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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.

We previously demonstrated the feasibility of generating therapeutic numbers of cytotoxic T lymphocyte (CTL) clones expressing a CD20-specific scFvFc:CD3zeta chimeric T cell receptor (cTCR), making them specifically cytotoxic for CD20+ B lymphoma cells. However, the process of generating and expanding he CTL clones was laborious, the CTL clones expressed the cTCR at low surface density, and they exhibited suboptimal proliferation and cytotoxicity. To improve the performance of the CTLs in vitro and in vivo, we engineered "second-generation'' plasmid constructs containing a translational enhancer (SP163) and CD28 and CD137 costimulatory domains in cis with the CD3zeta intracellular signaling domain of the cTCR gene. Furthermore, we verified the superiority of generating genetically modified polyclonal T cells expressing the second-generation cTCR rather than T cell clones. Our results demonstrate that SP163 enhances the surface expression of the cTCR; that the second-generation cTCR improves CTL activation, proliferation, and cytotoxicity; and that polyclonal T cells proliferate rapidly in vitro and mediate potent CD20-specific cytotoxicity. This study provides the preclinical basis for a clinical trial of adoptive T cell immunotherapy for patients with relapsed CD20+ mantle cell lymphoma and indolent lymphomas.

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.

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