U.S. patent application number 16/145688 was filed with the patent office on 2019-01-31 for polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods.
The applicant listed for this patent is The Rockefeller University. Invention is credited to Yoshikatsu Kaneko, Falk Nimmerjahn, Jeffrey Ravetch.
Application Number | 20190031737 16/145688 |
Document ID | / |
Family ID | 38581601 |
Filed Date | 2019-01-31 |
United States Patent
Application |
20190031737 |
Kind Code |
A1 |
Ravetch; Jeffrey ; et
al. |
January 31, 2019 |
Polypeptides With Enhanced Anti-Inflammatory And Decreased
Cytotoxic Properties And Relating Methods
Abstract
The invention provides a polypeptide containing at least one IgG
Fc region, wherein said at least one IgG Fc region is glycosylated
with at least one galactose moiety connected to a respective
terminal sialic acid moiety by a .alpha. 2,6 linkage, and wherein
said polypeptide having a higher anti-inflammatory activity as
compared to an unpurified antibody.
Inventors: |
Ravetch; Jeffrey; (New York,
NY) ; Nimmerjahn; Falk; (Erlangen, DE) ;
Kaneko; Yoshikatsu; (Niigata City, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Rockefeller University |
New York |
NY |
US |
|
|
Family ID: |
38581601 |
Appl. No.: |
16/145688 |
Filed: |
September 28, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15629056 |
Jun 21, 2017 |
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16145688 |
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15288764 |
Oct 7, 2016 |
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15629056 |
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13896070 |
May 16, 2013 |
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15288764 |
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12447204 |
Feb 18, 2010 |
8470318 |
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PCT/US2007/072771 |
Jul 3, 2007 |
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13896070 |
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PCT/US2007/008396 |
Apr 3, 2007 |
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12447204 |
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PCT/US2006/041791 |
Oct 27, 2006 |
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PCT/US2007/072771 |
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60789384 |
Apr 5, 2006 |
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60734196 |
Nov 7, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 47/68 20170801; C07K 16/00 20130101; C07K 2317/52 20130101;
C07K 16/18 20130101; A61P 29/00 20180101; C07K 2317/71 20130101;
C07K 2317/41 20130101; G01N 33/6854 20130101; C07K 16/06 20130101;
A61P 37/00 20180101; C07K 2317/76 20130101; C12P 21/005
20130101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C12P 21/00 20060101 C12P021/00; C07K 16/06 20060101
C07K016/06; C07K 16/18 20060101 C07K016/18; G01N 33/68 20060101
G01N033/68; A61K 47/68 20170101 A61K047/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] The Research leading to the present invention was supported
in part, by National Institutes of Health Grant No. AI 034662.
Accordingly, the U.S. Government has certain rights in this
invention.
Claims
1. An isolated polypeptide containing at least one IgG Fc region,
having altered properties compared to an unpurified antibody
preparation, wherein sialylation of the isolated polypeptide is
higher than the sialylation of the unpurified antibody
preparation.
2. The isolated polypeptide of claim 1, wherein said at least one
IgG Fc region is glycosylated with at least one galactose moiety
connected to a respective terminal sialic acid moiety by a .alpha.
2,6 linkage, and wherein said polypeptide having a higher
anti-inflammatory activity as compared to an unpurified antibody
preparation.
3. The isolated polypeptide of claim 1 or 2, wherein said at least
one IgG Fc region is glycosylated with at least one galactose
moiety connected to a respective terminal sialic acid moiety by a
.alpha. 2,6 linkage, and wherein said polypeptide having a reduced
binding to an Fc activating receptor as compared to an unpurified
antibody preparation.
4. The isolated polypeptide of claim 3, wherein the Fc activating
receptor is selected from the group consisting of Fc.gamma.RIIA,
Fc.gamma.RIIC and Fc.gamma.RIIIA.
5. The polypeptide of any one of claims 1-4 comprising a human
IgG1, IgG2, IgG3 or IgG4 Fc region, said polypeptide having a
higher content of the at least one galactose moiety connected to
the respective terminal sialic acid moiety by a .alpha. 2,6 linkage
as compared to an unpurified antibody.
6. The polypeptide of any one of claims 1-5, having an increased
anti-inflammatory activity in vitro.
7. The polypeptide of any one claims 1-6, having an increased
anti-inflammatory activity in vivo.
8. The polypeptide of any one of claims 1-7, derived from a
naturally occurring antibody source.
9. The polypeptide of any one of claims 1-7, derived from a
recombinant antibody source.
10. The polypeptide of any one of claims 1-7, wherein said
unmodified antibody comprises IVIG.
11. The polypeptide of any one of claims 1-7, derived from a cell
line having an enhanced activity of creating .alpha.2,6 linkages
between at least one galactose moiety and a respective terminal
sialic acid in a protein's polysaccharide chain.
12. The polypeptide of any one of claims 1-11, modified by
treatment with .alpha.2-6 sialyltransferase.
13. The polypeptide of any one of claims 1-12, which is
purified.
14. A pharmaceutical formulation comprising the polypeptide of any
one of claims 1-13 in combination with a suitable carrier or
diluent.
15. A method of modulating properties of a polypeptide comprising
an Fc region comprising altering the sialylation of the
polysaccharide chain of the Fc region.
16. A method of claim 15, wherein said properties comprise a higher
anti-inflammatory activity than an unpurified antibody.
17. The method of claim 15 or 16, wherein the step of altering
sialylation comprises: providing an unpurified source of the
polypeptide containing at least one Fc region, said unpurified
source of the polypeptide containing at least one Fc region
comprising a plurality of the polypeptides containing at least one
Fc region having a polysaccharide chain comprising a terminal
sialic acid connected to a galactose moiety through a .alpha. 2,6
linkage, and a plurality of the polypeptides containing at least
one Fc region lacking a polysaccharide chain comprising a terminal
sialic acid connected to a galactose moiety through the .alpha. 2,6
linkage; and increasing the ratio of the plurality of the
polypeptides containing at least one Fc region having the
polysaccharide chain comprising the terminal sialic acid connected
to the galactose moiety through the .alpha. 2,6 linkage to the
plurality of the polypeptide containing at least one Fc region
lacking the polysaccharide chain comprising the terminal sialic
acid connected to the galactose moiety through the .alpha. 2,6
linkage.
18. The method of any one of claims 15-17, wherein said unpurified
source of the polypeptide containing at least one Fc region
comprises a human unpurified IgG antibody.
19. The method of any one of claims 15-18, wherein the unpurified
source of the polypeptide containing at least one Fc region is
provided from expressing a vector comprising a nucleic acid
sequence in an expression system, wherein said nucleic acid
sequence is translated into an IgG antibody.
20. The method of claim 19, wherein the expression system comprises
modified host expression systems capable of N-linked glycosylation
selected from the group consisting of bacterial, fungal, plant,
vertebrate and invertebrate expression systems, and any
combinations thereof.
21. The method of any one of claims 15-20, wherein the step of
increasing the ratio of the plurality of the polypeptides
containing at least one Fc region having the polysaccharide chain
comprising the terminal sialic acid connected to the galactose
moiety through the .alpha. 2,6 linkage to the plurality of the
polypeptide containing at least one Fc region lacking the
polysaccharide chain comprising the terminal sialic acid connected
to the galactose moiety through the .alpha. 2,6 linkage is achieved
through a removal of the polypeptides containing at least one Fc
region lacking the polysaccharide chain comprising the terminal
sialic acid connected to the galactose moiety through the .alpha.
