novyi-NT Spores Research Articles

Anaerobic bacterial metabolism responsive microspheres for bacterial embolization cancer therapy

Anaerobic bacteriolytic cancer therapy, whether delivered locally or systemically, frequently encounters challenges related to limited colonization within hypoxic pockets of central tumors and activation of innate immunity. Herein we have developed trans-arterial bacteria embolization therapy using bacterial embolic microspheres. C. novyi-NT spores loaded calcium alginate embolic microspheres demonstrated C. novyi-NT metabolites-mediated microsphere degradation, releasing vegetative C. novyi-NT bacterial in hypoxic condition. Transcatheter directed bacterial microsphere embolization therapy occludes tumor feeding vessels with infused bacterial embolic microspheres and enhances tumoral hypoxia. Notably, anaerobic bacterial metabolism responsive microsphere-bacterial embolization therapy achieved a complete tumor response with enhanced tumor-specific bacterial delivery and colonization, resulting in cancer cell killing across the entire tumor. In vivo tumor response and immunological profiling revealed that bacterial embolization uniquely enhances anti-cancer response, effectively engaging direct anaerobic bacterial oncolysis and adaptive and innate immune responses in a cooperative manner.

Therapeutic Potential of Clostridium novyi NT in Cancer: Current Knowledge and Future Perspectives.

Resistance to conventional antitumour therapies and Hypoxia in patients with advanced solid tumours are two major reasons for the failure of conventional anti-tumour therapies. Therefore, it is important to find a new therapeutic method that can overcome these problems. An attenuated anaerobic bacterium, Clostridium novyi- NT, could target Hypoxic and Necrotic areas of tumours causing tumour lysis and stimulating a host anti-tumour immune response. To the best of our knowledge, the combination of bacterial anti-tumour therapy, chemotherapy, radiotherapy and immunotherapy may promote tumour regression, inhibit metastasis and develop a new strategy for the treatment of solid tumours. However, the possible molecular mechanisms of the combined therapies are still the biggest challenge. This review provides an overview of the history of bacterial cancer therapy and the development of a non-lethal strain of Clostridium novyi. Below is a precise definition of Hypoxic conditions in solid tumour tissue. To understand the anticancer effect of Clostridium novyi- NT spores, possible cell death mechanisms were summarised by the enzyme phospholipase C (nt01cx0979), which is secreted by Clostridium novyi- NT spores after germination in tumour tissue. The function of Clostridium novyi- NT spores in stimulating the host immune system to elicit anti-tumour responses was reviewed. Then, the results of anti-tumour combination therapies based on Clostridium novyi- NT spores were compiled. Identifying the molecular mechanisms of Clostridium novyi- NT in treating tumours and inducing cell death in invasive cancer cells, ultimately leading to tumour regression, may develop promising clinical strategies in the combined treatment of solid tumours.

A design of experiments screen reveals that Clostridium novyi-NT spore germinant sensing is stereoflexible for valine and its analogs

Although Clostridium novyi-NT is an anti-cancer bacterial therapeutic which germinates within hypoxic tumors to kill cancer cells, the actual germination triggers for C. novyi-NT are still unknown. In this study, we screen candidate germinants using combinatorial experimental designs and discover by serendipity that D-valine is a potent germinant, inducing 50% spore germination at 4.2 mM concentration. Further investigation revealed that five D-valine analogs are also germinants and four of these analogs are enantiomeric pairs. This stereoflexible effect of L- and D-amino acids shows that spore germination is a complex process where enantiomeric interactions can be confounders. This study also identifies L-cysteine as a germinant, and hypoxanthine and inosine as co-germinants. Several other amino acids promote (L-valine, L-histidine, L-threonine and L-alanine) or inhibit (L-arginine, L-glycine, L-lysine, L-tryptophan) germination in an interaction-dependent manner. D-alanine inhibits all germination, even in complex growth media. This work lays the foundation for improving the germination efficacy of C. novyi-NT spores in tumors.

Multifunctional porous microspheres encapsulating oncolytic bacterial spores and their potential for cancer immunotherapy.

