NZ627002B2 - Non-sialylated anti-inflammatory polypeptides - Google Patents
Non-sialylated anti-inflammatory polypeptides Download PDFInfo
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- NZ627002B2 NZ627002B2 NZ627002A NZ62700212A NZ627002B2 NZ 627002 B2 NZ627002 B2 NZ 627002B2 NZ 627002 A NZ627002 A NZ 627002A NZ 62700212 A NZ62700212 A NZ 62700212A NZ 627002 B2 NZ627002 B2 NZ 627002B2
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- polypeptide
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- isolated polypeptide
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Abstract
Disclosed is an isolated polypeptide comprising a modified sequence that is at least 75% identical to an IgG Fc region with the sequences of SEQ ID No: 1 or 2, and has a substitution at a position corresponding to F241 of SEQ ID No: 1 according to the Kabat numbering system, wherein the modified sequence is free of sialylation and the polypeptide has an anti-inflammatory activity that is higher than that of a parent polypeptide, wherein the sequences are as defined in the complete specification. uence is free of sialylation and the polypeptide has an anti-inflammatory activity that is higher than that of a parent polypeptide, wherein the sequences are as defined in the complete specification.
Description
NON-SIALYLATEDANTI-INFLAMMATORYPOLYPEPTIDES
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority of U.S. Provisional Application No. 61/577,361,
filed on December 19, 201 1. The content of the application is incorporated herein by
reference in its entirety.
GOVERNMENT INTERESTS
The invention disclosed herein was made, at least in part, with Government
support under Grant No. NIH AI035875 from the National Institutes of Health.
Accordingly, the U.S. Government has certain rights in this invention.
FIELD OF INVENTION
This invention relates to anti-inflammatory agents, compositions, and methods for
treating inflammatory disorders.
BACKGROUND
Inflammatory disorders, including autoimmune diseases, are disorders involving
abnormal activation and subsequent migration of white blood cells to affected areas of the
body. These conditions encompass a wide range of ailments that affect the lives of
millions of people throughout the world. Although various treatments are presently
available, many possess significantly side effects or are not very effective in alleviating all
symptoms. Thus, there are needs for anti-inflammatory agents for treating inflammatory
disorders and needs for methods of identifying and evaluating such agents.
Immunoglobulin G (IgG) has long been appreciated to mediate both pro- and anti
inflammatory activities through interactions mediated by its Fc fragment. 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. It has been proposed
that glycosylation of IgG is crucial for regulation of cytotoxicity and inflammatory
potential of IgG. For example, it has been suggested that anti-inflammatory activity of
IVIG is a property of the Fc fragment and its linked glycan, requiring terminal a.2,6 sialic
acid linkages, indicating a combined requirement for the specific polypeptide backbone
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and glycan structure for immunosuppression. (Anthony, et al., 2008, Science 320: 373-376
and ).
However, only a minor population of IgG in IVIG have glycans terminating in
α2,6 sialic acids (sFc) and the anti-inflammatory activity. As a result, for the suppression
of autoantibody triggered inflammation in a variety of clinical settings, one has to
administer IVIG at high doses (1-2g/kg), to enrich sialylated IgGs, or otherwise to increase
the sialylation of IgGs (US Application Nos. 20080206246, and 20090004179, and
Nimmerjahn et al. Annu Rev Immunol 26, 513-533 (2008)).
The present invention addresses and meets the above-mentioned needs by
identifying sialylation–free anti-inflammatory polypeptides.
It is a further alternative object of the present invention to provide one or more of:
an isolated polypeptide, a method for making a polypeptide, a polypeptide, an isolated
nucleic acid, an expression vector, an isolated host cell, a method of producing a
polypeptide, a pharmaceutical formulation, a method of treating an inflammatory disease
in non-human subjects and/or a use of a polypeptide which addresses and meets the above-
mentioned needs. The foregoing objects should be read disjunctively with the further
alterative object of at least providing the public with a useful choice.
SUMMARY
This invention relates to agents, such as polypeptides and antibodies, and methods
for treating inflammatory disorders, e.g., autoimmune diseases.
Accordingly, one aspect of this invention features an isolated polypeptide
comprising a modified sequence that is at least 75% (e.g., any number between 75% and
100%, inclusive, e.g., 70 %, 80%, 85%, 90%, 95%, 99%, and 100%) identical to an IgG
Fc region. The modified sequence is free of sialylation and the polypeptide has an anti-
inflammatory activity that is higher than that of a parent polypeptide. The parent
polypeptide can comprise the IgG Fc region, such as the sequence of SEQ ID NO: 1 listed
below. In some embodiments, the polypeptide has ability to bind to DC-SIGN, and to
bind to hFc γRIIA or RIIB. In one embodiment, the isolated polypeptide has an ability to
-5 4 -1
bind to hFc γRIIA or RIIB at a K of 2x10 M or lower (i.e., K of 5.0 x 10 M or
higher). Preferably, the modified sequence has a FA241 mutation. The modified
sequence can be at least 75% (e.g., any number between 75% and 100%, inclusive, e.g.,
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304124433
70%, 80%, 85%, 90%, 95%, 99%, and 100%) identical to SEQ ID NO: 2. In some
examples, the modified sequence comprises or consists essentially of SEQ ID NO: 2.
In another aspect, the invention provides a method for making a polypeptide
having an anti-inflammatory activity. The method includes, among others, steps of
providing a parent polypeptide having the sequence of an IgG Fc region or a first nucleic
acid sequence encoding the parent polypeptide; and modifying the parent polypeptide to
obtain a modified polypeptide so that the modified polypeptide is free of sialylation and
mimics the structural of a sialylated form of the IgG Fc region. The modifying step can be
conducted by modifying the first nucleic acid sequence to obtain a second nucleic acid
encoding the modified polypeptide. The invention also provides a polypeptide made by
the just-described method.
In a third aspect, the invention features an isolated nucleic acid comprising a
sequence encoding the polypeptide described above; an expression vector comprising the
nucleic acid; and a host cell comprising the nucleic acid. The invention also features a
method of producing a polypeptide. The method includes culturing the host cell in a
medium under conditions permitting expression of a polypeptide encoded by the nucleic
acid, and purifying the polypeptide from the cultured cell or the medium of the cell.
In a fourth aspect, the invention features a pharmaceutical formulation comprising
(i) the polypeptide or nucleic acid described above, and (ii) a pharmaceutically acceptable
carrier.
In a fifth aspect, the invention provides a method of treating an inflammatory
disease. The method includes administering to a subject in need thereof a therapeutically
effective amount of the above-described polypeptide or nucleic acid encoding the
polypeptide. Also provided is use of the polypeptide or nucleic acid in the manufacture of
a medicament for treating an inflammatory disease. The invention also features an
isolated polypeptide, nucleic acid, expression vector, host cell, composition, or method for
treating an inflammatory disease substantially as shown and described herein.
In a particular aspect the invention provides an isolated polypeptide comprising a
modified sequence that is at least 75% identical to an IgG Fc region with the sequences of
SEQ ID No: 1 or 2, and has a substitution at a position corresponding to F241 of SEQ ID
No: 1 according to the Kabat numbering system, wherein the modified sequence is free of
sialylation and the polypeptide has an anti-inflammatory activity that is higher than that of
a parent polypeptide.