2,6 linkage.
22. The method of claim 21, wherein said removal is achieved by
physical or chemical methods.
23. The method of claim 21 wherein said removal is achieved by a
method selected from the group consisting of HPLC, lectin affinity
chromatography, high pH anion exchange chromatography, and any
combination thereof.
24. The method of claim 23, wherein the lectin affinity
chromatography is performed using a lectin having a lower affinity
to .alpha.2,6 linkages than to .alpha.2,3 linkages between the
galactose moiety and the terminal sialic acid.
25. The method of any one of claims 15-24, wherein the step of
increasing the ratio of the plurality of the polypeptides
containing at least one Fc region having the polysaccharide chain
comprising the terminal sialic acid connected to the galactose
moiety through the .alpha. 2,6 linkage to the plurality of the
polypeptide containing at least one Fc region lacking the
polysaccharide chain comprising the terminal sialic acid connected
to the galactose moiety through the .alpha. 2,6 linkage is achieved
through an enrichment of said unpurified source of the polypeptide
containing at least one Fc region having the polysaccharide chain
comprising the terminal sialic acid connected to the galactose
moiety through the .alpha. 2,6 linkage.
26. The method of claim 25 wherein said enrichment is achieved by a
method selected from the group consisting of HPLC, lectin affinity
chromatography, high pH anion exchange chromatography, and any
combination thereof.
27. The method of claim 26, wherein the lectin affinity
chromatography is performed using a lectin having a higher affinity
to .alpha.2,6 linkages than to .alpha.2,3 linkages between the
galactose moiety and the terminal sialic acid.
28. The method of any one of claims 25-27 wherein said enrichment
is achieved by a chemical reaction with an enzyme creating .alpha.
2,6 linkages between the carbohydrate attached to the polypeptide
containing least one Fc region and a terminal sialic acid.
29. The method of claim 28, wherein the enzyme is .alpha.-(2,6)
sialyltransferase.
30. A method of treating an inflammatory disease comprising
administering to a patient a therapeutically effective dose of the
polypeptide of any one of claims 1-14.
31. The method of claim 30, wherein the inflammatory disease is
selected from the group consisting of arthritis, thrombocytopenia,
and nephritis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation patent application
of U.S. patent application Ser. No. 12/447,204, filed on Feb. 18,
2010, which is a National Stage filing under 35 U.S.C. .sctn.
371(c) of International Application Serial No. PCT/US07/72771 filed
Jul. 3, 2007. PCT/US07/72771 is a continuation-in-part patent
application of PCT Patent Application Number PCT/US07/08396, filed
on Apr. 3, 2007, which claims the benefit of U.S. Provisional
Patent Application No. 60/789,384, filed on Apr. 5, 2006, both of
which are incorporated herein by reference. PCT/US07/72771 also
claims the benefit of PCT Patent Application Number PCT/US06/41791,
filed on Oct. 27, 2006, and U.S. Provisional Patent Application No.
60/734,196, filed on Nov. 7, 2005, both of which are also
incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to novel method for designing
therapeutic polypeptides for treatment of inflammatory
diseases.
BACKGROUND
[0004] Although cellular receptors for immunoglobulins were first
identified nearly 40 years ago, their central role in the immune
response was only discovered in the last decade. They are key
players in both the afferent and efferent phase of an immune
response, setting thresholds for B cell activation and antibody
production, regulating the maturation of dendritic cells and
coupling the exquisite specificity of the antibody response to
effector pathways, such as phagocytosis, antibody dependent
cellular cytotoxicity and the recruitment and activation of
inflammatory cells. Their central role in linking the humoral
immune system to innate effector cells has made them attractive
immunotherapeutic targets for either enhancing or restricting the
activity of antibodies in vivo.
[0005] The interaction of antibodies and antibody-antigen complexes
with cells of the immune system effects a variety of responses,
including antibody dependent cell-mediated cytotoxicity (ADCC) and
complement dependent cytotoxicity (CDC), phagocytosis, inflammatory
mediator release, clearance of antigen, and antibody half-life
(reviewed in Daron, Annu Rev Immunol, 15, 203-234 (1997); Ward and
Ghetie, Therapeutic Immunol, 2, 77-94 (1995); Ravetch and Kinet,
Annu Rev Immunol, 9, 457-492 (1991)), each of which is incorporated
herein by reference).
[0006] Antibody constant domains are not involved directly in
binding an antibody to an antigen, but exhibit various effector
functions. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies or immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant
regions that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. Of the various human immunoglobulin classes, human
IgG1 and IgG3 mediate ADCC more effectively than IgG2 and IgG4.
[0007] Papain digestion of antibodies produces two identical
antigen binding fragments, called Fab fragments, each with a single
antigen binding site, and a residual "Fc" fragment, whose name
reflects its ability to crystallize readily. The Fc region is
central to the effector functions of antibodies. The crystal
structure of the human IgG Fc region has been determined
(Deisenhofer, Biochemistry, 20, 2361-2370 (1981), which is
incorporated herein by reference). In human IgG molecules, the Fc
region is generated by papain cleavage N-terminal to Cys, 226.
[0008] IgG has long been appreciated to mediate both pro- and
anti-inflammatory activities through interactions mediated by its
Fc fragment. Thus, while Fc-FcyR interactions are responsible for
the pro-inflammatory properties of immune complexes and cytotoxic
antibodies, intravenous gamma globulin (IVIG) and its Fc fragments
are anti-inflammatory and are widely used to suppress inflammatory
diseases. The precise mechanism of such paradoxical properties is
unclear but it has been proposed that glycosylation of IgG is
crucial for regulation of cytotoxicity and inflammatory potential
of IgG.
[0009] IgG contains a single, N-linked glycan at Asn.sup.297 in the
CH2 domain on each of its two heavy chains. The covalently-linked,
complex carbohydrate is composed of a core, biantennary
penta-polysaccharide containing N-acetylglucosamine (GIcNAc) and
mannose (man). Further modification of the core carbohydrate
structure is observed in serum antibodies with the presence of
fucose, branching GIcNAc, galactose (gal) and terminal sialic acid
(sa) moieties variably found. Over 40 different glycoforms have
thus been detected to be covalently attached to this single
glycosylation site. Fujii et al., J. Biol. Chem 265, 6009 (1990).
Glycosylation of IgG has been shown to be essential for binding to
all FcyRs by maintaining an open conformation of the two heavy
chains. Jefferis and Lund, Immune.l Lett. 82, 57 (2002), Sondermann
et al., J. Mol. Biol. 309, 737 (2001). This absolute requirement of
IgG glycosylation for FcyR binding accounts for the inability of
deglycosylated IgG antibodies to mediate in vivo triggered
inflammatory responses, such as ADCC, phagocytosis and the release
of inflammatory mediators. Nimmerjahn and Ravetch, Immunity 24, 19
(2006). Further observations that individual glycoforms of IgG may
contribute to modulating inflammatory responses has been suggested
by the altered affinities for individual FcyRs reported for IgG
antibodies containing or lacking fucose and their consequential
affects on cytotoxicity. Shields et al., J. Biol. Chem. 277, 26733
(2002), Nimmerjahn and Ravetch, Science 310, 1510 (2005). A link
between autoimmune states and specific glycosylation patterns of
IgG antibodies has been observed in patients with rheumatoid
arthritis and several autoimmune vasculities in which decreased
galactosylation and sialylation of IgG antibodies have been
reported. Parekh et al., Nature 316, 452 (1985), Rademacher et al.,
Proc. Natl. Acad. Sci. USA 91, 6123 (1994), Matsumoto et al., 128,
621 (2000), Holland et al., Biochim. Biophys. Acta December 27;
[Epub ahead of print] 2005. Variations in IgG glycoforms have also
been reported to be associated with aging and upon immunization,
although the in vivo significance of these alterations have not
been determined. Shikata et al., Glycoconj. J. 15, 683 (1998),
Lastra, et al., Autoimmunity 28, 25 (1998).
