A phase 2 study of orally administered live biotherapeutic salmonella-IL2 with FOLFIRINOX for stage IV pancreatic cancer

Abstract

Salmonella-IL2 is an attenuated Salmonella Typhimurium strain carrying the human gene for IL-2. When orally administered in preclinical trials, the bacterium colonizes tumors and locally releases IL-2, triggering immunologically-mediated tumor cell killing without untoward side effects. A non-randomized, phase 2 study evaluated the combination of Salmonella-IL2 with standard of care (SOC) chemotherapy where patients received Salmonella-IL2 plus FOLFIRINOX (FFX). Overall survival (OS), progression-free survival (PFS), safety, and biomarker data in each arm were studied. In total, 34 patients (30 in the trial, 4 via EAP) were enrolled: 26 received Salmonella-IL2 with FOLFIRINOX. Those patients who received more than five doses of Salmonella-IL2 with FOLFIRINOX (n = 20) had a mPFS of 15 months while the mOS was 20.3 months. Even though there were no complete responses, the partial response rate and the overall response rate was 70.0%. In addition, 41 serious adverse events were noted and attributed to SOC chemotherapy agents but none to Salmonella-IL2. Addition of Salmonella-IL2 to FOLFIRINOX is associated with increased mPFS and mOS when compared to previously reported outcomes with FOLFIRINOX alone in the literature. A multicenter, randomized, phase 3 trial is warranted. ClinicalTrials.gov identifier: NCT04589234.

Introduction

More than 66,000 people in the USA developed pancreatic cancer in 2024 [1]. Half of such patients will present with distant metastatic disease (mPDAC), for which the current standard of care (SOC) is either a four drug regimen (FOLFIRINOX or NALIRIFOX) or the two-drug regimen of Gemcitabine with nab-Paclitaxel (GEM/nabP) [2, 3]. A recent meta-analysis of these first line chemotherapeutic strategies evaluated seven trials using data for 2,581 patients that accumulated during more than a decade of clinical trials [4]. This analysis revealed that median progression free (PFS) and overall survival (OS) for the FOLFIRINOX and NALIRIFOX regimens were 7.3 or 7.4 months, and 11.7 or 11.1 months, respectively. For patients treated withGEM/nabP, median PFS was 5.7 months, and median OS was 10.4 months. In addition, a recent survey of clinical trials of novel approaches to treating metastatic pancreatic cancer revealed that most trials have been either terminated, suspended, withdrawn or simply are not presently recruiting [5]. Recent advances across multiple biological domains support growing optimism for the treatment of historically refractory cancers, such as metastatic pancreatic ductal adenocarcinoma. Progress in targeting KRAS signaling and tumor invasion pathways, including matrix metalloproteinase-2 inhibition, has revealed actionable vulnerabilities that challenge longstanding therapeutic nihilism in PDAC. [6],In parallel, emerging insights into non-coding RNA regulation and metabolic reprogramming highlight the potential to exploit transcriptional and metabolic dependencies across aggressive solid tumors [7–9]. Collectively, these advances support biologically informed, multi-mechanistic strategies and provide a strong conceptual rationale for innovative immunometabolic approaches, such as microbial-based cancer therapies in mPDAC.

In recent years, microbial-based cancer therapeutics have become attractive adjuncts for cancer therapy because of their intrinsic immune-enhancing and cancer-targeting properties [10, 11]. Salspera has developed an attenuated strain of Salmonella Typhimurium that is non-toxic, orally administered, and specifically engineered to carry the human gene for IL-2, which has a C-terminal truncation but retains biological activity (renamed Salmonella-IL2 or Saltikva) [12]. Salmonella-IL2 triggers immunologically mediated tumor cell killing within tumor microenvironments [13–15]. Significant anti-tumor effects of this bacterial strain have been demonstrated to date in preclinical models of metastatic colorectal cancer, pancreatic cancer, neuroblastoma, and osteosarcoma [12–19]. Moreover, a large mammal trial that we conducted in companion dogs with pulmonary metastases from osteosarcoma demonstrated a 22% complete response rate and a significant increase in disease-free survival in dogs who received Salmonella-IL2 plus standard of care Doxorubicin, when compared to the response in dogs given Doxorubicin alone. [20] Lastly, a phase I, single dose, dose escalation trial in humans demonstrated significant elevation of NK and NK-T cell populations after patients with metastatic GI cancers received Salmonella-IL2. [21].

