Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative approach for relapsed hematological malignancies. After 5 years of transplantation, the long-term survival of young (≤20 years) and adult patients with acute lymphoblastic leukemia (ALL) has been shown to range from 25 to 65% and from 20 to 50%, respectively . Donor-derived T and natural killer (NK) cells emerge after transplantation and control leukemia mainly through the graft-versus-leukemia (GVL) effect. However, disease recurrence remains the main cause of failure; a limitation that points to the need of developing treatment strategies in the context of pretransplantation or posttransplantation intervention to improve the GVL effect in higher risk patients. Since therapy intensification in the treatment of hematological malignancies seems to have reached a plateau in efficacy, it is highly likely that the development of innovative approaches, such as targeted therapy by small molecule inhibitors, monoclonal antibodies (mAbs)-based immunotherapy and cell-based strategies potentiated by gene therapy [2,3][2,3], will be needed to ameliorate the overall survival of leukemia patients.
Adoptive cell therapy (ACT) using genetically engineered T cells has proven successful in curing both lymphomas and acute and chronic leukemias. Thus, in the near future, it will most likely become an important part of consolidation therapy together with traditional treatments . In particular, cytotoxic T cells engineered to express chimeric antigen receptors (CARs) on their surface have been shown to recognize specific antigens on tumor cells and exert an antitumor response. Strategies to reduce relapse using CARs rely on the use of T cells that can be collected from either the patient, in an autologous or allogeneic post-HSCT setting, or a donor. This review describes the latest evidence supporting the effectiveness of donor-derived CAR-redirected T-cell therapy.
INTEGRATING TRANSPLANTATION WITH IMMUNOTHERAPY BY CD19-TARGETED-T CELLS
T cell-mediated tumor recognition is known to play a pivotal role in leukemia control. However, donor lymphocytes do not always eradicate an established leukemia resulting in the failure of HSCT to cure a significant number of patients.
One contributing factor beneath relapse is the development of different mechanisms that lead to cancer escape through inhibition of effective antitumor immune responses. ACT by infusion of T lymphocytes has emerged as an attractive option to overcome tolerance and eliminate cancer cells relapsing after HSCT. The proof-of-principle of this strategy relies on clinical data showing the evidence of persistent and complete response mainly due to the GVL effect, in the context of allogeneic HSCT, and donor lymphocyte infusion (DLI), in the case of relapse after transplantation. In this regard, the DLI approach showed a limited efficacy in producing a long-lasting GVL effect in acute leukemia or resulted into a life-threatening graft-versus-host disease (GvHD) associated with the infusion of high number of T cells . In our institution, escalating doses of DLI, starting from 1 × 106 in the human leukocyte antigen (HLA)-identical setting, 5 × 105 in the HLA-matched setting and 1 × 105 in the HLA-mismatched setting, were used in the case of minimal residual disease (MRD) positivity after HSCT in ALL patients. Despite the absence of clinical evidence of GvHD following DLI, all the patients relapsed .
Encouraging results have also been achieved using cytotoxic T cells modified by gene transfer. In particular, viral vector-mediated gene transfer of tumor-specific CARs was proven successful in the treatment of B-lineage neoplasms [6▪▪], making this approach one of the most promising therapeutic options in the context of ACT against cancer. CARs are artificial T-cell receptors generated by joining the heavy and light chain variable regions of a monoclonal antibody, expressed as a single-chain Fv (scFv) molecule, to the cytoplasmic T-cell receptor (TCR) signaling and costimulatory domains . The advantages of this approach lie in the ability of CAR-redirected T cells to recognize antigens in a non-HLA-restricted manner, circumventing the HLA-downregulation tumor escape mechanism. Furthermore, these cells are unlimited to specific haplotypes with respect to classical cytotoxic cells expressing TCR as unique activating receptor. As compared to the mAb approach, CAR-modified T cells confer better biodistribution, immunological memory and persistence overtime. In addition, the design of second-generation and third-generation CARs, carrying one or even more additional costimulatory molecules in the signaling domain, has further improved the persistence of CAR-targeted T cells in a potential hostile tumor milieu . Indeed, initial clinical trials aimed at translating CAR-T cell therapy into the clinic revealed lack of persistence and poor efficacy of effector populations that had been modified to express first-generation CAR molecules . Recently, CD19-specific second-generation CAR T cells, optimized by inserting additional costimulatory molecules, have been shown to control the disease and induce remission in patients relapsed, refractory pre-B-cell ALL [6 ▪▪ ,10,11 ▪ ][6 ▪▪ ,10,11 ▪ ][6 ▪▪ ,10,11 ▪ ]. However, the increased efficacy profile was associated with severe adverse events such as B-cell aplasia − with consequent need of immunoglobulin supplementation − cytokine storm and tumor lysis syndrome induced by CAR-targeted T cells .
