Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors

Abstract

Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date.

Figure 1. VSV-expressed cDNA libraries

a. The ASEL VSV-expressed cDNA library contains cDNA from normal human prostate cloned into VSV in direct, or reverse, orientation. b. Human prostate specific genes^–, but not the melanocyte-specific TRP-2, detected by PCR in the original human prostate plasmid library (lane 1) and in the VSV-cDNA plasmid library (2). rtPCR from cDNA of HT1080 cells infected with ASEL (MOI 0.1) (3) compared to uninfected cells (4) (predicted size for hPSCA shown with an arrow). c. BHK cells infected with ASEL Direct (1) or Reverse (2), or with control viruses (lanes 3&4) (MOI ~10) assayed for human PSA by Western Blot. Lane 5, uninfected BHK cells; Lane 6, 10⁴ human prostate LnCap cells. d. BHK cells infected with 10 fold dilutions of ASEL virus (1–6) assayed by rtPCR for PSA or human GAPDH. No PSA-specific signal was detected at dilutions lower than 1:100 of the original virus stock. (Expression of GFP from 100 pfu of VSV-GFP could be detected by this assay). +Positive/*negative for PCR upon nested PCR. e. Splenocytes infected with VSV-GFP or VSV-cDNA libraries from cells which did, or did not, express OVA (MOI 0.1), co-cultured with naive OT-I T cells and assayed for IFN-γ. Lane 1, splenocytes alone; (2–4), splenocytes infected with VSV-GFP, without OT-I (2), with OT-I (3) or with irrelevant T cells; (5&6), splenocytes infected with VSV-cDNA library from B16ova cells in Direct (5) or Reverse (6) orientation with OT-I; (7&8), splenocytes infected with VSV-cDNA library from B16 cells (no OVA) in Direct (7) or Reverse (8) orientations with OT-I. (9), OT-I activated by SIINFEKL peptide. (10–13), splenocytes infected with VSV-ova at MOI 0.01 (10), 0.1 (11), 1.0 (12) and 10 (13). (14), OT-I (no splenocytes, no VSV).

Figure 2. Intra-prostatic injection of ASEL induces autoimmunity

a. Prostate weights of mice injected intra-prostatically with PBS, VSV-GFP or ASEL 2 or 10 days after injection (n=3). b-d. Histology of prostates 10 days after intra-prostatic injection of PBS (b) or ASEL (c,d). Scale bars, 100µm. e,f. 10 days following intra-prostatic injection of PBS, or 10⁷pfu VSV-GFP or ASEL (6 mice per group) splenocytes from each mouse co-cultured with lysates of TC2 cells were assayed for IFN-γ (e) or IL-17 (f). Mean values of cytokine from two ELISA wells per sample are shown for each mouse.

Figure 3. Intravenous injection of ASEL has anti-tumor efficacy

a. Prostate weights 60d following 10⁷ pfu i.v. of ASEL (n=5) or VSV-GFP (n=7). b. Mean levels of IL-17 secreted by splenocytes from these mice co-cultured with lysates of B16 melanoma, TC2 prostate, normal mouse prostate or pancreas. c. Survival of mice bearing 7d TC2 tumors (n=7–8) injected intra-tumorally or intravenously with 10⁷pfu of VSV-GFP, ASEL, or heat inactivated VSV-GFP (days 7,9,11). d,e. Survival of mice bearing 7d TC2 (d) or B16 (e) tumors injected intravenously with ASEL, or with a VSV-cDNA library from human melanoma cells (Altered Self Melanoma Epitope Library, ASMEL) (days 7,9,11). f. Cumulative percentages of mice cured of 7d TC2 tumors when administered 3, 6 or 9 injections of ASEL or VSV-GFP i.t. or i.v. every other day. g. IL-17 secreted by splenocytes from the three mice cured of TC2 tumors by 9 intra-tumoral injections of ASEL in f., as well as from 3 mice treated similarly with VSV-GFP, co-cultured with lysates of B16, TC2, normal mouse prostate or pancreas. h. Survival of mice bearing 7d TC2 tumors (n=7/8), mock depleted, or depleted of CD4+, CD8+ or NK cells, injected intravenously with ASEL on days 7,9,11,14,16,18,21,23,25.

Figure 3. Intravenous injection of ASEL has anti-tumor efficacy

a. Prostate weights 60d following 10⁷ pfu i.v. of ASEL (n=5) or VSV-GFP (n=7). b. Mean levels of IL-17 secreted by splenocytes from these mice co-cultured with lysates of B16 melanoma, TC2 prostate, normal mouse prostate or pancreas. c. Survival of mice bearing 7d TC2 tumors (n=7–8) injected intra-tumorally or intravenously with 10⁷pfu of VSV-GFP, ASEL, or heat inactivated VSV-GFP (days 7,9,11). d,e. Survival of mice bearing 7d TC2 (d) or B16 (e) tumors injected intravenously with ASEL, or with a VSV-cDNA library from human melanoma cells (Altered Self Melanoma Epitope Library, ASMEL) (days 7,9,11). f. Cumulative percentages of mice cured of 7d TC2 tumors when administered 3, 6 or 9 injections of ASEL or VSV-GFP i.t. or i.v. every other day. g. IL-17 secreted by splenocytes from the three mice cured of TC2 tumors by 9 intra-tumoral injections of ASEL in f., as well as from 3 mice treated similarly with VSV-GFP, co-cultured with lysates of B16, TC2, normal mouse prostate or pancreas. h. Survival of mice bearing 7d TC2 tumors (n=7/8), mock depleted, or depleted of CD4+, CD8+ or NK cells, injected intravenously with ASEL on days 7,9,11,14,16,18,21,23,25.

