Anti-tumor efficacy of plasmid encoding emm55 in a murine melanoma model


Emm55 is a bacterial gene derived from Streptococcus pyogenes (S. pyogenes) that was cloned into a plasmid DNA vaccine (pAc/emm55). In this study, we investigated the anti-tumor efficacy of pAc/emm55 in a B16 murine melanoma model. Intralesional (IL) injections of pAc/emm55 significantly delayed tumor growth compared to the pAc/Empty group. There was a significant increase in the CD8+ T cells infiltrating into the tumors after pAc/emm55 treatment compared to the control group. In addition, we observed that IL injection of pAc/emm55 increased antigen-specific T cell infiltration into tumors. Depletion of CD4+ or CD8+ T cells abrogated the anti-tumor effect of pAc/emm55. Combination treatment of IL injection of pAc/emm55 with anti-PD-1 antibody significantly delayed tumor growth compared to either monotherapy. pAc/emm55 treatment combined with PD-1 blockade enhanced anti-tumor immune response and improved systemic anti-tumor immunity. Together, these strategies may lead to improvements in the treatment of patients with melanoma.

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Bacillus Calmette–Guerin


Dendritic cell








Programmed death receptor-1




Tumor-infiltrating lymphocytes


Toll-like receptor


Talimogene laherparepvec


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We would like to thank Charles James for technical assistance. This work has been supported in part by the Flow Cytometry and the Analytic Microscopy Core Facility at the H. Lee Moffitt Cancer Center & Research Institute, an NCI designated Comprehensive Cancer Center (P30-CA076292).


This work was supported by a sponsored research agreement from Morphogenesis, Inc. to the H Lee Moffitt Cancer Center and Research Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We wish to acknowledge the Donald A Adam Comprehensive Melanoma Research Center at Moffitt Cancer Center. KK was supported by a Phi Beta Psi sorority award.

Author information




Shari Pilon-Thomas and Joseph Markowitz conceived and planned the experiments; Brittany Bunch, Krithika Kodumudi, Ellen Scott, Jennifer Morse, and Amy Weber carried out the experiments; Brittany Bunch, Krithika Kodumudi, Anders E Berglund Shari Pilon-Thomas, and Joseph Markowitz analyzed and interpreted the data; Brittany Bunch and Krithika Kodumudi wrote the manuscript with support from Shari Pilon-Thomas and Joseph Markowitz; Shari Pilon-Thomas and Joseph Markowitz supervised the project.

Corresponding authors

Correspondence to Shari Pilon-Thomas or Joseph Markowitz.

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Conflict of interest

JM is the PI of an institutional grant from Morphogenesis for clinical trial activities. JM receives support from the Donald A. Adam Comprehensive Melanoma Research Center at Moffitt Cancer Center. Unrelated to this paper, JM was a member of an Array Biopharma Advisory Board in 2018 and is an advisory board member for Newlink Genetics. JM was also the recipient of a career enhancement program award under Melanoma Skin Cancer SPORE P50 CA158536 and the Institutional Research Grant number 17–173-22 from the American Cancer Society. JM received funding from Navigate BP and is currently funded by Jackson Laboratories for work unrelated to this paper. Moffitt Cancer Center has licensed Intellectual Property related to the proliferation and expansion of tumor-infiltrating lymphocytes (TILs) to Iovance Biotherapeutics. SPT is an inventor on such Intellectual Property. SPT participates in sponsored research agreements with Provectus Biopharmaceuticals, Iovance Biotherapeutics, Intellia Therapeutics, and Myst Therapeutics that are not related to this research. Dr. Pilon-Thomas has received research support that is not related to this research from the following entities: American Cancer Society -Leo and Anne Albert Charitable Foundation Research Scholar Grant (RSG-16–117-01-LIB), State of Florida Bankhead-Coley Cancer Research Program (7BC08), NIH-NCI (U01 CA244100-01 and R01 CA239219-01A1), V Foundation, and Swim Across America. Additionally, Dr. Pilon-Thomas is a co-Investigator on NIH-NCI (U54 CA193489-01A1 and R01 CA241559) research support, which is not related to this research. The other authors declare that they have no conflict of interest

Ethical approval

All experiments with mice were performed in compliance with the principles, and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals and protocols were approved after review by the Institutional Animal Care and Use Committee at the University of South Florida (Tampa, FL). The University of South Florida Comparative Medicine is fully accredited by AAALAC International as program #000434, is managed in accordance with the Guide for the Care and Use of Laboratory Animals, the Animal Welfare Regulations, the PHS Policy, the FDA Good Laboratory Practices, and the IACUC Principles and Procedures of Animal Care and Use, has an assurance #D16-00589 (A4100-01) on file with OLAW/PHS, and maintains registration #58-R-0015 with USDA/APHIS/AC.

Human and animal rights

C57BL/6 mice were obtained from Charles River (Frederick, MD). OT-I and MyD88 knock-out mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

Cell line authentication

B16 melanoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). M05 melanoma cells were generously provided by Dr. Kenneth Rock (Dana-Farber Cancer Institute). B16 and M05 cells were verified for the lack of microbial contamination including mycoplasma by IDEXX BioAnalytics. Cell lines were expanded and cryopreserved according to the culture and cryopreserving conditions recommended by American Type Culture Collection.

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Bunch, B.L., Kodumudi, K.N., Scott, E. et al. Anti-tumor efficacy of plasmid encoding emm55 in a murine melanoma model. Cancer Immunol Immunother 69, 2465–2476 (2020).

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  • Melanoma
  • emm55
  • Streptococcus pyogenes
  • Bacterial protein
  • Intralesional injection