ABSTRACT:CD70 has emerged as a promising target in acute myeloid leukemia (AML), and we have previously demonstrated the potency of an optimized CD70-targeted ligand-based chimeric antigen receptor (CAR). However, here, we identify in vivo CD70 antigen escape as a limitation of single-antigen targeting. Combination targeting of CD70 and CD33 may overcome AML antigen heterogeneity. We hypothesized that modifying our CD70 CAR platform to secrete a bispecific T-cell engaging antibody molecule (TEAM) targeting CD33 (7033) would create a therapeutic window whereby AML heterogeneity could be addressed without increasing tissue toxicity. We found that CD33 TEAMs mediated specific cytotoxicity across AML cell lines, including CD33 or CD70 single-antigen knockout tumors. 7033 CAR T cells eradicated tumor in an in vivo mixed tumor model of CD70 antigen escape and outperformed the previously optimized CD70 CAR in a patient-derived xenograft. In vivo gene expression profiling of CAR T cells revealed enhanced 7033 CAR T-cell pathway scoring for persistence, activation, and T-cell receptor signaling. Additionally, CD33 TEAMs successfully redirected T cells isolated from patients with AML to activate, secrete cytokines, and kill tumor targets despite exposure to substantial prior cytotoxic therapies. In summary, our findings demonstrate the feasibility of our 7033 CAR to overcome AML heterogeneity and leverage the bystander T cells of patients; this approach warrants further study in patients with this dire clinical need.

Chimeric antigen receptor (CAR) T cells are highly effective in haematological malignancies1. However, progressive loss of CAR T cells contributes to relapse in many patients2-4. Here we performed in vivo loss-of-function CRISPR screens in CAR T cells targeting B cell maturation antigen to investigate genes that influence CAR T cell persistence and function in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR library-edited T cells in vitro and at early and late time points in vivo to track the performance of gene-modified CAR T cells from manufacturing to survival in tumours. The screens revealed context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, whereas loss of PTPN2, ZC3H12A and RC3H1 conferred early growth advantages to CAR T cells in vivo. Notably, we identified cyclin-dependent kinase inhibitor 1B (encoded by CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late time points in vivo. CDKN1B ablation increased CAR T cell proliferation and effector function, significantly enhancing tumour clearance and overall survival. Our findings reveal differing effects of gene perturbation on CAR T cells over time and in different environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma and underscore the potential of in vivo screening for identifying genes to enhance CAR T cell efficacy.

Supplementary Figure S5. Intratumoral multiplex RNA-ISH IF imaging and analysis of CAR-TEAM treated mice.

AbstractPurpose: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell–engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). Experimental Design: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. Results: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. Conclusions: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

Supplementary Figure S7. Superior anti-tumor effect of meso^FAP compared to the combination of meso-CAR and FAP-CAR or meso^CD19.

Background : Loss or down-regulation of CD19 on B-cell lymphomas is a well described resistance mechanism to CD19-directed chimeric antigen receptor (CAR) T-cell therapies. Loss of CD58, ligand for CD2 on T-cells, has recently been identified as another mechanism of resistance to CD19-directed CAR-T cells. CD2 is a costimulatory receptor on T-cells, present in the immune synapse and important for T-cell activation and adhesion to antigen-presenting cells. We report here findings of a novel CD19-directed CAR design incorporating a secreted trispecific immune effector cell engager (TRIEC) consisting of three single chain variable fragments (scfv) targeting CD79b, CD2, and CD3 connected by glycine(4)serine linkers. Methods : Tumor models of resistant malignant B-cell lines were made through modulation or knockout of CD19 expression, with or without concomitant knockout of CD58 in Jeko-1 (Mantle Cell Lymphoma), Nalm6 (Acute Lymphoblastic Leukemia), and Raji (Burkitt lymphoma) cell lines. Primary human T-cells were transduced with a lentivirus encoding for second generation CD19 CAR and TRIEC molecule, CD19 CAR alone, or CD19 CAR with null TRIEC absent CD2 binding (CD2 scfv replaced with EGFR scfv). Results : CD19-directed second generation CAR T cells demonstrate reduced cytotoxicity and lower levels of cytotoxic cytokines when exposed to CD19low, CD19low/CD58KO, and CD19KO B-cell tumor lines compared to CD19/CD58 unmodified parental B-cell lines in vitro, and progressive decrement in tumor clearance of CD19low, CD19low/CD58KO, and CD19KO B-cell tumors in vivo. TRIEC molecules show specific binding of CD79b, CD2 (TRIEC only), EGFR (null TRIEC only) and CD3 antigen targets as assessed by anti-glycine(4)serine linker antibody. Untransduced T-cells co-cultured with Jeko-1 cells demonstrate increased cytolysis in the presence of TRIEC-containing supernatant vs null supernatant (p<0.0001). CD19 CAR, CD19 CAR + null TRIEC, and CD19 CAR + TRIEC constructs demonstrate equivalent activation and proliferation in the absence of tumor. CD19 CAR/TRIEC constructs demonstrate superior in vitro cytotoxicity against parental (p=.004), CD19low/58KO (p=.0011), and CD19KO (p=.0002) Jeko-1 and Raji 19low/58KO (p<.0001) cell lines compared to CD19 CAR and CD19 CAR/null TRIEC constructs. Conclusion : Harnessing CD2 signaling through incorporation of a first-in-class secreted trispecific CD79b/CD2/CD3 TRIEC enhances killing of CAR-resistant lymphoma models. This is the first use of a cell-secreted trispecific CD2/CD3 T-cell engaging molecule (TRIEC). CD19 CARs secreting TRIECs are effective against CD19 low/negative and CD58KO B-cell lines. Incorporation of CD2 binding into the TRIEC molecule increases cytotoxicity without eliciting nonspecific activation or unstimulated proliferation of CAR-T cells in the absence of tumor. in vivo studies are ongoing to evaluate CAR + TRIEC efficacy in NOD-Scid-common gamma chain knockout (NSG) mice engrafted with CAR-resistant B-cell tumors. Incorporation of CD2 signaling into CAR-T cells represents an attractive addition to circumvent tumor-intrinsic resistance mechanisms to current CD19-directed CAR-T cell therapies.

