Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient-derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a human stroma-patient-derived xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrates greater resistance to targeted therapy and chemotherapy and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on recruitment of TAMs and tumor immune exclusion to impact to response to immunologic therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stroma-mediated immunosuppression. Combined, our data confirm a critical role for human stroma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing.

Abstract: Chronic lymphocytic leukemia (CLL) presents challenges in treatment despite advancements in targeted therapies, often facing resistance or relapse. Chronic inflammation plays a significant role in CLL biology, with heightened inflammatory responses and immune dysfunction. Elevated levels of inflammatory cytokines support this notion. Activating signaling pathways such as NF-κB, Phosphoinositide 3-kinase delta (PI3Kδ), and MAPK via B-cell receptors and CD40 confers advantages to leukemic lymphocytes. Our research focuses on the proinflammatory protein S100A9 in CLL progression. We previously described that patients with CLL release exosomes containing S100A9 during disease progression, correlating with NF-κB activation. S100A9, known for its role in autoimmune diseases and cancers, modulates the antitumor immune response by influencing myeloid-derived suppressor cells. Receptors for S100A9 include Toll-like receptor 4, receptor for advanced glycation end products, and extracellular matrix metalloproteinase inducer (EMMPRIN). We identified a novel molecular mechanism involving the S100A9-EMMPRIN interaction in CLL using primary cells and an in vivo CLL mouse model (Eμ-TCL1). Additionally, we developed an Eμ-TCL1/S100A9–/– mouse model and explored pharmacological targeting of S100A9 in a patient-derived xenograft model, highlighting S100A9 as a promising therapeutic target in CLL with potential clinical applications.

Abstract: An unmet clinical need in chronic lymphocytic leukemia (CLL) is emerging due to the rapidly expanding group of patients with double refractory (Bruton's tyrosine kinase- and B-cell lymphoma 2-inhibitor) disease. So far, autologous T-cell–based therapies, including chimeric antigen receptor (CAR) T cells, have limited success in CLL, which has been attributed to an acquired CLL-mediated T-cell dysfunction and subset skewing toward effector cells at the expense of memory formation. T-cell responses rely on dynamic metabolic processes, particularly mitochondrial fitness. Although mitochondrial disruptions have been observed in solid tumor–infiltrating lymphocytes, their impact on T-cell immunity in lymphoproliferative disorders is unknown. Recent findings indicate that mitochondrial mass in CAR T cells correlates with CLL clinical outcomes. This prompted our investigation into the mitochondrial fitness in CLL T cells. Integrated metabolic and functional analyses revealed impaired, depolarized mitochondria across all T-cell subsets in untreated patients with CLL, leading to further ex vivo and in vivo mouse studies on the underlying signaling alterations. Multiomics profiling of transcriptome and epigenome revealed significant alterations in mitochondrial signaling, diminished adenosine monophosphate-activated protein kinase and autophagy activity, and upregulated glycolysis coupled with hyperactivation of Akt. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway during CLL T-cell culture induced metabolic reprogramming, enhancing mitochondrial activity, expression of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha, and memory differentiation. Underscoring clinical relevance, supplementation with the PI3Kδ inhibitor idelalisib during CAR T-cell manufacturing improved persistence and long-term leukemia-free remissions in an immunocompetent murine model. Our study suggests that modulating the abnormal CLL T-cell metabolism can enhance the efficacy of autologous T-cell therapies.

Chimeric antigen receptor (CAR) T cells have been largely unsuccessful in the treatment of non-hematologic malignancies, in part due to immunosuppressive networks at solid tumor beds and a paucity of viable targets. We previously showed that conventional 41BB/CD3ζ-based second-generation CAR T cells targeting OR5V1, an olfactory receptor with negligible expression in vital organs but present in various epithelial tumors, demonstrate therapeutic efficacy in models of gynecologic malignancies. To enhance metabolic superiority and antitumor effectiveness, we sought to generate a novel CD3ζ-less CAR containing the transmembrane and intracellular domains of Natural killer group 2 member D (NKG2D), which is expressed on the surface of natural killer (NK) cells, NKT cells and CD8 cells.