To determine whether adjunctive administration of Salmonella-IL2 is beneficial in patients receiving standard of care chemotherapy for stage IV pancreatic adenocarcinoma, a first of its kind phase II non-randomized clinical trial was performed where an orally administered bacterium was used with intent to treat. The primary outcome variables in this phase II study were overall survival, progression-free survival and response rates. Secondary outcome data were obtained using biomarker analyses and safety profile detection.

Methods

Trial design and monitoring

A Health Canada and local IRB approved, non-randomized, open label, two-arm study in patients with Stage IV adenocarcinoma of the pancreas was conducted at the Segal Cancer Centre of the Jewish General Hospital (JGH), McGill University in Montreal. The study comprised of two arms; Arm One: Salmonella-IL2 with FOLFIRINOX and Arm Two: Salmonella-IL2 with Gemcitabine/nab Paclitaxel. Salmonella-IL2 was orally administered concomitantly with the administration of the prescribed chemotherapy according to arm of enrollment. Specifically, 10 [9] colony forming units (cfu) of attenuated Salmonella-IL2 were administered orally after ingestion of a gastric acid neutralizing agent and followed with 200 ml of an isotonic crystalloid fluid. Dose escalation of the study drug was not permitted. Arm One: patients with stage IV pancreatic cancer were administered the FOLFIRINOX regimen given as first line chemotherapy in the first line. FOLFIRINOX was administered every two weeks and Salmonella-IL2 was administered three days after each cycle of FOLFIRINOX. Arm Two: patients with stage IV pancreatic cancer were administered Gemcitabine/nab Paclitaxel regimen given as first line chemotherapy in the first line. Gemcitabine/nab Paclitaxel was administered every four weeks on days one, eight, and fifteen; while Salmonella-IL2 was administered every two weeks. Dosing modification and “chemo holidays” were permitted at the discretion of treating oncologists. Routine blood work (complete blood counts, serum electrolytes, and hepatic function testing), CT scans, and biomarker analysis were performed at least every 3 months. Patients were followed biweekly to monitor for progress while on treatment and for the assessment of toxicity.

Primary outcome variables were overall survival and progression-free survival (as determined by CT imaging using RECIST 1.1 criteria) [22]. Biomarker data, adverse events (AEs) and Serious Adverse Events (SAEs) were tabulated secondary outcomes [23]. This study was conducted in accordance with the principles of the Declaration of Helsinki and the International Council for Harmonization guidelines for Good Clinical Practice [24]. Trial oversight was provided by the sponsor (Salspera Inc), the principal investigators (Gerald Batist, MD and Petr Kavan, MD) and their team at the JGH, an independent clinical trial monitor (Exactis, Montreal, QC, Canada), and an independent Data Safety and Monitoring Committee (DSMC). All patients provided written informed consent.

Patients

Eligible adults (> 18 years of age) had histologically confirmed diagnosis of stage IV pancreatic adenocarcinoma with radiographically confirmed distant metastatic disease measurable by Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1) criteria [22]. All patients had an Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 1. There were no minimum life expectancy requirements to enroll in the trial. Patients who were receiving other investigational agents or who had brain metastases were not enrolled in this trial. Full inclusion and exclusion criteria are provided in the protocol (Supplementary Appendix available upon request). Current standard of care practice for patients with stage IV pancreatic cancer is to offer one of two chemotherapeutic regimens (FOLFIRINOX or Gemcitabine/nab Paclitaxel) [2, 3]. Assignment to treatment arms was based on the clinician’s judgment of a patient’s likely tolerance to treatment. In addition to recruited study patients, Salspera also provided the study drug to four patients with stage IV pancreatic cancer via an Expanded Access Program (EAP). These patients were treated per the FOLFIRINOX Arm of the study protocol, met formal inclusion criteria for the trial, and were included in the study analysis. Per protocol, their CT scans were read using RECIST 1.1 criteria and all other aspects of the clinical trial protocol were followed for these EAP patients. Comprehensive molecular profiling was not standard-of-care at the trial site during this Phase 2 study, so BRCA/HRR and MSI/dMMR results were not uniformly available for reporting. Only the four EAP patients had genomic determination, and one of these patients had a BRCA mutation.

