Abstract Dysregulated signaling via the epidermal growth factor receptor (EGFR)-family is believed to contribute to the progression of a diverse array of cancers. The most common variant of EGFR is EGFRvIII, which results from a consistent and tumor-specific in-frame deletion of exons 2–7 of the EGFR gene. This deletion generates a novel glycine at the junction and leads to constitutive ligand-independent activity. This junction forms a novel shared tumor neo-antigen with demonstrated immunogenicity in both mice and humans. A 21-amino acid peptide spanning the junctional region was selected, and then one or five copies of this 21-AA neo-peptide were incorporated into live-attenuated Listeria monocytogenes-based vaccine vector. These vaccine candidates demonstrated efficient secretion of the recombinant protein and potent induction of EGFRvIII-specific CD8+ T cells, which prevented growth of an EGFRvIII-expressing squamous cell carcinoma. These data demonstrate the potency of a novel cancer-specific vaccine candidate that can elicit EGFRvIII-specific cellular immunity, for the purpose of targeting EGFRvIII positive cancers that are resistant to conventional therapies.

Citation: Zebertavage L, Bambina S, Shugart J, Alice A, Zens KD, Lauer P, et al. (2019) A microbial-based cancer vaccine for induction of EGFRvIII-specific CD8+ T cells and anti-tumor immunity. PLoS ONE 14(1): e0209153. https://doi.org/10.1371/journal.pone.0209153 Editor: Stephen J. Turner, Monash University, Australia, AUSTRALIA Received: May 19, 2018; Accepted: December 2, 2018; Published: January 2, 2019 Copyright: © 2019 Zebertavage et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper. Funding: This work was supported by funding from Susan G. Komen® CCR12226309 (KSB), NIH R01CA182311 (MJG and MRC) and R21AI126151 (MJG and MRC), the Kuni Foundation (MRC), and the Providence Portland Medical Foundation (KSB). Additional funding was provided by Aduro Biotech in the form of salaries to authors PL and BH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: P. Lauer and B. Hanson have ownership interest (including patents) in Aduro Biotech. Patents relevant to this manuscript held by authors include Patent# US20070207170A1 (Lauer), US9161974B2 (Bahjat), WO2007022511A2 (Bahjat), WO2012068360A1 (Lauer, Bahjat), US10105427B2 (Lauer, Hanson). This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors declare that no competing interests exist.

Introduction The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase critical for cell growth and survival. Overexpression of EGFR is frequently associated with human cancers including breast cancer, non-small cell lung cancer, ovarian cancer and malignant glioma [1–3]. Approximately 40% of glioblastoma multiforme (GBM) patients have tumors that overexpress EGFR [2]. Of these cases, approximately 70% also express a mutant form of the EGFR [4]. The most common of these mutations is EGFR variant III (EGFRvIII), resulting from a deletion of 267 amino acids spanning exons 2–7 of the EGFR gene [5]. This alteration of the ligand-binding domain of EGFR alters the ability of the receptor to bind to its canonical ligands and produces low-level constitutive signaling activity [6]. Unlike wild-type (wt) EGFR, EGFRvIII can form both homodimers as well as heterodimers with wt-EGFR and Her2, thus, EGFRvIII signals may differ from those elicited by wt-EGFR [4]. These atypical signaling pairs could explain why tumors expressing EGFRvIII are unusually resistant to the effects of tyrosine kinase inhibitors [7] and anti-EGFR antibodies such as cetuximab [8]. In addition to promoting proliferation, EGFRvIII expression up-regulates the anti-apoptotic molecule Bcl-x L and has been shown to mediate resistance to chemotherapeutic agents such as paclitaxel and cisplatin [9]. The presence of EGFRvIII also facilitates the STAT3-dependent induction of HIF-1α and promotes cell motility, invasion and metastasis [10]. Furthermore, expression of EGFRvIII by a subset of cells within a solid tumor can promote survival of EGFRvIII-negative cells via the IL-6/LIF/gp130-dependent induction of wt-EGFR [11]. These findings correlate with clinical data demonstrating that EGFRvIII expression in the presence of EGFR amplification is an indicator of a poor survival