Raghuveer Ramachandra

Abstract A36: CA-170, an oral small molecule PD-L1 and VISTA immune checkpoint antagonist, promotes T cell immune activation and inhibits tumor growth in pre-clinical models of cancer

The clinical success of antibody-mediated immune checkpoint blockade therapies has transformed the cancer therapy paradigm by demonstrating that durable antitumor immune responses and long-term remissions may be achieved in a subset of patients across a diverse range of cancers. However, the majority of patients fail to respond to antibody therapies targeting single immune checkpoint pathways and antibodies exhibit a long in vivo half-life (>15-20 days with >70% target occupancy for months) which may contribute to the emergence of immune-related adverse events. Additionally, antibody therapies must be administered by intravenous infusion in a hospital or clinic which places an additional burden on patients who may have mobility challenges. Thus, there is a significant opportunity for a novel immune checkpoint therapy that can address the shortcomings associated with the current antibody therapies. CA-170 is a small molecule, orally bioavailable antagonist of the PD-L1, PD-L2 and VISTA/PD-1H immune checkpoint pathways which is currently undergoing Phase I clinical testing. In preclinical safety studies conducted in rodents and non-human primates, orally administered CA-170 shows no signs of toxicity when dosed up to 1000 mg/kg for 28 consecutive days. CA-170 exhibits an oral bioavailability of approximately 40% and Citation Format: Adam S. Lazorchak, Troy Patterson, Yueyun Ding, Pottayil G. Sasikumar, Naremaddepalli S. Sudarshan, Nagaraj M. Gowda, Raghuveer K. Ramachandra, Dodheri S. Samiulla, Sanjeev Giri, Rajesh Eswarappa, Murali Ramachandra, David Tuck, Timothy Wyant. CA-170, an oral small molecule PD-L1 and VISTA immune checkpoint antagonist, promotes T cell immune activation and inhibits tumor growth in pre-clinical models of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A36.

Debio 0617B Inhibits Growth of STAT3-Driven Solid Tumors through Combined Inhibition of JAK, SRC, and Class III/V Receptor Tyrosine Kinases.

Tumor survival, metastases, chemoresistance, and escape from immune responses have been associated with inappropriate activation of STAT3 and/or STAT5 in various cancers, including solid tumors. Debio 0617B has been developed as a first-in-class kinase inhibitor with a unique profile targeting phospho-STAT3 (pSTAT3) and/or pSTAT5 in tumors through combined inhibition of JAK, SRC, ABL, and class III/V receptor tyrosine kinases (RTK). Debio 0617B showed dose-dependent inhibition of pSTAT3 in STAT3-activated carcinoma cell lines; Debio 0617B also showed potent antiproliferative activity in a panel of cancer cell lines and in patient-derived tumor xenografts tested in an in vitro clonogenic assay. Debio 0617B showed in vivo efficacy by inhibiting tumor growth in several mouse xenograft models. To increase in vivo efficacy and STAT3 inhibition, Debio 0617B was tested in combination with the EGFR inhibitor erlotinib in a non–small cell lung cancer xenograft model. To evaluate the impact of in vivo STAT3 blockade on metastases, Debio 0617B was tested in an orthotopic tumor model. Measurement of primary tumor weight and metastatic counts in lung tissue demonstrated therapeutic efficacy of Debio 0617B in this model. These data show potent activity of Debio 0617B on a broad spectrum of STAT3-driven solid tumors and synergistic activity in combination with EGFR inhibition. Mol Cancer Ther; 15(10); 2334–43. ©2016 AACR.

Abstract 4861: Oral immune checkpoint antagonists targeting PD-L1/VISTA or PD-L1/Tim3 for cancer therapy

