As promised in our latest article “Current and future directions in CAR-T and TCR-T cell…”, which focused on therapies with the use of modified “classical” T cells against solid tumors, we are initiating a new series of articles that will explore novel approaches and their advantages in cellular immunotherapies.
The high effectiveness of the adoptive immunotherapy in the treatment of human hematologic cancer has pushed current medicine into modern times. Adoptive cell transfer therapies are predominantly based on the transfer of autologous modified T cells. The reason behind sourcing T cells from the patient himself is to prevent the risk of adverse reactions like graft-versus-host disease (GvHD) and host allorejection. Unfortunately, the time needed for T cell isolation, their ex vivo modification and expansion may be too long, particularly for patients with rapidly progressive disease or those with an insufficient amount of the peripheral blood T cells . Therefore, researchers have been exploring different allogeneic approaches for some time, examining their potential effectiveness and safety for the patient – which we would like to briefly cover.
Rapid progress in genome modification has opened up new avenues in the design of cellular immunotherapies for cancer, including the development of “off-the-shelf” immune effector cells, transduced with a chimeric antigen receptor (CAR) or specific TCR construct (Fig. 1.). The development of a readily available source of effector cells derived from allogeneic donors is an attractive approach. However, the use of allogeneic cells carries the risk of GVHD and/or allorejection as a consequence of inappropriate matching of donor and recipient HLA molecules. Two main strategies are now being explored to avoid these adverse reactions and to develop a safe/“universal” source of allogeneic cell products: (i) genome editing of TCR and/or HLA class I expression in T cells and (ii) seeking for naturally occurring T cell subtypes or other populations across human lymphocytes that will not require genome editing for safe application.
Using allogeneic T cells in the adoptive immunotherapy carries the risk of GvHD as a consequence of donor TCRs recognizing the peptides presented by the host HLAs as antigenically foreign. On the other hand, the risk of allorejection is caused by the host TCRs recognizing donor pHLAs. These adverse reactions can be avoided by genetically impairing TCR and/or HLA class I expression in allogeneic T cells (Fig. 2). Disrupting the DNA sequences in the constant regions of α or β subunits of the TCR and/or HLA locus of MHC gene complex is achieved by using gene-editing techniques like: transcription activator-like effector nuclease (TALEN) [3-7] , zinc finger nuclease [8-10](, meganuclease  , megaTAL  and CRISPR/Cas9 system [13-15] . Because it is enough to target the T-cell receptor α-chain constant (TRAC) locus to inactivate TCR function, at present, this approach is the most frequently used. It is important to note that the multiplex genome editing technique using the one-shot CRISPR system allows additional modifications (Fig. 2), such as loss of immune checkpoints, including programmed cell death 1 (PD-1) , or FasL or both PD-1 and cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression .
All of the above approaches were carefully tested, mostly on animal models, and besides providing anti-leukemic effects, they were proven to reduce alloreactivity while improving resistance to spontaneous apoptosis and immunosuppression. The TALEN-modified cell therapy was tested in a small clinical trial in which two infants with relapsed refractory CD19+ B-cell acute lymphoblastic leukemia received allogeneic anti-CD19/TCR−/CD52− CAR-T cells. After a single-dose infusion of previously modified T cells, remission was achieved at 28 days in both infants, and CAR-T cells persisted until the health conditions of the children improved enough to allow successful allogeneic stem cell transplantation . TALEN were also used to disrupt the TRAC locus to generate universal allogeneic CAR-T cells against the tumor-associated antigen CS1, which are currently tested in relapsed and refractory multiple myeloma patients (NCT04142619). In another ongoing clinical trial (NCT04150497), the TALEN method was used to generate universal anti-CD22 CAR-T cells to treat patients with relapsed or refractory B-cell acute lymphoblastic leukemia.
The one-shot CRISPR-Cas9 (a revolutionary gene-editing technology) is another method being tested in ongoing clinical trials. Stadtmauer et al. (2020) demonstrated for the first time in a human clinical trial (NCT03399448) the safety and feasibility of this method in an autologous setting . The potential therapeutic effect of allogeneic T cells modified by the CRISPR method is being currently tested in ongoing clinical trials in patients with B-cells leukemia and lymphoma (NCT03166878, NCT04035434), multiple myeloma (NCT04244656) and renal cell carcinoma (NCT04438083). All these studies, although at the beginning of development, are promising off-the-shelf therapies that avoid GvHD responses and improve the persistence of infused allogeneic T cells in the recipient.
An alternative approach to generate “off-the-shelf” products is to search for naturally existing immune cell types with a favorable safety profile. These “universal” donor immune cells do not induce GvHD, so they do not require extensive genome modification and have the potential to attack different types of tumor cells. Natural Killer (NK) cells are a perfect example, however, other T cell subtypes or populations, including virus specific memory T cells, CD1- and MR1-restricted T cells as well as γδ T cells are also promising cell types to be used in cancer immunotherapies.
As opposed to T cells, NK cells do not express TCR responsible for GvHD development and thus do not require TCR deletion . In addition, high cytotoxic potential of NK cells against tumor cells goes along with the lack of IL-1 and IL-6 cytokines production, important mediators for cytokine-release syndrome [21, 22].
Unfortunately, the production of CAR-NK cells faces several practical problems compared to CAR-T cells, for example smaller NK cell numbers in donor blood, limited ex vivo expansion capacity, lower transduction efficiency, and loss of NK cell cytotoxic activity . Despite these difficulties, in a recent clinical trial (NCT03056339), NK cells isolated from cord blood and modified in vitro into anti-CD19 CAR-NK cells, showed high efficiency in patients with hematological malignancies, (64% of complete responses) without manifestation of major toxic effects .
Conventional αβ-TCR T cells selected in the thymus are strongly restricted to self MHC and are a major pool of T cells circulating in the human body . The polyclonality and a huge variety of TCR receptors allow T cells to recognize a wide repertoire of antigens that protect us not only from rapidly evolving pathogens, but also are responsible for maintaining tolerance. The TCR diversity is not constant during T cell differentiation and gets reduced upon antigen specific activation and clonal expansion such as in the course of T cell response to viral infection.
The effectiveness of allogeneic memory virus-specific T cells has been proven in many clinical trials , and most importantly, it has been observed to have a very limited GvHD, which makes these cells a potential universal candidate for the cancer treatment . Indeed, the use of memory virus-specific T cells as a universal starting platform for transduction of CAR or TCR is currently being investigated in phase I and II trials. For example, infusion of anti-CD19 CAR modified virus specific T cells in B-acute lymphoblastic leukemia patients led to cytotoxic elimination of B cells and complete remission in 2 out of 8 patients (NCT00840853).
In a study on preventing acute myeloid leukemia recurrence in patients after allogeneic hematopoietic cell transplantation, the relapse-free survival was 100% at a median of 44 months (a control group patients had 54% relapse-free survival) following infusion of virus-specific T cells expressed a Wilms tumor antigen-1 specific TCR (NCT01640301) .
Which of the improvements in the treatment of hematological and non-haematological patients using allogeneic cells described here will find further practical application is difficult to predict, so please stay connected and follow our blog post on novel therapy types. In our next article, we will continue on the allogeneic approach and describe the potential use of other unconventional subsets of T cells, such as iNKT cells, MR1-restricted T cells (invariant mucosa-bound T cells) and γδ-TCR T cells as “universal” donors in cancer immunotherapy.