Abstract
Background
Chimeric antigen receptor (CAR) T cells have achieved remarkable responses in patients with hematological malignancies; however, the potential of this therapeutic platform for ...solid tumors like glioblastoma (GBM) has been limited, due in large part to the targeting of single antigens in a heterogeneous disease. Strategies that allow CAR T cells to engage multiple antigens concomitantly may broaden therapeutic responses and mitigate the effects of immune escape.
Methods
Here we have developed a novel, dual-specific, tandem CAR T (TanCART) cell with the ability to simultaneously target both EGFRvIII and IL-13Rα2, two well-characterized tumor antigens that are frequently found on the surface of GBM cells but completely absent from normal brain tissues. We employed both standard immunological assays and multiple orthotopic preclinical models including patient-derived xenograft to demonstrate efficacy of this approach against heterogeneous tumors.
Results
Tandem CAR T cells displayed enhanced cytotoxicity in vitro against heterogeneous GBM populations, including patient-derived brain tumor cultures (P < .05). Compared to CAR T cells targeting single antigens, dual antigen engagement through the tandem construct was necessary to achieve long-term, complete, and durable responses in orthotopic murine models of heterogeneous GBM, including patient-derived xenografts (P < .05).
Conclusions
We demonstrate that TanCART is effective against heterogeneous tumors in the brain. These data lend further credence to the development of multi-specific CAR T cells in the treatment of GBM and other cancers.
Abstract
Targeting solid tumors with CAR T cells has proven to be more difficult due to their heterogenous target antigen expression, antigen escape, and due to their hostile tumor microenvironment ...(TME). Mesothelin represents a promising surface tumor antigen, since it has been associated with tumor invasion and is highly expressed on various cancer types, including pancreatic adenocarcinoma. As clinical trials of mesothelin targeting CAR have yet to show efficacy, we hypothesized that tumor stromal cells such as cancer-associated fibroblasts (CAFs) may play a role in this resistance. To provide a more favorable TME for CAR T cells, we generated a bicistronic lentiviral vector encoding a mesothelin CAR along with a secreted T cell engaging molecule (TEAM) that targets fibroblast activation protein (FAP), which is expressed by CAFs. We termed our constructs CARTEAM, and in this case, mesoFAP. We have assessed the activation and proliferative capacity of these CARTEAM through in vitro assays. We showed that TEAMs secreted by CAR T cells bind their appropriate target antigen by adding supernatant from CARTEAM to target cells expressing FAP. In a co-culture assay using a transwell system we demonstrated the cytotoxic effect of secreted TEAM interacting and recruiting bystander T cells against CAFs. In a real-time cell analysis (RTCA) co-culture assay with a pancreatic cancer cell line (AsPC1) and FAP expressing CAFs, we showed cell death of AsPC1 upon CAR recognition and cell death of CAFs through TEAM-mediated recruitment of bystander T cells and CART cells. We show in these co-culture systems, mimicking tumor and TME, that our CARTEAM construct is superior in the elimination of both cancer cell line and CAF, in comparison to control constructs, including mesothelin targeting CAR T cells (meso CAR) and meso CAR T cells secreting an unspecific CD19 TEAM (mesoCD19). We also used acoustic force microscopy to evaluate the additive effect of the TEAM molecule secreted to binding to tumor cells by mesothelin CAR T cells. In vivo experiments of subcutaneously injected tumor cells admixed with CAFs, show superior tumor control when treated with CARTEAM in comparison to control constructs. Based on these data, we demonstrate both the effective in vitro elimination of CAFs and pancreatic cancer cells through the application of CARTEAM and control of pancreatic tumor growth in vivo. Our studies provide a deeper insight into a dual targeting strategy using a novel CAR T cell secreting a TEAM against pancreatic cancer and its tumor microenvironment.
