cytotoxic activity against human cancer cells
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SITC 2018Annual MeetingNovember 10, 2018Washington, D.C.Presented:November 10, 2018
DeepTM IL-15 Primed multi-targeted T cells demonstrate potent antigen-specific cytotoxic activity against human cancer cells
Shawn P. Carey, Beth Pearce, Darren Smith, Pengpeng Cao, Christine McInnis, Amy Shaw, Jonas Bruun, Fabio Fachin, Becker Hewes, Jonathan B. Fitzgerald, Thomas L. Andresen, Andy Rakestraw Torque Therapeutics, Cambridge, MA
BackgroundAdoptive transfer of tumor-directed T cells has demonstratedencouraging clinical efficacy in some hematological and solidtumors. However, widespread success of such therapies hasbeen limited by (1) single-epitope targeting by geneticallymodified TCR and CAR T cell therapies and (2)insufficient support of transferred T cell survival and function.To direct immune activation in the tumor microenvironment,Torque has developed the Deep-PrimedTM T cell therapyplatform in which cytotoxic T lymphocytes (CTLs)simultaneously targeting multiple tumor associated antigens(TAA) are primed with immune-stimulatory drugs tethered totheir surface to provide localized and sustained support totumor-directed T cells. This study evaluates cancer cell-directed cytotoxicity by multi-target T cells with and withoutDeepTM IL-15, which is a cell-associated crosslinked multimerof human IL15-Fc.
MethodsCytotoxic T lymphocytes directed against cancer cellsexpressing TAA including MART-1 or PRAME were generatedfrom healthy donors using Torque’s modular TAA-primingapproach SlipstreamTM (See Figure 2 below and poster P272).
The effect of Deep IL-15 priming on bulk and antigen-specificT cell expansion was evaluated. CTL-mediated cytotoxicitywas assessed using a panel of partially HLA-matched humancancer cells with diverse TAA expression. Cytotoxic activitywas compared with and without Deep IL-15, and time courseanalysis of CTL function including expansion, activation, andcytotoxicity was used to provide mechanistic understandingof Deep IL-15 impact on anti-cancer cell activity by CTLs.
Results
Conclusions• Tumor-directed T cells generated using Torque’s modular TAA-priming approach elicit potent cytotoxicity against cancer cellsexpressing multiple TAA including PRAME and MART1.
• Deep IL-15 enhances the potency of anti-cancer cell activity byincreasing antigen-specific cytotoxicity at low E:T ratios andextended time points.
• By acting as a prolonged cell-tethered source of autocrine IL-15,Deep IL-15 enhances T cell survival and proliferation both priorto and following antigen exposure.
• Clinical trials evaluating TRQ15-01, Torque’s Deep IL-15 Primedmulti-targeted T cells, will initiate later this year.
Multi-target T cells demonstrate potent antigen-specific cytotoxic activity against human cancer cells
Figure 1. Deep IL-15 provides autocrine cytokine stimulation. Deep IL-15 is amultimer of human IL15-Fc monomers. IL15-Fc monomers consist of two subunits, eachconsisting of an effector attenuated IgG-Fc fused with an IL-15 receptor α-sushi-domainnoncovalently bound to a molecule of IL-15. Deep IL-15 Primed T cells are generated via aloading process in which cells are co-incubated with Deep IL-15 at high concentrations.
Surfacemodifier
Cleavable crosslinker
IL15-Fc Deep IL-15
T-cell
Deep IL-15
Deep IL-15Primed T Cell
HumanIgG-Fc
Collect cellsIsolate
monocytesMature into
dendritic cellsProprietary
dendritic cellsGenerate
T cellsTAA-specific
Primed T cells
TAA peptidesApheresis Multi-target T cell generation and priming
Figure 2. Slipstream™ Cell Manufacturing platform is a proprietary, high-efficiency T cell manufacturing platform engineered to operate as modularcompact factories. Slipstream production is semi-automated and fully closed, whicheliminates contamination risk between transfers and can dramatically reduce staffingrequirements and the factory footprint.