Historically, CAR T-cell therapy has been developed using patient-derived T-cell products and consistent manufacturing procedures alongside clinical results obtained by three independent laboratories [6 ▪▪ ,11 ▪ ,13][6 ▪▪ ,11 ▪ ,13][6 ▪▪ ,11 ▪ ,13]. In these studies, autologous CAR T cells were used to overcome the risk of GvHD while exhibiting a potent antitumor activity in both relapsed and refractory B-ALL patients either after or before HSCT. CAR T-cell therapy involves the stimulation of the patient's apheresis with either the OKT3 anti-CD3 mAb [11 ▪ ,13][11 ▪ ,13], or CD3/CD28-coated beads [6 ▪▪ ,10][6 ▪▪ ,10], and subsequent transduction of T cells using retroviral or lentiviral vectors. CAR T cells are then differentiated and infused upon fludarabine and cyclophosphamide conditioning chemotherapy aimed at depleting patient T lymphocytes. With this system, a complete response in 70–90% of patients and stable remission, associated with the persistence of CAR T cells, were achieved. All these studies included patients with blinatumomab-refractory disease and who had previously undergone HSCT, and no detectable GvHD was reported. Despite being derived from the patient, these T cells should be regarded as allogeneic in the posttransplantation setting, as of donor origin. The fact that in some cases patients experienced GvHD after transplantation, but not upon CAR T-cell infusion, suggests that CAR T cells might be tolerized in vivo.
Although patient-derived CAR T-cell therapy has led to encouraging results, there still remain several critical issues that need to be addressed in order to broaden its application into the clinic. For instance, in many patients, T cells isolated from their own blood to be used in existing experimental therapies can lead to a suboptimal CAR immunotherapy product because of previous chemotherapy, bone marrow transplantation or comorbidities. Also, the cost and feasibility of ex-vivo T-cell expansion and modification can hamper a broader application of CAR T-cell therapy. As a result, this therapeutic option remains restricted only to a limited numbers of patients. Moreover, chemotherapy is known to determine lymphopenia, a severe depletion of T cells, and, in some cases, can lead to selective expansion of regulatory T cells . Thus, these variables can contribute to the generation of suboptimal CAR T-cell products in terms of total cell number and fitness. Finally, the sustainability of a patient-specific T-cell product-based therapy, which strictly depends on timeliness and manufacturing expenses, has raised reasonable concerns with regard to the complexity and availability of this type of therapy . In an attempt to solve these limitations, donor-derived CAR T cells have recently been acknowledged as a valid alternative to current immunotherapies.
IMMUNE-MEDIATED INTERVENTION BY USING DONOR-DERIVED CHIMERIC ANTIGEN RECEPTOR T CELLS POSTALLOGENEIC TRANSPLANTATION
Behaving as long-lasting drugs, CAR-redirected immune cells have the potential of controlling active diseases in patients who have relapsed after HSCT by reducing leukemia infiltration and prolonging event-free survival. The adaptation of CAR T-cell therapy to a pure allogeneic setting through donor-derived T cells has been recently evaluated by distinct groups, whose goal was to define whether the cells of donor origin can be considered safe or not. In a dose-escalating study, donor-derived-second generation CD19 CAR-modified virus-specific T cells (VSTs), specific for cytomegalovirus, Epstein−Barr virus and adenovirus, were infused in eight patients relapsed or at high risk of relapse after HSCT. T-cell infusion was carried out in the absence of preconditioning regimens, even though these pretreatments are known to improve the persistence of CAR T cells. The T-cell product manufacturing process required a time period of 40 days and the final surface expression of CAR molecules ranged from 20 to 50%. Despite the infusions being well tolerated with no signs of GvHD, the expansion of infused cells was fairly modest and resulted in only a transient objective response in two out of six patients relapsed post-HSCT , suggesting that donor-derived CD19 CAR VST infusion, in the absence of preconditioning, is not sufficient to produce a long-lasting antitumor activity.
