ap-1903-reagent and Graft-vs-Host-Disease

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.

Topics: Adolescent; Caspase 9; Child; Child, Preschool; Female; Flow Cytometry; Genes, Transgenic, Suicide; Graft vs Host Disease; Haplotypes; Hematopoietic Stem Cell Transplantation; Humans; Lymphoproliferative Disorders; Male; Middle Aged; Organic Chemicals; Real-Time Polymerase Chain Reaction; T-Lymphocytes; Young Adult

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892.

Topics: Adolescent; Aspergillosis; Aspergillus fumigatus; Caspase 9; Child; Child, Preschool; Enzyme Induction; Female; Follow-Up Studies; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Immunotherapy, Adoptive; Lymphoma, Large-Cell, Anaplastic; Male; Myelodysplastic Syndromes; Organic Chemicals; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; T-Lymphocytes; Time Factors; Transgenes; Transplantation, Homologous; Treatment Outcome; Virus Diseases

Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct.. We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch.. Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence.. The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.).

Topics: Adolescent; Apoptosis; Caspase 9; Child; Child, Preschool; Female; Gene Transfer Techniques; Genes, Transgenic, Suicide; Graft vs Host Disease; Humans; Immunotherapy, Adoptive; Leukemia; Male; Organic Chemicals; Recurrence; Stem Cell Transplantation; T-Lymphocytes; Tacrolimus Binding Proteins

Safety switches are becoming relevant for the clinical translation of T-cell-based immunotherapies. In patients receiving an allogeneic hematopoietic stem cell transplant, the inducible caspase-9 gene (iC9) safety switch expressed by donor-derived T lymphocytes efficiently controls acute graft versus host disease (GvHD). However, in vivo elimination of iC9-T cells by the chemical inducer of dimerization (CID) that activates the iC9 protein is incomplete. To study this effect, we characterized the clonal diversity and dynamics of vector insertion sites (VIS) in iC9-T cells pre- and post-CID administration in four patients who developed GvHD. We identified 3,203 VIS among four patients and followed their in vivo clonal dynamics up to 161 days post-CID. VIS were categorized by their proximity to host genome elements, gene associations, and cis-modulatory relationship to mapped promoters. We found that VIS are preferentially located near open chromatin and promoter regions; furthermore, there was no evidence for selection bias among VIS surviving the CID treatment. The majority of iC9-T cells with high normalized VIS copy number at the time of GvHD onset were eliminated by CID, while iC9-T cells detectable post-CID generally have low normalized VIS copy number. We propose that suboptimal iC9 transgene expression is responsible for the incomplete elimination of iC9-T cells and illustrate here by simple model how cis-modulatory influences of local genome context and T-cell receptor activation status at time of CID treatment contribute to stochastic sparing of iC9-T cells.

Topics: Caspase 9; Chromatin; Genome, Human; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Immunotherapy; Organic Chemicals; Promoter Regions, Genetic; T-Lymphocytes; Transgenes; Transplantation, Homologous; Virus Integration

Activation of the inducible caspase 9 (iC9) safety gene by a dimerizing drug (chemical inducer of dimerization (CID) AP1903) effectively resolves the symptoms and signs of graft-versus-host disease (GvHD) in haploidentical stem cell transplant (HSCT) recipients. However, after CID treatment, 1% of iC9-T cells remain and can regrow over time; although these resurgent T cells do not cause recurrent GvHD, it remains unclear whether repeat CID treatments are a safe and feasible way to further deplete residual gene-modified T cells should any other adverse effects associated with them occur. Here, we report a patient who received an infusion of haploidentical iC9-T cells after HSCT and subsequently received three treatments with AP1903. There was a mild (grade 2) and transient pancytopenia following each AP1903 administration but no non-hematological toxicity. Ninety five percent of circulating iC9-T cells (CD3(+)CD19(+)) were eliminated after the first AP1903 treatment. Three months later, the residual cells had expanded more than eightfold and had a lower level of iC9 expression. Each repeated AP1903 administration eliminated a diminishing percentage of the residual repopulating cells, but elimination could be enhanced by T-cell activation. These data support the safety and efficiency of repeated CID treatments for persistent or recurring toxicity from T-cell therapies.

