Enthusiasm with results of early phase trials using chimeric-antigen-receptor (CAR)-T cells targeting CD19 have led to fast approval of this novel immunotherapy for the treatment of acute lymphoblastic leukemia and diffuse large B-cell lymphoma, and to an explosion of clinical trials with such cells. Despite potential for long-term immune surveillance by CAR-T cells, many patients treated on these trials are referred to a consolidative hematopoietic stem cell transplantation, as are all patients responding to CAR-T cells in a study we conducted. Overall, paucity of long-term data and lack of randomized trials focusing on consolidative HSCT impact clinical evidence. Nevertheless, limited T cell persistence and inherent leukemia resistance mechanisms have led us, as well as others, to this clinical decision making, and are hereby reviewed.
Chimeric antigen receptors (CARs) use a non-MHC restricted recognition site (usually a monoclonal-antibody derived ScFv) fused to one or more T cell activation domains, leading to antigen-specific T cell activation when transduced in T cells [1, 2]. CAR-T cells lead the immunotherapeutic revolution for hematologic malignancies, with autologous T cells transduced with a chimeric receptor targeting a cell surface membrane on the tumor lead to outstanding remission rates in B-cell malignancies, and two products have so far been FDA approved for CD19 targeting [3,4,5].
Since 2016, we have initiated a clinical trial of locally produced CD19 CAR-T cells to treat relapsed and refractory B-cell malignancies, using a CAR containing a CD28 costimulatory domain packaged in a retroviral construct (NCT02772198). Patients referred to CD19 CAR T cells have relapsed following or were resistant to several previous therapies. Most patients had a prior hematopoietic stem cell transplant (HSCT), with no significant graft-versus-host disease (GVHD) present at enrollment, and without active immunosuppression. Despite donor chimerism, the risk for GVHD from these T cells was minimal. The most common diagnosis of pediatric patients referred to CAR-T cells was acute lymphoblastic leukemia (ALL), and the remission rate of patients with ALL was 90%, the majority being MRD-negative by PCR for leukemia-specific immunoglobulin rearrangements . Our practice is to recommend a second (or third) HSCT for patients who respond to CAR-T cell therapy unless they suffer from significant comorbidities. Patients with a prior HLA-matched transplant are candidates for haploidentical procedures. Here, I will discuss the reasons leading to our clinical recommendation, by reviewing the data regarding HSCT following CD19 CAR-T cells.
Role of HSCT in ALL
HSCT serves as a consolidative therapy for acute leukemia, leading to cure in many patients with relapsed or very high risk disease. The therapeutic effect of HSCT is combined of direct cytotoxicity from the chemo-radiotherapy administered in the conditioning regimen, along with an immune effect termed graft-versus-leukemia (GVL). Patients transplanted with a lower disease burden have better outcomes . Moreover, patients with mild to moderate GVHD have higher rates of long-term remissions, attributed to an immunologic response against host targets [8, 9]. The major effectors of GVL in ALL are T cells, but most of the antigens targeted by these allogeneic T cells remain unknown. Loss of antigen presenting mechanisms is a known mechanism of leukemic resistance to the GVL effect . CAR-T cells may offer a similar immune-monitoring long-term effect against leukemia, but here a specific known antigen is being targeted . Durable immunologic control following CAR T cells may result in prolonged remission or cure, if
CAR effector cells are durable and functional; and
CAR target is essential for the leukemia.
Durability of effector CAR+ cells
Three major determinants were so far correlated with improved persistence of CD19 CAR-T cells in patients: the conditioning regimen, disease burden, and CAR costimulatory domain.
A lymphodepleting conditioning regimen is used by most groups prior to CAR-T cell infusion, and usually consists of fludarabine (dose range, 75–120 mg/m2) and cyclophosphamide (dose range, 900–2400 mg/m2). This regimen was shown to be superior in adult patients to cyclophosphamide alone, enabling longer durability of effector cells, improved expansion, and better remission rates . A similar trend was seen in pediatric patients with ALL treated with CAR-T cells , confirming this combination for conditioning in the majority of centers administering CAR-T cells to date.
High target burden of >15% CD19 + cells in the bone marrow was found to improve B-cell aplasia and CAR durability in pediatric patients with ALL treated with CD19-41BB CAR . This was not reported by other groups.
