Jinjuan Wang

Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells

Adoptive immunotherapy with T cells expressing a tumor-specific chimeric T-cell receptor is a promising approach to cancer therapy that has not previously been explored for the treatment of lymphoma in human subjects. We report the results of a proof-of-concept clinical trial in which patients with relapsed or refractory indolent B-cell lymphoma or mantle cell lymphoma were treated with autologous T cells genetically modified by electroporation with a vector plasmid encoding a CD20-specific chimeric T-cell receptor and neomycin resistance gene. Transfected cells were immunophenotypically similar to CD8(+) effector cells and showed CD20-specific cytotoxicity in vitro. Seven patients received a total of 20 T-cell infusions, with minimal toxicities. Modified T cells persisted in vivo 1 to 3 weeks in the first 3 patients, who received T cells produced by limiting dilution methods, but persisted 5 to 9 weeks in the next 4 patients who received T cells produced in bulk cultures followed by 14 days of low-dose subcutaneous interleukin-2 (IL-2) injections. Of the 7 treated patients, 2 maintained a previous complete response, 1 achieved a partial response, and 4 had stable disease. These results show the safety, feasibility, and potential antitumor activity of adoptive T-cell therapy using this approach. This trial was registered at www.clinicaltrials.gov as #NCT00012207.

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

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.

Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma.

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a “suicide gene” relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20+ malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned.

Mathematical Modeling of Chimeric TCR Triggering Predicts the Magnitude of Target Lysis and Its Impairment by TCR Downmodulation

We investigated relationships among chimeric TCR (cTCR) expression density, target Ag density, and cTCR triggering to predict lysis of target cells by cTCR+ CD8+ T human cells as a function of Ag density. Triggering of cTCR and canonical TCR by Ag could be quantified by the same mathematical equation, but cTCR represented a special case in which serial triggering was abrogated. The magnitude of target lysis could be predicted as a function of cTCR triggering, and the predicted minimum cTCR density required for maximal target lysis by CD20-specific cTCR was experimentally tested. cTCR density below ∼20,000 cTCR/cell impaired target lysis, but increasing cTCR expression above this density did not improve target lysis or Ag sensitivity. cTCR downmodulation to densities below this critical minimum by interaction with Ag-expressing targets limited the sequential lysis of targets in a manner that could be predicted based on the number of cTCRs remaining. In contrast, acute inhibition of lysis of primary, intended targets (e.g., leukemic B cells) due to the presence of an excess of secondary targets (e.g., normal B cells) was dependent on the Ag density of the secondary target but occurred at Ag densities insufficient to promote significant cTCR downmodulation, suggesting a role for functional exhaustion rather than insufficient cTCR density. This suggests increasing cTCR density above a critical threshold may enhance sequential lysis of intended targets in isolation, but will not overcome the functional exhaustion of cTCR+ T cells encountered in the presence of secondary targets with high Ag density.