[0010] Accordingly, there is a need for the development of methods
for the generation of polypeptides that would account for the
disparate observations of IVIG properties in vivo.
SUMMARY OF INVENTION
[0011] The present invention fills the foregoing need by providing
such methods and molecules. In one aspect, the invention provides
an isolated polypeptide containing at least one IgG Fc region,
having altered properties compared to an unpurified antibody
preparation, wherein sialylation of the isolated polypeptide is
higher than the sialylation of the unpurified antibody preparation.
In one embodiment, the isolated polypeptide containing at least one
IgG Fc region is glycosylated with at least one galactose moiety
connected to a respective terminal sialic acid moiety by a .alpha.
2,6 linkage, and wherein said polypeptide having a higher
anti-inflammatory activity as compared to an unpurified antibody.
In one embodiment the isolated polypeptide containing at least one
IgG Fc region is glycosylated with at least one galactose moiety
connected to a respective terminal sialic acid moiety by a .alpha.
2,6 linkage, and wherein said polypeptide having a reduced binding
to an Fc activating receptor as compared to an unpurified antibody
preparation. In a further embodiment the Fc activating receptor is
selected from the group consisting of Fc.gamma.RIIA, Fc.gamma.RIIC
and Fc.gamma.RIIIA.
[0012] In another aspect, the instant invention provides a
pharmaceutical formulation comprising a polypeptide containing at
least one Fc region having a higher anti-inflammatory activity, in
combination with a suitable carrier or diluent.
[0013] A method of modulating properties of a polypeptide
comprising an Fc region comprising altering the sialylation of the
polysaccharide chain of the Fc region.
[0014] In one embodiment the method comprises: providing an
unpurified source of the polypeptide containing at least one Fc
region, said unpurified source of the polypeptide containing at
least one Fc region comprising a plurality of the polypeptides
containing at least one Fc region having a polysaccharide chain
comprising a terminal sialic acid connected to a galactose moiety
through a .alpha. 2,6 linkage, and a plurality of the polypeptides
containing at least one Fc region lacking a polysaccharide chain
comprising a terminal sialic acid connected to a galactose moiety
through the .alpha. 2,6 linkage; and increasing the ratio of the
plurality of the polypeptides containing at least one Fc region
having the polysaccharide chain comprising the terminal sialic acid
connected to the galactose moiety through the .alpha. 2,6 linkage
to the plurality of the polypeptide containing at least one Fc
region lacking the polysaccharide chain comprising the terminal
sialic acid connected to the galactose moiety through the .alpha.
2,6 linkage.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-1C are illustrations of MALDI-Tof analysis of
SNA.sup.+ FC linkages showing the footprint histogram of the
enriched galactose-sialic acid structures with in vivo
anti-inflammatory activity (FIG. 1A) as compared to histograms from
sialic acid linkage standards, .alpha.2,3 sialyllactose (FIG. 1B)
and .alpha.2,6 sialyllactose (FIG. 1C).
[0016] FIGS. 2A-2D show: (A) glycan Maldi-T of MS analysis of IVIG
Fc fragments; (B) hypergalactosylation verified by comparing
relative band intensity ratios of terminal galactose as measured by
ECL and coomassie loading controls; (C) in vitro sialylation using
either .alpha.2,6 sialyltransferase ("ST6Gal") or .alpha.2,3
sialyltransferase ("ST3Gal") and confirmed by lectin blotting for
.alpha.2,6 linkages with SNA (top) or .alpha.2,3 linkages with ECL
(middle) and coomassie (bottom); and (D) the ability of in vitro
sialylated Fc to inhibit inflammation in mice.
[0017] FIGS. 3A and 3B show IVIG treated with linkage specific
sialidases (SAs), and the digestion verified by lectin blotting
(FIG. 3A) and the effect of specific removal of sialic acid
moieties on inflammation in mice (FIG. 3B).
DETAILED DESCRIPTION
[0018] The inventors have surprisingly found that the cytotoxic and
anti-inflammatory response of the IgG Fc domain results from the
differential sialylation of the Fc-linked core polysaccharide. The
cytotoxicity of IgG antibodies is reduced upon sialylation;
conversely, the anti-inflammatory activity of IVIG is enhanced. IgG
sialylation is shown to be regulated upon the induction of an
antigen-specific immune response, thus providing a novel means of
switching IgG from an innate, anti-inflammatory molecule in the
steady-state, to a adaptive, pro-inflammatory species upon
antigenic challenge. The Fc-sialylated IgGs bind to a unique
receptor on macrophages that in turn upregulates an inhibitory
Fc.gamma. receptor (Fc.gamma.R) thereby protecting against
autoantibody-mediated pathology. See, generally, Ravetch and
Nimmerjahn, J. Experim. Medicine 24(1): 11-15 (2007).
[0019] Accordingly, the instant disclosure provides an advantageous
strategy of creating and selecting IgGs with desired cytotoxic and
anti-inflammatory potential.
Definitions
[0020] Throughout the present specification and claims, the
numbering of the residues in an immunoglobulin heavy chain is that
of the EU index as in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991), which is expressly
incorporated herein by reference. The "EU index as in Kabat" refers
to the residue numbering of the human IgG1 EU antibody.
[0021] The term "native" or "parent" refers to an unmodified
polypeptide comprising an Fc amino acid sequence. The parent
polypeptide may comprise a native sequence Fc region or an Fc
region with pre-existing amino acid sequence modifications (such as
additions, deletions and/or substitutions).
[0022] The term "polypeptide" refers to any fragment of a protein
containing at least one IgG Fc region, including, without
limitation, fully functional proteins, such as, for example,
antibodies, e.g., IgG antibodies.
[0023] The term "Fc region" is used to define a C-terminal region
of an immunoglobulin heavy chain. The "Fc region" may be a native
sequence Fc region or a variant Fc region. Although the boundaries
of the Fc region of an immunoglobulin heavy chain might vary, the
human IgG heavy chain Fc region is usually defined to stretch from
an amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof.
[0024] The "CH2 domain" of a human IgG Fc region (also referred to
as "C.gamma.2" domain) usually extends from about amino acid 231 to
about amino acid 340. The CH2 domain is unique in that it is not
closely paired with another domain. Rather, two N-linked branched
carbohydrate chains are interposed between the two CH2 domains of
an intact native IgG molecule. It has been speculated that the
carbohydrate may provide a substitute for the domain-domain pairing
and help stabilize the CH2 domain (Burton, Mol Immunol, 22, 161-206
(1985), which is incorporated herein by reference).
[0025] The "CH3 domain" comprises the stretch of residues
C-terminal to a CH2 domain in an Fc region (i.e., from about amino
acid residue 341 to about amino acid residue 447 of an IgG).
[0026] The term "hinge region" is generally defined as stretching
from Glu216 to Pro230 of human IgG1 (Burton (1985). Hinge regions
of other IgG isotypes may be aligned with the IgG1 sequence by
placing the first and last cysteine residues forming inter-heavy
chain S--S bonds in the same positions.
