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Controlled Formation of CpG Oligonucleotide Multimers

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Technology Summary

Unmethylated cytosine-phosphodiester-guanine (CpG) oligonucleotides (ODNs) act as immunostimulants to cells that express Toll-like receptor 9 (TLR9), such as B cells, macrophages, dendritic cells, and monocytes. CpG ODNs can be used as an adjuvant to improve a vaccine’s humoral and cellular immune responses in a host. CpG ODNs tend to form troublesome G-tetrads in solution during manufacture and storage, leading to polymorphisms, aggregation, and precipitation that limits clinical applications.

FDA researchers developed a platform technology to functionalize a CpG ODN with a thermolytic substituent that prevents undesirable CpG-tetrad aggregation in solution during manufacture or storage, while alternatively enabling CpG-tetrad formation inside the cell which enhances activity. Use of FDA’s thermolytic substituent technology on an CpG ODN can delay the formation of G-tetrads until they are internalized by their target immune cells where multimerization is required. The effect of modifying an CpG ODN with thermolytic substituents was tested on D35 (Pro-D35 modified with thermolabile substituents), a well-characterized ODN that activates the innate immune system and enhances the T-cell effector mechanism. The thermolytic substituents reduced G-tetrad formation in potassium free solutions, while allowing tetrad formation when potassium salt concentrations mimicked those of an intracellular environment. Pro-D35 did not differ in cellular uptake or biological activity compared to unmodified D35, as shown by increased cytokine production and DC maturation. Pro-D35 demonstrated clinical potential in rhesus macaques challenge studies with Leishmania major. The FDA thermolytic CpG ODN technology can be used to develop immunotherapeutic oligonucleotides with improved storage stability and therapeutic efficacy.

Potential Commercial Applications Competitive Advantages
  • Adjuvant
  • Vaccine development
  • Stabilizes CpG ODNs for manufacture/storage (reduced G-tetrad formation)
  • Effective cellular uptake and controlled multimerization
  • Demonstrated clinical potential in primate studies

Development Stage: in vitro data; in vivo data; preclinical primate studies

Inventors: Daniela Verthelyi, Serge Beaucage, Andrzej Grajkowski


“Design and development of thermolytic DNA oligonucleotide prodrugs.” Ann N Y Acad Sci. 2005 Nov;1058:26-38. PMID: 16394123

“Thermolytic CpG-containing DNA oligonucleotides as potential immunotherapeutic prodrugs.” Nucleic Acids Res. 2005 Jun 21;33(11):3550-60. Print 2005. PMID: 15972797

“Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates.” Nucleic Acids Res. 2006;34(22):6488-95. Epub 2006 Nov 27. PMID: 17130156

Intellectual Property:

US Pat: US 9,809,824 B2, issued 11/7/2017

Product Area:  adjuvant; vaccine development

FDA Reference No: E-2004-024

Licensing Contact:
FDA Technology Transfer Program
Email: FDAInventionlicensing@fda.hhs.gov

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