U.S. patent application number 10/701887 was filed with the patent office on 2004-05-13 for methods for making antibody fragments and compositions resulting therefrom.
This patent application is currently assigned to Zyomyx, Inc.. Invention is credited to Nock, Steffen, Wilson, David S., Wu, Jiangchun.
Application Number | 20040091957 10/701887 |
Document ID | / |
Family ID | 25381391 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040091957 |
Kind Code |
A1 |
Nock, Steffen ; et
al. |
May 13, 2004 |
Methods for making antibody fragments and compositions resulting
therefrom
Abstract
Improved methods for making antibody fragments, preferably
F(ab').sub.2 fragments from various classes and subclasses of
antibodies is described. Pretreatment of antibodies with
deglycosylases or cellular inhibition of glycosylation during
expression, yields antibodies having improved susceptibility
towards protease cleavage, preferably pepsinolysis, which yields
F(ab').sub.2 antibody fragments. Compositions resulting from such
methods are further disclosed.
Inventors: |
Nock, Steffen; (Redwood
City, CA) ; Wilson, David S.; (Hayward, CA) ;
Wu, Jiangchun; (Fremont, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Zyomyx, Inc.
Hayward
CA
|
Family ID: |
25381391 |
Appl. No.: |
10/701887 |
Filed: |
November 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10701887 |
Nov 4, 2003 |
|
|
|
09882814 |
Jun 14, 2001 |
|
|
|
Current U.S.
Class: |
435/68.1 ;
530/387.1 |
Current CPC
Class: |
C07K 2317/41 20130101;
C07K 2317/54 20130101; Y10S 530/866 20130101; C07K 16/065
20130101 |
Class at
Publication: |
435/068.1 ;
530/387.1 |
International
Class: |
C12P 021/06; C07K
016/18 |
Claims
1. A method for preparing a F(ab').sub.2 fragment from a
glycosylated antibody, said method comprising the steps of: (i)
providing said glycosylated antibody, said glycosylated antibody
having a hinge region having one or more protease cleavage sites
located-within said hinge region, one or more non-hinge regions
adjacent said hinge region, said non-hinge region(s) having one or
more oligosaccharide groups attached thereto, said oligosaccharide
group(s) causing said protease cleavage site(s) within said hinge
region to be resistant to a proteolysis treatment; (ii) exposing
said glycosylated antibody to a deglycosylation treatment, said
deglycosylation treatment cleaving said oligosaccharide group(s)
attached to said non-hinge region(s) to form a partially or wholly
deglycosylated antibody having a hinge region cleavable by said
proteolysis treatment; and, (iii) exposing said partially or wholly
deglycosylated antibody to said proteolysis treatment to cause
proteolytic cleavage of said hinge region cleavable by said
proteolysis to form said F(ab').sub.2 fragment.
2. The method of claim 1 wherein said glycosylated antibody is a
plurality of glycosylated antibodies.
3. The method of claim 2 wherein at least some of said glycosylated
antibodies are polyclonal.
4. The method of claim 2 wherein said glycosylated antibodies are
monoclonal.
5. The method of claim 1 wherein said glycosylated antibody is
either an IgG.sub.1 or IgG.sub.2b glycosylated antibody.
6. The method of claim 5 wherein said IgG.sub.1 or IgG.sub.2b
antibody is from a rodent derived hybridoma cell culture or
ascites.
7. The method of claim 1 wherein said glycosylated antibody is
derived from the group consisting of rat, mouse, rabbit, goat,
sheep, lamb, chicken, or horse.
8. The method of claim 1 wherein said proteolysis is achieved
wholly or partly from protease treatments including components
selected from the list consisting of pepsin, proteases that cleave
pepsin substrates, papain, papain preactivated with cysteine, and
ficin.
9. The method of claim 1 wherein said proteolysis is achieved by a
protease capable of producing F(ab').sub.2 fragments from said
deglycosylated antibodies.
10. The method of claim 1 wherein said deglycosylase treatment
contains a glycosidase combination selected from the group
consisting of PNGase F, endo-O-glycosylase, sialidase A, PNGase
F/endo-O-glycosylase, PNGase F/sialidase A, PNGase
F/endo-O-glycosylase/sialidase A, endo-O-glycosylase/sialidase
A.
11. The method of claim 1 wherein said non-hinge regions comprise
the Fc and the Fab regions of said glycosylated antibody.
12. A method for preparing F(ab').sub.2 fragments comprising the
steps of: (i) growing a hybridoma cell that normally produces
glycosylated antibodies having a hinge region with one or more
protease cleavage sites located within said hinge region, one or
more non-hinge regions adjacent said hinge region, and one or more
oligosaccharide groups being attached to at least one of said
non-hinge regions by said hybridoma cell through glycosylation,
said oligosaccharide groups causing said hinge regions to be
resistant to a proteolysis treatment; (ii) administering to said
hybridoma cell an inhibitor of said glycosylation effective to
inhibit glycosylation of said antibodies to produce one or more
unglycosylated antibodies lacking said oligosaccharides within at
least one non-hinge region to cause said hinge region to be prone
to said proteolysis treatment; and, (iii) exposing said
unglycosylated antibodies to said proteolysis treatment, wherein
said unglycosylated antibodies' hinge regions are cleaved to form
said F(ab').sub.2 fragments from said unglycosylated
antibodies.
