Advances in Adoptive Cell Therapy Using Induced Pluripotent Stem Cell-Derived T Cells

Abstract

Adoptive cell therapy (ACT) using chimeric antigen receptor (CAR) T cells holds impressive clinical outcomes especially in patients who are refractory to other kinds of therapy. However, many challenges hinder its clinical applications. For example, patients who undergo chemotherapy usually have an insufficient number of autologous T cells due to lymphopenia. Long-term ex vivo expansion can result in T cell exhaustion, which reduces the effector function. There is also a batch-to-batch variation during the manufacturing process, making it difficult to standardize and validate the cell products. In addition, the process is labor-intensive and costly. Generation of universal off-the-shelf CAR T cells, which can be broadly given to any patient, prepared in advance and ready to use, would be ideal and more cost-effective. Human induced pluripotent stem cells (iPSCs) provide a renewable source of cells that can be genetically engineered and differentiated into immune cells with enhanced anti-tumor cytotoxicity. This review describes basic knowledge of T cell biology, applications in ACT, the use of iPSCs as a new source of T cells and current differentiation strategies used to generate T cells as well as recent advances in genome engineering to produce next-generation off-the-shelf T cells with improved effector functions. We also discuss challenges in the field and future perspectives toward the final universal off-the-shelf immunotherapeutic products.

Keywords: T cells; adoptive cell therapy; cancer immunotherapy; chimeric antigen receptor; induced pluripotent stem cells; off-the-shelf T cells; tumor infiltrating lymphocytes.

Figure 1

Generation of iPSC-derived T cells from different somatic cell sources. Non-T cell sources contain germline TCR gene, upon T cell differentiation, the iPSC-derived T cells express random TCR. These T cells can be used for studying normal T cell development and disease modeling. For applications in ACT, the exogenous TCR can be introduced to the iPSCs. Upon T cell differentiation, the transgenic TCR generates the CD3 signal, which then leads to allelic exclusion and inhibition of endogenous TCR rearrangement; therefore, the iPSC-derived T cells express the transgenic TCR to target specific antigens. Alternatively, peripheral blood T cells can serve as a cell source for iPSC generation. T cell has the rearranged TCR gene, which is retained throughout the reprogramming and differentiation process. For applications in ACT, T-iPSCs can be engineered with CAR to enhance tumor specificity, or the antigen-specific T cell clone can be used for reprogramming to generate the antigen-specific T cells.

Figure 2

Developmental markers during T cell differentiation and strategies to generate iPSC-derived T cells. The initial step of hematopoietic differentiation can be achieved by various protocols, including feeder-free protocols such as monolayer system, co-culture with mouse stromal cells and EB formation. During this step, the mesodermal (ME) cells expressing Brachyury and KDR are generated. The ME cells are committed further to HE, which express KDR, CD31, CD34 and CD144. During EHT process, CD43⁺ HSPC emerges from the HE layers. Specification of T cell lineage requires Notch signaling, which can be provided through co-culture with mouse stromal cells such as OP9-DL1 or OP9-DL4. Co-culture of iPSC-derived multipotent HSPCs with these cells in 2D or 3D system efficiently generates mature T cells with phenotypes CD8⁺ CD4^- TCR⁺ and CD3⁺. Alternatively, the Notch signals can be provided through a coating matrix mixture of retronectin and recombinant DL4 protein.

Figure 3

Engineered T-iPSC-derived T cells for next-generation ACT. Genome editing technologies can be used to eliminate the endogenous TCR to reduce the risk of graft-versus-host-disease (GvHD) or HLA molecules to reduce the risk of immune rejection for allogeneic use, or to introduce CAR to specifically target cancer cells. However, the conventional CAR with three ITAM motifs generates higher CD3 signals than endogenous TCR and results in altered T cell differentiation of iPSCs. CD19-1XX CAR construct is the novel CAR construct with mutated second and third ITAM motifs to reduce the CD3 signal. Apart from CAR, the iPSC-derived T cells can be modified to express MR1-restricted TCR to target a wide range of cancer cells. Other strategies to enhance cytotoxic activity and persistence include the expression of hnCD16 and IL-7 RF.