Chimeric antigen receptor T cell (CAR T) therapy has demonstrated unprecedented therapeutic activity in hematologic malignancies. However, generating potent clinical responses against solid tumors remains a challenge for CAR T therapy. As the field strives to improve the therapeutic efficacy of CAR T cells with novel target antigens and enhanced potency, the risks of on-target toxicity pose a major barrier to progress. To address these challenges, we have developed engineered CAR T cells to target solid tumors through AND logic gates, where CAR expression is conditionally induced by a transcription factor released from a priming receptor (PrimeRTM) upon binding to the PrimeR antigen. The AND gate limits off-tumor toxicity as it requires both CAR and PrimeR antigen expression in the tumor microenvironment. To ensure PrimeR expression and signal transduction upon antigen binding, while minimizing residual ‘‘leaky’’ CAR induction in the absence of PrimeR antigen, we screened hundreds of PrimeR binders using both arrayed and pooled strategies. In an arrayed strategy, we engineered T cells from four donors in multiwell plates using CRISPR-mediated, non-viral, site-specific integration of logic gates bearing a variable PrimeR binder and a fixed MSLN CAR. In addition, we employed a pooled screening strategy, where we engineered T cells from two independent donors with a pool containing a subset of >300 of the same logic gates. Engineered T cells from both strategies were co-cultured with cell lines bearing either both CAR and PrimeR antigens or a single antigen, in order to evaluate fidelity and on-target functionality. In the arrayed setting, on-target functionality was quantified based on the levels of CAR induction, cytokine secretion, T cell activation, and target cell killing in the presence of both antigens, while fidelity was assessed based on the absence of these activity signals in the presence of a single antigen. In the pooled setting, sorting based on functional markers was performed and sequencing was used to quantify the relative abundance of cells with each logic gate in different sorted populations. On-target activity and circuit fidelity were then quantified based on enrichments in different sorted populations. Results from the pooled and arrayed screens were highly concordant. We combined the screen readouts to nominate a small set of PrimeR binders that exhibited both high fidelity and on-target functionality. We confirmed the desired characteristics of these binders with targeted arrayed screens in additional conditions as well as in in-vivo models. We have applied both screen strategies to select a small set of leads from hundreds of candidate PrimeR binders in the context of a logic-gated MSLN CAR. As pooled and arrayed screens come with different sets of limitations and advantages, both serve as important tools for the effective selection of receptors in the development of novel cell therapies. Citation Format: Li Wang, Sofia Kyriazopoulou Panagiotopoulou, Rona Harari-Steinfeld, Dasmanthie De Silva, Michelle Tan, Laura Lim, Angela Boroughs, Cate Sue, Jon Chen, Jamie Thomas, Mary Chua, Ed Yashin, Christine Shieh, Ryan Fong, Sophie Xu, Grace Zheng, Brendan Galvin, Aaron Cooper, Tarjei Mikkelsen, Nicholas Haining. High throughput screening strategies in the development of logic gated cell therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5329.

