U.S. patent application number 15/629119 was filed with the patent office on 2017-11-09 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 | 20170320935 15/629119 |
Document ID | / |
Family ID | 38581601 |
Filed Date | 2017-11-09 |
United States Patent
Application |
20170320935 |
Kind Code |
A1 |
Ravetch; Jeffrey ; et
al. |
November 9, 2017 |
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; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Rockefeller University |
New York |
NY |
US |
|
|
Family ID: |
38581601 |
Appl. No.: |
15/629119 |
Filed: |
June 21, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14624483 |
Feb 17, 2015 |
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15629119 |
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13336199 |
Dec 23, 2011 |
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14624483 |
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12013212 |
Jan 11, 2008 |
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13336199 |
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11957015 |
Dec 14, 2007 |
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12013212 |
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PCT/US2007/072771 |
Jul 3, 2007 |
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11957015 |
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PCT/US2007/008396 |
Apr 3, 2007 |
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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 47/68 20170801;
C07K 16/18 20130101; C07K 2317/41 20130101; G01N 33/6854 20130101;
C07K 16/00 20130101; A61P 29/00 20180101; C07K 2317/76 20130101;
C12P 21/005 20130101; C07K 16/06 20130101; C07K 2317/71 20130101;
A61K 2039/505 20130101; C07K 2317/52 20130101; A61P 37/00
20180101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; G01N 33/68 20060101 G01N033/68; C12P 21/00 20060101
C12P021/00; C07K 16/06 20060101 C07K016/06; C07K 16/18 20060101
C07K016/18 |
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.-36. (canceled)
37. A method for preparing a pharmaceutical preparation comprising
a recombinant IgG antibody having increased anti-inflammatory
activity, the method comprising: expressing, in a host cell, a
recombinant IgG antibody; subjecting the expressed recombinant IgG
antibody to one or more purification steps to produce a preparation
comprising the recombinant IgG antibody; treating the composition
comprising the recombinant IgG antibody with an alpha-(2,6)
sialylatransferase and a donor of sialic acid to increase the
fraction of recombinant IgG antibody in the composition having
alpha-2,6-linked N-acetylneuraminic acid on N-glycans of the Fc
region of the recombinant IgG antibody thereby preparing a
composition enriched for sialylated recombinant IgG antibody,
wherein the enriched composition has increased anti-inflammatory
activity relative to the un-enriched composition, and processing
the composition enriched for sialylated recombinant IgG antibody to
produce a pharmaceutical preparation, wherein the processing
comprises combining the recombinant IgG antibody with a
physiologically acceptable carrier, excipient or stabilizer,
thereby preparing a pharmaceutical preparation comprising a
recombinant IgG antibody having increased anti-inflammatory
activity.
38. The method of claim 37, wherein the recombinant IgG antibody is
a monoclonal antibody.
39. The method of claim 37, wherein the recombinant IgG antibody is
chimeric antibody.
40. The method of claim 37, wherein the recombinant IgG antibody is
a humanized antibody.
41. The method of claim 37, where the composition enriched for
sialylated recombinant IgG antibody to affinity chromatography to
produce a composition further enriched for sialylated recombinant
IgG antibody.
42. The method of claim 37, wherein the host cell is a mammalian
cell.
43. The method of claim 42, wherein the mammalian cell line is a
CHO cell.
44. The method of claim 37, wherein the purification step comprises
ion-exchange chromatography or affinity chromatography.
40. The method of claim 37, wherein the composition enriched for
sialylated recombinant IgG antibody exhibits an increased
protective effect in a K/B.times.N serum-induced arthritis mouse
model relative to the un-enriched composition.
41. The method of claim 37, wherein the host cell harbors a
recombinant sialyltransferase.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of patent application
Ser. No. 14/624,483, filed on Feb. 17, 2015, which is a
continuation of patent application Ser. No. 13/336,199, filed on
Dec. 23, 2011, which is a continuation of patent application Ser.
No. 12/013,212, filed on Jan. 11, 2008, which is a continuation of
patent application Ser. No. 11/957,015, filed on Dec. 14, 2007,
which claims the benefit of continuation patent application of PCT
Patent Application Number PCT/US Serial No. 07/072771, filed on
Jul. 3, 2007 and is also a continuation-in-part of patent
application of PCT Patent Application Number PCT/US Serial No.
07/008396, filed on Apr. 3, 2007, which claims the benefit of U.S.
Provisional Patent Application No. 60/789,384, filed on Apr. 5,
2006, and U.S. Provisional Patent Application No. 60/734,196, filed
on Nov. 7, 2005, all of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The present invention relates to a 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 (GlcNAc) and
mannose (man). Further modification of the core carbohydrate
structure is observed in serum antibodies with the presence of
fucose, branching GlcNAc, 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. 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 one aspect, the isolated polypeptide is derived from a
recombinant source.
[0013] 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.
[0014] In another aspect, the invention provides a method of
modulating properties of a polypeptide comprising an Fc region
comprising altering the sialylation of the polysaccharide chain of
the Fc region.
[0015] 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 a 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 a 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 a 2,6
linkage.
[0016] In yet another embodiment the invention provides a method of
treating an inflammatory disease comprising administering to a
subject in need thereof a therapeutic composition comprising a
plurality of isolated polypeptides, each containing at least one
IgG Fc region, wherein a first portion of the respective Fc regions
comprises respective carbohydrate chains having galactose moieties
connected to respective terminal sialic acid moieties by 2,6
linkage; a dose of the therapeutic composition is smaller than a
dose of a second composition which comprises a plurality of
isolated polypeptides, each containing at least one IgG Fc region,
having a second portion of the respective Fc regions comprising
respective carbohydrate chains having galactose moieties connected
to respective terminal sialic acid moieties by 2,6 linkage; and
either the first portion is greater than the second portion,
whereby the dose of the therapeutic composition and the dose of the
second composition suppress inflammation to substantially the same
extent, or the first portion is greater than the second portion,
whereby the therapeutic composition suppresses inflammation to
substantially a greater extent than an equal dose of the second
composition.
[0017] In another aspect, the invention provides a method for
controlling the properties of an Fc-containing molecule, comprising
altering the sialylation of the oligosaccharides in the Fc region.
In different embodiments, the sialylation of the Fc region is
increased or decreased.
[0018] In another aspect, the invention provides methods of
treating diseases comprising administering to a patient in need
thereof a therapeutically effective amount of a protein comprising
Fc region with altered oligosaccharide sialylayion. In different
embodiments, the sialylation is increased or decreased. The disease
may be selected from oncology-related disorders and diseases or
conditions associated with inflammation. In different embodiments,
the disease or condition is rheumatoid arthritis or inflammatory
bowel disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1A-1C are footprint histograms of MALDI-TOF analysis
of SNA.sup.+ FC linkages, where the footprint histogram of enriched
galactose-sialic acid structures with in vivo anti-inflammatory
activity (1A) was compared to histograms from sialic acid linkage
standards, a 2-3 sialyllactose (1B) and a 2-6 sialyllactose
(1C).