13. The method of claim 12 wherein said hybridoma cell is part of a
hybridoma cell culture or ascites.
14. The method of claim 12 wherein said hybridoma cell is a
plurality of hybridoma cells.
15. The method of claim 12 wherein said hybridoma cell is part of a
monoclonal or polyclonal hybridoma cell line.
16. The method of claim 14 wherein said hybridoma cells are from
the same hybridoma cell line.
17. The method of claim 14 wherein said hybridoma cells are from
different hybridoma cell lines.
18. The method of claim 12 wherein said inhibitor of said
glycosylation contains bacitracin or tunicamycin.
19. A method for preparing F(ab').sub.2 fragments comprising the
steps of: (i) providing a hybridoma cell line that normally
produces glycosylated antibodies having a hinge region with one or
more protease cleavage sites located within said hinge region, one
or more non-hinge regions adjacent said hinge region, and one or
more oligosaccharide groups being attached to at least one of said
non-hinge regions by said hybridoma cell through glycosylation,
said oligosaccharide groups causing said hinge regions to be
resistant to a proteolysis treatment; (ii) altering said hybridoma
cell line to inhibit glycosylation of said antibodies within said
non-hinge regions to produce one or more unglycosylated antibodies
such that said unglycosylated antibodies are suceptible to
proteolysis treatment; (iii) causing said hybridoma cell line to
produce said unglycosylated antibodies; and, (iv) exposing said
unglycosylated antibodies to said proteolysis treatment to cleave
said unglycosylated antibodies to produce said F(ab').sub.2
fragments.
20. The method of claim 19 wherein said hybridoma cell is part of a
hybridoma cell culture or ascites.
21. The method of claim 19 wherein said altered cells are either
permanently or transiently altered.
22. An F(ab').sub.2 composition comprising: one or more
F(ab').sub.2 fragments, or derivative therefrom, produced by the
method selected from the group consisting of the methods of claim
1, claim 12, and claim 19.
23. The composition of claim 22 wherein said F(ab').sub.2 fragments
are an active ingredient of an antitoxin or anti-venom
medicament.
24. An immunoglobulin composition comprising: one or more
aglycosylated or deglycosylated immunoglobulins, said aglycosylated
or deglycosylated immunoglobulins being formed by preventing the
attachment of one or more oligosaccharides to said immunoglobulin,
or effecting the removal of an attached oligosaccharide from said
immunoglobulin by exposure to one or more deglycosylases, or both
by preventing attachment to and removing one or more
oligosaccharides from said immunoglobulin, wherein at least one of
said one or more aglycosylated or deglycosylated immunoglobulins
becomes cleavable by a protease which cleaves said aglycosylated or
deglycosylated immunoglobulins at a position to form F(ab').sub.2
fragment(s) from said aglycosylated or deglycosylated
immunoglobulins as a result of said immunoglobulin being
aglycosylated or deglycosylated.
25. A kit for making F(ab').sub.2 fragments from one or more
immunoglobulins, at least one of said immunoglobulins having one or
more oligosaccharides attached thereto that inhibit protease
activity that converts said immunoglobulins into F(ab').sub.2
fragments comprising: a deglycosylation composition containing one
or more deglycosylase enzymes or chemicals capable of removing or
reducing some or all of said oligosaccharides; and, a protease
composition containing one or more proteases capable of reacting
with said immunoglobulin produces F(ab').sub.2 fragments from said
deglycosylated antibodies.
26. The kit of claim 25, further comprising a purification medium
for purifying said F(ab').sub.2 fragments from non-F(ab').sub.2
fragments of said immunoglobulin or from uncleaved
immunoglobulin.
27. The kit of claim 25, further comprising instructions for
carrying out the method selected from the group consisting of the
methods of claim 1, claim 12, or claim 19.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] 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, New York,
pp. 2.7.1-2.7.12.
[0003] 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.
[0004] Harris, L. J., Larson, S. B., Hasel, K. W. and McPherson, A.
(1997). Refined structure of an intact IgG.sub.2a monoclonal
antibody. Biochemistry 36, 1581-1597.
[0005] 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.
[0006] Kim, H., et al. (1994). O-Glycosylation in hinge region of
mouse immunogloblulin G.sub.2b. J. Biol. Chem. 269,
12345-12350.
[0007] Laemmli, U. K. (1970). Cleavage of structural proteins
during the assembly of the head of the bacteriophage T4. Nature
227, 680-5.
[0008] Lamoyi, E. and Nisonoff, A. (1983). Preparation of
F(ab').sub.2 fragments from mouse IgG of various subclasses. J.
Immunol. Methods 56, 235-243.
[0009] Mariani, M., Cauragra, M., Tarditi, L. and Seccariani, E.
(1991). A new enzymatic method to obtain high-yield F(ab').sub.2
suitable for clinical use from mouse IgG.sub.1. Mol Immunol. 28,
69-77.
[0010] 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.
[0011] Nisonoff, A, Wissler, F. C., Lipman, L. N. and Woemley, D.
