Sabrina Basso

Cell Therapy of Stage IV Nasopharyngeal Carcinoma With Autologous Epstein-Barr Virus–Targeted Cytotoxic T Lymphocytes

PURPOSE Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-related malignancy expressing EBV antigens that are possible targets of cell therapy, including latent membrane protein 2 (LMP2). We conducted a clinical trial of EBV-targeted cell therapy with autologous virus-specific cytotoxic T lymphocytes (CTLs) for NPC refractory to conventional treatments. PATIENTS AND METHODS Ten patients with EBV-related stage IV NPC in progression after conventional radiotherapy and chemotherapy received intravenously autologous EBV-specific CTLs reactivated and expanded ex vivo from peripheral blood lymphocytes through stimulation with EBV-transformed autologous B-lymphoblastoid cell lines (LCL). Toxicity, specific cellular immune responses, and clinical tumor responses were evaluated. RESULTS EBV-specific CTLs could be generated in all patients and were predominantly CD3+/CD8+ T lymphocytes displaying specific killing of autologous EBV-LCL, autologous NPC cells as well as autologous targets bearing the EBV antigen LMP2. Patients received two to 23 infusions of EBV-specific CTLs that were well tolerated with the exception of grade 1 to 2 inflammatory reactions at the tumor site in two cases. Control of disease progression was obtained in six of 10 patients (two with partial response and four with stable disease). Analysis of interferon-gamma-producing cells demonstrated an increased frequency of EBV-specific immunity, with appearance of LMP2-specific responses in four patients, of whom three had clinical benefit. CONCLUSION Cell therapy with EBV-targeted autologous CTLs is safe, induces LMP-2-specific immunologic responses, and is associated with objective responses and control of disease progression in patients with stage IV NPC resistant to conventional treatments.

Preemptive Therapy of EBV-Related Lymphoproliferative Disease after Pediatric Haploidentical Stem Cell Transplantation

The treatment of Epstein‐Barr virus (EBV)‐related post‐transplant lymphoproliferative disease (PTLD) after hematopoietic stem cell transplantation (HSCT) is still unsatisfactory. We conducted a prospective trial to evaluate the impact of routine EBV surveillance and preemptive treatment with the anti‐CD20 monoclonal antibody rituximab on the development of PTLD in pediatric recipients of extensively T‐cell depleted HSCT from an HLA‐haploidentical relative. Twenty‐seven patients were included in the surveillance program, 12 developed EBV DNA positivity, with 8 of 12 presenting with sustained viral DNA levels requiring treatment with rituximab. Treatment was well tolerated, and induced clearance of EBV DNA in all patients. However, 4/8 patients showed a new increase in EBV load, coincident with the emergence of CD20−/CD19+ B cells in peripheral blood, accompanied by overt PTLD in 3 patients. The latter cleared PTLD after receiving donor EBV‐specific cytotoxic T‐lymphocytes (CTLs), and persist in remission at a median 30‐month follow‐up. EBV‐specific T‐cell frequency, undetectable at time of EBV DNA positivity, was restored by T‐cell therapy to levels comparable with controls. We conclude that preemptive therapy with rituximab is safe, but only partly effective in haplo‐HSCT recipients. Patients who progress to PTLD under rituximab treatment can be rescued permanently by infusion of EBV‐specific CTLs.

Prospective monitoring of polyomavirus BK replication and impact of pre-emptive intervention in pediatric kidney recipients.

Polyoma BK virus (BKV)‐associated nephropathy (PVAN) is a relevant cause of poor renal allograft survival. In a prospective analysis, we monitored BKV DNA in blood and urine samples from 62 consecutive pediatric kidney recipients. In patients with BKV replication, we analyzed the impact of reduction of maintenance immunosuppression on viral load kinetics and PVAN in patients with BKV replication. BKV‐specific immunity was concomitantly evaluated on blood samples of viremic patients, by measuring the frequency of BKV‐specific interferon‐γ‐producing and cytotoxic T cells, and BKV IgG antibody levels. At a median follow‐up of 24 months, BK viruria was observed in 39 of 62 patients, while BK viremia developed in 13 patients (21%). In all viremic patients, immunosuppression reduction resulted in the clearance of viremia, and prevented development of PVAN, without increasing the rate of acute rejection or causing graft dysfunction. As a consequence of immunosuppression adjustment, an expansion of BKV‐specific cellular immunity was observed that coincided with viral clearance. We conclude that treating pediatric kidney transplant patients pre‐emptively with immunosuppression reduction guided by BKV DNA in blood is safe and effective to prevent onset of PVAN. BKV‐specific cellular immunity may be useful to guide this intervention.

