Pre-immature dendritic cells (PIDC) pulsed with HPV16 E6 or E7 peptide are capable of eliciting specific immune response in patients with advanced cervical cancer

Cervical cancer cell lines used in this trial were either from the ATCC (Mannassas, VA) including the ME180, Caski and MS751 or established at the NCI (Bethesda, MD) like the Cav cervical cancer cell line. All cells were tested in our lab for HLA and HPV typing. Caski, MS751 and Cav are all HLA-A2 positive cell line while ME180 is an A2 negative cell line. Caski harbors the HPV16 genome while MS751 and the Cav harbor the HPV18 genome (low and high number of copies respectively). All cells grew in RPMI supplemented with 10% FCS, Penicillin, L-Glutamine and Sodium Pyruvate.

The cervical cancer cell lines mentioned above were used as targets in the cytotoxic T lymphocyte (CTL) assay. The cytolytic activity was measured by using the 4-hour 51Cr release assay. Lymphocytes were prepared from PBMC. These cells were selected and expanded in vitro in the presence of 1–20 μM of E6 peptide with 5 IU/ml of IL-2 (Chiron, Emeryville, CA) added on day 3 for 7 days. This 7 day cycle was repeated one more time, after which the cells were harvested and tested for specific cytolytic activity against E6 or E7 peptide pulsed autologous APC’s (EBV transformed B cells) or tumor cell lines harboring the HPV genome. Target cells were washed with RPMI medium and labeled with 200 μCi ⁵¹Cr sodium dichromate in the presence or absence of 10 μM of E6 or E7 peptide for 2 hours, after which target cells were washed and plated in 96 well round bottomed plates. Effectors were added to labeled targets at the desired effector to target ratio. The plates were centrifuged at 500 rpm for 5 min., and incubated at 37°C for 4 hours. Supernatants were harvested using a Skatron supernatant harvesting device, and the samples counted on a gamma counter. The percent specific lysis was determined, on triplicate samples, by the following formula:

A result was considered positive with a two fold increase or more in lytic units pre-immunization. If there was no detectable pre-immunization specific lysis, a post immunization lysis of greater than 10% above the non-peptide was considered positive, with an Effector:Target (E:T) ratio specified at 50:1. If an E:T ratio of 50:1 was not tested, the next highest ratio was used. In some patients we established CTL and tumor cell lines for future use in studies of antigen specificity.

ELISPOT assays were performed at the Laboratory of Cell-Mediated Immunity, SAIC-Frederick (CLIA-certified lab). Two frozen normal donor controls with known responsive values were run with each assay to assure quality control of the assay results. For all assays, at least one of the two controls was within 2 standard deviations of the laboratory-generated means for CMV and CEF. All assays were performed on 7–8 day in vitro stimulated PBMCs (100 K/well) as the effectors and peptide-pulsed autologous PBMCs (100 K/well) as the antigen presenting cells (APCs). When possible, PBMCs from the earliest time point were used as the APCs. However, if this was not possible, the pulsed PBMCs were assayed alone to make sure they were not producing any spots. Briefly, the day before assay setup, 96-well polyvinylidene fluoride (PVDF) membrane, HTS opaque plates (Millipore, Billerica, Massachusetts, MSIPS4W10) were coated overnight with a 1:100 dilution of anti-human IFN-γ capture antibody (1 mg/mL, Mabtech Inc., Mariemont, OH, Cat# 3420-3-1000) in Dulbecco’s phosphate buffered saline (DPBS) at room temperature. Antibody-coated plates were washed four times in DPBS the next day and blocked with 5% human AB ELISPOT medium at 37°C for approximately 2 hours. 1 × 105in vitro stimulated PBMCs and 1 × 105 autologous, peptide-pulsed PBMCs were plated per well. The plates were incubated for 18–20 hours at 37°C. The next day, the plates were manually washed six times with 0.05% Tween 20 in DPBS, followed by a 2-hour incubation at room temperature with a 1:2000 dilution of the biotinylated secondary antibody, anti-human IFN-γ (1 mg/mL Mabtech Inc., Mariemont, OH, Cat# 3420-6-1000) in DPBS/1% bovine serum albumin/0.05% Tween. After incubation and four washes in DPBS to remove excess antibody, a 1:3000 dilution of streptavidin alkaline phosphatase (Mabtech, Mariemont, OH, Cat# 3310–10) in DPBS/1% bovine serum albumin, was added to each well for 1 hour at room temperature followed by 4 manual washes in DPBS. Finally, the BCIP/NPT substrate, 100 μl/well, (KPL, Gaithersburg, Maryland, Cat# 50-81-08) was added for 7–10 minutes, resulting in the development of spots. The reaction was stopped by washing three times in distilled water. Plates were dried overnight and the spots were visualized and counted using the ImmunoSpot Imaging Analyzer system (Cellular Technology Ltd., Cleveland, OH). ELISPOT results were expressed as the “number of spots per 10⁶ responder cells” after subtracting background spots obtained in wells of effectors with non-pulsed PBMCs. For each subject, PBMCs obtained before and after vaccination were analyzed in the same assay to avoid inter-assay variability. A post-immunization fold increase >2 compared to pre-immunization was considered a positive response.

FACS analysis was done on the PIDCs before vaccine administration to document their phenotype. The data was further confirmed using PBMCs from healthy donors. Prior to the GM-CSF incubation; the PIDCs were all monocytes expressing CD14 and they did not express any of the antigen presenting cell surface markers. After two days in culture in the presence of GM-CSF, and pulsing for two hours with the HPV16 E6 or E7 peptide, in spite of the persistent expression of CD14 at slightly lower levels (Figure 1A), CD14 positive cells (both from PIDCs and PBMCs) were found to express antigen presenting cell associated surface markers (CD80, CD86, CD1a and Mannose receptor (CD206)) (Figure 1B-E). Furthermore, there was up regulation in HLA-DR (Figure 1F).

To test whether E6 protein is processed and presented in the proper context of MHC class I and to test the ability of T-cells generated by HPV16 E6 to cross react with an internally processed HPV18 E6, we tested the ability of post vaccination PBMCs to lyse established HLA-A2 positive cervical cancer cell lines harboring either the HPV16 or HPV18 genome. We found that these primed T-cells were able to lyse Caski cells (HLA-A2 and HPV16 positive), Cav cell line (HLA-A2 with a high number of copies of HPV18) and, to a lesser extent, the MS751 cell line (HLA-A2 harboring lower number of copies of HPV18) but not the HLA-A2 and HPV negative cervical cancer cells (ME180). We were able to see specific lysis in those cell lines and the lysis was higher in cell lines that carry higher number of copies of HPV (Figure 2). Similarly, the effector cells generated through the E7 vaccine were tested against tumor cells either expressing the HPV16 E7 genome or HPV16 E7 negative cell line. We were able to detect lysis of the E7 positive cell lines and not the E7 negative ones. Furthermore, effector cells were able to lyse only the HLA-A2 positive cell lines and not the HLA-A2 negative ones (data not shown).

Immunological responses were evaluated in 28 out of 32 treated patients (Tables 1 and 2). Three patients were excluded from the analysis since they received only one vaccine due to disease progression (2A, 9A, 6B), and one patient (14B) was not tested for an immune response due to lack of sufficient cell numbers to reproduce the results. Seventeen out of the 28 (61%) evaluated patients had positive immune responses by either ELISPOT, 51Cr release assay, or both [10/16 (63%) in arm A, and 7/12 (58%) in arm B]. The immune response data for both ELISPOT and 51Cr release assay are presented in Additional file 1: Figures S1 and Additional file 2: Figure S2.