prognosis in GBM [12], and that EGFRvIII expression independently correlates with poor prognosis in patients with gross-total resection (>95%) surviving ≥1 year [13]. Therefore, EGFRvIII-expressing cells have a selective survival advantage over those expressing only wt-EGFR, and this advantage may become more pronounced after treatment. Previous studies have shown that patients with EGFRvIII-expressing cancers have spontaneously developed humoral and cellular immune responses against EGFRvIII, suggesting that EGFRvIII serves as an immunogenic neo-antigen [14]. A 13 amino acid peptide from the unique splice junction of EGFRvIII (LEEKKGNYVVTDH), referred to as PEPvIII, has been used to vaccinate humans with EGFRvIII-expressing GBM. A phase 2 clinical trial of rindopepimut, a peptide vaccine containing PEPvIII conjugated to the carrier protein keyhole limpet hemocyanin (KLH), administered with adjuvant GM-CSF was performed in newly diagnosed GBM patients treated by gross total resection, radiation and temozolomide who had no radiographic evidence of progression. Humoral immune responses to EGFRvIII were observed in 6 of 14 immunized patients, while 3 of 17 showed a positive delayed type hypersensitivity (DTH) response. The median overall survival for patients treated with vaccine in combination with temozolomide was 26.0 months from the time of histologic diagnosis, versus 15.0 months for a matched cohort receiving only temozolomide [15]. However, a follow-up randomized, double-blind phase 3 trial of 745 patients (405 with minimal residual disease and 338 with significant residual disease, following maximal surgical resection and chemoradiation) treated with rindopepimut and temozolomide found no significant difference between patients receiving the investigational vaccine and patients treated with KLH and temozolomide. Intriguingly, in the small number of post-treatment samples obtained, EGFRvIII was lost equivalently in both the rindopepimut and control-treated patients [16]. Taken together, these results support the utility of EGFRvIII as an immunotherapeutic target, but suggest a vaccine with improved potency relative to PepvIII-KLH may achieve the desired outcome. Intracellular microbes elicit a robust CD8+ T cell response in immunocompetent hosts, a response necessary to kill infected cells and prevent microbial replication. With the goal of eliciting a similar response, live attenuated versions of these intracellular microbes are being explored as vectors for cancer vaccines. Listeria monocytogenes (Lm) is a ubiquitous Gram-positive facultative intracellular bacterium typically found in soil and food that is nonpathogenic to immune competent individuals. A live-attenuated double deleted Listeria (LADD Lm) vaccine platform has been developed and tested in several early-stage clinical trials [17, 18]. The LADD vaccine strain has complete deletions of two virulence genes: actA, required for intracellular motility and cell-to-cell spread and internalin B (inlB), required for direct hepatocyte invasion via the InlB-c-Met interaction [19]. The combination of the two deletions in the LADD platform results in a 1000x attenuation compared to WT Lm and limits liver toxicity by eliminating direct hepatocyte invasion and ActA-mediated cell-to-cell spread into hepatocytes from infected liver-resident Kupffer cells [20]. Importantly, the live-attenuated vaccine vector elicits a potent innate and adaptive immune response. Vaccine-induced inflammation also promotes APC maturation, antigen processing and presentation, and T cell expansion, resulting in a robust antigen-specific CD8+ T cell response [21]. In combination with our ability to construct Listeria that express tumor associated antigens, these inherent immunogenic properties make attenuated Listeria an attractive candidate for microbe-based cancer vaccines. Here, we describe the design of an EGFRvIII-expressing LADD Lm strain and demonstrate its efficacy in a preclinical tumor model. We demonstrate that this vaccine yields orders of magnitude higher EGFRvIII-specific CD8+ T cell responses compared to PepvIII-KLH in vivo, and effectively protects against EGFRvIII-expressing tumor challenge, indicating its potential for translation to treat EGFRvIII-expressing tumors in human trials.

