Ii-Key Hybrid Research Articles

Subgroup efficacy evaluation of the AE37 HER2 vaccine in breast cancer patients in the adjuvant setting.

3088 Background: AE37 is a Ii-Key hybrid of the HER2 peptide AE36 (HER2776-790), which stimulates peptide-specific T cells. We have completed the active phase of a prospective, randomized, multi-center, phase II trial of the AE37 vaccine in the adjuvant setting. The primary analysis, performed after a median follow up (f/u) of 25 months (mo), did not show a significant difference in disease free survival (DFS) between vaccinated and control patients (pts). However, demonstrating the efficacy of cancer vaccines may require more time than other therapies, especially in malignancies with relatively late recurrences like breast cancer. Here, we present updated efficacy data after extended f/u in subgroups of pts stratified by clinicopathologic characteristics. Methods: Clinically disease-free, node positive or high-risk node negative pts with any level of HER2 expression were randomized to receive AE37 + GM-CSF (VG) or GM-CSF alone (CG) following standard of care therapy. Pts received 6 monthly intradermal inoculations during the primary vaccine series (PVS) followed by 4 boosters administered every 6 mo. Kaplan Meier and log rank analyses were performed from the time of the first inoculation in pts who completed at least the PVS, according to stage, node status, tumor size, HER2 expression and ER/PR status. Results: There were no clinicopathologic differences between groups in the 298 enrolled pts (VG = 153, CG = 145). The vaccine is safe and well tolerated. After a median f/u of 55 mo, there was a trend toward improved DFS in the VG among stage IIB/III pts (VG, n = 73, DFS 82% vs CG, n = 61, 67%, HR = 0.48, p = 0.06) and those with low HER2 expression (HER2 LE, VG, n = 68, 89% vs CG, n = 66, 51%, HR = 0.47, p = 0.1). Improved DFS in the VG was documented in patients with both stage IIB/III disease and HER2 LE (VG, n = 39, 90% vs CG, n = 38, 32%, HR 0.3, p = 0.02) and triple negative (TNBC) pts (VG, n = 21, 89% vs CG, n = 21, 0%, HR 0.26, p = 0.05). Conclusions: The AE37 vaccine is safe and well tolerated and has statistically significant efficacy in stage IIB/III pts with HER2 LE and in TNBC pts. This justifies further evaluation in a phase III study enrolling stage IIb/III pts not eligible for trastuzumab treatment and the very high risk TNBC group. Clinical trial information: NCT00524277.

Primary analysis of a prospective, randomized, single-blinded phase II trial evaluating the HER2 peptide AE37 vaccine in breast cancer patients to prevent recurrence

AE37 is the Ii-Key hybrid of the MHC class II peptide, AE36 (HER2 aa:776-790). Phase I studies showed AE37 administered with granulocyte macrophage colony-stimulating factor (GM-CSF) to be safe and highly immunogenic. A prospective, randomized, multicenter phase II adjuvant trial was conducted to evaluate the vaccine's efficacy. Clinically disease-free node-positive and high-risk node-negative breast cancer patients with tumors expressing any degree of HER2 [immunohistochemistry (IHC) 1-3+] were enrolled. Patients were randomized to AE37 + GM-CSF versus GM-CSF alone. Toxicity was monitored. Clinical recurrences were documented and disease-free survival (DFS) analyzed. The trial enrolled 298 patients; 153 received AE37 + GM-CSF and 145 received GM-CSF alone. The groups were well matched for clinicopathologic characteristics. Toxicities have been minimal. At the time of the primary analysis, the recurrence rate in the vaccinated group was 12.4% versus 13.8% in the control group [relative risk reduction 12%, HR 0.885, 95% confidence interval (CI) 0.472-1.659, P = 0.70]. The Kaplan-Meier estimated 5-year DFS rate was 80.8% in vaccinated versus 79.5% in control patients. In planned subset analyses of patients with IHC 1+/2+ HER2-expressing tumors, 5-year DFS was 77.2% in vaccinated patients (n = 76) versus 65.7% in control patients (n = 78) (P = 0.21). In patients with triple-negative breast cancer (HER2 IHC 1+/2+ and hormone receptor negative) DFS was 77.7% in vaccinated patients (n = 25) versus 49.0% in control patients (n = 25) (P = 0.12). The overall intention-to-treat analysis demonstrates no benefit to vaccination. However, the results confirm that the vaccine is safe and suggest that vaccination may have clinical benefit in patients with low HER2-expressing tumors, specifically TNBC. Further evaluation in a randomized trial enrolling TNBC patients is warranted.