In solid tumors, CAR T cell efficacy is limited by off-tumor toxicity, poor persistence, and suppression by the tumor microenvironment (TME). To address these challenges we have engineered AB-1015, an integrated circuit T cell (ICT cell) intended for the treatment of ovarian cancer. The AB-1015 transgene cassette encodes an “AND” logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition and a dual shRNA-miR to resist TME suppression and improve ICT cell function and persistence. The AB-1015 DNA cassette is inserted into the T cell genome at a defined novel genomic site via CRISPR-based gene editing. The AB-1015 logic gate consists of a priming receptor that induces expression of an anti-mesothelin (MSLN) CAR upon engagement of a ALPG/P (alkaline phosphatase germ-line/placental). The dual-antigen specificity of the logic gate was assessed in mice engrafted with MSLN+ and ALPG/P+MSLN+ K562 tumors established on contralateral flanks. AB-1015 ICT cells eliminated ALPG/P+MSLN+ tumors, while sparing tumors that lacked ALPG/P. To assess the ability of AB-1015 to mediate killing of MSLN+ tumor cells in the context of heterogeneous cultures, we utilized an admixed co-culture system where ALPG/P+ target cells were spiked into cultures that were otherwise MSLN+. AB-1015 was able to eliminate admixed co-cultures where as few as 5-15% of the target cells expressed ALPG/P.The AB-1015 also contains a dual shRNA-miR that targets FAS and PTPN2, two critical mediators of T cells survival and function. FASL, the cognate ligand for FAS receptor, is expressed on the surface of activated T cells and is significantly overexpressed in the ovarian cancer TME. In vitro, AB-1015 demonstrated resistance to FAS-mediated apoptosis. Knockdown of PTPN2, a phosphatase involved in T cell proliferation and functional persistence, resulted in enhanced AB-1015 ICT cell expansion during repetitive stimulation over a period of 14 days, as well as a 30-fold reduction in tumor outgrowth compared with logic gated T cells alone. In summary, AB-1015 ICT cells are specific for ALPG/P+MSLN+, demonstrate superior potency, expansion, and persistence compared with logic gated T cells alone, and are resistant to ovarian TME suppression. These results support further evaluation of AB-1015 as a novel therapy for indications including ovarian, fallopian tube, or primary peritoneal cancer. Citation Format: Stephen Santoro, Aaron Cooper, Natalie Bezman, Jun Feng, Kanika Chawla, Jennifer McDevitt, Tarjei Mikkelsen, Susie Jun, W. Nicholas Haining. AB-1015, a novel integrated circuit T cells containing an ALPG/MSLN logic gate and FAS/PTPN2 shRNA-miR, demonstrates specific and potent activity against ALPG/MSLN tumors [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 585.

BackgroundIn solid tumors, CAR T cell efficacy is limited by off-tumor toxicity and suppression by the tumor microenvironment (TME). AB-X is an integrated circuit T cell (ICT cell) intended for the treatment of ovarian cancer. AB-X includes a transgene cassette with two functional modules: 1) an ”AND” logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition; 2) a dual shRNA-miR to resist TME suppression and improve ICT cell function. The AB-X logic gate consists of a priming receptor that induces expression of an anti-mesothelin (MSLN) CAR upon engagement of a ALPG/P (alkaline phosphatase germ-line/placental). The dual shRNA-miR mediates downregulation of FAS and PTPN2. The AB-X DNA cassette is inserted into the T cell genome at a defined novel genomic site via CRISPR-based gene editing.MethodsDual-antigen specificity of the logic gate was assessed in mice harboring MSLN+ and ALPG/P+MSLN+ K562 tumors established on contralateral flanks. Potency was measured in a subcutaneous MSTO xenograft model. Logic-gated ICT cells were compared with MSLN CAR T cells in both models. In vitro, expansion of ICT cells with the FAS/PTPN2 shRNA-miR was evaluated in a 14 day repetitive stimulation assay (RSA). In vivo, expansion and potency were measured in the MSTO xenograft model. An in vitro FAS cross-linking assay was conducted to assess the impact of FAS knockdown on FAS-mediated apoptosis.ResultsLogic-gated ICT cells demonstrated specific activity against ALPG/P+MSLN+ tumors, but had no effect against MSLN+ tumors in the K562 in vivo specificity model. In addition, logic-gated ICT cells demonstrated greater in vivo potency than MSLN CAR T cells in the MSTO xenograft model. In our RSA, ICT cells containing the FAS/PTPN2 shRNA-miR had 8-fold greater expansion than the MSLN CAR T cells. Enhanced expansion was confirmed in vivo with ICT cells demonstrating >10-fold expansion in tumors and peripheral blood, enabling comparable growth inhibition in MSTO xenografts at less than one quarter the dose of the MSLN CAR T cells. Importantly, PTPN2 knockdown resulted in balanced expansion of all T cell subsets, including CD45RA+, CCR7+ memory cells. Lastly, ICT cells containing the FAS/PTPN2 shRNA-miR were resistant to FAS-mediated apoptosis.ConclusionsAB-X ICT cells specifically recognize ALPG/P+MSLN+ tumors, demonstrate superior potency, expansion, and persistence compared with MSLN CAR T cells, and are resistant to ovarian TME suppression. AB-X will be evaluated in clinical trials for treatment of platinum resistant/refractory ovarian cancer.AcknowledgementsWe would like to acknowledge all of our colleagues at Arsenal Biosciences, without whom this work would not have been possible.