Supplementary Figure S8. Target expression and individual analysis of patient-derived organoids (PDO) with matching CAFs.

Supplementary Figure S2. In vitro setup of CARTEAM to CAF:AsPC-1 with different ratios showing superiority of mesoFAP compared to CARs targeting mesothelin only.

Supplementary Figure S6. Human CAF-1 cells do not expand in vivo in NSG mice in the presence or absence of AsPC-1 or PDX1294 cells.

Supplementary Figure S4. Still frames from live cell microscopy videos demonstrating reduced edge dynamics of meso^FAP CAR T-cells compared to controls and target cell layer on flow chamber of acoustic microscopy.

Abstract Introduction: Antigen heterogeneity is a challenge for CAR-T cell therapy in solid tumors due to antigen escape when targeting a single antigen. In ovarian cancer (OC), mesothelin (meso) and Mucin16 (MUC16) are two of the most overexpressed antigens. Moreover, CA125, which is part of MUC16, is lost in some OC patients, but the ectodomain remains on the cell surface. Targeting both antigens (Meso and MUC16ecto) with a tandem-CAR configuration could overcome antigen heterogeneity to improve CAR-T cell efficacy in OC. Material and Methods: We designed a library of different CARs with tandem Meso and MUC16ecto scFv combinations with varying G4S linker lengths, fused to a CD8 hinge/transmembrane domain and 41BBz intracellular domain. A series of constructs was then tested in an NFAT-GFP reporter system in Jurkat cells to determine which configuration induced maximal T cell activation and antigen-binding capacity via flow cytometry. We identified the top 2 activators and binders, produced primary human tandem CAR-T (TanCAR) cells by LV transduction, and tested their cytotoxicity in vitroagainst dual antigen-expressing or single antigen-expressing tumor cells using luciferase killing assays and Incucyte. We measured the cytokine production with a Luminex® Multiplex Assay and used a z-Movi® Cell Avidity Analyzer to test the tandem scFv avidity towards the antigens. Also, we examined TanCAR activity against a mixed tumor model in NSG mice. Their in vitro and in vivo activity were compared to CAR T cells targeting either single antigen (SS1-anti-Meso or 4H11-anti-MUC16ecto). Results: We found that TanCAR1 and TanCAR3 as the configurations with the best activation and binding capacity in response to cognate antigen. TanCAR1 and TanCAR3 had superior in vitro cytotoxic capacities compared to SS1 or 4H11 in tumor models expressing both antigens and in mixed cultures of tumor cells expressing one, both, or neither target antigen. From this mixed culture, we analyzed the remaining tumor cell populations by flow cytometry and observed an overgrowth of the population not targeted by CAR. In response to tumor cells, tandem CARs also produced INFg, IL6, IL-18, IL-13, and GM-CSF regardless of whether one or two antigens were expressed. For further experiments, we pursued TanCAR1 due to its slightly better functionality compared to TanCAR3. TanCAR1 had the same avidity for cells expressing the single antigens as SS1 or 4H11 CARs, but an intermediate avidity for the double Ag-expressing cell line compared to the single CARs. Finally, we tested TanCAR1-T cells in an in vivo mixed tumor model, showing that they better controlled and reduced tumor growth compared to either SS1 or 4H11. Conclusion: Targeting Meso and MUC16ecto through a tandem CAR design allows for a better anti-tumor response compared to CAR T cells targeting a single antigen. This approach may combat antigen heterogeneity by preventing antigen escape. Citation Format: Diego Salas-Benito, Filippo Birocchi, Alexander Armstrong, Amanda A. Bouffard, Tamina Kienka, Felix Korell, Mark B. Leick, Trisha Berger, Marcela V. Maus. Tandem CAR-T cells against mesothelin and MUC16ectoto overcome tumor-antigen heterogenicity [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 3992.

