Ex vivo chimeric antigen receptor (CAR) T cell therapy has proven successful in patients with B cell hematologic malignancies. However, current approaches are limited by the requirement for personal manufacturing processes and by barriers such as limited efficacy against solid tumors, treatment-associated toxicities, insufficient CAR-T cell trafficking to the tumor microenvironment, on-target off-tumor effects, and tumor antigen escape. Here, we describe a novel delivery platform that overcomes many of these barriers by employing targeted lipid nanoparticles (LNPs) to reprogram circulating human T cells in vivo. Using a NANOBODYVHH (variable heavy domain of heavy chain)-based targeting moiety, we deliver mRNA encoding a novel CD22 CAR specifically to CD8+ cells, enabling transient functional CAR expression in vitro and in vivo. Our targeted LNP formulation allows for repeated dosing and minimizes mRNA expression in off-target cells. Furthermore, in a humanized Nalm6 tumor mouse model, non-stimulated T cells reprogrammed in vivo inhibit tumor cell growth. Our platform is a flexible and broadly applicable CAR-T treatment for hematologic malignancies, which promises to be adaptable to other diseases.

Introduction: CAR T cell therapy has reshaped treatment strategies for relapsed and refractory large B cell lymphoma (rrLBCL); however, further breakthroughs are needed to improve long-term survival. To address the resistant mechanisms of CAR T cell therapy, we conducted scRNAseq using intratumoral T cells collected from rrLBCL patients with post-CAR T relapse and found that CAR T cells acquired a dysfunctional state in the microenvironment at relapse. In vitro serial killing assay identified reduced interleukin-2 (IL2) signaling as an acquired dysfunctional state, and in vivo PDX disseminated model supported that engineered IL2 targeting IL2Rßγ enhanced tumor cell control. Acquired dysfunction in CD8+ CAR T cells at post-CAR T progression: scRNAseq was conducted using intratumoral T cells collected from rrLBCL patients who relapsed after CAR T cell therapy (n = 7) and from patients who never received CAR T cells (n = 5). CAR-positive cells were significantly enriched in dysfunctional CD8 clusters (CD8Dys) and proliferating effector CD8 clusters (CD8Pr-Eff). TCR clonotype from T cell-rich tumors revealed that CD8Dys and CD8Pr-Eff shared major TCR clones in CAR T cells, and trajectory analysis showed the path through CD8Pr-Eff to CD8Dys, suggesting that CAR T cell clones underwent clonal expansion and progressively acquired dysfunction in the tumor microenvironment. IL2 reduction as a potential mechanism of resistance: CAR T cells generated from 9 healthy donors (HD) and 10 patient donors (PD) were cocultured with EGFP-expressing GCB LBCL cell lines at 1:1 E:T ratio, and tumor cells were replenished every 2 days at the same input as on day 0. Monitoring EGFP intensity using Incucyte showed that PD CAR T cells exhibited less killing relative to HD CAR T cells after the 5th round of stimulation. Then, scRNAseq was conducted using day 0 HD and PD products and day 8 co-cultured T cells, and CytoSig analysis revealed significantly reduced IL2 expression in day 8 PD. IL2Rßγ agonist rescued CAR T cell dysfunction in vitro and in vivo: In the serial killing assay, supplementation with IL2Rßγ agonist pegenzileukin enhanced CAR T cell cytotoxicity even after the 5th round of stimulation in both HD and PD groups. For in vivo evaluation, a luciferase-expressing PDX cell line (PDX-CL) generated from CAR T refractory model was injected into NSG mice, and mice were treated with HD CAR T +/- pegenzileukin after tumor engraftment. All PDX-CL-bearing mice treated with CAR T cells achieved remission by bioluminescence imaging; however, mice treated without pegenzileukin relapsed early, whereas those treated with the combination showed sustained remission. T cell level in peripheral blood 10 and 19 days after CAR T injection was higher in the combination group, suggesting pegenzileukin enhanced CAR T cell expansion and persistence. Conclusion: Acquired CAR T cell dysfunction in the tumor microenvironments contributes to CAR T cell refractoriness. Pegenzileukin prevents CAR T cell dysfunction and improves tumor control in vitro and in vivo. Citation Format: Kotaro Arita, Enyu Dai, Xubin Li, Irtiza N. Shiekh, Patrick K. Reville, Jared Henderson, Ashley Wilson, Christan Dillard, Kevin Bowman, Haopeng Yang, Fuliang Chu, R Andrew. Harkins, Nathan Pate, Katie Malley, Dinesh Bangari, Estela Rojas, Jason R. Westin, Sairah Ahmed, Sattva S. Neelapu, Francisco Vega, Rui Wang, Xiangming Li, Donald Shaffer, Linghua Wang, Michael R. Green. Non-alpha IL2R agonist improves CAR T cell therapy by preventing CAR T dysfunction in relapsed/refractory large B cell lymphoma [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A002.

