John S. Berry

Primary analysis of the prospective, randomized, phase II trial of GP2+GM-CSF vaccine versus GM-CSF alone administered in the adjuvant setting to high-risk breast cancer patients.

134 Background: GP2 is a HER2 derived, HLA-A2+-restricted immunogenic peptide designed to stimulate CD8+T cells to recognize tumor cells with any level of HER2 expression (IHC 1-3+). Accrual to a prospective, randomized, multi-center, phase II trial of the GP2 vaccine for prevention of breast cancer recurrence has completed. Here, the planned primary analysis of disease-free survival (DFS) is presented.nnnMETHODSnHLA-A2+ node positive or high-risk node negative breast cancer patients (pts) with any level of HER2 expression rendered disease-free by standard of care therapy (to include trastuzumab where appropriate) were randomized to receive GP2+GM-CSF (VG) or GM-CSF (CG) alone. Pts received 6 monthly inoculations (primary vaccine series = PVS) followed by 4 boosters administered every 6 months. The Kaplan Meier method was used for statistical analysis. The intention-to-treat (ITT) population is defined as the entire randomly assigned population. The per-treatment (PT) group excluded pts who recurred during the PVS or developed a second malignancy. A pre-specified subgroup analysis was performed based on HER2 expression level. HER2 overexpression (OE) is defined as IHC 3+or FISH >2.2.nnnRESULTSnWith 89 VG and 91 CG pts enrolled and vaccinated, there are no differences between groups with respect to age, node positivity, tumor size, grade, ER/PR status, and HER2 expression (p>0.05). The vaccine has been well tolerated with toxicities comparable between the VG and CG. Only one grade 3 local and systemic toxicity reaction has been reported in the VG. At 34 (1-60) month median follow-up, DFS was compared in the ITT (85% VG v 81% CG, p = 0.57) and PT (94% VG v 85% CG, p = 0.17) populations. In OE patients (51 VG and 50 CG) DFS was 94% VG v 89% CG, p = 0.86 (ITT) and 100% VG v 89% CG, p = 0.08 (PT).nnnCONCLUSIONSnGP2+GM-CSF is a novel vaccine that is safe and well tolerated. This phase II trial demonstrates potentially greater benefit in pts with HER2 OE tumors, in whom there have been no recurrences in the PT group. This may be due to synergism with trastuzumab therapy, thus justifying a phase III trial evaluating GP2 administered in the adjuvant setting to a HER2 OE population.nnnCLINICAL TRIAL INFORMATIONnNCT00524277.

The Noninvasive Carbon Dioxide Gradient (NICO2G) during Hemorrhagic Shock

ABSTRACT Hemorrhagic shock (HS) is a setting in which both pulmonary and cutaneous perfusion may be impaired. The goals of this study were to evaluate the relationship between end-tidal (etCO2), transcutaneous (tPCO2), arterial carbon dioxide (PaCO2) and lactate during lethal HS and to assess the effect of progressive HS on those variables and on a new variable, the noninvasive CO2 gradient ([NICO2G] or the difference between tPCO2 and etCO2). Ten consciously sedated swine were hemorrhaged, by means of a computerized exponential protocol, of up to 80% estimated blood volume for 20 min. End-tidal carbon dioxide, tPCO2, PaCO2, and lactate measurements were taken at baseline and every 5 min thereafter, that is, after 25%, 44%, and 62% total blood volume hemorrhage (TBVH) and at cardiac arrest. Cardiac arrest occurred on average at 67% TBVH. Data were analyzed by linear regression and one-way repeated-measures analysis of variance and are presented as means ± SD. Forty-nine paired measurements were made. There was no overall relationship between NICO2 variables and PaCO2: PaCO2 vs. tPCO2 (r2 = 0.002, P = 0.78); PaCO2 vs. etCO2 (r2 = 0.0002, P = 0.93). Rather, NICO2G increased at each level of blood loss: 4.0 ± 24.9 at baseline, 6.3 ± 35.7 at 25% TBVH, 25.0 ± 37.6 at 44% TBVH, 55.0 ± 33.9 at 62% TBVH, and 70.0 ± 33.2 at cardiac arrest (P < 0.05). Similarly, tPCO2 increased and etCO2 decreased at each level. Linear regression of NICO2G and lactate showed a better correlation than was observed for the other two variables: NICO2G, r2 = 0.58; tPCO2, r2 = 0.46; etCO2, r2 = 0.26. During HS, NICO2 monitors lose accuracy for approximating the PaCO2 but gain usefulness as hemodynamic monitors. Also, by combining data from two different organ systems, NICO2G demonstrated improved correlation with lactate than did either etCO2 or tPCO2 alone.