As the incidence of GvHD after DLI correlated with the amount of infused cells, Kochenderfer et al. sought to determine the antitumor response using a small number of donor-derived polyclonal-activated CD19 CAR T cells, ranging from 1 × 106 to 10 × 106. For this purpose, 10 patients affected by chronic lymphocytic leukemia, diffuse or mantle cell lymphoma, were treated with CAR cells after HSCT preceded by at least one standard DLI. Peripheral blood mononuclear cells (PBMCs) obtained from allogeneic transplant donor's apheresis were stimulated with OKT3, engineered by γ-retroviral vector, and differentiated for 8 days to achieve a transgene expression of approximately 60%. Because of safety concerns regarding GvHD, even in this trial no lymphocyte-depleting chemotherapy was performed. Three patients experienced regression of these malignancies and none developed GvHD. Although these findings suggest that CAR T cells exert antitumor activity in patients who were not subject to lymphocyte-depleting chemotherapy, it is likely that additional strategies relying on infusion of repeated doses and preconditioning will have to be devised in order to increase their efficacy.
Novel preclinical approaches have been recently evaluated in relapsed leukemias, such as CAR-redirected CD45RA-negative T cells, aimed to limit the risk of GvHD, and interleukin (IL)-12-secreting umbilical cord blood (UCB)-derived T cells. CD45RA-negative T cells transduced with CD19 CAR were effective in killing leukemic blasts, preserving memory response against pathogens while showing minimal alloreactivity in vitro and in vivo. UCB T cells modified to express both CD19 CAR and IL-12 were shown to specifically kill leukemic blasts and to enhance survival of tumor-bearing mice, providing a rationale for possible clinical applications in UCB transplant recipients .
As the standard protocol to modify effector T cells is based on clinical-grade recombinant viral vectors – being efficient and standardized, but with obvious issues pertaining to the manufacturing process − other approaches relying on nonviral methods have also been carried out with the goal to broaden the clinical application of CAR-targeted T-cell therapy. In fact, viral vector manufacturing is time-consuming, expensive and retains risks that need to be taken into account if the treatment will be translated into a clinical setting. In this regard, the MD Anderson Cancer has recently begun a Phase I and II trial using the nonviral Sleeping Beauty transposon and transposase system for production of CD19-specific CAR-modified T cells to treat patients with advanced B-cell malignancies. Both patient-derived and donor-derived CAR T cells have been used. The manufacturing process consists of 28 days of expansion and selection by repetitive stimulation with artificial antigen-presenting cells (e.g. K562 cells transfected to coexpress CD19, CD64, CD86, CD137L and membrane bound IL-15) and cytokines. Up to date, 42 patients have been enrolled. Three patients of the 21 treated after allogeneic HSCT remain in remission at a median of 5 months following the infusion. No toxicities, including GvHD, have been reported [20▪]. The implementation of T-cell production and modification could serve as a means to improve the therapeutic potential of nonviral-modified CAR T cells. An updated list of clinical studies with donor-derived CD19 CAR T cell after transplantation is shown in Table 1[16,17,20 ▪ ][16,17,20 ▪ ][16,17,20 ▪ ].
On the basis of feasibility and safety results obtained in a Phase I study with an allogeneic cytokine-induced killer (CIK) cell population in patients relapsing after HSCT (NCT01186809), our laboratory sought to optimize a clinical-grade stimulation protocol to generate and propagate CIK cells genetically modified to express CAR molecules through the Sleeping Beauty system. CIK cells are an effector CD3+ cell population with acquired non-major histocompatibility complex-restricted NK-like cytotoxicity, produced according to an easy clinical grade-validated protocol starting from a low amount of PBMCs in 18–21 days of culture and enriched in highly lytic CD3+ CD56+ cells. CIK cells have the great advantage of being characterized by negligible alloreactivity, minimal GvHD and intrinsic capability to reach leukemia-infiltrated tissues , thereby offering solutions for a safe evaluation of the allogeneic approach even after lymphocyte-depleting conditioning. The clinical feasibility of allogeneic CIK cells in adoptive cell therapy trials has been previously established by several independent laboratories .