Clostridium novyi-NT (C. novyi-NT) is an anaerobic bacterium that can be used for targeted cancer therapy because it germinates selectively in the hypoxic regions of tumor tissues. However, systemic administration of C. novyi-NT spores cannot effectively treat tumors because of the limited intratumoral delivery of active spores. In this study, we demonstrated that multifunctional porous microspheres (MPMs) containing C. novyi-NT spores have the potential for image-guided local tumor therapy. The MPMs can be repositioned under an external magnetic field, enabling precise tumor targeting and retention. Polylactic acid-based MPMs were prepared using the oil-in-water emulsion technique and then coated with a cationic polyethyleneimine polymer prior to loading with negatively charged C. novyi-NT spores. The C. novyi-NT spores delivered by MPMs were released and germinated in a simulated tumor microenvironment, effectively secreting proteins cytotoxic to tumor cells. In addition, the germinated C. novyi-NT induced immunogenic death of the tumor cells and M1 polarization of macrophages. These results indicate that MPMs encapsulated with C. novyi-NT spores have great potential for image-guided cancer immunotherapy.

Abstract PO-037: Development of an RGD CRISPR-modified Clostridium novyi NT spores as an intravenous oncotherapy

Abstract The efficacy of current oncotherapeutics is largely limited by an inability to access avascular tissues, which is in part responsible for forty years of stagnant pancreatic cancer statistics where the median survival remains a mere six months. Oncolytic bacteria such as Clostridium novyi-NT overcome this challenge with its ultrasensitive, innate affinity for hypoxic/necrotic areas found at the center of solid tumors and their metastases. While preclinical and clinical data from intratumoral injections of C. novyi-NT are promising, many tumors are inaccessible to such injections. Preclinical trials of analogous IV injections have uncovered other obstacles such as rapid clearance of C. novyi-NT by the immune system independent of septic complications. To mitigate rapid clearance, CRISPR/Cas9n was used to genetically modify a non-toxic form of C. novyi-NT to express a tumor targeting RGD peptide on the spore surface. Through this novel, first of its kind, methodology, spores with stronger affinity to a surface coated with the targeted binding partner of RGD, aVb3 integrin, have been generated. Importantly, there was no statistically significant difference in the genetically modified spore’s capacity for sporulation or germination when compared to unmodified C. novyi-NT spores, nor was a difference in lytic capacity observed, suggesting no relevant off-target effects from genomic modification. Biodistribution and efficacy of non-toxic RGD-modified spores was evaluated in an immunocompetent, syngeneic, pancreatic cancer murine model. Ongoing efforts to characterize the biodistribution and efficacy of the intravenously injected RGD-modified C. novyi-NT include the application of multiplex immunofluorescence, laser microdissection, and live, whole animal imaging. Supported as a pilot project by funds from NIH COBRE grant 1P20GM109024, Doctoral Dissertation Funds to KMD from NDSU, and by discretionary funds from investigators at UNMC. Citation Format: Kaitlin M. Dailey, Krysten Vance, Kyle McAndrews, Reed I. Jacobson, Jandro Delgado, Paige R. Johnson, Taylor M. Woolery, Megan Orr, Jiha Kim, Sanku Mallik, Kenneth W. Bayles, Michael A. Hollingsworth, Amanda E. Brooks. Development of an RGD CRISPR-modified Clostridium novyi NT spores as an intravenous oncotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-037.

Bacteriolytic Therapy: Tumor Microenvironment Targeting Nano–Bio Emulsion for Synergistic Combinational X‐Ray PDT with Oncolytic Bacteria Therapy (Adv. Healthcare Mater. 13/2020)

In article number 1901812 by Joonseok Lee, Dong-Hyun Kim, and co-workers, a tumor microenvironment targeting nano-bio therapeutic emulsion formulated with multi-functional nanoscintillators and anaerobic C. novyi-NT spores is developed for synergistic combinational cancer therapy. Nano-bio therapeutic emulsion is delivered to tumor sites under CT-image guidance, and effectively treats the normoxic peripheral and hypoxic central tumor region.

Tumor Microenvironment Targeting Nano-Bio Emulsion for Synergistic Combinational X-Ray PDT with Oncolytic Bacteria Therapy.