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In a second particular aspect the invention provides a method for making a
polypeptide having an anti-inflammatory activity the method comprising: providing a
parent polypeptide having the sequence of an IgG Fc region or a first nucleic acid
sequence enconding the parent polypeptide; and modifying the parent polypeptide to
obtain a modified polypeptide so that the modified polypeptide is free of sialylation
wherein the modifying step comprises introducing a substitution at a position
corresponding to F241 of SEQ ID No: 1 according to the Kabat numbering system.
In a third particular aspect the invention provides a polypeptide made by the
method described in the second particular aspect.
In a fourth particular aspect the invention provides an isolated nucleic acid
comprising a sequence encoding a polypeptide comprising a modified sequence that is at
least 75% identical to an IgG Fc region with the sequences of SEQ ID No: 1 or 2, and has
a substitution at a position corresponding to F241 of SEQ ID No: 1 according to the Kabat
numbering system, wherein the modified sequence is free of sialylation and the
polypeptide has an anti-inflammatory activity that is higher than that of a parent
polypeptide.
In a fifth particular aspect the invention provides an expression vector comprising
a nucleic acid as described in the fourth particular aspect, and/or an isolated host cell
comprising a nucleic acid as described in the fourth particular aspect.
In a sixth particular aspect the invention provides a method of producing a
polypeptide, comprising culturing the isolated host cell described in the fifth particular
aspect in a medium under conditions permitting expression of a polypeptide encoded by
the nucleic acid, and purifying the polypeptide from the cultured cell or the medium of the
cell.
In a seventh particular aspect the invention provides a polypeptide made by the
method described in the sixth particular aspect.
In an eighth particular aspect the invention provides a pharmaceutical formulation
comprising an isolated polypeptide comprising a modified sequence that is at least 75%
identical to an IgG Fc region with the sequences of SEQ ID No: 1 or 2, and has a
substitution at a position corresponding to F241 of SEQ ID No: 1 according to the Kabat
numbering system, wherein the modified sequence is free of sialylation and the
polypeptide has an anti-inflammatory activity that is higher than that of a parent
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304124433
polypeptide, or an isolated nucleic acid comprising a sequence encoding such a
polypeptide and a pharmaceutically acceptable carrier.
In a ninth particular aspect the invention provides a method of treating an
inflammatory disease in a non-human subject, comprising administering to the non-human
subject in need thereof a therapeutically effective amount of an isolated polypeptide
comprising a modified sequence that is at least 75% identical to an IgG Fc region with the
sequences of SEQ ID No: 1 or 2, and has a substitution at a position corresponding to
F241 of SEQ ID No: 1 according to the Kabat numbering system, wherein the modified
sequence is free of sialylation and the polypeptide has an anti-inflammatory activity that is
higher than that of a parent polypeptide, an isolated nucleic acid comprising a sequence
encoding such a polypeptide, or the pharmaceutical formulation described in the
immediately preceding paragraph.
In a tenth particular aspect the invention provides a use of an isolated polypeptide
comprising a modified sequence that is at least 75% identical to an IgG Fc region with the
sequences of SEQ ID No: 1 or 2, and has a substitution at a position corresponding to
F241 of SEQ ID No: 1 according to the Kabat numbering system, wherein the modified
sequence is free of sialylation and the polypeptide has an anti-inflammatory activity that is
higher than that of a parent polypeptide, an isolated nucleic acid comprising a sequence
encoding such a polypeptide, or a pharmaceutical formulation comprising the isolated
polypeptide or isolated nucleic acid described in this paragraph and a pharmaceutically
acceptable carrier.
The details of one or more embodiments of the invention are set forth in the
description below. Other features, objects, and advantages of the invention will be
apparent from the description and from the claims.
DESCRIPTION OF DRAWINGS
FIGs. 1a-c are diagrams and photographs showing that α2,6-linked sialic acid
conferred DC-SIGN binding activity to recombinant human IgG1 Fc.
FIGs. 2a-b are diagrams and photographs showing that disrupting Fc-glycan
interactions conferred DC-SIGN binding activity to recombinant human IgG1 Fc.
is as set of diagrams showing that the FA241 mutation in hIgG1 Fc
recapitulates anti-inflammatory activity of α2,6 sFc
FIGs. 4a-d are diagrams showing characterizing requirements for FA241 anti
inflammatory activity.
is a set of diagrams showing that FA241 mutation increased Fey receptor
binding.
FIGs. 6a-b are photographs showing IL-33 mRNA induction in bone marrow-
derived macrophages by FA241.
DETAILED DESCRIPTION
This invention is based, at least in part, on an unexpected discovery that non-
sialylated IgG Fc variants confer anti-inflammatory activity and mimic the effect of 2,6
sialylated Fc as anti-inflammatory mediators.
IgG and Fc Sialylation
IgG is the major serum immunoglobulin. It is a glycoprotein composed of two
identical heavy chains and two light chains, which in turn are composed of variable and
constant domain. IgG contains a single, N-linked glycan at Asn in the CH2 domain on
each of its two heavy chains. The covalently- linked, complex carbohydrate is composed
of a core, biantennary penta-polysaccharide containing N-acetylglucosamine (GIcNAc)
and mannose (man). Further modification of the core carbohydrate structure is observed
in serum antibodies with the presence of fucose, branching GIcNAc, galactose (gal) and
terminal sialic acid (sa) moieties variably found. Over 40 different glycoforms have thus
been detected to be covalently attached to this single glycosylation site (Fujii et al., J. Biol.
Chem. 265, 6009, 1990). Glycosylation of IgG has been shown to be essential for binding
to all FcyRs by maintaining an open conformation of the two heavy chains. Jefferis and
Lund, Immune. 1Lett. 82, 57 (2002), Sondermann et al, J. Mol. Biol. 309, 737 (2001). It
is believed that this 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.
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,
(1998).
As disclosed herein, certain non-sialylated IgG Fc variants surprisingly also confer
anti-inflammatory activity. Such variants, including the FA241 variant, represent species
within a larger genus of molecules that, by virtue of mimicking the structural and
biological properties of sialylated Fc, but do not require sialylation, can be developed as
anti-inflammatory therapeutics.
Polypeptides and Nucleic Acids
Polypeptides
As disclosed herein, this invention provides isolated polypeptides having
sequences of variants of human IgG Fc that lacks a polysaccharide chain having a terminal
sialic acid connected to a galactose moiety through the a2,6 linkage at the aforementioned
As . Such non-sialylated IgG Fc variants may be either derived from a naturally
occurring antibody or expressed in a cell line.
