MASTER Scaffolds for Rapid, Single-Step Manufacture and Prototyping of CAR-T cells

PROJECT SUMMARY
Despite the unprecedented clinical success of chimeric antigen receptor (CAR) T cell therapy, its widespread
application is limited by lengthy and labor-intensive ex vivo manufacturing procedures that result in: (i) high cost;
(ii) delays to infuse CAR cells to patients with rapidly progressing disease; and (iii) CAR cells with heterogeneous
composition and terminal differentiation, which limit their engraftment and persistence. There is a clear scientific
and medical need for approaches to improve CAR T cell production, including methods to reduce cell processing
times, reduce manufacturing costs, and reduce CAR cell differentiation. Recently, our labs collaboratively
developed a new technology for CAR cell production called MASTER (Multifunctional Alginate Scaffolds for T
cell Engineering and Release). MASTER consists of dry, macroporous alginate materials conjugated to αCD3
and αCD28 antibodies and encapsulating interleukin signaling. CAR generation with MASTER technology
involves seeding freshly isolated, non-activated patient PBMCs together with CAR-encoding retroviral vectors
and implanting scaffolds back into patients. Once implanted, MASTER mediates every step of the CAR
production process, thereby eliminating the current standard procedural steps of αCD3/αCD28 pre-activation,
viral transduction with spinoculation and interleukin-mediated CAR expansion. In vitro MASTER-generated CAR
cells demonstrate reduced cellular differentiation as compared to CAR cells generated with gold-standard,
“conventional” clinical protocols. In vivo MASTER-generated CAR cells demonstrate far superior in vivo cell
persistence, enhanced anti-tumor efficacy and far superior prevention of tumor growth after rechallenge. The
utility of this system is two-fold: 1) as a transformative therapeutic technology creating enhanced and affordable
CAR therapy for cancer care and 2) as a research tool enabling rapid development, prototyping and testing of
CAR therapeutic candidates. We have assembled a focused, multidisciplinary team comprised of an expert in
biomaterials and drug delivery (Brudno), an expert in viral engineering and protein production (Birnbaum), two
specialists in clinical CAR cell production (Chen, Roy) and a clinician focused on CAR cell therapies (Grover). In
this proposal we seek to further develop and validate MASTER scaffolds and the associated methods to make
them ready for widescale utilization by the research and clinical communities, including researchers in related
areas eager to work in the CAR field but deterred by the barriers to test CAR construct in vivo. Leveraging
transformative preliminary data that show that the shelf-stable MASTER scaffolds outperform conventional CAR
cells in preclinical mouse models of lymphoma, orthotopic pancreatic cancer, and metastatic lung and ovarian
tumors this proposal will validate MASTER scaffolds with a wide range of donors and at different scales, with
multiple viral vectors and CAR constructs and delineate the phenotype and function resulting from MASTER
production of CAR cells. The successful completion of these aims will propel our ultimate vision of low-cost and
tunable generation of CAR cells for both liquid and solid tumors and potentially beyond the oncology space.

Grant Number: 5R33CA281875-03
NIH Institute/Center: NIH
Principal Investigator: Yevgeny Brudno