scholarly journals Pharmacologic Control of CAR T Cells

2021 ◽  
Vol 22 (9) ◽  
pp. 4320
Author(s):  
Benjamin Caulier ◽  
Jorrit M. Enserink ◽  
Sébastien Wälchli
Keyword(s):  
T Cells ◽  
Small Molecules ◽  
Antigen Specificity ◽  
Car T Cells ◽  
Anti Cancer ◽  
Car T ◽  
Clinical Benefits ◽  
Response Profiles ◽  
Approved Drugs ◽  
Stimulation Of

Chimeric antigen receptor (CAR) therapy is a promising modality for the treatment of advanced cancers that are otherwise incurable. During the last decade, different centers worldwide have tested the anti-CD19 CAR T cells and shown clinical benefits in the treatment of B cell tumors. However, despite these encouraging results, CAR treatment has also been found to lead to serious side effects and capricious response profiles in patients. In addition, the CD19 CAR success has been difficult to reproduce for other types of malignancy. The appearance of resistant tumor variants, the lack of antigen specificity, and the occurrence of severe adverse effects due to over-stimulation of the therapeutic cells have been identified as the major impediments. This has motivated a growing interest in developing strategies to overcome these hurdles through CAR control. Among them, the combination of small molecules and approved drugs with CAR T cells has been investigated. These have been exploited to induce a synergistic anti-cancer effect but also to control the presence of the CAR T cells or tune the therapeutic activity. In the present review, we discuss opportunistic and rational approaches involving drugs featuring anti-cancer efficacy and CAR-adjustable effect.

Hematologic Malignancies: Plasma Cell Disorders

2017 ◽  
pp. 561-568 ◽  
Author(s):  
Madhav V. Dhodapkar ◽  
Ivan Borrello ◽  
Adam D. Cohen ◽  
Edward A. Stadtmauer
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
Cell Surface ◽  
Plasma Cell ◽  
Antigen Specificity ◽  
Immune Effector ◽  
Car T Cells ◽  
Car T

Multiple myeloma (MM) is a plasma cell malignancy characterized by the growth of tumor cells in the bone marrow. Properties of the tumor microenvironment provide both potential tumor-promoting and tumor-restricting properties. Targeting underlying immune triggers for evolution of tumors as well as direct attack of malignant plasma cells is an emerging focus of therapy for MM. The monoclonal antibodies daratumumab and elotuzumab, which target the plasma cell surface proteins CD38 and SLAMF7/CS1, respectively, particularly when used in combination with immunomodulatory agents and proteasome inhibitors, have resulted in high response rates and improved survival for patients with relapsed and refractory MM. A number of other monoclonal antibodies are in various stages of clinical development, including those targeting MM cell surface antigens, the bone marrow microenvironment, and immune effector T cells such as antiprogrammed cell death protein 1 antibodies. Bispecific preparations seek to simultaneously target MM cells and activate endogenous T cells to enhance efficacy. Cellular immunotherapy seeks to overcome the limitations of the endogenous antimyeloma immune response through adoptive transfer of immune effector cells with MM specificity. Allogeneic donor lymphocyte infusion can be effective but can cause graft-versus-host disease. The most promising approach appears to be genetically modified cellular therapy, in which T cells are given novel antigen specificity through expression of transgenic T-cell receptors (TCRs) or chimeric antigen receptors (CARs). CAR T cells against several different targets are under investigation in MM. Infusion of CD19-targeted CAR T cells following salvage autologous stem cell transplantation (SCT) was safe and extended remission duration in a subset of patients with relapsed/refractory MM. CAR T cells targeting B-cell maturation antigen (BCMA) appear most promising, with dramatic remissions seen in patients with highly refractory disease in three ongoing trials. Responses are associated with degree of CAR T-cell expansion/persistence and often toxicity, including cytokine release syndrome (CRS) and neurotoxicity. Ongoing and future studies are exploring correlates of response, ways to mitigate toxicity, and “universal” CAR T cells.