Various cancer therapies have been developed, but tumor recurrence with incomplete tumor killing and remaining tumor cells/tissues is frequent in monotherapies. Herein, a nano-bio therapeutic emulsion formulated with multifunctional nanoscintillators and anaerobic Clostridium novyi-NT spores for synergistic image-guided combinational cancer therapy is reported. MRI visible nanoscintillators (NSs) are synthesized with a NaGdF4 :Tb,Ce@NaGdF4 core/shell structure for an image-guided X-ray photodynamic therapy (PDT) of the normoxic peripheral tumor. An anaerobic oncolytic bacterium (C. novyi-NT) therapy is combined to treat the hypoxic central tumor tissues. Photosensitizer-coated NSs (PS-NSs) and C. novyi-NT spores are emulsified with clinically available ethiodized oil (Lipiodol) to be the nano-bio therapeutic emulsion and injected into the tumor with computed tomography image guidance. The distribution of nano-bio therapeutic emulsion, including PS-NSs and anaerobic C. novyi-NT spores in the tumor site, is confirmed by both X-ray and T1 -weighted magnetic resonance imaging. Following the image-guided X-ray PDT and anaerobic C. novyi- NT combination treatment, apoptotic cell death in cancer tissues, including both peripheral and central tumor regions, is significantly higher than in the control groups. This combination therapy approach using a nano-bio therapeutic emulsion is expected to overcome the limitations of conventional cancer therapy, resulting in increased cancer-therapeutic efficacy.

Iron-Oxide Nanocluster Labeling of Clostridium novyi-NT Spores for MR Imaging–Monitored Locoregional Delivery to Liver Tumors in Rat and Rabbit Models

PurposeTo label Clostridium novyi-NT spores (C. novyi-NT) with iron oxide nanoclusters and track distribution of bacteria during magnetic resonance (MR) imaging-monitored locoregional delivery to liver tumors using intratumoral injection or intra-arterial transcatheter infusion. Materials and MethodsVegetative state C. novyi-NT were labeled with iron oxide particles followed by induction of sporulation. Labeling was confirmed with fluorescence microscopy and transmission electron microscopy (TEM). T2 and T2* relaxation times for magnetic clusters and magnetic microspheres were determined using 7T and 1.5T MR imaging scanners. In vitro assays compared labeled bacteria viability and oncolytic potential to unlabeled controls. Labeled spores were either directly injected into N1-S1 rodent liver tumors (n = 24) or selectively infused via the hepatic artery in rabbits with VX2 liver tumors (n = 3). Hematoxylin-eosin, Prussian blue, and gram staining were performed. Statistical comparison methods included paired t-test and ANOVA. ResultsBoth fluorescence microscopy and TEM studies confirmed presence of iron oxide labels within the bacterial spores. Phantom studies demonstrated that the synthesized nanoclusters produce R2 relaxivities comparable to clinical agents. Labeling had no significant impact on overall growth or oncolytic properties (P >.05). Tumor signal-to-noise ratio (SNR) decreased significantly following intratumoral injection and intra-arterial infusion of labeled spores (P <.05). Prussian blue and gram staining confirmed spore delivery. ConclusionsC. novyi-NT spores can be internally labeled with iron oxide nanoparticles to visualize distribution with MR imaging during locoregional bacteriolytic therapy involving direct injection or intra-arterial transcatheter infusion.

Immune response to C. novyi-NT immunotherapy

Clostridium novyi-NT (CVN-NT) spores germinate in hypoxic regions of tumors and have successfully cured induced neoplasia in mouse models and resulted in objective tumor responses in naturally developing neoplasia in the dog. The objective of this pilot, descriptive, prospective, clinical investigation, was to evaluate and describe the immune response to CNV-NT spores to better understand which immune pathways might play a role in the response to this bacteriolytic immunotherapy. Intratumoral injection of CNV-NT spores result in increased phagocytosis and NK cell-like function after treatment. Intravenous injection of CNV-NT spores resulted in increased LPS-induced TNF-α production, LTA-induced IL-10 production and NK cell-like function post-treatment. Increased NK cell-like function was sustained to 28 (intratumoral) or 56 (intravenous) days post-treatment, and increased phagocytic function was sustained to 28 days post-treatment suggesting that CNV-NT spores induce longer-term immune cell function changes. Future investigations evaluating long-term immune system changes and associations between immune function and tumor remission rates should include evaluation of these pathways.

