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In vitro hydrolysis of NKTR-214 under physiological conditions generating NKTR-214 released IL-2 conjugates

Purpose: Aldesleukin, recombinant human IL2, is an effective immunotherapy for metastatic melanoma and renal cancer, with durable responses in approximately 10% of patients; however, severe side effects limit maximal dosing and thus the number of patients able to receive treatment and potential cure. NKTR-214 is a prodrug of conjugated IL2, retaining the same amino acid sequence as aldesleukin. The IL2 core is conjugated to 6 releasable polyethylene glycol (PEG) chains. In vivo, the PEG chains slowly release to generate active IL2 conjugates.Experimental Design: We evaluated the bioactivity and receptor binding of NKTR-214 and its active IL2 conjugates in vitro; the tumor immunology, tumor pharmacokinetics, and efficacy of NKTR-214 as a single agent and in combination with anti–CTLA-4 antibody in murine tumor models. Tolerability was evaluated in non-human primates.Results: In a murine melanoma tumor model, the ratio of tumor-killing CD8+ T cells to Foxp3+ regulatory T cells was greater than 400 for NKTR-214 compared with 18 for aldesleukin, supporting preferential activation of the IL2 receptor beta over IL2 receptor alpha, due to the location of PEG molecules. NKTR-214 provides a 500-fold greater exposure of the tumor to conjugated IL2 compared with aldesleukin. NKTR-214 showed efficacy as a single agent and provided durable immunity that was resistant to tumor rechallenge in combination with anti–CTLA-4 antibody. NKTR-214 was well tolerated in non-human primates.Conclusions: These data support further evaluation of NKTR-214 in humans for a variety of tumor types, adding to the repertoire of potent and potentially curative cancer immunotherapies. Clin Cancer Res; 22(3); 680–90. ©2016 AACR.

Supplemental Materials and Methods. ELISA study materials; NKTR-214 chemistry; Mapping of PEGylation sites; Identification of active released IL-2 conjugates; Binding affinity to IL-2 receptors; In vivo immune cell phenotyping, In vivo immune cell phenotyping; In vivo T cell antigen staining; In vivo depletion of immune cells; Hematology parameters for evaluation of immune markers in non-human primates. Supplemental Figure Legends S1-S4. Supplemental Tables S1 and S2. Table S1: Fold change of affinity to IL-2αβ and IL-2β for NKTR-214 and its released active IL-2 conjugates compared to aldesleukin; Table S2: Activation of Primary Cynomulgus Monkey and Human T Cells in Response to IL-2 and the Active Metabolite of NKTR-214

BackgroundCurrent clinical trials are using radiation therapy (RT) to enhance an antitumor response elicited by high-dose interleukin (IL)-2 therapy or immune checkpoint blockade (ICB). Bempegaldesleukin (BEMPEG) is an investigational CD122-preferential IL-2 pathway agonist with prolonged in vivo half-life and preferential intratumoral expansion of T effector cells over T regulatory cells. BEMPEG has shown encouraging safety and efficacy in clinical trials when used in combination with PD-1 checkpoint blockade. In this study, we investigated the antitumor effect of local RT combined with BEMPEG in multiple immunologically ‘cold’ tumor models. Additionally, we asked if ICB could further enhance the local and distant antitumor effect of RT+BEMPEG in the setting of advanced solid tumors or metastatic disease.MethodsMice bearing flank tumors (B78 melanoma, 4T1 breast cancer, or MOC2 head and neck squamous cell carcinoma) were treated with combinations of RT and immunotherapy (including BEMPEG, high-dose IL-2, anti(α)-CTLA-4, and α-PD-L1). Mice bearing B78 flank tumors were injected intravenously with B16 melanoma cells to mimic metastatic disease and were subsequently treated with RT and/or immunotherapy. Tumor growth and survival were monitored. Peripheral T cells and tumor-infiltrating lymphocytes were assessed via flow cytometry.ResultsA cooperative antitumor effect was observed in all models when RT was combined with BEMPEG, and RT increased IL-2 receptor expression on peripheral T cells. This cooperative interaction was associated with increased IL-2 receptor expression on peripheral T cells following RT. In the B78 melanoma model, RT+BEMPEG resulted in complete tumor regression in the majority of mice with a single ~400 mm3tumor. This antitumor response was T-cell dependent and supported by long-lasting immune memory. Adding ICB to RT+BEMPEG strengthened the antitumor response and cured the majority of mice with a single ~1000 mm3B78 tumor. In models with disseminated metastasis (B78 primary with B16 metastasis, 4T1, and MOC2), the triple combination of RT, BEMPEG, and ICB significantly improved primary tumor response and survival.ConclusionThe combination of local RT, BEMPEG, and ICB cured mice with advanced, immunologically cold tumors and distant metastasis in a T cell-dependent manner, suggesting this triple combination warrants clinical testing.

BackgroundHigh-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression. NKTR-214 has increased drug half-life, decreased toxicity, and increased CD8+T cell and natural killer cell stimulation compared with IL-2.MethodsAnimals bearing bilateral subcutaneous MCA-205 fibrosarcoma or CT26 colorectal tumors were treated with NKTR-214, RT, or combination therapy, and tumor growth of irradiated and abscopal lesions was assessed. Focal RT was delivered using a small animal radiation research platform. Peripheral and tumor-infiltrating immune phenotype and functional analyses were performed by flow cytometry. RNA expression profiling from both irradiated and abscopal lesions was performed using microarray.ResultsWe demonstrate synergy between RT of a single tumor and NKTR-214 systemic therapy resulting in dramatically increased cure rates of mice bearing bilateral tumors compared with RT or NKTR-214 therapy alone. Combination therapy resulted in increased magnitude and effector function of tumor-specific CD8+T cell responses and increased trafficking of these T cells to both irradiated and distant, unirradiated, tumors.ConclusionsGiven the increasing role of hypofractionated and stereotactic body RT as standard of care treatments in the management of locally advanced and metastatic cancer, these data have important implications for future clinical trial development. The combination of RT and NKTR-214 therapy potently stimulates systemic antitumor immunity and should be evaluated for the treatment of patients with locally advanced and metastatic solid tumors.

High dose interleukin-2 (IL-2) is active against metastatic melanoma and renal cell carcinoma, but treatment-associated toxicity and expansion of suppressive regulatory T cells (Tregs) limit its use in patients with cancer. Bempegaldesleukin (NKTR-214) is an engineered IL-2 cytokine prodrug that provides sustained activation of the IL-2 pathway with a bias to the IL-2 receptor CD122 (IL-2Rβ). Here we assess the therapeutic impact and mechanism of action of NKTR-214 in combination with anti-PD-1 and anti-CTLA-4 checkpoint blockade therapy or peptide-based vaccination in mice. NKTR-214 shows superior anti-tumor activity over native IL-2 and systemically expands anti-tumor CD8+ T cells while inducing Treg depletion in tumor tissue but not in the periphery. Similar trends of intratumoral Treg dynamics are observed in a small cohort of patients treated with NKTR-214. Mechanistically, intratumoral Treg depletion is mediated by CD8+ Teff-associated cytokines IFN-γ and TNF-α. These findings demonstrate that NKTR-214 synergizes with T cell-mediated anti-cancer therapies., Interleukin-2 can induce an anti-tumour response, but is associated with toxicity. Here, the authors demonstrate that an engineered interleukin-2 promotes intratumoral T regulatory cell depletion while enhancing effective anti-tumour CD8+ T cell responses that result in potent tumor suppression.