Study drug: salmonella-IL2

Salmonella-IL2 is an attenuated strain of S. enterica Typhimurium that is rendered avirulent by deletion of cya and crp genes required for cyclic-AMP maintenance of virulence. [25] In addition, this strain lacks the enzyme aspartate semialdehyde dehydrogenase (asd). Bacteria that lack this enzyme cannot synthesize diaminopimelic acid (DAP), an essential component of the bacterial cell wall, and thus cannot survive unless they carry a plasmid with the asd gene. We have constructed a plasmid (pIL2) by incorporating the cDNA sequence for a C-terminally truncated human IL-2 gene into plasmid pYA292, which also contains the asd gene and is therefore capable of rescuing this attenuated strain of S. enterica. Thus, if this engineered organism were to lose the plasmid, it would not be able to construct its bacterial cell wall and would quickly die. These circumstances result in stable plasmid maintenance without the need for antibiotic selection. In sum, our study agent is attenuated S. enterica with the pIL2 plasmid also known as Salmonella-IL2 (Saltikva).

Preclinical experimentation demonstrated that this engineered strain of Salmonella invades and colonizes tumor microenvironments [10, 15–19]. Orally administered Salmonella-IL2 carrying the IL-2 gene has been proven to produce biologically active IL-2 that is locally released in tumor microenvironments [12, 18]. The natural activity of IL-2 is to increase populations of NK and CD8 + cytotoxic T cells in tumor microenvironments as well as in the peripheral blood [26]. Both of these cell types have the unique property of indiscriminately killing cancer cells. We also have demonstrated in preclinical studies that Salmonella-IL2 significantly stimulates both a NK cell and CD8 + T cell population surge both within the tumor microenvironment and in the peripheral blood in preclinical studies. Both cell types are thought to impart a direct cancer cell kill and aid in the development of long-term anti-tumor memory. This has been observed experimentally. Salmonella infection alone (without the IL-2 gene) also appears to produce a bystander, immunologically mediated surge in various immune cell types, such as CD4 + T cells within the tumor microenvironment, further contributing to destruction of cancer cells but to a lesser effect than when the IL-2 gene is added to the attenuated Salmonella [9, 11, 15]. During investigations in nearly 4,000 mice administered Salmonella-IL2 using tumor models of metastatic colorectal cancer, primary breast cancer, primary neuroblastoma, metastatic osteosarcoma, or primary pancreatic cancer, no untoward effects of the drug were observed in treated mice [6, 8–15] .

The 10⁹ cfu dosing of Salmonella-IL2 was selected based on the outcomes of the Phase I trial where there were no adverse events at any dosing level. Thus, the highest dose was utilized for this Phase II trial. Moreover, the timing of administration of Salmonella-IL2 was based on extensive preclinical observation which demonstrated that maximal solid tumor colonization by Salmonella occurred when Salmonella-IL2 was administered 3 days after systemic chemotherapy was given. In addition, the dosing frequency of every 2 weeks was based on the preclinical observation that intratumoral bacterial cfu’s peak at 2 weeks in solid tumors and are nearly eliminated within 6 weeks. In the first patient with metastatic pancreas cancer treated with FOLFIRINOX plus Salmonella-IL-2, peripheral immune cellular populations were studied. Profiling of circulating immune cells indeed demonstrated a significant elevation in CD56^bright NK cells upon administration of Salmonella-IL2 when compared to pre-administration levels [27]. These findings were similar to what was observed in numerous preclinical experiments using various murine tumor models. The COVID pandemic restricted our ability to perform additional sampling and analyses in the present study.

Trial administration

Data were collected by investigators and a third-party clinical trial monitor (Exactis Innovations, Montreal, Quebec, Canada). Data were compiled by the clinical trial monitor and analyzed by the sponsor. An independent Data Safety and Monitoring Committee (DSMC) reviewed the clinical protocol and periodically reviewed the clinical trial data and gave permission to continue the trial to completion. All authors contributed to the interpretation of data and preparation of the manuscript. They vouch for the completeness of the data collected and the statistical analysis.