Citation Format: Marc Wehrli, Adam Kuo, Rebecca Larson, Irene Scarfò, Amanda Bouffard, Korneel Grauwet, Mark Leick, Andrea Schmidts, Stefanie Bailey, Tamina Kienka, Michael Kann, Sonika Vatsa, Harrison Silva, Kathleen Gallagher, Max Jan, Bryan Choi, David Ting, Marcela Maus. Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 569.
Anaplastic Large Cell Lymphoma (ALCL) is a clinical and biological heterogeneous disease including the ALK+ and ALK- systemic forms. While ALK+ ALCL are molecularly characterized and can be readily ...diagnosed, no specific markers and molecular events leading to ALK- ALCL transformation have been identified so far.
To discover biomarkers and/or genes potentially involved in ALK- ALCL pathogenesis, we applied the Cancer Outlier Profile Analysis (COPA) algorithm to a large gene expression profiling (GEP) data set including 249 cases of T-NHLs and normal T-cells. Among the top outliers, ERBB4 and COL29A1 genes were exclusively expressed in a subset of ALK- ALCL cases, and resulted highly correlated. Differential analysis comparing the expression profiles of ERBB4/COL29A1+ to ALK+ ALCL patients identified 48 genes up- and 37 down-regulated (qval < 0.0076). The most significantly over-represented functional category included transcriptional regulators. Gene-set-enrichment-analysis (GSEA) indicated that ERBB4/COL29A1+ samples shared activation of specific pathways such as inflammatory response, TNF-NF-κB, JAK-STAT, cytokine, and angiogenesis. RNA sequencing and 5’RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified two ERBB4 truncated transcripts (referred as I20ΔERBB4 and I12ΔERBB4), displaying Transcription Starting Sites (TSS) in intron 12 and 20, respectively. On the contrary, full length COL29A1 mRNA was expressed in ERBB4/COL29A1+ samples. RT-qPCR analysis performed on 170 T-NHL samples (51 PCTL-NOS, 44 ALK+ ALCL, and 75 ALK- ALCL) highlighted specific expression of ERBB4 aberrant transcripts in 25% of ALK- ALCL (18 out of 75). ERBB4+ patients showed higher expression of I20ΔERBB4 as compared to I12ΔERBB4 transcript. ERBB4 expression at protein level was confirmed by immunohistochemistry and Western Blotting on selected cases. Inspection of intronic regions spanning two hypothetical TSS revealed the presence of Human Endogenous Retrovirus (HERV) Long Terminal Repeats (LTR) displaying promoter activity, as demonstrated by luciferase assays.
In conclusion, we defined a new subclass of ALK- ALCL characterized by ectopic expression of ERBB4 and COL29A1 genes. To the best of our knowledge, ERBB4 truncated transcripts carrying intronic 5’UTR have never been described before. Further studies are required to address whether the expression of ERBB4 aberrant transcripts may contribute to the ALCL transformation and lymphoma maintenance. This information might lead to more rational therapeutic approaches for ERBB4+ ALCL patients.
No relevant conflicts of interest to declare.
Human induced pluripotent stem cells (iPSCs) provide a potentially unlimited resource for cell therapies, but the derivation of mature cell types remains challenging. The histone methyltransferase ...EZH1 is a negative regulator of lymphoid potential during embryonic hematopoiesis. Here, we demonstrate that EZH1 repression facilitates in vitro differentiation and maturation of T cells from iPSCs. Coupling a stroma-free T cell differentiation system with EZH1-knockdown-mediated epigenetic reprogramming, we generated iPSC-derived T cells, termed EZ-T cells, which display a highly diverse T cell receptor (TCR) repertoire and mature molecular signatures similar to those of TCRαβ T cells from peripheral blood. Upon activation, EZ-T cells give rise to effector and memory T cell subsets. When transduced with chimeric antigen receptors (CARs), EZ-T cells exhibit potent antitumor activities in vitro and in xenograft models. Epigenetic remodeling via EZH1 repression allows efficient production of developmentally mature T cells from iPSCs for applications in adoptive cell therapy.