On the basis of these findings, we coupled nonviral Sleeping Beauty-mediated transfection to CIK cell production in order to develop a more cost-effective and practical scale-up and manufacturing process than the viral transfection protocol. Using our nonviral Sleeping Beauty transposon system, we were able to achieve stable ectopic expression of anti-ALL CD19-specific and anti-AML CD123-specific CAR molecules in CIK cells as well as high levels of modification and efficient expansion of these CAR-redirected CIK cells. These latter ones showed specific cytotoxic activity, cytokine secretion and proliferation upon stimulation with AML and ALL blasts by CD123. CAR or CD19.CAR in vitro, respectively. In parallel with the in-vitro characterization, we prove their antitumor activity in vivo using a xenograft immuno-deficient mouse model (Magnani C.F. et al. unpublished data).
IMPLICATION FOR FUTURE CLINICAL PRACTICE
The use of allogeneic CD19-redirected T cells has paved the way for other complementary approaches in the context of HSCT. One of these is represented by the preemptive use of allogeneic CD19 CAR T cells upon early detection of leukemia relapse following HSCT by MRD stringent monitoring. Pulsipher et al.  have provided evidence that the analysis of risk factors and timing of relapse after allogeneic HSCT might constitute an important prognostic tool in leukemia patients requiring treatment with CD19-redirected T cells. Based on the identification of patients at high risk of relapse after transplant, presence of MRD pre-HSCT or post-HSCT and occurrence of GvHD, the period between day +55 and +200 after HSCT was found to be the optimal window for early immune intervention aimed at preventing relapse.
A second fascinating approach is represented by the use of CD19-redirected T cells as cellular transplant preconditioning whenever optimal disease eradication is not achieved. In line with this concept, the NCT02431988 trial COBALT by University College of London is planning to evaluate the efficacy of CAR19 T-cell therapy as an optimal bridge to allogeneic transplantation in patients with relapsed or resistant diffuse large B-cell lymphomas (DLBCLs). Specifically, the trial intends to evaluate the engraftment and efficacy of such therapy in terms of patients’ eligibility to allogeneic transplantation. Patients will receive preconditioning with intravenous fludarabine and cyclophosphamide prior to infusion of a single dose of CAR-modified T cells. An escalating dose protocol will be employed.
In light of the findings mentioned above, the creation of an off-the-shelf bank of CAR-redirected T cells – where these cells, which pose a minimal risk of inducing GvHD, are ready to be used by clinicians – is now regarded as a step toward eliminating the need of generating patient-specific T cells. In this regard, Torikai et al. reported the production of universal allogeneic tumor antigen-specific T cells from one donor, which then could be administered to several recipients. This remarkable result was achieved by genetically engineering CD19-specific-edited CAR T cells with designer zinc finger nucleases to inhibit expression of the endogenous αβ TCR. This protocol was shown to prevent a GvHD without compromising CAR-dependent functionality.
Relapse after HSCT still remains an unmet clinical need in patients affected by hematological diseases. In this regard, patients with B-cell malignancies after transplantation and posttransplantation have been successfully treated upon infusion of patient-derived CD19-targeted T cells. However, despite the promising results, some issues pertaining to toxicity and sustainability of the final CAR T-cell product have hindered a broad application into the clinic.
Here, we have reviewed the potential advantages of donor-derived CD19-redirected T-cell product in terms of feasibility and cell availability. In light of recent findings, donor-derived CAR T-cell therapy appears to be a safe and more feasible approach than patient-derived CAR T-cell therapy, with the potential of becoming a possible treatment for relapsed disease. Obviously, new experimental procedures aimed at optimizing feasibility of production/modification and clinical efficacy of allogeneic products, without posing a significant risk of GvHD, are likely to increase the range of application of CAR-based immunotherapy in the near future.
Financial support and sponsorship
This work was supported by grants from the AIRC Foundation (AIRC Molecular Clinical Oncology 5 per mille, 9962).
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
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