Endpoints

The primary endpoint was length of survival from the time of diagnosis and an objective response was defined as either a complete or partial response according to RECIST 1.1 criteria. Secondary endpoints included safety and systemic biomarker (CEA and CA19-9) responses. Patients alive at the time of data cutoff were censored at their last known date of follow-up as of March 1, 2025. PFS was defined as the time from treatment initiation to either radiologically confirmed disease progression per RECIST 1.1 criteria or death, whichever occurred first. These definitions match established oncology standards for survival endpoints and allow proper handling of censored observations. Adverse events were graded and categorized according to the National Cancer Institute Common Terminology Criteria for Adverse Events (v5.0) with attribution to the administered chemotherapy or study drug [23].

Statistical analysis

The Phase 2 trial was a small, signal-seeking study primarily intended to evaluate safety/feasibility and estimate preliminary efficacy; as such, the statistical framework was intentionally streamlined and focused on descriptive estimation. All statistical analyses were conducted in a pre-specified observational fashion. Survival distributions for OS and PFS were estimated using the Kaplan–Meier (KM) method, which provides a nonparametric estimate of the survival function and accommodates right-censored data without assuming an underlying survival distribution. Median survival times were reported with their corresponding 95% confidence intervals (CIs). Kernel density estimation plots of the survival data were constructed to visually support the observed survival. To quantify the relative risk of death or progression, hazard ratios (HRs) and their 95% CIs were estimated using Cox proportional hazards regression.

Results

Patients

As of the data cutoff date of March 1, 2025, 37 patients were enrolled in the clinical study. Twenty-six patients were enrolled in the Salmonella-IL2 plus FOLFIRINOX arm. Three withdrew from the study shortly after enrolling, and one patient was non-compliant with administration of the study drug, so that 22 patients remained for full analyses. With the four Expanded Access Program patients, a total of 26 patients remained in the Salmonella-IL2 plus FOLFIRINOX arm. Of these 26 patients, 20 patients received > 5 doses of Salmonella IL-2.

Eleven patients were enrolled in the Salmonella-IL2 plus Gemcitabine/nab Paclitaxel arm; three of them failed their screen entrance to the study. Thus, eight patients remained in the Salmonella-IL2 plus Gemcitabine/nab Paclitaxel arm. Table 1 details patient demographics. Enrolled patients tended to be older in the Gemcitabine/nab Paclitaxel arm but in all other noted characteristics, the participants tended be similar.

Table 1.

Demographics of SSP20-006. Arm One- Salmonella-IL2 + FOLFIRINOX; Arm Two – Salmonella-IL2 + Gemcitabine/nab Paclitaxel. Arm One also includes 4 Expanded Access Patients

	Arm One (N = 26)	Arm Two (N = 11)	Total (N = 37)
Race
N	26	11	37
Black	1 (4.2%)	1 (9.1%)	2 (5.7%)
Chinese	1 (4.2%)	0 (0.0%)	1 (2.9%)
South Asian—East Indian, Pakistani, Sri Lankan…	1 (4.2%)	0 (0.0%)	1 (2.9%)
White or Caucasian	23 (88.5%)	10 (90.9%)	33 (89.2%)
Age at consent
N	26	11	37
Mean ± SD	58.7 ± 11.2	68.0 ± 7.0	61.6 ± 10.9
Mean [CI 95%]	58.7 [54.0, 63.4]	68.0 [63.3, 72.7]	61.6 [57.9, 65.4]
Median (P25, P75)	61.5 (52.2, 65.5)	66.0 (63.5, 72.0)	63.0 (57.5, 69.0)
Min—Max	32.0—74.0	56.0—82.0	32.0—82.0
Sex at birth
N	26	11	37
Female	13 (50.0%)	6 (54.5%)	19 (51.4%)
Male	13 (50.0%)	5 (45.5%)	18 (48.6%)

Clinical activity and efficacy

Salmonella-IL2 with FOLFIRINOX

A therapeutic effect was observed in those patients who received five or more doses of Salmonella-IL2 plus FOLFIRINOX (n = 20). The median progression-free survival (mPFS) observed was 15 months (Fig. 1) while the median overall survival (mOS) observed was 20.3 months (Fig. 2). Kernel density estimation plots of the survival data visually support the observed increase in both mPFS and mOS (Fig. 3). This graphical evidence qualitatively reinforces the quantitative message in the data. Objectively, the partial response rate was 70.0%, there were no complete responses, and thus the overall response rate was 70.0%. Moreover, at one year, 65% of the patients were alive, and 40% were alive at two years. (Table 2).

