Antigen (Ag) escape is a frequent mechanism of relapse after CAR-T therapy, even though only ∼1% of leukemic and ∼0.1% of lymphoma cells are Ag⁻ at baseline. In this study, we modeled extreme Ag heterogeneity (>20%) to define how Fas/FasL-dependent bystander killing contributes to tumor clearance. Across patient cohorts, Fas expression predicted survival after CD19 CAR-T therapy, particularly in CD19-low disease. In both murine and human systems, Fas-dependent bystander killing required Ag stimulation and cell contact, operated within a defined therapeutic window, and could eradicate large fractions of Ag⁻ tumors in vivo . Pharmacologic potentiation with inhibitor of apoptosis protein antagonists or genetic stabilization of CAR-T membrane-bound FasL enhanced bystander killing but simultaneously induced CD4⁺ T cell fratricide, which was rescued by CAR-T Fas knockout. Importantly, Fas sensitization also enabled bispecific antibody-redirected T cells to mediate bystander killing in resistant tumors. Finally, targeting tumor-associated macrophages triggered Fas-dependent clearance of neighboring tumor cells. These findings establish Fas-mediated bystander killing as a generalizable and therapeutically actionable axis to prevent Ag escape and broaden the scope of targeted T cell therapies.

Supplementary Figures S1-S5

Transfer does not induce homeostatic inhibition on CD4+ T-cells

Immunotransplant modulates serum cytokines levels

Immunotransplant requires double checkpoint blockade

BACKGROUND:Cancer immunotherapies are generally effective in patients whose tumors contain a priori primed T-cells reactive to tumor antigens (TA). One approach to prime TA-reactive T-cells is to administer immunostimulatory molecules, cells, or pathogens directly to the tumor site, that is, in situ vaccination (ISV). We recently described an ISV using Flt3L to expand and recruit dendritic cells (DC), radiotherapy to load DC with TA, and pattern recognition receptor agonists (PRRa) to activate TA-loaded DC. While ISV trials using synthetic PRRa have yielded systemic tumor regressions, the optimal method to activate DCs is unknown.METHODS:To discover optimal DC activators and increase access to clinical grade reagents, we assessed whether viral or bacterial components found in common pathogen vaccines are an effective source of natural PRRa (naPRRa). Using deep profiling (155-metric) of naPRRa immunomodulatory effects and gene editing of specific PRR, we defined specific signatures and molecular mechanisms by which naPRRa potentiate T-cell priming.RESULTS:We observed that vaccine naPRRa can be even more potent in activating Flt3L-expanded murine and human DCs than synthetic PRRa, promoting cross-priming of TA-reactive T-cells. We developed a mechanistically diverse naPRRa combination (BCG, PedvaxHIB, Rabies) and noted more potent T-cell cross-priming than with any single naPRRa. The naPRRa triplet-as part of Flt3L-primed ISV-induced greater intratumoral CD8 T-cell infiltration, T-cells reactive to a newly defined tumorous neoantigen, durable tumor regressions.CONCLUSIONS:This work provides rationale for the translation of pathogen vaccines as FDA-approved clinical-grade DC activators which could be exploited as immune-stimulants for early phase trials.

Transfer induces homeostatic inhibition

Homeostatic activation and inhibition require JAK3-signaling

Immunotherapies directly enhancing anti-tumor CD8+ T cell responses have yielded measurable but limited success, highlighting the need for alternatives. Anti-tumor T cell responses critically depend on antigen presenting dendritic cells (DC), and enhancing mobilization, antigen loading and activation of these cells represent an attractive possibility to potentiate T cell based therapies. Here we show that expansion of DCs by Flt3L administration impacts in situ vaccination with oncolytic Newcastle Disease Virus (NDV). Mechanistically, NDV activates DCs and sensitizes them to dying tumor cells through upregulation of dead-cell receptors and synergizes with Flt3L to promote anti-tumor CD8+ T cell cross-priming. In vivo, Flt3L-NDV in situ vaccination induces parallel amplification of virus- and tumor-specific T cells, including CD8+ T cells reactive to newly-described neoepitopes, promoting long-term tumor control. Cross-presenting conventional Type 1 DCs are indispensable for the anti-tumor, but not anti-viral, T cell response, and type I IFN-dependent CD4+ Th1 effector cells contribute to optimal anti-tumor immunity. These data demonstrate that mobilizing DCs to increase tumor antigen cross-presentation improves oncolytic virotherapy and that neoepitope-specific T cells can be induced without individualized, ex vivo manufactured vaccines.

