Elsevier

Vaccine

Volume 27, Issue 30, 19 June 2009, Pages 3975-3983
Vaccine

The first clinical use of a live-attenuated Listeria monocytogenes vaccine: A Phase I safety study of Lm-LLO-E7 in patients with advanced carcinoma of the cervix

https://doi.org/10.1016/j.vaccine.2009.04.041Get rights and content

Abstract

Invasive carcinoma of the cervix (ICC) is the second most common cancer in women worldwide. Lm-LLO-E7 vaccine is a live-attenuated Listeria monocytogenes (Lm) that secretes the HPV-16 E7 antigen fused to a non-hemolytic fragment of the Lm protein listeriolysin O (LLO). In this Phase I trial, the safety of Lm-LLO-E7 was assessed in 15 patients with previously treated metastatic, refractory or recurrent ICC. Patients received 1 of 3 dose levels of Lm-LLO-E7 (1 × 109 CFU, 3.3 × 109 CFU or 1 × 1010 CFU) as an intravenous infusion, followed by a second dose 3 weeks later. All patients experienced a flu-like syndrome which responded to non-prescription symptomatic treatment. Severe (grade 3) adverse events related to Lm-LLO-E7 were reported in 6 patients (40%), but no grade 4 adverse events were observed. At the highest dose some patients had severe fever and dose limiting hypotension. By the end of the study protocol, 2 patients had died, 5 had progressed, 7 had stable disease and 1 qualified as a partial responder. This study shows for the first time that a live-attenuated Lm is safe to be administered to late stage ICC patients.

Introduction

Invasive carcinoma of the cervix (ICC) is the second most common cancer in women worldwide with over 450,000 new cases and 230,000 deaths annually, most of them occurring in developing countries [1]. Persistent infection with high-oncogenic risk human papillomavirus (HR-HPV) types is recognized as a necessary, but not sufficient, cause of cervical cancer [2], [3], [4]. HPVs 16 and 18 are the most prevalent types in malignant lesions, accounting for over 70% of ICC and over 50% of high-grade precursor lesions [5]. Although the advent of prophylactic HPV vaccines may have a significant impact on the incidence of ICC in the future, the worldwide implementation of such vaccines remains a challenge, particularly in developing countries [6]. Additionally, these vaccines are not intended to treat pre-existing HPV infections and associated malignancies, which require therapeutic vaccines, mostly targeting the E6 and E7 HPV oncoproteins [7].

Listeria monocytogenes (Lm) is a food-borne gram-positive bacterium that can occasionally cause disease in humans. Listeriosis is an uncommon infection, primarily affecting elderly individuals, newborns, pregnant women and immunocompromised individuals [8]. In addition to strongly activating innate immunity and inducing a cytokine response that enhances antigen-presenting cell (APC) function, Lm has the ability to replicate in the cytosol of APCs after escaping from the phagolysosome, which requires the virulence factor listeriolysin O (LLO) protein [9], [10]. This unique intracellular life cycle allows antigens secreted by Lm to be processed and presented in the context of both MHC class I and II molecules, resulting in potent cytotoxic CD8+ and Th1 CD4+ T-cell-mediated immune responses [10], [11].

Lm has been extensively investigated as a vector for cancer immunotherapy in pre-clinical models [12], [13], [14], [15], [16]. Lm-LLO-E7 is a recombinant live-attenuated Lm that secretes the antigen HPV-16 E7 fused to a non-hemolytic LLO [16]. Previous studies have shown that genetically fusing an antigen to LLO enhances the immunogenicity of tumor-associated antigens, resulting in a better therapeutic efficacy against established tumors [15], [16], [17].

Immunization of mice with Lm-LLO-E7 induces regression of established tumors expressing E7 and confers long-term protection [16]. Moreover, it is able to overcome immunological tolerance and impair the development and severity of autochthonous tumors in an E7 transgenic mouse model [18]. The therapeutic efficacy of Lm-LLO-E7 correlates with its ability to induce E7-specific tumor-infiltrating CTLs, mature dendritic cells, reduce the number of intratumoral regulatory CD4+ CD25+ T cells and inhibit tumor angiogenesis [19].

Lm has also a number of inherent advantages as a vaccine vector. The bacterium grows very efficiently in vitro without special requirements and it lacks LPS, which is a major toxicity factor in gram-negative bacteria, such as Salmonella[20]. Genetically attenuated Lm vectors also offer additional safety as they can be readily eliminated with antibiotics, in case of serious adverse effects and unlike some viral vectors, no integration of genetic material into the host genome occurs. Although the potential of Lm as a vaccine vector has been proven in pre-clinical studies, the feasibility of using live-attenuated Lm vectors as a cancer immunotherapeutic in humans has not been demonstrated yet. In a previous study, healthy volunteers were given orally escalating doses of an actA/plcB-deleted Lm strain without serious adverse effects or long-term sequelae [21]. However, no previous studies with Lm vectors have been done in cancer patients, who are the target population for Lm-LLO-E7 clinical use. Moreover, the highly attenuated Lm-LLO-E7 vector is given intravenously. We report herein the first clinical use and safety of a live-attenuated Lm vector to treat patients with advanced ICC, who had failed prior chemotherapy, radiotherapy, and/or surgery.

Section snippets

Construction of the Lm-LLO-E7 vaccine

Construction of Lm-LLO-E7 was previously described [16]. Briefly, the prfA-defective Lm strain XFL-7, which is derived from the streptomycin-resistant wild-type Lm 10403S strain [22], was transformed with the multicopy plasmid pGG55. This plasmid contains an expression cassette with the HPV-16 E7 gene fused to a truncated hly gene that encodes the first 441 residues of LLO. Additionally, pGG55 contains a mutated copy of the prfA gene that partially restores the virulence of XFL-7 and it is

Pre-clinical toxicity evaluation of Lm-LLO-E7

The MTD for IV administration of Lm-LLO-E7 in BALB/c female mice was 2.8 × 107 CFU. In the group that received 2.8 × 109 CFU, splenomegaly was observed in 2 out of 5 mice on day 7 after injection and it was defined as an AE. No deaths occurred at the doses tested. A dose of 2.8 × 107 CFU corresponds to a human dose of 7.8 × 1010 by body mass or 7.5 × 109 by surface area, a range that is consistent with the clinical doses used in this clinical trial. Importantly, an IV dose of 2.8 × 107 CFU of Lm-LLO-E7 was

Discussion

To our knowledge, this is the first clinical report demonstrating safety and feasibility on using a live-attenuated Lm vaccine in patients with cancer. Intravenous administration of Lm-LLO-E7 was consistently associated with a flu-like syndrome in all patients, characterized by pyrexia, chills, headache, tachycardia, hypotension, nausea and vomiting. These AE were acute and transient in most patients, resolving in the first 12 h after infusion. While well tolerated in the lower two dosage

Acknowledgments

The authors wish to acknowledge Dr. Yvonne Paterson from the University of Pennsylvania for her insight and guidance in the use of live Listeria monocytogenes vaccines, and Drs. Benigno Figueroa and Jorge Herrera-Rodriguez for their clinical work in Mexico. Dr. Pavle Vukojevc and Ms. Jelena Tasic for their essential work in the oversight of this clinical trial. This study was funded by Advaxis, Inc.

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