Fig. 1.

Median Progression-Free Survival (mPFS) of study compliant and Expanded Access Program patients who received greater than five doses of Salmonella-IL2 with FOLFIRINOX (Treatment) (n = 20) demonstrating a mPFS of 15 months. Legend: Treatment (Blue line)—Salmonella-IL2 with FOLFIRINOX. mPFS-median Progression-Free Survival; mOS-median Overall Survival

Fig. 2.

Median Overall Survival (mOS) of study compliant and Expanded Access Program patients who received greater than five doses of Salmonella-IL2 with FOLFIRINOX (Treatment) (n = 20) demonstrating a mOS of 20.3 months. Legend: Treatment (Blue line)—Salmonella-IL2 with FOLFIRINOX. mPFS-median Progression-Free Survival; mOS-median Overall Survival

Fig. 3.

Kernel density estimation plots of the survival data visually support the distribution of PFS and OS observed, illustrating that a higher proportion of patients in the Treatment Group (Salmonella-IL2 + FOLFIRINOX) experienced longer overall and progression-free survival than one would expect from the historical literature. mPFS-median Progression-Free Survival; mOS-median Overall Survival

Table 2.

Efficacy Analysis of Stage 4 PC Patients Treated with Salmonella-IL2 in Combination with FOLFIRINOX, including with and without EAP Patients. EAP- Expanded Access Program, FFX: FOLFIRINOX; mOS: median overall survival; mPFS: median progression-free survival; ORR: overall response rate; PRR: partial response rate; Yr: year. **Clopper-Pearson binomial method was used to compute the 95% confidence intervals (CI)

	Salmonella-IL2 + FFX mPFS (months)	Salmonella-IL2 + FFX mOS (months)	Response Rates (95% CI)**	% Surviving 1 and 2 years
All patients who received > 5 doses, including EAP patients (n = 20)	15 (Range 3–33 months)	20.3 (Range 5.3–44.1 months)	PRR: 70% (45.5–87.9%) CRR: 0 (0–13.9%) ORR: 70%	1 Yr Survival: 65% 2 Yr Survival: 40%
All patients including ≤ 5 doses, including EAP patients (n = 25)	15 (Range 3–33 months)	18.6 (Range: 2.1–44.1 months)	PRR: 56% (34.9–75.6%) CRR: 0 (0–13.7%) ORR: 56%	1 Yr Survival: 52% 2 Yr Survival: 32%
Compliant patients, no EAP patients, including ≤ 5 doses (n = 21)	12 (Range 3–32 months)	13.8 (Range 2.1–35.3 months)	PRR:47.6% (25.7–70.2%) CRR: 0 (25.7–70.2%) ORR: 47.6%	1 Yr Survival: 42.9% 2 Yr Survival: 19%
Compliant patients who received > 5 doses, No EAP patients (n = 16)	13.5 (Range 3–32 months)	18.6 (Range 5.3–35.3 months)	PRR: 62.5% (35.4–84.8%) CRR: 0 (0–16.9%) ORR: 62.5%	1 Yr Survival: 56.2% 2 Yr Survival: 25%

When examining all patients enrolled in the Salmonella-IL2 plus FOLFIRINOX arm of the trial and were compliant with the clinical study regimen, excluding the Expanded Access Program patients but only including those who received greater than five doses of Salmonella-IL2 (n = 16), the mPFS was 13.5 months while the mOS was 18.6 months for a PRR of 62.5%. (Table 2) Analysis of all compliant patients who received FOLFIRINOX plus Salmonella-IL2 including those EAP patients (n = 25), the mPFS was 15 months and the mOS was 18.6 months. Lastly, analysis of all compliant patients enrolled into the trial and excluding EAP patients (n = 21), the mPFS was 12.0 months compared to 5.8 months and the mOS was 13.8 months. (Table 2) Partial response rate (PRR), complete response rate (CRR) and overall response rate (ORR) is reported in Table 2 at a 95% Confidence Interval.