CAR T-cells have set a new standard of clinical activity in patients with hematologic malignancies but there are several barriers to broader patient access. Currently, the genetic modification of patients' T cells to produce CAR-T cell therapies is carried out ex vivo before infusing the cells back into the patient, using methods that are complex and hinder widespread use. Here we share a targeted lipid nanoparticle (LNP) encapsulating mRNA to reprogram circulating human T-cells in vivo, designed to overcome the significant limitations of current CAR T therapy. The LNP consists of a proprietary ionizable lipid formulation, developed for efficient transfection of T cells and improved tolerability, containing an mRNA encoding a second-generation CD22 CAR. Transfection of human primary T cells was only observed with the addition of a NANOBODY® VHH targeting CD8 to the surface of the LNP. CAR expression was measured for multiple days in up to 80% of CD8+ T cells, with minimal loss of viability and no nonspecific activation. In a whole-blood assay, CAR expression was limited to CD8+ cells (T cells and NK cells), with no CAR expression observed in CD4+ T cells, B cells, or granulocytes. LNP transfected T cells are capable of antigen specific killing. In a serial transfection and rechallenge experiment, cultured T cells were able to maintain cytotoxicity against repeat challenges of Nalm 6 cells but only with additional LNP treatments. The repeated transfected and cellular challenge did not induce exhaustion or disfunction. The in vivo CAR LNP system also efficiently transfected T cells from lymphoma patient PBMCs. Overall levels of in vitro transfection were generally increased in lymphoma patients and the resulting CAR expressing cells were active. CAR expression was greatest in central memory and effector memory T cell phenotypes and decreased in naïve T cells. High-dimensional Spectral Flow profiling identified increased populations of Tem cells in both DLBCL and follicular lymphoma patients, potentially explaining the increased levels of CAR expression. In both healthy donors and lymphoma patient samples, treatment with CAR LNPs significantly reduced B cells, showing the activity of the transfected cells. The capabilities of the LNP system have also been demonstrated in a PBMC humanized mouse model. LNP dosing resulted in expression of CAR in over 80% of circulating human CD8 T cells 6 hours after a single dose. CAR expression was also observed after 4 serial doses, with no substantial loss of CAR expression. Expression of a CAR eliminated any remaining B cells from the PBMC population without severe systemic toxicities. Taken together, we have demonstrated that this targeted LNP system is capable of specific, re-dosable expression of an active CAR that is tolerable for multiple transfections. These are the necessary capabilities to fundamentally alter patient access to CAR-T therapies. Andrew J. Sawyer, Viktor Lemgart, William Kuhlman, Aaron Griset, Mir Ali, Jennifer Richards, Angela Hadjipanayis, Emiko Desvaux, Seth Garren, Chris Hoefler, Haley Nguyen, Laura Strauss, Jing Jiao, Brandon Quido, Laura Powers, Allison Caron, Eyoung Shin, Virna Cortez-Retamozo, Austin Boesch, Dharini Shah, Tiffany Le, Rasika Kunden, Samantha Stewart, Fazila Nasimi, Sampa Maiti, Olga Lihoradova, Christian Mueller, Christopher Borges, Carla Lawendowski, Ulrik Nielsen, Valeria Fantin, Daryl Drummond, Donald Shaffer. Efficient generation of active CAR-T cells from healthy donors and lymphoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6119.

