Abstract Metastatic castration-resistant prostate cancer (mCRPC) represents an aggressive, treatment-refractory stage of prostate cancer with limited treatment options and poor prognosis. Chimeric antigen receptor (CAR)-T cell therapy has demonstrated success in hematological malignancies, but its efficacy in solid tumors is limited by tumor microenvironment (TME) barriers and tumor cell heterogeneity. In this study, we applied a single-cell enrichment-free liquid biopsy platform to monitor disease progression and CAR-T cell response in mCRPC patients enrolled in a Phase 1 clinical trial (NCT03873805). Using fluorescent whole-slide imaging (fWSI), we analyzed peripheral blood (PB) and bone marrow aspirate (BMA) from eight patients (four responders and four nonresponders) collected longitudinally before, during, and after therapy. Two key findings emerged: 1) lymphodepletion mobilized circulating tumor cells (CTCs) from bone marrow into PB, altering compartment-specific cellularity regardless of response, and 2) clearance of clonal CTCs after CAR-T cell infusion occurred in responders but not in nonresponders. Single-cell analyses further revealed that PB and BMA captured distinct CTC subtypes, underscoring the complementary value of both compartments for monitoring. This multi-omic analysis leverages high-resolution single-cell liquid biopsies to characterize circulating rare cells, such as CTCs and their subtypes, to correlate them with clinically observed responses to CAR-T cell therapy in mCRPC. Citation Format: Doanna Minh Pham, Stephanie Nicole Shishido, Saul J. Priceman, Tanya B. Dorff, Peter Kuhn. Single-cell liquid biopsy profiling in mCRPC receiving PSCA-targeted CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3746.

Background Pre-metastatic niches composed of mainly myeloid cells are recognized as critical for tumor metastasis. However, whether adaptive immune cells also play an important role in pre-metastatic niche formation remains to be explored.Methods CD4+ T cell accumulation in tumor-free lung tissues from mice bearing subcutaneous mouse tumors was detected by immunofluorescence/confocal microscopy. Tumor-conditioned media (TCM) from MB49-S1pr1high mouse bladder tumor cells or ID8 ovarian tumor cells were administered to tumor-free mice to induce pre-metastatic niche formation. We used mice lacking functional Signal Transducer and Activator of Transcription 3 (STAT3) in T cells and Il17a‒/‒ mice to investigate the roles of STAT3 and interleukin (IL)-17. In vivo time-course experiments were performed to assess whether CD4+ T cell clusters contribute to CD11b+ pre-metastatic clusters. CD4+ T cell migration and chemokine receptor expression assays were employed to identify tumor factors driving CD4+ T cell recruitment. A co-culture system with human MRC-5 lung fibroblasts, healthy donor-derived CD4+ T cells, myeloid cells, and TCM derived from human cancer cells was used to evaluate CD4+ T cell-driven fibroblast activation and IL-17A dependency for myeloid cell migration. Microscopic analyses were performed to confirm CD4+ T cell clusters in tumor-free lymph node tissues from patients with prostate cancer and postmortem lung and liver specimens from patients with ovarian cancer.Results We demonstrate that CD4+ T cells accumulate in tumor-free lungs and promote tumor metastasis in mouse models. CD4+ T-cell pre-metastatic niche formation requires STAT3, which regulates Th17 CD4+ T cells. TCM drives IL-6-dependent CCR4/CCR6 upregulation on naive CD4+ T cells. CD4+ T cell clusters contribute to myeloid cell accumulation, and ablating STAT3 in T cells abrogates both T cell and myeloid cluster formation. IL-17 inhibition reduces myeloid lung infiltration. In human co-cultures, CD4+ T cells amplify TCM-induced fibroblast pre-metastatic niche-like activation and myeloid recruitment in an IL-17A-dependent manner. CD4+ and IL-17+ or p-STAT3+ clusters were also detected in non-metastatic tissues from patients with several cancers.Conclusions CD4+ T cells form pre-metastatic niches through the STAT3-IL-17 axis, contributing to myeloid cell cluster formation, in part through amplifying fibroblast pre-metastatic niche-like activation. STAT3 and IL-17 in CD4+ T cells therefore are important for pre-metastatic niche formation and metastasis.

Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment of hematological malignancies. However, its application in treating solid tumors has encountered significant obstacles. This review explores the current state of CAR-T cell therapy for solid tumors, highlighting challenges including the pronounced heterogeneity of tumor antigens and the immunosuppressive tumor microenvironment. We explore a range of preclinical and clinical strategies to enhance the efficacy of solid tumor CAR-T cells. These strategies encompass engineering chimeric receptors that can simultaneously target multiple antigens expressed by tumor cells, as well as implementing combination therapies and armored CAR-T cells to overcome existing limitations. While encouraging advancements using solid tumor CAR-T cell therapies have been seen, addressing intrinsic challenges remains a significant endeavor. Ongoing investigation of these innovative strategies is essential for the successful application of CAR-T cells in the context of solid tumors.

Tumor-infiltrating lymphocyte (TIL) therapy, which involves extracting, expanding, and reinfusing immune cells to target cancer cells, has shown promise in melanoma treatment, but requires optimization for broader efficacy. The success of TIL therapy depends on the recognition of tumor-associated antigens, but neoantigen-reactive T-cells are often rare and exhausted in less immunogenic malignancies. Isolating T cells enriched in neoantigen reactivity prior to in vitro expansion and reinfusion may improve the response rates. To this end, our proprietary Specific Neo-Antigen Peptides (SNAP™) technology platform improves the accuracy of neoantigen prediction and validation by combining advanced computational modelling and PepSeq, a high-throughput screen for the physical credentialing of putative neoantigens based on their affinity to bind patient-specific HLA class II proteins. This approach allows for the education and enrichment of TILs (SNAP-TILs) with personalized, predefined, highly immunogenic neoantigens prior to expansion. Using the SNAP platform, we consistently achieved, on average, a SNAP-TIL product comprising 96% CD3+ cells, with a mixture of 75% effector and 23% central memory cells. SNAP-TILs exhibited greater efficacy and selectivity in immune infiltration than TIL, which was expanded by the rapid expansion protocol alone using ex vivo models. SNAP-TIL was also reactive in highly and poorly immunogenic tumors, with 70% and 50% tumor growth inhibition in melanoma and pancreatic patient-derived xenograft models, respectively. This study demonstrates the novel benefit of our Personalized Neoantigen Pipeline approach, potentially providing a durable antitumor immune response for a larger proportion of cancer patients.

Systematic whole-protein screening and comprehensive profiling of antigen-specific CD4+ T cells are crucial for advancing vaccine design and cancer immunotherapies, yet remain technically challenging. Here, we present a high-throughput platform that utilizes large-scale class II single-chain trimer libraries to detect antigen-specific CD4+ T cells, while simultaneously profiling their antigen specificity, TCRα/β sequences, MHC restriction, whole transcriptomes, and patient/timepoint origins at single-cell resolution. Upon rigorous platform validation, we screened the full SARS-CoV-2 spike receptor binding domain in a longitudinal cohort of 22 participants, identifying 2,188 antigen-specific CD4+ T cells and showing key metrics defining the immunogenicity of class II-restricted viral antigens. We further extended the platform to whole-protein screening of HPV-16 E6/E7 in a cohort of precancerous patients, indicating HPV-specific CD4 TCRs that, upon extensive characterization, demonstrate strong therapeutic potential. By integrating high-throughput antigen screening with high-dimensional, multi-modal cellular characterization, our approach provides detailed insight into CD4+ T cell immunity, potentially guiding vaccine design and next-generation TCR-based cancer immunotherapies.

Myeloid derived suppressor cells (MDSCs) are key players in the immune-suppressed tumor microenvironment (TME) and significantly contribute to immune checkpoint inhibition (ICI) resistance, making them favorable targets for cancer immunotherapy. Epigenetic reprogramming of MDSCs using histone deacetylase (HDAC) inhibitors shows promise to sensitize the TME to ICIs. However, the molecular mechanism of HDAC inhibition in MDSCs has yet to be elucidated. Murine and human MDSC models treated with Entinostat revealed that the long non-coding RNA Malat1 downregulates pSTAT3 and decreases MDSC-mediated suppression of T cell proliferation. Through HDAC inhibitor screens, we identified HDAC1 as preferentially regulating Malat1 expression, STAT3 activation, and MDSC suppression. We also show that HDAC1 inhibition increases MDSC apoptosis by shifting pro-vs. anti-apoptotic signals and increases G0/G1 cell cycle arrest via decreasing G1-S transition cyclin-CDK complexes. Collectively, our findings provide a multi-pronged mechanism of HDAC inhibition in MDSCs that inform the development of future rational combination therapies. One Sentence Summary:HDAC1 inhibition in MDSCs increases Malat1 , decreases pSTAT3, induces apoptosis/cell cycle arrest, and decreases suppression of T cells.

