The interleukin (IL)-23/T helper (Th)­17 axis plays a critical role in autoimmune diseases, and there is an increasing number of biologic therapies that target IL-23 and IL-17. The transcription factor retinoic acid receptor-related orphan nuclear receptor γt (RORγt) is important for the activation and differentiation of Th17 cells and thus is an attractive pharmacologic target for the treatment of Th17-mediated diseases. A novel series of pyrazinone RORγ antagonists was discovered through hybridization of two distinct screening hits and scaffold hopping. The series offers attractive potency and selectivity in combination with favorable druglike properties, such as metabolic stability and aqueous solubility. Lead optimization identified a clinical candidate, compound (S)-11 (BI 730357), for the treatment of autoimmune diseases.

Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner.

Topoisomerase inhibitors are potent DNA damaging agents with great potential as anti-cancer drugs for a wide range of solid tumors. However, dose-limiting toxicities such as myelosuppression and gastric toxicity have prevented them from reaching their full clinical potential. Targeting topoisomerase inhibitors with antibodies (i.e. antibody-drug conjugates; ADCs) may enhance the therapeutic window of these agents, but this approach typically limits applicability to a small subset of patients with tumors expressing the target antigen. We recently developed the alphalexTM tumor-targeting platform to overcome the limitations of ADC-based therapeutic strategies. Rather than targeting a specific antigen, alphalexTM consists of a unique variant of pH-Low Insertion Peptide, (pHLIP®; references 1-3) which targets the low pH environment of the tumor, a universal feature characteristic of all tumors due to the Warburg effect. These alphalexTM conjugates form an alpha helix only in low pH conditions, allowing for insertion of the peptide within the cancer cell membrane, delivery of C-terminal warheads across the membrane, and subsequent intracellular release of the agent via glutathione reduction of the linker, thereby allowing for tumor-specific intracellular delivery in an antigen-independent manner. We report the discovery and development of CBX-12, an alphalexTM conjugate of the potent topoisomerase inhibitor, exatecan. CBX-12 provides additional proof of mechanism to the alphalexTM platform by displaying remarkable tumor-targeting properties in preclinical models. CBX-12 displays enhanced stability in plasma in vivo, undergoing only 0.003% warhead release over 30 hours in circulation and demonstrating exquisite selectivity for tumor over normal tissues in mouse tumor models. Notably, CBX-12 allows for efficient delivery of exatecan into tumors due to a highly optimized cleavable linker, allowing CBX-12 to display extraordinary efficacy in a HER2-negative tumor model in an antigen-independent manner. At 10 mg/kg, CBX-12 treatment almost completely suppressed growth of human colorectal tumors in mice, with complete sparing of bone marrow. In contrast, in animals dosed with the equimolar free exatecan (1.15 mg/kg) there was substantial tumor growth accompanied by neutropenia and weight loss. This superior profile of CBX-12 allow us to greatly enhance efficacy relative to dosing equimolar amounts of unconjugated exatecan, which causes significant, dose-limiting bone marrow toxicity. We have demonstrated that CBX-12 is both safe and has potent anti-tumor activity in preclinical models, and we plan to rapidly move forward with the clinical development of CBX-12 as our lead candidate. References 1. Rather than targeting a specific antigen, alphalexTM includes a pHLIP® peptide. pHLIP® peptides are a family of pH-Low Insertion Peptides that target acidic cell surfaces. pHLIP® was developed at Yale University and the University of Rhode Island and is exclusively licensed to pHLIP, Inc. 2. Wyatt LC et al. Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotechnol. 2017;35(7):653-664. 3. Wyatt LC et al. Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors. PNAS. 2018;115(12):E2811-E2818. Citation Format: Robert J. Aiello, Sophia Gayle, Jane Bechtold, Patricia Bourassa, Johanna Csengery, Ketaki Deshpande, Kelli Jones, Lori Lopresti-Morrow, Robert Maguire, Dan Marshall, Hunter Moore, Timothy Paradis, Laurie Tylaska, Qing Zhang, Robert Volkmann, Ranjit S. Bindra, Peter M. Glazer, Vishwas Paralkar. CBX-12: A low pH targeting alphalex™-exatecan conjugate for the treatment of solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6249.

