Background
Lung cancer is the leading cause of cancer death in the world [
1]. Despite the recent development of novel treatment modalities for patients with non-small cell lung cancer (NSCLC), survival rates are still unsatisfactory [
2]. Furthermore, although molecular-targeted drugs are expected to cause fewer serious adverse events associated with the use of cytotoxic chemotherapeutic agents, but still cause some [
3,
4]. Therefore, the development of more effective and less toxic therapeutic modalities is eagerly awaited. In this regard, cancer immunotherapy is considered to be a promising option with minimum toxicity, but its effectiveness has not yet been proven to be superior to the presently available treatments. However, several ongoing clinical trials that are administering vaccines, such as MAGE-A3 or BLP25 for lung cancer as an adjuvant treatment or in a maintenance setting after standard chemotherapy, seem to be very promising [
5,
6]. Although these lung cancer trials have involved the administration of a single vaccine, a combination of multiple peptide vaccines has also been used in several types of solid cancer [
7,
8].
We have previously identified novel cancer-testis antigens, including up-regulated lung cancer 10 (URLC10; also called lymphocyte antigen 6 complex locus K [LY6K]) [
9], TTK protein kinase (TTK) [
10] and the cell division cycle associated gene 1 (CDCA1) [
11], that were found to be expressed at very high levels in lung cancer using the genome-wide cDNA microarray method. We have also previously reported peptide vaccines that target VEGFR1 [
12] and VEGFR2 [
13]. To induce a higher level of cytotoxic T lymphocytes (CTLs), also known as cytotoxic T cells, that have direct cancer cell killing activity or block the blood supply to cancer cells, we attempted to combine the peptides derived from cancer-testis antigens, as well as those designed to induce an anti-angiogenic effect to achieve an effective response in patients with advanced NSCLC. In the current study we report on the safety of combination therapy involving multiple peptides and a possible improvement in patient prognosis.
Methods
Study design
We performed two phase I clinical trials using two different combinations of peptide vaccines. In the first trial, we administered peptides derived from URLC10, TTK, VEGFR1 and VEGFR2, and in the second trial we administered peptides derived from URLC10, CDCA1, VEGFR1 and VEGFR2. All peptides were restricted to HLA-A*2402. Fifteen HLA-24-positive patients with NSCLC who failed to respond to the standard therapy were enrolled in the three patient/dose/cohort phase I trial involving 0.5, 1 or 3 mg/body for each peptide (for trial 1), or 1 or 3 mg/body for each peptide (for trial 2). The clinical characteristics and treatment information for all patients enrolled in the study are summarized in Table
1. Vaccines were administered weekly and the sites of vaccination were rotated weekly. Administration was by subcutaneous injection into the patient’s axillary region after mixing with incomplete Freund’s adjuvant (IFA) Montanide ISA 51, SEPPIC until progression of the disease was observed, or until the patient declined the continuation of the vaccine treatment. Immunological responses were evaluated by means of INF-gamma ELISPOT assays. Every measurable lesion was evaluated using response evaluation criteria in solid tumors (RECIST) 1.0, and the toxicities caused by the vaccination therapy were assessed using Common Terminology Criteria for Adverse Events (CTCAE) version 3. These studies were approved by the ethical committee of Fukushima Medical University (trial 1 approval number: 554; trial 2 approval number: 810) and were registered with ClinicalTrials.gov (trial 1:
NCT00633724; trial 2:
NCT00874588). Written informed consent was obtained from all individuals. The trials were carried out in accordance with the Helsinki declaration on experimentation on human subjects.