Supplementary Video S9. UTD T cells on AsPC1 + CAF

In 2 complementary Letters to Blood, Karschnia et al and Graham et al provide new insights into the neurological toxicities that are observed with B-cell maturation antigen–directed chimeric antigen receptor T-cell treatment for multiple myeloma, identifying a frequency of immune effector cell–associated neurotoxicity syndrome (ICANS) that exceeds 40%. Severe ICANS is identified in 8% of patients in this real-world series. Outcomes were generally favorable, although the authors describe rare, late Parkinsonism-like hypokinetic movement disorders (also known as movement and neurocognitive toxicities) post-ICANS in 2 patients.

Tumors evolve together with the tumor microenvironment (TME) and reshape it towards immunosuppression. Immunostimulating cytokines can be used to revert this state leading to effective antitumor immune responses, but their exploitation as anticancer drugs has been hampered by severe toxicity associated with systemic administration. Local, TME-targeted delivery of immune activating cytokines can deploy their antitumoral function more effectively than systemic administration while, at the same time, avoiding exposure of healthy organs and limiting toxicity. Here, we review different gene and cell therapy platforms developed for tumor-directed cytokine delivery highlighting their potential for clinical translation.

Background Blockade of the vascular endothelial growth factor (VEGF) pathway represents a long-standing strategy for anti-tumor activity, with drugs employing this approach for a variety of solid tumor types. It is also known that VEGF directly drives T cell exhaustion and dysfunction.1 However, systemic VEGF blockade causes significant cardiovascular and renal toxicity and limits the dose of these medications, restricting their maximal theoretical benefit. In an updated analysis of our previously published single cell RNA sequencing data from infusion products of Tisagenlecleucel,2 we discovered expression of VEGF and VEGFR1 on CAR-T cell infusion products. Building on this observation, we found that our previously reported CD703 and Mesothelin CAR-T cells produce VEGF, as detected by ELISA, and upregulate VEGFR1 upon activation (p<.0001), suggesting active signaling along this pathway may impact CAR-T cell biology. Methods To improve the therapeutic window of VEGF blockade and mitigate putative VEGF mediated T cell dysfunction, we generated CD70 and Mesothelin targeted CAR-T cells that secrete a VEGF-blocking single chain variable fragment (scFv, 70VEGF and MesoVEGF respectively). Results 70VEGF and MesoVEGF completely abrogated VEGF signaling in a HEK293 reporter assay (p<0.0001) and blocked all detectable VEGF by ELISA during CAR-T production (p<.0001) compared to CD19 scFv expressing control constructs (7019 and Meso19). 70VEGF and MesoVEGF demonstrated superior activation (by CD69 expression) and expansion in both short term plate bound antigen assays and long term repetitive stimulation assays with CD70 or Mesothelin expressing K562 targets. 70VEGF and MesoVEGF also mediated superior cytotoxicity and CAR-T expansion against RCC, AML, NSCLC, and ovarian cancer cell lines in vitro compared to 7019 and Meso19. Preliminary data suggest that neither CRISPR-Cas9 VEGFA knockout of 7019 (7019,VEGFKO) nor exogenous administration of anti-VEGF scFv alone were sufficient to phenocopy the proliferative advantage of 70VEGF when co-cultured with tumor cells; however, when exogenous anti-VEGF scFv was added to 7019,VEGFKO CAR-T cells the performance was similar to 70VEGF, arguing that a combination of intracrine and exogenous VEGF modulate CAR-T cell function. Nanostring gene expression profiling revealed distinct expression programs between MesoVEGF and Meso19 including lower apoptosis of the former. In vivo experiments with AML PDX, orthotopic RCC, and ovarian/lung cancer metastatic models are ongoing and will be reported at the conference. Conclusions Targeted delivery of a VEGF blocking scFv is a promising therapeutic strategy that augments CAR-T cell function in a tumor and target agnostic fashion and warrants further development. Acknowledgements *These first authors contributed equally References Kim C, Jang M, Kim Y. VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers. Sci Immunol. 2019;4(41) Haradhvala N, Leick M, Maurer K. Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma. Nature Medicine. 2022;(9):1848–1859 Leick M, Silva H, Scarfò S. Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia, Cancer Cell. 2022;40(5):494–508. Ethics Approval All care and conducted experiments on mice were carried out with protocols approved by the MGH Institutional Animal Care and Use Committee.

