【论文】WT1肽肿瘤疫苗对晚期胸腺上皮性恶性肿瘤的免疫作用
WT1 peptide-based immunotherapy for advanced thymic epithelial malignancies
Yusuke Oji1 , Masayoshi Inoue2, Yoshito Takeda3, Naoki Hosen4, Yasushi Shintani5, Manabu Kawakami6 Takuya Harada1, Yui Murakami1, Miki Iwai1, Mari Fukuda7, Sumiyuki Nishida3, Jun Nakata8, Yoshiki Nakae6, Satoshi Takashima3, Yoshiaki Shirakata3, Hiroko Nakajima7, Kana Hasegawa7, Hiroshi Kida3, Takashi Kijima9, Soyoko Morimoto8, Fumihiro Fujiki7, Akihiro Tsuboi8, Eiichi Morii10, Satoshi Morita11, Junichi Sakamoto12, Atsushi Kumanogoh3, Yoshihiro Oka4, Meinoshin Okumura5, Haruo Sugiyama7
Keywords: thymic carcinoma, thymoma, WT1, WT1 peptide vaccine
doi: 10.1002/ijc.31253
ABSTRACT
Thymic epithelial tumors are rare malignancies, and no optimal therapeutic regimen has been defined for patients with advanced disease. Patients with advanced thymic epithelial tumors, which were resistant or intolerable to prior therapies, were eligible for this study. Patients received 9 mer-WT1-derived peptide emulsified with Montanide ISA51 adjuvant via intradermal administration once a week as a monotherapy. After the 3 month-protocol treatment, the treatment was continued mostly at intervals of 2 to 4 weeks until disease progression or intolerable adverse events occurred. Of the 15 patients enrolled, 11 had thymic carcinoma (TC) and 4 had invasive thymoma (IT). Median period from diagnosis to the start of treatment was 13.3 and 65.5 months for TC and IT, respectively. No patients achieved a complete or partial response. Of the 8 evaluable TC patients, 6 (75.0%) had stable disease (SD), and 2 had progressive disease (PD). Of the 4 evaluable IT patients, 3 (75.0%) had SD, and 1 (25.0%) had PD. Median period of monotherapy treatment was 133 and 683 days, in TC and IT patients, respectively. No severe adverse events occurred during the 3 month-protocol treatment. As adverse events in long responders, thymoma-related autoimmune complications, pure red cell aplasia and myasthenia gravis occurred in two IT patients. Cerebellar hemorrhage
developed in a TC patient complicated with Von Willebrand disease. Induction of WT1-specific immune responses was observed in the majority of the patients. WT1 peptide vaccine immunotherapy may have anti-tumor potential against thymic malignancies.
Introduction
Thymic epithelial tumors are rare tumors of the thymus, which consist of thymomas and thymic carcinomas. Thymomas have an annual incidence of 0.15 in the USA1 and 0.32 cases in the Netherlands2 per 100,000 person-years.
Thymic carcinoma is a very rare disease that accounts for 10-15% of all thymic epithelial tumors.3 Surgery is the only therapeutic option for this condition.
However, thymic malignancies are often diagnosed in the advanced disease stage because of the lack of evident symptoms during their initiation and early progression. Palliative chemotherapy is the main therapeutic approach for unresectable or recurrent disease after radical resection. Based on the clinical evaluations of several chemotherapeutic regimens such as ADOC (cisplatin, doxorubicin, vincristine, and cyclophosphamide),4,5 VIP (etoposide, ifomide, and cisplatin)6, and CODE (cisplatin, vincristine, doxorubicin, and etoposide)7, anthracycline and platinum antitumor agent-based regimens (such as ADOC) are currently the choice for treatment of advanced thymic malignancies.
However, optimal medical management remains undefined.8 The WT1 gene was originally isolated as the gene responsible for a kidney neoplasm, Wilms’ tumor,9 and has been considered a tumor suppressor.
However, we and others have reported that WT1 is overexpressed in leukemia10 and various other types of solid tumors including lung,11 breast,12 colon,13 pancreatic14 cancers, and glioblastoma.15 It plays several oncogenic roles including involvement in cancer cell growth,16 resistance to apoptosis,17 enhancement of cell migration,18 and tumor vascularization.19 Furthermore, WT1 is one of the most promising targets for cancer immunotherapy20-23 and WT1-targeting cancer immunotherapies have been developed as novel therapeutic strategies.24-27 We have conducted multiple clinical trials to show the potential of WT1 peptide-based immunotherapy as a monotherapy,24, 28 as well as in combination with chemotherapy.29, 30 However, it remains unclear whether WT1 peptide-based cancer immunotherapy could induce anti-tumor, WT1-specific immune responses and have clinical potential as a therapeutic option for thymic epithelial malignancies. In the present study, we report results from a phase II study of WT1 peptide vaccine cancer immunotherapy in patients with advanced thymic epithelial malignancies.