L. (1960). Separation of univalent fragments from the bivalent
rabbit antibody molecule by reduction of disulfide bonds. Arch.
Biochem. Biophys. 89, 230-244.
[0012] Parham, P. (1983). On the fragmentation of monoclonal
IgG.sub.1, IgG.sub.2a, and IgG.sub.2b from BALB/c mice. J. Immunol.
131, 2895-2902.
[0013] 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.
[0014] Rousseaux, J., Rousseaux-Prevost, R. and Bazin, H. (1983).
Optimal conditions for the preparation of Fab and F(ab').sub.2
fragments from monoclonal IgG of different rat IgG subclasses. J.
Immunol. Methods 64, 141-146.
[0015] 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
[0016] 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.
[0017] 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').sub.2 fragment separated from the Fc
domain. An F(ab').sub.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').sub.2 is also a useful intermediate in the production of
monomeric, chemically tagged Fab monomers because F(ab').sub.2s 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.
[0018] 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,
2a, 2b and 3. Certain, and often important, members of antibody
classes 1 and 2b are recalcitrant to yielding F(ab').sub.2
fragments from pepsinolysis treatment. Even if pepsin cleaves such
antibodies, it often does not give good yields or yields different
non-F(ab').sub.2 products. Thus many important IgGs cannot be
efficiently converted to Fab dimers. Because IgG, class is the most
common for monoclonal antibodies used in biotechnology, there is a
need for reliable, universal methods for converting whole IgG.sub.1
and other pepsin resistant antibodies to intact F(ab').sub.2
antibody fragments.
[0019] Methods for the preparation of F(ab').sub.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').sub.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').sub.2 fragments is by
pepsinolysis, which is generally efficient for most antibodies from
the mouse IgG.sub.2a and IgG.sub.3 subclasses, but not generally
efficient for those from the IgG.sub.2b or IgG.sub.1, the latter
being the most common.
[0020] Many others have reported poor yields of F(ab').sub.2
fragments by treating mouse IgG.sub.1 antibodies with pepsin under
standard conditions (37.degree. 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.sub.1 antibodies appeared
to be completely resistant to pepsinolysis. Numerous alternatives
to pepsinolysis have been described for generating F(ab').sub.2
fragments from IgG.sub.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').sub.2s
from antibodies with uniform, predictable results. Thus, there is a
need for a universal method for preparing F(ab').sub.2 antibody
fragments from whole antibodies, especially those from IgG.sub.1
and IgG.sub.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
[0021] The invention provides methods for making F(ab').sub.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.
[0022] In one aspect, the invention provides a method for preparing
a F(ab').sub.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 one or more oligosaccharide groups attached
thereto, where the oligosaccharide group(s) cause the protease
cleavage site(s) within the hinge region to be resistant to a
proteolysis treatment. The glycosylated antibody or antibodies are
then exposed to a deglycosylation treatment, the deglycosylation
treatment cleaving the oligosaccharide group(s) attached to the
non-hinge region(s) to form a partially or wholly deglycosylated
antibody having a hinge region cleavable by the proteolysis
treatment. The partially or wholly deglycosylated antibody or
antibodies are then exposed to the proteolysis treatment to cause
proteolytic cleavage of the hinge region cleavable by the
proteolysis to form the F(ab').sub.2 fragment.
[0023] Certain preferred embodiments may have at least one of the
following features such as; the glycosylated antibody being a
plurality of glycosylated antibodies, at least some of the
glycosylated antibodies being polyclonal, the glycosylated
antibodies being monoclonal, the glycosylated antibody being either
an IgG.sub.1 or IgG.sub.2b glycosylated antibody, the IgG.sub.1 or
IgG.sub.2b antibody being from a rodent-derived hybridoma cell
culture or ascites, the glycosylated antibody being derived from
the group consisting of rat, mouse, rabbit, goat, sheep, lamb,
chicken, or horse, the proteolysis being achieved wholly or partly
from protease treatments including components selected from the
list consisting of pepsin, proteases that cleave pepsin substrates,
papain, papain pre-activated with cysteine, and ficin, the
proteolysis being achieved by a protease capable of producing
F(ab').sub.2 fragments from the deglycosylated antibodies, the
deglycosylase treatment containing a glycosidase combination
selected from the group consisting of PNGase F, endo-O-glycosylase,
sialidase A, PNGase F/endo-O-glycosylase, PNGase F/sialidase A,
PNGase F/endo-O-glycosylase/sialidase A,
endo-O-glycosylase/sialidase A, and/or the non-hinge regions
comprising the Fc and the F(ab') regions of the glycosylated
antibody.
[0024] Another aspect of the invention provides for a method for
preparing F(ab').sub.2 fragments. The method includes the steps of
growing a hybridoma cell that normally produces glycosylated
antibodies where the glycosylated antibodies have a hinge region
with one or more protease cleavage sites located within the hinge
region, one or more non-hinge regions adjacent the hinge region,
and one or more oligosaccharide groups being attached to at least
one of the non-hinge regions by the hybridoma cell through
glycosylation, the oligosaccharide groups causing the hinge regions
to be resistant to a proteolysis treatment. The hybridoma cell or
cells are administered an inhibitor of the glycosylation effective
to inhibit glycosylation of the antibodies to produce one or more
unglycosylated antibodies lacking the oligosaccharides within at
least one non-hinge region to render the hinge region prone to the
proteolysis treatment. The unglycosylated antibodies are exposed to
the proteolysis treatment so that the unglycosylated antibodies'
hinge regions are cleaved to form the F(ab').sub.2 fragments from
the unglycosylated antibodies.
