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.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Programmed death receptor-1
Agarwala SS (2016) The role of intralesional therapies in melanoma. Oncology (Williston Park) 30(5):436–441
Andtbacka RH, Kaufman HL, Collichio F, Amatruda T, Senzer N, Chesney J et al (2015) Talimogene laherparepvec improves durable response rate in patients with advanced melanoma. J Clin Oncol 33(25):2780–2788. https://doi.org/10.1200/JCO.2014.58.3377
Lippey J, Bousounis R, Behrenbruch C, McKay B, Spillane J, Henderson MA et al (2016) Intralesional PV-10 for in-transit melanoma—a single-center experience. J Surg Oncol 114(3):380–384. https://doi.org/10.1002/jso.24311
Liu H, Innamarato PP, Kodumudi K, Weber A, Nemoto S, Robinson JL et al (2016) Intralesional rose bengal in melanoma elicits tumor immunity via activation of dendritic cells by the release of high mobility group box 1. Oncotarget 7(25):37893–37905. https://doi.org/10.18632/oncotarget.9247
Toomey P, Kodumudi K, Weber A, Kuhn L, Moore E, Sarnaik AA et al (2013) Intralesional injection of rose bengal induces a systemic tumor-specific immune response in murine models of melanoma and breast cancer. PLoS ONE 8(7):e68561. https://doi.org/10.1371/journal.pone.0068561
Wiemann B, Starnes CO (1994) Coley's toxins, tumor necrosis factor and cancer research: a historical perspective. Pharmacol Ther 64(3):529–564
St Jean AT, Zhang M, Forbes NS (2008) Bacterial therapies: completing the cancer treatment toolbox. Curr Opin Biotechnol 19(5):511–517. https://doi.org/10.1016/j.copbio.2008.08.004
Bohle A, Schuller J, Knipper A, Hofstetter A (1990) Bacillus Calmette–Guerin treatment and vesicorenal reflux. Eur Urol 17(2):125–128
Rosevear HM, Lightfoot AJ, O'Donnell MA, Griffith TS (2009) The role of neutrophils and TNF-related apoptosis-inducing ligand (TRAIL) in bacillus Calmette–Guerin (BCG) immunotherapy for urothelial carcinoma of the bladder. Cancer Metastasis Rev 28(3–4):345–353. https://doi.org/10.1007/s10555-009-9195-6
Kresowik TP, Griffith TS (2009) Bacillus Calmette–Guerin immunotherapy for urothelial carcinoma of the bladder. Immunotherapy 1(2):281–288. https://doi.org/10.2217/1750743X.1.2.281
de Jong SD, Basha G, Wilson KD, Kazem M, Cullis P, Jefferies W et al (2010) The immunostimulatory activity of unmethylated and methylated CpG oligodeoxynucleotide is dependent on their ability to colocalize with TLR9 in late endosomes. J Immunol 184(11):6092–6102. https://doi.org/10.4049/jimmunol.0802442
Chikh G, de Jong SD, Sekirov L, Raney SG, Kazem M, Wilson KD et al (2009) Synthetic methylated CpG ODNs are potent in vivo adjuvants when delivered in liposomal nanoparticles. Int Immunol 21(7):757–767. https://doi.org/10.1093/intimm/dxp044
Mahfouz M, Hashimoto W, Das Gupta TK, Chakrabarty AM (2007) Bacterial proteins and CpG-rich extrachromosomal DNA in potential cancer therapy. Plasmid 57(1):4–17. https://doi.org/10.1016/j.plasmid.2006.11.001
Pilon-Thomas S, Li W, Briggs JJ, Djeu J, Mule JJ, Riker AI (2006) Immunostimulatory effects of CpG-ODN upon dendritic cell-based immunotherapy in a murine melanoma model. J Immunother 29(4):381–387. https://doi.org/10.1097/01.cji.0000199199.20717.67
Gantier MP, Tong S, Behlke MA, Irving AT, Lappas M, Nilsson UW et al (2010) Rational design of immunostimulatory siRNAs. Mol Ther 18(4):785–795. https://doi.org/10.1038/mt.2010.4