Recent successes in achieving highly durable clinical responses with antibodies to immune checkpoint receptors such as CTLA4 and PD1 have transformed the outlook for cancer therapy. While these antibody-based therapies show impressive clinical activity, they suffer from the shortcomings including the need to administer by intravenous injection, failure to show response in majority of patients and immune-related adverse events (irAEs) due to the breaking of immune self-tolerance. Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for months may be factors contributing to irAEs observed. We sought to discover and develop small molecule immune checkpoint antagonists capable of targeting PD-L1 and another immune checkpoint pathway. We reasoned that such therapeutic agents will be amenable for oral dosing, likely show greater response rate due to dual antagonism and allow better management of irAEs due a shorter pharmacokinetic profile. A focused library of compounds mimicking the interaction of checkpoint proteins was designed and synthesized. Screening and analysis of the resulting library led to the identification of hits capable of functional disruption of the checkpoint protein(s) signaling depending upon the pockets of sequence similarity of interacting proteins. Further optimization resulted in compounds targeting PD-L1/VISTA or PD-L1/TIM-3 with desirable physico-chemical properties and exposure upon oral administration.. The ability of compounds to disrupt specific immune checkpoint pathways was confirmed though functional studies. Identified lead compounds exhibit potent activity when tested in assays to rescue lymphocyte proliferation and effector functions inhibited by respective ligands/proteins. In a panel of functional assays, the selected lead compounds showed selectivity against other immune checkpoint pathways including CTLA4, LAG3 and BTLA. Lead compounds exhibited sustained immune PD in vitro and in vivo suggesting that drug efficacy may extend beyond drug clearance. Lead compounds exhibited significant efficacy in syngeneic pre-clinical tumor models of melanoma, breast carcinoma and colon cancers upon once a day oral dosing. In repeated dose toxicity studies, the most advanced compound, AUPM-170, a dual antagonist of PD-L1 and VISTA, was well tolerated at >100x of the efficacious doses. The data demonstrating the inhibition of PD-L1 and another immune checkpoint pathway (VISTA or Tim3) resulting in activation of T cells and anti-tumor activities support further development of these orally bioavailable agents. IND-enabling studies with one of the lead compounds, AUPM-170, are underway towards advancing it to the clinic. Citation Format: Pottayil Sasikumar, N S Sudarshan, Nagaraj Gowda, D S Samiulla, Raghuveer Ramachandra, T Chandrasekhar, Sreenivas Adurthi, Jiju Mani, Rashmi Nair, Sharad Singh, Amit Dhudashia, Nagesh Gowda, Murali Ramachandra. Oral immune checkpoint antagonists targeting PD-L1/VISTA or PD-L1/Tim3 for cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4861.

A novel peptide therapeutic targeting PD1 immune checkpoint with equipotent antagonism of both ligands and a potential for better management of immune-related adverse events

Recent advances in achieving highly durable clinical responses via inhibition of immune checkpoint proteins including CTLA-4 and PD1 have revolutionized the outlook for cancer therapy. However, along with impressive clinical activity (response rate of ~25% with either anti-CTLA-4 or anti-PD1 as single agent, but > 50% with a combination), severe immune-related adverse events (irAEs) due to the breaking of immune self- tolerance (25-30% with anti-CTLA-4 and up to 15-17% with anti-PD1) are becoming increasingly evident. Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for months are likely contributing to severe irAEs observed in the clinic with antibodies targeting immune checkpoint proteins. Our efforts are therefore focused on developing immune checkpoint blockers with potent anti-tumor activity but with a shorter pharmacokinetic profile as a strategy to better manage severe irAEs. Flexibility in adjusting the drug exposure because of a shorter t½ could also be advantageous for use in combination with other checkpoint modulators or anti-cancer agents. Peptide antagonist AUR-012, constructed with elements from human PD-1, displayed equipotent antagonism towards PD-L1 and PD-L2 with potent activity in rescue of lymphocyte proliferation and effector functions. Rescue of proliferation of immune cells analyzed upon stimulation with anti-CD3/anti-CD-28 indicated a complete rescue of CD4+ and CD8+ T cells. Interestingly, the proliferation of CD4+, Foxp3+ T cells was completely abolished with AUR-012 treatment indicating a complete suppression of regulatory T cells. Sustained activation of circulatory immune cells and their ability to secrete IFN-γ up to 72 h indicate that pharmacodynamic effects persist even after the clearance of AUR-012 in animal models, thus supporting a dosing interval of up to 3 days. In models of melanoma, breast, kidney and colon cancers, AUR-012 showed efficacy in inhibition of both primary tumor growth and metastasis. Additionally, anti-tumor activity of AUR-012 in a pre-established CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4+ and CD8+ T cells, and a reduction in PD1+ T cells (both CD4+ & CD8+) in tumor and blood. In 14-day repeated dose toxicity studies, AUR -012 was well tolerated at 100x of the efficacious doses. These findings demonstrating equipotent antagonism of both PD-L1 and PD-L2 signaling and the observed correlation between anti-tumor activities with the modulation of specific T-cell populations support further development of AUR-012 in the clinic.