[0025] Certain preferred embodiments of the invention may include
the hybridoma cell being a part of a hybridoma cell culture or
ascites, the hybridoma cell being a plurality of hybridoma cells,
the hybridoma cell being part of a monoclonal or polyclonal
hybridoma cell line, the hybridoma cells being from the same
hybridoma cell line, the hybridoma cells being from different
hybridoma cell lines, and/or the inhibitor of the glycosylation
contains bacitracin or tunicamycin.
[0026] Another aspect of the invention provides a method for
preparing F(ab').sub.2 fragments. The method includes the steps of
providing a hybridoma cell line that normally produces glycosylated
antibodies, the glycosylated antibodies having a hinge region with
one or more protease cleavage sites located within the hinge
region, one or more non-hinge regions adjacent the hinge region,
and one or more oligosaccharide groups being attached to at least
one of the non-hinge regions by the hybridoma cell through
glycosylation, where the oligosaccharide groups cause the hinge
regions to be resistant to a proteolysis treatment. The hybridoma
cell line or lines are then altered to inhibit glycosylation of the
antibodies within the non-hinge regions to produce one or more
unglycosylated antibodies such that the unglycosylated antibodies
are susceptible to proteolysis treatment and caused to produce the
unglycosylated antibodies. The unglycosylated antibodies are then
exposed to the proteolysis treatment to cleave the unglycosylated
antibodies to produce the F(ab').sub.2 fragments.
[0027] Certain preferred embodiments may have the hybridoma cell
being part of a hybridoma cell culture or ascites, and/or the
altered cells being either permanently or transiently altered.
[0028] In another aspect, the invention provides for an
F(ab').sub.2 composition comprising: one or more F(ab').sub.2
fragments, or derivative therefrom, produced by a method selected
from the methods disclosed above. In certain embodiments, the
F(ab').sub.2 fragments are an active ingredient of an anti-toxin or
anti-venom medicament.
[0029] Yet another aspect of the invention provides for an
immunoglobulin composition comprising: one or more aglycosylated or
deglycosylated immunoglobulins, the aglycosylated or deglycosylated
immunoglobulins being formed by preventing the attachment of one or
more oligosaccharides to the immunoglobulin, or effecting the
removal of an attached oligosaccharide from the immunoglobulin by
exposure to one or more deglycosylases, or both by preventing
attachment to and removing one or more oligosaccharides from the
immunoglobulin. At least one of the one or more aglycosylated or
deglycosylated immunoglobulins becomes cleavable by a protease
which cleaves the aglycosylated or deglycosylated immunoglobulins
at a position to form F(ab').sub.2 fragment(s) from the
aglycosylated or deglycosylated immunoglobulins as a result
[0030] Still yet another aspect of the invention provides for a kit
for making F(ab').sub.2 fragments from one or more immunoglobulins,
at least one of the immunoglobulins having one or more
oligosaccharides attached thereto that inhibit protease activity
that converts the immunoglobulins into F(ab').sub.2 fragments
comprising: a deglycosylation composition containing one or more
deglycosylase enzymes capable of removing some or all of the
oligosaccharides; and, a protease composition containing one or
more proteases capable of reacting with the immunoglobulin produces
F(ab').sub.2 fragments from the deglycosylated antibodies. In
certain preferred embodiments, the kit further comprises a
purification medium for purifying the F(ab').sub.2 fragments from
non-F(ab').sub.2 fragments of the immunoglobulin or from uncleaved
immunoglobulin, and/or the kit further comprises instructions for
carrying out the method selected from the group consisting of the
methods disclosed above.
[0031] These and other aspects and embodiments thereof of the
invention will become apparent to one skilled in the art by way of
reading the specification and drawings below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 depicts a gel image demonstrating how deglycosylation
improves pepsinolysis of IgG.sub.1 conversion to F(ab').sub.2
fragments.
[0033] FIG. 2 depicts a time-course of pepsinolysis of IgG.sub.1
and IgG.sub.2b subclasses.
[0034] FIG. 3 depicts further examples of the effect of PNGase
F-treatment on the pepsinolysis of IgG.sub.1 antibodies.
[0035] FIG. 4 depicts results from treatment of a polyclonal IgG
population from a non-immunized mouse with PNGase F, followed by a
pepsinolysis time-course.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0036] The term "antibody" means one or more antibodies. Included
in the term antibodies are immunoglobulins, whether natural or
partially or wholly produced artificially, e.g. recombinant. An
antibody may be monoclonal or polyclonal. The antibody may, in some
cases, be a member of one, or a combination immunoglobulin classes,
including: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG
class, however, are preferred in the present invention such as
IgG.sub.1 and IgG.sub.2b subclasses. The present invention
contemplates, in some preferred embodiments, providing methods for
making immunoglobulins, without regard to origin, cleavable by
pepsin or pepsin-like treatments resulting in F(ab').sub.2
fragments, where such immunoglobulins are otherwise not cleavable
by pepsin or pepsin-like treatments to yield F(ab').sub.2
fragments. For example, chicken immunoglobulins, IgY, may be made
cleavable by pepsin or pepsin-like treatments when subjected to the
methods of the present invention to produce F(ab').sub.2 fragments
therefrom.
