Drug Evaluation For reprint orders, please contact: [email protected] HER2 peptide nelipepimut-S (E75) vaccine (NeuVax™) in breast cancer patients at risk for
The HER2 peptide nelipepimut-S (E75) vaccine (NeuVax™) in breast cancer patients at risk for recurrence: correlation of immunologic data with clinical response
Nelipepimut-S (formerly known as E75) is an immunogenic peptide from the HER2 Erika J Schneble1, John S protein that is highly expressed in breast cancer. The NeuVax™ (Galena, OR, USA) Berry1, Francois A Trappey1, vaccine, nelipepimut-S plus granulocyte–macrophage colony-stimulating factor, is Guy T Clifton1, Sathibalan 2 designed for the prevention of clinical recurrences in high risk, disease-free breast Ponniah , Elizabeth Mittendorf3 & George E cancer patients. Although cancer vaccines such as NeuVax represent promising Peoples*,1 approaches to cancer immunotherapy, much remains to be elucidated regarding their 13AN !NTONIO -ILITARY -EDICAL #ENTER mechanisms of action: particularly given that multiple cancer vaccine trials have failed $EPARTMENT OF 'ENERAL 3URGERY &ORT to demonstrate a correlation between immunologic data and clinical outcome. Here, 3AM (OUSTON 48 53! we briefly discuss our clinical trial experience with NeuVax focusing on immunologic #ANCER 6ACCINE $EVELOPMENT response data and its implication on how the immune system may be affected by this ,ABORATORY 5NIFORMED 3ERVICES 5NIVERSITY OF THE (EALTH 3CIENCES peptide vaccine. Most importantly, we demonstrate the potential capability of certain "ETHESDA -$ 53! immunologic assays to predict clinical benefit in our trials. 35NIVERSITY OF 4EXAS -$ !NDERSON #ANCER #ENTER (OUSTON 48 53! Keywords: BREAST CANCER s % s (%2 PEPTIDE VACCINE s IMMUNOTHERAPY s NELIPEPIMUT 3 !UTHOR FOR CORRESPONDENCE 4EL &AX Background breast cancer patients decreasing recurrence GEORGEPEOPLES HOTMAILCOM The HER2/neu proto-oncogene, a member rates by approximately 50% [6–8]. of the EGF receptor family, is expressed in Recent data show that patients with a a variety of malignancies including breast HER2-overexpressing tumor treated with cancer. Historically, HER2 overexpression trastuzumab have a much better prognosis (immunohistochemistry [IHC] 3+ and/or than HER2 low-to-intermediate (IHC 1–2+) FISH >2.2]) has been associated with a poor patients not treated with trastuzumab [9]. How- prognosis. Given that the overexpression of ever, trastuzumab is limited in that it has only HER2 is associated with more aggressive been shown to be efficacious in the 20–30% subtypes of breast cancer and other adeno- of patients with tumors of the highest level of + carcinomas, HER2 is an attractive immuno- HER2 expression (IHC 3 ) [10] . HER2 low- logic target for both cell-mediated immunity, to-intermediate patients represent over 50% of including cytotoxic T cells, and antibody- breast cancer patients [10,11]. Thus, there exists mediated immunotherapy approaches [1,2]. a need to develop other adjuvant approaches Currently, in addition to traditional thera- to treat this HER2 low-to- intermediate- pies of surgery, chemotherapy, radiation and expressing population while avoiding the tox- endocrine therapy, a small percentage of icities associated with trastuzumab and other breast cancer patients benefit from trastu- HER2-targeted therapies. zumab and other HER2-targeted therapies An ideal immunologic therapy would including pertuzumab [3,4] and trastuzumab induce immunologic memory, making clinical emtasine [5]. Trastuzumab, a mono clonal response long-acting and sustainable. Trastu- antibody targeting the highest level of zumab and other monoclonal antibodies are HER2-overexpressing tumor cells, provides limited by passive mechanism of actions such as a proven disease-free survival (DFS) benefit ADCC. Without full activation of the immune to high-risk node- negative and node-positive system, there is little to no immunologic part of
10.2217/IMT.14.22 Immunotherapy (2014) 6(5), 519–531 ISSN 1750-743X 519 Drug Evaluation Schneble, Berry, Trappey et al.
