Sara Mastaglio

IL-7 and IL-15 allow the generation of suicide gene–modified alloreactive self-renewing central memory human T lymphocytes

Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (T(EM)) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (T(CM)) phenotype. To this, we generated suicide gene-modified T(CM) lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene-modified T(CM) cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene-modified T cells cultured with IL-7 and IL-15 persisted, differentiated in T(EM) cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene-modified T(CM) cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer.

Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors

Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients’ bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II–IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus–mycophenolate–ATG-F–rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.

Improving the safety of cell therapy with the TK-suicide gene.

While opening new frontiers for the cure of malignant and non-malignant diseases, the increasing use of cell therapy poses also several new challenges related to the safety of a living drug. The most effective and consolidated cell therapy approach is allogeneic hematopoietic stem cell transplantation (HSCT), the only cure for several patients with high-risk hematological malignancies. The potential of allogeneic HSCT is strictly dependent on the donor immune system, particularly on alloreactive T lymphocytes, that promote the beneficial graft-versus-tumor effect (GvT), but may also trigger the detrimental graft-versus-host-disease (GvHD). Gene transfer technologies allow to manipulate donor T-cells to enforce GvT and foster immune reconstitution, while avoiding or controlling GvHD. The suicide gene approach is based on the transfer of a suicide gene into donor lymphocytes, for a safe infusion of a wide T-cell repertoire, that might be selectively controlled in vivo in case of GvHD. The herpes simplex virus thymidine kinase (HSV-TK) is the suicide gene most extensively tested in humans. Expression of HSV-TK in donor lymphocytes confers lethal sensitivity to the anti-herpes drug, ganciclovir. Progressive improvements in suicide genes, vector technology and transduction protocols have allowed to overcome the toxicity of GvHD while preserving the antitumor efficacy of allogeneic HSCT. Several phase I-II clinical trials in the last 20 years document the safety and the efficacy of HSV-TK approach, able to maintain its clear value over the last decades, in the rapidly progressing horizon of cancer cellular therapy.

Adoptive immunotherapy with genetically modified lymphocytes in allogeneic stem cell transplantation

Hematopoietic stem cell transplantation from a healthy donor (allo‐HSCT) represents the most potent form of cellular adoptive immunotherapy to treat malignancies. In allo‐HSCT, donor T cells are double edge‐swords: highly potent against residual tumor cells, but potentially highly toxic, and responsible for graft versus host disease (GVHD), a major clinical complication of transplantation. Gene transfer technologies coupled with current knowledge on cancer immunology have generated a wide range of approaches aimed at fostering the immunological response to cancer cells, while avoiding or controlling GVHD. In this review, we discuss cell and gene therapy approaches currently tested in preclinical models and in clinical trials.

IL-7 receptor expression identifies suicide gene-modified allospecific CD8+ T cells capable of self-renewal and differentiation into antileukemia effectors.

In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy-that is, the genetic induction of a conditional suicide phenotype into donor T cells-allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk(+) T cells contained HA-1- and H-Y-specific CD8(+) cytotoxic T cells (CTL) precursors. Thymidine kinase-positive HA-1- and H-Y-specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.

Tracking genetically engineered lymphocytes long-term reveals the dynamics of T cell immunological memory

Antigen exposure and differentiation phenotype influence long-term persistence of memory T cells after hematopoietic stem cell transplant. Committing T cells to memory Adoptive cell transfer is an increasingly successful therapy for a variety of diseases; however, little is known about what regulates the survival of these cells in humans. Now, Oliveira et al. leverage patients who have received genetically modified hematopoietic stem cells to track T cells over time. They found labeled effector memory, central memory, and stem memory T cells 2 to 14 years after infusion in all patients. Antigen recognition was critical in driving persistence and expansion. The clones that survived long-term appeared to initiate preferentially from central and stem cell memory T cell populations. These data suggest that the original phenotype of infused cells may influence long-term persistence of adoptively transferred cells. Long-lasting immune protection from pathogens and cancer requires the generation of memory T cells able to survive long-term. To unravel the immunological requirements for long-term persistence of human memory T cells, we characterized and traced, over several years, T lymphocytes genetically modified to express the thymidine kinase (TK) suicide gene that were infused in 10 patients after haploidentical hematopoietic stem cell transplantation (HSCT). At 2 to 14 years after infusion and in the presence of a broad and resting immune system, we could still detect effectors/effector memory (TEM/EFF), central memory (TCM), and stem memory (TSCM) TK+ cells, circulating at low but stable levels in all patients. Longitudinal analysis of cytomegalovirus (CMV)– and Flu-specific TK+ cells indicated that antigen recognition was dominant in driving in vivo expansion and persistence at detectable levels. The amount of infused TSCM cells positively correlated with early expansion and with the absolute counts of long-term persisting gene-marked cells. By combining T cell sorting with sequencing of integration (IS), TCRα and TCRβ clonal markers, we showed that T cells retrieved long-term were enriched in clones originally shared in different memory T cell subsets, whereas dominant long-term clonotypes appeared to preferentially originate from infused TSCM and TCM clones. Together, these results indicate that long-term persistence of gene-modified memory T cells after haploidentical HSCT is influenced by antigen exposure and by the original phenotype of infused cells. Cancer adoptive immunotherapy might thus benefit from cellular products enriched in lymphocytes with an early-differentiated phenotype.