Abstract Understanding disease development, progression and response to treatment remains elusive for most types of cancer. This is due both to the genetic complexity seen in tumors as well as differences in how the surrounding cells and immune system respond to the developing tumor. To fully understand all aspects of disease progression and response to treatment, a systems level approach is needed. We have developed a platform that allows for high resolution multi-omics analysis of the tumor and its microenvironment. This platform is built upon a microfluidic system that enables the production of hundreds of thousands to millions of partitioned barcoded reactions. On this platform we generate Linked-Reads that allow for the detailed resolution of complex structural variant events. To obtain more detailed information about tumor heterogeneity and clonal structure, we also perform single cell DNA analysis that allows for the detection of sub-megabase copy number variants at the single cell level.The platform also provides detailed resolution of the cells occupying the microenvironment surrounding the tumor. Using a combination of single cell gene expression and enriched V(D)J B and T cell repertoire analysis, we identify cells constituting a tumor, as well as the immune and nonimmune cells surrounding the tumor. We also provide single cell profiling of the paired antigen receptor chains for both B and T cells. As a demonstration, we have performed this analysis on three types of tumor samples: metastatic melanoma (MM), primary colorectal cancer (CRC) and primary clear cell renal carcinoma (CCRC). We are able to identify a clonal expansion in the CCRC sample and determine there is no expansion in the CRC sample. The combination of these approaches allows for unparalleled characterization of disease development, progression and response. Citation Format: Sarah Garcia, Rajiv Bharadwaj, Stéphane Boutet, Claudia Catalanotti, Valeria Giangerra, Josephine Lee, Jessica Terry, Stephen Williams, Grace X. Zheng, Tarjei Mikkelsen, Michael Schnall-Levin, Ben Hindson, Deanna M. Church. Identifying genetic variation and cellular heterogeneity with a comprehensive cancer analysis toolkit [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 281.

Abstract Understanding the complex interactions between malignant, stromal and infiltrating lymphocyte cell types within the tumor microenvironment is a critical component of personalized cancer treatments. We describe an approach that couples single-cell transcriptional profiling of tumors with high-resolution receptor profiling of tumor infiltrating lymphocytes (TILs). Using a fully integrated, droplet-based system for 5' single cell RNA sequencing (scRNA-seq), we simultaneously profiled the transcriptome and immune repertoire of the same cells from a primary colorectal cancer (CRC) tumor and a primary non-small cell lung cancer (NSCLC) tumor. Each tumor varied in type and proportion of its cellular components, noticeably in the proportion of TILs. The tumor cells in the CRC were mostly epithelial in nature, with the TILs comprising 21% of the total cells sequenced. This lymphocyte population consisted of both T (5% CD4+, 3% CD8+) and, interestingly, B cells (5% CD19+), with a number of plasma B cells (IGH high, CD138+) also identified. The NSCLC tumor had a high immune cell infiltrate, with lymphocytes comprising 43% of cells (19% T, 23% B, and 5% plasma B cells) indicating a robust adaptive immune response. To examine these cells further, CD45+ cells were subject to scRNA-seq. When compared to the unenriched sample, the enriched cell population had a similar B to T cell ratio with a reduction in the proportion of plasma B cells, likely due to exclusion of mature plasma B cells by the sort (47% T (44% CD4+, 29% CD8+), 42% B, and 2% plasma B cells). To fully characterize the B cell infiltrate we applied targeted 5' scRNA-seq to obtain full length, paired, B cell receptor heavy and light chain sequences. Despite the presence of plasma B cells in the CRC tumor, limited clonal expansion was observed. In the NSCLC tumor, clonal expansion of B cells was observed in the unenriched tumor sample but these clonotypes were not present in the CD45+ population, suggesting the clonally expanded cells are in the mature plasma B cell population. We also applied a targeted scRNA-seq approach to obtain full length paired T cell receptor alpha and beta sequences, however, no