Vaccine-specific T-cell proliferation in response to a dual peptide cancer vaccine in breast and ovarian cancer patients

Meeting abstractsnnHER2 is a commonly expressed tumor-associated antigen in breast (BrCa) and, therefore, an attractive target for immunotherapy. We have investigated HER2-derived peptides as vaccines mixed with GM-CSF to include GP2 (a HLA-A2 and HLA-A3 restricted, CD8+ eliciting epitope) and AE37

Abstract 4670: Delayed urticarial reactions in the phase II trial of HER2/neu peptide vaccines + GM-CSF vs. GM-CSF alone in high risk breast cancer patients.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnWe are conducting a phase II trial evaluating two HER2-specific vaccines (AE37 and GP2) in the adjuvant setting for the prevention of breast cancer (BC) recurrence. We have observed the development of delayed urticarial reactions (DURs) in several patients after inoculation. In this report, we characterize the DURs and analyze immunologic responses (IR) in this population.nnAfter completion of standard of care therapy, disease-free, node-positive or high-risk node-negative BC patients (pts) with any level of HER2 expression (IHC1-3+) were randomized to receive either a peptide+GM-CSF (VG) or GM-CSF (CG). Pts receive 6 monthly intradermal inoculations during the primary vaccine series (PVS) followed by four boosters (B) every 6 mos. IR were measured by local reaction (LR) and delayed type hypersensitivity (DTH) pre (R0) and post-PVS (R6).nnTen (4.3%) of a total 231 initiated pts have had a DUR; 5 in VG (vDUR) (3AE37, 2 GP2), and 5 in CG (cDUR). Median time to onset of symptoms was the same for cDUR/vDUR (median 8 days (d); range, 0-17 d). All DUR pts described generalized pruritis/hives, with two also having difficulty swallowing/globus. Symptoms resolved with IV/oral steroids (n=4), oral steroids only (n=3), or antihistimines (n=3). Duration ranged from 4 d-4 mos with a median of 21 d. vDUR pts had a longer median duration of symptoms than cDUR pts (105 d v 9 d, p=0.02). Four pts (3 vDUR, 1 cDUR) had recurrent symptoms that resolved after treatment similar to above. The majority of events (9) occurred between B1 and B3, but one event did occur after the first inoculation. All DUR pts have received no further inoculations.nnComparing pts who have had a DUR to those who have not (noDUR), there were no significant differences in age, tumor size/grade, HER2 expression, or ER/PR status. 70% of DUR pts have environmental allergies. No other risk factor has been identified. vDUR pts had weaker IR than VG noDUR (LR- R0: 21v36mm, p=0.14; R6: 30v57mm, p=0.17) (DTH - R0: 0.6v2mm, p=0.25; R6: 17v25mm, 0.21). cDUR pts had stronger IR than CG noDUR (LR - R0: 46v1mm, p=0.005; R6: 93v50mm, p=0.01) (DTH - R0: 9.1v2.3mm, p=0.003; R6: 15.3v5mm, p=0.03). There have been no recurrences in the DUR group compared to 11.4% in the noDUR group (p=0.26).nnDUR has occurred infrequently with no long-term sequelae. DURs have occurred evenly between vDUR and cDUR indicating GM-CSF is the likely cause. Stronger IR in the cDUR group suggest that these ptss immune system may have a predisposition toward a more robust response to GM-CSF. Less robust DTH/LR as well as longer symptom duration in the vDUR suggest the vaccine may potentiate non-specific systemic immune activation in some pts. We plan to further characterize DUR in our ongoing trial.nnCitation Format: Alfred F. Trappey, John S. Berry, Timothy J. Vreeland, Diane F. Hale, Alan K. Sears, Guy Clifton, Sathibalan Ponniah, Michael Papamichail, Sonia A. Perez, Elizabeth Mittendorf, George E. Peoples. Delayed urticarial reactions in the phase II trial of HER2/ neu peptide vaccines + GM-CSF vs. GM-CSF alone in high risk breast cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4670. doi:10.1158/1538-7445.AM2013-4670