Abstract CT209: Correlation of robust local reactions prompting GM-CSF dose reduction to clinical response in a phase II trial of the AE37+GM-CSF HER2 peptide vaccine

Abstract Background: We are conducting a phase II clinical trial of the HER2 peptide vaccine AE37+GM-CSF for prevention of breast cancer recurrence in disease-free, node-positive or high-risk node-negative patients (pts), who have completed standard of care therapy. AE37, an Ii-Key hybrid of the HER2-derived peptide AE36 (aa:776-790), is an MHC Class II epitope capable of stimulating tumor reactive CD4+ helper T-cells. Previous investigation of intradermal inoculation with E75 (HER2 aa:369-377) +GM-CSF vaccine in phase I/II trials showed no recurrences at 24 months among the 18% of pts requiring a GM-CSF dose reduction due to robust local reactions (LR). Phase I/II studies have shown the safety, immunogenicity, and potential clinical benefit of AE37 + GM-CSF. Here, we examine the relationship between GM-CSF dose reduction and clinical recurrence rate (RR) in pts receiving AE37+GM-CSF. Methods: Patients with any level of HER2 expression (IHC1-3+) were enrolled and randomized to receive 6 monthly intradermal inoculations of AE37+GM-CSF or GM-CSF alone (controls) during the primary vaccination series, then four booster vaccinations administered every 6 months. Enrollment has been completed and pts continue with their vaccination series. LRs are measured after each inoculation, and pts with a LR measuring ≥ 100 × 100mm have their GM-CSF dose reduced on subsequent inoculations. Local and systemic toxicity are being monitored and clinical recurrences documented. Proportional RR are compared using a chi-square or Fisher exact test as appropriate. Results: Of 301 enrolled pts, 154 were randomized to the vaccinated arm (VG) and 147 randomized to the control arm (CG). The groups are well-matched for clinicopathologic characteristics. Toxicities have been almost exclusively grade 1 and 2. Study-wide, 18.9% of pts required dose reduction (CG 15.6%, VG 22.1%; p = 0.19). In vaccinated pts, the RR in the dose reduced group (DR) was 5.9% (2/34) versus the non-dose reduced group (NDR) RR of 14.2% (17/120) (p = 0.25). Among controls, the DR RR was 17.4% (4/23) vs the NDR RR of 12.9% (16/124) (p = 0.74). Comparing all DR pts, the relative risk of recurrence was reduced by 66% in the VG DR compared to the CG DR (5.9% versus 17.4%; p = 0.21). Conclusions: In a randomized phase II trial of the AE37+GM-CSF vaccine, pts who required a dose reduction of GM-CSF due to robust LR trend toward a lower recurrence rate. The overall dose reduction rate is similar to that in E75 trials (18.9% vs 17.9%) and occurred more frequently in the VG than CG. While dose reduction is seen in both the VG and the CG, the clinical benefit is only seen in the VG suggesting that while the LR may be a result of the GM-CSF, the specificity provided by the peptide is required for clinical benefit. Furthermore, these data suggest that GM-CSF should be dosed to produce large LR to enhance the benefit of peptide vaccines in the adjuvant setting. Citation Format: Julia M. Greene, Erika J. Schneble, Jennifer K. Litton, Jonathon Martin, Alfred F. Trappey, John S. Berry, Timothy J. Vreeland, Diane F. Hale, Guy T. Clifton, Alexandros Ardavannis, Michael Papamichail, Sonia Perez, Sathibalan Ponniah, Elizabeth A. Mittendorf, George E. Peoples. Correlation of robust local reactions prompting GM-CSF dose reduction to clinical response in a phase II trial of the AE37+GM-CSF HER2 peptide vaccine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT209. doi:10.1158/1538-7445.AM2015-CT209

The efficacy of chimeric vaccines constructed with PEP-1 and Ii-Key linking to a hybrid epitope from heterologous viruses

The heterologous epitope-peptide from different viruses may represent an attractive candidate vaccine. In order to evaluate the role of cell-permeable peptide (PEP-1) and Ii-Key moiety from the invariant chain (Ii) of MHC on the heterologous peptide chimeras, we linked the two vehicles to hybrid epitopes on the VP2 protein (aa197–209) of the infectious bursal disease virus and HN protein (aa345–353) of the Newcastle disease virus. The chimeric vaccines were prepared and injected into mice. The immune effects were measured by indirect ELISA. The results showed that the vehicle(s) could significantly boost immune effects against the heterologous epitope peptide. The Ii-Key-only carrier induced more effective immunological responses, compared with the PEP-1 and Ii-Key hybrid vehicle. The carrier–peptide hybrids all showed strong colocalization with major histocompatibility complex (MHC) class II molecules compared with the epitope-peptide (weakly-binding) after co-transfection into 293T cells. Together, our results lay the groundwork for designing new hybrid vaccines based on Ii-Key and/or PEP-1 peptides.

Correlation of HER2/neu antibody response to clinical response in a Phase II trial of the ae37+gm-csf her2 peptide vaccine

Meeting abstracts We are conducting a Phase II clinical trial of the HER2 peptide vaccine AE37+GM-CSF for prevention of breast cancer recurrence in disease-free, node-positive or high-risk node-negative patients, who have completed standard of care therapy. AE37, an Ii-Key hybrid of the HER2/ neu

Primary analysis of the prospective, randomized, single-blinded phase II trial of AE37 vaccine versus GM-CSF alone administered in the adjuvant setting to high-risk breast cancer patients.

638 Background: AE37 is the Ii-Key hybrid of the HER2 peptide AE36 (HER2 aa:776-790). It is a MHC class II epitope capable of stimulating CD4+ helper T-cells. We have completed accrual to a prospec...

Booster inoculations of the AE37 peptide vaccine enhance immunological responses in a phase II study.