Dimeric IgA (dIgA) can move through cells via the IgA/IgM polymeric immunoglobulin receptor (PIGR), which is expressed mainly on mucosal epithelia. Here, we studied the ability of dIgA to target commonly mutated cytoplasmic oncodrivers. Mutation-specific dIgA, but not IgG, neutralized KRASG12D within ovarian carcinoma cells and expelled this oncodriver from tumor cells. dIgA binding changed endosomal trafficking of KRASG12D from accumulation in recycling endosomes to aggregation in the early/late endosomes through which dIgA transcytoses. dIgA targeting of KRASG12D abrogated tumor cell proliferation in cell culture assays. In vivo, KRASG12D-specific dIgA1 limited the growth of KRASG12D-mutated ovarian and lung carcinomas in a manner dependent on CD8+ T cells. dIgA specific for IDH1R132H reduced colon cancer growth, demonstrating effective targeting of a cytoplasmic oncodriver not associated with surface receptors. dIgA targeting of KRASG12D restricted tumor growth more effectively than small-molecule KRASG12D inhibitors, supporting the potential of this approach for the treatment of human cancers.

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains unclear. Using single-cell RNA or T-cell receptor (TCR) sequencing of 32 619 CD3+CD4+ and CD26+/CD7+ and 29 932 CD3+CD4+ and CD26-/CD7- lymphocytes from the peripheral blood of 7 patients with CTCL, coupled to single-cell ATAC-sequencing of 26,411 CD3+CD4+ and CD26+/CD7+ and 33 841 CD3+CD4+ and CD26-/CD7- lymphocytes, we show that tumor cells in S e ' zary syndrome and mycosis fungoides (MF) exhibit different phenotypes and trajectories of differentiation. When compared to MF, Se ' zary cells exhibit narrower repertoires of TCRs and exhibit clonal enrichment. Surprisingly, we identified >= 200 mutations in hematopoietic stem cells from multiple patients with S e ' zary syndrome. Mutations in key oncogenes were also present in peripheral S e ' zary cells, which also showed the hallmarks of recent thymic egression. Together our data suggest that CTCL arises from mutated lymphocyte progenitors that acquire TCRs in the thymus, which complete their malignant transformation in the periphery.

Objective. To demonstrate that shared antibody responses in endometriosis and endometriosis-associated ovarian cancer spontaneously antagonize malignant progression and can be leveraged to develop future immu-notherapies.Methods. B cells from cyopreserved clear cell ovarian carcinoma (CCC, n = 2), endometrioid ovarian carci-noma (EC, n = 2), and endometriomas (n = 2) were isolated, activated, and EBV-immortalized. Antibodies were purified from B cell supernatants and used for screening arrays containing most of the human proteome. Targets were prioritized based on accessibility (transmembrane or secreted proteins), expression in endometri-osis and cancer, and concurrent IgA and IgG responses. We focused on antibodies targeting tumor-promoting syndecan binding protein (SDCBP) to demonstrate anti-tumor activity. Immunoblots and qPCR were performed to assess SDCBP expression in ovarian cancer and endometriosis cell lines and tumor samples. Recombinant IgG4 was generated using the variable heavy and light chains of dominant B cell receptors (BCRs) reacting against the extracellular domain of SDCBP, and used in in vivo studies in human CCC-and high-grade serous ovarian carci-noma (HGSOC)-bearing immunodeficient mice. Results. Nine accessible proteins detected by both IgA and IgG were identified in all samples -including SDCBP, which is expressed in ovarian carcinomas of multiple histologies. Administration of alpha-SDCBP IgG4 in OVCAR3 (HGSOC), TOV21G and RMG-I (CCC) tumor-bearing mice significantly decreased tumor volume compared to control irrelevant IgG4.Conclusions. Spontaneous antibody responses exert suboptimal but measurable immune pressure against malignant progression in ovarian carcinomas. Using tumor-derived antibodies for developing novel immuno-therapeutics warrants further investigation.(c) 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Supplementary Figure from Olfactory Receptor OR2H1 Is an Effective Target for CAR T Cells in Human Epithelial Tumors