memory. Cancer vaccines are a form of active immu- Laboratory & clinical studies notherapy where the body’s own immune system is uti- Nelipepimut-S has been studied in multiple labora- lized to target and kill tumor cells. The appeal of this tory and clinical studies [13,14,20–27]. The ability for active immunotherapeutic strategy includes the ability nelipepimut -S to stimulate CTL to lyse HER2- expressing to generate a sustained T-cell response and immunologic targets in the preclinical setting was established in both memory with minimal toxicity. More specifically, vac- ex vivo/in vitro studies of ovarian and breast tumor cells cines target tumor-associated antigens (TAAs) that are lines [13,27] and animal models (Table 1) [28,29]. Pharmoki- recognized by human cytotoxic T lymphocytes (CTLs), netic studies within animal models were not performed which are critical for the removal of tumor cells in vivo. given the small peptide dose used and rapid peptide HER2, the most studied TAA in breast cancer, has been clearance. As such, typical pharmokinetic parameters shown to elicit a specific immune response. Identification (e.g., absorption, plasma protein binding, distribution, of HLA-A2 as a restriction element in HER2-expressing metabolism and excretion) were not established. cancers [12] led to the discovery of CTL-eliciting epi- In addition to administering nelipepimut-S with GM- topes, nelipepimut-S and GP2 [13,14]. Nelipepimut-S, a CSF, other vaccine formulations utilizing the peptide have nine-amino-acid peptide derived from the extracellular included: loading the peptide onto autologous dendritic HER2 protein (KIFGSLAFL, HER2 p369-377) [13], is cells [20,24], inclusion into longer peptides to elicit both + the most studied HER2-derived peptide to date [15] . CTL and CD4 helper T-cell response [21,22], and use of a single peptide with different immuno adjuvants [23,25,26]. Introduction to NeuVax™ (Galena, OR, USA) All Phase I clinical trials utilizing GM-CSF as an immu- NeuVax™ (Galena, OR, USA), a vaccine comprised of noadjuvant revealed nelipepimut-S-specific CTL expan- nelipiminut-S and the hematopoietic growth factor, gran- sion [23,25,31]. However, despite the effective stimulation ulocyte–macrophage colony-stimulating factor (GM- of a nelipepimut-S-specific T-cell response, little was CSF), holds promise as an effective adjuvant therapy known from the initial trials regarding antitumor activ- for women with low-to-intermediate HER2 expression. ity secondary to the fact that these trials enrolled patients GM-CSF is known to induce proliferation, maturation with advanced disease (Table 2) [23,25,26]. and migration of dendritic cells [16,17], and it is intended Our group has extensively studied the nelipepimut-S to enhance nelipepimut-S-specific immunity through peptide as a vaccine but with a shift of focus from the augmentation of antigen presentation to CTLs [1,16,17]. treatment of late-stage disease to the prevention of disease Nelipepimut-S binds HLA-A2 and HLA-A3 [18,19] mol- recurrence in high-risk patients after standard-of-care ecules (expressed in ∼60–75% of the general population) therapy. In our Phase I/II clinical trials, we enrolled both on antigen presenting cells that then stimulate CTLs to node-positive and high-risk node-negative breast cancer recognize and lyse HER2-expressing tumor cells. patients who have been rendered clinically free of disease
Table 1. Selected preclinical studies of the ability for nelipepimut-S to stimulate cytotoxic T lymphocytes to lyse HER2-expressing targets. Study (year) Study design Immunologic response/study conclusion Ref.
Fisk et al. (1995) Fresh cell lines from ovarian s First described nelipepimut-S (KIFGSLAFL, HER2/neu, p369-377) [13] cancer pts s Identified common immunogenic epitopes of HER2 that were recognized by isolated CD3+CD4+CD8+ ovarian-specific CTL lines s Nelipepimut-S epitope from tumor-associated lymphocytes from HLA-A2+ tumor-associated lymphocytes dominantly recognized by all CTL lines tested
Lustgarten et al. In vitro/in vivo animal model s Nelipepimut-S capable of stimulating potent CTL response that lysed [28] (1997) tumor cells in vivo s Nelipepimut-S dose-dependent HER2 tumor-killing effect by nelipepimut-S-specific CTL in this mouse model
Disis et al. (1998) In vitro/in vivo animal model; s No dose-limiting toxicity on necropsy [29] nelipeminut-S-containing s No evidence of autoimmunity HER2-derived peptides plus s Significant T-cell response detected GM-CSF or IFA
Anderson et al. Ten healthy human donors s Nelipepimut-S-specific cytolytic activity in healthy donors when [30] (2000) presented on autologous DCs elicited in 50% of healthy donors #4, #YTOTOXIC 4 LYMPHOCYTE $# $ENDRITIC CELL '- #3& 'RANULOCYTEnMACROPHAGE COLONY STIMULATING FACTOR )&! )NCOMPLETE &REUNDS ADJUVANT PTS 0ATIENTS
520 Immunotherapy (2014) 6(5) future science group HER2 peptide nelipepimut-S (E75) vaccine (NeuVax™) in breast cancer patients at risk for recurrence Drug Evaluation
Table 2. Selected clinical studies examining the ability of nelipepimut-S to stimulate cytotoxic T lymphocytes. Study (year), phase, Patient sample Clinical response Toxicity/immunogenic response Ref. design size
Zaks and Rosenberg Four pts Not disclosed s Peptide-specific CTLs detected in post- but not pre- [26] (1998), nelipepimut-S (breast, ovarian immunization blood plus IFA and colorectal s CTLs failed to react with HER2+ tumor cells cancer)