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•Stroma-free differentiation of iPSCs into T cells expressing diverse TCRs•EZH1 repression generates mature EZ-T cells similar to peripheral blood αβ T cells•EZ-T cells can give rise to memory-like T cells upon activation•CAR EZ-T cells display enhanced antitumor activity in vitro and in vivo
Jing et al. combine a stroma-free differentiation system with EZH1-repression-mediated epigenetic reprogramming to generate developmentally mature iPSC-derived CAR T cells with enhanced antitumor activities.
472
Background: Relapsed/refractory clear cell renal cell carcinoma (ccRCC) progressing after treatment with CPI and VEGF inhibitor remains an area of unmet need. We have developed AB-2100, an ...autologous, integrated circuit T (ICT) cell engineered to include three new features: a sequential “AND” logic gate that requires the IO presence of two antigens in the tumor microenvironment (TME) to trigger T cell killing; a shRNA-miR module to enhance resistance to suppressive TME via constitutive knockdown of FAS and TGFBR2; and a constitutive synthetic pathway activator (SPA) that increases STAT3 signaling for enhanced T cell cytotoxicity and expansion. Methods: A previous clinical study of CA9-specific CAR-T cell therapy was limited by on-target, off-tumor toxicity. To overcome this, AB-2100 includes a sequential “AND” logic gate that consists of a priming receptor (PrimeR) targeting PSMA, and a CA9-targeted CAR that is upregulated upon PrimeR engagement with PSMA expressed on the tumor neovasculature. A series of assays were performed to assess the specificity and potency of AB-2100: dual-antigen specificity of the logic gate was assessed in vitro and in vivo against CA9+ and PSMA+CA9+ tumors; vascular priming was modeled by co-culturing AB-2100 cells with PSMA-expressing endothelial cells and CA9+ tumor cells; a FAS cross-linking assay was conducted to assess the impact of FAS knockdown; the enhanced anti-tumor activity conferred by TGFBR2 shRNA and SPA modules were assessed in a 786-O xenograft model; and AB-2100 potency was measured in a subcutaneous renal A498 xenograft. Results: AB-2100 selectively kills tumors that express both CA9 and PSMA, and not tumors that express CA9 alone, as assessed by in vitro cytotoxicity against single or dual antigen expressing tumor cell lines and by a dual flank xenograft model. Furthermore, we confirmed that co-culture with PSMA-expressing endothelial cells was sufficient to upregulate CA9 CAR expression and enable tumor cell killing. Finally, AB-2100 containing both shRNA-miR and SPA modules demonstrated enhanced anti-tumor activity in xenograft RCC models. Conclusions: These data demonstrate that AB-2100 selectively targets tumors co-expressing PSMA and CA9, and can overcome multiple suppressive mechanisms in the TME. These results support the evaluation of AB-2100 in the clinic for the treatment of advanced or metastatic ccRCC.
Abstract
One of the most promising candidates for Chimeric Antigen Receptor (CAR) T cell therapy beyond CAR19 is treatment of multiple myeloma with CAR T cells targeting B cell maturation antigen ...(BCMA). However, current reports of BCMA CAR in the clinic have a median progression free survival of 11.8 months, suggesting that targeting BCMA alone may not be sufficient. Evidence suggests patients treated with BCMA-targeted therapies may be thwarted by BCMA negative relapse. We have developed a novel second generation CAR T cell against another multiple myeloma target: transmembrane activator and CAML interactor (TACI). Mice were immunized against TACI and we designed a new scFv targeting TACI based on the resulting antibodies. Anti-TACI CAR T cells are cytotoxic in vitro and in vivo against multiple myeloma. To overcome single antigen loss, we designed bispecific tandem scFv CAR T cells targeting BCMA and TACI. These two antigens share a natural ligand, A Proliferation-Inducing Ligand (APRIL), which has also been used to design a dual-targeting CAR called TriPRIL. We show that these dual targeting CARs, based on tandem scFv or natural ligand design, have similar efficacy against wild type multiple myeloma models. However, this changes in the context of single antigen loss. We have characterized the proliferative (population doublings) and activation capability (CD69) of these CARs, as well as their memory (CCR7, CD45RA) and exhaustion phenotype (PD-1, TIM-3, LAG-3), with long term exposure to single antigen. Our studies show that sensitivity to antigen density differs between the tandem bispecifics and the natural ligand CAR. These data provide insight into how structural differences between dual-targeting CAR T cells affects their function.