Treatment of EBV-Related Post-Renal Transplant Lymphoproliferative Disease with a Tailored Regimen Including EBV-Specific T Cells

The treatment of EBV‐associated post‐transplant lymphoproliferative disease (PTLD) poses a considerable challenge. Efforts have been made to define regimens based on combination of the available therapeutic agents, chosen and tailored on a patient‐by‐patient basis, with the aim of augmenting event‐free patient and graft survival. Recently, autologous EBV‐specific cytotoxic T‐lymphocytes (CTL) have proved effective in enhancing EBV‐specific immune responses and reducing viral load in organ transplant recipients with active infection. We investigated the use of a tailored combined approach including autologous EBV‐specific CTL for the treatment of EBV‐related PTLD developing after pediatric kidney transplantation.

Polyomavirus BK-specific immunity after kidney transplantation.

Failure to mount or maintain a protective immune response may influence the development of polyomavirus BK (BKV)-associated nephropathy (PVAN). However, limited data are so far available on BKV-specific immunity after kidney transplantation. BKV-specific cellular immune response was retrospectively analyzed in kidney recipients with or without BKV infection/reactivation by measuring the frequency of interferon (IFN)-gamma-secreting cells in peripheral blood. Patients with BKV-active infection and good renal function (n=6) had a mean BKV-specific lymphocyte frequency 2 log lower than healthy controls and in the same range as BKV-seropositive recipients without active infection (n=7). Patients with PVAN (n=5) revealed undetectable levels of BKV-specific cells. However, two patients from the latter cohort treated with immunosuppression reduction showed the emergence of specific immunity, with IFN-gamma production in the same range as healthy controls. Our preliminary data suggest that lack of protective immunity toward BKV may favor the occurrence of BKV active infection and influence the progression to PVAN.

Dendritic cells pulsed with polyomavirus BK antigen induce ex vivo polyoma BK virus-specific cytotoxic T-cell lines in seropositive healthy individuals and renal transplant recipients

Polyoma BK virus (BKV)-associated interstitial nephritis has emerged as a relevant complication of immunocompromise after kidney transplantation, leading to reduced survival of the renal allograft. The limitations of current antiviral treatment and the high probability of rejection in kidney graft recipients when control of viral replication is attempted by reduction of immunosuppression warrant further efforts to develop alternative therapeutic tools. Cellular immunotherapy has proved to be a successful approach for prevention and/or treatment of other viral complications in the immunocompromised host. For assessing the feasibility of translating this strategy to the prevention of BKV-associated disease, a procedure for ex vivo reactivation of BKV-specific cytotoxic T cells (CTL) was developed from BKV-seropositive healthy donors and allograft recipients through stimulation with dendritic cells pulsed with inactivated BKV. The CTL lines thus obtained showed BKV specificity, as an efficient lysis of BKV-infected targets was accompanied by little or no reactivity against mock-infected autologous or allogeneic targets. In vitro killing of allogeneic BKV-infected targets, likely as a result of populations of TCRgammadelta+/CD3+ displaying MHC class I unrestricted cytotoxicity, was also displayed. Application of this culture system may allow a preemptive therapy approach to BKV-related complications in transplant recipients, based on CTL treatment guided by BKV DNA levels.

T-cell lines specific for peptides of adenovirus hexon protein and devoid of alloreactivity against recipient cells can be obtained from HLA-haploidentical donors.

Human adenovirus (HAdV) infection may cause life-threatening complications in recipients of hematopoietic stem cell transplantation (HSCT), the highest risk being observed in children given T-cell depleted haploidentical allografts. The effectiveness of pharmacologic therapy for HAdV infection is suboptimal. Recently, cell therapy was demonstrated to offer a unique opportunity to restore antiviral immune surveillance, leading to clearance of infection and prevention/treatment of disease. However, infusion of insufficiently selected HAdV-specific T cells in haplo-HSCT may increase the risk of graft-versus-host disease. We conducted scale-up experiments to validate a method of in vitro culture to expand T cells specific for HAdV from donor peripheral blood mononuclear cells (PBMC), based on stimulation with a pool of five 30-mer peptides derived from HAdV5 hexon protein, for use in recipients of haplo-HSCT. A total of 21 T-cell lines that included a majority of CD4+ T lymphocytes, were generated. Nineteen of the 21 T-cell lines proliferated specifically against HAdV. The 2 nonspecific, and 3 T-cell lines with lower specific activity, included a median of 48% CD8+ T cells. The 19 HAdV-specific T-cell lines showed a median 357-fold decrease in alloreactivity, compared with proliferation of noncultured donor PBMC in response to recipient PBMC, only 4/19 T-cell lines showing residual alloreactivity. Our data indicate that HAdV-specific CD4+ T-cell lines with efficient in vitro antiviral response and low/undetectable alloreactivity against recipient targets may be expanded from PBMC of most human leukocyte antigen-haploidentical HSCT donors after stimulation with HAdV hexon protein-derived peptides. These T cells may be safely employed for adoptive treatment of HAdV complications.

