A “prime-­‐pull” immunotherapy approach using a len6viral vector and intratumoral TLR4 agonist redirects cytotoxic T cells Tina C. Albershardt, AJ Parsons, P Berglund, JH ter Meulen
#P213 Immune Design, SeaEle, WA TILs
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Prime-­‐pull with ZVexTM and GLAASTM inhibits tumor growth B16OVA Tumor Growth 1000 Ctrl 10 2
38.0 10 2
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<Pacific Blue-A>: CD8a
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0.92 2
GLA 800 ZVex ZVex + GLA D0 600 ZVex + GLA D7 400 200 0
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Methods 1.  B6 mice were implanted with 1 x 105 B16OVA cells, s.c., in the flank. 2.  Prime: Tumor-­‐bearing mice were immunized with ZVexTM when tumors measured > 20 mm3. 3.  Pull: Intratumoral injecEon of GLAASTM was started at the same Eme (D0) or 7 days (D7) post-­‐vector immunizaEon and conEnued once every 3-­‐4 days. 4.  Mice were sacrificed 18 days post-­‐tumor challenge to harvest tumors. Tumor-­‐infiltraEng lymphocytes were isolated on a 1:1 cushion of Histopaque®-­‐1083 and stained with MHC-­‐I/SIINFEKL pentamers, CD3, CD8, CD4, CD25, and FOXP3 anEbodies for flow cytometry analysis. Error bars represent mean ± SEM. 10 3
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6
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53.0 10 4
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10 3
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<Pacific Blue-A>: CD8a
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Local administraEon of GLAASTM recruits CTLs to the tumor. 50K
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Recruited CTLs lyse tumor cells decreasing tumor burden. 83.7 150K
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Tumor-Infiltrating Lymphocytes
GLAASTM Splenocytes
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ZVexTM-­‐primed CD8 T cells are recruited to the tumor by intratumoral injec6on of GLAASTM CD25 The clinical efficacy of tumor-­‐specific effector T cells is limited by their proper trafficking to the site of the tumor and the locally immunosuppressive environment. Strategies to improve homing to tumors and to enhance acEvity of immune effector cells are needed to unlock the potenEal of acEve cancer immunotherapy. AdopEng the “prime-­‐pull” strategy developed by Shin and Iwasaki against the spread of infecEous herpes simplex virus 2 (Nature, 2012), tumor-­‐
bearing mice were immunized with the lenEviral vector, ZVexTM, to generate tumor anEgen-­‐specific effector and memory CD8 T cells within the peripheral Essue. Mice were then given intratumoral administraEon of the TLR4 agonist, GLAASTM (glucopyranosyl adjuvant system), which induces T cell homing chemokines CXCL9 and CXCL10, thereby recruiEng cytotoxic T cells to the tumor. We show here that this prime-­‐pull strategy increased tumor-­‐infiltraEng T cells, which provided effecEve anE-­‐tumor acEvity. Intratumoral injecEon of GLAASTM effecEvely directs systemically induced CD8 T cells to the site of the tumor. Because GLAASTM also sEmulates anEgen presentaEon and maturaEon of dendriEc cells, the prime-­‐pull approach may be an effecEve way to overcome two major barriers to acEve cancer immunotherapy. Results GL
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4 7 11 Days Post-­‐Tumor Challenge 14 18 Methods 1.  Mice were implanted with 1 x 105 tumor cells, s.c., in the flank. 2.  Prime: Tumor-­‐bearing mice were immunized with ZVexTM when tumors measured > 20 mm3. 3.  Pull: Intratumoral injecEon of GLAASTM was started at the same Eme (D0) or 7 days (D7) post-­‐vector immunizaEon and conEnued once every 3-­‐4 days. 4.  Tumor growth was measured 2-­‐3 Emes per week. Tumor volume was calculated based on a modified ellipsoid formula: length x width2 x π/6. Error bars represent mean ± SEM.