Disis et al. (1999) and 64 pts (breast, Not disclosed s 92% of patients develop T-cell immunity to HER2 peptide [21,22] Disis et al. (2002), ovarian and and 68% to HER2 protein domain subdominant HER2- NSCLC) s Epitope spreading in 84% of patients derived epitope plus s 38% of patients maintained immunity to HER2 protein at GM-CSF 1 year
Brossart et al. (2000), Ten pts Not disclosed s No side effects noted [32] pilot, peptide loaded (advanced s Major CTL response in vivo was induced with the HER2- onto autologous DCs breast and derived nelipepimut-S and the MUC1-derived M1.2 ovarian cancer) peptide, which lasted >6 months s Patient immunized with the HER2-derived peptides, MUC1-specific T lymphocytes were induced after seven immunizations, suggesting that antigen spreading in vivo may occur after successful immunization with a single tumor antigen
Knutson et al. Six pts; Not disclosed s Minimal toxicity [23] (2002), Phase I, stage III/IV s Nelipepimut-S-specific precursors developed in two nelipepimut-S breast (4)/ out of four evaluable patients and the responses were peptide plus GM-CSF ovarian (2) short-lived and not detectable at 5 months after the final cancer vaccination
Murray et al. 14 pts; stage Not disclosed s Vaccine found to be safe, no grade 3/4 toxicities [25] (2002), Phase I, IV breast (13) s Four out of the eight patients showed CTL-mediated lytic nelipepimut-S plus and ovarian (1) activity GM-CSF cancer pts
Kono et al. (2002), Nine pts One out of nine s No serious adverse side effects [24] Phase I, peptide (gastric cancer) pts with decreased s HER2 peptide-specific recognition demonstrated in six loaded onto tumor marker; one out of nine patients after immunization, whereas no autologous DCs out of nine with HER2 peptide-specific recognition was present prior to stabilization of immunization disease status ×3 s Peptide-specific delayed-type hypersensitivity response months occurred in three out of nine patients
Peoples et al. 53 pts; early- DFS VG versus CG: s Minor toxicity only (one grade 3–4%) [31] (2005), Phase I, stage NP breast 85.7 versus 59.8% s Nelipepimut-S-specific CTL expansion exhibited in all pts nelipepimut-S plus cancer post- at 22 months s DTH VG versus CG (33 vs 7 mm; p < 0.01) GM-CSF standard-of- (p < 0.19) care therapy
Peoples et al. 186 pts: early- VG versus CG s Mild local/systemic toxicities [34] (2008), Phase I/II, stage breast recurrence s 74% positive postvaccine DTH with an average induration nelipepimut-S plus cancer post- (5.6 vs 14.2%, of VG versus CG (14.0 ± 1.4 mm vs 2.1 ± 0.5 mm, GM-CSF standard-of- p = 0.04) at p < 0.0001) care therapy 20 months; s Nelipepimut-S-specific CTL proliferation peak levels after (NP = 95; (8.3 vs 14.8%) at the third or fourth dose in 65% of patients NN = 91) 26 months; VG s Statistically significant increase in median percentage VG = 101, with increased nelipepimut-S-CTLs pre- to post-vaccination CG = 85 time to recurrence s 43% pts maintained significant residual immunity (11 vs 8 months) (dimer > 0.5) 6 months postvaccination #' #ONTROL GROUP #4, #YTOTOXIC 4 CELL LYMPHOCYTE $# $ENDRITIC CELL $&3 $ISEASE FREE SURVIVAL $4( $ELAYED TYPE HYPERSENSITIVITY '- #3& 'RANULOCYTEn MACROPHAGE COLONY STIMULATING FACTOR )&! )NCOMPLETE &REUNDS ADJUVANT -5# -UCIN .. .ODE NEGATIVE .0 .ODE POSITIVE .3#,# .ON SMALL CELL LUNG CANCER PTS 0ATIENTS 6' 6ACCINE GROUP
future science group www.futuremedicine.com 521 Drug Evaluation Schneble, Berry, Trappey et al.
Table 2. Selected clinical studies examining the ability of nelipepimut-S to stimulate cytotoxic T lymphocytes (cont.). Study (year), phase, Patient sample Clinical response Toxicity/immunogenic response Ref. design size
Mittendorf et al. 182 pts: 106 VG versus CG DFS subset analyses (VG vs CG): [33] (2012), Phase I/II, (VG), 76 (CG) 24-month DFS s Lymph node-positive (90.2 vs 79.1%; p = 0.13) nelipepimut-S plus 94.3 versus 86.8% s HER2 IHC 1+−2+ (94.0 vs 79.4%; p = 0.04) GM-CSF (p = 0.08) s Grade 1 or 2 tumors (98.4 vs 86.0%; p = 0.01) s Optimally dosed (97.3 vs 86.8%; p = 0.08) #' #ONTROL GROUP #4, #YTOTOXIC 4 CELL LYMPHOCYTE $# $ENDRITIC CELL $&3 $ISEASE FREE SURVIVAL $4( $ELAYED TYPE HYPERSENSITIVITY '- #3& 'RANULOCYTEn MACROPHAGE COLONY STIMULATING FACTOR )&! )NCOMPLETE &REUNDS ADJUVANT -5# -UCIN .. .ODE NEGATIVE .0 .ODE POSITIVE .3#,# .ON SMALL CELL LUNG CANCER PTS 0ATIENTS 6' 6ACCINE GROUP
after surgery, chemotherapy and radiation as indicated modalities could provide insight into a vaccine’s specific + (Table 2) [31,33,34]. In the Phase I/II trials, HLA-A2/A3 immunologic mechanisms of action as well as early infor- breast cancer patients with any level of HER2 expression mation regarding clinical efficacy. However, the optimal were enrolled into the vaccine group while HLA-A2/A3- method to monitor activity of these peptide vaccines is patients were followed prospectively as a control group. not clear. HLA-A2/3 has never been reported as an independent Multiple failed cancer vaccine trials (e.g., Theratope, prognostic factor in breast cancer. Furthermore, a com- Stimuvax and Canvaxin) [40–42] have supplied infor- parison of control patients from a separate randomized mation regarding the endogenous immune response to trial of adjuvant vaccines found no difference in survival malignancy. Particularly within the context of mela- between HLA-A2-positive and -negative patients [35]. noma research, past cancer vaccine trials have assessed The vaccine group received 4–6 monthly inoculations of efficacy