Salmonella-IL2 with gemcitabine/nab paclitaxel

Of the eight patients who received Salmonella-IL2 with Gemcitabine/nab Paclitaxel, one patient received only two doses of the study drug, so seven patients were analyzed in this group. The mPFS was 6 months and the mOS was 8.6 months for patients in this study arm (data not shown). Given the small numbers of enrolled patients in this arm, conclusions regarding effectiveness were not possible. In addition, we learned that Gemcitabine possesses anti-microbial activity, thus is not the ideal candidate to be combined with any bacterial based immunotherapeutic [28].

Biomarker analysis

CA19-9 and CEA levels were drawn on every study participant every 3 months. CA19-9 was the more predominant biomarker that demonstrated elevation when compared to CEA for the diagnosis of pancreatic cancer. Specifically, a downward trend was observed with treatment with Salmonella-IL2 with FOLFIRINOX for both biomarkers and rose accordingly with disease progression (data not shown).

Safety

There were 41 serious adverse events (SAE) reported during the trial. Of these 41 events, only one (dehydration) was initially attributed to use of the study drug. On secondary review, the principal investigators and the DSMC concluded that this case of dehydration would likely be more attributable to the chemotherapy rather than to the study drug. (Table 3).

Table 3.

Serious Adverse Events: 41 serious adverse events were reported. Only 1 (dehydration) in one patient was initially attributed to the study drug, Salmonella-IL2. Secondary review by the Principal Investigator and the Data Safety and Monitoring Committee concluded that, similar to all other Serious Adverse Events, attribution to the study drug was not appropriate but rather to FOLFIRINOX or Gemcitabine/nab Paclitaxel administration

#	SAE description	Occurence
1	Abdomina; pain	3
2	Ascites	1
3	Bilary Infection	1
4	Bowel Obstruction	1
5	Bilary Obstruction	1
6	Cholangitis	2
7	Dehydration	2
8	Diarrhea	1
9	Fatigue	1
10	Febrile Neutropenia	2
11	Fever	5
12	Hematemesis	1
13	Jaundice	1
14	Mucisitis	1
15	Nausea	2
16	Neutropenia	1
17	Neutropenia Sepasis	1
18	Pancolitis	1
19	Peripheral Sensory Neuropathy	1
20	Probable cholangitis	1
21	Pulmonary Embolism	3
22	Sepasis	2
23	Severe Pain Radiating to the Back	1
24	Syncope	1
25	Tachycardia	1
26	Vomiting	3
	Total	41

Discussion

Stage IV pancreatic cancer is characterized by an extremely poor prognosis and alternatives to standard of care therapies are desperately needed. Currently, the standard of care is either use of a four-drug chemotherapeutic regimen (FOLFIRINOX) or a two-drug regimen (Gemcitabine/nab Paclitaxel). [2, 3] In 2011, Conroy, et al. reported a comparison of FOLFIRINOX and Gemcitabine alone, finding a significant increase in both OS and PFS for FOLFIRINOX, a result that ultimately led to the addition of nab Paclitaxel to Gemcitabine [29]. As previously stated, a recent meta-analysis of seven trials encompassing a total of 2,581 patients revealed a median PFS that ranged from 5.7–7.4 months and a median OS that ranged from 10.4–11.7 months regardless of the drug regimen used to treat metastatic pancreatic cancer [4]. Given that there have been no clinically significant improvements of metastatic pancreatic cancer therapies over the last 15 years, the critical need for innovation in therapeutic strategies is obvious. The present non-randomized, open label, two arm phase II clinical study demonstrated an increase in both median progression-free survival and median overall survival when patients were administered at least 5 doses of Salmonella-IL2 was given with FOLFIRINOX when compared to the historical pancreatic literature. Recruitment into the FOLFIRINOX arm was better than for the Gemcitabine/nab Paclitaxel arm and it is not possible to draw conclusions about the impact of adjunctive Salmonella-IL2 use in the latter arm. The fact that gemcitabine has anti-microbial properties may have played a role in the lack of response in this arm. [28].