Abstract B007: Improved disease-free survival in endometrial and ovarian cancer patients with low folate binding protein expression after treatment with the E39 peptide vaccine in a Phase I/IIa trial

Introduction: One of the most encouraging examples of targeted therapy for cancer is trastuzumab, but its success is dependent on levels of expression of its target, HER2. We have found that HER2 expression levels also have a significant impact on the efficacy of HER2-directed peptide vaccines. Analogous to HER2 in breast cancer, Folate Binding Protein (FBP) is over-expressed on ovarian and endometrial cancer cells (up to 80 - 90-fold higher) and increased FBP expression is associated with aggressive disease. As a result, multiple FBP-directed therapies are being developed. We are investigating E39 + GM-CSF, which is an HLA-A2-restricted FBP-derived peptide vaccine used to prevent recurrence in disease-free endometrial and ovarian cancer patients (pts) after standard of care (SOC), multi-modality therapy. We have shown that E39 is safe, effectively generates E39-specific immune responses, and may improve DFS when optimally dosed in a phase I/IIa trial.1 Little is known about the effects of FBP expression levels on FBP-directed therapies, including our E39 vaccine. Purpose: Here, we report clinical outcomes of patients based on FBP expression levels from a phase I/IIa trial of the E39+GM-CSF vaccine given for the prevention of recurrence in disease-free endometrial and ovarian cancer patients. Methods: Disease-free, HLA-A2+ pts were vaccinated (VG), while HLA-A2- pts were followed as untreated controls (CG). The VG received 6 monthly inoculations of E39+GM-CSF, including either 100, 500, or 1000mcg of peptide and 250mcg of GM-CSF. FBP expression testing was performed by immunohistochemistry and the results were graded 0-4+ based on the percentage of positively staining cells. Patient9s tumors were then categorized as low expression (FBPlo) if scored 0-1+ or high expression (FBPhi) if 2-4+. The pts were monitored for evidence of clinical recurrence through the SOC follow-up by their treating oncology team. Demographics, FBP expression and disease-free survival (DFS) were analyzed using appropriate statistical tests. Results: Thirty-eight enrolled pts underwent FBP expression testing (CG n = 20; VG n = 18). There were no clinicopathologic differences between groups (p≥0.1). Nineteen pts were found to be FBPlo (CG, n = 11; VG, n = 8) and 19 were FBPhi (CG, n = 9; VG, n = 10). Median follow up was 16.3 months. There was no significant difference in overall DFS between the CG and the VG (34.6% vs. 34.6%, p = 0.208). In FBPlo pts, there was improved DFS in the VG vs. CG (85.7% vs. 17.5%, p = 0.01) while there was no such difference in FBPhi pts (VG:13.9% vs. CG:44.4%, p = 0.83). Though groups were small, there was a dose-dependent effect on DFS; pts receiving 1000mcg (n = 4) had improved DFS compared to the Conclusion: This phase I/IIa trial has previously demonstrated that E39 is well-tolerated, elicits a strong, dose-dependent in vivo immune response and may improve DFS when properly dosed. This focused analysis based on FBP expression level revealed a DFS benefit in FBPlo, but not FBPhi, endometrial and ovarian cancer pts treated with E39. This may be due to immunotolerance from significantly higher endogenous exposure to the FBP antigen. This is concordant with findings in our trials of HER2-directed peptide vaccines in breast cancer pts. These findings warrant further study as they may have important implications regarding the target population for future E39 peptide vaccine trials. 1. Jackson DO, et al. Interim analysis of a phase I/IIa trial assessing E39+GM-CSF, a folate binding protein vaccine, to prevent recurrence in ovarian and endometrial cancer patients. In Press. Citation Format: Kaitlin M. Peace, Diane F. Hale, Timothy J. Vreeland, Doreen O. Jackson, Julia M. Greene, John S. Berry, IV, Alfred F. Trappey, Garth S. Herbert, Guy T. Clifton, Mark O. Hardin, Kathleen M. Darcy, Chad A. Hamilton, G. Larry Maxwell, George E. Peoples. Improved disease-free survival in endometrial and ovarian cancer patients with low folate binding protein expression after treatment with the E39 peptide vaccine in a Phase I/IIa trial [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B007.