Identification of Small Molecule Modulators to Enhance the Therapeutic Properties of Chimeric Antigen Receptor T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4712-4712
Author(s):  
Jonathan Rosen ◽  
Betsy Rezner ◽  
David Robbins ◽  
Ian Hardy ◽  
Eigen Peralta ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Small Molecules ◽  
Small Molecule ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Adoptive cellular therapies using engineered chimeric antigen receptor T cells (CAR-T cells) are rapidly emerging as a highly effective treatment option for a variety of life-threatening hematological malignancies. Small molecule-mediated modulation of T cell differentiation during the in vitro CAR-T manufacturing process has great potential as a method to optimize the therapeutic potential of cellular immunotherapies. In animal models, T cells with a central or stem memory (TCM/SCM) phenotype display enhanced in vivoefficacy and persistence relative to other T cell subpopulations. We sought to identify small molecules that promote skewing towards a TCM/SCM phenotype during the CAR-T manufacturing process, with associated enhanced viability, expansion and metabolic profiles of the engineered cells. To this end, we developed a high-throughput functional screening platform with primary human T cells using a combination of high-content immunophenotyping and gene expression-based readouts to analyze cells following a high-throughput T cell culture platform that represents a scaled-down model of clinical CAR-T cell production. Multicolor flow cytometry was used to measure expansion, cell viability and the expression levels of cell surface proteins that define TCM cells (e.g., CCR7, CD62L and CD27) and markers of T cell exhaustion (e.g., PD1, LAG3, and TIM3). In parallel, a portion of each sample was evaluated using high content RNA-Seq based gene expression analysis of ~100 genes representing key biological pathways of interest. A variety of known positive and negative control compounds were incorporated into the high-throughput screens to validate the functional assays and to assess the robustness of the 384-well-based screening. The ability to simultaneously correlate small molecule-induced changes in protein and gene expression levels with impacts on cell proliferation and viability of various T cell subsets, enabled us to identify multiple classes of small molecules that favorably enhance the therapeutic properties of CAR-T cells. Consistent with results previously presented by Perkins et al. (ASH, 2015), we identified multiple PI3K inhibitors that could modify expansion of T cells while retaining a TCM/SCM phenotype. In addition, we identified small molecules, and small molecule combinations, that have not been described previously in the literature that could improve CAR-T biology. Several of the top hits from the screens have been evaluated across multiple in vitro (e.g., expansion, viability, CAR expression, serial restimulation/killing, metabolic profiling, and evaluation of exhaustion markers) and in vivo (e.g., mouse tumor models for persistence and killing) assays. Results from the initial screening hits have enabled us to further refine the optimal target profile of a pharmacologically-enhanced CAR-T cell. In addition, we are extending this screening approach to identify small molecules that enhance the trafficking and persistence of CAR-T cells for treating solid tumors. In conclusion, the approach described here identifies unique small molecule modulators that can modify CAR-T cells during in vitro expansion, such that improved profiles can be tracked and selected from screening through in vitro and in vivo functional assays. Disclosures Rosen: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Hardy:Fate Therapeutics: Employment, Equity Ownership. Peralta:Fate Therapeutics: Employment, Equity Ownership. Maine:Fate Therapeutics: Employment, Equity Ownership. Sabouri:Fate Therapeutics: Employment, Equity Ownership. Reynal:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Foster:Fate Therapeutics: Employment, Equity Ownership. Borchelt:Fate Therapeutics: Employment, Equity Ownership. Meza:Fate Therapeutics: Employment, Equity Ownership. Thompson:Juno Therapeutics: Employment, Equity Ownership. Fontenot:Juno Therapeutics: Employment, Equity Ownership. Larson:Juno Therapeutics: Employment, Equity Ownership. Mujacic:Juno Therapeutics: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

CAR T Cells and Other Cellular Therapies for Multiple Myeloma: 2018 Update

2018 ◽  
pp. e6-e15 ◽  
Author(s):  
Adam D. Cohen
Keyword(s):  
T Cells ◽  
T Cell ◽  
Residual Disease ◽  
Checkpoint Inhibitors ◽  
Antigen Specificity ◽  
Cell Repertoire ◽  
Cellular Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Cellular therapies are a rapidly evolving approach to myeloma treatment, which bring a unique mechanism of action with the potential to overcome drug resistance and induce long-term remissions. Two primary approaches are being studied: non–gene-modified strategies, which rely on the endogenous anti-myeloma T-cell repertoire, and gene-modified strategies, which introduce a new T-cell receptor (TCR) or a chimeric antigen receptor (CAR) to confer novel antigen specificity. CAR T cells show the greatest activity to date. Multiple antigen targets, including B-cell maturation antigen (BCMA), CD19, CD38, CD138, and SLAMF7, are being explored for myeloma, and BCMA has emerged as the most promising. Preliminary data from four phase I studies of BCMA CAR T cells, each using a different CAR construct, that involved 90 evaluable patients with relapsed/refractory disease have been reported. These data show response rates of 60% to 100%, including minimal residual disease (MRD)-negative complete remissions, at effective doses (> 108 CAR-positive cells) after lymphodepleting conditioning. Response durability has been more variable, likely related to differences in CAR T-cell products, lymphodepleting regimens, patient selection criteria, and/or underlying biology/prognostic factors. In the two most recent studies, however, most patients remained progression free with median follow-up time of 6 to 10 months; some ongoing remissions lasted more than 1 year. Toxicities are similar to those from CD19 CAR T cells and include cytokine release syndrome and neurotoxicity that is reversible but can be severe. Multiple BCMA CAR T-cell studies are ongoing. Future directions include combinations with immunomodulatory drugs, checkpoint inhibitors, or other CAR T cells, as well as use of gene-edited cellular products to enhance the safety and efficacy of this approach.

