Enhancing the Antigen-Sensitivity of the CD22 CAR through Modulation of the Affinity and Linker-Length of the Single-Chain Fragment Variable
Authors: Kohler M. Eric , Walsh Zachary , Degolier Kole , Fry Terry J. 
Published: November 2020

The advent of chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL). CD19 directed CAR T cells have demonstrated the ability to induce complete remissions in up to 90% of r/r ALL patients. Despite this remarkable upfront success, relapse after CAR T cell therapy remains a major obstacle to long term remissions. A major mechanism for relapse after CD19-directed CAR T cell therapy is the recurrence of antigen-negative ALL cells. In recent years, CD22 CAR T cell therapy has emerged as an effective salvage therapy for patients with CD19-negative ALL. In a phase I clinical trial, CD22 CAR T cells were able to induce remission in up to 80% of patients with CD19-negative ALL. Patients achieving remission, who did not undergo a consolidative hematopoietic stem cell transplant, were found to be at high risk of relapse due to downregulation of the CD22 antigen below the threshold required for effective CD22 CAR T cell activity. Thus, strategies to increase the antigen-sensitivity of CD22 CAR T cells have the potential to enhance the induction and duration of remission in ALL patients.
As the properties of a CAR that influence sensitivity to antigen are not well defined, we began by testing the impact of increasing the affinity of the single-chain fragment variable (scFv) for the CD22 antigen. T cells from healthy donors were activated and transduced with a second-generation, 4-1BB CAR containing either the standard affinity (SA)-m971 scFv used in the prior clinical trial, or a high affinity (HA) scFv generated by affinity maturation of the m971 scFv. SA- and HA-CD22 CAR T cells were evaluated in vitro and in vivo against clones of the pre-B ALL cell line, NALM6, which express CD22 at wild type levels (CD22WT), sub-physiologic levels (CD22Lo), supra-physiologic levels (CD22Hi) or in which CD22 was deleted (CD22Neg). We found that the amount of CD22 expressed on the leukemia cells resulted in dose-dependent expression of activation markers, such as CD69 and CD25 (p&lt;0.05) on CD22 CAR T cells. Similarly, CAR T cell functions, such as the secretion of interferon-gamma (IFNg, p&lt;0.0001) and interleukin-2 (IL-2, p&lt;0.0001) as well as cytotoxic degranulation (p&lt;0.0001) were all significantly impacted by the amount of CD22 on the surface of NALM6. A similar pattern of antigenic dose-response was seen in the signaling of CAR T cells, with phosphorylation of ERK reflecting the level of CD22 antigen (p&lt;0.001) and correlating with the increased in vivo efficacy of the CAR T cells against CD22WT NALM6, relative to CD22Lo NALM6. Increasing the affinity of the CD22 CAR did not impact the in vivo efficacy against CD22WT NALM6 at either a therapeutic or subtherapeutic dose, however, HA-CD22 CAR T cells significantly prolonged the survival of NSG mice with CD22Lo NALM6, relative to SA-CD22 CAR T cells (p&lt;0.01). The enhanced activity of HA-CD22 CAR T cells against CD22Lo leukemia did not correlate with improved in vitro functionality, as the HA-CD22 CAR T cells surprisingly demonstrated lower IL-2 secretion (p&lt;0.01), lower proliferation (p&lt;0.05) and diminished in vitro lysis of CD22Lo NALM6 (p&lt;0.05), relative to SA-CD22 CAR T cells. ERK phosphorylation, however, was significantly increased in HA-CD22 CAR T cells (p&lt;0.01) and was the only in vitro marker which correlated with the enhanced in vivo activity seen with the affinity-matured CAR.
Previous clinical experience has demonstrated the importance of using a short linker (consisting of a single G4S sequence) between the heavy and light chains of the m971 scFv, therefore we next evaluated the impact of linker length on the activity of the HA-CD22 CAR. HA-CD22 CARs were generated with either a short- or long-linker (G4S x1 vs G4S x3, respectively) and evaluated in vitro and in vivo. While the short linker improved proliferation in vitro, there was no significant impact of linker length on cytokine production or lysis of CD22Lo NALM6. In a xenograft model, HA-CD22 CAR T cells with the long-linker demonstrated slower progression of CD22Lo leukemia and significantly prolonged survival of NSG mice with CD22WT leukemia relative to HA-CD22 CAR T cells with the short-linker (p&lt;0.01).
Taken together, these studies suggest that increasing the affinity of a scFv is a promising strategy for enhancing CAR sensitivity to low levels of target antigen, with the potential to decrease post-CAR T cell relapses due to antigen downregulation.

Disclosures
No relevant conflicts of interest to declare.