SAM pocket and in the gap occupied by the substrate peptide. The aryl ring of tetrahydroisoquinoline appears to form a π-aryl lamination interaction with the amino group of cinefungin. The hydrogen bond is formed between the hydroxyl group of compound C and the Leu437 backbone and Glu244. Hydrogen bond interactions are also formed between the amide of the pyrimidine ring and the backbone NH group of Phe580. Terminal piperidine acetamide is present on the solvent exposed surface without obvious critical contact. Collectively, this structure supports a noncompetitive, temperamental and competitive suppression mechanism with SAM.

In order to determine whether Compound C is a reversible inhibitor of PRMT5 / MEP50 and further explore the inhibition mechanism, affinity selective mass spectrometry (ASMS) was used to measure the binding of Compound C to various PRMT5 / MEP50 complexes. Positive binding could be detected in the binary complex containing PRMT5 / MEP50 with SAM, cinefungin or SAH and for the dead-end tertiary complex of PRMT5 / MEP50: H4 peptide: SAH or cinefungin. Since ASMS will not be able to detect irreversibly bound Compound C, these results are consistent with the reversible binding mechanism. Upon competition with a 10-fold excess of H4 peptide, the binding of Compound C was reduced in the PRMT5 / MEP50: H4 peptide: cinefungin complex. Binding of compound C was not detected at all in the PRMT5 / MEP50: H4 peptide complex or PRMT5 / MEP50 alone, suggesting that the SAM binding pocket needs to be occupied for compound C binding. These results are best suited for inhibition mechanisms that are competitive with SAM and competitive with H4 peptides.

Selectivity of Compound C was assessed in a panel of enzymes including type I and type II PRMT and lysine methyltransferase (KMT). PRMT9 / FBXO11, another type II PRMT and the only PRMT lacking the THW loop, was not included due to the lack of functional enzyme assay. Compound C did not inhibit any of the 19 enzymes on the methyltransferase selectivity panel with IC ₅₀ values> 40 μM, which resulted in> 4000 fold selectivity for PRMT5 / MEP50 (FIG. 6). Also selectivity for PRMT5 / MEP50 over other methyltransferases was observed for the PRMT5 tool compound (Compound B, Compound F and Compound E) used in the Biology section herein.

In summary, Compound C is a potent, selective and reversible inhibitor of the PRMT5 / MEP50 complex with IC ₅₀ 8.7 ± 5 nM. The crystal structure and ASMS binding data of PRMT5 / MEP50 complexed with Compound C are consistent with the SAM non-competitive protein substrate competitive mechanism.

biology

summary

PRMT5 is overexpressed in many human cancers and implicated in multiple cancer-related pathways. There is strong evidence for the use of PRMT5 inhibitors as therapeutic strategies in MCL as well as breast and brain cancers. To understand the category of PRMT5 inhibitor antiproliferative activity, Compound C was profiled in various in vitro and in vivo tumor models using 2D and 3D growth assays.

The identity of genes and pathways affected by PRMT5 inhibition is important for understanding the mechanism of PRMT5 inhibitors required for indication prioritization, discovery of predictive biomarkers, and design of rational combinatorial studies. Several in vitro mechanism studies have been performed to evaluate the biology of the response to PRMT5 inhibition. Arginine methylation levels of multiple PRMT5 substrates were assessed to monitor Compound C activity against PRMT5 in cells and xenograft tumors. RNA-sequencing of multiple cell lines was performed to evaluate the effect of Compound C on gene expression, splicing, and other molecular mechanisms and pathways regulated by PRMT5 activity. P53 pathway activity was monitored in cell lines treated with PRMT5 inhibitors.

Finally, compound C activity was tested in several xenograft models of MCL and breast cancer to assess the efficacy of PRMT5 inhibition in preclinical cancer models and to evaluate the molecular mechanisms and potential biomarkers of the response.

Cell line sensitivity

To assess the antiproliferative activity of PRMT5 inhibition in various tumor types, Compound C was profiled in 2D and 3D in vitro assays using an extensive panel of cancer cell lines and patient-derived tumor models. First, compound C was evaluated in a panel of cancer cell lines in a 2D 6 day growth / kill assay (FIG. 7). Cell lines representing tumor types where PRMT5 activity has been reported to regulate key pathways and / or cell growth and survival (eg lymphoma and MCL, glioma, breast cancer and lung cancer cell lines) were selected. Overall, the majority of tested cell lines showed gIC ₅₀ values of less than 1 μM, while the most sensitive lymphoma cell lines (mantle cell lymphoma and diffuse versus B-cell lymphoma cell lines) had gIC ₅₀ values in the single digit nM range.

Compound C induces cytotoxic responses in a subset of diffuse versus B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), glioblastoma, breast cancer and bladder cancer cell lines at concentrations above 100 nM in a 6-day growth / kill assay (FIG. 8, negative Y _min -T0 value). Overall, MCL and DLBLC cell lines showed the strongest cytotoxic response. The majority of breast cancer cell lines had low Y _min -T0 values, suggesting that PRMT5 inhibition leads to full growth inhibition in the breast cancer model, while the remaining cell lines showed partial cytostatic response (positive Y _min- T0 value).

Antiproliferative activity of PRMT5 inhibition was further tested on a large cancer cell line screen (240 cell lines, 10-day 2D growth assay) performed with the PRMT5 tool molecule (Biochemistry of Compound C and Compound B in FIG. 9, FIG. 4). Red / cell activity comparison). Overall, the majority of cell lines showed gIC ₅₀ values lower than 1 μM. Tumor types with median gIC ₅₀ <100 nM include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), Hodgkin's lymphoma (HL), multiple myeloma (MM), breast, glioma, kidney, melanoma and ovarian cancer It was. These data suggest that PRMT5 inhibitors exhibit a broad range of antiproliferative activity against various heme and solid tumor types.

A similar broad range of anti-growth effects were observed by the PRMT5 tool compound in a panel of patient-derived tumor models and cell lines (n = 73) in the soft agar 3D colony formation assay (FIG. 10). Relative growth IC ₅₀ values of less than 1 μM were observed in 37% of models including tumors of non-small cell lung cancer (NSCLC), breast, melanoma, colon and glioma. Tumor types with the lowest median IC ₅₀ value were large cell lung cancer, breast, kidney and glioma.

Overall, these data demonstrate that PRMT5 inhibitors have potent antiproliferative activity in various solid and blood cancer models. The following indications were selected for further investigation based on the observed activity in the study, literature hypothesis and potential for clinical development:

MCL 및 DLBCL (PRMT5 억제에 대한 강력한 항증식성 및 세포독성 반응)

MCL and DLBCL (potent antiproliferative and cytotoxic responses to PRMT5 inhibition)

유방암 (세포주에서 낮은 gIC₅₀ 값 및 완전한 성장 억제 및 환자-유래 모델의 패널에서의 콜로니 형성 검정에서 낮은 IC₅₀ 값)

Breast cancer (cell line ₅₀ from the low gIC value and complete growth inhibition and patient-low IC ₅₀ value in the assay of colony formation in the derived model panel)

교모세포종 (콜로니 형성 검정에서 낮은 IC₅₀ 값).