Interleukin-2 (IL-2) is a component of most protocols of adoptive cell transfer (ACT) therapy for cancer, but is limited by short exposure and high toxicities. NKTR-214 is a kinetically-engineered IL-2 receptor βγ (IL-2Rβγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rβγ. We report that ACT supported by NKTR-214 increases the proliferation, homing and persistence of anti-tumor T cells compared to ACT with IL-2, resulting in superior antitumor activity in a B16-F10 murine melanoma model. The use of NKTR-214 increases the number of polyfunctional T cells in murine spleens and tumors compared to IL-2, and enhances the polyfunctionality of T and NK cells in the peripheral blood of patients receiving NKTR-214 in a phase 1 trial. In conclusion, NKTR-214 may have the potential to improve the antitumor activity of ACT in humans through increased in vivo expansion and polyfunctionality of the adoptively transferred T cells., Adoptive cell transfer (ACT) of T cells for tumor treatment often requires IL-2 administration. Here, the authors show that a modified IL-2 cytokine (NKTR-214) can outperform IL-2 in a melanoma mouse model.

BackgroundA number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the ‘Cancer Immunity Cycle’. Anti-PD-1 checkpoint blockade (CPB) therapy is active on one of these pathways and reinvigorates anticancer T cell immunity, leading to long-term responses in a limited fraction of patients with cancer. We have previously shown that neoantigens-based adenovirus vectored vaccine in combination with anti-PD-1 further expands pre-existing anticancer immunity and elicits novel neoantigen-specific T cells thereby increasing efficacy to 50% of tumor clearance in mice. Here we added a third component to the CPB plus vaccine combination, which is able to modify the suppressive tumor microenvironment by reducing the number of tumor-infiltrating regulatory T cells (Tregs), as strategy for improving the therapeutic efficacy and overcoming resistance.MethodsThe antitumor efficacy of anti-PD-1, neoantigen vaccine and Treg modulating agents, either Bempegaldesleukin (BEMPEG: NKTR-214) or an anti-CTLA-4 mAb with Treg-depleting activity, was investigated in murine tumor models. We evaluated tumor growth in treated animals, neoantigen-specific T cells in tumors, tumor-infiltrating lymphocytes (TILs) and intratumoral Tregs.ResultsThe addition of BEMPEG or anti-CTLA-4 to the combination of vaccine and anti-PD-1 led to complete eradication of large tumors in nearby 100% of treated animals, in association with expansion and activation of cancer neoantigen-specific T cells and reduction of tumor-infiltrating Tregs.ConclusionThese data support the notion that the integrated regulation of three steps of the cancer immunity cycle, including expansion of neoantigen-specific T cells, reversal of the exhausted T cell phenotype together with the reduction of intratumoral Tregs may represent a novel rationally designed drug combination approach to achieve higher cure rates.

Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug. When administered in vivo, the PEG chains slowly release, creating a cascade of increasingly active IL2 protein conjugates bound by fewer PEG chains. The 1-PEG-IL2 and 2-PEG-IL2 species derived from NKTR-214 are the most active conjugated-IL2 species. Free-IL2 protein is undetectable in vivo as it is eliminated faster than formed. The PEG chains on NKTR-214 are located at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαβγ, reducing its ability to bind and activate the heterotrimer. The IL2Rαβγ complex is constitutively expressed on regulatory T cells (Tregs). Therefore, without the use of mutations, PEGylation reduces the affinity for IL2Rαβγ to a greater extent than for IL2Rβγ, the receptor complex predominant on CD8 T cells. NKTR-214 treatment in vivo favors activation of CD8 T cells over Tregs in the tumor microenvironment to provide anti-tumor efficacy in multiple syngeneic models. Mechanistic modeling based on in vitro and in vivo kinetic data provides insight into the mechanism of NKTR-214 pharmacology. The model reveals that conjugated-IL2 protein derived from NKTR-214 occupy IL-2Rβγ to a greater extent compared to free-IL2 protein. The model accurately describes the sustained in vivo signaling observed after a single dose of NKTR-214 and explains how the properties of NKTR-214 impart a unique kinetically-controlled immunological mechanism of action.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Purpose: Aldesleukin, recombinant human IL2, is an effective immunotherapy for metastatic melanoma and renal cancer, with durable responses in approximately 10% of patients; however, severe side effects limit maximal dosing and thus the number of patients able to receive treatment and potential cure. NKTR-214 is a prodrug of conjugated IL2, retaining the same amino acid sequence as aldesleukin. The IL2 core is conjugated to 6 releasable polyethylene glycol (PEG) chains. In vivo, the PEG chains slowly release to generate active IL2 conjugates. Experimental Design: We evaluated the bioactivity and receptor binding of NKTR-214 and its active IL2 conjugates in vitro; the tumor immunology, tumor pharmacokinetics, and efficacy of NKTR-214 as a single agent and in combination with anti–CTLA-4 antibody in murine tumor models. Tolerability was evaluated in non-human primates. Results: In a murine melanoma tumor model, the ratio of tumor-killing CD8+ T cells to Foxp3+ regulatory T cells was greater than 400 for NKTR-214 compared with 18 for aldesleukin, supporting preferential activation of the IL2 receptor beta over IL2 receptor alpha, due to the location of PEG molecules. NKTR-214 provides a 500-fold greater exposure of the tumor to conjugated IL2 compared with aldesleukin. NKTR-214 showed efficacy as a single agent and provided durable immunity that was resistant to tumor rechallenge in combination with anti–CTLA-4 antibody. NKTR-214 was well tolerated in non-human primates. Conclusions: These data support further evaluation of NKTR-214 in humans for a variety of tumor types, adding to the repertoire of potent and potentially curative cancer immunotherapies. Clin Cancer Res; 22(3); 680–90. ©2016 AACR.