Abstract 1650: Targeting CD47- SIRPα interaction by novel peptide-based antagonists

Background: Cluster of differentiation (CD47) is a trans-membrane glycosylated protein which is upregulated in several cancers. Increased expression of CD47 on tumor cells is associated with immune evasion and cancer progression. CD47 through its interaction with signal regulatory protein alpha (SIRPα), a cell-surface molecule on macrophages inhibits phagocytosis of tumor cells. Disrupting CD47-SIRPα interactions by monoclonal antibodies targeting CD47 and recombinant SIRPα proteins have been used as therapeutic strategies for treating cancer. Our objective was to discover and develop peptide/peptidomimetic based CD47 antagonists for disrupting CD47-SIRPα interactions. Methods: Through rational design based on crystal structure of CD47/SIRPα interacting interface, we designed peptides having potential to disrupt CD47-SIRPα interactions. FACS based cellular binding assay was developed to assess the binding affinity of CD47 antagonists. SIRPα protein labelled with fluorescent dye was incubated with Jurkat T cells expressing high levels of CD47 in the presence/absence of peptides. Binding affinity was measured by decrease in fluorescence. Functional activity of the peptides was evaluated in a FACS-based phagocytosis assays, in which tumor cells were incubated with human/mouse macrophages in the presence/absence of CD47 antagonists. Results: We identified CD47 antagonists demonstrating disruption of CD47-SIRPα interaction in a cellular binding assay. These peptides significantly inhibited phagocytosis of different tumor cells by macrophages. The lead CD47 antagonist displaying good ADME properties including moderate oral bioavailability was evaluated in a B16F10 syngeneic mouse tumor model. The lead CD47 antagonist inhibited primary tumor growth as well tumor metastasis to lungs. Biomarker characterization and efficacy studies in additional tumor models are ongoing. Citation Format: Pottayil G. Sasikumar, Chennakrishnareddy Gundala, Nagaraj M. Gowda, Sudarshan S. Naremaddepalli, Archana Bhumireddy, Rashmi Nair, Wesley Roy Balasubramanian, Anirudha Lakshminarasimhan, Samiulla S. Dodheri, Kiran Aithal, Raghuveer K. Ramachandra, Girish Daginakatte, Murali Ramachandra. Targeting CD47- SIRPα interaction by novel peptide-based antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1650. doi:10.1158/1538-7445.AM2017-1650

Abstract A72: Novel NAMPT inhibitors for the treatment of Pancreatic cancer

Pancreatic cancer is reported to be dependent on NAD salvage pathway for its growth and survival. Nicotinamide phosphoribosyl transferase (NAMPT), an enzyme that catalyzes the rate limiting step of NAD biosynthesis is over expressed in a number of cancers. Inhibition of NAMPT with first generation inhibitors has been demonstrated to result in anti-tumor efficacy in preclinical models. Clinical development of first generation NAMPT inhibitors has been hindered because of their poor pharmacological profile, high cytochrome inhibition and possibly mechanism-based toxicities. Therefore, our objective was to develop NAMPT inhibitors with the “best-in-class” profile with strategies for overcoming mechanism-based toxicities. Utilizing structure-guided drug design including determination of co-crystal structures and SAR-based approaches, we have identified a novel chemical series of inhibitors of NAMPT. Optimization of the series for transient target inhibition as a result of reduced binding strength coupled with desirable pharmacokinetic profile to minimize mechanism based toxicity resulted in identification of AU-4869 as the lead compound. AU-4869 showed potent cross-species activity and reduced strength of binding in comparison with first generation NAMPT inhibitors. Anti-proliferative activity of AU-4869 correlated well with NAD depletion in a pancreatic cancer cell line. The anti-proliferative activities were rescued in NAPRT-proficient cell lines with the addition of nicotinic acid due to the NAMPT independent salvage pathway for biosynthesis of NAD, confirming the mechanism of action through NAD depletion. AU-4869 exhibited desirable drug-like properties including solubility, permeability, metabolic stability, lack of CYP & hERG inhibition and pharmacokinetic exposure upon oral dosing. At well-tolerated doses, AU-4869 exhibited superior efficacy at MTD doses in mice xenograft models as compared to first generation inhibitors. Addition of nicotinic acid improved the tolerability of AU-4869 and reversed the effects of mechanism based toxicity in Rodents. Anti-tumor activities of AU-4869 in the presence of nicotinic acid in NAPRT-deficient pancreatic cancer models are currently being evaluated in preclinical models. Citation Format: Dinesh Chikanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Narasimha Rao, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra, Shekar Chelur, Chetan Pandit, Murali Ramachandra. Novel NAMPT inhibitors for the treatment of Pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A72.

