Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors

Abstract

Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity towards B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs (ITAM)-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared to standard first and second generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors.

High-Affinity GD2-Specific CAR T Cells Induce Fatal Encephalitis in a Preclinical Neuroblastoma Model

“…20, with 14- to 17-hour effector/target coincubations. Nontransduced T cells, or T cells transduced with an irrelevant CAR (e.g., anti-CD19), were included in all experiments to control for alloreactivity between the T-cell donor and the SY5Y tumor line, which is minimal in our experience (perhaps owing to the generally low baseline expression of MHC molecules in the tumor line; ref.…”

Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors

“…(47, 57) A recently described MSLN CAR construct was generated to provide the DAP12 killer immunoglobulin-like receptor with signaling activation that includes the ITAM domain. (58) Preclinical experiments demonstrated that T cells engineered with this CAR displayed increased potency in vivo associated with retention of CAR expression, compared to second-generation MSLN CARs comprising either CD28 or 4-1BB signaling domains. (58) Other costimulatory domains have been tested in combination with other antigens, including FcRγ, OX40, DAP10, and CD27.…”

“…(58) Preclinical experiments demonstrated that T cells engineered with this CAR displayed increased potency in vivo associated with retention of CAR expression, compared to second-generation MSLN CARs comprising either CD28 or 4-1BB signaling domains. (58) Other costimulatory domains have been tested in combination with other antigens, including FcRγ, OX40, DAP10, and CD27. (3, 56, 59) Choosing an appropriate costimulation domain is essential to sustain CAR T-cell activity and calibrate T-cell persistence.…”

Can Targeting Stroma Pave the Way to Enhanced Antitumor Immunity and Immunotherapy of Solid Tumors?

“…The majority of these studies utilized adoptive transfer of FAP-specific chimeric antigen receptor expressing (FAP-CAR) T cells. FAP-CAR have been generated using single chain Fv regions based on the sequence of three different monoclonal antibodies (mAbs) to FAP, two of which, 73.3 (25) and MO36 (105), inhibited tumor growth in multiple tumor models and exhibited little if any toxicity unless administered repeatedly or expressed in hyperfunctional T cells (25,28,104). The loss of antitumor activity of 73.3 FAP-CAR T cells in FAP-deficient mice established the specificity of these CAR–T cells and demonstrated that the antitumor activity was dependent on expression of FAP by host stromal cells.…”