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Background: NKTR-262 is a novel therapeutic, which delivers intratumoral engagement of the TLR 7/8 pathway, promoting an immune stimulatory environment and local injection site tumor antigen release. When NKTR-262 is administered in combination with NKTR-214, a CD122-biased cytokine agonist currently in clinical trials as a monotherapy and in combination with nivolumab, the combined effect of innate immune stimulation and enhanced antigen presentation with sustained T cell activation leads to systemic tumor immunity. Materials and methods: Ten subcutaneous syngeneic mouse tumor models with diverse histologies were assessed for NKTR-262 and NKTR-214 combination treatment efficacy. Once established, tumors were treated with a single peritumoral dose of NKTR-262, while NKTR-214 was administered i.v. on q9dx3 schedule. A subset of tumor models were inoculated bilaterally to assess abscopal effect of the combination treatment. Regression of injected tumors and the abscopal effect in contralateral untreated tumors was assessed by tumor size measurements. In bilateral models immune cell activation in both tumors was assessed by flow cytometry. Cytokine induction was measured in plasma and tumors in select models. Results: Combination treatment with NKTR-262 and NKTR-214 showed efficacy in all tested tumor models. Efficacy varied from significant tumor growth inhibition to up to 100% complete responses in multiple models. Synergistic efficacy was demonstrated in select models where single agent NKTR-262 or NKTR-214 activity was compared to the combination treatment. Immune cell phenotyping showed that combining NKTR-262 with NKTR-214 induced a two-step immune response in treated and abscopal tumors. At early timepoints, accumulation of activated granulocytes correlated with tumor cell death and dendritic cell activation. The innate response was followed by selective tumor infiltration by CD8 T cells and a reduction of immunorepressive cells. Single agent treatment showed only a subset of the cellular changes observed in the combination. Immune cell activation was shown to correlate with immune stimulatory cytokine release in NKTR-262 treated tumors. Conclusions: We present a designed combination therapy that mimics a natural immune response by activating a broad immune cell network in multiple nonclinical tumor models independent of tissue origin. Combining NKTR-262 and NKTR-214 engages the entire immune activation cascade required for systemic tumor clearance from local tumor antigen production to a sustained systemic T cell response. Unlike treatments that stimulate downstream components of select immune pathways without eliciting systemic tumor immunity, a comprehensive anti-tumor immune activation by coordinated engagement of innate and adaptive immune cells may increase the success of immune therapy for patients. Citation Format: Saul Kivimae, Marlene Hennessy, Rhoneil Pena, Yolanda Kirksey, Wildaliz Nieves, Phi Quatch, Janet Cetz, Zhongxu Ren, Haiying Cai, BoLiang Deng, Wen Zhang, John L. Langowski, Christie Fanton, Neel K. Anand, Werner Rubas, Steve Doberstein, Jonathan Zalevsky. Comprehensive antitumor immune activation by a novel TLR7/8 targeting agent NKTR-262 combined with CD122-biased immunostimulatory cytokine NKTR-214 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3755.