In one embodiment, the Fc region includes one or more substitutions of the hlgGl
amino acid sequence. While not limited thereto, exemplary IgGl Fc regions are provided
as follows:
Fc of hlgGl (starting from amino acid 210 in Kabat system):
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 1; F241 and F243 are underlined)
Fc of hlgGl FA241:
KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVALFPPKPKDTLMI SRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVE ESNGQPEN
NYKT TPPVLD S DGSFFLYSKL TVDKSRWQQGNVF S C SVMHEALHNHYTQKS L S L SPGK
(SEQ I D NO:2)
Fc ofhlgGl FA243:
KVDKRVEPKS CDKTHTCPPCPAPELLGGP SVFLAPPKPKDTLMI SRTPEVTCVVVDVS HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVL TVLHQDWLNGKEYKCKVSNKALP
AP I E K I SKAKGQPREPQVYTLPP SRDEL TKNQVS L TCLVKGFYP S DIAVEWE SNGQPEN
NYKT TPPVLD S DGSFFLYSKL TVDKSRWQQGNVF S C SVMHEALHNHYTQKS L S L SPGK
(SEQ I D NO:3)
The terms "peptide," "polypeptide," and "protein" are used herein interchangeably
to describe the arrangement of amino acid residues in a polymer. A peptide, polypeptide,
or protein can be composed of the standard 20 naturally occurring amino acid, in addition
to rare amino acids and synthetic amino acid analogs. They can be any chain of amino
acids, regardless of length or post-translational modification (for example, glycosylation
or phosphorylation). The peptide, polypeptide, or protein "of this invention" include
recombinantly or synthetically produced versions having the particular domains or
portions that bind to DC-SIGN, FcyRIIA, and FcyRIIB. The term also encompasses
polypeptides that have an added amino-terminal methionine (useful for expression in
prokaryotic cells).
An "isolated" polypeptide or protein refers to a polypeptide or protein that has
been separated from other proteins, lipids, and nucleic acids with which it is naturally
associated. The polypeptide/protein can constitute at least 10% (i.e., any percentage
between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%, 95%,
and 99%) by dry weight of the purified preparation. Purity can be measured by any
appropriate standard method, for example, by column chromatography, polyacrylamide
gel electrophoresis, or HPLC analysis. An isolated polypeptide/protein described in the
invention can be purified from a natural source, produced by recombinant DNA
techniques, or by chemical methods. A functional equivalent of IgG Fc refers to a
polypeptide derivative of IgG Fc, e.g., a protein having one or more point mutations,
insertions, deletions, truncations, a fusion protein, or a combination thereof. It retains
substantially the activity of the IgG Fc, i.e., the ability to bind to the respective receptor
and trigger the respective cellular response. The isolated polypeptide can contain SEQ ID
NO: 2. In general, the functional equivalent is at least 75% (e.g., any number between
75% and 100%, inclusive, e.g., 70 %, 80%, 85%, 90%, 95%, and 99%) identical to SEQ
ID NO: 2.
The "percent identity" of two amino acid sequences or of two nucleic acids is
determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA
87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA
90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST
nucleotide searches can be performed with the NBLAST program, score=100, wordlength-
12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences homologous to the protein
molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can
be utilized as described in Altschul et al, Nucleic Acids Res. 25(17):3389-3402, 1997.
When utilizing BLAST and Gapped BLAST programs, the default parameters of the
respective programs (e.g., XBLAST and NBLAST) can be used.
The amino acid composition of the polypeptide described herein may vary without
disrupting the ability of the polypeptide to bind to the respective receptor and trigger the
respective cellular response. For example, it can contain one or more conservative amino
acid substitutions. A "conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a similar side chain. Families
of amino acid residues having similar side chains have been defined in the art. These
families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic
side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-
branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid
residue in, e.g., SEQ ID NO: 2, is preferably replaced with another amino acid residue
from the same side chain family. Alternatively, mutations can be introduced randomly
along all or part of the sequences, such as by saturation mutagenesis, and the resultant
mutants can be screened for the ability to bind to the respective receptor and trigger the
respective cellular response to identify mutants that retain the activity as descried below in
the examples.
A polypeptide as described in this invention can be obtained as a recombinant
polypeptide. To prepare a recombinant polypeptide, a nucleic acid encoding it (e.g.,
FA241, SEQ ID NO: 2) can be linked to another nucleic acid encoding a fusion partner,
e.g., glutathione-s-transferase (GST), 6x-His epitope tag, or M13 Gene 3 protein. The
resultant fusion nucleic acid expresses in suitable host cells a fusion protein that can be
isolated by methods known in the art. The isolated fusion protein can be further treated,
e.g., by enzymatic digestion, to remove the fusion partner and obtain the recombinant
polypeptide of this invention.
Nucleic Acids
Another aspect of the invention features an isolated nucleic acid comprising a
sequence that encodes the polypeptide or protein described above. A nucleic acid refers to
a DNA molecule (e.g., a cDNA or genomic DNA), an RNA molecule (e.g., an mRNA), or
a DNA or RNA analog. A DNA or RNA analog can be synthesized from nucleotide
analogs. The nucleic acid molecule can be single-stranded or double-stranded, but
preferably is double-stranded DNA. An "isolated nucleic acid" refers to a nucleic acid the
structure of which is not identical to that of any naturally occurring nucleic acid or to that
of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers,
for example, (a) a DNA which has the sequence of part of a naturally occurring genomic
DNA molecule but is not flanked by both of the coding sequences that flank that part of
the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid
incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a
manner such that the resulting molecule is not identical to any naturally occurring vector
or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a
fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d)
a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a
fusion protein. The nucleic acid described above can be used to express the fusion protein
of this invention. For this purpose, one can operatively linked the nucleic acid to suitable
regulatory sequences to generate an expression vector.
A vector refers to a nucleic acid molecule capable of transporting another nucleic
acid to which it has been linked. The vector can be capable of autonomous replication or
integrate into a host DNA. Examples of the vector include a plasmid, cosmid, or viral
vector. The vector includes a nucleic acid in a form suitable for expression of the nucleic
acid in a host cell. Preferably the vector includes one or more regulatory sequences
operatively linked to the nucleic acid sequence to be expressed.
A "regulatory sequence" includes promoters, enhancers, and other expression
control elements (e.g., polyadenylation signals). Regulatory sequences include those that
direct constitutive expression of a nucleotide sequence, as well as tissue-specific
regulatory and/or inducible sequences. The design of the expression vector can depend on
such factors as the choice of the host cell to be transformed, the level of expression of
protein or RNA desired, and the like. The expression vector can be introduced into host
cells to produce a polypeptide of this invention. A promoter is defined as a DNA
sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A
strong promoter is one which causes mRNAs to be initiated at high frequency.
Any polynucleotide as mentioned above or a biologically equivalent
polynucleotide available to the artisan for the same intended purpose may be inserted into
an appropriate expression vector and linked with other DNA molecules to form
"recombinant DNA molecules" expressing this receptor. These vectors may be comprised
of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors
include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial
chromosomes and other forms of episomal or integrated DNA. It is well within the
purview of the artisan to determine an appropriate vector for a particular use.
A variety of mammalian expression vectors may be used to express the above-
mentioned IgG Fes in mammalian cells. As noted above, expression vectors can be DNA
sequences that are required for the transcription of cloned DNA and the translation of their
mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a
variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells.
Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-
yeast or bacteria-animal cells. An appropriately constructed expression vector should
contain: an origin of replication for autonomous replication in host cells, selectable
markers, a limited number of useful restriction enzyme sites, a potential for high copy
number, and active promoters. Expression vectors may include, but are not limited to,
cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
Commercially available mammalian expression vectors which may be suitable, include but
are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCTneo (Promega),
pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs),
pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl
(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV- 1(8-2) (ATCC
37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo
(ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC
37565).
Also within the scope of this invention is a host cell that contains the above-
described nucleic acid. Examples include E. coli cells, insect cells (e.g., using baculovirus
expression vectors), yeast cells, or mammalian cells. See e.g., Goeddel, (1990) Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.