NKTR-214 is a CD122-biased cytokine agonist designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand CD8 T and natural killer cells (NK) over Tregs in the tumor. We evaluated the tumor immunology, biodistribution and anti-tumor activity of NKTR-214 combined with ACT in the pmel-1 ACT/B16F10 melanoma tumor model. NKTR-214+ACT provides a robust and durable anti-tumor response compared to IL-2+ACT with less frequent dosing in the aggressive B16F10 model. NKTR-214+ACT led to significant tumor growth inhibition at day 14 compared with IL-2+ACT, 174mm3 vs 484mm3 tumor volume, respectively (p Citation Format: Giulia Parisi, Justin Saco, Felix Bergara, Paige Krystofinski, Ruixue Zhang, Cristina Puig Saus, Siwen Hu-Lieskovan, Begonya Comin-Anduix, Anna Wu, Deborah H. Charych, Antoni Ribas. Enhanced expansion and tumor targeting of adoptively transferred T cells with 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 3566.

3085Background: BXCL701 targets DPP8/9 and stimulates migration and cytotoxicity of T and NK cells in addition to targeting fibroblast activator protein (FAP) which forms an immunological barrier t...

5582Background: NKTR-214 is a biased agonist of the IL2Rbg (CD122) pathway that activates and mobilizes CD8 T and NK cells into the tumor microenvironment. The PARP inhibitor rucaparib has demonstr...

The adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) has been shown to induce effective anti-tumor response. However, tumors frequently relapse after an initial response. Another strategy towards stimulating the immune system is the use of high-dose interleukin-2 (IL-2) to target the IL-2 receptor (IL2R), leading to immune cell expansion. However, clinically approved IL-2 expands both tumor-killing CD8+ effector T cells (CD8T) as well as regulatory T cells (Tregs) through binding the IL-2Rβγ and IL-2Rαβγ complexes, respectively. Tregs in the tumor lead to immune suppression, which hampers the antitumoral response. NKTR-214 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. We used the pmel-1 ACT/B16 melanoma tumor model to test the anti-tumor activity of NKTR-214 and evaluate its effects on tumor-specific TCR transgenic T cells. On Day 0 (D0) C57BL/6 mice were implanted with B16-F10 mouse melanoma cells and lymphodepleted with 500 cGy on D6. On D7, mice were treated with either the combination of ACT (T lymphocytes activated in vitro with 1 μg/ml gp100) plus NKTR-214 (0.8 mg/kg, q9dx3, i.v.) or with C57/B6 T cells plus PBS (vehicle control). The tumors of the vehicle control mice (n=12) rapidly grew to the 1500mm3 endpoint in 12 days post-treatment, versus 35 days for the NKTR-214 group (n=12) with only 1 out of 12 mice reaching endpoint. Bioluminescence imaging was used to visualize the in vivo distribution and tumor-homing of antigen-specific T cells. Interestingly, the reporter T cells were retained in the spleen until D7 and could be seen migrating to the tumor at D9 reaching peak of bioluminescence at day 12, a delayed time point compared to the 5 days usually observed in mice treated with standard IL-2. The signal persisted in the NKTR-214 + ACT group until D20 versus D7 in the vehicle-control animals. These data suggest that NKTR-214 + ACT is well tolerated and provides a robust anti-tumor response in the aggressive B16F10 model. Treatment with NKTR-214 + ACT robustly mobilizes T cells into the tumor where they durably persist. The robust and long-lasting effect of NKTR214 supports its potential use in combination with cell-based therapeutics. Citation Format: Giulia Parisi, Justin Saco, Siwen Hu-Lieskovan, Ruixue Zhang, Paige Krystofinski, Cristina Puig Saus, Deborah H. Charych, Antoni Ribas. Antitumor activity of NKTR-214 in combination with pmel-1 ACT in an aggressive murine melanoma model [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 2671. doi:10.1158/1538-7445.AM2017-2671

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

The purpose of this study was to investigate therapeutic and mechanistic synergies between single-dose radiotherapy and systemic administration of NKTR-214. NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol. NKTR-214 is 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 Regulatory CD4 T cells. Preclinical models demonstrated NKTR-214 preferentially expands effector CD8+ T cells and NK cells within the tumor resulting in marked tumor growth suppression as a single-agent and in combination with checkpoint inhibitors. A phase I/II trial is in progress to evaluate NKTR-214 safety and efficacy in an outpatient setting. Radiation therapy can induce antigen-release and epitope spreading, while NKTR-214 can active and expand antigen-specific effector populations. We hypothesized that the combination of systemic NKTR-214 and local radiotherapy would generate better therapeutic responses than either treatment alone. In this study, we evaluated the combination of systemic NKTR-214 treatment with single fraction high-dose radiotherapy (20 Gy) in multiple murine models. We used flow cytometry, multi-spectral histology, and whole tumor mRNA profiling to investigate local, systemic and potential abscopal immune responses. We used Nur77-GFP reporter mice to enable detection of T cell receptor ligation in vivo and to evaluate the effects of NKTR-214+RT on tumor-reactive T cells. The results from these studies indicate that the combination of NKTR-214 and radiotherapy is synergistic, providing significantly better anti-tumor responses than either monotherapy. Consistent with previous observations, NKTR-214 alone induces expression of a wide range of activation markers expressed by CD4 and CD8 T cells as well as NK cells in the blood, lymph nodes and tumor. The combination of radiotherapy and NKTR-214 was found to have several unique effects including a significant increase (>75%) in the absolute numbers of lymphocytes in the peripheral blood, increased expression of activation markers (CD25, PD-1) by CD8 T cells in the blood and tumor, and increased density of tumor-infiltrating NK cells. Evaluation of tumor infiltrating lymphocytes (TIL) in Nur77-GFP reporter mice revealed that the combination of NKTR-214+RT resulted in a higher frequency of recently activated (Nur77-GFP+) CD8 T cells in treated (irradiated) and abscopal (non-irradiated) tumors. Whole tumor mRNA profiling and multi-spectral histology of these tumors is being assessed to identify key differences in the tumor-microenvironment that may help to define the underlying mechanism of action. Taken together, these data provide evidence of synergy between localized radiotherapy and systemic NKTR-214 treatment via an expansion of activated, tumor-specific CD8 T cells. Citation Format: Michael J. Mcnamara, Melissa Kasiewicz, Ian Hilgart-Martiszus, Ute Hoch, Deborah H. Charych, William L. Redmond. NKTR-214 synergizes with radiotherapy to drive tumor regression [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 1604. doi:10.1158/1538-7445.AM2017-1604