[0020] FIG. 2 summarizes experiments demonstrating that enrichment
of a 2,6 linkages between sialic acid and galactose improves
anti-inflammatory properties of IVIG Fc fragments.
[0021] FIGS. 3A and 3B are a group of photographs (3A) and a
diagram (3B) demonstrating that removal of a 2,6 linkages between
sialic acid and galactose attenuates anti-inflammatory properties
of IVIG Fc fragments.
[0022] FIG. 4 demonstrates that reduced cytotoxicity does not
depend on the linkage between galactose and sialic acid.
[0023] FIGS. 5A and 5B are a group of photographs (5A) and a
diagram (5B) demonstrating that the in vivo anti-inflammatory
activity of the 2,6 sialylated IgG Fc is solely a property of the
IgG Fc glycan.
[0024] FIGS. 6A-6D illustrate the hierarchy of antibody-isotype
mediated effector functions in vivo. Shown are the B16-F10 lung
metastasis and platelet depletion models in C57BL/6 mice
(mean.+-.SEM). (A and B) Mice were injected with B16-F10 melanoma
cells followed by injection of TA99-isotype switch variants or
control antibodies (200 .mu.g per injection) on days 0, 2, 4, 7, 9
and 11. Mice were sacrificed 15 days after tumor cell injection and
the number of surface lung metastasis was evaluated. Asterisk
indicates P<0.0001; double asterisks P<0.01. (C and D) Mice
were injected with 4 .mu.g of 6A6-antibody switch variants and
platelet counts were determined at the indicated time points (C).
(D) The platelet count 4 hours after injection of the 6A6 isotype
variants is shown as the percentage of the total platelet count
before antibody injection.
[0025] FIGS. 7A and 7B illustrate that IgG2a-mediated effects are
independent of the complement cascade in vivo (mean+/-SEM). (A)
Mice deficient for complement receptor 2 (CR2-/-) or complement
component C3 or C4 (C4-/-) were injected with B16-F10 melanoma
cells and treated with 100 .mu.g of the TA99-IgG2a antibody per
injection. After 15 days animals were sacrificed and lung surface
metastasis count was determined. Asterisk indicates P<0.0001. B)
C57BL/6, .gamma.-chain-/-, CR2-/-, and C4-/- mice were injected
with 4 .mu.g of the 6A6-IgG1, -IgG2a, or IgG2b antibody isotypes
and platelet counts were determined before and hours after antibody
injection (Nimmerjahn, et al., (2005). Shown is the platelet count
4 hours after antibody injection relative to the platelet count
before injection percent. The experiments were done twice with 3-4
animals per group.
[0026] FIGS. 8A-8E illustrate the Fc.gamma. receptor dependence of
antibody isotype-mediated effector functions (A and B). Mice
deficient for the common .gamma.-chain (.gamma.-/-), or a chains of
activation Fc.gamma.-receptor I (Fc.gamma.RI-/-) or III
(Fc.gamma.RIII-/-) were injected with B16-F10 melanoma cells and
treated with 100 .mu.g of the TA99-IgG2a antibody or PBS (mock) as
described. At day 15 after tumor cell injection mice were
sacrificed, lungs were prepared (A) and the number of lung surface
metastasis was quantified (B) (mean+/-SEM). Asterisk indicates
P<0.0001. The experiment was performed twice with 5 mice per
group. (C and D) Fc.gamma.RI-/- mice were injected with B16-F10
melanoma cells and treated with the TA99-IgG2a antibody at 100
.mu.g per injection. At days 0, 2 and 4 after tumor cell injections
mice were injected with 200 .mu.g of an Fc.gamma.RIV blocking
antibody or an isotype matched control antibody. Lungs were
prepared at day 15 after tumor cell injection (C) and lung surface
metastasis were quantified (D) (mean+/-SEM). Asterisk indicates
P<0.001. (E) The indicated mouse strains were injected with 4
.mu.g of the 6A6-isotype switch variants and the platelet count was
determined 4 hours after injection of the respective antibody
variants (mean+/-SEM). To block immune complex binding to
Fc.gamma.RIV mice were injected with 200 .mu.g of an
Fc.gamma.RIV-blocking antibody (Nimmerjahn, F., et al., (2005)).
Shown is the relative platelet count 4 hours after antibody
injection in percent. Experiments were performed twice with 4-6
mice per group.
[0027] FIGS. 9A-9C illustrate differential isotype specific
negative regulation by the inhibitory receptor Fc.gamma.RIIB (A and
B). C57BL/6 wild-type or Fc.gamma.-receptor IIB deficient
(Fc.gamma.RIIB-/-) mice were injected with B16-F1O melanoma cells
and treated with TA99-IgG1 or IgG2a isotype switch variants (100
.mu.g per injection). Lungs were prepared on day 15 after tumor
cell injection. The experiment was done twice with 5 mice per
group; representative lungs are shown in (A) and the quantification
in (B). Asterisk indicates P<0.0001; double asterisk indicates
P<0.05. (C) C57BL/6 or Fc.gamma.RIIB-/- mice were injected with
2 .mu.g of the indicated 6A6-antibody isotype variants and platelet
counts were determined before and 4 hours after antibody injection.
Shown is the increase in platelet depletion for the different
antibody isotypes in Fc.gamma.RIIB-/- mice compared to wildtype
animals. Shown is one representative out of three experiments with
5 mice per group.
[0028] FIGS. 10A-10C illustrate that the enhancement of the (A/I)
ratio of modified antibodies increases their efficacy. (A) Shown is
the fold increase in association constants (K.sub.A) for the
complement component C1q and Fc.gamma.R-receptors I-IV in binding
to fucose-containing TA99-IgG1, -IgG2a and -IgG2b isotypes compared
to fucose-deficient TA99 isotype switch variants. (B and C) C57BL/6
mice were injected with B16-F10 melanoma cells and treated with
TA99-IgG2b containing fucose or TA99-IgG2b deficient in fucose;
Shields, R. L., et al., J Biol Chem 277, 26733-40 (2002); Shinkawa,
T., et al., J Biol Chem 278, 3466-73. (2003); and Niwa R., et al.,
Cancer Res 64, 2127-33. (2004)) (50 .mu.g per injection). Lungs
were prepared at day 15 after tumor cell injection. One
representative lung out of 4 animals per group (B) and the
quantification of the lung surface metastasis count is shown (C).
Asterisk indicates P<0.0001.
[0029] FIG. 11 illustrates the effect of reducing sialic acid
content on the in vivo cytotoxicity of an antibody. The effect of
sialic acid residues in Asn-297 linked sugar side chains on
antibody dependent cytotoxicity in vivo is described. Mice (n=4)
were injected intravenously with 4 .mu.g of the respective 6A6-IgG1
antibodies and platelet counts were determined before and 4 hours
after antibody injection. Shown is the platelet depletion in
percent 4 hours after injection of the antibody variants.