Abstract Mechanism of action studies are imperative to translating oncology therapeutics into the clinic informing testable hypotheses and potential combination partners. This often involves large and time-consuming mouse studies to test a small number of compounds in an attempt to show statistically significant benefit. Likewise, testing multiple compounds and combination partners in these systems is limited by time, cost, and a desire to reduce the number of animals used in biological research. To overcome these limitations, we utilized an implantable microdevice (IMD) developed by KiburMed to measure intra-tumoral drug responses and to differentiate the mechanism of action of multiple cytokine agents in parallel in the MC38 mouse model. After sizable tumor growth in mice and implantation of the IMD device with 9 different therapeutic candidates, MC38 mice were taken down at different time points (24 hour, 4 days,7 days) to assess response. Local tumor response in the study was measured by cyclical immunofluorescence for deep cellular response phenotyping with a panel of 32 markers for comprehensive immune cell phenotyping. Among the candidate murine surrogate compounds in the device, SAR445877 (anti-PD-1-IL-15 mutein) was tested as a monotherapy, as a combination partner with anti-PDL1 and compared against anti-PD1 monotherapy. With further validation of this novel platform, results from this work are aligned with previous studies (1) showing a murine surrogate for SAR445877 in combination with anti-PDL1 increases immune cell populations more than either single agent alone. These analyses show CD3, CD4, and CD8 were significantly increased in SAR’877 murine surrogate + anti-PDL1 compared to SAR’877 alone or anti-PD1 alone. In addition, T cells could infiltrate at a distance away from treatment release region, well into tumor core. Other significant changes in immune cell markers include increases in CD45+, CD11b, CD11c, CD20 and CD27. In conclusion, we have shown that multiple oncology therapeutics can be screened simultaneously in a small number of mice using an implantable microdevice and data obtained with this technology is consistent with that obtained from traditional mouse tumor experiments. (1) Preclinical characterization of SAR445877, an anti-PD-1 antibody-IL-15 mutein fusion protein with robust anti-tumor efficacy as monotherapy and in combination with PD-L1 blockade. Marie Bernardo; Yu-an Zhang; Dan Lu; Stella Martomo; Fatima Menas; Chen Zhu; Raymond Perez; Jeegar Patel; Donald Shaffer; Xiangming Li, Cancer Res (2023) 83 (7_Supplement): 2972. *This work was supported and funded by Sanofi. Citation Format: Xiangming Li, Julien Tessier, Joon Sang Lee, Seth Garren, Virna Cortez-Retamozo, Meenu Sharma, Angela Jankowski, Erik Zarazinski, Ann Fiore, Oliver Jonas, Colin Brenan, Donald Shaffer, Angela Hadjipanayis. Spatiotemporal tumor immune modulation by localized delivery of cancer therapeutics using an implantable microdevice [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 2053.

Abstract During persistent viral infections and cancer, antigen-specific T cells become exhausted, express elevated levels of inhibitory receptors and gradually lose their functional potential. Immune checkpoint blockade can restore function of exhausted T cells to result in tumor clearance, however, only a minority of cancer patients see durable tumor control. Immune-stimulatory cytokines can augment the antitumor efficacy of checkpoint blockade, but their clinical use is marred by substantial toxicity. SAR445877 (SAR’877, formerly KD050), is a novel antibody-cytokine fusion protein consisting of Fc silenced human anti-PD-1 IgG1 fused to a mutated interleukin-15 (IL-15)/IL-15 receptor alpha sushi domain fusion, which can cis-activate direct cytokine stimulation to effector T cells and alleviate off target binding and the resultant toxicity. Here, we leveraged existing models of chronic antigen exposure to characterize the potential of SAR’877 to restore activity of exhausted T cells.C57BL/6 mice exposed to lymphocytic choriomeningitis virus (LCMV) Clone 13 develop persistent viral infection that results in development of dysfunctional, exhausted T cells. We tested the SAR’877 murine surrogate molecule (mKD050) using the LCMV Clone-13 (CL13) infection model for its ability to promote T cell function in vitro, in vivo, and to control viral infection. Treatment of exhausted T cells with mKD050 in vitro was able to increase expansion and cytokine production of antigen-specific CD8 T cells which was superior to anti-PD-1 antibody or non-targeting antibody-IL15/IL-15Ra fusion protein (mntKD050). Moreover, in vivo treatment of CL13 infected mice with mKD050 increased the numbers and functional output of antigen-specific CD4 and CD8 T cells compared to anti-PD-1 or mntKD050 treatments. Further, mKD050 increased the frequency of antigen-specific TCF1+TIM-3- stem-like CD8 T cells, which are critical to maintaining the T cell pool during chronic viral infection and cancer. The enhanced T cell function observed following treatment with mKD050 translated to hastened clearance of CL13. Finally, the hastened CL13 clearance observed following mKD050 treatment required an intact CD4 T cell compartment. Given the robust activity of mKD050 in reinvigorating dysfunctional T cells in a mouse model of chronic viral infection, we utilized the MIMIC CD8 T cell exhaustion model to explore whether SAR’877 could rescue the function of exhausted human T cells. SAR’877 stimulated proliferation and functional activity of exhausted human CD8 T cells in vitro. SAR’877 more potently induced proliferation and IFNγ and granzyme B production compared to anti-PD-1 and non-targeted IL15 mutein alone. Together, these results demonstrate that the PD-1 targeted IL15 mutein SAR’877 rescues the functional activity of chronically stimulated, exhausted T cells. Citation Format: Isaraphorn Pratumchai, Marie Bernardo, Kristi Lynn Marquardt, Chen Zhu, Fatima Menas, Roberto Carrio, Tony Byers, Donald Shaffer, Xiangming Li, John Ross Teijaro. SAR445877, an anti-PD-1 antibody-IL-15 mutein fusion protein restores function to exhausted T cells [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 4063.

