Cell Therapy For Solid Tumors Research Articles

CAR‐T‐cell products in solid tumors: Progress, challenges, and strategies

AbstractT cells engineered to express chimeric antigen receptors (CARs) with specificity toward tumor cells have led to promising outcomes in patients with hematological malignancies. Nevertheless, the application of CAR‐T cell therapy in solid tumors encounters several obstacles. These include tumor heterogeneity and immune escape, T cell exhaustion and restricted infiltration into the tumors that affect the therapeutic efficacy of CAR‐T cells, as well as “on‐target, off‐tumor” toxicities that can lead to severe and even fatal adverse events. In recent years, clinical trials and new approaches of CAR‐T cell therapy for solid tumors have made certain progress. Here, we update the outcomes of related clinical trials and summarize engineered strategies aiming to improve the efficacy and therapeutic safety of CAR‐T cells based on experimental and clinical studies.

Abstract 6656: Revolutionizing solid tumor treatment: Viral protein-targeted CAR-T with oncolytic virus

Abstract Major challenges for CAR-T cell therapy in solid tumors include various limitations due to the issue of appropriate antigen selection, tumor heterogeneity, and the immune-suppressive tumor microenvironment (TME). To overcome these hurdles, we’ve developed a novel therapy, CLM-202 which is a combination therapy of oncolytic Newcastle disease virus (NDV) and NDV F protein-targeted CAR-T cell therapy (NDV F CAR-T). After NDV infects tumor cells, NDV envelope protein F is expressed on the tumor cell surface. NDV F CAR-T recognizes NDV F protein and kills the tumor cells. This approach can be applied to patients with various indications, regardless of the presence of the target protein. Using NDV to deliver antigens minimizes the risk of tumor recurrence due to tumor heterogeneity. Furthermore, it mitigates off-target toxicity by targeting oncolytic virus-derived proteins, not human proteins. Oncolytic NDV also triggers a potent immune response within the tumor microenvironment, thereby altering the immunosuppressive conditions of the tumor and improving CAR-T efficacy. Therefore, CLM-202 can be a multipurpose solution for solid tumors. We have developed a second-generation CAR-T cell therapy, NDV F CAR-T, which can be used in combination with an oncolytic virus known as NDV. NDV F CAR-T is designed to specifically target the F protein expressed when cells are infected by NDV. It incorporates a single-chain variable fragment (scFv) derived from an antibody that can bind specifically to the F protein of NDV-infected cells. To assess the activity of NDV F CAR-T in tumor cells against a variety of solid tumors as indications, we conducted several experiments. We initially confirmed the expression of the F protein in NDV-infected tumor cells. We also conducted cytotoxicity tests to confirm NDV F CAR-T's ability to induce death in NDV-infected tumor cell lines. Furthermore, to assess the efficacy of NDV F CAR-T in an in vivo model, we established an orthotopic mouse model. In this model, we administered NDV via intratumoral injection, followed by the single-dose administration of NDV F CAR-T to perform the efficacy test. We have confirmed that NDV infects all solid tumor cell lines representing different indications and induces the expression of the F protein in infected tumor cells. Furthermore, we have observed that NDV infection in these diverse tumor cells results in robust tumor cell-killing activity by NDV F CAR-T and the subsequent activation of CAR-T. Moreover, in an orthotopic mouse model, we have demonstrated that the administration of NDV followed by NDV F CAR-T induces potent tumor growth inhibition. In summary, our research proposes a novel therapeutic strategy, CLM-202 that utilizes NDV F CAR-T and NDV to overcome the limitations of CAR-T cell therapy in solid tumors, including tumor heterogeneity and immune-suppressive tumor microenvironments. This approach aims to effectively treat a wide range of indications. Citation Format: Soyoung Choi, Kiwan Kim, Sangeun Lee, Jeongeun Choi, Song-Jae Lee, Yong Gu Lee, Jeonghun Kim, Hoyoung Lee, Kisoon Kim, Man-Seong Park, Seong-Won Song. Revolutionizing solid tumor treatment: Viral protein-targeted CAR-T with oncolytic virus [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 6656.

