TRAC Gene Research Articles

Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression

Despite the remarkable success of CAR T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous IFN-γ promoter for transgenic IL-15 expression. We demonstrate that IFN-γ expression is tightly regulated by TCR signaling. By introducing IRES-IL15 into the 3’-UTR of the IFN-γ gene via HDR-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen-stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Lastly, as part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.

A first-in-human phase 1, multicenter, open-label study of CB-012, a next-generation CRISPR-edited allogeneic anti-CLL-1 CAR-T cell therapy for adults with relapsed/refractory acute myeloid leukemia (AMpLify).

TPS6586 Background: CAR-T cell therapies have shown significant clinical benefit in treating adults with many hematologic malignancies. In acute myeloid leukemia (AML), a challenge in the development of CAR-T cell therapies has been the limitation of suitable target antigens since many are also expressed on hematopoietic stem cells (HSCs). The target antigen C-type lectin-like molecule-1 (CLL-1) has emerged as an attractive therapeutic target due to its expression on AML mature blasts and leukemic stem cells and its absence on HSCs. CB-012 is an allogeneic CAR-T cell therapy that targets CLL-1. CB-012 is derived from healthy donor T cells by using Cas12a CRISPR hybrid RNA-DNA (chRDNA) technology to introduce 5 genome edits: (1) TRAC gene knockout (KO), which eliminates T cell receptor expression to reduce the risk of graft-versus-host disease, (2) site-directed insertion of a fully human anti-CLL-1 CAR into the TRAC locus, (3) KO of PD-1 to enhance antitumor activity by reducing T cell exhaustion, (4) β2-microgobulin ( B 2M) gene KO, which eliminates HLA class I presentation to mitigate host T cell-mediated rejection, and (5) site-directed insertion of a B2M–HLA-E-peptide fusion into the B 2M locus to reduce NK cell-mediated rejection. In murine xenograft models of AML, CB-012 significantly reduced the tumor burden and increased the survival of mice bearing CLL-1+PD-L1+ tumors. Methods: CB-012 is being evaluated in a multicenter, Phase 1 clinical trial in patients with relapsed/refractory (r/r) AML. A 3+3 dose escalation design is being utilized with the primary objectives to determine the safety and tolerability of CB-012 and the recommended Phase 2 dose (RP2D). An expansion phase will follow dose escalation at the recommended dose(s) for expansion. Additional objectives include preliminary antitumor activity and pharmacokinetics. Key inclusion criteria include nonproliferative disease defined as ≤25% blasts in the bone marrow and peripheral blood, ECOG performance status ≤1, and adequate organ function. After receiving lymphodepletion therapy with cyclophosphamide (750 mg/m2/d) and fludarabine (30 mg/m2/d) administered concurrently for 3 days followed by a 2-day break, patients receive a single-dose infusion of CB-012 and are followed for safety and efficacy. The dose escalation portion of the study is actively enrolling patients. The AMpLify trial is registered at clinicaltrials.gov (NCT06128044). Clinical trial information: NCT06128044 .

Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome.

Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype. To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence. In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments. Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies. We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro. This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.

BCMA/CD47-directed universal CAR-T cells exhibit excellent antitumor activity in multiple myeloma

BackgroundBCMA-directed autologous chimeric antigen receptor T (CAR-T) cells have shown excellent clinical efficacy in relapsed or refractory multiple myeloma (RRMM), however, the current preparation process for autologous CAR-T cells is complicated and costly. Moreover, the upregulation of CD47 expression has been observed in multiple myeloma, and anti-CD47 antibodies have shown remarkable results in clinical trials. Therefore, we focus on the development of BCMA/CD47-directed universal CAR-T (UCAR-T) cells to improve these limitations.MethodsIn this study, we employed phage display technology to screen nanobodies against BCMA and CD47 protein, and determined the characterization of nanobodies. Furthermore, we simultaneously disrupted the endogenous TRAC and B2M genes of T cells using CRISPR/Cas9 system to generate TCR and HLA double knock-out T cells, and developed BCMA/CD47-directed UCAR-T cells and detected the antitumor activity in vitro and in vivo.ResultsWe obtained fourteen and one specific nanobodies against BCMA and CD47 protein from the immunized VHH library, respectively. BCMA/CD47-directed UCAR-T cells exhibited superior CAR expression (89.13-98.03%), and effectively killing primary human MM cells and MM cell lines. BCMA/CD47-directed UCAR-T cells demonstrated excellent antitumor activity against MM and prolonged the survival of tumor-engrafted NCG mice in vivo.ConclusionsThis work demonstrated that BCMA/CD47-directed UCAR-T cells exhibited potent antitumor activity against MM in vitro and in vivo, which provides a potential strategy for the development of a novel “off-the-shelf” cellular immunotherapies for the treatment of multiple myeloma.Graphic

Abstract 3618: High-avidity BCMA CAR and high-affinity, non-cleavable CD16 Fc receptor incorporated in off-the-shelf CAR T cells promote multi-antigen targeting and durable anti-tumor cytotoxicity in the treatment of multiple myeloma

