Cloning and expansion of antigen-specific T cells using iPS cell technology: development of “off-the-shelf” T cells for the use in allogeneic transfusion settings
Recent advances in adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) have led to moderate therapeutic anti-cancer effects in clinical trials. However, a critical issue, namely that CTLs collected from patients are easily exhausted during expansion culture, has yet to be solved. To address this issue, we have been developing a strategy which utilizes induced pluripotent stem cell (iPSC) technology. This strategy is based on the idea that when iPSCs are produced from antigen-specific CTLs, CTLs regenerated from such iPSCs should show the same antigen specificity as the original CTLs. Pursuing this idea, we previously succeeded in regenerating melanoma antigen MART1-specific CTLs, and more recently in producing potent CTLs expressing CD8αβ heterodimer. We are now developing a novel method by which non-T derived iPSCs are transduced with exogenous T cell receptor genes. If this method is applied to Human Leukocyte Antigen (HLA) haplotype-homozygous iPSC stock, it will be possible to prepare “off-the-shelf” T cells. As a first-in-human trial, we are planning to apply our strategy to relapsed acute myeloid leukemia patients by targeting the WT1 antigen.
KeywordsAdoptive immunotherapy iPS cells CD8αβ T cells WT1 antigen
Cancer immunotherapy; current status and recent issues
Cancer immunotherapy has evolved remarkably during the past several years. Immune checkpoint blockade drugs, such as Ipilimumab (anti-CTLA-4 monoclonal antibody (mAb)) and pembrolizumab (anti-PD-1 mAb), have shown therapeutic effects leading to their subsequent approval for the treatment of some types of cancers [1, 2, 3]. However, their therapeutic efficacy reaches only 20–30%, and side effects such as autoimmune reactions and other immune-related adverse events are frequently observed .
As another strategy, the therapeutic delivery of CTLs directly into patients has also shown good results. For example, Rosenberg and colleagues have demonstrated that administration of ex vivo expanded tumor-infiltrating lymphocytes (TILs) was effective in melanoma patients . Transfer of T cell receptor (TCR) genes into patients’ peripheral T cells has achieved good clinical outcomes as well. For example, NY-ESO1 antigen-specific TCR gene therapy has been shown to be effective in melanoma, synovial sarcoma, and multiple myeloma [6, 7, 8], indicating that targeting a single antigen can be effective for some types of cancer. In addition, T cell-enforced expression of chimeric antigen receptor (CAR), an engineered receptor molecule which combines an antibody recognition domain and cytoplasmic signaling domains, has shown enormous efficacy in the treatment of B cell leukemia .
In both TCR and CAR engineering, peripheral T cells are transduced by a retrovirus, bringing about the risk of tumorigenicity due to the random integration of a transfected gene into the genome. Moreover, in the autologous setting it would be costly to produce T cells, and in some cases it will be difficult to obtain potent CTLs from patients.
Cloning and expansion of T cells using reprogramming technology
Based on this concept, several groups at Riken Research Center for Allergy and Immunology including ours launched an initial project in 2004, prior to the invention of iPSC technology. At that time, we utilized the nuclear transfer technology for the reprogramming of T cells, and the “clone mouse” and ESCs were successfully produced from murine NKT cells [10, 11].
After the invention of iPSC technology, we came to use it since it is much easier and more efficient than nuclear transfer technology. Groups around H. Koseki and S.I. Fujii in Riken continued to focus on NKT cells, while our group’s effort turned towards the regeneration of CTLs with the aim to target tumor antigens.
Regeneration of CTLs utilizing iPSC technology
At first, we produced iPSCs from human CTLs specific for melanoma antigen MART1 . As a cell source, we obtained long-term cultured MART1-specific CTLs established from a melanoma patient by the National Institute of Health (NIH) (Fig. 1b, c). We then regenerated CTLs from MART1-T-iPSCs and found that almost all regenerated T cells expressed TCR specific for MART1 antigen (Fig. 1d).
