Julia M. Greene

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. METHODS HLA-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. RESULTS With 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). CONCLUSIONS GP2+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. CLINICAL TRIAL INFORMATION NCT00524277.

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

BACKGROUND Folate binding protein(FBP) is an immunogenic protein over-expressed in endometrial(EC) and ovarian cancer(OC). We are conducting a phase I/IIa trial of E39 (GALE 301)+GM-CSF, an HLA-A2-restricted, FBP-derived peptide vaccine to prevent recurrences in disease-free EC and OC patients. This interim analysis summarizes toxicity, immunologic responses, and clinical outcomes to date. METHODS HLA-A2+ patients were vaccinated(VG), and HLA-A2- or -A2+ patients were followed as controls(CG). Six monthly intradermal inoculations of E39+250mcg GM-CSF were administered to VG. Demographic, safety, immunologic, and recurrence rate(RR) data were collected and evaluated. RESULTS This trial enrolled 51 patients; 29 in the VG and 22 in the CG. Fifteen patients received 1000mcg E39, and 14 received <1000mcg. There were no clinicopathologic differences between groups(all p = 0.1). E39 was well-tolerated regardless of dose. DTH increased pre- to post-vaccination (5.71.5 mm vs 10.33.0 mm, p = 0.06) in the VG, and increased more in the 1000mcg group (3.82.0 mm vs 9.53.5 mm, p = 0.03). With 12 months median follow-up, the RR was 41% (VG) vs 55% (CG), p = 0.41. Among the 1000mcg patients, the RR was 13.3% vs 55% CG, p = 0.01. Estimated 2-year DFS was 85.7% in the 1000mcg group vs 33.6% in the CG (p = 0.021). CONCLUSIONS This phase I/IIa trial reveals that E39+GM-CSF is well-tolerated and elicits a strong, dose-dependent in vivo immune response. Early efficacy results are promising in the 1000 mcg dose cohort. This study proves the safety and establishes the dose of E39 for a larger prospective, randomized, controlled trial in HLA-A2+ EC and OC patients to prevent recurrence.

Preclinical testing of a novel personalized cancer vaccine for all solid tumors and all patients

Meeting abstracts A variety of autologous tumor/dendritic-cell (DC) vaccines have been pursued. Our prior autologous tumor/DC fusion (dendritoma) vaccine demonstrated clinical benefit in metastatic melanoma; however, dendritoma production is difficult and not scalable for commercialization. We

Tumor lysate particle loaded dendritic cell vaccine: preclinical testing of a novel personalized cancer vaccine

AIM We developed a novel approach to efficiently deliver autologous tumor antigens to the cytoplasm of dendritic cells (DC) using yeast cell wall particles (YCWP). MATERIALS AND METHODS Loading of YCWP, leakage of protein from loaded YCWP and cytoplasmic delivery of YCWP content was assessed using fluorescent-tagged experiments. Spectrophotometric analysis compared the epitope-specific T-cell responses following antigen presentation via YCWP versus exogenous loading. The in vivo effectiveness of tumor lysate (TL) particle loaded DC (TLPLDC) vaccine was assessed using murine melanoma models. RESULTS In fluorescence-tagged experiments, YCWP efficiently delivered antigen to the cytoplasm of DC. TLPLDC loading was more effective than conventional exogenous loading of DC. Finally, in murine melanoma models, TLPLDC outperformed an analogous dendritoma vaccine. CONCLUSION The TLPLDC vaccine is commercially scalable and holds the potential of producing personalized vaccines.

Initial phase I/IIa trial results of an autologous tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine in patients with solid tumors

INTRODUCTION Tumor vaccines use various strategies to generate immune responses, commonly targeting generic tumor-associated antigens. The tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine is produced from DC loaded with autologous tumor antigens, creating a patient-specific vaccine. Here, we describe initial phase I/IIa trial results. METHODS This trial includes patients with any stage solid tumors, ECOG ≤1, and >4 months life-expectancy. A personalized vaccine is created using 1 mg of tumor and 120 ml blood (to isolate DC). Primary vaccination series (PVS) is four monthly inoculations. Patients are followed per standard of care (SOC). Endpoints include safety and tumor response (RECIST v1.1). RESULTS 44 patients were enrolled and vaccinated consisting of 31 late stage patients with residual/measurable disease, and 13 disease-free patients after SOC therapies. While 4 patients progressed before completing the PVS, 12/31 (39%) demonstrated clinical benefit (2 complete responses, 4 partial responses, 6 stable disease). In the adjuvant setting, 46% of late stage patients remain disease free at a median of 22.5 months. CONCLUSIONS The TLPLDC vaccine is scalable, generates a personalized DC vaccine, and requires little autologous tumor tissue and few DC. The vaccine is safe, with primarily grade 0-2 toxicities, and nearly 40% clinical benefit rate in varied tumors, warranting further study. TRIAL REGISTRATION ISRCTN81339386, Registered 2/17/2016.

