A feasibility study of cyclophosphamide, trastuzumab, and an allogeneic GM-CSF-secreting breast tumor vaccine for HER2+ metastatic breast cancer

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccines are bioactive, but limited by disease burden and immune tolerance. Cyclophosphamide augments vaccine activity in tolerant neu mice and in patients with metastatic breast cancer. HER2-specific monoclonal antibodies (mAb) enhance vaccine activity in neu mice. We hypothesized that cyclophosphamide-modulated vaccination with HER2-specific mAb safely induces relevant HER2-specific immunity in neu mice and patients with HER2+ metastatic breast cancer. Adding both cyclophosphamide and the HER2-specific mAb 7.16.4 to vaccination maximized HER2-specific CD8+ T-cell immunity and tumor-free survival in neu transgenic mice. We, therefore, conducted a single-arm feasibility study of cyclophosphamide, an allogeneic HER2+ GM-CSF-secreting breast tumor vaccine, and weekly trastuzumab in 20 patients with HER2+ metastatic breast cancer. Primary clinical trial objectives were safety and clinical benefit, in which clinical benefit represents complete response + partial response + stable disease. Secondary study objectives were to assess HER2-specific T-cell responses by delayed type hypersensitivity (DTH) and intracellular cytokine staining. Patients received three monthly vaccinations, with a boost 6 to 8 months from trial entry. This combination immunotherapy was safe, with clinical benefit rates at 6 months and 1 year of 55% [95% confidence interval (CI), 32%-77%; P = 0.013] and 40% (95% CI, 19%-64%), respectively. Median progression-free survival and overall survival durations were 7 months (95% CI, 4-16) and 42 months (95% CI, 22-70), respectively. Increased HER2-specific DTH developed in 7 of 20 patients [of whom 4 had clinical benefit (95% CI, 18-90)], with a trend toward longer progression-free survival and overall survival in DTH responders. Polyfunctional HER2-specific CD8+ T cells progressively expanded across vaccination cycles. Further investigation of cyclophosphamide-modulated vaccination with trastuzumab is warranted.

Trial registration: ClinicalTrials.gov NCT00399529.

©2014 American Association for Cancer Research.

Figures

Figure 1. HER-2-specific MAbs combined with HER-2-specific vaccination augment tumor-free survival and HER-2-specific CD8+ T cell levels in neu mice

Neu mice (10 per group) were challenged with NT-2.5 tumor cells, and vaccinated 3 days later. A single dose of cyclophosphamide (CY) was given one day prior to vaccination in selected mice; weekly HER-2-specific MAb (7.16.4) or nonspecific IgG was also started one day prior to vaccination. (A) Vaccination + 7.16.4. (B) Vaccination + CY. (C) Vaccination + CY + 7.16.4. (D) Splenic T cells were isolated from neu mice at the end of treatment (3-5 per group). CD8+ T cells were isolated and incubated with NT2.5B7.1 target cells for IFNγ ELISPOT analysis. Circles represent individual mice; bars represent the average value. neuGM=3T3 cells that secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) and express rat HER-2 (neu vaccine); GM=3T3 cells that secrete GM-CSF (control vaccine).

Figure 2. Study schema

Patients received up to four vaccination cycles. Cyclophosphamide (CY) was given on day −1, and vaccine on day 0. Trastuzumab (T) was given weekly during active vaccine cycles, and timed to coincide with CY administration on day −1. During the interim period between cycles 3 and 4, trastuzumab could be given weekly or every 3 weeks as per the standard of care. GM-CSF levels and immunity were measured as indicated. CY=cyclophosphamide; T=trastuzumab; DTH=delayed-type hypersensitivity to HER-2-derived peptides. Skin biopsies on days +3 and +7 were from the largest vaccine site.

Figure 3. Progression-free survival (PFS) and overall survival (OS) of vaccinated patients

(A) Kaplan-Meier curves of the percent PFS and OS in months from the time of eligibility determination (n=20). (B) Kaplan-Meier curves of PFS stratified by DTH-positive and DTH-negative research subjects. (C) Kaplan-Meier curves of OS stratified by DTH-positive and DTH-negative research subjects. DTH=delayed-type hypersensitivity.

Figure 4. Vaccination elicits HER-2-specific CD8+ T-cell responses in MBC patients as measured by intracellular cytokine staining (ICS)

(A) HER-2-specific CD8+ T cells that secrete IFNγ, tumor necrosis factor-α(TNF-α), or interleukin-2 (IL-2) are detectable by ICS in vaccinated patients. A representative patient is shown. (B) The percentage of HER-2-specific CD8+ T cells that secrete IFNγ, TNF-α, or IL-2 increase across the vaccination cycles. The connected points represent the mean and standard deviation of values across the patient population, with three replicates per patient. (C) The proportion of polyfunctional HER-2-specific CD8+ T cells that secrete more than one cytokine increases across the vaccination cycles.

Figure 5. Markers of immune suppression in the peripheral blood decrease after vaccination

(A) CD4+FoxP3+ Tregs decrease across the vaccination cycles. (B) CD4+ FoxP3+ Tregs decrease from baseline after one vaccination cycle, p=0.043. (C) CD4+FoxP3+ CTLA-4+ Tregs decrease from baseline after one vaccination cycle, p=0.01. (D) HLA-DRneg/linlo/CD33+/CD11b+ MDSCs, p<0.0001.