. 2014 Aug 13;6(249):249ra111.

doi: 10.1126/scitranslmed.3008982.

Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses

Nicholas J Roberts^#¹, Linping Zhang^#², Filip Janku^#³, Amanda Collins^#², Ren-Yuan Bai^#⁴, Verena Staedtke^#^{4

5}, Anthony W Rusk⁶, David Tung², Maria Miller², Jeffrey Roix², Kristen V Khanna⁶, Ravi Murthy⁷, Robert S Benjamin⁸, Thorunn Helgason³, Ariel D Szvalb⁹, Justin E Bird¹⁰, Sinchita Roy-Chowdhuri¹¹, Halle H Zhang², Yuan Qiao¹, Baktiar Karim¹², Jennifer McDaniel¹³, Amanda Elpiner¹⁴, Alexandra Sahora¹⁵, Joshua Lachowicz¹⁶, Brenda Phillips¹⁷, Avenelle Turner¹⁸, Mary K Klein¹⁹, Gerald Post¹³, Luis A Diaz Jr^{1

20}, Gregory J Riggins⁴, Nickolas Papadopoulos¹, Kenneth W Kinzler¹, Bert Vogelstein¹, Chetan Bettegowda^{1

4}, David L Huso¹², Mary Varterasian², Saurabh Saha^#², Shibin Zhou^#¹

Affiliations

¹ The Ludwig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical Institute at The Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 21287, USA.
² BioMed Valley Discoveries Inc., 4520 Main Street, Kansas City, MO 64111, USA.
³ Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
⁴ Department of Neurosurgery, The Johns Hopkins Medical Institutes, Baltimore, MD 21231, USA.
⁵ Department of Neurology, The Johns Hopkins Medical Institutes, Baltimore, MD 21231, USA.
⁶ Animal Clinical Investigation LLC, 4926 Wisconsin Avenue, NW Washington, DC 20016, USA.
⁷ Department of Interventional Radiology, Division of Diagnostic Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
⁸ Department of Sarcoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
⁹ Department of Infectious Diseases, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹⁰ Department of Orthopedic Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹¹ Department of Pathology, Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
¹² Department of Molecular and Comparative Pathobiology, The Johns Hopkins University, Baltimore, MD 21205, USA.
¹³ The Veterinary Cancer Center, 129 Glover Avenue, Norwalk, CT 06850, USA.
¹⁴ VCA Great Lakes Veterinary Specialists, 5035 Richmond Road, Bedford Heights, OH 44146, USA.
¹⁵ The Oncology Service, Friendship Hospital for Animals, 4105 Brandywine Street, NW, Washington, DC 20016, USA.
¹⁶ BluePearl Veterinary Partners, 410 West 55th Street, New York, NY 10019, USA.
¹⁷ Veterinary Specialty Hospital of San Diego, 10435 Sorrento Valley Road, San Diego, CA 92121, USA.
¹⁸ Veterinary Cancer Group of Los Angeles at City of Angels Veterinary Specialty Center, 9599 Jefferson Boulevard, Culver City, CA 90232, USA.
¹⁹ Southern Arizona Veterinary Specialty and Emergency Center, 141 East Fort Lowell, Tucson, AZ 85705, USA.
²⁰ The Swim Across America Laboratory at Johns Hopkins, Baltimore, MD 21231, USA.

^# Contributed equally.

PMID: 25122639
PMCID: PMC4399712
DOI: 10.1126/scitranslmed.3008982

Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses

Nicholas J Roberts et al. Sci Transl Med. 2014.

. 2014 Aug 13;6(249):249ra111.

doi: 10.1126/scitranslmed.3008982.

Authors

Affiliations

¹ The Ludwig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical Institute at The Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 21287, USA.
² BioMed Valley Discoveries Inc., 4520 Main Street, Kansas City, MO 64111, USA.
³ Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
⁴ Department of Neurosurgery, The Johns Hopkins Medical Institutes, Baltimore, MD 21231, USA.
⁵ Department of Neurology, The Johns Hopkins Medical Institutes, Baltimore, MD 21231, USA.
⁶ Animal Clinical Investigation LLC, 4926 Wisconsin Avenue, NW Washington, DC 20016, USA.
⁷ Department of Interventional Radiology, Division of Diagnostic Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
⁸ Department of Sarcoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
⁹ Department of Infectious Diseases, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹⁰ Department of Orthopedic Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹¹ Department of Pathology, Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
¹² Department of Molecular and Comparative Pathobiology, The Johns Hopkins University, Baltimore, MD 21205, USA.
¹³ The Veterinary Cancer Center, 129 Glover Avenue, Norwalk, CT 06850, USA.
¹⁴ VCA Great Lakes Veterinary Specialists, 5035 Richmond Road, Bedford Heights, OH 44146, USA.
¹⁵ The Oncology Service, Friendship Hospital for Animals, 4105 Brandywine Street, NW, Washington, DC 20016, USA.
¹⁶ BluePearl Veterinary Partners, 410 West 55th Street, New York, NY 10019, USA.
¹⁷ Veterinary Specialty Hospital of San Diego, 10435 Sorrento Valley Road, San Diego, CA 92121, USA.
¹⁸ Veterinary Cancer Group of Los Angeles at City of Angels Veterinary Specialty Center, 9599 Jefferson Boulevard, Culver City, CA 90232, USA.
¹⁹ Southern Arizona Veterinary Specialty and Emergency Center, 141 East Fort Lowell, Tucson, AZ 85705, USA.
²⁰ The Swim Across America Laboratory at Johns Hopkins, Baltimore, MD 21231, USA.

