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AUTL presented three posters at ASGCT over the last two days. Most relevant near term to AUTL is the CCR poster, as AUTO6NG (GD2), which is scheduled to enter the clinic this year, contains an IL-7-CCR. The poster noted that gene expression from an IL-7-CCR-GD2CAR-T was largely identical to both an IL-2 and IL-12-CCR-GD2CAR-T. Notably in mice a GM-CSF-CCR-GD2CAR-T produced the greatest CAR-T expansion, persistence and overall survival.
In addition to a lack of cytokine support in the solid tumour microenvironment, FasL expression could trigger killing of activated CAR-T since these cells will express Fas. AUTL described a Fas-CD40 construct that neutralised FasL induced apoptosis while maintaining CAR-T function. Controlled activation of CAR and or cytokine secretion may be advantageous in solid tumours and AUTL describes a two component system based on minocycline. For a CAR, minocycline inactivates the CAR while for IL-12, use of minocycline leads to secretion.
Cytokine support of cell therapy has most often required systemic dosing of cytokines. A chimeric chemokine receptor with constant heavy and light chain domains from an IgG are fused to the transmembrane and intracellular domains of the chosen cytokine receptor. An IL-2 CCR drove significant T-cell expansion in the absence of cytokine starvation CCR control
Of the top fifty genes activated by exogenous IL-2, forty-four were activated by IL-2-CCR. Truncation of the IL2R beta chain resulted in either increased or diminished T cell expansion. Substitution of IL-7 for IL-2, increased T-cell expansion, while IL-18 increased IFNy secretion over IL-2. Substitution of GM-CSF for IL-2 increased myeloid cell expansion.
Eighteen CCR-GD2 CARs were evaluated against a GD2 CAR control and while most preserved CAR-T effector function, the IL-12 and IL-7 CCR's drove greater CAR-T expansion and production of IFNy. Gene expression changes for IL-2-CCR and IL-7-CCR and IL-7-CCR and IL-12-CCR were identical.
In a GD-2 colon cancer model using murinised components, CCR-IL-7 or CCR-GM-CSF GD2 CAR-T, demonstrated improved tumour control compared to GD2 CAR-T; CAR-T expansion was significantly higher for the GM-CSF-CCR and correlated with a pronounced increase in overall survival.
In a metastatic melanoma model similar observations were made and at day fourteen, only GM-CSF-CCR-GD2 CAR-T were detected.
FAS: Fas ligand is expressed by most cancers, regulatory T-cells, endothelial cells, myeloid-derived suppressor cells and cancer-associated fibroblasts. Activated T-cells including CAR-T constitutively express Fas and engagement of FasL leads to CAR-T death via apoptosis. Truncated non signalling Fas rescued T-cells from Fas mediated apoptosis, but significantly diminished CAR-T proliferation and efficacy
Adding the intracellular pro-survival domain of a TNF receptor rescued CAR-T proliferation in the presence of FasL; seventeen different receptors were evaluated including a short of BCMA, 41BB, CD27, CD40 and Fn14.
Against GD2 FasL+ target cells, GD2 CAR-T exhibited a high degree of apoptosis which was reversed in the presence of Fas truncated or Fas-receptors. While all GD2 CAR-T with one of five shortlisted Fas combinations were equipped against GD2+ target cells, with repeat stimulation, Fas-CD40 retained the highest level of activity, correlating with greater retention of a central memory phenotype.
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