With the remarkable success of antibodies focusing on PD-1/PD-L1, the immune checkpoint blockade approach has established itself as a cornerstone to cancer therapy. While PD-1/PD-L1 antibodies primarily focus on T cells to achieve antitumor efficacy, other cells in the tumor microenvironment such as myeloid cells, including MDSCs, also play a role in immune evasion, thus contributing to the lack of response in 70-80% of patients. To overcome the immune resistance induced by MDSCs, V-domain Ig suppressor of T-cell activation (VISTA) expressed predominantly on myeloid cells and tumor-infiltrating lymphocytes is considered as an ideal target. Recent findings also support the role of VISTA pathway in clearance of apoptotic bodies and prevention of autoimmunity. VISTA is reported to mediate immune suppression through homophillic interaction as well as interaction with V-Set and immunoglobulin domain containing 8 (VSIG8). We sought to discover and develop an orally available, small-molecule VISTA antagonist targeting both VISTA and VSIG8 pathways. Unlike antibodies targeting VISTA in early clinical trials, an oral agent potentially offers the convenience and flexibility to adjust the dose and schedule to address any emergent adverse events and ease of combination therapy. Since VISTA belongs to B7 family and the extracellular Ig domain of VISTA shares significant sequence homology with the B7 family ligands PD-L1 and PD-L2, a focused library of compounds mimicking the interaction of checkpoint proteins of B7 family 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 lead compounds targeting both VISTA and VSIG8 signaling pathways with desirable drug-like properties including good oral bioavailability. Potent functional activity comparable to that obtained with an anti-VISTA or anti-VSIG8 antibody in rescuing effector functions was observed with the lead compound along with selectivity against other immune checkpoint proteins. An advanced lead compound exhibited sustained immune PD in tumor-bearing animals including desirable impact on myeloid and T cells in both circulation and tumor. The advanced lead compound also exhibited significant efficacy in syngeneic preclinical tumor models of melanoma and colon cancers upon once-a-day oral dosing with excellent tolerability. Further development of the oral VISTA antagonist is under way towards advancing it to the clinic. Citation Format: Pottayil G. Sasikumar, Sudarshan S. Naremaddepalli, Raghuveer K. Ramachandra, Nagesh Gowda, Manikyala Rao Yerramsetti, Srinivasa Rao Bandireddy, Sreenivas Adurthi, Jiju Mani, Rashmi Nair, Amit A. Dhudashia, Samiulla S. Dodheri, Nagaraj M. Gowda, Murali Ramachandra. Functional antagonism of VSIG8-mediated immune suppression by oral VISTA agents [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B006.

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.

Antibodies targeting PD-1 and PD-L1 towards stimulation of T cells have revolutionized cancer therapy because of the highly durable response in multiple cancer indications. Although macrophages and other myeloid immune cells offer much promise as effectors of cancer immunotherapy, efforts to modulate them for therapeutic benefit have gained momentum only in the last few years. The CD47/signal regulatory protein alpha (SIRPα) axis is a critical regulator of myeloid cell activation and serves as an immune checkpoint for macrophage-mediated phagocytosis. Because of its frequent upregulation in several cancers, CD47 contributes to immune evasion and cancer progression. Disruption of CD47-SIRPα interaction is now being evaluated as a therapeutic strategy for cancer with the use of monoclonal antibodies targeting CD47 or SIRPα, and engineered receptor decoys. In view of the requirement for the intravenous dosing for all reported CD47 targeting agents, we sought to discover and develop an orally available small-molecule CD47 antagonist. An oral CD47 agent potentially offers the convenience, flexibility to adjust the dose and schedule to address any emergent adverse events, and ease of combination therapy. Through rational design based on the crystal structure of CD47/SIRPα interacting interface, we generated a series of peptides capable of disrupting CD47-SIRPα interactions. Relative binding affinities of the peptides were determined in a newly established cellular assay that utilizes a SIRPα-derived peptide as the probe. The high-affinity peptides were further truncated to identify the shortest peptide pharmacophore. The elements of this pharmacophore were incorporated into nonpeptidic small-molecule scaffolds, resulting in lead compounds disrupting CD47/SIRPα interaction. The lead CD47 antagonists induced phagocytototic activity of human macrophages to a similar extent as commercially available anti-CD47 antibodies. Further optimization of these leads resulted in compounds with desirable physicochemical properties and good oral bioavailability. An advanced lead CD47 antagonist inhibited primary tumor growth (~90%TGI) in a mouse syngeneic model of B-cell lymphoma upon twice-a-day oral dosing. Biomarker characterization and efficacy studies in additional tumor models are ongoing. These findings support further development of these orally bioavailable agents for use in the clinic. Citation Format: Pottayil G. Sasikumar, Chennakrishnareddy Gundala, Wesley R. Balasubramanian, Sudarshan S. Naremaddepalli, Archana Bhumireddy, Sandeep S. Patil, Amit A. Dhudashiya, Vijaysai Rayavarapu, Anirudha Lakshminarasimhan, Dodheri S. Samiulla, Kiran Aithal, Girish Daginakatte, Murali Ramachandra. Potent antitumor activity of a novel and orally available small-molecule antagonist targeting the CD47/SIRPα pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B007.

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

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