Deep IL-15 promotes extended T cell activation, expansion, and survival upon antigen exposure
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Figure 7. (A) Deep IL-15 drives enhanced expansion and activation of MART1 –targeted T cells (76% specificity by tetramer staining) upon antigen exposure. T cells +/-Deep IL-15 cultured alone and in 1:1 co-culture with MART1neg target cells (RPMI6666)or MART1pos target cells (RPMI6666 loaded with MART126-35 peptide). (B) Deep IL-15promotes activation (CD25+) and prevents apoptosis (Annexin-V+) in MART1 – targetedT cells on day 5 of co-culture with MART1pos target cells.
Deep IL-15 acts as a cell-tethered source of autocrine IL-15 to support T cell expansion
Figure 5. (A) Deep IL-15 loading drives dose-dependent expansion of multi-TAA –targeted T cells. Cell expansion quantified by AUC of cell number over 10 days. (B) DeepIL-15 remains cell-associated over many days, driving more persistent T cell expansionthan soluble IL-15. Expansion following media exchanges on indicated daysdemonstrates persistent function of cell-associated Deep IL-15 versus soluble IL-15.
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Deep IL-15 loading induces dose-dependent T cell expansion over a
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Figure 6. (A) Deep IL-15 promotes survival of MART1-specific T cells (HLA-A2/MART26-35-PE tetramer+) in culture over 5 days. T cells were loaded with Deep IL-15or mock-loaded. Plot illustrates effect of Deep IL-15 on survival of antigen-specific cellsfrom 3 donor lots of MART1 – targeted T cells. (B) Schematic of T cell–cancer cell co-cultures used to evaluate how Deep IL-15-mediated support of antigen-specific cellsimpacts antigen response. (C) Deep IL-15 loading promotes maximum T cell activationin antigen-positive (RPMI6666 loaded with MART126-35 peptide, MeWo, SKMEL5) co-cultures initiated at Day 0 or Day 2. Dashed lines indicate frequency of MART1-specificT cells at co-culture start for mock-treated (gray) and Deep IL-15 Primed (blue) T cells.
Deep IL-15 promotes survival of antigen-specific cells to maintain antigen responsiveness of T cells
Deep IL-15 enhances multi-target T cell cytotoxic activity against human cancer cells
Figure 3. (A) PRAME – targeted T cells trained against a cassette of PRAME peptidesexhibit multi-epitope specificity. (B) MART1 – targeted T cells (HLA-A2/MART26-35-PEtetramer+) elicit antigen-restricted cytotoxicity against MART1neg RPMI6666 pulsed withMART126-35 peptide over 5-day co-culture. (C) PRAME – targeted T cells (Lot 1; blue)and MART1 – targeted T cells (Lot 2; orange) demonstrate cytotoxicity against partiallyHLA-matched antigen-positive solid tumor cells in a short-term (6 hr) cytotoxicity assay.
PRAME–targeted T cellsMART1–targeted T cells
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Figure 4. (A) Deep IL-15 prolongs cytotoxicity by PRAME – targeted T cells, resulting inenhanced potency by day 4 of co-culture. (B) Time course analysis shows that Deep IL-15Primed T cells prevent target cancer cell proliferation at later time points following earlycytotoxicity measured using a standard 6 hr assay. All cytotoxicity data shown is at 10:1total T cell : target ratio.
Total T cell and antigen-specific T cell : target cell ratios
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Illustration of total T cell and antigen-specific T cell : target cell ratios based upon antigen-specificity of lots
Total T cell : target cell ratio
Total T cell : target cell ratio
Cytotoxicity by PRAME – targetedT cells on day 4 of co-culture
Deep IL-15 enhances cancer cell-directed cytotoxicity by
multi-target T cells
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Cell-associated Deep IL-15 promotes
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Deep IL-15 Primed T cells prevent cancer cell outgrowth at
later time points, resulting in improved cytotoxic potency