Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2001-06-14
2004-04-13
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S070210, C530S387100, C530S866000
Reexamination Certificate
active
06720165
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the fields of antibodies and fragments thereof, immunology, biological and chemical assay development, drug discovery, medical diagnostics and treatments, and proteomics.
RELATED REFERENCES
Andrew, S. M, and Titus, J. A. (1997). Purification and Fragmentation of Antibodies. In
Current Protocols in Immunology,
edited by Coligan, J. W., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M. and Strober, W., John Wiley & Sons, N.Y., pp. 2.7.1-2.7.12.
Gorini, G., Medgyesi, G. A. and Doria, G. (1969). Heterogeneity of mouse myeloma gamma-G globulin as revealed by enzymatic proteolysis. J. Immunol. 103, 1132-1142.
Harris, L. J., Larson, S. B., Hasel, K. W. and McPherson, A. (1997). Refined structure of an intact IgG
2a
monoclonal antibody. Biochemistry 36, 1581-1597.
Hindley, S. A. et al. (1993). The interaction of IgG with Fc-gamma-RII: involvement of the lower hinge binding site as probed by NMR. Biochem. Soc. Trans. 21, 337S.
Kim, H., et al. (1994). O-Glycosylation in hinge region of mouse immunogloblulin G
2b
. J. Biol. Chem. 269, 12345-12350.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680-5.
Lamoyi, E. and Nisonoff, A. (1983). Preparation of F(ab′)
2
fragments from mouse IgG of various subclasses. J. Immunol. Methods 56, 235-243.
Mariani, M., Cauragra, M., Tarditi, L. and Seccariani, E. (1991). A new enzymatic method to obtain high-yield F(ab′)
2
suitable for clinical use from mouse IgG
1
. Mol Immunol. 28, 69-77.
Milenic, D. E., Esteban, J. M., Colcher, D. (1989). Comparison of methods for the generation of immunoreactive fragments of a monoclonal antibody (B72.3) reactive with human carcinomas. J. Immunol. Methods 120, 71-83.
Nisonoff, A, Wissler, F. C., Lipman, L. N. and Woernley, D. L. (1960). Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds. Arch. Biochem. Biophys. 89, 230-244.
Parham, P. (1983). On the fragmentation of monoclonal IgG
1
, IgG
2a
, and IgG
2b
from BALB/c mice. J. Immunol. 131, 2895-2902.
Parham, P. (1986). Preparation and purification of active fragments from mouse monclonal antibodies. In
Handbook of Experimental Immunology
, Vol. 1: Immunochemistry (D. M. Wier, ed.) pp14.1-14.23. Blackwell Scientific, Oxford.
Rousseaux, J., Rousseaux-Prevost, R. and Bazin, H. (1983). Optimal conditions for the preparation of Fab and F(ab′)
2
fragments from monoclonal IgG of different rat IgG subclasses. J. Immunol. Methods 64, 141-146.
Yamaguchi, Y., Kim, H., Kato, K., Masuda, K., Shimada, I. and Arata, Y. (1995). Proteolytic fragmentation with high specificity of mouse immunoglobulin G: Mapping of the proteolytic cleavage sites in the hinge region. J. Immunol. Methods 181, 259-267.
BACKGROUND OF THE INVENTION
Antibodies, and in particular, antibody fragments, are heavily utilized in diagnostic, therapeutic, and biological research applications. Often there are substantial advantages to using antibody fragments that are produced by proteolysis of IgGs.
Full size IgG antibodies have three domains, each of approximately 50 kd molecular weight, the three domains comprising two identical “Fab” (antigen binding) fragments, and an Fc (crystallizable domain). It is often advantageous to remove the Fc domain from the antibody prior to use to yield, as in the case of pepsin cleavage, a F(ab′)
2
fragment separated from the Fc domain. An F(ab′)
2
maintains the binding characteristics of a full size IgGs despite its loss of the Fc domain. The Fc domain can invoke a variety of undesired biological effector functions that can interfere with the therapeutic or diagnostic uses of the antibodies, thus removal of the Fc region has substantial value. The F(ab′)
2
is also a useful intermediate in the production of monomeric, chemically tagged Fab monomers because F(ab′)
2
s are held together by 1-3 disulfide bonds between the heavy chains. Mild chemical reduction of such disulfide bonds may result in the formation of monomeric Fab fragments having cysteines available for reacting with chemical labels or reactive surfaces.