Emergence of BCR-ABL–specific cytotoxic T cells in the bone marrow of patients with Ph + acute lymphoblastic leukemia during long-term imatinib mesylate treatment

Imatinib mesylate has been demonstrated to allow the emergence of T cells directed against chronic myeloid leukemia cells. A total of 10 Philadelphia chromosome-positive acute lymphoblastic leukemia patients receiving high-dose imatinib mesylate maintenance underwent long-term immunological monitoring (range, 2-65 months) of (p190)BCR-ABL-specific T cells in the bone marrow and peripheral blood. (p190)BCR-ABL-specific T lymphocytes were detected in all patients, more frequently in bone marrow than in peripheral blood samples (67% vs 25%, P < .01) and resulted significantly associated with lower minimal residual disease values (P < .001), whereas absent at leukemia relapse. Specific T cells were mainly effector memory CD8(+) and CD4(+) T cells, producing interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 (median percentage of positive cells: 3.34, 3.04, and 3.58, respectively). Cytotoxic subsets able to lyse BCR-ABL-positive leukemia blasts also were detectable. Whether these autologous (p190)BCR-ABL-specific T cells may be detectable under other tyrosine-kinase inhibitors, expanded ex vivo, and exploited for immunotherapy remains to be addressed.

T-cell therapy for EBV-associated nasopharyngeal carcinoma: preparative lymphodepleting chemotherapy does not improve clinical results

BACKGROUND We and others have demonstrated that adoptive cell therapy with Epstein-Barr virus (EBV)-specific autologous cytotoxic T lymphocytes (CTLs) may control disease progression in patients with EBV-associated nasopharyngeal carcinoma (NPC). With the aim of favoring in vivo T-cell expansion, we optimized our cell therapy approach by administering higher doses of EBV-specific CTLs, following lymphodepleting chemotherapy. PATIENTS AND METHODS Eleven patients with EBV-related NPC in whom conventional treatment failed have been enrolled. Patients received nonmyeloablative lymphodepleting chemotherapy consisting of cyclophosphamide and fludarabine. Two doses of autologous EBV-specific CTLs were subsequently infused, 2 weeks apart. Study end points were feasibility and clinical outcome. RESULTS All patients enrolled completed the treatment and were assessable for analysis. The median dose of CTLs per infusion was 3.7 × 10(8). Therapy was well tolerated, with no severe adverse events ascribable to either chemotherapy or cell therapy. Disease control (defined as either tumor regression or disease stabilization lasting >4 months) was obtained in 6 of 11 patients, in keeping with previously published results. CONCLUSIONS Our data confirm that EBV-specific CTL therapy is safe and associated with antitumor activity in patients with advanced NPC. The use of lymphodepleting chemotherapy before high-dose CTL infusion did not enhance the clinical benefit observed in our previous series.

Generation and ex vivo expansion of cytotoxic T lymphocytes directed toward different types of leukemia or myelodysplastic cells using both HLA-matched and partially matched donors

OBJECTIVE Successful priming and in vitro expansion of anti-leukemia cytotoxic T lymphocytes (CTL) are preliminary conditions for designing approaches of adoptive immunotherapy in patients with hematological malignancies undergoing allogeneic hematopoietic stem cell transplantation (HSCT). In this study, we evaluated the possibility of generating and expanding in vitro CTL directed toward different types of either leukemia or myelodysplastic cells, using both HLA-matched and partially matched donors. PATIENTS AND METHODS Eleven donor/recipient pairs were enrolled; donor-derived dendritic cells, pulsed with patient blast cells, were used to generate CTL. RESULTS Anti-leukemia CTL lines were successfully obtained from 10 of 11 donors. After repeated rounds of stimulation, CTL lines showed, along with an increase in cytotoxic activity, a variable but continuous expansion of cultured cells. In order to increase the magnitude of CTL expansion, two anti-leukemia CTL lines were further stimulated using allogeneic feeder cells, anti-CD3, and low doses of interleukin-2 (IL-2). This stimulation gave rise to 150-fold to 270-fold expansion of the absolute number of cultured cells. Most cultures showed either absent or low reactivity of anti-leukemia CTL against patient non-leukemia cells. Three anti-leukemia CTL lines displayed a more pronounced cytotoxicity against nonmalignant recipient cells, which was always lower than that observed against leukemia blasts (LB). Spectratyping analysis of the TCR-Vbeta subfamilies revealed a preferential expansion of oligoclonal populations that persisted in CTL lines following repeated rounds of stimulation. CONCLUSIONS Results provide the biological background for designing protocols of adoptive immunotherapy for the control of minimal residual disease in patients with hematological malignancies given HSCT.