T-cell Receptor Alpha-chain Locus Research Articles

A CD3 Fusion Receptor (CD3-FR) Uniquely Enables Compatibility of Allogeneic CAR-T and -NK Cells with T Cell Engagers to Enhance Antitumor Function and Limit Antigen Escape

Chimeric antigen receptor (CAR) T cells have shown remarkable clinical success in the treatment of many malignancies. However, certain challenges remain, including broad patient access and durability of response. We have shown that a scalable manufacturing platform, where genetically edited induced pluripotent stem cell (iPSC) master cell lines are created to serve as a renewable starting material for the derivation of uniformly engineered CAR T or NK cells, can facilitate broad patient access in an off-the-shelf manner. To improve durability of response, often plagued by antigen heterogeneity found in cancer, we are pursuing several combination strategies, merging cell therapy with therapeutic agents such as checkpoint blockade therapy and monoclonal antibodies to increase anti-tumor activity and multi-antigen targeting of cancer. T cell engagers have also shown remarkable advancements in the treatment of various cancer types, but they are also afflicted by their own challenges and would equally benefit from combination strategies. Unfortunately, T cell engagers are not compatible with allogeneic adoptive cell therapy, as the cells used in the allogenic setting do not express the CD3 molecule used for interaction with the engagers, as the T cell receptor (TCR) that supports CD3 surface expression, is either not expressed in NK cells or has been ablated in T cells to prevent graft-versus-host disease. To investigate the anti-tumor synergy between off-the-shelf cell therapy and T cell engagers, we developed a novel CD3ε fusion receptor (CD3-FR) to uniquely support the expression of a functional CD3 on TCR-less allogeneic T and NK cells and to enable compatibility between allogeneic cell therapy and T cell engagers. Initially, CD3-FR constructs with various co-stimulating endodomains were confirmed to elicit T cell engager-dependent NFAT activity in TCR alpha chain (TRAC) knockout (T-KO) Jurkat cells. Next, iPSCs already containing a CAR inserted into the TRAC locus were engineered to contain CD3-FR. CAR+ CD3-FR+ iPSCs were differentiated into CAR-iT cells, uniformly expressing both modalities (>95% CAR+, >90% CD3ε+, TCR not detected). To demonstrate that CD3-FR+ CAR-iT cells can elicit T cell engager-dependent antitumor activity, CAR antigen (1° Ag) negative but T cell engager (2° Ag) positive target cells were used in various cytotoxicity assays. As expected, control CAR-iT cells failed to kill the 1° Ag negative tumors despite addition of T cell engager targeting 2° Ag. On the other hand, CD3-FR+ CAR-iT cells showed potent activity toward target cells but only in the presence of the T cell engager (>80% cytolysis). Furthermore, when comparing the expression of activation markers in CD3-FR+ CAR-iT cells in co-cultures with or without the T cell engager, we observed an increased proportion of CD69+ (61% vs. 28%) and CD25+ (37% vs. 2%) cells in the CD3-FR+ CAR-iT cell co-cultures containing the T cell engager. Cytokine production was also tested, with increased TNF (5-fold) and IFNγ (7-fold) measured in supernatants from CD3-FR+ co-cultures upon the addition of T cell engager, further demonstrating T cell engager-dependent functionality and specificity of the CD3-FR construct. To test the ability of CD3-FR CAR-iT cells to mitigate 1° Ag escape, we used a target cell population heterogenous for the CAR-specific target (1° Ag, 50% positive) but homogenous for EpCAM (2° Ag, 100% positive). After three days, the standard CAR-iT cells failed to control tumor cell growth due to antigen escape. In contrast, CD3-FR+ CAR-iT cells exhibited robust control (80% cytolysis) of the 1° Ag heterogenous targets in the presence of αEpCAM, indicating mitigation of 1° Ag escape. Lastly, CD3-FR was edited into iPSC-derived NK (iNK) cells to demonstrate compatibility of CD3-FR+ NK cells with T cell engagers. An xCelligence-based killing assay was performed in the presence of a T cell engager, where CD3-FR+ iNK cells showed superior target cell killing (~80% lysis) after 96 hours compared to parental iNK cells (~30% lysis), demonstrating the potential for synergy between T cell engagers and NK cells (Figure 1). Taken together, these studies present a novel opportunity to combine T cell engager technology with off-the-shelf CAR-iT or iNK cell products, uniquely merging two powerful therapeutics whose combination was previously underappreciated in improving cancer therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering for Cell Therapy Development

