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Safety and Immunogenicity of the PRAME Cancer Immunotherapeutic in Patients with Resected Non–Small Cell Lung Cancer: A Phase I Dose Escalation Study

Open AccessPublished:August 18, 2016DOI:https://doi.org/10.1016/j.jtho.2016.08.120

      Abstract

      Introduction

      Adjuvant platinum-based chemotherapy is standard treatment for surgically resected stage II to IIIA NSCLC, but the relapse rate is high. The preferentially expressed antigen of melanoma (PRAME) tumor antigen is expressed in two-thirds of NSCLC and offers an attractive target for antigen-specific immunization. A phase I dose escalation study assessed the safety and immunogenicity of a PRAME immunotherapeutic consisting of recombinant PRAME plus proprietary immunostimulant AS15 in patients with surgically resected NSCLC (NCT01159964).

      Methods

      Patients with PRAME-positive resected stage IB to IIIA NSCLC were enrolled in three consecutive cohorts to receive up to 13 injections of PRAME immunotherapeutic (recombinant PRAME protein dose of 20 μg, 100 μg, or 500 μg, with a fixed dose of AS15). Adverse events, predefined dose-limiting toxicity, and the anti-PRAME humoral response (measured by enzyme-linked immunosorbent assay) were coprimary end points. Anti-PRAME cellular responses were assessed.

      Results

      A total of 60 patients were treated (18 received 20 μg of PRAME, 18 received 100 μg of PRAME, and 24 received 500 μg of PRAME). No dose-limiting toxicity was reported. Adverse events considered by the investigator to be causally related to treatment were grade 1 or 2, and most were injection site reactions or fever. All patients had detectable anti-PRAME antibodies after four immunizations. The percentages of patients with PRAME-specific CD4-positive T cells were higher at the dose of 500 μg compared with lower doses. No predefined CD8-positive T-cell responses were detected.

      Conclusion

      The PRAME immunotherapeutic had an acceptable safety profile. All patients had anti-PRAME humoral responses that were not dose related, and 80% of those treated at the highest dose showed a cellular immune response. The dose of 500 μg was selected. However, further development was stopped after negative results with a similar immunotherapeutic in patients with NSCLC.

      Keywords

      Introduction

      Lung cancer is the leading cause of cancer deaths worldwide, with most cases being of the NSCLC histological tumor type.
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      We investigated the safety and immunogenicity of escalating doses of a recombinant PRAME protein (recPRAME, GSK, Rixensart, Belgium) administered with a fixed dose of the proprietary immunostimulant AS15 (referred to as the PRAME immunotherapeutic) in patients with PRAME-positive pathological stage IB to IIIA NSCLC after complete surgical resection. Here we report safety and immunogenicity data measured 3 weeks after the fourth administration of the PRAME immunotherapeutic that led to dose selection according to predefined rules.

      Methods

      The open-label, phase I study was conducted in 22 centers in Germany, France, Italy, Poland, the Russian Federation, and the United States (ClinicalTrials.gov identifier NCT01159964). The study protocol was approved by institutional review boards at each participating center. Written informed consent was obtained from each patient before the performance of any study-specific procedures.
      Coprimary objectives were to document and characterize for each dose of the PRAME immunotherapeutic tested the potential dose-limiting toxicities (DLTs) and the anti-PRAME humoral immune response. Secondary objectives included evaluation of the overall safety profile and cell-mediated antigen-specific immune (CMI) responses.

      Patients

      Patients were 18 years of age or older with pathological stage IB to IIIA NSCLC after complete (R0) surgical resection. The resection had to be at least a lobectomy. Patients were allowed to receive adjuvant platinum-based chemotherapy before enrollment. The tumor had to be PRAME-positive as determined on the basis of formalin-fixed paraffin-embedded tissue samples by quantitative reverse-transcriptase polymerase chain reaction at a central laboratory (Response Genetics, Inc., Los Angeles, CA).
      • Lerut E.
      • Van Poppel H.
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      • Gruselle O.
      • Coche T.
      • Therasse P.
      Rates of MAGE-A3 and PRAME expressing tumors in FFPE tissue specimens from bladder cancer patients: potential targets for antigen-specific cancer immunotherapeutics.
      Protocol-defined inclusion and exclusion criteria are provided in the Supplementary Methods.