Abbreviations: SA, sialic acid
[0030] FIGS. 12A and 12B illustrate factors that influence
Fc-receptor dependent activities of antibody isotypes. FIG. 12A
indicates individual antibody isotypes have different affinities
for activating and inhibitory Fc receptors (see text). Red arrows
indicate preferential interactions of the indicated antibody
isotypes with cellular Fc-receptors; black arrows indicate lower
affinity interactions. In the case of IgG2a the broken red arrow
indicates that the interaction might be blocked as Fc.gamma.RI is
continuously occupied with monomeric IgG2a. The table summarizes
the actual A/I ratios based on the affinities of the individual
Fc-receptors for the respective antibody isotypes (Nimmerjahn et
al., 2005). FIG. 12B indicates the ratio of activating to
inhibitory Fc-receptors on immune cells such as DCs, macrophages
and neutrophils is regulated by exogenous factors. Cytokines like
IL-4, IL-10 or TGF-f3 upregulate Fc.gamma.RIIB thereby setting high
thresholds for cell activation, whereas inflammatory mediators
downregulate the inhibitory and upregulate the activating
Fc-receptors. For therapeutic approaches Fc.gamma.RIIB mediated
inhibition might be circumvented by using Fc.gamma.RIIB-blocking
antibodies.
DETAILED DESCRIPTION
[0031] 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). The inventors
have further surprisingly discovered that the anti-inflammatory
response depends on the nature of the linkage between galactose and
sialic acid moieties. The observation that the anti-inflammatory
activity of IVIG is dependent on a precise glycan structure on the
Fc further supports the model that the inventors have previously
advanced (Y. Kaneko, F. Nimmerjahn, J. V. Ravetch, Science 313, 670
(2006); F. Nimmerjahn, J. V. Ravetch, J Exp Med 204, 11 (2007))
that a specific lectin receptor, and not a canonical Fc receptor,
is involved in this pathway. The data underlying this invention
support a model in which binding of the 2,6 sialylated Fc to its
cognate lectin receptor expressed on a population of regulatory
myeloid cells results in the trans upregulation of the inhibitory
IgG Fc on effector macrophages, located at sites of inflammation,
such as the inflamed joint, thus raising the threshold required for
cytotoxic IgGs to engage activation FcRs and trigger inflammatory
responses (F. Nimmerjahn, J. V. Ravetch, Science 310, 1510
(2005)).
[0032] Accordingly, the instant disclosure provides an advantageous
strategy of creating and selecting IgGs with desired cytotoxic and
anti-inflammatory potential.
Definitions
[0033] 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.
[0034] 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).
[0035] The term "polypeptide" refers to any fragment of a protein
containing at least one IgG Fc region and fragments thereof,
including, without limitation, fully functional proteins, such as,
for example, antibodies, e.g., IgG antibodies. When a polypeptide
of the invention is compared to an unpurified antibody preparation,
such a preparation is typically a blood sample, serum sample,
and/or IVIG sample, derived from a mammal, e.g., a human donor. The
preparation may be unfractionated or partially fractionated but
typically comprises only about 2-4% sialylated Fc containing
proteins. Compositions of the invention enriched or formulated to
have immunosuppressive activity typically comprise at least about
5% sialylated Fc containing proteins or more (e.g., 5-10%, 10-30%,
30-50%, 50-100% or ranges or intervals thereof).
[0036] 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.
[0037] The "CH2 domain" of a human IgG Fc region (also referred to
as "Cy2" 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).
[0038] 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).
[0039] 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.
[0040] 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 a chain thereof) which is responsible for
binding an Fc region. One exemplary binding domain is the
extracellular domain of an FcR chain.
[0041] 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. The
term also includes Fc fragments provided the fragment contains at
least one amino acid residue that is glycosylated or suitable for
glycosylation as described herein.
[0042] 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.
[0043] 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.
[0044] 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 5th Ed. each of which is
incorporated herein by reference).
[0045] "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.
[0046] "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.
[0047] 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.
[0048] 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. As mentioned above in the
BACKGROUND section, IgG contains a single, N-linked glycan at
Asn.sup.297 in the CH2 domain on each of its two heavy chains. The
N-linked glycan structure can end with no galactose, one galactose,
or two galactoses, referred as G0, G1, or G2. In a sialylated
antibody, a sialic acid (S) is linked to a galactose (G) with the
formation of an .alpha.-linkage between the two saccharides. Once
both galactoses are linked to sialic acids, the sialylated antibody
has a glycoform with 2 galactoses (G2) linked with 2 sialic acids
(S2), i.e., a G2S2 sialylated glycoform.
[0049] "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.
[0050] 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.
[0051] 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.
[0052] 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).
[0053] "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.
[0054] The polypeptides of the instant invention may be
recombinantly produced, for example, from a cDNA, such as, for
example SEQ ID NO: 1. The polypeptides of different embodiments
include Fc regions or functional fragments thereof.
[0055] 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.
Host Expression Systems
[0056] 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 293T (ATCC CRL-11268)), 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.
Therapeutic Formulations
[0057] 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).
[0058] 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.
[0059] 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).
[0060] 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.
[0061] 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 .gamma. 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.
Creation of Sialylated Polypeptides Containing at Least One IgG Fc
Region.
[0062] 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).
[0063] 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.
[0064] 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).
[0065] 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.
[0066] 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
.alpha.-(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).
[0067] 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.
[0068] 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.
[0069] 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 (3rd 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.
[0070] 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.
[0071] 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.
[0072] 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).
[0073] 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
[0074] 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.
[0075] 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.
[0076] 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 Fc.gamma.-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
uI/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.
[0077] A 5-10 fold reduction in binding affinity was observed for
the sialylated forms of these antibodies to their respective
activating Fc.gamma.Rs 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, Fc.gamma.RIV, when compared to IgG1
binding to its activation receptor Fc.gamma.RIII, the effect of
sialylation was to generate a binding affinity for IgG2b for its
activation receptor Fc.gamma.RIV 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 Fc.gamma.R111 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
Fc.gamma.Rs and thus reduced their in vivo cytotoxicity.
[0078] 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. 203(3):789-97 (2006).
[0079] A common mechanism for this anti-inflammatory activity was
proposed involving the induction of surface expression of the
inhibitory Fc.gamma.RIIB molecule on effector macrophages, thereby
raising the threshold required for cytotoxic IgG antibodies or
immune complexes to induce effector cell responses by activation
Fc.gamma.R triggering. Nimmerjahn and Ravetch, Immunity 24, 19
(2006).
Example 2. Asialylation of IVIG Decreases the Anti-Inflammatory
Effect of IVIG in Mouse Arthritis Model
[0080] Mice
[0081] C57BL/6 and NOD mice were purchased from the Jackson
Laboratory (Bar Harbor, Me.). Fc.gamma.RIIB.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/B.times.N mice. Female mice at 6-10 weeks of age were
used for all experiments and maintained at the Rockefeller
University animal facility.
