Although adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells has achieved unprecedented response rates in patients with certain hematological malignancies, this therapeutic ...modality is still far from fulfilling its remarkable potential, especially in the context of solid cancers. Antigen escape variants, off-tumor destruction of healthy tissues expressing tumor-associated antigens (TAAs), poor CAR-T cell persistence, and the occurrence of functional exhaustion represent some of the most prominent hurdles that limit CAR-T cell ability to induce long-lasting remissions with a tolerable adverse effect profile. In this review, we summarize the main approaches that have been developed to face such bottlenecks, including the adapter CAR (AdCAR) system, Boolean-logic gating, epitope editing, the modulation of cell-intrinsic signaling pathways, and the incorporation of safety switches to precisely control CAR-T cell activation. We also discuss the most pressing issues pertaining to the selection of co-stimulatory domains, with a focus on strategies aimed at promoting CAR-T cell persistence and optimal antitumor functionality.
Among the many oncology therapies, few have generated as much excitement as CAR-T. The success of CAR therapy would not have been possible without the many discoveries that preceded it, most notably, ...the Nobel Prize-winning breakthroughs in cellular immunity. However, despite the fact that CAR-T already offers not only hope for development, but measurable results in the treatment of hematological malignancies, CAR-T still cannot be safely applied to solid tumors. The reason for this is, among other things, the lack of tumor-specific antigens which, in therapy, threatens to cause a lethal attack of lymphocytes on healthy cells. In the case of hematological malignancies, dangerous complications such as cytokine release syndrome may occur. Scientists have responded to these clinical challenges with molecular switches. They make it possible to remotely control CAR lymphocytes after they have already been administered to the patient. Moreover, they offer many additional capabilities. For example, they can be used to switch CAR antigenic specificity, create logic gates, or produce local activation under heat or light. They can also be coupled with costimulatory domains, used for the regulation of interleukin secretion, or to prevent CAR exhaustion. More complex modifications will probably require a combination of reprogramming (iPSc) technology with genome editing (CRISPR) and allogenic (off the shelf) CAR-T production.
Stem and progenitor cells hold great promise for regenerative medicine and gene therapy approaches. However, transplantation of living cells entails a fundamental risk of unwanted growth, potentially ...exacerbated by CRISPR-Cas9 or other genetic manipulations. Here, we describe a safety system to control cell proliferation while allowing robust and efficient cell manufacture, without any added genetic elements. Inactivating TYMS, a key nucleotide metabolism enzyme, in several cell lines resulted in cells that proliferate only when supplemented with exogenous thymidine. Under supplementation, TYMS−/−-pluripotent stem cells proliferate, produce teratomas, and successfully differentiate into potentially therapeutic cell types such as pancreatic β cells. Our results suggest that supplementation with exogenous thymidine affects stem cell proliferation, but not the function of stem cell-derived cells. After differentiation, postmitotic cells do not require thymidine in vitro or in vivo, as shown by the production of functional human insulin in mice up to 5 months after implantation of stem cell-derived pancreatic tissue.
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A genetic modification in stem cells makes their proliferation possible only under thymidine supplementation. This method is proposed to reduce the risk of uncontrolled cell proliferation for stem cell-based therapies.
Immune modulation has become a central element in many cancer treatments, and T cells genetically engineered to express chimeric antigen receptors (CAR) may provide a new approach to cancer ...immunotherapy. Autologous CAR T cells that have been re-directed toward tumor-associated antigens (TAA) have shown promising results in phase 1 clinical trials, with some patients undergoing complete tumor regression. However, this T-cell therapy must carefully balance effective T-cell activation, to ensure antitumor activity, with the potential for uncontrolled activation that may produce immunopathology. An inducible Caspase 9 (iCasp9) "safety switch" offers a solution that allows for the removal of inappropriately activated CAR T cells. The induction of iCasp9 depends on the administration of the small molecule dimerizer drug AP1903 and dimerization results in rapid induction of apoptosis in transduced cells, preferentially killing activated cells expressing high levels of transgene. The iCasp9 gene has been incorporated into vectors for use in preclinical studies and demonstrates effective and reliable suicide gene activity in phase 1 clinical trials. A third-generation CAR incorporating iCasp9 re-directs T cells toward the GD2 TAA. GD2 is over-expressed in melanoma and other malignancies of neural crest origin and the safety and activity of these GD2-iCAR T cells will be investigated in CARPETS and other actively recruiting phase 1 trials.
