Subject characteristics for both studies are shown in . For ANZ-100, 6 subjects with CRC, 2 with PDA, and one with melanoma received a single dose of either 106, 3×107, or 3×108 cfu of Lm. Their median age was 60 (range 49 to 71). The median number of prior therapies was 4. For CRS-207, 5 subjects with mesothelioma, 7 with PDA, 3 with NSCLC, and 2 with ovarian cancer received at least one infusion of CRS-207 (6 subjects at 1×108, 4 subjects at 3×108, 6 subjects at 1×109, one subject at 1×1010 cfu). Their median age was 61 (range 40 to 79). The median number of prior therapies was 4.
Treatment-Related Toxicities and Dose Limiting Toxicity Events
A detailed description of treatment related grade ≥ 2 toxicities for both studies is provided in Supplemental Table 1
. ANZ-100 was well-tolerated at all dose levels. The most frequent AEs of any grade were transient grade ≤ 3 lymphopenia (9 pts, 100%), grade ≤ 3 hyperglycemia (8 pts, 89%), hypophosphatemia (5 pts, 56%), and fever (7 pts, 78%). No DLTs were observed and ANZ-100 was well tolerated up to the maximum planned dose.
CRS-207 was also well tolerated. The most frequent AEs of any grade were transient lymphopenia (17 pts, 100%), hypophosphatemia (6 pts, 35%), transaminitis (7 pts, 41%), fever (9 pts, 53%), chills/rigors (9 pts, 53%), nausea (9 pts, 53%), fatigue (6 pts, 35%), and hypotension (6 pt, 35%). All of these AEs were grade ≤ 2 except for transient ≥ grade 3 lymphopenia and hypophosphatemia, one grade 3 transaminitis, and one grade 3 fever. The first dose cohort received 1×108 cfu of CRS-207 at 3 week intervals for four doses. Two AEs occurred in subjects dosed in Cohort 1 (1×108 cfu) which met the initial protocol-defined criteria of DLTs. One subject experienced transient grade 3 hypophosphatemia 4 hours following the 2nd infusion of CRS-207. A second subject experienced a grade 3 temperature approximately 22 hours after receiving the first infusion. The subject was treated with acetaminophen and temperature returned to baseline within 24 hours. DLT criteria were amended to allow for transient grade 3 hypophosphatemia and fever. After transient grade 4 lymphopenia was identified in the 1×109 cohort, grade 4 lymphopenia was considered as a DLT only if it persisted for more than 4 days. Following dosing of 3 subjects in Cohort 2 (1×109 cfu) without reaching a DLT, one subject was dosed in Cohort 3 at 1×1010 cfu dose. This subject experienced a grade 2 cytokine release syndrome requiring aggressive fluid resuscitation. Due to the nature of the event, this was considered a DLT and the dose level was considered too toxic for further recruitment. The MTD was determined to be 1×109 cfu. To better characterize toxicities, additional subjects were enrolled into cohort 2 (1×109 cfu, n=3+3=6). Manageable hypotension and modest elevations in liver function tests (LFTs) were observed. An intermediate dose of 3×108 cfu was also added. Four subjects were enrolled into this cohort before the study was terminated. Manageable hypotension and transient LFT elevations (3 of 4 subjects) was also observed at the new dose level. One subject had elevations up to 11× ULN after a second dose in the absence of any changes in bilirubin. The LFTs improved without intervention. The subject was not re-dosed.
In general, subjects treated at all dose levels experienced symptoms that might be expected from a cytokine release-like syndrome from a bacteremia. While not all subjects experienced the same events, a constellation of symptoms was common. Lm was administered IV over a 2 hour period. Subjects typically had a temperature peak at 2–4 hours, sometimes associated with rigors, nausea, headaches, dehydration, and dry mouth. Mild hypotension was self-correcting or corrected with IV fluids. The most consistent laboratory abnormalities were transient, self-correcting electrolyte abnormalities and lymphopenias with nadirs at 4 hour post infusion. The degree of lymphopenia was dose-dependent and the most significant hypotension occurred at the highest dose level. The transient, self-correcting nature suggests that these abnormalities are the result of electrolytes and lymphocytes transiently shifting out of the blood compartment. Overall review of the safety data from the trial did not identify any significant toxicity with Lm or Lm-mesothelin that was not reversible or unexpected from either previous studies in cynomologus monkeys or based on mechanism of action.
Shedding and clearance
ANZ-100 was not detected in the blood, stool, urine, or sputum specimens collected at any time point. Lm suspected to be CRS-207 was detected in blood cultures of four subjects. In 2 of the subjects, the cultures were negative by 24 hours. An ovarian cancer subject who received a dose of 1×108 cfu had positive cultures at 4 and 24 hours after the first IV infusion. Subsequent blood cultures 4 days after dosing were negative. Blood cultures taken after her second infusion were negative. Lm suspected to be CRS-207 was detected in blood cultures of a mesothelioma subject receiving 1×109 cfu at day 4 after the second IV infusion. Subsequent cultures were negative. All remaining blood, stool, or urine specimens collected throughout the study for all subjects were negative.