Abstract 3611: A patient-derived organoid platform to assess the effectiveness of γδTCR cell therapy in solid tumors

Abstract Recent advances in cancer immunotherapy have positively impacted the life expectancy of patients for an extensive range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. HUB developed an innovative alternative, building on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when embedded into the extracellular matrix. Patient-derived organoids are generated from healthy and malignant tissues, and they recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity and morphological and functional traits. Cell therapies have achieved positive results in treating haematological cancers. Recently, the unique tumour-targeting properties of γδ T cell receptors have been leveraged to develop safer and more effective strategies. HUB and Gadeta joined efforts to explore the therapeutic potential of GDT002 - i.e. αβ T cells engineered with a Gadeta proprietary new γδTCR - to target solid cancers. A preclinical basket trial was performed using patient-derived organoids from several tumour types (colorectal, breast, head and neck, ovarian cancers) cocultured with GDT002. The results showed broad reactivity of GDT002 against solid tumor PDOs. Interestingly, GDT002 was particularly efficient at killing ovarian cancer-derived PDOs, allowing Gadeta to move GDT002 to clinical trials for this indication confidently. This study reveals that our tumor organoid co-culture with engineered T cells platform holds significant value for the preclinical development of cellular therapies. Citation Format: Andrea Bisso, Rene Overmeer, Sjoerd Baardman, Chris Coomans, Cesar Oyarce, Sylvia F. Boj. A patient-derived organoid platform to assess the effectiveness of γδTCR cell therapy in solid tumors [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 3611.

Abstract 49: LYL119, an investigational ROR1-targeted CAR T-cell product incorporating four novel reprogramming technologies designed for effective cell therapy for solid tumors

Abstract Effective solid tumor cell therapy requires new strategies to improve T-cell activation, persistence, and durable function. We developed four complementary T-cell reprogramming technologies to enhance chimeric antigen receptor (CAR) T-cell therapy in solid tumors: 1) overexpression of the activator protein 1 (AP-1) family transcription factor c-Jun to delay T-cell exhaustion and improve antitumor activity; 2) nuclear receptor subfamily 4A member 3 (NR4A3) gene knockout (KO) to further delay exhaustion and enhance functionality; 3) Epi-RTM manufacturing protocols to preserve stem-like characteristics; and 4) Stim-RTM technology, a novel activating reagent to improve product potency compared with standard reagents. LYL119 is an investigational ROR1-targeted CAR T-cell product that combines these technologies to create potent and durable CAR T cells. Healthy or NSCLC patient donor T cells were manufactured with the Epi-R protocols, activated with Stim-R or a standard reagent, and transduced with a vector encoding a ROR1 CAR and c-Jun. The NR4A3 gene or a control gene was edited using SpyFiTM Cas9 nuclease (Aldevron®). Cytotoxicity, cytokine production, phenotype, and single-cell transcriptomic and epigenetic profiles were evaluated in vitro after antigen restimulation assays designed to promote exhaustion. CAR T cell activity was evaluated in vivo using a ROR1+ NSCLC xenograft mouse model. Research and clinical scale LYL119 products achieved ~90% genome editing efficiency at the NR4A3 target gene resulting in a 13-fold protein reduction compared to non-edited CAR T cells. LYL119 exhibited superior cytotoxicity and cytokine production upon antigen restimulation across 7 different ROR1+ solid tumor cell lines compared to CAR T cells that lacked one or more reprogramming technologies. After repeated rounds of tumor cell killing, LYL119 displayed reduced surface expression of exhaustion-related receptors (e.g. TIM-3) and higher expression of stemness-related markers (e.g. CD127) compared to non-edited CAR T cells. Furthermore, transcriptomic analysis revealed global downregulation of exhaustion-related gene signatures and retention of unique cell subsets characterized by upregulation of memory and effector-associated gene signatures. LYL119 exhibited robust antitumor efficacy in vivo across a 10-fold dose range, including a very low dose of 1 × 105 CAR T cells. Lastly, LYL119 derived from NSCLC patient donor T cells also demonstrated enhanced cytotoxicity in vitro compared to control CAR T cells. These nonclinical data suggest LYL119, which combines c-Jun overexpression, NR4A3 KO, Epi-R protocols, and Stim-R technology, can limit exhaustion, maintain stem-like features, and has the potential to provide effective and durable CAR T-cell antitumor activity in patients with ROR1+ solid tumors. Citation Format: Viola C. Lam, Aileen Li, Meritxell Galindo Casas, Jessica Barragan, Christina Cheung, Jessica Briones, Esha Afreen, Grant Vavra, Jia Lu, Purnima Sundar, Rowena Martinez, Candace Sims, Shobha Potluri, Omar Ali, Alexander S. Cheung, Rachel C. Lynn. LYL119, an investigational ROR1-targeted CAR T-cell product incorporating four novel reprogramming technologies designed for effective cell therapy for solid tumors [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 49.

Abstract 3992: Tandem CAR-T cells against mesothelin and MUC16ectoto overcome tumor-antigen heterogenicity

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.