Materials and methods Antibodies, cells and reagents Murine squamous cell carcinoma (SCCVII) cells [22] (generously provided by Walter T. Lee, Duke Cancer Institute, Durham NC) were grown in 10% RPMI-1640 with L-glutamine (ThermoFisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated FBS (Atlas Biologicals, Fort Collins, CO), MEM Eagle Nonessential Amino Acid Solution, Penicillin-Streptomycin, L-glutamine, HEPES Buffer (Lonza, Basel, Switzerland), and Sodium Pyruvate Solution (ThermoFisher Scientific). Antibodies for flow cytometry include anti-mouse CD8α-PerCP-Cy5.5 (clone 5H10, ThermoFisher Scientific), CD4-FITC (clone RM4-5, eBioscience, San Diego, CA), CD154/CD40L-PE (clone MR1, eBioscience), anti-mouse IFNγ-APC (clone XMG1.2, eBioscience) and TNF-PE-Cy7 (clone MP6-XT22, BD Biosciences, Franklin Lanes, NJ). Peptides for restimulation were synthesized by A&A Labs (San Diego, CA). H-2Kk MHC-tetramers incorporating the defined EEKKGNYV peptide (EGFRvIII murine epitope) were obtained from the NIH Core Facility at Emory University, (Atlanta, GA). Animal models Female C57BL/6, BALB/c, SJL and C3H/HeJ mice aged 5–8 weeks were obtained from Jackson Laboratories (Bar Harbor, ME) for use in these experiments. Animal protocols were approved by Providence Health & Services IACUC (Animal Welfare Assurance No. A3913-01). L. monocytogenes construction, growth and vaccination All strains were based on the previously described parental LADD Lm (ΔactAΔinlB) strain [17]. A 21-AA sequence (PASRALEEKKGNYVVTDHGSC) that overlaps the novel junction created by the 267-AA deletion in EGFRvIII was selected as the immunogenic peptide. Two EGFRvIII 20-40 -expressing constructs were designed, one with a single copy of the peptide and one with five copies. EGFRvIII 20-40 was flanked by peptides predicted to facilitate cleavage of the construct by the immunoproteasome. The fusion protein included a C-terminal OVA 257-264 (SIINFEKL) tag. The expression cassette was codon optimized for Lm and cloned downstream of an actA promoter in-frame with the 100 N-terminal amino acids of the actA gene. The expression cassette was cloned into a derivative of the pPL2 integration vector and stably integrated at the tRNAArg locus of the bacterial chromosome of vaccine platform strain as described previously [23]. To assess protein expression, vaccine strains were infected into the mouse dendritic cell line DC2.4 and cell lysates were harvested for western blotting using an antibody raised to the mature amino terminus of ActA as described [24]. To assess in vivo immunogenicity, BHI broth was inoculated with a single colony from a BHI agar plate and grown overnight at 37°C. Stationary phase cultures were split the next morning and allowed to return to midlog phase before dilution and immunization. All doses were confirmed by plating vaccination material. Lm-EGFRvIII or control Lm-Ova [25] were administered to mice at a dose of 1x105-1x107 CFU retro-orbital IV, depending on mouse strain. For comparison, groups of mice were vaccinated by subcutaneous injection of 50μg KLH-PEPvIII (generously provided by Celldex Therapeutics) along with 2μg murine recombinant GM-CSF (R&D Systems, Minneapolis, MN). Peptide stimulation, intracellular cytokine staining and flow cytometry To enumerate EGFRvIII-specific T cells in following treatment, spleens were first dissociated using a 70μm cell strainer (ThermoFisher Scientific) and syringe. Red blood cells were lysed with ACK Lysing Buffer (Lonza) and the resultant splenocytes were washed three times in PBS, counted and diluted to 1x106 viable cells/100μl. These cells were then stimulated with 1μg peptide and 1μl GolgiPlug (Becton Dickinson) for four hours, washed 3X with PBS and surfaced stained with anti-mouse CD8α-PerCP-Cy5.5 and CD4-FITC. Following the protocol for the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with GolgiPlug (Becton Dickinson), cells were then fixed, permeabilized and frozen at -80°C for future analysis. Upon thawing, cells were washed and stained with anti-mouse IFNγ-APC, TNF-PE-Cy7, and CD40L PE. Samples were analyzed using the BD LSRII flow cytometer (Becton Dickinson). T2 peptide binding assay Kk-expressing T2 cells (generously provided by Peter Cresswell, Yale University) are a Tap-deficient cell line that cannot assemble MHCI for presentation on the cell surface unless provided with exogenous MHC-binding peptides. The cells were incubated overnight with the indicated concentrations of peptide. Cells were washed, stained with an anti-Kk antibody (eBiosciences) and acquired using an LSR II flow cytometer. Plasmids and transfection MSCV-XZ066-EGFRvIII was a gift from Alonzo Ross [26] (Addgene plasmid #20737) and pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE was a gift from Hans Clevers [27] (Addgene plasmid #32702). SCCVII cells were transfected using Lipofectamine 2000 Transfection Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer protocol and selected in 2μg/ml puromycin as well as through three cycles of fluorescence-assisted cell sorting (FACS) for high endogenous GFP and RFP expression, respectively, to generate SCCVII-EGFRvIII and SCCVII-control cells, respectively. In vivo tumor assays For tumor protection studies, female C3H mice were vaccinated with 1x105 CFU of Lm-EGFRvIIIx5 or Lm-OVA at day -21 and again at day -7 relative to tumor challenge. At day 0, animals were injected subcutaneously with 2x106 tumor cells (SCCVII-EGFRvIII or SCCVII-Vector) on the hind flank according to group. For dual flank experiments, female C3H mice were given simultaneous challenge of SCCVII-EGFRvIII and SCCVII-Vector on opposing hind flanks. Starting at day 7, tumor progression was monitored on both flanks with calipers to the endpoint of 12mm maximum diameter for either tumor, at which point the animal was euthanized. For long-term protection experiments, female C3H mice were vaccinated with 1x105 CFU of Lm-EGFRvIIIx5 or Lm-Ova at day -21 and again at day -7 relative to tumor challenge. At day 0, mice were injected subcutaneously with SCCVII-EGFRvIII or SCCVII-vector control on the hind flank according to group. For therapeutic studies, at d0 mice were injected subcutaneously with SCCVII-EGFRvIII as above and treated with Lm-EGFRvIIIx5 or vehicle control on d3. Tumor size was monitored from day 7 with calipers to the endpoint of 12mm maximum diameter, at which point the animal was euthanized. Statistics Data were analyzed and graphed using Prism (GraphPad Software, La Jolla, CA). Individual data sets were compared using Student’s T-test and analysis across multiple groups was performed using ANOVA with individual groups assessed using Tukey’s comparison. Overall survival of groups was compared using log rank test for differences in Kaplan-Meier survival curves.