Background: Immunotherapy is an attractive option for brain tumor therapy if a robust infiltrative T cell response can be elicited in the tumor. NKTR-214 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. To assess the potential activity of single agent NKTR-214 in GBM, we used an orthotopic rat glioblastoma survival model. Methods:NKTR-214 was administered at 0.1 or 0.3 mg/kg q2w iv to Sprague-Dawley rats starting 2 or 7 days (D2, D7) post (p)-implantation of 106 C6 glioma cells into the right striatum. The model requires euthanasia by ~D14 due to tumor burden. Brain tumors were characterized by magnetic resonance (MR) and immunohistochemistry (IHC) for infiltration of CD4+ and CD8+ T cells and for retention of PEG polymer in brain tumor. Results: Compared to rats receiving vehicle (n = 15), survival was significantly prolonged after NKTR-214 treatment (n = 43, mean 17.2 vs. 10.0 days (P < 0.001) with 15% of rats across all groups alive and tumor-free at Day 50 when the study was terminated. Both doses were equally effective and well tolerated. Surprisingly, treated rats bearing large MR-detectable D7 tumors survived significantly longer compared to rats bearing microscopic D2 tumors (6/21 or ~30% versus 0/21 or 0% respectively at D50, mean 23.1 vs. 12 days, P < 0.004). Concordantly, CD8+ T cells in D7 tumors were significantly increased after NKTR-214 therapy compared to vehicle and D2 tumors, while CD4+ remained low with no significant difference between groups. PEG polymer was detected in the tumor at least 72 hours p-injection. Conclusions: NKTR-214 is well tolerated, prolongs survival and induces immunological activity in the brain when administered to rats harboring orthotopic GBM. While there was no significant dose dependence, a marked increase in survival was observed when larger D7 tumors were treated with NKTR-214 compared to microscopic D2 tumors, associated with increased intratumoral CD8+ T cells. Levels of CD4+ were unchanged, consistent with the mechanism of CD-122 biased activation of the IL2 pathway. While requiring further study, it is intriguing that the increased sensitivity of larger tumors also corresponds to onset of angiogenesis and rapid tumor growth in this model. NKTR-214 is currently being evaluated in an outpatient Phase 1 / 2 clinical trial for the treatment of solid tumors. The results presented suggest a potential role for NKTR-214 in the treatment of patients afflicted with GBM. Citation Format: Lawrence Recht, Seema Nagpal, Taichang Jang, Milton Merchant, Irene Choi, Ute Hoch, Deborah Charych. Single agent NKTR-214, a biased IL2 pathway agonist, increases immune cell infiltrates in brain tumors and prolongs survival in rodent (rattus) glioblastoma (GBM) [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 1598. doi:10.1158/1538-7445.AM2017-1598

Layer systems composed of polydiacetylene lipids are known to undergo an irreversible colorimetric transition from blue to red upon exposure to an external trigger (e.g., heat, pH change, specific ligand−target interaction). These optical transitions offer numerous possibilities toward the development of molecule-based sensory materials. Polymerization of the diacetylene systems usually occurs only in a highly ordered phase as a topochemical reaction, if the diacetylenic units are packed in an optimal orientation. A novel system based on hydrazide derivatives of single-chain diacetylene lipids is described. These materials show an unusual aggregation and polymerization behavior in organic solution, in contrast to the parent carboxylic acids. In addition, these hydrazide lipids undergo an unprecedented reversible color change (blue/red) in polymerized vesicles when the pH of the surrounding aqueous medium is cycled between acidic and basic conditions. This unusual behavior is attributed to the unique hydro...

Directed crystallization of calcite and CdS was studied on polymerized 10,12 pentacosadiynoic acid (p-PDA) template. Precise lattice match between calcite a axis and the carboxylates periodicity along the linear polymer backbone, and stereochemical fit between the titled monolayer and inclined carbonate at the (0 1 2) of calcite, induce total control of calcite nucleation. Polymer crystalline domains can be several hundreds of micrometers in dimension. Consequently, the templated calcite crystals are all coaligned within this range. p-PDA film with CdS nucleated on it, produces a selected area electron diffraction pattern which is a superimposition of 4 different projection of CdS FCC structure. All the projections have a common (2 2 0) diffraction spot, suggesting alignment along this direction. This suggests that crystallites can in fact be in any orientation with only one crystal axis aligned with the substrate. This represents a unique example of template crystallization where limited control is manifested. The p-PDA film is particularly suitable for template nucleation studies due to its rigidity and large domains. This results in well-defined crystal orientation over the extent of the substrate domains.

A blue to red color change is induced on addition of phospholipase A2 to modified PDA vesicles 1 (PDA=polydiacetylene). This bathochromic transition results from chemical modification of the vesicles by hydrolysis of the enzyme substrate embedded in the PDA matrix. Addition of a known phospholipase inhibitor or removal of Ca2+ ions suppresses the color change, which suggests the potential for applications in high-throughput screening assays.

Von Blau nach Rot andert sich die Farbe modifizierter PDA-Vesikel 1 bei der Zugabe von Phospholipase A2 (PDA = Polydiacetylen). Diese bathochrome Verschiebung ist die Folge einer chemischen Modifizierung der Vesikel durch die Hydrolyse des in die PDA-Matrix eingebetteten Enzymsubstrats (schematisch gezeigt). Zugabe eines bekannten Phospholipase-Inhibitors oder Entfernung der Ca2 +-Cofaktoren unterdrucken die Farbanderung. Damit sollte das System auch fur das schnelle Screening einer grosen Zahl an Testsubstanzen geeignet sein.

Polymerized thin films based on polydiacetylenes (PDAs) undergo distinct color transitions that lend themselves to applications in biosensing, surface modification, nonlinear optics, and molecular electronics. The mechanism of the thermochromic blue to red color transition of PDA thin films was investigated at the molecular level using atomic force microscopy and at the macroscopic level with visible absorption and Fourier transform infrared spectroscopy. The thermochromic transition temperature is found to be between 70 and 90 °C. At the molecular level, the ordering of the film increases at the thermochromic transition and remains ordered up to temperatures well above the transition (e.g., 130 °C). No evidence for previously suggested entanglement or disordering of the alkyl side chains is observed. The pendant side chains rearrange from a partially disordered configuration characteristic of the blue film,to a well-ordered close-packed hexagonal arrangement in the red form. The rearrangment of the pendant side chains is linked to the formation of the red phase PDA.

A comparative study of the frictional properties of alkanethiols and alkylsilanes as a function of chain length is presented. The monolayers were produced by self-assembly on Au(111) and mica, respectively. The same tip was used for all the experiments, and freshly cleaved mica was used as a reference. For both types of films, the frictional forces depend strongly on the number of carbon atoms in the alkane chain (CH3−(CH2)n-1−R). Thiols and silanes give rise to similar frictional force for the same n when n > 11, while for n < 11 the behavior is markedly different; the silanes exhibit higher friction, larger than that for the thiols by a factor of ∼3 for n = 6. The increase in friction is attributed to the increased disorder that occurs when going from a thiol to a silane anchor or when decreasing n. It is proposed that disorder favors the increase of the number and type of low-energy modes (kinks, bending, distortions) that are available for excitation and energy dissipation.