      Treatment Regimen

      The composition of the PRAME immunotherapeutic (recPRAME plus AS15) is provided in the Supplementary Methods. The PRAME immunotherapeutic was administered intramuscularly into the deltoid muscle or thigh, alternating sides for each dose.
      Escalating doses of recPRAME (20 μg, 100 μg, and 500 μg) combined with a fixed dose of AS15 were evaluated in three consecutive cohorts. A maximum of 13 injections of the PRAME immunotherapeutic were to be administered according to the following schedule: the first five injections were given at 3-week intervals followed by eight injections at 12-week intervals. Patients were actively followed for an additional year for safety and clinical outcomes. This regimen has been used for the investigation of other immunotherapeutics, including the melanoma-associated antigen A3 (MAGE-A3) immunotherapeutic in patients with NSCLC.
      • Vansteenkiste J.F.
      • Cho B.C.
      • Vanakesa T.
      • et al.
      Efficacy of the MAGE-A3 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small-cell lung cancer (MAGRIT): a randomised, double-blind, placebo-controlled, phase 3 trial.
      Escalation to each dose level occurred when all 15 planned patients had initiated treatment with the lower dose and when at least three patients had received at least four injections. Dose escalation procedures are provided in the Supplementary Methods.

      Assessment of Safety

      All adverse events (AEs) occurring throughout the study until 30 days after the last administration of the study product were recorded. Serious adverse events (SAEs) related to study treatment, DLTs, onset of autoimmune disease, and pregnancy outcomes were recorded for 1 year after administration of the last study treatment. Disease recurrences and deaths due to disease were not considered SAEs. AE severity was graded according to the Common Terminology Criteria for Adverse Events, version 4.0.

      National Cancer Institute, US National Institutes of Health. Common terminology criteria for adverse events (CTCAE) v4.0. http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. Accessed July 2, 2015.

      AEs were coded to the preferred term level using the Medical Dictionary for Regulatory Activities.

      Medical Dictionary for Regulatory Activities. Welcome to MedDRA. http://www.meddra.org. Accessed July 2, 2015.

      A DLT was defined as any of the following AEs considered related or possibly related to administration of the PRAME immunotherapeutic: (1) a grade 3 or higher AE (myalgia, arthralgia, headache, fever, rigors/chills, or fatigue persisting had to have persisted for 48 hours despite therapy to be considered a DLT), (2) a grade 2 or higher allergic reaction occurring within 24 hours after injection of PRAME immunotherapeutic, (3) any decrease in renal function with a creatinine clearance less than 40 mL/min, and (4) any symptomatic and confirmed adrenal insufficiency. Renal and adrenal AEs were included because PRAME is expressed at low levels in normal kidney and adrenal tissues.
      At each visit, blood and urine samples were collected for evaluation of a variety of routine hematologic, biochemical, and coagulation parameters. Of note, measurement of serum cortisol level and renal function tests and urinalysis were performed at each visit, and an antinuclear antibody test was performed at every second visit.
      A data and safety monitoring committee and internal safety review team reviewed the safety data, the clinical relevance of any DLT event, and its relationship to the study treatment.

      Immunogenicity

      Humoral Immunity

      Anti-PRAME antibody concentrations were measured before administration of the first dose, 3 weeks after dose 2, and 3 weeks after dose 4.
      Anti-PRAME immunoglobulin G (IgG) antibodies were measured by enzyme-linked immunosorbent assay as described in the Supplementary Methods. A humoral immune response was defined as a postimmunization anti-PRAME antibody concentration equal to or above the clinical cutoff value (12 EU/mL, defined from 102 healthy donors) in initially seronegative patients (seroconversion) and a twofold or greater increase in postimmunization anti-PRAME antibody concentrations in initially seropositive patients.

      Cell-Mediated Immunity

      CMI response was measured before the first dose and 3 weeks after dose 4, as described in the Supplementary Methods. Briefly, the presence and functionality of the PRAME-reactive T-cell response was assessed by stimulation of peripheral blood mononuclear cells in an in vitro multiple-well assay.
      PRAME T-cell immunogenicity (characterized by detection and quantification of T cells producing both interferon-γ [IFN-γ] and tumor necrosis factor-α [TNF-α] in an in vitro assay) cutoff scores for a positive response were defined from a panel of healthy donors (n = 23, cutoff of 2.68 for CD4-positive T-cell analysis and 1.15 for CD8-positive T-cell analysis). A patient was considered a CD4-positive or CD8-positive T-cell responder if the ratio of immunogenicity scores between a positive postimmunization sample and its corresponding baseline was 4 or greater. Frequencies of PRAME-specific T cells were estimated on the basis of quantification of defined positive wells in the in vitro assay. Frequency cutoffs were defined from healthy donors as 6.32 × 10-6 for CD4 positivity and 1.9 × 10-6 for CD8 positivity.