Abstract An effective cancer therapy requires both killing cancer cells and targeting tumor-promoting pathways or cell populations within the tumor microenvironment (TME). We purposely search for molecules that are critical for multiple cell types in the TME and identified nuclear receptor subfamily 4 group A member 1 (NR4A1) as one such molecule. NR4A1 has been shown to promote the aggressiveness of cancer cells and maintain the immune suppressive TME. Using genetic and pharmacological approaches, we establish NR4A1 as a valid therapeutic target for cancer therapy. Importantly, we have developed the first-of-its kind proteolysis-targeting chimera (PROTAC, named NR-V04) against NR4A1. NR-V04 effectively degrades NR4A1 within hours of treatment in vitro and sustains for at least 4 days in vivo, exhibiting long-lasting NR4A1-degradation in tumors and an excellent safety profile. NR-V04 leads to robust tumor inhibition and sometimes eradication of established melanoma tumors. At the mechanistic level, we have identified an unexpected novel mechanism via significant induction of tumor-infiltrating (TI) B cells as well as an inhibition of monocytic myeloid derived suppressor cells (m-MDSC), two clinically relevant immune cell populations in human melanomas. Overall, NR-V04-mediated NR4A1 degradation holds promise for enhancing anti-cancer immune responses and offers a new avenue for treating various types of cancer such as melanoma. Citation Format: Lei Wang, Yufeng Xiao, Yuewan Luo, Rohan Master, Jiao Mo, Myung-Chul Kim, Yi Liu, Chandra Maharjan, Urvi Patel, Xiangming Li, Donald Shaffer, Guertin Kevin, Haoyang Zhuang, Emily Moser, Keiran Smalley, Daohong Zhou, Guangrong Zheng, Weizhou Zhang. PROTAC mediated NR4A1 degradation as a novel strategy for cancer immunotherapy [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 2469.

An effective cancer therapy requires both killing cancer cells and targeting tumor-promoting pathways or cell populations within the tumor microenvironment (TME). We purposely search for molecules that are critical for multiple tumor-promoting cell types and identified nuclear receptor subfamily 4 group A member 1 (NR4A1) as one such molecule. NR4A1 has been shown to promote the aggressiveness of cancer cells and maintain the immune suppressive TME. Using genetic and pharmacological approaches, we establish NR4A1 as a valid therapeutic target for cancer therapy. Importantly, we have developed the first-of-its kind proteolysis-targeting chimera (PROTAC, named NR-V04) against NR4A1. NR-V04 effectively degrades NR4A1 within hours of treatment in vitro and sustains for at least 4 days in vivo, exhibiting long-lasting NR4A1-degradation in tumors and an excellent safety profile. NR-V04 leads to robust tumor inhibition and sometimes eradication of established melanoma tumors. At the mechanistic level, we have identified an unexpected novel mechanism via significant induction of tumor-infiltrating (TI) B cells as well as an inhibition of monocytic myeloid derived suppressor cells (m-MDSC), two clinically relevant immune cell populations in human melanomas. Overall, NR-V04-mediated NR4A1 degradation holds promise for enhancing anti-cancer immune responses and offers a new avenue for treating various types of cancer.

