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Glycoengineering

Chinese hamster ovary (CHO) cells or yeast cells used in the production of protein based biopharmaceuticals can be glycoengineered in order that a more human, afucosylated, glycoprofile is produced. Glycoengineering endeavors to generate a correct human, optimized glycosylation profile to give the highest therapeutic efficiency.

Afucosylated monoclonal antibodies (mAbs) are engineered so that the oligosaccharides in the Fc region of the antibody do not contain any fucose sugar units. The absence of the core fucose sugar results in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC, which is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies) action at significantly lower dosed concentrations but with much higher efficiency compared with their traditional fucosylated counterparts. [1]

When present at similar concentrations to the fucosylated variant, the afucosylated mAb can exhibit a 40-100 fold increase in ADCC function. [2]

This is due to a higher effector cell affinity for the afucosylated mAb over the omnipresent inhibiting serum immunoglobulin G (IgG) species.  Conversely, fucosylated mAbs possess a lower effector cell affinity over serum IgG. [3-5]

Therapeutic antibody-induced ADCC in human blood

Figure 1: Therapeutic antibody-induced ADCC in human blood. Therapeutic antibodies show the same antigen binding activity irrespective of core fucosylation of the Fc.

(a)   Non-fucosylated antibodies overcome the competition with serum IgG to bind to the effector cells through much higher binding affinity to FcγRIIIa than serum IgG, and thereby induce high ADCC.
(b)   Fucosylated antibodies fail to recruit effector cells effectively due to low binding affinity to the FcγRIIIa.
(c)   The high ADCC of non-fucosylated antibodies is inhibited by the fucosylated counterparts through the competition for binding to the antigen on target cells.expiration. [1]

Fragment Crytallizable (Fc) Region Modification

An alternative approach is where the pharmacokinetic profile is improved via increased plasmatic half-life.  The Fc region of the protein biopharmaceutical is very slightly modified, typical variation of 2-3 amino acids.  This very subtle change in the chemical structure can yield relative half-life increases 4- and 5-fold, as has been witnessed during pharmacokinetic studies of Bevacizumab (Xtend-VEGF) and Cetuximab (Xtend-EGFR) and their unmodified counterparts respectively. [6]

Increasing antibody affinity

Figure 2: Increasing antibody affinity to FcRn promotes half-life extension in cynomolgus monkeys.

(a)   Log-linear serum concentration versus time profiles of anti-VEGF (Bevacizumab) antibodies in cynomolgus monkeys. All antibodies were administered via single 60 minute intravenous infusion at 4 mg/kg and serum antibody concentrations were determined using a VEGF antigen down immunoassay. Results are shown as mean ± standard error (N = 2 for Bevacizumab and N = 3 for variants).
(b)   Log-linear serum concentration versus time profiles of anti-EGFR antibodies in cynomolgus monkeys. Monoclonal antibodies were administered via single 30 minute intravenous infusion at 7.5 mg/kg and serum antibody concentrations were determined using an EGFR antigen-down immunoassay. Results are shown as mean of N = 2 animals per test article. [6]

Improved half-life
Figure 3: Improved half-life translates into greater in vivo efficacy.
(a)   Log-linear serum concentration versus time profiles of anti-VEGF antibodies in hFcRn mice. All antibodies were administered via single intravenous bolus at 2 mg/kg, and serum antibody concentrations were determined using a human immunoglobulin recognition immunoassay. Results are plotted as mean ± standard error (N = 6).
(b)   Log-linear serum concentration versus time profiles of anti-EGFR antibodies in hFcRn mice. The study design was identical to that described in panel (a) except that serum concentrations were measured with an EGFR antigen down immunoassay.
(c)   Xenograft study in hFcRn/Rag1−/− mice comparing activity of native IgG1 and Xtend variant versions of Bevacizumab against established SKOV-3 tumors. Tumor volume is plotted versus day post tumor cell injection. Antibodies were dosed 5 mg/kg every 10 days starting on day 35 (indicated by the arrows). N=8 mice/group. * p= 0.028 at 84 days.
(d)   Xenograft study in hFcRn/Rag1−/− mice comparing activity of anti-EGFR antibodies against established A431 tumors. Tumor volume is plotted versus day post tumor cell injection. Antibodies were dosed 5 mg/kg every 10 days starting on day 10 (indicated by the arrows). N=9 mice/group. * p= 0.005 at 35 days. [6]
 

[1] Satoh. M. et al, Production of therapeutic antibodies with controlled fucosylation, mAbs 1:3, 230-236; May/June 2009

[2] Beck A, Wurch T, Bailly C, Corvaia N. Strategies and challenges for the next generation of therapeutic antibodies. Nat Rev Immunol. 2010;10:345–352

[3] Kanda Y, Yamada T, Mori K, Okazaki A, Inoue M, Kitajima-Miyama K, et al. Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharide: the high-mannose, hybrid, and complex-types. Glycobiology 2007; 17:104-18

[4] Satoh M, Iida S, Shitara K. Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies. Expert Opin Biol Ther 2006; 6:1161-73

[5] Iida S, Misaka H, Inoue M, Shibata M, Nakano R, Yamane-Ohnuki N, et al. Nonfucosylated therapeutic IgG1 antibody can evade the inhibitory effect of serum immunoglobulin G on antibody-dependent cellular cytotoxicity through its high binding to FcγRIIIa. Clin Cancer Res 2006; 12:2879-87

[6] Desjarlais J. R., Enhanced antibody half-life improves in vivo activity, Nat Biotechnol. 2010 February ; 28(2): 157–159

 
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