Radiation therapy (RT) can prime and boost systemic anti-tumor effects via STING activation, resulting in enhanced tumor antigen presentation and antigen recognition by T cells. It is increasingly recognized that optimal anti-tumor immune responses benefit from coordinated cellular (T cell) and humoral (B cell) responses. However, the nature and functional relevance of the RT-induced immune response are controversial, beyond STING signaling, and agonistic interventions are lacking. Here, we show that B and CD4+ T cell accumulation at tumor beds in response to RT precedes the arrival of CD8+ T cells, and both cell types are absolutely required for abrogated tumor growth in non-irradiated tumors. Further, RT induces increased expression of 4-1BB (CD137) in both T and B cells; both in preclinical models and in a cohort of patients with small cell lung cancer treated with thoracic RT. Accordingly, the combination of RT and anti-41BB therapy leads to increased immune cell infiltration in the tumor microenvironment and significant abscopal effects. Thus, 4-1BB therapy enhances radiation-induced tumor-specific immune responses via coordinated B and T cell responses, thereby preventing malignant progression at unirradiated tumor sites. These findings provide a rationale for combining RT and 4-1bb therapy in future clinical trials.

Supplementary Data from Treatment of Chronic Lymphocytic Leukemia with a Hypomethylating Agent Induces Expression of NXF2, an Immunogenic Cancer Testis Antigen