3095 Background: We are conducting a multicenter randomized phase II trial of AE37, the Ii-Key hybrid peptide of HER2 776-790 (AE36). The purpose of the study is to determine if the AE37 vaccine can prevent recurrence in disease-free conventionally treated node-positive (NP) and high-risk node-negative (NN) breast cancer patients at significant risk for recurrence. Since clinical efficacy is anticipated to occur as the result of long lasting memory immune responses induced by vaccination, repeated booster inoculations were scheduled as part of the trial. Here we present data on immune responses in patients who received boosters up to 24 months after completion of the primary vaccination series (PVS). Methods: The trial is enrolling NP or high-risk NN patients with any degree of HER2 expression (IHC 1-3+ or FISH > 1.2) rendered disease-free following standard of care therapy. The vaccine group (VG) received AE37+GM-CSF and control group (CG) GM-CSF alone in 6 monthly i.d. inoculations followed by boosters administered every 6 months x 4. Immunologic responses were assessed in vivo by dermal reactions at the inoculation site, and in vitro, against the AE36 peptide, with proliferation and IFN-γ ELISPOT assays. Results: 25 patients in the VG and 23 in the CG have completed their boosters. After the last booster (BRC24), 100%, 54% and 54% in the VG (vs. 9%, 18% and 27% in the CG) responded by dermal reaction, proliferation and IFN-γ ELISPOT, respectively. Mean dermal reactions (orthogonal mean in mm) in vaccinated patients was 25.9±3.13 at completion of the PVS (R6) and increased to 35.47±4.35 at BRC24 (p=0.01). VG patients increased their proliferation response (stimulation index, SI) to AE36 from 0.97±0.046 at baseline (R0) before vaccination to 2.27±0.57 at R6 (p=0.0003) which was maintained until BRC24 (SI 2.21±0,33, p<0.0001). The number of IFN-γ specific spots/106 PBMC increased from 26.88±12.36 at R0 to 40.35±17.02 (p=0.07) at R6, up to 62±16.82 (p=0.0076) at BRC24. Conclusions: Our data demonstrate that AE37 vaccine boosters enhance the immune responses against HER elicited during the PVS, thus sustaining long lasting immunity, a prerequisite for possible clinical efficacy which is currently being evaluated. Clinical trial information: NCT00524277.

From bench to bedside: The use of the li-Key technology to improve helper peptides for clinical use in cancer vaccines.

2508 Background: Work involving peptide vaccines has shown that peptides containing MHC Class II epitopes, which elicit CD4+ T cell responses, may play a role in potentiating an immune response. The Ii-Key peptide (amino acids 77-80 of the immune-regulatory Ii protein), when covalently linked to an MHC Class II epitope, can induce conformational change in the epitope binding groove, increasing CD4+ T cell stimulation up to 250 fold. Here we present an update of results from clinical trials evaluating this novel technology in an adjuvant breast cancer vaccine targeting HER2/neu. Methods: We reviewed our trials investigating AE37, a hybrid peptide created by the addition of the Ii-Key peptide (LRMK) to AE36 (GVGSPYVSRLLGICL), an MHC Class II-binding peptide from the intracellular domain of the HER2 protein. We have completed a phase I study and are currently conducting a randomized phase II trial of the AE37 peptide + GM-CSF in the adjuvant treatment of disease-free breast cancer patients with any level of HER2 expression (IHC 1-3+ or FISH>1.2). Results: Phase I data showed the vaccine to be safe and effective in raising anti-HER2 immunity. Importantly, even the cohort of patients given AE37 without GM-CSF showed significant increases in both in vivo and in vitro immune responses. To date, we have enrolled 201 patients to our phase II trial (Vaccine (VG)=103, Control (CG)=98). Toxicity has been minimal (99% of local and systemic toxicities grade ≤2). VG patients have shown significant increases in both in vivo and in vitro responses to both AE36 and AE37, with consistently stronger responses to the AE37 hybrid peptide than the native AE36. With a median f/u of 22 months, Kaplan Meier projections estimate recurrence rates of 10.3% in the VG compared to 18% in the CG; a 43% risk reduction. Conclusions: The AE37 peptide vaccine appears to be effective in eliciting a strong immune response and possibly preventing breast cancer recurrence. These results provide an important proof of concept and suggest that additional studies evaluating Ii-Key hybrid peptide vaccines are warranted, whether in the field of immunotherapy or more traditional vaccines.

An assessment of disease features and immune response in breast cancer patients that did not recur after receiving HER2 peptide, AE37 vaccine in a randomized phase II trial.