Panobinostat is an oral pan histone-deacetylase inhibitor used in the treatment of relapsed and refractory multiple myeloma. Previously published studies of panobinostat demonstrated synergy with bortezomib but included few patients exposed to newer agent combinations (ie, panobinostat plus daratumumab or carfilzomib). Here, we report outcomes of panobinostat-based combinations at an academic medical center among patients whose disease had been heavily pretreated with modern agents. We retrospectively analyzed 105 patients with myeloma treated with panobinostat at The Mount Sinai Hospital in New York City between October 2012 and October 2021. These patients had a median age of 65 (range 37-87) and had received a median of 6 prior lines of therapy while in 53% the disease was classified as triple class refractory and in 54% the disease had high-risk cytogenetics. Panobinostat was most commonly utilized at 20 mg (64.8%) as part of a triplet (61.0%) or quadruplet (30.5%). Aside from steroids, panobinostat was most commonly administered in combination with lenalidomide, pomalidomide, carfilzomib, and daratumumab in descending order of frequency. Among the 101 response-evaluable patients, the overall response rate was 24.8%, clinical benefit rate (≥minimal response) was 36.6%, and median progression-free survival was 3.4 months. Median overall survival was 19.1 months. The most common toxicities ≥grade 3 were hematologic, primarily neutropenia (34.3%), thrombocytopenia (27.6%), and anemia (19.1%). Panobinostat-based combinations produced modest response rates in patients with heavily pretreated multiple myeloma, over half of whom had triple-class refractory disease. Panobinostat warrants continued investigation as a tolerable oral option for recapturing responses in patients whose disease has progressed after receipt of standard-of-care therapies.

AbstractT cell–based therapies have induced cancer remissions, though most tumors ultimately progress, reflecting inherent or acquired resistance including antigen escape. Better understanding of how T cells eliminate tumors will help decipher resistance mechanisms. We used a CRISPR/Cas9 screen and identified a necessary role for Fas–FasL in antigen-specific T-cell killing. We also found that Fas–FasL mediated off-target “bystander” killing of antigen-negative tumor cells. This localized bystander cytotoxicity enhanced clearance of antigen-heterogeneous tumors in vivo, a finding that has not been shown previously. Fas-mediated on-target and bystander killing was reproduced in chimeric antigen receptor (CAR-T) and bispecific antibody T-cell models and was augmented by inhibiting regulators of Fas signaling. Tumoral FAS expression alone predicted survival of CAR-T–treated patients in a large clinical trial (NCT02348216). These data suggest strategies to prevent immune escape by targeting both the antigen expression of most tumor cells and the geography of antigen-loss variants.Significance:This study demonstrates the first report of in vivo Fas-dependent bystander killing of antigen-negative tumors by T cells, a phenomenon that may be contributing to the high response rates of antigen-directed immunotherapies despite tumoral heterogeneity. Small molecules that target the Fas pathway may potentiate this mechanism to prevent cancer relapse.This article is highlighted in the In This Issue feature, p. 521