Learning from Clostridium novyi-NT: How to defeat cancer.

Side effects associated with conventional anticancer therapies have prompted the new idea of solid tumor treatment strategy. One of them is using bacteria explored as potential antitumor agents over more than one century. Notably, the ideal therapy is a specifical target to tumors with limited toxicity. Here, we take "Clostridium novyi" for the search keyword in the PubMed from 2000 to 2015 and describe that C. novyi-NT spores act as "Trojan horse" for bacteriolytic therapy. This therapy is based on the fact that the live and attenuated obligate anaerobic bacteria are capable of binary fission selectively in anoxic areas of solid tumors and direct tumoricidal effects. Our succinct review mainly concentrates on the potential mechanisms of combination bacteriolytic therapy, an effective and safe tumor therapy with the help of C. novyi-NT. Importantly, C. novyi-NT spores were shown to induce solid tumor regression and exhibit the property to initiate an immune response. Therefore, C. novyi-NT spores should be an effective and safe tumor therapy.

Abstract No. 419 - CT-guided intra-tumoral injection of gold-coated Clostridium novyi-NT spores for bacteriolytic therapy in solid tumors

Bacteriolytic therapy using anaerobic bacteria is considered as a promising approach for the treatment of solid tumors. To improve therapeutic efficacy while potentially reducing toxicity, we demonstrate an approach to label Clostridium novyi-NT (C. novyi-NT) spores with branched gold nanoparticles (BGNP) thus permitting intra-procedural X-ray CT visualization of spore delivery to solid tumors upon intra-tumoral injection. C. Novyi-NT spores were kindly provided by BioMed Valley Discoveries (Kansas City, MO, USA). BGNP were synthesized in an aqueous solution of sodium cholate and then the BGNP were modified with 4-mercaptobenzoic acid to render negatively charge. BGNP-coated spores were prepared using a simple electrostatic deposition method. In vitro phantom and in vivo (PC-3 human prostate cancer mice model) CT studies were performed using a micro-CT imaging system (NanoPET/CT®, Mediso Ltd-Bioscan Inc., Hungary-US). Successful preparation of BGNP-coated C. Novyi-NT spores was confirmed by various physico-chemical characterization methods. In the PC3 mouse model, BGNP-coated spores were locally injected into the hypoxic center of each tumor guided with CT confirmation of intra-tumoral delivery. The therapeutic efficacy of these CT-guided infused BGNP-coated spores demonstrated in immunohistochemical analyses such as H&E and TUNEL staining. The infused BGNP-coated spores exhibited strong therapeutic efficacy consistent with that observed for native spores suggesting that the innate anti-tumoral function was preserved. Our BGNP-coating on the C. Novyi-NT spores have the potential to not only facilitate CT image-guided bacteriolytic procedures but also to serve as a novel BGNP-mediated combinational therapeutic delivery platform.

Abstract No. 21FEATURED ABSTRACT - Iron oxide labeled Clostridium novyi-NT spores for image-guided bacteriolytic therapy

