Design and Assessment of Novel Anti-CD30 CARs with Human Antigen-recognition Domains.

Chimeric antigen receptors are artificial fusion proteins that incorporate antigen-recognition domains and T-cell signaling domains. CD30 is a cell-surface protein expressed on Hodgkin lymphoma (HL), some T-cell lymphomas, and some B-cell lymphomas. CD30 has a restricted expression pattern in normal cells, so CD30 has good potential as a clinical target for CAR T cells. We compared 3 different anti-CD30 CAR designs incorporating a single-chain variable fragment derived from the 5F11 fully-human monoclonal antibody. 5F11-28Z has hinge, transmembrane, and costimulatory domains from CD28 and a CD3-zeta. T-cell activation domain. 5F11-CD828Z has hinge and transmembrane domains from CD8-alpha, a CD28 costimulatory domain, and a CD3-zeta T-cell activation domain. 5F11-CD8BBZ is identical to 5F11-CD828Z except for the replacement of the CD28 moiety with a 4-1BB moiety. We found that T cells expressing 5F11-CD8BBZ had lower levels of CD30-specific degranulation and cytokine release compared with CD28-containing CARs. When compared to the CD28-containing CARs, T cells expressing 5F11-CD8BBZ had higher levels of nonspecific functional activity including degranulation, cytokine release, and proliferation when stimulated with CD30-negative target cells. We established tumors in nod-scid common gamma-chain (NSG) mice and treated the tumors with T cells expressing different CARs. T cells expressing 5F11-28Z were most effective at eradicating tumors. T cells expressing 5F11-CD828Z had intermediate effectiveness, and T cells expressing 5F11-CD8BBZ were least effective. CD30+ T cells are lost from cultures of T cells containing 5F11-28Z-expressing T cells. This indicated the killing of CD30+ T cells by the 5F11-28Z-expressing T cells. Despite this, the number of T cells in the cultures consistently accumulated to numbers needed for use in a clinical trial. Based on all in vitro and murine experiments comparing the different CARs, we selected 5F11-28Z for further development, and we have initiated a clinical trial testing 5F11-28Z T cells.