The fibroblast growth factor (FGF) pathway promotes tumor growth and angiogenesis in many solid tumors. Although there has long been interest in FGF pathway inhibitors, development has been complicated: An effective FGF inhibitor must block the activity of multiple mitogenic FGF ligands but must spare the metabolic hormone FGFs (FGF-19, FGF-21, and FGF-23) to avoid unacceptable toxicity. To achieve these design requirements, we engineered a soluble FGF receptor 1 Fc fusion protein, FP-1039. FP-1039 binds tightly to all of the mitogenic FGF ligands, inhibits FGF-stimulated cell proliferation in vitro, blocks FGF- and vascular endothelial growth factor (VEGF)-induced angiogenesis in vivo, and inhibits in vivo growth of a broad range of tumor types. FP-1039 antitumor response is positively correlated with RNA levels of FGF2, FGF18, FGFR1c, FGFR3c, and ETV4; models with genetic aberrations in the FGF pathway, including FGFR1-amplified lung cancer and FGFR2-mutated endometrial cancer, are particularly sensitive to FP-1039-mediated tumor inhibition. FP-1039 does not appreciably bind the hormonal FGFs, because these ligands require a cell surface co-receptor, klotho or β-klotho, for high-affinity binding and signaling. Serum calcium and phosphate levels, which are regulated by FGF-23, are not altered by administration of FP-1039. By selectively blocking nonhormonal FGFs, FP-1039 treatment confers antitumor efficacy without the toxicities associated with other FGF pathway inhibitors.

Background : Sensitive and selective molecular recognition is important throughout biology. Certain organisms and toxins use specific binding at the cell surface as a first step towards invasion. A new series of biomolecular materials, with novel optical and interfacial properties, have been designed to sense molecular recognition events. These polymers, the diacetylenic lipids, have previously been shown to undergo chromatic transitions in response to virus binding to the surface of the material. Results : Gangliosides that specifically bind cholera toxin, heat-labile Escherichia coli enterotoxin and botulinum neurotoxin were incorporated into a matrix of diacetylenic lipids, 5–10% of which were derivatized with sialic acid. The lipids were self-assembled into Langmuir-Blodgett layers and polymerized with ultraviolet irradiation, yielding a polydiacetylene membrane with a characteristic blue color into which the ganglioside is non-covalently incorporated. When toxin is added, the polymerized membrane turns red. The response is specific and selective, and can be quantified by visible absorption spectrophotometry. Conclusions : Polydiacetylenic lipid membranes offer a general ‘litmus test' for molecular recognition at the surface of a membrane. A concentration of 20 ppm of protein could be detected using polymerized thin films. The speed, sensitivity and simplicity of the design offers a new and general approach towards the direct colorimetric detection of a variety of different molecules.

Background: NKTR-214 is a novel immunostimulatory cytokine which provides a sustained and biased activation signal through the heterodimeric IL-2R receptor (IL2Rbg or CD122), resulting in significant expansion of CD8+ T and NK cells relative to regulatory T cells in the tumor microenvironment. As a single agent in preclinical tumor models, NKTR-214 significantly increases the intratumoral CD8T/Treg ratio (>400). Here we examine activity and pharmacodynamic effects of NKTR-214 alone and combined with immune checkpoint blockade in both a murine tumor model and in a human in vitro co-culture system. Methods: Mice bearing subcutaneous CT26 colon tumors were treated with single agent NKTR-214 (q9d), murine anti-CTLA-4 or anti-PD-1 (twice-weekly), or combinations of these agents. Serum cytokines were assessed by multiplex ELISA (Quansys). T cell clonality and infiltration were assessed in tumors 7 days after treatment initiation, with TCR V and J beta usage determined by ImmunoSEQ (Adaptive Biotechnologies). Effects on human cytokine and chemokine expression were examined by the BioMAP Combo ELECT (DiscoverX) in vitro system utilizing human fibroblasts or endothelial cells, PBMCs and either HT29 colon adenocarcinoma or HT1299 NSCLC cell lines. 1-PEG-IL2, the most active species of the NKTR-214 prodrug, or Keytruda (anti-PD-1) was added over a range of concentrations alone or in combination, with biomarkers assessed 48 hours later. Results: In tumor-bearing mice, NKTR-214 led to increases in serum IFNg as well as increases in the chemokine MCP-1 (CCL2), a chemoattractant of CD4 T, CD8 T and NK cells. Single-agent NKTR-214 led to significant tumor growth inhibition and 10% tumor free animals, while single-agents anti-CTLA-4 or anti-PD-1 lacked efficacy. Combination of NKTR-214 with checkpoint blockade was synergistic and led to greater tumor-free animals (67% in combination with anti-CTLA-4, 90% with anti-PD-1). NKTR-214 combined with anti-PD-1 was also more efficacious than the combination of anti-CTLA-4 and anti-PD-1. TCR repertoire analysis demonstrated superior increases in TIL clonality and infiltration with NKTR-214 compared to either anti-CTLA-4 or anti-PD-1 alone. NKTR-214 combined with either mode of checkpoint inhibition led to significant increases in both parameters, with the greatest effect occurring when combined with anti-PD-1. In the human BioMAP system, NKTR-214 active species led to significant increases in granzyme B, IFNg, IL-6, IL-17A and TNFa, while these markers were only modestly induced by Keytruda alone. When NKTR-214 was combined with Keytruda, additive and synergistic increases in these factors were found. In addition, the combination significantly decreased uPA in the fibroblast-HT29 system and MDC in the endothelial-HT1299 system, both factors associated with tumor progression. Conclusions: NKTR-214 delivers sustained signaling of the IL-2 pathway. Analysis of multiple immune markers in mice and human tumor-immune cell cultures provide mechanistic rationale for the increased T and NK cell activation in the tumor microenvironment after treatment with NKTR-214. When combined with anti-PD-1, significant increases in T cell receptor clonality, tumor infiltration and immune activation biomarkers are achieved - a significant finding, given that concomitant increase in clonality and infiltration has been correlated with favorable responses in the clinic. In vitro studies with human cells demonstrate the potentiation of immune activation markers associated with anti-tumor effects in combination with anti-PD-1, further suggesting these findings may be translatable from mouse to human. NKTR-214 is currently in a Phase 1 clinical trial to evaluate the pharmacokinetics, pharmacodynamics and activity in an outpatient setting. Citation Format: John L. Langowski, Murali Addepalli, Laurie VanderVeen, Rhoneil Pena, Ravikumar Nutakki, Yolanda Kirksey, Ute Hoch, Jonathan Zalevsky, Stephen K. Doberstein, Deborah H. Charych. The CD122-biased immunostimulatory cytokine NKTR-214 combined with checkpoint blockade leads to mobilization of antitumor immunity and synergistic activity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B057.

Biological matrices can direct the absolute alignment of inorganic crystals such as calcite. Cooperative effects at an organic-inorganic interface resulted in similar co-alignment of calcite at polymeric Langmuir-Schaefer films of 10,12-pentacosadiynoic acid (p-PDA). The films nucleated calcite at the (012) face, and the crystals were co-aligned with respect to the polymer's conjugated backbone. At the same time, the p-PDA alkyl side chains reorganized to optimize the stereochemical fit to the calcite structure, as visualized by changes in the optical spectrum of the polymer. These results indicate the kinds of interactions that may occur in biological systems where large arrays of crystals are co-aligned.