scholarly journals Modular Chimeric Antigen Receptor Systems for Universal CAR T Cell Retargeting

2020 ◽  
Vol 21 (19) ◽  
pp. 7222
Author(s):  
Ashley R. Sutherland ◽  
Madeline N. Owens ◽  
C. Ronald Geyer
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activity ◽  
Cellular Immunotherapy ◽  
Antigen Receptors ◽  
Antigen Specificity ◽  
Precise Control ◽  
Car T Cells ◽  
Multiple Antigen ◽  
Car T

The engineering of T cells through expression of chimeric antigen receptors (CARs) against tumor-associated antigens (TAAs) has shown significant potential for use as an anti-cancer therapeutic. The development of strategies for flexible and modular CAR T systems is accelerating, allowing for multiple antigen targeting, precise programming, and adaptable solutions in the field of cellular immunotherapy. Moving beyond the fixed antigen specificity of traditional CAR T systems, the modular CAR T technology splits the T cell signaling domains and the targeting elements through use of a switch molecule. The activity of CAR T cells depends on the presence of the switch, offering dose-titratable response and precise control over CAR T cells. In this review, we summarize developments in universal or modular CAR T strategies that expand on current CAR T systems and open the door for more customizable T cell activity.

scholarly journals A Deep Insight Into CAR-T Cell Therapy in Non-Hodgkin Lymphoma: Application, Opportunities, and Future Directions

2021 ◽  
Vol 12 ◽  
Author(s):  
Faroogh Marofi ◽  
Heshu Sulaiman Rahman ◽  
Muhammad Harun Achmad ◽  
Klunko Nataliya Sergeevna ◽  
Wanich Suksatan ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
B Cell ◽  
Molecular Mechanisms ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Anti Cancer ◽  
Car T

Non-Hodgkin’s lymphoma (NHL) is a cancer that starts in the lymphatic system. In NHL, the important part of the immune system, a type of white blood cells called lymphocytes become cancerous. NHL subtypes include marginal zone lymphoma, small lymphocytic lymphoma, follicular lymphoma (FL), and lymphoplasmacytic lymphoma. The disease can emerge in either aggressive or indolent form. 5-year survival duration after diagnosis is poor among patients with aggressive/relapsing form of NHL. Therefore, it is necessary to understand the molecular mechanisms of pathogenesis involved in NHL establishment and progression. In the next step, we can develop innovative therapies for NHL based on our knowledge in signaling pathways, surface antigens, and tumor milieu of NHL. In the recent few decades, several treatment solutions of NHL mainly based on targeted/directed therapies have been evaluated. These approaches include B-cell receptor (BCR) signaling inhibitors, immunomodulatory agents, monoclonal antibodies (mAbs), epigenetic modulators, Bcl-2 inhibitors, checkpoint inhibitors, and T-cell therapy. In recent years, methods based on T cell immunotherapy have been considered as a novel promising anti-cancer strategy in the treatment of various types of cancers, and particularly in blood cancers. These methods could significantly increase the capacity of the immune system to induce durable anti-cancer responses in patients with chemotherapy-resistant lymphoma. One of the promising therapy methods involved in the triumph of immunotherapy is the chimeric antigen receptor (CAR) T cells with dramatically improved killing activity against tumor cells. The CAR-T cell-based anti-cancer therapy targeting a pan–B-cell marker, CD19 is recently approved by the US Food and Drug Administration (FDA) for the treatment of chemotherapy-resistant B-cell NHL. In this review, we will discuss the structure, molecular mechanisms, results of clinical trials, and the toxicity of CAR-T cell-based therapies. Also, we will criticize the clinical aspects, the treatment considerations, and the challenges and possible drawbacks of the application of CAR-T cells in the treatment of NHL.