[0082] Serum was prepared as described previously (Bruhns, et al.,
Immunity 18, 573 (2003)). Briefly, serum is separated from blood
collected from the K/B.times.N mice (6-12 weeks old). Several weeks
of serum collection were pooled together and frozen in aliquots to
be used in all the experiments described here. One intravenous
injection of 1.5.times. diluted K/B.times.N serum (4 .mu.l of
pooled K/B.times.N serum per gram of mouse) induced arthritis.
Arthritis was scored by clinical examination. Indices of all four
paws are added: 0 [unaffected], 1 [swelling of one joint], 2
[swelling of more than one joint], and 3 [severe swelling of the
entire paw]. IVIG is injected 1 hr before K/B.times.N serum
injection. Some mice received 5 .mu.g of platelet depleting
6A6-IgG2b antibody, and platelet counts were determined at 0, 4,
and 24 hours post treatment using an Advia 120 haematology system
(Bayer). All experiments were done in compliance with federal laws
and institutional guidelines and have been approved by the
Rockefeller University (New York, N.Y.).
[0083] Antibodies and Soluble Fc Receptors
[0084] 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.
203(3):789-97 (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).
[0085] 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 K.times.N serum, an IgG 1 immune
complex-mediated inflammatory disease model. De-sialylation with
neuraminidase abrogated the protective effect of the IVIG
preparation in the K.times.N 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
[0086] Preparation of IVIG with an Increased Content of Sialic
Acid
[0087] 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.
[0088] These sialic acid enriched fractions were tested for
protective effects in the K.times.N 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 bi-antennary, 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 Fc.gamma.RIIB 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
[0089] 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.
[0090] 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
[0091] Murine Model for Goodpasture's Disease
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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
[0097] Sequential Maldi-Tof analysis of SNA+(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
[0098] 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 G0 (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 a 2-6 sialyltransferase ("ST6Gal") or a 2-3
sialyltransferase ("ST3Gal") and confirmed by lectin blotting for a
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 a 2-6 sialylated Fcs (black triangles) or 0.66 mg
a 2-3 sialylated Fcs (red triangles). 1 hour later, 0.2 ml of
K/B.times.N 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
[0099] 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 a 2-6
linkages in IVIG (left lane), and a 2-3 SA tx IVIG (center lane),
but not in a 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/B.times.N sera. As shown in FIG. 3B, footpad swelling was
observed in mice administered K/B.times.N 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 (squares) were not protected from footpad
swelling.
Example 9. Reduced Cytotoxicity does not Depend on the Nature of
Linkage Between Sialic Acid and Galactose
[0100] The inventors have previously demonstrated that sialylation
of the N-linked glycan associated with the Fc domain of IgG
resulted in reduced FcR binding, leading to a reduction in the A/I
ratio (Kaneko, et al., Science 313, 670 (2006)), a value derived
from the affinity constants for an IgG Fc binding to individual
activating (A) or inhibitory (I) IgG Fc receptors. This ratio has
been shown to be predictive of the in vivo cytotoxicity for a
specific IgG Fc (F. Nimmerjahn, J. V. Ravetch, Science 310, 1510
(2005)). Fc sialylation thus reduced the cytotoxicity of IgG
antibodies in the induced thrombocytopenia model as well as in in
vitro models of ADCC (Kaneko, et al., Science 313, 670 (2006),
Scallon, et al., Mol. Immunol 44, 1524 (2007)). The inventors,
therefore, set out to determine if this reduction in FcR binding
and cytotoxicity was influenced by the sialic acid-galactose
linkage. A monoclonal anti-platelet IgG2b antibody previously shown
to lead to platelet consumption was sialylated in vitro as
described above and tested for in vivo activity. Both terminal 2,3
and 2,6 in vitro sialylated IgG Fc reduced the cytotoxicity of this
anti-platelet antibody, 6A6-IgG2b, in an in vivo model of
thrombocytopenia (FIG. 4), consistent with previous studies
(Kaneko, et al., Science 313, 670 (2006), Scallon, et al., Mol.
Immunol 44, 1524 (2007)). Thus, the effect of Fc sialylation on the
cytotoxicity of an IgG antibody is not dependent on the specificity
of the linkage to the penultimate galactose.
[0101] In contrast, the anti-inflammatory activity of the
sialylated IgG Fc fragment (a property which the inventors have
shown to be independent of the canonical IgG Fc receptors (F.
Nimmerjahn, J. V. Ravetch, Science 310, 1510 (2005); F. Nimmerjahn,
J. V. Ravetch, J Exp Med 204, 11 (2007)) displayed a clear
preference for the 2,6 sialic acid-galactose linkage, as seen in
FIG. 3B.
[0102] These results further support the inventors' previous
observations that the anti-inflammatory property of IVIG is
mediated through a distinct pathway that does not involve binding
to canonical Fc.gamma.Rs, which is in sharp contrast to previously
accepted models (Park-Min et al., Immunity 26, 67 (2007); Siragam
et al., Nat Med 12, 688 (2006)).
Example 10: In Vivo Anti-Inflammatory Activity of the 2,6
Sialylated IGG Fc is Solely a Property of the IGG Fc Glycan
[0103] To fully demonstrate that the in vivo anti-inflammatory
activity of the 2,6 sialylated IgG Fc is solely a property of the
IgG Fc glycan and not the result of other components that might be
found in the heterogeneous, IVIG Fc preparations, the
anti-inflammatory activity of sialylated IVIG Fc was recapitulated
using a homogeneous, recombinant human IgG1 Fc substrate (rFc),
derived from a cDNA (SEQ ID NO. 1) expressed in 293T cells. The
purified recombinant human IgG1 Fc fragment was glycan engineered
in vitro, as described above, by .beta.1,4 galactosylation,
followed by 2,6 sialylation (FIG. 5A). The preparation was purified
and characterized by lectin blotting and MALDI-TOF analysis (FIG.
5A) before in vivo analysis. Glycosylation was confirmed by lectin
blotting for terminal galactose with ECL (top panel), .alpha.2,6
sialic acid with SNA (middle panel), and coomassie loading controls
are shown in the bottom panel.
[0104] Mice were administered IVIG, SNA+ IVIG Fcs, or sialylated
rFc (2,6ST rFc) 1 hour prior to K/B.times.N sera, and footpad
swelling was monitored over the next several days. As seen in FIG.
5B, the 2,6 sialylated recombinant human IgG1 Fc fragment
demonstrated comparable anti-inflammatory activity to that obtained
with either IVIG-derived sialic-enriched Fc fragments (SNA+ IVIG
Fc) or in vitro 2,6 sialylated IVIG-derived Fc fragments (2,6ST
IVIG Fc). Mean and standard deviation of clinical scores of 4-5
mice per group are plotted; *denotes p<0.05 as determined by
Kruskal-Wallis Anova followed by Dunn's post hoc.
[0105] Each of these preparations was active at 30 mg/kg, as
compared to the 1,000-2,000 mg/kg required for native IVIG (Table
1).