Background: While immune checkpoint blockade is a promising therapeutic approach, combination with agents that modulate complementary pathways may improve responses. Interleukin-2 (IL-2) immunotherapy leads to long-term responses in a small percentage of cancer patients, but systemic toxicity limits its use. In addition, IL-2 expands T regulatory cells, antagonizing antitumor immunity. NKTR-214 is a novel CD122-biased immunostimulatory cytokine which combines biased activation of the IL-2R beta receptor, greatly favoring activation of effector over regulatory T cells in the tumor microenvironment, with improved pharmacokinetics and tolerability compared to Proleukin® (aldesleukin) in non-clinical models. Here we examine the efficacy and mechanism of NKTR-214 combined with checkpoint blocking antibodies in murine tumor models. Methods: Mice bearing subcutaneous B16, LLC, CT26 or EMT6 tumors were treated with single agent NKTR-214 (q9d), aldesleukin (bid or qd), murine anti-CTLA-4 or anti-PD-1 (twice-weekly), or combinations of these agents. Tumor volume was measured before, during and after treatment. Immune cells were profiled by flow cytometry; CD8 or NK requirements for efficacy were assessed by in vivo depletion through serial anti-CD8 or anti-asialo-GM1 antibody injections, respectively. Antitumor memory and specificity was assessed in complete responders by challenging with EMT6 or CT26 followed by observation with no additional test article treatments. Results: In the B16 model, superior single-agent efficacy was achieved by NKTR-214 with a 10-fold lower dose than aldesleukin, yet significantly increased CD8/Treg ratio in the tumor (>400). In the EMT6 model, while both aldesleukin and NKTR-214 synergized with anti-CTLA-4, a greater percentage of complete responders were consistently observed with NKTR-214 (73% versus 44%). Antitumor immunity induced by the combination required both NK and CD8 T cell activity, and was durable and specific with mice remaining tumor-free after re-challenge with an EMT6 but not CT26 implant. Finally, NKTR-214 combined with anti-PD-1 provided superior activity in the CT26 model compared with the combination of anti-CTLA-4 and anti-PD-1 (90% versus 60% tumor-free, respectively). Conclusions: NKTR-214 enables access to the potent IL-2 pathway, providing a mechanism of action complementary to checkpoint inhibition. Favorable pharmacokinetics of NKTR-214 allows sustained tumor exposure and dosing schedules commensurate with antibody therapies. The nonclinical results support the ongoing Phase 1 trial of NKTR-214 in patients with solid tumors. Citation Format: John L. Langowski, Rhoneil Pena, Yolanda M. Kirksey, Murali K. Addepalli, Ute Hoch, Jonathan Zalevsky, Stephen K. Doberstein, Deborah H. Charych. Durable antitumor activity of the CD122-biased immunostimulatory cytokine NKTR-214 combined with immune checkpoint blockade. [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 558.

The preparation, structure, and mechanical stability of self-assembled monolayers formed by octadecyltriethoxysilane (OTE) on mica have been studied by atomic force microscopy. The nanometer scale morphology of the films (3-D clusters, pinholes, etc.) is compared for various preparation methods and correlated with their macroscopic wettability. High-resolution images of the atomically smooth monolayer (1.5 A root mean square roughness) reveal the existence of only short range order. By applying a load above 10 nN with sharp Si{sub 3}N{sub 4} tips (radius < 300 A), the film could be removed leaving 25 A deep holes. Using the same tip, thiol monolayers on gold could be displaced at loads 5 nN, although, in this case, the displacement was reversible. In contrast to the case of OTE, films formed by octadecyldimethylmethoxysilane (ODMS) showed the presence of only 3-D clusters with poor adhesion to the mica substrate. On the basis of these results, we conclude that the mechanical strength of the films formed by OTE is due to siloxane cross-linking between molecules rather than to chemical bonding to the mica substrate. 13 refs., 5 figs.

11545Background: NKTR-214 is an immune-stimulatory cytokine designed to provide a controlled, sustained, and CD122-biased receptor signal through the heterodimeric IL2 receptor pathway (IL2Rbg) res...

Direct visualization of the self-assembling and liquid crystalline texture formation in glycine-modified diacetylene (Gly-DA) Langmuir monolayers at room temperature is achieved using Brewster angle microscopy (BAM). The striped (smectic) texture appears directly after spreading the monolayer at the air-water interface The spatial period of this texture has been estimated to be in the range 0.015-0.005 mm, which is in agreement with the range predicted theoretically for this phase. When pressure is applied, the width of the strips decreases and, finally, the striped texture disappears. Another texture appears in the pressure range 30-35 mN/m: the well-defined pseudo-focal-conic texture, which points to a disordered hexagonal columnar mesophase. In addition, direct visualization of the Gly-DA polymerization under UV radiation exposure via BAM is achieved. We observe focal-conic texture reminiscent of smectic C phase. 18 refs., 3 figs.

Background: At the time of ovarian cancer platinum resistance, standard therapies have limited activity. New anti-cancer drug combinations may lead to improved clinical outcomes. Activity of topoisomerase I (Top I) inhibitors or poly(ADP-ribose) polymerase (PARP) inhibitors have been reported in ovarian cancer and have demonstrated potentiation in other models. Objective: To investigate the efficacy of the Top I inhibitor etirinotecan pegol (NKTR-102) and the PARP inhibitor rucaparib (CO-338) as single agents and in combination in patient-derived xenograft (PDX) models of ovarian cancer. Methods: Five ovarian cancer PDX models in SCID mice were treated for 28 days with NKTR-102 (10 mg/kg/q7d IV), rucaparib (150 mg/kg/qd PO), or combination NKTR-102 plus rucaparib at the same doses and schedules. Of the five PDX models, four were platinum resistant and one was a platinum-sensitive positive control. Each treatment arm had a total of 10 mice and animals were observed daily for body weight and general condition. Tumor size was assessed weekly by ultrasound. The primary endpoint was change in tumor cross-sectional area (cm2) from pretreatment baseline to final tumor burden. Western blot was performed to investigate potential biomarkers of response. Results: Compared to vehicle-treated controls, statistically significant improvement (p Conclusion: NKTR-102 and rucaparib are well tolerated in mice and demonstrate activity in platinum-resistant and platinum-sensitive ovarian cancer PDX models. Combination therapy demonstrated a trend towards decreased tumor size when compared to single agent activity during treatment. Current efforts are focused on extending the time on study for the PDX models evaluated to determine if statistical superiority of the NKTR-102/rucaparib combination can be established. Citation Format: Caroline C. Nitschmann, Rachel M. Hurley, Marc A. Becker, Xiaonan Hou, Ute Hoch, Thomas C. Harding, Deborah H. Charych, Scott H. Kaufmann, Paul Haluska, Saravut J. Weroha. Antineoplastic activity of Top I inhibitor etirinotecan pegol (NKTR-102) and PARP inhibitor rucaparib (CO-388) in platinum-resistant high-grade serous BRCA WT ovarian cancer PDX models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B50.