The costimulatory domain of the CAR is by far the most significant determinant of CAR-T cell persistence to date. First generation CARs showed poor persistence and expansion when tested in patients, which were improved by adding costimulatory constructs in the CAR . In second generation CARs, the use of 41BB (CD137) as a costimulatory domain induces less exhaustion and a tendency toward central memory phenotype of CAR T cells, leading to superior results compared with CD28-based CARs in preclinical models [15, 16]. CD19 CAR-T cells with a 41BB domain used by several groups showed prolonged persistence: The 6 month probability of 41BB-based CD19 CAR persistence in the study conducted at the Children’s Hospital of Philadelphia was 68% , in comparison with lack of detectable CAR-T cells beyond 68 days in children treated at the National Cancer Institute with a CD28-based CAR .
Overall, the durability of effector CAR T cells may be achieved in some patients, reported to have detectable CAR + cells several years after therapy. Nevertheless, this still cannot be anticipated a-prior. Preclinical models using stem-memory T cells  or more physiologic control of the transduced CAR  may further improve the CAR durability.
Is CD19 an essential target on ALL?
CD19, a B-cell receptor co-receptor, was thought to be essential for ALL, and thus ideal as a CAR target . Forms of B-cell precursor ALL not expressing CD19 were first observed in clinical trials of immunotherapy targeting this antigen, in ~20–40% of patients [13, 17, 21]. Similar to other forms of anti-cancer therapy, continuous exposure to immunotherapy may induce resistance through evolutionary stress. Thus, higher rates of CD19-negative phenotypes are seen in trials with longer persisting CAR T cells (Table 1), but even short-term targeting may result in sufficient pressure to induce this form of relapse.
Interestingly, in many cases an isoform of CD19 is still expressed, while the targeting epitope in exon 2 of CD19 is lost secondary to mutations or alternative splicing, thus providing still some signaling . The CD19 isoform lacking exon 2 is found in many leukemia upfront by mRNA , but rarely does it lead, prior to CD19-selective pressure, to loss of antigen expression. Currently, prediction based on exon splicing cannot be made prior to cell therapy.
Another form of CD19 loss is lineage switch, in which a B-cell phenotype ALL relapses as a myeloid leukemia [24, 25]. Leukemia is known as a clonal disease, and relapse can arise from the major leukemic clone at diagnosis, but also from ancestral clones . Lineage switch has been mostly observed in MLL-rearranged ALL [25, 27], but also in BCR-ABL1 + ALL , ZNF384-rearranged , and other leukemia subtypes. The two former groups are thought to arise from an hematopoietic stem cell, thus relapse may occur from different ancestral stages which are not committed to B-cell lineage and CD19 expression is essential to their lineage program. Different immunoglobulin rearrangements seen in MLL-r ALL and BCR-ABL1 + ALL after lineage switch following CD19 directed therapies serves as additional evidence to support this. Thus, these two groups of leukemia may have increased risk of relapse even in the presence of persisting CD19 CAR T cells, and may require additional therapy.
HSCT following CAR T cells
Outcome reports in CD19 CAR T-cell trials focus on hematologic remission, which nowadays is insufficient in ALL, and should be replaced by an MRD-negative response and long-term survival. This is further noted in clinical data from CAR trials: all patients in our trial who had an MRD-positive CR relapsed , as has been reported by others . The method of MRD detection may be also of significance: despite all pediatric patients achieving CR being MRD-negative by flow cytometry, Gardner et al. report that by next-generation sequencing only 17/27 were found MRD-negative following CAR-T cells . Similar results were reported from adults , but this has yet to be correlated with long-term survival. Rising MRD after CAR-T cells induced remission was used as an indication for HSCT by some investigators in the ELIANA trial . Nevertheless, MRD detection has its limitations, and relapses occur even after MRD-negative remissions.
Reports on HSCT following CAR-T cells are varied, and are summarized in Table 1. Park et al. report long-term data of adults with ALL treated with CD28-based short-lived CD19 CAR, with a median durability of 14 days. The median follow-up in the study was 29 months. Seventeen patients underwent an allo-HSCT, with no benefit in event-free survival whether patients who entered CR were transplanted or not . Of note, despite lower relapse rates after HSCT, the transplant related mortality of this small cohort is relatively high (35%). The pediatric branch from the NCI reported different results following a CD28-based short-lived CAR: A total of 28 patients achieved flow-cytometry based MRD-negative CR following CAR-T cells. After a median follow-up of 19 months, only two of 21 patients consolidated with an HSCT had relapsed, compared to 6 out of 7 patients who did not have a subsequent transplantation. The group calculated a hazard ratio of 16.9 for relapse if not having an HSCT following an MRD-negative response to CD28-based CAR T cells .
