Arina Lazareva

Genome editing of allogeneic T cells can provide “off-the-shelf” alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor α chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator–like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3’ long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 10 6 to 2.0 × 10 6 CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy.

Introduction: Adequate immune reconstitution post-HSCT is crucial for the success of transplantation, and can be affected by both patient- and transplant-related factors.Areas covered: A systematic literature search in PubMed, Scopus, and abstracts of international congresses is performed to investigate immune recovery posttransplant. In this review, we discuss the pattern of immune recovery in the post-transplant period focusing on the impact of stem cell source (bone marrow, peripheral blood stem cells, and cord blood) on immune recovery and HSCT outcome. We examine the impact of serotherapy on immune reconstitution and the need to tailor dosing of serotherapy agents when using different stem cell sources. We discuss new techniques being used particularly with cord blood and haploidentical grafts to improve immune recovery in each scenario.Expert opinion: Cord blood T cells provide a unique CD4+ biased immune reconstitution. Initial studies using targeted serotherapy with cord grafts showed improved immune recovery with limited alloreactivity. Two competing haploidentical approaches have developed in recent years including TCRαβ/CD19 depleted grafts and post-cyclophosphamide haplo-HSCT. Both approaches have comparable survival rates with limited alloreactivity. However, delayed immune reconstitution is still an ongoing problem and could be improved by modified donor lymphocyte infusions from the same haploidentical donor.

ABSTRACT: CD19-negative relapse is a leading cause of treatment failure after chimeric antigen receptor (CAR) T-cell therapy for acute lymphoblastic leukemia. We investigated a CAR T-cell product targeting CD19 and CD22 generated by lentiviral cotransduction with vectors encoding our previously described fast-off rate CD19 CAR (AUTO1) combined with a novel CD22 CAR capable of effective signaling at low antigen density. Twelve patients with advanced B-cell acute lymphoblastic leukemia were treated (CARPALL [Immunotherapy with CD19/22 CAR Redirected T Cells for High Risk/Relapsed Paediatric CD19+ and/or CD22+ Acute Lymphoblastic Leukaemia] study, NCT02443831), a third of whom had failed prior licensed CAR therapy. Toxicity was similar to that of AUTO1 alone, with no cases of severe cytokine release syndrome. Of 12 patients, 10 (83%) achieved a measurable residual disease (MRD)-negative complete remission at 2 months after infusion. Of 10 responding patients, 5 had emergence of MRD (n = 2) or relapse (n = 3) with CD19- and CD22-expressing disease associated with loss of CAR T-cell persistence. With a median follow-up of 8.7 months, there were no cases of relapse due to antigen-negative escape. Overall survival was 75% (95% confidence interval [CI], 41%-91%) at 6 and 12 months. The 6- and 12-month event-free survival rates were 75% (95% CI, 41%-91%) and 60% (95% CI, 23%-84%), respectively. These data suggest dual targeting with cotransduction may prevent antigen-negative relapse after CAR T-cell therapy.

2] For example, in the ELIANA study, 41% and 53% of patients demonstrated grade 3 to 4 thrombocytopenia and neutropenia, respectively, persisting beyond day 30. Impaired hematological recovery after CAR T-cell therapy can lead to susceptibility to opportunistic infections, bleeding, and/or transfusion dependence, causing significant morbidity and mortality. Pre-CAR T-cell therapy lymphodepletion causes cytopenia that generally recovers in most patients within a month after CAR T-cell therapy. However, a proportion of patients develop persistent cytopenia, particularly neutropenia, associated with bone marrow (BM) hypoplasia that can persist for several months after CAR T-cell therpay. We hypothesized that for those patients who have undergone allogeneic SCT before CAR T-cell therapy and develop persistent cytopenia after CAR-T cell therapy, an unconditioned CD34 1 -selected stem-cell boost (SCB) from the SCT donor could be used to improve the cytopenia, similar to its use in those who have poor graft function after SCT. A cohort of 101 pediatric and young adults from 2 centers in the United Kingdom with r/r B-ALL were treated with CAR T-cell therapy from May 2016 through December 2021. Of those patients, 2 were not evaluable for assessment of cytopenias after day 128 of CAR infusion (1 died on post-infusion day 4, and 1 had a morphological relapse on day 28). Of the 99 evaluable patients, 52 had undergone SCT before CAR therapy and 23 (44.2%) of them developed grade 3 to 4 cytopenia. Of the 47 patients who did not undergo an SCT, 23 (48.9%) developed grade 3 to 4 cytopenia. The difference in the rate of cytopenia between the 2 groups was not significant (P 5 .5999).