A live-attenuated Listeria vaccine (ANZ-100) and a live-attenuated Listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: phase I studies of safety and immune induction

Abstract

Purpose: Listeria monocytogenes (Lm)-based vaccines stimulate both innate and adaptive immunity. ANZ-100 is a live-attenuated Lm strain (Lm ΔactA/ΔinlB). Uptake by phagocytes in the liver results in local inflammatory responses and activation and recruitment of natural killer (NK) and T cells, in association with increased survival of mice bearing hepatic metastases. The Lm ΔactA/ΔinlB strain, engineered to express human mesothelin (CRS-207), a tumor-associated antigen expressed by a variety of tumors, induces mesothelin-specific T-cell responses against mesothelin-expressing murine tumors. These two phase I studies test ANZ-100 and CRS-207 in subjects with liver metastases and mesothelin-expressing cancers, respectively.

Experimental design: A single intravenous injection of ANZ-100 was evaluated in a dose escalation study in subjects with liver metastases. Nine subjects received 1 × 10(6), 3 × 10(7), or 3 × 10(8) colony-forming units (cfu). CRS-207 was evaluated in a dose-escalation study in subjects with mesothelioma, lung, pancreatic, or ovarian cancers. Seventeen subjects received up to 4 doses of 1 × 10(8), 3 × 10(8), 1 × 10(9), or 1 × 10(10) cfu.

Results: A single infusion of ANZ-100 was well tolerated to the maximum planned dose. Adverse events included transient laboratory abnormalities and symptoms associated with cytokine release. Multiple infusions of CRS-207 were well tolerated up to 1 × 10(9) cfu, the determined maximum tolerated dose. Immune activation was observed for both ANZ-100 and CRS-207 as measured by serum cytokine/chemokine levels and NK cell activation. In the CRS-207 study, listeriolysin O and mesothelin-specific T-cell responses were detected and 37% of subjects lived ≥15 months.

Conclusions: ANZ-100 and CRS-207 administration was safe and resulted in immune activation.

Figure 1. ANZ-100 administration results in lymphocyte and natural killer (NK) cell declines in the peripheral blood suggesting margination into tissue and induces NK cell activation as evidenced by CD38 expression on CD3⁻CD16/56⁺ cells

A, Measurement of lymphocytes in the peripheral blood shows transient lymphocyte declines following ANZ-100 administration. B, Measurement of NK cells in the peripheral blood shows transient NK cell declines following ANZ-100 administration. Legend refers to cohort dose level. C, CD38 expression on NK cells by cohort dose level. Ratio of peak CD38 MESF value between 48h and 96h post dosing and pre-dose value. A significant upregulation of CD38 was noted for all dose levels (p = 0.0008). There appears to be a slight dose-dependent trend but it was not statistically significant (p = 0.1238). D, Histogram showing CD38 expression on NK cells after ANZ-100 administration in subject 001–005.

Figure 2. ANZ-100 induction of chemokines/cytokines is dose dependent and favors the induction of Th1 cytokines

A, MCP-1 chemokine expression presented by cohort dose level. Ratio calculated from peak chemokine value 2 hours post-dose compared to mean chemokine value of two pre-dose measurements. At the highest dose level of 3×10⁸ cfu, a significant induction of MCP-1 was observed (p = 0.0006). B, MIP-1β chemokine expression presented by cohort dose level. Ratio calculated from peak chemokine value 2 hours post-dose compared to mean chemokine value of two pre-dose measurements. At the highest dose level of 3×10⁸ cfu, a significant induction of MIP-1β was observed (p = 0.0002). C, IFN-γ cytokine expression after ANZ-100 administration in subjects at the 3×10⁸ dose level. D, IL-12p70 cytokine expression after ANZ-100 administration in subjects at the 3×10⁸ dose level.

Figure 3. CRS-207 induction of chemokines/cytokines is present at all dose levels tested

A, MCP-1 chemokine expression presented by cohort dose level after dose 1 and 2. B, MIP-1β chemokine expression presented by cohort dose level after dose 1 and 2. C, IP-10 chemokine expression presented by cohort dose level after dose 1 and 2. D, Cytokine expression 24 hours after first dose of CRS-207 presented by cohort dose level. The data for the single subject with a dose of 1×10¹⁰ cfu is included in the plot for reference.

Figure 4. CRS-207 induces both listeriolysin-O (LLO)-specific and mesothelin-specific T cell responses

A, LLO-specific T cell responses were analyzed using IFN-γ Elispot. B, Mesothelin-specific T cell responses were analyzed using IFN-γ Elispot. *T cell responses were considered positive when the frequency of specific responses were ≥ 1 in 10⁵ PBMCs (LLO Elispot) or CD8⁺ PBL (mesothelin Elispot) and increased by at least 2-fold compared to baseline. Final T cell responses are reported. The maximal response to a single best peptide is reported. The boxed patient identification numbers represent subjects who lived ≥ 15 months.