Abstract Immune cell therapy has proven highly effective for the treatment of multiple myeloma (MM). However, key challenges remain that include disease relapse, limited patient access, and inability to effectively combine with existing standard-of-care therapies. Rapid progress in the development of off-the-shelf, multiplexed-engineered, induced pluripotent stem cell (iPSC)-derived cell therapies enables large-scale manufacture of immune cells incorporating multiple novel synthetic controls of cell function to improve cell fitness, enhance cell function, and enable synergistic combination with existing effective therapies such as CD38-targeted antibody (mAb) therapy. We have developed an iPSC-derived chimeric antigen receptor T (CAR-iT) cell therapy that uniquely leverages elements of both adaptive and innate immunity by incorporating a BCMA-targeted CAR (BCMA-CAR) derived from a scFv domain exhibiting high-binding affinity in the low nanomolar range, and a high-affinity, non-cleavable CD16 (hnCD16) Fc receptor to enable antibody-dependent cellular cytotoxicity with mAb therapy. The genetic deletion of CD38 gene was also incorporated to eliminate the possibility for CD38-mediated fratricide, and genetic deletion of TRAC gene was introduced to remove the potential risk of graft-versus-host disease in an allogeneic setting. These CAR iT cells, which were generated from a clonally-derived iPSC line, demonstrated homogenous expression of each genetic edit (>95% BCMA-CAR and hnCD16; <1% CD38 and TCR surface expression). Using a stringent, disseminated xenograft mouse model of multiple myeloma, MM.1S, which was allowed to achieve complete systemic engraftment during the initial 4 days, treatment with BCMA-CAR iT cells demonstrated comparable tumor cell clearance (p=0.0024 vs. vehicle at Day 17) to primary BCMA-CAR T cells (p=0.002 vs. vehicle). In combination with a CD38-targeted mAb to exploit hnCD16 and biallelic CD38 KO, BCMA-CAR iT cells are capable of dual-antigen targeting to address antigen escape and promote durable tumor control. To this end, a single dose of BCMA-CAR iT cells combined with daratumumab exhibited near complete TGI for the duration of the study (p<0.0001 vs. vehicle at Day 37). Together, these studies demonstrate CAR iT cells incorporating a high-avidity BCMA CAR and high-affinity, non-cleavable CD16 Fc receptor can uniquely leverage elements of both adaptive and innate immunity and can be combined with CD38-targeted mAb to potentially outcompete primary BCMA CAR T cells. As these CAR iT cells can be administered off-the-shelf, key challenges associated with current immune cell therapy, such as patient access and inability to synergize with standard-of-care therapies, can be addressed for the treatment of relapsed/refractory MM. Citation Format: John Reiser, Alison O'Connor, Bryan Hancock, Spas Markov, Brian Groff, Alma Gutierrez, Miguel Meza, Mark Jelcik, Yijia Pan, Alex Garcia, Bobby Goulding, Matthew Denholtz, Tom Lee, Ramzey Abujarour, Ryan Bjordahl, Armin Rehm, Raedun Clarke, Jode Goodridge, Bahram Valamehr. High-avidity BCMA CAR and high-affinity, non-cleavable CD16 Fc receptor incorporated in off-the-shelf CAR T cells promote multi-antigen targeting and durable anti-tumor cytotoxicity in the treatment of multiple myeloma [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 3618.

Towards Automated Engineering of Donor-Derived T Lymphocytes into CRISPR/Cas9-Mediated CAR T Cells in a Closed-System

Chimeric antigen receptor (CAR)-expressing T cells have revolutionized immunotherapy of cancer. However, some patients derive no benefit from approved CARTcell therapies due to limitations such as toxicity and use of autologous cells which are sometimes unfit and risk manufacturing failures. Additional or more effective CARTcell therapies are needed. Harnessing the endogenous homology-directed DNA repair pathway via precision genome editing based on Cas9RNP/rAAV6, we and others have demonstrated the feasibility of targeting transgene(s) integration into specific gene site(s) including their incorporation under endogenous regulatory elements. Using this strategy, we aimed to engineer healthy donor-derived CAR T cells from αβ+ T lymphocytes. For enhanced safety, we metabolically reprogrammed the CAR T cells by inactivating the UMPS gene needed for uridine biosynthesis. The UMPS-/-CAR T cells become dependent on exogenous uridine thereby serving as a cell control mechanism. We hypothesized that donor-derived αβ+ T lymphocytes can be engineered using the Cas9RNP-rAAV6 strategy in an automated closed-system into controllable CAR T cells. We inactivated TRAC and UMPS gene loci in αβ+T lymphocytes using Lonza4D nucleofector. Electroporated cells were simultaneously transduced with CD19CAR-containing rAAV6 DNA vector. We obtained TRAC gene knockout up to 97% with knock-in up to 89%. Disruption of the UMPS gene would transform the cells to being auxotrophic and depend on uridine supply for viability and growth. To evaluate the CART cells' antitumor potency, we cocultured the CD19CART cells with GFP-expressing Nalm6 leukemia cell lines. The CD19CART cells eradicated the leukemia cells within 72 hours. To mimic relapse, we re-challenged cocultured CART with Nalm6 cells and the tumor cells were again depleted within 72 hours. Expectedly, IL-2 production was elevated in CD19CART cells challenged with Nalm6. Next, we tested the ability to control the cytotoxicity of the CD19CART cells by coculturing them with Nalm6 cells in separate groups with/without exogenous uridine. Interestingly, the CD19CART cells cocultured without uridine eradicated the target cells within 72 hours but lost their potency upon subsequent challenge (Figure 1). In contrast, CD19CAR T cells supplied with uridine remained potent and repeatedly eradicated target cells indicating the ability to turn off the potency of CART cells with uridine supply/withdrawal. To establish an automated closed-system for CART cells, we adopted the Miltenyi CliniMACS Prodigy with in-line electroporator (Elpo) for process engineering. First, we determined optimized parameters for electroporating αβ+ T lymphocytes on Elpo(Table1). Cells electroporated with these settings were (>80% viability) comparable to untreated cells. We first engineered CART cells with Elpo/Miltenyi reagents in an open system(i.e.cultivated Elpo-electroporated cells in culture vessels/incubators). We obtained 95% of TRAC and UMPS gene knockouts and up to 90% CD19CAR transgene knock-in to the TRAC locus - an efficiency slightly higher than using Lonza 4D nucleofector. To scale up the Cas9-based T cell engineering into a single automated process on the CliniMACS Prodigy, the Cas9 and guide RNA reagents would be scaled up 1000fold. To make the process cost-effective, we tested AZD7468, a small molecule DNAPKcs inhibitor. We optimized/reduced the amounts of reagents upto 90% less of Cas9RNP used without any significant reduction in gene editing outcomes. AZD7648 reduced the MOI needed for viral transduction by 50% while maintaining 80% or higher levels of transgene knock-in efficiency. Next, we commenced pilot studies to automate the CAR T cell engineering process in closed-system. Currently, we have achieved an automated TRAC gene knockout of over 90% and CAR transgene knock-in of slightly >10%. Further optimization studies are underway to improve automated gene knock-in efficiency. We have engineered donor-derived αβ+ T lymphocytes into potent/controllable CD19CART cells. The UMPS-deficient CD19CART cells lose antileukemia activity without uridine. We show that metabolic reprogramming can be combined with precision genome editing to generate controllable CART cells. Finally, our data demonstrate the feasibility of automating CRISPR/Cas9-mediated engineering of CAR T cells within closed-system.

