T-cell Receptor Knockout Research Articles

Abstract 6787: Evaluation of CAR-T cytotoxicity in solid tumors: A screening workflow using 3D patient-derived tumoroids

Abstract Introduction: Over the last decade, chimeric antigen receptor T cell (CAR-T)-based immunotherapy has achieved unprecedented clinical benefits for the treatment of hematological malignancies. However, application of CAR-T therapy to solid tumors remains challenging, in part due to tumor antigen heterogeneity and microenvironmental complexity present in these tumors. Therefore, selecting physiologically-relevant models is critical for validation of the on-target efficacy and off-target effect of CAR-T therapy. Emerging 3D patient-derived tumoroid models better recapitulate the morphological organization, mutational status, and gene expression levels of patient tumors. Hereby, we propose a screening platform to evaluate mesothelin (MSLN)-targeted CAR-T therapy for solid tumors using patient-derived tumoroids. Methods: T cells were isolated from peripheral blood mononuclear cells of healthy human donors. Meso3 (region III of MSLN) or CD19 CARs (served as control) were knocked-in (KI) to activated T cells using electroporation-based CRISPR/Cas9 system followed by CAR Cassette containing adeno-associated virus (AAV) transduction. T-cell receptor (TCR) knockout (KO) and CAR-KI efficiency were validated by flow cytometry. Patient-derived tumoroids with high and low MSLN expression were co-cultured with various effector-to-target ratios of Meso3 CAR, CD19 CAR and Control (TCR KO without CAR KI) T cells. Cytotoxicity was evaluated by caspase-based live cell imaging. Key cytokine secretion was measured using a multiplexing platform 2-3 days post co-culture. Results: T-cell electroporation of CRISPR/Cas9 yielded a high KO efficiency (>90%) of TCR as well as high AAV-mediated CAR cassette-KI efficiency (>70%). The killing efficiency of Meso3 CAR-T cells with different effector to target ratios was successfully captured in a ratio-dependent manner via live cell imaging and analysis platform in the MSLN-high tumoroids, while minimum killing was observed in MSLN-low tumoroids. As expected, control and CD19 CAR-T cells showed no killing towards MSLN-high or -low tumoroids, which do not express CD19. Moreover, the observed killing ability correlated with secretion of cytokines associated with T cell-mediated cytotoxicity. Conclusion: These findings demonstrated the ability of 3D patient-derived models as a screening platform for validations of CAR-T cell therapy in vitro. This platform has potential advantages over commonly used impedance-based and flow cytometry-based killing assay since it better captures the 3D architecture and microenvironment of the tumor origin and allows real-time quantification. With the ability of accurately detecting on and off-target killing, quick turn-around time, and cost-effectiveness compared to in vivo systems, this platform allows for high-throughput screening workflows for CAR-T cell therapies. Citation Format: Xiaoyu Yang, Maryam Mafreshi, Garrett Wong, Vivek Chandra, Colin Paul, Chris Yankaskas, Brittany Balhouse, Jason Sharp, Matt Dallas, Erik Willems, David Kuninger. Evaluation of CAR-T cytotoxicity in solid tumors: A screening workflow using 3D patient-derived tumoroids [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 6787.

Examining the Relationship Between Gamma Delta T Cells and Sensory Peripheral Nerves in the Skin

Abstract Wound repair involves a complex system of immune cells and mechanisms to induce proper healing of a laceration. The transmission of pain signals and immune functions occur simultaneously, suggesting a collaboration between immune and nervous systems. Gamma delta (γδ) T cells are present in the epidermis and dermis where they participate in wound repair by producing growth factors, cytokines, and chemokines. Dermal γδ T cells are adjacent to nerves in the skin. We set out to determine whether sensory peripheral nerve endings and γδ T cells exhibit crosstalk during wound repair. Using immunofluorescent microscopy we found that peripheral neurons and γδ T cells are in close proximity in both the epidermis and the dermis suggesting a transmission of signals. To determine whether γδ T cell dysfunction impacts neurons, we examined non-wounded and wounded skin derived from lean versus diet-induced obese (DIO) mice. There was less interaction between γδ T cells and sensory peripheral nerves in both nonwounded and wounded DIO mouse skin. To determine whether the lack of γδ T cells impacts peripheral nerve repair and wound healing responses, we compared wildtype and T-cell receptor knockout (TCRd −/−) mice. TCRd −/−mice did not exhibit any differences in the number of sensory peripheral nerves in nonwounded skin. However, there were fewer sensory peripheral nerves surrounding the wound edge in mice lacking γδ T cells. Together this data indicates that γδ T cells regulate how sensory peripheral nerves innervate a wound, but are not required for neuronal development. Understanding the mechanism that regulate the interdependent relationship between γδ T cells and sensory peripheral nerves could further our understanding of how to improve wound repair.