To develop iron-oxide labeled Clostridium novyi-NT spores for MRI visualization during bacteriolytic therapy. Studies have demonstrated that both direct and intravenous delivery of spores can effectively treat solid tumors. MRI visualization of spore delivery should be valuable for intra-procedural optimization of tumor-directed administration procedures and early prediction of treatment outcomes. Vegetative state C. novyi NT were incubated for 24 hours with iron oxide particles coupled with rhodamine B (Ocean Nanotech, Inc.) and then placed in cooked meat medium to induce sporulation. Resulting spores underwent viability studies comparing germination rates as measured by optical density. Confocal microscopy was used to evaluate labeling efficacy and transmission electron microscopy (TEM) to confirm iron oxide uptake. MRI phantoms were constructed by mixing 5 x 10^8 spores with 250µL of agarose. T2*-weighted images of the phantoms were acquired using a 7T MR scanner. Direct injections of iron-oxide labeled spores in 6 Sprague Dawley rats with N1-S1 liver tumors were performed for in vivo visualization with MRI. Animals were survived for 72 hours to confirm germination of bacteria, visualized through gram stain on histology. Labeled spores demonstrated a similar germination rate as control spores at 24, 48, and 72 hours of incubation. Fluorescence microscopy demonstrated labeling efficacy of 100% and TEM showed diffuse deposition of iron oxide within the spores. T2*-weighted imaging showed reduced signal in the labeled spores phantom (SNR 9.46 ± 0.45) compared to unlabeled controls (SNR 22.3 ± 1.93). Labeled spores could be visualized with MRI immediately after direct injection and also at 3 days follow-up. Vegetative state bacteria were visible on histology in all successfully treated tumors. C. novyi NT spores can be efficiently labeled with iron oxides by inducing sporulation in labeled vegetative state bacteria. These methods have direct applications for image-guided bacteriolytic therapy and are permitting ongoing comparisons between IV and direct injection administration routes as well as the investigation of intra-arterial catheter-directed alternatives.

284 - Phase I clinical study of intratumoral injection of oncolytic Clostridium novyi-NT spores in patients with advanced cancers

284 - Phase I clinical study of intratumoral injection of oncolytic Clostridium novyi-NT spores in patients with advanced cancers

Branched Gold Nanoparticle Coating of Clostridium novyi-NT Spores for CT-Guided Intratumoral Injection.

Branched gold nanoparticle (BGNP)-coated Clostridium novyi-NT (C. novyi-NT) spores are developed for computed tomography (CT)-guided bacteriolytic tumor therapy. The BGNP-coated spores are successfully injected into a tumor site under CT image guidance. As a result, a strong antitumor effect is observed in a PC3 prostate tumor-bearing mouse model.

P1.09 - Phase I clinical study of intratumoral injection of Clostridium novyi-NT spores in patients with advanced cancer

ABSTRACT Background: Intratumoral injection of Clostridium novyi-NT (attenuated strain of Clostridium) induced a microscopically precise, tumor-localized response in a rat orthotopic brain tumor model and in companion dogs bearing spontaneous solid tumors. Furthermore, intratumoral injection can plausibly induce an immune mediated abscopal effect in non-injected tumor sites. Methods: A standard 3 + 3 dose escalation Phase 1 study of a single intratumoral injection of C.novyi-NT was designed to determine the DLTs, MTD and preliminary activity in adult patients with advanced cancers and superficially located tumors (1-12 cm in size). The SOFA score and temperature are used to guide antibiotic usage. Results: To date, a total of 5 patients (women, n = 3; men, n = 2) with advanced sarcomas (leiomyosarcoma, n = 2; chondrosarcoma, n = 1; carcinosarcoma, n = 1; angiosarcoma, n = 1), median age 54 years, have received an intratumoral injection of 1 x104 (n = 3) and 3 x104 spores (n = 2). Evidence of germination has been noted in 3 patients and has consisted of pain in the injected tumor, fever, elevated WBC with a left shift, an elevated C-reactive protein, and necrosis and gas pockets in the injected tumor on radiographic imaging. Treatment-related grade 3 adverse events include leukocytosis, pathologic fracture, pain and respiratory insufficiency. The most significant morbidity was observed in a patient with a large volume of tumor destruction leading to a pathologic fracture of the necrotic right proximal humerus, which required an aggressive orthopedic surgical procedure. Of note histopathology demonstrated extensive tumor necrosis with small foci of residual tumor cells during surgery. Two patients had between 22-24% shrinkage in the injected tumor and an overall response of stable disease at 2 months. Because exploratory cytokine analysis suggests the potential for a systemic response and because abscopal responses were seen in the companion dog studies, the clinical protocol has been amended to include biopsies of both injected and non-injected tumors pre and post C.novyi-NT injection. Conclusions: Intratumoral injection of Clostridium novyi-NT is feasible and has lead to significant destruction of injected tumor masses in the first two doses studied.