To produce a polypeptide of this invention, one can culture a host cell in a medium under
conditions permitting expression of the polypeptide encoded by a nucleic acid of this
invention, and purify the polypeptide from the cultured cell or the medium of the cell.
Alternatively, the nucleic acid of this invention can be transcribed and translated in vitro,
e.g., using T7 promoter regulatory sequences and T7 polymerase.
All of naturally occurring IgG Fes, genetic engineered IgG Fes, and chemically
synthesized IgG Fes can be used to practice the invention disclosed therein. IgG Fc
obtained by recombinant DNA technology may have the same amino acid sequence as
[FA241] SEQ ID NO: 2) or an functionally equivalent thereof. The term "IgG Fc" also
covers chemically modified versions. Examples of chemically modified IgG Fc include
IgG Fes subjected to conformational change, addition or deletion of a sugar chain, and IgG
Fc to which a compound such as polyethylene glycol has been bound.
One can verify the efficacy of a polypeptide/protein thus-made using an animal
model, such as a transgenic mouse, as described below. Any statistically significant
increase in in vivo expression of IL-33 basophils or expression of the FcyRIIB receptor on
effector macrophages indicates the polypeptide/protein is a candidate for treating the
disorders mentioned below. In one embodiment, the above described assays may based on
measurement of a binding to DC-SIGN protein or DC-SIGN cells. The art is replete
with various techniques available to the artisan that will be suitable to measuring the
ability of a compound to a DC-SIGN or to DC-SIGN cells and related changes in
expression of a gene regulated by the DC-SING pathway, such as IL-33. The artisan will
be capable of mixing and matching these various research tools without undue
experimentation. Once purified and tested by standard methods or according to the assays
and methods described in the examples below, non-sialylated IgG Fc variants can be
included in pharmaceutical composition for treating inflammatory disorders.
As used herein, "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.
As used herein, "antibody fragments", may 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 CHI 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.
As used herein, the term "Fc fragment" or "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 avariant 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.
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 75 or 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.
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 FcyRI, FcyRII, and FcyRIII subclasses,
including allelic variants and alternatively spliced forms of these receptors. FcyRII
receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ primarily in the
cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
FcyRIIB 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).
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).
Compositions
Within the scope of this invention is a composition that contains a suitable carrier
and one or more of the agents described above, such as the non-sialylated IgG Fc variants.
The composition can be a pharmaceutical composition that contains a pharmaceutically
acceptable carrier or a cosmetic composition that contains a cosmetically acceptable
carrier.
The term "pharmaceutical composition" refers to the combination of an active
agent with a carrier, inert or active, making the composition especially suitable for
diagnostic or therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable carrier,"
after administered to or upon a subject, does not cause undesirable physiological effects.
The carrier in the pharmaceutical composition must be "acceptable" also in the sense that
it is compatible with the active ingredient and can be capable of stabilizing it. One or
more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an
active compound. Examples of a pharmaceutically acceptable carrier include, but are not
limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a
composition usable as a dosage form. Examples of other carriers include colloidal silicon
oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.
The above-described composition, in any of the forms described above, can be
used for treating disorders characterized by inflammation. An effective amount refers to
the amount of an active compound/agent that is required to confer a therapeutic effect on a
treated subject. Effective doses will vary, as recognized by those skilled in the art,
depending on the types of diseases treated, route of administration, excipient usage, and
the possibility of co-usage with other therapeutic treatment.
A pharmaceutical composition of this invention can be administered parenterally,
orally, nasally, rectally, topically, or buccally. The term "parenteral" as used herein refers
to subcutaneous, intracutaneous, intravenous, intrmuscular, intraarticular, intraarterial,
intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any
suitable infusion technique.
A sterile injectable composition can be a solution or suspension in a non-toxic
parenterally acceptable diluent or solvent. Such solutions include, but are not limited to,
1,3-butanediol, mannitol, water, Ringer's solution, and isotonic sodium chloride solution.
In addition, fixed oils are conventionally employed as a solvent or suspending medium
(e.g., synthetic mono- or diglycerides). Fatty acid, such as, but not limited to, oleic acid
and its glyceride derivatives, are useful in the preparation of injectables, as are natural
pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil,
polyoxyethylated versions thereof. These oil solutions or suspensions also can contain a
long chain alcohol diluent or dispersant such as, but not limited to, carboxymethyl
cellulose, or similar dispersing agents. Other commonly used surfactants, such as, but not
limited to, TWEENS or SPANS or other similar emulsifying agents or bioavailability
enhancers, which are commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms also can be used for the purpose of formulation.
A composition for oral administration can be any orally acceptable dosage form
including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions.
In the case of tablets, commonly used carriers include, but are not limited to, lactose and
corn starch. Lubricating agents, such as, but not limited to, magnesium stearate, also are
typically added. For oral administration in a capsule form, useful diluents include, but are
not limited to, lactose and dried corn starch. When aqueous suspensions or emulsions are
administered orally, the active ingredient can be suspended or dissolved in an oily phase
combined with emulsifying or suspending agents. If desired, certain sweetening,
flavoring, or coloring agents can be added.
Pharmaceutical compositions for topical administration according to the described
invention can be formulated as solutions, ointments, creams, suspensions, lotions,
powders, pastes, gels, sprays, aerosols, or oils. Alternatively, topical formulations can be
in the form of patches or dressings impregnated with active ingredient(s), which can
optionally comprise one or more excipients or diluents. In some preferred embodiments,
the topical formulations include a material that would enhance absorption or penetration of
the active agent(s) through the skin or other affected areas. The topical composition is
useful for treating inflammatory disorders in the skin, including, but not limited to eczema,
acne, rosacea, psoriasis, contact dermatitis, and reactions to poison ivy.
A topical composition contains a safe and effective amount of a dermatologically
acceptable carrier suitable for application to the skin. A "cosmetically acceptable" or
"dermatologically-acceptable" composition or component refers a composition or
component that is suitable for use in contact with human skin without undue toxicity,
incompatibility, instability, allergic response, and the like. The carrier enables an active
agent and optional component to be delivered to the skin at an appropriate
concentration(s). The carrier thus can act as a diluent, dispersant, solvent, or the like to
ensure that the active materials are applied to and distributed evenly over the selected
target at an appropriate concentration. The carrier can be solid, semi-solid, or liquid. The
carrier can be in the form of a lotion, a cream, or a gel, in particular one that has a
sufficient thickness or yield point to prevent the active materials from sedimenting. The
carrier can be inert or possess dermatological benefits. It also should be physically and
chemically compatible with the active components described herein, and should not
unduly impair stability, efficacy, or other use benefits associated with the composition.
The topical composition may be a cosmetic or dermatologic product in the form known in
the art for topical or transdermal applications, including solutions, aerosols, creams, gels,
patches, ointment, lotion, or foam.
Treatment Methods
The described invention provides methods for treating in a subject an inflammatory
disorder. The term "inflammatory disorder" refers to a disorder that is characterized by
abnormal or unwanted inflammation, such as an autoimmune disease. Autoimmune
diseases are disorders characterized by the chronic activation of immune cells under non-
activating conditions. Examples include psoriasis, inflammatory bowel diseases (e.g.,
Crohn's disease and ulcerative colitis), rheumatoid arthritis, psoriatic arthritis, multiple
sclerosis, lupus, type I diabetes, primary biliary cirrhosis, and transplant.
