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The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.

Objective Memory stem T (Tscm) cells are long-lived, self-renewing T cells that play a relevant role in immunologic memory. This study was undertaken to investigate whether Tscm cells accumulate in rheumatoid arthritis (RA). Methods The polarization and differentiation profiles of circulating T cells were assessed by flow cytometry. Antigen-specific T cells were characterized by staining with major histocompatibility complex class II tetramers. The T cell receptor (TCR) repertoire was analyzed by high-throughput sequencing using an unbiased RNA-based approach in CD4+ T cell subpopulations sorted by fluorescence-activated cell sorting. Results We analyzed the dynamics of circulating Tscm cells (identified as CD45RA+CD62L+CD95+ T cells) by flow cytometry in 27 RA patients, 16 of whom were also studied during treatment with the anti-tumor necrosis factor (anti-TNF) agent etanercept. Age-matched healthy donors were used as controls. CD4+ Tscm cells were selectively and significantly expanded in RA patients in terms of frequency and absolute numbers, and significantly contracted upon anti-TNF treatment. Expanded CD4+ Tscm cells displayed a prevalent Th17 phenotype and a skewed TCR repertoire in RA patients, with the 10 most abundant clones representing up to 53.7% of the detected sequences. CD4+ lymphocytes specific for a citrullinated vimentin (Cit-vimentin) epitope were expanded in RA patients with active disease. Tscm cells accounted for a large fraction of Cit-vimentin-specific CD4+ cells. Conclusion Our results indicate that Tscm cells, including expanded clones specific for relevant autoantigens, accumulate in RA patients not exposed to biologic agents, and might be involved in the natural history of the disease. Further analysis of Tscm cell dynamics in autoimmune disorders may have implications for the design and efficacy assessment of innovative therapies.

Tumor-infiltrating lymphocytes (TILs) may express different inhibitory receptors (IRs) on the cell surface that are exploited by cancer cells to evade the immune attack. Thus, exhausted T cells display an unresponsive functional profile and fail to kill tumor cells. TIM-3 and LAG-3 represent highly expressed inhibitory receptors in exhausted T cells infiltrating solid tumors. We recently showed that a large fraction of bone marrow infiltrating CD8+ cells co-express multiple inhibitory receptors, including TIM-3, in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplantation. These observations advocate for strategies to generate innovative tumor-specific T cell products resistant to inhibitory signals and able to persist long-term in treated patients. To reach these objectives we developed a strategy to simultaneously redirect T cell specificity by TCR gene transfer and permanently disrupt IRs by CRISPR/Cas9 system in long-living memory stem T cells (TSCM) for adoptive cell therapy. Primary T cells were activated with CD3/CD28-conjugated beads and cultured with low doses of IL7 and IL15, to preserve their TSCM phenotype, and electroporated with Cas9/gRNA ribonucleoproteins (RNPs) targeting a coding sequence of TIM-3,LAG3, 2B4 and of the TCR α and β chain constant region (TRAC and TRBC1/2) genes. Each gene was edited alone or in combination in a multiplex approach. The efficiencies of NHEJ-mediated inactivation of each gene were assessed by cytofluorimetric analysis and confirmed at molecular level by surveyor assay or ddPCR. In order to avoid mispairing between the endogenous TCR and the tumor-specific TCR, we simultaneously inactivated the constant regions of α and β chain of the endogenous TCR genes. Efficiency of TRAC and/or TRBC1/2 gene disruption was 98%, when measured as % of CD3neg cells. TCR knocked-out cells could be efficiently (70-85%) transduced with a lentiviral vector encoding for TCR specific for an HLA-A2 restricted peptide from NY-ESO1. We then focused on IR gene inactivation. Single gene editing produced up to 98%, 93% and 63% genetic knock-out of the TIM-3 , the LAG-3 and the 2B4 genes, respectively. We finally combined one IR inactivation with TCR gene editing in a single protocol. We obtained an average of 68% and 69% of TIM3neg and LAG3neg NY-ESO1-TCR redirected T cells, respectively, and more than 90% of edited cells showed a TSCM phenotype. When tested in functional assays, TCR-IR-edited TSCM cells proved highly effective and specific in killing HLA-A2+ NY-ESO1+ multiple myeloma cells. Of notice, TIM-3neg and LAG-3neg cells proved more effective in producing IFNg, TNFa and CD107a, once challenged with tumor cells. In conclusion, by combining the versatility of multiplex gene editing by CRISPR/Cas9 with culture conditions designed to engineer TSCM cells, we can generate innovative tumor-specific cellular products redirected against tumor antigens and resistant to inhibitory signals. Ongoing in vivo evaluation of tumor-specific IR disrupted memory stem T cells will be reported and discussed. Disclosures Bonini: Intellia Therapeutics: Research Funding.

NA A major hurdle to the field of allogeneic hematopoietic stem cell transplantation (HSCT) is the lack of validated biomarkers of protective immunity against opportunistic infections, including CMV reactivation syndromes. This issue is particularly relevant to alternative-donor settings, for example, HLA-haploidentical HSCT (haplo-HSCT) and cord blood transplantation, where aggressive GvHD prophylaxis results in a profound and long-lasting state of immune incompetence. Despite the introduction of routine post-transplant viral monitoring and preemptive antiviral therapy, CMV reactivation syndromes remain a major cause of transplant related mortality. Accordingly, serological evidence of prior CMV infection is still one of the main negative prognostic factors in HSCT. Different studies have demonstrated that a faster recovery of CMV-specific T-cell responses associates with a reduced CMV reactivation incidence in HSCT from HLA-identical sibling and matched unrelated donors. Given its clinical and biological peculiarities, it is currently unknown whether these observations are directly translatable to the haplo-HSCT setting. In this study, while aiming to follow the recovery of CMV-specific T-cell responses after haplo-HSCT, we unexpectedly found that protective levels of CMV immunity were associated with a reduced incidence of severe infections overall.

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR beta- and alpha-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

The extensive exploitation of the antitumor effect of donor lymphocytes infused after allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is limited by the risk of graft-versus-host disease (GvHD). To overcome this limitation, we investigated the therapeutic potential of donor lymphocytes engineered with the suicide gene thymidine kinase of herpes simplex virus (TK) in 23 patients experiencing recurrence of hematologic malignancies after allo-HSCT. Long-term follow-up of infused patients included analysis of engraftment of genetically engineered lymphocytes, in vivo assessment of antitumor effect, and control of GvHD by ganciclovir. All 17 patients evaluable for engraftment and graft-versus-leukemia (GvL) had circulating TK+ cells detectable beginning at a median time of 18 days. Eleven patients (65%) experienced a substantial clinical benefit resulting in 6 (35%) complete remissions and 5 (29%) partial responses. The antitumor effect tightly correlated with the in vivo received ganciclovir, resulting in elimination of TK+ cells and effective and selective treatment of GvHD. Immunization against HSV-TK was observed in 7 patients but did not preclude an effective GvL. These data validate the feasibility, safety, and efficacy of TK+ cells in the context of allografting and represent the basis for a broader application of this technology. The extensive exploitation of the antitumor effect of donor lymphocytes infused after allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is limited by the risk of graft-versus-host disease (GvHD). To overcome this limitation, we investigated the therapeutic potential of donor lymphocytes engineered with the suicide gene thymidine kinase of herpes simplex virus (TK) in 23 patients experiencing recurrence of hematologic malignancies after allo-HSCT. Long-term follow-up of infused patients included analysis of engraftment of genetically engineered lymphocytes, in vivo assessment of antitumor effect, and control of GvHD by ganciclovir. All 17 patients evaluable for engraftment and graft-versus-leukemia (GvL) had circulating TK+ cells detectable beginning at a median time of 18 days. Eleven patients (65%) experienced a substantial clinical benefit resulting in 6 (35%) complete remissions and 5 (29%) partial responses. The antitumor effect tightly correlated with the in vivo received ganciclovir, resulting in elimination of TK+ cells and effective and selective treatment of GvHD. Immunization against HSV-TK was observed in 7 patients but did not preclude an effective GvL. These data validate the feasibility, safety, and efficacy of TK+ cells in the context of allografting and represent the basis for a broader application of this technology.

The use of retroviral vectors in gene therapy has raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. We have analyzed the consequences of retroviral transduction in T cells from leukemic patients treated with allogeneic stem cell transplantation and donor lymphocytes genetically modified with a suicide gene (HSV-TK). Retroviral vectors integrate preferentially within or near transcribed regions of the genome, with a preference for sequences around promoters and for genes active in T cells at the time of transduction. Quantitative transcript analysis shows that one fifth of these integrations affect the expression of nearby genes. However, transduced T cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions, and immune repertoire in vivo , with no evidence of clonal selection up to 9 yr after administration. Analysis of integrated proviruses in transduced cells before and after transplantation indicates that integrations interfering with normal T cell function are more likely to lead to clonal ablation than expansion in vivo . Despite the potentially dangerous interactions with the T cell genome, retroviral integration has therefore little consequence on the safety and efficacy of T cell transplantation.

Background: Alveolar macrophages (AMs) are more efficient antigen-presenting cells in allergic individuals than in nonatopic subjects. Objective: We studied whether this difference may be correlated to increased expression of membrane costimulatory molecules, such as the B7 molecules (CD80 and CD86). Methods: Eleven subjects with allergic asthma sensitized to Dermatophagoides pteronyssinus and 5 healthy nonatopic volunteers underwent bronchoalveolar lavage, and the costimulatory molecule expression on AMs was evaluated. Peripheral blood T cells, either freshly isolated or as established D pteronyssinus -specific cell lines, were cultured with autologous monocytes or AMs as antigen-presenting cells. In vitro allergen-induced proliferation and cytokine production were evaluated in the presence of B7-blocking reagents. Results: Allergic individuals had a significantly higher proportion of AMs expressing the CD80 molecule than control subjects (28.5% ± 14.8% vs 1.4% ± 1.2%; P P > .1). In a large proportion of the asthmatic subjects we studied, AMs were presenting soluble antigens (tetanus toxoid and streptolysin-O) to freshly isolated T cells more efficiently than AMs from nonatopic control subjects. Finally, both T-cell proliferation and cytokine production of D pteronyssinus- specific established T-cell lines were inhibited by a CD80-blocking antibody in a dose-dependent manner. Conclusion: Costimulation by means of CD80 expressed by AMs is probably involved in the amplification of the allergen-specific T-lymphocyte response in the airways of asthmatic subjects. (J Allergy Clin Immunol 1999;103:1136-42.)