[0027] The term "binding domain" refers to the region of a
polypeptide that binds to another molecule. In the case of an FcR,
the binding domain can comprise a portion of a polypeptide chain
thereof (e.g., the .alpha. chain thereof) which is responsible for
binding an Fc region. One exemplary binding domain is the
extracellular domain of an FcR chain.
[0028] A "functional Fc region" possesses at least a partial
"effector function" of a native sequence Fc region. Exemplary
"effector functions" include C1q binding; complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor; BCR), etc. Such effector
functions generally require the Fc region to be combined with a
binding domain (e.g., an antibody variable domain) and can be
assessed using various assays as herein disclosed, for example.
[0029] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. A "variant Fc region" as appreciated by one of ordinary
skill in the art comprises an amino acid sequence which differs
from that of a native sequence Fc region by virtue of at least one
"amino acid modification." Preferably, the variant Fc region has at
least one amino acid substitution compared to a native sequence Fc
region or to the Fc region of a parent polypeptide, e.g., from
about one to about ten amino acid substitutions, and preferably
from about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide, and more preferably at least about
90% homology therewith, more preferably at least about 95% homology
therewith, even more preferably, at least about 99% homology
therewith.
[0030] The term "altered glycosylation" refers to a polypeptide, as
defined above, be it native or modified, in which the carbohydrate
addition to the heavy chain constant region is manipulated to
either increase or decrease specific sugar components. For example,
polypeptides, such as, for example, antibodies, prepared in
specific cell lines, such as, for example, Lec2 or Lec3, may be
deficient in the attachment of sugar moieties such as fucose and
sialic acid.
[0031] The terms "Fc receptor" or "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. In one
embodiment of the invention, FcR is a native sequence human FcR. In
another embodiment, FcR, including human FcR, binds an IgG antibody
(a gamma receptor) and includes receptors of the Fc.gamma.RI,
Fc.gamma.RII, and Fc.gamma.RIII subclasses, including allelic
variants and alternatively spliced forms of these receptors.
Fc.gamma.RII receptors include Fc.gamma.RIIA (an "activating
receptor") and Fc.gamma.RIIB (an "inhibiting receptor"), which have
similar amino acid sequences that differ primarily in the
cytoplasmic domains thereof. Activating receptor Fc.gamma.RIIA
contains an immunoreceptor tyrosine-based activation motif (ITAM)
in its cytoplasmic domain. Inhibiting receptor Fc.gamma.RIIB
contains an immunoreceptor tyrosine-based inhibition motif (ITIM)
in its cytoplasmic domain (see review in Daron, Annu Rev Immunol,
15, 203-234 (1997); FcRs are reviewed in Ravetch and Kinet, Annu
Rev Immunol, 9, 457-92 (1991); Capel et al., Immunomethods, 4,
25-34 (1994); and de Haas et al., J Lab Clin Med, 126, 330-41
(1995), Nimmerjahn and Ravetch 2006, Ravetch Fc Receptors in
Fundemental Immunology, ed William Paul 5.sup.th Ed. each of which
is incorporated herein by reference).
[0032] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to an in vitro or in vivo cell-mediated reaction in which
cytotoxic cells that express FcRs (e.g., monocytic cells such as
natural killer (NK) cells and macrophages) recognize bound antibody
on a target cell and subsequently cause lysis of the target cell.
In principle, any effector cell with an activating Fc.gamma.R can
be triggered to mediate ADCC. One such cell, the NK cell, expresses
Fc.gamma.RIII only, whereas monocytes, depending on their state of
activation, localization, or differentiation, can express
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Ravetch and Bolland, Annu Rev
Immunol, (2001), which is incorporated herein by reference.
[0033] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least one type of an activating Fc receptor, such as,
for example, Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, and neutrophils, with PBMCs and NK cells being
preferred. The effector cells may be isolated from a native source
thereof, e.g., from blood or PBMCs as described herein.
[0034] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired biological activity.
[0035] The phrase "sialic acid content" of an antibody refers both
to the total number of sialic acid residues on an Fc region of a
heavy chain of an antibody and to the ratio of sialylated
antibodies to asialylated antibodies in an unpurified antibody
preparation, unless the phrase is in a context clearly suggesting
that another meaning is intended.
[0036] "Antibody fragments", as defined for the purpose of the
present invention, comprise a portion of an intact antibody,
generally including the antigen binding or variable region of the
intact antibody or the Fc region of an antibody which retains FcR
binding capability. Examples of antibody fragments include linear
antibodies; single-chain antibody molecules; and multispecific
antibodies formed from antibody fragments. The antibody fragments
preferably retain at least part of the hinge and optionally the CH1
region of an IgG heavy chain. More preferably, the antibody
fragments retain the entire constant region of an IgG heavy chain,
and include an IgG light chain.
[0037] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler and Milstein, Nature, 256, 495-497 (1975), which is
incorporated herein by reference, or may be made by recombinant DNA
methods (see, e.g., U.S. Pat. No. 4,816,567, which is incorporated
herein by reference). The monoclonal antibodies may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352, 624-628 (1991) and Marks
et al., J Mol Biol, 222, 581-597 (1991), for example, each of which
is incorporated herein by reference.
[0038] In other embodiments of the invention, the polypeptide
containing at least one IgG Fc region may be fused with other
protein fragments, including, without limitation, whole proteins. A
person of ordinary skill in the art will undoubtedly appreciate
that many proteins may be fused with the polypeptide of the present
invention, including, without limitation, other immunoglobulins,
especially, immunoglobulins lacking their respective Fc regions.
Alternatively, other biologically active proteins or fragments
thereof may be fused with the polypeptide of the present invention,
as described, for example, in the U.S. Pat. No. 6,660,843, which is
incorporated herein by reference. This embodiment is especially
advantageous for delivery of such biologically active proteins or
fragments thereof to cells expressing Fc receptors. Further,
different markers, such as, for example, GST tag or green
fluorescent protein, or GFP, may be used.
[0039] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (see U.S. Pat. No. 4,816,567; Morrison
et al., Proc Natl Acad Sci USA, 81, 6851-6855 (1984); Neuberger et
al., Nature, 312, 604-608 (1984); Takeda et al., Nature, 314,
452-454 (1985); International Patent Application No.
PCT/GB85/00392, each of which is incorporated herein by
reference).
[0040] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
residues are those of a human immunoglobulin sequence. The
humanized antibody optionally also will comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin. For further details, see Jones et al.,
Nature, 321, 522-525 (1986); Riechmann et al., Nature, 332, 323-329
(1988); Presta, Curr Op Struct Biol, 2, 593-596 (1992); U.S. Pat.
No. 5,225,539, each of which is incorporated herein by
reference.
[0041] The polypeptides containing at least one IgG Fc region
include those in which specific amino acid substitutions, additions
or deletions are introduced into a parental sequence through the
use of recombinant DNA techniques to modify the genes encoding the
heavy chain constant region. The introduction of these
modifications follows well-established techniques of molecular
biology, as described in manuals such as Molecular Cloning
(Sambrook and Russel, (2001)). In addition, the polypeptides with
at least one Fc region will include those polypeptides which have
been selected to contain specific carbohydrate modifications,
obtained either by expression in cell lines known for their
glycosylation specificity (Stanley P., et al., Glycobiology, 6,
695-9 (1996); Weikert S., et al., Nature Biotechnology, 17,
1116-1121 (1999); Andresen D C and Krummen L., Current Opinion in
Biotechnology, 13, 117-123 (2002)) or by enrichment or depletion on
specific lectins or by enzymatic treatment (Hirabayashi et al., J
Chromatogr B Analyt Technol Biomed Life Sci, 771, 67-87 (2002);
Robertson and Kennedy, Bioseparation, 6, 1-15 (1996)). It is known
in the art that quality and extent of antibody glycosylation will
differ depending on the cell type and culture condition employed.