To date, immunotherapeutic strategies have found limited success in achieving either meaningful overall or progression-free survival in advanced pancreatic cancer [30, 31]. Bacterial based immunotherapy potentially represents a novel paradigm shift in treating malignancies [10]. The unique propensity to colonize a solid tumor microenvironment and essentially function as an in situ bioreactor that produces the immune modulating protein IL-2 in a non-toxic manner is especially attractive for pancreatic cancer. Salmonella-IL2 appears to be consistently successful in both preclinical experimentation and observational clinical studies. A multifactorial impact has been observed in tumor cell destruction with this treatment strategy. Multiple preclinical tumor models (pancreatic cancer, colorectal carcinoma, breast cancer, and sarcoma) consistently demonstrate robust tumor colonization by Salmonella-IL2, a release of IL-2 into the tumor microenvironment, and subsequent immune cell mediated tumor cell killing. [14–18].

In addition, in a recent phase I trial in humans with metastatic GI cancers who were administered only one dose of Salmonella-IL2, a statistically significant increase in peripheral blood NK and NK-T cell populations after Samonella-IL2 administration was observed. [21] Furthermore, preclinical experiments using this attenuated strain of Salmonella without the IL-2 gene demonstrated colonization and growth within the tumor microenvironment as it initiated a bystander immune-mediated tumor destruction [15–19]. Generation of IL-2 by the engineered organism further enhances the observed immune-mediated tumor destruction. Lastly, these rapidly growing bacteria appear to compete for nutrient resources and consequently may essentially starve cancer cells [32]. Preclinical investigations also demonstrated a synergy with Salmonella-IL2 and chemotherapy [33]. Specifically, it has been shown that an equivalent tumor cell kill can be achieved in a preclinical murine model of breast cancer when maximum tolerated dosing (MTD) of Doxorubicin was compared to a 75% reduction in the Doxorubicin dose, but with Salmonella-IL2 added to that therapy agent.

Anecdotally, we observed that patients in the present clinical trial who were administered modified dosing of FOLFIRINOX (because of either chemo holidays, and/or eliminating oxaliplatin, and/or reduced dosing of 5-FU and/or Irinotecan) rather than MTD FOLFIRINOX experienced less serious adverse events and fared better in both progression-free survival and overall survival. These intriguing associations were unexpected and should be studied further.

Similar to experiences with other immune therapies, a minimum number of doses is required to observe a therapeutic effect of Salmonella-IL2 [34, 35]. It was determined that greater than five doses were required to observe sustained increases in both progression-free survival and overall survival. Unlike most other immunotherapies, no serious adverse events were observed in our observational clinical trial. Importantly, in this Phase 2 trial, one patient experienced persistent diarrhea and underwent stool testing, which was negative for Salmonella, and no clinical late-onset Salmonella infections were identified during the protocol-defined follow-up period. In addition, in the preceding Phase 1 clinical trial, periodic stool cultures were obtained prospectively, and Salmonella shedding was not demonstrated, providing supportive evidence that clinically meaningful shedding is unlikely under the tested conditions. [21].

Multiple efforts to implicate immune-based therapies in the treatment of pancreatic.

cancer have to date shown very limited success [36]. However, more recent studies using targeted mRNA vaccine therapies in combination with FOLFIRINOX in the adjuvant setting for early-stage resectable pancreatic cancer, demonstrated that immune-based therapeutics can indeed have a beneficial impact in some patients [37]. In these studies, the clinical benefits are associated with the demonstration of both specific antibody and T cell immune responses to the antigens. While this approach appears to be limited to a minority of pancreatic cancer patients with surgically resectable early-stage disease and requires a very burdensome process to isolate specific tumor antigens that can be used to create the personalized mRNA vaccine, it does demonstrate that immune-based therapies can be effective even in pancreas cancer. In this preliminary study of the more common presentation of this tragic disease, our immune-based approach, again in combination with FOLFIRINOX, has yielded startling clinical outcomes. Salmonella-IL2 indiscriminately targets solid cancers, and importantly its use does not require a costly, patient-specific manufacturing process. [10, 12–21].

Limitations and future directions

This Phase II study has several limitations inherent to its design. The non-randomized, single-arm approach limits control of confounding variables and introduces potential selection bias. Inclusion of patients treated under an Expanded Access Program may have added heterogeneity; however, all patients received an identical standard-of-care chemotherapy backbone, and incorporation of these patients enabled efficient generation of real-world evidence in a disease with limited single-center accrual.