Objectives: To develop chimeric antigen receptor (CAR) T cells targeting the olfactory receptor family 5 subfamily V member 1 (OR5V1) for the treatment of ovarian cancer. Methods: OR5V1 expression was quantitated by real-time quantitative polymerase chain reaction (RT-QPCR) and western blot analysis of 17 normal tissues and 75 ovarian cancers of various histologies. The sequence of an antibody fragment that recognizes the extracellular domain of OR5V1 was predicted from the amino acid sequence of OR5V1. Focusing on this antibody fragment, potential linear B cell epitopes were predicted. The peptides were biotinylated and tetramerized using fluorescently labeled streptavidin. Immortalized B cells derived from 10 human high-grade serous ovarian cancers reacting with the tetramer were fluorescence-activated cell sorting (FACS) sorted and subjected to 10x single cell B cell receptor sequencing. The sequences of the variable heavy and variable light chains of a dominant clone representing 54% of the population were identified and used to predict the sequence of a single-chain variable fragment (scFv) interacting with OR5V1. We designed the CAR construct using the olfactory receptor signal peptide, followed by the OR5V1 scFv, linked by a glycine/serine spacer, followed by a CD8a hinge, and transmembrane domains, and intracellular domains of co-stimulatory 4-1BB and CD3ζ. The construct was cloned into a retroviral vector, and human T cells were retrovirally transduced to express the OR5V1 CAR. The OR5V1 -specific cytotoxic killing of HeLa cells with endogenous expression of OR5V1 was demonstrated in vitro using luciferase assays and in vivo using an immunodeficient mouse model. Differences between the means of experimental groups were calculated using a two-tailed unpaired Student’s t-test or two-way ANOVA with multiple comparisons. A p-value of <0.05 was considered statistically significant. Results: OR5V1 is expressed in ovarian cancers of multiple histologies, with expression in normal human tissues limited to the testis. There was no cytotoxic killing by OR5V1 CAR T cells of healthy human adipocytes, hepatocytes, or neurons. OR5V1 CAR T cells demonstrated cytotoxicity against HeLa cells, with nearly 100% specific killing in a dose-dependent manner, compared to mock transduced T cells (p=0.0002). Administration of OR5V1 CAR T cells in HeLa tumor-bearing mice significantly abrogated tumor growth, while mock transduced T cells were unable to prevent accelerated malignant progression (p<0.0001). Tumors treated with OR5V1 CAR T cells demonstrated not only reduced tumor volumes (p<0.0001) and weights (p=0.0023) but also significant central necrosis compared to those treated with mock transduced T cells. Conclusions: This study characterizes the expression pattern of OR5V1 in normal tissues and ovarian tumors of multiple histologies. We also demonstrate the therapeutic efficacy of targeting OR5V1-expressing tumors with CAR T cells re-directed against OR5V1, with a likely admissible toxicity profile. Objectives: To develop chimeric antigen receptor (CAR) T cells targeting the olfactory receptor family 5 subfamily V member 1 (OR5V1) for the treatment of ovarian cancer. Methods: OR5V1 expression was quantitated by real-time quantitative polymerase chain reaction (RT-QPCR) and western blot analysis of 17 normal tissues and 75 ovarian cancers of various histologies. The sequence of an antibody fragment that recognizes the extracellular domain of OR5V1 was predicted from the amino acid sequence of OR5V1. Focusing on this antibody fragment, potential linear B cell epitopes were predicted. The peptides were biotinylated and tetramerized using fluorescently labeled streptavidin. Immortalized B cells derived from 10 human high-grade serous ovarian cancers reacting with the tetramer were fluorescence-activated cell sorting (FACS) sorted and subjected to 10x single cell B cell receptor sequencing. The sequences of the variable heavy and variable light chains of a dominant clone representing 54% of the population were identified and used to predict the sequence of a single-chain variable fragment (scFv) interacting with OR5V1. We designed the CAR construct using the olfactory receptor signal peptide, followed by the OR5V1 scFv, linked by a glycine/serine spacer, followed by a CD8a hinge, and transmembrane domains, and intracellular domains of co-stimulatory 4-1BB and CD3ζ. The construct was cloned into a retroviral vector, and human T cells were retrovirally transduced to express the OR5V1 CAR. The OR5V1 -specific cytotoxic killing of HeLa cells with endogenous expression of OR5V1 was demonstrated in vitro using luciferase assays and in vivo using an immunodeficient mouse model. Differences between the means of experimental groups were calculated using a two-tailed unpaired Student’s t-test or two-way ANOVA with multiple comparisons. A p-value of <0.05 was considered statistically significant. Results: OR5V1 is expressed in ovarian cancers of multiple histologies, with expression in normal human tissues limited to the testis. There was no cytotoxic killing by OR5V1 CAR T cells of healthy human adipocytes, hepatocytes, or neurons. OR5V1 CAR T cells demonstrated cytotoxicity against HeLa cells, with nearly 100% specific killing in a dose-dependent manner, compared to mock transduced T cells (p=0.0002). Administration of OR5V1 CAR T cells in HeLa tumor-bearing mice significantly abrogated tumor growth, while mock transduced T cells were unable to prevent accelerated malignant progression (p<0.0001). Tumors treated with OR5V1 CAR T cells demonstrated not only reduced tumor volumes (p<0.0001) and weights (p=0.0023) but also significant central necrosis compared to those treated with mock transduced T cells. Conclusions: This study characterizes the expression pattern of OR5V1 in normal tissues and ovarian tumors of multiple histologies. We also demonstrate the therapeutic efficacy of targeting OR5V1-expressing tumors with CAR T cells re-directed against OR5V1, with a likely admissible toxicity profile.