Materials and Methods
WT1 peptide vaccine immunotherapy WT1 peptide vaccine immunotherapy was performed under the approval of the Ethical Review Board of Osaka University Faculty of Medicine.
This trial was registered at the UMIN Clinical Trials Registry as Umin000002001. Eligibility criteria included the following: histologically confirmed thymic carcinoma or thymoma not amenable to potentially curative therapies, WT1 protein expression in tumor cells, HLA-A*24:02 positivity, age range of 16 to 85 years, and adequate organ function.
The Good Manufacturing Practice (GMP)-grade, 9-mer modified WT1-derived peptide (mWT1-235, 235–243 a.a., CYTWNQMNL; Peptide Institute, Ibaraki, Osaka, Japan and Multiple Peptide Systems, San Diego, CA, USA), was used for immunization.
Patients who met the criteria for the clinical study were intradermally injected with 3 mg of mWT1-235 peptide emulsified with Montanide ISA51 adjuvant (Sepic, Paris, France) once a week for 12 consecutive weeks. After the 3-month protocol treatment, WT1 peptide vaccine immunotherapy was continued until disease progression or intolerable adverse events were observed. Trials were registered as Umin000002001 and then UMIN000015997 at the UMIN Clinical Trials Registry.
Patient Assessment
The antitumor effect was assessed by determining the response of target lesions on computed tomography (CT) scans according to the Response Evaluation Criteria in Solid Tumors (RECIST) v1.0. When slowing or inhibition of tumor progression was observed during the protocol treatment period, after the 3 month protocol WT1 peptide vaccination was further continued, mostly at intervals of two to four weeks. All patients were given the best supportive care after dropout from the WT1 peptide vaccine clinical trial as determined by their responsible oncologist. The characteristics of patients involved in this trial are summarized in Table 1. Safety was assessed by monitoring and recording of adverse events, vital signs, clinical chemistries, hematology, and urinalysis. Adverse events were graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v3.0.
Monitoring of WT1-specific immune responses
WT1 delayed-type hypersensitivity (DTH) skin test was performed every month during the protocol treatment as previously reported.31 Briefly, 10 μg of WT1 peptide, which was used as the test drug, solved in saline and a saline alone control were intradermally injected in the forearm of the skin. DTH-positivity was defined as erythema ≧2 mm in diameter measured post 48 h injection.
Production of IgG antibody against WT1 peptide was assessed using ELISA as described previously31 with minor modifications. The WT1 a.a. 235–252 peptide with the additional sequence PKK at its C terminus to increase its solubility (CMTWNQMNLGATLKGVAAPKK) was synthesized as the capture antigen for WT1-235 IgG antibody by PH Japan (Hiroshima, Japan). Briefly, 0.2 μg of modified peptide was covalently linked to each well of 96-well plates using the peptide coating kit from Takara (Shiga, Japan) according to the manufacturer's instructions. After blocking, patient sera diluted at 1:100 were added to each peptide-coated or non-coated well for overnight incubation at 4°C. All samples were measured in duplicate. Bound WT1 peptide IgG antibody was detected using horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG antibody and HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology) as second- and third- antibodies, respectively. The absorbance at 450 nm was measured. WT1-235 IgG antibody levels were defined as [average absorbance values of peptide-coated wells] – [absorbance value of corresponding non-coated well]. Samples were scored as positive when the WT1-235 IgG antibody level was 0.05 or more than 0.05. This cutoff level was determined from the value of mean + 2SD of 40 healthy individuals
Immunohistochemistry
Immunohistochemical analysis was performed to examine the expression of WT1 protein in tumor cells as previously reported32 with minor modifications. After deparaffinization, the sections were antigen retrieved in 10 mM Tris-HCl (pH 9.0) containing 1 mM EDTA. These sections were incubated with a monoclonal WT1 antibody 6F-H2 (Dako Cytometry, Glostrup, Denmark) (dilution, 100:1) or isotype control mouse IgG1 at 4°C overnight and then reacted with Envision kit/HRP (Dako Cytomation) at room temperature for 30 min. After incubation with 0.7% H2O2 solution, immunoreactive WT1 protein was visualized using DAB solution (Dako Cytometry). The sections were then counterstained with hematoxylin. Samples were scored as positive when more than 10% of tumor cells were stained with 6F-H2 antibody in either their cytoplasm or nucleus.