Anti-CD4 antibodies have been documented in about 10-20% of HIV-infected patients. This autoimmune response could be triggered by increased CD4 processing and unveiling of hidden (cryptic) epitopes. Multiple markers of exposure to HIV have been described in exposed uninfected individuals. Here, we investigated the mechanisms underlying the generation of anti-CD4 antibodies in a cohort of 54 seronegative exposed uninfected individuals. We identified anti-CD4 antibodies above normal levels in 16 of 47 (34%) exposed uninfected subjects. The fine specificity of these antibodies was different in this cohort when compared with those found in HIV+ patients. This suggested the possibility of different mechanisms underlying the generation of anti-CD4 antibodies in these two groups. Indeed, in exposed uninfected subjects, we found circulating CD4 T cells specific for gp120, but not for CD4. In contrast, HIV-1-seropositive patients had peripheral blood T cells specific for both molecules. Noncovalent binding of gp120 to soluble CD4 enhanced activation of gp120-specific T lymphocytes in exposed uninfected subjects, but not in HIV+ subjects. Moreover, gp120-specific T cells isolated from exposed uninfected, but not from HIV+, subjects provided help for anti-CD4 antibody production by B cells pulsed with CD4-gp120 complex. We conclude that gp120-specific T cells are present in exposed uninfected individuals, and can provide intermolecular help for anti-CD4 antibody production. This mechanism is distinct from that found in HIV-1-seropositive patients and may play a protective role against HIV-1 infection in vivo.

Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (T-CM) phenotype and to high interleukin (IL)-7R alpha expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC-expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC-expanded T cells modified with a suicide gene in the setting of haplo-HSCT. Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (T(CM)) phenotype and to high interleukin (IL)-7Rα expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC-expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC-expanded T cells modified with a suicide gene in the setting of haplo-HSCT. "Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (T(CM)) phenotype and to high interleukin (IL)-7Rα expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC-expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC-expanded T cells modified with a suicide gene in the setting of haplo-HSCT."

Accumulating experimental and clinical evidence has been obtained over recent years in support of the notion that the immune system has the potential to cure cancer. The most convincing example is the graft versus leukaemia effect observed after allogeneic haematopoietic stem cell transplantation. In the autologous setting, however, the isolation and expansion of naturally occurring tumour-specific T cells is a challenging task. Cancer antigens are often self-antigens and cancer-specific T cells are thus subject to selective mechanisms of central and peripheral tolerance. The significant advances in gene-transfer technologies developed over the last decade have offered new tools to overcome these limitations. Natural T cells can be genetically modified to generate high numbers of ‘supernatural’ tumour-reactive T cells from virtually every cancer patient. Supernatural T cells may express clonal receptors providing new specificities, factors increasing T-cell performance or safety factors enabling their elimination in case of toxicity. Technological improvements applied to novel concepts of T-cell biology and oncogenesis will allow to simultaneously equip T cells with different transgenes and expand a real ‘army’ of lymphocytes trained to selectively eradicate cancer cells.

Background Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality. Methods In a phase I-II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per mu L or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124. Findings From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17-66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34-127) from transplantation and 23 days (13-42) from infusion. Ten patients developed acute GVHD (grade I-IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25-73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure. Interpretation Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation. Background Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality. Methods In a phase I-II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per mu L or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124. Findings From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17-66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34-127) from transplantation and 23 days (13-42) from infusion. Ten patients developed acute GVHD (grade I-IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25-73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure. Interpretation Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation. Background Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality.Methods In a phase I-II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per mu L or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124.Findings From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17-66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34-127) from transplantation and 23 days (13-42) from infusion. Ten patients developed acute GVHD (grade I-IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25-73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure.Interpretation Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation.

Background. Haploidentical family donors represent the ideal solution to offer to every patient with high risk leukemia the potential cure of hematopoietic stem cell transplantation. Extensive application of haploidentical transplantation (haplo-SCT) has been limited by high rate of late transplant related mortality (TRM) and relapse associated with the delayed immune reconstitution (IR) secondary to the procedures of profound T-cell depletion required for severe graft-vs-host-disease (GvHD) prevention. Methods. In a haplo-SCT phase I-II multicenter, open, non-randomized trial sponsored by MolMed SpA, we infused donor lymphocytes genetically engineered to express the suicide gene herpes simplex thymidine kinase (TK-DLI) to induce early IR, while selectively controlling GvHD. We enrolled 54 patients (pts) -median age 51- with high-risk hematologic malignancies. Thirty-two patients were in remission at transplantation. Results. After myeloablative conditioning regimen, 50 pts received a median 11×10^6/ kg CD34+ and 1.1×10^4/kg CD3+ after Clinimacs CD34+ selection. Median time to engraftment of neutrophils > 1.0 ×10^9/L and platelets > 50 ×10^9/L was 14 days. TK cells could always be prepared in the appropriate timeframe, and no drop out secondary to inadequate cell manipulation occurred. Twenty-eight pts received TK-DLI at a dose of CD3+ cells of 0.9–40 ×10^6/kg: 22 pts obtained prompt IR with CD3+>100/mcl at day +75 (median) from haplo-SCT and day +23 from TK-DLI. Eleven pts developed GvHD (10 acute GvHD grade I-IV and 1 chronic GvHD) that was always abrogated by the suicide gene induction; no progression from acute GvHD to chronic GvHD and no GvHD-related death or long-term complication occurred. No acute or chronic adverse event related to the gene transfer procedure was observed during extended follow-up. With a median follow-up of 178 days (range 2–1821), the 3-year TRM in intention to-treat (ITT) analysis was 40% with last mortality event at d+166. Significantly, the cumulative infectious mortality was 10% in TK-treated immune-reconstituted patients. Immune reconstitution obtained with TK-cells infusion correlated with rapid development of a wide T-cell repertoire and detection of high frequencies of T-cells specific for opportunistic pathogens. Initially, TK-cells represented the predominant T cell component in reconstituting patients and showed a effector memory phenotype, an oligoclonal repertoire and a persistent ganciclovir sensitivity. At later times, untransduced cells progressively expanded and became the prevalent circulating lymphocyte population. This reconstitution kinetic was observed only in patients with TK-cell engraftment, suggesting a critical homeostatic contribution of TK-cells early after transplant. One year after TK cells infusion the repertoire and distribution of T cell subsets completely normalized. In ITT, the median leukemia-free survival (LFS) for patients transplanted with advanced chemoresistant disease was at d+169, while patients in remission at the time of transplantation showed an overall survival in ITT of 51% at 1 year. Conclusions. This multicenter trial confirm the safety and potential benefit in improving survival of the gene transfer technology integrated with a standard therapeutic procedure such as allogeneic SCT. The conditional benefit for patients treated with TK cells was remarkable as indicated by a complete abrogation of late infectious mortality provided by the fast and effective immune reconstitution. In the future, this technology based on genetic engineering of activated donor lymphocytes with higher allo-reactive potentialwill potentially impact also the transplant outcome of patients with refractory disease.

Gene therapy is a promising therapeutic strategy for genetic and acquired hematologic diseases. With the improvements in gene transfer and expression, factors affecting safety and efficacy of gene therapy can now be evaluated to establish the best clinical benefit-to-risk ratio. The induction of immune responses against gene therapy components is one of the potential limitations. We studied the occurrence of such event in 23 patients treated with donor lymphocyte infusions DLIs), with lymphocytes transduced to express the HSV-TK suicide gene for relapse of hematologic malignancies occurring after allogeneic hematopoietic stem cell transplantation (HSCT). The suicide gene was used to selectively control graft-versus-host disease (GvHD). Seven patients given infusions late after HSCT developed an immune response against the transgene. Immunization involved appearance of thymidine kinase (TK)-specific CD8(+) effectors and required a level of immunocompetence at the time of TK-DLI that can be achieved only several months after transplantation. This did not prevent graft-versus-leukemia (GvL) effect of the TK-DLI, since 5 of 7 immunized patients maintained the complete remission achieved prior to immunization. We suggest that appropriate study designs taking into account the immune suppression of the patient and time-kinetics of GvL mediated by TK-transduced donor lymphocytes may allow the full exploitation of TK-DLI. Gene therapy is a promising therapeutic strategy for genetic and acquired hematologic diseases. With the improvements in gene transfer and expression, factors affecting safety and efficacy of gene therapy can now be evaluated to establish the best clinical benefit-to-risk ratio. The induction of immune responses against gene therapy components is one of the potential limitations. We studied the occurrence of such event in 23 patients treated with donor lymphocyte infusions DLIs), with lymphocytes transduced to express the HSV-TK suicide gene for relapse of hematologic malignancies occurring after allogeneic hematopoietic stem cell transplantation (HSCT). The suicide gene was used to selectively control graft-versus-host disease (GvHD). Seven patients given infusions late after HSCT developed an immune response against the transgene. Immunization involved appearance of thymidine kinase (TK)-specific CD8(+) effectors and required a level of immunocompetence at the time of TK-DLI that can be achieved only several months after transplantation. This did not prevent graft-versus-leukemia (GvL) effect of the TK-DLI, since 5 of 7 immunized patients maintained the complete remission achieved prior to immunization. We suggest that appropriate study designs taking into account the immune suppression of the patient and time-kinetics of GvL mediated by TK-transduced donor lymphocytes may allow the full exploitation of TK-DLI.

