A phase I study of combination vaccine treatment of five therapeutic epitope-peptides for metastatic colorectal cancer; safety, immunological response, and clinical outcome

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in industrialized countries [1]. In the last decade, the combined regimens of multiple anticancer drugs have been applied and markedly improved the survival of patients with CRC at stages III and IV [2]. However, many patients often face to progression of the diseases due to chemo-resistance.

Recent development in genome-based technologies has enabled us to obtain comprehensive gene expression profiles of malignant cells compared with normal cells [3]. By applying cDNA microarray technology coupled with laser micro-dissection, we had identified three oncoantigens, ring finger protein 43 (RNF43) [4], 34 kDa-translocase of the outer mitochondrial membrane (TOMM34) [5], and KOC1 (IMP-3; IGF-II mRNA binding protein 3) [6, 7], as targets for development of cancer peptide vaccines for CRC. An oncoantigen was defined as a molecule with high immunogenicity in our immune system and with oncogenic function that plays a critical role in the growth of tumor cells. Since the oncoantigen is essential for the cell growth, the probability of immune escape of cancer cells by reducing or lacking these proteins is expected to be low [8, 9]. In addition, these three molecules are specifically expressed in cancer cells, the risk of autoimmune reactions by vaccine treatment using the peptides derived from these proteins is expected to be minimum [4‐6].

Although immunotherapy using tumor infiltrating cells (TIL) or vaccine treatment have been expected as a promising modality to treat cancer, recent reports have indicated several mechanisms in tumor tissues to protect cancer cells from immune attacks [10]. For example, the limitation of antitumor effects of cytotoxic T lymphocytes (CTL) was explained by tumor cell heterogeneity; a subset of tumor cells revealed downregulation or loss of expressions of human leukocyte antigen (HLA) or targeted antigen proteins [11, 12]. The growth of solid neoplasms always accompanies with neovascularization [13] which is associated with the expression of vascular endothelial growth factor receptor 1 (VEGFR1) [14] and VEGFR2 [15]. These two molecules were highly expressed in tumor vascular endothelial cells. Hence, our vaccine treatment including the peptides derived from VEGFR1 and VEGFR2 is also able to target neovascular endothelial cells, suppress neovascurization, and then reduce the energy and oxygen supply into the tumor tissues.

In this study, since an HLA-A*2402 allele is the most common HLA-A allele in the Japanese population with the allelic frequency of approximately 60% [16], we selected five HLA-A*2402-restricted peptides derived from RNF43, TOMM34, KOC1, VEGFR1, and VEGFR2 for the clinical trial. The purpose of this study was to evaluate the safety and biological responses of these five peptides. Additionally, we compared the immunological responses of the separate injections of each of five peptides with those of the single injection of a cocktail of five peptides. We here demonstrate the safety of these peptides and a promising result of our cocktail treatment of five peptides for the improvement of prognosis of advanced CRC.

From February 2007 to March 2009, nineteen CRC patients were enrolled in this study and received vaccine treatment. One patient (case 7) who refused continuation of the vaccine therapy after a single administration of peptides was excluded from this analysis. Characteristics of eighteen patients are summarized in Table 2. Most of the patients except two cases had received chemotherapy regimens including fluorouracil, irinotecan or oxaliplatin prior to the vaccine treatment; one (case 11) of the two exceptions was due to pelvic abscess after the low anterior resection. The other exception (case 16) was because this patient refused chemotherapy. Another patient (case 18) had refused surgical treatment because she was afraid of the high complication risk of the second operation of the recurrent tumor in the pelvis. We provided vaccination with the written informed consent, but she accepted to receive curative resection after 8 weeks of vaccination. This case was included in the immunological analysis, but excluded from the analysis of survival.

We performed phase I study using five novel epitope peptides, including three peptides derived from three oncoantigens as well as two peptides targeting VEGFR1 and VEGFR2, for colorectal cancer. Vaccinations of five peptides in three different doses as well as cocktail treatment of five peptides in metastatic CRC patients were well tolerated without any severe systemic adverse events. Although two patients revealed ulceration at the skin injection site, they were able to continue the vaccine treatment. Hence, the safety of our study was confirmed as previous reports using the same class of peptides [21, 22, 25].