Abstract 5491: Mapping ovarian cancer spatial organization uncovers immune evasion drivers at the genetic, cellular, and tissue level

Abstract Immune exclusion and evasion are central barriers to the success of immunotherapies and cell therapies in solid tumors. Here we applied single cell spatial and perturbational transcriptomics alongside clinical,histological, and genomic profiling to elucidate immune exclusion and evasion in high-grade serous tubo-ovarian cancer (HGSC). Using high-plex spatial transcriptomics we profiled more than 1.3 million cells from 95 tumors and 60 patients, revealing generalizable principles in HGSC tumor tissue organization.Our data demonstrates that effector T cells resist stroma-mediated trapping and sequestration. However, upon infiltration into the tumor, T cells, as well as Natural Killer (NK) cells, preferentially co-localize only with a subset of malignant cells that manifest a distinct transcriptional cell state. The latter consists of dozens of co-regulated genes and is repressed under various copy number alterations. Performing CRISPR Perturb-seq screens in ovarian cancer cells, we identified functionally diverse genetic perturbations - including knockout of the insulin sensing repressor PTPN1 and the epigenetic regulator ACTR8 - that de-repress the proposed immunogenic malignant cell state identified in patients and indeed sensitize ovarian cancer cells to T cell and NK cell cytotoxicity. Taken together, our study uncovered a profound connection between somatic genetic aberrations, malignant cell transcriptional dysregulation, and immune evasion at the cellular and tissue level, allowing us to identify targets that reprogram malignant cell states as an avenue to unleash anti-tumor immune responses. Citation Format: Karmen Aguirre, Christine Yiwen-Yeh, Olivia Laveroni. Mapping ovarian cancer spatial organization uncovers immune evasion drivers at the genetic, cellular, and tissue level [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 5491.

Abstract 6328: Preclinical development of CAR T cells with antigen-inducible IL-18 enforcement to treat GD2-positive solid cancers

Abstract Cytokine-engineering is a promising strategy to overcome limitations of CAR T cell therapy in solid tumors. We have developed a novel investigational medicinal product, consisting of CAR T cells directed against ganglioside GD2 along with CAR-inducible secretion of IL-18, to enforce their activation response and effector functions in the microenvironment of solid cancers. Our lentiviral all-in-one vector combines constitutive expression of a 14.G2a antibody-derived GD2-specific, 4-1BB-costimulated CAR with inducible nuclear factor of activated T cells (NFAT)-driven expression of the human IL-18 transgene. A control construct contains EGFP in the place of IL-18 (GD2EGFPCART). Lentiviral transduction of T cells from various individual donors with both preclinical and clinical batches of virus resulted in CAR surface expression in >40% of T cells. GD2IL18CART contained effector memory and central memory T cell phenotypes at similar frequencies as the control. CAR engagement by immobilized anti-idiotype antibody or by GD2-positive tumor cells selectively induced IL-18 release, with production of IL-18 strictly depending on GD2 antigen engagement. GD2IL18CART responded to interaction with GD2-positive tumor cells with superior IFN-γ, TNF-α and IL-2 cytokine release and superior target cytolysis compared to the control CAR T cells without inducible IL-18, whereas IL-6 was secreted at similar concentrations. In an NSG xenograft model of disseminated GD2-positive tumors, GD2IL18CART had superior in vivo anti-tumor activity than GD2EGFPCART, with eradication of GD2-positive tumor xenografts as detected by bioluminescence imaging while the control achieved only limited extension of progression-free survival. Finally, we established GMP-compliant manufacturing of GD2IL18CART that is feasible and efficient at clinical scale, using an automated protocol and device. Three validation runs confirmed robust expansion of CAR+ T cells to final cell numbers of 3.5x109 CAR+ cells (median; range 3.4 to 3.7x109), with consistent transduction efficiencies of >54% at the in-process control time-point and at harvest. GD2IL18CART from clinical-scale manufacturing runs responded to interaction with GD2 with strictly antigen-dependent secretion of both IL-18 and IFN-γ and efficiently lysed various GD2-positive tumor targets. In conclusion, we assembled all prerequisites for clinical evaluation of an IL-18 cytokine enhanced, GD2-specific CAR T cell product for the treatment of patients with a variety of solid tumors, recruited on the basis of GD2 immunofluorescence pre-screening (EU CT 2022-501725-21-00). Besides addressing the safety of GD2IL18CART therapy in pediatric and adult patients as the primary endpoint, the study will allow to gain substantial knowledge on the biological effects of IL-18 in the TME of the individual solid tumors. Citation Format: Lena Fischer-Riepe, Sareetha Kailayangiri, Katharina Zimmermann, Rita Pfeifer, Michael Aigner, Bianca Altvater, Sascha Kretschmann, Simon Voelkl, Jordan Hartley, Celine Dreger, Katja Petry, Andreas Bosio, Angelika von Doellen, Wolfgang Hartmann, Holger Lode, Dennis Goerlich, Andreas Mackensen, Melanie Jungblut, Axel Schambach, Hinrich Abken, Claudia Rossig. Preclinical development of CAR T cells with antigen-inducible IL-18 enforcement to treat GD2-positive solid cancers [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 6328.

