Ex Vivo Generation of Human T-Cell Progenitors from Cord Blood CD34+ Cells Can Accelerate Immune Reconstitution after Umbilical Cord Blood Transplantation in Adult Patient with Hematologic Malignancies
Authors: Forcade Edouard , Huynh Anne , Magrin Elisa , Diana Jean-Sebastien , Schwartz Eden , Aussel Clotilde , Gabrion Aurélie , Maulet Axelle , Girardot Alice , Ternaux Brigitte , Tuchmann-Durand Caroline , Baek Jimni , Lhermitte Ludovic , Touzart Aurore , Asnafi Vahid , Bouazza Naim , Bauquet Aurelie , Soheili Shabi , Negre Olivier , André Isabelle , Treluyer Jean-Marc , Hermine Olivier , Cavazzana Marina 
Published: December 2024

Introduction: Allogeneic hematopoietic stem cell transplantation (AlloHSCT) remains a standard treatment for high risk hematologic malignancies. Umbilical cord blood (UCB) grafts are often used for patients without HLA- matched sibling donor (MSD) or an appropriate unrelated donor (10/10 UD) as they allow for greater HLA incompatibility due to cord blood-derived T cells being antigen naïve. This HLA disparity increases the risk of acute and chronic graft-versus-host disease (GVHD), and is associated with post- transplant immunodeficiency with increased risk of infections and relapse. The risk of infection and non- relapse mortality (NRM) is significantly higher in UCB transplantations (UCBT) compared to MSD, 10/10 MUD (matched-unrelated donor) transplantations, or haplo-identical HSCT, attributed to delayed immune reconstitution affecting various immune cell populations like CD4 and CD8 T cells, Treg, NK, iNKT, pDC and others. Consequently, UCB transplants are now less frequently used, with haplo-identical AlloHSCT emerging as an alternative for ethnical minorities or rare haplotypes. Previous reports (PMID: 32622752) showed that greater T cell recovery at day 100 correlates with improved post UCBT outcomes.
We hypothesized that a rapid establishment of T-cell-mediated immunity after a partially HLA-compatible UCBT could reduce infection and relapse risks without increasing GVHD. We developed a protocol using ex vivo- committed Human T Lymphoid Progenitor cells (HTLPs) that seed the thymus immediately after transplantation, accelerating the production of mature and polyclonal T cells.
Methods: The safety and efficacy of HTLPs (characterized by CD7 surface marker) to accelerate immune reconstitution were evaluated in a phase I/II multicentric and single-arm clinical trial after a single UCBT in adults with hematologic malignancy, adding back HTLPs generated from a second UCB (NCT04707300). The drug product (DP) consists of the cell suspension obtained after 7 days of ex-vivo CD34+ cell culture in contact with the fusion protein DL-4, Retronectin® and a combination of cytokines (PMID: 34117371), to generate HTLPs. After a conditioning regimen, without serotherapy, HTLPs were injected intravenously before unmanipulated UCB HSCT on Day0. To assess safety, dose-limiting toxicity (DLT) and efficacy, we planned a dose escalation, starting from 0.5x106 CD7+ cells/kg body weight, in order to evaluate the incidence of grade III-IV GVHD related to HTLPs, if any. The presence of &gt; 50/mm3 CD4+ T cells at two consecutive measures within four months post HSCT has been set as the efficacy threshold. Thymic regeneration was evaluated by tracking Recent Thymic Emigrant (RTE, CD4+CD31+CD45RA+) cells, imaging, and repertoire analysis.
Results: We assessed the safety of the DP at starting dose of 0.5x106 CD7+cells/kg (n=2) and at 0.9 x106 CD7+ cells/kg (n=2). Patients underwent UCBT for anaplastic lymphoma (n=2), high risk acute myeloid (n=1) and acute lymphoblastic leukemia (n=1), with a median differentiation around 88% and proliferation index of 14. No or low CD3+ cells (&lt;5000 cells/kg) were detectable in the DP after differentiation.
Primary engraftment was confirmed for all patients and importantly no early toxicity due to HTLPs has been reported, reassuring the safety of the procedure. Chimerism analysis on whole blood at day 30 after transplantation showed a full donor chimerism for the four patients. One patient died from severe acute GVHD linked to the T-cell expansion of unmanipulated UCB, confirmed by SNP chimerism on T-cells. All other patients had significant CD4+ T-cell expansion post-transplantation compared to the historical cohort and achieved a median count of 230/mm3 CD4+ T-cells within four months post HSCT. Notably, all patients exhibited an early increase in RTE within three months, and qualitative assessment of T-cell diversity through analysis of repertoire. More interestingly, a 63-year-old patient restored a higher thymic function, suggesting regenerative properties of HTLPs on thymic epithelium.
Conclusions: First dosing of HTLPs in the first human clinical trials using UCB confirmed the safety and the reproducibility of GMP manufacturing. Thus, HTLPs may offer an exciting perspective for improving immune reconstitution in HSCT.