The average numbers of specific CTL induction against these five peptides after 12 vaccinations of the doses of 0.5 mg, 1.0 mg, and 3.0 mg were calculated to be 2.0, 2.3, and 3.2 peptides/patient, respectively. In this study, the maximum tolerated dose was not observed. Moreover, there was no sign of immunosuppression due to excessive administration at the doses of 3.0 mg. Although the number of the patients enrolled in this study was very limited, the higher dose of peptide seems to induce peptide-specific CTLs more effectively than the lower dose. Hence, we would like to the recommended dose of each peptide for further study to be 3.0 mg.

We also attempted vaccinations of the 5-peptide cocktail that was also well tolerated without any serious systemic toxicity. Although there were some concerns that the mixture of multiple peptides in a single injection might reduce the immune response due to the different affinity of each peptide to an HLA molecule, we observed that the average numbers of peptide-specific CTL inductions per patient was 3.5 within 3 courses of vaccinations. Our results demonstrated that the cocktail of multiple peptides is non-inferiority to the separate injection of each of multiple peptides. A previous report described that the frequency of CTL induction specific to vaccinated peptides in advanced CRC was approximately 33% [26]. However, we observed the peptide-specific CTL induction in more than 60% of the patients after the 12-week of vaccination at the dose of 3.0 mg/week, indicating that our peptide epitopes might have higher immunogenicity than peptides used previously.

Among the 18 patients we evaluated clinically, one patient showed the complete response and six patients kept stable conditions for 4–7 months. Hence, the response rate (RR) was calculated to be 5.6% and the disease control rate (DCR) to be 38.9%. One review article reported that the RR of active cancer vaccines for CRC was 0.9% and the DCR was 11.1% [26]. Hence, our clinical data has also implied the possible higher immunogenicity of our vaccines.

Although our peptide vaccination did not show obvious superiority in the aspect of RR or DCR, compared to the worldwide standard therapies for CRC [27, 28], the median overall survival time (MST) of our patients was 13.5 months which seemed longer than other presently-available standard therapies; for example, the MST was 6.1 months and 6.2 months in a phase III trial of Cetuximab [27] or Panitumumab [28] in patients with chemotherapy-refractory metastatic CRC, respectively. As US Food and Drug Administration described in “The guidance for therapeutic cancer vaccine”, this kind of vaccine treatment usually takes a few to several months to show clinical benefit due to the time lag to induce the sufficient number of effecter cells and is expected to demonstrate delayed effects [24]. The relation between peptide specific responses and OS was shown in Figure 2. Patients who revealed the CTL induction to two or more peptides within 3 courses (MST: 13.5 months) trended to survive longer (P = 0.086) than patients with CTL induction to one or no peptide (MST: 3.7 months). Moreover, patients with CTL induction against three or more peptides within 6 courses of the vaccination (MST: 27.8 months) had significantly longer survival (P = 0.032) than the remaining patients (MST: 3.7 months), suggesting the more CTLs were induced, the better prognosis was expected and supporting the use of multiple peptides for advanced cancer patients. Similarly, local reactions at peptides-injected sites could be possibly a good predictive biomarker(s) for longer survival; Patients with grade 2 local skin reactions at injected site of vaccine survived significantly (P = 0.001) longer (MST: 29.5 months) than patients with grade 1 reactions (MST: 4.4 month). These data suggested that the monitoring of the CTL responses and the skin reactions might become good predictive markers during the treatment for the efficacy of vaccination. Although it is very difficult at present, the prior selection of patients who are likely to respond well and induce CTLs effectively to vaccination is also very important. The presence of a higher number of infiltrated T cells in tumor microenvironment was suggested as a predictive biomarker for the response to immunotherapies and the selection of patients with the better treatment outcome of vaccinations [29].

In conclusion, although the number of patients in this early-phase trial is very limited, our peptides vaccine therapy was demonstrated to be safe, effectively induce peptide-specific immune responses and possibly improve the prognosis of advanced colorectal cancer. It is certain that although we need to verify this preliminary result by a much larger double-blind study, we believe that further stages of clinical trials should be worth doing.

This study indicated that the combination of five novel peptides can induce strong peptide specific immune responses in the group who received the dosage of 3.0 mg, and that the single injection of “the peptide cocktail” is likely to induce the similar immune responses to the multiple injections of “each peptide”, and that the overall survival of patients treated with our peptides was prolonged obviously after 6 months from the first vaccination, which has implied the delayed response of the vaccination. Moreover, the induction of peptide specific immune responses had significant relevance to longer survival. Although we need to verify this preliminary result by a much larger double-blind study, we believe that these findings surely lead to the novel therapeutic strategy for advanced colorectal cancer.