Periodic Reporting for period 1 - CARiPSCTcells (Generation of safe and efficient, off-the-shelf, chimeric antigen receptor (CAR)-engineered T cells for broad application)
Periodo di rendicontazione: 2015-07-01 al 2017-06-30
The successful and promising clinical results of adoptive T cell therapies for cancer dictate for further advancements, which would broaden their applicability for more patients and for more diseases. Current approaches require the isolation expansion and reinfusion of tumor infiltrating lymphocytes (TILs), the labor-intensive ex-vivo generation and expansion of tumor antigen-specific T cell lines or the genetic engineering of autologous T cells with tumor antigen specific TCRs, and most recently with Chimeric Antigen Receptors (CARs). Yet, there are still limitations, which impede the progress and broader use of T cell therapies. In many cases the autologous T cell isolation and expansion could be problematic or impossible (e.g immunosuppressed patients after chemotherapy, immune-deficient patients presenting with malignancies). Also, the isolation and ex vivo manipulation of autologous cells requires processing time, which can be critical for the patient’s health. In addition, the existing ex vivo T-cell expansion protocols push T cells to a terminal differentiated effector state at the cost of their regenerative capacity and resulting in exhausted, less persistent cellular products. The development of broadly applicable cellular therapeutics, which have been manufactured, functionally validated and banked in advance, and can be applied beyond HLA histocompatibility limitations would improve the consistency and availability and reduce the cost of adoptive T cell therapy. Towards this future goal, this project explored the feasibility of a novel strategy for generating unlimited, “off the shelf”, safe, antigen-specific T lymphocytes with optimized features across histocompatibility barriers.We specifically focus on developing a universally applicable adoptive therapy for Multiple Myeloma (MM). This malignant disease of antibody producing plasma cells is the 2nd most common hematological malignancy, and accounts for 1.4 % of all cancers and for 1.8 % of all cancer mortality worldwide. Despite four decades of drug innovation MM remains incurable by means of chemotherapy and/or autologous stem cell transplantation.
Work progress and achievements so far:
We have previously reported in a proof-of-concept study (Themeli et al. Nat Biotechnol 2013) that genetic engineering of T-cell derived induced pluripotent stem cells (TiPSC) with Chimeric Antigen Receptors (CARs) can be an efficient strategy to concomitantly harness the unlimited availability of induced pluripotent stem cells and direct the specificity and functional potential of TiPSC-derived T cells. Based on this technology, we aimed in this study to further investigate novel stem cell genetic engineering strategies in order to obtain in vitro, unlimited, safe and broadly applicable T cells. We proposed to target MM with a novel inducible CD38-targeting CAR (CD38CAR). In addition, we aimed to simultaneously knockout the expression the endogenous T cell receptor (TCR) and the HLA antigens on the CAR-engineered TiPSCs (CARTiPSC) in order to extend the applicability of CARTiPSC-derived T cells across HLA-barriers.
Objective 1: We generated CAR constructs from human anti-CD38 antibodies. CD38CART cells effectively eradicate human MM cell lines and MM tumors in vitro and in vivo (Drent et al. Mol Ther 2017). We attempted to improve the safety of TiPSC-derived CD38CART cells by using a doxycycline (Dox)-controlled expression of the CAR with a Tet-on system, thus, the expression of the CAR could be induced by the administration of Dox. Using the CRISPR/Cas9-mediated gRNA-guided genome editing technology, we targeted both TRAC (TCRa chain) alleles of TiPSCs in order to generate TCR knockout TiPSC lines. 3 TCR-knockout clonal lines were selected, which expressed the CD38CAR upon Dox treatment. Inducible CD38-TiPSC were further successfully differentiated to mature CD8ab T cells.
Objective 2: For this obective we modified the HLA class I expression of inducible CD38-TiPSCs which were generated in objective 1. We generated b2m-/- inducible (ind)CD38-TiPSCs using the CRISPR/Cas9 system. The generated b2m-/- indCD38-TiPSCs could be efficiently differentiated towards CD8 CD38CAR-T cells in vitro.
Overall results: We have succeeded in generating an unlimited source of non-alloreactive, low-immunogenic inducible CD38CAR-T cells. These cells can recognize and kill MM cells in vitro.
We have previously reported in a proof-of-concept study (Themeli et al. Nat Biotechnol 2013) that genetic engineering of T-cell derived induced pluripotent stem cells (TiPSC) with Chimeric Antigen Receptors (CARs) can be an efficient strategy to concomitantly harness the unlimited availability of induced pluripotent stem cells and direct the specificity and functional potential of TiPSC-derived T cells. Based on this technology, we aimed in this study to further investigate novel stem cell genetic engineering strategies in order to obtain in vitro, unlimited, safe and broadly applicable T cells. We proposed to target MM with a novel inducible CD38-targeting CAR (CD38CAR). In addition, we aimed to simultaneously knockout the expression the endogenous T cell receptor (TCR) and the HLA antigens on the CAR-engineered TiPSCs (CARTiPSC) in order to extend the applicability of CARTiPSC-derived T cells across HLA-barriers.
Objective 1: We generated CAR constructs from human anti-CD38 antibodies. CD38CART cells effectively eradicate human MM cell lines and MM tumors in vitro and in vivo (Drent et al. Mol Ther 2017). We attempted to improve the safety of TiPSC-derived CD38CART cells by using a doxycycline (Dox)-controlled expression of the CAR with a Tet-on system, thus, the expression of the CAR could be induced by the administration of Dox. Using the CRISPR/Cas9-mediated gRNA-guided genome editing technology, we targeted both TRAC (TCRa chain) alleles of TiPSCs in order to generate TCR knockout TiPSC lines. 3 TCR-knockout clonal lines were selected, which expressed the CD38CAR upon Dox treatment. Inducible CD38-TiPSC were further successfully differentiated to mature CD8ab T cells.
Objective 2: For this obective we modified the HLA class I expression of inducible CD38-TiPSCs which were generated in objective 1. We generated b2m-/- inducible (ind)CD38-TiPSCs using the CRISPR/Cas9 system. The generated b2m-/- indCD38-TiPSCs could be efficiently differentiated towards CD8 CD38CAR-T cells in vitro.
Overall results: We have succeeded in generating an unlimited source of non-alloreactive, low-immunogenic inducible CD38CAR-T cells. These cells can recognize and kill MM cells in vitro.
Adoptive CAR-T cell therapy for cancer has emerged as potentially curative alternative with breakthrough results from clinical trials initiated in the USA. The development of ‘off the shelf’ applicable immunotherapeutic tools will lift immunotherapy from an individual basis and will allow the availability of controlled, validated and safe immunotherapeutics for a broad patient population. Multiple myeloma is the second most common hematologic malignancy and thus, a broadly applicable adoptive T cell immunotherapy would be of benefit for many patients. This project will lay the foundation of a new strategy for the broad application of iPSC-derived T cells, not only for targeting MM, but also for all CAR based T cell therapies, since the results obtained from our studies could be translated also to other malignancies. The protocols and therapeutic cells generated in this study can be the basis of a new GMP graded production process, in which large batches of CARTiPSC-derived T cells will be generated and applied in early clinical phase I/II trials to test their safety and efficacy.