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Phase I Clinical Trial of HER2-Specific Immunotherapy with Concomitant Hamilton et al. Journal of Translational Medicine 2012, 10:28 http://www.translational-medicine.com/content/10/1/28 RESEARCH Open Access Phase I clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibtion Erika Hamilton1, Kimberly Blackwell1, Amy C Hobeika2, Timothy M Clay3, Gloria Broadwater4, Xiu-Rong Ren5, Wei Chen5, Henry Castro3, Frederic Lehmann3, Neil Spector1, Junping Wei6, Takuya Osada6 and H Kim Lyerly2* Abstract Background: Patients with HER2-overexpressing metastatic breast cancer, despite initially benefiting from the monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib, will eventually have progressive disease. HER2-based vaccines induce polyclonal antibody responses against HER2 that demonstrate enhanced anti-tumor activity when combined with lapatinib in murine models. We wished to test the clinical safety, immunogenicity, and activity of a HER2-based cancer vaccine, when combined with lapatinib. Methods: We immunized women (n = 12) with metastatic, trastuzumab-refractory, HER2-overexpressing breast cancer with dHER2, a recombinant protein consisting of extracellular domain (ECD) and a portion of the intracellular domain (ICD) of HER2 combined with the adjuvant AS15, containing MPL, QS21, CpG and liposome. Lapatinib (1250 mg/day) was administered concurrently. Peripheral blood antibody and T cell responses were measured. Results: This regimen was well tolerated, with no cardiotoxicity. Anti-HER2-specific antibody was induced in all patients whereas HER2-specific T cells were detected in one patient. Preliminary analyses of patient serum demonstrated downstream signaling inhibition in HER2 expressing tumor cells. The median time to progression was 55 days, with the majority of patients progressing prior to induction of peak anti-HER2 immune responses; however, 300-day overall survival was 92% (95% CI: 77-100%). Conclusions: dHER2 combined with lapatinib was safe and immunogenic with promising long term survival in those with HER2-overexpressing breast cancers refractory to trastuzumab. Further studies to define the anticancer activity of the antibodies induced by HER2 vaccines along with lapatinib are underway. Trial registry: ClinicalTrials.gov NCT00952692 Keywords: HER2, Antitumor immunity, Immunization, Breast cancer Introduction year. Lapatinib, a potent reversible inhibitor of HER2 The human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor (EGFR) tyrosine overexpressed in 20-30% of breast cancers, is associated kinases [3], in conjunction with chemotherapy, increases with more aggressive tumor behavior [1]. Treatment time to progression in these patients [4]. Unfortunately, with combinations of the anti-HER2 antibody trastuzu- responses to lapatinib are generally short-lived, and pro- mab and chemotherapy lengthens survival in patients gression remains a significant clinical problem. with metastatic HER2-overexpressing breast cancer [2]. Intriguingly, the overexpression of HER2 persists in However, progressive disease typically occurs within one trastuzumab and lapatinib-refractory tumors [5,6], and thus, targeting HER2 with cancer immunotherapy is a * Correspondence: [email protected] potentially effective strategy. A variety of vaccines tar- 2Department of Surgery, Division of General Surgery, Duke University geting HER2, based on proteins, peptides, modified Medical Center, Durham, NC, USA Full list of author information is available at the end of the article tumor cells, viral vectors, pDNA and dendritic cells © 2012 Hamilton et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hamilton et al. Journal of Translational Medicine 2012, 10:28 Page 2 of 9 http://www.translational-medicine.com/content/10/1/28 (DC) have been developed. Results from phase I and II been discontinued no sooner than four and three weeks, studies of HER2-targeting cancer vaccines [7] have respectively, before the first ASCI administration. Initi- demonstrated that HER2 is immunogenic, and that ally,priorlapatinibwasnotpermitted;however,this immune responses against HER2 may be associated with severely limited enrollment and therefore, an amend- an improved clinical outcome [8-13]. ment was made to permit prior and ongoing lapatinib One protein-based vaccine, dHER2 Antigen-Specific use. Known autoimmune disease, immunosuppressive Cancer Immunotherapeutic (ASCI) a recombinant therapies or HIV, significant cardiovascular disease or HER2 protein, including a truncated intracellular arrhythmias were exclusionary criteria. domain (ICD) and the complete extracellular domain (ECD), combined with the immunological