Genomic loss of patient-specific HLA in acute myeloid leukemia relapse after well-matched unrelated donor HSCT

To the editor: Allogeneic hematopoietic stem cell transplantation (HSCT) can grant long-term control and cure of acute myeloid leukemia (AML) thanks to the antitumor effect of the transplanted immune system. Still, relapse remains an open issue: in the haploidentical setting, we and others

Posttransplantation cyclophosphamide and sirolimus for prevention of GVHD after HLA-matched PBSC transplantation

To the editor: Graft-versus-host disease (GVHD), both acute and chronic, is still a leading cause of nonrelapse mortality after transplantation.[1][1] High-dose, posttransplantation cyclophosphamide (PTCy) is an attractive approach for in vivo allodepletion across the HLA barrier in allogeneic

Cytokine-induced killer cells engineered with exogenous T-cell receptors directed against melanoma antigens: enhanced efficacy of effector cells endowed with a double mechanism of tumor recognition.

Cytokine-induced killer (CIK) cells consist of a heterogeneous population of polyclonal T lymphocytes displaying NK phenotype and HLA-unrestricted cytotoxic activity against a broad range of tumors. We sought to determine whether transduction of CIK cells with T cell receptor (TCR) genes specific for tumor-associated antigens could generate effector cells endowed with a double mechanism of tumor recognition. HLA-A2-restricted TCR-transduced (TD) CIK directed against the melanoma antigens Mart1 and NY-ESO1 were generated by lentiviral transduction and successfully expanded over a 3-4-week period. TD-CIK cells were both CD3(+)/CD56(-) and CD3(+)/CD56(+) (31±8% and 59±9%, respectively), indicating that both major histocompatibility complex (MHC)-restricted T cells and MHC-unrestricted CIK could be targeted by lentiviral transduction. At the end of the culture, the majority of both unmodified and TD-CIK displayed an effector memory phenotype, without considerable expression of replicative senescence and exhaustion markers. Functionally, TD-CIK specifically recognized tumor cells expressing the relevant antigen as well as maintained their MHC-unrestricted tumor activity. The cytotoxic activity of TD-CIK against HLA-A2(+) melanoma cell lines was significantly higher than the untransduced counterparts at a low effector:target ratio (cytotoxic activity of TD-CIK was from 1.9- to 4.3-fold higher than untransduced counterparts). TD-CIK were highly proficient in releasing high amount of IFN-γ upon antigen-specific stimulation and were able to recognize primary melanoma targets. In conclusion, we showed that (1) the reproducibility and simplicity of CIK transduction and expansion might solve the problem of obtaining adequate numbers of potent antitumor effector cells for adoptive immunotherapy; (2) the presence of both terminal effectors as well as of less differentiated progenitors might confer them long survival in vivo; and (3) the addition of an MHC-restricted antigen recognition allows not only targeting tumor surface antigens but also a wider range of cytoplasmic or nuclear antigens, involved in tumor proliferation and survival. TD-CIK cells with a double mechanism of tumor recognition are an attractive and alternative tool for the development of efficient cell therapeutic strategies.

209. TCR Gene Editing in a Single Step of T Cell Activation To Redirect T Cell Specificity and Prevent GvHD

Transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach for cancer adoptive immunotherapy. Yet, TCR gene transfer into mature T cells results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. Thus, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to α and β chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes (Provasi et al, Nat. Med. 2012). While successful, the complete editing requires multiple manipulation steps involving repeated cell activation cycles and transductions. To reduce the duration and complexity of cell product generation, we recently developed a ‘single TCR editing’ (SE) procedure, based on the disruption of the endogenous TCR α chain only followed by the transfer of the tumor specific TCR genes. This SE method generates redirected T cells fully devoid of their natural TCR repertoire in a single round of cell activation. We validated the SE protocol exploiting an HLA-A2 restricted TCR specific for NY-ESO-1 (expressed by a considerable proportion of high risk multiple myeloma). The SE strategy allowed rapid production of high numbers of tumor specific T cells enriched for an early differentiation phenotype. When fucntionaly tested (co-culture, γ-IFN and 51Cr release) against the U266 myeloma cell line (NY-ESO-1+HLA-A2+), all NY-ESO-1 redirected T cells showed a strikingly high killing activity. However, when we assess the alloreactive potential of the different redirected T cells in mixed lymphocyte reactions, we observed that the allogeneic lysis by SE T cells was markedly lower (p=0.05) than that of conventional TCR transfer cells (TR). These results were validated in NSG mice, where the genetically modified T cells were infused after the engraftment of the U266 myeloma. All animals treated with tumor specific T cells were completely myeloma-free at the time of sacrifice, demonstrating the powerful anti-tumor potential of the NY-ESO-1 redirected T cells. However, the overall survival of mice treated with TR vs SE cells was 26% vs 100% respectively (p<0,001) corresponding with a significant difference in acute and chronic GvHD occurrence (71% vs 0%, p<0,001). Consistently, histopathological analysis of human T cell infiltration in the organs revealed a significantly higher score in mice treated with TR cells (p<0.001). The relative simplicity of the SE protocol enables rapid generation of highly performing tumor specific T cells, fully devoid of their endogenous TCR repertoire, and thus incapable of participating in GvHD. Such single TCR edited cells thus potentially represent a further advance in adoptive immunotherapy for cancer.