Background T-cell based immunotherapies such as CAR-T, bispecific mAb, transgenic T cells and checkpoint blockade have profound efficacy in multiple tumor types but share a common limitation – target antigen (Ag) escape.1 2 One approach to address this limitation has been therapy directed at a ‘parallel’ target (e.g. CD22 after CD19 loss), however, these lineage markers are frequently lost together.3 Here, we describe an alternate, broadly applicable, approach: potentiating fasL/fas-signaling to increase localized bystander killing of Ag-tumor cells and thereby prevent Ag escape. Methods We used a CRISPR/Cas9 library to screen for tumor expressed molecules that inhibit or facilitate T-cell killing. We then evaluated one candidate -fas- using murine transgenic T cells, murine and human CAR-T cells, bispecific mAb redirected PBMC, and tumoral RNAseq data from a large CAR-T clinical trial. Results GFP-specific (JEDI) CD8 T cells were co-cultured with on-target (GFP+) and bystander (mCherry+) lymphoma cells that had been transfected with a CRISPR/Cas9 library; this screen revealed several tumor-expressed candidate molecules inhibiting or facilitating T-cell killing. Notably, we observed a marked dependence on fas for on-target tumor killing and then, surprisingly, an exquisite dependence on fas for localized bystander tumor killing. (figure 1).Because bystander tumor killing appeared critically fas-dependent, we hypothesized that potentiating fas-signaling might increase bystander killing. An in vitro screen of small molecules that modulate fas-pathway revealed several candidates, including inhibitors of histone deacetylases (HDAC), inhibitors of apoptosis proteins (IAP) and Bcl-2 family members in murine and human systems (figure 2). To validate these candidates, we demonstrated that HDACi increased GFP-specific T cell killing of both on-target and bystander lymphoma cells, in a completely fas-dependent manner (figure 3). Similarly, using a bispecific antibody-based system, we demonstrated increased, fas-dependent, T cell killing of both on-target and bystander human lymphoma cells with inhibitors of IAP and bcl-2 family members (e.g. MCL1). Conclusions T-cell mediated tumor killing can be potentiated with fas pathway modulators. This augmentation improves both fas-dependent Ag+ and Ag-tumor cell death. Further studies of modulating the fas pathway alongside T-cell based immunotherapies are needed as potential treatments to prevent antigen escape and improve patient outcomes. Acknowledgements We thank the flow cytometry core facility, microscopy core facility, and the CCMS animal facility at ISMMS. Ethics Approval The studies were approved by The Mount Sinai Institutional Review Board. References Zaretsky J, Garcia-Diaz A, Shin D, et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J Med 2016: 375(9); 819–20. Majzner R, Mackall C. Tumor antigen escape from CAR T-cell therapy. Cancer Discov 2018;8(10):1219–1226. Jacoby E, Nguyen S, Fountaine T, et al. CD19 CAR immune pressure induces B-precusor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity. Nat Commun 2016; 7:12320.

Abstract BACKGROUND In situ vaccination (ISV) against lymphoma can be achieved with Flt3-ligand treatment to recruit dendritic cells (DC), radiotherapy to load DC with tumor antigens, and intratumoral injection of a pattern-recognition- receptor agonist (PRRa) to activate antigen-loaded DC. While clinical trials of ISV using synthetic PRRa have yielded remissions, the optimal approach to activate DC is unknown. We hypothesize that ‘natural’ PRRa, such as the attenuated pathogens in common prophylactic vaccines, could target multiple PRR, leading to more robust activation of DC as compared to synthetic PRRa. METHODS 20 FDA-approved vaccines (BCG, MMR, etc.) were screened in vitro, for their effect on DC phenotype and function. DC tumor antigen cross-presentation was assessed using CRISPR gene-edited β2m−/−GFP-lymphoma cells and a GFP-specific CD8 T cell system. Mechanisms of immune activation were interrogated using MyD88, TRIF, MAVS, and IFNAR knockout mice, as well as a library of CRISPR-edited PRR-null Raw264 macrophages. Potent vaccines functioning through distinct mechanisms were evaluated in vivo in an ISV using the A20 murine lymphoma model. RESULTS Several vaccines induced robust DC activation, cross-presentation and increases in T cell activation, proliferation, and tumor killing. Some vaccines were more effective than synthetic PRRa in activating DCs to induce a T cell response. Distinct PRR and signaling pathways were engaged by different vaccines. In vivo, vaccine combination therapy induced tumor regression in a majority of animals. CONCLUSIONS Prophylactic vaccines, used alone or in combination, are effective DC activators and can be repurposed for use in ISV, with immediate translation into the clinic.