Identification of Healthy Donor Phenotypic and Functional Signatures That Predict Allogeneic, Hypoimmune CD19-Directed CAR T Cell Potency

Allogeneic, hypoimmune (HIP) CAR T cells may provide off-the-shelf CAR T cells that allow for expanded access for patients, overcome manufacturing failures, and provide an efficacious alternative to CAR T cells manufactured from heavily pre-treated T cells. However, donor T cells are the primary source of variability during production of allogeneic HIP CAR T cells, and may influence clinical efficacy. Previously, we reported a series of T cell functional assays that differentiate T cell potency as a means to select high-quality CAR T cell donors for clinical trials. Here, we explore donor phenotypic and functional signatures that are associated with desirable pre-clinical attributes for the generation of allogeneic HIP CD19-directed CAR T cells. Human T cells from healthy donors were obtained by leukapheresis and used to generate allogeneic HIP CD19-directed CAR T cells by disrupting B2M, CIITA, and TRAC genes using Cas12b nuclease, and lentiviral transduction to overexpress CD47 and to express the CD19 CAR.Phenotype profiling (PBMC subsets, memory subsets, viability, exhaustion, and activation markers) was assessed on apheresis, CD4 or CD8 enriched subsets and post-CAR manufacturing. Generated CAR T cells were evaluated in vitro by repetitive tumor challenge (3x) performed over a 14-day period using the IncuCyte platform. Bulk cytokine analysis was measured by MesoScale Discovery (MSD). In vivo efficacy was evaluated using a systemic NALM-6 challenge in immune-deficient NSG mice at HIP CD19 CAR T cell doses of 5e5 and 5e6 cells per animal. Gene expression profiling was assessed on pre-production, post-CAR T cell manufacturing, and post-serial tumor challenge CD4+ and CD8+ T cells using the Nanostring nCounter CAR T panel. Functional and phenotypic attributes were used for weighted donor performance scoring. To do this, cell functional assays were defined to produce single, quantitative indices to enable the classification of CAR T cells based on functional performance. We assessed differences in CAR T cell performance and rank scored donor performance. Associations between donor attributes, phenotype/ functional T cell attributes and ranked donor performance were evaluated using Spearman's correlations. Correlative comparison of phenotype and donor attributes in relation to weighted donor performance scores indicated that cellular subtypes at leukapheresis correlate with CAR T performance. Gene expression profiling shows that outperforming donors upregulate expression of mucosal associated invariant T (MAIT) cell canonical TCRb chain TRBV28. Outperforming donors have increased expression of Tc1/Th1 and Tc17/Th17 associated cytokines: IFNg, Il-17A, Il-17F, Il23, Il22, and have reduced expression of Tc2/Th2 associated cytokine Il5. Outperforming donors also showed reduced expression of MHC class II of alpha and beta heterodimers (HLA-DQA1 and HLA-DQB1). The information collected across phenotypic and functional comparisons will be compared with clinical performance.

Universal Anti-CD7 CAR-T Cells Targeting T-ALL and Functional Analysis of CD7 Antigen on T/CAR-T Cells.

Chimeric antigen receptor T (CAR-T) cell therapy initiates new methods and turns the scale of clinical treatment on relapsed/refractory acute T lymphoblastic leukemia (T-ALL). In this study, we generated the second-generation CD7-targeting CAR-T cells with a new antigen-binding single-chain variable fragment sequence and made it universal via CRISPR-based knockout of TRAC and CD7 genes (termed UCAR-T). The CD7 UCAR-T cells can efficiently proliferate and lyse T-ALL tumor cell in vitro, along with prominent proinflammatory cytokines secretion. A Jurkat-based xenograft mouse model further verified the superior cytotoxicity of the UCAR-T cells in vivo. During the UCAR-T construction, we observed a CD4/CD8 ratio shift among CD7-/- T/CAR-T cells, which motivated us to further analyze the effects of CD7 antigen on T/CAR-T cells. We sorted out CD7+/- T or anti-CD19 CAR-T cells after partially CD7 knockout and performed functional, phenotypic detection, as well as translational analysis. CD7-/- CAR-T cells tended to be CD8 negative and showed slightly better cytotoxicity at long-term assay. RNA-seq further confirmed an elevation of activated CD4 memory cell subpopulation. However, limited distinction on crucial regulatory genes and pathways was revealed, suggesting the safety and feasibility of UCAR-T application as well as the potential translational rather than transcriptional regulation of CD7 antigen.

CLL-1-Targeted Allogeneic CAR-T Cells Exhibit High on-Target Specificity and Potent Cytotoxicity in Preclinical Models of Acute Myeloid Leukemia

Background: CLL-1 is a compelling therapeutic target for acute myeloid leukemia (AML) as it is highly expressed on AML tumor cells and leukemic stem cells, but is not expressed on hematopoietic stem cells. An allogeneic anti-CLL-1 CAR-T cell therapy (CB-012) is in development for relapsed or refractory (r/r) AML. The CAR was generated with a fully human scFv targeting CLL-1 that was selected from a panel of scFvs. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology to leverage both checkpoint disruption and immune cloaking for potentially improved antitumor activity. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to reduce graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding to prolong antitumor activity. This multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012. In vitro and in vivo studies evaluated specificity for antigen binding, antigen-dependent activity, and preclinical safety assessments. Results: CB-012 CAR-T cells express a fully human anti-CLL-1 scFv-containing CAR construct and demonstrate potent antigen-dependent cytotoxic activity in human AML cell line co-cultures. In AML xenograft models, a single-dose administration of CB-012 CAR-T cells exerted robust tumor control, leading to significant prolongation of survival. Cell binding studies suggested that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity when examined in the context of a collection of cell types representing vital tissues. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated specific interaction with human CLL-1, without detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised mouse models. Conclusion: CB-012 demonstrated highly specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was further demonstrated by screening in an unbiased protein-binding study and no adverse safety signals were observed in murine models. These data support advancing the development of CB-012 into a first-in-human clinical trial for patients with r/r AML.