Abstract 339: Identification of novel covalent inhibitors of K-Ras G12C that are efficacious in a xenograft model of NSCLC

KRAS is the frequently mutated isoform in RAS driven cancers. The G12C mutation is more predominantly associated with various tumor types over other changes in K-Ras. Although direct targeting of RAS is very challenging, it is possible to selectively target G12C mutant K-Ras using a covalent approach. Mutant specific covalent inhibitors with high selectivity against wild type K-Ras and other GTPases are expected to lead to efficacy with a very high degree of tolerability. Here, we report identification of lead compounds from two distinct chemical series that selectively target K-Ras G12C. Molecular modeling based on the reported crystal structures aided in the identification of these compounds. Covalent binding of the lead compounds to K-Ras G12C was demonstrated by MALDI-TOF. Lead compounds were potent in selectively inhibiting proliferation of cell lines with K-Ras G12C mutation but not with wild type K-Ras. The anti-proliferative activity of the lead compounds correlated well with their potency in a cellular mechanistic assay. Lead compounds from both series exhibited excellent drug-like properties including solubility, metabolic stability, permeability lack of CYP inhibition and desired exposure in pharmacokinetic studies. In a xenograft model of NSCLC, the lead compound demonstrated dose-dependent tumor growth inhibition with excellent tolerability upon oral dosing. In summary, we have identified a novel, potent and selective K-Ras G12C inhibitor with optimized drug-like properties including oral bioavailability and efficacy in a NSCLC derived xenograft model. Toxicity evaluation is ongoing towards progressing the lead compound to the clinic. Citation Format: Leena Khare Satyam, Dinesh Chikkanna, Aswani K. G, Vinayak V. Khairnar, Sreekanth Reddy, Vakkapatla Durgaprasad, Kowju Radhakrishna, Sunil K. Panigrahi, Anuradha Ramanathan, Kumari Mahasweta, Anirudha Lakshminarasimhan, Narasimha R. K, Vinutha R, Sreevalsam Gopinath, Suryakant Kumar, Mubarak H. Shah, Raghuveer Ramachandra, Kiran A. B, Chetan Pandit, Murali Ramachandra. Identification of novel covalent inhibitors of K-Ras G12C that are efficacious in a xenograft model of NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 339.

Abstract 3070: Potent and selective inhibition of CDK7 by novel covalent inhibitors

Background: Phosphorylation of the RNA polymerase II (RNAPII) in C-terminal domain (CTD) by Cyclin-dependent kinase 7 (CDK7) is an important step in cellular transcription process. Hence pharmacological modulation of CDK7 kinase activity is considered as an interesting approach to treat cancers that critically dependent on transcription to maintain their oncogenic state. Experimental procedures: Multiple series of novel covalent CDK7 inhibitors were identified by SBDD approach based on the binding mode of known CDK7 inhibitors to find early hits. Iterative medicinal chemistry efforts were performed to identify several lead compounds by optimizing the initial hits to achieve good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile. Summary: Highly potent ATP competitive covalent inhibitors of CDK7 from two distinct chemical series were identified. They show time-dependent inhibition of CDK7 enzyme activity as a proof of covalent binding and exhibit potent anti-proliferative activity in cell lines derived from various tumor types. CDK7 modulation by these compounds was also confirmed by monitoring cellular pS5RNAPII levels. Representative compounds from each series showed very good selectivity profile in broad kinase (332) panel. Lead molecules were identified based on excellent drug-like properties (solubility, permeability and good oral bioavailability). Tolerability and efficacy studies in rodent xenograft models are ongoing with selected leads to test their impact on tumor growth inhibition and to determine therapeutic window by oral administration. Conclusion: We have identified novel and selective CDK7 covalent inhibitors from two distinct chemical series with optimized drug-like properties including oral bioavailability. These compounds are being evaluated for anti-tumor activity in mouse xenograft models. Citation Format: Leena Khare Satyam, Ramulu Poddutoori, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Raghuveer Ramachandra, Manoj Kumar Pothuganti, Shilpa S. Nayak, Nandish C, Chandranath Naik, Ravindra MV, Madhu B. Dabbeeru, Nagaraju A, Mahankali B, Thomas Antony, Chetan Pandit, Shekar Chelur, Girish Daginakatte, Susanta Samajdar, Murali Ramachandra. Potent and selective inhibition of CDK7 by novel covalent inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3070.

