Abstract Introduction: The development, physiology function, and pathogenesis of the tissues are intricately orchestrated by the spatial organization of diverse cell populations. Proteins are the fundamental components within individual cells that control every function. Therefore, by integrating both information on cellular location and molecular profiling, spatial proteomics has emerged as an indispensable field for understanding mechanisms governing tissue homeostasis, disease progression, and therapeutic responses. Here, I introduce a spatial proteomics method designed to target selective cell phenotypes within definite tissue niches for in-depth spatial biology analysis. Method: In this approach, we developed a sequential pipeline highlighted with immunofluorescence staining, microscopic photobleaching, fluorescence cell sorting, and liquid chromatography-mass spectrometry (LC-MS) analysis. The process begins with staining 400-μm-thick tissue macrosections with fluorescence-conjugated antibodies to visualize specific cell types of interest. We employed two different fluorescence-conjugated antibodies of the same clone (e.g., Alexa488-anti-CD11c and Alexa647-anti-CD11c antibodies) for the staining on every targeted cell (e.g., CD11c+ immune cells). We then introduced “photobleaching barcodes” using a confocal microscope to photobleach/exhaust either one of the fluorescence signals stained on cells by exposing it to the matched laser. This allows us to create differentiable fluorescence barcodes that label cells at distinct regions, incorporating cell spatial information into our fluorescence detection. After tissue dissociation, barcoded cells were sorted into different groups which were also classified by their original locations in the macrosection. Finally, protein extraction and LC-MS analysis were conducted on the collected cells to enable comprehensive spatial proteomic analysis. Results: The initial application in investigating dendritic cell (DC) subsets in mouse spleen during lipopolysaccharide (LPS)-induced inflammation reveals significant proteome differences among three splenic DC subsets, categorized by their locations in or outside spleen T-cell zones as well as control DCs. Our result aligns with previously published proteome data in splenic DCs, underscoring the feasibility and reliability of our technology. Currently, we are in the process of evaluating breast cancer cell heterogeneity in human biopsy specimens using our approach to profile the tumor-immune microenvironment. Conclusion: Spatial proteomic findings from both applications are poised to discover potential drug targets that benefit treatment efficacy for inflammatory diseases and breast cancer. This technology features deep protein profiling for cell-type-specific spatial proteomics and is designed for broad applications across diverse tissue specimens in various diseases. Citation Format: Yi-Chien Wu, Elie Abi Khalil, Samuel Weng, Steve Lee. Tissue-niche-based and cell-type-selective in-depth proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3772.

Intratumoral IRIF imaging demonstrates similar patterns for GFP-IBD (green) and TagRFP-IBD (red) foci in tumor at 24 h after irradiation with 6 Gy

Intratumoral IRIF imaging demonstrates similar patterns for GFP-IBD (green) and TagRFP-IBD (red) fluorescence in tumor at 3 h after irradiation with 6 Gy.Control tumor

Three-dimensional imaging of irradiated MCF7 tumor xenografts with dual fluorescent reporters for IRIF (TagRFP, red) and shRNAmir expression (ZsGreen, green)

Time-lapse live cell imaging of caspase-3/7 activation in irradiated Panc02 cells treated with PUGNAc or alloxan

Supplementary Methods and Supplementary Figure, Movie and Table Legends

intratumoral imaging of Hoechst 33342 stained nuclei (blue), GFP-IBD (green) and TagRFP-IBD (red) reporters in untreated control tumor xenograft.

S1. Validation of TagRFP-IBD as IRIF reporter in vitro. S2. Effects of radiation on MCF7 cells with altered O-GlcNAcylation. S3. Targeting O-GlcNAc modification by small molecule inhibitors affects kinetics of IRIF resolution in different cell lines. S4. Cellular apoptosis assays using fluorescent dyes and a caspase substrate probe. S5. Alloxan potentiates senescence in Panc02 cells in a radiation dose-dependent manner. S6. Blocking O-GlcNAcylation in vivo by inducing shOGT or alloxan treatment qualitatively reduces EZH2 immunoreactivity after irradiation.

Heatmap and Reactome pathway proteomic data

O-GlcNAc proteomic hits of significance

Objectives: Adolescents and young adults (AYA) who experience patient-centered care are more likely to speak with their provider about their contraceptive concerns and select and maintain a contraceptive method – yet most do not receive such care. The objective of this study was to develop and evaluate the Tangible Tool, a decision aid to support patient-centered contraceptive counseling among AYA.

