Brian Philip

A highly compact epitope-based marker / suicide gene for easier and safer T-cell therapy

A compact marker/suicide gene that utilizes established clinical-grade reagents and pharmaceuticals would be of considerable practical utility to T-cell cancer gene therapy. Marker genes enable measurement of transduction and allow selection of transduced cells, whereas suicide genes allow selective deletion of administered T cells in the face of toxicity. We have created a highly compact marker/suicide gene for T cells combining target epitopes from both CD34 and CD20 antigens (RQR8). This construct allows selection with the clinically approved CliniMACS CD34 system (Miltenyi). Further, the construct binds the widely used pharmaceutical antibody rituximab, resulting in selective deletion of transgene-expressing cells. We have tested the functionality of RQR8 in vitro and in vivo as well as in combination with T-cell engineering components. We predict that RQR8 will make T-cell gene therapy both safer and cheaper.

Comparison of different suicide-gene strategies for the safety improvement of genetically manipulated T cells.

Use of adoptive T-cell therapy (ACT) is increasing; however, T-cell therapy can result in severe toxicity. Consequently, several suicide-gene strategies that allow selective destruction of the infused T cells have been described. We compared effectiveness of four such strategies in vitro in Epstein Barr virus (EBV)-cytotoxic T lymphocytes (CTLs). Herpes simplex virus thymidine kinase (HSV-TK), human inducible caspase 9 (iCasp9), mutant human thymidylate kinase (mTMPK), and human CD20 codon optimized genes were cloned in frame with 2A-truncated codon optimized CD34 (dCD34) in a retroviral vector. Codon-optimization considerably improved CD20 expression. EBV-CTLs could be efficiently transduced in all constructs, with transgene expression similar to the control vector containing dCD34 alone. Expression was maintained for prolonged cultures. Expression of the suicide genes was not associated with alterations in immunophenotype, proliferation, or function of CTLs. Activation of HSV-TK, iCasp9, and CD20 ultimately resulted in equally effective destruction of transduced T cells. However, while iCasp9 and CD20 effected immediate cell-death induction, HSV-TK-expressing T cells required 3 days of exposure to ganciclovir to reach full effect. mTMPK-transduced cells showed lower T-cell killing all time points. Our results suggest that the faster activity of iCasp9 might be advantageous in treating certain types of acutely life-threatening toxicity. Codon-optimized CD20 has potential as a suicide gene.

Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies

Mature T cell cancers are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy (normal) T cells. Unlike B cell depletion, pan–T cell aplasia is prohibitively toxic. We report a new targeting strategy based on the mutually exclusive expression of T cell receptor β-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identify an antibody with unique TRBC1 specificity and use it to demonstrate that normal and virus-specific T cell populations contain both TRBC1+ and TRBC2+ compartments, whereas malignancies are restricted to only one. As proof of concept for anti-TRBC immunotherapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T cells, which recognized and killed normal and malignant TRBC1+, but not TRBC2+, T cells in vitro and in a disseminated mouse model of leukemia. Unlike nonselective approaches targeting the entire T cell population, TRBC-targeted immunotherapy could eradicate a T cell malignancy while preserving sufficient normal T cells to maintain cellular immunity.

A Rapamycin activated Caspase 9 based suicide gene

Engineered T cell therapies show considerable promise in the treatment of refractory malignancies. Given the ability of engineered T cells to engraft and persist for prolonged periods along with unpredicted toxicities, incorporation of a suicide gene to allow selective depletion after administration is desirable. Rapamycin is a safe and widely available immunosuppressive pharmaceutical that acts by heterodimerization of FKBP12 with the FRB fragment of mTOR. The apical caspase caspase 9 is activated by homodimerization through its CARD domain. We developed a rapamycin-induced caspase 9 suicide gene. First, we showed that caspase 9 could be activated by a two-protein format with replacement of the CARD domain with both FRB and FKBP12. We next identified an optimal compact single-protein rapamycin caspase 9 (rapaCasp9) by fusing both FRB and FKBP12 with the catalytic domain of caspase 9. Functionality of rapaCasp9 when co-expressed with a CD19 CAR was demonstrated in vitro and in vivo.

717. UCART19, an Allogeneic “Off-the-Shelf” Adoptive T-Cell Immunotherapy Against CD19+ B-Cell Leukemias

Autologous T-cells engineered to express chimeric antigen receptors (CARs) that target specific tumor antigens are known to be of high potential in treating different kinds of cancer. However, they must be generated on a “per patient” basis, thereby limiting the population of patients that could benefit from this approach. In particular, immune homeostasis may be affected in heavily pre-treated patients, such that autologous T-cells may be low in number, not fully functional, or unable to expand, thereby restricting the amount of cells that could be manufactured. The use of allogeneic T-cells isolated from healthy third party donors could constitute an easy-to-scale-up alternative, producible in advance, with potential for standardized quality controls, better batch consistency, and immediate availability for administration to a larger number of patients. In this context, we have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CD19-CAR+ T-cell–based frozen products. Our platform involves the use of transcription activator-like effector nucleases (TALEN), which mediate the simultaneous inactivation of two genes through genome editing. The knockout of the TCR alpha gene eliminates TCR expression and is intended to abrogate the donor T-cells potential for graft-versus-host disease (GvHD), while knocking out the CD52 gene makes donor T-cells resistant to the lymphodepleting agent alemtuzumab. In addition, our T-cells are engineered to co-express the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab.We have obtained proof-of-concept for the application of this approach by manufacturing TCR/CD52-deficient RQR8+ and cD19-CAR+ T-cells (UCART19) using a good manufacturing practice-compatible process and have demonstrated that the resulting UCART19 cells were functional using in vitro assays. Furthermore we have demonstrated the ability of UCART19 cells to engraft into an orthotopic human CD19+ lymphoma xenograft immunodeficient mouse model. UCART19 cells exhibited antitumor activity equivalent to that of standard CD19 CAR T-cells. We also demonstrated that UCART19 cells did not mediate alloreactivity in a xeno-GvHD mouse model. Finally, the effectiveness of the rituximab-induced depletion mechanism of RQR8+ cells was shown in an immunocompetent mouse model. This valuable dataset supports the development of allogeneic CAR T-cells, and UCART19 will be investigated in an exploratory, first-in-human, clinical trial where refractory/relapsed CD19+ B-cell leukemia patients are to be enrolled.

Addition of the C-terminus of CD6 to a chimeric antigen receptor enhances cytotoxicity and does not compromise expression

T cells expressing chimeric antigen receptors (CARs) are a promising new cancer immunotherapy that has now reached the clinic. CARs are synthetic receptors that redirect T cells towards a tumour‐associated antigen and activate them through various fused signalling regions, for example derived from CD3ζ, 4‐1BB or CD28. Analysis of the optimal combination of CAR components including signalling domains is not yet comprehensive and may vary with the particular application. The C‐terminus of the T‐cell surface receptor CD6 is critical for its co‐stimulatory effects and signals through two phospho‐tyrosine motifs that bind to the intracellular adaptor proteins GADS and SLP‐76. Addition of the C terminus of CD6 did not compromise CAR expression, showing it was a stable moiety that can be used independently of the native receptor. A third‐generation CAR containing 4‐1BB, CD3ζ and the C terminus of CD6 (4‐1BBz‐CD6) enhanced interferon‐γ release and cytotoxicity when compared with the second‐generation 4‐1BB CD3ζ (4‐1BBz) CAR. The CD6 C terminus is a valuable addition to potential components for modular design of CARs to improve effector function, particularly cytotoxicity.