Table 1
Patient clinical characteristics
1 | 54/M | Recurrence | AD | LN, bone | 2 | L, T, R1, R2 | 0.5 | 5th (PLT, RT) |
2 | 48/M | IIIB | AD | PM, effusion | 2 | L, T, R1, R2 | 0.5 | 5th (PLT) |
3 | 65/M | Recurrence | AD | PM | 2 | L, T, R1, R2 | 0.5 | 6th (PLT, EGFR-TKI) |
4 | 58/M | IV | AD | Primary, bone | 2 | L, T, R1, R2 | 1 | 4th (PLT) |
5 | 60/M | IV | AD | Primary, LN | 1 | L, T, R1, R2 | 1 | 3rd (PLT) |
6 | 47/M | IV | AD | Primary, LN, ADR | 0 | L, T, R1, R2 | 1 | 3rd(PLT, RT) |
7 | 40/M | IIIA | AD | Primary, LN | 1 | L, T, R1, R2 | 3 | 3rd(PLT) |
8 | 69/M | Recurrence | SQ | PM | 1 | L, T, R1, R2 | 3 | 3rd(PLT, RT) |
9 | 65/M | Recurrence | AD | Dissemination | 0 | L, T, R1, R2 | 3 | 2nd(PLT, RT) |
10 | 57/M | Recurrence | PLEO | LN | 1 | L, C, R1, R2 | 1 | 3rd(PLT, RT) |
11 | 55/F | IIIB | AD | Primary, LN, effusion | 2 | L, C, R1, R2 | 1 | 5th(PLT, EGFR-TKI) |
12 | 62/M | Recurrence | AD | PM | 1 | L, C, R1, R2 | 1 | 2nd(PLT) |
13 | 68/F | IV | AD | Primary, bone, effusion | 2 | L, C, R1, R2 | 3 | 2nd(PLT) |
14 | 39/F | IV | NSCLC | Primary, liver, bone | 2 | L, C, R1, R2 | 3 | 2nd(PLT, RT) |
15 | 61/M | Recurrence | AD | PM, LN | 1 | L, C, R1, R2 | 3 | 5th(PLT, RT, EGFR-TKI) |
Patient eligibility
Patients with an advanced or a recurrent non-small cell lung cancer who failed to respond to the standard therapy were enrolled in these two trials. Eligibility criteria were as follows: (1) patients who had an HLA-A*2402 allele evaluated using DNA genotyping; (2) adequate bone-marrow, cardiac, pulmonary, hepatic and renal functions including a white blood cell count of 1500-15000/mm3, a platelet count of >75 000/mm3, total bilirubin of < three times that of the institutional normal upper limit, levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase of < three times that of the institutional normal upper limits, and levels of creatinine of < two times the institutional normal upper limit; (3) no other therapy for lung cancer within 4 weeks prior to the initiation of the trial; (4) an ECOG performance status of 0–2; and (f) an age of ≥20 years. The exclusion criteria for patients participating in the two clinical trials were as follows: (1) pregnancy (including women of childbearing potential); (2) breast feeding; (3) bleeding disorder; (4) infections requiring antibiotics treatment; (5) concomitant treatment with steroid or immunosuppressant; and (6) decision of unsuitableness by principal investigator or physician-in-charge.
Peptides
The amino acid sequences of the peptides used were RYCNLEGPPI (URLC19-177), VYGIRLEHF (CDCA1-56), SYRNEIAYL (TTK-567), TLFWLLLTL (VEGFR1-770) and RFVPDGNRI (VEGFR2-169); these were expected to bind to an HLA-A24 molecule. These peptides were synthesized as GMP grade as described elsewhere [
10‐
13]. The purity (>97%) and identity of the peptides were determined using analytical high-performance liquid chromatography and mass spectrometry analysis, respectively. Peptides were dissolved in dimethyl-sulfoxide at the concentration of 20 mg/ml and stored at −80°C.