Discussion We demonstrate that potent EGFRvIII-specific CD8+ T cell responses can be elicited using LADD, an attenuated L. monocytogenes strain, as the vaccine vector. Using this approach we were able to define an EGFRvIII-specific CD8+ T cell epitope in C3H mice and demonstrate control of EGFRvIII-expressing tumors in immunocompetent mice. LADD Lm-based vectors are not susceptible to the same vector-specific neutralizing immunity that limits viral vaccine vectors [28] and this represents a novel potent candidate vaccine for patients with EGFRvIII-expressing tumors. Previous reports using EGFRvIII-targeted vaccines have failed to demonstrate an EGFRvIII-specific T cell response in mice that could be measured by ICS or ELISpot [29–31]. When compared with results from previous pre-clinical studies using recombinant protein or peptide-pulsed dendritic cells, we observe a significantly more potent in vivo T cell response to our EGFRvIII-expressing vaccine. In addition to using a potent, live microbial vaccine vector, we identified a novel Kk-restricted epitope, EEKKGNYV, within the EGFRvIII 20-40 immunogen. Using this defined class I-restricted peptide, we were able to determine the magnitude and quality of the EGFRvIII-specific CD8+ T cell response to Lm-EGFRvIII and the previously described PepvIII-KLH conjugate. In addition, we were able to test the efficacy of the vaccine candidate using a squamous cell carcinoma cell line, engineered to express full-length EGFRvIII, which was syngeneic to the C3H mouse strain and therefore able to present the EEKKGNYV peptide on H2k. This direct presentation was not possible on prior tumor models that were tested in mice expressing MHC class I H2d haplotypes [29] or in human cells in immunosuppressed animals [30] and in these models tumor control was associated with antibody rather than T cell responses [31]. Targeting T cell responses to EGFRvIII rather than antibody responses is particularly relevant in view the failure of the phase III PepvIII-KLH conjugate trial [16] and the promising results of a recent phase 1 trial infusing EGFRvIII-specific chimeric antigen receptor (CAR) T cells into recurrent glioblastoma patients. In this latter study, autologous T cells were engineered to express an EGFRvIII-binding CAR signaling through CD3ζ and the 4-1BB costimulation domain and then infused back into patients. These T cells were observed to effectively traffic to tumors and significantly decreased EGFRvIII expression [32]. In patients, the PepvIII-KLH conjugate did generate a detectable antibody response to EGFRvIII, and the clinical data suggested that antibody responses correlated with clinical activity [16]. We demonstrate that PEPvIII does generate an antigen-specific T cell response to the neoantigen within EGFRvIII in C3H mice, and this vaccine had been shown to generate CD8 T cell-mediated tumor control in murine models [31]. While one of the strengths of the Listeria platform is that repeated vaccination is not limited by neutralizing antibody responses, antibody responses may be elicited following Listeria infection and contribute to T cell immunity [33]. Further studies are necessary to determine whether CAR T cell transfer or endogenous vaccination yields superior results in clinical settings, and whether antibodies contribute to tumor control by Lm-EGFRvIII. EGFRvIII-expressing cancer cells frequently represent a subclonal population of the tumor, and as discussed above, in the clinical studies tumor expression of EGFRvIII was lost equivalently in both the rindopepimut and control-treated patients [16]. While this suggests that the cancer cells have a potential path to immune escape, the EGFRvIII+ cancer cells are more resistant to chemotherapeutic agents such as paclitaxel and cisplatin [9] and a subclonal population of EGFRvIII+ cells can promote survival of EGFR wt neighbors [11]. Therefore, while successful clearance of EGFRvIII+ cancer cells would not be expected to affect EGFR wt neighbors, it has the potential to render the tumor significantly more susceptible to conventional therapies as part of combination treatments. Moreover, a strong tumor-specific T cell response has the potential to dramatically change the inflammatory environment of the tumor, and support epitope spreading to common mutations shared between EGFRvIII+ and EGFR wt neighbors. Thus the selectivity and specificity of the EGFRvIII epitope has the potential to immunoedit the tumor to a more treatable state, and provide a focus for further immune control of residual disease. Given our experience constructing and producing L. monocytogenes-based vaccines, data with this vector in previous phase I clinical trials, and the demonstrated immunogenicity of EGFRvIII 20-40 -expressing LADD Lm, we believe Lm-EGFRvIII to be an ideal candidate for testing in patients with EGFRvIII-expressing cancers. The studies will include validation of the EGFRvIII neo-eptiope as a target for CD8 T cells in patients. As such, a phase 1 study testing ADU-623, a Listeria vaccine of similar construction expressing EGFRvIII-NY-ESO-1, is currently ongoing in patients with recurrent grade 3/4 glioblastomas (ClinicalTrials.gov identifier: NCT01967758).

Acknowledgments This work was supported by funding from Susan G. Komen CCR12226309 (KSB), NIH R01CA182311 (MJG and MRC) and R21AI126151 (MRC and MJG), the Kuni Foundation (MRC), and the Providence Portland Medical Foundation (KSB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the NIH Tetramer Core Facility (contract HHSN272201300006C) for provision of Kk-EEKKGNYV tetramers.