Li K, Qu S, Chen X, Wu Q, Shi M (2017) Promising targets for cancer immunotherapy: TLRs, RLRs, and STING-mediated innate immune pathways. Int J Mol Sci. https://doi.org/10.3390/ijms18020404
Shi M, Chen X, Ye K, Yao Y, Li Y (2016) Application potential of toll-like receptors in cancer immunotherapy: systematic review. Medicine (Baltimore) 95(25):e3951. https://doi.org/10.1097/MD.0000000000003951
Bendelac A, Medzhitov R (2002) Adjuvants of immunity: harnessing innate immunity to promote adaptive immunity. J Exp Med 195(5):F19–23. https://doi.org/10.1084/jem.20020073
Krieg AM (2003) CpG motifs: the active ingredient in bacterial extracts? Nat Med 9(7):831–835. https://doi.org/10.1038/nm0703-831
Ramiya VK, Jerald MM, Lawman PD, Lawman MJ (2014) Autologous tumor cells engineered to express bacterial antigens. Methods Mol Biol 1139:243–257. https://doi.org/10.1007/978-1-4939-0345-0_21
Glikin GC, Finocchiaro LM (2014) Clinical trials of immunogene therapy for spontaneous tumors in companion animals. Sci World J 2014:718520. https://doi.org/10.1155/2014/718520
Brown EL, Ramiya VK, Wright CA, Jerald MM, Via AD, Kuppala VN, Hazell WS, Lawman PD, Lawman MJ (2014) Treatment of metastatic equine melanoma with a plasmid DNA vaccine encoding Streptococcus Pyogenes EMM55 protein. J Equine Vet Sci 34:704–708. https://doi.org/10.1016/j.jevs.2013.11.012
Kodumudi KN, Siegel J, Weber AM, Scott E, Sarnaik AA, Pilon-Thomas S (2016) Immune checkpoint blockade to improve tumor infiltrating lymphocytes for adoptive cell therapy. PLoS ONE 11(4):e0153053. https://doi.org/10.1371/journal.pone.0153053
Kodumudi KN, Weber A, Sarnaik AA, Pilon-Thomas S (2012) Blockade of myeloid-derived suppressor cells after induction of lymphopenia improves adoptive T cell therapy in a murine model of melanoma. J Immunol 189(11):5147–5154. https://doi.org/10.4049/jimmunol.1200274
Vohra N, Verhaegen M, Martin L, Mackay A, Pilon-Thomas S (2010) TNF-alpha-treated DC exacerbates disease in a murine tumor metastasis model. Cancer Immunol Immunother 59(5):729–736. https://doi.org/10.1007/s00262-009-0793-5
Pilon-Thomas S, Nelson N, Vohra N, Jerald M, Pendleton L, Szekeres K et al (2011) Murine pancreatic adenocarcinoma dampens SHIP-1 expression and alters MDSC homeostasis and function. PLoS ONE 6(11):e27729. https://doi.org/10.1371/journal.pone.0027729
Kotera Y, Shimizu K, Mule JJ (2001) Comparative analysis of necrotic and apoptotic tumor cells as a source of antigen(s) in dendritic cell-based immunization. Cancer Res 61(22):8105–8109
Ibrahim-Hashim A, Abrahams D, Enriquez-Navas PM, Luddy K, Gatenby RA, Gillies RJ (2017) Tris-base buffer: a promising new inhibitor for cancer progression and metastasis. Cancer Med 6(7):1720–1729. https://doi.org/10.1002/cam4.1032
Elso CM, Roberts LJ, Smyth GK, Thomson RJ, Baldwin TM, Foote SJ, Handman E (2004) Leishmaniasis host response loci (lmr13) modify disease severity through a Th1/Th2-independent pathway. Genes Immun 5(2):93–100. https://doi.org/10.1038/sj.gene.6364042
Kaufman HL, Kim DW, DeRaffele G, Mitcham J, Coffin RS, Kim-Schulze S (2010) Local and distant immunity induced by intralesional vaccination with an oncolytic herpes virus encoding GM-CSF in patients with stage IIIc and IV melanoma. Ann Surg Oncol 17(3):718–730. https://doi.org/10.1245/s10434-009-0809-6