Immunotherapy with T cells expressing the chimeric antigen receptor (CAR) specific for the CD19 antigen (CD19.CAR-Ts) is a very effective treatment in B cell lymphoid malignancies. However, B cell ...aplasia and cytokine release syndrome (CRS) secondary to the infusion of CD19.CAR-Ts remain significant drawbacks. The inclusion of safety switches into the vector encoding the CAR is seen as the safest method to terminate the effects of CD19.CAR-Ts in case of severe toxicities or after achieving long-term sustained remissions. By contrast, the complete elimination of CD19.CAR-Ts when CRS occurs may jeopardize clinical responses as CRS and antitumor activity seem to concur. We have demonstrated, in a humanized mouse model, that the inducible caspase-9 (iC9) safety switch can eliminate CD19.CAR-Ts in a dose-dependent manner, allowing either a selective containment of CD19.CAR-T expansion in case of CRS or complete deletion on demand granting normal B cell reconstitution.
Savoldo and colleagues show in a humanized mouse model that the inclusion of the iC9 safety switch provides a unique regulation of CD19.CAR-Ts, ranging from on-demand ablation for normal B cell reconstitution to their contained expansion in case of toxicities, without complete elimination for sustained antitumor activity.
Gene-modified cellular therapies carry inherent risks of severe and potentially fatal adverse events, including the expansion of alloreactive cells or malignant transformation due to insertional ...mutagenesis. Strategies to mitigate uncontrolled proliferation of gene-modified cells include co-transfection of a suicide gene, such as the inducible caspase 9 safety switch (ΔiC9). However, the activation of the ΔiC9 fails to completely eliminate all gene-modified cells. Therefore, we tested a two suicide gene system used independently or together, with the goal of complete cell elimination. The first approach combined the ΔiC9 with an inducible caspase 8, ΔiC8, which lacks the endogenous prodomain. The rationale was to use a second caspase with an alternative and complementary mechanism of action. Jurkat cells co-transduced to co-express the ΔiC8, activatable by a BB homodimerizer, and the ΔiC9 activatable by the rapamycin analog sirolimus were used in a model to estimate the degree of inducible cell elimination. We found that both agents could activate each caspase independently, with enhanced elimination with superior reduction in cell regrowth of gene-modified cells when both systems were activated simultaneously. A second approach was employed in parallel, combining the ΔiC9 with the RQR8 compact suicide gene. RQR8 incorporates a CD20 mimotope, targeted by the anti-CD20 monoclonal antibody rituxan, and the QBend10, a ΔCD34 selectable marker. Likewise, enhanced cell elimination with superior reduction in cell regrowth was observed when both systems were activated together. A dose-titration effect was also noted utilizing the BB homodimerizer, whereas sirolimus remained very potent at minimal concentrations. Further
in vivo
studies are needed to validate these novel combination systems, which may play a role in future cancer therapies or regenerative medicine.
Gene edited and engineered cell-based therapies are a promising approach for treating a variety of disorders, including cancer. However, the ability of engineered cells to persist for prolonged ...periods along with possible toxicity raises concerns over the safety of these approaches. Although a number of different one-dimensional suicide systems have been incorporated into therapeutic cell types, the incorporation of a two-layered suicide system that allows controlled killing of therapeutic cells at different time points is needed. In this study, we engineered a variety of therapeutic cells to express two different kill switches, RapaCasp9 and HSV-TK and utilized Rapamycin and Ganciclovir respectively to activate these kill switches. We show that the function of both RapaCasp9 and HSV-TK molecules is preserved and can be activated to induce apoptosis detected early (24 h) and late (48 h) post-activation respectively, with no toxicity. In vivo, we show the eradication of a majority of cells after treatment in subcutaneous and orthotopic models. Furthermore, we demonstrate how both suicide switches work independently and can be activated sequentially for an improved killing, thus ensuring a failsafe mechanism in case the activation of a single one of them is not sufficient to eliminate the cells. Our findings highlight the reliability of the double suicide system, effective on a variety of cells with different biological characteristics, independent of their anatomic presence.