[0037] The term "antibody fragment" refers to one or more
derivatives of an antibody that is less than full-length.
Preferably, the antibody fragment retains at least a significant
portion of the full-length antibody's specific binding ability.
Examples of antibody fragments include in certain circumstances,
but are not limited to Fabs and F(ab').sub.2s.
[0038] A "F(ab').sub.2" fragment is an antibody fragment, for
example, one essentially equivalent to that obtained from certain
pepsin cleavable immunoglobulins (typically IgG) by digestion with
pepsin at about pH 4.0-4.5.
[0039] A "Fab'" fragment is an antibody fragment essentially
equivalent to that obtained by reduction of the disulfide bridge or
bridges joining the two heavy chain pieces in the F(ab').sub.2
fragment.
[0040] An "Fab" fragment may be an antibody fragment essentially
equivalent to that obtained by digestion of immunoglobulins
(typically IgG) with the enzyme papain.
[0041] A protease is an enzyme capable of cleaving a protein
substrate into smaller polymeric or even in some instances,
monomeric units. Papain and pepsin are but two examples of
proteases. Of particular importance to the present invention are
proteases that act on substrates similar to those of the substrates
for various forms of pepsin because pepsin, in certain situations,
cleaves certain antibodies in a manner that produces highly useful
F(ab').sub.2 antibody fragments. The present invention contemplates
that other proteases may also produce F(ab').sub.2 fragments from
certain whole antibodies in a manner different than that of pepsin,
but like pepsin, are affected by the presence of oligosaccharides
adjacent the protease substrate site which yields F(ab').sub.2
antibody fragments. In certain embodiments, the protease used is a
protease capable of producing F(ab').sub.2 fragments from wholly,
or partially deglycosylated antibodies, preferably an antibody
whose N-glycosyl attached oligosaccharides are wholly or partially
removed.
[0042] Pepsinolysis is the activity of cleaving a protein with
pepsin or a pepsin-like treatment to produce two or more
sub-components from the protein. With respect to antibodies,
pepsinolysis yields in certain circumstances, F(ab').sub.2
fragments. Pepsin-like treatment contemplates that other proteases
or protease-like processes, whether enzymatic, catalytic, including
use of catalytic antibodies, or chemical in nature, which may or
may not act on the same substrate or in the same manner as pepsin,
may also produce F(ab').sub.2 fragments from certain antibodies and
may also benefit from the methods of deglycosylation or
aglycosylation described herein.
[0043] Glycosidases are, in some embodiments, enzymes that catalyze
the hydrolysis of N-glycosidic or O-glycosidic linkages between
sugar units, or between sugars and non-sugar units such as amino
acids, including amino acids such as asparagine, serine, and
threonine, in polypeptides, and in particular in antibodies. A
particularly preferred example of a glycosidase is peptide:
N-glycosidase (PNGase F) that cleaves the sugar from an asparagine
sidechain, leaving aspartic acid. Other embodiments employ
different glycosidases that cleave one or more saccharides from
certain other amino acids. Yet other embodiments provide that some
or all of the attached saccharides are partly or completely
degraded resulting in partially or completely deglycosylated
antibodies, where such antibodies become cleavable by pepsin or
pepsin-like treatments which then result in the production of
F(ab').sub.2 fragments. Glycosidases may, for example, include
exoglycosylases, endoglycosylases, combinations of
exoglycosylase(s) and endoglycosylase(s), and/or may include
sialidases, fucosidases, mannosidases, galactosidases, and
xylosidases, for example.
[0044] Deglycosylation means, in certain embodiments, the removal
of one or more attached oligosaccharides from a protein, and in
particular, antibody, structure. Deglycosylation may be achieved by
enzymatic treatment, including natural or recombinant enzymes in
natural, concentrated, or purified forms. Deglycosylation may occur
within the antibody cells during growth of the antibodies, or may
occur outside such cells during growth by glycosidases produced by
such cells or different cells or from enzymes added to the growth
media. Deglycosylation may also be achieved by certain chemical
procedures or catalytic procedures, including catalytic antibodies,
provided such methods do not destroy the binding abilities of the
resulting F(ab').sub.2 fragments. Deglycosylation may be complete,
partial, or a combination of both, such that the resulting antibody
or antibodies become cleavable by pepsin or pepsin-like treatments
as a result of such deglycosylation. Deglycosylation treatments may
be combined with other strategies, such as aglycosylation,
described below, to work in concert to make antibodies become
cleavable by pepsin or pepsin-like treatments to produce
F(ab').sub.2s.
[0045] Aglycosylation means the interruption or prevention of
processes that would otherwise produce proteins, and in particular,
antibodies, that are glycosylated at one or more position within
such proteins or antibodies. This includes processes that reduce
the size or amount of branching, or otherwise alter the composition
of the glycosylation sites on proteins so as to render them more
susceptible to proteolysis by pepsin or to other pepsin-like
treatments. Such interruption may arise by exposure to
glycosylation inhibitors such as with bacitracin or tunicamycin, or
by genetic inhibition of glycosylation including knock-out mutants
of such glycosylases or other upstream metabolic components.