Hypoimmune, Allogeneic CD22-Directed CAR T Cells That Evade Innate and Adaptive Immune Rejection for the Treatment of Large B Cell Lymphoma Patients That Are Relapsed/Refractory to CD19-Directed CAR T Cell Therapy

Despite the success of CD19CAR therapy against large B-cell lymphoma, up to 60% of CD19-directed CAR T cell treated patients relapse after, or are refractory to, treatment. Of the relapsed/refractory population a significant subset of patients, around 30%, have CD19-negative tumor and a short median survival prognosis - necessitating the implementation of rapid intervention strategies (Atilla, Transl Oncol 2022; Spiegel, Blood 2021). Sana is developing “off-the shelf” allogeneic CD22-directed CAR T cells that may enable rapid intervention and induce clinically meaningful activity for these CD19 CAR T cell relapsed/refractory patients. Here we describe methods to create hypoimmune (HIP), allogeneic CD22-directed CAR T cells using a clinically validated CD22CAR that: 1) support adaptive immune cell evasion via B2M and CIITA gene disruption, 2) promote persistence and innate immune cell evasion by overexpression of CD47, and 3) mitigate GvHD via TRAC gene disruption, (Hu, Nat Commun 2023). HIP, allogeneic CD22-directed CAR T cells (CD22 HIP CAR T cells) were derived from healthy donor CD8 + and CD4 + T cells that were gene-edited to disrupt the B2M, CIITA, and TRAC (TCR) genes and transduced to overexpress CD47 and express a second-generation, clinically validated CD22-directed CAR. Gene-edited Mock T cells and unedited, allogeneic CD47-CD22CAR T cells were generated as donor-matched controls. The CD22 HIP CAR T cells were evaluated for immune evasive activity using a previously described in vitro xCelligence cytotoxicity assay (Hu, Nat Commun 2023). The T cells were further evaluated in vitro for dose-dependent short-term and long-term cytolytic activity analysis via luciferase or IncuCyte assay, respectively. Correlate cytokine responses were performed by meso-scale discovery analysis and T cell expansion by IncuCyte classification. Systemic NALM and RAJI parental or CD19 antigen knockout tumor models (IV injection) were executed in a NSG or NSG-SGM3 mouse background. xCelligence analysis confirmed that CD22 HIP CAR T cells were protected from NK and macrophage recognition; whereas Mock control T cells that lack HLA expression succumbed to NK cell and macrophage killing within 5-hours of the 50-hour assay due to the absence of CD47 protection. Furthermore, CD22 HIP CAR T cells elicited cytotoxicity and robust cytokine responses (e.g. IFNγ, IL-2) in a dose-dependent manner comparable to allogeneic CD47-CD22CAR T cells when cultured with CD22 +/CD19 + or CD22 +/CD19 - NALM, RAJI, or K562 targets. The CD22 HIP CAR T cell and allogeneic CD47-CD22CAR T cell control produced low level IFNγ (<400pg/mL) and Granzyme B (<100pg/mL) when cultured alone or with CD22 - tumor targets. These basal levels of IFNγ and Granzyme B did not have long-term cytolytic effects on CD22 - tumor cells, nor was any significant level of IL-2 produced by CD22 HIP CAR T cells cultured alone, or in the presence of CD22-negative tumor targets. Indeed, CD22 HIP CAR T cell proliferation was dependent upon CD22 + tumor co-culture. NALM and RAJI CD19 knockout in vivo tumor models demonstrated that CD22 HIP CAR T cells significantly reduced flux (Day 21 AUC vs Mock: NALM and RAJI (p=0.0001)) and prolonged survival (Median vs Mock: p<0.004). Furthermore, CD22 HIP CAR T cells elicited comparable, dose-dependent antitumor activity relative to allogeneic CD47-CD22CAR T cell controls in a systemic NALM tumor model. Our data provide evidence that CD22 HIP CAR T cells elicit robust antitumor activity in an antigen and dose-dependent manner. Furthermore, these studies demonstrate that CD22 HIP CAR T cells are agnostic to CD19 expression and selectively elicit pharmacologic activity against, or in response to, CD22 + tumor. Results also demonstrate that the HIP approach to generate allogeneic CAR T cells that evade innate and adaptive immune rejection can be expanded as a platform to generate CAR T cells against target antigens alternative to CD19. Collectively, the data suggest that CD22 HIP CAR T cells display a combination of antigen-specific pharmacologic activity and immune evasion that support their progression into human clinical studies for the treatment of CD19 CAR T cell-refractory patients.

Preliminary Results of Nathali-01: A First-in-Human Phase I/IIa Study of UCART20x22, a Dual Allogeneic CAR-T Cell Product Targeting CD20 and CD22, in Relapsed or Refractory (R/R) Non-Hodgkin Lymphoma (NHL)

