Pottayil Govindan Nair Sasikumar

Magnetic CLEAR supports for solid-phase synthesis of peptides and small organic molecules

Paramagnetic versions of the CLEAR supports were prepared by entrapment of magnetite particles during suspension polymerization of allylamine, trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate, and trimethylolpropane trimethacrylate. Swelling studies in a broad range of solvents showed comparable swelling of magnetic beads and regular CLEAR beads. The application of the magnetic beads as the support in solid-phase synthesis of peptides (Leu-enkephalinamide, substance P, acyl carrier protein (65–74) amide, and amyloid β-protein (34–42) amide) and small organic affinity ligands based on the s-triazine scaffold was demonstrated. Adequate stability of the beads and retention of their magnetic properties were observed throughout the synthesis steps.

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.

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.

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 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

Small-Molecule Immune Checkpoint Inhibitors Targeting PD-1/PD-L1 and Other Emerging Checkpoint Pathways

Advances in harnessing the immune system for cancer treatment have been spectacular in recent years as witnessed by the approval of a number of antibodies targeting the PD-1/PD-L1 immune checkpoint pathway spanning an expanding list of indications. However, it is well recognized that while these antibodies show impressive clinical activity, they suffer from shortcomings including the failure to show response in a majority of patients, their need to be administered by intravenous injection, and immune-related adverse events due to the breaking of immune self-tolerance. Small-molecule-based therapeutic approaches offer the potential to address the shortcomings of these antibody-based checkpoint inhibitors. In the first part of this review, we discuss the rationale for small-molecule-based checkpoint therapy followed by efforts on the discovery of small-molecule-based approaches targeting the PD-1/PD-L1 axis and other immune checkpoint pathways. In the latter part of the article, we describe small-molecule inhibitors simultaneously targeting two non-redundant checkpoint inhibitor pathways as an approach to improve the response rate. A brief review of the progress of an oral small-molecule checkpoint inhibitor currently in clinical development is presented at the end.

Abstract A96: First-in-class orally available immune checkpoint antagonists for cancer therapy

Highly durable clinical responses observed with antibodies to immune checkpoint receptors such as CTLA4 and PD1 have revolutionized the outlook of cancer therapy. However, while these antibodies show impressive clinical activity, they suffer from the shortcomings including the need to administer by intravenous injection and immune-related adverse events 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 are likely contributing to irAEs observed. Herein we report the discovery of the first-in-class small molecule PD-L1 antagonists that are amenable for oral dosing to achieve potent anti-tumor activity but with a shorter pharmacokinetic profile as a strategy to better manage irAEs. A focused library of compounds mimicking the interaction of PD1 with PD-L1 was designed and synthesized. Screening and analysis of the resulting library led to the identification of compounds capable of functional disruption of the PD-L1 signaling. Further optimization of the initial hits resulted in compounds with desirable physico-chemical properties and exposure upon oral administration. Disruption of the PD1-PDL1 interaction by lead compounds was confirmed in binding assays. Potent activity comparable to that obtained with an anti-PD1 antibody in rescuing lymphocyte proliferation and effector functions inhibited by PD-L1 was observed with lead compounds. In a panel of functional assays, the lead compounds showed selectivity against other immune checkpoint pathways including CTLA4, TIM3, LAG3 and BTLA. In syngeneic pre-clinical models of melanoma and colon cancers, significant efficacy comparable to that observed with an anti-PD1 antibody in inhibition of both primary tumor growth and metastasis was noted upon once a day oral dosing. In a 14-day repeated dose toxicity studies, the lead compound was well tolerated at >100x of the efficacious doses. The findings demonstrating the inhibition of PD-L1 pathway 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 are underway towards advancing it to the clinic. Citation Format: Pottayil G. Sasikumar, Sudarshan S. Naremaddepalli, Nagaraj Gowda, Sreenivas Adurthi, Samiulla Dhodheri, Amit Dhudashia, Raghuveer Ramachandra, Murali Ramachandra. First-in-class orally available immune checkpoint antagonists for cancer therapy. [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 A96.