Gene-editing technology has revolutionized molecular therapeutics, enabling DNA-engineering-based approaches to treat disease. Despite this, development of medicines using gene editing has been hampered by technological, immunological, and legal limitations. We described previously the discovery of novel type II and type V gene-editing systems from metagenomic data, the characterization of these systems in vitro, and a demonstration of their activity in immortalized cell lines. Here, we substantially advance this work with three separate, novel gene-editing systems, demonstrating their utility for cell therapy development. We express and purify the nuclease components of both type II and type V effectors and show that all three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain constant region occurred in up to 95% of cells, both copies of the TCR beta-chain constant region in up to ~90% of cells, and beta-2 microglobulin in up to 95% of cells. Simultaneous double knock-out of TRAC and TRBC was obtained at an equal frequency. Gene editing with our systems had no effect on T cell viability. Further, we use our novel gene-editing systems to exploit homology-dependent DNA repair to integrate a CAR construct into the TCR alpha-chain locus (in up to ~60% of T cells), and demonstrate high-level CAR expression and antigen-dependent CAR-T cytotoxicity. Such robust editing activity at the TCR loci will permit efficient engineering and manufacture of allogeneic chimeric antigen receptor- (CAR) and TCR-based cell therapies.We next applied our novel gene-editing tools to NK cells and B cells. We achieved almost 100% gene disruption at the CD38 locus in NK cells and integrated a chimeric antigen receptor into the NK cell genome at a frequency of ~40%. Such CAR-NK cells displayed robust CAR-directed cellular cytotoxicity. B cell editing occurred in approximately 80% of target cells with successful transgene integration.Cas9 is notorious for tolerating mismatches and bulges in its target, resulting in high levels of unwanted DNA double-strand break formation. In contrast, interrogation of our gene editing systems using GUIDE-seq reveals no or very few off-target sites even at doses greatly in excess of those required for high-frequency gene editing. Finally, as our systems are taken from microbes found in environmental samples rather than from human pathogens, we expect pre-existing immunity to our nucleases will be quite rare. In all, we show that three new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development. DisclosuresNo relevant conflicts of interest to declare.

Gene Editing of TRAC Locus Utilizing Megatal Nucleases Increases Expression of Transgenic TCRs Delivered Via Lentiviral Vector-Mediated Gene Transfer

