Glycosylation and the Demands of Antibody Engineering - Roy Jefferis examines precise questions about how sugars drive antibody function. - BioPharm International


Glycosylation and the Demands of Antibody Engineering
Roy Jefferis examines precise questions about how sugars drive antibody function.

BioPharm International
Volume 20, Issue 10

KJM: Will changing the site of glycosylation from ASN 297 to another position have a radical effect on antibody performance?

RJ: Yes, but in a negative way. In one series of experiments, the glycosylation site of IgG1 was removed and an IgA glycosylation site was introduced. This resulted in a total loss of biological function (ADCC and complement fixation). This reflects the fact that the oligosaccharide of IgG makes multiple non-covalent interactions with the protein surface and determines the precise three-dimensional structure required for activation of ADCC and other responses.

KJM: Why is Herceptin not effective in all cases of breast cancer? Is it because some patients lack receptors which bind the antibody?

RJ: Yes, in part. Herceptin has specificity for a molecule (Her 2) expressed on the surface of breast cancer cells. However, the level of expression determines the amount of antibody that can bind and consequently the efficacy of kill. In the UK, patients are rated for receptor density on their cancer cells on a 1 to 5 scale. If they fall into the 1–2 range, they simply won't get treated because the probability of response is so small. We hope that the next generation of non-fucosylated Herceptin drugs will extend the range of efficacy. There are a number of other biological considerations related to the biological heterogeneity of the tumor cells.

KJM: Antibodies seem to work well in some situations as cancer therapeutics, but they often fail. Is it because resistant cell populations arise? If the therapy is bound to eventually fail, how can the immense cost be justified?

RJ: It must be appreciated that tumor cells are very heterogeneous, both between and within patients. Some cancers are more aggressive than others; some may have their origin in a precursor cell which is undifferentiated and won't respond to the drug, but will continue to throw off mature tumor cells.

Although the overall benefit of a particular therapy may seem to be marginal to an outside observer, for an individual cancer patient the value of the life extension is immeasurable. The National Health Service in the UK struggles with the question and refers to the National Institute for Clinical Excellence, which provides an independent evaluation of clinical data on experimental drugs and makes recommendations on a cost–benefit basis.

KJM: It has been stated in the press that the devastating side effects in patients treated with the anti-CD28 recombinant monoclonal antibody, which elicited a catastrophic cytokine cascade in a Phase 1 trial, "could not have been predicted." Why could the effects not have been predicted? Can they now be predicted?

RJ: Opinions are divided among the experts. The government appointed Expert Group concluded:

"Although Fc receptors for IgG4, the particular antibody type of TGN1412, are thought to be rare, such cross-linking activity of TGN1412 in vivo cannot be excluded on current evidence."

This statement is in error; IgG4 is fully capable of engaging and activating FcγRI and the non-fucosylated form activates FcγRIII. There is high homology but not identity between human and non-human primate IgGs and FcRs.

KJM: Would it make sense to design aglycosylated antibodies, devoid of effector functions, for use as carriers of low molecular weight toxins, to deliver a poison to a cell?

RJ: Yes, and this is already underway.

KJM: How far are we from engineering yeast and bacterial strains that effectively glycosylate proteins?

RJ: These developments are well advanced. "Knock-out" and "knock-in" technology has been applied to plants, moss, yeast, etc., to suppress non-human glycoforms and generate human glycoforms. It is hoped that these vehicles will allow production of selected homogeneous antibody glycoforms and reduce upstream costs. Downstream costs may not be reduced and new protocols for the removal of host proteins will have to be developed.

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