Abstract
Though CAR T cell therapy has had success treating hematologic malignancies, these treatments have shown only modest efficacy in solid tumors. Little is known about the necessary molecular ...components required for CAR T cell cytotoxicity, especially in the context of solid tumors. We investigated the role of IFNgR signaling on solid tumors and discovered across tumor types (including glioblastoma, pancreatic, ovarian and lung) loss of IFNgR1 signaling resulted lower CAR T cell cytotoxicity in vitro and that this was irrespective of target antigen (including endogenous EGFR, IL13Ra2, mesothelin, and exogenous CD19). We also validated this in orthotopic xenograft models of glioblastoma and pancreatic cancer in vivo. CAR T cells exposed to wildtype (WT) or IFNgR1 KO tumor had similar transcriptional profiles, but the tumor cells had different signatures in response to CAR T cell co-culture. Further investigation showed marked upregulation of cell adhesion pathways in WT cells compared to IFNgR1 KO. We utilized microscopy with acoustic force and discovered CAR T cells had lower cell binding avidity to cells lacking IFNgR1 compared to WT cells. Following CAR T cell exposure, Intercellular Adhesion Molecule 1 (ICAM-1) was strongly upregulated at both the transcriptional and protein levels in WT cells but not IFNgR1 KO cells. We overexpressed ICAM-1 on IFNgR1 KO tumor cells and observed that CAR T cell binding avidity and cytotoxicity was restored to that of WT levels. This work highlights the importance of CAR T cell binding avidity and adhesion for optimal cytotoxicity. Better understanding of CAR T cell function will inform future construct design for successful therapies against solid tumors.
RCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.
Abstract CAR T cell activity in solid tumors is limited by off-tumor toxicity and functional suppression from the tumor microenvironment (TME). To address these challenges, we have developed AB-1015, ...an autologous ICT cell product intended for use in ovarian cancer. AB-1015 incorporates two functional modules: an” AND” logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition, and a dual shRNA-miR targeting FAS and PTPN2 to resist TME suppression and to improve ICT cell function. The AB-1015 logic gate consists of a priming receptor (PrimeR) against ALPG/P and an inducible anti-MSLN CAR that is upregulated upon PrimeR engagement. At basal state, only a small percentage of AB-1015 cells have detectable CAR present at low MFI. The percentage of AB-1015 cells that express CAR, as well as the CAR MFI, increase in response to increasing levels of ALPG. This unique feature of the logic gate aims to increase the tumor specificity of AB-1015, given that ALPG/P and MSLN are not co-expressed in the normal tissues. To functionally test the dual-antigen specificity, AB-1015 cells were co-cultured with K562 cells engineered with only one target antigen (ALPG or MSLN), both antigens (ALPG and MSLN), or neither antigen. AB-1015 demonstrated potent killing of K562-ALPG/MSLN cells, and minimal activity against K562 cells expressing only MSLN. In addition, AB-1015 showed over 100-fold increase in IFNg production when co-cultured with K562-ALPG/MSLN cells, compared to co-culture with K562-MSLN cells that do not express ALPG. The dual-antigen specificity of the logic gate was further assessed in vivo using a dual flank tumor xenograft model where one tumor expressed both ALPG and MSLN, and the contralateral tumor expressed MSLN alone. Compared with the RNP group, the constitutive anti-MSLN CAR demonstrated tumor reduction on both flanks. In contrast, the activity of AB-1015 was specific to the ALPG+MSLN+ tumor. The anti-tumor activity of AB-1015 