Invented by Frederick R. Taylor, Ellen Garber, Biogen MA Inc
The Biogen MA Inc invention works as followsThe invention provides a way to produce aglycosylated polypeptides containing Fc, such as antigens, with the desired effector function. The invention also includes aglycosylated anti-bodies produced by the method, as well as ways to use such antibodies as therapeutics.
Background for Antibodies with altered effector functions and methods of making the same
The immune response is the mechanism that allows the body to defend itself from foreign substances which invade and cause infection or disease. This mechanism relies on antibodies that are produced or given to the host’s ability to bind antigens through their variable region. The constant region of the Fc domain or the constant region of the antibody is often used to target the antigen for destruction once it is bound.
For instance, one activity performed by the Fc region of the antibody is binding complement proteins that can help lyse the target antigen (for example, a pathogen in the cell). The Fc region can also bind to FcRs on the surface immune cells or effector cells. These cells have the capability to trigger additional immune effects. These immune effects can include the release of immune activaters, regulation of antibodies production, endocytosis and phagocytosis as well as cell killing. These responses can be crucial to the efficacy and safety of an antibody in some cases, but they may also cause unwanted side effects. A common example of a side effect caused by effectors is an acute fever. This occurs when inflammatory cytokines are released. “Another example is the deletion of antigen bearing cells over a long period of time.
The effector function can be avoided using fragments of antibodies that lack the Fc region, e.g. Fab?2, single chain antibodies (sFv), but these fragments are less stable, have only one antigen-binding site, instead of two, and they’re more difficult to purify.
There are currently only a few ways to reduce an antibody’s effector function while still retaining its other useful attributes. Mutating amino acids involved in effector-binding interactions on the antibody surface is one way to reduce the effector function. Some mutations reduce effector function but residual activity is usually retained. These mutations may also make the antibody immune.
Another way to reduce the effector function of antibodies is to remove the sugars linked to specific residues within the Fc region. This can be done by, for example, altering or deleting the residue to which the sugar is attached, removing sugars by enzyme, producing the antibody using cells that have been cultured with a glycosylation inhibitor, or expressing the antigen in cells incapable of glycosylating proteins. The foregoing approaches do not eliminate effector functions, but they leave them in the form complement-dependent Fc receptor binding and complement-dependent cytolytic function. It is important to further reduce the effector function in order to ensure complete ablation.
Accordingly, there is a need for an improved way to make aglycosylated antigens with altered or decreased effector functions.
The invention resolves the above problems of glycosylated antibody, or any Fc-containing proteins, by providing improved ways to produce aglycosylated binding antigen proteins, such as aglycosylated IgG, and aglycosylated IgG, with only minimal modifications. The invention relates to a method of introducing an amino-acid alteration in a first position, which leads to a reduced glycosylation at a second or different position. The first amino acids can be modified in a way that the side chain chemistry is desirable, allowing it to be linked to, for instance, an additional functional moiety such as a blocker, detectable, diagnostic or therapeutic moiety. The antigen-binding polypeptides resulting from aglycosylation, such as aglycosylated IgG antibodies, have, for instance, an altered or reduced function. “The polypeptides of the invention provided a significant decrease in unwanted effector function compared to other conventional methods of aglycosylating Fc region.
The invention, according to the inventor, has many advantages, including, but not limited to the following:
Accordingly, according to one aspect of the invention, it provides a variant polypeptide or polypeptide containing an Fc-region, wherein the Fc-region has a modified amino acid with a preferred side chain chemical, and a residue second amino acid that is glycosylated less than a parent polypeptide.
In certain embodiments, side chain chemistry from the first amino acid can be covalently coupled to an additional moiety. This moiety could, for instance, be a blocking, detectable, diagnostic, or therapeutic moiety.
In one embodiment, the functional moieties is a blocking moie, which inhibits or stops glycosylation at the second residue of amino acids. The blocking moiety may also be used to block effector functions, such as by inhibiting binding of the Fc-region of the polypeptide with an Fc receptor, complement protein, or the like.
In a preferred embodiment the blocking moiety forms when the amino acid residue of the first is a Cysteine or has side chain chemistry containing a Thiol.
In certain embodiments, the amino acid first contains a cysteine or a mixed disulfide, adduct of cystine or a disulfide-linked cystine.
In another preferred embodiment, the blocker moiety is a PEG or PEG-maleimide.
In a similar embodiment, the PEG moiety can be attached to the side chain of the polypeptide if the first amino acid is cysteine.
In certain embodiments, the chemistry of the side chains is reduced in order to remove the disulfide, cystine adduct or mixed disulfide, and then the PEG moiety attached to the residue.
In another embodiment, the functional moieties is a detectable moieties, such as but not limited, a fluorescent or isotopic moieties.
In another embodiment, the diagnostic moiety is the functional moiety. This is the moiety that can reveal the presence of disease or disorder.
In another embodiment, the functional moiety is a therapeutic moiety such as, but not limited to, an anti-inflammatory agent, anti-cancer agent, anti-neurodegenerative agent, or anti-infective agent.
In another aspect, a variant polypeptide is a parent polypeptide that has an Fc region and a modified amino acid. The modified amino acid is spatially located such that reduced glycosylation is achieved at a second acid. In a preferred embodiment the variant polypeptide that is aglycosylated also has a reduced effector function as compared to parent polypeptide.
In a similar embodiment, the first modified amino acid is separated from the second by at least one amino acid position, such as by 2, 3, 4, 6, 7, 8, 9 or 10 positions of amino acids residues or more.
Click here to view the patent on Google Patents.