Topics: Caspase 9; Child; Dose-Response Relationship, Drug; Drug Administration Schedule; Genes, Transgenic, Suicide; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Lymphocyte Activation; Male; Organic Chemicals; Stem Cell Transplantation; T-Lymphocytes; Treatment Outcome

Anti-tumor cellular immunotherapies that implement a suicide gene system can limit potential undesirable effects. In a haplo-identical bone marrow transplant clinical trial, over 90% of iCaspase-9-expressing cells were eradicated after AP1903 exposure, and signs of graft-versus-host disease disappeared. Nevertheless, low numbers of genetically modified T cells survived this treatment. We studied genetically modified cell lines (GMCL) that carried a dual iCaspase-9/ΔCD19 DNA construct (ΔCD19=truncated CD19). With AP1903 exposure, a low percentage of cells (1.47±0.67%; n=5 replications) persisted in vitro. Repeated exposures to increasing AP1903 doses generated low (GMCLLR) and high AP1903-responders (GMCLHR), which expressed different levels of surface ΔCD19 and intracellular iCaspase-9. Compared with GMCLHR, GMCLLR exhibited higher methylation of 5'-long-terminal repeat (LTR) promoters, both in the number of sequences with at least one methylated CpG (16 vs 51.5%, respectively) and in the number of CpG islands (1.2 vs 8.9%, respectively). Four days of 5-azacytidine exposure reduced methylation and increased ΔCD19 and iCaspase-9 expression. Interestingly, LTR demethylation restored GMCLLR sensitivity to AP1903 by 24.3-fold (1.8 vs 43.8%) without affecting GMCLHR. We showed that 5'-LTR-methylation inhibited transgene expression and caused AP1903 hypo-responsiveness. Treating with a hypomethylating agent restored AP1903 sensitivity. This approach can be applied in further clinical trials to improve iCaspase-9 response if low response is detected.

Topics: Antigens, CD19; Azacitidine; Bone Marrow Transplantation; Caspase 9; DNA Methylation; Genes, Transgenic, Suicide; Genetic Therapy; Graft vs Host Disease; Humans; Jurkat Cells; Organic Chemicals; Transplantation, Homologous

Topics: Caspase 9; Female; Genes, Transgenic, Suicide; Graft vs Host Disease; Humans; Immunotherapy, Adoptive; Male; Organic Chemicals; T-Lymphocytes; Tacrolimus Binding Proteins

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. One strategy to treat GVHD is to equip donor T cells with a conditional suicide mechanism that can be triggered when GVHD occurs. The herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir system used clinically has several limitations, including immunogenicity and cell cycle dependence. An alternative switch based on chemically inducible apoptosis was designed and evaluated. A chimeric human protein was expressed comprising an extracellular marker (DeltaLNGFR), the Fas intracellular domain, and 2 copies of an FK506-binding protein (FKBP). Primary human T lymphocytes retrovirally transduced with this construct could be purified to homogeneity using immunomagnetic beads. Genetic integrity of the construct was ensured by redesigning repetitive sequences. Transduced T cells behaved indistinguishably from untransduced cells, retaining the ability to mount a specific antiallogeneic immune response. However, they rapidly underwent apoptosis with the addition of subnanomolar concentrations of AP1903, a bivalent "dimerizer" drug that binds FKBP and induces Fas cross-linking. A single 2-hour treatment eliminated approximately 80% of T cells, and multiple exposures induced further apoptosis. T cells were eliminated regardless of their proliferation state, suggesting that the AP1903/Fas system, which contains only human components, is a promising alternative to HSV-tk for treating GVHD.

Topics: Apoptosis; Bone Marrow Transplantation; Cross-Linking Reagents; Dose-Response Relationship, Drug; fas Receptor; Gene Rearrangement; Graft vs Host Disease; Humans; Immunomagnetic Separation; Lymphocyte Activation; Organic Chemicals; Recombinant Fusion Proteins; Retroviridae; T-Lymphocytes; Tacrolimus Binding Proteins; Time Factors; Transduction, Genetic; Transgenes