Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells

Abstract

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Figures

Figure 1 |. Clinical trial of CAR22 T cells in adults and children with relapsed ALL.

a, Schema of trial design. b-c, Expansion and persistence of CART22 in peripheral blood from treated b, children and c, adults. d, Outcomes after CART22 treatment. Green arrow represents a second CART22 infusion in pediatric patient 4.

Figure 2 |. scFv linker influences CAR surface membrane activity.

a, Schematic of CAR22-short and long constructs. b, Affinity and size measurements of purified CAR22-short and long scFvs. c, Predictive modeling of scFv structures. d-e, Composite confocal microscopy images of GFP-tagged d, CAR22-long and e, CAR22-short constructs expressed on human T cells.

Figure 3 |. CAR clustering leads to antigen-independent signaling.

a, Difference in phospho-peptide quantity in resting CAR22-short compared to CAR22-long T cells. Proteins with >1.5-fold difference are shown. b, Upregulated transcriptional programs in resting CAR22-short compared to CAR22-long T cells. c, Heatmap of relative phospho-peptide quantities in short versus long scFv linker versions of CAR19, CAR22 and CAR33 T cells.

Figure 4 |. Functional characterization of CAR22-engineered T cells.

Quantification of a, F-actin (three independent experiments, n=1000 measurements per group) and b, perforin (four independent experiments, n=2400 measurements per group) polarization in CAR T cells engaged with Nalm6. c, Nalm6 survival over time during in vitro co-culture with control (un-engineered), CAR22-long or short T cells. Secretion of d, IFNγ, e, IL-2 and f, TNFα over time during co-culture. g, Leukemia progression over time in xenograft mice bearing Nalm6 treated with control, CAR22-long or short T cells (representative of 4 replicate experiments, n=4–7 mice per condition; see Supplementary Figure 1 for individual animal responses and Supplementary Figure 2 for experimental replicates). h, Quantification of CAR T cells in animal peripheral blood on day 15 after T cell transfer, and i, animal survival over time. *P<0.05, **P<0.001, ***P<0.0001, ****P<0.00001. Statistics reflect differences between CAR22-short and long T cells.

Figure 5 |. Development of a novel CD22 CAR with potent pre-clinical activity.

a, Pipeline for development and evaluation of new CD22-f2-short CAR. b, Affinity and size of purified CD22-f2-long and short scFvs. c, Expression of CD22 CARs on primary T cells. d, Measurement of secreted IFNγ by CD22-engineered T cells after 24h exposure to CD22+ target cells. e, Progression of Nalm6 disease burden in xenograft mice treated with CD22-f2-short and long T cells (Representative of 4 replicate experiments, n=4–7 mice per condition; see Supplementary Figure 5 for individual animal responses and Supplementary Figure 6 for experimental replicates). f, Survival of Nalm6-bearing xenograft mice after treatment with m971 or CD22-f2 CAR T cells. Data are presented as mean values +/− standard error of the mean (S.E.M.) Statistics reflect differences between CAR22-short and long T cells.