adjuvant Treatment/Monitoring AS15,containingMPL,QS21,CpGandliposome,was The dHER2 ASCI containing 500 μg of recombinant evaluated in two early phase clinical studies of patients dHER2 protein (a truncated intra-cellular sequence with HER2-overexpressing breast cancer (NCT (ICD) and the complete extracellular sequence (ECD)), 00058526 and NCT 00140738) [14]. In both studies, the reconstituted in the AS15 adjuvant, a liposomal formu- data showed that dHER2 immunizations were well toler- lation containing MPL, QS21 and CpG, was adminis- ated, consistently immunogenic at the 500 μgdoseand tered intramuscularly every 2 weeks for 6 that clinical activity (including prolonged stable disease) administrations (See Figure 1 depicting one cycle). Up was associated with antibody and T cell responses. to three total cycles of immunization were permitted One important observation from the prior dHER2 unless discontinued due to progression of disease. Clini- ASCI studies was that the polyclonal antibody-contain- cal tumor evaluations were conducted at baseline and at ing serum from immunized patients had functional the end of the cycles (e.g., week 12, week 26). Lapatinib activity against signaling pathways mediated by HER2. (1250 mg) was taken by mouth daily. Specifically, incubation of breast cancer cell lines with Cardiac monitoring consisted of a 12-lead ECG at serumfromtwoimmunizedpatientsdemonstratedan screening, and at week 12, week 26, and follow-up visit impact on molecular pathways resembling that of trastu- #1 and a MUGA evaluation of ejection fraction (EF) at zumab [14]. Because clinical trials have demonstrated screening, week 6, 12, 20, 26, follow-up visits #1 and #4. that combinations of lapatinib and trastuzumab lead to enhanced clinical activity and combined effects on sig- Analysis of anti-HER2 antibody binding by ELISA naling pathways [15], there has been interest in combin- Serum was collected pre- (week 0) and every 2 weeks ing the polyclonal anti-HER2 serum with trastuzumab while on study. 96-well plates were coated with HER2 and indeed, increased apoptosis of human HER2-overex- ICD protein (5 μg/well) or HER2 ECD protein (5 μg/ pressing breast cancer cells was observed when lapatinib well) and incubated with 100 μl of serially diluted was combined with HER2-specific polyclonal antisera patient serum or trastuzumab. Plates were then incu- generated from rabbits immunized with dHER2 ASCI bated with mouse anti-human IgG conjugated alkaline [16]. We therefore hypothesized that the lapatinib would phosphatase (AP) (Sigma) and developed using p-nitro- enhance the anti-signaling activity of the polyclonal Abs phenyl phosphate (Sigma). Absorbance (415 nm) was induced by the dHER2 vaccine in humans. First, it was read using a BioRad microplate reader. Titers were necessary to establish that the induction of anti-HER2 defined as the greatest dilution at which the mean antibodies by the dHER2 vaccine was not affected by absorbance was at least twice negative control. lapatinib and this was the primary purpose of this study. ELISPOT analysis Methods IFN-g ELISPOT assays (Mabtech Inc., Cincinnati, OH) Patients were performed according to the manufacturer’s instruc- Patients provided consent under a protocol approved by tions as previously described 16]. Briefly, PBMC the Duke University Medical Center Institutional Review (250,000 cells/well) were added to each well and stimu- Board. Enrollment requirements were age 18 or older, lated overnight with HER2 ICD or HER2 ECD overlap- stage IV HER2- overexpressing (HER2 3+ or FISH +) ping peptide mixes (1 μg/ml) to detect HER2-specific breast cancer, documented disease progression or responses, or CMV peptide mix (2.6 μg/ml), HIV pep- relapse following at least one prior standard therapy tide mix (2.6 μg/ml) (both from BD Bioscience) and a containing trastuzumab, ECOG status of 0 or 1, ade- mixture of PMA (50 ng/ml) and Ionomycin (1 μg/ml) as quate hematologic counts, hepatic and renal function controls. Results were normalized to the number of and an LVEF of 50% or greater. Concurrent bisphospho- spots per 1 × 106 PBMC. Based on our previously nates and hormonal therapy were permitted. Prior che- reported experience [17], a positive response was motherapy and/or trastuzumab were required to have defined as an increase by ≥ 40 spots from baseline. Hamilton et al. Journal of Translational Medicine 2012, 10:28 Page 3 of 9 http://www.translational-medicine.com/content/10/1/28 Figure 1. Interval between injections 2 weeks 2 weeks Continue treatment in case of CR, PR, SD or MR at TE Daily dose of lapatinib dHER2 + AS15 ASCI 1234 5 6 7 8 9 101112
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