Background: UCART20x22 is a dual targeted allogeneic CAR T-cell product candidate generated from healthy donor T-cells. Donor-derived T-cells are transduced with a bicistronic lentiviral vector to express CARs targeting CD20 and CD22. TALEN ® gene editing technology is used to inactivate the TRAC and CD52 genes to minimize graft-versus-host disease (GvHD) and allow for the use of alemtuzumab (CLLS52, an anti-CD52 monoclonal antibody) in the lymphodepletion (LD) regimen, respectively. UCART20x22 previously demonstrated robust and sustained activity in vitro and in vivo (Aranda-Orgilles B, et al. Cancer Immunol Res 2023). Here we present the initial preliminary results from the NatHaLi-01 trial (NCT05607420), the first-in-human Phase 1/2a dose-finding and expansion study evaluating UCART20x22 in R/R B-cell NHL. Methods: The primary endpoints are safety, tolerability, and determining the recommended phase 2 dose of UCART20x22. Additional endpoints include anti-lymphoma activity and cellular kinetics of UCART20x22. Key eligibility criteria are age 18-80y, lymphoma expression of CD20 and/or CD22 by local assessment, and ≥ 2 prior treatment regimens including autologous CD19 CAR T-cell therapy (CAR19), if eligible. After LD with FCA (fludarabine 30 mg/m 2 × 3d, cyclophosphamide 0.5g/m 2 × 3d, CLLS52 12 mg on D1, 24 mg on D2, D3), a single infusion of UCART20x22 is administered at a flat dose level ([DL1] 50 x 10 6 cells; [DL2] 150 x 10 6 cells; and [DL3] 450 x 10 6 cells). Results: As of 01 July 2023, 3 pts were enrolled and treated at DL1. Pt 1 is a 76yo female with double-expressor diffuse large B-cell lymphoma (DLBCL) relapsed after R-CHOP, radiation therapy, and polatuzumab vedotin with bendamustine/rituximab (BR) and was considered ineligible for CAR19 due to recent high dose bendamustine. Pt 2 is a 65yo female with R/R triple-hit lymphoma transformed from follicular lymphoma who was previously treated with radiation therapy, BR, dose-adjusted R-EPOCH, and two separate CAR19 treatments. Pt 3 is an 18yo female with R/R DLBCL transformed from marginal zone lymphoma who previously received chemoimmunotherapy, venetoclax, ibrutinib, BR, CAR19, obinutuzumab, glofitamab, tafasitamab, lenalidomide, and an experimental epigenetic modifier. Cytokine release syndrome (CRS) occurred in all patients. Pt 1 had G1 CRS and received tocilizumab (toci) x3 and dexamethasone (dex) x1. Pt 2 had G2 CRS and received toci and dex x1. Pt 3 had G1 CRS and received toci x1. All pts' CRS resolved with treatment. No immune effector cell associated neurotoxicity (ICANS) or GvHD was observed. There were no UCART20x22 dose limiting toxicities (DLTs), and there was 1 CLLS52 SAE and 1 DLT. The SAE was G3 CMV reactivation and was managed with IV antivirals. The DLT was in Pt 2, who entered the study with G1/2 cytopenias and a hypocellular bone marrow and had prolonged bone marrow aplasia after treatment thought to be due to cumulative chemotherapy exposure. This pt proceeded to allogeneic hematopoietic stem cell transplant (HSCT) from a matched donor while in complete remission from UCART20x22 treatment. Responses were assessed per Lugano criteria beginning on D28. At D28, all 3 pts responded with 2 CRs and 1 PR. Host lymphocytes remained suppressed through D28 for all pts. UCART20x22 expansion was observed by flow cytometry in 3/3 pts and was initially detected on D7 followed by peaks between D9-14. Peak expansion ranged from 16-682 cells/μL and was predominantly CD8 T-cells. Ferritin levels and increases in serum cytokines, including IFNγ, TNFα, IL-2, and IL-6, correlated closely with UCART20x22 expansion. Summary: Treatment with UCART20x22 at DL1 has been safe and well-tolerated, with no UCART20x22 DLTs or ICANS, and CRS was <G3 in all pts. There was 1 DLT of prolonged bone marrow aplasia in a pt with impaired hematopoietic reserve at study entry. UCART20x22 was active at DL1, with all 3 pts responding, including 2 CRs in pts who had previously received CAR19. UCART20x22 expansion was observed in all pts. These initial data support the continued study of UCART20x22 in R/R NHL, and updated results will be presented.

NATHALI‐01: A PHASE 1/2A TRIAL OF UCART20X22, AN ALLOGENEIC DUAL CAR T‐CELL THERAPY FOR PATIENTS WITH RELAPSED/REFRACTORY B‐CELL NON‐HODGKIN LYMPHOMA (NHL)