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor characterized by a strongly immunosuppressive tumor microenvironment (TME) that represents a barrier also for the development of effective immunotherapies. The possibility to revert this hostile TME by immunoactivating cytokines is hampered by the severe toxicity associated with their systemic administration. Here, we exploited a lentiviral vector–based platform to engineer hematopoietic stem cells ex vivo with the aim of releasing, via their tumor-infiltrating monocyte/macrophage progeny, interferon-α (IFN-α) or interleukin-12 (IL-12) at the tumor site with spatial and temporal selectivity. Taking advantage of a syngeneic GBM mouse model, we showed that inducible release of IFN-α within the TME achieved robust tumor inhibition up to eradication and outperformed systemic treatment with the recombinant protein in terms of efficacy, tolerability, and specificity. Single-cell RNA sequencing of the tumor immune infiltrate revealed reprogramming of the immune microenvironment toward a proinflammatory and antitumoral state associated with loss of a macrophage subpopulation shown to be associated with poor prognosis in human GBM. The spatial and temporal control of IL-12 release was critical to overcome an otherwise lethal hematopoietic toxicity while allowing to fully exploit its antitumor activity. Overall, our findings demonstrate a potential therapeutic approach for GBM and set the bases for a recently launched first-in-human clinical trial in patients with GBM.

The immunosuppressive tumor microenvironment is a major hurdle for cancer therapy. Our lab previously developed a cell and gene therapy strategy for the tumor-targeted delivery of IFN-alpha based on the transduction of hematopoietic stem/progenitor cells (HSPC) with lentiviral vectors (LV) expressing the IFN-alpha gene under the control of the TIE2 enhancer/promoter. The specificity of this strategy is ensured by the restricted expression of IFN-alpha in a population of HSPC-derived tumor-infiltrating macrophages expressing the TIE2 receptor (TEM), which are recruited at the tumor site. TEM-mediated delivery of IFN-alpha has shown efficacy in different tumor models of both solid and hematopoietic origin, including a model of human glioma in immunodeficient mice. New data obtained in a more relevant immunocompetent mouse model of orthotopic glioma closely reproducing several features of the human disease show dramatic tumor inhibition in mice treated with IFN-alpha gene therapy. We are currently characterizing the contribution of IFN-induced antiangiogenic and modulatory activity on the innate and adaptive immune system underlying such response. To further improve the therapeutic index of our strategy, we are developing inducible strategies to superimpose a timer and a rheostatic switch on the amount of IFN-alpha secreted in the tumor microenvironment. Indeed, whereas a sustained cytokine output could ensure efficacy and long-term protection from tumor recurrence, it may raise concerns for long-term effects, especially in case of cancer eradication. By fusing a destabilizing domain (DD) to a protein of interest (POI), the former can confer its instability to the latter. This destabilization can be rescued in a reversible and dose-dependent manner with the addition of a small molecule specifically binding to the DD. To apply this technology to our strategy, we have designed and in vitro tested different fusion proteins of IFN-alpha (DD-IFN-alpha) with or without the addition of flexible or cleavable linkers and selected them for their capacity to be stabilized in presence of their specific ligand in vitro. Through this approach, we have identified fusion proteins with low basal activity and high fold induction upon ligand treatment. These novel regulated forms of IFN-alpha are functional and their specific activities are comparable to the wild type. Based on preliminary evidence of in vivo ligand-regulated IFN release, we are now testing the safety and efficacy of our new platforms in inducing antitumor responses in melanoma, colon, and glioma models of cancer. This abstract is also being presented as Poster B31. Citation Format: Filippo Birocchi, Melania Cusimano, Anna Ranghetti, Tiziano Di Tomaso, Barbara Costa, Peter Angel, Nadia Coltella, Luigi Naldini. Development of chimeric forms of IFN-alpha for “on demand” in vivo cancer gene therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR13.