Phase 1 Trial of Image-Guided Oncolysis by Clostridium Novyi-NT Spore Inoculation: Early Technical Insights

Purpose Hypoxic environments, as occurs in tumors, are favorable for Clostridium subspecies to germinate. Clostridium novyi-NT (C. novyi-NT) induces a microscopic & tumor-confined lysis after intratumoral (IT) injection in rat orthotopic brain tumor models & spontaneous solid tumors in dogs, with the commonest toxicity being the traditional accompanying symptoms of bacterial infections. A first-in-man phase 1 study selecting therapy-refractory solid tumors palpable or identifiable under imaging guidance and amenable to percutaneous injection of C. novyi-NT spores is being conducted. Tumor environment hypoxia is dynamic, spatially heterogenic, and not evaluable using clinical imaging technology, thereby rendering a priori localization of the optimal IT location for spore inoculation impossible. In order to compensate for this unknown parameter, we employed a staged, multifocal IT delivery process to theoretically increase the likelihood of spore deposition within a milieu conducive for germination. We present our initial experience with C. novyi-NT spore delivery adopting this approach. Material and Methods Advanced solid tumor cancer patients with at least 1 injectable tumor >1 cm are being enrolled in 5 dose-escalating cohorts to receive single 3-cc IT injections of 1E4, 3E4, 1E5, 3E5, or 1E6 C. novyi-NT spores. Results Four patients have been treated (3 in cohort 1, 1 in cohort 2). Median tumor diameter measured at point of guide needle access was 5.4 cm (2.3-8.9 cm). Sites included intermuscular upper extremity and subcutaneous fat abdominal lesion (2 each), which were uneventful; in toto delivery of the agent was accomplished. Two patients demonstrated clinical evidence of germination within 72 hours corroborated by imaging. Pathology confirmed oncolysis. The liquefied components of the tumor were managed with evacuation via percutaneous drainage and pre-specified protocol-mandated multi-antimicrobial therapy. Conclusions Clostridium novyi-NT spores can be delivered using existing imaging technologies and devices producing clinical evidence of germination and confirming the capability of inducing oncolysis in human neoplastic tissues.

ET-58 * THE GENETICALLY ENGINEERED CLOSTRIDIUM NOVYI-NT IS EFFICACIOUS IN THE TREATMENT OF INTRACRANIAL GLIOBLASTOMAS

Glioblastomas (GBM) exhibit a remarkable histopathologic heterogeneity with extensive necrotic regions intermixed with cell proliferation that generate local hypoxia and thus, limit the effectiveness greatly of traditional cancer therapies. The spore-forming bacterium Clostridium spp., however, has the ability to precisely target and lyse tumor cells in hypoxic environments. Here we show that Clostridium novyi-NT (C. novyi-NT), an engineered strain devoid of the lethal alpha-toxin, induces a microscopically tumor-localized response in orthotopically implanted syngeneic F98 and human xenograft 060919 rat GBM models. Both, intravenous and intratumoral, injections of C. novyi-NT spores resulted in germination within 24 hours and a rapid fall in luciferase activity, an indicator of tumor burden, in both animal models. C. novyi-NT germination was evidenced by the appearance of vegetative forms of C. novyi-NT bacteria. Strikingly, C. novyi-NT precisely localized to the tumor and micro-invasive tumor islands buried in the normal brain parenchyma, sparing adjacent normal cells only a few microns away. This bacterial biosurgery led to a significant survival advantage in these extremely aggressive rat models. Brain edema and increased intracranial pressure as a result of C. novyi-NT infection was common and medically managed while brain abscess formation was not clearly observed with appropriate use of antibiotics. Abscessation, however, is a potential side effect and can develop in human patients possibly requiring neurosurgical abscess drainage, a routine clinical procedure. In summary, C. novyi-NT can act as a tool to precisely eradicate GBM and significantly extend survival. Despite concerns on treatment-related toxicity, bacterial C. novyi-NT treatment may have a place in selected GBM patients in the future given the lack of alternative therapy options and dismal prognosis.

Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses.