[0046] Oligosaccharides are typically polymeric sugar molecules,
including monomeric sugars, and in some instances containing one or
more interrupting monomeric units that are not sugars.
Oligosaccharides may include other substitutions attached thereto.
The term oligosaccharide, in certain embodiments, is
interchangeable with the term carbohydrate which includes
monomeric, and/or polymeric carbohydrates. In certain embodiments,
antibodies have a conserved glycosylation site, for example on
mammalian IgGs in the Fc region (CH.sub.2 domain) is at a single
asparagine (Asn 297 according to the numbering system used in
Edelman et al, Proc. Natl. Acad. Sci. USA 63:78-85, 1969.) The
invention provides removing oligosaccharides from antibodies at
conserved, preferably known conserved, positions within the
antibody.
[0047] Hybridomas are cells made from, for example,
non-antibody-secreting cultured myeloma cells with normal B cells
from the spleen of an immunized mouse. The fusion of a myeloma cell
from a line that has lost the ability to secrete immunoglobulin
with a B cell known to secrete a particular antibody results in a
remarkable hybrid cell that produces the antibody made by its
B-cell component but retains the capacity of its myeloma component
to multiply indefinitely.
[0048] General methods employed by the present invention may be
found, for example, in Current Protocols in Immunology (1997), John
Wiley & Sons, Inc., herein incorporated by reference in its
entirety for all purposes, and for the purpose of providing general
methods employed by the present invention.
[0049] One aspect of the invention provides for methods for
overcoming problems associated with converting mouse IgG molecules,
and most importantly IgG.sub.1 and IgG.sub.2b antibodies, to
dimeric Fab fragments. In one embodiment, the invention provides
for treating the IgG molecules with reagent(s) that remove N-linked
or O-linked oligosaccharides from the IgGs before pepsinolysis to
produce deglycosylated antibodies that are more susceptible to
cleavage by pepsin to form the dimeric Fab fragment.
Deglycosylation improves the pepsinolysis of, for example,
important classes of mouse IgGs such as the pepsin-resistant
IgG.sub.1s and some pepsin-resistant IgG.sub.2bs.
[0050] In preferred embodiments, treating IgGs with commercially
available peptide: N-glycosidase (PNGase F) is sufficient to
convert the IgGs to a form readily cleavable by pepsin or other
F(ab').sub.2 protease to produce F(ab').sub.2 fragments. There are
a variety of other methods, including chemical or enzymatic methods
that could also be used either to remove the carbohydrate groups,
or to deplete glycosylated antibodies from mixtures of glycosylated
and non-glycosylated antibody populations. The invention covers any
such methods for removing oligosaccharides from antibodies or
preventing glycosylation, but in particular, the use of PNGase F or
other enzymes to remove the relevant carbohydrate groups is
particularly preferred.
[0051] According to certain preferred embodiments of the invention,
IgG.sub.1 molecules are rendered pepsin-sensitive by treatment with
peptide: N-glycosidase F (PNGase F), or other enzymes capable of
removing N-linked oligosaccharides. The invention provides methods
for converting mouse IgG.sub.1 antibodies resistant to pepsinolysis
absent deglycosylation or aglycosylation treatment to a form that
is efficiently cleaved by pepsin under standard reaction
conditions. Other embodiments provide for the removal of
oligosaccharides, preferably N-linked carbohydrate groups, from
IgG.sub.2b molecules which are otherwise resistant to pepsinolysis
or other F(ab').sub.2-producing protease treatments to increase the
yield of F(ab').sub.2 fragments.
[0052] This invention describes a method for overcoming the
problems associated with converting mouse IgG molecules, and most
importantly IgG.sub.1 and IgG.sub.2b antibodies, to dimeric Fab
fragments. The invention provides in certain, preferred
embodiments, methods for pre-treating IgG molecules with reagent(s)
that remove N-linked oligosaccharides from the IgGs. The resulting
deglycosylated antibodies are more susceptible to cleavage by
pepsin to form the dimeric Fab fragment. Such deglycosylation
improves the pepsinolysis of at certain members of at least two
classes of mouse IgGs-IgG.sub.1 and IgG.sub.2b.
[0053] In particularly preferred embodiments, treating IgGs with
commercially available peptide: N-glycosidase (PNGase F) converts
IgGs to readily pepsinolysis cleavable molecules which yield
F(ab').sub.2 fragments. There are a variety of other methods,
chemical or enzymatic, that could also be used either to remove the
carbohydrate groups, or to deplete glycosylated antibodies from
mixtures of glycosylated and non-glycosylated antibody
populations.