TABLE-US-00001 TABLE 1 Different dosages of Fc fragment containing
preparations result in the same extent of inflammation suppression
in arthritis model. IVIG SNA.sup.+ SNA.sup.+ 2,3ST 2,6ST 2,6ST IVIG
prep IVIG Fc IVIG IVIG Fc IVIG Fc IVIG Fc rFc Dose 1 g/kg 0.33 g/kg
0.1 g/kg 0.033 g/kg 0.033 g/kg 0.033 g/kg 0.033 g/kg Amount/ 20 mg
6.66 mg 2 mg 0.66 mg 0.66 mg 0.66 mg 0.66 mg mouse injection
Example 11
In Vivo Activity of IgG Subclasses Dependant on Fc.gamma.R
Specificity
[0106] To address the role of individual Fc.gamma.Rs to the in vivo
activities of specific IgG subclasses a series of antibodies were
constructed for two defined epitopes, in which the V.sub.H regions
of the cloned hybridoma recognizing either the melanosome gp75
antigen (TA99 family) or anti-platelet integrin antigen (6A6
family) were grafted onto the C57BL/6-derived G1, 2a, 2b or 3
constant regions and co-expressed with the appropriate light chains
in 293 T cells (Nimmerjahn et al., Immunity 23, 41-51 (2005);
Vijayasaradhi et al., J. Exp Med 171, 1375-80 (1990); and Clynes et
al., Proc Natl Acad Sci USA 95, 652-6 (1998)). These recombinant
antibodies were purified and tested for binding affinity to their
cognate antigen (Table 2) and to soluble, recombinantly expressed
Fc.gamma.R I, II, III or IV by surface plasmon resonance or to
transfected cells expressing a heterologous Fc receptor. Switching
IgG constant regions did not affect the binding affinity of the
resultant antibodies to their respective antigens (Table 2).
TABLE-US-00002 TABLE 2 Affinities of TA99-antibody switch variants
for gp75 KA(1/M) KD(M) TA99-IgG1 .sup. 2.7 .times. 10.sup.9 * 3.8
.times. 10.sup.-10 TA99-IgG2a 1.6 .times. 10.sup.9 6.1 .times.
10.sup.-10 TA99-IgG2b 1.8 .times. 10.sup.9 5.7 .times. 10.sup.-10
TA99-IgG3 1.5 .times. 10.sup.9 6.6 .times. 10.sup.-10 * Antibody
affinities were determined by surface plasmon resonance (SPR)
analysis (Nimmerjahn et al., (2005)). A soluble version of the
extracellular domain of gp75 was injected at a flow rate of 30
.mu.l/min over the immobilized antibody variants and association
and dissociation constants were calculated. Each data point
represents the mean of five experiments performed in duplicates at
different concentrations with a SE below 5%. Id.
[0107] In contrast, specific differences in binding affinity of
each subclass to specific Fc.gamma.Rs were observed, as shown in
Table 3. For example, IgG1 bound with 10-fold higher affinity to
the inhibitory receptor Fc.gamma.RIIB than to its activation
counterpart, Fc.gamma.RIII, while IgG2a and 2b displayed the
reverse pattern, binding with 10-fold higher affinity for the
activation receptor Fc.gamma.RIV than to the inhibitory receptor
Fc.gamma.RIIB. IgG3 did not bind to any of the known Fc.gamma.Rs.
The ratio of activation to inhibitory binding (A/I), as shown in
Table 2, thus can differ by as much as 2 orders of magnitude
between IgG subclasses and FcRs.
TABLE-US-00003 TABLE 3 Affinities of Fc.gamma.-receptors for
antibody isotypes sFc.gamma.RIIB sFc.gamma.RIII sFc.gamma.RIV
sFc.gamma.RI A/I IgG1(+fucose) 3.33 .times. 10.sup.6 0.31 .times.
10.sup.6 -/- -/- 0.1 IgG1(-fucose) 1.32 .times. 10.sup.6 0.51
.times. 10.sup.6 -/- -/- 0.4 IgG1(-SA) 4.00 .times. 10.sup.6 0.50
.times. 10.sup.6 -/- -/- 0.1 IgG1(+SA) 0.39 .times. 10.sup.6 0.07
.times. 10.sup.6 -/- -/- 0.2 IgG2a(+fucose) 0.42 .times. 10.sup.6
0.68 .times. 10.sup.6 2.9 .times. 10.sup.7 1.6 .times. 10.sup.8
69** IgG2a(-fucose) 3.34 .times. 10.sup.6 1.54 .times. 10.sup.6
3.06 .times. 10.sup.8 1.8 .times. 10.sup.8 92** IgG2b(+fucose) 2.23
.times. 10.sup.6 0.64 .times. 10.sup.6 1.7 .times. 10.sup.7 -/- 7**
IgG2b(-fucose) 1.0 .times. 10.sup.7 1.06 .times. 10.sup.6 2.03
.times. 10.sup.8 -/- 20** IgG3 -/- -/- -/- -/- -/- *Numbers
represent the affinity (K.sub.A) of the indicated antibody isotypes
to the indicated soluble Fc.gamma.-receptors (sFc.gamma.R) as
measured by surface plasmon resonance analysis (Nimmerjahn et al.,
(2005)). A/I is the ratio of the affinity of the activating
(sFc.gamma.RIII or sFc.gamma.RIV, respectively) to the inhibitory
receptor Fc.gamma.R-RIIB. A double asterisk indicates the ratio of
sFc.gamma.RIV to sFc.gamma.RIIB; -/- indicates no detectable
binding. Each data point represents the mean of five experiments
performed in duplicates at different concentrations with a SE below
5%. +/- SA indicates antibodies enriched or depleted for sialic
acid sugar residues. Id.
Materials and Methods:
[0108] Mice: C57BL/6 and C57BL/6-129SF2/J mice were obtained from
the Jackson Laboratory (Bar Harbor, Me.). .gamma..sup.-/-,
Fc.gamma.RIIB.sup.-/- and Fc.gamma.RIII.sup.-/- mice were generated
in our laboratory and backcrossed for 12 generations to the C57BL/6
background. Fc.gamma.RI.sup.-/- 129/B6 mice were generously
provided by Dr. Hogarth (The Austin Research Institute, Victoria,
Australia). Fc.gamma.RI/III.sup.-/- mice were generated in our
laboratory by crossing Fc.gamma.RI-/- with Fc.gamma.RIII-/- mice
and subsequent selection for double knockout animals. CR2-/-, C3-/-
and C4-/- knockout mice were provided by Michael Carroll (CBR
Institute for Biomedical Research, Harvard Medical School). Female
mice at 2 to 4 months 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.).
[0109] Cell culture: 293T, CHO-K1, B16-F10 and YB2/0 cells were
cultured according to ATCC guidelines.