Abstract Metastatic castration-resistant prostate cancer (mCRPC) remains an incurable and immunologically cold solid malignancy. Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is highly expressed in over 85% of mCRPC tumors and represents an attractive therapeutic target. Although chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic cancers, its efficacy in solid tumors, including prostate cancer, has been limited by the immunosuppressive tumor microenvironment (TME) and heterogeneous antigen expression. Interleukin-12 (IL-12) has the potential to overcome these barriers by activating and recruiting immune cells into tumors and promoting epitope spreading to counter antigen heterogeneity. While armored CAR-T cells engineered to produce IL-12 have been developed, further refinement is needed to optimize both potency and safety. Autologous IL-12-producing T cell therapy has previously shown clinical activity in melanoma, but systemic toxicity constrained its use, indicating that IL-12 secreted by CAR-T cells, though locally produced, can still diffuse into circulation. Here, we present STEAP1-directed CAR-T cells engineered to conditionally secrete a collagen-binding domain-IL-12 fusion protein (CBD-IL-12) upon antigen engagement. We demonstrate that fusing IL-12 to a CBD markedly enhances its retention within prostate tumors while limiting systemic spread in mice. As a result, intra-tumoral levels of IFN-γ, CXCL9, and GM-CSF remained comparably high to those induced by IL-12, yet without associated elevations in serum alanine aminotransferase (ALT) or off-target T cell infiltration in healthy organs. Flow cytometry revealed increased infiltration of T cells, NK cells, and cross-presenting dendritic cells, together with reduced monocytic myeloid-derived suppressor cells, following treatment with CBD-IL-12-expressing STEAP1 CAR-T cells. Immunohistochemistry and spatial transcriptomic analysis confirmed increased immune infiltrates and activation of IL-12 pathway and antigen processing and presentation by major histocompatibility complex-I in the CBD-IL-12 CAR-T treated tumor. Further, tertiary lymphoid structure-related chemokine and chemokine receptors including cxcr4, cxcr5, cxcl12 and cxcl13 as well as co-stimulatory molecules such as cd80, cd86, cd40 and tnfsf4 were upregulated in the tumor. When combined with anti-PD-1 and anti-CTLA-4 antibodies, CBD-IL-12 armored CAR-T cells eradicated established prostate tumors in mice without preconditioning. The CAR-T therapy generated durable anti-tumor immune memory to STEAP1 and other antigens. Our findings suggest that CBD fusion can localize potent but toxic immunomodulators such as IL-12 to the tumor site, offering a promising strategy to improve the safety and effectiveness of CAR-T therapies for solid tumors. Citation Format: Koichi Sasaki, Vipul Bhatia, Yuta Asano, Jakob Bakhtiari, Pooja Kaur, Chuyi Wang, Takumi Matsuo, Olivier Dubois, Po-Chuan Chiu, Donny Gun, Charanjit Singh, Ioanna Panagi, Laurine Noblecourt, Maria Nikolaidi, Truman Chong, Gerardo Javier, Saul J. Priceman, Aude G. Chapuis, John K. Lee, Jun Ishihara. Collagen-binding IL-12-armored STEAP1 CAR-T cells for advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1524.

