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CRM197 is a non-toxic mutant of diphtheria toxin, currently used as a carrier protein for polysaccharides and haptens to make them immunogenic. There is some dispute about the toxicity of CRM197, with evidence that it is toxic to yeast cells and some mammalian cell lines.

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. CRM197 is widely used as a carrier protein for conjugate vaccines. A potential 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. A summary of the uses and properties of CRM197 has been published. CRM197, like diphtheria toxin, is a single polypeptide chain of 535 amino acids (58.4 kDa) consisting of two subunits (linked by disulfide bridges).

Manufacturing

The gene for CRM197 has been cloned into Corynebacterium diphtheriae, the bacterium that produces the native toxin. 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 Sigma-Aldrich. 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.

Three companies have succeeded at manufacturing CRM197 as a recombinant protein. Ligand's wholly-owned subsidiary, Pelican (previously Pfenex), a San Diego–based developer of the Pelican Expression Technology™ production platform, produces the protein ("PeliCRM™") in India at Serum Institute of India Pune 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. Recombinant CRM197 is also made in low-mutation Clean Genome® E. coli by Scarab Genomics LLC where transport of CRM197 into the bacterial cell periplasm enhances its stability and proper folding.

Uses

CRM197 is used as a carrier protein in a number of approved conjugate vaccines. Hibtiter™, a vaccine to protect against Haemophilus influenzae 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. A further example of a vaccine currently in use that is a CRM197 conjugate is the meningitis ACWY vaccine, Menveo, produced by GlaxoSmithKline. In addition, CRM197 made in the Pelican Expression Technology™ platform is used in Merck's VAXNEUVANCE and Serum Institute's Pneumosil.

CRM197 possesses a binding site for EGF receptor heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family. As this receptor is overexpressed on cancer cells, there have been efforts to use CRM197 as an anti-cancer therapy. 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.

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.

Preclinical studies have shown that CRM197 is also suitable for conjugation and presentation of peptide epitopes, a vaccinal approach that could have applications in Streptococcal infection, cancer, or Alzheimer's disease therapy.

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. One of these mutants, called CRM197, interested researchers because its lack of toxicity suggested a better starting material for diphtheria vaccine than the wild-type protein, and the protein was found to enhance the immunogenicity of bacterial polysaccharides. 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. The meningococcal vaccine Menveo, from Novartis, is four Neisseria meningitidis polysaccharides plus CRM197. This vaccine gained FDA approval in 2010.

References

1. Bröker M, Costantino P, DeTora L, McIntosh ED, Rappuoli R (July 2011). "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 (4): 195–204. doi:10.1016/j.biologicals.2011.05.004. PMID 21715186.
2. "CRM197 Product Details". Reagentproteins.com. Retrieved 3 December 2015.
3. Qiao J, Ghani K, Caruso M (March 2008). "Diphtheria toxin mutant CRM197 is an inhibitor of protein synthesis that induces cellular toxicity". Toxicon. 51 (3): 473–477. doi:10.1016/j.toxicon.2007.09.010. PMID 17988701.
4. Malito E, Bursulaya B, Chen C, Lo Surdo P, Picchianti M, Balducci E, et al. (April 2012). "Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197". Proceedings of the National Academy of Sciences of the United States of America. 109 (14): 5229–5234. Bibcode:2012PNAS..109.5229M. doi:10.1073/pnas.1201964109. PMC 3325714. PMID 22431623.
5. ^ Shinefield HR (June 2010). "Overview of the development and current use of CRM(197) conjugate vaccines for pediatric use". Vaccine. 28 (27): 4335–4339. doi:10.1016/j.vaccine.2010.04.072. PMID 20452430.
6. Rappuoli R (September 1983). "Isolation and characterization of Corynebacterium diphtheriae nontandem double lysogens hyperproducing CRM197". Applied and Environmental Microbiology. 46 (3): 560–564. Bibcode:1983ApEnM..46..560R. doi:10.1128/aem.46.3.560-564.1983. PMC 239316. PMID 6416165.
7. NYSE MKT: PFNX
8. "Menveo Group A, C, W135 and Y conjugate vaccine - Summary of Product Characteristics (SPC) - (eMC)". www.medicines.org.uk. Retrieved 2016-10-01.
9. Mitamura T, Higashiyama S, Taniguchi N, Klagsbrun M, Mekada E (January 1995). "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 Journal of Biological Chemistry. 270 (3): 1015–1019. doi:10.1074/jbc.270.3.1015. PMID 7836353.
10. Buzzi S, Rubboli D, Buzzi G, Buzzi AM, Morisi C, Pironi F (November 2004). "CRM197 (nontoxic diphtheria toxin): effects on advanced cancer patients". Cancer Immunology, Immunotherapy. 53 (11): 1041–1048. doi:10.1007/s00262-004-0546-4. PMC 11032786. PMID 15168087. S2CID 20025369.Dateoka S, Ohnishi Y, Kakudo K (June 2012). "Effects of CRM197, a specific inhibitor of HB-EGF, in oral cancer". Medical Molecular Morphology. 45 (2): 91–97. doi:10.1007/s00795-011-0543-6. PMID 22718294. S2CID 24111518.
11. Caro-Aguilar I, Ottinger E, Hepler RW, Nahas DD, Wu C, Good MF, et al. (March 2013). "Immunogenicity in mice and non-human primates of the Group A Streptococcal J8 peptide vaccine candidate conjugated to CRM197". Human Vaccines & Immunotherapeutics. 9 (3): 488–496. doi:10.4161/hv.23224. PMC 3891704. PMID 23249976.
12. Tobias J, Jasinska J, Baier K, Kundi M, Ede N, Zielinski C, Wiedermann U (February 2017). "Enhanced and long term immunogenicity of a Her-2/neu multi-epitope vaccine conjugated to the carrier CRM197 in conjunction with the adjuvant Montanide". BMC Cancer. 17 (1): 118. doi:10.1186/s12885-017-3098-7. PMC 5301331. PMID 28183282.
13. Vingtdeux V, Zhao H, Chandakkar P, Acker CM, Davies P, Marambaud P (January 2017). "A modification-specific peptide-based immunization approach using CRM197 carrier protein: Development of a selective vaccine against pyroglutamate Aβ peptides". Molecular Medicine. 22: 841–849. doi:10.2119/molmed.2016.00218. PMC 5263057. PMID 27900387.
14. Uchida T, Gill DM, Pappenheimer AM (September 1971). "Mutation in the structural gene for diphtheria toxin carried by temperate phage". Nature. 233 (35): 8–11. doi:10.1038/newbio233008a0. PMID 4999827.
15. Anderson P (January 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". Infection and Immunity. 39 (1): 233–238. doi:10.1128/IAI.39.1.233-238.1983. PMC 347931. PMID 6600444.
16. "Pfizer Receives FDA Approval for Prevnar 13™ for the Prevention of Invasive Pneumococcal Disease in Infants and Young Children". Pfizer.com. February 24, 2010. Archived from the original on 8 December 2015. Retrieved 3 December 2015.
17. Cooper B, DeTora L, Stoddard J (January 2011). "Menveo®): a novel quadrivalent meningococcal CRM197 conjugate vaccine against serogroups A, C, W-135 and Y". Expert Review of Vaccines. 10 (1): 21–33. doi:10.1586/erv.10.147. PMID 21162617. S2CID 13478399.

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