SUMMARY Chimeric antigen receptor (CAR) T-cell therapy has been a breakthrough for relapsed or refractory large B-cell lymphoma (rrLBCL). However, suboptimal CAR T-cell activity can lead to therapeutic failure and dismal outcome. Using single cell RNA-sequencing of rrLBCL tumors, we identify a prominent population of clonally expanded dysfunctional CAR+ CD8 T-cells indicative of ongoing tumor cell engagement, proliferation, and dysfunction at the time of progression from CAR T-cell therapy. Furthermore, we show that rrLBCL patient-derived CAR T-cells are more prone to dysfunction and loss of cytotoxicity compared to healthy donor-derived CAR T-cells. Using both antigen-driven and CAR-driven models of T-cell dysfunction, we show that pegenzileukin, a non-alpha IL2 agonist, can prevent T-cell dysfunction. In both in vitro and in vivo CAR T-cell models, pegenzileukin improved T-cell expansion and tumor control. This provides pre-clinical rational for use of pegenzileukin in combatting T-cell dysfunction, a central mechanism of CAR T-cell failure. HIGHLIGHTS Tumor-infiltrating CD8 CAR T-cells show clonal expansion and dysfunction at the time of progression. rrLBCL patient-derived CAR T-cells are more prone to dysfunction compared to healthy-donor-derived CAR T-cells. Pegenzileukin, a non-alpha IL2 agonist, rescues antigen– and CAR-driven CD8 T-cell dysfunction and improves CAR T-cell responses in vivo.

Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. A major unmet medical need in immuno-oncology (IO) is extending the benefits of these therapies to patients who never experience tumor regression (primary resistance) or who derive initial benefit followed by tumor progression (acquired resistance). To improve clinical benefits of anti-PD-1 antibody, we generated SAR445877 (formerly KD050), which is an antibody cytokine fusion molecule that combines a human anti-PD-1 antibody with a mutated fusion of human IL15 and human IL15Rα sushi domain. SAR445877 binds to human PD-1 and inhibits immunosuppressive signaling through the PD-1/PD-L1 pathway. The IL15-IL15Rα fusion contains a single amino acid mutation that confers reduced potency of IL2/15Rβγ stimulation relative to wild type IL-15, reduces binding to IL-2Rβ expressing HEK 293 cells, (EC50: >108 nM vs 4.05nM) and decreases pSTAT5 activation in HEK blue IL-2 reporter assay by more than 5-fold. Through its IL15-IL15Rα fusion moiety, SAR445877 stimulates cells expressing IL2/15Rβγ with much weaker activity than wild type IL-15 when measured by CTLL-2 cell proliferation in vitro (EC50: 398.8nM vs 1.68nM). Fusion of IL-15 to anti-PD-1 antibody mediates cis-activation of human PD-1+ cells, including both PD-1+ HEK blue reporter cell line and human primary T cells, with higher potency compared to non-targeted IL-15. This cis-activation could result in expansion and activation of antigen-specific CD8+ T cells with improved activity in the tumor microenvironment compared to non-targeted IL-15. Antitumor efficacy of SAR445877 was explored in the CT26 colorectal cancer model using the clinical candidate. Transgenic mice expressing human PD-1 and PD-L1 were implanted with CT26 tumors expressing human PD-L1 and treated with SAR445877. SAR445877 displayed a more potent antitumor efficacy than pembrolizumab. Further, treatment with SAR445877 increased the CD8/CD4 T cell ratio and significantly increased the percentage of effector memory CD8 T cells in tumors. In anti-PD-L1 antibody resistant Pan02 syngeneic tumor model, a murine surrogate molecule produced a remarkable antitumor activity and was well tolerated. Combination treatment of murine surrogate with anti-mPD-L1 displayed greater antitumor activity than single agent treatment (TGI = 99.30% vs 78.78%). In summary, SAR445877 can target potency-reduced IL-15 to PD-1+ T cells and induce cis-activation of T cells. SAR445877 exhibited potent anti-tumor efficacy in PD-1 resistant preclinical model with good tolerability. These results support further evaluation of SAR445877 in a phase I clinical trial (ClinicalTrials.gov #NCT05584670). Citation Format: Marie Bernardo, Yu-an Zhang, Dan Lu, Stella Martomo, Fatima Menas, Chen Zhu, Raymond Perez, Jeegar Patel, Donald Shaffer, Xiangming Li. Preclinical characterization of SAR445877, an anti-PD-1 antibody-IL-15 mutein fusion protein with robust anti-tumor efficacy as monotherapy and in combination with PD-L1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2972.