Adoptive cell therapies used to treat advanced prostate cancer are being developed to target several tumor-associated antigens, including prostate-specific membrane antigen (PSMA). Chimeric antigen receptor (CAR) T cell therapy using the single chain variable fragment (scFv) derived from the humanized murine mAb clone, J591, as the antigen-binding domain has shown promising anti-tumor activity. However, it has also been associated with macrophage activation syndrome and other unwanted toxicities, highlighting the need for more specific and human-derived antigen-binders with optimized construct designs for improved safety and efficacy. Here, we optimize a human scFv-based PSMA-targeted CAR (hPSMA-CAR) with highly selective PSMA targeting. We further introduce a membrane-bound IL-12 (mbIL12) molecule, which enhances potency with increased T cell expansion, IFNy production and anti-tumor cell activity in vitro . Using two clinically-relevant bone-metastatic prostate cancer models, we show that mbIL12-engineered hPSMA-CAR T cells drive potent in vivo anti-tumor responses. In summary, we have developed a promising therapeutic that has potential to promote safe and effective treatment of advanced PSMA+ prostate cancer.

177 Lu-PSMA-617 (Pluvicto TM , Lu-177 RLT) is an FDA-approved targeted radioligand therapy (RLT) for metastatic castration-resistant prostate cancer (mCRPC), but its durability of response to this singular approach poses a challenge to the field. Chimeric antigen receptor (CAR) T cell therapy has revolutionized clinical practice for hematological malignancies, but its clinical development for solid tumors, including mCRPC, has been encumbered by antigen heterogeneity and the immunosuppressive tumor microenvironment (TME). Here, we evaluate the therapeutic combination of Lu-177 RLT and PSCA-CAR T cells to overcome these barriers. In human xenograft and mouse syngeneic prostate cancer models with homogeneous or heterogeneous antigen expression, the sequential administration of Lu-177 RLT, cyclophosphamide (Cy), and PSCA-CAR T cells improves tumor control and prolongs survival compared to monotherapies. Mechanistically, Lu-177 RLT alone or with Cy remodels the TME by promoting pro-inflammatory myeloid responses and activating endogenous T cells, while enhancing CAR T cell activation and effector function. We additionally evaluated 225 Ac-PSMA-617 RLT as an emerging approach in combination with CAR T cells and observed anti-tumor responses, supporting its potential as an alternative RLT partner. These findings support RLT as an immune priming strategy to enhance CAR T cell therapy and provide a rationale for clinical translation of this combination in mCRPC. One Sentence Summary:Combining 177 Lu-PSMA-617 radioligand therapy with PSCA-CAR T cells improves tumor control and survival in prostate cancer models by overcoming the antigen heterogeneity and reshaping the immunosuppressive tumor microenvironment.

Endothelial cells (ECs) are crucial in cancer development and progression, partly by regulating tumor angiogenesis and immune modulation. As a key component of the RNA-induced silencing complex, Argonaute 1 (AGO1) regulates tumor biology, yet the specific function of AGO1 within ECs in the tumor microenvironment remains undefined. Here, we investigated the effects of endothelial-specific AGO1 knockout (EC-AGO1-KO) on tumor vascularization and immune regulation in a mouse syngeneic breast cancer model induced by E0771 cells. EC-AGO1-KO mice exhibited significantly reduced tumor burden compared to their wild-type (WT) littermates, accompanied by reduced vascularization and enhanced immune cell infiltration. Histological and single-cell RNA sequencing analyses revealed increased infiltration of CD8⁺ T cells and macrophages in EC-AGO1-KO tumors, indicative of an immunostimulatory microenvironment. In vitro, AGO1 knockdown in mouse ECs co-cultured with E0771 tumor cells led to higher levels of Cxcl10 and Vcam1 expression, suggesting a pro-inflammatory and leukocyte-recruiting effect. Together, these findings identify endothelial AGO1 as a key regulator of tumor vasculature and immune homeostasis in breast cancer, suggesting that targeting endothelial AGO1 may represent a novel therapeutic strategy to modulate tumor vasculature while enhancing anti-tumor immunity.