Abstract 5511: Immune masking strategies to extend the pharmacokinetics of allogeneic cell therapies

Abstract The dramatic success of CAR T cell therapies in the treatment of certain B cell malignancies has led to a wave of novel cell therapies for cancer. All approved engineered cell therapies are derived from autologous T cells. This has posed barriers for patient access to qualified treatments that include a lengthy production time, inconsistent product characteristics, uncertain manufacturability, and a high cost of manufacturing. To address these obstacles, allogeneic “universal” cell therapies, derived from healthy donors or differentiated pluripotent cells are engineered and expanded in large quantities for off-the-shelf treatment. In allogeneic CAR T cell therapy, T cell receptor (TCR) knockout is required to prevent graft-versus-host disease of alloreactive T cells targeting against host tissue. Allogeneic cells may have a very limited half-life following infusion, however, as they are rapidly targeted by the patient’s own immune system causing host-versus-graft disease. Gene editing is used to increase the persistence of allogeneic T cells by enabling them to evade host T cell or natural killer (NK) cell surveillance. A conventional method of preventing host T cell rejection is to knockout (KO) the β-2 microglobulin (β2M) to diminish the expression of MHC class I protein. To further minimize rejection, overexpression of nonclassical MHC class I protein, HLA-E, is engineered into allogeneic T cells to evade host NK cell rejection. Strategies to improve allogenic CAR T cell persistence through immune evasion have implications for developing a therapeutic product consisting of both CAR T and CAR NK cells. Here we show the use of different methods to investigate the effectiveness of the immune evasion strategies. We found that the effectiveness of HLA-E expression in the suppression of NK cell rejection is highly correlated with the expression of CD94/NKG2A on the host NK cells. Host NK cells with low CD94/NKG2A expression will rapidly recognize and kill allogeneic CAR-T cells even when HLA-E is overexpressed. As it is not possible to control CD94/NKG2A expression in patient NK cells, we sought to identify alternative factors to allow allogeneic cells to evade rapid immune recognition without requiring long term suppression of immune response. To mitigate the donor-to-donor variation on alloreactivity, we developed a strategy to identify NK ligands that can effectively diminish host NK activation in the presence of allogeneic CAR T cells with TCR and B2M gene KOs. A platform using K562 cell line was developed to screen a wide variety of NK inhibitory peptides and synthetic ligands to identify their inhibitory activities. To date, we have identified several synthetic proteins that demonstrate enhanced NK inhibitory function. These synthetic proteins can be used to complement, or to replace HLA-E and enable more extended persistence of an allogeneic cell product in a broader patient population. Citation Format: Meriam Vejiga, Don Wang, Guangnan Li, Kyle Pratt, Phung Gip, James Trager, Ivan Chan. Immune masking strategies to extend the pharmacokinetics of allogeneic cell therapies [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 5511.

Genome-wide off-target analyses of CRISPR/Cas9-mediated T-cell receptor engineering in primary human T cells.

ObjectivesExploiting the forces of human T cells for treatment has led to the current paradigm of emerging immunotherapy strategies. Genetic engineering of the T‐cell receptor (TCR) redirects specificity, ablates alloreactivity and brings significant progress and off‐the‐shelf options to emerging adoptive T‐cell transfer (ACT) approaches. Targeted CRISPR/Cas9‐mediated double‐strand breaks in the DNA enable knockout or knock‐in engineering.MethodsHere, we perform CRISPR/Cas9‐mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T‐cell products. Whole‐genome sequencing was performed to analyse whether CRISPR/Cas9‐mediated DNA double‐strand break at the TCR locus is associated with off‐target events in human primary T cells.ResultsTCRα chain and TCRβ chain knockout leads to high on‐target InDel frequency and functional knockout. None of the predicted off‐target sites could be confirmed experimentally, whereas whole‐genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low‐frequency off‐target events genome‐wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low‐frequency InDels. Therefore, off‐target events are unlikely to be caused by the CRISPR/Cas9 engineering.ConclusionThe combinatorial approach of whole‐genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9‐mediated TCR knockout without potentially harmful exonic off‐target effects.

