Anti-angiogenic activity of VXM01, an oral T-cell vaccine against VEGF receptor 2, in patients with advanced pancreatic cancer: A randomized, placebo-controlled, phase 1 trial

Abstract

VEGFR-2 is expressed on tumor vasculature and a target for anti-angiogenic intervention. VXM01 is a first in kind orally applied tumor vaccine based on live, attenuated Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2. We here studied the safety, tolerability, T effector (Teff), T regulatory (Treg) and humoral responses to VEGFR2 and anti-angiogenic effects in advanced pancreatic cancer patients in a randomized, dose escalation phase I clinical trial. Results of the first 3 mo observation period are reported. Locally advanced or metastatic, pancreatic cancer patients were enrolled. In five escalating dose groups, 30 patients received VXM01 and 15 placebo on days 1, 3, 5, and 7. Treatment was well tolerated at all dose levels. No dose-limiting toxicities were observed. Salmonella excretion and salmonella-specific humoral immune responses occurred in the two highest dose groups. VEGFR2 specific Teff, but not Treg responses were overall increased in vaccinated patients. We furthermore observed a significant reduction of tumor perfusion after 38 d in vaccinated patients together with increased levels of serum biomarkers indicative of anti-angiogenic activity, VEGF-A, and collagen IV. Vaccine specific Teff responses significantly correlated with reductions of tumor perfusion and high levels of preexisting VEGFR2-specific Teff while those showing no antiangiogenic activity had low levels of preexisting VEGFR2 specific Teff, showed a transient early increase of VEGFR2-specific Treg and reduced levels of VEGFR2-specific Teff at later time points - pointing to the possibility that early anti-angiogenic activity might be based at least in part on specific reactivation of preexisting memory T cells.

Keywords: VEGFR2; anti-angiogenic treatment; cancer immunotherapy; oral vaccination; pancreatic cancer.

Figure 1.

(A). Preexisting VEGFR2-specific T effector cells detected immediately at d0 in N = 19 VXM01 and 10 placebo patients with significant increased spot counts in test wells compared to negative control wells as determined by IFNγ Elispot assay. Frequencies of VEGFR2-specific T cells are indicated for each patient by individual dots as difference in mean spot numbers between wells containing VEGFR2 and neg. control antigens. (B). T cell response of one representative VXM01 patient before (d0) and at subsequent time points after the vaccination against negative control antigens (huIg), recall antigens (CMV+Adenov.), VEGFR2 and MUC1 as assessed by IFNγ Elispot analysis. Total spots per well (1 × 10⁵ TC/well) are shown in (C), the respective fold change between mean spots in VEGFR2 compared to Ig containing negative control wells of this patient are shown in (D). Error bars show mean + SEM of triplicate wells.

Figure 2.

Cumulative T cell responses against recall antigens, VEGFR2 and MUC1 (A) or VEGFR2 only (B-D) in vaccinated VXM01 (black circles) or placebo patients (white circles). Antigen specific T cell responses are shown for both groups as fold increase of IFNγ spots in test wells over neg. control wells (A, B), or fold change of VEGFR2 specific T cell response at different time points after vaccination (as determined by fold change of test wells over neg. control wells) over respective values before vaccination (d0) (C, D). Cumulative analysis of all collected samples is shown in the left figure panel, while cumulative data of those patients who completed the observation period of 3 mo are shown in the right panel. Individual courses of VEGFR2 T cell responses over time are shown in D. Mean and SEM are depicted by error bars. *; a statistical trend toward higher increases of VEGFR2 specific T cell responses after 38 d in the vaccinated group compare to the placebo group was detected by two sided student's t-test. (A, B) completed time course placebo group n = 8 and VXM01 n = 19. In C and D placebo n = 7, VXM01 n = 18 (patient 10701 und 10702 excluded due to false positive d0 response).

Figure 3.