Abstract 51: OPB-101: An optimized mesothelin-specific CAR T cell product expressing CD8-targeted IL-2/15 and engineered to resist T cell exhaustion

Abstract T cell therapies have had modest efficacy in solid tumors due to their failure to proliferate following infusion. We have designed a mesothelin (MSLN) CAR T product (OPB-101) which includes a novel promoter (OP1), an optimized CAR, a safety switch, and a CD8α-targeted IL-2/15 designed cytokine to promote T cell expansion and improve efficacy in solid tumors. Human anti-MSLN binders were optimized for the target using our OUTSPACERTM library. CAR evaluation was conducted using in vitro coculture assays and validated NSG tumor xenograft models. OUTSMARTTM IL-2/15 was designed to avoid regulatory T cell activation by ablating IL-2Rα binding and re-targeted to CD8α to enhance CD8+ T cell and NK stimulation. EGFRoptTM was engineered for improved sensitivity to EGFR-targeted immunotherapies. Using 2A polypeptides, all transgenes were expressed with an antigen-dependent, inducible “stim-on” promoter (OP1) to regulate CAR expression, cytokine production, and resistance to exhaustion. The efficacy of the OPB-101 construct was tested in stringent exhaustion assays. In vivo efficacy was assessed with mice engrafted with s.c. H1650 tumors that were treated by i.v. injection of mock, CAR only or OPB-101 T cells at doses ranging from 0.25 × 106 to 2 × 106 cells. Tumor volume and weight were measured for 80 days. T cell levels and phenotype were measured by peripheral blood (PB) draws and intratumoral analysis (IT; day 14). OPB-101 efficiently killed MSLN+ tumor cell lines in repeat challenge and spheroid assays, and inducibly produced the IL-2/15 cytokine. IL-2/15 expression did not result in antigen-independent CAR T cell proliferation but extended survival in conditions without exogenous cytokine support. In vivo efficacy studies showed tumor control at 2 × 106 cells in the CAR only condition and modest anti-tumor efficacy at the 1 × 106 dose. In contrast, OPB-101 demonstrated complete and durable tumor elimination at the lowest dose of 0.25 × 106 cells (OPB-101 versus CAR only at 1 × 106 cells, p < 0.0001 and p < 0.0005 for donor 1 and 2, respectively). OPB-101 efficacy was associated with robust expansion in the PB (>100-fold) within IT (>5-fold) compared to the CAR only condition. OPB-101 PB and IT T cells showed reduced levels of PD-1, TIGIT and CD39 compared to CAR only suggesting resistance to exhaustion. Finally, studies conducted in tumor-free mice showed that OPB-101 expansion in vivo was dependent on the presence of tumor. In summary, OPB-101 using a regulated promoter (OP1) to dynamically express an optimized MSLN CAR, EGFRoptTM, and a CD8-targeted IL-2/15 cytokine enhances anti-tumor efficacy against solid tumors by promoting CAR expansion and persistence while limiting T cell exhaustion. These technologies allow for complete elimination of solid tumors at a low treatment dose (i.e., <250K cells) and may pave the way to more effective CAR T cell therapies for solid tumors. Citation Format: Rupesh Amin, Howell Moffett, Kevin Haworth, Jerry Chen, Jason Yokoyama, Allan Wang, Leah Tait, Maria Steele, John Crowl, Thaddeus Davenport, Joseph DeSautelle, Jared Hammer, Willimark Obenza, Vanessa Montoya, Robin Kirkpatrick, Laura Baker, Tina Tan, Kristie Shirley, Bradley Hammerson, Robert Langan, David Clausen, Paul Sample, Brian Weitzner, Shujun Yuan, Marc Lajoie, Scott Boyken, Aaron Foster. OPB-101: An optimized mesothelin-specific CAR T cell product expressing CD8-targeted IL-2/15 and engineered to resist T cell exhaustion [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 51.