Other examples of inflammatory disorders that can be treated by the methods of
this invention include asthma, myocardial infarction, stroke, inflammatory dermatoses
(e.g., dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria,
necrotizing vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, eosinophilic
myositis, polymyositis, deraiatomyositis, and eosinophilic fasciitis), acute respiratory
distress syndrome, fulminant hepatitis, hypersensitivity lung diseases (e.g.,
hypersensitivity pneumonitis, eosinophilic pneumonia, delayed-type hypersensitivity,
interstitial lung disease (ILD), idiopathic pulmonary fibrosis, and ILD associated with
rheumatoid arthritis), and allergic rhinitis. Additional examples also include myasthenia
gravis, juvenile onset diabetes, glomerulonephritis, autoimmune throiditis, ankylosing
spondylitis, systemic sclerosis, acute and chronic inflammatory diseases (e.g., systemic
anaphylaxia or hypersensitivity responses, drug allergies, insect sting allergies, allograft
rejection, and graft-versus-host disease), and Sjogren's syndrome.
A "subject" refers to a human and a non-human animal. Examples of a non-human
animal include all vertebrates, e.g., mammals, such as non-human mammals, non-human
primates (particularly higher primates), dog, rodent (e.g., mouse or rat), guinea pig, cat,
and rabbit, and non-mammals, such as birds, amphibians, reptiles, etc. In one
embodiment, the subject is a human. In another embodiment, the subject is an
experimental, non-human animal or animal suitable as adisease model.
A subject to be treated for an inflammatory disorder can be identified by standard
diagnosing techniques for the disorder. Optionally, the subject can be examined for the
level or percentage of one or more of cytokines or cells atest sample obtained from the
subject by methods known in the art. If the level or percentage is at or below a threshold
value (which can be obtained from a normal subject), the subject is a candidate for
treatment described herein. To confirm the inhibition or treatment, one can evaluate
and/or verify the level or percentage of one or more of the above-mentioned cytokines or
cells in the subject after treatment.
"Treating" or "treatment" refers to administration of a compound or agent to a
subject who has a disorder with the purpose to cure, alleviate, relieve, remedy, delay the
onset of, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state
secondary to the disorder, or the predisposition toward the disorder.
An "effective amount" or "therapeutically effective amount" refers to an amount of
the compound or agent that is capable of producing a medically desirable result in a
treated subject. The treatment method can be performed in vivo or ex vivo, alone or in
conjunction with other drugs or therapy. A therapeutically effective amount can be
administered in one or more administrations, applications or dosages and is not intended to
be limited to a particular formulation or administration route.
The agent can be administered in vivo or ex vivo, alone or co-administered in
conjunction with other drugs or therapy, i.e., a cocktail therapy. As used herein, the term
"co-administration" or "co-administered" refers to the administration of at least two agents
or therapies to a subject. In some embodiments, the co-administration of two or more
agents/therapies is concurrent. In other embodiments, a first agent/therapy is administered
prior to a second agent/therapy. Those of skill in the art understand that the formulations
and/or routes of administration of the various agents/therapies used may vary.
In an in vivo approach, a compound or agent is administered to a subject.
Generally, the compound or agent is suspended in a pharmaceutically- acceptable carrier
(such as, for example, but not limited to, physiological saline) and administered orally or
by intravenous infusion, or injected or implanted subcutaneously, intramuscularly,
intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically,
intratracheally, or intrapulmonarily.
The dosage required depends on the choice of the route of administration; the
nature of the formulation; the nature of the patient's illness; the subject's size, weight,
surface area, age, and sex; other drugs being administered; and the judgment of the
attending physician. Suitable dosages are in the range of 0.01-100 mg/kg. Variations in
the needed dosage are to be expected in view of the variety of compounds/agents available
and the different efficiencies of various routes of administration. For example, oral
administration would be expected to require higher dosages than administration by i.v.
injection. Variations in these dosage levels can be adjusted using standard empirical
routines for optimization as is well understood in the art. Encapsulation of the compound
in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) can
increase the efficiency of delivery, particularly for oral delivery.
Example 1: Methods and Materials
This example describes general methods and materials used in Examples 2-7.
Mice
Wild-type C57BL/6 mice were purchased from Jackson Laboratories. SIGNR1
mice were provided by A. McKenzie. CDllc-DC-SIGN transgenic mice were provided
by T. Sparwasser. hDC-SIGN BAC transgenic mice in a SIGNRl background were
generated in inventors' laboratory as previously described. KRN TCR C57BL/6 mice
(gifts from D. Mathis and C. Benoist) were bred with NOD mice to generate K/BxN mice.
Blood from K/BxN mice (6-12 weeks of age) were collected and serum containing
arthritogenic antibodies pooled together. Passive transfer of 200 K/BxN serum by i.v.
injection to naive mice (8-12 weeks of age) induced arthritis. Inflammation was scored 0-
3 for each paw and added together for a total clinical score per individual mouse.
Recombinant Fc preparation
IDEC-114, a recombinant source of full-length human IgGl monoclonal antibody,
was digested with papain overnight at 37°C to cleave the Fab and Fc fragments.
Following digest, the reaction was stopped by the addition 2.5 mg/mL iodoacetamide. To
separate cleaved fragments from undigested antibody, samples were passed over a HiPrep
26/60 S-200HR size-exclusion column (GE HEALTHCARE). The Fc fragment was
subsequently purified with protein G agarose beads. Sample purity was verified by
coomassie brilliant blue staining of SDS-polyacrylamide gels. Alternatively, recombinant
Fes were produced by the transient transfection of human IgGl Fc-expressing plasmids
into 293T cells followed by ammonium sulfate precipitation of supernatant fractions and
protein G purification. The genetic sequence coding for the Fc region of human IgGl was
amplified from 420 IgGl by standard PCR protocols and ligated into pSecTag2
(INVITROGEN). Point mutations were introduced into the Fc coding sequence by
standard site-directed mutagenesis techniques and verified by DNA sequencing. PCR
primers for the Phe to Ala substitution at position 241 (FA241) were
'-ggggaccgtcagtcgccctcttccccccaa-3' (SEQ ID NO: 4) and
'- ttggggggaagagggcgactgacggtcccc-3' (SEQ ID NO: 5).
Protein expression and purity was confirmed by immunoblotting with anti-human Fc
antibodies and/or coomassie brilliant blue staining of SDS-polyacrylamide gels.
Two-step in vitro sialylation reaction
Following purification, 10-50 mg/mL Fc fragments were buffer exchanged into a
galactosylation reaction buffer (50mM MOPS, pH 7.2; 20mM MnCl ) and incubated
overnight at 37°C with 50 mg UDP-galactose and 0.75 U pi,4-galatosyltransferase.
Galactosylation was confirmed by lectin blots using ECL to recognize terminal galactose
residues. Galactosylated Fes were then buffer exchanged into a sialylation reaction buffer
(100 mM MOPS, 0.2 mg/mL BSA, 0.5% Triton X-100, pH 7.4) and incubated overnight
at 37°C with 50 mg CMP-sialic acid and 0.75 U a2,6-sialyltransferase. Sialylation was
confirmed by lectin blots using SNA to recognize terminal sialic acid residues with an a2-
6 linkage.