      Dose Selection Criteria

      The dose was selected on the basis of safety and immunogenicity data. A specific dose was considered adequate if no more than two cases of DLT were reported at any time among the 15 patients in each cohort and if the dose showed anti-PRAME antibody responses of 70% or more (≥11 of 15 patients) after four immunizations. If more than one dose level satisfied both the safety and humoral immune response criteria, selection of the dose would also take into account CMI responses. If the best immunological dose could not be determined by applying these criteria, the highest dose with acceptable safety and immunogenicity was to be selected.

      Statistical Analysis

      All statistical analyses were performed using SAS software version 9.2 (SAS, Inc., Cary, NC). The study was descriptive and no comparative tests were performed. The total treated cohort included all patients enrolled into the study who had received at least one injection of PRAME immunotherapeutic. The according-to-protocol cohort for immunogenicity included all patients who met the eligibility criteria, complied with the protocol-defined procedures, had received at least the first four PRAME immunotherapeutic doses, and had completed the study visit 3 weeks after the fourth immunization. Geometric mean antibody concentrations were calculated for anti-PRAME IgG antibodies.

      Results

      From July 12, 2010, to October 17, 2011, 357 patients were screened. Of the 342 patients with a valid test result, 198 (57.9%) had a PRAME-positive tumor and 60 were enrolled into the study (Fig. 1). The data lock point (DLP) for dose selection was on 28 February 2012. The overall mean age of patients in the total treated population was 62.8 years. Squamous cell carcinoma and adenocarcinoma were the most frequent histological tumor types in each cohort (Table 1). Approximately 40% of patients in each cohort had received prior platinum-based adjuvant chemotherapy.
      Figure 1
      Figure 1Patient flow through the study until 3 weeks after the fourth immunization. Missing means no tumor sample received by the laboratory. Inconclusive means invalid test result or quantity not sufficient for testing. Numbers of patients who completed the study until 3 weeks after the fourth immunization: 15 in cohort 1, 17 in cohort 2, and 22 in cohort 3. PRAME, preferentially expressed antigen of melanoma; SAE, serious adverse event; AE, adverse event.
      Table 1Demographic and Disease Characteristics (Total Treated Cohort)
      CharacteristicsCohort 1 (20 μg)

      n = 18
      Cohort 2 (100 μg)

      n = 18
      Cohort 3 (500 μg)

      n = 24
      Age at screening, y
       Mean ± SD61.3 ± 7.5562.3 ± 8.1264.1 ± 8.16
       Median61.062.064.5
       Range46–7150–7648–78
      Sex
       Female4210
       Male141614
      ECOG PS
       0131113
       15711
      T category
       T1A011
       T1B011
       T2A81112
       T2B226
       T3834
      N category
       N012816
       N1577
       N2131
      Stage
       IB7610
       IIA177
       IIB704
       IIIA353
      Histopathological type
       Adenocarcinoma5510
       Adenocarcinoma, bronchioloalveolar200
       Adenosquamous carcinoma101
       Large cell carcinoma111
       Squamous cell carcinoma81112
       Other110
      Platinum-based adjuvant chemotherapy
       Yes (no)8 (10)7 (11)9 (15)
      ECOG PS, Eastern Cooperative Oncology Group performance status.
      At the time of the DLP, 365 doses of PRAME immunotherapeutic were administered across the three cohorts, all patients had received at least one dose, 45 of 60 patients continued to receive treatment, and the highest number of doses administered to any patient was 10.
      After the DLP, issues impacting the electronic data capture system used in this study (the [email protected] Web system) were detected between April and May 2013. The issues related to incorrect signatory display and the audit trail. There was no impact on data integrity or on the analyzed data. The technical root cause was identified and corrective actions were taken. There was no impact on subject safety. Submission of this paper was delayed until all issues were fully resolved.