with immune surrogate end points but have NeuVax during the primary vaccine series. A voluntary rarely been able to correlate this data to clinical benefit booster program was initiated, and patients electing to or prevention of tumor progression [43]. In 2013, Hail- participate were administered booster inoculations every emichael et al. specifically addressed clinical trials where 6 months out to 5 years post-primary vaccination series. CTL proliferation occurs without an associated benefi- These Phase I/II NeuVax clinical trials demonstrated that cial tumor response [44]. Here, incomplete Freund’s adju- the vaccine has minimal toxicity and is capable of stimu- vant (IFA), the most commonly used peptide vaccine lating peptide-specific T-cell expansion, even within the adjuvant to date, was assessed. IFA, a water-in-oil emul- context of low-level antigen expression [31,33,34,36] and sion, acts slowly to release antigen to the immune system after multiple booster doses [37]. Perhaps most impor- to enhance immunogenicity of vaccines. In a series of tantly, dose-responsive clinical benefit was shown. In experiments using a gp100 peptide plus IFA vaccine in the combined trials of node-positive and node-negative a mouse model, the authors observed a T-cell trap with patients, 60-month DFS was 89.7% in the vaccine group sequestration of CTL at the vaccination site. The seques- versus 80.2% in the control group (p = 0.08), although tered T cells underwent deletion without evidence of only 35% of patients were optimally dosed secondary immunologic memory and subsequent hyporesponsive- to trial design. The 5-year DFS approached significance ness. This diminished response occurred even in vacci- with 94.7% surviving disease free in the optimally dosed nation of B cell-deficient and CD4 knockout mice, thus patients in comparison to 80.2% in the control group eliminating B cells and Tregs as causative sources [44]. (p = 0.05) [38]. Interestingly, superior clinical benefit was Interestingly, vaccine trials utilizing IFA have failed to also shown in HER2 low-to-intermediate-expressing reveal objective clinical benefit [43], which, based on the patients versus controls (88.1 vs 77.5%, p = 0.16) [38]. work by Hailemichael et al., one may hypothesize is due Patients with low-to-intermediate HER2 expression will to IFA-associated CTL sequestration, dysfunction and thus serve as the vaccination population for the ongoing deletion [44]. Phase III clinical trial. CTL degradation within a T-cell trap provides some explanation as to the lack of observed clinical response Correlating immune & clinical responses despite the demonstration of CTL proliferation in An integral component of cancer vaccine trials is in vivo response to vaccines. The Phase I clinical trials of neli- and ex vivo immune response monitoring as a means to pepimut-S plus adjuvant [23,25,26,31] revealed E75-specific predict reaction to vaccination and potential clinical ben- CTL proliferation with associated antitumor activity in efit [39]. Current assays are designed to assess the type and all but the trial utilizing IFA [26]. As discussed above, magnitude of T-cell response over multiple time points our Phase I/II trials [31,33,34,36] evaluating nelipepimut-S throughout the trial. Theoretically, immune-monitoring have utilized GM-CSF (as opposed to IFA) as the
522 Immunotherapy (2014) 6(5) future science group HER2 peptide nelipepimut-S (E75) vaccine (NeuVax™) in breast cancer patients at risk for recurrence Drug Evaluation immunoadjuvant. As will be discussed below, we have response was assessed. In the node-negative trial, a pre- been able to correlate nelipepimut-S-specific immune vaccination DTH was measured as well and compared response and increased DFS. Such ability to correlate with the postvaccination DTH [34]. immune response with clinical benefit has alluded past Using the Luminex® assay (Luminex Molecular cancer vaccine trials set in the metastatic setting or in Diagnostics Inc., ON, Canada) to delineate cytokine patients with residual tumor burden. However, our trial profiles in our vaccinated patients, significant differ- design differs from previous studies in that we have ences were elucidated between vaccinated and unvac- administered the vaccine as an adjuvant therapy to high- cinated patients. Specifically, monocyte chemotactic risk, disease-free patients. Just as vaccination site T-cell protein-1 (MCP-1), a chemokine associated with stimu- trap is attributed to long-lasting adjuvants such as IFA, lating tumor-associated macrophages, angiogenesis and T cells may also be sequestered at the site of extensive metastasis [46], showed the most significant elevation tumor burden with its hostile microenvironment. Utiliz- postvaccination (p = 0.003) [47]. However, as opposed ing a vaccine in the setting of minimal to no residual to the metastatic setting where increased MCP-1 levels disease and with a rapidly degrading adjuvant such as are implicated in tumor progression, high MCP-1 levels GM-CSF, such as in our Phase I/II trials, avoids these act as favorable prognostic indicators within the context limitations. of an adjuvant cancer vaccine. An association between MCP-1 and pre-existing HER2 immunity has also been Immunogenic testing demonstrated [48–50]. NeuVax vaccination correlated Our group has performed extensive immunologic stud- with the largest MCP-1 increase in higher-risk patients ies to elucidate the specific immunologic