Towards gene therapy for EBV-associated posttransplant lymphoma with genetically modified EBV-specific cytotoxic T cells.

Epstein-Barr virus (EBV)-associated posttransplant lymphoma (PTLD) is a major cause of morbidity/mortality after hematopoietic stem cell (SCT) or solid organ (SOT) transplant. Adoptive immunotherapy with EBV-specific cytotoxic lymphocytes (CTLs), although effective in SCT, is less successful after SOT where lifelong immunosuppression therapy is necessary. We have genetically engineered EBV-CTLs to render them resistant to calcineurin (CN) inhibitor FK506 through retroviral transfer of a calcineurin A mutant (CNA12). Here we examined whether or not FK506-resistant EBV-CTLs control EBV-driven tumor progression in the presence of immunosuppression in a xenogeneic mouse model. NOD/SCID/IL2rγ(null) mice bearing human B-cell lymphoma were injected with autologous CTLs transduced with either CNA12 or eGFP in the presence/absence of FK506. Adoptive transfer of autologous CNA12-CTLs induced dramatic lymphoma regression despite the presence of FK506, whereas eGFP-CTLs did not. CNA12-CTLs persisted longer, homed to the tumor, and expanded more than eGFP-CTLs in mice treated with FK506. Mice receiving CNA12-CTLs and treated with FK506 survived significantly longer than control-treated animals. Our results demonstrate that CNA12-CTL induce regression of EBV-associated tumors in vivo despite ongoing immunosuppression. Clinical application of this novel approach may enhance the efficacy of adoptive transfer of EBV-CTL in SOT patients developing PTLD without the need for reduction in immunosuppressive therapy.

Optimization of methodology for production of CD25/CD71 allodepleted donor T cells for clinical use.

BACKGROUND AIMS: Immunotherapy with allodepleted donor T cells improves immunity after T cell-depleted hematopoietic stem cell transplantation. We developed a methodology for selective depletion of alloreactive T cells after activation with host antigen-presenting cells by targeting T cells up-regulating CD25 and CD71. Combined depletion of these cells yields a pool of allodepleted donor T cells with antiviral properties with minimal capacity to cause graft-versus-host disease. METHODS: Mature dendritic cells were irradiated and used to stimulate donor peripheral blood mononuclear cells for 4 days. The co-culture was stained with anti-CD71-biotin followed by CliniMACS CD25 and Anti-Biotin Reagents (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany) before depletion on the CliniMACS Plus (Miltenyi Biotec GmbH). Residual alloreactivity was tested by flow cytometry, a secondary mixed lymphocyte reaction and limiting dilution analysis, and specific anti-viral immunity with pentamer staining. The large-scale protocol was tested under current good manufacturing practice conditions in five donor-recipient pairs of human leukocyte antigen-matched volunteer donors. RESULTS: We developed a closed-system methodology using cell differentiation bags for cell culture and the COBE2991 Cell Processor (CaridianBCT, Lakewood, CO, USA). We also validated an anti-CD71-biotin generated for ex vivo clinical use. In five large-scale runs, the depleted fraction demonstrated excellent viability (99.9%), minimal residual expression of CD3/CD25 and CD3/CD71 (<0.2%) and passed tests for Mycoplasma, endotoxin, bacterial and fungal sterility. In secondary mixed lymphocyte reaction assays, the median response to host after allodepletion was 0%, whereas responses to third-party peripheral blood mononuclear cells were preserved (median, 105%; range 37%-350%). Limiting dilution analysis assays also demonstrated a reduction in response to host (median, -1.11 log) with preservation of third-party responses, and testing with human leukocyte antigen-restricted pentamers showed that populations of Epstein-Barr virus-specific and cytomegalovirus-specific CD8(+) T cells were retained after depletion. CONCLUSIONS: We optimized a protocol for the combined immunomagnetic depletion of alloreactive CD25/CD71 T cells under current good manufacturing practice conditions and tested the efficacy in five donor-recipient pairs.

Functional characterization of alloreactive T cells identifies CD25 and CD71 as optimal targets for a clinically applicable allodepletion strategy.