625 Background: In a phase I trial of AE37, the Ii-Key hybrid of HER2 derived peptide AE36 (776-790), administered with immunoadjuvant GMCSF demonstrated the vaccine to be safe and capable of stimulating CD4+helper T cells with HER2 specific anti-tumor activity. Here we present analysis of immune markers and patient feature that may impact recurrence from an ongoing prospective, randomized, single-blinded Phase IIb trial of AE37+GMCSF v GMCSF alone in adjuvant high risk breast cancer (BC) patients. Methods: After completion of standard therapy; disease-free, node positive or high risk node negative BC patients (pts) were randomized to receive either AE37+GMCSF or GMCSF in 6 monthly intradermal inoculations. Immunologic responses were measured using [3H]-thymidine incorporation assay (in vitro), delayed-type hypersensitivity (DTH) reactions (in vivo) and T regulatory cells (Tregs). Among those vaccinated recurrent pts (VR) are compared to non recurrent (VNR) pts. Results: We have vaccinated 109 pts with 8.3% recurrence rate at 2 year median follow up. VR v VNR were younger (44 v 50 yo p=0.11), had higher grade (67% v 44% p=0.32), more ER/PR- (44% v 38% p=0.75), larger tumors (89% v 50% p=0.06), and node positive (89% v 70% p=0.37). No difference for HER2 status (IHC 3+, 44% v 49% p=0.64). Both VR and VNR responded to vaccine though the mean DTH and proliferative stimulation index was approximately 10% less in VR pts (18 v 20 p=0.73; 1.96 v 2.2 p=0.77 respectively). The most predictive measure was change in Tregs with VR pts less likely to decrease their Tregs levels (50% v 76% p=0.17) after vacination and more likely experience increased Tregs (17% v 8% p=0.48). A decrease in Tregs had an inverse trend towards recurrence (p=0.17). Conclusions: Preliminarily, it appears most pts immunologically respond to vaccine though slightly less for VR in most assays. The changes of Tregs appear to correlate best with disease recurrence. Whether this reflects an association with disease status or a failure of the vaccine is yet to be seen. These levels may become important in predicting risk for clinical recurrence in future vaccine trials.

Abstract LB-218: Immune response assessment in a phase II trial of AE37 HER2 peptide vaccine

Abstract Introduction: AE37 is the Ii-Key hybrid of the HER2 derived peptide AE36 (776-790). A phase I trial administering AE37 with the immunoadjuvant GMCSF demonstrated the vaccine to be safe and capable of stimulating CD4+helper T cells with HER2 specific anti-tumor activity. Here we present an analysis of our immunologic testing of our prospective, randomized, single-blinded, phase II trial of the AE37+GMCSF vs. GMCSF alone for the prevention of breast cancer recurrence. Methods: After completion of indicated standard therapy; disease-free, node positive or high risk node negative breast cancer patients were randomized to receive either AE37+GMCSF (VG) or GMCSF (CG) in 6 monthly intradermal inoculations. Patients were enrolled with any level of HER2 expression (IHC 1+ 2+ or 3+). Specific immunologic responses to both AE36 and AE37 were evaluated. In vitro responses were measured using [3H]-thymidine incorporation assay and in vivo responses using delayed-type hypersensitivity (DTH) reactions. T regulatory cells (Tregs) were measured throughout vaccination series. Data was analyzed using Pearson chi-squared tests. Results: To date 217 patients have enrolled (VG=109, CG=108). HER2 over-expression was present in 54 (49.5%) VG and 51 (47.2%) CG, p=0.783. VG had 59 (51.4%) ER positive and CG had 58 (53.7%, p=0.985). The in vitro proliferation responses with a stimulation index (SI) ≥2 were classified as high responders VG 36 (33.0%) vs CG 8 (7.4%, p<0.001), SI 1.5-2 were classified as low responders VG 19 (17.4%) vs CG 16 (14.8%, p=0.600) or SI <1.5 as non responders VG 54(49.5%) vs CG 84(77.8%, p<0.001). The in vivo DTH reactions measured in 149 (VG n=86, CG n=63) were stratified as responders with ≥5mm (VG 74(86.0%) vs CG 17(27.0%) p<0.0001) or non responders with <5mm (VG 12 (14.0%) vs CG 46(73.0%) p<0.001). Tregs responses were measured in 107 patients (VG n=56, CG=51) and categorized according to percent change from pre-vaccination baseline as an increase >110% (5(8.9%) VG vs 10 (19.6%) CG, p=0.112), no change 90-110% (10 (17.9%) VG vs 13 (25.5%) CG, p=0.337), or decrease <90% (41 (73.2%) VG vs 28 (54.9%), p=0.048). Conclusion: VG patients had significant immunologic responses compared to CG. VG had a statistical decrease in Tregs compared to CG. Monitoring immunologic tests and Tregs throughout the vaccination process may stratify patients into responders and non responders and thus assist in identifying patients that will have recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-218. doi:1538-7445.AM2012-LB-218

P1-13-01: An Update of a Phase II Trial of the HER2 Peptide AE37 Vaccine in Breast Cancer Patients To Prevent Recurrence.