mechanisms by with lower initial serum MCP-1 levels. This suggests which NeuVax confers clinical benefit. Again, as opposed that the larger the MCP-1 response, the greater the to clinical trials intended to treat later-stage patients, our vaccine-induced, peptide-specific immunity [51]. Thus, group has been able to correlate magnitude of peptide- cytokine profiling may identify patients with low initial specific immune response with clinical benefit [33]. We serum MCP-1 levels who are most likely to benefit from have previously outlined the immunologic monitoring vaccination. methods used in these trials including delayed-type Despite the presence of an immune response to Neu- hypersensitivity (DTH) reaction, dimer assay, ELISPOT Vax, the interplay between malignancy and the immune assay, assessment of regulatory T cells, cytokine evalua- system is a complex process where tumors are able to tion, epitope spreading and circulating tumor cell (CTC) escape immune recognition. The fact that most TAAs quantification as surrogates for immunologic response to are also self-antigens tolerated by the immune system is NeuVax [15] . a major obstacle in cancer immunotherapy. A subpopu- Phase I/II NeuVax clinical trials assessed ex vivo and lation of regulatory CD4+ T cells, Tregs, are implicated in vivo response by evaluating nelipepimut-S-specific in the process of immune response downregulation and CTL expansion and DTH, respectively. Peptide-specific escape from immune recognition [52]. Identification of CTL expansion was measured by the dimer assay. Briefly, the regulatory CD4+CD25+ T-cell population, appreci- nelipepimut-S peptide-loaded dimers are mixed with the ated for prevention of autoimmunity and regulation of patient’s peripheral blood sample. This sample is ana- inflammatory reactions, as well as implicated in sup- lyzed by flow cytometry to quantify the number of CD8+ pressing immune response against certain malignancies, T cells specifically bound to the peptide dimer molecules enhanced the understanding of CD4+ T cells’ impor- [45]. The assay is run on blood without any ex vivo manip- tance in immune response regulation [53,54]. Our group ulation and/or stimulation. Of note, vaccinated HER2 analyzed Treg levels in a subset of vaccinated patients. low-to-intermediate-expressing patients have displayed Here, postvaccination Treg levels significantly decreased larger maximum immunologic responses compared with despite an overall increase in levels of circulating CD4+ overexpressed patients (p = 0.04), an intriguing finding T cells [55]. given the reduced mortality of low-expressor patients Having shown that vaccination with nelipepimut-S compared with controls (p = 0.08) [36]. DTH is assessed plus GM-CSF can elicit clonal expansion of nelipepimut- by injecting 100 μg of nelipepimut-S in 0.5 ml of normal S-specific CTL by dimer assay, we next sought to deter- saline (without GM-CSF) 1 month after completion of mine whether vaccination with this single peptide was the vaccine series. Normal saline is injected as a paral- capable of inducing epitope spreading [56]. Although the lel volume control. 48–72 h later, the area of erythema mechanism of epitope spreading is not fully understood, and/or induration that develops in response to this injec- this phenomenon refers to the natural spread of immu- tion is measured in two dimensions and recorded as nity from one portion of an immunogenic protein to the orthogonal mean. In both of our node-positive and other areas within the same antigen (intra-antigenic) or node-negative early-phase trials, a postvaccination DTH to other antigens (interantigenic) [22]. We examined this
future science group www.futuremedicine.com 523 Drug Evaluation Schneble, Berry, Trappey et al.
phenomenon in 44 patients enrolled in the early stage patients. All patients with node-positive and 95% of the NeuVax clinical trials. Intra-antigenic epitope spreading node-negative patients showed nelipeminut-S- specific to GP2, a second MHC class I epitope derived from the clonal expansion, although node-negative patients HER2 protein, was observed in 100% of node-positive showed only moderate expansion to this subdominant and 85% of node-negative patients [56]. Of note, inter- epitope. This high percentage of epitope spreading in antigenic spreading to E41, an epitope from the folate clinically disease-free patients suggests the presence of binding protein antigen, was also shown in 63% of tested occult disease [56].
4.00
3.50
3.00
2.50
2.00
R6 dimer (mdi) 1.50
1.00 Recurrence above the mean 1/30 (3%) Mean R6 dimer = 0.63 mdi p = 0.09 0.50 Recurrence below the mean 8/56 (14%)
0.00 Patient
1. 0 96.7% ee 76.9% 0.9 85.7%
0.8 Log rank p = 0.12
0.7 R6 above the mean n = 30 Median f/u: action surviving disease fr R6 below the mean 60 months Fr n = 26 0.6 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Time (months)
Figure 1. Post-vaccination (R6) dimer levels in vaccinated patients and disease-free survival benefit of vaccine patients with a R6 dimer above the mean. (A) R6 dimer levels in vaccinated patients. Recurrences are shown in red. The mean R6 dimer in the vaccine group is 0.63 mdi ± 0.08. Of the 30 patients with an R6 dimer above the mean, only one recurred, compared with eight of the 56 below the mean (p = 0.09). (B) Disease-free survival benefit of vaccine patients with a R6 dimer above the mean (p = 0.12), a 76.9% relative risk reduction. f/u: Follow-up; mdi: Mean dimer index.