Objectives: Abortion stigma is pervasive in the US. While abortion itself does not cause mental illness, abortion stigma may worsen psychological responses to the procedure. Few intervention studies have addressed abortion stigma. Psychological research demonstrates improved coping through cognitive restructuring and reconstructing personal narratives. With a theoretical framework grounded in cognitive therapy, this study tests whether a narrative intervention reduces abortion stigma.

Abstract The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. Implications: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

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.

Abstract Background: Immunotherapy offers the potential for durable responses in a growing list of cancer indications. NKTR-214 is comprised of the cytokine IL2 conjugated o multiple PEG units. Upon in vivo administration some of these PEG molecules are released to leave activated IL2-conjugates that bind selectively at the IL2 receptor beta subunit (IL2Rβ). The active IL-2-conjugates favor proliferation of tumor-killing CD8+ memory T cells (CD8T) over immunosuppressive regulatory T cells (Treg) in the tumor microenvironment (Charych, JCO 2013, 31:15, Suppl. 1).We have also shown that NKTR-214 results in sustained exposure of drug in the tumor and robust immune system activation that translates between rodents and monkeys at safe doses, (AACR; Mol Cancer Ther 2013;12 (11 Suppl): Abstract B296). The receptor selectivity and improved pharmacokinetic profile leads to substantially improved single-agent efficacy and safety over the original IL-2 cytokine. Combinations of different checkpoint blockade antibodies have shown great promise clinically and in preclinical tumor models, highlighting the importance of targeting multiple immune activation pathways. Here we examine the combination of NKTR-214 with anti-CTLA4 antibody in murine breast and colon tumor models to explore the interaction of two mechanisms of action: checkpoint inhibition and direct CD8T cell activation via the IL-2 receptor beta. Methods: Female BALB/c mice bearing established tumors implanted with CT-26 (Murine Colon Carcinoma) or EMT6 (Murine Mammary Carcinoma) cells were treated with single agent NKTR-214, murine anti-CTLA-4 antibody or the two agents in combination. NKTR-214 was administered at 0.8 mg/kg i.v. and anti-CTLA-4 at 100 μg/mouse i.p. Results: In both the EMT6 breast and the CT-26 colon models, the combination of NKTR-214 with anti-CTLA-4 yielded significantly better tumor growth inhibition compared to either agent dosed alone. In the EMT6 model 10/12 animals were tumor free by day 20 in the combination arm and stayed tumor free until the end of study (60 days). Similarly in the CT-26 model, 8/12 animals were tumor free by day 25 in the combination arm and stayed tumor free until the end of study (day 60). Single agent anti CTLA-4 or NKTR-214 did not show significant tumor inhibition in either model. No toxicities were observed when NKTR-214 was combined with anti CTLA-4 antibody. Conclusions: NKTR-214 is a highly differentiated immunotherapy with a new mechanism of action that shows efficacy both as a single agent (as previously shown in a mouse melanoma model) and in combination. Combining NKTR-214 mediated T cell activation with CTLA-4 blockade is highly synergistic in murine models of cancer and holds the promise for durable responses in patients. Citation Format: Steve Lee, Murali Addepalli, Ute Hoch, Rhoneil Pena, Payal Shirsat, Yolanda Kirksey, Stephen K. Doberstein, Deborah Charych, Seema Kantak. Synergy between an engineered cytokine, NKTR-214, and CTLA-4 blockade in murine colon and breast tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5032. doi:10.1158/1538-7445.AM2014-5032