T-cell receptor (TCR) transgenic T cell therapies have demonstrated efficacy in late stage and refractory oncologic malignancies, including melanoma and synovial cell sarcoma. However, these responses are generally either short in duration or occur in a minority of patients. One of the limitations of transgenic TCR therapies may be related to the mispairing of the introduced transgenic TCR alpha and beta chains with the endogenous TCR chains, resulting in non-productive TCR transgenic T cells. Using genome editing, endogenous TCR genes can be eliminated, potentially reducing mispairing with a subsequent increase in the activity of the transgenic TCR. It is essential that therapeutic genome editing be achieved with sufficient specificity to minimize off-target editing events. MegaTALs, derived by fusing a DNA binding array to an engineered meganuclease, can be iteratively refined in response to off-target detection, achieving a genome-wide increase in on-target versus off-target discrimination. To this end, we developed a megaTAL targeting the T-cell receptor alpha chain (TRAC) locus and identified a prominent off-target site using unbiased double strand break detection methods coupled with targeted amplicon sequencing. Optimization of this megaTAL using a combination of protein engineering approaches resulted in a reagent achieving >90% editing of the TRAC locus in primary human T cells and the off-target site was eliminated. The TRAC specific megaTAL resulted in a 92% reduction in CD3 surface expression a surrogate marker of TCR expression (5.6±0.4% vs 97.6±0.2%, p<0.0001). To further evaluate this nuclease we transduced primary human T cells with a lentiviral vector encoding an anti-NY-ESO-1 transgenic TCR. Expression of the transgenic TCR was followed by staining for the TCR Vbeta13.1 protein and we observed 74% transduction efficiency accounting for Vbeta13.1 usage in the endogenous TCR repertoire (79.4±2.6% vs 5.1±0.7%, p<0.0001). Of the transduced Vbeta13.1 positive cells, 64.3% were positive for the transgenic TCR as determined by tetramer staining, suggesting 35.7% of transduced cells exhibit TCR mispairing with components of the endogenous TCR. In parallel, T cells were treated with a combination of megaTAL and TCR vector transduction. Transgenic TCR expression in TRAC megaTAL edited T cells restores CD3 surface expression in all Vbeta13.1-positive transduced cells (74.0±2.4% vs 5.6±0.4%, p < 0.0001). In addition, 96% of the TRAC edited Vbeta13.1 positive cells were now positive for the transgenic TCR as shown by the tetramer staining (95.5±1.0% vs 64.3±4.8%, p<0.01). Treatment of transgenic TCR expressing human T cells with the TRAC specific megaTAL resulted in a 31% reduction in mispairing of the transgenic TCR (4.4±1.0% vs 35.7±4.8%, p<0.01). Moreover, TRAC megaTAL treatment produced a 1.7-fold increase in the expression of the transgenic TCR in tetramer positive cells (gMFI 6292±662 vs 3800±200, p <0.05). TRAC edited TCR-transgenic T cells exhibit a similar phenotypic profile as un-edited TCR transgenic T cells and TRAC megaTAL editing does not result in a significant impact on T cell expansion, CD4 to CD8 T cell ratios, T cell memory subsets, or lentiviral integration during manufacturing. These results demonstrate that the megaTAL architecture facilitates the iterative refinement of nuclease specificity and activity, generating exquisitely selective, single-component reagents, and highlight the potential of megaTAL driven TRAC gene editing to further improve expression of transgenic TCRs. DisclosuresMagee:bluebirdbio: Employment, Equity Ownership. Diaconu:bluebirdbio: Employment, Equity Ownership. Pogson:bluebirdbio: Employment, Equity Ownership. Gupta:bluebirdbio: Employment, Equity Ownership. Jensen-Smith:bluebirdbio: Employment, Equity Ownership. Collette:bluebirdbio: Employment, Equity Ownership. Chavkin:bluebirdbio: Employment, Equity Ownership. Pasackow:bluebirdbio: Employment, Equity Ownership. Edwards:bluebirdbio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Astrakhan:bluebird bio: Employment, Equity Ownership. Certo:bluebird bio: Employment, Equity Ownership. Jarjour:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership, Patents & Royalties.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Although chimeric antigen receptor T (CAR-T) cell therapy achieves high remission rates, challenges (e.g., toxicity management and relapse prevention) remain. The major risks are cytokine release syndrome and related neurological toxicity. The influence of the CAR surface density on the efficacy/safety of CAR-T cell therapy and the factors determining CAR density were not elucidated comprehensively. Here, we discovered that the use of the MND promoter increased the transduction rate and reduced the CAR surface density. Additionally, MND-driven CAR-T cells had prolonged antileukemia activity in a mouse model. In an initial dual-armed anti-CD19 CAR-T cell pilot study (ClinicalTrials.gov: NCT03840317), eight and six subjects were infused with MND and EF1α promoter-driven autologous CAR-T cells (3 × 105 CAR-T cells/kg), respectively. MND subjects developed mild fever and lower interferon gamma (IFN-γ) concentrations than in the EF1A19 group. All but one subject in each cohort reached minimal residual disease (MRD)-negative complete remission after the first month of evaluation. These results represent the first comprehensive study on the promoter-driven modulation of CAR-T cell functionality. These findings encourage further evaluation of the potential of the MND promoter to drive CAR-T cells as a broadly applicable cellular product for anticancer immunotherapy.

Abstract IA22: Targeting CARs to the TRAC locus enhances T-cell potency

Abstract Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. The most successful CARs used to date are those targeting CD19, which offer the prospect of complete remissions in patients with chemorefractory/relapsed B cell malignancies. CARs are typically transduced into patient T cells using γ-retroviral or other randomly-integrating retroviral vectors (RVs), which may result in variegated CAR expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific modifications in human primary cells, including site-specific transgene integration into the CCR5 and AAVS1 loci in T lymphocytes. Using CRISPR/Cas9 to edit human T cells, we have established conditions yielding efficient target gene disruption and CAR insertion in a single step. We have found that directing a CD19 CAR to the human T-cell receptor (TCR) alpha chain (TRAC) locus not only results in efficient and uniform CAR expression in human peripheral blood T cells, but, remarkably, also enhances T-cell potency, vastly outperforming that of conventionally generated CAR T cells. We further show that CAR gene expression under the control of the TCR alpha promoter minimizes tonic signaling and allows effective regulation of CAR expression. These findings provide important insights into CAR immunobiology and highlight the potential of CRISPR/Cas9 genome editing to advance immunotherapies. Citation Format: Michel Sadelain, Justin Eyquem. Targeting CARs to the TRAC locus enhances T-cell potency [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA22.

Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.

Expression of a second receptor rescues self-specific T cells from thymic deletion and allows activation of autoreactive effector function.

Allelic exclusion at the T-cell receptor alpha chain locus is incomplete resulting in the generation of T cells that express two T-cell receptors. The potential involvement of such T cells in autoimmunity has been suggested [Padovan, E., Casorati, G., Dellabona, P., Meyer, S., Brockhaus, M. & Lanzavecchia, A. (1993) Science 262, 422-424; Heath, W. R. & Miller, J. F. A. P. (1993) J. Exp. Med. 178, 1807-1811]. Here we show that expression of a second T-cell receptor can rescue T cells with autospecific receptors from thymic deletion and allow their exit into the periphery. Dual receptor T cells, created by constitutive expression of two transgenic T-cell receptors on a Rag1-/- background, are tolerant to self by maintaining low levels of autospecific receptor, but selfreactive effector function (killing) can be induced through activation via the second receptor. This opens the possibility that T cells carrying two receptors in the periphery of normal individuals contain putatively autoreactive cells that could engage in autoimmune effector functions after recognition of an unrelated environmental antigen.

T‐Cell Receptor Alpha Haplotype influences Vα Epitope Expression on both Cortisone‐Resistant Thymocytes and Lymph Node T Cells

We have previously described a mouse strain congenic for the T-cell receptor alpha chain locus (Tcra). The strain, B10.D2.C-Tcraa/Bo, carries the Tcraa haplotype of BALB/c instead of the Tcrab haplotype normally found in B10.D2. By comparing B10.D2.C-Tcraa and B10.D2 mice, we now show that the Tcra haplotype influences the frequencies of V alpha 8 epitope expression on T cells detected by the novel B21.14 monoclonal antibody (MoAb). This finding agrees well with our previous observation that Tcra haplotype influences the frequencies of expression of two other V alpha 8 epitopes, detected by the KT50 and KT65 MoAbs. The specificities of the three V alpha 8-specific MoAbs were compared by scatter diagram analysis of staining frequencies obtained in individual mice. The B21.14 and KT50 MoAbs appear to have very similar specificities; these two MoAbs stain slightly more alpha beta T cells than the KT65 MoAb. Investigations with another novel MoAb, the V alpha 2-specific B20.1, revealed that the Tcra haplotype also influences the frequency of V alpha 2 epitope expression. The effect of the Tcra haplotype on V alpha 2 epitope expression was evident not only among lymph node T cells but also among mature, cortisone-resistant thymocytes. Thus, the influence of the Tcra haplotype is imprinted already in the thymus. The most likely explanations for Tcra haplotype dependency of V alpha epitope expression are differing numbers of V alpha-gene segment subfamily members and/or allelic polymorphism. The significance of Tcra haplotype-dependent V alpha epitope expression is discussed.

A Tcra congenic mouse: V alpha epitope expression is influenced by both Tcra haplotypes and background genes.

A T-cell receptor alpha chain locus (Tcra) congenic mouse is described. The Tcraa haplotype of BALB/c (donor strain) was bred on to B10.D2 (background strain, Tcrab haplotype) by using a Bgl I Tcra-C restriction fragment length polymorphism. Tcraa/b heterozygous offspring from the eleventh backcross generation were brother-sister mated to obtain Tcra-Ca homozygous animals. The resulting congenic line, B10.D2.C-Tcraa/Bo carries a recombination between the Tcra and the hr loci; thus, the transferred differential segment is the centromeric 18-27 cM of the BALB/c chromosome 14. Analysis with a multitude of Tcra-V and Tcrd-V probes demonstrates that the complete Tcraa haplotype is contained within this differential segment. Lymph node T cells of BALB/c (Tcraa) B10.D2 (Tcrab) and B10.D2.C-Tcraa were stained with anti-V alpha 8 (KT50, KT65), anti-V alpha 3.2 (RR3-16) and anti-V alpha 11.1 and 2 (RR8-1) monoclonal antibodies. We find that the frequencies of V alpha epitope expression are highly Tcra haplotype-dependent even though an influence of background genes is also observed. Thus, Tcra-V germline differences may possibly influence the T cell repertoire, in addition to the already well known positive and negative thymic selections. Tcra haplotype does not influence the frequencies of V beta utilization. However, BALB/c mice have fewer V beta 11+ T cells than B10.D2 and B10.D2-Tcraa, therefore, the BALB/c genome must harbor a V beta 11 deleting gene(s) in addition to those described so far.