In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK+ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK+ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK+ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK+ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK+ lymphocytes were more potent than EM TK+ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK+ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT. In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK+ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK+ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK+ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK+ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK+ lymphocytes were more potent than EM TK+ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK+ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT.

The outcome of haplo-SCT is limited by delayed immune reconstitution resulting in a high rate of late mortality and relapse. Here, we report results of a phase II multicenter trial (MM TK007) of early add-backs of donor lymphocytes genetically engineered to express the herpes simplex thymidine kinase (TK) suicide gene after haplo-SCT in inducing immune reconstitution and selective control of GvHD by ganciclovir. Twentysix advanced age pts (median age 51, 17–63) were transplanted for high risk leukemia; disease status at SCT was CR1 (8), CR2 (7), refractory (11). A median of 12.2x106/kg (7.3–16.8) CD34+ selected (Clinimacs) and 1x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning. 24/26 pts engrafted with a median time of 14 d (8–21) for ANC >1.0x109/l and 13 d (11–24) for plt >50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-add-back. Sixteen pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42 and 13 pts obtained CD3+ >100/mcl at a median time of 91 d (61–127) from SCT and 24 d (14–42) from TK-DLI. Transduced cells were documented ex vivo in all pts and represented a median of 48% (10–90) of CD3+ cells. Five pts developed acute GvHD, (grade I to IV) that was always completely abrogated by ganciclovir. In patients in CR at time of SCT who were alive at d +42 and received add-backs of Tk cells, OS rate was 46% at 800 days (intention-to-treat analysis: 38% OS at 800 days post-SCT). Of significance, the cumulative incidence of TRM and relapse showed a 40% probability of mortality with a median time of death of 90 days and last event at day +166. This figure indicate that TK cell add-backs abolish late mortality after CD34+ SCT in adults. In patients in relapse at time of HSCT, a median OS of 201 days was obtained in ITT, with a significant advantage on expected survival without transplantation (60 d) and superior results as compared to haplo EBMT registry (80 d). The 2-year estimation of events of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from late infectious mortality after haplo-SCT. We believe that these results are due to the rapid development of a wide T cell repertoire obtained by TK cell infusions. Immunological follow-up showed Th1/Tc1 effector memory T cells, with a wide TCR repertoire in the first 3 months after SCT in all patients. High frequencies of T cells specific for CMV (median: 35 and 93 spots/105 cells with CMV-infected donor and host fibroblasts) and EBV (median: 58 and 41 spots/105 cells with donor and host EBV-LCL) were detected by gIFN ELISpot at time of immunereconstitution, and correlated with complete control of viral infections. Normalization of the T cell repertoire was documented by spectratype, immune-phenotype for naïve and memory T cell subsets and gIFN ELIspot 6 months after treatment. A phase III randomized multicentric study will start in 2006.

Gene transfer into T lymphocytes is currently being tested for the treatment of lymphohematologic disorders. We previously showed that suicide gene transfer into donor lymphocytes infused to treat leukemic relapse after allogeneic hematopoietic stem cell transplantation allowed control of graft-versus-host disease. However, the T-cell receptor (TCR) activation and sustained proliferation required for retroviral vector transduction may impair the half-life and immune competence of transduced cells and reduce graft-versus-leukemia activity. Thus, we tested lentiviral vectors (LVs) and stimulation with cytokines involved in antigen-independent T-cell homeostasis, such as interleukin 7 (IL-7), IL-2, and IL-15. Late-generation LVs transduced efficiently nonproliferating T cells that had progressed from G0 to the G1 phase of the cell cycle on cytokine treatment. Importantly, IL-2 and IL-7, but not IL-15, stimulation preserved physiologic CD4/CD8 and naive-memory ratios in transduced cells with only minor induction of some activation markers. Functional analysis of immune response to cytomegalovirus (CMV) showed that, although CMV-specific T cells were preserved by all conditions of transduction, proliferation and specific killing of autologous cells presenting a CMV epitope were higher for IL-2 and IL-7 than for IL-15. Thus, LV transduction of IL-2 or IL-7 prestimulated cells overcomes the limitations of retroviral vectors and may significantly improve the efficacy of T-cell–based gene therapy.

We have previously shown that the infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase (HSV-tk) gene is an efficient tool for controlling graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect. In addition to the GVL effect, the administration of donor HSV-tk(+) cells could have a clinical impact in promoting immune reconstitution after T-cell-depleted stem cell transplantation (SCT). To explore this hypothesis, we have investigated whether in vitro polyclonal activation, retroviral transduction, immunoselection, and expansion affect the immune competence of donor T cells. We have observed that, after appropriate in vitro manipulation, T cells specific for antigens relevant in the context of SCT are preserved in terms of frequency, expression of T-cell receptor, proliferation, cytokine secretion, and lytic activity. A reduction in the frequency of allospecific T-cell precursors is observed after prolonged T-cell culture, suggesting that cell manipulation protocols involving a short culture time and high transduction efficiency are needed. Finally, the long-term persistence of HSV-tk(+) cells was observed in a patient treated in the GVL clinical trial, and a reversion of the phenotype of HSV-tk(+) cells from CD45RO(+) to CD45RA(+) was documented more than 2 years after the infusion. Based on all this evidence, we propose a clinical study of preemptive infusions of donor HSV-tk(+) T cells after SCT from haploidentical donors to provide early immune reconstitution against infection and potential immune protection against disease recurrence. (C) 2003 by The American Society of Hematology. We have previously shown that the infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase (HSV-tk) gene is an efficient tool for controlling graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect. In addition to the GVL effect, the administration of donor HSV-tk(+) cells could have a clinical impact in promoting immune reconstitution after T-cell-depleted stem cell transplantation (SCT). To explore this hypothesis, we have investigated whether in vitro polyclonal activation, retroviral transduction, immunoselection, and expansion affect the immune competence of donor T cells. We have observed that, after appropriate in vitro manipulation, T cells specific for antigens relevant in the context of SCT are preserved in terms of frequency, expression of T-cell receptor, proliferation, cytokine secretion, and lytic activity. A reduction in the frequency of allospecific T-cell precursors is observed after prolonged T-cell culture, suggesting that cell manipulation protocols involving a short culture time and high transduction efficiency are needed. Finally, the long-term persistence of HSV-tk(+) cells was observed in a patient treated in the GVL clinical trial, and a reversion of the phenotype of HSV-tk(+) cells from CD45RO(+) to CD45RA(+) was documented more than 2 years after the infusion. Based on all this evidence, we propose a clinical study of preemptive infusions of donor HSV-tk(+) T cells after SCT from haploidentical donors to provide early immune reconstitution against infection and potential immune protection against disease recurrence. (C) 2003 by The American Society of Hematology.

The genetic transfer of tumor-specific T cell receptors (TCRs) into mature T lymphocytes enables T cell specificity to be redirected towards cancer cells, however the transfer of novel TCRs into polyclonal T cells, while overcoming tolerance barriers, may be limited by factors intrinsic to TCR biology. Specifically, the tumor-specific a and b TCR chains are expressed in lymphocytes that already bear an endogenous TCR on their cell surfaces. Gene-modified cells thus express at least two different TCRs that compete for binding to the CD3 complex, resulting in mutual TCR dilution and reduced avidity. Furthermore, since TCRs are heterodimers, the a and b chains of the endogenous TCR have the potential to mispair with the respective α and β chains of the transgenic TCR to produce a new hybrid TCR, with unpredictable and potentially harmful specificity. This represents a major concern in TCR transfer adoptive immunotherapy, both in autologous and allogeneic settings. To permanently eliminate the expression of the endogenous TCR and the risk of mispairing, our group recently developed a TCR gene editing approach. This technique is based on the transient transfer of zinc-finger nucleases (ZFN) to induce DNA double strand breaks in the constant regions of the endogenous TCR a and/or b chain genes, leading to permanent gene disruption. Upon lentiviral transfer of a tumor-specific TCR, such fully TCR-edited T cells express only the exogenous tumor-specific TCR transgenes at high levels (Provasi, Genovese et al., Nature Medicine 2012). While the complete editing procedure (both a and b TCR chains) currently requires multiple manipulation steps, ‘single TCR editing’, based on the ZFN-mediated knock-down of a single endogenous TCR chain (a or b) followed by the introduction of the tumor-specific TCR, enables the generation of redirected T cells devoid of their natural TCR repertoire during a single round of T cell activation, improving the feasibility of the clinical translation of this approach. This might be particularly useful to reduce the risk of GvHD after allogeneic hematopoietic stem cell transplantation. We exploited a HLA-A2 restricted TCR specific for NY-ESO-1, a cancer testis antigen expressed by solid tumors and hematological malignancies, to directly compare the safety and efficacy profile of unedited TCR transferred T cells (TR), single TCR edited (SE) lymphocytes and completely TCR edited (CE) T cells. We observed that gene editing does not detectably affect the phenotype, function or proliferative potential of engineered lymphocytes. Our protocols ensured the maintenance of the early differentiated memory phenotype, with enrichment in central memory and CD45RA+/CD62L+/CD95+ memory stem T (TSCM) cells. Upon lentiviral transfer of the NY-ESO-1-specific TCR, we observed significantly higher levels of the tumor-specific TCR expression, evaluated as NY-ESO-1 specific dextramer binding, in edited versus transferred T cells (relative fluorescence intensity to untransduced cells: CE: 37; SE: 31; TR: 19). Edited T cells were more efficient than unedited-TCR transferred T cells in killing NY-ESO-1-pulsed cell lines (half maximal effective peptide concentration in a 51Cr release assay: 310, 210, 186 nM for TR, SE and CE T cells respectively) and NY-ESO-1+ myeloma cell lines naturally processing the antigen. Importantly our SE and CE T cells displayed no activity against NY-ESO-1- targets. Importantly, in NSG mice, NY-ESO-1 redirected single edited and complete edited T cells completely eliminated an NY-ESO1+ HLA-A2+, WT1- myeloma cell line, that, on the contrary, expanded in bone marrow in the presence of WT1-redirected CE T cells. Our results demonstrate that the TCR single editing approach is effective in redirecting T cell specificity as evidenced by the potent anti-tumor effect observed while potentially eliminating the risk of GvHD associated with the infusion of donor-derived lymphocytes. Moreover, the relative speed and simplicity of the TCR single editing protocol should facilitate its clinical application to patients with hematological malignancies. Disclosures: Reik: Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

