Immune checkpoint inhibitors have transformed cancer therapy, yet many patients fail to achieve durable responses due to insufficient T cell reinvigoration. Cytokines offer promise for enhancing immunotherapy, but their clinical use is limited by toxicity and a narrow therapeutic index. Immunocytokines, engineered fusion proteins combining antibody specificity with cytokine activity, aim to overcome these challenges by targeting cytokine delivery to immune cells or the tumor microenvironment. We describe SAR445877 (SAR’877), a potentially novel PD-1–targeted immunocytokine that fuses a high-affinity anti–PD-1 antibody with a detuned IL-15/IL-15Rα sushi domain complex. SAR’877 blocks PD-1/PD-L1 and PD-1/PD-L2 interactions while selectively delivering IL-15 signals to PD-1 + T cells, enhancing proliferation and activation of antigen-experienced CD8 + and CD4 + T cells and NK cells, while minimizing systemic inflammation. Mechanistically, SAR’877 activates STAT5 signaling in PD-1 + lymphocytes and restores effector function in exhausted T cells. In preclinical models, a murine surrogate of SAR’877 accelerated viral clearance and induced robust antitumor immunity by expanding cytotoxic CD8 + T cells and promoting Th1 polarization. Notably, SAR’877 outperformed anti–PD-1 plus untargeted IL-15, highlighting the therapeutic potential of targeted IL-15 delivery. These findings position SAR’877 as a promising next-generation immunotherapy with enhanced efficacy and reduced cytokine-associated toxicities.

Mechanism of action studies are imperative for translating oncology therapeutics into the clinic informing testable hypotheses and potential combination partners. One class of oncology therapeutics, immune-stimulatory cytokines can augment the antitumor efficacy of checkpoint blockade, but further translational work is required to understand beneficial treatment effects versus unwanted immunotoxicities. SAR445877 (SAR’877, formerly KD050), is a novel antibody-cytokine fusion protein consisting of an 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 effector T cells by biasing IL-15 stimulation to T cells expressing PD-1. Here we used immunohistochemistry (IHC), flow cytometry and spatial transcriptomics to characterize SAR’877 treatment effects on residual tumors in a B16-OVA mouse model. Mice were treated with isotype control, anti-mPD-1 (anti-murine PD-1), mntSAR’877 (mIgG2a-mutmIL15) or mSAR’877 (murine surrogate anti-mPD-1-mutmIL15). The mSAR’877 treated animals showed regression of tumors, decreased necrosis and increased tumor immune cell infiltration compared to isotype control, mntSAR’877, and anti-mPD-1. IHC performed on residual tumors treated with mSAR’877 displayed a further decrease of PD-L1+ cells compared to anti-mPD-1 or mntSAR’877. Spatial transcriptomic profiling using the GeoMx DSP instrument revealed significant infiltration of innate immune cells (NK, monocytes, and neutrophils) and adaptive immune cells (CD8 T and CTL cells) into the tumor leading-edge compartment when compared to anti-mPD-1. CD8 T cell and NK cell increases were orthogonally validated by IHC and flow cytometry. Pathway analyses showed these changes were also associated with an upregulation of cytotoxic pathways when compared to controls. Together, these data elucidated the mechanism of anti-PD-1/IL15 showing contributions of NK and CD8 T cells associated with anti-tumor activity observed in mice. These findings suggest that mSAR’877 shows superior efficacy to single agent PD-1 or untargeted IL-15 in mouse tumor models. Note: all authors are employed by Sanofi and may hold stock and/or stock options Julien Tessier, Joon Sang Lee, Ingeborg M. Langohr, Caroline Morel, Xiangming Li, Seth Garren, Virna Cortez-Retamozo, Dinesh Bangari, Donald Jackson, Donald Shaffer, Angela Hadjipanayis. Multiomics preclinical characterization of SAR445877, an anti-PD-1 antibody-IL-15 mutein fusion protein with robust anti-tumor efficacy as monotherapy [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 6064.

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.