Results
WT1 protein expression in thymic malignancies
The expression of WT1 protein in tumor cells was immunohistochemically examined as a patient selection criteria. WT1 protein was overexpressed in 11 (84.6％) of 13 TC and four (80.0％) of five IT. These findings show that WT1
protein was overexpressed in the majority of thymic epithelial malignancies.
Patient characteristics
Between September 2009 and June 2014, 15 patients were enrolled; 11 had thymic carcinoma (TC), and four had invasive thymoma (IT). (Fig. 1) The patient demographics and disease characteristics are summarized in Table 1. All
patients had advanced disease. The majority of patients were stage IVb at the start of WT1 peptide vaccine immunotherapy. Of 11 TC patients, 10 had received previous chemotherapy. The remaining one (Pt. 7) had received radiotherapy alone because he had complications with a hemostatic defect, Von Willebrand disease. All of the IT patients (n=4) had received previous chemotherapy.
Fig.1 Trial profile.
118x93mm (300 x 300 DPI)
Median number of months from diagnosis to the start of WT1 peptide vaccine was 13.3 and 65.5 in TC and IT patients, respectively. No patients had complications stemming from autoimmune-related disorders at the start of WT1 peptide vaccine immunotherapy.
Protocol administration and efficacy
Nine of 11 TC patients completed the protocol treatment. Two went off the protocol treatment because of disease progression, (Pt. 11) and treatment-unrelated death (Pt. 10). Two of the nine TC patients (Pts. 1 and 7)
had no target lesion according to RECIST criteria. Of the 8 evaluable patients, 6 (75.5 %) had stable disease (SD) at the end of the 3 month protocol treatment. (Table 2) After the 3 month protocol treatment, WT1 peptide vaccine
immunotherapy was continued until disease progression or intolerable adverse events. The median number of days of treatment with WT1 peptide vaccine as a monotherapy was 133 (20- 1,024) in TC patients.
Clinical responses during the period of 3 months protocol treatment were evaluated according to RECIST criteria v1.1. WT1-specific immune responses were evaluated in eleven patients who completed the protocol treatment.
Duration of WT1 peptide vaccine indicates the period in which patients were treated with WT1 peptide vaccine as a monotherapy. NE, not evaluable. TC, Thymic carcinoma; IT, invasive thymoma.
All the IT patients completed the protocol treatment. Of the four patients, three (75.0 %) had stable disease (SD) at the end of the protocol treatment. (Table 2) The remaining patient (Pt. 12) had a large pericardial tumor, which grew progressively despite prior chemotherapy regimens with ADOC, CBDCA plus PTX, or AMR, each administered at different time points. This patient was scored as PD at the end of the protocol treatment because of a growing mediastinal lymph node. However, the pericardial tumor was stabilized during the treatment with WT1 peptide vaccine. WT1 peptide vaccine immunotherapy was continued for the three IT patients until disease progression or intolerable adverse events. The median number of days of treatment with WT1 peptide vaccine as a monotherapy was 683 (112-1,015) in the four IT patients.
Cases
A 59-year-old male patient (Pt. 3) had a recurrent thymic carcinoma (squamous cell carcinoma) with multiple lung, right kidney, and multiple bone metastases after surgical resection and irradiation. Although he had had been treated with two different chemotherapy regimens: eight courses of carboplatin (CBDCA) plus paclitaxel (PTX) and, then four courses of weekly docetaxel (DTX), the recurrent disease progressively grew under the treatment.
Therefore, he enrolled in the clinical trial. The disease had been stabilized for 4.5 months of treatment with WT1 peptide vaccine alone in association with induction of WT1-specific cellular immune responses as shown by positive WT1-DTH skin test (Figs. 2A and 2C).
A 53-year-old male patient (Pt. 14) with a pleural disseminated thymoma (type B1, stage IVb) was treated with WT1 peptide vaccine immunotherapy. His prior therapies consisted of chemotherapy (ADOC) and ion beam radiotherapy. After the protocol treatment, WT1 peptide vaccine was continued at intervals of every 2 to 3 weeks. His tumors regressed after stabilization during the course of treatment (Figs. 2B and 2C).
