Using virally expressed melanoma cDNA libraries to identify tumor-associated antigens that cure melanoma

Abstract

Multiple intravenous injections of a cDNA library, derived from human melanoma cell lines and expressed using the highly immunogenic vector vesicular stomatitis virus (VSV), cured mice with established melanoma tumors. Successful tumor eradication was associated with the ability of mouse lymphoid cells to mount a tumor-specific CD4(+) interleukin (IL)-17 recall response in vitro. We used this characteristic IL-17 response to screen the VSV-cDNA library and identified three different VSV-cDNA virus clones that, when used in combination but not alone, achieved the same efficacy against tumors as the complete parental virus library. VSV-expressed cDNA libraries can therefore be used to identify tumor rejection antigens that can cooperate to induce anti-tumor responses. This technology should be applicable to antigen discovery for other cancers, as well as for other diseases in which immune reactivity against more than one target antigen contributes to disease pathology.

Figure 1

Validation of the altered self melanoma epitope VSV-cDNA library. (a) BHK cells were screened for expression of gp100 and TRP2, PSA, or GAPDH by rtPCR after infection with no virus (lane 1), the ASMEL (MOI ~1) (lane 2) or VSV-GFP (lane 3). Lane 4, cDNA from the human LnCap prostate cancer cell line. (b) 10⁶ C57BL/6 splenocytes infected 24 h previously with either VSV-GFP, ASMEL or a VSV-cDNA library derived from normal human prostate tissue (MOI ~10) were co-cultured with either naive Pmel or OT-I T cells (effector/target ratio of 5:1) 4 and 28 h after virus infection. Supernatants were assayed 24–48 h later for IFN-γ, to detect transfer of expression of the hgp100_25–33 KVPRNQDWL peptide from the virus to infected splenocytes and subsequent presentation to the Pmel T cells. (c) Lane 1, splenocytes alone (no virus, no T cells); lane 2, splenocytes infected with VSV-GFP with added Pmel T cells; lane 3, splenocytes infected with the ASMEL with added OT-I T cells, which recognize the irrelevant SIINFEKL epitope of the OVA antigen; lane 4, splenocytes infected with ASMEL with added Pmel T cells, which recognize the hgp100_25–33 KVPRNQDWL epitope; lane 5, splenocytes infected with VSV-cDNA from normal human prostate with added Pmel T cells; lane 6, naive Pmel alone (no splenocytes, no virus). Error bars, s.d.

Figure 2

Intravenous ASMEL cures established B16 melanomas. (a) Mice bearing B16 tumors established for 5 d were either mock depleted (IgG), or were depleted of CD4⁺ or CD8⁺ T cells. Mice were then administered nine intravenous injections of the ASMEL or VSV-GFP (10⁷ pfu/injection) on days 8–10, 15–17 and 22–24. Survival with time is shown. (b,c) Six months after treatment with nine intravenous injections of the ASMEL, the only overt symptoms of autoimmune vitiligo were a whitening of the whiskers (b) and tails (b,c) compared to age-matched mice treated with PBS. (d) Pooled lymphoid cells (10⁶/well) from mice treated with nine intravenous injections of the ASMEL were infected 24 h later with ASMEL or VSV-GFP (MOI ~10). The cultures were replenished 24 h later by an additional 10⁶ lymphoid cells with a further round of virus infection 24 h after that. Cultures were stimulated 48 h after the final infection with virus, with either prostate tumor TC2 or melanoma B16 cell lysates and 48 h later, supernatants were assayed for IL-17 by ELISA. Lanes 1–4, lymphoid cells from ASMEL-treated mice treated with VSV-GFP and no tumor lysate (1); VSV-GFP and B16 tumor lysate (2); ASMEL and B16 tumor lysate (3) or ASMEL and TC2 tumor lysate (4) (ref. 40). Lanes 5–7, lymphoid cells from ASMEL-treated mice depleted of CD4 (5), CD8 (6) or NK cells (7) and treated in vitro with ASMEL and B16 tumor lysate. Error bar, s.d.

Figure 3

VSV functions as an hsp70-mediated adjuvant. (a) Expression of hsp70 was assayed by rtPCR from cDNA prepared from pooled lymphoid cells (10⁴/well) of C57BL/6 mice 24 h after infection with either VSV-GFP or ASMEL at increasing MOI as shown. (b) Expression of hsp70 by rtPCR from pooled lymphoid cells (10⁴/well) of C57BL/6 mice at 24, 36 or 48 h after infection with either VSV-OVA, VSV-GFP or ASMEL at MOI of 10. (c) Pooled lymphoid cells (10⁴/well) from mice treated with nine intravenous injections of ASMEL were infected 24 h later with ASMEL or VSV-GFP at various MOI and either in the presence, or absence, of added recombinant hsp70 (10 μg/ml). Representative wells were assayed by rtPCR 24 h later, for hsp70 expression. Similarly treated triplicate wells were replenished with an additional 10⁴ lymphoid cells and treated with a further round of virus infection 24 h later. Forty-eight hours following this final infection with virus, cultures were stimulated with melanoma B16 cell lysates and 48 h later supernatants were assayed for IL-17 by ELISA. (d) Expression of hsp70 was assayed by rtPCR from cDNA prepared from pooled lymphoid cells (10⁴/well) of TLR7^−/− (lane 1), MyD88^−/− (2), TLR4^−/− (3) or C57BL/6 (4) mice 24 h after infection with the ASMEL at an MOI of 10. (e) Pooled lymphoid cells (10⁴/well) from C57BL/6, MyD88^−/−, TLR4^−/− or TLR7^−/− mice treated with nine intravenous injections of ASMEL were infected 24 h later with ASMEL at an MOI of 10 either in the absence (–rhsp70), or presence (+rhsp70), of added recombinant hsp70 (10 μg/ml). Representative wells were assayed 24 h later, by rtPCR for hsp70 expression. Similarly treated triplicate wells were replenished with an additional 10⁴ lymphoid cells and reinfected with a further round of virus infection 24 h later. Forty-eight hours after this final infection with virus, cultures were stimulated with melanoma B16 cell lysates and 48 h later supernatants were assayed for IL-17 by ELISA.