was established in an intraperitoneal OVCAR3 ovarian xenograft model that resembles high-grade serous ovarian cancer histology. AB-1015 demonstrated potent anti-tumor activity as demonstrated by a decrease in bioluminescent signal from the tumors treated with AB-1015. To further increase the stringency of our preclinical models, we engineered the subcutaneous MSTO xenograft model to express FASL. In this model, conventional anti-MSLN CAR T cells failed to control tumor outgrowth. In contrast, AB-1015 resists FASL suppression via knockdown of FAS on the ICT surface. As a result, AB-1015 is capable of completely clearing these otherwise difficult-to-treat tumors in this model. In summary, maximal AB-1015 effector function depends on PrimeR engagement with ALPG/P and CAR binding to MSLN. AB-1015 demonstrates superior potency compared with conventional anti-MSLN CAR T cells and is resistant to ovarian TME suppression in preclinical studies. Based on these promising preclinical data, AB-1015 is being studied in a phase I clinical trial (NCT05617755) for patients with platinum-resistant ovarian cancer. Citation Format: Jun Feng, Jasper Williams, Hongruo Yun, Dina Polyak, James Zhang, Michelle Nguyen, Irene Scarfo, Jessica Fuhriman, Aaron Cooper, Jennifer McDevitt, Stephen Santoro. AB-1015, an Integrated Circuit T (ICT) cell therapy containing an ALPG/MSLN logic gate and FAS/PTPN2 shRNA-miR, for the treatment of ovarian cancer abstract. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B080.
Abstract
T cells expressing chimeric antigen receptors (CARs) have demonstrated remarkable clinical benefit in certain hematological malignancies albeit with side effect such as cytokine release ...syndrome, on Target off tumor response and limited efficacy in solid tumors. Here, we present the preclinical evaluation of a novel T cell engineering platform designed to overcome potential hurdles of CARs and other T cell receptor modalities. Specifically, we have generated novel T Cell Receptor Fusion Constructs (TRuC™s) that fuse binder domains to subunits of the T cell receptor. These constructs when introduced using lentiviral technology, integrate into endogenous TCR, reprograming T cells to target tumor antigen in a non-major histocompatibility complex (MHC) restricted fashion and harnessing the full spectrum of TCR signaling. TRuC™ variants were constructed by recombinant fusion of an scFv or sdAb to various TCR subunits via a flexible linker sequence. Likewise, CD28ζ and 41BBζ CARs were generated using the same binders for side-by-side comparison. We demonstrated that TRuC™ variants can effectively reprogram T cells to recognize tumor surface antigens in a non-MHC-restricted fashion. TRuC™s are distinct from CARs in their ability to activate T cells through the entire TCR without additional costimulatory domains. In vitro, TRuC™ T cells were equally potent as CAR T cells in eliminating tumor cells. Compared to CAR-T cells, TRuC-T cells produced lower cytokine levels and proliferated less. Despite these difference, TRuC™ T cells were more efficient in clearing tumors in subcutaneous Raji and MSTO-211H mesothelioma models. Our findings support the development of TRuC-T cells for the treatment of solid tumors.
Citation Format: Ekta Patel, Jian Ding, Nikolaus Thorausch, Janani Krishnamurthy, Rashmi Choudhary, Solly Weiler, Bonnie Le, Patrick Tavares, Adam Zieba, Justin Quinn, Yan Wang, Wolfgang Schamel, Irene Scarfo, Marcela Maus, Patrick Baeuerle, Dainel Getts, Robert Hofmeister. Characterization of a novel class of engineered (TCR) fusion constructs (TRuCTMs) aimed to treat solid tumors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3584.
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