Supplementary Data from HER2-Specific T Cells Target Primary Glioblastoma Stem Cells and Induce Regression of Autologous Experimental Tumors

Background We previously developed a saline-formulated mixture of 4 synthetic messenger ribonucleic acids (mRNAs) encoding the cytokines IL-12, IFN-α, GM-CSF, and IL-15 sushi (IL-15-IL-15Rα receptor fusion) that promoted systemic immunity and tumor eradication upon intratumoral administration in multiple mouse tumor models. Here, we explored whether stabilization of the mRNAs through a lipoplex (LPX) formulation could confer increased expression of the encoded cytokines to result in improved antitumor efficacy. To this end, we developed a pH-responsive amphoteric LPX by mixing preformed liposomes with mRNA in aqueous/aqueous solutions. The resulting complex was designed to destabilize under neutral pH in circulation, leading to a limited exposure in non-target organs following intratumoral administration. Methods Biological activity of LPX-formulated mRNA-encoded cytokines or reporter genes were compared to that of naked mRNA in vivo using C57BL/6 and BALB/c mice harboring TC-1, B16F10 and CT26 tumors, respectively. Following intratumoral administration, cytokines were analyzed in serum and organs by ELISA and luciferase expression was followed by bioluminescent imaging. Anti-tumor efficacy was assessed by tumor growth inhibition and survival of CT26-tumor bearing mice and expansion of tumor-reactive T cells were analyzed by flow cytometry. Abscopal effects were assessed in C57BL/6 mice bearing B16F10 subcutaneous tumors on both flanks. Results In vivo studies demonstrated a favorable tumor-to-liver translation ratio of luciferase reporter mRNA formulated with the LPX, supporting the hypothesis that the LPX would destabilize in circulation. The LPX formulation further increased target expression of each of the four encoded cytokines in tumors ranging from 7 to 47-fold compared to saline solution in B16F10-tumor bearing animals. Intratumoral administration of LPX-mRNA to mice with subcutaneous CT26 colorectal tumors significantly inhibited tumor growth, with stronger tumor growth inhibition when compared to treatment with saline-formulated mRNA. This increased antitumor activity was correlated with stronger pharmacodynamic responses, including expansion of T cells reactive against the tumor antigen gp70. C57BL/6 mice bearing B16F10 subcutaneous tumors on both flanks received an intratumoral injection of LPX-mRNA on one side. Treatment resulted in significant inhibition of tumor growth and prolonged survival. Antitumor efficacy was further enhanced when combined with anti-PD-1 antibody, resulting in complete tumor regression in a subset of mice, consistent with an abscopal response. Conclusions Together, these results demonstrate the potent anticancer activity of a novel LPX-formulated mRNA mixture. Ethics Approval Animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) and in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 354 Seven days following exposure to SAR444245, exhausted CD8 T cells demonstrated enhanced IFN-γ and TNF-α secretion, decreased co-inhibitory molecule expression, and increased proliferation compared to cells not exposed to SAR444245. CD8 T cells exposed to SAR444245 on the day of initial activation, followed by repetitive antigen exposure, proliferated and maintained IFN-γ similar to acutely stimulated CD8 T cells. Luminex cytokine array analysis of treated CD8 T cells demonstrated robust expression of cytokines and effector molecules, and increased polyfunctionality. In short term assays, SAR444245 induced T cell expansion without significant increase in cytotoxicity. However, in long-term assays mimicking chronic CAR stimulation and exhaustion, addition of SAR444245 to CAR T-cells resulted in enhanced CAR T-cell proliferation through day 8, and enhanced control of tumor cells through repeated additions. In an in vivo Raji-Luc model, the combination of SAR444245 with CD19/CD22 CAR T cells demonstrated enhanced in vivo CAR T expansion and sustained anti-tumor efficacy compared to CAR T cells alone. Summary/Conclusion: SAR444245 treatment of exhausted antigen-specific or CAR T cells restored functionality and alleviated T cell dysfunction in vitro and in vivo. These data suggest that SAR444245 rescue of CAR T cell exhaustion through maintenance of CAR T cell proliferative capacity, cytotoxicity, and polyfunctionality and provide rationale for future clinical study of SAR444245 with CAR T cells. HemaSphere | 2023;7(S3) EHA2023 Hybrid Congress Copyright Information: (Online) ISSN: 2572-9241 © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open access Abstract Book distributed under the Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) which allows third parties to download the articles and share them with others as long as they credit the author and the Abstract Book, but they cannot change the content in any way or use them commercially. Abstract Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx.Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 355