Background: While immune checkpoint blockade is a promising therapeutic approach, combination with agents that modulate complementary pathways may improve responses. Interleukin-2 (IL-2) immunotherapy leads to long-term responses in a small percentage of cancer patients, but systemic toxicity limits its use. In addition, IL-2 expands T regulatory cells, antagonizing antitumor immunity and resulting in a poorer clinical outcome. NKTR-214 is a novel CD122-biased immunostimulatory cytokine which combines biased activation of the IL-2R beta receptor subunit, greatly favoring activation of effector over regulatory T cells, with improved pharmacokinetics and tolerability compared to Proleukin in non-human models. Here we examine the efficacy and mechanism of NKTR-214 combined with anti-CTLA-4 in murine tumor models. Methods: Mice bearing subcutaneous EMT6 mammary tumors were treated with NKTR-214 q9d, murine anti-CTLA-4 or anti-PD-1 twice-weekly, or both in combination. Immune cell profiling was assessed by flow cytometry following treatment. CD8 or NK cells were depleted in vivo by serial anti-CD8 or anti-asialo-GM1 antibody injections, respectively. Antitumor memory and specificity was assessed in complete responders by challenging with EMT6 or CT26 colon carcinoma implants with no additional treatment. Results: While NKTR-214 and anti-CTLA-4 were not as efficacious individually, their combination was synergistic and well-tolerated with 83% of test animals tumor-free. Combination treatment increased NK and memory effector CD8 cells in both tumor and spleen. Antitumor immunity by the combination was durable and specific as 70% and 100% of mice remained tumor-free after challenge with a second and third EMT6 implant, but not after a subsequent CT26 implant. NKTR-214 combined with anti-PD-1 also proved synergistic with 40% of animals remaining tumor free following treatment. In vivo depletion of either CD8 effector or NK cells abrogated efficacy suggesting both contribute to the response. Conclusions: The mechanism of NKTR-214 antitumor immunity is complementary to checkpoint inhibition. Favorable pharmacokinetics of NKTR-214 allows sustained tumor exposure and dosing schedules commensurate with other therapies. This new therapeutic combination of NKTR-214 and checkpoint inhibition may similarly enable durable responses in humans. Citation Format: John L. Langowski, Seema S. Kantak, Rhoneil Pena, Yolanda Kirksey, Murali Addepalli, Steve Lee, Ute Hoch, Deborah H. Charych, Stephen K. Doberstein. Antitumor activity of the CD122-biased immunostimulatory cytokine combined with immune checkpoint blockade requires innate and adaptive immunity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B015.

The mild conditions and specificity of biological catalysts are attractive incentives for their use in the formation of surfaces with well-defined chemical functionality. Herein, we describe the synthesis, characterization, and enzymatic modification of a functionalized polymeric bilayer assembly. The assembly is composed of a self-assembled monolayer of octadecylsilane and a Langmuir-Blodgett monolayer of polydiacetylene functionalized with the dipeptide phenylalanine-alanine (Phe-Ala). We demonstrate via X-ray photoelectron spectroscopy surface analysis that the surface-bound Phe-Ala dipeptide is a substrate for specific cleavage by the enzyme subtilisin BPN[prime]. In-situ surface transformations via enzymatic synthesis or cleavage offer an alternative to chemical treatments of organic thin films. 28 refs., 4 figs.

Pressure-area diagrams and Brewster angle microscopy of osmium(II)-tris-4,7-diphenylphenanthroline perchlorate (Os(DPP)3) monolayers at the air/water interface (0.05 M HClO4) revealed the existence of 2-D solid aggregates. Their compression produces structurally homogenous films of densely packed Os(DPP)3 centers. Electrochemical experiments carried out directly at the air/water interface using “line” electrodes generated diffusion controlled current due to the lateral electron hopping on the 2-D network of the Os(DPP)3 centers. Voltammetric current was interpreted in terms of the unimolecular rate constant of electron transfer (k1 ≈ 5 × 108 s−1) involving neighboring donor acceptor sites. In subsequent experiments, addition of small quantities of a polar solvent (acetonitrile, benzonitrile, nitrobenzene, dimethylformamide or N-methylformamide) to the subphase led to a complete resolvation of the Os(DPP)3 monolayer and resulted in changes in the electron transfer rate constant. Analysis of these data in view of the Marcus theory showed a good correlation between the observed nuclear frequency factor and the inverse longitudinal relaxation time of the solvents used in the solvent exchange experiments.

Detection of receptor-ligand interactions is generally accomplished by indirect assays such as enzyme-linked immunosorbent assay. A direct colorimetric detection method based on a polydiacetylene bilayer assembled on glass microscope slides has been developed. The bilayer is composed of a self-assembled monolayer of octadecylsilane and a Langmuir-Blodgett monolayer of polydiacetylene. The polydiacetylene layer is functionalized with an analog of sialic acid, the receptor-specific ligand for the influenza virus hemagglutinin. The sialic acid ligand serves as a molecular recognition element and the conjugated polymer backbone signals binding at the surface by a chromatic transition. The color transition is readily visible to the naked eye as a blue to red color change and can be quantified by visible absorption spectroscopy. Direct colorimetric detection by polydiacetylene films offers new possibilities for diagnostic applications and screening for new drug candidates or binding ligands.

Biological membranes provide numerous functions for the survival of cells, ranging from molecular recognition to signal transduction and energy conversion. The spacial organization of proteins, enzymes, glycoproteins, and glycolipids in the membrane is provided by the lipid bilayer matrix. The lipids of the membrane are small molecules which have the common characteristic of having both a hydrophilic and a hydrophobic moiety. In aqueous media, they are entropically driven to self-organize in bimolecular sheets (see the article by Alper in this issue). These sheets, in addition to providing a fluid matrix for a wide variety of biomolecules, also serve to impede the flow of molecules across the membrane.

A purposefully designed microelectrode is positioned at the air-water interface where it functions as a one-dimensional line electrode, probing the dynamics of lateral processes in Langmuir monolayers. This report focuses on the comparison of fluidity and the mechanisms of lateral charge transport in two monolayer systems consisting of transition metal complexes. Octadecylferrocene monolayers are liquid-like where diffusivity of the individual molecules decreases with surface concentration. Electrochemical measurements reveal a liquid/gas phase transition and a value of critical temperature. Osmium tris(diphenylphenanthroline) perchlorate forms aggregates on the water surface. Lateral charge transport in this case is only possible when the monolayer is compressed above 6 mN m , at which point electron hopping becomes a viable mechanism of charge propagation. Further compression of the monolayer leads to an increase in the apparent diffusion coefficient, an effect related to the increasing extent of electroactivity of the osmium sites in the monolayer. These measurements are interpreted in terms of the first order rate constant of electron hopping.