Protective T cell receptor identification for orthotopic reprogramming of immunity in refractory virus infections

Viral infections cause life-threatening disease in immunocompromised patients and especially following transplantation. Tcell receptor (TCR) engineering redirects specificity and can bring significant progress to emerging adoptive Tcell transfer (ACT) approaches. Tcell epitopes are well described, although knowledge is limited on which TCRs mediate protective immunity. In this study, refractory adenovirus (AdV) infection after hematopoietic stem cell transplantation (HSCT) was treated with ACT of highly purified Hexon5-specific Tcells using peptide major histocompatibility complex (pMHC)-Streptamers against the immunodominant human leukocyte antigen (HLA)-A∗0101-restricted peptide LTDLGQNLLY. AdV was successfully controlled through this oligoclonal ACT. Novel protective TCRs were isolated exvivo and preclinically engineered into the TCR locus of allogeneic third-party primary Tcells by CRISPR-Cas9-mediated orthotopic TCR replacement. Both TCR knockout and targeted integration of the new TCR in one single engineering step led to physiological expression of the transgenic TCR. Reprogrammed TCR-edited Tcells showed strong virus-specific functionality such as cytokine release, effector marker upregulation, and proliferation capacity, as well as cytotoxicity against LTDLGQNLLY-presenting and AdV-infected targets. In conclusion, exvivo isolated TCRs with clinical proven protection through ACT could be redirected into Tcells from naive third-party donors. This approach ensures that transgenic TCRs are protective with potential off-the-shelf use and widened applicability of ACT to various refractory emerging viral infections.

Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR

AbstractAnti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Transgenic lymphocytes targeting minor histocompatibility antigens for post-transplant immunotherapy

Background & Aim The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Methods, Results & Conclusion Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis. To prevent off-target effects caused by the mispairing of endogenous and recombinant TCR chains, the endogenous TCR knockout was made using gRNA/Cas9 ribonucleoprotein complexes. Transgenic alpha and beta-chain constant domains were modified to be resistant to Cas9 cleavage and to form more stable TCR. Antigen-specificity was measured by binding to MHC-tetramers loaded with the MiHA peptide and by TCR-mediated activation of the Jurkat 76 triple parameter reporter cell line upon antigen stimulation. Most of the generated cell lines were specific to the desired antigen, and none were activated in response to irrelevant peptides presented in the same HLA. After analysis of cross-reactivity to allogeneic HLA on the panel of Epstein-Barr virus-transformed lymphoblastoid cell lines, the best receptors would be selected for further therapeutic application. Adoptive transfer of MiHA-specific genetically modified T cells holds great promise for the treatment of leukemia relapse after allo-HSCT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis.

A T-cell reporter platform for high-throughput and reliable investigation of TCR function and biology.

ObjectiveTransgenic re‐expression enables unbiased investigation of T‐cell receptor (TCR)‐intrinsic characteristics detached from its original cellular context. Recent advancements in TCR repertoire sequencing and development of protocols for direct cloning of full TCRαβ constructs now facilitate large‐scale transgenic TCR re‐expression. Together, this offers unprecedented opportunities for the screening of TCRs for basic research as well as clinical use. However, the functional characterisation of re‐expressed TCRs is still a complicated and laborious matter. Here, we propose a Jurkat‐based triple parameter TCR signalling reporter endogenous TCR knockout cellular platform (TPRKO) that offers an unbiased, easy read‐out of TCR functionality and facilitates high‐throughput screening approaches.MethodsAs a proof‐of‐concept, we transgenically re‐expressed 59 human cytomegalovirus‐specific TCRs and systematically investigated and compared TCR function in TPRKO cells versus primary human T cells.ResultsWe demonstrate that the TPRKO cell line facilitates antigen‐HLA specificity screening via sensitive peptide‐MHC‐multimer staining, which was highly comparable to primary T cells. Also, TCR functional avidity in TPRKO cells was strongly correlating to primary T cells, especially in the absence of CD8αβ co‐receptor.ConclusionOverall, our data show that the TPRKO cell lines can serve as a surrogate of primary human T cells for standardised and high‐throughput investigation of TCR biology.