[0054] Another aspect of the invention provides for kits for making
F(ab').sub.2 fragments from one or more immunoglobulins, at least
one of the immunoglobulins having one or more oligosaccharides
attached thereto that inhibit protease activity that converts the
immunoglobulins into F(ab').sub.2 fragments comprising: a
deglycosylation composition containing one or more deglycosylase
enzymes or chemicals capable of removing or reducing some or all of
the oligosaccharides; and, a protease composition containing one or
more proteases capable of reacting with the immunoglobulin produces
F(ab').sub.2 fragments from the deglycosylated antibodies. In
preferred embodiments, the kit further comprises a purification
medium for purifying the F(ab').sub.2 fragments from
non-F(ab').sub.2 fragments of the immunoglobulin or from uncleaved
immunoglobulin, and/or further comprising instructions for carrying
out the method selected from the group consisting of the methods
disclosed above.
[0055] The invention further provides for medicaments which employ
as active ingredients, F(ab').sub.2 fragments, such medicaments
including, for example, anti-toxin remedies and anti-venom
remedies.
[0056] The invention, in another aspect, provides for intermediate
stage immunoglobulins, preferably antibodies, which have been
partially or wholly deglycosylated, aglycosylated, or both, to make
such immunoglobulins cleavable by an F(ab').sub.2 producing
protease to form F(ab').sub.2 fragments from such
immunoglobulins.
[0057] All references cited herein are incorporated by reference in
their entirety for all purposes and any stated purpose as if each
reference were incorporated by reference in its entirety as such
where individually cited.
EXAMPLES
Antibodies
[0058] MAB9647 (raised against human IL-8) was produced by Covance,
Inc. (Princeton, N.J.) from mouse ascites fluid using the hybridoma
cell line HB-9647 from ATCC (Manassas, Va.) and was protein
G-purified. MAB3.1 was raised against human IL-3 by BD Pharmingen
(Franklin Lakes, N.J.) and was protein G-purified from tissue
culture supernatent. MAB206 was raised against human IL16 by
R&D Systems (Minneapolis, Minn.) and was protein A-purified
from mouse ascites fluid from clone 6708.111 (catalog number
MAB206). MAB6002 raised against the human IgG Fc region was
produced by Covance, and obtained from mouse ascites fluid using
the hybridoma cell line CRL-1788 from ATCC and was protein
G-purified. MAB 6001 raised against the human IgG1 Fc region was
produced by Covance from mouse ascites fluid using the hybridoma
cell line CRL-1755 from ATCC and was protein G-purified.
Antibody Deglycosylation
[0059] Individual glycosidases or combinations thereof, were
prepared as follows: 50U/ml PNGase F alone, and a combination of 50
U/ml PNGase F with 0.012 U/ml endo-o-glycosylase, and 0.05 U/ml
Sialidase A. (Enzymes were obtained from Prozyme of San Leandro,
Calif. or New England Biolabs, Beverly, Mass. In the above
situations, the Prozyme unit definitions are used. For all cases
below for the PNGase, the unit definition was established by New
England Biolabs.) Reaction cocktails contained 1 mg/ml IgG in a 50
mM Na.sub.2PO.sub.4, pH 7.0 buffer, and were reacted at about
37.degree. C. for 36 hours. Deglycosylation treatments may also be
carried out using 1-4 mg/ml antibody in 50 mM NaPO.sub.4, pH of
about 7.5, and 10-20 U/.mu.l PNGase F. PNGase F is purified from
Flavobacterium meningosepticum and has an apparent molecular weight
of about 36,000 Daltons. PNGase F cleaves between the innermost
GlcNAc and asparagine residues of high mannose, hybrid, and complex
oligosaccharides from N-linked glycoproteins. One unit of PNGase is
defined by New England Biolabs as being the amount of enzyme
required to remove >95% of the carbohydrate from 10 .mu.g of
denatured RNase B in 1 hour at 37.degree. C. in a total reaction
volume of 10 .mu.l (65 NEB units=1 IUB milliunit).
[0060] Endo-O-glycosidase (O-glycopeptide
endo-D-galactosyl-N-acetyl-alpha- -galactosaminohydrolase, EC
3.2.1.97) cleaves unsubstituted Galbeta(1-3)GalNAcalpha
disaccharides attached to the serine or threonine residues of
glycoproteins or glycopeptides. Substitutions such as sialic acid,
galactose, fucose or N-acetylglucosamine may first be removed with
the appropriate exoglycosidase prior to treatment with
Endo-O-Glycosidase. Typically, a neuraminidase such as Sialidase A
is used to remove sialic acid. One unit of Endo-O-Glycosidase is
defined as the amount of enzyme required to produce 1 umole of
p-nitrophenol in 1 min at 37.degree. C. pH 5
from-nitrophenyl-2-acetamido-2-deoxy-3-O-(beta--
D-galactopyranosyl)-alpha-D-galactopyranoside. Sialidase A
(N-acetylneuraminate glycohydrolase, EC 3.2.1.18) cleaves all
non-reducing terminal sialic acid residues from complex
carbohydrates and glycoproteins. The relative cleavage rates for
different linkages are: alpha(2-6)>alpha(2-3)>alpha(2-8),
alpha(2-9). In addition, Sialidase A will cleave branched sialic
acids (linked to an internal residue). This property makes it
unique among sialidases. High concentrations of enzymes and
prolonged incubation times may be required for cleaving branched
residues. One unit of Sialidase A is defined as the amount of
enzyme required to produce 1 .mu.mole of methylumbelliferone in 1
minute at 37.degree. C. pH 5 from
2'-(4-methylumbelliferyl)-alpha-D-N-a- cetylneuraminic acid.
