Small molecule potent IRAK4 inhibitors from a novel bicyclic heterocycle class were designed and synthesized based on hits identified from Aurigene’s compound library. The advanced lead compound, CA-4948, demonstrated good cellular activity in ABC DLBCL and AML cell lines. Inhibition of TLR signaling leading to decreased IL-6 levels was also observed in whole blood assays. CA-4948 demonstrated moderate to high selectivity in a panel of 329 kinases as well as exhibited desirable ADME and PK profiles including good oral bioavailability in mice, rat, and dog and showed >90% tumor growth inhibition in relevant tumor models with excellent correlation with in vivo PD modulation. CA-4948 was well tolerated in toxicity studies in both mouse and dog at efficacious exposure. The overall profile of CA-4948 prompted us to select it as a clinical candidate for evaluation in patients with relapsed or refractory hematologic malignancies including non-Hodgkin lymphoma and acute myeloid leukemia.

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Expression of recombinant proteins in mammalian cells is useful for obtaining products with normal posttranslational modifications. We describe a simple and economical method for the production of milligram levels of proteins in murine fibroblasts. Retroviral or LIPOFECTAMINE TM (Gibco Laboratories) transduction was employed to generate stable murine-fibroblast producer cells. Confluent cultures of stable fibroblast clones were maintained for up to 1 month in 0.5 % serum. Culture medium was collected every 2-3 days and polyhistidine-tagged proteins were purified by ammonium sulphate precipitation and Ni 2+ -nitrilotriacetic acid affinity chromatography. Highly pure, active, glycosylated recombinant proteins, including human β-glucuronidase, mouse β-glucuronidase, aminopeptidase N (CD13) and a single-chain antibody-enzyme fusion protein, were obtained with yields of 3-6 mg/l of culture medium. Fc-tagged proteins were also produced and purified in a single step by Protein A affinity chromatography with yields of 6-12 mg/l. The techniques described here allow simple and economical production of recombinant mammalian proteins with post-translational modifications.

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

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.