Introduction: Autologous CAR T-cell therapies have been transformative in the treatment of selected blood cancers. Despite this remarkable success, long term studies on patients treated with CD19 or CD22 CAR T-cells revealed the emergence of relapses often due to antigen loss. The therapeutic options after CAR T-cell relapses are limited, underscoring the urgent need to develop novel therapies to improve current survival rates. In addition, there is a need to develop allogeneic “off-the-shelf” therapies that are readily available at the time of treatment decision and that overcome the development limitations of current autologous approaches. To address these challenges, UCART20x22, the first allogeneic dual CAR T-cell product candidate targeting two well-validated antigens in B-cell malignancies, CD20 and CD22, was generated from normal healthy donor T cells using a bicistronic lentiviral construct to express both CARs. TALEN® gene editing technology was used to inactivate the TRAC and CD52 genes to both minimize Graft-vs-Host Disease and allow for the use of alemtuzumab (an anti-CD52 monoclonal antibody) in the lymphodepleting regimen. UCART20x22 displays strong activity against tumor cell lines with diverse CD20/CD22 antigen combinations, as well as increased activity against cells presenting both targets simultaneously using in vitro cytotoxic and proliferation assays. The activity of UCART20x22 cells persists against tumor cells expressing both antigens (CD20, CD22) or only one. In a pre-clinical in vivo model carrying subcutaneous lymphoma tumors expressing different antigen combinations in a single mouse, UCART20x22 cells provide efficient in vivo clearance of tumor cells expressing one or two antigens in a dose dependent manner. Furthermore, in primary NHL patient samples expressing diverse CD22 and CD20 antigen levels, UCART20x22 displays robust and specific cytotoxic activity and IFN-γ release in all tested combinations. Methods: NatHaLi-01 (NCT05607420) is a Phase1/2a open-label dose-finding and dose-expansion study to evaluate the safety, expansion, persistence, and clinical activity of UCART20x22 in subjects with relapsed or refractory B-Cell NHL. Primary endpoints are safety, tolerability, and determining the MTD/RP2D of UCART20x22. Additional endpoints are anti-lymphoma activity and describing the expansion and trafficking of UCART20x22. Eligibility criteria include age 18‒80y, lymphoma cell expression of either or both CD20 and CD22, and ≥ 2 prior treatment regimens including autologous CD19 CAR T-cell therapy if eligible. After lymphodepletion with FCA (fludarabine 30 mg/m2 × 3d, cyclophosphamide 0.5g/m2 × 3d, alemtuzumab 12 mg on D1, 24 mg on D2, D3), patients will receive a single infusion of UCART20x22 at a flat dose level ([DL]; DL1-50 × 106 cells, DL2-150 × 106 cells, and DL3-450x106 cells). The trial is currently open for enrollment. The research was funded by: Cellectis, S.A. Keywords: aggressive B-cell non-Hodgkin lymphoma, cellular therapies, ongoing trials Conflicts of interests pertinent to the abstract. E. Bachy Honoraria: Roche, Takeda, Kite/Gilead, BMS, Novartis, Pfizer, Incyte, ADC Therapeutics Research funding: Amgen, BMS Other remuneration: Kite/Gilead, BMS, Novartis, Pfizer A. Ramakrishnan Consultant or advisory role Novartis, AztraZeneca, Genmab, Janssen A. Alfonso Consultant or advisory role BMS, Syros, Jazz Pharma, Astellas Honoraria: Novartis, BMS, Abbvie, Jazz Pharma, Janssen Research funding: Astra Zeneca P. A. Riedell Consultant or advisory role AbbVie, Genmab, ADC Therapeutics, Pharmacyclics, Novartis, BMS, Kite/Gilead, Nurix Therapeutics, Nektar Therapeutics, Takeda, Intellia Therapeutics, Sana Biotechnology, BeiGene, Janssen, and CVS Caremark Honoraria: Novartis Research funding: BMS, Kite/Gilead, Novartis, MorphoSys, CRISPR Therapeutics, Calibr, Xencor, Fate Therapeutics, Roche, and Tessa Therapeutics Other remuneration: Kite Pharma G. Cartron Consultant or advisory role Roche, BMS, Abbvie, Onwards therapeutics, MedXcell, MabQi Honoraria: Roche, BMS, Jansen, Gilead, Novartis, Abbvie Educational grants: Roche P. Barba Honoraria: Allogene, Amgen, BMS, Kit/Gilead, Incyte, Jazz Pharmaceuticals, Miltenyi Biomedicine, Nektar Novartis and Pierre Fabre S. Meadows Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. A. LaCroce Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. D. Thomas Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. C. Poirot Employment or leadership position: Cellectis, S.A. Stock ownership: Cellectis, S.A. K. J. Newhall Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. D. J. Lee Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. M. G. Frattini Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. J. Abramson Consultant or advisory role AbbVie, Astra-Zeneca, BeiGene, BMS, Caribou Biosciences, Cellectar, Century Therapeutics, Epizyme, Genentech, Genmab, Incyte, Interius, Janssen, Kite Pharma, Kymera, Lilly, Morphosys, Mustang Bio, Ono Pharma, Regeneron, Takeda

Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

Efficient and Specific Multi-Locus Editing of Allogeneic CAR T Cells for Hypoimmunity during Large Scale Manufacture Using Cas12b

Introduction: Allogeneic CAR T cell therapies may offer key advantages over autologous therapies including choice of starting cells, better manufacturing control, and lower cost. However, the manufacture of these cells imposes a high demand on genome engineering specificity and efficiency, given the dual needs for product safety and yield. This challenge is amplified for newer generations of allogeneic CAR T cell designs that may offer improved performance but require more edits to produce, which may increase the potential of unintended genome modification and insufficient yield of fully engineered product. Here we have tested a novel editing platform, Cas12b, for the ability to mediate efficient and specific disruption of three genes (B2M, CIITA, and TRAC) during large scale manufacturing of CAR T cells with immune evasive properties and abrogated graft-vs-host response. We have characterized the resulting cell product for on-target modification efficiency, type and levels of unintended genome modification, and potency for tumor clearance in a Nalm6 humanized mouse model, with the aim of demonstrating suitability for translation to human studies. Methods: Our manufacturing strategy to generate hypoimmune CAR T cells introduces five genetic modifications, using Cas12b for disruption of B2M, CIITA, and TCR expression, and lentiviral transduction to overexpress CD47 and introduce a CD19 CAR. T cells obtained from healthy donors were first transduced with lentiviral vector and then edited simultaneously to disrupt the B2M, CIITA and TRAC genes via electroporation of Cas12b mRNA and appropriate guides. Edited and purified cells were characterized via flow cytometry for efficiency of each genetic modification. Cells were further characterized for knockout levels via next generation sequencing (NGS) as well as vector copy number per diploid genome (VCN). Cells were characterized for viability, proliferation, and efficacy in a Nalm6 mouse model of tumor clearance. An initial assessment of chromosomal translocations was performed by G banding karyotype analysis. To assess editing specificity, each Cas12b guide was evaluated by Cas-OFFinder and an unbiased oligonucleotide duplex capture assay in T cells from multiple donors under conditions of high on-target modification. Candidate off-target sites were then examined for evidence of cleavage via PCR amplification from the manufactured cell product followed by NGS. Results: Our manufacturing process yielded clinically relevant scales of cell product having > 85% B2M knockout, > 90% CIITA knockout, and < 1% of residual TCR expressing cells. VCN levels were < 5. Cell viability and proliferation were minimally impacted by the gene editing process. Cells exhibited robust anti-tumor properties with doses of 7 x 106 hypoimmune CD19 CAR T cells (per animal) providing efficient killing in a Nalm6 mouse model. Initial karyotype analysis identified low levels of translocation between on-target sites with overall translocation levels otherwise not significantly affected compared to unedited cells. Our characterization of Cas12b in edited T cells across multiple donors revealed high specificity, consistent with previous reports (Nat Commun 10 212), with studies to date having yielded no evidence of cleavage at any candidate off-target site. Summary: Cas12b-mediated multi-locus editing of allogeneic CAR T cells during large scale manufacture resulted in a high yield product with potent tumor clearance capabilities. Initial analysis of editing specificity across multiple donors and engineering runs identified no off-target sites, and early karyotype assessments indicated an overall level of chromosomal translocations consistent with prior experiences in the field. In companion in vivo studies, these hypoimmune CD19 CAR T cells have been shown to efficiently evade host immune response (see posters by Hu and Duback). Together, these data demonstrate a profile for potency, genome integrity, and manufacturing yield that supports progressing these cells into human studies.