Adoptive transfer of bone marrow-derived macrophages
Bone marrow cells were flushed from tibias and femurs of DC-SIGNtg or
SIGNRl mice, and seeded in non-tissue culture treated 10-cm plates in RPMI 1640
growth media supplemented with 10% FBS, 1% Pen/Strep, IL-3 (5 ng/mL,
PEPROTECH), and M-CSF (5 ng/mL, PEPROTECH). Following overnight incubation at
37°C, non-adherent cells were recovered and transferred to non-tissue culture treated 10-
cm plates with IL-3/M-CSF-supplemented RPMI growth media, and cultured for 5-7 days
at 37°C. Mature macrophages were trypsinized and plated in 6-well plates at a density of
2xl0 cells/well, and allowed to attach overnight. The next day macrophages were pulsed
with the indicated recombinant Fc preparation for 30 min at 37°C. The cells were
recovered, washed with cold PBS, and lxlO cells were administered i.v. to wild-type
C57BL/6 mice. One hour post-injection, recipient mice were challenged with K/BxN sera.
Expression and purification of soluble human DC-SIGN
A plasmid containing the cDNA sequence of the extracellular domain (ECD) of
human DC-SIGN was provided by K. Drickamer. The coding sequence for DC-SIGN
ECD was modified to introduce an N-terminal strep tag by standard PCR techniques and
ligated to pET28b(+). pET28b-strepDCSIGN was transformed into E. coli strain
BL21/DE3 and grown in 3 L of TB growth medium at 37°C until bacteria culture reached
OD 0.7-0.8. Protein expression was induced by the addition of 100 mg/L of IPTG and
cultures incubated at 37°C for 3.5 h. Bacteria were pelleted by centrifugation at 4000xg
for 10 min at 4°C. Bacteria pellets were resuspended in 10 mM Tris-HCl, pH 7.8, and
lysed by sonication. Inclusion bodies were pelleted by centrifugation at 10,000xg for 15
min at 4°C and solubilized in 100 mL of 6 M guanidine-HCl; 100 mM Tris-HCl, pH 7.8;
0.2% TRITON X-100. Particulate matter was removed by centrifugation at 20,000xg for
min at 4°C, and the supernatant fraction was dialyzed against 250 mM NaCl; 25 mM
Tris-HCl, pH 7.8; 25 mM CaCl . After dialysis, insoluble precipitates were removed by
centrifugation at 20,000xg for 30 min at 4°C, and the supernatant fraction applied to a
strep-tactin resin (NOVAGEN) to pull down strep-tagged DC-SIGN ECD. Bound
proteins were eluted from resin with elution buffer supplied by manufacturer
(NOVAGEN). Fractions were analyzed by SDS-PAGE and positive fractions combined
and loaded onto a mannose-agarose column to select for active receptors. DC-SIGN ECD
was eluted with 250 mM NaCl; 25 mM Tris-HCl, pH 7.8; 5 mM EDTA. Fractions were
analyzed by SDS-PAGE.
Surface plasmon resonance
To determine the interaction between various recombinant Fc preparations to
soluble hDC-SIGN or hFcyRs, steady-state affinity measurements were recorded on a
Biacore T100 sensor. Receptors, diluted to 20-50 μg/mL in NaOAc pH 5.0, were
immobilized on CM5 chips at high density (2000 RU) by standard amine coupling. For
hDC-SIGN interactions, injections were performed at aflow rate of 20 μΕ/ηη with
commercially available HBS-P+ buffer adjusted to pH 9.0 and supplemented with 2 mM
CaCl and 500 mM NaCl. For hFcyRs interactions, injections were performed at aflow
rate of 20 μΕ/ηη with commercially available HBS-EP+ buffer. Surfaces were
regenerated with a short pulse of 50 mM NaOH. K values were calculated after
subtraction of background binding to a control flow cell using Biacore Evaluation
software.
RT-PCR
Total RNA was extracted from bone marrow-derived macrophages using RNeasy
Mini kit (QIAGEN). One microgram of total RNA was used to analyze IL-33 mRNA
expression by RT-PCR using OneStep RT-PCR kit (QIAGEN). Expression of GAPDH
served as a loading control. PCR primers for mIL-33 were 5'-gaagatcccaacagaagacc-3'
(SEQ ID NO: 6) and 5'-ttccggaggcgagacgtcac-3'(SEQ ID NO: 7); and mGAPDH were 5'-
gccgcctggagaaacctgc-3' (SEQ ID NO: 8) and 5'-tgaggtccaccaccctgttg-3' (SEQ ID NO: 9).
The PCR conditions were 94°C for 30 s; 55°C for 30 s; 72°C for 60 sx 35 cycles (IL-33)
or 25 cycles (GAPDH).
Example 2: a2,6-linked sialic acid conferred DC-SIGN binding activity to recombinant
human IgGl Fc
A minor population of antibodies in IVIG preparations suppresses autoantibody-
induced inflammation. These antibodies, containing terminal a2,6-linked sialic acid on
the Fc glycan, mediate an anti-inflammatory response by binding to SIGNR1 on marginal
zone macrophages or its human orthologue, DC-SIGN, on myeloid cells.
To study the interaction of sFc to DC-SIGN, a soluble form of the extracellular
domain of DC-SIGN (DC-SIGN ECD) was purified from bacteria and immobilized on
CM5 chips. sFc was prepared from full-length IDEC-114 antibodies and sialylated in
vitro (Figure lb), and was specifically bound to DC-SIGN-conjugated surface (Figure la).
Steady-state affinity measurements were carried out and the K value for this interaction
was calculated as ~1.3xl0 M (Figure l ). In contrast, an asialylated glycoform of
IDEC-114 Fc showed no binding activity to DC-SIGN suggesting that sialylation induces
a conformational change in on the Fc backbone to reveal a DC-SIGN binding site.
As shown in Figure la, it was found that recombinant cc2,6-sFc bound to soluble
DC-SIGN as measured by surface plasmon resonance (SPR). Fes were prepared by
papain cleavage of full-length human monoclonal IgGl antibody (IDEC-114) followed by
in vitro galactosylation and sialylation reactions. The SPR sensorgrams for antibody
binding to immobilized DC-SIGN are shown for sialylated and galactosylated glycoforms
of hlgGl Fes as described above in Example 1. It was found that Fc concentrations
flowed over DC-SIGN ECD range from 3-0.8 μΜ . As shown in Figure lb, lectin blot
with SNA confirmed attachment of sialic acid with 2,6-linkage on Fc (top panel);
coomassie stained loading controls was shown in bottom panel. Steady-state K
measurement of sFc binding to DC-SIGN (as shown in a.) was calculated by Biacore
Evaluation software.
Example 3 Mutations disrupting Fc-glycan interactions conferred DC-SIGN binding
activity to recombinant human IgGl Fc
The core oligosaccharide chain attached to Asn makes extensive noncovalent
interactions with the amino acid backbone of the Fc. Conformational changes in the Fc
induced by different sugar residues attached to the core glycan are mediated by these
protein-carbohydrate interactions. Alanine substitutions that abolish key contact points
between the Fc backbone and glycan residues appear to impart DC-SIGN binding activity.