In this work, an ELISA for the quantitative determination of IgG anti-CD4 autoantibodies was validated and utilized in the follow-up of two cohorts of HIV-1-exposed seronegative subjects. A serum with an arbitrarily assigned concentration of 100,000 units/ml was used as a reference, and the detection limit, inter- and intraassay variability, and analytical recovery were calculated. The study subjects included adults sexually exposed to HIV-1-infected partners and the newborns of HIV-1+ mothers who seroreverted by 18 months of age. Some of these individuals were studied over an 18- to 24-month period. The detection limit of the assay was 2000 AU/ml. Intra- and interassay variability was, respectively, 3.92 and 3.90%. Analytical recovery in an assay in which a fixed amount of anti-CD4 antibodies was added to different samples was 98%. A proportion of adults (16 of 47, 34.0%) and babies (12 of 27, 44.4%) had significantly higher concentrations of anti-CD4 antibodies. Among them, 8 adults maintained the same concentration as that found in the first determination; on the other hand, 12 babies born to seronegative mothers showed a significant increase in the concentration of anti-CD4 antibodies during their first months of life. In conclusion, anti-CD4 antibodies can be measured using a validated ELISA. They represent a serologic trait that is quantitatively conserved in HIV-1-exposed seronegative adult individuals and is actively acquired by newborns to HIV+ mothers.

Donor lymphocyte infusion (DLI) plays a crucial role in promoting immune reconstitution and anti-tumor activity in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). The efficacy of DLI is, however, limited by the occurrence of graft-versus-host disease (GvHD). We showed that the infusion of lymphocytes transduced by a retroviral vector expressing a suicide gene (HSV-TK) and a surface marker (|[Delta]|LNGFR) allows to control GvHD in 100% of the cases, while preserving anti-tumor and antiviral activities in 65% and 83% of the patients. Expansion (up to 40% of circulating cells) and long-term persistence (>10 years) of transduced cells was observed in >40 patients treated with HSV-TK+ DLI in the context of HLA-identical and haplo-identical HSCT. No acute or chronic adverse or toxic effect due to the gene transfer procedure was observed in these patients, who were treated with a total of >1011 cells generated by >60 independent transductions. Analysis of gene expression profiles showed that

Abstract 4191 Introduction: HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a readily available therapeutic option for patients suffering from high-risk hematological malignancies who lack an HLA-compatible donor or for whom there is no time to find one. To avoid GVHD, haplo-HSCT has been classically performed under conditions of strict T-cell depletion, in the form of CD34-selected (“naked”) grafts. Unfortunately, naked haplo-HSCT is followed by a profound state of immune incompetence, which associates with high non-relapse mortality (NRM) rates, mainly due to opportunistic infections. Over the last decade, different strategies have been developed in order to speed-up immune reconstitution after haplo-HSCT, including the delayed infusion of donor T cells (DLI), the selective depletion of T-cell subsets from the graft or the use of an unmanipulated graft followed by novel strategies of immune suppression. Aim: To analyze in depth the early phenomena of immune reconstitution following haplo-HSCT in order to find early surrogate biomarkers of NRM. Results: From 2004 to 2010, we prospectively studied multiple parameters of T-cell immune reconstitution in 89 pts treated at our Center with haplo-HSCT. Time-points of analysis were pre-transplant, at day 30, day 90 and day 180 post-transplant. Underlying diseases included: high-risk AML 53 pts (60%), ALL 11 pts (12%), CLL 2 pts (2%), tyrosine kinase inhibitors-resistant CML 4 pts (5%), Hodgkin lymphoma 6 pts (7%), NHL 2 pts (2%), MDS with high International Prognostic Scoring System 7 pts (7%), other 4 pts (4%). Eighteen patients (20%) were given prophylactic suicide gene-modified DLI starting at day 30 post-transplant, while 64 patients (80%) received an unmanipulated graft followed by rapamycin until day 100. Overall, the incidence of grade III-IV acute GvHD was as low as 12%. Chronic extensive GvHD developed in 28% of pts. Compared with historical naked haplo-HSCT, the recovery of T-cell counts was accelerated: at day 90, median CD3+ cells were 378 per μL (0–2817), CD4+ 127 (0–804), CD8+ 173 (0–1922). Higher T-cell counts, however, did not clearly associate with lower NRM rates. After the initial expansion of effector memory cells (CD45RA−/CD62L−) in patients given DLI (P95%) pts that did not reactivate the virus at later time points. Strikingly, while in pts with 1000 spots/mL, this was 0% (P Conclusions: These results clearly indicate that the early reconstitution of T-cell immunity to CMV after haplo-HSCT does not only protect from subsequent viral reactivation, but also surrogates for lower NRM rates. Moreover, they warrant the investigation of a CMV-specific IFN-γ ELISPOT cut-off value of 1000 spots/mL as a predictive biomarker in larger, multicenter series. Disclosures: No relevant conflicts of interest to declare.

Abstract 667 Transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal CD8+ T cells is an attractive strategy for targeted cancer immunotherapy. However, the successful implementation of this approach is limited by technical and safety issues, including inefficient gene transfer, unstable transgene expression, exhaustion of gene-modified cells and most importantly, the unpredictable results of mispairing between the endogenous and exogenous TCR chains. Indeed, co-expression of the endogenous and exogenous TCR in the same cell not only reduces cell-surface expression of the introduced tumor-specific TCR, but also drives the potential for these gene-modified T cells to acquire autoreactive specificities. Such TCR mispairing has been shown to result in autoreactive T cells in animal models [Bendle et al., Nat Med. 2010]. To partially overcome these limitations, we have developed a novel strategy based on zinc finger nucleases (ZFNs) that permits editing of T cell specificity at the DNA level, combining the disruption of the endogenous TCR chain genes with the transfer of a tumor-specific TCR. Two sets of ZFNs were designed targeting the constant regions of the α (TRAC-ZFN) and β (TRBC-ZFN) TCR chain genes, respectively. We transiently delivered these ZFNs into primary T lymphocytes activated with anti-CD3 and anti-CD28 antibody-conjugated beads and cultured with low doses of IL-7 and IL-15, to promote the survival and expansion of the ZFN-modified cells. ZFN delivery into activated T lymphocytes abrogated expression of the CD3/TCR complex on the cell surface (% of CD3neg cells with TRAC-ZFN: 34%±11 and with TRBC-ZFN: 30%±9). No phenotypic differences were observed in CD3pos and CD3neg lymphocytes, which displayed a similar CD4/CD8 ratio while displaying an early T-cell differentiation phenotype, as evidenced by high expression of CD62L, CD27, CD28 and IL-7Rα markers. Sorted CD3neg cells proved stable in culture (demonstrating that ZFN exposure was well tolerated), and did not respond to TCR-dependent stimulation with the mitogen PHA, as expected for cells carrying a disrupted TCR gene. CD3neg cells were efficiently transduced with a lentiviral vector encoding a tumor-specific exogenous TCR chain, resulting in the restoration of cell surface translocation of CD3, thus facilitating the selective expansion of TCR-transduced cells by polyclonal stimulation. To demonstrate the antitumor activity of these modified cells we selected an HLA-A2 restricted, codon-optimized cysteine-modified TCR specific for the Wilms' tumor antigen 1 (WT1). To achieve complete editing of T cell specificity, we established a protocol that sequentially disrupted the endogenous TCR α and β chains with high efficiency (averages: 36% and 18%), followed by lentiviral transfer of the tumor-specific TCR α and β chains (average efficiencies: 65% and 25%). This procedure resulted in a population of TCR-edited lymphocytes encoding only the tumor-specific TCR that, in the absence of competition from the endogenous receptor, was expressed at high physiological levels. Accordingly, TCR-edited lymphocytes were superior to conventional TCR-transferred cells in promoting specific recognition of WT1-expressing targets, including primary leukemias, and most importantly, were devoid of residual endogenous TCR reactivity including alloreactivity. Finally, in a humanized GvHD model, we showed that, at a variance with 100% of mice infused with unmanipulated T cells, and 80% of mice receiving TCR-transferred lymphocytes developing lethal GvHD, no GvHD was observed upon infusion of matched TCR-edited cells, despite robust T cell engraftment rates across all groups. These data demonstrate that the successful genetic re-programming of T cell specificity in primary lymphocytes results in a functionally superior target specific killing activity and thus has the potential to greatly improve the safety and therapeutic benefit of cancer immunotherapy, without triggering its potentially negative effects. (Provasi and Genovese: equal contribution). Disclosures: Liu: Sangamo Biosciences: Employment. Reik:Sangamo Biosciences: Employment. Chu:Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Zhang:Sangamo Biosciences: Employment. Bordignon:Molmed: Employment. Holmes:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Bonini:Molmed: Consultancy.

