We have demonstrated the feasibility of engineering allogeneic T cells with two distinct safety mechanisms, selective allodepletion and suicide gene-modification. In combination, these modifications should enable addback of substantial numbers of T cells with anti-viral and anti-tumor activity, even after haploidentical transplantation. Our data show that the suicide gene, iCasp9, functions efficiently (>90% apoptosis after treatment with dimerizer) and that down-modulation of transgene expression that occurred with time was rapidly reversed upon T cell activation, as would occur when alloreactive T cells encountered their targets. We also showed that ΔCD19 was a suitable selectable marker that enabled efficient and selective enrichment of transduced cells to >90% purity, and that these manipulations had no discernable effects on the immunological competence of the engineered T cells with retention of antiviral activity, and regeneration of a CD4+CD25+Foxp3+ population with Treg activity.
Given that the overall functionality of suicide genes depends on both the suicide gene itself and the marker used to select the transduced cells, translation into clinical use requires optimization of both components, and of the method used to couple expression of the two genes. The two most widely used selectable markers in clinical practice each have drawbacks. Neomycin phosphotransferase (neo
) encodes a potentially immunogenic foreign protein and requires a 7-day culture in selection medium, which not only increases the complexity of the system, but is also potentially damaging to virus-specific T cells26
. The most widely used surface selection marker is ΔLNGFR, but recent report of leukemia in a mouse model raises concerns regarding its oncogenic potential27
, despite its apparent clinical safety 28
. Furthermore, ΔLNGFR selection is not widely available, because it is used almost exclusively in gene therapy. A number of alternative selectable markers have been suggested. CD34 has been well-studied in vitro29,30
, but the steps required to optimize a system designed primarily for selection of rare hematopoietic progenitors, and more critically, the potential for altered in vivo T cell homing31
, make CD34 sub-optimal for our purpose. As an alternative marker, we used ΔCD19, since clinical grade CD19 selection is readily available as a method for B-cell depletion of stem cell autografts. We demonstrated that ΔCD19 enrichment could be performed with high purity and yield and, more importantly, the selection process had no discernable effect on subsequent cell growth and functionality.
The effectiveness of suicide gene activation in ΔCD19-selected iCasp9 cells compared very favorably to that of neo
- or ΔLNGFR-selected cells transduced to express the HSVtk gene. The earlier generations of HSVtk constructs provided 80-90% suppression of 3
and showed similar reduction in killing efficiency upon extended in vitro culture33
, but were nonetheless clinically efficacious. Complete resolution of both acute and chronic GVHD has been reported with as little as 80% in vivo reduction in circulating gene-modified cells. These data supported the hypothesis that transgene down-modulation seen in vitro is unlikely to be an issue because activated T cells responsible for GVHD will upregulate suicide gene expression and will therefore be selectively eliminated in vivo14
. Whether this effect is sufficient to allow retention of virus- and leukemia-specific T cells in vivo remains to be tested in a clinical setting. By combining in vitro selective allodepletion prior to suicide gene modification, we hope that the need to activate the suicide gene mechanism will be significantly reduced, thereby maximizing the benefits of addback T cell.
The high efficiency of iCasp9-mediated suicide seen in vitro has been replicated in vivo. In a SCID mouse-human xenograft model, more than 99% of iCasp9-modified T cells were eliminated after a single dose of dimerizer18
. Safety testing has been performed on healthy human volunteers using AP1903, which has extremely close functional and chemical equivalence to AP20187, and would be used in a proposed clinical application. Maximal plasma level of 10 to 1275ng/ml AP1903 (equivalent to 7 to 892nM) was attained over a 0.01mg/kg to 1.0mg/kg dose range administered as a 2-hour intravenous infusion34
. There were no significant adverse effects. After allowing for rapid plasma redistribution, the concentration of dimerizer used in vitro remains readily achievable in vivo.
We believe iCasp9 will prove to have several advantages over HSVtk, including the ability to use ganciclovir and related drugs without causing inadvertent T cell destruction, and a reduced risk of destructive immune responses against transduced cells since the iCasp9 is almost entirely human-derived. Although both our selectable marker (ΔCD19) and our suicide gene (iCasp9) are of human origin, one potential limitation of our approach is that linkage of expression of the selectable marker to the suicide gene uses a 2A-like cleavable peptide which is of non-human origin. However, this product is only 20 amino acids long, and is therefore less likely to be immunogenic than entirely non-human proteins such as neo or HSVtk.
It is essential to define the culture conditions that are optimal for maintaining the immunological competence of suicide gene-modified T cells, since phenotype, repertoire and functionality can all be affected by the stimulation used for polyclonal T cell activation35,36
, the method for selection of transduced cells26
, and duration of culture37
. The addition of CD28 co-stimulation and the use of cell-sized paramagnetic beads to generate gene modified-cells that more closely resemble unmanipulated PBMC in terms of CD4:CD8 ratio, and expression of memory subset markers including lymph node homing molecules CD62L and CCR7, have been proposed as methods for improving the in vivo functionality of gene-modified T cells35,36
. CD28 co-stimulation has also been reported to increase the efficiency of retroviral transduction and expansion35,36
. Interestingly however, we found that the addition of CD28 co-stimulation had no impact on transduction of allodepleted cells (data not shown), and the degree of cell expansion seen in our study was higher compared to the anti-CD3 alone arm in these studies. Furthermore, iCasp9-modified allodepleted cells retained significant anti-viral functionality, and approximately one fourth retained CD62L expression. There was also regeneration of CD4+
regulatory T cells. The allodepleted cells we used as starting material for T cell activation and transduction may have been less sensitive to the addition of anti-CD28 antibody as co-stimulation. CD25-depleted PBMC / EBV-LCL co-cultures contained T cells and B cells that already express CD86 at significantly higher level than unmanipulated PBMC (data not shown) and may themselves provide co-stimulation. In addition, it has been reported that depletion of CD25+
regulatory T cells prior to polyclonal T cell activation with anti-CD3 enhances the immunological competence of the final T cell product37
. In order to minimize the effect of in vitro culture and expansion on functional competence, we used a relatively brief culture period whereby cells were expanded for a total of 8 days post-transduction with CD19-selection being performed on day 4.
Finally, we have demonstrated that we can scale up production sufficiently to treat adult patients at doses of up to 107 cells/kg: allodepleted cells can be activated and transduced at 4×107cells per flask, and a minimum of 8-fold return of CD19-selected final cell product can be obtained on day 8 post-transduction, to produce at least 3×108 allodepleted gene-modified cells per original flask. The increased culture volume is readily handled in additional flasks or bags.
The allodepletion and iCasp9-modification we describe should significantly improve the safety of adding back T cells, particularly after haploidentical stem cell allografts. This should in turn enable greater dose-escalation, with a higher chance of producing a hoped for anti-leukemia effect. We will explore this possibility in a clinical study of our modified allodepleted T cells.