Versatile strategy for controlling the specificity and activity of engineered T cells

Abstract

The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR–T-cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as “switch” molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a “universal” anti-FITC–directed CAR-T cell. Optimization of this CAR–switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR–T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy.

Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies

“…Alternatively, the large adjacent loop domain of the CD20 antigen may create a steric hindrance for binding of the IgG4m sCAR. We observed a similar effect supporting this hypothesis in our work with site-specific FITC conjugation on the anti-CD22 antibody m971 (45). In this case, the m971 antibody has a membrane proximal epitope on CD22 (39) and several large modular domains that may sterically preclude binding.…”

“…The increase in sCAR-T-cell activity observed when the sCAR hinge was shortened from 45 amino acids to 12 amino acids supports this hypothesis. A similar relationship was observed in the accompanying publication which reports a sCAR-T cell specific for FITC and a FMC63-based switch molecule that is site-specifically modified with FITC (45). FITC conjugation sites near the antigen-binding interface afforded better activity than sites distal to the binding interface.…”

“…FITC conjugation sites near the antigen-binding interface afforded better activity than sites distal to the binding interface. Moreover, we have shown, in that study, that nonspecific conjugation of switches with FITC did not provide sufficient in vivo activity to clear Nalm-6 tumor (45). Collectively, these studies underscore the importance of switch structure on optimizing immunological synapse geometry, which directly impacts sCAR-T-cell activity.…”

“…In addition, this added control over sCAR-T-cell activity may allow engineered cells to be turned off after tumor elimination to allow healthy B cells to repopulate in leukemia and lymphoma patients who are in remission. Indeed, in the companion article we show that switch discontinuation can lead to the restoration of the peripheral blood B cells in a surrogate mouse model using anti-FITC CAR-T cells and FITC labeled anti-murine CD19 switches (45). Finally, we expect that the ability of sCAR-T cells to target more than one antigen in vivo by codelivery of CD19 and CD20 switches may be an effective method to prevent relapse as a result of antigen-loss escape mutations in patients (11), and may provide improved efficacy in the treatment of cancers with heterogeneous antigen expression.…”

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

“…Finally, this single universal CAR can be used for heterogeneous tumors with distinct antigens (or reoccurring tumors with altered antigen expression) by simply using multiple switch molecules. Thus, this platform (which also includes a recombinant version using peptide neoepitopes) should decrease the acute and chronic toxicity associated with this promising new cell therapy, simplify its application, and allow it to be used with both liquid and solid tumors (Ma et al 2016).…”