These results show the clinical potential of WT1 peptide vaccine immunotherapy for thymic epithelial malignancies.
Adverse event
All patients developed local erythema and swelling of grade 1 at the vaccination sites. Grade 3 hyponatremia developed after 10 vaccinations in an IT patient (Pt. 13) who also suffered from appetite loss and general fatigue due to disease progression. Hemoglobin levels decreased from grade 1 to 2 in a TC patient (Pt.9) who had disease progression at the end of protocol treatment. No other hematological and/or non-hematological adverse events of grade 2 or more than 2 were observed during the protocol treatment.
Cerebellar hemorrhage of grade 4 developed on day 1,241 of the treatment after 59 vaccinations in a TC patient (Pt. 7) who suffered complications with Von Willebrand disease. Thymoma-related autoimmune complications developed in two IT patients. An IT patient developed pure red cell aplasia (PRCA) of grade 3 on day 1,015 of the treatment after 66 vaccinations (Pt. 14). Another IT patient developed myasthenia gravis (MG) of grade 2 on day 799 of treatment after 38 vaccinations (Pt. 15). These results indicate WT1 peptide vaccine immunotherapy is well tolerated as a therapy for TC but may need further investigation for its association with autoimmune-related complications in IT.
Induction of WT1-specific Immune responses
WT1-DTH positivity and WT1 peptide IgG antibody production during the period of protocol treatment were examined to analyze the induction of WT1-specific immune responses. Both WT1-DTH and WT1 IgG antibody responses were negative at the start of WT1 peptide vaccine immunotherapy in all patients enrolled in the trial. Of nine evaluable TC patients, six (66.7 %) and five (55.6 %) became positive for WT1-DTH and WT1 IgG antibody, respectively, during 3 months of WT1 peptide vaccine treatment. Of four IT patients, all four and one (25.0 %) became positive for WT1-DTH and WT1 peptide IgG antibody, respectively. (Table 2) Twelve of 13 patients who completed the 3 month-protocol treatment showed either positive WT1-DTH or WT1 peptide IgG production. Two TC and one IT patient had both positive WT1-DTH and WT1 peptide IgG antibody responses. These results indicate that WT1 peptide vaccine elicited WT1-specific immune responses in the majority of the patients with thymic epithelial tumors.
Discussion
In the present study, we conducted a phase II clinical trial of WT1 peptide vaccine immunotherapy for advanced thymic epithelial malignancies, and showed that: 1) the target molecule, WT1 protein was overexpressed in the
majority of thymic malignancies, 2) vaccination with WT1 peptide induced WT1-specific immune responses, and 3) WT1 peptide immunotherapy had clinical potential with a stable disease rate of 75.0% and 75.0% in TC and IT
patients, respectively, at 3 months.
As far as we know, this is the first report of WT1-targeting immunotherapy for thymic epithelial malignancies. The patients in the present study had advanced diseases, which were resistant or intolerable to prior therapies. Stable disease rates for these advanced thymic malignancies were 75.0% and 75.0%, in TC and IT, respectively, at 3 months of WT1 peptide vaccine immunotherapy. The median number of days of WT1 peptide vaccine as a monotherapy was 133 and 683 in TC and IT patients, respectively. The clinical outcomes observed in the present study indicate that WT1 peptide vaccine immunotherapy has clinical potential for the treatment of thymic malignancies. Furthermore,
immunohistochemical analysis showed that the target molecule, WT1 protein was overexpressed in the majority (≧ 80%) of both TC and IT. Taken together, WT1 peptide vaccine immunotherapy may be a novel therapeutic option
applicable to the majority of thymic epithelial malignancies.
The thymus is a specialized primary lymphoid organ of the immune system. Immune abnormalities such as auto-immune-related complications33,34 and susceptibility to infections have been reported in thymomas.35,36 Therefore, we examined whether WT1 peptide vaccine could elicit anti-tumor WT1-specific immune responses in the patients with thymic malignancies. WT1-DTH became positive in the majority (76.9%) of the patients treated. Production of WT1 peptide IgG antibody was also observed but with less frequency (46.2%). These results show that WT1-specific immune responses were induced in the majority of patients with thymic malignancies. However, the majority of the patients in the study either had positive WT1-DTH or WT1 peptide IgG antibody production alone and not both. We previously reported that the combination of positive DTH to WT1 peptide and WT1-235 IgG antibody production was a better predictor of prolonged PFS and OS, compared to either of them alone, in patients with recurrent glioblastoma.31 The results presented in the present study show that WT1 peptide vaccine failed to induce both of the immune responses in the majority of patients enrolled in the trial. These findings indicate that the induction of WT1-specific immune responses by the current WT1 peptide vaccine is unsatisfactory in patients with thymic malignancies, which led us to believe that a more robust induction of WT1-specific anti-tumor immune responses is required for achievement of better clinical outcomes in thymic epithelial malignancies treated with WT1 peptide vaccine immunotherapy.