Abstract A044: Initial phase I/IIa trial results of an autologous tumor lysate + yeast cell wall particles + dendritic cells vaccine (TLPLDC) in patients with solid tumors

Introduction: The majority of tumor vaccines (vax) target tumor-specific/associated antigens with a variety of delivery systems/immunoadjuvants. As opposed to testing an “off the shelf” vax to treat a subset of patients (pts), our approach is tailored to the specific pt but applicable to all pts, all tumors. Our vax uses autologous dendritic cells (DC) loaded with the full range of tumor antigens from a pt9s cancer. Our particle loading system is highly efficient, reducing the need for large tumor volume, high numbers of DC or long culture times. The autologous tumor lysate (TL) is loaded into yeast cell wall particles (YCWP) that are naturally and efficiently taken up into isolated autologous DC. These autologous TL, particle-loaded, DC (TLPLDC) can be produced in 48hrs. Here, we describe the results of the TLPLDC vax in the first 20 pts treated. Methods: We are conducting an ongoing open label phase I/IIa trial enrolling pts with any solid tumor and treating them with autologous TLPLDC specifically created in our phase I trial center under regulatory oversight. Enrollment criteria include pts with all solid tumors of all stages, with ECOG status ≤1, and >4 months life expectancy. Exclusion criteria are chronic immunosuppression or viral infection and pregnancy. DC are isolated from the pt9s peripheral blood monocytes from a 120ml blood draw; cytokines are added to generate immature monocyte-derived DC. A minimum of 1 mg frozen tumor is required from surgery or biopsy. Tumor cells are lysed by multiple freeze/thaw cycles in buffer to produce TL. YCWP are prepared by NaOH digestion of all non-cell wall material from Saccharomyces cerevisiae, followed by washings. These hollow YCWP are loaded with concentrated TL solution via absorption, then freeze dried. Tetanus helper peptide and CpG oligonucleotides are similarly loaded into the YCWP as immunoadjuvants. TLPLDC are generated by incubating TL-loaded YCWP in the presence of DC and cytokines. After phagocytosis, final TLPLDC are frozen in single dose (1x106 cells/dose) vials. After QA/QC testing and lot-release, pts receive 4 monthly intradermal inoculations and then boosters every 6 months for as long as doses last. Trial endpoints are safety and efficacy using CTCAE and RECIST criteria, respectively. Results: To date, 20 pts have been enrolled to the TLPLDC trial with a median follow-up of 7 mos. The study population is 60% male, 90% Caucasian, 95% stage III/ IV disease (dz), with a median age of 58 years. Tumor types include chondrosarcoma, melanoma, astrocytoma, glioblastoma, Ewing9s sarcoma, and renal cell, ovarian, breast, esophageal, pancreatic, squamous cell, bladder, and small cell lung carcinomas. Of the 20 pts, 17 had measurable dz on initiation of treatment, and 3 were vaccinated adjuvantly. Twelve patients completed treatment, 6 are in treatment, and 2 are consented but not initiated. The 12 pts who have completed treatment received a median of 4 inoculations, and no grade ≥3 toxicities have been noted. Flu-like symptoms have been seen in 20%. Of the 17 pts with measurable dz, 10 have completed treatment with 6 demonstrating clinical benefit (1 CR, 2 PR, 3 SD). In the 3 adjuvant pts, 2 have completed treatment, and all 3 remain dz-free at a median follow- up of 10 months. Conclusions: Current results from this ongoing phase I/ IIa trial indicate that the autologous TLPLDC vax is well-tolerated and safe. The vax appears to have clinical activity in a variety of solid tumors to include melanoma with a complete response. Based on our prior studies with a similar vax and these initial results, TLPLDC may be most efficacious in the minimal residual dz setting; therefore, an adjuvant randomized phase IIb trial in stage III/IV (resected) melanoma pts has been initiated. Citation Format: Julia M. Greene, Diane F. Hale, Erika J. Schneble, Xianzhong Yu, Pauline Nichol, Sook Yin, Thomas Wagner, George E. Peoples. Initial phase I/IIa trial results of an autologous tumor lysate + yeast cell wall particles + dendritic cells vaccine (TLPLDC) in patients with solid tumors. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A044.

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 abstracts Folate 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 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