Activated T cells from umbilical cord blood armed with anti‐CD3 × anti‐CD20 bispecific antibody mediate specific cytotoxicity against CD20+ targets with minimal allogeneic reactivity: a strategy for providing antitumor effects after cord blood transplants

Umbilical cord blood transplantation (CBT) has emerged as a viable option for patients with hematologic and nonhematologic malignancies who do not have an HLA-matched sibling or matched unrelated donor.1–4 The advantages of CBT include rapid access to a donor and a greater tolerance for HLA-disparity due to the naivety of the newborn’s immune system. Outcomes with CBT are comparable to transplant with bone marrow or peripheral blood (PB) stem cells with an equal or lesser incidence of acute and chronic graft-versus-host disease (GVHD).2 Unfortunately, CBT has been limited by a greater incidence in transplant-related mortality from opportunistic infections, primarily due to delays in neutrophil engraftment and immune reconstitution.5,6 Thus, strategies to improve engraftment and immune reconstitution, while decreasing relapse rates, may significantly improve outcomes after CBT. The number of total nucleated and CD34+ cells have been identified as the most critical variables that predict engraftment and outcomes after CBT.7 To date, CBT has been successfully employed in children, but its application has been limited in adults by insufficient number of stem cells in a single cord blood (CB) unit.8 Strategies that have been employed in an effort to circumvent the limitation of cell dose include the use of multiple CB units and coinfusion of an ex vivo expanded CB unit.3,7 While transplantation with ex vivo expanded CB cells has failed to enhance engraftment in clinical studies, both preclinical and clinical studies have shown that the cotransplantation of two or more CB units significantly improves the rate of engraftment over a single CB transplant when an insufficient cell dose of hematopoietic stem cells (HSCs) is administered.3,7,9,10 Furthermore, patients receiving double-CB transplants have similar rates of acute and chronic GVHD, transplant-related mortality, and disease-free and overall survival when similar cell doses are compared between single- and double-CB transplants.11,12 These findings support umbilical CB as a worthy stem cell source for allogeneic stem cell transplant (alloSCT) and warrant further investigation into methods to optimize engraftment and immune reconstitution. Strategies that can boost immunity and outcomes after CBT include adoptive transfer of activated T cells or tumor primed T cells.13 Even though CB contains significantly higher absolute numbers of T, NK, and B lymphocytes than adult PB,14,15 CB T cells fundamentally differ from “naive” adult T cells due to a relative Th2 bias with fewer CB T cells expressing HLA-DR and CCR-5 activation markers.16 Moreover, a higher rate of CB T cells progress through cell cycle and enter apoptosis compared with adult blood, indicating high cell turnover.16 In vitro apoptosis of CB T lymphocytes can be prevented by cytokines signaling through the common γ-chain cytokine receptor family including IL-2, IL-4, IL-7, and IL-15.17–19 Circulating neonatal T cells express higher levels of the IL-7 receptor α-chain (CD127) than adult naive T cells.19 IL-7 is involved in thymocyte development at a stage preceding the T-cell receptor rearrangement.20 In contrast to IL-7, IL-15 induces the differentiation of CD8 T lymphocytes in vitro.21 Studies by Szabolcs and colleagues13,22 have reported significantly enhanced T-cell expansion with IL-7, along with IL-2 and CD3/CD28 costimulation. In addition, IL-7 promotes the preservation of a polyclonal T-cell receptor repertoire and a surface phenotype that favors lymph node homing and lacks alloreactivity while reducing the activation-induced cell death. Infusion of expanded CB T cells may alleviate posttransplant lymphopenia and qualitative T-cell defects until immune reconstitution is established. In an earlier study,23 we showed that anti-CD3–activated murine splenocytes could enhance survival in lethally irradiated (9 Gy) 6- to 8-week-old female BDF1 (C57BL6 × DBA, H2b/H2d) mice after transplantation of syngeneic HSC-containing splenocytes. There was a significant enhancement in overall survival after transplant in mice given dose-limiting numbers of unmanipulated splenocytes. The infused anti-CD3–activated murine splenocyte population consisted of 77.6% CD3+ cells. More recently, Hexner and coworkers24 showed that T cells can enhance hematopoietic engraftment by stimulating stem-cell differentiation in human xenografts in immunodeficient mice. Consistent with these findings, immune reconstitution is significantly delayed in patients who receive T-cell-depleted alloSCT. These studies strongly suggest that T cells play an important role in facilitating engraftment and/or hematopoiesis. A graft-versus-leukemia (GVL) effect after alloSCT plays a major role in eradicating residual disease after chemotherapy with both myeloablative and nonmyeloablative conditioning regimens.25 Although GVHD provides the benefit of overlapping GVL effect, it is restricted by its toxicity and high mortality rate. We hypothesize that CB anti-CD3–activated T cells (CBATCs) can be expanded ex vivo, armed with bispecific antibodies (BiAbs) and redirected to tumor targets while providing help for engraftment. In this study, we show that CBATCs and CBATCs armed with anti-CD3 × anti-CD20 BiAb exhibit significantly low alloreactivity and produce low levels of cytokines and chemokines yet mediate effective target-specific killing.