Redirection of Genetically Engineered CAR-T Cells Using Bifunctional Small Molecules

2015 ◽  
Vol 137 (8) ◽  
pp. 2832-2835 ◽  
Author(s):  
Min Soo Kim ◽  
Jennifer S. Y. Ma ◽  
Hwayoung Yun ◽  
Yu Cao ◽  
Ji Young Kim ◽  
...  
Keyword(s):  
T Cells ◽  
Small Molecules ◽  
Genetically Engineered ◽  
Car T Cells ◽  
Car T

scholarly journals Getting CD19 Into Shape: Expression of Natively Folded “Difficult-to- Express” CD19 for Staining and Stimulation of CAR-T Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
Elisabeth Lobner ◽  
Anna Wachernig ◽  
Venugopal Gudipati ◽  
Patrick Mayrhofer ◽  
Benjamin Salzer ◽  
...  
Keyword(s):  
T Cells ◽  
Car T Cells ◽  
Car T ◽  
Stimulation Of

TREM1/Dap12-based CAR-T cells show potent antitumor activity

Immunotherapy ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1043-1055 ◽  
Author(s):  
Bing Chen ◽  
Min Zhou ◽  
Hai Zhang ◽  
Chen Wang ◽  
Xiaocui Hu ◽  
...  
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Car T Cells ◽  
Car T ◽  
Clinical Benefits ◽  
Chimeric Immunoreceptor

Aim: Chimeric antigen receptor-engineered T (CAR-T) cells have gained huge success in treating hematological malignancies, yet the CD3ζ-based CAR-T therapies have not shown comparable clinical benefits in solid tumors. We designed an alternative chimeric immunoreceptor in which a single-chain variable fragment was fused to the transmembrane-cytoplasmic domains of triggering receptor expressed on myeloid (TREM1), which may show potent antitumor activity. Methods: To generate TREM1/DNAX activation protein of 12 kDa (Dap12)-based CAR-T cells, TREM1 along with DAP12 was transduced into T cells. Results: TREM1/Dap12-based CAR-T cells showed more lysis in vitro and a similar antitumor effect in mouse models compared with CD19BBζ CAR-T cells. Conclusion: In this study, we designed a TREM1/Dap12-based CAR, which was not reported previously and demonstrated that TREM1/Dap12-based CAR-T cells had potent antitumor activity in vitro and in vivo.

scholarly journals FT819: Translation of Off-the-Shelf TCR-Less Trac-1XX CAR-T Cells in Support of First-of-Kind Phase I Clinical Trial

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4434-4434
Author(s):  
Chiawei Chang ◽  
Sjoukje Van Der Stegen ◽  
Mandal Mili ◽  
Raedun Clarke ◽  
Yi-Shin Lai ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Cell Differentiation ◽  
Cancer Center ◽  
Antigen Specificity ◽  
Car T Cells ◽  
Differentiation Protocol ◽  
Car T