Abstract 2052 T cell engineering against tumor antigens aims at ameliorating current immunotherapeutic strategies. To date, however, the suboptimal persistence of the transferred cells represents a serious limitation of this approach. The most appropriate T cell subset to be infused should ensure optimal in vivo persistence and yet appropriate anti-tumor activity. Here we report that culturing highly purified naïve T (TN) cells with beads conjugated to anti-CD3 and anti-CD28 antibodies, allows the retrieval of a novel post-mitotic CD45RA+ CD62L+ CCR7+ T cell population, which requires IL-7 and IL-15 for expansion and maintenance. This population is highly proliferative and sensitive to RV and LV transduction, expresses low levels of γIFN and cytotoxic molecules and is best defined as IL-7Rα+ CXCR4+ c-kit+ CCR5− HLA-DR− PD-1−. When infused in immunodeficient mice, genetically manipulated and in vitro expanded TN proved superior engraftment and longer persistence than transduced central memory (TCM) cells, and xenoreactivity comparable to that of unmanipulated lymphocytes. Engineered TN, but not TCM, maintained engraftment and xenoreactivity in serial transplantation experiments, indicating unique self-renewal abilities. Given the great potentials of this novel TN-derived cell population for immune-gene therapy, we further characterized it by molecular profiling. The gene expression signature is typical of antigen-experienced lymphocytes and classifies these cells between naturally occurring TN and TCM lymphocytes. Because of this and of the self-renewal abilities displayed in vivo, we termed them as precursor to TCM (TpreCM). We next sought to identify the natural counterpart of this TpreCM population in healthy donors, exploiting some of the markers present in the TpreCM signature, such as CD95 which is expressed by all memory T subsets but not by TN, and we selected the pp65 protein of cytomegalovirus (CMV) as a model antigen. CMV persistent infection induces a T-cell response that is maintained throughout life, indicating that a self-renewing memory T cells are generated. We studied the phenotype of CMV pp65-specific CD8+ T cells in seropositive donors and identified antigen-specific CD45RA+CD62L+. Among CD45RA+CD62L+ cells CMV-specific cells were enriched for CD45ROdim and CD95+ lymphocytes, which represent bona fide TpreCM. To functionally characterize natural TpreCM, we sorted CD45RA+CD62L+ cells according to their CD95 expression and challenged them with increasing doses anti-CD3 antibody, with or without a costimulatory signal. We found that among CD45RA+CD62L+ cells, only CD95+ lymphocytes were responsive to TCR-triggering alone, while CD95− cells required costimulation to proliferate. In conclusion, we identified a novel memory T cell subset, and identified conditions able to gene-modify and expand these memory lymphocytes while preserving their functional characteristics. Exploitation of these concepts might improve cancer adoptive immunotherapy. Disclosures: Bonini: MolMed: Consultancy.

Manipulation required to expand or engineer tumor-specific lymphocytes often induces terminal differentiation, resulting in poor in vivo efficacy. We hypothesized that initial targeting of Naive T cells (TN) could overcome this limitation. We activated and efficiently transduced FACS-sorted TN, TCM and TEM cells by viral vectors. Activation of TN with anti CD3/CD28 beads resulted in a predominant population of highly proliferative CD45RA+CD62L+CCR7+ T cells, that did not produce γIFN nor cytotoxic molecules and required IL7 and IL15 for their generation and maintenance. Manipulated-TN were post-mitotic, and expressed CD45RO, CD122, CXCR3 and CD95. Compared to manipulated-TCM and -TEM, naïve-derived T cells expressed higher levels of CD127, c-Kit and CXCR4 and lower levels of CCR5, HLA-DR and PD-1. To verify their differentiation potential, TN TCM and TEM were transduced to express a WT1-specific TCR and stimulated with WT1 peptide. In this context, TN expanded at higher numbers, differentiated into both CD45RA-CD62L+ and CD45RA-CD62L- cells and generated a mixed population of CD127± cells. When infused in NOD/Scid mice, manipulated TN showed a degree of xenoreactivity comparable to that of unmanipulated lymphocytes. In contrast to manipulated TCM cells, manipulated TN cells engrafted in secondary and tertiary recipients indicating that persistence, self-renewal abilities and expansion potential are preserved in this novel T cell subset after genetic manipulation.

The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction. RI Ambrosi, Alessandro/H-4424-2011

Abstract 3764 T cell receptor (TCR) gene-transfer is an attractive strategy for the adoptive immunotherapy of tumors. However, the full potential of this approach is limited by a number of technical hurdles including inefficient gene transfer, unstable transgene expression, exhaustion of gene-modified cells and most importantly, co-expression of the endogenous and exogenous tumor-specific TCR in the same cell. The co-expression of endogenous and exogenous TCR causes not only reduced cell-surface expression of the introduced tumor-specific TCR, but also the potential for T cells to acquire autoreactive specificities due to mispairing between the different TCR chains. Mispaired TCR have been shown to be autoreactive and potentially harmful in animal models. To address these limitations, we developed a novel strategy based on zinc finger nucleases (ZFNs) that allows for the first time the editing of T cell specificity at the DNA level, by combining the disruption of the endogenous TCR chain genes with the transfer of a tumor-specific TCR. We first stimulated PBL with anti-CD3 and anti-CD28 antibody-conjugated beads, and cultured these cells in low dose IL-7/IL-15 to preserve early differentiated T cells. We transiently expressed ZFNs targeting the constant region of the TCR alpha or beta chain genes in activated T lymphocytes using a chimeric Ad5/F35 vector, thus promoting the genetic disruption of the endogenous TCR. Lymphocytes targeted by each set of ZFNs abrogated expression of the CD3/TCR complex on the cell surface. CD3neg cells could be expanded in culture with IL7 and IL15 and expressed differentiation markers typical of central memory T cells (CD62L, CD127, CD27 and CD28), indistinguishably from unmodified T cells. Sorted CD3neg cells proved stable in culture and permissive to lentiviral transduction. Indeed, introduction of exogenous TCR chains on a Lentivirus restored the expression and functionality of the CD3/TCR complex and allowed selective expansion of TCR-transduced cells by stimulation via the TCR complex. As a model TCR, we selected an HLA-A2 restricted, codon-optimized cysteine-modified TCR specific for the Wilms' tumor antigen 1 (WT1), which is expressed by several solid tumors and leukemias and contributes to the uncontrolled proliferation of cancer cells. For a complete editing of T cell specificity, we established a protocol that sequentially disrupted the endogenous TCR chains followed by lentiviral transfer of the WT1-specific TCR. This procedure resulted in a population of TCR-edited lymphocytes encoding only the tumor-specific TCR that, in the absence of competition, was expressed at high and physiological levels. Accordingly, TCR-edited lymphocytes were superior to conventional TCR-transferred cells in promoting specific recognition of WT1-expressing targets, including primary leukemias, and most importantly, were devoid of residual endogenous reactivity including alloreactivity. These data demonstrate that the successful genetic re-programming of T cell specificity in primary lymphocytes results in a functionally superior target specific killing activity and thus has the potential to greatly improve the safety and therapeutic benefit of cancer immunotherapy. (Provasi and Genovese: equal contribution). Disclosures: Reik: Sangamo: Employment. Liu:Sangamo: Employment. Chu:Sangamo: Employment. Bordignon:Molmed: Employment. Holmes:Sangamo: Employment. Gregory:Sangamo: Employment. Bonini:Molmed: Consultancy.

Abstract 1471 Differentiation stage of adoptively transferred T cells has a critical impact on the success of immune-gene therapy. Ex vivo T cell manipulation often induces their terminal differentiation, resulting in poor persistence and activity of transferred cells. We previously showed that costimulation and culture with γ-chain cytokines generates gene modified T cells with a functional central memory (TCM) phenotype superior to effector/effector memory (TEM) counterparts for expansion potential and antitumor activity. Here we investigated the consequence of initial targeting of selected T cell subpopulations. We activated and efficiently transduced FACS-sorted T Naïve (TN), TCM and TEM cells by viral vectors. In contrast to TCM and TEM, TN had a greater expansion potential and more sustained expression of IL7-Rα. Strikingly, manipulation of TN resulted in a predominant population of post-mitotic lymphocytes expressing the CD45RA+CD62L+CCR7+ naïve phenotype. These cells expressed markers common to early differentiated cells (CD27, CD28, CD127 and Bcl2) and markers proper of memory lymphocytes (CD45RO, CD122 and CXCR3). Post-mitotic TN produced lower levels of IFNg and Granzyme A, expressed higher levels of c-Kit and CXCR4, and lower levels of HLA-DR, CCR5 and PD1 than memory counterparts. To verify their self-renewal and differentiation potential upon antigen stimulations, TN, TCM and TEM were transduced to express a WT1-specific TCR. Upon multiple stimulations TN expanded at higher numbers and were unique in the ability to generate a mixed population of CD127+/CD127- lymphocytes. When infused in immunodeficient mice, transduced TN proved higher engraftment and persistence potential than memory counterparts, reconstituted a mixed CD45RA±CD62L± phenotype and were significantly higher xenoreactive. These results suggest that gene transfer into TN lymphocytes might increase the efficacy of cancer immunotherapy. Disclosures: Bonini: MolMed: Consultancy.