Figure 4. Sub optimal vaccination induces immune escape variants

a-d. H&E staining of tumors (1.0cm diameter) from mice bearing 7d TC2 tumors treated i.v. with PBS (a,c) or ASEL (b,d) (days 7,9,11) (typically d20 for PBS (TC2) or d50 for ASEL (TC2R)). Scale bars, 100µm. e,f. Three TC2R tumors from mice treated with ASEL (1–3), one tumor from a mouse treated with PBS (4), as in a-d. above, and in vitro cultured TC2 cells (5) analyzed by rtPCR for murine prostate specific genes PSCA, PSMA and STEAP (e) or for N-Cadherin, SLUG or SNAIL^{, , ,} (f). *Cultured TC2 cells positive for N-Cadherin by nested PCR and weakly positive by Western Blot at lower levels than detected in TCR2 tumors.

Figure 5. TC2R tumors can be treated with a second vaccination

a. The IEEL contained cDNA from three TCR2 tumors cloned into VSV. b. Western blot for murine N-Cadherin from BHK cells infected with VSV (1), IEEL Reverse (2) or Direct (3) (MOI ~10). Equal loading confirmed by ß-actin probing. c. Survival of mock, or VSV-GFP,-vaccinated mice bearing 7d TC2 tumors treated with three i.v. injections of VSV-GFP or ASEL, either as viral supernatant (10⁷pfu) or as virus pre-loaded onto CD8+ T cells [T(ASEL)]. d. Mice bearing 7d TC2 tumors were treated i.v. with VSV-GFP or ASEL (days 7,9,11). On days 25,27,29 mice initially treated with ASEL received i.v. IEEL virus pre-loaded onto CD8+ T cells [T(IEEL)]. e. VSV-vaccinated mice bearing 7d TC2 tumors were injected intravenously with VSV-GFP, ASEL or IEEL (10⁷pfu) or with virus pre-loaded onto CD8+ T cells [T(ASEL)/T(IEEL)] (d 7,9,11). On days 20,22,24, surviving mice were treated i.v. with T cells loaded with IEEL [T(ASEL)/T(IEEL)] or ASEL [T(ASEL)/T(ASEL)]. f-i. Splenocytes from mice which either did (f,g) or did not (h,i) reject TC2/TC2R tumors following i.v. ASEL + T(IEEL) (d) or T(ASEL) + T(IEEL) (e) were co-cultured with lysates of TC2, TC2R, B16, normal mouse prostate or pancreas and assayed for (f,g) IL-17 or (h,i) IFN-γ. Results are from three survivor mice (f,g) or two mice which succumbed to TC2R tumors (h,i).

Figure 5. TC2R tumors can be treated with a second vaccination

a. The IEEL contained cDNA from three TCR2 tumors cloned into VSV. b. Western blot for murine N-Cadherin from BHK cells infected with VSV (1), IEEL Reverse (2) or Direct (3) (MOI ~10). Equal loading confirmed by ß-actin probing. c. Survival of mock, or VSV-GFP,-vaccinated mice bearing 7d TC2 tumors treated with three i.v. injections of VSV-GFP or ASEL, either as viral supernatant (10⁷pfu) or as virus pre-loaded onto CD8+ T cells [T(ASEL)]. d. Mice bearing 7d TC2 tumors were treated i.v. with VSV-GFP or ASEL (days 7,9,11). On days 25,27,29 mice initially treated with ASEL received i.v. IEEL virus pre-loaded onto CD8+ T cells [T(IEEL)]. e. VSV-vaccinated mice bearing 7d TC2 tumors were injected intravenously with VSV-GFP, ASEL or IEEL (10⁷pfu) or with virus pre-loaded onto CD8+ T cells [T(ASEL)/T(IEEL)] (d 7,9,11). On days 20,22,24, surviving mice were treated i.v. with T cells loaded with IEEL [T(ASEL)/T(IEEL)] or ASEL [T(ASEL)/T(ASEL)]. f-i. Splenocytes from mice which either did (f,g) or did not (h,i) reject TC2/TC2R tumors following i.v. ASEL + T(IEEL) (d) or T(ASEL) + T(IEEL) (e) were co-cultured with lysates of TC2, TC2R, B16, normal mouse prostate or pancreas and assayed for (f,g) IL-17 or (h,i) IFN-γ. Results are from three survivor mice (f,g) or two mice which succumbed to TC2R tumors (h,i).

Figure 6. Immunogenicity of Altered Self/Self Libraries

a. Survival of mice bearing 7d TC2 tumors (n=7–8) injected intravenously with 10⁷ pfu VSV-GFP, ASEL, or SEL (days 7,9,11). b-e. IL-17 secreted from lymph node cells/splenocytes infected with (b) VSV-GFP, (c) no virus, (d) ASEL or (e) SEL (MOI 1) for 2 weeks and co-cultured with lysates of TC2, B16, normal mouse prostate, pancreas or PBS.