Abstract 697: Enhanced tumor control and survival in solid tumor models following combined low dose radiotherapy and T cell therapy

Abstract Background: Solid tumors remain a significant challenge in cancer therapy due to their resistance to conventional treatments. Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising approach for treating solid tumors, but its effectiveness is often limited by the poor infiltration of CAR-T cells into the tumor microenvironment. Thus, enhancing a robust infiltration of chimeric antigen receptor T cells into solid tumors is essential for an effective antitumor response and therapeutic success. Methods: In this study, we investigated the combined effects of multiple doses of low-dose radiotherapy (LD-RT) and CAR T-cell therapy on tumor growth and survival using two models of NSG mice implanted with gastric carcinoma (GSU) or pancreatic adenocarcinoma (CAPAN-2) cells. The engineered CAR-T cells were designed to target guanyl cyclase-C (GCC) or mesothelin, respectively. Furthermore, our study employed atomic force microscopy (AFM) in a small cohort to investigate the impact of LD-RT and CAR-T cell therapy on tumor stiffness and plasticity. Results: The synergistic effect of combining low-dose radiotherapy (LD-RT) with T-cell therapy resulted in enhanced tumor control and prolonged survival in mice with tumors, surpassing the outcomes observed with either monotherapy alone. This combined treatment holds promise for improving responses in patients with solid tumors by leveraging LD-RT to enhance the intratumoral infiltration of effector T cells. Our findings suggest that the nanomechanical signature may be a potential biomarker for LD-RT combined CAR T cell therapy in solid tumors. Conclusions: Our findings substantially contribute to treating solid tumors using cell therapies, highlighting the potential for improved infiltration of adoptively transferred T cells following radiotherapy (RT), which can transform existing therapeutic strategies for solid tumors while minimizing toxicity. Citation Format: Nahum Puebla, Gitika Srivastava, Sara Nizzero, Natalie Wall Fowlkes, Hampartsoum Barsoumian, Yun Hu, Carola Leuschner, Thomas Riad, Marko Loparic, Marija Plodinec, James Welsh. Enhanced tumor control and survival in solid tumor models following combined low dose radiotherapy and T cell therapy [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 697.

Abstract 38: AB-2100, a PSMA-inducible CA9-specific CAR T cell product for the treatment of ccRCC provides long-term tumor responses in preclinical mouse model

Abstract Common challenges of CAR-T cell therapies in solid tumors, such as clear cell renal cell carcinoma (ccRCC), include insufficient therapeutic potency and lack of tumor specificity. We have developed AB-2100, an autologous integrated circuit T (ICT) cell product, generated via CRISPR-mediated knock-in of a single transgene into a safe-harbor locus. AB-2100 encodes a transcriptionally regulated sequential AND gate that comprises a priming receptor (PrimeR) specific for PSMA and an inducible CAR targeting CA9 antigen, which is widely expressed on local and metastatic lesions. AB-2100’s sequential AND logic-gate confers tumor-specific activity by priming off of PSMA-expressing tumor vasculature to induce CA9 CAR expression. This unique feature of the logic gate is intended to increase the safety profile of AB-2100 given that PSMA and CA9 are predicted to have limited co-expression in normal tissues. Additional functionality includes short-hairpin RNAs (shRNA) against Fas and TGFBR designed to prevent tumor-mediated resistance, and a synthetic pathway activator (SPA) that drives constitutive STAT3 signaling and enhanced T cell cytotoxicity and expansion. Mechanism of action studies demonstrate that AB-2100 can prime off of PSMA-expressing endothelial cells and induce tumor-specific killing of CA9 tumor cells, leading to the eradication of ccRCC targets in vitro. AB-2100 also exhibited selective killing of dual antigen expressing tumors in vivo using a dual-flank subcutaneous xenograft model. Preclinical xenograft studies also demonstrated that TGFBR shRNA and SPA modules enhanced antitumor activity of ICTs. When AB-2100 potency was evaluated in the subcutaneous A498 xenograft model, treatment with AB-2100 resulted in complete and durable anti-tumor responses. In summary, preclinical data demonstrate that AB-2100 selectively targets tumors co-expressing PSMA and CA9, and can overcome multiple suppressive mechanisms in the tumor microenvironment. These results support the evaluation of AB-2100 in the clinic for the treatment of advanced or metastatic ccRCC. Citation Format: Suchismita Mohanty, Jeremy Chen, Alma Gomez, Angela Boroughs, Irene Scarfo, Laura Lim, Kevin Dang, Marvin Chew, Rakesh Sudhakah, Michelle Nguyen, Thomas J. Gardner, Beatriz Millare, James Zhang, Darrian Moskowitz, Stanley Zhou, Neroli H. Xie, Nickolas Attanasio, Amanda Fearon, Ivan Chan, Vibhavari Sail, Vince Thomas, Jennesa Smith, Jennifer McDevitt, Levi Gray-Rupp, Alba Gonzalez, Christopher Murriel, W. Nicholas Haining. AB-2100, a PSMA-inducible CA9-specific CAR T cell product for the treatment of ccRCC provides long-term tumor responses in preclinical mouse model [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 38.