[0110] Antibodies and recombinant proteins: The 6A6 and TA99
antibody isotype switch variants and soluble Fc.gamma.-receptors
and gp75 were produced by transient transfection of 293T cells and
subsequent purification from culture supernatants as described
(Nimmerjahn, F., et al., (2005)). For generation of TA99 antibody
variants lacking fucose YB2/0 cells were stably transfected with
the respective TA99 heavy and light chains. Fucose content of
antibodies was verified by immunoblotting with biotinylated aleuria
aurantia lectin (Vector laboratories) followed by detection with
streptavidin-AP (Roche). Antibodies enriched or depleted for sialic
acid residues were generated by affinity chromatography with
sambucus nigra lectin (Vector laboratories). Sialic acid content
was verified by immunoblotting with biotinylated sambucus nigra
lectin (Vector laboratories). Purified C1q was from Calbiochem. The
Fc.gamma.RIV-blocking antibody 9E9 has been described before Id. A
hamster IgG1 anti-TNP antibody was used as an isotype control
antibody (Pharmingen).
[0111] Surface plasmon resonance (SPR) analysis: A Biacore 3000
biosensor system was used to assay the interaction of soluble mouse
Fc.gamma.-receptors I, II, III and IV and soluble gp75 with the
indicated antibody isotypes. Additionally, soluble human
Fc.gamma.-receptors IIA (131H-allele), IIB and IIIA (158F-allele)
were used to measure the affinity to human IgG antibody isotypes.
Antibodies or BSA as a control protein were immobilized at high and
low densities to flow cells of CM5 sensor chips (Biacore) by
standard amine coupling as suggested by the manufacturer. Soluble
Fc.gamma.-receptors were injected at 5 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 10 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. As described for soluble human Fc-receptors a 1:1 Langmuir
binding model closely fitted the observed sensorgram data and was
used in all experiments Id. Alternatively, soluble Fc-receptors
were immobilized to sensor chips with the same result described
above.
[0112] In vivo model systems: The platelet depletion model:
Experiments were performed essentially as described before. Id.
Briefly, mice were injected intravenously with 4 .mu.g of the
recombinant 6A6 antibody isotype switch-variants diluted in 200
.mu.l of PBS. Alternatively, mice were injected with 2 .mu.g of the
6A6 antibody variants to study Fc.gamma.RIIB-mediated negative
regulation of antibody functions in vivo. Platelet counts before
injection and at indicated time points after injection were
determined by blood collection (40 .mu.l) from the retro-orbital
plexus and measuring platelet counts of a 1:10 dilution in PBS/5%
BSA in an Advia 120 haematology system (Bayer). To block
Fc.gamma.RIV in vivo mice were injected 30 minutes before
administration of the 6A6 antibody variants with 200 .mu.g of the
blocking Fc.gamma.RIV antibody 9E9 or with 200 .mu.g of a hamster
isotype control antibody (Pharmingen).
[0113] The B16-F10 lung metastasis model: Experiments were
performed as described (Vijayasaradhi et al., J Exp Med 171,
1375-80. (1990); and Clynes et al., Proc Natl Acad Sci USA 95,
652-6. (1998)) with minor modifications. Mice were injected with
5.times.10.sup.5 B16-F10 tumor cells intravenously and either left
untreated or were injected with the indicated amounts of isotype
control (Sigma) or TA99-isotype switch variants on days 0, 2, 4, 7,
9, 11 intraperitoneally. To block Fc.gamma.RIV in vivo mice were
injected with 200 .mu.g of the 9E9 or the respective hamster
isotype control antibody on days 0, 2, and 4 intravenously. On day
15 after tumour cell injection mice were sacrificed and lungs were
analyzed for the presence of surface metastasis by an investigator
blinded for the experimental setup.
[0114] Statistical analysis: The paired Student's t-test was used
for determining significance of the results.
Example 12
A/I Ratios of Antibody Variants Predictive of In Vivo Biological
Activity
[0115] To determine how these differences in binding affinities
relate to in vivo biological activity, the ability of these
antibodies to mediate tumor clearance or platelet depletion was
investigated. As seen in FIG. 6, both TA99 (FIGS. 6A and B) and 6A6
(FIGS. 6C and D) with IgG2a constant regions display enhanced tumor
or platelet clearance, respectively, as compared to these
antibodies with IgG1 constant regions. IgG2a and 2b are equivalent
in their ability to mediate platelet clearance, while IgG2a results
in enhanced tumor ADCC in the metastatic melanoma model as compared
to IgG2b. The hierarchy of activity for the IgG subclasses is thus
IgG2a.gtoreq.IgG2b>IgG1>>IgG3. The mechanism of this
differential activity was determined by repeating these experiments
in specific activating Fc.gamma.R or complement deficient strains.
No differences in in vivo activity were observed for IgG1, 2a or 2b
in complement deficient strains (C4, C3 or CR1/2) (FIG. 7). In
contrast, IgG1, 2a and 2b were all dependent on activating
Fc.gamma.R expression, since activity was abrogated in the common
.gamma. chain deficient background (FIGS. 8A, B and E). While IgG2a
activity could result from its ability to bind with high affinity
to Fc.gamma.RI, intermediate affinity to Fc.gamma.RIV or low
affinity to Fc.gamma.R III, only Fc.gamma.RIV binding was relevant
to its in vivo activity (FIGS. 8C, D and E). Similarly, IgG2b
activity was Fc.gamma.RIV dependent and Fc.gamma.RIII independent
(FIG. 8E). In contrast, IgG1 mediated effector activity was
exclusively Fc.gamma.RIII dependent (FIG. 8E).
[0116] The balance of activation to inhibitory receptor expression
has been shown to determine the threshold for IgG mediated effector
cell triggering (Ravetch and Lanier, Science 290, 84-9 (2000)). The
binding affinities of IgG subclasses to the inhibitory receptor
vary by a factor of 10, suggesting that a differential dependence
of the subclasses on the inhibitory effect of Fc.gamma.RIIB might
be observed. As seen in FIG. 9, IgG1 displays the greatest
enhancement in activity in mice lacking the inhibitory receptor in
both the tumor clearance and platelet depletion models (FIG. 9A-C),
while IgG2a shows the smallest enhancement in both models. The
magnitude of IgG2b enhancement in Fc.gamma.RIIB deficient strains
differs in the two models, showing significant enhancement in the
tumor clearance model and minimal enhancement in the platelet
depletion model. This difference is likely due to the intermediate
A/I ratio of this receptor rendering it more sensitive to the
levels of surface expression of Fc.gamma.RIIB on the specific
effector cells mediating the in vivo responses. Since different
populations of effector cells are responsible for the biological
responses in the two models, the IgG2b data support our previous
observations that RIIB levels are minimal on splenic macrophages,
the cell type responsible for platelet clearance (Nimmerjahn et
al., Immunity 23, 41-51. (2005); and Samuelsson et al., Science
291, 484-6. (2001)), and higher on alveolar macrophages, the
relevant effector cells in the metastatic melanoma model
(Shushakova et al., J Clin Invest 110, 1823-30 (2002)). These
results demonstrate the predictive value of the A/I ratio in
determining the contribution of inhibitory signaling to in vivo
activities. A high A/I ratio, as found for IgG2a, renders the
antibody essentially insensitive to differences in Fc.gamma.RIIB
expression on different effector cell populations, while a low A/I
ratio, as found for IgG1, maximizes the role of Fc.gamma.RIIB. For
antibodies with intermediate A/I ratios, like IgG2b, the in vivo
activity will be determined by the specific effector cell involved
in the response, reflecting the differences in Fc.gamma.RIIB levels
and its regulation by the cytokine milieu. This difference in
Fc.gamma.RIIB dependence for IgG1 and IgG2 may reflect the
biological roles of these subclasses in vivo, insuring that the
most abundant subclass, IgG1, is under tight regulation by an
inhibitory receptor, thus preventing effector cell activation in
the absence of a second signal that down regulates Fc.gamma.RIIB
and lowers the threshold for activation. Such second signals are
provided by pro- and anti-inflammatory cytokines and chemokines
which have been demonstrated to alter the levels of surface
expression of activation or inhibitory receptors (Shushakova
(2002)). In contrast, the role of inhibitory receptor expression on
IgG2a potency, and to a lesser extent, IgG2b, is less significant,
reflecting the effector bias of T.sub.H1 cytokines which induce
both IgG2a switching and Fc.gamma.RIV expression.