Graded strength of treatment-associated VEGFR2 responses (as determined by the difference between IFNγ spots in test wells and neg. control wells) in vaccinated (A, C black circles) or placebo patients (B, C open circles based on the fold increase over d0 defining no increase as grade 0, increase < 3 fold as grade 1, increase >3 fold to <5 fold as grade 2 and increase >5 fold as grade 3. In A and B Each dot represents a value of one individual patient. In (C) cumulative data are represented as mean, SEM are depicted by error bars (n = 30 vaccinated and n = 11 placebo).

Figure 4.

Regulatory T cell response in VXM01 and placebo patients. Frequencies of total Treg among CD4⁺ T cells were determined throughout the observation period in PBMC by flow cytometry on the basis of simultaneous CD3, CD4, CD25, and FoxP3 expression and lack of CD127 expression (A) and separately plotted for vaccinated (all data VXM01 n = 30; completed time course VXM01 n = 25) and placebo (all data vxm01 n = 12; completed time course n = 10) patients (B). C+D. The presence and suppressive activity of VEGFR2 reactive Treg as determined by a functional Treg specificity assay is shown for one representative patient (C), depicting the relative VEGFR2 specific Tcon suppression as percentage reduction of the Tcon proliferation compared to wells containing Treg that were stimulated with negative control antigen (IgG). (D) Cumulative data show the average VEGFR2-specific Treg suppression of Tcon proliferation as the mean and SEM for all obtained samples in the vaccinated (VXM01) or placebo group (left panel) and for those patients who completed the entire observation period (right panel).

Figure 5.

Antiangiogenic activity of VXM01. (A–C) Assessment of tumor perfusion K_trans by MRI-based recording (A; representative picture) of contrast signal enhancement. (B) Cumulative data of tumor perfusion in vaccinated (VXM01) and placebo patients showing the mean and SEM for those patients who provided at least d0 and d38 levels (left panel) and for those who completed the entire observation period (right panel). Individual d0 and d38 values of the latter group are shown in (C). The p value indicates an overall significant reduction of K_trans levels between d0 and d38 as calculated by wilcoxon test. (D, E) Cumulative data showing the mean and SEM values of collagen IV (D) and VEGF-A (E) serum levels in the vaccinated (VXM01) or placebo group for those patients who provided at least d0 and d38 levels (left panel) and for those who completed the entire observation period (right panel). (F). Highly significant correlation between the change of tumor perfusion between d0 and d38 in VXM01 treated patients who completed the whole observation period and the respective strength of vaccine induced VEGFR2 specific T cell response at day 21 (determined as fold increase over d0).

Figure 6.

(A–D) Assessment of VEGFR2 specific T cell responses in tumor perfusion responders and in non-responders. Cumulative T cell responses against VEGFR2 in vaccinated VXM01 patients (black circles) and placebo (open circles) recording VEGFR-2 specific Teff response as fold change over neg. control (A), VEGFR2 specific Treg activity (B) and the change of VEGFR2 specific Teff response over time (fold change over d0) (C). Patients who showed a reduced or stable tumor perfusion (delta K_trans < 0.0) between d0 and d38 are shown in the left figure panel while patients showing increased tumor perfusion after 38 d are shown in the right figure panels, respectively. Only patients who completed the entire observation period were included. Respective cumulative data of the whole placebo group (open circles) are shown for comparison. (D) Significantly increased levels of preexisting VEGFR2 specific Teff cells in VXM01 patients showing reduced or stable tumor perfusion 38 d after vaccination. The p value indicates the statistical difference between tumor perfusion responders and non-responders as determined by two-sided student's t test. (E-F) Cumulative T cell responses against VEGFR2 in vaccinated VXM01 patients (black circles) or placebo patients (open circles) with high preexisting VEGFR2 specific Teff levels (above median of the entire patient group) (left panel) or low levels of preexisting VEGFR2 specific Teff (below median) (right panel) recording Mean+SEM of VEGFR2 specific Teff response as fold change over negative control (E) or the change of VEGFR2 specific Teff response over time (fold change over d0). (F). Only patients who completed the entire observation period were included.