The transfer of high-avidity T cell receptors (TCR) isolated from tumor-specific lymphocytes into polyclonal T cells is an attractive strategy for the adoptive immunotherapy of tumors. However, three major hurdles hinder the effectiveness of this strategy: i) inefficient gene transfer and unstable transgene expression, ii) exhaustion of gene-modified cells, and iii) mispairing of endogenous and exogenous TCR chains. To solve these issues, we used a bi-directional lentiviral vector (LV) expressing a codon-optimized, cysteine-modified TCR for the Wilms tumor antigen 1 (WT1) to genetically modified human central memory T lymphocytes (TCM). TCM have been reported superior to effectors in mediating long-lasting anti-tumor responses in vivo. Transduced cells showed high (∼16%) and stable (70 days in culture) levels of WT1-pentamer binding and cytotoxic activity towards WT1+ primary AML leukemic blasts. To further enhance the safety profile and potency of modified TCM, we developed a platform to genome edit TCM based on Zinc Finger Nucleases (ZFN). In order to ensure stable and consistent transgene expression, we demonstrated the targeted insertion of the transgene into a predefined genomic locus (CCR5) by homologous recombination in up to 6% of treated TCM, with concurrent inactivation of 18% of the targeted CCR5 gene by non-homologous end joining (NHEJ). These findings prompted us to address the major technological hurdle of TCR gene transfer: the co-expression of endogenous and tumor specific TCRs in the same cell. Indeed, this result in competition for the CD3 co-receptor molecules, leading to TCR dilution, and in TCR mispairing, giving rise to unpredictable specificities that are potentially autoreactive. To solve these issues, we developed ZFN specific for the endogenous TCR β chain gene to promote the disruption of TCR β by NHEJ. We obtained functional inactivation of this gene in up to 7% of TCM. Targeted lymphocytes failed in expressing the CD3/TCR complex on cell surface, and could be sorted as CD3neg. CD3neg proved stable in culture, maintained a TCM phenotype and were resistant to further TCR-mediated triggering. For a complete editing of T cell specificity, the WT1-specific TCR was transferred with high efficiency (>20%) by LV on CD3neg cells. WT1-TCR rescued CD3 surface expression and was expressed in edited lymphocytes at higher levels to that observed upon conventional TCR gene transfer. Accordingly, TCR-edited cells proved superior to cells undergoing conventional TCR gene transfer in promoting killing of relevant targets, and in recognizing low (10 nM) peptide concentrations. Overall, these data show the feasibility of rapid and efficient editing of TCM to generate safe and functional tumor-specific T cells and may open new perspectives to cancer adoptive immunotherapy. (PG and EP: equal contribution) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2937.

Abstract 4090 Poster Board III-1025 Allogeneic hematopoietic transplantation (allo-HCT) is the only curative option for patients affected by high-risk acute myeloid leukemia (AML). This is largely due to the ability of allogeneic immune system to eradicate leukemic stem cells (LSC). However, the fact that some patients still relapse after allo-HCT, suggests that strategies to increase LSC targeting by donor T cells are needed. For this purpose, we exploited the unique ability of myeloid blasts to differentiate into leukemic dendritic cells (LDC). We observed that a short (48h) exposure to calcium ionophore A23187 and IL-4 is able to induce LDC differentiation in 14/16 (86%) of AML that we studied, both de novo and secondary. Importantly, despite phenotypic and functional changes indicative of differentiation into DC-like cells, the process was accompanied by the maintenance of disease markers such as CD34 and CD117. Moreover, LDC maintained the expression of the oncogenic protein WT1, which is a putative LSC antigen. Thanks to these favourable characteristics, LDC proved to be superior to the original blasts in expanding leukemia-reactive T lymphocytes both in the autologous and allogeneic HCT setting (on average, 5-fold expansion of blasts-stimulated T cells vs 95-fold expansion of LDC-stimulated T cells, SEM=2,7 and 67,7 respectively, p=0,01). We observed that the level of T-cell expansion directly correlate with the percentage of LDC obtained upon treatment with A23187 and IL-4. Most importantly, LDC proved to be more potent than blasts in expanding central memory T lymphocytes (TCM), which are known to confer superior anti-tumor immunity (on average, 29% of TCM upon stimulation with blasts vs 53% TCM upon stimulation with LDC, SEM=7,2 and 5,7 respectively, p=0,01). LDC-expanded T lymphocytes were able to efficiently recognize and kill leukemic blasts in vitro (on average, 953 specific spots of IFN-g/50'000 effectors at E:T ratio of 10:1 -SEM=120- and 29% of specific killing at E:T ratio of 50:1 -SEM=7,4-). Importantly, analysis of different HLA-settings and different targets of patient origin, suggests that LDC can expand T lymphocytes with specificities against multiple antigens expressed by the original leukemia. In particular, we observed the expansion of WT-1 specific T cells upon LDC stimulation. Finally, when infused in NOD/Scid mice transplanted with the original leukaemia, LDC-stimulated T lymphocytes were able to induce long-term complete remissions (>16 weeks) in all mice analyzed, suggesting that this approach may be active against leukemic stem cells. These results show for the first time that LDC-stimulated human T cells could exert a strong GvL activity in vivo. Disclosures: Bordignon: Molmed Spa: Employment.

Abstract 374 The transfer of a T cell receptor (TCR) from a high-avidity tumor-specific lymphocyte to polyclonal lymphocytes as been proposed as an attractive approach to overcome the major limitations of adoptive immunotherapy of cancer, such as the difficulty in expanding rare, high-avidity tumor-specific CTLs from cancer patients, in conditions that can preserve their function and prevent exhaustion. However, complete exploitation of this approach is limited by technical and safety issues, like the low and transient transgene expression, unpredictable pairing of the exogenous and endogenous TCR chains and poor survival and expansion potential of gene-modified effector lymphocytes. We developed a strategy aimed at overcoming these limitations. First, to increase TCR expression and facilitate proper TCR pairing we used a codon-optimized high-avidity TCR, specific for an HLA-A2-restricted peptide from the oncogenic Wilms tumor antigen 1 (WT1), modified with point mutations to introduce cysteines into the constant regions of the α and β chains. We cloned genes encoding the TCR in third generation lentiviral vectors under the control of a bi-directional PGK or a bi-directional EF1α promoter. Second, to increase the expected life span and expansion potential of gene modified cells, we transduced lymphocytes upon activation with anti-CD3 and anti-CD28 antibody-conjugated beads (bCD3/CD28) and culture with low doses of IL-7/IL-15. Human T lymphocytes were efficiently transduced by both vectors. However, PGK promoter was superior to EF1α in sustaining stochiometric expression of WT1-specific TCR chains, at levels appropriate for efficient HLA-A2/WT1 pentamer binding (16%), for up to 70 days, in the absence of further T cell stimulation. The phenotype of transduced cells was consistent with early T cell differentiation (CD62L+, CD28+CD27+, IL7Rα+, IL-2+ γIFN±), a phenotype associated with long-term persistence of adoptively transferred lymphocytes. Most importantly, TCR transduced cells were able to specifically produce γIFN and exhibited cytotoxic activity against WT1+HLA-A2+ primary leukemic blasts from AML patients. Third, to further improve the safety of the strategy and ensure predictable levels of transgene expression, we developed, for the first time, a protocol for site-specific integration of transgenes into primary T lymphocytes by exploiting zinc finger nucleases (ZFNs). Delivery in primary T lymphocytes of ZFNs targeting the CCR5 gene, in association with a GFP expression cassette flanked by CCR5 homology arms, resulted in efficient (6%) site-specific integration of GFP into the CCR5 locus. Extensive molecular analysis on sorted gene-modified cells and clones confirmed site-specific integration and showed a biallelic CCR5 disruption in 87% of GFP+ clones, due to a combined site-specific integration in one CCR5 allele, and non-homologous end joining (NHEJ) repair in the other allele. Finally, we exploited ZFNs to address the major limitation of TCR gene transfer, represented by the co-expression in the same cell of the endogenous and tumor-specific TCR. Such co-expression results in a dilution of tumor-specific TCR, thus reducing the efficacy of gene-modified cells, and might result in autoreactive specificities due to misparing between endogenous and exogenous α and β TCR chains. To this purpose, we used a new set of ZFNs specific for the constant region of the TCR β chain and knock down the expression of endogenous TCR in 20% of Jurkat cell line and 3.5% of primary human lymphocytes. These results show that a complete editing of T cell specificities can be achieved in human lymphocytes. (E.P. and P.G. equally contributed to this work). Disclosures: Gregory: Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment.

The potency of cellular adoptive immunotherapy against cancer has been revealed by persistent and complete clinical responses obtained with allogeneic hemopoietic cell transplantation (allo-HSCT) followed by the adoptive transfer of donor T lymphocytes and by initial clinical responses observed with the adoptive transfer of tumor specific cytotoxic T lymphocytes (CTLs) in cancer patients. Major hurdles limiting adoptive T cell therapy relate to toxicity (i.e. graft-versus-host disease-GvHD in allo-HSCT) and efficacy (i.e. difficulty in expanding rare, high-avidity tumor-specific CTLs in conditions that can preserve their function and prevent exhaustion). The transfer of the T cell receptor (TCR) from high-avidity tumor-specific CTLs to polyclonal lymphocytes may overcome these difficulties, but is still limited by low and transient transgene expression, unpredictable pairing of the exogenous and endogenous TCR chains and poor survival and expansion potential of gene-modified effector lymphocytes. To overcome these limitations, we cloned genes encoding a high-avidity TCR specific for an HLA-A2-restricted peptide from the oncogenic Wilms tumor antigen 1 (WT1126-135), in a third generation lentiviral vector under the control of a bi-directional PGK or a bi-directional EF1α promoter. To increase TCR expression and facilitate appropriate TCR pairing, we used a codon-optimized TCR, modified with point mutations to introduce cysteines into the constant regions of the α and 7β chains. Human T lymphocytes were efficiently transduced by both vectors, following activation with anti-CD3 and anti-CD28 antibody-conjugated beads (bCD3/CD28) and culture with low doses of IL-7/IL-15. However, the PGK promoter was superior to EF1α in sustaining stochiometric expression of WT1-specific TCR chains, at levels appropriate for efficient HLA-A2/WT1 pentamer binding (16%), for up to 50 days, in the absence of further T cell stimulation. Additionally, we observed that a phenotype consistent with early (naïve and central memory) T cell differentiation (CD45RA−/+CD62L+, CD28+CD27+, IL7Ra+, IL-2+ γIFN±) was preserved in TCR-modified lymphocytes generated in these culture conditions. Sorted naïve (CD45RA+/CD62L+) and central memory (CD45RA−/CD62L+) lymphocytes were efficiently transduced by TCR-LV and maintained the original T cell phenotype. Accordingly, TCR-modified lymphocytes showed excellent survival and expansion capacity, and, upon antigenic stimulation mediated high WT1-specific γIFN production and cytotoxic activity. To improve the safety of the strategy, we attempted sitespecific integration of transgenes using of the ZFN technology. Adenoviral transfer of a set of ZFN specific for the putative safe-harbor locus CCR5 coupled with integrase defective lentiviral vectors carrying the donor DNA flanked transgene, enables efficient site-specific integration in human lymphocytes resulting in stable transgene expression. Analyses of sorted gene-modified cells is currently ongoing. Site-specific integration of an optimized, leukemia-specific TCR into both naïve and central memory lymphocytes may represent an effective method for the generation of robust engineered tumor-specific CTLs.