Chimeric antigen receptor (CAR) T cell therapy has limited efficacy against solid tumors such as prostate cancer due to the immunosuppressive tumor microenvironment (TME). Combining CAR T cells with existing therapies that remodel the TME and promote endogenous immune responses, such as radiation therapy and chemotherapies, may strengthen antitumor responses. Here, we assessed the potency of combining focal radiotherapy (RT), cyclophosphamide (Cy) preconditioning, and prostate stem cell antigen (PSCA)-CAR T cells against syngeneic prostate cancer models. Focal RT alone increased T cell and dendritic cell infiltration and activation in the irradiated tumor. Furthermore, the combination of all three therapies was critical for enhanced antitumor responses and survival across multiple subcutaneous, bone-metastatic, and multifocal disease models. This combination, in the irradiated TME and tumor-draining lymph nodes (tdLN), led to greater antigen presentation by myeloid cells and endogenous T cell activation and cytotoxicity. Our study demonstrates the potency of combining focal RT with PSCA-CAR T cells, significantly improving therapeutic responses in the irradiated tumor and contributing to a more robust systemic immune response against metastatic burden in prostate cancer.

Aging is the greatest risk factor for breast cancer, and although epithelial cells are the source of carcinomas, epithelial changes alone do not fully explain cancer susceptibility. Fibroblasts and macrophages are key stromal constituents around the cells of origin for cancer in breast tissue. With age, macrophages surrounding terminal ductal lobular units (TDLUs) become increasingly immunosuppressive. CD105+ fibroblasts intercalate within TDLUs, drive luminal differentiation, and give rise to immunosuppressive cancer-associated fibroblasts in other tissues. We propose that differences in fibroblasts are a crucial component of the stroma that shapes cancer susceptibility. Primary peri-epithelial fibroblast cultures were established from prophylactic and reduction mammoplasties from 30 women ranging in age from 16 to 70 years and from BRCA1 mutation carriers. Growth characteristics, transcriptional profiles, differentiation potential, and secreted proteins were profiled for fibroblast subtypes from diverse donors. Co-cultures with fibroblasts, macrophages, and T cells were used to ascertain the functional role played by CD105+ fibroblasts in immune cell modulation. We found that peri-epithelial CD105+ fibroblasts are enriched in older women as well as women who carry BRCA1 mutations. These CD105+ fibroblasts exhibit robust adipogenesis and secrete factors related to macrophage polarization. Macrophages cocultured with fibroblasts better maintain or enhance polarization states than media alone. CD105+ fibroblasts increased expression of immunosuppressive macrophage genes. CD105+ fibroblasts supported anti-inflammatory macrophage-mediated suppression of T cell proliferation, whereas CD105− fibroblasts significantly reduced the suppressive effect of anti-inflammatory macrophages on T cell proliferation. Establishment of a coculture system to dissect the molecular circuits between CD105+ fibroblasts and macrophages that drive immunosuppressive macrophage polarization has broad utility in understanding mammary gland development and events that precede cancer initiation. CD105+ fibroblasts and macrophages may coordinate to suppress immunosurveillance and increase breast cancer susceptibility.

Chimeric antigen receptor (CAR)-T cell immunotherapy, effective in blood cancers, shows limited success in solid tumors, such as prostate, pancreatic, and brain cancers due, in part, to an immunosuppressive tumor microenvironment (TME). Immunosuppression affects various cell types, including tumor cells, macrophages, and endothelial cells. Conventional murine-based models offer limited concordance with human immunology and cancer biology. Therefore, we have developed a human "tumor-on-a-chip" (TOC) platform to model elements of immunosuppression at high spatiotemporal resolution. Our TOC features an endothelial cell-lined channel that mimics features of an in vivo capillary, such as cell attachment and extravasation across the endothelium and into the TME. Using 70 kDa dextran and fluorescence-recovery-after-photobleaching (FRAP), we confirmed physiologic interstitial flow velocities (0.1-1 μm s-1). Our device demonstrates that tumor-derived factors can diffuse in the opposite direction of interstitial flow to reach the endothelium up to 200 μm away, and at concentrations as high as 20% of those at the tumor margin. M2-like immunosuppressive macrophages and endothelial cells affect prostate tumor cell growth, clustering, and migration. M2-like macrophages also induce PD-L1 and inhibit ICAM-1 gene expression on the adjacent endothelium in a pattern that limits CAR-T cell extravasation and effector function. This observation is abrogated in the presence of the anti-PD-L1 drug atezolizumab. These results provide mechanistic insight for in vivo observations showing limited CAR-T cell extravasation and effector function in solid tumors. Furthermore, they point to a specific role of M2 macrophages in driving CAR-T cell migration into and within the TME and could prove useful in the development of novel therapies to improve solid tumor CAR-T cell therapies.