      Safety

      DLT and Study Withdrawals

      No case of DLT was reported.
      Five patients were withdrawn from study treatment before the fifth immunization (see Fig. 1), including three who were withdrawn for safety reasons. One patient in cohort 1 was withdrawn on account of a related SAE. In this 69-year-old male with underlying myopericarditis after pneumonectomy, hypertension, atrial fibrillation, and dyspnea, acute pulmonary edema (grade 2) developed on the day after dose 2 and 7 days after reduction of the furosemide dose and lasted for 1 day. Two patients in cohort 3 were withdrawn: one owing to an SAE (transitional cell carcinoma considered unrelated to treatment) and one because of a nonserious AE.

      AEs

      Between dose 1 and the DLP, at least one AE (related or unrelated) was reported by 78% of patients (14 of 18) in cohort 1, by 83% (15 of 18) in cohort 2, and by 100% (24 of 24) in cohort 3. All but three of the reported AEs were grade 1 or grade 2. There were 46 patients (12 each in cohorts 1 and 2 and 22 in cohort 3) who reported treatment-related AEs, all of which were grade 1 or 2 (Table 2). The most frequently reported related AEs were injection site reactions including pain, erythema, and edema. Other frequent related AEs included pyrexia, fatigue, chills, and asthenia. No potential immune-mediated disease was reported.
      Table 2Summary of Treatment-Related Adverse Events Reported at Least Twice in Any Group (Any Grade) from Dose 1 until the Data Lock Point, by Maximum Grade (Total Treated Cohort)
      Adverse EventCohort 1

      n = 18
      No. patients with at least one administered dose.
      Cohort 2

      n = 18
      No. patients with at least one administered dose.
      Cohort 3

      n = 24
      No. patients with at least one administered dose.
      Grade 1Grade 2Grade 1Grade 2Grade 1Grade 2
      n
      No. patients reporting the adverse event at least once.
      n
      No. patients reporting the adverse event at least once.
      n
      No. patients reporting the adverse event at least once.
      n
      No. patients reporting the adverse event at least once.
      n
      No. patients reporting the adverse event at least once.
      n
      No. patients reporting the adverse event at least once.
      Injection site reaction4583136
      Pyrexia436271
      Fatigue12141
      Chills21311
      Asthenia123
      Headache32
      Arthralgia112
      Influenza-like illness12
      Myalgia3
      Pain in extremity2
      Note: See Supplementary Table 1 for all treatment-related adverse events from dose 1 until the data lock point, by maximum grade (total treated cohort).
      a No. patients with at least one administered dose.
      b No. patients reporting the adverse event at least once.
      Two deaths occurred during the study, both on account of progression of NSCLC. One of these patients died while still undergoing the study treatment, whereas the other died after having been withdrawn from the study treatment because of disease recurrence. Five SAEs were reported, including one (acute pulmonary edema) that was considered possibly related to the treatment and led to withdrawal of the patient from the study (see earlier and Supplementary Table 1).

      Abnormal Laboratory Test Results

      There were three cases of grade 3 abnormal laboratory parameters, none of which were reported as AEs: hyponatremia (an increase from a sodium level of 131 mmol/L at screening to 127 mmol/L at the time of the sixth immunization [ongoing at the DLP] but no hyperkalemia) developed in one patient (in cohort 2), and two patients (in cohort 3) had increased γ-glutamyl transferase levels present at screening that remained unchanged during the study.
      The abnormal laboratory test results reported as AEs were one case of grade 1 decreased creatinine clearance (in cohort 2) that developed 21 days after dose 2, resolved after 35 days, and was considered unrelated to treatment; one case of grade 1 decreased blood cortisol level (in cohort 3) that developed on the day of dose 3, was considered unrelated to treatment, and resolved after 11 days; and one case of grade 1 increased creatinine clearance (in cohort 3) that developed on the day of dose 2, was considered treatment related, and had an unknown duration.