524 Immunotherapy (2014) 6(5) future science group HER2 peptide nelipepimut-S (E75) vaccine (NeuVax™) in breast cancer patients at risk for recurrence Drug Evaluation
In order to further investigate the presence of occult inconsistent assay conditions, CTC hold potential as disease, we have used the CellSearch® system (Veri- an extremely sensitive, clinically relevant surrogate dex, NJ, USA) to quantify and phenotype CTCs to monitor vaccine response in clinically disease-free [57]. Whereas ex vivo phenotypic (dimer/multimer) patients. In the metastatic setting, substantial numbers and functional (ELISPOT, cytokine flow cytometry) of CTC have been identified in up to 70% of patients T-cell assays are limited by lower sensitivity given the [58]. Here, CTC burden is shown to predict treatment potential for interfering background cytokines and efficacy, progression-free and overall survival prior to
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4.00 )
3.00 Recurrence above the mean 1/26 (4%) dimer (mdi 2.00
Mean max. dimer change = 1.08 mdi Delta max. 1. 00 p = 0.06
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-2.00 Patient
1. 0 96.2% ee
0.9 80.4%
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0.7 Delta max. dimer above the mean, n = 26 Median f/u: action surviving disease fr Delta max. dimer below 60 months Fr the mean, n = 30 0.6 0.00 10.00 20.00 30.00 40.0050.00 60.00 Time (months)
Figure 2. Mean dimer change in vaccinated patients and disease-free survival benefit of vaccine patients with a maximum dimer change above the mean. (A) Mean dimer change in vaccinated patients. The difference between baseline and max. mdi was available in 56 HER2 underexpressing, vaccine group patients. Of the 26 patients above the mean difference (1.08 mdi ± 0.17), one recurred, compared with six clinical recurrences in the 30 patients below the mean (p = 0.06). (B) Disease-free survival benefit of vaccine patients with a maximum dimer change above the mean (p = 0.07), a 80.4% relative risk reduction. f/u: Follow-up; Max.: Maximum; mdi: Mean dimer index.
future science group www.futuremedicine.com 525 Drug Evaluation Schneble, Berry, Trappey et al.
and at any point after initiation of systemic therapy for the entire vaccine group. At 60 months, 3% of the [59–61]. Data from our pilot study evaluating CTC patients above the mean recurred, while 14% recurred revealed quantifiable levels of CTC in peripheral blood below the mean (p = 0.09), a 76.9% relative risk reduc- samples both pre- and post-vaccination [62]. Expand- tion (Figure 1A & 1B). No clinical recurrence occurred in ing this pilot study, data was collected in 25 patients patients with HER2 low-to-intermediate expression with within the NeuVax trial. CTC were present in 76% of a mean difference in dimer levels ranked in the top third. patients (mean: 4.8 ± 1.0 CTC/20 ml of blood) with Examining the mean dimer change from R0 to R6, the 68% of patients exhibiting at least two CTCs. CTC mean dimer level was also compared. Here, the greatest appeared to decrease from pre- to post- vaccination. Six dimer change also demonstrated a potential DFS advan- months after the primary vaccination series, nine out of tage and 80.4% relative risk reduction (Figure 2A & 2B). ten patients exhibited stable or decreasing CTC levels. Together, these analyses suggest that patients exhibiting Following NeuVax booster administration, there was a robust ex vivo immune responses have lower recurrence >50% reduction in CTC/20 ml (pre- 1.1 ± 0.5 vs post- rates suggesting E75-specific CTL clonal expansion as a 0.4 ± 0.2, p = 0.14) in a matched cohort [63]. Although valid biomarker predicting response in patients treated initial data is promising, correlating CTC with traits to with NeuVax [66]. include HLA expression levels, immunologic response From this initial analysis, our group advanced to and DFS remains challenging secondary to low CTC a logistical regression model to determine the best detection levels. The specific clinical significance has immune response parameters for predicting disease yet to be determined and will require larger studies to recurrence after completion of the primary vaccina- make meaningful correlations. However, recent studies tion series [67]. Here, the immunologic data from all suggest that even a single CTC detected by the Cell- vaccinated patients were examined. Subgroup analysis Search system in the adjuvant setting has prognostic was performed to include HER2 low-to-intermedi- significance [64,65]. ate-expressing and nonboosted patients as well. The logistical regression model was performed with back- Biomarkers associated with NeuVax response wards elimination of ex vivo/in vivo tests to predict As discussed above, multiple modalities are available for the chance of recurrence (Table 3). The odds ratio and immunologic monitoring in cancer vaccine trials. Impor- the area under the curve from the receiver operating tantly, our group has been able to associate immune characteristic curves was reported for statistical analy- response magnitude with the clinical benefit of Neu- sis (Figure 3). Results revealed that patients lacking Vax™ [66]. We examined the relationship between ex vivo pre-existing immunity, or a low R0 dimer level, have immunologic response and clinical recurrence after improved DFS after NeuVax. 