Autophagy is the cellular process by which cytoplasmic contents are degraded and recycled through a lysosomal pathway. In cancer, autophagy can contribute to both tumor promotion and tumor suppression, and emerging evidence supports a role in resistance to immunotherapy. We investigated whether inhibition of autophagy with a small molecule inhibitor of the class III phosphoinositide 3-kinase Vps34 could stimulate anti-tumor immune responses and potentiate the efficacy of immune checkpoint blockade (ICB). Treatment with the Vps34 inhibitor (Vps34i) inhibited autophagy and was weakly cytotoxic to B16F10, MC38, EMT6 and CT26 mouse syngeneic tumor cell lines. In certain cancers, autophagy has been linked to lysosomal degradation of MHC I proteins, thereby downregulating expression and contributing to immune evasion. Notably, we observed minimal upregulation of MHC I surface expression on a subset of mouse and human cell lines. We further explored whether autophagy inhibition would result in release of any soluble immune stimulatory factors from mouse tumor cell lines. Inhibition of Vps34 markedly increased the secretion of several proinflammatory chemokines, including CCL5 and CXCL10. Increased chemokine secretion was also observed in a panel of human cancer cell lines, including NCI-H2009 lung cancer and PC-3 prostate cancer, confirming that the response is observed in human cancer cell lines derived from different tissue origins. As the chemokines CCL5 and CXCL10 are critical to the development of robust anti-tumor immunity, we next tested whether Vps34i treatment could potentiate the anti-tumor efficacy of ICB. We explored the activity of Vps34i as a single agent and in combination with anti-PD-1 antibody in three different syngeneic tumor models, including EMT6, CT26 and MC38. No single agent activity was observed in these models, however, combination of anti-PD-1 and Vps34i significantly inhibited the growth of CT26 tumors compared to control. Anti-tumor efficacy of combination treatment was not significantly different compared to single agents, and no complete tumor regressions were observed. No significant increases in CCL5 and CXCL10 were observed in the serum of combination treated mice, potentially explaining the lack of robust efficacy. Our results suggest that inhibition of autophagy promotes the secretion of proinflammatory cytokines from mouse and human cancer cells in vitro. As the autophagy pathway exhibits cross talk with other major cell signaling pathways, the tumor contexture may be a critical determinant of the role of autophagy in anti-tumor immune responses. Future experiments will explore which tumor contexts are sensitive to combination of autophagy inhibition and ICB. Citation Format: Marie Bernardo, Yu-an Zhang, Martin Graf, Jane Cheng, Fangxian Sun, Virna Cortez-Retamozo, Sukhvinder Sidhu, Eladio Marquez, Donald Jackson, Jack Pollard, Timothy R. Wagenaar, Donald Shaffer. Inhibition of the autophagy protein Vps34 induces tumor cell secretion of proinflammatory chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5590.

BackgroundSAR444245 is a non-alpha IL-2 Synthorin TM molecule designed with a site-specific non-natural amino acid serving as a bioconjugation site for a single PEG. The non-natural amino acid is positioned to enable the PEG bioconjugation to obscure block binding to the IL-2 alpha receptor, while retaining near-native affinity with the intermediate affinity βγ IL-2 receptor. The non-alpha features of SAR444245 minimize activation of immune suppressive regulatory CD4+ T cells, while retaining activity on CD8+ T cells and NK cells expressing the IL-2 βγ receptors. NK cells exert anti-tumor activity through antibody dependent cellular cytotoxicity (ADCC) of IgG antibodies as well as antibody independent mechanisms.MethodsHere, we utilized a panel of human primary PBMC based immunoassays and transcriptomic analysis to evaluate whether SAR444245 may improve ADCC function of IgG1 anti-tumor target antibodies.ResultsWe characterized the ability of SAR444245 to enhance the cytolytic function of NK cells towards the prototypic NK target cell K562 as well as to modulate NK cell ADCC in combination with EGFR or CD20-targeting antibodies. In vitro assays demonstrated that SAR444245 can activate NK cells, promote NK cell proliferation and improve cytotoxicity of NK cells against K562 cells and across a panel of human EGFR and CD20 positive cell lines. In PBMC based ADCC assays with 1ug/ml of antibody, SAR444245 improved ADCC function maximally by 9-fold for an anti-EGFR antibody and at 5-fold for an anti-CD20 antibody. SAR444245 exhibited dose-dependent enhancement of NK cell ADCC function. Notably, this activity was observed in cell lines expressing varying levels of EGFR and CD20. SAR444245 treatment was associated with dose dependent increases in NK cell degranulation and IFN-γ production. Transcriptomic profiling revealed that SAR444245 had broad effects on NK cell biology leading to changes in inhibitory and activating receptors.ConclusionsIn summary, these results indicate that SAR444245 can enhance the cytolytic activity of NK cells and enhance the ADCC effect of tumor-directed antibodies by activating NK cells.