Using longer-lived 41BB-based CAR for the treatment of adult ALL, Turtle et al. report that most relapse events occurred in the absence of fludarabine in the conditioning regimen, resulting in rapid loss of effector cells . Following a combined fludarabine and cyclophosphamide conditioning longer durability was shown, and 10 of 17 patients achieving remission were referred to HSCT: eight were alive and in remission, one had a CD19 + relapse, and one died in remission. Four of the seven patients not transplanted were alive in remission, one had relapsed and two died in CR. The median follow-up of this group is relatively shorter.
In a pediatric study with 41BB-based CARs, Gardner et al. report that 11 out of 40 patients underwent an HSCT after entering a remission with CAR-T cells, and 2 of the 11 relapsed, both with CD19 positive disease. Sixteen of the 29 patients who were in CR without a subsequent HSCT had relapsed: 7 with CD19-negative blasts, and 9 still expressing CD19 on leukemia cells . A later report of that study found improved outcome if an HSCT is performed after CAR T cells in two subpopulations: patients with rapid loss of CAR T cells, and patients who have not been transplanted before . Eight patients reported to have been transplanted after achieving remission in the ELIANA trial, using 41BB-based CARs, for loss of effector cells, rising MRD or physician discretion. Four of them were with ongoing remission after HSCT and the clinical outcome of the other four is unknown . In our trial, only four patients who responded to CAR T cells did not undergo HSCT, two relapsed and two others completed recently their treatment and are attempting targeted therapy as maintenance to control their leukemia: one with BCR:ABL + ALL after 3 prior HSCT, and one with severe prior transplant morbidity attempting JAK inhibitors. Two of the 14 patients who underwent HSCT relapsed with CD19 + disease.
Overall, as shown in Table 1, studies with CD28-based short-term CAR T cells have a higher tendency of referral to HSCT compared to studies with 41BB-based CAR T cells. However, with limited data of trials, differences in median follow-up and no design focusing on HSCT, no evidence-based recommendation can be made.
Long-term remissions have been observed following CD19 CAR T cells in ALL, with durable persistence of CAR-T cells. Nevertheless, the presence of long-lived CAR-T cells cannot be assured upfront, and loss of effector cells may occur after several weeks/months in the majority of patients, depending on the conditioning given and the CAR construct. Moreover, increasing observations of CD19 expression loss after CD19 CAR T cells are concerning despite ongoing CAR persistence, and may be related to the origin of the leukemic cells. As leukemia is a heterogenous group of disorders arising from different cells and with different properties, in several leukemia subgroups, targeting of CD19 may not suffice long-term. Deeper states of response, detected by newer technologies for MRD, will aid determining the risk of relapse following CAR-T cells.
New trials of double targeting CARs have launched in the past year, and long-term data is much anticipated. Currently, given the limited data demonstrating lower relapse rates in patients transplanted after achieving remission with CD19 CAR-T cells, our practice is to offer an HSCT to any patient who does not suffer from severe comorbidities when the cells have limited durability. We reason that patients who had a prior HSCT had relapsed due to an insufficient GVL effect, and recommend using a different HLA-matched or HLA-mismatched donor. With increased safety and availability of haplo-HSCT, this is feasible in most patients. Nevertheless, evidence-based results should rely on future randomized trials, in which patients who enter an MRD-negative remission should either undergo HSCT or be followed for ongoing CAR-T cell persistence.
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Publication of this supplement was sponsored by Gilead Sciences Europe Ltd, Cell Source, Inc., The Chorafas Institute for Scientific Exchange of the Weizmann Institute of Science, Kiadis Pharma, Miltenyi Biotec, Celgene, Centro Servizi Congressuali, Almog Diagnostic.
Conflict of interest
The author has received lecture fees from Novartis, Israel, and received grant support from the Dotan Center for Hematologic Malignancies.
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Jacoby, E. The role of allogeneic HSCT after CAR T cells for acute lymphoblastic leukemia. Bone Marrow Transplant 54, 810–814 (2019). https://doi.org/10.1038/s41409-019-0604-3