We have previously reported that a WT1-protein derived, 16-mer helper peptide, WT1-332, could elicit Th1-type CD4+T-cell responses37 and bind to multiple HLA class II molecules.38 Moreover, HLA-DPB1*05:01-restricted, WT1-332-specific TCR-transduced CD4+T cells exhibited not only Th1-type cytokine production in response to WT1-332 peptide, but also cytotoxic activity against HLA-DPB1*05:01-positive, WT1-expressing human leukemia cells through the granzyme B/perforin pathway.39 Since HLA class II molecules are expressed in the majority of thymic malignancies (data not shown), combinatorial vaccination with helper-epitope peptide, WT1-332 and CTL-epitope peptide or peptides will not only enhance WT1-specific CD8+T cell-mediated CTL responses but also induce CD4+T cell-mediated ones, which will ultimately lead to better disease control. Future clinical research is needed to examine the efficacy of combining WT1 peptide-based therapy of WT1-332 helper peptide and WT1 CTL-epitope peptides.
Autoimmune diseases such as MG and PRCA are well-known complications of thymomas.40, 41 Their frequencies are reported to be approximately 40% and 5%, respectively. Therefore, whether cancer immunotherapy could enhance pre-existing autoimmune responses in its application for thymoma therapy is an essential question. Since PD-1 and its ligand, PD-L1, are expressed in 23% and 23~64% of thymomas,42,43 an immune checkpoint inhibitor PD-1 antibody could be tested as a novel therapeutic strategy for thymoma. However, because of their nature, immune checkpoint inhibitors can cause a variety of immune related adverse events. Reportedly, MG developed after administration of a PD1 inhibitor shortly after the initiation of the treatment in cancer patients without history of thymoma.44,45 Since immune checkpoint inhibitors enhance pre-existing immune responses, they can theoretically promote autoimmune complications in thymoma patients. In the present clinical trial, two thymoma cases developed autoimmune-related complications after treatment for a length of more than two years with WT1 peptide vaccine. Immune responses induced by the WT1 peptide vaccine are principally WT1-specific and are unlikely to be responsible for such autoimmune-related complications. In fact, no autoimmune-related complications have developed in more than 800 WT1 peptide vaccine-treated cases with malignancy other than thymoma. Therefore, autoimmmune complications observed in the WT1 vaccine-treated thymoma patients are considered to be disease-specific and can be explained by the development of pre-existing autoimmune responses, which could be enhanced by the absence of chemotherapy-induced immunosuppression during the WT1 peptide vaccine treatment. However, it could be postulated that the WT1 peptide vaccine contributes to the development of such autoimmune-related complications through nonspecific activation of the immune system including pre-existing autoimmune responses. Taken ogether, the association of WT1 peptide vaccine with the development of autoimmune-related complications in patients with thymoma should be further examined in future clinical trials.
Acknowledgements
This work was supported in part by a Grants-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (T15K184460, T264621790). We thank Dr Seiji Hayashi (Kinki National Hospital, Osaka, Japan) and Dr Hiroshi Matsuoka (Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Graduate School of Medicine, Hyogo, Japan) for their great support as Effect
Safety Evaluation Committee members for the clinical study. We also thank Tomoe Umeda, Nobuko Toda, Akiko Ohta, and Sae Hayashi (Departtments of Cancer Immunotherapy, Cancer Immunology, and Functional Diagnostic
Science, Osaka University Graduate School of Medicine, Japan) for their support for the study. We thank Dr Masayuki Takeda (Department of Medical Oncology, Faculty of Medicine, Kinki University, Japan) and Dr Hiroya Egawa
(Internal Medicine, Hiroshima City Asa Citizens Hospital, Japan) for their support for the management of patients. We would like to thank Editage (www.editage.jp) for English language editing.
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