3082 Background: Durability of response is the hallmark of immunotherapy but only a fraction of patients experience this prolonged benefit. Recent studies suggest more patients may benefit from combining agents with complementary immunological mechanisms. NKTR-214 is an engineered form of IL-2 that directly activates cytotoxic T cells by targeting the IL-2 receptor beta subunit and exhibits a pharmacokinetic profile that is more like an antibody than a cytokine. Anti-CTLA-4 and anti-PD1 are antibodies that block negative regulation of T cells. Here we show that combining either antibody with NKTR-214, provides significant tumor growth inhibition in resistant mouse models. Methods: For combination studies, BALB/c mice bearing established CT-26 (Colon Carcinoma) or EMT6 (Mammary Carcinoma) tumors were treated with single agent NKTR-214, murine anti-CTLA-4 antibody, murine anti-PD-1 or the two agents in combination. Results: NKTR-214 exhibits a plasma and tumor exposure that is 600-fold and 500-fold greater respectively, than an equivalent dose of the original IL-2 cytokine. The optimized PK profile allows q9d dosing schedule instead of bid; the latter being typical for cytokines. In both the EMT-6 and the CT-26 models, the combination of NKTR-214 with anti-CTLA-4 provided 10/12 and 8/12 tumor-free animals respectively, up to 40 days after the last dose and showed clear synergy compared to either agent alone. The combination with anti-PD1 was also synergistic and showed tumor regression in 5/10 animals. Single agent administration did not show any significant tumor growth inhibition in these models. The combinations were well tolerated with no body weight loss or other clinical signs. Conclusions: NKTR-214 directly stimulates cytotoxic T cells and is therefore complementary to the mechanism of checkpoint inhibition using antibodies. The favorable pharmacokinetics of NKTR-214 enables dosing schedules that are more like an antibody allowing convenient combination with other antibodies. Combining NKTR-214 mediated T cell activation with CTLA-4 or PD1 blockade is synergistic in murine models of cancer and holds the promise for durable responses in humans.

Abstract Introduction: Microtubule inhibitors are a mainstay of cancer therapy despite limitations in efficacy and tolerability. Such deficiencies are typical of cytotoxic drugs and a variety of strategies have been investigated to improve them, including the use of polymer conjugates. For example, etirinotecan pegol (EP, formerly NKTR-102) is a topoisomerase I inhibitor-polyethylene glycol (PEG) conjugate engineered by attaching irinotecan molecules to PEG using a biodegradable linker. EP has been studied in multiple clinical oncology studies and showed significantly higher response rates and decreased bone marrow toxicity in Phase 2 compared to historical data for irinotecan. EP is currently in a Phase 3 trial (BEACON; NCT01492101) for metastatic breast cancer. We here report a novel PEG-taxane conjugate, PEG-TX1, which exhibits improved tumor growth inhibition compared to a recently approved taxane, cabazitaxel (CBZ). We have explored the basis for the improved preclinical activity, and show increased intratumoral exposure of the released active taxane (TX1) following administration of PEG-TX1, compared with that achieved with CBZ. Methods: Tumor growth inhibition was examined using subcutaneous NCI-H460 tumor xenografts in nu/nu athymic mice. Test articles were administered intravenously via tail vein, q7dx3, at the maximum tolerated dose with a study endpoint of 2000 mm3. In PK/PD experiments to determine tissue and plasma concentrations of CBZ, TX1, and PEG-TX1, a single dose of test article was administered intravenously, and samples were collected at various time points to 14 d. Drug concentrations in tissue and plasma were quantified using LC/MS. Results: In the NCI-H460 xenograft model, PEG-TX1 (administered q7dx3) delayed tumor growth by 336%, producing 90% partial and 10% complete tumor regressions. In comparison, CBZ delayed tumor growth by 213% and produced only 40% partial tumor regressions and 0% complete regressions. Following a single administration of PEG-TX1 vs. CBZ, significantly greater tumor growth inhibition was observed for PEG-TX1. This improved activity correlated well with increased tumor accumulation of PEG-TX1 and TX1 than was achieved for CBZ. Throughout the study, the concentration of TX1 was at least 10-fold higher in tumor tissue than in plasma or all other tissues examined. CBZ did not show such a sustained increase in tumor exposure relative to other tissues. Conclusions: We demonstrate that PEG-TX1, a PEG-taxane prodrug utilizing Nektar's proprietary polymer conjugation technology, achieved superior tumor growth suppression in a murine xenograft model. Tumor growth inhibition is consistent with the enhanced concentration of taxane detected in tumors following administration of PEG-TX1. These data further demonstrate the utility of polymer conjugation to modify the biodistribution of drug conjugates and thereby enhance the therapeutic properties of an active pharmacophore. Citation Format: Dennis G. Fry, Christine Brew, Steve Lee, Ute Hoch, Wen Zhang, Antoni Kozlowski, Fadil Dahhani, Nagabhusan Tangudu, Stephen D. Harrison, Jennifer Riggs-Sauthier. A new polymer conjugated taxane shows improved efficacy in tumor xenograft models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2465. doi:10.1158/1538-7445.AM2013-2465