Vaccine-Induced Memory CD8+ T Cells Provide Clinical Benefit in HER2 Expressing Breast Cancer: A Mouse to Human Translational Study

Abstract

Purpose: Immune-based therapy for metastatic breast cancer has had limited success, particularly in molecular subtypes with low somatic mutations rates. Strategies to augment T-cell infiltration of tumors include vaccines targeting established oncogenic drivers such as the genomic amplification of HER2. We constructed a vaccine based on a novel alphaviral vector encoding a portion of HER2 (VRP-HER2).

Patients and methods: In preclinical studies, mice were immunized with VRP-HER2 before or after implantation of hHER2⁺ tumor cells and HER2-specific immune responses and antitumor function were evaluated. We tested VRP-HER2 in a phase I clinical trial where subjects with advanced HER2-overexpressing malignancies in cohort 1 received VRP-HER2 every 2 weeks for a total of 3 doses. In cohort 2, subjects received the same schedule concurrently with a HER2-targeted therapy.

Results: Vaccination in preclinical models with VRP-HER2 induced HER2-specific T cells and antibodies while inhibiting tumor growth. VRP-HER2 was well tolerated in patients and vaccination induced HER2-specific T cells and antibodies. Although a phase I study, there was 1 partial response and 2 patients with continued stable disease. Median OS was 50.2 months in cohort 1 (n = 4) and 32.7 months in cohort 2 (n = 18). Perforin expression by memory CD8 T cells post-vaccination significantly correlated with improved PFS.

Conclusions: VRP-HER2 increased HER2-specific memory CD8 T cells and had antitumor effects in preclinical and clinical studies. The expansion of HER2-specific memory CD8 T cells in vaccinated patients was significantly correlated with increased PFS. Subsequent studies will seek to enhance T-cell activity by combining with anti-PD-1.

Figure 1.. Immunogenicity and efficacy of VRP-HER2 in HER2-transgenic mouse model.

Mice were vaccinated with 1×10⁷ IU in the footpad. Two weeks post-vaccination spleen and serum was taken and analyzed. (N=4 mice/group) A.) Splenocytes were stimulated with empty VRP vector or a pool of HER2 peptides and IFN-γ producing cells were analyzed by ELISPOT. B.) Serum was analyzed for HER2-specific antibodies using a cell-based ELISA. Absorbances are corrected for signal seen in a negative control plate. C.) MM3MG-HER2 tumor cells were implanted into the mammary fat pad of mice. Preventative vaccines were given 2 weeks prior to implantation and therapeutic vaccines were given 1 day post tumor implantation. Mice that completely cleared tumors were rechallenged on the opposite side in the mammary fat pad on day 35. Tumors were measured biweekly. (n=5 mice/group) D.) Splenocytes from mice in (C) were stimulated with a pool of HER2 peptides and IFN-γ producing cells were analyzed by ELISPOT. E.) Splenocytes from mice in (C) following rechallenge were incubated with brefeldin A alone or a mixture of HER2 peptides and CD107a/b antibodies to measure degranulation by flow cytometry. Cells were pregated on live CD45+ CD8+ CD44hi prior to analysis of CD107a/b expression. F.) Serum from mice in (C) was analyzed for HER2-specific antibodies using a cell-based ELISA.

Figure 2.. Identification of expanded memory CD8 T cell population following vaccination using CYTOF.

PBMCs were collected pre-vaccination and 6 weeks post the first vaccination. Cells were stimulated with a pool of HER2 peptides and stained for analysis by CYTOF mass cytometry. SPADE analysis was performed and clusters of cells were identified using surface markers. A. viSNE plots showing expression of select cell surface markers by all cells (Top, blue) or the memory CD8 T cell SPADE cluster that was expressing significantly more perforin post-vaccination (Bottom, orange). B. Heatmap of expression of all surface markers by each SPADE cluster. C. Change in the percent of perforin expression by cells in the identified memory CD8 T cell SPADE cluster from HER2 peptide restimulation PBMCs post-vaccination for each individual patient.

Figure 3.. Induction of poly-functional VRP and HER2-specific antibodies following vaccination.

A.) Patient serum pre and post VRP-HER2 vaccination was tested for VRP vector specific antibodies in a VRP neutralization assay. VRP vector expressing a control transgene was incubated with patient serum and plated with Vero cells. Expression of transgene was determined by staining of cells with fluorochrome labeled antibody for transgene and analyzed by flow cytometry. B and C.) Serum was analyzed for HER2-specific antibodies using a cell-based ELISA. Cells expressing full length HER2 (B) or cells expressing a mutated form of HER2 that is not bound by Trastuzumab or Pertuzumab (C) were used. Absorbances are corrected for signal seen in a negative control non HER2 expressing plate. Several dilutions were assessed and the maximum observed fold change in the level of HER2-specific antibody signal post-vaccination for each patient is reported. If values were below the level of detection for a particular patient, they are not shown. D.) CEM.NKR cell line expressing HER2 (targets) were incubated with serum from patients vaccinated with VRP-HER2 and normal donor PBMC (effectors) at an E:T ratio of 1:25 in an ADCC assay for 2-4 hours. Cells were stained with annexinV and 7-AAD to determine total lysis of CEM.NKR-HER2 targets. Shown as percent change in maximum killing resulting from serum pre verses post VRP-HER2 vaccination. E.) Individual killing results for patients 4, 8, and 11 from (D) are shown. F.) internalization of HER2 was measured using a Mars1-tagged form of HER2. Background signal from non-HER2 expressing cells was subtracted. Level of Mars1 signal was then normalized to untreated cells. VIA= Vaccine-induced antibodies. Individual results from patients 8, 15, and 19 are shown. *p<0.05

Figure 4.. Expansion of perforin expressing memory CD8 T cell population is significantly associated with improved progression free survival.

A. Progression free and overall survival curves for each cohort. Number at risk for each timepoint is shown below. B. Progression free and overall survival of patients grouped according to change in perforin expression (Decrease, dotted; increase, solid) by memory CD8 T cells following vaccination.