Abstract Although chimeric antigen receptor (CAR)-expressing T cells have proven success in hematologic malignancies, their effectiveness in solid tumors has been largely unsuccessful thus far. We found that some olfactory receptors are expressed in a variety of solid tumors of different histologic subtypes, with a limited pattern of expression in normal tissues. Quantification of OR2H1 expression by qRT-PCR and Western blot analysis of 17 normal tissues, 82 ovarian cancers of various histologies, eight non–small cell lung cancers (NSCLCs), and 17 breast cancers demonstrated widespread OR2H1 expression in solid epithelial tumors with expression in normal human tissues limited to the testis. CAR T cells recognizing the extracellular domain of the olfactory receptor OR2H1 were generated with a targeting motif identified through the screening of a phage display library and demonstrated OR2H1-specific cytotoxic killing in vitro and in vivo, using tumor cells with spontaneous expression of variable OR2H1 levels. Importantly, recombinant OR2H1 IgG generated with the VH/VL sequences of the CAR construct specifically detected OR2H1 protein signal in 60 human lung cancers, 40 ovarian carcinomas, and 73 cholangiocarcinomas, at positivity rates comparable with mRNA expression and without OR2H1 staining in 58 normal tissues. CRISPR/Cas9-mediated ablation of OR2H1 confirmed targeting specificity of the CAR and the tumor-promoting role of OR2H1 in glucose metabolism. Therefore, T cells redirected against OR2H1-expressing tumor cells represent a promising therapy against a broad range of epithelial cancers, likely with an admissible toxicity profile.

e14507 Background: We hypothesized that tumor-infiltrating B cells in endometriosis and endometriosis-associated ovarian cancer can be used to identify novel, targetable antigen domains to inhibit the progression of ovarian carcinomas. We aimed to identify targets that are spontaneously recognized by B-cell-derived antibodies within ovarian clear cell carcinoma (CCC), endometrioid carcinoma (EC), and endometriosis and to determine the preclinical anti-cancer efficacy of a candidate antibody that recognizes the extracellular domain of an identified target. Methods: B cells from freshly dissociated CCC (n = 2), EC (n = 2), and endometriomas (n = 2) were isolated, activated, and EBV-immortalized. Antibodies were purified from B cell supernatants and used for screening in a proteome array. Targets were prioritized based on accessibility (transmembrane or secreted proteins), expression in endometriosis and cancer, and concurrent IgA and IgG responses. Tumor-promoting syndecan binding protein (SDCBP) was identified and SDCBP-reactive B cells were FACS-sorted and subjected to single cell B cell receptor sequencing to determine the variable heavy and light chain sequences of enriched clonotypes. We then generated a recombinant IgG4 antibody targeting SDCBP with the dominant VH/VL matching sequences. Immunoblots and qPCR were performed to assess SDCBP expression in ovarian cancer cell lines and tumor samples. In vivo studies compared anti-tumor potential of the α-SDCBP IgG4 with controls using immunodeficient mouse models of human CCC and high-grade serous ovarian carcinoma (HGSOC). Results: Nine accessible proteins were detected by both IgA and IgG in all samples, including SDCBP. SDCBP is expressed in ovarian cancer cell lines and tumor samples of multiple histologies, including CCC, EC, and HGSOC. Administration of α-SDCBP IgG4 in TOV21G (CCC) tumor-bearing mice significantly decreased tumor volume compared to non-antigen-specific irrelevant IgG4 (iIgG4, p = 0.002) and vehicle (0.001), and correspondingly trended toward decreased tumor weight compared to vehicle (p = 0.06). Likewise, administration of α-SDCBP IgG4 in OVCAR3 (HGSOC) tumor-bearing mice significantly decreased tumor volume compared to iIgG4 (p = 0.03) and trended toward decreased tumor weight (p = 0.06). Conclusions: An α-SDCBP IgG4 has demonstrated anti-tumor efficacy in SDCBP+ CCC and HGSOC, and SDCBP-targeted therapy for endometriosis and associated malignant conditions, as well as HGSOC, warrants further investigation.