Abstract A043: Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro

Abstract Overall survival of pediatric B-precursor ALL patients reached 90% in recent years. However, the outcome for refractory or relapsed children remains very poor. Anti-CD19 chimeric antigen receptor T-cells (CD19-CAR) showed significant antileukemic activity in relapsed and refractory B-precursor ALL. Especially in children, isolation of a suitable T-cell amount for autologous CAR T-cell manufacturing can be challenging due to low blood volume, low T-cell counts and clinical condition. In this case, the adoptive transfer of CAR T-cells from an unmatched healthy third-party donor provides a promising strategy. In order to prevent life-threatening graft-versus-host disease, a knockout (KO) of the endogenous T-cell receptor (TCR) has to be performed. Here, we generated CD19-CARs with a CRISPR/Cas9 mediated TCR KO, which remain highly functional and show strongly reduced alloreactivity compared to conventional CAR T-cells introduced into third-party T-cells. T-cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and activated via anti-CD3/anti-CD28 stimulation. Retroviral transduction of a second generation anti-CD19 CAR (containing CD3zeta and 4-1BB stimulatory domains) was performed, followed by CRISPR/Cas9 mediated KO of the T-cell receptor beta chain via electroporation. After eleven days of expansion in the presence of IL-7 and IL-15, cells were purified for TCR KO-CD19-CAR T-cells via magnetic separation. Finally, the cell product was analyzed for cellular characteristics, functionality and alloreactivity by flow cytometry. A mean transduction rate of 37% for CD19-CARs and 40% for TCR KO-CD19-CARs was reached as well as a mean TCR KO rate of 78%. Both CD19-CARs as well as TCR KO-CD19-CARs showed suitable amounts of CD4- (45% vs. 33%) and CD8-T-cells (37% vs. 48%). The phenotype of CD19-CARs and TCR KO-CD19-CARs were comparable with mainly central memory (CM) (38% vs. 40%) and effector memory (EM) (57% vs. 51%) T-cells. The expansion of TCR KO-CD19-CARs was significantly reduced compared to conventional CD19-CARs (54-fold vs. 109-fold). This effect was not mediated by the loss of the TCR, but due to electroporation procedure. While CD19-CARs with or without TCR KO showed almost no background expression of the activation marker CD25 (2% vs 1%), contact with CD19-expressing targeT-cells resulted in a comparable upregulation of CD25 in both groups (95% vs. 94%). Co-culture with a CD19-expressing targeT-cell line led to an increased Interferon-γ secretion compared to unstimulated CARs, which was not significantly altered by the TCR KO (17% CD19-CAR vs. 14% TCR KO-CD19-CAR). CD19-dependent proliferative capacity of CAR T-cells was not influenced by loss of the TCR, as in both cases 97% of the T-cells proliferated after antigen recognition. Both CD19-CARs as well as TCR KO-CD19-CARs showed high, antigen-specific killing of 86% vs. 87% of the CD19-expressing targeT-cells at a 1:1 effector to target ratio. To evaluate the alloreactive potential of those T-cells, T-cells were co-cultured with irradiated PBMCs pooled from six different donors. 20% of TCR-expressing T-cells showed proliferation upon contact with non-HLA-matched PBMCs, whereas T-cells with a TCR KO showed almost no proliferation (<3%), demonstrating significantly reduced alloreactivity of TCR KO T-cells. CD19-CAR T-cells lacking the endogenous TCR show a balanced CD4 to CD8 ratio and high proportion of the favorable CM T-cell phenotype. TCR KO-CD19-CARs remain highly functional and show similar activation, cytotoxicity, proliferative capacity and cytokine secretion as conventional CD19-CARs upon antigen recognition. T-cells with TCR KO do not mediate an alloreactive response to non-HLA-matched PBMCs and therefore are promising candidates for the generation of CAR T-cells derived from nonmatched healthy third-party donors or for use of donor-derived CAR T-cells after haploidentical stem cell transplantation. Citation Format: Dana Stenger, Tanja Stief, Theresa Käuferle, Semjon Manuel Willier, Felicitas Rataj, Kilian Schober, Ramin Lotfi, Beate Wagner, Dirk H. Busch, Sebastian Kobold, Franziska Blaeschke, Tobias Feuchtinger. Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A043.

Non-viral and viral delivery solutions for next generation cell therapy

Abstract The successes of chimeric antigen receptor (CAR) T cells in treating blood cancers have highlighted the cell therapy era. However, the difficulty of delivering molecules into immune cells has been an obstacle to more rapid advancement. Here we present an innovative large-scale Lentivirus (LV) production system as a solution to lower the cost and time of viral production. On the other hand, the next generation cell therapy will rely heavily on gene editing, especially in a safer non-viral integration manner. We have demonstrated that our novel non-viral all-in-one electroporation method provides high efficiency of gene knock-in in primary T cells. The new LV production system was developed for the clinical grade production of lentiviral vectors (LVVs) on a large-scale serum-free suspension platform. This technology employs a newly developed propriety set of GMP reagents comprising of culture media, suspension cells, transfection reagent and boosting enhancers. The system is able to deliver greater than 1 × 108 (TU/mL) functional titer with un-concentrated LVVs. For CRISPR/Cas9 gene editing in primary T cells, we were able to reach more than 90% knockout efficiency for most genes we tested, including T cell receptor (TCR), with Cas9 RNP electroporation using Neon Transfection System. More importantly, gene knock-in efficiency can be reached to greater than 30% with all-in-one electroporation, which delivers Cas9 RNP and donor DNA in one reaction using our newly developed electroporation buffer. Moreover, TCR knockout in addition to a knock-in at another locus can also be done in a single electroporation using our all-in-one method, which is ideal for developing next generation CAR and TCR-T cells.