Pepsinolysis
[0061] After carrying out deglycosylation reactions, antibodies are
buffer-exchanged into 20 mM NaOAc, pH 4.5. PNGase was optionally
present during pepsinolysis. Pepsinolysis conditions were 30% v/v
pepsin agarose (settled bed volume, beads washed in 20 mM NaOAc, pH
4.5), about 0.5 to 2 mg/ml IgG, 20 mM NaOAc, 260 mM KCl, 0.1%
Triton-X-100, pH 4.5. Reactions were incubated at 37.degree. C.
with agitation for stated periods of time. After each time point,
the slurry was loaded onto a centrifugal filter device (Millipore
UFC30HVNB, Bedford, Mass.) and spun in a microcentrifuge (12,000 g
for 2 minutes). The resulting filtrate was diluted 1:1 with
non-reducing protein loading buffer (62.5 mM Tris HCL, 25%
glycerol, 2% SDS, 0.01% Bromphenol Blue) and loaded onto a 4-20%
gradient SDS-polyacrylamide gel (Product number 161-1123, Bio Rad,
Hercules, Calif.). SDS-PAGE was performed according to Laenmrli
(1970).
F(AB').sub.2 Reduction and Alkykation
[0062] The products of the pepsin cleavage were exchanged by
dialysis into 0.1M Na.sub.2PO.sub.4, 5 mM EDTA, pH 6.0, and then
treated with 20 mM 2-mercaptoethylamine (MEA) for 90 minutes at
37.degree. C. The MEA was then removed by dialyzing for 6 hours at
4.degree. C. against 0.1 M Na.sub.2PO.sub.4, 5 mMEDTA, pH 6.0,
using a Sephadex G-25 column (PD-10, Amersham-Pharmacia,
Piscataway, N.J.). The reduced Fab' was then treated with 20 mM
maleimide-activated biotin (Pierce product No. 21901) or
N-ethylmaleimide (NEM) for 2 hours at room temperature, and the
unincorporated biotin-maleimide or NEM was then removed by gel
filtration (Superdex-75 resin, Amersham-Pharmacia). Antigen-binding
activity of the biotinylated F(ab') fragments was confirmed by
Surface Plasmon Resonance (SPR) using streptavidin-coated chips
(Biocore 3000, Uppsala, Sweden).
Streamlined Deglycosylation/Pepsinolysis
[0063] A preferred method for producing F(ab').sub.2 fragments
includes the following steps. Antibodies (1-4 mg/ml) in 25 mM
Na.sub.2PO.sub.4, pH 7.5 is treated with 5U/.mu.l PNGase F (New
England BioLabs unit definition and reagent) for 4 or more hours.
Pepsinolysis is then carried out using a pepsinolysis reaction
containing 30% by volume pepsin agarose beads (volume of settled
matrix bed, washed in 20 mM NaOAc, pH 4.5), 20% by volume 5.times.
pepsinolysis buffer (163 mM NaOAc, 1M KCl, 0.5% Triton-X-100, pH
3.5) and 50% by volume of the abovementioned deglycosylation
reaction. Hence, there is no need for buffer exchange or PNGase F
removal. The resulting cocktail typically has a pH of about 4.5.
The pepsinolysis is carried out for about 1-14 hours, depending on
which antibody is used. Eight hours is usually optimal.
Data
[0064] FIG. 1 depicts a gel image demonstrating how deglycosylation
improves pepsinolysis of IgG, conversion to F(ab').sub.2 fragments.
A 17 hour time-course of MAB9647 after treatment with a cocktail of
three enzymes that remove both N- and O-linked carbohydrate groups
("3 Enzymes"), or that remove only N-linked carbohydrates ("PNGase
F"), or a control reaction with no glycosidase. The positions of
full length IgG and the F(ab').sub.2 fragments are shown. The
number of hours of pepsinolysis is shown at the top of each
lane.
[0065] FIG. 2 depicts a time-course of pepsinolysis of IgG.sub.1
and IgG.sub.2b subclasses. The time (in hours) of pepsin-treatment
is shown at the top of each lane, after treatment with PNGase F or
a control reaction with no glycosidase. Panel (a) demonstrates the
behavior of MAB947, an IgG.sub.1 subclass antibody, and panel (b)
shows a similar result for MAB6001, of subclass IgG.sub.2b. Panel
(c) shows the result of converting the F(ab').sub.2 fragments from
MAB9647 and MAB6001 into monmeric Fab' fragments by treatment with
2-mercaptoethylamine followed by alkylation with
N-ethylmaleimide.
[0066] FIG. 3 depicts further examples of the effect of PNGase
F-treatment on the pepsinolysis of IgG.sub.1 antibodies. Three IgG
molecules were either treated with PNGase F or no glycosidase, and
then exposed to pepsin for various times (MAB3.1: 8 hours; MAB206:
10 hours; and MAB6002: 5 hours).
[0067] FIG. 4 depicts results from treatment of a polyclonal IgG
population from a non-immunized mouse with PNGase F, followed by a
pepsinolysis time-course. The time in hours is shown above each
lane.
* * * * *