Several classes of IgG antibodies exist having differences based on the sequence of the heavy chain. Consequently, different classes have different susceptibilities to proteolysis by pepsin. Mouse-derived monoclonal antibodies include four IgG subclasses:
1
,
2
a
,
2
b
and
3
. Certain, and often important, members of antibody classes
1
and
2
b
are recalcitrant to yielding F(ab′)
2
fragments from pepsinolysis treatment. Even if pepsin cleaves such antibodies, it often does not give good yields or yields different non-F(ab′)
2
products. Thus many important IgGs cannot be efficiently converted to Fab dimers. Because IgG
1
class is the most common for monoclonal antibodies used in biotechnology, there is a need for reliable, universal methods for converting whole IgG
1
and other pepsin resistant antibodies to intact F(ab′)
2
antibody fragments.
Methods for the preparation of F(ab′)
2
fragments by pepsinolysis have been described which produce antibody fragments that retain full binding activity but do not possess the effector functions conferred by the Fc domain. See Nisonoff et al., and Andrew and Titus. However, as discussed herein, these methods are of limited use depending, in part, on the type and source of antibody used as a starting material. F(ab′)
2
fragments may be selectively reduced to Fab fragments having free cysteines in the linker region (Nisonoff et al.) This allows Fab fragments to be labeled or attached to solid supports or labels through a region of the protein that is distal to the antigen-binding site. The most common method for generating F(ab′)
2
fragments is by pepsinolysis, which is generally efficient for most antibodies from the mouse IgG
2a
and IgG
3
subclasses, but not generally efficient for those from the IgG
2b
or IgG
1
, the latter being the most common.
Many others have reported poor yields of F(ab′)
2
fragments by treating mouse IgG
1
antibodies with pepsin under standard conditions (37° C., pH 4.5), and such procedures typically also produce several other cleavage products as well (See Gorini et al.; Laymoyi and Nisonoff; Parham; Mariani et al.; and, Andrew and Titus.) About 50% of the IgG
1
antibodies appeared to be completely resistant to pepsinolysis. Numerous alternatives to pepsinolysis have been described for generating F(ab′)
2
fragments from IgG
1
molecules, including the use of papain (under slightly reducing conditions), V8 protease, or ficin, for example. See generally Parham; Milenic et al.; Mariana et al.; Yamaguchi et al.; and, Andrew and Titus, however, each of these failed to provide a reliable method for preparing F(ab′)
2
s from antibodies with uniform, predictable results. Thus, there is a need for a universal method for preparing F(ab′)
2
antibody fragments from whole antibodies, especially those from IgG
1
and IgG
2b
subclasses. There is also a need for a method for converting other immunoglobulins from other species such as chickens and their IgY antibodies. The invention disclosed herein addresses these, and other needs as discussed below and as will become apparent to one of ordinary skill in the art reading this disclosure and subsequent claims.
SUMMARY OF THE INVENTION
The invention provides methods for making F(ab′)
2
antibody fragments from antibodies, in particular, antibodies that have one or more oligosaccharide groups attached to regions of the antibody other than the hinge region.
In one aspect, the invention provides a method for preparing a F(ab′)
2
fragment from a glycosylated antibody. The method includes the steps of providing a glycosylated antibody where the glycosylated antibody has a hinge region having one or more protease cleavage sites located within the hinge region, and one or more non-hinge regions adjacent the hinge region, the non-hinge region(s) having on
Nock Steffen
Wilson David S.
Wu Jiangchun
Saunders David
Townsend and Townsend / and Crew LLP
Zyomix, Inc.
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