Preclinical Evaluation of Allogeneic Anti-Bcma Chimeric Antigen Receptor T Cells with Safety Switch Domains and Lymphodepletion Resistance for the Treatment of Multiple Myeloma

Multiple myeloma (MM) is a hematological disease of plasma B cells that remains incurable despite the availability of numerous therapies. The plasma cell-specific expression of the TNF superfamily receptor BCMA may allow targeting of normal and malignant plasma cells. Genetically engineered chimeric antigen-receptor T cells (CAR T) have shown tremendous promise in the treatment of several hematological diseases, including MM. However, conventional autologous CAR T therapies use patient-derived T cells and the logistics of on-demand CAR T manufacture limits their availability to a broad patient pool.Here we describe the preclinical evaluation of an allogeneic CAR T therapy targeting BCMA that has the potential for a readily available, off-the-shelf therapy for MM and other malignancies expressing BCMA. Human T cells were transduced with recombinant lentiviral vectors encoding three BCMA CAR candidates designed with fully human anti-BCMA scFvs, CD8a transmembrane domains and the intracellular signaling domains of 4-1BB and CD3zeta. All CAR T efficiently killed BCMA-expressing multiple myeloma cell lines (KMS12BM, MM1.S, Molp-8 and OPM-2), but not BCMA-negative REH cells in vitro and in vivo. Whereas 2 of the 3 candidates exhibited target-independent cytokine production, accelerated T cell differentiation and reduced target cell-induced expansion in vitro, the third candidate did not exhibit this scFv-induced autoactivation and was chosen as the lead molecule.Due to the allogeneic nature of this T cell therapy, the possibility of graft-versus-host (GvH) reactions can be a safety concern. We applied Cellectis’ know-how and TALEN® technology for the gene inactivation of the T cell receptor (TCR) alpha chain to significantly reduce the probability for TCR-mediated GvH reactions and found that TCR knockout did not affect CAR T activity in vitro or in vivo. Furthermore, we incorporated intra-CAR rituximab-recognition domains into the CAR molecule to enable depletion of CAR T cells from patients when necessary. We found that this modified CAR retained anti-BCMA CAR T activity and enabled CAR T depletion by rituximab.Another aspect of allogeneic CAR T therapies is the rejection of the CAR T by host-versus-graft (HvG) reactions. Lymphodepletion prior to CAR T infusion enhances CAR T efficacy in autologous CAR T trials and may also prevent anti-CAR HvG reactions in allogeneic therapy settings. Engineering lymphodepletion resistance into CAR T cells could therefore enable sustained lymphodepletion for enhanced allogeneic CAR T persistence and efficacy. CD52 is expressed on all lymphocytes and administration of the anti-CD52 antibody alemtuzumab for prolonged lymphodepletion is an approved treatment for multiple sclerosis. TALEN®-mediated knockout of CD52 protected BCMA CAR T from alemtuzumab-induced cytotoxicity and did not alter BCMA CAR T anti-tumor activity.Taken together these results support allogeneic BCMA CAR T as an off-the-shelf adoptive immunotherapy for the treatment of multiple myeloma and other BCMA-positive malignancies. DisclosuresBoldajipour:Pfizer: Employment. Galetto:Cellectis SA: Employment. Sommer:Pfizer Inc.: Employment. Pertel:Pfizer Inc.: Employment. Valton:Cellectis Inc.: Employment. Park:Pfizer Inc.: Employment. Gariboldi:Cellectis SA: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Dong:Pfizer Inc.: Employment. Boucher:Pfizer Inc.: Employment. Van Blarcom:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Rajpal:Pfizer Inc.: Employment. Smith:Cellectis SA: Employment. Kuo:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.

T cell receptor knockout efficiency utilizing an engineered meganuclease is influenced by stimulation conditions.