Despite repeated associations between T cell infiltration and outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that the hallmarks of tumor recognition in ovarian cancer-infiltrating T cells are primarily restricted to tissue-resident memory (TRM) cells. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3+CD8+CD103+CD69+ TRM cells and immunohistochemistry of 122 high-grade serous ovarian cancers shows that only progenitor (TCF1low) tissue-resident T cells (TRMstem cells), but not recirculating TCF1+ T cells, predict ovarian cancer outcome. TRMstem cells arise from transitional recirculating T cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes that eventually become exhausted. Therefore, ovarian cancer is indeed an immunogenic disease, but that depends on ∼13% of CD8+ tumor-infiltrating T cells (∼3% of CD8+ clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM-like cells. Our results define the signature of relevant tumor-reactive T cells in human ovarian cancer, which could be applicable to other tumors with unideal mutational burden.

BACKGROUND. Mycosis fungoides/Sezary syndrome (MF/SS) are the most common subtypes of cutaneous T cell lymphoma. The patients (pts) with early-stage MF usually have an indolent course and are managed with skin directed therapies. In contrast, pts with advanced-stage disease, have poor prognosis and a median overall survival between 2 and 5 years. Over the past 20 years only six novel biological/targeted agents (bexarotene, denileukin diftitox, vorinostat, romidepsin, brentuximab vedotin, mogamulizumab) have been FDA approved for systemic therapy of relapsed/refractory MF/SS however, none render curative approach. Overall response rate across all agents is 30% with CR rate of only 10%. Classical chemotherapy has no advantage over less toxic novel targeted agents in MF/SS. The head-to-head comparison of active therapeutic agents in prospective randomized trials is mostly absent and systemic therapy sequencing strategies are usually derived from personal experience, expert opinion, or retrospective reviews. The biomarkers directing the selection of targeted agents for individual patients are not available or reliable. In this study, we tested sensitivity of autologous lymphoma cells obtained from peripheral blood to various therapeutic agents and combinations ex vivo, in order to predict response to tested agents in vivo and develop more sophisticated, personalized, cost-effective, and clinical approach for treatment of pts with advanced stage MF/SS. METHODS. A 384-well plate is coated with poly-L-ornithine to help anchor the cells and the CTCL cells collected from the patients at Moffitt Cancer Center are thawed and incubated over-night at 37C. The next day, isolated CTCL cells are stained with CytoLight Rapid Green Reagent from Essen Bioscience. Once the dye is neutralized and washed out from the cells CytoxRed from Essen Bioscience is added to the media with cells. The cells are then distributed in a 384-well plate. Next, the drugs are diluted and added to the plate. There is a 5-time serial dilution of the drug and if it is a drug combination, one drug is serial diluted and the second drug is constant. After plating, the cells are incubated at 37C for 30min to let them rest and equilibrate. The EVOS M7000 (Life Technologies) is used using the GFP and Texas Red cubes to detect live (green) and dead (red) cells at 0h, 6h, 12h, 18h and 24h. When analyzing the data, the Celleste 5 (Life Technologies) program is used, specifically the Live/Dead application. The intensity threshold is kept constant throughout the entire experiment. The total number of live cells are used to calculate the viability of the cells at each time point. All the time points are done in triplicates and media with DMSO is used as the control. RESULTS. Out of 11 MF/SS pts samples treated with single agent and/or combinatorial drugs, 4 pts samples had no appreciable responses to single or combination treatments. In this study, response to treatment is deemed favorable if there is a decrease of 50% or more in viable cells in the course of the experiment. Seven pts samples did have responses against either a single drug or a combination of drugs. Out of the 7 pts that did respond, the most prevailing treatment was the combination of Venetoclax and Duvelisib with 6 out of 7 pts samples having a response to this combination. The next noteworthy response was seen with Romidepsin alone in 4 out of 7 pts. CONCLUSION. Our observations on MF/SS pts samples thus far demonstrates various sensitivities to treatments for individual pts. This may account for genetic and epigenetic changes which highlight the unmet need for personalization of treatment and sensitivity screening for each patient, with the decisive goal to offer a personalized and efficacious treatment modalities.