5-year follow-up of the prospective, completed Phase I/ Immunologic response of HER2 low-to-interme- II NeuVax clinical trials [34,38]. Ex vivo immune response diate expressing patients is of importance based on was assessed for E75-specific CTL clonal expansion by the DFS benefit shown in the Phase I/II trials. Those the dimer assay prevaccination (R0), after the primary patients with minimal pre-existing HER2-specific vaccination series (R6) and 6 months after completing immunity who experience a robust T-cell response after the primary vaccination series (RC6). In vivo immune vaccination are less likely to recur than those who have response was assessed by DTH reactions to nelipepimut- pre-existing immunity to HER2 or have a small T-cell S at baseline and post-primary vaccine series. Of the 195 response. In HER low-to-intermediate-expressing patients enrolled, 187 evaluable patients were available patients, a single variable, R0 dimer, tended to predict for analysis; 108 in the vaccine group and 79 in the con- recurrence with an odds ratio of 1.9; meaning patients trol group. R6 dimer assays were available for 86 patients with an R0 level of nelipepimut-S specific CTL greater in the vaccine group. R6 dimer levels of individual than 1.19 were nearly twice as likely to recur. Here, patients were compared with the mean R6 dimer level the initial dimer value at R0 had a negative predictive
Table 3. Logistic regression model associating the immunologic variables R0, RC6 and delayed-type hypersensitivity with increased recurrence. Sample (n) Variables in the optimal model R0 odds ratio (p-value) Sensitivity, specificity AUC Vaccine group (n = 93) R0, RC6 2.36 (0.09) 75%, 73% 0.71 HER2 low-to-intermediate expression R0 1.90 (0.19) 80%, 83% (R0 < 1.19) 0.72 (IHC 1 or 2+, FISH <2.2; n = 58) Unboosted (n = 52) R0, post-PVS DTH 3.37 (0.12) 85%, 70% 0.76 !5# !REA UNDER THE CURVE $4( $ELAYED TYPE HYPERSENSITIVITY )(# )MMUNOHISTOCHEMISTRY 063 0RIMARY VACCINE SERIES
526 Immunotherapy (2014) 6(5) future science group HER2 peptide nelipepimut-S (E75) vaccine (NeuVax™) in breast cancer patients at risk for recurrence Drug Evaluation
A Vaccinated patients
RC6 dimer + +
R0 dimer + +
0.25 0.5 1248 Odds ratio
B HER2 low-expressor patients C Unboosted patients
Post-PVS DTH + +
R0 dimer ++ R0 dimer + +
0.25 0.5 12480.25 0.5 124816 32 Odds ratio Odds ratio
Figure 3. Forest plot depiction of the logistic regression model analysis. Both pre- and post-vaccination dimer, R0 and RC6, are included in the optimal regression model in vaccinated patients. A single variable, the prevaccination dimer (R0), predicted increased chance of recurrence among multiple modeling groups. (A) Vaccinated patients, (B) HER2 low-expressor patients and (C) unboosted patients. DTH: Delayed-type hypersensitivity; PVS: Primary vaccine series. value of 97% and the sensitivity and specificity were The promise of NeuVax exists in the prevention of 80 and 83%, respectively. Interestingly, the R0 dimer breast cancer recurrence by stimulation of the active was the dominant variable in multiple modeling groups immune system. Extensive immunologic monitoring demonstrating that a low pre-existing immunity is the of the NeuVax clinical trials has revealed a correlation most important factor in predicting clinical benefit. of clinical outcome with the magnitude of immune This analysis may explain why Neuvax works best in response measured across multiple modalities. Spe- HER2 low-to-intermediate-expressing patient with less cifically, R0 dimer holds potential as a predictive bio- HER2 antigen exposure. marker. Exhibition of a low R0 dimer indicates a lack of pre-existing immunity. Data suggest that patients Conclusion with a low R0 dimer have improved DFS with those In the context of clinical trials, the immune monitoring demonstrating a more robust response recurring less of a vaccine administered in the adjuvant setting differs often. This suggests that induction, rather than ampli- than that in metastatic setting. In past trials conducted fication, of an anti-HER2 immune response confers within the metastatic setting, CTL proliferation in a survival advantage. The clinical utility lies in the response to vaccination failed to show a correlation with ability of an initial R0 dimer assessment to identify clinical response. An explanation may be the sequester- patients who will benefit from the vaccine prior to ing of CTL away from a patient’s peripheral circulation inoculation. This holds important implications in towards the hostile environment of an existing tumor future trial design. burden. Within the context of adjuvant therapies, sam- Confirmation of NeuVax’s clinical utility will require a pling peripheral blood mononuclear cells brings new large, multicenter, randomized, double-blind trial. Mov- significance to real-time immune monitoring. As a short- ing forward, the Phase III adjuvant trial (Clinicaltrials. lived vaccination delivered to patients without significant gov identifier: NCT01479244) entitled ‘PRESENT: disease burden, NeuVax may avoid trafficking of T cells Prevention of Recurrence in Early-Stage, Node-Positive either to an existing tumor burden or sequestration at the Breast Cancer with Low-to- Intermediate HER2 Expres- vaccination sites where antigen-driven T-cell dysfunction sion with NeuVax Treatment’ is currently enrolling and deletion ensues. high-risk, node-positive patients with HER2 1+ and 2+
future science group www.futuremedicine.com 527 Drug Evaluation Schneble, Berry, Trappey et al.