Abstract Recent reports describing allogeneic chimeric antigen receptor (CAR) T cell therapies have generated tremendous excitement over the possibility of a universal CAR T platform. We are utilizing a highly engineered meganuclease designed to target the T cell receptor (TCR) alpha chain to eliminate expression of the TCR, preventing graft versus host disease following adoptive transfer. Meganuclease cleavage efficiency is highly dependent on stimulation of T cells prior to delivery of the meganuclease. To determine optimal activation conditions for TCR knockout and cell yield, T cells were activated with Dynabeads® Human T cell Activator CD3/CD28, Immunocult™ CD3/CD28 and CD3/CD28/CD2 T Cell Activators, and MACS® GMP TransAct CD3/CD28 reagent prior to nucleofection. T cells were stained prior to and following nucleofection to identify cell activation markers and cytokine receptors (CD69, IL2R, IL7R, IL15R, IL21R). Knockout efficiency was determined by staining for CD3 expression. Our results indicate that Dynabead-stimulated T cells expressed the highest levels of activation markers at 18 hours post-stimulation, and that Dynabeads® and Immunocult™ CD3/CD28/CD2 reagents both promote robust T cell proliferation and support the highest TCR knockout efficiency following our protocol. Furthermore, expression of cytokine receptors varies both during stimulation and post-nucleofection, suggesting an optimal cocktail of cytokines may promote T cell proliferation. Future studies will focus on further optimization of T cell stimulation conditions that may promote TCR knockout and expansion of T cells post-nucleofection.

394. Generation of a Novel Allogeneic CAR T Cell Platform Utilizing an Engineered Meganuclease and AAV Donor Template to Achieve Efficient Disruption of T Cell Receptor Expression and Simultaneous Homology-Directed Insertion of a CD19 CAR

Chimeric antigen receptor (CAR) T cell therapies have achieved dramatic results treating hematological malignancies by redirecting the specificity of T cells to target CD19 expressing cancer cells. Clinical trials reporting these impressive results have used autologous therapies, which pose significant manufacturing, logistical, and cost issues, complicating the implementation of such therapies on a larger scale. Furthermore, CAR T cells are typically generated by using Lenti- or Retro- viral vectors, resulting in random integration of the CAR gene, heterogeneous expression of the CAR on the cell surface, and the potential for insertional mutagenesis. We have developed a platform to address these issues by using a streamlined process to insert a CAR gene into the T cell receptor (TCR) alpha chain locus of T cells obtained from healthy donors using a highly optimized engineered meganuclease. The resulting gene-edited allogeneic CAR T cells do not express an endogenous TCR and therefore should not be capable of eliciting graft versus host disease upon adoptive transfer. We first produced and validated an engineered meganuclease targeting TCRα, and developed a scalable process for electroporating T cells with meganuclease mRNA that routinely results in greater than 60% knockout of the TCR, as measured by flow cytometry. To test our gene knock-in approach, we treated cells with our meganuclease to induce a double-strand break (DSB) in TCRα. We then transduced these cells using an AAV6 vector containing a donor DNA template consisting of a second generation CD19 CAR driven by an exogenous promoter and flanked by homology arms to the target site in the TCRα sequence. Homology directed repair of the DSB utilizing this donor template results in insertion of the CD19 CAR at this site in the genome. Incorporating this process into a 14 day procedure for generation and expansion of TCR knockout CAR T cells, we successfully generated a large population of TCR knockout cells with >70% of those cells stably expressing the CD19 CAR. Importantly, these CAR T cells proliferated when co-cultured with CD19+ target cells, released cytokines including IFN-γ and IL-2, and exhibited potent cytotoxic activity against CD19+ target cells. Studies to confirm in vivo antitumor activity are currently being conducted. In summary, we describe a novel, highly efficient, and scalable method to produce allogeneic CAR T cells in a streamlined process using an engineered meganuclease in combination with an AAV6 donor template.

αβ T cells and a mixed Th1/Th17 response are important in organic dust-induced airway disease

BackgroundOrganic dust exposure in agricultural environments induces an inflammatory response that attenuates over time, yet repetitive dust exposures result in chronic lung diseases. Animal models resembling this chronic lung inflammatory response have been developed, yet the underlying cellular mechanisms are not well defined. ObjectiveBecause mice repetitively exposed to organic dust extracts (DE) display increased CD3+ T cell lung infiltrates, we sought to determine the phenotype and importance of these cells. MethodsMice received swine confinement DE repetitively for 3 weeks by established intranasal inhalation protocol. Studies were conducted with peptidoglycan (PGN) because it is a major DE component in large animal farming environments and has shared similar biologic effects with DE. Enumeration of T cells and intracellular cytokine profiles were conducted by flow cytometry techniques. Whole lung homogenate cytokines were analyzed by multiplex immunoassay. T cell receptor (TCR) αβ knockouts were used to determine the functional importance of αβ-expressing T cells. ResultsDE increased lung-associated CD3+CD4+ T cells and interleukin (IL)-17 (but not IL-4, interferon [IFN]-γ, IL-10) producing CD4+ T cells. PGN treatment resulted in increased IL-17 and IFN-γ producing CD4+ T cells and IFN-γ producing CD8+ T cells. Both DE and PGN augmented expression of cytokines associated with Th1 and Th17 polarization in lung homogenates. DE-induced lung mononuclear aggregates and bronchiolar compartment inflammation were significantly reduced in TCR knockout animals; however, neutrophil influx and alveolar compartment inflammation were not affected. ConclusionStudies demonstrated that DE and PGN exposure promote a Th1/Th17 lung microenvironment and that αβ-expressing T cells are important in mediating DE-induced lung pathologic conditions.