Species of Clostridium bacteria are notable for their ability to lyse tumor cells growing in hypoxic environments. We show that an attenuated strain of Clostridium novyi (C. novyi-NT) induces a microscopically precise, tumor-localized response in a rat orthotopic brain tumor model after intratumoral injection. It is well known, however, that experimental models often do not reliably predict the responses of human patients to therapeutic agents. We therefore used naturally occurring canine tumors as a translational bridge to human trials. Canine tumors are more like those of humans because they occur in animals with heterogeneous genetic backgrounds, are of host origin, and are due to spontaneous rather than engineered mutations. We found that intratumoral injection of C. novyi-NT spores was well tolerated in companion dogs bearing spontaneous solid tumors, with the most common toxicities being the expected symptoms associated with bacterial infections. Objective responses were observed in 6 of 16 dogs (37.5%), with three complete and three partial responses. On the basis of these encouraging results, we treated a human patient who had an advanced leiomyosarcoma with an intratumoral injection of C. novyi-NT spores. This treatment reduced the tumor within and surrounding the bone. Together, these results show that C. novyi-NT can precisely eradicate neoplastic tissues and suggest that further clinical trials of this agent in selected patients are warranted.

Evaluation of Clostridium novyi–NT spores in dogs with naturally occurring tumors

To establish the maximum tolerated dose of Clostridium novyi-NT spores in tumor-bearing dogs and evaluate spore germination within tumors and tumor response. 6 client-owned dogs. A standard dose-escalation study was planned, with maximum tolerated dose defined as the highest dose at which 0 or 1 of 6 dogs had dose-limiting toxicoses (DLT). Dogs received 1 dose of C. novyi-NT spores i.v.. Toxicoses were graded and interventions performed according to specific guidelines. Grade 3 or higher toxicosis or any toxicosis combination that substantially affected patient status was considered DLT. Clinical response was measured by use of response evaluation criteria in solid tumors at 28 days. The first 2 dogs had DLT. The dose was decreased. Two of the next 4 dogs had DLT; therefore, dose administration was stopped because the study endpoint had been reached. The most common toxicosis was fever (n = 6 dogs). Two dogs developed abscesses (1 within a nasal carcinoma and 1 splenic abscess) attributable to C. novyi-NT infection; both required surgical intervention. Clostridium novyi-NT was cultured from 1 of 6 tumors. Five dogs were available for response assessment (4 had stable disease; 1 had progressive disease). Results indicated that C. novyi-NT can germinate within tumors of dogs. Toxicosis, although common and sometimes severe, was manageable with treatment. Further studies in dogs with superficial tumors may allow for continued dose escalation and provide information for use in clinical trials in veterinary and human oncology.

Bacteriolytic therapy of experimental pancreatic carcinoma

To investigate the effectiveness of Clostridium novyi (C. novyi)-NT spores for the treatment of established subcutaneous pancreatic tumor in the syngeneic, immunocompetent Panc02/C57Bl/6 model. C. novyi-NT spores were applied intravenously to animals carrying established pancreatic tumors of three different sizes. Systemic immune responses in peripheral blood and spleen were examined by flow cytometry. Supplementary, cytotoxic activity of lymphocytes against syngeneic tumor targets was analyzed. Application of spores identified, that (1) small tumors (< 150 mm(3)) were completely unaffected (n = 10); (2) very large tumors (> 450 mm(3)) responded with substantial necrosis followed by shrinkage and significant lethality most likely due to tumor lysis syndrome (n = 6); and (3) an optimal treatment window exists for tumors of approximately 250 mm(3) (n = 21). In this latter group, all tumor-bearing animals had complete tumor regression and remained free of tumor recurrence. In subsequent tumor rechallenge experiments a significant delay in tumor growth compared to the initial tumor cell inoculation was observed (tumor volume at day 28: 197.8 +/- 87.3 mm(3) vs 500.1 +/- 50.9 mm(3), P < 0.05). These effects were accompanied by systemic activation of immune response mechanisms predominantly mediated by the innate arm of the immune system. The observed complete tumor regression is encouraging and shows that immunotherapy with C. novyi-NT is an interesting strategy for the treatment of pancreatic carcinomas of defined sizes.