Cecilia Barese

The TGF-β/SMAD pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia.

Natural killer (NK) cells are key components of the innate immune system, providing potent antitumor immunity. Here, we show that the tumor growth factor-β (TGF-β)/SMAD signaling pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia (ALL). We characterized NK cells in 50 consecutive children with B-ALL at diagnosis, end induction and during maintenance therapy compared with age-matched controls. ALL-NK cells at diagnosis had an inhibitory phenotype associated with impaired function, most notably interferon-γ production and cytotoxicity. By maintenance therapy, these phenotypic and functional abnormalities partially normalized; however, cytotoxicity against autologous blasts remained impaired. We identified ALL-derived TGF-β1 to be an important mediator of leukemia-induced NK cell dysfunction. The TGF-β/SMAD signaling pathway was constitutively activated in ALL-NK cells at diagnosis and end induction when compared with healthy controls and patients during maintenance therapy. Culture of ALL blasts with healthy NK cells induced NK dysfunction and an inhibitory phenotype, mediated by activation of the TGF-β/SMAD signaling pathway, and abrogated by blocking TGF-β. These data indicate that by regulating the TGF-β/SMAD pathway, ALL blasts induce changes in NK cells to evade innate immune surveillance, thus highlighting the importance of developing novel therapies to target this inhibitory pathway and restore antileukemic cytotoxicity.

Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma

Purpose Transforming growth factor-β (TGF-β) production in the tumor microenvironment is a potent and ubiquitous tumor immune evasion mechanism that inhibits the expansion and function of tumor-directed responses; therefore, we conducted a clinical study to discover the effects of the forced expression of a dominant-negative TGF-β receptor type 2 (DNRII) on the safety, survival, and activity of infused tumor-directed T cells. Materials and Methods In a dose escalation study, eight patients with Epstein Barr virus-positive Hodgkin lymphoma received two to 12 doses of between 2 × 107 and 1.5 × 108 cells/m2 of DNRII-expressing T cells with specificity for the Epstein Barr virus-derived tumor antigens, latent membrane protein (LMP)-1 and LMP-2 (DNRII-LSTs). Lymphodepleting chemotherapy was not used before infusion. Results DNRII-LSTs were resistant to otherwise inhibitory concentrations of TGF-β in vitro and retained their tumor antigen-specific activity. After infusion, the signal from transgenic T cells in peripheral blood increased up to 100-fold as measured by quantitative polymerase chain reaction for the transgene, with a corresponding increase in the frequency of functional LMP-specific T cells. Expansion was not associated with any acute or long-term toxicity. DNRII-LSTs persisted for up to ≥ 4 years. Four of the seven evaluable patients with active disease achieved clinical responses that were complete and ongoing in two patients at > 4 years, including in one patient who achieved only a partial response to unmodified tumor-directed T cells. Conclusion TGF-β-resistant tumor-specific T cells safely expand and persist in patients with Hodgkin lymphoma without lymphodepleting chemotherapy before infusion. DNRII-LSTs can induce complete responses even in patients with resistant disease. Expression of DNRII may be useful for the many other tumors that exploit this potent immune evasion mechanism.

Toward a Rapid Production of Multivirus-Specific T Cells Targeting BKV, Adenovirus, CMV, and EBV from Umbilical Cord Blood

Umbilical cord blood (CB) has emerged as an effective alternative donor source for hematopoietic stem cell transplantation. Despite this success, the prolonged duration of immune suppression following CB transplantation and the naiveté of CB T cells leave patients susceptible to viral infections. Adoptive transfer of ex vivo-expanded virus-specific T cells from CB is both feasible and safe. However, the manufacturing process of these cells is complicated, lengthy, and labor-intensive. We have now developed a simplified method to manufacture a single culture of polyclonal multivirus-specific cytotoxic T cells in less than 30 days. It eliminates the need for a live virus or transduction with a viral vector, thus making this approach widely available and GMP-applicable to target multiple viruses. The use of overlapping PepMixes as a source of antigen stimulation enable expansion of the repertoire of the T cell product to any virus of interest and make it available as a third party “off the shelf” treatment for viral infections following transplantation.