As shown in Figure 2A, FA241 and FA243 mutations exhibit DC-SIGN binding
activity without in vitro enzymatic processing. Apparent K values range from 6x10 ' M
for FA241 to 3x10 M for FA243. Previous reports indicate that these mutations increase
sialylation of antibodies, presumably by making the glycan more accessible to
glycosyltransferases, when expressed in mammalian cells. To verify this, a lectin blot was
performed to determine if FA241 and FA243 binding to DC-SIGN is due to increased
sialylation of transiently expressed proteins. As shown in Figure 2B, SNA blots did not
detect terminal sialic acid residues in purified FA241 and FA243 indicating that DC-SIGN
interaction was independent of sialic acid modification.
More specifically, residues F241, F243, D265, and R301 along the amino acid
backbone of IgGl Fc were replaced with alanine to disrupt noncovalent interactions with
oligosaccharide residues. Fes were expressed and purified from 293T cells and analyzed
for DC-SIGN binding activity with surface plasmon resonance as described above. It was
found Fes bearing mutations FA241 or FA243 exhibited increased affinity to DC-SIGN
relative to affinity measurements for sFc (Figure la). Lectin blots with ECL (Figure lb,
middle panel) and SNA (top panel) were carried out to determine terminal sugar moieties
on Fes purified from 293T cells. As a positive control for sialylated Fes, FA241 was
sialylated in vitro as described in Figure la. Coomassie stained loading controls shown in
bottom panel of Figure lb.
Example 4 FA241 mutation in hlgGl Fc recapitulated anti-inflammatory activity of 26
If the FA241 and FA243 mutations mimic the DC-SIGN binding activity of sFc,
assays were carried out to examine if these mutations could replicate the anti
inflammatory activity of sFc in vivo. Age- and sex-matched SIGNR1 and hDC-
SIGN /SIGNRr mice were challenged with arthritogenic K/BxN sera and treated with
sFc, FA241, or FA243 at an effective dose of 0.033 g g. Consistent with previous
findings, sFc suppressed footpad swelling in DC-SIGN mice but not in SIGNRl .
Similarly, FA241 demonstrated comparable anti-inflammatory activity to that of sFc in
hDC-SIGN /SIGNRr mice. Mice administered FA243 showed no reduction in joint
inflammation. These findings suggest that recombinant Fes bearing an F A mutation
(FA241) recapitulates the DC-SIGN binding and anti-inflammatory activity of sFc in the
absence of sialic acid modification.
As showing in Figure 3, hDC-SIGN /SIGNRl (white squares) and SIGNR1
(black squares) mice were administered 0.7 mg/mouse of sFc, FA241, or FA243 by i.v.
injection. Mice were subsequently challenged with K/BxN sera 1 h later. Footpad
swelling was monitored and scored over several days. As previously reported, anti
inflammatory activity of sFc is DC-SIGN-dependent (left panel). FA241 also suppressed
arthritic inflammation in K/BxN challenged mice in a DC-SIGN-dependent manner.
FA243 did not reduce significantly footpad swelling at day 6. Means and SEM of clinical
scores of 4-5 mice per group are plotted on Day 6.
Example 5 Characterizing requirements for FA241 anti-inflammatory activity
To identify the determinants for the anti-inflammatory activity of FA241, bone
marrow-derived macrophages (ΒΜΜΦ) from CDllc.DC-SIGN and SIGNR1 mice
were stimulated with FA241 or other Fc preparations and transferred to WT C57BL/6
recipient mice challenged with K/BxN sera.
Briefly, bone marrow-derived macrophages from CDllc.DC-SIGN and SIGNR1
mice were cultured in IL-3 (5 ng/mL) and M-CSF (5 ng/mL) for 5-7 days. As shown
Figure 4, DC-SIGN ΒΜΜΦ were pulsed with 0.5 mg/mL asialylated (black bars) or
sialylated (white bars) glycoforms of indicated Fc preparations. Fc-treated ΒΜΜΦ were
transferred to WT C57BL/6 recipient mice followed by K/BxN challenge. As shown in
Figure 4b SIGNRl (black bars) and DC-SIGN (white bars) ΒΜΜΦ were pulsed with
0.5 mg/mL of indicated Fc preparation and transferred to WT C57BL/6 recipient mice
followed by K BxN challenge. Similarly, DC-SIGN ΒΜΜΦ were pulsed with either 0.5
mg/mL of FA241 or deglycosylated FA241 (Figure 4c, white bars) or PBS (Figure 4c
black bar) and transferred to WT C57BL/6 recipient mice followed by K/BxN challenge.
FA241 was deglycosylated with PNGase F and glycan removal confirmed by lectin
blotting. DC-SIGN ΒΜΜΦ were pulsed with indicated asialylated Fc preparation or
PBS (Figure 4d, black circle) and transferred to WT C57BL/6 recipient mice followed by
K/BxN challenge. In all cases, footpad swelling was monitored and scored over several
days. Means and SEM of clinical scores of 4-5 mice per group are plotted. *P < 0.05, as
determined by an analysis of variance (ANOVA) test, followed by a Tukey post hoc test.
As shown in Figure 4A, asialylated or sialylated FA241 preparations were equally
effective at suppressing joint inflammation compared to sFc. WT Fc preparations,
however, required a2,6-linked sialic acid since DC-SIGN ΒΜΜΦ pulsed with asialylated
WT Fc did not transfer protection to recipient mice. Corresponding with results showed in
Figure 3, both sFc and FA241 required DC-SIGN expression on ΒΜΜΦ to transfer
protection (Figure 4B). Though FA241 does not require sialic acid to transfer protection,
deglycosylation with PNGase F abrogated the anti-inflammatory properties of FA241
(Figure 4C) suggesting that the Fc glycan is still necessary. Furthermore, to show that the
observed anti-inflammatory activity is specific for the F iA mutation, DC-SIGN ΒΜΜΦ
were pulsed with Fes bearing alternative mutations that do not confer enhanced DC-SIGN
binding. Only FA241- stimulated ΒΜΜΦ protected K/BxN challenged recipient mice.
Example 6 FA241 mutation increased Fey receptor binding
If an alanine substitution at position 241 induces a conformational change in the
Fc, then perhaps affinity towards human Fey receptors will be altered. It was previously
reported that sialylation reduces the affinity of IgGs to FcyRs, consequently attenuating
ADCC activity in vivo.
Recombinant IgGl Fc's binding to soluble FcyRs was measured by surface
plasmon resonance (SPR). Fes were prepared in the manner described above. The SPR
sensorgrams for antibody binding to immobilized hFcYRIIA and hFcyRIIB are shown
in Figure 5 for asialylated and sialylated glycoforms of WT hlgGl Fc and for asialylated
FA241 Fc.
As shown in Figure 5, asialylated glycoforms of WT Fc bound to hFcyRIIA and
RUB with an observed K value of ~2-3xl 0 M. sFc, however, did not appear to bind
either hFcyRIIA or RUB. Surprisingly, FA241 appears to bind both hFcyRIIA and RUB
with an order of magnitude greater affinity (K = ~2xl0 M).