Supplementary Figure from IgA-Dominated Humoral Immune Responses Govern Patients' Outcome in Endometrial Cancer

Abstract The role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we report that concomitant accumulation of T, B and plasma cells at tumor beds predicts better survival. However, only B cell markers predict survival specifically in high-grade endometrioid type and serous tumors. Accordingly, immune protection is associated with class-switched IgA and, to a lesser extent, IgG. Notably, expression of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA are superior predictors of outcome, and correlate with defects in methyl mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drives inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and ER stress pathways, while thwarting DNA repair mechanisms. Therefore, coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer, and therefore the potential for effective immunotherapies. Supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687 and R01CA211913), and from Cancer Center Support Grant (CCSG) CA076292

Phosphatidylinositol-3 kinase (PI3K) delta signaling is essential for CLL B cell survival and proliferation. Several PI3K inhibitors are approved for treatment of CLL, but PI3K inhibitor-induced alterations in T cell function have been correlated with immune-related adverse events (irAEs). Zandelisib is a novel selective PI3Kδ inhibitor in clinical development with an intermittent dosing schedule for treatment of B cell malignancies. Here, we investigated the dynamic immunomodulatory properties of zandelisib alone or in combination with BTK inhibitor ibrutinib in normal human T cells, as well as the impact of continuous dosing of zandelisib on effector and regulatory immune cells, irAEs, and survival in a preclinical CLL murine model. Normal human T cells treated ex vivo with zandelisib, ibrutinib, or both were assessed for phenotype and function. A CLL model was initiated by transferring leukemic EμTCL1 splenocytes into syngeneic wildtype recipients. CLL-bearing mice were treated with vehicle or zandelisib (50 mpk bid or 100 mpk qd) for 3 weeks (n=5 per group). Antitumor efficacy, T cell and myeloid subsets in blood, bone marrow and spleen were analyzed. Survival was also observed in a separate cohort of mice treated with zandelisib (100 mpk qd). T cell proliferation was impaired by zandelisib (250nM) and ibrutinib (100 nM) alone, with the combination further reducing T cell proliferation. Zandelisib decreased expression of suppressive markers PD-1, CTLA-4, GITR and CD39 on iTregs; however, no further decrease was observed when zandelisib was combined with ibrutinib. Zandelisib also reduced normal T cell activation and expression of immune checkpoints PD-1 and CTLA-4 on CD4+ T cells and dose dependently inhibited T cell memory differentiation ex vivo, increasing the naïve/central to effector memory ratio. Combination with ibrutinib did not further affect T cell activation, checkpoint expression or memory differentiation. In the CLL murine model, no meaningful difference was seen between bid or qd administration of zandelisib in the reduction of tumor burden (CD19+ CD5+ CLL cells). Zandelisib treatment reduced activated T cells, antigen-experienced T cells, and Tregs. Increased naïve/central to effector memory ratio was observed in treated mice, and PD-1 was downregulated on memory T cells, indicating less T cell exhaustion. Zandelisib decreased macrophages in the spleen but did not impact myeloid-derived suppressor cells. Zandelisib treatment improved survival of CLL-bearing mice significantly when compared to vehicle group (median survival of zandelisib group 140 days vs. 105 days vehicle). In conclusion, zandelisib treatment reduced Tregs, prevented terminal memory differentiation, and T-cell exhaustion—features that have been shown to permit CLL immune evasion—demonstrated antitumor efficacy, and improved overall survival in a preclinical CLL model. Citation Format: Kamira Maharaj, Melanie Mediavilla-Varela, John J. Powers, Sandra Wiley, Angimar Uriepero, Wael Gamal, Kun Jiang, Eva Sahakian, Javier Pinilla-Ibarz. Efficacy and immune profiling of PI3K delta inhibitor zandelisib (ME-401) in a preclinical chronic lymphocytic leukemia (CLL) model [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 5496.