Glioblastoma (low IC ₅₀ value in colony formation assay).

Lymphoma Biology

As mentioned above, Compound C elicited a potent cytotoxic response in mantle cells and a subset of diffuse large B-cell lymphoma cell lines (FIGS. 7-8). Since PRMT5 is frequently overexpressed in MCL and plays an important role in the MCL pathways (such as Cyclin D1 and p53), compound C activity and mechanisms have been evaluated in several cellular mechanism studies. Compound C efficacy was evaluated in two xenograft models of mantle cell lymphoma.

Cell Mechanism Data (Lymphoma)

SDMA Suppression

PRMT5 is responsible for the majority of cell symmetric arginine dimethylation. To better understand the biological mechanisms that correlate PRMT5 inhibition to the anticancer phenotype, substrates were identified using SDMA antibodies that recognize a subset of symmetrically dimethylated cellular proteins at arginine residues. The identity of the protein detected by the SDMA antibody was determined by immunoprecipitation and mass-spectrometry analysis (Methylscan ™) using SDMA antibody in Z138 cell lysate (from control and PRMT5 inhibitor treated cells). Among the SDMA containing proteins, the majority are factors involved in cell splicing and RNA processing (SmB, Lsm4, hnRNPH1, etc.), transcription (FUBP1) and translation, which highlights the role of PRMT5 as an important regulator of cellular RNA homeostasis. .

SDMA antibodies were then used in Western and ELISA assays to determine compound C dependency inhibition of methylation. First, Z138 MCL cells (Compound C gIC ₅₀ 2.7 nM, gIC ₉₅ 82 nM and gIC ₁₀₀ 880 nM, cytotoxic response in 6-day growth / kill assay, FIG. 7-8) were treated with increasing concentrations of Compound C. On day 1 and day 3 the cellular IC ₅₀ of SDMA inhibition was determined (FIG. 11). SDMA ELISA revealed a time-dependent change in SDMA levels with IC ₅₀ values of 4.79 nM on day 3 and EC ₅₀ on day 1 and day 7.3 and 2.35, respectively (FIG. 11, Panel A). Complete inhibition of SDMA was observed at concentrations above 19 nM (EC ₉₀ ) on day 3. Complete growth inhibition in Z138 cells was observed at gIC ₉₅ (82 nM) to gIC ₁₀₀ (880 nM), concentrations above EC ₉₀ of SDMA inhibition (in a 6-day growth / kill assay). These data suggest that PRMT5 activity needs to be> 90% inhibited in order to trigger full growth inhibition and cytotoxicity in Z138 cells.

To assess whether inhibition of SDMA levels predicts cell growth responses to Compound C, SDMA IC ₅₀ values were determined in a panel of MCL cell lines. SDMA IC ₅₀ values were in the range of 0.3-14 nM in panels of five MCL cell lines (FIG. 11, Panel B) (sensitive Z138, Granta-519, Maver-1 and moderately resistant Mino, and Jeko-1). 7-8), suggesting that SDMA is not a response marker but a marker of PRMT5 activity that can be used to monitor PRMT5 inhibition in a susceptibility and resistance model.

Gene Expression Profiling of Lymphoma Cell Lines

PRMT5 methylates histones and proteins involved in RNA processing, so PRMT5 inhibition is expected to have a significant effect on cellular mRNA homeostasis. To further decipher the cellular mechanisms that are regulated by PRMT5 and contribute to the cellular response to PRMT5 inhibitors, global gene expression changes were evaluated in lymphoma models susceptible to PRMT5 inhibition. To explain the gene expression changes occurring in lymphoma cell lines upon treatment with PRMT5 inhibitors, four susceptible lymphoma cell lines (two MCL cell lines-Z138 and Granta-519 and two DLBCL cell lines-DOHH2 and RL) were RNA-sequenced. Profiled by analysis.

First, gene expression changes were evaluated in lymphoma cell lines treated with increasing concentrations of PRMT5 tool molecules for 2 and 4 days (FIG. 12). The effect on RNA expression was time- and dose-dependent, with 48 genes commonly regulated across four lymphoma cell lines. These data demonstrate that PRMT5 inhibition triggers expression changes in hundreds of genes, and a subset of these changes is common for all four sensitive lymphoma cell lines tested. The relevance of these genes in the mechanism of cellular response to PRMT5 inhibition is being evaluated.

SDMA and Gene Expression Changes

To confirm gene expression changes found by RNA-seq experiments, qPCR analysis of the expression of a subset of genes was performed (genes with robust changes and genes involved in the p53 pathway). Z138 cells were treated with increasing doses of Compound C for 2 and 4 days, RNA was isolated and analyzed by qPCR. 13 shows a representative dose-response curve in the left panel and summarizes gene expression EC ₅₀ values (day 4) in the right panel. Overall, all 11 genes tested showed time- and dose-dependent expression changes, EC ₅₀ values ranged from 22 to 332 nM and median gene expression EC ₅₀ was 212 nM. Importantly, the median EC ₅₀ value of gene expression corresponds to Compound C concentration that causes maximum inhibition of cell methylation at Z138 (as measured by SDMA antibody ELISA, FIG. 11), indicating that almost complete inhibition of PRMT5 activity is due to genes. Suggests that it is required to establish a change in the expression program. These data highlight the association between the degree of PRMT5 inhibition and changes in gene expression and growth phenotype, both of which require nearly complete inhibition of PRMT5 activity.

PRMT5 Suppression and Splicing

The effects of PRMT5 inhibition on cell splicing were studied because PRMT5 methylated the spliceosome subunit and PRMT5 inhibition attenuated arginine methylation of many proteins involved in splicing. Changes in RNA splicing were evaluated in the lymphoma RNA-seq dataset described above.

There are several molecular mechanisms by which cell splicing can be regulated (FIG. 14, Panel A), where maintenance of introns (B) typically results in changes in gene expression, while exon skipping or alternative splice sites Use induces isotype conversion (A, CE). PRMT5 tool compound treatment resulted in a dose- and time-dependent increase in intron maintenance in all lymphoma cell lines tested (FIG. 14, Panel B). Interestingly, splicing factor map analysis enriches in introns where a subset of splicing factor binding sites is maintained across all four cell lines, including hnRNPH1 (methylated directly by PRMT5), hnRNPF, SRSF1, and SRSF5. This suggests that the effect of PRMT5 on cell splicing may depend on the methylation of multiple components (Sm and hnRNP proteins) of the spliceosome apparatus. PRMT5 inhibition also induced isotype conversion (alternative splicing) in lymphoma cell lines (FIG. 15, Panel A), and 34 genes showed consistent alternative splicing changes across all cell lines tested (FIG. 15, panels B and C).