Antigen-specificity is one of the hallmarks of the adaptive immune system, and CD8 T cells are particularly crucial for eliminating malignant cells with aberrant expression of tumor-associated self-antigens or neoantigens. We established a high-throughput pooled CRISPR/Cas9 screen for functionally assessing cancer cell genes involved in modulating the cytotoxic activity of antigen-specific CD8 T cells. As validation of our approach, we identified many genes known to be altered in cancers refractory to immunotherapy, including MHC component B2m and the checkpoint molecule Cd274. Interestingly, the cell surface death receptor Fas was identified as a strong hit in mediating the killing of antigen-positive lymphoma and was further verified with breast cancer, despite the availability of other cytotoxic effectors such as perforin/granzymes. Using a 2-antigen system, we surprisingly observed that Fas was also mediating the killing of neighboring GFP-negative cancer cells by T cells highly specific for a GFP epitope. This phenomenon was not dependent on MHC Class I expression but was potentiated by interferon gamma, which is known to upregulate Fas expression. Even for T cells that were previously primed, this bystander cytotoxicity required the presence of neighboring antigen-positive cells, suggesting a very hyperlocal and temporally regulated off-target effect that was not dependent on natural killer cells in vivo. We believe this phenomenon to be crucial to the durable remissions seen with highly antigen-specific approaches such as CAR-T cell therapy. Modulating this pathway may be a viable approach for preventing immune escape of rare antigen-loss variants or treating heterogeneous tumors with no clear single target antigen.

Indolent non-Hodgkin's lymphomas (iNHLs) are incurable with standard therapy and are poorly responsive to checkpoint blockade. Although lymphoma cells are efficiently killed by primed T cells, in vivo priming of anti-lymphoma T cells has been elusive. Here, we demonstrate that lymphoma cells can directly prime T cells, but in vivo immunity still requires cross-presentation. To address this, we developed an in situ vaccine (ISV), combining Flt3L, radiotherapy, and a TLR3 agonist, which recruited, antigen-loaded and activated intratumoral, cross-presenting dendritic cells (DCs). ISV induced anti-tumor CD8(+) T cell responses and systemic (abscopal) cancer remission in patients with advanced stage iNHL in an ongoing trial (NCT01976585). Non-responding patients developed a population of PD1(+) CD8(+) T cells after ISV, and murine tumors became newly responsive to PD1 blockade, prompting a follow-up trial of the combined therapy. Our data substantiate that recruiting and activating intratumoral, cross-priming DCs is achievable and critical to anti-tumor T cell responses and PD1-blockade efficacy.

Abstract T-cell transfer into lymphodepleted recipients induces homeostatic activation and potentiates antitumor efficacy. In contrast to canonical T-cell receptor–induced activation, homeostatic activation yields a distinct phenotype and memory state whose regulatory mechanisms are poorly understood. Here, we show in patients and murine models that, following transfer into lymphodepleted bone marrow transplant (BMT) recipients, CD8+ T cells undergo activation but also simultaneous homeostatic inhibition manifested by upregulation of immune-checkpoint molecules and functional suppression. T cells transferred into BMT recipients were protected from homeostatic inhibition by PD-1/CTLA4 dual checkpoint blockade (dCB). This combination of dCB and BMT—”immunotransplant”—increased T-cell homeostatic activation and antitumor T-cell responses by an order of magnitude. Like homeostatic activation, homeostatic inhibition is IL7/IL15-dependent, revealing mechanistic coupling of these two processes. Marked similarity in ex vivo modulation of post-BMT T cells in mice and patients is promising for the clinical translation of immunotransplant (NCT03305445) and for addressing homeostatic inhibition in T-cell therapies. Significance: For optimal anticancer effect, T-cell therapies including chimeric antigen receptor T-cell, tumor-infiltrating lymphocyte, and transgenic T-cell therapies require transfer into lymphodepleted recipients and homeostatic activation; however, concomitant homeostatic inhibition mitigates T-cell therapies' efficacy. Checkpoint blockade uncouples homeostatic inhibition from activation, amplifying T-cell responses. Conversely, tumors nonresponsive to checkpoint blockade or BMT are treatable with immunotransplant. See related commentary by Ansell, p. 1487. This article is highlighted in the In This Issue feature, p. 1469