A First-in-Human Phase 1, Multicenter, Open-Label Study of CB-010, a Next-Generation CRISPR-Edited Allogeneic Anti-CD19 CAR-T Cell Therapy with a PD-1 Knockout, in Patients with Relapsed/Refractory B Cell Non-Hodgkin Lymphoma (ANTLER Study)

Background: Autologous CAR-T cell therapies have shown significant benefit in the treatment of adults with relapsed/refractory B cell non-Hodgkin lymphoma (r/r B-NHL). However, these CAR-T cell therapies have inherent challenges including functional deficiencies in the patient's T cells that could yield an inconsistent or impaired product. In addition, the extended time and complexity in manufacturing the product can limit patient access and potentially require bridging therapy. CB-010 is an allogeneic, off-the-shelf anti-CD19 CAR-T cell therapy derived from healthy donor T cells; it is the lead program in Caribou's allogeneic CAR-T cell therapy platform. Patients (pts) receiving CB-010 do not require leukapheresis or bridging therapy. A next-generation CRISPR-Cas9 genome-editing technology developed at Caribou, which uses Cas9 and CRISPR hybrid RNA-DNA (chRDNA) guides to significantly reduce off-target editing, was implemented to generate 3 genome edits in the manufacture of CB-010: (i) knockout of the TRAC gene to eliminate TCR expression to reduce the risk of graft-versus-host disease (GvHD), (ii)site-specific insertion of a CD19-specific CAR into the TRAC locus, and (iii) knockout of the gene encoding PD-1, designed to limit premature CAR-T cell exhaustion and enhance antitumor activity. Statistically significant preclinical survival benefit across B-NHL subtypes and longer-duration efficacy in vivo compared to similar CAR-T cells without a PD-1 knockout support the clinical evaluation of CB-010. Methods and Study Design: CB-010 is being investigated in a multicenter, Phase 1 clinical trial in pts with r/r B-NHL. A 3+3 dose escalation design followed by expansion at the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) is being utilized. Primary objectives are to determine the safety and tolerability of CB-010 and the RP2D. Additional key objectives include preliminary antitumor activity and pharmacokinetics (PK). After serially receiving lymphodepletion therapy with cyclophosphamide (60mg/kg/day for 2 days) and fludarabine (25mg/m2/day for 5 days), pts receive a single dose infusion of CB-010 and are followed for safety and efficacy. Initial antitumor activity, as well as time to progression, overall survival, and disease response as measured by Cheson criteria will be evaluated. The study is actively enrolling patients. Cohort 1 at an initial single dose of 40 million CAR-T cells has been completed successfully with 6 patients. Dose escalation of CB-010 is ongoing, and patients are being enrolled in cohort 2 to receive a single dose of 80 million CAR-T cells. The ANTLER Phase 1 trial is registered on clinicaltrials.gov (NCT04637763).

Abstract 551: UCART20x22: First allogeneic dual CAR T-cell therapy for the treatment of B-cell malignancies

Abstract Autologous CAR T-cell therapies have been transformative in the treatment of selected blood cancers. Despite this remarkable success, common mechanisms of resistance, such as tumor antigen escape, tumor heterogeneity and weaker CAR activity with low levels of antigen, emphasize the need to further optimize CAR T-cell therapies. In addition, there is a need to develop allogeneic “off-the-shelf” therapies that are readily available at the time of treatment decision. To address these challenges, we generated UCART20x22, the first allogeneic dual CAR T-cell targeting two well-validated antigens in B-cell malignancies, CD20 and CD22. Using in vitro cytotoxic and proliferation assays, we demonstrated that UCART20x22 displays strong activity against tumor cell lines with diverse CD20/CD22 antigen combinations, as well as increased activity against cells presenting both targets simultaneously. The specific activity of dual UCART20x22-cells persists overtime against tumor cells expressing both antigens (CD20, CD22) or only one. We also developed a pre-clinical model carrying subcutaneous lymphoma tumors expressing different antigen combinations in one single mouse. In this model, dual CAR T-cells provide efficient in vivo clearance of tumor cells expressing one or two antigens (CD20 and/or CD22) in a dose dependent manner, starting at a low dose of 1x106 CAR T-cells. Using a disseminated model of lymphoma with different combinations of antigen-expressing tumor cells, we demonstrated robust efficacy and persistence of UCART20x22 cells in the bone marrow. Furthermore, we used primary Non-Hodgkin Lymphoma patient samples expressing diverse CD22 and CD20 antigen levels to demonstrate that UCART20x22 displays robust and specific cytotoxic activity as well as IFNγ release in all tested combinations. Besides efficiently targeting two commonly expressed antigens in B-cell malignancies, UCART20x22 incorporates TALEN® mediated TRAC and CD52 specific gene editing to prevent Graft-vs-Host Disease and improve persistence in the presence of alemtuzumab (an anti-CD52 monoclonal antibody that can be used as part of a lymphodepleting regimen). These attributes allow the production of allogeneic CAR T-cells from healthy individuals that can be administered at the time of treatment decision. In summary, we show an efficient first in class allogeneic dual CAR T-cell product candidate with demonstrated in vitro and in vivo properties to overcome antigen escape in B-cell malignancies. Citation Format: Beatriz Aranda-Orgilles, Isabelle Chion-Sotinel, Steven Grudman, Ben Mumford, Chantel Dixon, Roman Galetto, Agnes Gouble, Laurent Poirot. UCART20x22: First allogeneic dual CAR T-cell therapy for the treatment of B-cell malignancies [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 551.