Immunosuppressive microenvironments, the lack of immune infiltration, and antigen heterogeneity pose significant challenges for chimeric antigen receptor (CAR)-T cell therapies to tackle solid tumors. CAR-T cells were armed with immunostimulatory payloads, such as interleukin-12 (IL-12), to overcome this issue, but faced intolerable toxicity during clinical development. Here, we show that collagen-binding domain-fused IL-12 (CBD-IL-12) was retained within syngeneic murine prostate tumors, after secretion from CAR-T cells targeting human six transmembrane epithelial antigen of the prostate 1 (STEAP1). This led to equivalently high intratumoral interferon-γ levels without hepatotoxicity and infiltration of T cells into non-target organs, compared with unmodified IL-12. Both innate and adaptive immune compartments were dramatically activated and recognized diverse tumor antigens after CBD-IL-12 CAR-T cell treatment. Combination immunotherapy of CBD-IL-12 CAR-T cells and immune checkpoint inhibitors eradicated large tumors in an established prostate cancer model, without pre-conditioning chemotherapy. The therapy generated anti-tumor immunological memory. CBD-fusion to potent yet toxic payloads of CAR-T therapy may remove obstacles to their clinical translation towards elimination of solid tumors. ### Competing Interest Statement K.S., Y.A., A.G.C, J.K.L. and J.I. are inventors on a provisional patent application covering the technology described in this work. K.S. and J.I. are inventors on international patent applications covering CBD-IL-12 protein therapy. J.K.L. is an inventor on International Patent Applications related to STEAP1 CAR-T cells. J.K.L. holds equity in, serves on the scientific advisory board of, and receives research funding from PromiCell Therapeutics. J.K.L. is a consultant for Lyell Immunopharma. J.I. is a founder and shareholder in Arrow Immune Inc. J.I. is a scientific advisor of Libo Pharma Corp.

Adipose tissue inflammation contributes to obesity-induced insulin resistance. However, increasing evidence shows that high BMI (obesity) is not an accurate predictor of poor metabolic health in individuals. The molecular mechanisms regulating the metabolically activated M1 macrophage phenotype in the adipose tissues leading to insulin resistance remain largely unknown. Although the Janus Kinase (Jak)/signal transducer and activator of transcription 3 (Stat3) signaling in myeloid cells are known to promote the M2 phenotype in tumors, we demonstrate here that the Jak2/Stat3 pathway amplifies M1-mediated adipose tissue inflammation and insulin resistance under metabolic challenges. Ablating Jak2 in the myeloid compartment reduces insulin resistance in obese mice, which is associated with a decrease in infiltration of adipose tissue macrophages (ATMs). We show that the adoptive transfer of Jak2-deficient myeloid cells improves insulin sensitivity in obese mice. Furthermore, the protection of obese mice with myeloid-specific Stat3 deficiency against insulin resistance is also associated with reduced tissue infiltration by macrophages. Jak2/Stat3 in the macrophage is required for the production of pro-inflammatory cytokines that promote M1 macrophage polarization in the adipose tissues of obese mice. Moreover, free fatty acids (FFAs) activate Stat3 in macrophages, leading to the induction of M1 cytokines. Silencing the myeloid cell Stat3 with an in vivo siRNA targeted delivery approach reduces metabolically activated pro-inflammatory ATMs, thereby alleviating obesity-induced insulin resistance. These results demonstrate Jak2/Stat3 in myeloid cells is required for obesity-induced insulin resistance and inflammation. Moreover, targeting Stat3 in myeloid cells may be a novel approach to ameliorate obesity-induced insulin resistance.