Poly (ADPribose) polymerase inhibitors (PARPi) have shown great promise in the treatment of cancer, however, their current clinical use has been largely limited to homologous recombination-deficient (HRD) tumors. While these drugs are efficacious as monotherapies, durable responses beyond 12 months are uncommon and their activity against HRD-negative tumors is limited. These findings have prompted great interest in combining PARPi’s with chemotherapy to increase the duration of response in HRD-positive tumors and expand the activity of these drugs against HRD-negative tumors which comprise a much greater fraction of the total number of cancer cases each year. While such combinations are highly active against tumors independent of HRD status, they are also extremely cytotoxic against bone marrow cells. As such, dose reductions ranging from 5- to 20-fold are required when PARPi’s are combined with chemotherapy which has resulted in favorable safety profiles but limited efficacy. Tumor-targeting strategies could overcome this efficacy barrier but the technologies developed thus far are limited by numerous issues, including: (a) lack of universal, non-saturating tumor-targeting mechanisms which limit their use to specific tumor types and their corresponding antigens, (b) inability to deliver therapeutically relevant levels of drug(s) directly into tumor cells, and (c) insufficient tumor penetration leading to sub-optimal tumor exposure. Herein, we report the development of alphalex, a novel tumor-targeted drug delivery platform which allows the efficient and selective delivery of PARPi’s into tumors with significant normal-tissue sparing. Tumor-targeting is achieved using a pH- sensitive peptide which forms an alpha-helix in cellular membrane under low pH conditions associated with the tumor microenvironment. Under these conditions, the peptide translocates across cancer cell membranes to deliver the PARPi directly into the cytoplasm. We demonstrate that alphalex peptide-based conjugates can be combined safely and effectively with both cytotoxic chemotherapies and radiation therapy (RT) and that this approach can be used to selectively kill both HRD-positive and –negative tumors with significant sparing of the bone marrow. The safety and efficacy of the approach was demonstrated in both engineered and patient-derived xenografts (PDXs) in vivo using two FDA-approved PARPi’s across two independent laboratories. These data highlight an entirely new approach to apply PARPi’s against solid tumors independent of HRD status. Furthermore, our approach can be applied to a diverse range of DNA repair inhibitors for potent and selective chemo/radio-sensitization in a tissue-agnostic manner. Citation Format: Vishwas Paralkar, Robert J. Aiello, Dan Marshall, Johanna Csengery, Patricia Bourassa, Qing Zhang, Brett S. Robinson, Lori Lopresti-Morrow, Jane Bechtold, Laurie Tylaska, Timothy Paradis, Gregory Slaybaugh, Hanna Visca, Anna Moshnikova, Dhammika Weerakkody, Oleg Andreev, Yana K. Reshetnyak, Donald Engelman, Ranjit Bindra, Peter Glazer, Per Hellsund. Targeting solid tumor acidic microenvironment with an alphalex PARP inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2981.

e14664 Background: Poly(ADP-ribose)polymerase inhibitors (PARPi’s) are a promising new class of anti-cancer agents, but their clinical application has largely been limited to tumors with homologous recombination deficiency (HRD), such as those with BRCA1/2 mutations. One strategy to target HRD-negative tumors with PARPi’s is to combine them with chemotherapy, although clinical trials indicate that dose-limiting toxicities are a major barrier to achieving synergistic efficacy with these combinations. Methods: We sought to test the hypothesis that HRD-negative cancers can be effectively treated with tumor-targeted PARPi’s in combination with chemotherapy, using our recently developed alphalex platform. This platform allows small molecule anti-cancer agents to penetrate cell membranes only at the low pH associated with the tumor microenvironment and tumor cells, directly delivering drugs to tumors while sparing normal tissue. We tested whether alphalex PARPi-conjugates in combination with chemotherapy could selectively kill cancers independent of HRD status, using a range of in vitro and in vivo tumor models. Results: We conjugated a diverse range of structurally unique PARPi’s using the alphalex platform, and demonstrated that these molecules are delivered directly into tumor cells in a pH-dependent manner. We observed significant reductions in PARylation activity and exquisite synergy with DNA damaging agents in vitro. We then demonstrated that alphalex-PARPi conjugates in combination with both temozolomide (TMZ) and irinotecan induce significant tumor cell killing in HRD-negative tumors in vivo. Importantly, we found that our tumor-targeting approach significantly reduced normal tissue toxicity, with almost complete sparing of the bone marrow relative to TMZ alone. Conclusions: The alphalex platform enables PARPi combinations with clinically relevant chemotherapies, as a means to target HRD-negative cancers without significant bone marrow toxicity. Based on these successful proof-of-concept data, we are now performing IND-enabling studies for an alphalex PARPi conjugate (CBX-11), and we anticipate initiating a Phase I clinical trial in January 2020 in solid tumors independent of HRD status.