Figure 4

Optimization of ACT_eff cells for broad delivery into widespread metastases (melanoma and others). Bioengineering of CD8⁺ or CD4⁺ ACT_eff cells with vastly improved capacity for homing into widespread, metastatic tissues is now possible by combinatorially leveraging and integrating new glycoengineering and genetic engineering technologies with the latest knowledge on immune cell homing and cancer metastatic circuitries. As shown, suboptimal and/or minimal native glycosylation of CD44 and PSGL-1 on ACT_eff cells could be compensated for using a (1) cell-extrinsic GPS approach with requisite α1,3FT (eg, FTVI or others) in generation of (2) E-selectin-binding CD44-sLe^X (HCELL) and E/P-selectin-binding PSGL-1-sLe^X (CLA) homing determinants. GPS may advantageously generate additional, unidentified selectin-binding glycoprotein and glycolipid homing determinants (not shown). Consequent bimolecular association of HCELL with VLA-4 via a Rap/Rac signaling mechanism, or of PSGL-1 with VLA-4 (not shown), would activate VLA-4 adhesion to VCAM-1 via a ‘step-2 bypass’. (3–4) Cell-intrinsic creation of HCELL and CLA is shown, whereby viral transduction or transfection of mod-RNA, cDNA, or CRISPR-based platforms encoding α1,3FT (eg, FTVI or others) would result in its cytoplasmic translation, insertion into the golgi compartment, and heightened synthesis of sLe^X-selectin-binding moieties on CD44 and PSGL-1 (and possibly other glycoproteins and glycolipids, not shown) transiting the secretory pathway. Genetically introduced (5) CXCR1 or CXCR2, normally low or absent on ACT_eff cells, would prime Gα_i signaling and homing responses when bound by cognate chemokines, CXCL1 or CXCL8, expressed by melanoma cells (or by other cancer types). (6) Genetic overexpression of α_Vβ₃ or Mac-1 (α_Mβ₂), also normally absent or low on ACT_eff cells would, when rendered fully active potentially by (7) HCELL/PSGL-1 ‘step-2 bypass’ biomolecular association or by (8) CXCR1/CXCR2 chemokine receptor signaling, bind a plethora of diverse tumor endothelial adhesive proteins as shown. (9) Lesional targeting and homing specificity could be improved through positive selection and/or genetic overexpression of multiple different TCR, TCR_gm, or CAR receptors (and co-stimulators) recognizing diverse TA’s and with capacities to activate integrins as shown. (10) Preconditioning regimens applied either prior to and/or following ACT_eff cell infusion could synergistically enhance trafficking capabilities through augmentation of tumor endothelial or ACT_eff cell pro-homing determinants, including adhesion molecules, chemokines and chemokine receptors, and TA. Incorporation of inducible-suicide genes (to limit ACT_eff-associated cytokine storms and inflammation), immune checkpoint blockers, and inhibitors of immune-evasive mechanisms could vastly improve immunotherapeutic outcomes in advanced cancer patients with widespread metastases.