Introduction: NKTR-214 is a biologic prodrug currently in a Phase 1 / 2 clinical trial in patients with solid tumors, as a single agent and in combination with anti-PD1. It is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Tregs. Here we describe a mechanistic mathematical model that quantifies conjugated aldesleukin (IL-2) species that are generated from NKTR-214, their sustained exposure, and biased receptor pharmacology in rodent models. Methods: A mechanistic mathematical model, using ordinary differential equations (ODE), was developed using Matlab® programming language to describe the dynamics of NKTR-214 PEG release, clearance, distribution, and receptor occupancy in vivo. The model was conditioned using experimental data for in vitro binding of unconjugated IL-2 and active conjugated IL-2 derived from NKTR-214 to IL-2 receptors using surface plasmon resonance, in vitro PEG release data, and mouse pharmacokinetics data. Simulations were performed to 1) quantify the concentration-time profiles of the various conjugated IL-2 species derived from NKTR-214 and contribution of each of them to the NKTR-214 mechanism of action, and 2) compare occupancy at IL-2Rβγ and IL-2Rαβγ receptors after administration of therapeutic doses of NKTR-214 and aldesleukin. Results: After NKTR-214 administration, PEG chains are released slowly from the prodrug and active conjugated IL-2 species gradually increase and reach maximum concentrations about one day post-dose, followed by sustained exposure for up to a week. Compared to an equivalent dose of aldesleukin, the active conjugated IL-2 species derived from NKTR-214 achieve a 26-fold higher area under the curve (AUC) of IL-2Rβγ occupancy, and a 0.34-fold lower AUC of IL-2Rαβγ occupancy. The significant difference is due to the combined effects of slow release of active conjugated IL-2 species from NKTR-214 and their favorable binding kinetics towards IL-2Rβγ. Aldesleukin, even when simulated at repeated daily doses or constant infusion, is incapable of increasing the receptor occupancy at IL-2Rβγ without simultaneously increasing the receptor occupancy at IL-2Rαβγ in this model. Conclusions: The mechanistic model demonstrated how NKTR-214’s intrinsic design enables both biased receptor pharmacology and sustained exposure in vivo. Such biased receptor binding could not be achieved by a sustained delivery of aldesleukin. Citation Format: Samira Khalili, Aleksandrs Odinecs, Deborah H. Charych, Vidula Dixit, Peter Kirk, Thomas Chang, John Langowski, Werner Rubas, Steve Doberstein, Jonathan Zalevsky, Michael A. Eldon, Ute Hoch. Mechanistic modeling of a new kinetically-controlled CD122 agonist for cancer immunotherapy: NKTR-214 pharmacokinetics, pharmacodynamics, and receptor pharmacology [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 1617. doi:10.1158/1538-7445.AM2017-1617

Introduction: Talazoparib (BMN 673) is a highly potent and specific PARP1/2 inhibitor (PARPi) that has demonstrated significant clinical activity in patients with germline BRCA mutation ovarian and breast cancer, as well as in patients with small cell lung cancer. Non-clinically, combination of PARPi, including talazoparib, with Topoisomerase 1 (Top1) inhibitors, such as irinotecan, has shown synergy in BRCA1 mutant MX-1 model (Shen et al. 2013; Hoch et al., NCI-AACR-EORTC 2014). Etirinotecan pegol (NKTR-102, pegylated irinotecan) is a long-acting Top1 inhibitor, designed to provide extended drug exposure to the tumor. In preclinical models, NKTR-102 has improved efficacy and tolerability over irinotecan. We therefore hypothesized that the combination of talazoparib with NKTR-102 would be well tolerated and harness the full synergistic potential of combined Top1 and PARP inhibition. Method: In tolerability studies, three dose levels of talazoparib (0.1, 0.2 and 0.3 mg/kg, QDx14) given with NKTR-102 (10 and 50 mg/kg irinotecan-equivalent dose, iv. Q7Dx2), or irinotecan (10, 30 and 60 mg/kg, ip, Q7Dx2) were evaluated in non-tumor-bearing nude mice (Balb/c, n = 4 per group). In efficacy studies, multiple dose levels of a single agent (i.e., talazoparib, NKTR-102, or irinotecan) as well as corresponding dose combinations were evaluated in NCI-H1048 and HT-29 xenograft tumors with wildtype BRCA status (n = 8-10 per group). Tumor volume and body weight were measured twice weekly. Results: For talazoparib and NKTR-102, all combination doses were tolerated, the maximum mean body weight loss reached 6.2% in the