Overall, changes in splicing of hundreds of genes have been observed, highlighting that the PRMT5 effect on splicing is specific to a limited number of RNAs rather than overall. One possible explanation for this specificity may be that PRMT5 directly modulates RNA binding of certain splicing factors such as hnRNPH1. The role of alternative splicing changes in the mechanism of action of PRMT5 inhibitors is under investigation, and one particular example is discussed in the sections below.

MDM4 Splicing and Activation of the p53 Pathway

PRMT5 knockout or knockdown has been reported to lead to MDM4 isotype conversion, which leads to inactivation of MDM4 ubiquitin ligase activity against p53 (described in the Background section). PRMT5 inhibition induced activation of the p53 pathway in four lymphoma cell lines tested in RNA-seq experiments (GSEA). To understand whether p53 activation is associated with MDM4 isotype conversion, MDM4 alternative splicing was analyzed. MDM4 isotype conversion was observed in all four lymphoma cell lines. Next, changes in MDM4 splicing were confirmed in panels of four MCL cell lines by RT-PCR (FIG. 16, Panels A, Z138, JVM-2 and MAVER-1 MCL cell lines are sensitive to Compound C). Whereas REC-1 is the most resistant MCL cell line). In Z138 and JVM-2 cells (both p53 wild type), Compound C induced MDM4 isotype conversion. In MAVER-1 and REC-1 cells (both p53 mutants), the basal expression of the MDM4 long form was low / undetectable and therefore MDM4 isotype conversion could not be detected. Subsequently, p53 and p21 (or pK target gene, CDKN1A) protein expression increased in JVM-2 and Z138 cells (FIG. 16, Panel B). Importantly, in Z138 cells, 200 nM Compound C and 5 μM MDM2 inhibitor (Nutlin-3) treatment increased p53 and p21 expression to similar levels. These data suggest that PRMT5 inhibition regulates MDM4 splicing in cell lines expressing high levels of MDM4 long isotype and induces p53 pathway activity in p53 wild type cell lines. The role of the p53 pathway in the biology of the response of p53 wild-type MCL cells to PRMT5 inhibition is being evaluated.

In addition, the dose-response of changes in MDM4 splicing, SDMA inhibition and p53 expression in Z138 cells treated with increasing concentrations of Compound C were evaluated to assess the relationship between PRMT5 inhibition, MDM4 splicing and p53 activation ( Figure 17, Panels A and B). SDMA levels were undetectable in compounds C at concentrations above 8 nM by Western blot. At the same time, changes in MDM4 splicing and p53 / p21 protein expression in Compound C at concentrations above 8 nM were evident. These results suggest that PRMT5 activity needs to be substantially inhibited (SDMA levels not detectable by Western) before changes in gene splicing and subsequent pathway activity occur (MDM4 / p53 / p21). do.

These data suggest that PRMT5 inhibition activates wild type p53 through the regulation of MDM4 splicing. This mechanism may be useful in cancer types where p53 is not frequently mutated, such as heme and pediatric malignancies. In lymphoma models, PRMT5 inhibition leads to significant (GSEA analysis) and relatively rapid activation of the p53 pathway, which is likely to contribute to the growth / killing phenotype observed in cell lines treated with PRMT5 inhibitors. Knockdown / rescue experiments will be used to further assess the role of the MDM4 / p53 pathway in PRMT5 inhibitor induced cell responses.

MDM4 isotype expression and p53 mutations are potential predictive biomarkers of response to PRMT5 inhibition in MCL. In the panel of MCL cell lines, the only two wild type p53 cell lines, Z138 and JVM-2, were the most sensitive cell lines (the only two MCL cell lines showing cytotoxicity in the lowest gIC ₅₀ value and 6-day growth / kill assays). In both cell lines, Compound C treatment induced MDM4 isotype conversion and p53 pathway activation. We exclude further evaluation of the p53 predictive biomarker hypothesis due to the limited number of MCL cell lines and the extremely low success rate of establishing the primary MCL model. The p53 pathway may be important for the biology of the response of p53 wild-type cells to PRMT5 inhibitors, but our data indicate that PRMT5 inhibition causes antiproliferative effects in the absence of functional p53 (eg Maver-1 cell line). There is a strong emphasis on the importance of other pathways that can also drive efficacy.

Mantle Cell Lymphoma: Comparison and Combination Activity of Compound C with Ibrutinib.

Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, has recently been approved for use in MCL with an unprecedented overall response rate of nearly 70 percent in relapse / refractory conditions (Wang ML, et al. N Engl J Med. 2013 Aug 8; 369 (6): 507-16). However, the majority of patients treated with ibrutinib do not achieve complete remission and the median progression-free survival is approximately 14 months. To understand whether Compound C can be used for ibrutinib resistant MCL, Compound C and ibrutinib susceptibility were evaluated in a 6-day growth / kill assay (FIG. 18, Panel A). Cell lines with low Compound C gIC ₅₀ values (Z-138, Maver-1 and JVM-2) are resistant to ibrutinib, while ibrutinib sensitive cell lines (Mino, Jeko-1) are only intermediate to Compound C. It is susceptible to the extent (FIG. 18, Panel A). These data suggest that the activity profiles of ibrutinib and Compound C do not overlap and the ibrutinib resistant MCL model is sensitive to PRMT5 inhibition. In addition, the combination of the PRMT5 inhibitor and ibrutinib showed synergistic antiproliferative activity in the majority of the tested MCL cell lines (combination index (CI) <1) (FIG. 18, panels B and C), which are two compounds. Suggests that the combination of may provide increased therapeutic benefit. These data indicate that PRMT5 inhibitors can be used in an ibrutinib resistant MCL patient population and that combinations of PRMT5 inhibitors and ibrutinib can be explored in both ibrutinib refractory and sensitive situations.

Efficacy in Mantle Cell Lymphoma Model

To test whether the efficacy observed in in vitro growth / kill assays in lymphoma cell line models applies to in vivo situations, compound C efficacy studies were conducted in xenograft models of mantle cell lymphomas (sensitive Z138 and Maver-1 cell lines). Was performed. First, the therapeutic effect of Compound C treatment on tumor growth was tested in a 21-day efficacy study in a Z-138 MCL xenograft model. Tumors in all Compound C dose groups ranged from at least 40% TGI in the lowest dose group (25 mg / kg BID) to as high as> 90% in the upper 100 mg / kg BID dose group compared to the vehicle sample. Significant differences in body weight and volume were shown (no weight loss was observed in all groups in all efficacy studies presented, FIG. 19, panel A). PD analysis of tumors with SDMA Western showed that all dose groups had a reduction of more than 70% of the methyl mark ranging from as high as> 98% in the upper dose group (FIG. 19, Panel B).