Preliminary report of a clinical trial supporting the sequential use of an attenuated E39 peptide (E39') to optimize the immunologic response to the FBP (E39+GM-CSF) vaccine

Meeting abstractsnnFolate Binding Protein (FBP) is overexpressed in breast, endometrial, and ovarian cancers. E39 (FBP191-199, EIWTHSYKV) is an HLA-A2 restricted FBP peptide vaccine already shown to generate significant in vivo immunologic response (IR) in a Phase I/IIa trial in endometrial and

Abstract OT3-1-09: Combination immunotherapy with trastuzumab and the HER2 vaccine E75 (nelipepimut-S) in high-risk HER2+ breast cancer patients to prevent recurrence

Background: In an adjuvant phase II trial, the HER2-derived nelipepimut-S (E75) + GM-CSF vaccine (Neuvax) has been shown to reduce breast cancer recurrence. Preclinical testing of the combination of trastuzumab (Tz) and nelipepimut-S has shown synergistic cytolysis against HER2 expressing cancer cells. In pilot phase II data in HER2+ patients (pts), 55 pts dosed with CD8-eliciting HER2 derived peptide vaccines sequentially after treatment with Tz resulted in no recurrences at 36 months median follow-up compared with a 16% recurrence rate in 34 randomized controls treated with Tz without vaccine (p=.012). Based on these data, we have designed a trial to evaluate the ability of the combination of Tz and the E75 vaccine concurrently to prevent recurrence in pts with high-risk, HER2+ breast cancer. Trial Design: This study will be a multicenter, prospective, randomized, single-blinded, phase II trial evaluating adjuvant Tz + NeuVax (E75+GM-CSF) vs. Tz + GM-CSF alone in high-risk HER2+ (IHC 3+ and/or FISH >2.2) breast cancer pts. High-risk pts include: 1) those that did not achieve a pathologic complete response (pCR) after neoadjuvant chemotherapy and HER2- targeted therapy or 2) those treated with upfront surgery that are node positive (> 4+ LN or 1-3+ LN if hormone receptor negative). Pts must be HLA-A2 /A3+ to be eligible (E75 is HLA-A2/A3-restricted) with ECOG performance status 0-1. Pts will be enrolled after completing standard of care multi-modal therapy but prior to the 3rd dose of Tz maintenance therapy (monotherapy). Pts will be randomized 1:1 to receive either NeuVax or GM-CSF alone which will be administered as six monthly intradermal inoculations concurrently with Tz therapy. Pts will then receive four booster inoculations of either NeuVax or GM-CSF every 6 months. The primary efficacy endpoint is to compare disease-free survival (DFS) between treatment arms. Secondary objectives will include evaluation of local and systemic toxicity, distant recurrence free survival, and in vivo/in vitro immunologic responses. From previously published experience with Tz, we expect a recurrence rate of 20% in Tz (plus GM-CSF) treated pts and anticipate that the combination of Tz with E75+GM-CSF will reduce this recurrence rate to 5%. In order to show statistical difference between these recurrence rates, we plan to enroll 50 pts per treatment arm (100 total) with a type I error rate of 5% and 80% power to detect the primary endpoint. Trial accrual is anticipated to begin in September of 2014, with a two year period for trial enrollment followed by a three year follow-up period. Conclusion: We hypothesize that combination adjuvant immunotherapy with Tz and NeuVax will result in a greater reduction in breast cancer recurrence than Tz therapy alone. We have designed a prospective, randomized, single-blinded, phase II trial evaluating the efficacy of this immunotherapy combination in high-risk HER2+ breast cancer pts to test this hypothesis. Contact Information: This trial is funded by a DoD grant to EAM with matching funds from Galena Biopharma and is being conducted with the assistance of the academic CRO, Cancer InCITe, LLC. Citation Format: Beth A Mittendorf, Erika J Schneble, Nuhad K Ibrahim, Julia M Greene, John S Berry, Alfred F Trappey, Guy T Clifton, Jarrod P Holmes, Sathibalan Ponniah, George E Peoples. Combination immunotherapy with trastuzumab and the HER2 vaccine E75 (nelipepimut-S) in high-risk HER2+ breast cancer patients to prevent recurrence [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT3-1-09.