Example 13
Modified Antibody with a Lower Amount of Fucose and a Greater A/I
Ratio Compared to Unmodified Antibody
[0117] The relationship between the A/I ratio of IgG subclasses and
in vivo activity was further tested using modified IgG constant
regions. FcR binding to IgG is dependent on the presence of
N-linked glycosylation at position 297; deglycosylation abrogates
all FcR binding (Krapp, J Mol Biol 325, 979-89 (2003)). However,
selective removal of specific carbohydrates, such as fucose, has
been suggested to modify human IgG1 binding to human Fc.gamma.RIII
and thus to NK cell mediated ADCC in vitro (Shields, R. L., et al.,
J Biol Chem 277, 26733-40. (2002); T. Shinkawa et al., J Biol Chem
278, 3466-73 (2003); and Niwa et al., Cancer Res 64, 2127-33
(2004)). Fucose-deficient TA99-IgG1, 2a and 2b were prepared and
their binding to Fc.gamma.RI, II, III and IV was compared. C1q or
antigen binding was not affected by the lack of fucose as described
before (Shields (2002)). However, as shown in FIG. 10 and Table 3,
fucose deficient antibodies differed in their binding affinities to
their cognate Fc.gamma.Rs, with TA99-IgG1 with or without fucose
displaying minimal differences in binding to FcRIIB and III, while
IgG2a and 2b fucose deficient antibodies bound with an order of
magnitude higher affinity to FcRIIB and FcRIV as compared to fucose
sufficient antibodies. These differences in binding affinities
resulted in altered A/I ratios that were most pronounced for IgG2b
(FIG. 10 and Table 3) and translated into significantly enhanced in
vivo activity for IgG2b. This selective effect of de-fucosylation
on FcR binding further illustrates the specificity of IgG
subclasses in their interactions with individual FcRs and the
predictive value of the A/I ratio in determining in vivo
activity.
Example 14
Modified Antibody With A Lower Amount Of Sialic Acid
[0118] The role of sialic acid residues in antibody sugar side
chains was investigated by injecting mice with 6A6-IgG1 antibody
variants enriched or depleted for sialic acid residues and
measuring antibody mediated platelet depletion. Antibodies enriched
for sialic acid in their sugar side chains displayed strongly
reduced affinity for both the activating FcRIII and inhibitory
FcRIIB (Table 3). Consistent with this loss in overall affinity for
Fc-receptors and the low A/I ratio of 0.18, this antibody had a
severely impaired in vivo activity and mediated only minimal
platelet depletion (FIG. 11).
Example 15
Human Antibodies Display Differential A/I Ratios
[0119] To investigate if human antibody isotypes also display
differential A/I ratios soluble versions of human
Fc.gamma.-receptors were prepared and their affinity for human IgG
antibody isotypes was measured by surface plasmon resonance
analysis. As shown in Table 4 human FcRs also have differential A/I
ratios for individual human IgG antibody isotypes.
TABLE-US-00004 TABLE 4 Affinities of Human FcRs to Human IgG
Isotypes A/I sFcRIIB sFcRIIA sFcRIIIA IIA/IIB IIIA/IIB IgG1 7.8
.times. 10.sup.4 2.5 .times. 10.sup.5 4.3 .times. 10.sup.5 3.1 5.5
IgG1-fucose 7.8 .times. 10.sup.4 2.4 .times. 10.sup.5 6.0 .times.
10.sup.6 3.1 76 IgG2 3.0 .times. 10.sup.4 1.4 .times. 10.sup.5 1.4
.times. 10.sup.4 4.5 0.4 IgG2-fucose 2.6 .times. 10.sup.4 1.3
.times. 10.sup.5 1.4 .times. 10.sup.5 5.3 5.7 IgG4 4.8 .times.
10.sup.4 3.5 .times. 10.sup.4 -/- 0.7 n.a. * Numbers represent the
affinity (K.sub.A) of the indicated antibody isotypes to the
indicated soluble Fc.gamma.-receptors (sFc.gamma.R) as measured by
surface plasmon resonance analysis (Nimmerjahn et al., (2005)). A/I
is the ratio of the affinity of the indicated activating to the
inhibitory receptor Fc.gamma.RIIB. -/- indicates no detectable
binding; n.a. indicated not applicable. Each data point represents
the mean of five experiments performed in duplicates at different
concentrations with a SE below 5%.
[0120] In contrast to the single chain inhibitory Fc-receptor,
activating Fc.gamma.Rs (with the exception of human Fc.gamma.RIIA)
cannot transmit activating signals in the absence of an accessory
chain, the common gamma chain (.gamma.-chain), that carries an ITAM
motif required for triggering cell activation. Fc.gamma.RI,
Fc.gamma.RIII and Fc.gamma.RIV are dependent on .gamma.-chain
expression; thus deletion of this receptor subunit leads to the
functional loss of all activating Fc-receptors and several other
non-FcR-related proteins such as PIR-A and NK cell cytotoxicity
receptors (Moretta et al., Annu Rev Immunol 19, 197-223 (2001);
Ravetch, (2003)).