Background. Chronic graft-versus-host disease (cGvHD) is a common complication of allogeneic hemopoietic cell transplantation (allo-HCT). The pathogenesis of cGvHD is poorly understood. In cGvHD, the homeostasis of B lymphocytes is perturbed, as demonstrated by the production of autoantibodies. B-cell depletion with monoclonal antibodies (mAb) interferes with autoantibody production and ameliorates signs and symptoms of cGvHD. In mouse models, cGvHD and autoantibodies associate with the long-term persistence of host B cells after allo-HCT (Sylvain Perruche et al., Transplantation 2006). It has been postulated that host B cells may present alloantigens to donor T cells and, in turn, receive help for autoantibody production. This could be crucial to the pathogenesis of cGvHD. Aim. To investigate whether the long-term persistence of host B lymphocytes is associated with cGvHD and autoantibodies in humans. Patients and methods. We recruited 13 consecutive patients with active cGvHD (4 mild, 5 moderate, 4 severe according to NIH classification) with a median time of onset of 6 months (range 3–36) from HLA-identical sibling (9 patients) and HLA-matched unrelated (4) allo-HCT. As controls, we chose 10 patients that underwent HLAidentical sibling (2), HLA-matched unrelated (5) or haploidentical (3) allo-HCT and never experienced cGvHD. In the two groups, we studied: circulating autoantibodies, including anti-nuclear (ANA), anti-DNA, anti-extractable nuclear antigen, anti-beta2 glycoprotein, anti-neutrophil cytoplasm, anti-thyroid, anti-mytocondria antibodies, rheumatoid factor, absolute numbers of T (CD3+, CD4+, CD8+), conventional B (CD19+), B1 (CD5+/CD19+) and NK cells (CD16+/CD56+) in the graft and in the peripheral blood, microchimerism by short-tandem repeats (STR) on B, T and myeloid cells purified by immunomagnetic cell sorting (sensitivity 0,01%). Results. Patients with cGvHD had high-titer circulating ANA (>1:160) more frequently than controls (54% versus 10%, P Conclusions. This study indicates that autoantibody production during cGvHD does not associate with long-term persistence of host B cells in humans. Moreover, it suggests that the early depletion of donor B lymphocytes in vivo may be effective for GvHD prophylaxis

Adoptive immunotherapy of leukemia aims at eradicating the disease through the transfer of leukemia-reactive lymphocytes. Donor lymphocytes infusions (DLI) after allogeneic hemopoietic cell transplantation (allo-HCT) have shown to cure chronic myelogenous leukemia (CML). Unfortunately, when applied to acute leukemias, the DLI approach is far less successful. Strategies to increase the efficacy of adoptive immunotherapy for the cure of acute leukemias are therefore urgently needed. To this aim, blasts from acute myeloid leukemias (AML) can be differentiated in vitro into dendritic cells (leukemic DC or LDC) that directly present leukemic antigens to T lymphocytes. This is commonly achieved by culturing AML blasts with cytokines such as GM-CSF, IL-4 and TNF-α for 7–14 days. LDC can be further induced to functional maturation by exposure to CD40L. The efficacy of these protocols is, however, suboptimal, since a large proportion of AML sub-types do not differentiate into LDC. The addition of calcium ionophore (A23187) has been shown to rescue some of the cases. The aim of this study is to identify the characteristics associated with the ability of blast from AML to differentiate into functionally mature LDC. To this, we studied a cohort of 17 consecutive AML patients. AML blasts were induced to differentiate into LDC either by GM-CSF, IL-4 and TNF- α or by cytokines and A23187. LDC were further matured with CD40L. LDC differentiation was defined by expression of MHC class II, costimulatory (CD80, CD86, CD40, CD70) and adhesion molecules (CD54, CD58). The overall efficacy of LDC differentiation by cytokines was 20%. Maturation of LDC induced by cytokines failed, as ascertained by the lack of up-regulation of CD83 and the chemokine receptor CCR7. Upon exposure of AML blasts to cytokines and A23187, we observed LDC differentiation in more than 90% of the cases. LDC differentiation occurred in 24–48 hrs and was associated with the maintenance of disease markers expression (c-kit, CD34). Interestingly, blasts derived from AML secondary to myelodysplastic syndromes directly differentiated into mature LDC. On the other hand, LDC derived from primary AML were insensitive to the maturative stimulus CD40L. Accordingly, LDC from secondary AML were the most potent inducers of allogeneic T-cell proliferation. The efficacy of the combination of calcium ionophore and cytokines in differentiating myelodysplastic blasts into LDC provides the rational for novel adoptive immunotherapeutic approaches to treat high risk AML. Furthermore, the differential ability of primary leukemic blasts and blasts secondary to myelodysplastic syndromes to rapidly differentiate into functionally mature LDC suggest the existence of molecular factors committing AML cells to the DC lineage. The identification of such factors and their exploitation, for example by gene transfer, will enable the generation of LDC from all cases of AML for the stimulation of the immune system against leukemia.

T-cell depletion of allogeneic hematopoietic stem cell graft (HSCT) represents the most powerful approach to prevent graft-versus-host disease (GvHD), thus allowing to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. We hypothesized that early add-back of suicide-gene transduced donor lymphocytes (TK cells) to leukemic patients undergoing haploidentical HSCT (haplo-HSCT) could provide early immune-reconstitution and selective control of GvHD. In a phase II clinical trial (protocol MMTK007), 17 of 29 enrolled pts, (median age 52), received add-backs of 10^7/kg TK cells 42 days after haplo-HSCT. TK cells engraftment, observed in 14 patients, was necessary and sufficient for a rapid and effective immunereconstitution (IR), with a median of 144 (101–336) CD3+, 59 (28–149) CD4+ and 86 (52–279) CD8+ cells/mcl at day 100 after HSCT. Accordingly, engraftment of TK cells was tightly correlated with clinical outcome: while 3/3 pts who failed TK cells engraftment died of infections, only 1/14 TK engrafted patients died from infections (last event at day +166). As shown in Table I, the immune repertoire of treated patients improved significantly at 6 months post transplant and normalized completely in 12 months. High numbers of T cell precursors specific for CMV and EBV were detected at immune reconstitution (median of 86 and 69 gIFN specific spots/10^5 PBMC respectively) and predicted subsequent freedom from viral reactivation (p=0.002). Six pts developed acute (GvHD), (grade I to IV) that was always completely abrogated by the suicide system. Overall survival of TK cells treated patients is 50% at three years. These results indicate that TK-DLI abolish late mortality after CD34+ haplo-SCT in adults. A phase III multicentric study will start in 2007 to validate prospectively the advantage of TK-DLI in haplo-SCT.

Retroviral vectors have raised safety concerns for the genotoxic risk associated with their uncontrolled insertion into the human genome. The occurrence of T-cell leukemia in patients treated by gene therapy for X-SCID has been correlated with insertional activation of a T-cell proto-oncogene. However, no such event has been reported in other pre-clinical or clinical trials, suggesting the existence of disease- and/or transgene-specific risk factors in the X-SCID case. We have analyzed the integration pattern of a retroviral vector expressing a suicide gene (HSV-TK) and a surface marker (ΔLNGFR) in T-lymphocytes from patients treated with donor lymphocyte infusion (DLI) after hematopoietic stem cell transplantation for therapy of acute and chronic leukemia. The suicide gene allows to control graft-versus-host disease, a major complication of DLI, while preserving anti-tumor and antiviral activities. Proviral integrations were analyzed by sequencing the vector-genome junctions in T-cell DNA obtained from 4 different patients up to 9 years after treatment. Over 85% of proviral integrations occurred within transcription units, with a preference for first introns and upstream promoter regions. Over 75% of intragenic integrations were in genes active in T cells at the time of transduction, and ~4% occurred at recurrent genomic locations (hot spots). Microfluidic real-time PCR in RNA extracted from individual T-cell clones harboring proviral integrations in proximity of transcription start sites showed that one fifth of these integrations affect the expression of nearby genes. However, transduced T-cell populations maintain remarkably stable gene expression profiles, phenotype, biological functions and immune repertoire in vivo, with no evidence of clonal selection up to nine years after administration. Analysis of transduced cells before and after transplantation indicates that integrations interfering with normal T-cell function are more likely to lead to clonal ablation than expansion. Despite the potentially dangerous interactions with the T-cell genome, retroviral integration has therefore little consequences on the safety and efficacy of T-cell transplantation.