Abstract Introduction AE37 is the Ii-Key hybrid of the HER2−derived peptide AE36 (HER2:776–790). A phase I trial administering AE37 with the immunoadjuvant GM-CSF demonstrated the vaccine to be safe and capable of stimulating CD4+helper T-cells with HER2−specific anti-tumor activity. Here we present an update of our prospective, randomized, single-blinded, phase II trial of the AE37 vaccine for the prevention of breast cancer recurrence in disease-free, high risk patients. Methods After completion of standard therapy, disease-free, node positive or high risk node negative breast cancer patients were randomized to receive either AE37+GM-CSF (vaccine) or GM-CSF alone (control) in six monthly intradermal inoculations. Patients were enrolled with any level of HER2 expression, (IHC 1+ 2+ or 3+). Specific immunologic responses to both AE36 and AE37 were evaluated in all patients at pre-determined intervals: before (RO), mid-series (R3), upon completion (R6), and at six (RC6) and 12 (RC12) months after completion of the vaccine series. In vitro responses were measured using the [3H]-thymidine incorporation assay and in vivo responses using delayed-type hypersensitivity (DTH) reactions. The trial's primary endpoint is disease recurrence. Results To date, 215 patients have enrolled (vaccine=92, control=123). 99% of local and systemic toxicities were ≤grade 2 or less. There were no grade 4–5 local or systemic toxicities and no difference between toxicity profiles of vaccine and control groups. Vaccine patients exhibited a statistically significant increase from baseline in AE36 and AE37 proliferative responses at each time point, including maintenance of this response up to 12 months post-vaccination (AE36 (cpm): R0=0, R3=1335, R6=1242, RC6=1586, RC12=1360; AE37: R0=0, R3=2859, R6=2300, RC6=3235, RC12=3279, p<0.001) while there have been no proliferative changes for control patients (AE36: R0=91, R3=95, R6=97, RC6=126, RC12=48; AE37: R0=291, R3=399, R6=319, RC6=103, RC12=0). Vaccine patients also had statistically significant increases in DTH reactions to both AE36 and AE37 (AE36 (mm): R0=0, R6=15, RC6=15, RC12=15; AE37: R0=0, R6=24, RC6=17, RC12=20 p=<0.001) while controls had no response (AE36: R0, R6, RC6, RC12=0; AE37: R0, R6, RC6, RC12=0). With a median follow up of 17 months, breast cancer recurrences were reduced by 42% in vaccine patients compared to control patients (7.6% vs. 13.2%, p=0.15). In an analysis of patients with low HER2 expression (IHC 1 or 2+), vaccine patients experienced a 49% reduction in recurrence compared to controls (9.5% vs. 18.6%, p=0.16) with no reduction seen in HER2 over-expressing patients (6.0% vs. 7.9%, p=0.49). Conclusions The AE37 vaccine is safe and well tolerated with only mild toxicity, which is attributable to the GM-CSF immunoadjuvant. The AE37 vaccine elicits strong peptide specific in-vivo and ex-vivo immune responses, which are maintained for 12 months after completion of the vaccine series. While the number of recurrences are still low, the recurrence rate appears to decrease in vaccinated patients. Administration of the AE37 vaccine may reduce the risk of breast cancer recurrence with the greatest benefit in patients with low levels of HER2 expression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-13-01.

Recognition of Ii-Key/MHC Class II Epitope Hybrids Derived from Proinsulin and GAD Peptides by T Cells in Type 1 Diabetes

In order to determine whether the Ii-Key technology can enhance the presentation of specific epitopes associated with type 1 diabetes, we have designed and synthesized a series of Ii-Key/proinsulin and GAD epitope hybrid peptides. Peptides of proinsulin and GAD shown to be recognized by CD4+ T cells of type 1 diabetes patients have been selected from the literature and modified with Ii-Key. A total of 23 Caucasian type 1 diabetes subjects and 17 normal subjects as controls were included in the study. Reactive T cells were identified using an IFN-γ ELISPOT assay. We selected 5 proinsulin and 5 GAD epitopes. Regarding the activity of the proinsulin Ii-Key hybrids, 3 out of 15 patients (20%) demonstrated a positive response to one or more Ii-Key hybrid peptides compared to no responders in the control subjects. Two out of 8 patients demonstrated a positive response to one or more Ii-Key/GAD65 hybrids. Proinsulin Ii-Key hybrids and peptides were recognized only by DR3/DR4 0302+ve diabetic patients. Control subjects showed no detectable response to stimulation with Ii-Key hybrids or peptides, neither for proinsulin nor GAD65. We have now shown that the use of Ii-Key-modified MHC class II epitopes, derived from proteins associated with insulin-secreting cells, can detect the presence of specifically activated CD4+ T helper cells with greater sensitivity than unmodified epitopes in the standard ELISPOT assay. The use of these technologies may be of use in identifying patients at the earliest stages of type 1 diabetes.

Abstract LB-414: Comparison of in vitro and in vivo immunologic responses in a prospective, randomized, single-blinded phase II trial evaluating the HER-2/neu peptide vaccines GP2 and AE37 in breast cancer patients