The absence of a concurrent control arm increases susceptibility to temporal confounding. Nevertheless, this signal-seeking design is appropriate for early-phase evaluation, as outcomes associated with first-line mFOLFIRINOX in metastatic pancreatic ductal adenocarcinoma are well-characterized across clinical trials and real-world datasets, providing a robust external benchmark for contextual interpretation.

Direct confirmation of intratumoral bacterial colonization and localized IL-2 release was not performed. Mandatory tumor biopsies were not incorporated because of ethical, logistical, and feasibility considerations in patients with metastatic disease, where repeated or deep tissue sampling carries meaningful risk and could have adversely affected enrollment. Despite this limitation, mechanistic plausibility is supported by extensive preclinical data demonstrating tumor-selective Salmonella colonization, localized IL-2 expression, and immune activation, as well as by the observed clinical activity in the absence of systemic IL-2-associated toxicity.

Prospective immune profiling was planned but was largely precluded by COVID-19-related operational constraints, including restricted access to correlative laboratories, biospecimen shipping challenges, and patient safety considerations limiting serial blood sampling. Consequently, immune phenotyping was performed in only the first treated patient [27].

The modest sample size (n = 37) further limits statistical power and generalizability. The sample size was intentionally selected to balance ethical considerations, feasibility, and the need to detect a clinically meaningful efficacy signal relative to well-characterized historical outcomes. The addition of Expanded Access Program patients to the formal study patients might be considered a limitation, but given the limited number of patients with stage IV pancreatic cancer at a single site, “real-world” evidence generated from these added patients created an opportunity to analyze the study drug in an accepted, efficient, and cost effective manner [38]. Accordingly, results are interpreted as descriptive and hypothesis-generating, with uncertainty reflected by confidence intervals rather than formal hypothesis testing. Importantly, the magnitude and consistency of observed clinical activity across efficacy endpoints, together with a favorable safety profile, support advancement to a larger, adequately powered, multicenter Phase 3 trial designed to confirm efficacy, validate mechanism of action through immunologic assessments and intratumoral evidence of Salmonella, assess generalizability across broader patient populations, and provide definitive estimates of treatment effect.

This clinical trial represents the first evaluation of a genetically engineered bacterial therapeutic delivering an immune modulator in combination with standard-of-care chemotherapy for metastatic pancreatic ductal adenocarcinoma. Despite the limitations inherent to an early-phase study, the observed clinical activity is encouraging. Accordingly, the planned Phase 3 program will consist of a multisite, randomized controlled trial designed to support Biologics License Application-enabling development, incorporating enhanced microbial safety surveillance, including scheduled stool cultures supplemented by symptom-triggered testing and standardized reporting of microbial-related adverse events.

The Phase 3 study will be robustly powered to confirm efficacy and will include mandatory tumor biopsies, integrated molecular profiling, and comprehensive assessment of both systemic and intratumoral immune activation to directly link biological effects with clinical outcomes. In parallel, based on the observed activity in this historically treatment-refractory disease, additional Phase II studies are planned to evaluate this therapeutic approach across other solid tumor indications.

Author Contribution

All authors contributed to the interpretation of data and preparation of the manuscript. They vouch for the completeness of the data collected and the statistical analysis.

Funding

The study was collaboratively designed by the clinical trial sponsor, Salspera, and the principal investigators, Drs. Batist and Kavan. Salspera provided comprehensive funding for the trial, including regulatory approvals, study execution, data acquisition, and both statistical analysis and interpretation. To ensure scientific rigor and minimize bias, data analysis and interpretation were independently conducted by two separate statisticians, each funded by Salspera. Preparation of the manuscript and the decision to submit to the Cancer Immunology, Immunotherapy were jointly made by all of the authors reflecting a shared commitment to transparency and the advancement of immuno-oncology research.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Conflicts of interest

Dr. Daniel Saltzman is a Professor of Surgery and Pediatrics and the Chief of Pediatric Surgery at the University of Minnesota. Dr. Saltzman is also the co-founder and chief medical officer of Salspera. Salspera is a biotechnology company centered around bacterial based immunotherapeutics and hold the patents for Salmonella-IL2. This conflict has been extensively disclosed and is managed by the Office of Conflict Administration at the University of Minnesota.