Donor-recipient incompatibility for human leukocyte antigen (HLA) ligands of killer cell immunoglobulin-like receptors (KIRs) in haploidentical hematopoietic stem cell transplantation (HSCT), has been associated with a selective graft versus leukemia (GvL) effect mediated by donor-derived alloreactive natural killer (NK) cells expressing KIRs whose ligands are missing in the recipient. In this study, we show that NK cells arising from hematopoietic stem cell progenitors after transplantation into haploidentical recipients, acquire a receptor repertoire that is compatible with patient-specific tolerance due to engagement of patient HLA ligands by inhibitory NK receptors. Using four-color immunofluorescence with monoclonal antibodies (mAbs) specific for the receptors CD94/NKG2A, KIR2DL1/2DS1, KIR2DL2/2DL3/2DS2 and KIR3DL1, we have analyzed NK receptor reconstitution kinetics in eleven adult patients affected by acute myeloid (n=9) or lymphoblastic (n=2) leukemia, who underwent HSCT from a KIR ligand matched (n=5) or mismatched (n=6) haploidentical family donor, using high doses (median 12.5x106/kg) of purified CD34+ progenitors. Nine patients achieved long-term (>150 days) complete remission of disease, independently from disease status at time of transplantation, and, importantly, from the presence (n=5) or absence (n=4) of donor NK alloreactivity. Within the first two months after transplantation, the vast majority (96% at 30 days, 86% at 60 days; SD 2% and 11%, respectively) of NK cells arising in the patients expressed the inhibitory receptor CD94/NKG2A, whose ligand HLA-E is ubiquitously expressed by cells positive for classical HLA class I molecules including leukemic blasts. As shown by mAb inhibition studies, lysis of patient-derived phytohemagglutinin-activated T cell blasts by these early arising NK cells was specifically inhibited by engagement of CD94/NKG2A. KIR expression was restored with variable kinetics in the later post-transplantation phase (3–9 months). Interestingly, however, during this period, NK cells devoid of CD94/NKG2A were found to express at least one KIR specific for an HLA ligand present in the patient, suggesting functional silencing of NK cells arising in the later phases after transplantation by acquisition of specific KIRs. Taken together, these data challenge current broad view on putative antileukemic effect of alloreactive NK cells reconstituting from haploidentical donor CD34+ cells and suggest that optimal exploitation of NK alloreactivity for GvL requires the presence of NK cells matured in the context of the donor’s rather than the recipient’s HLA repertoire. Ultimately, these findings provide a rationale for emerging clinical evidence in favor of efficacy of NK-based immunotherapy with mature donor NK cells.

Suicide gene therapy (SGT) is a powerful approach to exploit anti-host reactivity following allogeneic hemopoietic cell transplantation (allo-HCT) for a full graft-versus-leukemia (GvL) effect, while controlling graft-versus-host disease (GvHD). This is accomplished through genetic modification of donor lymphocytes with a suicide gene. The herpes simplex thymidine kinase (TK) suicide gene converts at a cellular level the pro-drug ganciclovir (GCV) into tri-phosphate toxic derivatives, thence conferring selective sensitivity. Clinical studies as well as animal models have substantiated the concept that a time-wise GCV administration is able to actually separate the therapeutic GvL effect from life-threatening GvHD. Genetic modification of lymphocytes with TK is currently pursued through retroviral vectors (RV). In vitro RV genetic modification requires proliferation, which is easily accomplished by polyclonal stimulation. Polyclonal stimulation with anti-CD3 antibodies (aCD3) has been show to reduce anti-host reactivity of gene-modified lymphocytes. This possibly limits the impact of the suicide gene strategy in allo-HCT. In this study we tackled the rules governing anti-host reactivity of TK+ human lymphocytes. We found that TK+ lymphocytes generated with aCD3 are mainly CD45RA−CCR7− effector memory (EM) cells (84,6±6,6%). Upon re-stimulation they produce interferon-γ, perforin B and granzyme A, but fail to up-regulate CD40L. EM TK+ lymphocytes have a mixed phenotype in regard to CD28/CD27 co-expression and displayed a limited ability to engraft and cause GvHD in a xenogeneic model using conditioned NOD/scid mice (take: 11%). In sharp contrast, TK+ lymphocytes generated with novel protocols taking advantage of anti-CD3 and anti-CD28 antibodies conjugated to para-magnetic cell-sized beads are enriched for CD45RA−CCR7+ central memory (CM) cells (65,3±6,2%) that are able to produce IL-2 and to strongly up-regulate CD40L. CM TK+ lymphocytes are homogenously CD28+CD27+ (91,1,3±2,5%). When infused in conditioned NOD/scid mice CM TK+ lymphocytes persistently engrafted and caused lethal GvHD in a significant fraction of mice (take: 55%, P=0,0017 vs EM TK+ cells). GCV administration to diseased animals resulted in the elimination of TK+ cells in blood and in target organs. Treated animals were rescued with survival up to 120 days (P=0,009 vs saline-treated mice). These results demonstrate that CM TK+ lymphocytes retain significant anti-host reactivity and provide a clue to their in vitro generation. CM TK+ lymphocytes are promising candidates for safe and effective allo-HCT for the cure of hematological malignancies.

Haplo-identical stem cell transplantation (haplo-SCT) is a therapeutic option for pts with high risk hematologic malignancies lacking an HLA matched donor. The full exploitation of haplo-SCT is limited by delayed immune reconstitution and prolonged risk of life-threatening infections. We previously identified in an academic haplo-SCT trial, effective doses of donor lymphocytes genetically engineered to express a suicide gene (the herpes simplex thymidine kinase (TK-DLI) as an effective tool for preventing disease relapse and promoting immune reconstitution. In case of GvHD, TK-DLI could be selectively eliminated by ganciclovir. A phase II multicenter trial (MM TK007), aimed at verifying the therapeutic activity of early add-backs of TK+ cells after haplo-SCT in inducing a stable immune reconstitution, started in 2002. 20 pts were recruited and transplanted for AML (16, 8 of whom post MDS), ALL (2), advanced NHL and HD (2); disease status at SCT was CR1(5), CR2(7), refractory(8). Median time from diagnosis to SCT was 394 d (95-3752), median age was 52 (17–62). A median of 12.3x106/kg (7.3–15.5) CD34+ selected cells (Clinimacs) and 1.05x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning regimen. GvHD prophylaxis was never administered post-SCT. 18/20 pts engrafted with a median time of 13 d (8–21), for ANC >1.0x109/l and 13 d (11–24) for plt >50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-DLI. The statistical end-point of the study was to achieve immune reconstitution in 7/18 treated pts. So far, 10 pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42. Although this study will continue up to 18 treated pts, the primary end-point has been already achieved since 7/9 evaluable pts obtained CD3+ >100/μl at a median time of 76 d (61–99) from SCT and 21 d (14–31) from TK-DLI. Transduced cells were documented ex vivo in all pts; median counts at peak of immune reconstitution were 483 CD3+/μl, 304 CD8+/μl, 130 CD4+/μl. Prompt immune reconstitution resulted in complete control of viral infections and virtually eliminated late infections frequently reported after haplo-SCT. In 1 pt a grade II biopsy-proven acute GvHD, involving skin and liver was observed and completely abrogated by ganciclovir (10 mg/kg/day) in the absence of immunosuppressive drugs. One pt relapsed at d 300; overall, 6 pts are alive in CR at d +423, +363, +317, +255, +109, +104. The preliminary results of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from infectious mortality after haplo-SCT while providing an effective and selective treatment for GvHD. The potential of this strategy in preventing disease relapse is promising and will remain the main focus of the long-term follow-up.

Donor lymphocyte infusions (DLI) play a crucial role in promoting immune reconstitution and anti-tumor activity in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). The efficacy of DLI is, however, limited by the occurrence of graft-versus-host disease (GvHD). In three different clinical trials, we showed that the infusion of lymphocytes transduced by a retroviral vector expressing a suicide gene (HSV-TK) and a surface marker (ΔLNGFR) allows to control GvHD in 100% of cases, while preserving anti-tumor and antiviral activities. In >40 patients treated with TK-cells in the context of HLA identical and haplo-identical HSCT, we observed consistent expansion (up to 40% of circulating cells) and long-term persistence (>10 years) of transduced cells. No acute or chronic adverse or toxic effect due to the gene transfer procedure was observed in these patients, who were treated with a total of >1011 cells generated by >60 independent transductions. Analysis of vector integration sites was preformed by LM-PCR on lymphocytes obtained up to 10 years after treatment from 4 patients, and selected for ΔLNGFR expression. 60% of the sequences met our validity criteria and were unambiguously mapped onto the human genome by Ensembl BLAST analysis. Transduced T-cells were highly polyclonal, with vector integrations occurring preferentially within genes (52%), and particularly within the first intron (25%). Quantitative PCR analysis of selected integrations was performed to follow the dynamics of individual T-cell clones during time. Microchip analysis of the gene expression profile showed that 22,000 genes (0.9%) were differentially expressed in ΔLNGFR+ vs. ΔLNGFR- T-cells from two different patients, suggesting that no significant perturbation was induced by retroviral transduction or HSV-TK/ΔLNGFR expression in human lymphocyte populations. Interestingly, these data also show the substantial biological identity of T-cell population generated by HSCT and those administered as DLI. Finally, the influence of proviral integration on the expression of targeted genes was studied in individual T-cell clones transduced in vitro or obtained ex vivo from treated patients by quantitative gene expression analysis.

6515 Background: Haplo-identical stem cell transplantation (haplo-SCT) is a therapeutic option for patients with high risk hematologic malignancies lacking an HLA matched donor. The full exploitation of haplo-SCT is limited by delayed immune recovery, resulting in prolonged risk of life-threatening infections. Infusion of small numbers of donor T cells, aimed at providing immune reconstitution, results in severe forms of graft-versus-host-disease (GvHD). We previously showed that infusion of donor lymphocytes (DLI) expressing the herpes simplex virus thymidine kinase (Tk) is an efficient tool for controlling GvHD and preserving anti-tumor activity. Methods: We designed a dose-finding clinical protocol of Tk-DLI for immune reconstitution and relapse prevention after haplo-SCT. Eight pts with high risk hematologic malignancies who underwent haplo-SCT received escalating doses (10e6–10e7/kg) of Tk-DLI from day +42. Results: Transduced cells were documented ex vivo in all evaluable pts. While no CD3+ cells we...

Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.