Abstract BACKGROUND: We are conducting a prospective, randomized, single-blinded phase II trial evaluating the HER-2/neu peptide vaccines AE37 and GP2 in the adjuvant setting to prevent breast cancer recurrence. AE37 is the Ii-Key hybrid of the HER-2/neu peptide AE36 (HER-2/neu aa:776–790) and is capable of stimulating CD4+ helper T-cells with anti-tumor activity. GP2 is an HLA-A2+ -restricted immunogenic peptide from the HER-2/neu protein (HER-2/neu aa: 654–662) which is capable of stimulating CD8+ cytotoxic T-lymphocytes with the ability to recognize and destroy HER-2/neu-expressing tumor cells. Here we present data comparing the immunological profiles between these CD4 and CD8 eliciting vaccines METHODS: Node positive or high-risk node negative breast cancer patients with any level of HER-2/neu expression rendered disease-free by standard treatments were enrolled. Patients were HLA-typed at enrollment. HLA-A2+ patients were placed in the GP2 arm and randomized to either GP2 + GM-CSF (immunoadjuvant) or GM-CSF alone. HLA-A2− patients were placed in the AE37 arm and randomized to either AE37 + GM-CSF or GM-CSF alone. Vaccinations were given in six monthly intradermal inoculations. In vitro (3H-thymidine proliferation for AE37, HLA-A2:immunoglobulin dimer for GP2) and in vivo (delayed-type hypersensitivity (DTH)) immune responses were measured before (R0), mid-series (R3), upon completion (R6), and at six (RC6) and twelve (RC12) months after completion of the vaccine series. RESULTS: We have enrolled 247 patients (AE37=171, 87 vaccine and 84 control) (GP2=76, 41 vaccine and 35 control). Proliferative responses to both AE37 and AE36 were increased from baseline in the vaccine group at all time points (AE37: R0=0.98, R3=3.29, R6=3.38, RC6=3.64, RC12=3.68, p<0.001; AE36: R0=0.95, R3=1.92, R6=2.12, RC6=2.18, RC12=1.80, p<0.001) while they did not change for the control group. The population of circulating GP2-specific CD8+ T-cells measured in the dimer assay increased from baseline in the vaccine group at all time points (R0=1.00, R3=1.61, R6=1.85, RC6=1.95, RC12=1.93, p<0.05) with no change in the control group. DTH responses increased in both AE37 and GP2 vaccinated patients from pre- to post-vaccination (AE37: 2.7 vs 30.2 mm, p<0.001; AE36: 2.2 vs 16.3 mm, p<0.001; GP2: 3.5 vs 29.4 mm, p<0.001) while there was no significant change in the control group. CONCLUSIONS: In a novel prospective, randomized, single-blinded phase II vaccine trial of the AE37 and GP2 peptide vaccines with a GM-CSF only control group, breast cancer patients vaccinated with either a CD4 or CD8 eliciting peptide vaccine exhibit high levels of HER-2/neu-specific in vitro and in vivo immune responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-414. doi:10.1158/1538-7445.AM2011-LB-414

Abstract LB-413: Circulating regulatory T cells (CD4+CD25high CD127low) decrease in breast cancer patients after vaccination with a modified HER-2/neu HLA class II peptide (AE37) vaccine

Abstract BACKGROUND: Regulatory T cells (TReg) are elevated in breast cancer patients with the number of TReg cells being inversely associated with clinical outcome. We are conducting a prospective, randomized phase II trial of the HER-2/neu peptide vaccines GP2 and AE37. GP2 is an HLA-A2+ -restricted peptide (HER-2/neu aa: 654–662) capable of eliciting a CD8+ cytotoxic T cell response directed against HER-2/neu expressing tumors. AE37 is the Ii-Key hybrid of the HER-2/neu peptide AE36 (HER-2/neu aa:776–790) and is capable of stimulating CD4+ helper T-cells with anti-tumor activity. Given the ability of AE37 to activate CD4+ helper T cells, concern exists that unwanted stimulation of TReg cells may occur with vaccination; although, this was not seen in our phase I trial. We have sought to confirm this finding by evaluating peripheral TReg cell levels in patients from our larger phase II breast cancer vaccine trial. METHODS: Node positive or high-risk node negative breast cancer patients with any level of HER-2/neu expression rendered disease-free by standard treatments were enrolled. HLA-A2+ patients were placed in the GP2 arm and randomized to either GP2 + GM-CSF (immunoadjuvant) or GM-CSF alone. HLA-A2− patients were placed in the AE37 arm and randomized to either AE37 + GM-CSF or GM-CSF alone. Vaccinations were given as six monthly intradermal inoculations. CD4+CD25high CD127low (CD127low) TReg cell levels were measured before (R0), mid-series (R3), upon completion (R6), and at six (RC6) and twelve (RC12) months after completion of the vaccine series using antibody staining and flow cytometry analysis. p values were calculated using the Student's t-test. RESULTS: TReg cells were measured in 65 patients in the AE37 arm (34 vaccine, 31 adjuvant) and 33 patients in the GP2 arm (17 vaccine, 16 adjuvant). Patients receiving the AE37 vaccine exhibited a decrease in CD127low TReg cells from baseline at all measured timepoints (R0=4.2, R3=3.7, p<0.01; R6=3.7, p=0.01; RC6=3.5, p<0.01; RC12=3.7, p<0.00001) while the randomized adjuvant patients demonstrated no decrease at any time point except from R0 to R3 (R0=4.3, R3=3.6, p=0.02; R6=4.1, p=0.58; RC6=3.9, p=0.06; RC12=3.9, p=0.21). In the GP2 arm, CD127low TReg cell levels did not change for either GP2 vaccinated patients (R0=3.6, R3=3.7, p=0.47; R6=4.1, p=0.67; RC6=4.3, p=0.56; RC12=4.3, p=0.16) or in randomized adjuvant patients (R0=4.4, R3=4.0, p=0.58; R6=4.4, p=0.95; RC6=3.8, p=0.25; RC12, p=0.59). CONCLUSIONS: Administration of a CD4 T cell-eliciting HER-2/neu peptide (AE37) vaccine to breast cancer patients induced a sustained decrease in circulating CD127low TReg cells. No such decrease was seen in randomized adjuvant-treated patients. Use of the CD8-eliciting, GP2 vaccine did not change circulating TReg cell levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-413. doi:10.1158/1538-7445.AM2011-LB-413

Corrections: 2009 San Antonio Breast Cancer Symposium Abstracts

Corrections: 2009 San Antonio Breast Cancer Symposium Abstracts

Effect of a novel II-key hybrid HER2/neu peptide (AE37) vaccine with GM-CSF as compared to GM-CSF alone on levels of regulatory T-cell (Treg) populations.