high dose group (0.3 mg/kg talazoparib plus 50 mg/kg NKTR-102). For talazoparib and irinotecan, MTD was observed at 0.2 mg/kg BMN 673 plus 30 mg/kg irinotecan. In efficacy studies, single-agent NKTR-102 had dose-dependent anti-tumor activity against NCI-H1048 tumor, while talazoparib showed limited activity with QDx14 dosing schedule. Combination of talazoparib (0.2 or 0.3 mg/kg, QDx14) with NKTR-102 (10 mg/kg, Day 1) exhibited significantly greater anti-tumor effect than either single agent alone. In contrast, combination of talazoparib (0.2 mg/kg, QDx14) with irinotecan (30 mg/kg, Day 1) only had moderate combination effect in the NCI-H1048 model. In HT-29 model, additive anti-tumor effect was also observed for talazoparib + NKTR-102, but not for talazoparib + irinotecan under the same experimental condition. Conclusion: Our data indicate that the combination of NKTR-102 and talazoparib is better tolerated than that of irinotecan and talazoparib, and results in stronger combination anti-tumor activity. The results support potential development of the combination in human studies. Citation Format: Ying Feng, Yuqiao (Jerry) Shen, Leonard E. Post, Steve Doberstein, Deborah Charych, Ute Hoch. Combination of PARP inhibitor talazoparib with etirinotecan pegol exhibits synergistic anti-tumor effect in non-BRCA preclinical cancer models. [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 C54.

Anillin is a conserved component of the contractile ring that is essential for cytokinesis, and physically interacts with three conserved cleavage furrow proteins, F-actin, myosin II and septins in biochemical assays. We demonstrate that the Drosophila scraps gene, identified as a gene involved in cellularization, encodes Anillin. We characterize defects in cellularization,pole cell formation and cytokinesis in a series of maternal effect and zygotic anillin alleles. Mutations that result in amino acid changes in the C-terminal PH domain of Anillin cause defects in septin recruitment to the furrow canal and contractile ring. These mutations also strongly perturb cellularization, altering the timing and rate of furrow ingression. They cause dramatic vesiculation of new plasma membranes, and destabilize the stalk of cytoplasm that normally connects gastrulating cells to the yolk mass. A mutation closer to the N terminus blocks separation of pole cells with less effect on cellularization, highlighting mechanistic differences between contractile processes. Cumulatively, our data point to an important role for Anillin in scaffolding cleavage furrow components, directly stabilizing intracellular bridges, and indirectly stabilizing newly deposited plasma membrane during cellularization.

To identify novel protein therapeutics we have utilized an integrated screening system in which a collection of full-length human cDNA clones, encoding virtually all secreted proteins and receptors, was assembled. Soluble secreted proteins were expressed in a high-throughput format using human cells as the expression host and tested in high-throughput cell-based assays. Through this approach, a novel cytokine, FPT025, was identified in a human monocyte proliferation assay. FPT025 has no apparent sequence homology to known cytokines or any other genes and is expressed in human spleen, skin, brain, and other tissues. The purified recombinant FPT025 protein stimulated human primary monocyte proliferation and/or survival. FPT025 protein specifically bound to human primary monocytes and activated ERK1/2 phosphorylation in primary monocytes as well as in a human monocytic cell line, THP-1. FPT025 promoted formation of the myeloid lineage colonies, CFU-M, in a human bone marrow colony formation assay and enhanced proliferation of cells with myeloid cell surface markers from human monocytes. To identify the receptor of FPT025, a collection of extracellular domains (ECD) of transmembrane proteins was screened for their ability to block FPT025 activation of monocyte proliferation. In this screen, we found that FPT025 binds to the M-CSF receptor (M-CSFR) with high affinity. Furthermore, we showed that the binding of FPT025 to M-CSFR was specific and could be competed by M-CSF. The soluble ECD of M-CSFR inhibited both the binding of FPT025 to M-CSFR and the activity of FPT025 in monocyte proliferation. Therefore, FPT025 functions as a novel ligand of the M-CSF receptor and participates in the regulation of myeloid lineage differentiation, proliferation, and survival.