      Immunogenicity

      Two patients (one in cohort 1 and one in cohort 2) were seropositive for anti-PRAME IgG antibodies at baseline. Neither had received prior chemotherapy. All patients, except one in cohort 1 and two in cohort 3 (two of these patients had not received prior chemotherapy), were seropositive after two doses, and all were seropositive and had a humoral response (see “Methods”) at the post–dose 4 assessment (Fig. 2). Anti-PRAME antibodies were higher after dose 4 than after dose 2 in all cohorts. Seropositivity rates and geometric mean antibody concentrations after four immunizations were similar in patients who had or had not received prior platinum-based chemotherapy (Fig. 2).
      Figure 2
      Figure 2Seropositivity rates and geometric mean antibody concentrations (GMCs) for preferentially expressed antigen of melanoma (PRAME) immunoglobulin G (IgG) antibodies (according-to-protocol cohort for immunogenicity). N is number of patients with available results, n/% is number/percentage of patients with concentrations above the cutoff, vertical lines indicate 95% confidence intervals, dotted line shows assay cutoff (12 EU/mL). Abbreviations: Pre, before dose 1; Post II, 3 weeks after the second dose; Post IV, 3 weeks after the fourth dose; Post IV CT, patients who had received platinum-based chemotherapy before study enrollment; Post IV no CT, patients who had not received prior chemotherapy.
      After four doses, the numbers of patients with PRAME-specific CD4-positive T cells (TNF-α–positive/IFN-γ–positive) immunogenicity scores equal to or above the cutoff were five of 10 in cohort 1, seven of 10 in cohort 2, and 14 of 15 in cohort 3 (Table 3). There was a tendency toward a better induction of anti-PRAME CD4+ T cells with increasing recPRAME dose, with the highest estimated frequencies of PRAME-specific CD4-positive cells observed in cohort 3. Similar immunogenicity scores among individuals who had or had not received prior chemotherapy were observed (Fig. 3). Taking baseline immunogenicity scores into account, after four immunizations the percentages of patients with a PRAME-specific CD4-positive T-cell response were 33% in cohort 1, 60% in cohort 2, and 80% in cohort 3 (see Table 3).
      Table 3PRAME-Specific CD4-Positive and CD8-Positive T Cells (TNF-α–Positive/IFN-γ–Positive) Immunogenicity Score, Cellular Response, and Frequency before Treatment and after Dose 4 (According-to-Protocol Cohort)
      Immunogenicity Score
      See Supplementary Data for definition of immunogenicity score.
      (≥Cutoff)
      Response Rates to PRAME
      Response rate means that the ratio of immunogenicity scores positive postimmunization sample over baseline) is 4 or greater.
      Frequency above the Cutoff
      Frequencies of PRAME-specific T cells were estimated on the basis of quantification of defined positive wells in the in vitro assay (cutoff = 6.32 × 10-6 for CD4-positive cells and 1.9 × 10-6 for CD8-positive cells).
      Baseline

      n/N (%)
      After Dose 4

      n/N (%)
      (≥Cutoff and 4× Baseline)

      n/N (%)
      Baseline

      n/N (%)
      After Dose 4

      n/N (%)
      CD4 cells TNF-α (+), IFN-γ (+) (cutoff = 2.68)
       Cohort 10 of 11 (0%)5 of 10 (50%)3 of 9 (33%)0 of 11 (0%)6 of 10 (60%)
       Cohort 22 of 11 (18%)7 of 10 (70%)6 of 10 (60%)1 of 11 (9%)7 of 10 (70%)
       Cohort 35 of 16 (31%)14 of 15 (93%)12 of 15 (80%)2 of 16 (13%)14 of 15 (93%)
      CD8 cells TNF-α (+), IFN-γ (+) (cutoff = 1.15)
       Cohort 12 of 11 (18%)1 of 9 (11%)0 of 8 (0%)0 of 11 (0%)0 of 9 (0%)
       Cohort 20 of 11 (0%)0 of 10 (0%)0 of 10 (0%)0 of 11 (0%)0 of 10 (0%)
       Cohort 31 of 16 (6%)1 of 13 (8%)0 of 13 (0%)0 of 16 (0%)1 of 13 (8%)
      Note: See the Supplementary Data for details of the methods of assessment of cell-mediated immunity responses. N is number of patients in the according-to-protocol cohort at each visit (for response rates, N is the number of patients with preimmunization and postimmunization results), and n is the number of responders (column 4) or number with immunogenicity score (column 5) or frequency (column 6) above the defined cutoff.
      PRAME, preferentially expressed antigen of melanoma; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ.
      a See Supplementary Data for definition of immunogenicity score.
      b Response rate means that the ratio of immunogenicity scores positive postimmunization sample over baseline) is 4 or greater.
      c Frequencies of PRAME-specific T cells were estimated on the basis of quantification of defined positive wells in the in vitro assay (cutoff = 6.32 × 10-6 for CD4-positive cells and 1.9 × 10-6 for CD8-positive cells).
      Figure 3
      Figure 3Preferentially expressed antigen of melanoma (PRAME)-specific CD-positive T-cell (tumor necrosis factor-α–positive/interferon-γ–positive) immunogenicity scores before treatment and after dose 4 (according-to-protocol cohort for immunogenicity). Vertical lines indicate 95% confidence intervals, dotted line shows cutoff (2.68). See for details of the derivation of cutoffs and methods. Pre, before dose 1; post IV, 3 weeks after the fourth dose; Post IV CT, patients who had received platinum-based chemotherapy before study enrollment; Post IV no CT, patients who had not received prior chemotherapy.
      After four doses, the numbers of patients with PRAME-specific CD8-positive T cells (TNF-α–positive/IFN-γ–positive) immunogenicity scores equal to or above the cutoff were one of 14 in cohort 1, zero of 13 in cohort 2, and one of 14 in cohort 3. No CD8-positive T-cell responder could be identified (see Table 3).

