SAXS‐enabled Macromolecular Engineering at SIBYLS

Abstract

Macromolecular engineering provides novel opportunities in many fields. However, progress can be slow when structural analysis relies on low‐throughput structural techniques. Employing unique and high‐throughput X‐ray scattering (SAXS) tools, we have engineered a Self‐Assembling Protein Nanoparticle to display Antibodies (SAPNA) with application in CAR T Cell therapy. Adoptive CAR T Cell therapy is a highly effective, but extremely expensive new cancer treatment with costs in the range of ~$1 million per patient (hospital fees included). The therapy requires >100 million CAR T cells per infusion per patient. A key step in the CAR T manufacturing process is polyclonal expansion of the patient‐ or donor‐derived T cells. This is currently achieved through CD3/CD28 T cell receptor stimulation by agonist antibodies (either soluble or chemically linked to magnetic beads). We have shown our engineered SAPNA solution is highly effective relative to existing technologies at expanding CAR T cells with potential application in fighting cancer. High‐Throughput SAXS (HT‐SAXS) was an integral technique that enabled structural insights into engineered constructs. A crucial step was monitoring macromolecular changes in response to mutations and solution conditions. We also utilized Size Exclusion Chromatography SAXS (SEC‐SAXS) to assess the antibody display, while providing important information on the structural dynamics of the antibodies themselves. Herein, we detail the SAXS platform (sibyls.als.lbl.gov) we used to rapidly converge on our functional SAPNA and SAPNA’s potential application in CAR T Cell therapy.Support or Funding InformationUS DOE‐BER, Integrated Diffraction Analysis Technologies (IDAT) program. Additional support from NIGMS project ALS‐ENABLE (P30 GM124169) and a High‐End Instrumentation Grant S10OD018483.