The formation of a non-linear diffusion zone of electroactive material in solution adjacent to a microelectrode is now a well-known phenomenon [ll. It is observed whenever at least one dimension of an electrode becomes small compared with the thickness of the diffusion layer. For example, a hemispherical diffusion zone develops in solution around a microdisk electrode and a hemicylindrical diffusion zone is known to form at microband electrodes [l]. (This statement reflects convenient approximations of the behavior of microdisk and microband electrodes by the current to hemisphere and hemicylinder respectively.1 These simple patterns also prevail when one examines a redox system in two-dimensional (2-D), space. However, in two dimensions, only one dimension of a working electrode is relevant and thus radial diffusion begins to dominate mass transport when the length of a line electrode becomes small compared with the diffusion layer thickness. We have recently described an electrochemical experiment in which redox species are confined to the air-water interface [2]. N-octadecyNferrocenecarboxamide) (C,,Fc), a water-insoluble and non-volatile surfactant, is spread at the water surface in a Langmuir trough. Its surface concentration can be controlled over a wide range by adjusting the position of a movable barrier which determines the area of water surface available to the monolayer assembly. The electrochemical experiments addressing the C,,Fc monolayer on the water surface are carried out with a one dimensional (1-D) microelectrode positioned in the plane of the air-water interface. The counter-electrode and reference electrode are immersed in the subphase behind the barrier as shown in Fig. 1. We showed that the 2-D nature of the surface monolayer and the effectively 1-D character of the microelectrode lead to the development of a linear diffusion layer in two dimensions against the line working electrode 121. The shape of the voltammetric curves is identical

A new electrochemical method is described to investigate the dynamics of lateral diffusion processes in Langmuir monolayers at the air/water interface. The key element of this technique is a gold microband electrode, which can be positioned in the plane of the water surface and which functions as a 1D electrode in contact with a 2D collection of electroactive, water insoluble molecules

Two novel electrochemical methods of investigation of lateral processes in monolayer and bilayer assemblies are described. The first involves an interdigitated micro-electrode array consisting of fifty pairs of 50 nm thick, 800 nm long, and 4 μm wide electrodes deposited on a glass surface. An amphiphilic bilayer consisting in part of the N-methyl,N'-octadecylbipyridyl molecules is self-assembled in the interelectrode gap. Translational diffusion of the electroactive amphiphile depends on the charge in the head group region and on the fluidity of the assembly controlled by the overall oxidation state of the octadecylbipyridyl. The second method involves 0.1 cm long, 50 nm wide gold micro-band electrodes positioned at the air/water interface and addressing surface monolayer of an octadecylferrocene amphiphile under controlled surface pressure conditions. Electrochemical measurements demonstrate that the lateral translational diffusion and the electron hopping involving ferrocene/ferrocenium sites are the two channels of the lateral charge transport

Background: IL-2 was the first cancer immunotherapy approved, producing durable responses in ∼10% of patients treated for metastatic melanoma and renal cancer. However, IL-2 has poor pharmacokinetics, is both an activator and suppressor of the immune system, and is associated with severe cardiovascular side effects of hypotension and vascular leak syndrome, limiting widespread use. NKTR-214 is a novel immunotherapeutic consisting of IL-2 conjugated at a defined region within the protein to 4-6 polyethylene glycol (PEG) chains. Once administered, PEG chains slowly release to generate active IL-2 conjugates biased towards activation of CD122, the IL-2 receptor beta subunit (IL-2Rβ), highly expressed on memory effector CD8+ T cells (CD8T). In the tumor microenvironment, NKTR-214 preferentially expands CD8T over regulatory T cells (Treg), thus tipping the balance in favor of immune activation with a CD8T/Treg >400. In syngeneic mouse tumor models, NKTR-214 demonstrated robust single agent efficacy, synergized with checkpoint inhibitor antibodies, and protected against tumor rechallenge. Here we describe the cardiovascular safety of NKTR-214 in monkeys. Methods: Cardiovascular function was assessed in telemeterized male monkeys after single IV bolus injections of 0.01, 0.03, and 0.1 mg/kg using a Latin square design with a 14-day washout period between doses. Telemetry recordings were collected from three critical 24 hr time periods post-dose. Arterial blood pressure was collected continuously, averaged every 5 min, and reported every 0.25 hour. Inflammatory cytokines (TNFα and IFNγ), immune system activation markers (sCD25, lymphocytes), and IL-2 conjugate exposure were analyzed by ELISA in blood collected by venipuncture at multiple timepoints post dose. Results: There were no ECG changes that were attributed to NKTR-214 treatment at doses up to 0.1 mg/kg/dose. sCD25 and total lymphocyte counts increased from baseline in a dose-responsive manner, producing a 24-fold and 4-fold increase in sCD25 and lymphocytes respectively at 0.1 mg/kg. Continuous monitoring of blood pressure for up to 104 hrs post-dose indicated the absence of hypotension at all dose levels. The inflammatory cytokines TNFα and IFNγ remained undetectable, suggesting no-associated cytokine release, despite the robust immune activation. Exposure to active IL-2 conjugate was high and sustained, exceeding that achieved with the HD-IL-2 regimen in patients. Conclusion: The results support the safety and tolerability of NKTR-214 at doses expected to activate the immune system and support the development of this agent for patients with intractable cancers. Citation Format: Chunmei Ji, Ute Hoch, Deborah H. Charych, Theresa D. Sweeney. NKTR-214, a novel CD122-biased immunostimulatory cytokine, does not cause hypotension in non-human primates despite greater cytokine exposure than achieved with high dose Interleukin-2 (HD-IL-2). [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 A95.

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.

We have developed a method for the rapid and unambiguous identification of sequences of hit compounds from one-bead-one-compound combinatorial libraries of peptide and peptoid ligands. The approach uses a cleavable linker that is hydrophilic to help reduce nonspecific binding to biological samples and allows for the attachment of a halogen tag, which greatly facilitates post-screening sequencing by tandem mass spectrometry (MS/MS). The linker is based on a tartaric acid unit, which, upon cleavage from resin, generates a C-terminal aldehyde. This aldehyde can then be derivatized with a bromine-containing amino-oxy compound that serves as an isotope tag for subsequent MS/MS analysis of y-ion fragments. We have applied this linker and method to the syntheses of a number of peptoids that vary in sequence and length and have also demonstrated single-bead sequencing of a peptoid pentamer. The linker is also shown to have very low levels of nonspecific binding to proteins.