Construct Design

• Binder selection and affinity tuning; scFv, VHH and ligand-based recognition
• Hinge and transmembrane choices and their effect on synapse geometry
• Costimulatory domain trade-offs: CD28 potency versus 4-1BB persistence
• Armoured CARs, logic gates, and safety switches such as iCasp9 and EGFRt

Gene Delivery

• Lentiviral and gammaretroviral vectors; vector copy number limits and insertional risk
• Non-viral delivery: Sleeping Beauty and piggyBac transposons
• Site-specific knock-in at the TRAC locus and why targeted integration matters
• Allogeneic strategies: TRAC and B2M editing to limit GvHD and rejection

Manufacturing

• Apheresis, selection and activation; starting-material variability
• Transduction, expansion platforms and closed-system processing
• Cryopreservation, cold chain and the practical limits of vein-to-vein time
• Release assays: identity, purity, potency, sterility, mycoplasma and endotoxin

Clinical

• Lymphodepletion rationale and dose-finding in cell therapy
• Cytokine release syndrome and ICANS: ASTCT consensus grading and intervention
• Tocilizumab and corticosteroid algorithms, and their effect on efficacy
• Mechanisms of relapse: antigen loss, T-cell exhaustion and poor persistence

Translation

• IND-enabling package and CMC comparability across process changes
• Designing a potency assay regulators will accept
• GMP requirements for cell therapy facilities and personnel
• Scale-out versus scale-up economics, and access in cost-constrained health systems