Abstract C190: Potent and selective inhibition of CDK7 by novel covalent inhibitors

Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacological modulation of CDK7 kinase activity is considered as an approach to treat cancer. Multiple series of CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR based approach. Early compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumor activity. We have identified compounds from two distinct chemical series that are highly potent in inhibiting CDK7 in biochemical assays. These inhibitors demonstrate time-dependent inhibition of CDK7 indicating covalent nature of binding. The compounds showed potent anti-proliferative activity in cell lines derived from various tumor types and this was accompanied by CDK7 modulation in cells as monitored by pS5RNAPII levels. They have excellent drug-like characteristics including solubility, permeability, metabolic stability and good oral bioavailability. In a broad panel of kinases (332 kinase), selected compounds from both series showed good selectivity profile. Tolerability and efficacy studies are ongoing with selected early leads to test their impact on tumor growth inhibition in xenograft models. We have identified novel and selective CDK7 covalent inhibitors from two series with desirable drug-like properties, which are being evauated for anti-tumor activity in xenograft models. Citation Format: Ramulu Poddutoori, Leena K. Satyam, Girish Daginakatte, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Raghuveer Ramachandra, Anirudha Lakshminarasimhan, Manoj Pothuganti, Shilpa Nayak, Nandish C, Chandranath Naik, Ravindra MV, Madhu Dabbeeru, Thomas Antony, Chetan Pandit, Murali Ramachandra, Shekar Chelur, Susanta Samajdar. Potent and selective inhibition of CDK7 by novel covalent inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C190.

Abstract 1231: Equipotent antagonism, transient immune activation and excellent antitumor efficacy with a peptide inhibitor of PD-1 immune check point pathway.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pioneering success of anti-CTLA4 antibody (Ipilimumab) and the impressive clinical data on agents that target PD-1 or its ligands have opened novel avenues in the area for caner immunotherapy. However, along with impressive clinical activity, immune-related toxicities have also been observed in significant (25-30% with anti-CTLA4 and up to 15% with anti-PD1) patient population. Sustained inhibition of PD-1 signaling as a result of a very long half-life (>15-20 days) and >70% target occupation for months are likely contributing to severe adverse effects observed in clinical trials with antibodies targeting PD-1 signaling. In the present study, we sought to characterize in detail a novel peptide antagonist of the PD1 signaling providing desired anti-tumor efficacy with shorter exposure and thus transient immune activation for effective management of severe adverse effects. Design of peptide to disrupt PD-1 signalling pathway was carried out by selecting individual fragments from PD-1 ectodomain reported to participate in binding. Sequences critical for ligand-receptor interaction were identified and combined in a non-linear fashion. The strategy resulted in a novel peptide, AUR-012 (29-mer), which displayed equipotent antagonism in disruption of PD1-PDL1/2 interaction and highly effective in restoration of proliferation and effector functions of mouse splenocytes and monkey and human PBMCs using species specific ligands. In preclinical models of melanoma, breast, kidney and colon cancers, AUR-012 showed superior showed superior efficacy compared to therapeutic agents currently used in the clinic in inhibition of both primary tumour growth and metastasis. Additionally, AUR-012 showed additive anti-tumor activity in a pre-established tumor models when combined with tumor vaccination or a chemotherapeutic agent such as cyclophosphamide known to induce “immunological cell death”. Anti-tumoral activity was correlated with increased drug distribution within the tumor while promoting intratumoral recruitment of CD4+ and CD8+ T cells. Furthermore, AUR -012 was well tolerated in 14 days repeated dose toxicity studies at 100x efficacious doses. The above findings demonstrating equipotent activity antagonism of both PD-L1 and PD-L2 signaling coupled with impressive efficacy in preclinical models support further development of AUR-012 for clinical use. Citation Format: Pottayil G. Sasikumar, Leena K. Satyam, Rajeev Shrimali, Raghuveer Ramachandra, Ketha A. Reddy, Adurthi Sreenivas, Amit Dhudashia, Dodderi S. Samiulla, Murali Ramachandra. Equipotent antagonism, transient immune activation and excellent antitumor efficacy with a peptide inhibitor of PD-1 immune check point pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1231. doi:10.1158/1538-7445.AM2013-1231