Maletzki C, Linnebacher M, Kreikemeyer B, Emmrich J (2008) Pancreatic cancer regression by intratumoural injection of live Streptococcus pyogenes in a syngeneic mouse model. Gut 57(4):483–491. https://doi.org/10.1136/gut.2007.125419
Schetters STT, Jong WSP, Horrevorts SK, Kruijssen LJW, Engels S, Stolk D, Daleke-Schermerhorn MH, Garcia-Vallejo J, Houben D, Unger WWJ, den Haan JMM, Luirink J, van Kooyk Y (2019) Outer membrane vesicles engineered to express membrane-bound antigen program dendritic cells for cross-presentation to CD8+ T cells. Acta Biomater. https://doi.org/10.1016/j.actbio.2019.04.033
Kim WS, Jung ID, Kim JS, Kim HM, Kwon KW, Park YM, Shin SJ (2018) Mycobacterium tuberculosis GrpE, a heat-shock stress responsive chaperone, promotes Th1-biased T cell immune response via TLR4-mediated activation of dendritic cells. Front Cell Infect Microbiol 27(8):95. https://doi.org/10.3389/fcimb.2018.00095
Takeda Y, Azuma M, Funami K, Shime H, Matsumoto M, Seya T (2018) Type I interferon-independent dendritic cell priming and antitumor T cell activation induced by a mycoplasma fermentans lipopeptide. Front Immunol 14(9):496. https://doi.org/10.3389/fimmu.2018.00496
Friedrich C, Mamareli P, Thiemann S, Kruse F, Wang Z, Holzmann B, Strowig T, Sparwasser T, Lochner M (2017) MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium. PLoS Pathog 13(5):e1006357. https://doi.org/10.1371/journal.ppat.1006357
Pilon-Thomas S, Mackay A, Vohra N, Mule JJ (2010) Blockade of programmed death ligand 1 enhances the therapeutic efficacy of combination immunotherapy against melanoma. J Immunol 184(7):3442–3449. https://doi.org/10.4049/jimmunol.0904114
Ozgun A, Sondak VK, Markowitz J (2016) Resistance patterns to anti-PD-1 therapy in metastatic melanoma. Chin Clin Oncol 5(6):75. https://doi.org/10.21037/cco.2016.08.01
Ribas A, Puzanov I, Dummer R, Schadendorf D, Hamid O, Robert C et al (2015) Pembrolizumab versus investigator-choice chemotherapy for ipilimumab-refractory melanoma (KEYNOTE-002): a randomised, controlled, phase 2 trial. Lancet Oncol 16(8):908–918. https://doi.org/10.1016/S1470-2045(15)00083-2
Weber JS, D'Angelo SP, Minor D, Hodi FS, Gutzmer R, Neyns B et al (2015) Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial. Lancet Oncol 16(4):375–384. https://doi.org/10.1016/S1470-2045(15)70076-8
Jia YY, Tan WJ, Duan FF, Pan ZM, Chen X, Yin YL et al (2017) A Genetically modified attenuated listeria vaccine expressing HPV16 E7 Kill tumor cells in direct and antigen-specific manner. Front Cell Infect Microbiol 7:279. https://doi.org/10.3389/fcimb.2017.00279
Markowitz J, Kodumudi K, De Aquino DB, Sondak VK, Pilon-Thomas S (2019) Trial in progress: First in human Phase I study using a plasmid DNA coding for Emm55 streptococcal antigen (IFx-Hu2.0) in patients with unresectable stage III or stage IV cutaneous melanoma [abstract]. Cancer Res 79(13 Suppl):Abstract nr CT119
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.
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
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.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
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). https://doi.org/10.1007/s00262-020-02634-4
- Streptococcus pyogenes
- Bacterial protein
- Intralesional injection