(For example, Patel et al., Biochem J, 285, 839-845 (1992)) have
reported that the content of sialic acid in antibody linked sugar
side chains differs significantly if antibodies were produced as
ascites or in serum-free or serum containing culture media.
Moreover, Kunkel et al., Biotechnol Prog, 16, 462-470 (2000) have
shown that the use of different bioreactors for cell growth and the
amount of dissolved oxygen in the medium influenced the amount of
galactose and sialic acid in antibody linked sugar moieties. These
studies, however, did not address how varying levels of sialic acid
residues influence antibody activity in vivo.
[0042] Host Expression Systems
[0043] The polypeptide of the present invention can be expressed in
a host expression systems, i.e., host cells, capable of N-linked
glycosylation. Typically, such host expression systems may comprise
bacterial, fungal, plant, vertebrate or invertebrate expression
systems. In one embodiment the host cell is a mammalian cell, such
as a Chinese hamster ovary (CHO) cell line, (e.g. CHO-K1; ATCC
CCL-61), Green Monkey cell line (COS) (e.g. COS 1 (ATCC CRL-1650),
COS 7 (ATCC CRL-1651)); mouse cell (e.g. NS/0), Baby Hamster Kidney
(BHK) cell line (e.g. ATCC CRL-1632 or ATCC CCL-10), or human cell
(e.g. HEK 293 (ATCC CRL-1573)), or any other suitable cell line,
e.g., available from public depositories such as the American Type
Culture Collection, Rockville, Md. Further, an insect cell line,
such as a Lepidoptora cell line, e.g. Sf9, a plant cell line, a
fungal cell line, e.g., yeast such as, for example, Saccharomyces
cerevisiae, Pichia pastoris, Hansenula spp., or a bacterial
expression system based on Bacillus, such as B. subtilis, or
Eschericiae coli can be used. It will be appreciated by one of
ordinary skill in the art that in some cases modifications to host
cells may be required to insure that N-linked glycosylation and
glycan maturation occur to result in a complex, biantennary sugar
as typically found on the Fc domain of human IgG.
[0044] Therapeutic Formulations
[0045] Therapeutic formulations comprising the polypeptides
containing at least one IgG Fc region can be prepared for storage
by mixing the polypeptides of the present invention having the
desired degree of purity with optional physiologically acceptable
carriers, excipients or stabilizers (see, e.g., Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the
form of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and include buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenyl, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptide;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0046] The formulations herein may also contain more than one
active compound as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0047] The active ingredients may also be entrapped in a
microcapsule prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0048] In preferred embodiments, the formulations to be used for in
vivo administration are sterile. The formulations of the instant
invention can be easily sterilized, for example, by filtration
through sterile filtration membranes.
[0049] Sustained-release preparations may also be prepared.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
modified antibody, which matrices are in the form of shaped
articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (see, e.g., U.S. Pat. No. 3,773,919), copolymers of
L-glutamic acid and y ethyl-L-glutamate, non-degradable
ethylene-vinyl acetate, degradable lactic acid-glycolic acid
copolymers such as the LUPRON DEPOT.TM. (injectable microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide
acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such
as ethylene-vinyl acetate and lactic acid-glycolic acid enable
release of molecules for over 100 days, certain hydrogels release
proteins for shorter time periods. When encapsulated antibodies
remain in the body for a long time, they may denature or aggregate
as a result of exposure to moisture at 37.degree. C., resulting in
a loss of biological activity and possible changes in
immunogenicity. Rational strategies can be devised for
stabilization depending on the mechanism involved. For example, if
the aggregation mechanism is discovered to be intermolecular S--S
bond formation through thio-disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from
acidic solutions, controlling moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
[0050] Creation of sialylated polypeptides containing at least one
IgG Fc region.
[0051] The polypeptides of the present invention can be further
purified or modified so that they have an increased amount of
sialic acid compared to unmodified and/or unpurified antibodies.
Multiple methods exist to reach this objective. In one method, the
source of unpurified polypeptides, such as, for example, IVIG, is
passed through the column having lectin, which is known to bind
sialic acid. A person of the ordinary skill in the art will
appreciate that different lectins display different affinities for
.alpha.2,6 versus .alpha.2,3 linkages between galactose and sialic
acid. Thus, selecting a specific lectin will allow enrichment of
antibodies with the desired type of linkage between the sialic acid
and the galactose. In one embodiment, the lectin is isolated from
Sambuccus nigra. A person of the ordinary skill in the art will
appreciate that the Sambuccus nigra agglutinin (SNA) is specific
for sialic acids linked to galactose or N-acetylgalactosamine by
.alpha.(2-6) linkages. Shibuya et al, J. Biol. Chem., 262:
1596-1601 (1987). In contrast, the Maakia amurensis ("MAA") lectin
binds to sialic acid linked to galactose by .alpha.(2-3) linkages.
Wang et al, J Biol Chem., 263: 4576-4585 (1988).
[0052] Thus, a fraction of the polypeptides containing at least one
IgG Fc region having a desired linkage between the galactose and
the sialic acid will be retained in the column while a fraction
lacking such linkage will pass through. The sialylated fraction of
the polypeptides containing at least one IgG Fc region can be
eluted by another wash with a different stringency conditions.
Thus, it is possible to obtain a preparation of the polypeptide of
the present invention wherein the content of sialic acid is
increased compared to the normal content. Further, one may employ
an enzymatic reaction with a sialyltransferase and a donor of
sialic acid as described, for example, in the U.S. Pat. No.
20060030521.
[0053] Suitable non-limiting examples of sialyltransferase enzymes
useful in the claimed methods are ST3Gal III, which is also
referred to as .alpha.-(2,3)sialyltransferase (EC 2.4.99.6), and
.alpha.-(2,6)sialyltransferase (EC 2.4.99.1).
[0054] Alpha-(2,3)sialyltransferase catalyzes the transfer of
sialic acid to the Gal of a Gal-.beta.-1,3GlcNAc or
Gal-.beta.-1,4GlcNAc glycoside (see, e.g., Wen et al., J. Biol.
Chem. 267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem.
256: 3159 (1991)) and is responsible for sialylation of
asparagine-linked oligosaccharides in glycopeptides. The sialic
acid is linked to a Gal with the formation of an .alpha.-linkage
between the two saccharides. Bonding (linkage) between the
saccharides is between the 2-position of NeuAc and the 3-position
of Gal. This particular enzyme can be isolated from rat liver
(Weinstein et al., J. Biol. Chem. 257: 13845 (1982)); the human
cDNA (Sasaki et al. (1993) J. Biol. Chem. 268: 22782-22787;
Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-1401) and
genomic (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) DNA
sequences are known, facilitating production of this enzyme by
recombinant expression.
[0055] Activity of .alpha.-(2,6)sialyltransferase results in
6-sialylated oligosaccharides, including 6-sialylated galactose.
The name ".alpha.-(2,6)sialyltransferase" refers to the family of
sialyltransferases attaching sialic acid to the sixth atom of the
acceptor polysaccharide. Different forms of
a-(2,6)sialyltransferase can be isolated from different tissues.
