Feeder Cells as Active Process Determinants in Adoptive Cell Therapy: Biological Imprinting, Critical Quality Attributes, and Feeder-Free Alternatives

Abstract

Adoptive Cell Therapy (ACT) manufacturing is commonly described as a sequence of isolation, activation, genetic modification, expansion, harvest, and formulation. Within this framework, feeder cells are frequently treated as ancillary reagents whose principal function is to increase cell number. This view is incomplete. Irradiated peripheral blood mononuclear cells, Epstein-Barr virus-transformed lymphoblastoid cells, engineered K562 derivatives, and other cellular stimulators create a dynamic signaling niche that integrates T-cell receptor or natural killer-cell receptor engagement, co-stimulation, adhesion, membrane-bound cytokines, soluble mediators, metabolic competition, and time-dependent loss of feeder viability. Consequently, feeder systems can imprint differentiation state, metabolic fitness, clonal composition, exhaustion susceptibility, trafficking competence, cytotoxicity, cytokine polyfunctionality, and in vivo persistence. In this review, feeder cells are reframed as active process determinants whose material attributes and operating parameters should be linked prospectively to product critical quality attributes. We compare feeder platforms used for tumor-infiltrating lymphocytes, conventional and engineered alpha-beta T cells, natural killer cells, gamma-delta T cells, and virus-specific T cells; examine mechanistic evidence for biological imprinting; and propose a risk-based analytical framework covering feeder identity, proliferative arrest, ligand density, residual feeder clearance, adventitious-agent control, and functional consistency. Feeder-free approaches, including anti-CD3/CD28 beads, soluble or degradable nanomatrices, ligand-presenting particles, membranederived stimulatory particles, cytokine-only systems, and engineered biomaterials, are then evaluated according to their ability to reproduce rather than
merely replace feeder-derived signals. We argue that successful process simplification requires a defined target product profile, mechanistically anchored comparability studies, and orthogonal potency assays. Treating feeder selection as an early process-design decision may improve manufacturing robustness, facilitate comparability, and reduce the risk that expansion yield is optimized at the expense of therapeutic fitness.