Anton Simeonov Ph.D. National Institute of Health

In this literature review, learn how researchers generated tumor-targeted human T lymphocytes from iPSCs for cancer therapy.

Adoptive T-cell therapy is a promising new technique in which T cells are collected from a cancer patient, genetically engineered to express chimeric antigen receptors specifically directed towards antigens on the patient’s tumor cells, and are then infused back into the patient. Unfortunately, the approach requires laborious cloning of antigen-specific T cells or the genetic engineering of autologous T cells from each individual patient and is limited to antigens for which patient-specific T cells can be detected, as well as the need to match T-cell specificity to the human leukocyte antigen (HLA) of the recipient patient in cases of orthologous grafts. A facile platform to produce large quantities of antigen-specific T lymphocytes thus represents an unmet need. Production of T lymphocytes from human induced pluripotent stem cells (iPSCs) in vitro is feasible, offering the potential for such scaled-up production.

In the present work, Themeli and colleagues* combine the aspect of unlimited availability of iPSCs with the ability to custom-tailor the specificity of a T cell through the chimeric antigen receptor technology to generate phenotypically defined, functional, and expandable T cells that are genetically targeted to a tumor antigen of interest (Figures A–F). Peripheral blood T lymphocytes from a healthy volunteer were transduced with two retroviral vectors each encoding two of the reprogramming factors KLF4, SOX2, OCT-4, and c-MYC to produce T-cell iPSCs. An iPSC clone was then stably transduced with a bicistronic lentiviral vector encoding a second-generation chimeric antigen receptor specific for CD19, along with an mCherry fluorescent marker.

Differentiation of 1928z CAR–engineered T-iPSCs into CD19-specific functional T lymphocytes. Figure A: The study concept. Peripheral blood lymphocytes are reprogrammed to pluripotency by transduction with retroviruses encoding c-MYC, SOX2, KLF4, and OCT-4 (Takahashi et al., Cell 2007;131:861–872). The resulting T-iPSCs are genetically engineered to express a CAR and are then differentiated into T cells that express both the CAR and an endogenous T-cell receptor. Figure B: In vitro T-lymphoid differentiation protocol. T-iPSCs were stably transduced with a bicistronic lentiviral vector encoding the 19–28z CAR and the fluorescent marker mCherry. mCherry+ CAR+ T-iPSCs are differentiated in three steps: (i) mesoderm formation (days 1–4), (ii) hematopoietic specification and expansion (days 5–10), and (iii) T-lymphoid commitment (days 10–30). Fluorescence microscopy images (below) show mCherry expression was maintained throughout the differentiation process. Scale bars, 100 μM.

Figures A and B

Figure C: Flow cytometric analysis of 1928z-T-iPSC–derived cells at day 30 of differentiation. Representative plots are of at least five independent differentiations. Figure D: 1928z-T-iPSC-T cells were seeded into cultures of 3T3 cells or 3T3 cells expressing CD19 (3T3-CD19). Co-cultures shown 24 h after T-cell seeding; formation of T-cell clusters and elimination of the 3T3-CD19 monolayer are visible. Scale bars, 100 mM. Figure E: Flow cytometric analysis of CD25 and CD69 expression on the surface of 1928z-T-iPSC-T cells 48 hours after exposure to 3T3 or 3T3-CD19 cells. Figure F: Luminex multiplex cytokine analysis of culture supernatant 24 hours after seeding of 1928z-T-iPSC-T cells on 3T3 or 3T3-CD19 cells. Data are presented as mean of two independent experiments ± SD. CAR, chimeric antigen receptor; iPSC, induced pluripotent stem cell; GFP, green fluorescent protein; BMP, bone morphogenic protein; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; SCF, stem cell factor; IL, interleukin; TCR, T-cell receptor; TNF, tumor necrosis factor; IFN, interferon.

Figures C, D, E, and F

The authors found that the generated T cells have the requisite properties of γδ T cells; moreover, the iPSC-derived T cells suppressed tumor growth in a mouse model in which animals were inoculated with human Burkitt lymphoma cells. Thus, the combined use of iPSCs and chimeric antigen receptor technologies can potentially deliver large quantities of T lymphocytes targeted to a chosen antigen.

Overall, the above approach appears to be promising, but as with all types of gene manipulations, the risk of insertional mutagenesis associated with the chimeric antigen receptor engraftment will need to be evaluated extensively.

*Abstract from Nature Biotechnology 2013, Volume 31: 928–933

Progress in adoptive T-cell therapy for cancer and infectious diseases is hampered by the lack of readily available, antigen-specific, human T lymphocytes. Pluripotent stem cells could provide an unlimited source of T lymphocytes, but the therapeutic potential of human pluripotent stem cell–derived lymphoid cells generated to date remains uncertain. Here we combine induced pluripotent stem cell (iPSC) and chimeric antigen receptor (CAR) technologies to generate human T cells targeted to CD19, an antigen expressed by malignant B cells, in tissue culture.

These iPSC-derived, CAR-expressing T cells display a phenotype resembling that of innate γδ T cells. Similar to CAR-transduced, peripheral blood γδ T cells, the iPSC–derived T cells potently inhibit tumor growth in a xenograft model. This approach of generating therapeutic human T cells “in the dish” may be useful for cancer immunotherapy and other medical applications.

Anton Simeonov, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Nature Biotechnology titled “Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy.” Authors of the paper are Themeli M, Kloss CC, Ciriello G, Fedorov VD, Perna F, Gonen M, and Sadelain M.

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