Next, the efficacy of Compound C was evaluated in the Maver-1 MCL xenograft model (FIG. 20). Tumors in all Compound C dose groups measured on day 18 were significant in volume ranging from minimum 50% TGI in the lowest dose group to as high as> 90% in the upper dose group compared to the vehicle sample. The difference was shown. PD analysis of tumors using SDMA showed that all dose groups had an 80-95% reduction in methyl marks.

These data demonstrate that Compound C treatment results in significant tumor growth inhibition (close to 100% TGI) in xenograft models of mantle cell lymphoma. Nearly complete inhibition of SDMA signal (> 90%) appeared to be required for maximum TGI (> 90%), suggesting that PRMT5 activity needs to be> 90% inhibited to obtain significant efficacy in tumors.

Lymphoma Biology Summary

PRMT5와 암 사이에 현재 설명된 가장 강력한 메카니즘 연관성은 MCL에서이다. PRMT5는 MCL에서 빈번하게 과다발현되고, 핵 구획에서 고도로 발현되며, 여기서 그것은 히스톤 메틸화의 수준을 증가시키고, 종양 억제 유전자의 하위세트를 침묵시킨다. 중요하게는, 대다수의 MCL 환자에서 전위되는 종양유전자인 시클린 D1은 PRMT5와 회합하고 cdk4-의존성 메카니즘을 통해 PRMT5 활성을 증가시킨다. PRMT5는 DNA 복제를 음성 조절하는 주요 유전자의 억제를 매개하여 시클린 D1-의존성 신생물 성장을 가능하게 한다. PRMT5 녹다운은 시클린 D1-의존성 세포 형질전환을 억제하여 종양 세포의 사멸을 야기한다. 이들 데이터는 MCL에서의 PRMT5의 중요한 역할을 강조하고, PRMT5 억제가 MCL에서의 치료 전략으로서 사용될 수 있다는 것을 시사한다.

The strongest mechanism association currently described between PRMT5 and cancer is in the MCL. PRMT5 is frequently overexpressed in MCL and highly expressed in nuclear compartments, where it increases the level of histone methylation and silences a subset of tumor suppressor genes. Importantly, cyclin D1, an oncogene translocated in the majority of MCL patients, associates with PRMT5 and increases PRMT5 activity through a cdk4-dependent mechanism. PRMT5 mediates the inhibition of key genes that negatively regulate DNA replication, enabling cyclin D1-dependent neoplasia growth. PRMT5 knockdown inhibits cyclin D1-dependent cell transformation resulting in death of tumor cells. These data highlight the important role of PRMT5 in MCL and suggest that PRMT5 inhibition can be used as a treatment strategy in MCL.

화합물 C는 현재까지 시험된 (6-일 성장/사멸 검정에서) 가장 감수성인 세포주에 속하는 MCL 세포주에서 성장을 억제하고 사멸을 유도한다. 시험된 MCL 세포주의 패널에서, 3종의 세포주는 gIC₅₀ <10 nM을 가졌고, 2종의 세포주는 gIC₅₀ ≤100 nM을 나타냈고, 1종의 세포주는 gIC₅₀ >1 μM을 가졌다. PRMT5 및 시클린 D1의 하류 표적에 대한 화합물 C 효과는 그것이 항성장 및 아폽토시스촉진 반응에 기여하는지 여부를 평가하기 위해 현재 조사 중이다.

Compound C inhibits growth and induces death in MCL cell lines belonging to the most sensitive cell lines tested to date (in the 6-day growth / kill assays). In the panel of the tested MCL cell lines of the three cell lines gIC ₅₀ <had a 10 nM, of two cell lines showed a gIC ₅₀ ≤100 nM, one type of cell line is gIC _50> had a 1 μM. Compound C effects on downstream targets of PRMT5 and cyclin D1 are currently being investigated to assess whether it contributes to anti-growth and apoptosis responses.

MCL 세포주에서 PRMT5 기질을 평가하기 위해 SDMA 항체 메틸스캔™을 사용하였다. 대다수의 SDMA 함유 단백질은 세포 스플라이싱 및 RNA 프로세싱 (SmB, Lsm4, hnRNPH1 등), 전사 (FUBP1) 및 번역에 수반되는 인자이며, 이는 세포 RNA 항상성의 중요한 조절인자로서 PRMT5의 역할을 강조한다. SDMA 항체를 추가로 MCL 세포주의 패널에서 PRMT5 억제를 평가하는데 사용하였고, 여기서 SDMA IC₅₀ 값은 감수성 및 저항성 모델에서 유사하였으며, 이는 SDMA가 반응의 마커가 아니라 PRMT5 억제의 마커라는 것을 시사한다.

SDMA Antibody Methylscan ™ was used to evaluate PRMT5 substrates in MCL cell lines. The majority of SDMA containing proteins are factors involved in cell splicing and RNA processing (SmB, Lsm4, hnRNPH1, etc.), transcription (FUBP1) and translation, highlighting the role of PRMT5 as an important regulator of cellular RNA homeostasis. SDMA antibodies were further used to assess PRMT5 inhibition in a panel of MCL cell lines, where SDMA IC ₅₀ values were similar in the sensitivity and resistance model, suggesting that SDMA is not a marker of response but a marker of PRMT5 inhibition.

화합물 C 처리는 RNA의 하위세트에서 스플라이싱 변화를 유도하였고, 특히 MDM4 이소형 전환이 MCL 및 DLBCL 세포주에서 관찰되었으며, 이는 PRMT5 억제가 MDM4 스플라이싱의 조절을 통해 p53 경로를 활성화시킨다는 것을 시사한다. 녹다운/구출 실험을 사용하여 PRMT5 억제제 유도된 세포 반응에서 MDM4/p53 경로의 역할을 추가로 평가할 것이다.

Compound C treatment induced splicing changes in a subset of RNA, in particular MDM4 isotype conversion was observed in MCL and DLBCL cell lines, suggesting that PRMT5 inhibition activates the p53 pathway through regulation of MDM4 splicing do. Knockdown / rescue experiments will be used to further assess the role of the MDM4 / p53 pathway in PRMT5 inhibitor induced cell responses.

MDM4 이소형 발현 및 p53 돌연변이는 MCL에서 PRMT5 억제에 대한 반응의 잠재적 예측 바이오마커이다. MCL 세포주 패널에서, 2종의 야생형 p53 세포주인 Z138 및 JVM-2가 가장 감수성인 세포주이었다 (최저 gIC₅₀ 값 및 6-일 성장/사멸 검정에서 세포독성을 나타내는 유일한 2종의 MCL 세포주).

MDM4 isotype expression and p53 mutations are potential predictive biomarkers of response to PRMT5 inhibition in MCL. In the panel of MCL cells, the two wild type p53 cell lines, Z138 and JVM-2, were the most sensitive cell lines (the only two MCL cell lines showing cytotoxicity in the lowest gIC ₅₀ value and 6-day growth / kill assays).