Figure 4

VSV-induced TGF-β masks the tumor-specific IL-17 recall response. (a,b) Lymphoid cell cultures from ASMEL-vaccinated mice were screened for IL-17 (a) or TGF-β (b) secretion after infection with 12 single viruses purified by limiting dilution from the parental, unselected ASMEL stock (ASMEL nos. 1–12), with representative viruses selected from the screen of Supplementary Figure 1a. VSV-cDNA clone no. 3 (encoding NRAS sequences), VSV-cDNA clone no. 7 (encoding TYRP1 sequences), VSV-cDNA clone no. 11 (encoding CYC1 sequences) or with VSV-GFP (all infections at MOI 10). (c) The IL-17 screening assay of a above was repeated but in the presence of TGF-β RII Fc chimera to inhibit TGF-β in the cultures^,. Error bars, s.d.

Figure 5

Tumor-specific immunity induced by VSV-cDNA libraries is mediated by combinations of antigens. (a) Lymphoid cell cultures from ASMEL-vaccinated mice were screened for IL-17 secretion after no infection (lane 13); or with infection with 10⁷ pfu of individual single viruses from the parental, unselected ASMEL stock (ASMEL nos. 1–6) (lanes 1–3, 5–7); with the selected viruses VSV-cDNA clone no. 3 (NRAS), VSV-cDNA clone no. 7 (TYRP1), VSV-cDNA clone no. 11 (CYC1) (lanes 9–11); or with VSV-GFP (lane 13); in addition, infections were performed with three-way combinations (3 × 10⁶ pfu of each virus) of ASMEL no. 1 + no. 2 + no. 3 (lane 4); ASMEL no. 4 + no. 5 + no. 6 (lane 8); or VSV-cDNA clones no. 3 + no. 7 + no. 11 (lane 12). Error bars (a–c), s.d. (b) The experiment in a was repeated in the presence of TGF-β RII Fc chimera to inhibit TGF-β in the cultures^,. (c) Pooled lymphoid cell cultures (10⁵/well) from naive C57BL/6 mice were infected every 2 d with the ASMEL or VSV-GFP over a 2-week period, with regular replenishment of the lymphoid cells. Lymphoid cell cultures were either left uninfected (lane 1); or infected with single viruses (10⁶ pfu) VSV-GFP (lane 2); ASMEL parental virus stock (3); VSV-cDNA clone no. 3 (NRAS) (4); VSV-cDNA clone no. 7 (TYRP1) (5), VSV-cDNA clone no. 11 (CYC1) (6); VSV-cDNA clone no. 12 (TRIM33) (7). In addition, lymphoid cell cultures were infected with two-way combinations of viruses: clone no. 3 (NRAS) + clone no. 7 (TYRP1) (8); clone no. 3 (NRAS) + clone no. 11 (CYC1) (9); clone no. 3 (NRAS) + clone no. 12 (TRIM33) (10); clone no. 7 + clone no. 11 (11); clone no. 7 + clone no. 12 (12); clone no. 11 + clone no. 12 (13). Lymphoid cell cultures were also infected with three-way combinations of viruses: clone no. 3 + clone no. 7 + clone no. 11 (14); clone no. 3 + clone no. 7 + clone no. 12 (15); clone no. 3 + clone no. 11 + clone no. 12 (16); clone no. 7 + clone no. 11 + clone no. 12 (17). Finally cultures were also infected with a four-way combination of clone no. 3 + clone no. 7 + clone no. 11 + clone no. 12 (18). Forty-eight hours following the final infection with virus, cultures were stimulated with B16 melanoma cell lysates for three consecutive days. Forty-eight hours later supernatants were assayed for IL-17 by ELISA. (d) Mice bearing 5-d-old established B16 tumors were treated (n = 6–8/group) with intravenous injections of VSV-GFP or VSV-expressing the melanoma-associated antigen hgp100 as described. (e) Mice bearing 5-d-old established B16 tumors were treated (n = 6–8/group) with nine intravenous injections on days 6–8, 13–15 and 20–22 of two-way combinations (total viral dose 10⁷ pfu/injection; 5 × 10⁶ pfu of each individual virus) of either VSV-cDNA clone no. 3 (NRAS) + clone no. 7 (TYRP1); clone no. 3 (NRAS) + clone no. 11 (CYC1); or clone no. 7 + clone no. 11. A fourth group was treated with a three-way combination (total viral dose 10⁷ pfu/injection; 3 × 10⁶ pfu of each individual virus) of VSV-cDNA clone no. 3 (NRAS) + clone no. 7 (TYRP1) + clone no. 11 (CYC1). Survival with time is shown.