Background Leukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4) is an immunoinhibitory protein expressed on the surface of myeloid cells that has been increasingly recognized as a therapeutic target of interest in immuno-oncology (IO). Upon binding its ligands, MHC I molecules (e.g. HLA-G/HLA-A), LILRB2 inhibits myeloid cell activation and promotes an M2-like (anti-inflammatory) state. LILRB2 was the first target prioritized from a macrophage discovery effort leading to the development of JTX-8064, a humanized monoclonal antibody that specifically binds to and antagonizes LILRB2. JTX-8064 has been shown to induce an M1-like (pro-inflammatory; anti-tumor) functional state in macrophages. Rodents do not express LILRB proteins limiting their usefulness as a model for preclinical study of JTX-8064. To overcome this limitation, we conducted an ex vivo human tumor histoculture study to assess the pharmacodynamic effects of LILRB2 antagonism. Protein and/or gene expression analysis of matched tumor samples enabled the discovery of predictive biomarkers associated with the induction of specific pharmacodynamic signatures in ex vivo-cultured human tumors in response to JTX-8064. Finally, tumor types were identified that had a high prevalence of these predictive biomarkers suggesting they may be priority indications for JTX-8064 therapy. Methods More than 100 fresh treatment-naïve human tumor samples obtained post-surgery from kidney, lung, and head and neck cancer were treated with JTX-8064 or isotype control antibody for 24 hrs in the histoculture system. RNA was isolated from tumors prior to any treatment as well as from JTX-8064 and isotype control treated samples. Gene expression was analyzed using the NanoString nCounter® and qPCR assays. Additional IHC analyses were performed on baseline untreated tumor samples. Results JTX-8064 was shown to induce pharmacodynamic responses to treatment significantly above isotype control indicative of macrophage polarization, IFNg-signaling, and T cell inflammation. To identify predictive biomarkers of pharmacodynamic response to JTX-8064, matched untreated samples were characterized by gene expression analysis and by IHC (CD8, CD163, and HLA-G proteins). Numerous LILRB2 pathway-related molecules (e.g. HLA-A, HLA-B, CD163, LILRB2) and gene signatures were found to be statistically significantly higher in the untreated kidney, head and neck, and lung cancer samples of matched pharmacodynamic responders compared to non-responders. Further bioinformatics analysis revealed additional cancer subtypes where these biomarkers are enriched. Conclusions These data will inform indication selection and combination strategies for JTX-8064 to maximize potential therapeutic benefit for patients with solid tumor malignancies.

Abstract B7-H4 (VTCN1, B7x, B7S1) is a transmembrane protein belonging to the B7 family of costimulatory proteins and has been shown to inhibit T cell proliferation, cytokine secretion, and cytotoxic lymphocyte (CTL) induction. B7-H4 expressed on tumor cells or macrophages has been associated with poor prognosis and impaired T cell function in renal cell and ovarian cancers. Here we show B7-H4 is abundantly expressed in human breast cancer with triple negative breast cancer (TNBC) having the highest overall B7-H4 mRNA expression. We developed a specific and sensitive immunohistochemistry (IHC) assay for evaluation of B7-H4 protein and quantified B7-H4 expression in 156 breast tumor samples. Approximately 70% of the breast tumor samples had detectable B7-H4 expression whereas none of the normal or benign breast tissues stained positive for B7-H4. Multiplex IHC and flow cytometry studies showed that the majority of B7-H4 expression was restricted to the tumor epithelial cells, the CD45+ immune cells were negative for B7-H4 expression. Interestingly none of the TNBC samples that were positive for B7-H4 showed detectable expression of PD-L1 suggesting that B7-H4 and PD-L1 checkpoint proteins may act in a mutually exclusive manner. To evaluate the role of B7-H4 on tumor immune evasion, we overexpressed murine or human B7-H4 on the mouse colon-26 (CT26) tumor cell line and injected these cells intravenously into Balb/c mice. By day 14 we observed significantly more tumors as well as larger percent tumor area in the lungs of mice given CT26 cells transduced with human or mouse B7-H4 as compared to vector control transduced cells. These data suggest B7-H4 expression in tumors can accelerate tumor growth in immune competent mice and that targeting B7-H4 may provide therapeutic benefit. Given the mutually exclusive expression patterns of B7-H4 and PD-L1 a B7-H4 targeting agent may provide particular benefit in those patients where current anti-PD-1/PD-L1 therapies are not effective. Citation Format: Shaffer DR, Nagashima K, Cortez-Retamozo V, Feldman I, Smith J, Zafari M, Larson R, Mabry R, Novorantseva T, Briskin M, Sathyanaryananan S. Mutually exclusive expression pattern of the immune co-inhibitory molecules B7-H4 and PD-L1 in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-11-07.