최근 몇년간, 이브루티닙의 임상 탐구는 MCL 치료에 대한 접근법을 극적으로 변화시켰다. 시험관내 데이터는 PRMT5 억제제가 이브루티닙 저항성 MCL 환자 집단에서 사용될 수 있다는 것 및 PRMT5 억제제와 이브루티닙의 조합물이 이브루티닙 불응성 및 감수성 상황 둘 다에서 탐구될 수 있다는 것을 나타낸다.

In recent years, Ibrutinib's clinical investigation has dramatically changed its approach to MCL treatment. In vitro data indicate that the PRMT5 inhibitor can be used in an ibrutinib resistant MCL patient population and that a combination of PRMT5 inhibitor and ibrutinib can be explored in both ibrutinib refractory and susceptible situations.

생체내 연구는 외투 세포 림프종의 이종이식 모델에서 화합물 C 처리가 유의한 종양 성장 억제 (100% TGI에 근접함)를 가져온다는 것을 입증한다. 종양에서 최대 효능 (TGI >90%)을 얻기 위해서는, PRMT5 활성의 거의 완전한 억제 (>90%)가 요구되는 것으로 보인다.

In vivo studies demonstrate that Compound C treatment results in significant tumor growth inhibition (close to 100% TGI) in xenograft models of mantle cell lymphoma. In order to achieve maximum efficacy (TGI> 90%) in tumors, it appears that almost complete inhibition of PRMT5 activity (> 90%) is required.

Breast cancer biology

Cell line screening data demonstrates that breast cancer cell lines are susceptible to PRMT5 inhibition and exhibit nearly complete growth inhibition in a 2D 6-day growth / kill assay (low Y _min- T0, FIGS. 7-9). Additionally, data from colony formation assays in panels of patient-derived (PDX) tumor models suggested that breast tumors belonged to the most sensitive tumors in the panel (based on Compound E relative IC ₅₀ values, FIG. 10). . Thus, breast cancer cell lines were evaluated in several growth / death and mechanism studies to assess the role and therapeutic potential of PRMT5 inhibition in breast cancer.

To understand PRMT5 inhibitor activity across different breast tumor subtypes, a panel of breast cancer cell lines was profiled in a 7-day growth assay using the PRMT5 tool compound (FIG. 21). PRMT5 inhibition attenuates cell growth with low IC ₅₀ values across the various subtypes of breast cancer cell lines tested. Median IC ₅₀ values were lowest in TNBC (triple negative breast cancer) cell lines compared to HER2 or hormone receptor (HR) positive cell lines.

In the 6-day growth / kill assay, the majority of breast cancer cell lines showed cytostatic effects. To assess whether long-term exposure to Compound C will affect the cytostatic versus cytotoxic properties of the response, the PRMT5 inhibitor was evaluated in a long-term growth / kill assay (FIG. 22). In SKBR3, MDA-MB-468 and MCF-7 cells, treatment with Compound C (as well as tool molecular compound B) induced a cytotoxic response upon prolonged exposure to the compound (7-10 days). In ZR-75-1 cells, the PRMT5 inhibitor triggered a cytostatic response at all time points (Days 3-12), while Z-138 (MCL, included as a control) cells at all time points in the assay (Day 3-day 10) showed significant total net cell death. These data suggest that PRMT5 inhibition induces net cell death (cytotoxic response) upon longer exposure (> 5 days) in a subset of breast cancer cell lines.

To test whether Compound C effects on cell growth were associated with changes in cell cycle distribution, propidium iodide FACS (fluorescence activated cell sorting) analysis was used to assess the effect of Compound C on the cell cycle. (Figure 23). Overall, FACS results are consistent with long-term proliferation data, indicating that long-term Compound C treatment in 3 of 4 breast cancer cell lines resulted in induction of cell death (increasing <2N) after 7-10 days of treatment. Prove it. In MCF-7 cells (p53 wild type), Compound C treatment induced the accumulation of cells of phase G1 (2N) and loss of cells from phase S (> 2N and <4N) on day 2 Cell death was followed on day 10 as evidenced by the accumulation of cells in sub-G1 phase (<2N). In ZR-75-1 cells (p53 wild type), Compound C had a minor effect on cell cycle distribution, where there was a decrease in G1 (2N) and an increase in> 4N cell fraction on days 7 and 10. MDA-MB-468 and SKBR-3 cell lines responded similarly to Compound C treatment with a decrease in G1 (2N) phase (day 7 or 10), an increase in G2 / M (4N), and> 4N DNA content This coincided with the accumulation of cells of subG1 (<2N), indicating cell death. These data suggest that PRMT5 inhibition affects the distribution of cells in the cell cycle and that phenotypic results depend on the cellular context.

To assess whether PRMT5 activity was equally inhibited in susceptible and resistant breast cancer cell lines, the level of SDMA in the cells after PRMT5 inhibitor treatment was measured (FIG. 24). Overall, the timing of SDMA reduction was similar for all cell lines tested (sensitive and resistant). Maximum inhibition of SDMA was observed on day 3. SDMA IC ₅₀ in MDA-MB-468 cells was 5.4 nM, which was similar to SDMA IC ₅₀ in Z138 cells. These data indicate that SDMA is a marker of PRMT5 catalytic activity and does not predict antiproliferative responses to PRMT5 inhibition. SDMA IC ₅₀ values are further evaluated in a panel of breast cancer cell lines.

Efficacy in an In Vivo Breast Cancer Model

Next, the efficacy of PRMT5 inhibition was evaluated in an in vivo model of breast cancer. First, MDA-MB-468, a triple negative breast cancer xenograft model, was treated with 100 mg / kg (QD and BID) and 200 mg / kg (QD) of Compound C (FIG. 25). Maximum tumor growth inhibition (TGI = 83%) was observed in the 100 mg / kg BID treated group, where SDMA inhibition exceeded 90%, whereas Compound C treatment was not effective in 100 mg / kg QD treated animals. , SDMA inhibition was less than 80%. These data suggest that SDMA levels need to be almost completely suppressed (> 90%) to identify significant TGI in an in vivo breast cancer xenograft model.

Breast Cancer Summary

유방암에서, 높은 PRMT5 발현 및 높은 PDCD4 (프로그램화된 세포 사멸 4) 수준은 전체적으로 불량한 생존을 예측한다.

In breast cancer, high PRMT5 expression and high PDCD4 (programmed cell death 4) levels overall predict poor survival.

유방암 세포주 및 유방암 환자-유래 모델은 2D 성장/사멸 및 콜로니 형성 검정에서 시험된 가장 감수성인 모델에 속하였다.

Breast cancer cell lines and breast cancer patient-derived models were among the most sensitive models tested in 2D growth / death and colony formation assays.

