CRM197

[1]CRM197 is a non-toxic mutant of diphtheria toxin, currently used as a carrier protein for polysaccharides and haptens to make them immunogenic.[2]

Description

CRM197 is a genetically detoxified form of diphtheria toxin. A single mutation at position 52, substituting glutamic acid for glycine, causes the ADP-ribosyltransferase activity of the native toxin to be lost. The structural basis for the lack of CRM197 toxicity has recently been elucidated.[3] CRM197 is widely used as a carrier protein for conjugate vaccines. An advantage of CRM197 over toxoided proteins is that, because it is genetically detoxified, it retains its full complement of lysine amines for conjugation. There is also evidence suggesting that, compared with tetanus toxoid, there is less carrier-induced suppression of the immune response, especially when there are many individual polysaccharides linked to the same carrier protein.[4] A summary of the uses and properties of CRM197 has been published.[4] CRM197, like diphtheria toxin, is a single polypeptide chain of 535 amino acids (58.4 kD) consisting of two subunits (linked by disulfide bridges).

Manufacturing

The gene for CRM197 has been cloned into Corynebacterium diphtheriae, the bacteria that produces the native toxin.[5] Like the wild type toxin, CRM197 is expressed as a secreted protein at relatively low yields (typically <100 mg/L). Corynebacterium expressed CRM197 is available from several sources, including List Laboratories and SigmaAldrich. The low yield and high cost of commercially available native CRM197 has led to efforts to produce CRM197 in other bacteria but this has proven a difficult task until recently.

Two companies have succeeded at manufacturing CRM197 as a recombinant protein. Pfenex, a San Diego-based developer of biosimilar therapeutics,[6] produces the protein in India using Pseudomonas fluorescens and various proprietary expression technologies for high yield. Fina BioSolutions LLC of Rockville, Maryland has achieved multi-gram/L expression of CRM197 in E. coli (“EcoCRMTM”) as an intracellular, properly folded soluble protein. Fina Biosolutions currently provides the protein for pre-clinical use.

Uses

CRM197 is used as a carrier protein in a number of approved conjugate vaccines. HibtiterTM, a vaccine to protect against Haemophius influenza type b, approved by the FDA in 1990, was the first conjugate vaccine to use CRM197 (the vaccine was discontinued in 2007). Pfizer's Prevnar, which in 2000 became the first pneumococcal conjugate vaccine to gain FDA approval, comprises polysaccharides from pneumococcal serotypes conjugated to CRM197.A larger number of clinical and pre-clinical conjugate vaccines using CRM197 as the carrier protein are being evaluated.

CRM197 possess a binding site for EGF receptor heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family.[7] As this receptor is overexpressed on cancer cells, there have been efforts to use CRM197 as an anti-cancer therapy.[8] The cancer immunotherapy company Imugene reported dramatic improvements in antibody titers from its B cell peptide cancer immunotherapy targeting HER2 when it used CRM197 as a carrier protein.[5]

CRM197 is being evaluated as a potential drug delivery fusion protein. The Swiss-based Turing Pharmaceuticals is working on CRM197 fusion constructs with therapeutic proteins of up to 1,000 amino acids in length.[6]

History

In 1971 Tsuyoshi Uchida, in the laboratory of Alwin Pappenheimer at Harvard, used nitroguanidine to create mutants of diphtheria toxin, which were called Cross Reacting Materials, or CRMs.[9] One of these mutants, called CRM197, interested researchers because its lack of toxicity suggested a better starting material for diptheria vaccine than the wild-type protein, and the protein was found to enhance the immunogenicity of bacterial polysaccharides.[10] The pharmaceutical company Wyeth took advantage of this immunogenicity in the 1990s when it conjugated seven polysaccharides from Streptococcus pneumoniae to CRM197 to create the original Prevnar vaccine which was FDA approved in February 2000. A 13-polysaccharides Prevnar was FDA-approved in 2010.[11] The meningococcal vaccine Menveo, from Novartis, is four Neisseria meningitidis polysaccharides plus CRM197. This vaccine gained FDA approval in 2010.[12]

References

  1. ↑ Broker, M., Costantino, P., DeTora, L., McIntosh, E. D., Rappuoli, R., Biochemical and biological characteristics of cross-reacting material 197 CRM197, a non-toxic mutant of diphtheria toxin: use as a conjugation protein in vaccines and other potential clinical applications. Biologicals, 39:195, 2011.
  2. ↑ "CRM197 Product Details". Reagentproteins.com. Retrieved 3 December 2015.
  3. ↑ Malito, Structural basis for lack of toxicity of the diptheria toxin mutant CRM197, PNAS 109: 5229, 2012
  4. 1 2 Shinefield, H. R., Overview of the development and current use of CRM(197) conjugate vaccines for pediatric use. Vaccine, 28:4335, 2010
  5. ↑ Rappuoli R., Isolation and characterization of Corynebacterium diphtheriae nontandem double lysogens hyperproducing CRM197., Appl Environ Microbiol. 46:560, 1983
  6. ↑ NYSE MKT: PFNX
  7. ↑ Mitamura, et al. Diphtheria Toxin Binds to the Epidermal Growth Factor (EGF)-like Domain of Human Heparin-binding EGF-like Growth Factor/Diphtheria Toxin Receptor and Inhibits Specifically Its Mitogenic Activity, The J. Bio Chem, 270, 1995
  8. ↑ Buzzi, et al. CRM197 (nontoxic diphtheria toxin): effects on advanced cancer patients., Cancer Immunol Immunother. 53:1041, 2004.; Dateoka, S. et al., Effects of CRM197, a specific inhibitor of HB-EGF, in oral cancer., 45:91, 2012
  9. ↑ Uchida T, Gill DM, Pappenheimer AM. (1 November 1971). "Mutation in the Structural Gene for Diphtheria Toxin carried by Temperate Phage β.". Nature New Biology (233): 8–11. doi:10.1038/newbio233008a0.
  10. ↑ Anderson P. (January 1, 1983). "Antibody responses to Haemophilus influenzae type b and diphtheria toxin induced by conjugates of oligosaccharides of the type b capsule with the nontoxic protein CRM197.". Infect Immun. 39 (1): 233–8. PMID 6600444.
  11. ↑ "Pfizer Receives FDA Approval for Prevnar 13™ for the Prevention of Invasive Pneumococcal Disease in Infants and Young Children". Pfizer.com. February 24, 2010. Retrieved 3 December 2015.
  12. ↑ Cooper B1, DeTora L, Stoddard J. (January 1, 2011). "Menveo®): a novel quadrivalent meningococcal CRM197 conjugate vaccine against serogroups A, C, W-135 and Y.". NExpert Rev Vaccines. 1 (1): :21–33. PMID 21162617.
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