Analysis of a T-cell tumor-specific breakpoint cluster at human chromosome 14q32.

Cytogenetic abnormalities involving chromosome 14 band q32 are consistently observed in human T-cell tumors. Patients with ataxia-telangiectasia (AT) are especially prone to development of these tumors, which frequently carry either inversion inv(14)(q11;q32) or translocation t(14;14) (q11;q32) chromosomes. We have previously shown that the cytogenetic breakpoints of one t(14;14)(q11;q32) chromosome and two inv(14)(q11;q32) chromosomes in T-cell tumors from AT and non-AT patients join the T-cell receptor alpha chain locus, at chromosome band 14q11, with a region(s) at 14q32 centromeric of the immunoglobulin heavy chain variable region (VH) gene IGHV. We now show that these two inv(14) breakpoints are linked by 2.1 kb of germ-line 14q32 DNA and that the three breakpoints define, by in situ hybridization analysis, a single locus at chromosome band 14q32.1 located about 15-20 million base pairs on the centromeric side of the IGH locus. Sequence analysis of the 14q32.1 breakpoint regions indicates that abnormal recombination does not universally result from mistaken V-D-J joining (D = diversity region; J = joining region). Therefore, we invoke a tumor selection model to describe the role of the 14q32.1 locus in tumor development.

Juxtaposition of the T-cell receptor alpha-chain locus (14q11) and a region (14q32) of potential importance in leukemogenesis by a 14;14 translocation in a patient with T-cell chronic lymphocytic leukemia and ataxia-telangiectasia.

We describe a t(14;14)(q11;q32) translocation in a patient with T-cell chronic lymphocytic leukemia and ataxia-telangiectasia (AT). By using a battery of joining (J)-segment probes from the T-cell receptor (TCR) alpha-chain locus TCRA, three distinct J alpha rearrangements were observed. One rearrangement reflected a normal TCRA variable (V) region V alpha-to-J alpha recombination. The second rearrangement was caused by the translocation even itself, which joined a DNA segment from 14q32 centromeric to the immunoglobulin heavy chain locus (IGH) and a J alpha gene located approximately 75 kilobases (kb) 5' of the TCRA constant region gene (C alpha). A third rearrangement involved a 17-kb internal deletion 3' to the translocation, a rearrangement within the J alpha locus that has been observed once before in a patient with AT. Analysis of these three rearrangements underscores the increase in aberrant locus-specific recombination in lymphocytes from patients with AT. Furthermore, these studies support the view that a growth-effecting gene is present in the 14q32 region that participates in the leukemogenic process.

Breakpoints in the human T-cell antigen receptor alpha-chain locus in two T-cell leukaemia patients with chromosomal translocations.

Specific chromosomal translocations have been observed in several human and animal tumours and are believed to be important in tumorigenesis. In many of these translocations the breakpoints lie near cellular homologues of transforming genes, suggesting that tumour development is partly due to the activation of these genes. The best-characterized example of such a translocation occurs in mouse plasmacytoma and human B-cell lymphoma, where c-myc, the cellular homologue of the viral oncogene myc, is brought into close proximity with either the light- or heavy-chain genes of the immunoglobulin loci, resulting in a change in the regulation of the myc gene. T-cell malignancies also have characteristic chromosomal abnormalities, many of which seem to involve the 14q11-14q13 region. This region has recently been found to contain the alpha-chain genes of the human T-cell antigen receptor. Here we determine more precisely the chromosome breakpoints in two patients whose leukaemic T cells contain reciprocal translocations between 11p13 and 14q13. Segregation analysis of somatic cell hybrids demonstrates that in both patients the breakpoints occur between the variable (V) and constant (C) region genes of the T-cell receptor alpha-chain locus, resulting in the translocation of the C-region gene from chromosome 14 to chromosome 11. As the 11p13 locus has been implicated in the development of Wilms' tumour, it is possible that either the Wilms' tumour gene or a yet unidentified gene in this region is involved in tumorigenesis and is altered as a result of its translocation into the T-cell receptor alpha-chain locus.