Enzyme-linked immunospot (ELISPOT) assay
Specific CTL response was measured using an ELISPOT assay following
in vitro sensitization. Frozen peripheral blood mononuclear cells (PBMCs) isolated from each patient were thawed, and the viability was confirmed to be more than 90%. 500,000 PBMC cells from each patient were cultured with 10 mg/ml of respective peptide and 100 IU/ml of IL-2 (Novartis, Emeryville, CA, USA) at 37°C for two weeks (each peptide was added to the culture medium on days 0 and 7). After CD4
+ cell depletion using a Dynal CD4-positive isolation kit (Invitrogen, Carlsbad, CA, USA), the IFN-γ ELISPOT assay was performed using a Human IFN-γ ELISpot PLUS kit (MabTech, Nacka Strand, Sweden) according to the manufacturers’ instructions. Briefly, HLA-A*2402-positive B-lymphoblast TISI cells (IHWG Cell and Gene Bank, Seattle, WA, USA) were incubated with 20 mg/ml of each peptide overnight, then the peptide in the media was washed out to prepare the peptide-pulsed TISI cells as stimulator cells. Prepared CD4-negative cells were cultured with the peptide-pulsed TISI cells (2 × 10
4 cells/well) at the ratio of responder cells and stimulator cells (R/S ratio) of 1:1, 1:2, 1:4 and 1:8 on 96-well plates at 37°C overnight. Non-peptide-pulsed TISI cells were used as negative controls. To confirm the IFN-γ productivity, responder cells (2.5 × 10
3 cells/well) were stimulated with PMA (66 ng/ml) and ionomycin (3 mg/ml) without stimulator cells overnight, and then applied to the IFN-γ ELISPOT assay. All ELISPOT assays were performed in triplicate wells. The plates were analyzed using the automated ELISPOT reader, ImmunoSPOT S4 (Cellular Technology Ltd, Shaker Heights, OH, USA) and ImmunoSpot Professional Software Version 5.0 (Cellular Technology Ltd). The number of peptide specific spots was calculated by subtracting the spot number in the control well from the spot number in well with peptide-pulsed TISI cells. Antigen specific CTL responses were classified into 4 groups (−, +, ++ or +++) according to a previously reported protocol [
14]. If the CTLs were indicated as +, we judged them as being positive in this study. The quality of our ELISPOT assay was ranked at the average level by the ELISPOT panel of Cancer Immunotherapy Consortium (CIC;
http://cvc.assaymgmt.webbasix.com).
Flow cytometrical analysis
The presence of CTLs with peptide-specific T cell receptor was analyzed using a FACS-CantoII (Becton Dickinson, San Jose, CA, USA), using VEGFR1 or VEGFR2-derived epitope peptide-MHC dextramer-PE (Immudex, Copenhagen, Denmark), CDCA1-derived epitope peptide-MHC pentamer-PE (ProImmune Ltd., Oxford, UK), or URLC10-derived epitope peptide-MHC tetramer-PE (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) according to the manufacturers’ instructions. HIV-derived epitope peptide (RYLRDQQLL)-MHC dextramer, pentamer or tetramer-PE was used as a negative control. Briefly, cells were incubated with the peptide-MHC dextramer, pentamer or tetramer-PE for 10 min at room temperature, and then treated with FITC-conjugated anti-human CD8 mAb, APC-conjugated anti-human CD3 mAb, PE-Cy7-conjugated anti-human CD4 mAb, and 7-AAD (BD Pharmingen, San Diego, CA, USA) at 4°C for 20 min.
Statistical analysis
Statistical analysis for correlation between clinical response and reaction at the injection site (RAI) was performed Fisher’s exact test. Overall survival rates were analyzed using the Kaplan-Meier method, and survival was measured in days from the first vaccination to death. Statistical significance of the survival period was analyzed using the log-rank test.
Discussion
Among the large number of therapeutic cancer vaccine trials for solid tumors being conducted worldwide, most involve the administration of a single vaccine [
15,
16]. For lung cancer, two large phase 3 trials using MAGE-A3 or BLP25 are expected to be very promising (ClinicalTrials.gov NCT00480025 and NCT01015443) [
5,
6]. However, single vaccine therapies in these trials may have some disadvantages as compared with treatment involving a mixture of multiple peptides derived from multiple proteins; one important factor is that antigen expression occurs in a relatively limited proportion of tumors. For example, the expression of MAGE-A3 has been reported in only 40% of cases [
17], and in only 24% of Japanese patients [
18]. The other important issue is the frequency of CTL induction, the rate of which largely depends on the nature of individual antigens. In fact, two lung cancer studies reported previously shown CTL induction in only 20-53% of the cases treated with vaccines [
6,
19]. In this regard as recently reported, treatment using multiple vaccine therapy has some advantages owing to the possibility that CTL induction may be higher for one or more antigens [
7,
8]. Further in renal cell cancer, clinical benefits have been shown lately using a multiple peptide vaccination named IMA901, and a phase 3 study is currently ongoing [
20]. In the present study, we have conducted a vaccine trial for lung cancer using multiple peptide vaccines, and observed that the specific CTL responses against one or more epitope peptides were very effective. In only two out of the 15 patients, no CTL induction was observed using any of the four peptides. Although we administered our vaccine treatment to the patients as a second line or later treatment, they achieved a median survival time of 398 days and a 1-year survival rate of 58.3%. Previous major second line trial data regarding NSCLC using a cytotoxic chemotherapeutic drug revealed a median survival time of about ~8 months and a 1-year survival rate of ~30% [
21]. Hence, we expect that our vaccine formulation may contribute to an improvement in the prognosis of patients with NSCLC, although further investigation of survival benefit using a larger number of patients is required.