The forkhead family transcription factor (FOXP3) is an essential regulator for the development of regulatory T cells (Tregs) and orchestrates both suppressive function and Treg lineage identity. ...Stable expression of FOXP3 enables Tregs to maintain immune homeostasis and prevent autoimmunity. However, under pro-inflammatory conditions, FOXP3 expression in Tregs can become unstable, leading to loss of suppressive function and conversion into pathogenic T effector cells. Therefore, the success of adoptive cell therapy with chimeric antigen receptor (CAR) Tregs is highly dependent on the stability of FOXP3 expression to ensure the safety of the cell product. To warrant the stable expression of FOXP3 in CAR-Treg products, we have developed an HLA-A2-specific CAR vector that co-expresses FOXP3. The transduction of isolated human Tregs with the FOXP3-CAR led to an increase in the safety and efficacy of the CAR-Treg product. In a hostile microenvironment, under pro-inflammatory and IL-2-deficient conditions, FOXP3-CAR-Tregs showed a stable expression of FOXP3 compared to Control-CAR-Tregs. Furthermore, additional exogenous expression of FOXP3 did not induce phenotypic alterations and dysfunctions such as cell exhaustion, loss of functional Treg characteristics or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs displayed an excellent ability to prevent allograft rejection. Furthermore, FOXP3-CAR-Tregs revealed coherent Treg niche-filling capabilities. Overexpression of FOXP3 in CAR-Tregs has thereby the potential to increase the efficacy and reliability of cellular products, promoting their clinical use in organ transplantation and autoimmune diseases.
•Modification of CAR vectors with an additional FOXP3 is an essential approach for clinical use.•Additional FOXP3 in CAR-Tregs does not cause cellular dysfunction.•CAR-Tregs with additional FOXP3 show neither an exhausted phenotype nor reduced Treg fitness.•Additional FOXP3 does not affect regulation of immune responses or niche filling capabilities in humanized mice.•Additional FOXP3 leads to stability under proinflammatory conditions and survival advantage under IL-2-deprivated conditions.
Building safety into CAR-T therapy Peters, Daniel T.; Savoldo, Barbara; Grover, Natalie S.
Human vaccines & immunotherapeutics,
12/2023, Letnik:
19, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Chimeric antigen receptor T cell (CAR-T) therapy is an innovative immunotherapeutic approach that utilizes genetically modified T-cells to eliminate cancer cells using the specificity of a monoclonal ...antibody (mAb) coupled to the potent cytotoxicity of the T-lymphocyte. CAR-T therapy has yielded significant improvements in relapsed/refractory B-cell malignancies. Given these successes, CAR-T has quickly spread to other hematologic malignancies and is being increasingly explored in solid tumors. From early clinical applications to present day, CAR-T cell therapy has been accompanied by significant toxicities, namely cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and on-target off-tumor (OTOT) effects. While medical management has improved for CRS and ICANS, the ongoing threat of refractory symptoms and unanticipated idiosyncratic toxicities highlights the need for more powerful safety measures. This is particularly poignant as CAR T-cell therapy continues to expand into the solid tumor space, where the risk of unpredictable toxicities remains high. We will review CAR-T as an immunotherapeutic approach including emergence of unique toxicities throughout development. We will discuss known and novel strategies to mitigate these toxicities; additional safety challenges in the treatment of solid tumors, and how the inducible Caspase 9 "safety switch" provides an ideal platform for continued exploration.
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