Antigen Sensitivity of CD22-Specific Chimeric TCR Is Modulated by Target Epitope Distance from the Cell Membrane

Abstract

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR+ CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR+ CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR+ CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.

Antibody-mediated B-cell depletion before adoptive immunotherapy with T cells expressing CD20-specific chimeric T-cell receptors facilitates eradication of leukemia in immunocompetent mice

“…Global lymphodepletion has been shown to increase T-cell survival, 16,17 but the effect of selective B-cell lymphodepletion before adoptive transfer of B-cell antigenspecific T cells has not been evaluated. Although several B cellassociated molecules have been targeted by cTCRs, including CD19, 18,19 CD20, [1][2][3] and CD22, 5 no studies have addressed the in vivo function of cTCR ϩ T cells in a model system in which both normal and neoplastic cells express the same target molecule. In this study we have targeted CD20 on both normal and leukemic B cells in immunocompetent mice.…”

“…[1][2][3][4] In vitro experimentation has shown that high expression density of CD20 on normal human B cells down-modulates cTCR molecules from the surface of CD20-specific cTCR ϩ T cells. 5 Downmodulation of canonical TCR has been associated with reduced sensitivity and effector functions, 6 suggesting cTCR downmodulation may limit target recognition. Persistent exposure to CD20 on B cells may also impair CD20-specific cTCR ϩ T-cell survival.…”

Chimeric antigen receptor modified T cell therapy for B cell malignancies

“…tumor cells [38], and CAR-mediated targeting of an epitope of CD22 located in close proximity to the tumor cell membrane was more efficient than targeting a more distally located epitope [39]. The density of target molecules on the tumor cell and of CARs on the T cell may also significantly impact CAR-modified T cell function [39,40]. These observations highlight the fact that CAR design is in evolution, and it is presently unclear if there are cardinal characteristics of CAR design that are necessary for optimal clinical efficacy of CAR-modified T cells.…”

“…For example, a ROR1-specific CAR incorporating a short extracellular spacer between the scFv and transmembrane domain was more effective than its long spacer counterpart in lysis of ROR1 ? tumor cells [38], and CAR-mediated targeting of an epitope of CD22 located in close proximity to the tumor cell membrane was more efficient than targeting a more distally located epitope [39]. The density of target molecules on the tumor cell and of CARs on the T cell may also significantly impact CAR-modified T cell function [39,40].…”

Immunotherapy for B-Cell Neoplasms Using T-Cells Expressing Chimeric Antigen Receptors : From Antigen Choice to Clinical Implementation

“…Other CARtargeting CD22 (CART22), CD23 (CART23), and CD70 (CART70) antigens as well as κ LC (CARTκ) were also developed showing promising results in in vitro and in vivo mouse tumour models. [57][58][59][60] With respect to the therapeutic potential of tested CAR, it is worthwhile to note that suboptimal cytotoxic effects of these genetically engineered T cells vary among targeted antigens. Although several explanations may be proposed, one possible reason is the difference in expression levels of the targeted antigens in malignant and normal B cells.…”