Targeting metabolism to improve CAR-T cells therapeutic efficacy.

Chimeric antigen receptor T (CAR-T) cell therapy achieved advanced progress in the treatment of hematological tumors. However, the application of CAR-T cell therapy for solid tumors still faces many challenges. Competition with tumor cells for metabolic resources in an already nutrient-poor tumor microenvironment is a major contributing cause to CAR-T cell therapy's low effectiveness. Abnormal metabolic processes are now acknowledged to shape the tumor microenvironment, which is characterized by increased interstitial fluid pressure, low pH level, hypoxia, accumulation of immunosuppressive metabolites, and mitochondrial dysfunction. These factors are important contributors to restriction of T cell proliferation, cytokine release, and suppression of tumor cell-killing ability. This review provides an overview of how different metabolites regulate T cell activity, analyzes the current dilemmas, and proposes key strategies to reestablish the CAR-T cell therapy's effectiveness through targeting metabolism, with the aim of providing new strategies to surmount the obstacle in the way of solid tumor CAR-T cell treatment.

CAR T cells redirected to B7-H3 for pediatric solid tumors: Current status and future perspectives

Despite intensive therapies, pediatric patients with relapsed or refractory solid tumors have poor outcomes and need novel treatments. Immune therapies offer an alternative to conventional treatment options but require the identification of differentially expressed antigens to direct antitumor activity to sites of disease. B7-H3 (CD276) is an immune regulatory protein that is expressed in a range of malignancies and has limited expression in normal tissues. B7-H3 is highly expressed in pediatric solid tumors including osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, neuroblastoma, and many rare tumors. In this article we review B7-H3-targeted chimeric antigen receptor (B7-H3-CAR) T cell therapies for pediatric solid tumors, reporting preclinical development strategies and outlining the landscape of active pediatric clinical trials. We identify challenges to the success of CAR T cell therapy for solid tumors including localizing to and penetrating solid tumor sites, evading the hostile tumor microenvironment, supporting T cell expansion and persistence, and avoiding intrinsic tumor resistance. We highlight strategies to overcome these challenges and enhance the effect of B7-H3-CAR T cells, including advanced CAR T cell design and incorporation of combination therapies.

Using Oncolytic Virus to Retask CD19-Chimeric Antigen Receptor T Cells for Treatment of Pancreatic Cancer: Toward a Universal Chimeric Antigen Receptor T-Cell Strategy for Solid Tumor.

Chimeric antigen receptor (CAR) T cells targeting the B-cell antigen CD19 are standard therapy for relapsed or refractory B-cell lymphoma and leukemia. CAR T cell therapy in solid tumors is limited due to an immunosuppressive tumor microenvironment and a lack of tumor-restricted antigens. We recently engineered an oncolytic virus (CF33) with high solid tumor affinity and specificity to deliver a nonsignaling truncated CD19 antigen (CD19t), allowing targeting by CD19-CAR T cells. Here, we tested this combination against pancreatic cancer. We engineered CF33 to express a CD19t (CF33-CD19t) target. Flow cytometry and ELISA were performed to quantify CD19t expression, immune activation, and killing by virus and CD19-CAR T cells against various pancreatic tumor cells. Subcutaneous pancreatic human xenograft tumor models were treated with virus, CAR T cells, or virus+CAR T cells. In vitro, CF33-CD19t infection of tumor cells resulted in >90% CD19t cell-surface expression. Coculturing CD19-CAR T cells with infected cells resulted in interleukin-2 and interferon gamma secretion, upregulation of T-cell activation markers, and synergistic cell killing. Combination therapy of virus+CAR T cells caused significant tumor regression (day 13): control (n = 16, 485 ± 20 mm 3 ), virus alone (n = 20, 254 ± 23 mm 3 , p = 0.0001), CAR T cells alone (n = 18, 466 ± 25 mm 3 , p = NS), and virus+CAR T cells (n = 16, 128 ± 14 mm 3 , p < 0.0001 vs control; p = 0.0003 vs virus). Engineered CF33-CD19t effectively infects and expresses CD19t in pancreatic tumors, triggering cell killing and increased immunogenic response by CD19-CAR T cells. Notably, CF33-CD19t can turn cold immunologic tumors hot, enabling solid tumors to be targetable by agents designed against liquid tumor antigens.