^# Contributed equally.

PMID: 25122639
PMCID: PMC4399712
DOI: 10.1126/scitranslmed.3008982

Abstract

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.

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Figures

**Fig. 1. Germinated *C. novyi*–NT bacteria within microscopic rat brain tumor lesions**
Gram stain showed vegetative *C. novyi*–NT bacteria (yellow arrowheads) localized in tumor (T) and stellate microinvasion (S) but not in normal brain tissue (Br) from F344 Fisher rats. (A) Interface of tumor and normal brain; scale bar, 30 μm. (B) Interface of tumor and normal brain; scale bar, 10 μm. (C) Interface of normal brain, tumor, and stellate microinvasion of neoplastic tissue; scale bar, 30 μm. (D) *C. novyi*–NT germination evident in stellate microinvasive lesion; scale bar, 10 μm.

**Fig. 2. Photographic and computed tomography images from dog 11-R01 showing a PR to *C. novyi*-NT therapy**
Images span pretreatment to day 70 after first intratumoral dose of *C. novyi*-NT spores. (A) Pretreatment image of the peripheral nerve sheath tumor. (B) Abscess formation on day 3 of the study, with extent confined to tumor. (C) Medical debridement after spontaneous abscess rupture and discharge of necrotic and purulent material allowed healing by second intention. (D) The wound had healed completely by day 70 of the study, and 77.6% reduction in the largest diameter of the tumor was noted. (E) Pretreatment computed tomography (CT) image, taken 4 days before first treatment showed extent of tumor (yellow circle) at the intersection of the pinna and cranium. (F) Posttreatment CT image on day 10 of the study showed almost complete debulking of tumor.

**Fig. 3. Photographic and CT images from dog 04-R03 showing a CR to *C. novyi*-NT therapy**
Images span pretreatment to day 60 after first intratumoral dose of *C. novyi*–NT spores. (A) Pretreatment image of the soft tissue sarcoma. (B) Tumor localized abscess formed on day 15 of the study, 1 day after a third dose of *C. novyi*–NT spores. (C) Tumor debulking was complete by day 27 of the study, and healthy granulation tissue had formed. (D) The wound had healed completely by day 60 of the study, and no residual tumor was noted (CR). (E) Pretreatment CT image, taken 5 days before first treatment, showing extent of tumor (yellow circle) on antebrachium. (F) Posttreatment CT image on day 62 of the study showing complete loss of tumor mass.

**Fig. 4. CT and MRI images from the human patient**
(A) Posttreatment CT with contrast on day 3 demonstrating evidence of intra- and extramedullary air collection. (B) Pretreatment MRI (T₁ with gadolinium contrast) of the right upper humerus showing a contrast enhancing mass involving the soft tissue and possibly adjacent bone. (C) Posttreatment MRI on day 4 demonstrating diminished contrast enhancement in the tumor mass compared to baseline. (D) Posttreatment MRI on day 29 showing a homogeneous non-enhancing mass consistent with ongoing necrosis. Tumor is highlighted with yellow arrowheads.

**Fig. 5. Extensive tumor necrosis in the human patient treated with *C. novyi*-NT spores**
(A and B) Pretreatment tumor biopsy showing viable tumor (leiomyosarcoma) cells; scale bars, 100 and 30 μm, respectively. (C and D) Posttreatment tumor biopsy, 4 days after intratumoral injection of *C. novyi*-NT spores, showing extensive necrosis of tumor cells; scale bars, 100 and 30 μm, respectively.

See this image and copyright information in PMC

Comment in

Immunotherapy: the treatment bug--fighting cancer with bacterial infection.
Killock D. Killock D. Nat Rev Clin Oncol. 2014 Oct;11(10):562. doi: 10.1038/nrclinonc.2014.151. Epub 2014 Sep 2. Nat Rev Clin Oncol. 2014. PMID: 25178634 No abstract available.
Therapeutics: Bacterial treatment for cancer.
Lokody I. Lokody I. Nat Rev Cancer. 2014 Oct;14(10). doi: 10.1038/nrc3827. Nat Rev Cancer. 2014. PMID: 25230884 No abstract available.

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Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses

Affiliations

Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses

Authors

Affiliations

Abstract

Figures

Comment in

References

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Research Materials