      Dose Selection

      All doses fulfilled the predefined criteria for dose selection in terms of safety and humoral immunogenicity. There was a tendency toward better induction of anti-PRAME CD4+ T cells with increasing antigen dose without a major increase in AE frequency. Thus, as defined per protocol, the dose of 500 μg was selected for further investigation.

      Discussion

      This phase I study was designed to select a recPRAME dose for further development on the basis of safety and immunogenicity criteria. We observed no cases of DLT, and the safety profile was consistent with that in a parallel study of the PRAME immunotherapeutic in patients with melanoma.

      Gutzmer R, Rivoltini L, Levchenko E, et al. Safety and immunogenicity of the PRAME cancer immunotherapeutic in metastatic melanoma: results of a phase I/II dose escalation study. ESMO Open. 2016;1:e000068.

      In all patients, the PRAME immunotherapeutic induced a humoral response that appeared to be independent of the dose level. Pretreatment anti-PRAME antibodies were observed in only two patients, although our sample size of 60 patients was too small to provide definitive estimates of baseline anti-PRAME antibody production in patients with resected PRAME-positive NSCLC tumors. Spontaneous humoral immune responses against PRAME have not been described.
      • Weber J.S.
      • Vogelzang N.J.
      • Ernstoff M.S.
      • et al.
      A phase 1 study of a vaccine targeting preferentially expressed antigen in melanoma and prostate-specific membrane antigen in patients with advanced solid tumors.
      As in our experience with the PRAME immunotherapeutic, few patients with NSCLC and melanoma expressing MAGE-A3 had baseline anti–MAGE-A3 antibodies.
      • Stockert E.
      • Jäger E.
      • Chen Y.T.
      • et al.
      A survey of the humoral immune response of cancer patients to a panel of human tumor antigens.
      • Vansteenkiste J.
      • Zielinski M.
      • Linder A.
      • et al.
      Adjuvant MAGE-A3 Immunotherapy in resected non-small-cell lung cancer: phase II randomized study results.
      By contrast, baseline antibodies to other tumor antigens such as NY-ESO-1 are more frequently detected (up to 23% for NY-ESO-1 baseline antibodies in NSCLC
      • Türeci O.
      • Mack U.
      • Luxemburger U.
      • et al.
      Humoral immune responses of lung cancer patients against tumor antigen NY-ESO-1.
      ). The ability of some patients to mount an immune response to tumor antigens may reflect differences in tumor biology, individual immune competence, and previous or ongoing natural responses against the tumor.
      The percentage of patients showing PRAME-specific CD4-positive responses appeared to be dose dependent and was observed to be highest in cohort 3. In a parallel study in patients with metastatic melanoma, the PRAME immunotherapeutic also induced humoral immune responses in all patients and a CD4-positive response in most patients.

      Gutzmer R, Rivoltini L, Levchenko E, et al. Safety and immunogenicity of the PRAME cancer immunotherapeutic in metastatic melanoma: results of a phase I/II dose escalation study. ESMO Open. 2016;1:e000068.