66 Background: The success of checkpoint blockade therapy is often dependent on CD8 T cell activation against tumor antigens. However, clinical benefit is only seen in a subset of patients, suggesting that there are other possibly targetable immunosuppressive pathways that are allowing the tumor to escape immune surveillance. Methods: A CD8 T cell that recognizes the EGFP200-208 peptide epitope allowed for the use of EGFP as a model tumor antigen while monitoring expression levels at the single cell resolution. Using a lymphoma line expressing EGFP or mCherry as our antigen-positive and -negative cancer models, we employed 3 screening strategies: 1) a forward genetics approach in which we selected for tumor cells that had naturally developed resistance to killing by CD8 T cells; 2) a reverse genetics approach that involved the use of pooled CRISPR libraries to identify knockout clones with a selection advantage or disadvantage when pressured by activated T cells; and 3) small molecule libraries, including FDA-approved drugs, to identify compounds that increased antigen-specific CD8 T cell killing. Results: Despite antigen recognition and early activation in response to the resistant tumor line, T cells failed to produce effector cytokines and underwent apoptosis in a PD-L1 and CTLA-4 independent manner. Candidate genes mediating this phenotype were derived from expression differences between the original susceptible tumor line and the immunoedited resistant tumor line. The pooled CRISPR approach was validated in a curated library by identifying genes with known roles in T cell-mediated killing and antigen presentation, such as Fas, B2m, and Tap1, as well as known suppressive molecules such as BTLA and PD-L1. LDL receptor expressed on the cancer cell emerged as a possible novel suppressor of T cells. Decitabine and 4-cinnolinethiol, among other small molecules, emerged as possible enhancers of CD8 T cell activity. Conclusions: We have identified several gene candidates as potentially novel and targetable checkpoint-like molecules, as well as small molecule compounds that may be able to enhance the anti-tumor activity of CD8 T cells. Efforts are ongoing in order to validate these targets and to screen larger libraries.

123 Background: In patients with low-grade lymphoma, in situ vaccination has yielded both partial and complete remissions in clinical trials. Though clinical responses have been observed with multiple pattern recognition receptor agonists (PRRa), the optimal immune stimulant is unknown. We hypothesize that natural PRRa, such as the attenuated pathogens or subunits found in common prophylactic vaccines, could target multiple PRR in a physiologically relevant context and lead to a more robust activation of dendritic cells (DCs) versus synthetic PRRa. Methods: 20 vaccines, including BCG, Typhim Vi, MMR-II, etc. were screened in vitro, where DC phenotype and function were evaluated by flow cytometry. Flt3L-mobilized DC ability to phagocytose, process, present, and cross-present soluble protein or tumor derived antigen, were assessed using CRISPR gene-edited, β2m(-/-) GFP-lymphoma cells and a novel GFP-specific (‘JEDI’) CD8 T cell system. Vaccine mechanism of immune activation was elucidated using a library of PRR-null macrophage cell lines. Potent vaccines were also evaluated in vivo in a Flt3L-primed in situ vaccination using the A20 murine lymphoma model. Results: Several vaccines induced robust DC activation and several showed significant increases in subsequent T cell activation, proliferation, and tumor killing, suggesting increased antigen processing and cross-presentation by DCs. Some vaccines, either as single agents or in combination, were significantly more effective than synthetic PRRa in activating DCs and inducing a T cell response. In vivo, vaccine combination therapies induced tumor regression in a majority of animals, suggesting synergistic immune activation. Conclusions: This data suggests prophylactic vaccines are effective clinical-grade DC activators and can be repurposed for use in the in situ vaccination maneuver, with immediate translation into the clinic. Additionally, by extensive in vitro evaluation in parallel with in vivo studies, this work aims to identify a predictive in vitro molecular immune signature that correlates closely with adjuvant efficacy in vivo.