화합물 C 처리는 6-일 성장/사멸 검정에서 완전한 성장 억제를 유발하였고, PRMT5 억제제에 대한 장기간 노출은 시험된 4종의 세포주 중 3종에서 세포 사멸을 유도하였다.

Compound C treatment resulted in complete growth inhibition in the 6-day growth / kill assay, and prolonged exposure to the PRMT5 inhibitor induced cell death in three of the four cell lines tested.

7-일 증식 검정에서, TNBC 세포주는 Her2 및 호르몬 수용체 양성 세포주보다 PRMT5 억제에 더 감수성이었다.

In the 7-day proliferation assay, TNBC cell lines were more sensitive to PRMT5 inhibition than Her2 and hormone receptor positive cell lines.

SDMA 수준은 PRMT5 억제제로 처리된 감수성 및 저항성 유방암 세포주에서 감소하였으며, 이는 SDMA가 반응의 마커가 아니라, PRMT5 활성의 마커라는 것을 시사한다.

SDMA levels decreased in sensitive and resistant breast cancer cell lines treated with PRMT5 inhibitors, suggesting that SDMA is not a marker of response but a marker of PRMT5 activity.

MDA-MB-468 이종이식 모델에서, 화합물 C 처리는 100 mg/kg BID 처리군에서 종양 성장 억제 (TGI= 83%)를 유발하였고, 여기서 SDMA 억제는 90%를 초과하였으며, 반면에 100 mg/kg QD 처리된 동물에서 화합물 C 처리는 효과적이지 않았고, SDMA 억제는 80% 미만이었다. 이러한 데이터는 생체내 유방암 이종이식 모델에서의 유의한 TGI를 확인하기 위해 SDMA 수준이 거의 완전히 억제 (>90%)될 필요가 있다는 것을 시사한다.

In the MDA-MB-468 xenograft model, Compound C treatment resulted in tumor growth inhibition (TGI = 83%) in the 100 mg / kg BID treated group, where SDMA inhibition exceeded 90%, while 100 mg / Compound C treatment was not effective in kg QD treated animals and SDMA inhibition was less than 80%. These data suggest that SDMA levels need to be almost completely suppressed (> 90%) to identify significant TGI in an in vivo breast cancer xenograft model.

종합적으로, 이들 데이터는 유방암, 특히 TNBC 하위유형에서의 잠재적 치료 전략으로서의 PRMT5 억제를 시사한다.

Overall, these data suggest PRMT5 inhibition as a potential therapeutic strategy in breast cancer, particularly in the TNBC subtype.

Gliomas (GBM) Biology

PRMT5 protein is frequently overexpressed in glioblastoma tumors, and high PRMT5 levels are strongly correlated with both high grade (grade IV) and poor survival in GBM patients (Yan F, et al. Cancer Res. 2014 Mar 15; 74 (6): 1752-65). PRMT5 knockdown attenuates the growth and survival of GBM cell lines and significantly improves survival in an ortho Gli36 xenograft model (Yan F, et al. Cancer Res. 2014 Mar 15; 74 (6): 1752-65). PRMT5 also plays an important role in normal mouse brain development through the regulation of neuronal progenitor cell growth and differentiation (Bezzi M, et al. Genes Dev. 2013 Sep 1; 27 (17): 1903-16).

Glioblastoma cell line model belongs to the most susceptible tumor type in the soft agar colony forming assay (FIG. 10). In the 2D, 6-day growth / kill CTG assay, GBM cell lines had gIC ₅₀ values ranging from 40-22000 nM, where the response was mostly cytostatic, with the exception of the SF539 cell line (FIGS. 7 and 8). In order to understand the effect of PRMT5 inhibition on cell growth and survival upon longer exposure to PRMT5 inhibitors, compound C activity was tested in a 2D, 14-day growth / kill CTG assay (FIG. 26). Overall, the nature of the cytostatic / cytotoxic response did not change upon exposure to the compound and SF539 was the only cell line that suffered apoptosis in response to PRMT5 inhibition.

Next, the effect on cellular methylation and p53 pathway was assessed by measuring SDMA, p53 and p21 protein levels and MDM4 splicing in GBM cells treated with PRMT5 inhibitors (FIG. 27). PRMT5 inhibition resulted in a decrease in SDMA signal regardless of its sensitivity to PRMT5 inhibition in all cell lines tested (FIG. 27, Panel B). Alternative MDM4 splicing was detected in all cell lines except SF539, which is a p53 mutant and has low basal expression of the long MDM4 isotype (FIG. 27, Panel A). p53 levels were increased in all cell lines, whereas induction of p21 protein was only observed in cell lines with wild type p53 (Z138 (MCL), U87-MG and A172 (GBM)). These data suggest that PRMT5 inhibitors can activate the p53 pathway in GBM models, potentially through inactivation of MDM4 activity, similar to the effects observed in lymphoma models. Importantly, GBM cell line susceptibility was not correlated with p53 mutation status, suggesting that additional mechanisms contribute to the growth inhibition phenotype induced by PRMT5 inhibition. Interestingly, PRMT5 inhibition induced a cytostatic response in wild type p53 GBM cell line. The role of p53 in the response of GBM cell lines to PRMT5 inhibition will be further tested in future studies. In addition, the effect of PRMT5 inhibition on cell cycle and neural differentiation in the GBM model is under investigation.

Gliomas Summary

PRMT5 단백질은 교모세포종 종양에서 빈번하게 과다발현되고, 높은 PRMT5 수준은 GBM 환자에서의 고등급 (등급 IV) 및 불량한 생존과 강한 상관관계가 있다.

PRMT5 protein is frequently overexpressed in glioblastoma tumors, and high PRMT5 levels are strongly correlated with high grade (grade IV) and poor survival in GBM patients.

교모세포종 세포주 모델은 연질 한천 콜로니 형성 검정에서 가장 감수성인 종양 유형에 속하였다.

Glioblastoma cell line models were among the most susceptible tumor types in the soft agar colony forming assay.

2D, 6- 및 14-일 성장/사멸 CTG 검정에서, PRMT5 억제에 대한 GBM 반응은 대부분 세포증식억제성이었다 (4종의 세포주 중 3종, 1종의 세포주는 세포독성 반응을 가졌음).

In 2D, 6- and 14-day growth / kill CTG assays, GBM responses to PRMT5 inhibition were mostly cytostatic (3 out of 4 cell lines, 1 cell line had a cytotoxic response).

PRMT5 억제는 시험된 모든 세포주에서 PRMT5 억제에 대한 그의 감수성과 관계없이 SDMA 신호의 감소를 유발하였다.

PRMT5 inhibition caused a decrease in SDMA signal regardless of its sensitivity to PRMT5 inhibition in all cell lines tested.

Additional Susceptible Tumor Types

Cell line and patient-derived model screening data suggest that PRMT5 inhibitors attenuate cell growth and survival in a wide range of tumor types (FIGS. 7-10).

