Immunoselected Tumor Antigens Predicts the Outcome of Gene Therapy with IL-12-Transduced Tumor Cell Vaccine
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Gene Therapy (1999) 6, 865–872 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt Cytotoxic T lymphocyte response against non- immunoselected tumor antigens predicts the outcome of gene therapy with IL-12-transduced tumor cell vaccine M Rodolfo, C Zilocchi, B Cappetti, G Parmiani, C Melani and MP Colombo Experimental Oncology D, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy The colon adenocarcinoma C26, carrying two endogenous related tumor antigens, whereas nonresponders had CTL tumor-associated antigens (TAA) recognized by CTL, has that recognized preferentially the FR␣ antigen. CD8 from been transduced with the gene coding for the human folate responder mice were characterized to release high levels receptor ␣ (FR␣) as an additional antigen in order to study of granulocyte–macrophage (GM)-CSF upon antigen the efficacy of vaccination against a tumor expressing mul- stimulation. Tumors obtained from mice that died despite tiple antigens. A dicistronic vector was used to transduce vaccination lost expression of the FR␣ transgene but main- the IL-12 genes to create C26/IL-12/FR␣ that has been tained expression of endogenous C26 antigens. Immuno- used as a cellular vaccine to treat mice bearing lung met- selection against FR␣ antigen was not observed in tumors astases of C26/FR␣. After vaccination mice were partially from non-vaccinated controls and from CD8-depleted vac- splenectomized and splenic lymphocytes frozen and used cinated mice. Down-regulation of FR␣ antigen expression retrospectively to study in vitro CD8 T cell response related was due, at least in part, to methylation of retroviral vector to the treatment outcome. Vaccination cured 50% of mice long terminal repeat promoter since FR␣ expression was and the effect was CD8 T cell dependent. Mice either cured partially restored, ex vivo, by treatment with 5-aza-2Ј- (responders) or not cured (nonresponders) by vaccination deoxy-cytidine (aza). These results indicate that CD8 T developed tumor-specific CTL. However, analysis of CTL cell-mediated immunoselection and production of GM-CSF specificity and pCTL frequencies revealed that responders are determining factors for the efficacy of tumor vaccines. had a predominant CTL activity against endogenous C26- Keywords: cytokine gene transfer; tumor immunity; CTL Introduction against immunodominant antigens, such as melanoma- associated MART-1/MelanA or Pmel17/gp100, can Active tumor immunotherapy by vaccination with mediate in vivo destruction of antigen-positive tumor irradiated neoplastic cells is expected to activate an cells but also select for antigen loss variants. It has been immune response against the entire repertoire of tumor- reported that during tumor progression, antigenic vari- associated antigens (TAA), a few of which have been ants can appear which may loose not only the HLA class 1,2 molecularly defined. Data have emerged that encour- I molecules, but also T cell epitopes.7–9 age the use of cellular-based vaccines for melanoma In this study we have analyzed the CD8 T cell response 3,4 patients. Active immunotherapy can be further associated with the therapeutic activity following IL-12- improved by genetic modification of tumor cells through transduced tumor cell vaccination. Considering that 5 insertion and expression of cytokine genes. In fact, pre- immunization may direct the immune response against clinical studies have shown that the injection of such an antigen that is therapeutically irrelevant or lost engineered tumor cells induces a local inflammatory because of immune selection, we have set up a model reaction leading to the activation of a systemic antitumor system in which vaccination therapy is directed to a memory response and, in some cases, to the cure of estab- tumor carrying more than one known antigen. This 6 lished tumors. enables testing which antigen is the relevant target of A major issue in the use of cell-based vaccines is the host-induced immune response but also the relationship role of the different antigens or subsets of antigens between immunodominance and immunoselection. We expressed by tumor cells in eliciting a T cell response used the C26 colon carcinoma which expresses as immu- determining tumor regression. In fact, CTL responses nodominant TAA the Ld restricted peptide gp70 (423– 431), called AH1, encoded by env gene of the endogenous ecotropic murine leukemia virus (MuLV) emv-1.10 In Correspondence: M Rodolfo, Experimental Oncology D, Istituto Nazionale addition, C26 express at least one additional, but as yet Tumori, via Venezian, 1-20133 Milan, Italy molecularly undefined, subdominant tumor rejection Received 12 August 1998; accepted 25 November 1998 antigen recognized by a CTL clone,11 TA9A (tumor anti- CD8 reactivity and clinical