Efficient Secretion of Murine IL-2 From an Attenuated Strain of Clostridium sporogenes, a Novel Delivery Vehicle for Cancer Immunotherapy

Abstract

Despite a history dating back to the 1800s, using Clostridium bacteria to treat cancer has not advanced beyond the observation that they can colonise and partially destroy solid tumours. Progress has been hampered by their inability to eradicate the viable portion of tumours, and an instinctive anxiety around injecting patients with a bacterium whose close relatives cause tetanus and botulism. However, recent advances in techniques to genetically engineer Clostridium species gives cause to revisit this concept. This paper illustrates these developments through the attenuation of C. sporogenes to enhance its clinical safety, and through the expression and secretion of an immunotherapeutic. An 8.6 kb sequence, corresponding to a haemolysin operon, was deleted from the genome and replaced with a short non-coding sequence. The resultant phenotype of this strain, named C. sporogenes-NT, showed a reduction of haemolysis to levels similar to the probiotic strain, C. butyricum M588. Comparison to the parental strain showed no change in growth or sporulation. Following injection of tumour-bearing mice with purified spores of the attenuated strain, high levels of germination were detected in all tumours. Very low levels of spores and vegetative cells were detected in the spleen and lymph nodes. The new strain was transformed with four different murine IL-2-expressing plasmids, differentiated by promoter and signal peptide sequences. Biologically active mIL-2, recovered from the extracellular fraction of bacterial cultures, was shown to stimulate proliferation of T cells. With this investigation we propose a new, safer candidate for intratumoral delivery of cancer immunotherapeutics.

Heterologous Gene Regulation in Clostridia: Rationally Designed Gene Regulation for Industrial and Medical Applications

“…No-toxic strain of C. sporogenes NCIMB 10696 was created previously by deleting the putative Streptolysin S (SLS) operon C.…”

“…However, most clostridia are completely benign . Beyond the domain of human health, solventogenic clostridia have been utilized in the industrial production of petrochemicals due to their ability to produce acetone–butanol–ethanol (ABE) by microbial fermentation (Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum), while cellulolytic, biofuel-producing strains, such as Clostridium cellulolyticum and Clostridium cellulovorans, are now attracting attention to satisfy the increasing demand for cleaner fuels. , Oncolytic strains, such as Clostridium sporogenes and Clostridium novyi, selectively germinate in the hypoxic/necrotic regions of tumors and their endogenous oncolytic activity can result in partial destruction of the tumor. , Subsequent studies have highlighted distinct advantages to using certain Clostridium species as a therapeutic vector in bacterial-mediated cancer therapy. − The addition, through genetic engineering, of clinically approved heterologous products, such as antibodies, proinflammatory cytokines, , and checkpoint inhibitors, could further increase their therapeutic value. An alternative approach is clostridia-directed enzyme prodrug therapy (CDEPT), which has seen significant advances in recent years. − Thus, demand for synthetic strains of these species has highlighted the need for genetic tools that will enable the precise control of synthetic products of industrial or therapeutic value.…”

“…In combination with different 5′ UTR sequences, this promoter-5′ UTR library was evaluated for gene expression in two Clostridium sensu stricto species: C. sporogenes-NT and C. butyricum DSM 10702 .…”

“…6 , 7 Subsequent studies have highlighted distinct advantages to using certain Clostridium species as a therapeutic vector in bacterial-mediated cancer therapy. 8 − 10 The addition, through genetic engineering, of clinically approved heterologous products, such as antibodies, 11 proinflammatory cytokines, 12 , 13 and checkpoint inhibitors, could further increase their therapeutic value. An alternative approach is clostridia-directed enzyme prodrug therapy (CDEPT), which has seen significant advances in recent years.…”

“…Strains and Plasmids Used in This Work supplemented with 12.5 μg/mL of chloramphenicol or on LB agar plates supplemented with 25 μg/mL of chloramphenicol. No-toxic strain of C. sporogenes NCIMB 10696 was created previously by deleting the putative Streptolysin S (SLS) operon 12. C. butyricum-type strain DSM 10702 was purchased from DSMZ-German Collection.…”