2565 Background: Regulatory T cells (Treg) inhibit effector T-cell function and prevent autoimmunity; they are recognized as obstacles to cancer immunotherapy. Tregs are predominantly CD4+, suggesting that MHC class II peptide vaccines may stimulate Tregs, decreasing vaccine efficacy. Additionally, results from recent trials have suggested that GM-CSF immunoadjuvant may decrease the efficacy of cancer vaccines or even be harmful. We addressed these issues in an ongoing phase II clinical trial in breast cancer (BCa) patients receiving the novel Ii-key MHC class II HER2/neu peptide vaccine (AE37). Methods: Disease-free, high-risk BCa patients enrolled after standard therapy and were randomized to 6 monthly inoculations of AE37+GM-CSF (peptide group; PA) or GM-CSF alone (adjuvant group; AA). Peripheral blood is drawn before, during, immediately after and 6 months after vaccination (R0, R3, R6, and RC6 respectively). Tregs were detected with CD4, CD25, CD127 and FoxP3 antibodies and flow cytometry. Patients are monitored for recurrence of BCa. Results: Of 150 post-vaccination patients, paired data are available in 57 (28 AA, 29 PA). CD4+ and CD8+ lymphocyte levels are similar in both groups and do not significantly change with vaccination. Treg (CD4+CD25hi) populations do not change in the AA (R0: 5.1±.3%; R3: 4.8±.3%; R6: 5.0±.3%; RC6: 5.2±.2%, p=NS) but decrease in the PA (R0: 5.1±.3%; R3: 4.5±.2%, p=0.004; R6: 4.7±.3%, p=NS; RC6: 4.3±.6%, p=0.01) with vaccination. Tregs as quantified by CD4+CD25hiCD127- cells also do not change in the AA but decrease in the PA (R0: 4.3±.3%; R3: 3.8±.2%, p=0.01; R6: 3.8±.3, p=NS; RC6: 3.8%±.7, p=0.04) with vaccination. Populations of CD4+FoxP3+ and CD25hiFoxP3+ cells do not significantly differ between groups or change with vaccination. At a median follow up of 13 mo., there have been 7.2% (6/83) recurrences in the AA and 2.9% (2/67) in the PA (p=0.30). Conclusions: The AE37 + GM-CSF vaccine decreases Tregs during and after completion of vaccination while GM-CSF alone does not alter Treg levels. This suggests that AE37 may allow the immune system to more effectively fight cancer and correlates with an observed decrease in recurrences. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Antigen Express

Interim Analysis of a Randomized Phase II Study of the Novel Ii-Key Hybrid HER2/Neu Peptide (AE37) Vaccine To Prevent Breast Cancer Recurrence: United States Military Cancer Institute Clinical Trials Group Study I-05.

Abstract BACKGROUND: CD4+ T helper peptides from HER2/neu have been evaluated in vaccine trials. The Ii-Key addition, a 4-amino-acid (LRMK) modification, increases vaccine potency when compared to unmodified class II epitopes. We present results of a prospective, randomized, single-blinded phase II clinical trial of the Ii-Key hybrid HER2/neu peptide (AE37) + GM-CSF immunoadjuvant vaccine versus GM-CSF alone in the adjuvant setting in disease-free, high risk breast cancer (BCa) patients to prevent recurrence.METHODS: Disease-free, high risk BCa patients who have completed standard adjuvant therapy were enrolled and randomized to receive six monthly inoculations of either 500 mcg of AE37 with 62.5 or 125 mcg of GM-CSF (Peptide group; PG) or 62.5 or 125 mcg of GM-CSF alone (adjuvant group; AG). Toxicity was assessed after each inoculation using National Cancer Institute Common Terminology Criteria for Adverse Events v3.0 (CTCAE). Immunologic response was monitored using delayed type hypersensitivity reactions (DTH) and 3H-thymidine proliferative assays for both hybrid AE37 (LRMK+HER2/neu:776-790) and AE36 (unmodified HER2/neu:776-790) peptides. Patients were clinically, radiographically, and pathologically monitored for recurrence of BCa.RESULTS: Thus far, 120 (49 PG, 71 AG) of the planned 200 patients have completed the primary series. The PG and AG have similar demographic/prognostic characteristics (Table 1). Toxicity profiles in the PG and AG were almost identical with no grade 4-5 local toxicities and no grade 3-5 systemic toxicities in either arm. Median DTH reaction to AE36 and AE37 increased significantly from baseline at 1 month after completion of the primary series in the PG group (AE36: 0.0±0.8 cm to 15.3 ±2.1 cm; AE37: 0.0±0.7 cm to 24.5±2.6 cm; p<0.0001) and did not change in the AG group (AE36: 0.0±0.5 cm to 0.0±1.4 cm; AE37: 0.0±0.7 cm to 0.0±1.6 cm; p>0.05). Median proliferation response to AE36 and AE37 increased significantly from baseline at 3, 6, and 12 months after the start of the vaccine series in the PG (p<0.015) and did not change significantly in the AG. At a median follow up of 13 months, there have been no (0.0%) recurrences in the PG (0/49) compared to 7.0% (5/71) in the AG (p=0.08).CONCLUSIONS: The modified peptide AE37 is safe with mild toxicities observed primarily due to the GM-CSF immunoadjuvant. AE37 elicits a strong HER2/neu-specific in-vivo and ex-vivo immune response to the modified and unmodified peptides. Importantly, the AE37 peptide vaccine may protect against BCa recurrences. peptideadjuvantp valueN=4971 Age (median)49520.06Node Positive75.5%62.1%0,16Grade 348.9%57.8%0.44Tumor ≥2 cm55.1%56.1%1ER/PR negative38.8%40.9%0.84HER2 over-expressor59.2%60.6%1 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3183. NOTE: This abstract was accepted for presentation at the Symposium after the Abstract Book went to press.