Full Biology Summary

화합물 C는 스플라이세오솜 성분, 히스톤, 전사 인자 및 키나제를 포함한 다양한 세포 단백질 상에서 대칭 아르기닌 디메틸화를 억제한다. 따라서, PRMT5 억제제는 전사, 스플라이싱 및 mRNA 번역에서의 변화를 포함한 다수의 메카니즘을 통해 RNA 항상성에 영향을 미친다.

Compound C inhibits symmetric arginine dimethylation on various cellular proteins, including the spliceosome component, histones, transcription factors and kinases. Thus, PRMT5 inhibitors affect RNA homeostasis through a number of mechanisms, including changes in transcription, splicing and mRNA translation.

PRMT5 억제는 유전자 발현 및 스플라이싱 변화를 유도하여 궁극적으로 p53의 유도를 가져온다. 화합물 C는 외투 세포 및 미만성 대 B-세포 림프종 뿐만 아니라 유방 및 신경교종 암 세포주 (지금까지 시험된 유일한 종양 유형)에서 p53 유비퀴틴 리가제인 MDM4의 이소형 전환을 유도하고, p53 단백질을 안정화시키고, p53 표적 유전자 발현 신호전달을 유도한다.

PRMT5 inhibition induces gene expression and splicing changes, ultimately leading to the induction of p53. Compound C induces isotype conversion of p53 ubiquitin ligase, MDM4, and stabilizes p53 protein in mantle cells and diffuse large B-cell lymphomas as well as breast and glioma cancer cell lines (the only tumor type tested so far) Induces target gene expression signaling.

화합물 C는 광범위한 고형 및 헴 종양 세포주에서 증식을 억제하고, 외투 세포 및 미만성 대 B-세포 림프종, 유방, 방광 및 신경교종 세포주의 하위세트에서 세포 사멸을 유도한다. 외투 세포 및 미만성 대 B-세포 림프종 세포주에서 가장 강력한 성장 억제가 관찰되었다. 외투 세포 림프종 및 유방암의 이종이식 모델에서 화합물 C 효능을 시험하였고, 여기서 그것은 종양 성장을 유의하게 억제하였다. 이들 데이터는 외투 세포 림프종, 미만성 대 B-세포 림프종, 유방암 및 뇌암에서의 치료 전략으로서의 화합물 C의 사용에 대한 강력한 근거를 제공한다.

Compound C inhibits proliferation in a wide range of solid and heme tumor cell lines and induces cell death in a subset of mantle cells and diffuse large B-cell lymphoma, breast, bladder and glioma cell lines. The strongest growth inhibition was observed in mantle cells and diffuse large B-cell lymphoma cell lines. Compound C efficacy was tested in xenograft models of mantle cell lymphoma and breast cancer, where it significantly inhibited tumor growth. These data provide a strong basis for the use of Compound C as a treatment strategy in mantle cell lymphoma, diffuse versus B-cell lymphoma, breast cancer and brain cancer.

Example 2

Combination

Survival benefits were determined for CT-26 (colon carcinoma) tumor model mice and A20 (lymphoma) tumor model mice treated with Compound C and anti-OX40 as a single agent and in combination. Mice were given vehicle or 126.1 mg / kg Compound C orally once daily for three weeks. Mice received 5 mg / kg of anti-OX40 (clone OX86) or the corresponding vehicle intraperitoneally twice a week for 21 days. Clone OX86 is a rat anti-mouse OX40 receptor antibody.

FIG. 28 is the mean in an A20 tumor model treated with the corresponding vehicle (Groups 1 and 3), Compound C (Group 6), anti-OX40 (Group 2) and the combination of Compound C with anti-OX40 (Group 11). Indicates survival.

FIG. 29 shows a CT-26 tumor model treated with the corresponding vehicle (Groups 1 and 3), Compound C (Group 6), anti-OX40 (Group 2) and the combination of Compound C with anti-OX40 (Group 11). Mean survival.

Treatment of A20 xenograft tumors with a combination of anti-OX-40 antibody and Compound C led to moderate survival benefits, highlighting the potential synergistic interactions between the two agents.

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<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> Description of Artificial Sequence: Synthetic 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 Ser 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> Description of Artificial Sequence: Synthetic polynucleotide <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> Description of Artificial Sequence: Synthetic 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> Description of Artificial Sequence: Synthetic polynucleotide <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 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> Description of Artificial Sequence: Synthetic 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 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> Description of Artificial Sequence: Synthetic polynucleotide <400> 24 gctagcacca ccatggagac agacacactc ctgttatggg tactgctgct ctgggttcca 60 gt; 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> Description of Artificial Sequence: Synthetic polynucleotide <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> Description of Artificial Sequence: Synthetic 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 Ser 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> Description of Artificial Sequence: Synthetic polynucleotide <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> Description of Artificial Sequence: Synthetic 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 Ser 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)

A combination of a type II protein arginine methyltransferase (type II PRMT) and an immunomodulator that is an anti-OX40 antibody or antigen binding fragment thereof. The combination of claim 1, wherein the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. The combination according to claim 1 or 2, wherein the type II PRMT inhibitor is a compound of formula (III) or a pharmaceutically acceptable salt thereof.

here

Represents a single or double bond;
R ¹ is hydrogen, R ^z , or —C (O) R ^z , wherein R ^z is optionally substituted C _1-6 alkyl;
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)-;
Each R is independently hydrogen or optionally substituted C _1-6 aliphatic;
Ar is a monocyclic or bicyclic aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein Ar is 0, 1, 2, 3, 4, or 5, as valency permits Are substituted with R ^y groups;
Each R ^y is independently halo, —CN, —NO ₂ , optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —OR ^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 ^B C (O) R ^A , -NR ^B C (O) N (R ^B ) ₂ , -NR ^B C (O) N (R ^B ) N (R ^B ) ₂ , -NR ^B C (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 ^B C (= NR ^B ) R ^B , -C (= S) R ^A , -C (= S) N (R ^B ) ₂ , -NR ^B C (= S) R ^A , -S (O) R ^A , -OS (O) ₂ R ^A , -SO ₂ R ^A , -NR ^B SO ₂ R ^A , or -SO ₂ N (R ^B ) ₂ ;
Each R ^A is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
Each R ^B is independently selected from hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two R ^{The B} group together with its intervening atoms form an optionally substituted heterocyclic ring;
R ⁵ , R ⁶ , R ⁷ and R ⁸ are independently hydrogen, halo, or optionally substituted aliphatic;
Each R ^X is independently selected from the group consisting of halo, —CN, optionally substituted aliphatic, —OR ′, and —N (R ″) ₂ ;
R ^' is hydrogen or optionally substituted aliphatic;
Each R ″ is independently hydrogen or an optionally substituted aliphatic, or two R ″ together with their intervening atoms form a heterocyclic ring;
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, as valency permits. The combination according to any one of claims 1 to 3, wherein the type II PRMT inhibitor is a compound of formula (X) or a pharmaceutically acceptable salt thereof.