Immunotherapy with allodepleted donor T cells (ADTs) improves immunity after T cell-depleted stem cell transplantation, but infection/relapse remain problematic. To refine this approach, we characterized the expression of surface markers/cytokines on proliferating alloreactive T cells (ATs). CD25 was expressed on 83% of carboxyfluorescein diacetate succinimidyl ester(dim) ATs, confirming this as an excellent target for allodepletion. Seventy percent of CD25(-) ATs expressed CD71 (transferrin receptor), identifying this as a novel marker to target ATs persisting after CD25 depletion. Comparison of residual alloreactivity after combined CD25/71 versus CD25 immunomagnetic depletion showed enhanced depletion of alloreactivity to host with CD25/71 depletion in both secondary (2 degrees) mixed lymphocyte reactions (P < .01) and interferon-gamma enzyme-linked immunospot assays (P < .05) with no effect on third-party responses. In pentamer/interferon-gamma enzyme-linked immunospot assays, antiviral responses to cytomegalovirus, Epstein-Barr virus, and adenovirus were preserved after CD25/71 allodepletion. CD25/71 ADTs can be redirected to recognize leukemic targets through lentiviral transfer of a chimeric anti-CD19zeta T-cell receptor. Finally, we have established conditions for clinically applicable CD25/71 allodepletion under European Union Good Manufacturing Practice conditions, resulting in highly effective, reproducible, and selective depletion of ATs (median residual alloreactivity to host in 2 degrees mixed lymphocyte reaction of 0.39% vs third-party response of 62%, n = 5). This strategy enables further clinical studies of adoptive immunotherapy with larger doses of ADTs to enhance immune reconstitution after T cell-depleted stem cell transplantation.

Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease.

Epstein-Barr virus (EBV)-driven posttransplantation lymphoproliferative disease (PTLD) is a serious complication of immunosuppression after either stem cell transplantation (SCT) or solid organ transplantation (SOT). Adoptive transfer of EBV-specific cytotoxic T lymphocytes (EBV-CTLs) is an effective prophylaxis and treatment for PTLD after SCT, but not for PTLD after SOT when pharmacologic immunosuppression cannot be discontinued. We report the generation of calcineurin (CN) mutants that render EBV-CTL resistant to the immunosuppressants tacrolimus (FK506) and cyclosporin A (CsA): mutant CNa12 confers resistance to CsA but not FK506, and mutant CNa22 confers resistance to FK506 but not CsA, whereas mutant CNb30 renders CTLs resistant to both calcineurin inhibitors. Untransduced EBV-CTLs do not proliferate in the presence of FK506/CsA. However, EBV-CTLs transduced with a retroviral vector coding for these mutants retain the ability to both proliferate and secrete normal levels of interferon-gamma in the presence therapeutic levels of FK506 (CNa12), CsA (CNa22), or both (CNb30). The cytotoxicity and phenotype of EBV-CTL lines were unaffected by expression of these mutant CNs. This approach should allow effective immunotherapy with EBV-CTLs in the SOT setting without risking the graft by reduction in immunosuppression, and represents a generic approach to improving immunotherapy in the face of immunosuppression.

Generation of trispecific cytotoxic T cells recognizing cytomegalovirus, adenovirus, and Epstein-Barr virus: an approach for adoptive immunotherapy of multiple pathogens.

Cytomegalovirus (CMV), adenovirus (Ad), and Epstein-Barr virus (EBV) are a major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Adoptive immunotherapy with donor-derived cytotoxic T cells (CTLs) directed against EBV or CMV prevents the clinical manifestations of these viruses. We have designed a protocol for the simultaneous generation of polyclonal CTL specific for CMV, Ad, and EBV, which could be used to restore immunity to multiple viruses after SCT. EBV-transformed lymphoblastoid cell lines (LCLs), transduced with an adenoviral vector carrying a transgene for the immunodominant CMV antigen pp65 (Ad5f35-pp65GFP), were used to stimulate peripheral blood mononuclear cells in 6 normal donors. We detected the simultaneous presence of CD8 CTL recognizing peptide epitopes from all 3 viruses by pentamer staining. Enzyme-linked immunospot assays demonstrated a median 29-fold (8 to 248), 47-fold (2 to 137), or 18-fold (5 to 29) increase in cells secreting interferon-gamma in response to CMV, adenoviral, or EBV antigens, respectively, compared with unmanipulated peripheral blood mononuclear cell, with concomitant loss of alloreactivity. The CTL lines showed cytotoxicity against autologous LCL alone and increased cytotoxicity to autologous LCLs pulsed with CMV pp65 peptides or infected with Ad. In summary, we have developed a protocol for the generation of CTL with trivirus specificity, enabling adoptive transfer of CTL recognizing multiple viruses to restore cellular immunity after SCT.