Vaccination with ENO1-DNA Prolongs Survival of Genetically Engineered Mice with Pancreatic Cancer

Gastroenterology. 2013 May;144(5):1098-106.

Cappello P and F. Novelli.

 

Abstract

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is an aggressive tumor, and patients typically present with late-stage disease; rates of 5-year survival after pancreaticoduodenectomy are low. Antibodies against a-enolase (ENO1), a glycolytic enzyme, are detected in more than 60% of patients with PDA, and ENO1-specific T cells inhibit the growth of human pancreatic xenograft tumors in mice. We investigated whether an ENO1 DNA vaccine elicits anti-tumor immune responses and prolongs survival of mice that spontaneously develop autochthonous, lethal pancreatic carcinomas.

METHODS: We injected and electroporated a plasmid encoding ENO1 (or a control plasmid) into KrasG12D/Cre mice (KC) and KrasG12D/Trp53R172H/Cre (KPC) mice when they were 4 weeks old (when pancreatic intraepithelial lesions are histologically evident). Anti-tumor humoral and cellular responses were analyzed by histology, immunohistochemistry, ELISAs, flow cytometry, and ELISpot and cytotoxicity assays. Survival was analyzed by Kaplan-Meier analysis.

RESULTS: The ENO1 vaccine induced antibody and a cellular responses and increased survival times by a median 138 days in KC mice and 42 days in KPC mice, compared with mice given the control vector. In histologic analysis, the vaccine appeared to slow tumor progression. The vaccinated mice had increased serum levels of anti-ENO1 immunoglobulin G, which bound the surface of carcinoma cells and induced complement-dependent cytotoxicity. ENO1 vaccination reduced numbers of myeloid-derived suppressor cells and T-regulatory cells, and increased T-helper 1 and 17 responses.

CONCLUSIONS: In a genetic model of pancreatic carcinoma, vaccination with ENO1 DNA elicits humoral and cellular immune responses against tumors, delays tumor progression, and significantly extends survival. This vaccination strategy might be developed as a neo-adjuvant therapy for patients with PDA.

 

Supplementary

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease and remains one of the most resistant cancers to traditional therapies. Historically, chemotherapy and radiotherapy has provided any significant benefit for survival in PDA patients and remain palliative cures. This holds true even with the introduction of molecularly-targeted agents, chosen on the basis of their involvement in pathways considered to be important in PDA development and progression 1. Recently, FOLFIRINOX (5-flourouracil, leucovorin, oxaliplatin and irinotecan) treatment has provided a limited survival advantage in advanced PDA2. Effective therapeutic strategies are therefore urgently needed to improve the PDA survival rate.

Immunotherapy has been considered to have potential value in many solid tumors but this promise has to be yet realized in pancreatic cancer. The few PDA-associated antigens which have been used to develop vaccines, namely CEA, Kras, MUC1 and gastrin, have already been tested in clinical trials and have been demonstrated to have little impact on survival, although some more recent vaccine trials seem to be promising. Similar results were obtained with single agent therapy blocking immune checkpoint molecules as cytotoxic T lymphocyte agent (CTLA)-4 and programmed cell death ligand 1 (PD-L1).

In this paper authors exploit a genetically engineered mouse model, spontaneously developing PDA due to the pancreatic-specific expression of mutated Kras alone (hereafter called KC) or in combination with mutated p53 (hereafter called KPC), to validate an immunotherapeutic strategy based on DNA vaccination. DNA plasmids can easily and safely deliver genes coding for tumor-associated antigens, to which potent immune responses can be induced. DNA vaccines have great advantages: they are stable, do not contain viral proteins (avoiding the induction of anti-viral response after repeated boosts) and have widespread application compared to other vaccines. In this study authors injected DNA plasmid into femoral muscle and applied two shots of electroporation (Figure 1) in order to enhance the plasmid uptake from muscle cells and resident antigen presenting cells. Electroporation, indeed, determines a homogenous long-lasting expression of injected DNA.