Long-term follow-up of adoptive transfer of autologous T cells expressing a chimeric antigen receptor (CAR) directed to CD19 antigen has demonstrated encouraging, durable clinical outcome in various B cell malignancies. However, to make such CAR-T cells available to a broader base and to reach a more diverse patient population, challenges associated with product consistency, cost of manufacture, precision genetic engineering and on-demand availability still need to be addressed. FT819 is a first-of-kind off-the-shelf CAR-T cell product candidate derived from a renewable master pluripotent cell line. FT819 comprises precise genetic engineering of multiple targeting events at the single cell level and is produced using a clonally-derived master cell bank (MCB) that serves as the starting material to support consistent and reproducible clinical manufacturing. The engineered features of FT819 include the targeted integration of a novel CD19 1XX-CAR into the T-cell receptor α constant (TRAC) locus to provide antigen specificity, enhanced efficacy and temporally-regulated CAR expression driven by an endogenous (TCR) promoter. Such features are designed to also eliminate the possibility of graft versus host disease (GvHD) by nullifying the TCR. To develop the MCB for FT819, αβ T cells were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently engineered to direct CD19 1XX-CAR into the TRAC locus with knockout of the TCR. To generate clonal lines, engineered iPSCs were sorted by flow cytometry for various markers and single cells were seeded into individual wells of feeder-free 96-well plates. Engineered iPSC clones were screened for integration of CAR into the TRAC locus by amplifying the genomic DNA flanking the homologous recombination site and confirmed by a SNP phasing assay. Clones were further screened for random integration of donor template by quantitative PCR and the CAR copy number was confirmed by droplet digital PCR. Genome stability of each clone was also confirmed by karyotype analysis. Overall, the described screening initiative surveyed 774 clones to select the ideal MCB for FT819. Utilizing our stage-specific T cell differentiation and expansion protocol, we demonstrated that T cells derived from the FT819 (FT819-iTs) expanded greater than 100,000-fold during the clinical manufacturing process and the cells expressed greater than 95% T lymphocyte markers such as CD45, CD7, intracellular CD3, and TRAC-regulated CAR. Further modifications to the T cell differentiation protocol resulted in enhanced expression of CD8 αβ from less than 25% to greater than 70% of the total population. In addition, expression of CD2, CD5, and CD27 was increased by approximately 5- to 20-fold. In vitro functional studies showed that FT819-iTs possess antigen specificity as confirmed by cytokine release and cytotoxic T lymphocytes (CTL) assays. Upon stimulation with a wild type acute lymphoblastic leukemia line, Nalm-6, FT819-iTs expressed 30% CD107a/b compared to 2% when stimulated by Nalm-6 CD19KO. In an in vitro CTL assay, greater than 80% of Nalm-6 WT cells were lysed with effector to target (E:T) ratio at 10:1 as compared to Nalm-6-CD19KO, which showed less than 10% lysis at the same E:T ratio. Finally, in an in vivo tumor model, FT819-iTs generated from our original and modified T cell differentiation protocols showed similar tumor burden control and prolonged survival rate when compared to primary CAR-T cells (days of survival >80days, p>0.1). In a more stringent in vivo model, FT819-iTs generated from the modified differentiation protocol demonstrated higher anti-tumor response and better animal survival rate compared to iTs from the original T cell differentiation protocol (Day 30 p<0.005). Small molecules are known to modulate cell functions and when treated with compound A, FT819-iTs further delayed tumor growth and increased anti-tumor potency when compared to DMSO treated group (Day 17, P<0.05). Collectively, the preclinical studies suggest that FT819 is a consistent and uniform off-the-shelf CAR T cell product candidate with the first-of-kind Phase 1 clinical trial for the treatment of B cell malignancies in an allogeneic setting study planned for 2020. Disclosures Chang: Fate Therapeutics: Employment. Van Der Stegen:Memorial Sloan Kettering Cancer Center: Employment. Mili:Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Lai:Fate Therapeutics: Employment. Witty:Fate Therapeutics: Employment. Lindenbergh:Memorial Sloan Kettering Cancer Center: Employment. Yang:Fate Therapeutics: Employment. Husain:Fate Therapeutics: Employment. Shaked:Fate Therapeutics: Employment. Groff:FATE THERAPEUTICS: Employment. Stokely:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Chu:Fate Therapeutics: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. ORourke:Fate Therapeutics: Employment. Gutierrez:Fate Therapeutics: Employment. Riviere:Juno Therapeutics: Consultancy, Equity Ownership, Research Funding; Fate Therapeutics: Consultancy; Memorial Sloan Kettering Cancer Center: Employment. Sadelain:Memorial Sloan Kettering Cancer Center: Employment; Fate Therapeutics: Consultancy, Patents & Royalties; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

scholarly journals Generation of Multiplexed Engineered, Off-the-Shelf CAR T Cells Uniformly Carrying Multiple Anti-Tumor Modalities to Prevent Tumor Relapse

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11
Author(s):  
Chiawei Chang ◽  
Eigen Peralta ◽  
Gloria Hsia ◽  
Bi-Huei Yang ◽  
Wen-I Yeh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fold Increase ◽  
Cell Receptor ◽  
Control Group ◽  
Antigen Specificity ◽  
Car T Cells ◽  
Car T ◽  
Specific Cytotoxicity