Abstract 5022: Peptide therapeutic inhibiting PD1 immune checkpoint ligands: Potential for greater anti-tumor immune response and better management of immune-related adverse events

Antibodies to immune checkpoint receptors have revolutionized the outlook of cancer therapy by achieving highly durable clinical responses. However, along with impressive clinical activity (response rate of ∼25% with either anti-CTLA4 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-CTLA4 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. The antibodies targeting PD-1 signaling pathway are either based on receptor (PD-1) or its ligands (PD-L1 & PDL2). It has also been reported that PDL1 also interacts with B7-1(CD80) resulting in negative T-cell regulation. Our efforts are therefore focused on (a) developing a first in class peptide therapeutic inhibiting all three reported interaction of PD-1 pathway to improve the anti-tumor immune response (b) developing immune checkpoint blockers with potent anti-tumor activity but with a shorter pharmacokinetic profile as a strategy to better manage severe irAEs. Peptide antagonist AUR-012, constructed with elements from human PD-1 receptor, displayed equipotent antagonism towards PD-L1 and PD-L2 with potent activity in rescue of lymphocyte proliferation and effector functions. Additionally crosslinking study to evaluate the effect of AUR-12 on B7.1 - PDL-1 interaction supports the finding that AUR-012 prevents the interaction of B7.1 with PD-L1. 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 and 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 inhibition of all PD-1/ PD-1 ligands mediated co-inhibitory signaling pathway 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. Citation Format: Murali Ramachandra, Pottayil G. Sasikumar, Rajeev K. Shrimali, Sreenivas Adurthi, Raghuvir Ramachandra, Leena K. Satyam, Amit A. Dhudashia, Samiulla Dhodheri, K B. Sunilkumar. Peptide therapeutic inhibiting PD1 immune checkpoint ligands: Potential for greater anti-tumor immune response and better management of immune-related adverse events. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5022. doi:10.1158/1538-7445.AM2014-5022

Abstract B294: Antitumor efficacy of AUR-012, a peptide antagonist of the PD1 immune checkpoint pathway, correlates well with the modulation of specific T-cell populations.

Recent breakthroughs in achieving highly durable clinical responses via immunotherapy to inhibit immune checkpoint proteins including CTLA4 and PD1 have revolutionized the outlook for cancer therapy. However, along with impressive clinical activity (response rate of ∼25% with either anti-CTLA4 or anti-PD1 as single agent, but more than 50% with a combination), immune-related toxicities due to the breaking of immune self-tolerance (25-30% with anti-CTLA4 and up to 15-17% with anti-PD1) are becoming increasingly visible. Sustained inhibition of the signaling as a result of a very long half-life (>15-20 days) and >70% target occupancy for months are likely contributing to severe adverse effects observed in clinical trials with antibodies targeting immune checkpoint proteins. Our efforts are therefore focused towards developing novel immune checkpoint blockers with potent antitumor activity but with a shorter pharmacokinetic profile as a strategy to better manage severe adverse effects. Herein we report our findings from the pharmacodynamic characterization of a novel peptide inhibitor of the PD1 signaling that displays desired pharmacokinetic and efficacy profile. Peptide antagonist AUR-012, constructed with elements from human PD-1 ectodomain, 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 cell subset populations analyzed in vitro using anti-CD3/anti CD-28 stimulation indicated that the functional capacity of T-cells has been reinvigorated by AUR-012 resulting in a complete rescue of CD4+ and CD8+ T cell proliferation in the presence of PD-L1. Similar effect in the rescue of proliferation was also observed for B-cells and NK 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 irrespective of the short half-life of the peptide. 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 tumor growth and metastasis. Additionally, antitumor 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 conclusion, the observed correlation between antitumor activities with the modulation of specific T-cell populations supports the use of immunophenotyping as a potential therapeutic biomarker strategy in the planned clinical studies with AUR-012. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B294. Citation Format: Murali Ramachandra, Pottayil G. Sasikumar, Rajeev K. Shrimali, Sreenivas Adurthi, Raghuveer Ramachandra, Leena K. Satyam, Amit A. Dhudashiya, Dodheri S. Samiulla, K B Sunilkumar. Antitumor efficacy of AUR-012, a peptide antagonist of the PD1 immune checkpoint pathway, correlates well with the modulation of specific T-cell populations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B294.

Magnetic CLEAR beads: supports for solid-phase synthesis and affinity capture

Paramagnetic versions of the CLEAR supports were prepared by direct incorporation of magnetite particles during suspension polymerization of allylamine, trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate, and trimethylolpropane trimethacrylate. Swelling studies in a broad range of solvents showed comparable swelling of composite beads and regular CLEAR beads. The application of the composite beads as the support in solid-phase synthesis of peptides and small organic affinity ligands is described. Adequate stability of the beads and retention of their magnetic properties were observed throughout the synthesis steps.