Franco Novelli-figure1

Crucial is the choice of target delivered by DNA plasmid. By Serological Proteome Analysis (SERPA) authors have identified a dozen antigens expressed by PDA, and specifically recognized by autoantibodies present in the sera of PDA patients. One of these antigens, α-enolase (ENO1), is recognized by over 60% of PDA patients. ENO1 is coded by the ENO1 gene, and is overexpressed in the cytoplasm of PDA cells as well as being membrane-expressed. When on cell surface ENO1 acts as a plasminogen receptor thus playing an important role in plasmin degradation, extracellular matrix disruption and cell invasion 3. Immune cells, bacteria and tumor cells use this system to invade tissues.

In this study, authors demonstrate that the ENO1 DNA vaccine significantly induced a specific immune response that prolonged survival of both PDA mouse models: from 336 to 474 days of age for KC mice, the longest overall survival ever reported, and from 203 to 245 days for KPC mice. ENO1 DNA vaccine elicited several protective immunological mechanisms. Firstly, high levels of anti-ENO1 IgG, which bound cell surface of PDA cells and mediated their killing through complement-dependent cytotoxicity (Figure 2). Second, an activation of specific Th1 and Th17 cells, and a large recruitment of CD3 cells into the tumor area. Th1/Th17 cytokines favor the switching to effector subclasses of anti-ENO-1 antibodies (Figure 2). Cytokines produced by Th1/Th17 cells such as IFNg and TNFa can also directly affect tumor cells by inducing cell death or senescence. An additional effect of the ENO1 DNA vaccine is the decrease in cells with suppressive activity typical of the tumor microenvironment, namely myeloid derived suppressive cells and T regulatory cells (Figure 2). Most importantly, the therapeutic efficacy of the ENO1 DNA vaccine was observed to be very promising when the administration protocol started at 8-9 months of age, a setting close to clinical condition.

Franco Novelli-2

Present data, therefore, indicate that it may be possible to design adjuvant therapies to elicit anti-ENO1 responses in resected patients to prevent recurrences, or to prolong survival of untreatable patients. The right drug combination could transform these encouraging preclinical results into an effective clinical protocol. A lot of evidence has shown that several anticancer agents, including classic chemotherapeutics and targeted compounds, stimulate tumor-specific immune responses either by inducing immunogenic cell death or by engaging immune effector mechanisms.

 

Reference:

1.         Vaccaro V, Gelibter A, Bria E, et al. Molecular and genetic bases of pancreatic cancer. Curr Drug Targets 2012;13:731-43.

2.         Conroy T, Desseigne F, Ychou M, et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med 2011;364:1817-25.

3.         Capello M, Ferri-Borgogno S, Cappello P, et al. alpha-enolase: a promising therapeutic and diagnostic tumor target. Febs J;278:1064-74.

 

Acknowlwdgments:  

This work was supported in part by grants from: the European Community, Seventh Framework Program European Pancreatic Cancer-Tumor-Microenvironment Network (EPC-TM-Net, no. 256974); Associazione Italiana Ricerca sul Cancro (AIRC) 5 x 1000 (no. 12182) and IG (no. 5548 and 11643); Ministero della Salute: Progetto Integrato Oncologia; Regione Piemonte: Ricerca Industriale e Sviluppo Precompetitivo (BIOPRO and ONCOPROT), Ricerca Industriale “Converging Technologies” (BIOTHER), Progetti strategici su tematiche di interesse regionale o sovra regionale (IMMONC), Ricerca Sanitaria Finalizzata, Ricerca Sanitaria Applicata; Ministero dell’Istruzione e della Ricerca (MIUR), Progetti di Rilevante Interesse Nazionale (PRIN 2009); University of Turin-Progetti di Ateneo 2011: Mechanisms of REsistance to anti-angiogenesis regimens THErapy (grant Rethe-ORTO11RKTW). P.C. was supported by a fellowship from Fondazione Italiana Ricerca sul Cancro (FIRC).

Contact:

Francesco Novelli, Center Research Medical Studies, Azienda Ospedaliera Città della salute e della Scienza di Torino, University of Turin, Via Cherasco 15 10125-Torino, Italy. Email: franco.novelli@unito.it

 

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