Peptide vaccines used in this trial included peptides that originated from VEGFR1 and VEGFR2 for targeting angiogenesis in tumors. Bevacizmab, an antibody targeting VEGF, has already been used to treat the advanced non-squamous type of NSCLC [
22]. Although anti-angiogenic therapy alone does not have sufficient efficacy to induce tumor shrinkage [
23], it may support the induction of a strong anti-tumor effect and/or contribute to improved patient survival when it is combined with other therapies [
24,
25]. Therefore, we considered that the combination of anti-angiogenic peptides with peptides derived from tumor-specific antigen-proteins may cause a synergistic clinical effect in patients with NSCLC. In addition, since HLA molecules are down-regulated in many types of advanced solid cancer including lung cancer [
26,
27], peptides targeting blood vessels in which HLA molecules are stably expressed should have some anti-tumor effect by reducing the blood supply to tumors.
In our vaccine trial, although we did not observe tumor shrinkage, we observed a possible survival benefit. “Clinical benefit without tumor shrinkage” is considered to be one of the characteristics of cancer vaccine treatment [
28]. In fact, the guidance for therapeutic cancer vaccines released from the Food and Drug Administration (FDA) in the United States that was released in 2011 (
http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/UCM278673.pdf) mentioned that therapeutic cancer vaccine treatment can provide a survival benefit without evident tumor shrinkage. The FDA guidance further commented that “clinical progression that is asymptomatic and/or is not likely to result in life-threatening complications with further progression (e.g., central nervous system (CNS) metastases or impending fractures from bony metastases) may not be sufficient reason for discontinuation of the administration of a cancer vaccine”. Accumulating evidence has indicated the necessity of establishing novel criteria for the evaluation of clinical response in immunotherapy such as immune-related response criteria (irRC) [
28]. Researchers have started using overall survival or relapse-free survival in recently conducted trials as endpoints in immunotherapy clinical trials.
Our data suggested that PS, CTL induction and pre-treatment serum CRP level might be potential predictive markers for vaccine treatment. Extensive and systematic approaches regarding biomarker discovery for vaccine therapy have been carried out [
29]. In addition, several prognostic factors possibly related to immunotherapy including clinico-pathological parameters or immunological parameters have been reported [
30]. Some previous studies have implicated PS and CTL as good prognostic factors [
31,
32] in line with our findings. However, although our study has suggested that patients with a higher CRP level (≥1.0 mg/ml) had significantly shorter survival times than those with a lower CRP level, the usefulness of CRP as a prognostic marker has been controversial [
33,
34].
The US FDA guidance also suggests that cancer vaccine should be administered to patients at an earlier stage, at which the immune system has not been heavily damaged by cytotoxic anti-cancer drugs. In this regard, administration of vaccine therapy should be more appropriate as an adjuvant treatment after surgery, or as an early phase treatment after relapse of the disease in combination with or without chemotherapy.
In summary, we conducted phase I trials with multiple peptide vaccines for patients with NSCLC. These vaccine treatments were well tolerated and prolongation of patient survival owing to vaccine treatment might be expected. We believe that vaccine treatment using multiple peptides is likely to be very promising, although this should be validated by further advanced-phase clinical trials.
Acknowledgements
The authors would like to thank Prof. Yusuke Nakamura, Dr. Takuya Tsunoda and Dr. Koji Yoshida at the Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, for their excellent advice and cooperation and for providing all of the peptides. The authors would also like to thank Ms. Kimura, Ms. Kikuta and Ms. I at the Department of Regenerative Surgery, Fukushima Medical University, Fukushima, JAPAN for providing excellent technical support and for the preparation of the vaccines used for vaccination.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HS participated as principle investigator of the study. HS, Mitsunori H, YS, TK, KI and MG participated in the design and coordination of the study, data acquisition and analysis and helped draft the manuscript. KT participated as the main coordinator and investigator regarding the immunological data analysis and evaluation. MF, TY, SM, NO, HY, TH, AY, JO and MH participated in the clinical data acquisition and evaluation, and helped draft the manuscript. All authors read and approved the final manuscript.