Concurrently, H. Nakauchi’s group introduced the Riken method and reported their success in regenerating CTLs specific for viral antigen  in the same issue as ours. Later that year, M. Sadelain’s group reported that CAR-expressing T cells were regenerated from T cell-derived iPSCs transduced with a CAR gene , and H. Wakao’s group succeeded in regenerating human MAIT cells from MAIT-derived iPSCs . Recently, two groups, S. Kaneko’s group and S. I. Fujii’s group, reported the regeneration of human NKT cells [16, 17].
Development of a method to induce potent CTLs expressing CD8αα heterodimer
Regeneration of CTLs specific for WT1 antigen
Regenerated CTLs were able to kill leukemia cells expressing endogenous WT1 antigen (data not shown). Regenerated CTLs were also able to prolong the survival in a xenograft leukemia mouse model where regenerated WT1-CTLs were infused after the inoculation of HL60 human leukemia cells (Fig. 3b).
We also investigated the safety of regenerated CTLs. It is known that when human peripheral T cells are infused into immunodeficient mice, mice will soon die due to graft-versus-host disease caused by xeno-reactive T cells present in the polyclonal T cell population. In contrast, in our approach regenerated T cells are monoclonal and thus thought to be basically safe. Indeed, when we infused CTLs into immunodeficient mice, no tissue damage was observed during a follow-up period of 6 months. We also checked the risk of tumorigenicity of infused cells; no leukemic change was observed in the infused mice during 6 months of follow-up (data not shown).
The TCR-iPSC method; another strategy toward the allogeneic transfusion setting
To examine whether this idea works in practice, we cloned WT1-specific TCR genes from CTLs regenerated from WT1-T-iPSCs and transduced them into iPSCs that had been originally derived from monocytes of an HLA-haplotype homozygous healthy volunteer (WT1-TCR-iPSCs). Using these WT1-TCR-iPSCs, we succeeded in regenerating CD8αβ CTLs which exhibited a growth potential and antigen-specific cytotoxic activity comparable to CD8αβ CTLs regenerated from T-iPSCs (data not shown).
We think that applying this method will dramatically expand the versatility of our strategy. We plan to utilize TCR genes whose efficacy and safety have been clinically tested and ensured. As a source of iPSCs, we plan to use those established by the iPSC stock project, which has been initiated in 2013 by the Center for iPS Cell Research and Application in Japan. The iPSC stock project has been preparing HLA-haplotype homozygous iPSCs. Thus, we are envisioning to use this system for preparing “off-the-shelf” T cells by mass production and cryopreservation of regenerated CTLs. Although this idea seems ideal, it presents a combination of regenerative medicine and gene therapy and is as such expected to face some difficulties with regard to regulatory approval.
In the present article, we have proposed a possible strategy to attack cancer by regenerative medicine. Since WT1 antigen is known to be expressed in various types of cancer, the very same CTLs used for the first clinical trial could be used for other types of cancer. We are also thinking of a strategy to target neo-antigens, where we will incorporate personalized medicine into our allogeneic T cell therapy.
We demonstrated that with the technology described in this article it is possible to produce potent CTLs that can kill tumor cells both in vitro and in vivo. However, we are aware that there are several limitations to the current culture procedures which need to be addressed. One point is that the regenerated CTLs are close to effector cells, as shown in our previous report , while it is preferable to use naïve or memory-type cells in adoptive T cell therapy. Another point is that, at present, we are using mouse-derived feeder cells (OP9 and OP9/DLL1 cells) for the production of T cells from iPSCs. Although the use of xenogeneic material during the early phase of the culture period might be acceptable for a small-scale clinical trial, it will be essential to perform all culture procedures under xeno-free conditions before this strategy can be implemented in routine clinical settings.
If this “off-the-shelf” T cell strategy can be realized, it will bring about a phenomenal breakthrough in cancer immunotherapy. Furthermore, we are expecting that this approach could be utilized not only for cancer but also for the treatment of other diseases related to immunity, such as infectious diseases, autoimmune diseases or allergies.