For example, one specific form of this enzyme, ST6Gal II, can be
isolated from brain and fetal tissues. Krzewinski-Recchi et al.,
Eur. J. Biochem. 270, 950 (2003).
[0056] In addition, a person of average skill in the art will
appreciate that cell culture conditions can be manipulated to
change the sialylation rate. For example, to increase the sialic
acid content, production rate is decreased and osmolality is
generally maintained within a lower margin suitable for the
particular host cell being cultured. Osmolality in the range from
about 250 mOsm to about 450 mOsm is appropriate for increased
sialic acid content. This and other suitable cell culture
conditions are described in, e.g., U.S. Pat. No. 6,656,466. Patel
et al., Biochem J, 285, 839-845 (1992) have reported that the
content of sialic acid in antibody linked sugar side chains differs
significantly if antibodies were produced as ascites or in
serum-free or serum containing culture media. Moreover, Kunkel et
al., Biotechnol. Prog., 16, 462-470 (2000) have shown that the use
of different bioreactors for cell growth and the amount of
dissolved oxygen in the medium influenced the amount of galactose
and sialic acid in antibody linked sugar moieties.
[0057] In another embodiment, host cells, such as, for example,
immortalized human embryonic retina cells, may be modified by
introducing a nucleic acid encoding a sialyltransferase such as,
for example, an .alpha.-2,3-sialyltransferase or an
.alpha.-2,6-sialyltransferase, operably linked to a promoter, such
as, for example, a CMV promoter. The .alpha.-2,3-sialyltransferase
may be the human .alpha.-2,3-sialyltransferase, known as SIAT4C or
STZ (GenBank accession number L23767), and described, for example,
in the U.S. Pat. No. 20050181359.
[0058] The nucleic acid encoding the sialyltransferase may be
introduced into the host cell by any method known to a person of
ordinary skill in the art. Suitable methods of introducing
exogenous nucleic acid sequences are also described in Sambrook and
Russel, Molecular Cloning: A Laboratory Manual (3.sup.rd Edition),
Cold Spring Harbor Press, N Y, 2000. These methods include, without
limitation, physical transfer techniques, such as, for example,
microinjection or electroporation; transfections, such as, for
example, calcium phosphate transfections; membrane fusion transfer,
using, for example, liposomes; and viral transfer, such as, for
example, the transfer using DNA or retroviral vectors.
[0059] The polypeptide containing at least one IgG Fc region may be
recovered from the culture supernatant and can be subjected to one
or more purification steps, such as, for example, ion-exchange or
affinity chromatography, if desired. Suitable methods of
purification will be apparent to a person of ordinary skill in the
art.
[0060] A person of ordinary skill in the art will appreciate that
different combinations of sialylation methods, disclosed above, can
lead to production of the polypeptides containing at least one IgG
Fc region with an extremely high level of sialylation. For example,
one can express the polypeptide containing at least one IgG Fc
region in the host cells overexpressing sialyltransferase, as
described above, and then further enrich the sialylated fraction of
these polypeptides by, for example, sialylating these polypeptides
in an enzymatic reaction followed by an affinity chromatography
using lectin-containing columns. Similarly, an enzymatic reaction
followed by affinity chromatography may be used for IVIG source of
the polypeptides containing at least one IgG Fc region.
[0061] To examine the extent of glycosylation on the polypeptides
containing at least one IgG Fc region, these polypeptides can be
purified and analyzed in SDS-PAGE under reducing conditions. The
glycosylzation can be determined by reacting the isolated
polypeptides with specific lectins, or, alternatively as would be
appreciated by one of ordinary skill in the art, one can use HPLC
followed by mass spectrometry to identify the glycoforms. (Wormald,
M R et al., Biochem 36:1370 (1997).
[0062] To describe the instant invention in more details, several
non-limiting illustrative examples are given below.
Examples
Example 1. IVIG with Increased Sialic Acid Content Exhibits
Decreased Cytotoxicity
[0063] To determine if specific glycoforms of IgG are involved in
modulating the effector functions of antibodies the role of
specific, Asn.sup.297-linked carbohydrates in mediating the
cytotoxicity of defined IgG monoclonal antibodies was explored. The
anti-platelet antibodies, derived from the 6A6 hybridoma, expressed
as either an IgG1, 2a or 2b switch variant in 293 cells as
previously described (6), were analyzed by mass spectroscopy to
determine their specific carbohydrate composition and structure.
These antibodies contain minimal sialic acid residues. Enrichment
of the sialic acid containing species by Sambucus nigra lectin
affinity chromatography yielded antibodies enriched 60-80 fold in
sialic acid content. Comparison of the ability of sialylated and
asialylated 6A6-IgG1 and 2b antibodies to mediate platelet
clearance revealed an inverse correlation between sialylation and
in vivo activity. Sialylation of 6A6 IgG antibodies resulted in a
40-80% reduction in biological activity.
[0064] To determine the mechanism of this reduction in activity
surface plasmon resonance binding was performed on these antibodies
for each of the mouse FcYRs and to its cognate antigen.
[0065] Surface plasmon resonance analysis was performed as
described in Nimmerjahn and Ravetch, Science 310, 1510 (2005).
Briefly, 6A6 antibody variants containing high or low levels of
sialic acid residues in their sugar side chains were immobilized on
the surface of CM5 sensor chips. Soluble Fcy-receptors were
injected at different concentrations through flow cells at room
temperature in HBS-EP running buffer (10 mM Hepes, pH 7.4, 150 mM
NaCl, 3.4 mM EDTA, and 0.005% surfactant P20) at a flow rate of 30
.mu.l/min. Soluble Fc-receptors were injected for 3 minutes and
dissociation of bound molecules was observed for 7 minutes.
Background binding to control flow cells was subtracted
automatically. Control experiments were performed to exclude mass
transport limitations. Affinity constants were derived from
sensorgram data using simultaneous fitting to the association and
dissociation phases and global fitting to all curves in the set. A
1:1 Langmuir binding model closely fitted the observed sensorgram
data and was used in all experiments.
[0066] A 5-10 fold reduction in binding affinity was observed for
the sialylated forms of these antibodies to their respective
activating FcyRs as compared to their asialylated counterparts,
while no differences in binding affinity for the antigen were
observed. Since IgG2b binds with a higher affinity to its
activation receptor, FcyRIV, when compared to IgG1 binding to its
activation receptor FcyRIII, the effect of sialylation was to
generate a binding affinity for IgG2b for its activation receptor
FcyRIV that was comparable to that of asialylated IgG1 binding to
its activation receptor FcYRIII. This effect of this quantitative
difference in activation receptor binding resulted in sialylated
IgG2b displaying an in vivo activity comparable to that of
asialylated IgG1. Similarly, sialylation of IgG1 reduces its
already low binding affinity for its activation receptor FcyRIII by
a factor of 7 thereby generating a physiologically inactive
antibody. Thus, sialylation of the Asn.sup.297 linked glycan
structure of IgG resulted in reduced binding affinities to the
subclass-restricted activation FcyRs and thus reduced their in vivo
cytotoxicity.
[0067] To determine the generality of the observation that
sialylation of the N-linked glycan of IgG was involved in
modulating its in vivo inflammatory activity, we next examined the
role of N-linked glycans on the anti-inflammatory activity of IVIG.