HLA and TCR Knockout by Zinc Finger Nucleases: Toward “off-the-Shelf” Allogeneic T-Cell Therapy for CD19+ Malignancies.

AbstractAbstract 3766Cell therapy by infusion of T cells can reconstitute immunity to combat pathogens and malignancies. However, the time required to manufacture T cells with the desired properties and in sufficient numbers ex vivo is often incompatible with the treatment window for patients. Furthermore, autologous T cells from patients with advanced disease may have compromised function and be tolerant to desired antigens. A potential solution would be an approach to infuse allogeneic T cells that avoids immune-mediated rejection caused by host T cells recognizing disparate major or minor histocompatibility antigens on the infused cells. To broaden the application of T cell therapy, we investigated whether HLA gene expression can be disrupted by designer zinc-finger nucleases (ZFNs). ZFNs comprise a zinc finger DNA binding domain designed to bind a specific DNA sequence fused to the cleavage domain of Fok I endonuclease. Since FokI dimerization is required to introduce a double strand break (DSB), we generated ZFN pairs that flank the intended DNA target sequences in the required spatial conformation. Cellular repair of the DSB by error-prone non-homologous end joining allows disruption of HLA gene expression. As an initial proof of concept experiment, transfection of ZFN pairs designed to target exon 3 of the HLA-A locus into the human kidney cell line HEK293 resulted in 10% genetic modification of the HLA-A loci. We generated clones of HEK293 cells that showed deletion or insertion mutations within the ZFN binding site of one or both HLA-A alleles leading to early termination of translation. These HLA-Anull HEK293 clones evaded HLA-A-restricted lysis by T cell clones, even after interferon-γ and TNF-α treatment was used to upregulate HLA expression. Since only transient expression of ZFNs is needed to disrupt a target gene, we tested the ability to disrupt HLA-A gene expression by electro-transfer of in vitro-transcribed ZFN mRNA into primary T cells. We show that a single administration of the mRNA encoding the ZFNs targeting HLA-A could render over 40% of primary T cells HLA-A negative. We enriched the HLA-Anull population by paramagnetic bead separation to obtain a pool of T cells >90% of which lack HLA-A expression. An attractive potential clinical application for HLAnull allogeneic T cells is to redirect their specificity independent of HLA via expression of a chimeric antigen receptor (CAR) targeting CD19. Thus, we eliminated HLA-A expression from CD19-specific CAR+ T cells and demonstrated that they (i) evade HLA-A-restricted lysis by T cell clones, and (ii) specifically lysed CD19+ tumor targets. Finally, to further improve this T cell product and eliminate potential deleterious immune mediated recognition by the endogenous T cell receptor (TCR) on allogeneic CAR+ T cells, we used ZFN pairs targeting the TCR α or the TCR β locus. Transient expression of these ZFNs resulted in permanent disruption of endogenous TCR expression and a highly enriched αβ TCRnull cell population could be generated by paramagnetic bead selection. These data support our plans to develop allogeneic T cells as “off-the-shelf” biologics that can be infused on demand as “drugs”. Disclosures:Reik:Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment.

Enterochromaffin cell hyperplasia and decreased serotonin transporter in a mouse model of postinfectious bowel dysfunction

Patients with postinfective irritable bowel syndrome and Trichinella spiralis-infected mice share many features including visceral hypersensitivity and disordered motility. We assessed enterochromaffin (EC) numbers and serotonin transporter (SERT) using National Institute of Health (NIH) female mice studied for up to 56 days post-T. spiralis infection. The effects of steroid treatment and the T-cell dependence of the observed responses were assessed by infection of hydrocortisone-treated or T-cell receptor knock out [TCR (betaxdelta) KO] animals. Enterochromaffin cell density in uninfected animals increased from duodenum 10.0 cells mm-2 (5.9-41.0) to colon 61.8. (46.3-162) cells mm-2 P<0.0001. Infection increased duodenal and jejunal counts which rose to 37.3 (22-57.7) cells mm-2 and 50.6 (7-110.8) cells mm-2, respectively, at day 14. Infection significantly reduced jejunal SERT expression, with luminance values falling from 61.0 (45.1-98.3) to a nadir of 11.6 (0-36.0) units at day 9, P<0.001. Specific deficiencies in all T cells reduced EC hyperplasia and abrogated infection-induced mastocytosis. Thus infection induced inflammation increases EC numbers, as has been reported in PI-IBS, and reduces SERT. This may increase mucosal 5HT availability and contribute to the clinical presentation of PI-IBS.