      In our study, signs of CD8-positive T-cell immunogenicity were detectable in a few patients and protocol-defined CD8-positive T cells responses were absent. By contrast, PRAME-specific CD8-positive T-cell immunogenicity was reported ex vivo in patients with leukemia.
      • Rezvani K.
      • Yong A.S.M.
      • Tawab A.
      • et al.
      Ex vivo characterization of polyclonal memory CD8+ T-cell responses to PRAME-specific peptides in patients with acute lymphoblastic leukemia and acute and chronic myeloid leukemia.
      Immunization of patients with acute myeloid leukemia with dendritic cells generated from autologous leukemic blasts induced specific CD8-positive T-cell responses (using enzyme-linked immunospot assay) against PRAME.
      • Li L.
      • Giannopoulos K.
      • Reinhardt P.
      • et al.
      Immunotherapy for patients with acute myeloid leukemia using autologous dendritic cells generated from leukemic blasts.
      CD8-positive cells are considered the main effector cells involved in direct killing of malignant cells, and adoptive cell transfer of tumor-infiltrating CD8-positive cells to patients with melanoma can mediate tumor regression.
      • Rosenberg S.A.
      • Yang J.C.
      • Sherry R.M.
      • et al.
      Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy.
      However, recent evidence points to an important and previously underestimated role of CD4-positive cells in facilitating CD8-positive cell activity and in direct killing of tumor cells.
      • Melief C.J.M.
      “License to kill” reflects joint action of CD4 and CD8 T cells.
      • Tomita Y.
      • Yuno A.
      • Tsukamoto H.
      • et al.
      Identification of promiscuous KIF20A long peptides bearing both CD4+ and CD8+ T-cell epitopes: KIF20A-epecific CD4+ T-cell immunity in patients with malignant tumor.
      In addition to influencing development of CD8-positive cell memory, the anticancer activities of CD4-positive cells include directly and indirectly activating cytotoxic killing and production of cytokines that impact tumor cell–aging mechanisms.
      • Melief C.J.M.
      “License to kill” reflects joint action of CD4 and CD8 T cells.
      Adoptive transfer of CD4-positive T cells along with CD8-positive T cells in mice appeared to improve and prolong therapeutic efficacy.
      • Church S.E.
      • Jensen S.M.
      • Antony P.A.
      • Restifo N.P.
      • Fox B.A.
      Tumor-specific CD4(+) T cells maintain effector and memory tumor-specific CD8(+) T cells.
      Thus, strong CD4-positive responses induced by the PRAME immunotherapeutic may point to the presence of enhanced antitumor activity. Furthermore, recent results of large trials evaluating immune checkpoint inhibitors and another cancer immunotherapeutic suggest that the induction of tumor-specific immune responses may be insufficient for improving clinical outcomes in the absence of releasing the immune blockade (Vansteenkiste et al., in press).
      • Ramnath N.
      • Dilling T.J.
      • Harris L.J.
      • et al.
      Treatment of stage III non-small cell lung cancer: diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines.
      • Arriagada R.
      • Bergman B.
      • Dunant A.
      • Le Chevalier T.
      • Pignon J.-P.
      • Vansteenkiste J.
      Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer.
      • Douillard J.-Y.
      • Rosell R.
      • De Lena M.
      • et al.
      Adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage IB-IIIA non-small-cell lung cancer (Adjuvant Navelbine International Trialist Association [ANITA]): a randomised controlled trial.
      • Strauss G.M.
      • Herndon 2nd, J.E.
      • Maddaus M.A.
      • et al.
      Adjuvant paclitaxel plus carboplatin compared with observation in stage IB non-small-cell lung cancer: CALGB 9633 with the Cancer and Leukemia Group B, Radiation Therapy Oncology Group, and North Central Cancer Treatment Group Study groups.
      • Scagliotti G.V.
      • Fossati R.
      • Torri V.
      • et al.
      Randomized study of adjuvant chemotherapy for completely resected stage I, II, or IIIA non-small-cell lung cancer.
      • Brahmer J.R.
      Harnessing the immune system for the treatment of non-small-cell lung cancer.
      • Hall R.D.
      • Gray J.E.
      • Chiappori A.A.
      Beyond the standard of care: a review of novel immunotherapy trials for the treatment of lung cancer.
      • Chen D.S.
      • Mellman I.
      Oncology meets immunology: the cancer-immunity cycle.
      • Mellman I.
      • Coukos G.
      • Dranoff G.
      Cancer immunotherapy comes of age.
      • Decoster L.
      • Wauters I.
      • Vansteenkiste J.F.
      Vaccination therapy for non-small-cell lung cancer: review of agents in phase III development.
      • Lynch T.J.
      • Bondarenko I.
      • Luft A.
      • et al.
      Ipilimumab in combination with paclitaxel and carboplatin as first-line treatment in stage IIIB/IV non-small-cell lung cancer: results from a randomized, double-blind, multicenter phase II study.
      • Brahmer J.R.
      • Tykodi S.S.
      • Chow L.Q.M.
      • et al.
      Safety and activity of anti-PD-L1 antibody in patients with advanced cancer.
      • Topalian S.L.
      • Hodi F.S.
      • Brahmer J.R.
      • et al.
      Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.
      • Pardoll D.M.
      The blockade of immune checkpoints in cancer immunotherapy.
      • Langer C.J.
      Emerging Immunotherapies in the treatment of non-small cell lung cancer (NSCLC): the role of immune checkpoint inhibitors.
      • Vansteenkiste J.F.
      • Cho B.C.
      • Vanakesa T.
      • et al.
      Efficacy of the MAGE-A3 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small-cell lung cancer (MAGRIT): a randomised, double-blind, placebo-controlled, phase 3 trial.
      As observed in the parallel study in metastatic melanoma, no patient had a CD8-positive T-cell response according to the predefined response criteria, although signs of T-cell immunogenicity were detected. Very low levels of antigen-specific CD8-positive cells induced by vaccination with a tumor antigen have been reported previously.
      • Carrasco J.
      • Van Pel A.
      • Neyns B.
      • et al.
      Vaccination of a melanoma patient with mature dendritic cells pulsed with MAGE-3 peptides triggers the activity of nonvaccine anti-tumor cells.
      Furthermore, active immunotherapy with recombinant proteins has not been associated with substantial CD8-positive T-cell responses.
      • Valmori D.
      • Souleimanian N.E.
      • Tosello V.
      • et al.
      Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming.
      Thus, although the sensitivity and specificity of T-cell assays will directly affect detection rates, it is likely that the weak T-cell responses we observed are due to low induction of antigen-specific CD8-positive responses by the recombinant protein. It is worth noting that at the time of the study, the role of CD8-positive T cells and checkpoint inhibition in anticancer responses was less well recognized than today. Our data raise the question whether a combination of the PRAME immunotherapeutic and a checkpoint inhibitor would have enhanced effects on cytotoxic T cells.
      CMI responses should be included as end points for future studies of cancer immunotherapeutics.
      An exploratory analysis showed no negative impact of prior chemotherapy on humoral or CMI responses.
      In conclusion, consistent with a parallel phase I dose escalation study in patients with metastatic melanoma, in this study conducted in patients with resected NSCLC, the PRAME immunotherapeutic dose of 500 μg was immunogenic in most patients with NSCLC with a clinically acceptable safety profile. This dose was selected for further evaluation in a randomized phase II study (NCT01853878). However, the study was stopped early when results of a large phase III study of another similar immunotherapeutic showed no benefit of treatment compared with placebo in patients with NSCLC.
      • Vansteenkiste J.F.
      • Cho B.C.
      • Vanakesa T.
      • et al.
      Efficacy of the MAGE-A3 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small-cell lung cancer (MAGRIT): a randomised, double-blind, placebo-controlled, phase 3 trial.