Adoptive T cell therapy for solid tumors: current landscape and future challenges.

Adoptive cell therapy (ACT) comprises different strategies to enhance the activity of T lymphocytes and other effector cells that orchestrate the antitumor immune response, including chimeric antigen receptor (CAR) T-cell therapy, T-cell receptor (TCR) gene-modified T cells, and therapy with tumor-infiltrating lymphocytes (TILs). The outstanding results of CAR-T cells in some hematologic malignancies have launched the investigation of ACT in patients with refractory solid malignancies. However, certain characteristics of solid tumors, such as their antigenic heterogeneity and immunosuppressive microenvironment, hamper the efficacy of antigen-targeted treatments. Other ACT modalities, such as TIL therapy, have emerged as promising new strategies. TIL therapy has shown safety and promising activity in certain immunogenic cancers, mainly advanced melanoma, with an exciting rationale for its combination with immune checkpoint inhibitors. However, the implementation of TIL therapy in clinical practice is hindered by several biological, logistic, and economic challenges. In this review, we aim to summarize the current knowledge, available clinical results, and potential areas of future research regarding the use of T cell therapy in patients with solid tumors.

Low-dose radiotherapy synergizes with iRGD-antiCD3-modified T cells by facilitating T cell infiltration

Background and purposePoor penetration of transferred T cells represents a critical factor impeding the development of adoptive cell therapy in solid tumors. We demonstrated that iRGD-antiCD3 modification promoted both T cell infiltration and activation in our previous work. Interest in low-dose radiotherapy has recently been renewed due to its immuno-stimulatory effects including T cell recruitment. This study aims to explore the synergistic effects between low-dose radiotherapy and iRGD-antiCD3-modified T cells. Materials and methodsFlow cytometry was performed to assess the expression of iRGD receptors and chemokines. T cell infiltration was evaluated by immunohistofluorescence and in vivo real-time fluorescence imaging and antitumor effects were investigated by in vivo bioluminescence imaging in the gastric cancer peritoneal metastasis mouse model. ResultsWe found that 2 Gy irradiation upregulated the expression of all three iRGD receptors and T-cell chemokines. The addition of 2 Gy low-dose irradiation boosted the accumulation and penetration of iRGD-antiCD3-modified T cells in peritoneal tumor nodules. Combining 2 Gy low-dose irradiation with iRGD-antiCD3-modified T cells significantly inhibited tumor growth and prolonged survival in the peritoneal metastasis mouse model with a favorable safety profile. ConclusionAltogether, we demonstrated that low-dose radiotherapy could improve the antitumor potency of iRGD-antiCD3-modified T cells by promoting T cell infiltration, providing a rationale for exploring low-dose radiotherapy in combination of other adoptive T cell therapies in solid tumors.

Enhancing CAR‐NK Cells Against Solid Tumors Through Chemical and Genetic Fortification with DOTAP‐Functionalized Lipid Nanoparticles

AbstractNatural killer (NK) cells are crucial in the innate immune response and show promise in cancer immunotherapy, but face challenges in activation and targeted gene delivery. In this study, bifunctional lipid nanoparticles (DLNPs) containing 1,2‐dioleoyl‐3‐trimethylammonium‐propane (DOTAP), designed to bolster the antitumor efficacy of chimeric antigen receptor‐modified NK (CAR‐NK) cells by facilitating activation and efficient CAR mRNA delivery are introduced. The DLNPs, created by functionalizing FDA‐approved LNP compositions, exhibit excellent mRNA encapsulation and colloidal stability. NK cells primed with DLNPs show increased cytotoxicity against cancer cells via extracellular signal‐regulated kinase/Mitogen‐Activated Protein Kinase pathway modulation and mitochondrial dynamics changes. The DLNPs enter NK cells predominantly through clathrin‐mediated endocytosis, boosting mRNA delivery and overcoming NK cells' resistance to genetic manipulation. CAR‐NK cells targeting glypican‐3, prevalent in hepatocellular carcinoma, show significant therapeutic effects in an orthotopic mouse model. These findings underscore the potential of DLNPs in enhancing CAR‐NK cell therapy for solid tumors, marking a significant stride in NK cell‐based cancer immunotherapy and broadening prospects for NK cell‐related disease interventions.