Mesothelin membranous staining was detected in all 7 available archived samples for subjects enrolled into the CRS-207 study. The extent of staining is listed in .
PBMC phenotype analysis after ANZ-100 administration
Peripheral blood was analyzed prior to treatment with ANZ-100, daily for 5 days following treatment, and then weekly for one month. Immune activation was determined by phenotypic analysis of NK cells (CD3−CD16/56+). Interestingly, there was a transient reduction in peripheral lymphocyte and NK cell numbers () that reached a nadir at day 2 following treatment, suggesting the possibility that ANZ-100 induced activation results in lymphocyte and NK cell margination from the peripheral blood to other compartments. A significant upregulation of the activation marker CD38 was noted on NK cells for all dose levels (p = 0.0008, ). There appears to be a dose-dependent trend but it was not statistically significant (p = 0.1238). This level increased at the 96-hour time point as shown for one patient ().
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
Cytokine/chemokine induction after ANZ-100 and CRS-207 administration
Serum samples were collected prior to and at 2 hours, 6 hours, and daily for 5 days following ANZ-100 administration and analyzed for the presence of MCP-1, MIP-1α, MIP-1β, LT-α, IP-10, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IFN-γ, and TNF-α. At the highest dose level of 3×108 cfu, a significant induction of cytokines and chemokines such as MCP-1 and MIP-1β was observed (p = 0.0006 and p = 0.0002, respectively, ). The response peaked at 2 hours after the completion of the 2-hour IV ANZ-100 infusion and returned to baseline within 48 to 72 hours. Subjects at the highest dose level also had a consistent induction of the TH1 cytokines IFN-γ and IL12p70 (). The upregulation of the CD38 activation marker suggests biologic activity of ANZ-100 at doses as low as 1×106 cfu. However, a more consistent induction of pro-inflammatory cytokines and chemokines was observed in subjects receiving the higher dose level of 3×108 cfu.
ANZ-100 induction of chemokines/cytokines is dose dependent and favors the induction of Th1 cytokines
In the CRS-207 study, chemokine induction was noted for all dose levels including the starting dose level of 1×108 cfu. For MCP-1, MIP1-ß, and IP-10, the pattern was consistently elevated for all dose levels (p = 0.0050, p < 0.0001, and p < 0.0001, respectively) and did not vary significantly across time points (). The degree of upregulation is less than that observed in the ANZ-100 study, which may be due to the fact that the levels were taken at 24 hours rather than at the expected 2-hour post infusion peak (). The induction of IL-10 (p = 0.049), IL-12p70 (p = 0.100), IL-6 (p = 0.059), and TNFα (p = 0.092) are higher in the 1×109 cfu dose level as compared to the 3×108 dose level (). There was no significant difference in the induction levels for IFN-γ (p = 0.375) or IL-8 (p = 0.171). The data for the single subject with a dose of 1×1010 cfu is included in the plot for reference.
CRS-207 induction of chemokines/cytokines is present at all dose levels tested
Detection of LLO-specific and mesothelin-specific T cell responses after CRS-207 administration
LLO-specific T cell responses were analyzed in 8 subjects with viable samples pre-treatment and post the second and fourth infusion of CRS-207 (). Final T cell responses are reported. Six of the subjects were positive for vaccine-induced Lm
-specific responses. The CEF-specific responses are provided in Supplemental Figure 3
. In the CRS-207 study, mesothelin-specific CD8+
T cell responses were induced in 6 of the 10 evaluable subjects ().
CRS-207 induces both listeriolysin-O (LLO)-specific and mesothelin-specific T cell responses
Efficacy and survival
While the CRS-207 study enrolled subjects with multiple disease types and was not powered to assess survival, thirty-seven percent of this Phase 1 patient population survived for ≥ 15 months, with 3 subjects alive as of October 14, 2010 (). Of the 6 “long-term” survivors, 3 had PDA, 2 had NSCLC, and 1 had mesothelioma. These 6 subjects had prior immunotherapy or subsequent local radiation. Five of 6 subjects received all 4 doses of CRS-207 and all 5 evaluable subjects demonstrated vaccine-induced Lm-specific responses. One subject had been discontinued from study after 1 dose due to a protocol violation and samples were not collected for immunological evaluation. In addition, 4 of the 5 evaluable subjects among the long-term survivors had stable disease by RECIST at day 91 (end of study). Eight out of 8 evaluable subjects in the group that survived < 15 months had progressive disease by day 91. All 5 evaluable subjects who lived ≥ 15 months developed LLO responses. One additional subject lived ≥ 15 months but was not tested because a post-treatment sample was not collected. Thus, the induction of LLO-specific T cell responses may serve as a biomarker of immune competency in future studies. In this small subset of 10 subjects with multiple histologic types of cancer, the induction of mesothelin-specific responses did not correlate with survival. The induction of mesothelin-specific T cell responses as a marker of response to CRS-207 requires further investigation in a larger study of more homogenous subjects. These data provide the rationale for further evaluation of this Lm-mesothelin vaccine in a Phase 2 study.
CRS-207 Clinical and Immune Response Summary