Ii-Key/HPV16 E7 hybrid peptide immunotherapy for HPV16+ cancers

Activation of antigen-specific CD4+ T cells is critical for vaccine design. We have advanced a novel technology for enhancing activation of antigen-specific CD4+ T helper cells whereby a fragment of the MHC class II-associated invariant chain (Ii-Key) is linked to an MHC class II epitope. An HLA-DR4-restricted HPV16 E7 epitope, HPV16 E7(8–22), was used to create a homologous series of Ii-Key/HPV16 E7 hybrids testing the influence of spacer length on in vivo enhancement of HPV16 E7(8–22)-specific CD4+ T lymphocyte responses. HLA-DR4-tg mice were immunized with Ii-Key/HPV16 E7(8–22) hybrids or the epitope-only peptide HPV16 E7(8–22). As measured by IFN-γ ELISPOT assay of splenocytes from immunized mice, one of the Ii-Key/HPV16 E7(8–22) hybrids enhanced epitope-specific CD4+ T cell activation 5-fold compared to the HPV16 E7(8–22) epitope-only peptide. We further demonstrated that enhanced CD4+ T cell activation augments the CTL activity of a H-2D b-restricted HPV16 E7(49–57) epitope in HLA-DR4+ mice using an in vivo CTL assay. Binding assays indicated that the Ii-Key/HPV16 hybrid has increased affinity to HLA-DR4+ cells relative to the epitope-only peptide, which may explain its increased potency. In summary, Ii-Key hybrid modification of the HLA-DR4-restricted HPV16 E7(8–22) MHC class II epitope generates a potent immunotherapeutic peptide vaccine that may have potential for treating HPV16+ cancers in HLA-DR4+ patients.

CD4+ T cells in antitumor immunity: utility of an Ii-Key HER2/neu hybrid peptide vaccine (AE37)

Background: Early clinical trials of HER2/neu-derived peptide vaccines indicate that they may be useful for preventing recurrence in breast cancer patients rendered disease-free after standard-of-care therapy. An effective vaccination strategy will probably require stimulation of T helper (Th) cells. AE37 is an HER2/neu-derived peptide that has been modified to enhance antigen-specific stimulation of Th cells by linkage of the Ii-Key moiety of the MHC class II-associated invariant chain (Ii protein). Objective: To review the literature regarding the role of a Th response in immunotherapy with a focus on this novel HER2/neu-derived AE37 peptide. Results/conclusion: Improved immuno-genicity of the AE37 Ii-key hybrid peptide has been demonstrated in animal models, ex vivo patient cells, and, most recently, in a Phase I clinical trial in breast cancer patients. Future clinical trials incorporating AE37 into a peptide vaccine strategy are warranted.

Results of the First Phase I Clinical Trial of the Novel Ii-Key Hybrid Preventive HER-2/neuPeptide (AE37) Vaccine

HER-2/neu is overexpressed in breast cancer and is the source of immunogenic peptides. CD4(+) T-helper peptides for HER-2/neu are being evaluated in vaccine trials. The addition of Ii-Key, a four-amino-acid LRMK modification, increases vaccine potency when compared with unmodified class II epitopes. We present the results of the first human phase I trial of the Ii-Key hybrid HER-2/neu peptide (AE37) vaccine in disease-free, node-negative breast cancer patients. The dose escalation trial included five dose groups, to determine safety and optimal dose of the hybrid peptide (100 microg, 500 microg, 1,000 microg) and granulocyte-macrophage colony-stimulating factor (GM-CSF; range, 0 to 250 microg). In the event of significant local toxicity, GM-CSF (or peptide in absence of GM-CSF) was reduced by 50%. Immunologic response was monitored by delayed-type hypersensitivity and [(3)H]thymidine proliferative assays for both the hybrid AE37 (LRMK-positive HER-2/neu:776-790) and AE36 (unmodified HER-2/neu:776-790). All 15 patients completed the trial with no grade 3 to 5 toxicities. Dose reductions occurred in 47% of patients. In the second group (peptide, 500 microg; GM-CSF, 250 microg), all patients required dose reductions, prompting peptide-only inoculations in the third group. The vaccine induced dose-dependent immunologic responses in vitro and in vivo to AE37, as well as AE36. The hybrid AE37 vaccine seems safe and well tolerated with minimal toxicity if properly dosed. AE37 is capable of eliciting HER-2/neu-specific immune responses, even without the use of an adjuvant. This trial represents the first human experience with the Ii-Key modification, and to our knowledge, AE37 is the first peptide vaccine to show potency in the absence of an immunoadjuvant.