clear T cell expansion was observed in either sample. These findings emphasize the importance of examining receptor sequences rather than relying on the presence of B or T cells alone to determine if a robust immune response is being mounted. The presence of tumor-infiltrating B lymphocytes has been linked to a favorable clinical outcome in some types of cancers. Identification of these cells, their subsets and high resolution profiling of their receptor sequences using scRNA-seq allows a novel insight into the adaptive immune response in tumors. This technology will allow better characterization of tumor heterogeneity and the adaptive immune response to the tumor microenvironment and will serve as a foundation for future research into tumor immunology and immunotherapy. Citation Format: Sarah E. Taylor, Stephane Boutet, Valeria Giangarrá, Grace X. Zheng, Alvaro M. Barrio, Luz Montesclaros, Josephine Lee, Samuel Marrs, Kevin J. Wu, Paul Ryvkin, Tarjei Mikkelsen, Deanna M. Church. Analyzing infiltrating B cell populations in the tumor microenvironment using single cell transcriptomics [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 1013.

Abstract Advances in single cell RNA quantification techniques have enabled comprehensive study of subpopulations of cells within a heterogeneous population. The application of single cell quantification techniques to oncology is helping to elucidate the complex variability in genetic and epigenetic interactions that occur within tumor cells and their microenvironment. However, current single cell RNA-sequencing methods are limited by their reliance on costly infrastructure and laborious experimental protocols. We developed the GemCode Platform, which combines microfluidics with molecular barcoding and custom bioinformatics software to enable 3’ mRNA counting from thousands of single cells. Here we utilized the GemCode Platform to profile primary cells from healthy donors and cancer patients. Cell lines and cancer samples were obtained from commercial sources. Single cells, reagents and a single gel bead containing barcoded oligonucleotides were encapsulated into picoliter-sized droplets using the 10X Genomics GemCode Platform. The platform achieved extremely high cell loading efficiency (> 50%), enabling the creation of libraries from precious samples. Lysis and barcoded reverse transcription of RNAs from single cells were performed inside each droplet. High quality next generation sequencing libraries were finished in a single bulk reaction. The GemCode software suite was utilized for processing, interactive analysis and visualization of single cell gene expression data. We demonstrated single cell behavior through mouse- and human cell mixing experiments with a low doublet rate of <1%, making the platform suitable for profiling of rare cancer cell populations. We profiled >40,000 peripheral blood mononuclear cells from healthy donors and detected all major subpopulations (i.e., B cells, CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, monocytes) in similar proportions to those previously reported in the literature. Notably, the high-throughput nature of the platform enabled resolution of finer sub-structures such as CD4+ effector memory cells and CD4+ central memory cells. Experiments comparing cells isolated from patients with hematologic malignancies (such as CLL, AML and CML) with whole bone marrow from healthy donors further demonstrate the power of single cell profiling for characterizing disease-associated changes in complex tissues. We demonstrate the ability to perform high-throughput gene expression profiling of mRNAs in single cells. The high-throughput platform enables detection of rare cells in a heterogeneous tumor population. Moreover, efficient cell loading enables analysis of clinically relevant sample types with limited cell input. An integrated single cell mRNA analysis will lead to novel insights into the molecular characteristics of individual cancer cells and provide targets for therapeutic intervention. Citation Format: Grace X.y. Zheng, Tarjei Mikkelsen, Jessica Terry, Phillip Belgrader, Paul Ryvkin, Ryan Wilson, Tobias D. Wheeler, Zachary Bent, Geoff McDermott, Solongo Ziraldo, Alexander Wong, Michael Schnall-Levin, Ben Hindson. Single cell mRNA quantification from 1000s of cells in healthy and malignant tumor samples using a high-throughput droplet-based system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 150.