Human parainfluenza virus-3 can be targeted by rapidly ex vivo expanded T lymphocytes

BACKGROUND AIMS Human parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients and currently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients but requires determination of T-cell antigens on targeted viruses. METHODS HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by interferon (IFN)-γ ELISpot, intracellular cytokine staining and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51Cr release and CD107a mobilization assays. Virus-specific T cells targeting six viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T cells was determined by immunomagnetic sorting for IFN-γ-producing cells. RESULTS HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T cells showed a CD4+ predominance (mean CD4:CD8 ratio 2.89) and demonstrated specificity for multiple HPIV3 antigens. The expanded T cells were polyfunctional based on cytokine production but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3+/CD45RA-/CCR7-) in responder cells. DISCUSSION HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4+ T cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of six-virus T cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune-compromised patients.

75. Development of an LMP-Specific T Cell Bank for Third Party Use as a Curative Strategy Post-Transplant Lymphoproliferative Disease After Solid Organ Transplant

EBV-associated tumors in the immune deficient host express type II and III latency antigens including latent membrane protein 1 (LMP1) and LMP2, which can serve as potential targets for immunotherapy. Several studies have documented the safety and efficacy of LMP-specific T cells for patients with EBV-associated malignancies, but clinical applications may be limited by the time to generate LMP-specific T cell products as well as the availability of an appropriate donor source. We hypothesize that the administration of “off the shelf” third party LMP-specific cytotoxic lymphocytes (LMP-CTLs) will rapidly restore EBV-specific T-cell immunity and prevent relapse in patients with post-transplant lymphoproliferative disease PTLD post solid organ transplant (SOT). To develop the third party T cell bank, we manufactured healthy donor-derived LMP-specific T cells from eligible donors with a wide range of HLA types in our good manufacturing practices (GMP) facility. This T-cell bank is for several clinical trials including a proposed multicenter Childrens Oncology Group (COG) trial (ANHL1522) for patients with PTLD after SOT. Currently, 15 LMP-specific T-cell products have been manufactured from healthy donors and released for third-party use using autologous monocytes and lymphoblastoid cell lines (LCL), transduced with an adenoviral vector expressing ΔLMP1 and LMP2. T-cell products were active against LMP2 (mean:172 SFU/1×10^5 cells; range:13-655), LMP1 (33; 1-322), and LCL (87; 0-424) as determined by IFN-γ ELISPOT assay. Epitope mapping of LMP-specific T cells using IFN-γ ELISPOT assay demonstrates that these products recognize a broad epitope repertoire within LMP1 and LMP2. At the time of cryopreservation, the T-cell products comprised a mean of 45% CD8+ T-cells, 35% CD4+ T-cells, and 9% NK cells. No B cells or monocytes were detected in the final products. Thus far, one patient with NK/T cell non-Hodgkin Lymphoma received third party LMP-specific T cells achieving a very good partial response. No infusion-related toxicities were observed, and LMP-specific T cells were detectable post-infusion. Thus, third party LMP-specific T cells appear to be a safe and promising therapeutic modality for patients with EBV-associated lymphomas, and a third party bank will make this therapeutic more readily available to patients with PTLD post-SOT.

685. Restoration of Antiviral Immunity Following Hematopoietic Stem Cell Transplantation with Virus-Specific T-Lymphocyte Therapy

Background: Adoptive immunotherapy using virus-specific cytotoxic T-lymphocyte (VST) products has been successful in restoring antiviral immunity after hematopoietic stem cell transplantation (HSCT). VST may be produced from a stem cell donor in 10-14 days using current rapid protocols, or banked, partially-HLA matched VST may be used as an “off the shelf” treatment. Objective: To evaluate the clinical efficacy of HSCT donor-derived VST and third-party VST for prevention or treatment of CMV, EBV, and adenovirus following HSCT. Design/Methods: VST were cultured from HSCT or third-party donors using a GMP-compliant rapid-manufacture protocol. VST were tested for specificity and non-alloreactivity via IFN-γ ELISpot and cytotoxicity assays. Patients were monitored for 45 days following infusion for toxicity and for up to 22 months for immune reconstitution. Results: Nineteen patients received VST on existing protocols, of which 15 products were derived from HSCT donors, and 6 were from partially matched, third-party donors. Sixteen patients were treated for CMV (N=11), EBV (N=5), and/or adenovirus infections(n=3), of which fourteen (88%) had partial or complete antiviral responses. Immune reconstitution against targeted viruses was seen using IFN-g ELISpot at a median time of 28 days following infusion. Three patients developed grade I-II GVHD within 45 days of VST infusion, all of which was treatment responsive. Sixteen of the 19 patients remain alive and free of active viral infection up to 22 months post-VST infusion. Conclusions: VST are effective for the restoration of antiviral following HSCT. Expansion of targeted viruses may further extend the utility of this therapy.