This purified IgG fraction obtained from the pooled serum of
5-10,000 donors, when administered intravenously at high doses (1-2
g/kg), is a widely used therapeutic for the treatment of
inflammatory diseases. Dwyer, N. Engl. J. Med. 326, 107 (1992).
This anti-inflammatory activity is a property of the Fc fragment
and is protective in murine models of ITP, RA and nephrotoxic
nephritis. Imbach et al., Lancet 1, 1228 (1981), Samuelsson et al.,
Science 291, 484 (2001), Bruhns et al., Immunity 18, 573 (2003),
Kaneko et al., J. Exp. Med. in press (2006).
[0068] A common mechanism for this anti-inflammatory activity was
proposed involving the induction of surface expression of the
inhibitory FcyRIIB molecule on effector macrophages, thereby
raising the threshold required for cytotoxic IgG antibodies or
immune complexes to induce effector cell responses by activation
FcyR triggering. Nimmerjahn and Ravetch, Immunity 24, 19
(2006).
Example 2. Asialylation of Ivig Decreases the Anti-Inflammatory
Effect of Ivig in Mouse Arthritis Model
[0069] Mice
[0070] C57BL/6 and NOD mice were purchased from the Jackson
Laboratory (Bar Harbor, Me.). FcyRIIB.sup.-/- mice were generated
in the inventors' laboratory and backcrossed for 12 generations to
the C57BL/6 background. KRN TCR transgenic mice on a C57BU6
background (K/B) were gifts from D. Mathis and C. Benoist (Harvard
Medical School, Boston, Mass.) and were bred to NOD mice to
generate K/BxN mice. Female mice at 8-10 weeks of age were used for
all experiments and maintained at the Rockefeller University animal
facility. All experiments were done in compliance with federal laws
and institutional guidelines and have been approved by the
Rockefeller University (New York, N.Y.).
[0071] Antibodies and Soluble Fc Receptors
[0072] 6A6 antibody switch variants were produced by transient
transfection of 293T cells followed by purification via protein G
as described. Nimmerjahn and Ravetch, Science 310, 1510 (2005).
Sialic acid rich antibody variants were isolated from these
antibody preparations by lectin affinity chromatography with
Sambucus nigra agglutinin (SNA) agarose (Vector Laboratories,
Burlingame, Calif.). Enrichment for sialic acid content was
verified by lectin blotting (see below). Human intravenous immune
globulin (IVIG, 5% in 10% maltose, chromatography purified) was
purchased from Octapharma (Hemdon, Va.). Digestion of human IVIG
was performed as described. Kaneko Y. et al., Exp. Med. in press
(2006). Briefly, IVIG was digested by 0.5 mg/ml papain for 1 hr at
37.degree. C., and stopped by the addition of 2.5 mg/ml
iodoasetamide. Fab and Fc resulting fragments were separated from
non-digested IVIG on a HiPrep 26/60 S-200HR column (GE Healthcare,
Piscataway, N.J.), followed by purification of Fc and Fab fragments
with a Protein G column (GE Healthcare) and a Protein L column
(Pierce, Rockford, Ill.). Fragment purity was checked by
immunoblotting using anti-human IgG Fab or Fc-specific antibodies.
(Jackson ImmunoResearch, West Grove, Pa.). Purity was judged to be
greater than 99%. The F4/80 antibody was from Serotec (Oxford, UK).
The Ly 17.2 antibody was from Caltag (Burlingame, Calif.). Sheep
anti-glomerular basement membrane (GBM) antiserum (nephrotoxic
serum, NTS) was a gift from M. P. Madaio (University of
Pennsylvania, Philadelphia, Pa.). Soluble Fc receptors containing a
C-terminal hexa-hisitidine tag were generated by transient
transfection of 293T cells and purified from cell culture
supernatants with Ni-NTA agarose as suggested by the manufacturer
(Qiagen).
[0073] IVIG was treated with neuraminidase and the composition and
structure of the resulting preparation was analyzed by mass
spectroscopy. No detectable sialic acid containing glycans remained
after neuraminidase treatment. These IgG preparations were then
tested for their ability to protect mice from joint inflammation
induced by passive transfer of KxN serum, an IgG 1 immune
complex-mediated inflammatory disease model. De-sialylation with
neuraminidase abrogated the protective effect of the IVIG
preparation in the KXN serum induced arthritis model. This loss of
activity was not the result of reduced serum half-life of the
asialylated IgG preparations or the result of changes to the
monomeric composition or structural integrity of the IgG. Removal
of all glycans with PNGase had a similar effect and abrogated the
protective effect of IVIG in vivo.
Example 3. Ivig Fraction with Enriched Sialic Acid Content
Decreases Inflammation in Mouse Arthritis Model
[0074] Preparation of IVIG with an Increased Content of Sialic
Acid
[0075] Since sialic acid appeared to be required for the
anti-inflammatory activity of IVIG, the basis for the high dose
requirement (1 g/kg) for this anti-inflammatory activity could be
the limiting concentration of sialylated IgG in the total IVIG
preparation. The IVIG was fractionated on an SNA-lectin affinity
column to obtain IgG molecules enriched for sialic acid modified
glycan structures.
[0076] These sialic acid enriched fractions were tested for
protective effects in the KxN serum transfer arthritis model as
compared to unfractionated IVIG. A 10 fold enhancement in
protection was observed for the SNA-binding fraction, such that
equivalent protection was obtained at 0.1 g/kg of SNA-enriched IVIG
as compared to 1 g/kg of unfractionated IVIG. The serum half-life
and IgG subclass distribution of the SNA enriched fraction was
equivalent to that of unfractionated IVIG. The effect of
sialylation was specific to IgG; sialylated N-linked glycoproteins
such as fetuin or transferrin with similar biantennary, complex
carbohydrate structures had no statistically significant
anti-inflammatory activity at equivalent molar concentrations of
IgG. Finally, the mechanism of protection of the sialylated IVIG
preparation was similar to unfractionated IVIG in that it was
dependent on FcyRIIB expression and resulted in the increased
expression of this inhibitory receptor on effector macrophages.
Example 4. The Increased Anti-Inflammatory Response of IVIG with
Increased Sialic Acid Content is Mediated by Sialylation of the
N-Linked Glycan on the FC Domain
[0077] Since the polyclonal IgG in IVIG may also contain O and N
linked glycans on the light chains or heavy chain variable domains
that can be sialylated, we confirmed that the increase in
anti-inflammatory activity of the SNA-enriched IgG preparation
resulted from increased sialylation of the N-linked glycosylation
site on the Fc. Fc fragments were generated from unfractionated and
SNA fractionated IVIG and tested for their in vivo activity. As
observed for intact IgG, SNA-purified Fc fragments were enhanced
for their protective effect in vivo when compared to Fc fragments
generated from unfractionated IVIG. In contrast, Fab fragments
displayed no anti-inflammatory activity in this in vivo assay.
Thus, the high dose requirement for the anti-inflammatory activity
of IVIG can be attributed to the minor contributions of sialylated
IgG present in the total preparation. Enrichment of these fractions
by sialic acid binding lectin chromatography consequently increased
the anti-inflammatory activity.
[0078] These results using passive immunization of IgG antibodies
indicated that the ability of IgG to switch from a pro-inflammatory
to an anti-inflammatory species is influenced by the degree of
sialylation of the N-linked glycan on the Fc domain.
