Enhancing the Tumor Selectivity of a Picornavirus Virotherapy Promotes Tumor Regression and the Accumulation of Infiltrating CD8+ T Cells

Abstract

Picornaviruses have emerged as promising cancer therapies due to their ability to drive cytotoxic cellular immune responses and for promoting oncolysis. These properties include preferential replication in tumor cells, the induction of strong innate and adaptive immune responses, and the ease with which their genomes can be manipulated. We have developed Theiler's murine encephalomyelitis virus (TMEV) as an immunotherapy vector that promotes strong adaptive immune responses to tumor antigens embedded within its genome. To further explore its usefulness as cancer therapy, we investigated whether direct intratumoral delivery of TMEV could promote tumor regression. We generated several picornavirus hybrids using substrains of TMEV that have unique immunopathologic characteristics, despite their extensive sequence homology. These hybrids exhibit a unique propensity to infect and replicate in melanoma. We have identified GD7-KS1, a virus that is particularly effective at replicating and infecting B16 melanoma in vitro and provides benefit as an oncolytic therapy in vivo after intratumoral injection. In addition, this virus promotes the mobilization and accumulation of CD8(+) T cells within treated tumors. Altogether, these findings demonstrate that picornavirus substrains can be used to rationally design virus hybrids that promote antitumor responses and add to the known strategies identified by us and others to further enhance the therapeutic potential of vectors used to treat cancer.

Figure 1. The use of Daniel's strain of TMEV as oncolytic therapy fails to inhibit outgrowth of breast cancer and melanoma

(A) Left: Average tumor index of TUBO breast tumors in Balb/c mice beginning on day of intratumoral injection of TMEV-DA (d. 9). Right: Individual tumor growth over time. No increase in survival due to virus therapy was observed by Kaplan Meier Analysis. Median survival: TMEV-DA – 19 days, Vehicle-Ctrl. – 34 days. (B) Left: Average tumor index of B16 tumors treated with intratumoral TMEV-DA (d.7). NS - Not significant at any timepoint tested (Students t-test). Right: Growth of individual tumors in TMEV-DA and vehicle control groups. No significant survival advantage by Kaplan Meier Analysis was observed. Median survival: TMEV-DA – 22 days, Vehicle-Ctrl. – 17 days.

Figure 2. Generation of chimeric viruses composed of genomic elements from the TMEV substrains Daniel's and GDVII

(A) Cloning strategy and map of DA/GDVII chimeric vectors. A 3 kilobase Kpn I and Stu I restriction fragment was used to generate an initial DA/GDVII vector. Site-directed mutagenesis and directional cloning were used to replace capsid flanking regions with wild-type TMEV-DA sequences. GD7-P1 contains the complete capsid structure of GDVII and all non-capsid encoding elements are derived from TMEV-DA. (B) Virus plaques generated after transfection of virus encoding vectors into BHK cells. (Right) Plaque measurements from each virus. (* Significant by ANOVA p<0.05).

Figure 3. GD7-KS1 demonstrates enhanced cytotoxicity towards and increased replication in melanoma

(A) Melanoma lines (B16, B16-OVA and B16-F10), breast cancer lines (TUBO and 4T1) and the thymic lymphoma line (EL4) were exposed to TMEV-DA or GD7-KS1 for 24 and 48 hours. At the given time points MTT assays were performed to assess percent survival. (B) Relative fold change in VP2 specific virus transcript levels assessed in melanoma, breast cancer and lymphoma at time 0 and 24 hours. Values are relative to uninfected control. (C) Growth kinetics of TMEV-DA and GD7-KS1 in the virus propagating cell line BHK and in B16 melanoma assessed at given time points and expressed as log₁₀ virus plaque forming units. Cells were infected at an MOI of 0.01. PFU/cell were calculated based on plating 10⁶ cells and the total virus yield from 2 mL of media (* Significant by t-test, p<0.05). Data points and standard deviation for A-C represent triplicate independent measurements.

Figure 4. GDVII capsid containing viruses delay tumor outgrowth and promote increased survival when used as oncolytic therapy

(A) Tumor bearing mice were treated with GD7-KS for six days beginning on day 8. Tumor index was calculated until overwhelming tumor burden was reached (n=5/group). (Right) Individual tumor growth curves through completion of the study. The median survival was 18 days for vehicle control and 23 days for GD7-KS1 treated animals (* Significant by Log-Rank, p=0.021). (B) Tumor bearing mice were treated with the GDVII capsid only virus GD7-P1 for 6 days beginning on day 7 and tumors were monitored as above (n=5/group). (Right) Tumor growth curves for individual animals. The median survival was 18 days for vehicle control and 25 days for the GD7-P1 treated group (* Significant by Log-Rank, p=0.003).

Figure 5. Intratumoral delivery of GD7-KS1 promotes the accumulation of CD8+ T-cells

(A) Tumor index of B16 bearing hosts treated with intratumoral vehicle or GD7-KS1. Tumors were measured until tumor harvest (* Significant by t-test, p<0.05). (B) Percentage (top) and absolute number of CD8+ and CD4+ T-cells recovered from B16 host tumors treated with vehicle or GD7-KS1 in (A) (Significant by t-test, p<0.05). (C) Tumor infiltrating CD45+ gated cells were assessed for CD8 and tetramer for VP2 antigen (* Significant by t-test, p<0.05).

Figure 6. GD7-KS1 promotes the activation of tumor specific CD8+ T-cells

(A) Tumor index of B16-OVA tumors implanted into B6 hosts treated with control vehicle alone or GD7-KS1 virus. Tumors were measured until harvest. (B) Tumors from (A) were dissociated and analyzed by FACS. Total CD45+ cells were gated and analyzed for the percentage of CD8+ and CD4+ cells within dissociated tumors. Ratio of CD8 cells to CD4 cells comparing control to GD7-KS1 treatment. (C) Percent of CD8+ T-cells specific for the tetramer H-2K^b-SIINFEKL. Cells within plot are from CD45+ gate. (D) Histogram of CD8 staining comparing vehicle to GD7-KS1 and mean fluorescence intensity of groups. * Significant by t-test, p<0.05.