The development of chimeric antigen receptor (CAR) T cell therapeutics is widely recognized as a significant advancement for the treatment of cancer. However, several obstacles currently impede the broad use of CAR T cells, including the inherent process variability, cost of manufacturing, the absolute requirement for precise and uniform genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity and tumor growth. Utilizing our previously described induced pluripotent stem cell (iPSC)-derived T (iT) cell platform, we illustrate here the unique ability to address these challenges by creating a consistent CAR iT cell product that can be repeatedly manufactured in large quantities from a renewable iPSC master cell bank that has been engineered to mitigate the occurrence of graft versus host disease (GvHD), antigen escape and tumor relapse. Utilizing our proprietary cellular reprogramming and engineering platform and stage-specific T cell differentiation protocol, we demonstrate that iPSC can be engineered at the single cell level to generate a fully characterized clonal iPSC line, which can then be accessed routinely to yield CAR iT cells in a highly scalable manufacturing process (&gt;100,000 fold expansion). Through bi-allelic targeting of a CAR into the T cell receptor alpha constant (TRAC) region, we generated CAR iT cells with uniform CAR expression (99.0 ± 0.5% CAR+) and complete elimination of T cell receptor (TCR) expression to avoid GvHD in the allogeneic setting. We elected to utilize the 1XX-CAR configuration, which has demonstrated superior anti-tumor performance relative to other CAR designs and when introduced into iT cells displayed enhanced antigen specificity (% specific cytotoxicity at E:T=10:1, antigen positive group: 86.4 ± 7.8; antigen null group: 8.9 ± 3.5). To enhance persistence without reliance on exogenous cytokine support, we engineered signaling-fusion complexes, including IL-7 receptor fusion (RF), into iPSC and studied its impact on iT phenotype, persistence, and efficacy. In vitro, IL-7RF clones demonstrated improved anti-tumor activity in a serial antigen dependent tumor challenge assay (Day 10, relative tumor counts, IL-7RF group: 1.95 ± 0.01; control group: 57.56 ± 4.55, P&lt;0.000001). In a preclinical in vivo model of disseminated leukemia, IL-7RF clones demonstrate enhanced tumor growth inhibition (Day 34, Log [BLI], IL-7RF group: 6.68 ± 1.93; control group: 9.99 ± 0.23, P=0.0143). We next investigated a unique strategy to incorporate multi-antigen targeting potential into anti-CD19 1XX CAR iT cells with the addition of a high-affinity non-cleavable CD16 (hnCD16) Fc receptor. The combination of hnCD16 with anti-CD19 1XX CAR culminated in iT cells capable of multi-antigen specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. Utilizing CD19 negative leukemia cells as targets, superior antibody-dependent cellular cytotoxicity (ADCC) was demonstrated by the combination of hnCD16 CAR iT and Rituximab (% specific cytotoxicity at E:T=1:1, hnCD16 group + Rituximab: 75.64 ± 2.12; control group + Rituximab: 16.98 ± 3.87, P&lt;0.001). To address T cell fitness, the role of CD38 knockout (KO) in T cells was investigated, which we have previously shown to mediate NK cell resistance to oxidative stress induced apoptosis. CD38 gene was disrupted at the iPSC stage to generate 1XX-CAR T cells that lack CD38 expression (% CD38+ population, CD38WT group: 69.67 ± 24.34; CD38KO group: 0.12 ± 0.11) and upon antigen mediated stimulation, CD38KO CAR iT cells showed higher percentages of degranulation (2.3-fold increase in CD107a/b), and IFNγ (4.1-fold increase) and TNFα (2.5-fold increase) production. Antigen specific in vitro tumor killing also was enhanced in CD38KO CAR iT cells (EC50, 3.2-fold decrease). Lastly, to avoid the potential host-mediated rejection, the inclusion of allogeneic defense receptor (ADR) which has been shown to significantly reduce host-mediated rejection will be discussed. Collectively, the described studies demonstrate that iPSCs are an ideal cellular source to generate large-quantities of uniformly multi-edited off-the-shelf CAR T cell products that include a best-in-class CAR design, enhanced product modalities, and complete elimination of TCR expression to avoid the potential of GvHD while maintaining high anti-tumor efficacy in allogeneic setting. Disclosures Hsia: Fate Therapeutics Inc.: Current Employment. Clarke:Fate Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Lee:Fate Therapeutics, Inc.: Current Employment. Robbins:Fate Therapeutics, Inc.: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Hanok:Fate Therapeutics, Inc.: Current Employment. Carron:Fate Therapeutics, Inc.: Current Employment. Navarrete:Fate Therapeutics, Inc.: Current Employment. ORourke:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Gentile:Fate Therapeutics, Inc.: Current Employment. Nguyen:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

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