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

Meeting abstractsnnWe 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

Preliminary results of the Phase IIa trial of a folate binding protein (FBP) adjuvant cancer vaccine (E39+GM-CSF) in ovarian and endometrial cancer patients to prevent recurrence

Meeting abstractsnnFBP (aka Folate Receptor-a) is an immunogenic protein that is over-expressed in breast, endometrial (EC) and ovarian cancer (OC). FBP expression in malignant cells is 20-80 fold higher compared to the limited distribution in normal cells. We have completed enrollment of a Phase

Abstract 2558: HLA-A2 and HER2 expression levels as clinical prognostic factors in breast cancer patients: implications for peptide cancer vaccine trials

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnIntroduction: Our group has been investigating HER2-derived peptide vaccines administered in the adjuvant setting to high risk breast cancer patients (pts) to prevent disease recurrence. Our group has previously shown that pts with HER2 low-expressing (LE) tumors are at higher risk of recurrence than over-expressing (OE) tumors in the trastuzumab era. Given peptide-based cancer vaccination require presentation by a specific HLA molecule, our group has also examined and found comparable disease recurrence rates between HLA-A2+ and HLA-A2- patients. Here, we report recurrence data based on HLA-A2 allele/HER2 expression sub-grouping in our unvaccinated, control pts.nnMethods: After completing standard of care therapy, high-risk, disease-free breast cancer pts with HER2 LE (IHC 1+, 2+) or OE (IHC 3+) tumors are eligible for enrollment in our phase II trial evaluating the HER2-derived peptide vaccines, AE37 (MHC Class II, HLA-non-restricted epitope) and GP2 (MHC Class I, HLA-A2+ restricted epitope). Patients are HLA-typed as A2+ or A2- then randomized to vaccine vs. adjuvant alone. Demographics between groups are compared using chi squared or fisher exact.nnResults: Thus far, 208 pts have been enrolled to the adjuvant alone control arms of the trial. Five pts were excluded from analysis (1 ineligible and 4 second malignancies) for a total of 203 evaluable pts: 88 HLA-A2+ (48 OE and 40 LE) and 115 HLA-A2- (48 OE and 67 LE). A2+ and A2- OE pts exhibited significant difference in respect to age (52 vs. 47, p=0.013) and tumor size (41.7% vs. 68.1% ≥ 2 cm, p=0.01) respectively.nnWith median follow-up of 39 months, recurrence rates were again compared between A2+ vs. A2- pts (11.4% vs. 11.3%, p=0.50) as well as HER2 subgroups (OE, 6.25% vs. LE, 15.8%, p=0.02). The latter difference was particularly prominent among the A2- pts (A2-/OE, 4.2% vs. 16.4%, p=0.03) but less so in the A2+ pts (A2+/OE, 8.3% vs. A2+/LE, 15%, p=0.26). With no recurrence difference between LE A2 subgroups (LE/A2+, 15% vs. LE/A2-, 16.4%, p=0.54), the OE subgroup comparison was notable for 50% increased disease recurrence in A2+ pts (A2+/OE, 8.3% vs. A2-/OE, 4.2%, p=0.33).nnConclusions: While there is no difference in recurrence between A2+ and A2- pts overall or among the HER2 LE subset, there does appear to be a difference in the HER2 OE subset. Compared to A2+/OE pts, the A2-/OE pts exhibit 50% decreased disease recurrence despite significantly younger age and larger tumor size. These findings need to be confirmed but are important given that many peptide vaccines target A2+ pts. Therefore, these results may impact future trial design.nnCitation Format: Erika J. Schneble, Alfred F. Trappey, Timothy J. Vreeland, John S. Berry, Diane F. Hale, Alan K. Sears, Guy T. Clifton, Sathibalan Ponniah, Elizabeth A. Mittendorf, George E. Peoples. HLA-A2 and HER2 expression levels as clinical prognostic factors in breast cancer patients: implications for peptide cancer vaccine trials. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2558. doi:10.1158/1538-7445.AM2014-2558