The combination according to any one of claims 1 to 4, wherein the type II PRMT inhibitor is Compound C or a pharmaceutically acceptable salt thereof.

The combination of any one of the preceding claims, wherein the immunomodulatory agent is an OX40 agonist. The method of claim 6, wherein the immunomodulatory agent is CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; At least one of CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR having up to two amino acid substitutions in said CDR. The combination is an anti-OX40 antibody or antigen binding fragment thereof. The variable heavy chain sequence of claim 6 or 7, wherein the immunomodulatory agent has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and at least 90% sequence to the amino acid sequence set forth in SEQ ID NO: 11 A combination that is an anti-OX40 antibody or antigen binding fragment thereof comprising a variable light sequence with identity. A combination of type II protein arginine methyltransferase (type II PRMT) with an immunomodulator, wherein the immunomodulator is an anti-OX40 antibody or antigen binding fragment thereof, and the type II PRMT inhibitor is Compound C or a pharmaceutically acceptable salt thereof ,

,
Immunomodulators include CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; At least one of CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR having up to two amino acid substitutions in said CDR. Anti-OX40 antibody or antigen-binding fragment thereof
Combination. A combination of type II protein arginine methyltransferase (type II PRMT) with an immunomodulator, wherein the immunomodulator is an anti-OX40 antibody or antigen binding fragment thereof, and the type II PRMT inhibitor is Compound C or a pharmaceutically acceptable salt thereof ,

,
An immunomodulatory agent comprises a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 -OX40 antibody or antigen-binding fragment thereof
Combination. Treating a cancer in a human in need thereof by administering the combination of any one of claims 1 to 10 together with at least one of a pharmaceutically acceptable carrier and a pharmaceutically acceptable diluent. A method of treating cancer in a human being in need thereof. A pharmaceutical composition comprising a therapeutically effective amount of a type II protein arginine methyltransferase (type II PRMT) inhibitor, and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulator that is an anti-OX40 antibody or antigen binding fragment thereof. A pharmaceutical composition comprising a therapeutically effective amount of a type II protein arginine methyltransferase (type II PRMT) inhibitor and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulatory agent, wherein the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof , Type II PRMT inhibitor is Compound C or a pharmaceutically acceptable salt thereof,

Immunomodulators include CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; A term comprising at least one of CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9 or each CDR, a direct equivalent having up to two amino acid substitutions in said CDR -OX40 antibody or antigen-binding fragment thereof
Pharmaceutical composition. A pharmaceutical composition comprising a therapeutically effective amount of a type II protein arginine methyltransferase (type II PRMT) inhibitor, and a second pharmaceutical composition comprising a therapeutically effective amount of an immunomodulatory agent, wherein the immunomodulatory agent is an anti-OX40 antibody or antigen binding fragment thereof And the type II PRMT inhibitor is Compound C or a pharmaceutically acceptable salt thereof,

An immunomodulatory agent comprises a variable heavy chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a variable light chain sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 -OX40 antibody or antigen-binding fragment thereof
Pharmaceutical composition. The pharmaceutical composition of claim 12, wherein the type II PRMT inhibitor is a protein arginine methyltransferase 5 (PRMT5) inhibitor or a protein arginine methyltransferase 9 (PRMT9) inhibitor. The pharmaceutical composition of claim 12 or 15, wherein the type II PRMT inhibitor is a compound of Formula (III) or a pharmaceutically acceptable salt thereof.

here

Represents a single or double bond;
R ¹ is hydrogen, R ^z , or —C (O) R ^z , wherein R ^z is optionally substituted C _1-6 alkyl;
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)-;
Each R is independently hydrogen or optionally substituted C _1-6 aliphatic;
Ar is a monocyclic or bicyclic aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, wherein Ar is 0, 1, 2, 3, 4 or 5, as valency permits Are substituted with R ^y ;
Each R ^y is independently halo, —CN, —NO ₂ , optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —OR ^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 ^B C (O) R ^A , -NR ^B C (O) N (R ^B ) ₂ , -NR ^B C (O) N (R ^B ) N (R ^B ) ₂ , -NR ^B C (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 ^B C (= S) R ^A , -S (O) R ^A , -OS (O) ₂ R ^A , -SO ₂ R ^A , -NR ^B SO ₂ R ^A , or -SO ₂ N (R ^B ) ₂ ;
Each R ^A is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
Each R ^B is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two R ^B The group together with its intervening atoms form an optionally substituted heterocyclic ring;
R ⁵ , R ⁶ , R ⁷ , and R ⁸ are independently hydrogen, halo, or optionally substituted aliphatic;
Each R ^X is independently selected from the group consisting of halo, —CN, optionally substituted aliphatic, —OR ′, and —N (R ″) ₂ ;
R ^' is hydrogen or optionally substituted aliphatic;
Each R ″ is independently hydrogen or an optionally substituted aliphatic, or two R ″ together with their intervening atoms form a heterocyclic ring;
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, as valency permits. The pharmaceutical composition of claim 12, wherein the type II PRMT inhibitor is a compound of Formula (X) or a pharmaceutically acceptable salt thereof.

18. The pharmaceutical composition of any one of claims 12 and 15-17, wherein the type II PRMT inhibitor is Compound C or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of any one of claims 12 and 15-18, wherein the immunomodulatory agent is an OX40 agonist. The method of claim 19, wherein the immunomodulatory agent is CDRH1 as set forth in SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO: 2; CDRH3 as set forth in SEQ ID NO: 3; CDRL1 as set forth in SEQ ID NO: 7; At least one of CDRL2 as shown in SEQ ID NO: 8 and / or CDRL3 as shown in SEQ ID NO: 9, or a direct equivalent of each CDR having up to two amino acid substitutions in said CDR. A pharmaceutical composition that is an anti-OX40 antibody or antigen binding fragment thereof. The variable heavy chain sequence of claim 19 or 20, wherein the immunomodulatory agent has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and at least 90% sequence to the amino acid sequence set forth in SEQ ID NO: 11 A pharmaceutical composition that is an anti-OX40 antibody or antigen binding fragment thereof comprising a variable light sequence with identity. A method of treating cancer in a human being in need thereof comprising administering to the human being in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 12-21. How to treat. The method of claim 22, wherein the Type II PRMT inhibitor and immunomodulatory agent are administered to the patient simultaneously, sequentially, in any order, systemically, orally, intravenously, and intratumorally. The method of claim 22 or 23, wherein the type II PRMT inhibitor is administered orally. The method of claim 22, wherein the cancer is melanoma, lymphoma or colon cancer. Use of a combination of any one of claims 1 to 10 for the manufacture of a medicament. Use of the combination of any one of claims 1 to 10 for the treatment of cancer.

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