[0121] The only IgG isotype that could consistently be assigned to
an individual activating Fc-receptor in vivo was IgG1. The deletion
of the low affinity receptor Fc.gamma.RIII abrogates IgG1 mediated
effector functions in various models like arthritis,
glomerulonephritis, IgG-dependent anaphylaxis, IgG mediated
hemolytic anemia and immunothrombocytopenia (Hazenbos et al.,
Immunity 5, 181-188 (1996); Meyer et al., Blood 92, 3997-4002
(1998); Fossati-Jimack et al., J Exp Med 191, 1293-1302 (2000); Ji
et al., Immunity 16, 157-168 (2002); Bruhns et al., Immunity 18,
573-581 (2003); Fuji et al., Kidney Int 64, 1406-1416 (2003);
Nimmerjahn et al., (2005)). Under many circumstances, such as host
response to viral or bacterial infections (Coutelier et al., J Exp
Med 165, 64-69 (1987); Schlageter and Kozel, Infect Immun 58,
1914-1918 (1990); Markine-Gorianyoff and Coutelier, J Virol 76,
432-435 (2002); Taborda et al, J Immunol 170, 3621-3630 (2003)),
and antibody-mediated cytotoxicity or antibody-based therapy (Kipps
et al., J Exp Med 161, 1-17 (1985); Fossati-Jimack et al., J Exp
Med 191, 1293-1302 (2000); Uchida et al., J Exp Med 199, 1659-1669
(2004); Nimmerjahn et al., (2005)) the most potent antibody
isotypes are of the IgG2a and IgG2b isotype. Therefore, a thorough
understanding of how these isotypes exert their function is
essential.
[0122] Considering the isotype specificities of the high affinity
Fc.gamma.RI (binding exclusively IgG2a) and the low affinity
Fc.gamma.RIII (binding IgG1, IgG2a, and IgG2b) (reviewed in Ravetch
and Kinet, Annu Rev Immunol 9, 457-492 (1991); Hulett and Hogarth,
Adv Immunol 57, 1-127 (1994)), these two receptors are likely
candidates responsible for IgG2a and IgG2b effector functions.
Although there is some suggestion that Fc.gamma.RI and III might
participate in a limited fashion in IgG2a-mediated effector
responses (Ioan-Facsinay et al., Immunity 16, 391-402 (2002);
Barnes et al., Immunity 16, 379-389, (2002)), the majority of
studies concluded that IgG2a and IgG2b triggered effects occur
independently of these two receptors, but in a gamma chain
dependent manner (Hazenbos et al., Immunity 5, 181-188 (1996);
Meyer et al., (1998); Fossati-Jimack et al., (2000); Uchida et al.,
(2004); Nimmerjahn et al., (2005)). Especially in the case of IgG2a
these results seem to be surprising as Fc.gamma.RI shows a high
affinity for this isotype (KA: 10.sup.8-10.sup.9 M.sup.-1).
However, the increased affinity allowed this receptor to bind
monomeric IgG2a as efficiently as immune complexes (ICs),
indicating that newly generated ICs would be expected to have only
limited access to Fc.gamma.RI (FIG. 12A).
[0123] Fc.gamma.RIV requires .gamma. chain for its surface
expression (Nimmerjahn, (2005)) and, as has been described for
other .gamma.-chain dependent Fc-receptors, cross-linking of
Fc.gamma.RIV by immune complexes induces activating signaling
pathways leading to sustained calcium flux (reviewed in Ravetch and
Bolland, (2001); Nimmerjahn et al., (2005)).
[0124] Even if several activating Fc-receptors with the same
isotype specificity are present on the same cell, only those
Fc-receptors will be engaged that show the optimal affinity for the
respective isotype (FIG. 12A). Therefore, IgG1 immune complexes
will only trigger Fc.gamma.RIII as it is the only activating
Fc-receptor that can bind IgG1 (Takai, (1994); Hazenbos et al.,
(1996); Meyer et al., (1998); Nimmerjahn et al., (2005)). IgG2a and
IgG2b, despite their ability to bind Fc.gamma.RI (in the case of
IgG2a) or Fc.gamma.RIII (in the case of IgG2a and 2b) will
functionally be dependent on Fc.gamma.RIV, as Fc.gamma.RI will be
occupied by monomeric IgG2a and the low affinity of RIII will not
result in productive engagement at normal serum concentration of
these isotypes. These same principles also apply for the human
system, where it has been shown that human Fc.gamma.RIIIA has a
higher affinity for IgG1 as compared to human Fc.gamma.RIIA. In
addition, the presence of allelic variants which show differential
affinities for the specific antibody isotypes further supports this
concept (Dijstelbloem et al., Trends Immunol, 22, 510-516
(2001)).
[0125] The present invention provides a mechanistic basis for the
observed variation in IgG subclass activity in both active and
passive vaccination and in the variable pathogenicity of the IgG
subclasses in autoimmune conditions. The selective Fc.gamma.R
binding affinities of the IgG subclasses, and not their ability to
fix complement, is predictive of the in vivo activity for cytotoxic
antibodies in models of tumor clearance, platelet and B cell
depletion (Uchida et al., J Exp Med 199, 1659-69. (2004); Clynes
and Ravetch (1995); Clynes (1998); Samuelsson (2001)). Similarly,
the biological consequences of modifications to IgG antibodies are,
in turn, dependent on their effects on specific FcR binding
affinities that result in changes to the ratio of activation to
inhibitory receptor affinities. These considerations will be
significant factors in the design of both antibody-based
immunotherapeutics and active vaccination protocols to insure
either the selective engineering of IgG Fc domains or induction of
IgG subclasses with optimal Fc.gamma.R activation to inhibitory
ratios.
[0126] 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.
Sequence CWU 1
1
111398DNAHomo sapiens 1atgggtgaca atgacatcca ctttgccttt ctctccacag
gtgtccagtc cgaggtgaag 60ctggatgaga ctggaggagg cttggtgcaa cctgggaggc
ccatgaaact ctcctgtgtt 120gcctctggat tcacttttag tgactactgg
atgaactggg tccgccagtc tccagagaaa 180ggactggagt gggtagcaca
aattagaaac aaaccttata attatgaaac atattattca 240gattctgtga
aaggcagatt caccatctca agagatgatt ccaaaagtag tgtctacctg
300caaatgaaca acttaagagt tgaagacatg ggtatctatt actgtacggg
ttcttactat 360ggtatggact actggggtca aggaacctca gtcaccgtga
gctcagcctc caccaagggc 420ccatcggtct tccccctggc accctcctcc
aagagcacct ctgggggcac agcggccctg 480ggctgcctgg tcaaggacta
cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 540ctgaccagcg
gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc
600agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat
ctgcaacgtg 660aatcacaagc ccagcaacac caaggtggac aagagagttg
agcccaaatc ttgtgacaaa 720actcacacat gcccaccgtg cccagcacct
gaactcctgg ggggaccgtc agtcttcctc 780ttccccccaa aacccaagga
caccctcatg atctcccgga cccctgaggt cacatgcgtg 840gtggtggacg
tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg
900gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac
gtaccgtgtg 960gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg
gcaaggagta caagtgcaag 1020gtctccaaca aagccctccc agcccccatc
gagaaaacca tctccaaagc caaagggcag 1080ccccgagaac cacaggtgta
caccctgccc ccatcccggg atgagctgac caagaaccag 1140gtcagcctga
cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag
1200agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 1260tccttcttcc tctacagcaa gctcaccgtg gacaagagca
ggtggcagca ggggaacgtc 1320ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacgcagaa gagcctctcc 1380ctgtctccgg gtaaatga 1398
* * * * *