The antiviral efficacy of the murine alpha-1 interferon transgene against ocular herpes simplex virus type 1 requires the presence of CD4(+), alpha/beta T-cell receptor-positive T lymphocytes with the capacity to produce gamma interferon.

Alpha/beta interferons (IFN-alpha/betas) are known to antagonize herpes simplex virus type 1 (HSV-1) infection by directly blocking viral replication and promoting additional innate and adaptive, antiviral immune responses. To further define the relationship between the adaptive immune response and IFN-alpha/beta, the protective effect induced following the topical application of plasmid DNA containing the murine IFN-alpha 1 transgene onto the corneas of wild-type and T-cell-deficient mice was evaluated. Mice homozygous for both the T-cell receptor (TCR) beta- and delta-targeted mutations expressing no alpha beta or gamma delta TCR (alpha beta/gamma delta TCR double knockout [dKO]) treated with the IFN-alpha 1 transgene succumbed to ocular HSV-1 infection at a rate similar to that of alpha beta/gamma delta TCR dKO mice treated with the plasmid vector DNA. Conversely, mice with targeted disruption of the TCR delta chain and expressing no gamma delta TCR(+) cells treated with the IFN-alpha 1 transgene survived the infection to a greater extent than the plasmid vector-treated counterpart and at a level similar to that of wild-type controls treated with the IFN-alpha 1 transgene. By comparison, mice with targeted disruption of the TCR beta chain and expressing no alpha beta TCR(+) cells (alpha beta TCR knockout [KO]) showed no difference upon treatment with the IFN-alpha1 transgene or the plasmid vector control, with 0% survival following HSV-1 infection. Adoptively transferring CD4(+) but not CD8(+) T cells from wild-type but not IFN-gamma-deficient mice reestablished the antiviral efficacy of the IFN-alpha 1 transgene in alpha beta TCR KO mice. Collectively, the results indicate that the protective effect mediated by topical application of a plasmid construct containing the murine IFN-alpha 1 transgene requires the presence of CD4(+) T cells capable of IFN-gamma synthesis.

The many faces of host responses to tuberculosis.

Tuberculosis remains today one of the top three fatal infectious diseases, together with acquired immune deficiency syndrome (AIDS) and malaria. During the last decade, 90 million new infections occurred, resulting in approximately 30 million deaths. Although there is currently effective chemotherapy, consisting of three specific drugs, this regimen must be continued for a period of at least 6 months, which in many cases, results in problems with compliance. Lack of compliance further impacts on the development of multidrug-resistant strains of the bacterium, which consequently raises the cost of treatment, making the expense of curing tuberculosis prohibitive in many developing countries. Despite the enormous numbers of people infected with this organism, it is estimated that only 10% of affected individuals show evidence of clinical symptoms. Many parameters, notably socio-economic factors, co-infection with human immunodeficiency virus (HIV) and genetic predisposition of the host, influence the susceptibility to disease. Much work has been invested to elucidate the biology of the interaction between Mycobacterium tuberculosis and its host, both in experimental animal models and in clinical studies. Here we review some of the latest developments in the understanding of the immune response required to control this pathogen. It is hoped that further progress in this field will lead to a more rational approach towards the development of an effective vaccine and novel chemotherapeutic agents.

γδ intraepithelial lymphocytes drive tumor necrosis factor‐α responsiveness to intestinal iron challenge: relevance to hemochromatosis

The dependence of intestinal epithelial cell (IEC) growth and differentiation on intraepithelial lymphocytes (IELs) expressing the gamma/delta (gamma delta) T-cell receptor (TCR), suggested a potential role for gamma delta + IELs in the regulation of iron absorption. We therefore examined the levels of hepatic iron and the IEL cytokine responses in C57BL/6J control and class I and TCR knockout lines (placed on a C57BL/6J genetic background) following the administration of supplemental dietary iron. The highest level of liver iron was found in the beta 2-microglobulin knockout (beta 2m-/-) mice followed by the TCR-delta knockout (TCR delta-/-) animals. TCR-alpha knockout (TCR alpha-/-) and control animals did not differ in their iron levels. Liver iron loading correlated inversely with the ability of the mice to generate an IEL tumor necrosis factor (TNF)-alpha response. These observations suggest a model in which IEC iron loading is communicated to IELs via the HFE class I protein. The result of this communication is the initiation of TNF-alpha release by gamma delta + IELs (sustained by macrophages and dendritic cells) contributing to the upregulation of ferritin expression and possibly to the normal maintenance of the IEC apoptotic pathway.