Use of traC Gene to Type the Incidence and Distribution of pXFAS_5235 Plasmid-Bearing Strains of Xylella fastidiosa subsp. fastidiosa ST1 in Spain.

Xylella fastidiosa (Xf) is a phytopathogenic bacterium with a repertoire of self-replicating genetic elements, including plasmids, pathogenicity islands, and prophages. These elements provide potential avenues for horizontal gene transfer both within and between species and have the ability to confer new virulence traits, including the ability to colonize new host plants. However, they can also serve as a ‘footprint’ to type plasmid-bearing strains. Genome sequencing of several strains of Xf subsp. fastidiosa sequence type (ST) 1 from Mallorca Island, Spain, revealed the presence of a 38 kb plasmid (pXFAS_5235). In this study, we developed a PCR-based typing approach using primers targeting the traC gene to determine the presence of pXFAS_5235 plasmid or other plasmids carrying this gene in a world-wide collection of 65 strains X. fastidiosa from different subspecies and STs or in 226 plant samples naturally infected by the bacterium obtained from the different outbreaks of Xf in Spain. The traC gene was amplified only in the plant samples obtained from Mallorca Island infected by Xf subsp. fastidiosa ST1 and from all Spanish strains belonging to this ST. Maximum-likelihood phylogenetic tree of traC revealed a close relatedness among Spanish and Californian strains carrying similar plasmids. Our results confirm previous studies, which suggested that a single introduction event of Xf subsp. fastidiosa ST1 occurred in the Balearic Islands. Further studies on the presence and role of plasmids in Xf strains belonging to the same or different subspecies and STs can provide important information in studies of epidemiology, ecology, and evolution of this plant pathogen.

Use of traC gene to type the incidence and distribution of pXFAS_5235 plasmid-bearing strains of X. fastidiosa subsp. fastidiosa ST1 in Spain

Use of traC gene to type the incidence and distribution of pXFAS_5235 plasmid-bearing strains of X. fastidiosa subsp. fastidiosa ST1 in Spain

A novel mutation in TRAC in a patient with abnormal newborn screening for severe combined immunodeficiency

Background: The T cell receptor (TCR)-α chain plays a key role in TCR structure and function. Biallelic mutations in TRAC, encoding the constant region of the TCR-α chain, obliterates TCR expression and results in immunodeficiency. TCR-α chain deficiency presents at infancy or childhood with repeated viral and bacterial infections, enlarged liver, spleen, and lymph nodes as well as autoimmune features and lymphoma (OMIM #615387). Aim: To broaden the genotypic and phenotypic spectrum of TCR-α chain deficiency. Methods: We present a case report of a patient with severe combined immunodeficiency (SCID) due to a novel autosomal recessive mutation in TRAC. Results: Our patient was identified at 13 days of life due to abnormal T cell receptor excision circle levels detected by newborn screening (NBS). Immune evaluation revealed profound lymphopenia, depressed responses to the mitogen PHA and a skewed T cell repertoire, all consistent with SCID. The patient was found to carry a novel homozygous mutation in the TRAC gene. Conclusion: A novel homozygous mutation in the TRAC gene caused profound T cell lymphopenia and aberrant in vitro mitogenic response, the hallmarks of SCID. Statement of Novelty: TCR-α chain deficiency is a rare and relatively new condition and not very well defined. We herein report a novel mutation in TRAC resulting in SCID.

DEVELOPMENT OF CRISPR/CAS9-BASED GENOME EDITING PLATFORM FOR THE GENERATION OF ALLOGENEIC CAR-T CELLS

<h3>Background</h3> Despite its remarkable clinical success, the autologous nature of chimeric antigen receptor (CAR)-T cell therapy restricts the access of patients to this treatment. Thus, one of the next frontiers in the field is to develop an allogeneic, ‘off-the-shelf' therapy. This can be achieved by knocking out the T cell receptor (TCR) from CAR-T cells using genome editing tools to prevent the development of graft versus host disease, an often fatal complication following the allogeneic transplant of T cells. In this study, we establish a methodology for depleting the TCR by knocking out the TRAC gene using CRISPR/Cas9 genome editing. <h3>Methods</h3> We tested two guide RNAs (gRNAs) sequences against the TRAC gene (TRAC1 and TRAC2) which were cloned into lentiviral vectors upstream the anti-CD19 CAR gene. Lentiviral particles were used to transduce Jurkat cells. These cells were used as a model for TCR depletion after electroporation of a plasmid for transient expression of a high-fidelity Cas9. <h3>Results</h3> After transduction, CAR+ Jurkat cells were enriched by magnetic selection to obtain homogeneous expression of CAR and TRAC gRNAs. Flow cytometry analysis after selection demonstrated that 96.3 and 97.7% of cells (TRAC1 and TRAC2, respectively) expressed CAR on the surface. Next, cells were electroporated with a plasmid encoding Cas9 and GFP using two different media: Solution V and Opti-MEM. After 24h of electroporation in Solution V, CAR-T cells displayed high viability (∼90%) regardless of the presence of Cas9-GFP plasmid. In Opti-MEM medium, however, electroporation induced high cell death rates (12.1-16.0% viability). Furthermore, the frequency of GFP+ cells after electroporation in Opti-MEM, was 1.6-fold (TRAC1) and 1.9-fold (TRAC2) lower compared to Solution V. To evaluate the efficiency of gRNAs in depleting the TCR, electroporated cells in Solution V were analyzed after 1, 3 and 8 days for CD3 expression. Notably, TRAC gRNA 1 led to a remarkable reduction in the frequency of CD3+ cells (from 91.6 to 7.41%). In contrast, this reduction was less pronounced for TRAC gRNA 2 (decrease of 21.8%). <h3>Conclusion</h3> These data demonstrate the feasibility of TCR depletion by knocking out the TRAC gene using CRISPR/Cas9 and establish the best gRNA sequence for this purpose, paving the groundwork for the establishment of a platform to produce ‘off-the-shelf' allogeneic CAR-T cells. Support: FAPESP grants 2013/08135-2, 2019/18672-1, 2019/18702-8 and 2020/02043-2.