response to tumor vaccines M Rodolfo et al 866 gen 9A). The antigenic repertoire of C26 cells was further increased by transducing the human gene coding for the folate receptor ␣ (FR␣), a gp38 binding protein which is overexpressed on human carcinomas especially of the ovary,12,13 which encodes for at least two different pep- tides recognized by CTL in the context of either Kd or DdLd MHC class I of BALB/c mice (our unpublished data). IL-12 was chosen because of its potent antitumor effects.14 We have previously reported that vaccination with irradiated C26 cells engineered to produce IL-12 were more active than C26 cells transduced with IL-2 in curing mice bearing C51, another colon carcinoma shar- ing the emv-1-derived TAA.15 We have now examined whether the antigen specificity and cytokine profile of CTL generated by IL-12 vaccine can be associated with tumor eradication or with progression of antigen loss variants. Results Active immunotherapy with C26/IL-12/FR␣ tumor cell is dependent on CD8+ T lymphocytes C26 cells engineered to produce IL-12 and to express the human FR␣ (C26/IL-12/FR␣) were used to treat mice bearing lung metastases of C26/FR␣. Cell vaccination, given s.c. biweekly for 2 weeks, cured 13 of 26 mice while all 29 untreated counterparts died of lung metastases within 50 days (Figure 1a, P Ͻ 0.0001 by log-rank test). IL-12 production determined the efficacy of the vaccine, since treatment with C26/FR␣ cells (not transduced with IL-12 genes) did not change mice survival significantly (Figure 1b). The therapeutic effect was dependent on host CD8 T cells. In fact, no cures or increase of survival time were observed in CD8-depleted, vaccinated mice (Figure 1c). In contrast, the effectiveness of C26/IL-12/FR␣ cell vaccination was not changed by depletion of CD4 lym- phocytes: in fact five of 13 vaccinated mice were cured while 13 of 13 controls died of metastatic disease (Figure 1d, P Ͻ 0.001). We then tested whether partial splenectomy performed at the end of vaccination therapy affected the treatment outcome. Figure 1e shows survival curves of controls and vaccinated mice that underwent hemisplenectomy 3 days after the last vaccine injection (day 16). Partial spleen Figure 1 Survival of mice bearing C26/FR␣ lung metastases after removal by surgery did not change the rate of survivors vaccination with C26/IL-12/FR␣ (a), or with C26/FR␣ cells (b), after CD8 (five of 10 cured mice, P Ͻ 0.01) or the survival time from (c) or CD4 (d) depletion, and after partial splenectomy (e). Mice injected that of non-splectomized mice reported in Figure 1a. This i.v. with 5 × 103 C26/FR␣ cells (day 0) were vaccinated with 3 × 106 150 surgical procedure enabled us to collect individual lym- Gy irradiated tumor cells injected s.c. on days 3, 6, 9 and 13. Treatment phocyte samples after vaccination and to analyze the with anti-CD4 and anti-CD8 mAbs were initiated on day 1 and repeated antitumor response in mice identified as responders or every 2 weeks. Partial splenectomy was performed on day 16. nonresponders on the basis of the therapeutic outcome. directed against FR␣-transduced BALB/c, HTG and 5R CTL from cured mice (responders) display a preferential fibroblast lines (indicating that FR␣ codes for antigenic recognition of C26-related TAA/s on C26/FR␣ cells than peptides associated with either H-2Dd or Kd, CTL from nonresponder mice respectively), while nontransduced HTG and 5R were not Lymphocytes from mice vaccinated with C26/IL-12/FR␣ lysed (Figure 2, upper panel). CTL activity was MHC were tested for CTL activity against a panel of target cells restricted as inhibited by mAbs to either CD3 or CD8 expressing different TAAs of the C26/FR␣ tumor. (data not shown). Untreated tumor-bearing controls did Cytolysis was directed against C26 and syngeneic F1- not show significant CTL activity. transformed fibroblasts pulsed with the AH1 peptide, the Splenocytes from mice cured (responders) or not cured immunodominant CTL antigen eluted from the C26 vari- (nonresponders) by C26/IL-12/FR␣ cell vaccination were ant CT26,10 but not against F1 cells not pulsed or pulsed then separately tested for CTL activity against the same with an irrelevant peptide (not shown). CTL were also panel of target cells. Although lymphocytes from CD8 reactivity and clinical response to tumor vaccines M Rodolfo et al 867 Figure 2 In vitro lytic activity of lymphocytes from mice treated with C26/IL-12/FR␣ vaccines and from untreated controls (upper panel), and from mice cured (responders) or not cured (nonresponders) (lower panel) against a panel of target cells expressing the different tumor antigens. Splenocytes obtained from four to six mice in each group were pooled and tested after 6 days MTLC with irradiated C26/FR␣ cells. responders and nonresponders lysed C26/FR␣ tumor anti-C26/FR␣ CTLp frequencies were measured (data not cells to the same extent, responders’ lymphocytes dis- shown).