Example 7 IL-33 mRNA induction in bone marrow-derived macrophages by FA241
sFc induce a T 2-dependent anti-inflammatory pathway that requires IL-4
secretion from an FcsRI leukocyte population, likely basophils, to upregulate FcyRIIB on
regulatory macrophages. Administration of IL-33 in vivo or in vitro stimulates basophils
to release stores of IL-4. IL-33 mRNA expression is upregulated in spleens of WT
C57BL/6 mice treated with sFc or IVIG, but not in SIGNRl mice. This suggests that
sFc may induce IL-33 expression in SIGNR1 or DC-SIGN cells. In this example, assays
were carried out and show that stimulating DC-SIGN ΒΜΜΦ with FA241 appears to
upregulate IL-33 expression.
More specifically, bone marrow-derived macrophages from CD1 lc.DC-SIGN and
SIGNRl mice were cultured in the manner described above. ΒΜΜΦ were seeded onto
12-well plates in serum-free RPMI media and allowed to adhere overnight at 37°C. Next
day, cells were pulsed with 0.5 mg/mL of indicated Fc in serum-free RPMI media for 1 h
(Figure 6a.) or 4 h (Figure 6b.) at 37°C. mRNA was harvested from cells at specified time
points and 1 g total RNA used for RT-PCR amplification of IL-33 mRNA (top panels).
GAPDH amplification served as a loading control (bottom panels). Plasmid co-expressing
DA265 Fc and human sialyltransferase (ST6Gall) were transfected into 293T cells
yielding highly sialylated recombinant Fc (ST6-DA265).
As shown in Figure 6, stimulating DC-SIGN ΒΜΜΦ with FA241 appears to
upregulate IL-33 expression. Despite greater basal expression levels of IL-33 compared to
DC-SIGN ΒΜΜΦ, SIGNR1 ΒΜΜΦ downregulated IL-33 mRNA expression in
response to FA241 treatment.
The foregoing example and description of the preferred embodiments should be
taken as illustrating, rather than as limiting the present invention as defined by the claims.
All publications cited herein are hereby incorporated by reference in their entirety. As will
be readily appreciated, numerous variations and combinations of the features set forth
above can be utilized without departing from the present invention as set forth in the
claims. Such variations are not regarded as a departure from the scope of the invention,
and all such variations are intended to be included within the scope of the following
claims.
SHR510611NZPR
303913701
Unless the context clearly requires otherwise, throughout the description and the
claims, the words “comprise”, comprising”, and the like, are to be construed in an
inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense
of “including, but not limited to”.
The reference to any prior art in the specification is not, and should not be taken as,
an acknowledgement or any form of suggestion that the prior art forms part of the
common general knowledge in New Zealand.
SHR510611NZPR
304074531
Claims (27)
1. An isolated polypeptide comprising a modified sequence that is at least 75% identical to an IgG Fc region with the sequences of SEQ ID No: 1 or 2, and has a substitution at a position corresponding to F241 of SEQ ID No: 1 according to the Kabat numbering system, wherein the modified sequence is free of sialylation and the polypeptide has an anti- inflammatory activity that is higher than that of a parent polypeptide.
2. The isolated polypeptide of claim 1, wherein the parent polypeptide comprises the IgG Fc region.
3. The isolated polypeptide of claim 1 or claim 2, wherein the IgG Fc region comprises the sequence of SEQ ID NO:1.
4. The isolated polypeptide of any one of claims 1-3, wherein the isolated polypeptide has an ability to bind to DC-SIGN.
5. The isolated polypeptide of any one of claims 1-4, wherein the isolated polypeptide has an ability to bind to hFc γRIIA or RIIB
6. The isolated polypeptide of any one of claims 1-5, wherein the isolated polypeptide has an ability to bind to hFc γRIIA or RIIB at a K of 2x10 M or lower (i.e., K 4 -1 of 5.0 x 10 M or higher).
7. The isolated polypeptide of any one of claims 1-6, wherein the modified sequence has a FA241 mutation.
8. The isolated polypeptide of any one of claims 1-7, wherein the modified sequence is at least 75% identical to SEQ ID NO: 2.
9. The isolated polypeptide of claim 8, wherein the modified sequence is at least 80% identical to SEQ ID NO: 2. SHR510611NZPR 304074531
10. The isolated polypeptide of claim 9, wherein the modified sequence is at least 90% identical to SEQ ID NO: 2.
11. The isolated polypeptide of claim 10, wherein the modified sequence is at least 95% identical to SEQ ID NO: 2.
12. The isolated polypeptide of claim 11, wherein the modified sequence is at least 99% identical to SEQ ID NO: 2.
13. The isolated polypeptide of claim 12, wherein the modified sequence comprises SEQ ID NO: 2.
14. The isolated polypeptide of claim 9, wherein the modified sequence consists essentially of SEQ ID NO: 2.
15. A method for making a polypeptide having an anti-inflammatory activity, the method comprising: providing a parent polypeptide having the sequence of an IgG Fc region or a first nucleic acid sequence encoding the parent polypeptide; and modifying the parent polypeptide to obtain a modified polypeptide so that the modified polypeptide is free of sialylation wherein the modifying step comprises introducing a substitution at a position corresponding to F241 of SEQ ID No: 1 according to the Kabat numbering system.
16. The method of claim 15, wherein the modifying step is conducted by modifying the first nucleic acid sequence to obtain a second nucleic acid encoding the modified polypeptide.
17. The method of claim 16, comprising culturing an isolated host cell comprising the second nucleic acid in a medium under conditions permitting expression of the polypeptide encoded by the second nucleic acid, and purifying the polypeptide from the cultured cell or the medium of the cell.
18. A polypeptide made by the method of any one of claims 15-17. SHR510611NZPR 304074531
19. A pharmaceutical formulation comprising (i) the polypeptide of any one of claims 1-14 and 18 , and (ii) a pharmaceutically acceptable carrier.
20. A method of treating an inflammatory disease in a non-human subject, comprising administering to the non-human subject in need thereof a therapeutically effective amount of the polypeptide of any one of claims 1-14 and 18 or a pharmaceutical formulation of claim 19.
21. Use of a polypeptide of any one of claims 1-14 and 18 or a pharmaceutical formulation of claim 19 in the manufacture of a medicament for treating an inflammatory disease.
22. An isolated polypeptide as claimed in claim 1, substantially as hereinbefore described with particular reference to any one or more of the examples, figures and/or sequence listing.
23. A method for making a polypeptide as claimed in claim 15, substantially as hereinbefore described with particular reference to any one or more of the examples, figures and/or sequence listing.
24. A polypeptide as claimed in claim 18, substantially as hereinbefore described with particular reference to any one or more of the examples, figures and/or sequence listing.
25. A method of producing a polypeptide as claimed in claim 17, substantially as hereinbefore described with particular reference to any one or more of the examples, figures and/or sequence listing.
26. A pharmaceutical formulation as claimed in claim 19, substantially as hereinbefore described with particular reference to any one or more of the examples, figures and/or sequence listing.
27. A method as claimed in claim 20, substantially as hereinbefore described with particular reference to any one or more of the examples, figures and/or sequence listing.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161577361P | 2011-12-19 | 2011-12-19 | |
US61/577,361 | 2011-12-19 | ||
PCT/US2012/068718 WO2013095966A1 (en) | 2011-12-19 | 2012-12-10 | Non-sialylated anti-inflammatory polypeptides |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ627002A NZ627002A (en) | 2016-08-26 |
NZ627002B2 true NZ627002B2 (en) | 2016-11-29 |
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