T cell dysfunction from chronic antigen stimulation limits the efficacy of T cell therapies. SAR444245 is a PEGylated, non-alpha IL2; PEG is attached to a novel amino acid such that it prevents binding to the alpha subunit of the IL-2 receptor, avoiding drug toxicities and expansion of regulatory T-cells, yet retains binding to the beta-gamma receptors that expand tumor-killing T cells. T-cell assays: CD8 T cells isolated from healthy donors were repetitively stimulated with antigen over 12 days to establish exhausted CD8 T cells. They were then exposed to SAR444245 for 7 days. Proliferation and co-inhibitory marker expression were measured as well as cytokine production by Luminex. CART cell assays: CD19 CAR T cells were generated from relapsed LBCL patient PBMCs obtained at the time of leukapheresis. Short term CAR T cell cytotoxicity was assessed by co-culture with 2 LBCL cell lines in the presence or absence of SAR444245 over 48 hours. Chronic CART stimulation was modelled in vitro by repeat addition of target tumor cells to CAR T cells every 48 hours over 8 days in the presence or absence of SAR444245. Live T cell and lymphoma cell counts as well as T-cell immunophenotyping were obtained at baseline and days 4 and 8. In vivo: Luciferase expressing Raji cells were injected into NSG mice. Tumor burden was monitored via BLI. Mice were either treated with anti-CD19/22 CAR T cells alone or in combination with SAR444245 (weekly treatment for 3 doses). Blood samples were obtained from mice on days 11 and 18 days post-CART infusion to immunophenotyping. Seven days following exposure to SAR444245, exhausted CD8 T cells demonstrated enhanced IFNγ and TNFα secretion, decreased co-inhibitory 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 IFNγ similar to acutely stimulated CD8 T cells. Cytokine 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 CART-cell proliferation through day 8, and enhanced control of tumor cells. In an in vivo Raji model, the combination of SAR444245 with CD19/CD22 CART cells demonstrated enhanced CART expansion and sustained anti-tumor efficacy compared to CAR T cells alone. SAR444245 treatment of exhausted CD8 or CAR T cells restored functionality and alleviated T cell dysfunction. These data suggest that SAR444245 rescue of T cell exhaustion through maintenance of T cell proliferative capacity, cytotoxicity, and polyfunctionality and provide rationale for future clinical study of SAR444245. Encore Abstract - previously submitted to EHA 2023 The research was funded by: Sanofi Keywords: Aggressive B-cell non-Hodgkin lymphoma, Cellular therapies, Immunotherapy Conflicts of interests pertinent to the abstract. A. Choi Employment or leadership position: Sanofi R. Carrio Employment or leadership position: Sanofi N. Pate Employment or leadership position: Sanofi K. Malley Employment or leadership position: Sanofi D. Bangari Employment or leadership position: Sanofi J. Gavigan Employment or leadership position: Sanofi C. Shi Employment or leadership position: Sanofi B. Liu Employment or leadership position: Sanofi T. Byers Employment or leadership position: Sanofi I. Sassoon Employment or leadership position: Sanofi M. Cucchetti Employment or leadership position: Sanofi R. Wang Employment or leadership position: Sanofi M. Agarwal Employment or leadership position: Sanofi G. Abbadessa Employment or leadership position: Sanofi E. Meibalan Employment or leadership position: Sanofi L. Powers Employment or leadership position: Sanofi J. Cao Employment or leadership position: Sanofi X. Ying Employment or leadership position: Sanofi K. Balko Employment or leadership position: Sanofi Q. Yu Employment or leadership position: Sanofi J. Jiao Employment or leadership position: Sanofi V. Cortez-Retamozo Employment or leadership position: Sanofi S. Sidhu Employment or leadership position: Sanofi D. Shaffer Employment or leadership position: Sanofi X. Li Employment or leadership position: Sanofi M. R Green Research funding: Sanofi

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.

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

Supplementary Figure 1. Systemic neutrophilia in tumor bearing mice. Supplementary Figure 2. Increased metastasis does not result from increased intravasation or post-extravasation events. Supplementary Figure 3. NK-mediated clearance of B16-F10-GFP+ cells. Supplementary Figure 4. Ly6G+ neutrophils are more abundant in spleens of 4T1 bearing mice. Supplementary Figure 5. Splenocyte conditioned medium activates endothelial cells and induces MMP-9 secretion.

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.

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies 1 . Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies 2 – 4 . Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2 + breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.

Abstract Current HER2-targeted therapies have markedly improved the outcome of cancer patients with HER2 overexpressing tumors. However, these patients may eventually relapse or develop treatment resistance. In addition, HER2-low patients that are not eligible for treatment constitute a significant portion of the breast cancer patients. To address these unmet needs, we have developed a novel HER2-targeting T cell engager, SAR443216. This is a trispecific antibody with binding sites for HER2, CD3 and CD28, and containing a mutated IgG4-Fc which lacks effector functions. CD28 binding contributes to T cell activation, including activation of IL-2 and NFκB pathways, as well as induction of anti-apoptotic protein, Bcl-xL. In the presence of HER2-positive cancer cells, SAR443216 is able to activate primary human CD4 and CD8 T cells, resulting in T cell proliferation and secretion of cytokines and granzyme B. Moreover, it has potent in vitro T cell-dependent cellular cytotoxicity (TDCC) against a panel of HER2-expressing cancer cell lines, including those that are HER2-low. The potency of in vitro TDCC is largely correlated with HER2 surface expression in the target cells. Finally, in a HER2-low breast cancer xenograft model, SAR443216 also exhibited significant anti-tumor activity in immuno-deficient NSG mice reconstituted with primary human T cells. Thus, SAR443216 represents a promising new drug for cancer patients with HER2-expressing tumors, including those who are currently ineligible for stand-of-care therapy. Citation Format: Wenwen Sha, Sri Vadde, Zhili Song, Edward Seung, Zhen Xing, Liqing Chen, Virna Cortez-Retamozo, Sukhvinder Sidhu, Dinesh Bangari, Lan Wu, Ronnie Wei, Zhi-yong Yang, Gary Nabel, Vasiliki Pelekanou, Michele Sanicola-Nadel, Serena Masciari, Dmitri Wiederschain, Lily Pao. SAR443216, a novel trispecific T cell engager with potent T cell-dependent cytotoxicity for HER2-low tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1825.