Example 5. Increase of Anti-Inflammatory Activity, Mediated by
Sialylation of IgG, Occurs During an Active Immune Response
[0079] Murine Model for Goodpasture's Disease
[0080] In this model, mice are first sensitized with sheep IgG
together with adjuvant and four days later injected with a sheep
anti-mouse glomerular basement membrane preparation (nephrotoxic
serum, NTS). Briefly, mice were pre-immunized intraperitoneally
with 200 .mu.g of sheep IgG (Serotec) in CFA, followed by
intravenous injection of 2.5 .mu.l of NTS serum per gram of body
weight four days later. Blood was collected from non-treated
control mice four days after the anti-GBM anti-serum injection, and
serum IgG was purified by Protein G (GE Healthcare, Princeton,
N.J.) and sepharose-bound sheep IgG column, generated by covalently
coupling sheep IgG on NHS-activated sepharose column (GE
Healthcare, Princeton, N.J.), affinity chromatography.
[0081] Pre-sensitization followed by treatment with NTS induces
mouse IgG2b anti-sheep IgG antibodies (NTN immunized). Kaneko Y. et
al., Exp. Med., 203:789 (2006). Mouse IgG2b antibodies are
deposited in the glomerulus together with the NTS antibodies and
result in an acute and fulminant inflammatory response by the IgG2b
mediated activation of FcyRIV on infiltrating macrophages. In the
absence of pre-sensitization inflammation is not observed,
indicating that the mouse IgG2b anti-sheep IgG antibodies are the
mediators of the inflammatory response.
[0082] To determine if active immunization resulting in
pro-inflammatory IgG is associated with a change in sialylation,
serum IgG and IgM from preimmune and NTS immunized mice were
characterized for sialic acid content by SNA lectin binding. Total
IgG sialylation was reduced on average by 40% in immunized mice as
compared to the unimmunized controls. The effect was specific for
IgG; sialylation of IgM was equivalent pre and post immunization.
This difference in sialylation was more pronounced when the sheep
specific IgG fraction from mouse serum was analyzed, showing a
50-60% reduction in sialylation compared to preimmune IgG.
[0083] These results were confirmed by MALDI-TOF-MS analysis.
Monosaccharide composition analysis was performed by UCSD
Glycotechnology Core Resource (San Diego, Calif.). Glycoprotein
samples were denatured with SDS and 2-mercaptoethanol, and digested
with PNGase F. The released mixed N-glycans were purified by
reversed-phase HPLC and solid-phase extraction, and then exposed
hydroxyl groups of the N-glycans were methylated. The resulting
derivatized saccharides were purified again by reversed-phase HPLC
and subject to MALDI-TOF-MS.
[0084] The analysis of the pre and post immunization IgGs confirmed
that the changes in the N-glycan structure were specific to the
terminal sialic acids moieties. The mouse IgG2b anti-sheep
antibodies that were deposited in the glomeruli, previously shown
to be responsible for engagement of the FcyRIV bearing,
infiltrating macrophages displayed reduced sialic acid content as
compared to the pre-immunized controls.
Example 6. Analysis of Linkages Between Sialic Acid and Galactose
in IVIG
[0085] Sequential Maldi-Tof analysis of SNA.sup.+ (Sambuccus Nigra
Agglutinin) IVIG Fc linkages was performed to determine the
structure of the sialylated IgG Fc fraction that was protective in
the ITP, RA and nephrotoxic nephritis models described above.
Glycan peaks generated in Maldi-TOF were isolated, further
fractionated, and reanalyzed until galactose-sialic acid structures
were obtained. The footprint histogram of the enriched
galactose-sialic acid structures with in vivo anti-inflammatory
activity (FIG. 1A) were compared to histograms from sialic acid
linkage standards, .alpha.2-3 sialyllactose (FIG. 1B) and
.alpha.2-6 sialyllactose (FIG. 1C). The signature peaks of the
standards are identified by arrows, shown by arrows for .alpha.2-3
(FIG. 1B) or .alpha.2-6 (FIGS. 1A and 1C), respectively, and
compared to the peaks obtained from the sample.
Example 7. Enrichment of IVIG Fc Fragments in .alpha.2,6 Linkages
by In Vitro Glycosylation Improves Anti-Inflammatory Properties of
IVIG
[0086] As shown in FIG. 2A, glycan Maldi-Tof MS analysis of IVIG Fc
fragments showed structures ending in no galactose (peak G0), one
galactose (peak G1), two galactose (peak G2), or in sialic acid
(indicated by a bracket entitled "Terminal sialic acid"). To
determine the in vivo activity of 2,3 or 2,6 sialylated IgG Fc,
samples were treated with sialidase, followed by galactose
transferase to convert the GO (no galactose) and G1
(single_galactose) to G2 (fully galactosylated) to increase
potential sialylation sites. As shown in FIG. 2B
hypergalactosylation was verified by comparing relative band
intensity ratios of terminal galactose as measured by ECL and
coomassie loading controls. In vitro sialylation was performed
(FIG. 2C) using either .alpha. 2-6 sialyltransferase ("ST6Gal") or
a 2-3 sialyltransferase ("ST3Gal") and confirmed by lectin blotting
for .alpha. 2-6 linkages with SNA (top) or .alpha.2-3 linkages with
ECL (middle) and coomassie (bottom). To evaluate the ability of in
vitro sialylated Fc to inhibit inflammation (FIG. 2D) mice received
either 0.66 mg of .alpha. 2-6 sialylated Fcs (black triangles) or
0.66 mg .alpha. 2-3 sialylated Fcs (red triangles). 1 hour later,
0.2 ml of K/BxN sera was administered, and the swelling of footpads
(clinical score) was monitored over the next seven days.
Anti-inflammatory activity was observed for the 2,6 sialylated IgG
Fc fragments but not for the 2,3 sialylated molecules. These
results are consistent with the data shown above and indicate that
a preferential linkage of 2,6 sialic acid-galactose is involved in
the anti-inflammatory activity of sialylated IgG.
Example 8. Removal of .alpha. 2-6 but not 2,3 Sialic Acid Linkages
Abrogates the Immunosuppressive Properties of IVIG
[0087] IVIG was treated with linkage specific sialidases (SAs), and
the digestion verified by lectin blotting (FIG. 3A). The top panel
shows positive Sambucus nigra lectin (SNA) staining for .alpha. 2-6
linkages in IVIG (left lane), and .alpha. 2-3 SA tx IVIG (center
lane), but not in .alpha. 2-3,6 SA tx IVIG (right lane). The middle
panel is a dot blot for .alpha.2-3 sialic acid linkages (MAL I),
displaying positive staining for the fetuin positive control only;
100 .mu.g protein are loaded per dot. The bottom panel shows
coomassie loading control. 10 .mu.g/lane are shown in the blot and
gel. To examine the effect of specific removal of sialic acid
moieties, mice were given 1 g/kg of IVIG preparations prior to 200
.mu.l of K/BxN sera. As shown in FIG. 3B, footpad swelling was
observed in mice administered K/BxN sera (white circles) over the
course of a week, as measured by clinical scoring. IVIG treated
mice showed minimal swelling (black triangles), as did mice treated
with .alpha.2-3 SA tx IVIG (white triangles), while mice receiving
.alpha.2-3,6 SA tx IVIG (red squares) were not protected from
footpad swelling.
[0088] All patent and non-patent publications cited in this
disclosure are incorporated herein in to the extent as if each of
those patent and non-patent publications was incorporated herein by
reference in its entirety. Further, even though the invention
herein has been described with reference to particular examples and
embodiments, it is to be understood that these examples and
embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be devised
without departing from the spirit and scope of the present
invention as defined by the following claims.
* * * * *