Chimeric Antigen Receptors With Mutated IgG4 Fc Spacer Avoid Fc Receptor Binding and Improve T Cell Persistence and Antitumor Efficacy

• Published in: Molecular therapy 2015-04, Vol.23 (4), p.757-768
• Main Authors: Jonnalagadda, Mahesh, Mardiros, Armen, Urak, Ryan, Wang, Xiuli, Hoffman, Lauren J, Bernanke, Alyssa, Chang, Wen-Chung, Bretzlaff, William, Starr, Renate, Priceman, Saul, Ostberg, Julie R, Forman, Stephen J, Brown, Christine E
• Format: Article
• Language: English
• Subjects: Animals; Antigens; Cells; Hematology; Immunoglobulin G - immunology; Immunotherapy; Ligands; Lymphocytes; Mice; Mutant Chimeric Proteins - metabolism; Mutation; Neoplasms, Experimental - immunology; Neoplasms, Experimental - therapy; Neutrophils; Original; Protein Binding; Receptors, Antigen - genetics; Receptors, Antigen - metabolism; Receptors, Fc - metabolism; Research centers; T-Lymphocytes - immunology
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1038/mt.2014.208

The success of adoptive therapy using chimeric antigen receptor (CAR)–expressing T cells partly depends on optimal CAR design. CARs frequently incorporate a spacer/linker region based on the constant region of either IgG1 or IgG4 to connect extracellular ligand-binding with intracellular signaling domains. Here, we evaluated the potential for the IgG4-Fc linker to result in off-target interactions with Fc gamma receptors (FcγRs). As proof-of-principle, we focused on a CD19-specific scFv-IgG4-CD28-zeta CAR and found that, in contrast to CAR-negative cells, CAR+ T cells bound soluble FcγRs in vitro and did not engraft in NSG mice. We hypothesized that mutations to avoid FcγR binding would improve CAR+ T cell engraftment and antitumor efficacy. Thus, we generated CD19-specific CARs with IgG4-Fc spacers that had either been mutated at two sites (L235E; N297Q) within the CH2 region (CD19R(EQ)) or incorporated a CH2 deletion (CD19Rch2Δ). These mutations reduced binding to soluble FcγRs without altering the ability of the CAR to mediate antigen-specific lysis. Importantly, CD19R(EQ) and CD19Rch2Δ T cells exhibited improved persistence and more potent CD19-specific antilymphoma efficacy in NSG mice. Together, these studies suggest that optimal CAR function may require the elimination of cellular FcγR interactions to improve T cell persistence and antitumor responses.