3076 Background: Checkpoint blockade has emerged as an effective strategy for enhancing the endogenous T cell response against tumor-associated antigens. Its clinical benefit is maximized when combined with other immunomodulators or therapeutic maneuvers that induce immunogenic cell death. In order to find additional targetable suppressors of T cell activity, we have created an easily manipulable model of cancer immunoediting. Use of previous transgenic CD8 T cell models (e.g. OT-1) are confounded by difficulty in assessing tumor antigen (e.g. OVA) levels independent of MHC expression. Methods: Balb/c-derived A20 lymphoma cells expressing EGFP (A20-GFP-R0) were cultured with JEDI T cells that recognize the EGFP200-208 peptide epitope presented on H-2Kd (Agudo et al., Nat Biotech 2015). This novel system permitted the flow sorting of rare ( < 0.1%) surviving cells based on antigen and MHC class I expression.Five iterations of this T-cell selective pressure allowed for the enrichment of JEDI-resistant clones (A20-GFP-R5) that exclude antigen-loss and antigen presentation-deficient variants, two common mechanisms of immune escape. Results: A20-GFP-R5 exhibit marked (~100-fold) resistance to killing by JEDI T cells, despite at-least-equivalent levels of MHC-I and GFP expression compared to A20-GFP-R0. T cell activation, as measured by surface expression of CD25 and CD69 as well as proliferation, is nearly identical in response to either R0 or R5, suggesting that there is no impairment of the TCR-peptide-MHC complex. However, we observe a severe deficiency in the production of effector cytokines TNFα and INFγ in T cells cultured with R5. This effect seems to be independent of the PD-1 signaling pathway, since R5 has similar levels of PD-L1 expression, and treatment with PD-1 blocking antibodies cannot rescue the exhausted T cell phenotype. Conclusions: By naturally selecting for a rare subpopulation of cancer cells that are resistant to killing by CD8 T cells, we have found a PD-L1-independentcheckpoint pathway utilized by cancer to evade the immune response. Whole exome and RNA-seq studies are ongoing in order to identify the resistance mechanism(s) and to assess their use as potential immunotherapy targets.

e14538 Background: Lymphomas comprise the 5thmost common cancers in the U.S. and the majority of these are incurable with standard chemo-immunotherapy, thus, novel, mechanistically distinct therapies are needed, such as immunotherapy. Two promising classes of immunotherapy are: checkpoint blockade (e.g. anti-PD1 and anti-CTLA-4 antibody) and adoptive T-cell transfer lymphocytes into lymphodepleted recipients (e.g. CARs and TILs). Methods: In a PDL1(+), CD80/86(+) murine lymphoma model, we have developed a novel therapy combining these approaches into 'checkpoint-blockade-primed immunotransplant' comprised of: -treatment of tumor-bearing animals (donors) with anti-PD-1 and anti-CTLA-4 antibodies -splenocyte and bone marrow harvest and transfer to lymphodepleted/myeloablated (9Gy TBI) recipient Results: The combined therapy immunotransplant maneuver results in superior anti-tumor immunity compared to either checkpoint blockade or syngeneic transplant individually. Transferred T cells significantly increase surface and intra-cellular PD1 and CTLA-4, respectively, in both CD4 and CD8 T cells. Treatment of both tumor-bearing donor and recipient with anti-PD1 and anti-CTLA-4 antibodies induces cure of the majority of recipients, in a CD8 and IFNγ-dependent manner, despite the finding that antibody therapy alone (without transplantation and T cell transfer) induces minimal anti-tumor effect. Herein, we have demonstrated that T cells exposed to checkpoint blockade and transfer into the lymphopenic host demonstrate greater: -response to common γ-chain cytokines (per in vitro STAT5 phosphorylation) -in vitro production of IFNγ and TNF production in response to exposure to cognate tumor antigen -in vivo proliferation after exposure to tumor antigen Conclusions: Overall the data suggest both that: lymphopenia-exposed T cells become activated by checkpoint blockade and checkpoint blockade-exposed T cells become more activated by common γ-chain cytokines associated with lymphopenia. These data guide the development of combination therapies such as immunotransplant which we demonstrate is uniquely capable of curing the majority of established tumors.