Glyco-Humanized Afucosylated Antibodies

Glyco-Engineered Antibodies

 
In the four subtypes of antibody IgG, monoclonal antibody drugs currently widely used in clinical trials are mainly IgG1 subtypes, with a small amount of IgG4 and IgG2 subtypes. An IgG1 molecule contains a Fab fragment and a Fc fragment that reacts with an antigen, and in Fc fragments there are two double-day linear complex polysaccharide structures linked to N297 site.
 
The IgG1 subtypes anti-cancer antibody drug can be used to play the role of cancer (ADCC activity) through its Fc part and the Fc receptor FcR (CD16) on the surface of the immune cells, especially the NK cells. , however, has those been approved cancer monoclonal antibody drug has low ADCC activity, because the patient's blood of natural IgG1 will compete with nonspecific antibody drug combination FcRγIIIa receptor on the surface of the immune cells, affect the ADCC of antibody drugs.
 
IgG1 type of anti-cancer antibodies drugs mainly through mediated cytotoxic effect (antibody - dependent cellular cytotoxicity, ADCC), complement mediated cytotoxic effect (complement - dependent cytotoxicity, CDC) and apoptosis (apoptosis) to attackandkill cancer cells.Although all three of these mechanisms contribute to the overall therapeutic effect, the contribution is different.
 
The two double antenna complex polysaccharide chains in Fc part of anti-cancer antibody drug producd by wild type CHO cells contain Fucose, while the existence of Fucose base will obstruct between antibodies and Fc receptor, thus affecting the antibody ADCC activity and anti-cancer effect. Studies have shown that [1], the affinity of monoclonal antibodies without Fucose and FcR (FcR) is significantly improved. In Lec13 mutant cells, there was a 50 fold increase in IgG, and a 100-fold increase in the effect of ADCC [2].No core fucose makes the Asn162 and Fc polysaccharide in FcRγIIIa receptor have a higher affinity.
 
Two monoclonal antibody drug lack of fucose have been approved for use in clinical treatment. In 2012 Japan approved Mogamulizumab to FDA for the treatment of adult T cell leukemia. In 2013, the United States approved Obinutuzumab to FDA for the treatment of chronic lymphatic cancer [4-5]. Currently more than 50 lack of fucose antibody drugs have been in clinical trials, for example, AstraZeneca's MEDI - 551 antibody against CD19 targets [6] and against IL - 5 r alpha targets in the treatment of asthma, chronic lung disease and high eosinophil syndrome Benralizumab [8], GlaxoSmithKline GSK2831781 for the treatment of autoimmune diseases [9], Seattle Genetics for CD40 targets of SEA - CD40 [10], KaloBios Pharmaceuticals in the treatment of leukemia KB004 [11], and so on.
 
At present, there are three kinds of technology to produce loss fucose monoclonal antibody in CHO cells: (A) excessive expression in the CHO cells in rats glycosylation enzyme β, 14 - N - acetylglucosaminyltransferase III (GnTIII) to prevent fucose transferred to Fc sugar chain; (B) knockout of the diphytase gene 8(Fut8) in the CHO cell by genetic engineering, to achieve the complete deletion of the fucose in the antibodies; (C) the addition of the rock algal analogues to the CHO cell culture medium to significantly reduce the incorporation rate of the fucose in the antibody. 
 
The company has successfully developed lack of fucose CHO cell strain and lack of fucose  GS - / - CHO cell strain, and many innovative and efficient antibody were expressed in the cell strain successfully.
 
Fut8-/- CHO cell lines and Fut8-/- gs-/ - CHO cell lines are the industrial production cells that endow the higher ADCC activity with the single antidrug.
 
The monoclonal drugs lack of fucose can not only significantly improve the drug efficacy, but also significantly reduce the toxicity and side effects of the drugs, and significantly reduce the production cost and improve the enterprise benefit.
 
 
Glycosylated antibody
 
As more and more therapeutic antibodies are mass-produced in CHO cells, the characteristics of engineered cell lines has important eggect on the activity of antibodies.
 
Although the glycosylation of CHO cells is very close to the glycosylation of human cells, there are some key differences. For example, the antibodies expressed by CHO cells have a large number of G0F sugar structures, a small amount of G1F and G2F structure, and the sialic acid at the end of the sugar chain is almost negligible. In addition, 1-20% of the antibody molecules in the CHO cell source are High Mannose, while the antibodies in the human source are very trace (<0.1%) [12]. The distribution and kinetics of the antibody were significantly affected by the absence of Galactose and high mannose at the end of the sugar chain. Antibodies without saliva acid can be combined with the Asialoglycoprotein Receptor (ASGPR) in the liver and removed from the blood. Antibodies with high Mannose can also be combined and quickly cleared by the liver's Mannose Receptor.
 
 
In addition, although CHO cells capable of glycoprotein of sialic acid modification, but cells lack of α-2, 6 - sialyltransferase, thus the antibodies produced in CHO cells only have sialic acid in α-2, 3 sites [14]. And human antibody have α-2, 6 sialic acid at the ends of the 
Antagen (Beijing) biotechnologies Co., LTD., have fixed-point integrates glycosylation gene in CHO cells - K1 genome by gene targeting technology, produced the antibodies with human saliva acid modified (figure 4 b).
 
We can customize the antibodies use the CHO-EFGS cell line that can be used to express the lack of the fucose and Sialylated antibody. And in lack of fucose, on the basis of the introduction of saliva acid, not only has no effect on the improve ADCC activity (figure 5), but also to screen out the molecular immunogenicity, increase the half-life in the body. The anti-tumor antibodies that are missing from the algal sugar can increase the ADCC activity of the tumor by 100 times in the same dose.
 
References:
  1. Satoh M, Iida S, Shitara K. "Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies". Expert Opin Biol Ther. 2006; 6: 1161–73.
  2. Shields RL et al.. “Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity”. J. Biol. Chem. 2002; 277:26733-40.
  3. Subramaniam, J; Whiteside G; McKeage K; Croxtall J. "Mogamulizumab: First Global Approval". Drugs. 2012; 72 (9): 1293–1298.
  4. https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s000lbl.pdf
  5. https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm488013.htm
  6. https://clinicaltrials.gov/ct2/show/NCT02200770?term=medi-551&recrs=ab&r... .
  7. http://www.bloodjournal.org/content/122/21/1810?sso-checked=true Blood, 2013;122:1810.
  8. https://clinicaltrials.gov/ct2/show/NCT02968914?term=afucosylated&rank=2
  9. https://clinicaltrials.gov/ct2/show/NCT02195349?term=afucosylated&rank=1
  10. https://clinicaltrials.gov/ct2/show/NCT02376699?term=SEA-cd40&rank=1
  11. https://clinicaltrials.gov/ct2/show/NCT01211691?term=KB004&rank=1
  12. Flynn GC, Chen X, Liu YD, et al. Naturally occurring glycan forms of human immunoglobulins G1 and G2. Mol Immunol 2010; 47:2074-82.
  13. Kanda Y, Yamada T, Mori K, et al. Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: The high-mannose, hybrid, and complex types. Glycobiology 2007; 17:104-18.
  14. Lee EU, Roth J, Paulson JC. Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2,6-sialyltransferase. J Biol Chem 1989; 264:13848-55.
  15. Anthony RM, Nimmerjahn F, Ashline DJ, et al. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 2008; 320:373-6.
  16. Washburn N, Schwab I, Ortiz D, et al. Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity. Proc Natl Acad Sci. 2015;112:E1297-306.

 

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