Dual controls for therapeutic cell activation or elimination
A cellular, cytoplasmic technique for dual control for therapeutic cellular activation or depletion
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Embodiment 1
[0516] Example 1: Construction and evaluation of caspase-9 suicide switch expression vector
[0517] Vector construction and confirmation of expression
[0518] Here we present a safety switch that can be stably and efficiently expressed in human T cells. The system consists of a human gene product with low immunogenic potential that has been modified to drug interact with a small molecule dimerizing agent capable of causing transduced T cells expressing the modified gene. Selective elimination of cells. Additionally, inducible caspase-9 maintains function in T cells overexpressing anti-apoptotic molecules.
[0519] An expression vector suitable for use as a therapeutic agent is constructed comprising a modified human caspase-9 activity fused to a human FK506 binding protein (FKBP) (eg, FKBP12v36). Caspase-9 / FK506 hybrid activity can be dimerized using small molecule drugs. Full-length, truncated and modified forms of caspase-9 activity are fused to the ligand-binding doma...
Embodiment 2
[0574] Example 2: Use of the iCasp9 suicide gene to improve the safety of allogeneic depleted (Allodepleted) T cells following haploidentical stem cell transplantation
[0575] Presented in this example are expression constructs and methods of using expression constructs to improve the safety of allogeneic depleted T cells following haploid identical stem cell transplantation. A retroviral vector encoding iCasp9 and a selectable marker (truncated CD19) was generated as a safety switch for donor T cells. Even after allogeneic depletion (using an anti-CD25 immunotoxin), donor T cells were efficiently transduced, expanded, and subsequently enriched to >90% purity by CD19 immunomagnetic selection. Engineered cells retain antiviral specificity and functionality and contain subclasses with modulated phenotypes and functions. Activation of iCasp9 with small molecule dimerizers rapidly produces >90% apoptosis. Although transgene expression is downregulated in resting T cells, iCasp9...
Embodiment 3
[0637] Example 3: CASPALLO-1 Phase Clinical Trial of Allogeneic Depleted T Cells Transduced with the Inducible Caspase-9 Suicide Gene Following Haploid Identical Stem Cell Transplantation
[0638] This example presents the use of figure 2 Results of a phase 1 clinical trial of an alternative suicide gene strategy shown in . Briefly, donor peripheral blood mononuclear cells were co-cultured with recipient irradiated EBV-transformed lymphoblastoid cells (40:1) for 72 hr, allogeneic depleted with CD25 immunotoxin, and then treated with iCasp9-carrying Retroviral supernatant transduction of suicide genes and a selectable marker (ΔCD19); ΔCD19 allows enrichment to >90% purity by immunomagnetic selection.
[0639] Examples of protocols for producing cell therapy products are provided herein.
[0640] source material
[0641] Up to 240 ml (in two collections) of peripheral blood were obtained from transplant donors according to established protocols. In some cases, depending on ...
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