Background Bispecific T cell engager (BiTE), such as blinatumomab, has demonstrated significant clinical success in treating hematological malignancies like B cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma. However, the application of BiTEs in solid tumors has proven challenging, primarily due to the lack of targetable tumor antigens and the immunologically “cold” nature of the tumor microenvironment, which limits immune system activation.Methods We developed a novel oncolytic virus (OV) platform by engineering a chimeric vaccinia virus to express either a truncated non-signaling CD19 antigen (CD19t) or truncated B cell maturation antigen (BCMAt) on the surface of infected tumor cells. Here, we advance a combinatorial platform using an OV to redirect CD19-targeted or BCMA-targeted T cell engagers (TCEs) to drive antitumor responses against multiple solid tumors.Results We found that OV-infected tumor cells in combination with TCEs significantly improved tumor cell killing against solid tumor models, with efficacy comparable to that of chimeric antigen receptor T cells. This combination approach enhanced antitumor responses using in vivo human tumor xenograft models and promoted more effective elimination of solid tumor cells than either therapy alone. Our studies highlight OVs combined with clinically approved TCEs as a readily translatable, tumor-agnostic, off-the-shelf strategy to effectively target solid tumors.Conclusions Our findings demonstrate that the combination of OV and TCEs offers a promising strategy to drive antitumor immune responses against solid tumors. This approach represents a novel and universal platform currently in phase 1 clinical trial combining TCE therapy with oncolytic virotherapy, overcoming antigen heterogeneity and immunological barriers for the effective treatment of solid tumors.

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy has demonstrated marked success in the control of hematological malignancies. However, CAR T cell therapy has been less successful when applied to solid tumors. This has been attributed to multiple factors including tumor infiltration, antigen heterogeneity, and the immunosuppressive tumor microenvironment. The hostile milieu and chronic exposure to antigen that CAR T cells face in the tumor microenvironment induces T cell exhaustion which is associated with an overall loss of therapeutic efficacy. Methods: Strategies for overcoming T cell exhaustion are central to improving CAR T cell efficacy in the treatment of solid tumors. The incorporation of survival-promoting proteins, such as cytokines, into CAR T cells have been used to generate a supportive local immune environment. Interleukin-2 (IL-2) is a cytokine that acts as a potent growth factor in T cells; however, therapeutic application of systemic IL-2 has been associated with severe toxicities and T cell exhaustion. To address this, we have developed membrane-tethered engineered cytokine agonists (MECAs) that anchor IL-2 and IL-2 partial agonists to the cell surface membrane of CAR T cells, restricting their signaling to the expressing cells to maximize autocrine signaling and prevent toxicity. Results: Here, we evaluate the influence of MECAs on the proliferation, exhaustion state, and cytotoxic function of CAR T cells. CAR T cells expressing the MECAs demonstrated improved cytotoxic function and proliferation with in vitro models. MECA expressing CAR T cells did not cause toxicity in control mice or in an orthotopic glioma model. Preliminary results from the toxicity study indicate improved in vivo antitumor activity of MECA expressing CAR T cell compared to CAR only. Conclusion: Exhaustion of antigen-specific T cells is a challenge in multiple cancer immunotherapy modalities, and the proposed MECAs have the potential to revolutionize immunotherapy for solid tumors as an effective and widely applicable solution. Citation Format: Diana Gumber, Saul Priceman, Christine J Brown, Leo Wang. Improving antitumor T cell therapy with membrane-tethered engineered cytokine agonists (MECAs) [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A122.

INTRODUCTION:The 12th Annual 2025 Coffey-Holden Prostate Cancer Academy (CHPCA) Meeting, "Deciphering Resistance: Beyond the Androgen Paradigm," was held at the University of California, Los Angeles (UCLA), Luskin Conference Center, in Los Angeles, CA, from June 19 to 22, 2025. METHODS:The CHPCA Meeting is a discussion-focused conference held annually by the Prostate Cancer Foundation (PCF), for in-depth academic analysis of emerging research with the greatest potential to drive new understandings and treatments for prostate cancer. The 2025 CHPCA Meeting included attendance by 79 academic investigators and 39 talks over 8 sessions. RESULTS:The session topics included: drug discovery in academia, non-apoptotic cell death mechanisms, understanding and overcoming treatment resistance, chromosomal instability (CIN) as a driver of metastasis and treatment resistance, targeting metastatic sites, immunotherapy sensitizers, and optimizing therapy delivery and biomedical engineering. DISCUSSION:This meeting report summarizes the presentations from the 2025 CHPCA Meeting. We hope that disseminating this information will directly contribute to novel research efforts and improved treatment strategies for patients with prostate cancer.