      Acknowledgments

      GlaxoSmithKline Biologicals S. A. (GSK) (Rixensart, Belgium) was the funding source and was involved in all stages of the study conduct and analysis. GSK also funded all costs associated with the development and publishing of this article. All authors had full access to the data. The authors thank the patients who participated in this study and their families. They also acknowledge the investigators and their clinical teams for their contribution to the study and their support and care of patients, in particular, Chiara Catania, Kimberly Costas, Mikl Fedianin, Oleg Gladkov, Tomasz Grodzki, Michael Guarino, Leszek Herjan, John Kucharczuk, Charles Marquette, Xavier Quantin, Christian Schulz, Axel Skubala, and Howard West. The authors thank the global and regional clinical operations, in particular, Isabelle Benoot and Frédéric Basyn, and safety teams of GSK Vaccines for their contribution to the study, the scientific writer for clinical protocol and clinical report writing, and the team of statisticians at GSK for the statistical analysis, in particular, Carline Vanden Abeele. The authors thank Response Genetics, Inc. (Los Angeles, CA) for its technical support. The authors thank Dr. Joanne Wolter (medical writer on behalf of GSK Vaccines) for assistance in preparing the first draft of the manuscript, and Dr. Sophie Timmery (XPE Pharma and Science) for coordination and editorial assistance.

      Supplementary Data

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