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.

Senescence is an important p53-controlled tumor suppressor program that not only opposes the proliferation of cancer cells but also promotes their immune-mediated clearance in certain contexts. In hepatocellular cancer, p53 induction promotes an innate immune cell-mediated clearance of senescent cells wherein natural killer (NK) cells seem to play the primary sentinel role. Whether NK cells also surveil cancer cells in other tumor types when p53 is activated to promote a senescence response is unknown. To identify the role that NK and other innate immune cell types have on the surveillance and destruction of lung adenocarcinoma cells, we developed an orthotopic transplantation model where p53 gene function could be restored to induce senescence after successful engraftment of tumor cells in the mouse lung. Contrary to precedent, we found that NK cells actually limited the efficient clearance of tumor cells from the mouse lung after p53 restoration. Instead, activation of p53 induced the infiltration of monocytes, neutrophils, and interstitial macrophages. Loss of NK cells further promoted expansion of these inflammatory cell types and tumor clearance after p53 restoration. These observations suggest that NK cell responses to p53 activation in lung adenocarcinoma is distinct from those found in other tumor types and that diverse innate immune cell populations may play context-dependent roles during tumor immune surveillance. Further, our data provide an impetus to understand the broader mechanisms that regulate cancer cell destruction by multiple cell types of the innate immune system and distinct cancer contexts.

Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy.

Bone marrow-derived myeloid cells can accumulate within tumors and foster cancer outgrowth. Local immune-neoplastic interactions have been intensively investigated, but the contribution of the systemic host environment to tumor growth remains poorly understood. Here, we show in mice and cancer patients (n = 70) that lung adenocarcinomas increase bone stromal activity in the absence of bone metastasis. Animal studies reveal that the cancer-induced bone phenotype involves bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh neutrophils, which exhibit cancer-promoting properties. Experimentally reducing Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth. These observations posit osteoblasts as remote regulators of lung cancer and identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven protumoral response.

Checkpoint blockade immunotherapies can be extraordinarily effective, but might benefit only the minority of patients whose tumors are pre-infiltrated by T cells. Here, using lung adenocarcinoma mouse models, including genetic models, we show that autochthonous tumors that lacked T cell infiltration and resisted current treatment options could be successfully sensitized to host antitumor T cell immunity when appropriately selected immunogenic drugs (e.g., oxaliplatin combined with cyclophosphamide for treatment against tumors expressing oncogenic Kras and lacking Trp53) were used. The antitumor response was triggered by direct drug actions on tumor cells, relied on innate immune sensing through toll-like receptor 4 signaling, and ultimately depended on CD8(+) T cell antitumor immunity. Furthermore, instigating tumor infiltration by T cells sensitized tumors to checkpoint inhibition and controlled cancer durably. These findings indicate that the proportion of cancers responding to checkpoint therapy can be feasibly and substantially expanded by combining checkpoint blockade with immunogenic drugs.

AbstractImmune cells promote the initial metastatic dissemination of carcinoma cells from primary tumors. In contrast to their well-studied functions in the initial stages of metastasis, the specific roles of immunocytes in facilitating progression through the critical later steps of the invasion–metastasis cascade remain poorly understood. Here, we define novel functions of neutrophils in promoting intraluminal survival and extravasation at sites of metastatic dissemination. We show that CD11b+/Ly6G+ neutrophils enhance metastasis formation via two distinct mechanisms. First, neutrophils inhibit natural killer cell function, which leads to a significant increase in the intraluminal survival time of tumor cells. Thereafter, neutrophils operate to facilitate extravasation of tumor cells through the secretion of IL1β and matrix metalloproteinases. These results identify neutrophils as key regulators of intraluminal survival and extravasation through their cross-talk with host cells and disseminating carcinoma cells.Significance: This study provides important insights into the systemic contributions of neutrophils to cancer metastasis by identifying how neutrophils facilitate intermediate steps of the invasion–metastasis cascade. We demonstrate that neutrophils suppress natural killer cell activity and increase extravasation of tumor cells. Cancer Discov; 6(6); 630–49. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561