tumors. Due to a potential relationship between HLA cancer cells. This enhanced CTL-mediated lysis type and prognostic factors, the Phase III study is only occurred regardless of the level of HER2 expression + + enrolling HLA-A2 and HLA-A3 patients to be ran- [69]. Subsequently, a pilot clinical study revealed lower domized to nelipepimut-S plus GM-CSF or GM-CSF recurrence rates in breast cancer patients receiving alone as the control group. The primary end point is adjuvant trastuzumab followed by vaccination with 36 month DFS [68]. a CD8-eliciting peptide vaccine compared with adju- Ultimately, combination, both active and passive, vant trastuzumab alone [70]. These findings prompted immunotherapy may portend the most significant con- the initiation of an ongoing Phase II clinical trial, tribution to the treatment of HER2-expressing breast Combination Immunotherapy with Herceptin and cancer patients. The synergistic mechanism of action the HER2 Vaccine NeuVax (Clinicaltrials.gov identi- between trastuzumab and nelipepimut-S was exam- fier: NCT01570036). This trial hopes to demonstrate ined in a preclinical study of trastuzumab- pretreated the clinical efficacy of combining the known benefits breast cancer cells, which were lysed more efficiently of proven passive immunotherapy with the promise of by E75-primed CTL compared with untreated breast active specific immunotherapy.
Executive summary Background s Cancer vaccines are a promising immunotherapeutic strategy given minimal toxicity and the ability to generate a sustained immunologic response with immunologic memory. s Vaccines work by targeting tumor-associated antigens that can be recognized by cytotoxic T lymphocytes (CTLs), critical for the removal of tumor cells in vivo. Introduction to NeuVax™ (Galena, OR, USA) s NeuVax™ (Galena, OR, USA) utilizes E75 or nelipepimut-S, a nine-amino-acid peptide derived from the HER2 protein (KIFGLSAFL, HER2 p369-377) as its immunologic focus. s NeuVax is administered to disease-free patients in the adjuvant setting after completion of standard-of-care therapy with concurrent endocrine therapy as indicated. s The vaccine administers nelipepimut-S in 6-monthly intradermal inoculations with granulocyte–macrophage colony-stimulating factor (GM-CSF) as an immunoadjuvant with booster inoculations every 6 months after the primary vaccine series. Laboratory & clinical studies s NeuVax shows promise in Phase I/II clinical trials although the mechanism of action, whether by inducing an immune response versus augmenting a pre-existing immune response, has been unknown. Correlating immune & clinical response s An integral component of clinical trials for cancer vaccines is in vivo and ex vivo immune response monitoring with multiple cancer vaccine studies failing to correlate peptide-specific CTL expansion with clinical benefit. s T-cell trafficking at vaccination and tumor sites may account for T-cell degradation and deletion leading to absence of immunologic memory and subsequent hyporesponsivness. s Vaccination models utilizing rapidly degrading adjuvant (GM-CSF) within the adjuvant setting may avoid the pitfalls of CTL sequestration. Immunologic testing s NeuVax immunogenicity has been assessed by multiple immunologic parameters to include delayed-type hypersensitivity, dimer assay, ELISPOT assay, Treg population assessment, cytokine evaluation, epitope spreading and CTC quantification. Biomarkers associated with NeuVax response s Recent correlation of immunogenic variables with DFS supports nelipepimut-S-specific CTL clonal expansion as a valid biomarker for predicting clinical recurrence after NeuVax administration. s Examination of nelipepimut-S-specific CTL clonal expansion data with clinical benefit suggests induction, rather than amplification, of an anti-HER2 immune response correlates with NeuVax’s efficacy. s Whereas a large tumor burden may sequester CTLs, in vitro immune monitoring may be more accurate within the adjuvant setting. Future perspective & conclusion s Confirmation of NeuVax’s clinical utility will require a large, multicenter, randomized trial. s The Phase III adjuvant trial (NCT01479244) titled ‘PRESENT: Prevention of Recurrence in Early-Stage, Node-Positive Breast Cancer with Low to Intermediate HER2 Expression with NeuVax Treatment’ is currently enrolling HLA-A2+ and/or HLA-A3+ node-positive patients with HER2 1+ and 2+ tumors. Enrollment will be complete in 2014. s The vaccine may also be clinically synergistic in combination with Herceptin®. A Phase II trial examining the clinical efficacy of trastuzumab plus NeuVax versus trastuzumab plus GM-CSF alone is currently enrolling.
528 Immunotherapy (2014) 6(5) future science group HER2 peptide nelipepimut-S (E75) vaccine (NeuVax™) in breast cancer patients at risk for recurrence Drug Evaluation
Financial & competing interests disclosure lNANCIAL INVOLVEMENT WITH ANY ORGANIZATION OR ENTITY WITH A '% 0EOPLES HAS INVENTOR RIGHTS TO NELIPEPIMUT 3 4HIS VACCINE lNANCIAL INTEREST IN OR lNANCIAL CONmICT WITH THE SUBJECT MAT HAS BEEN LICENSED FOR COMMERCIAL DEVELOPMENT (E IS ENTITLED TER OR MATERIALS DISCUSSED IN THE MANUSCRIPT APART FROM THOSE TO lNANCIAL PROCEEDS ASSOCIATED WITH THIS LICENSE PER &EDERAL DISCLOSED POLICY '% 0EOPLES ALSO CONSULTS IN THE DEVELOPMENT OF THE .O WRITING ASSISTANCE WAS UTILIZED IN THE PRODUCTION OF THIS VACCINE 4HE AUTHORS HAVE NO OTHER RELEVANT AFlLIATIONS OR MANUSCRIPT
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