Epigenetic reprogramming of CAR T cells for in vivo functional persistence against solid tumors.

Limited CAR T-cell expansion and persistence hinder therapeutic responses in solid cancer patients. To enhance the functional persistence of engineered T-cell therapies, we performed genetic disruption in human CAR T cells of SUV39H1, a histone 3 lysine 9 methyltransferase that promotes heterochromatin formation. This resulted in phenotypic CAR-T reprogramming that elicited optimal and sustained antitumor functionality. Single-cell transcriptomic (scRNA-seq) and chromatin accessibility (scATAC-seq) analyses of tumor-infiltrating CAR T cells showed early reprogramming into self-renewing, stem-like populations with decreased expression of dysfunction genes in all subpopulations. Moreover, we provided evidence that SUV39H1 inactivation elicits potent and durable functional persistence upon multiple tumor rechallenges. This opens a safe path to enhancing adoptive cell therapies for solid tumors.

CAR-T cell therapy targeting surface expression of TYRP1 to treat cutaneous and rare melanoma subtypes

A major limitation to developing chimeric antigen receptor (CAR)-T cell therapies for solid tumors is identifying surface proteins highly expressed in tumors but not in normal tissues. Here, we identify Tyrosinase Related Protein 1 (TYRP1) as a CAR-T cell therapy target to treat patients with cutaneous and rare melanoma subtypes unresponsive to immune checkpoint blockade. TYRP1 is primarily located intracellularly in the melanosomes, with a small fraction being trafficked to the cell surface via vesicular transport. We develop a highly sensitive CAR-T cell therapy that detects surface TYRP1 in tumor cells with high TYRP1 overexpression and presents antitumor activity in vitro and in vivo in murine and patient-derived cutaneous, acral and uveal melanoma models. Furthermore, no systemic or off-tumor severe toxicities are observed in an immunocompetent murine model. The efficacy and safety profile of the TYRP1 CAR-T cell therapy supports the ongoing preparation of a phase I clinical trial.

CCR10-mediated Enhancement of T Cell Trafficking for Improved Tumor Immunotherapy.

The effectiveness of adoptive T cell therapy for solid tumors remains suboptimal, partly attributed to insufficient T cell infiltration into the tumor site. A promising strategy involves directing T cells towards the tumor utilizing tumor-specific chemokine receptors. We analyzed chemokine receptor expression in activated T cells and chemokine expression in breast and lung cancer using The Cancer Genome Atlas (TCGA) data. Subsequently, we generated 1G4 T cell receptor-engineered T (TCR-T) cells with CCR10 and performed in vitro and in vivo efficacy tests. CCR10 exhibited insufficient expression in various human T cells. Analysis of TCGA RNA sequencing data revealed elevated expression of the chemokine CCL28, the corresponding chemokine for CCR10, in breast and lung cancer. Consequently, we generated CCR10-1G4 TCR-T cells. CCR10-1G4 dual expressing TCR-T cells exhibited comparable cellular cytotoxicity but increased mobility compared to 1G4 TCR-T cells in vitro. Furthermore, injecting CCR10-1G4 dual expressing TCR-T cells into a xenograft tumor model demonstrated enhanced in vivo trafficking and a greater reduction of tumor burden. This study highlights the potential of CCR10 for developing efficient adoptive T-cell treatments targeting solid tumors.

Elevating the potential of CAR-T cell therapy in solid tumors: exploiting biomaterials-based delivery techniques.

Chimeric antigen receptor (CAR) T cells exhibit promising progress in addressing hematologic malignancies. However, CAR-T therapy for solid tumors remains limited, with no FDA-approved CAR-T products available for clinical use at present. Primary reasons include insufficient infiltration, accumulation, tumor immunosuppression of the microenvironment, and related side effects. Single utilization of CAR-T cannot effectively overcome these unfavorable obstacles. A probable effective pathway to achieve a better CAR-T therapy effect would be to combine the benefits of biomaterials-based technology. In this article, comprehensive biomaterials strategies to break through these obstacles of CAR-T cell therapy at the tumor sites are summarized, encompassing the following aspects: 1) generating orthotopic CAR-T cells; 2) facilitating CAR-T cell trafficking; 3) stimulating CAR-T cell expansion and infiltration; 4) improving CAR-T cell activity and persistence; 5) reprogramming the immunosuppressive microenvironments. Additionally, future requirements for the development of this field, with a specific emphasis on promoting innovation and facilitating clinical translation, are thoroughly discussed.