U.S. patent application number 15/028917 was filed with the patent office on 2016-09-08 for sialylated glycoproteins.
This patent application is currently assigned to Momenta Pharmaceuticals Inc.. The applicant listed for this patent is MOMENTA PHARMACEUTICALS, INC.. Invention is credited to Chia Lin CHU, Leona E. LING, Laura RUTITZKY, Birgit C. SCHULTES, Lynn ZHANG.
Application Number | 20160257754 15/028917 |
Document ID | / |
Family ID | 52828588 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160257754 |
Kind Code |
A1 |
SCHULTES; Birgit C. ; et
al. |
September 8, 2016 |
SIALYLATED GLYCOPROTEINS
Abstract
Pharmaceutical preparations containing polypeptides having
particular sialylation patterns, and methods for the treatment of
immune-related thrombocytopenia with such preparations, are
described.
Inventors: |
SCHULTES; Birgit C.;
(Arlington, MA) ; CHU; Chia Lin; (Somerville,
MA) ; RUTITZKY; Laura; (Somerville, MA) ;
ZHANG; Lynn; (Acton, MA) ; LING; Leona E.;
(Winchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MOMENTA PHARMACEUTICALS, INC. |
Cambridge |
MA |
US |
|
|
Assignee: |
Momenta Pharmaceuticals
Inc.
Cambridge
MA
|
Family ID: |
52828588 |
Appl. No.: |
15/028917 |
Filed: |
October 14, 2014 |
PCT Filed: |
October 14, 2014 |
PCT NO: |
PCT/US2014/060363 |
371 Date: |
April 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61891778 |
Oct 16, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2848 20130101;
C07K 2317/21 20130101; A61K 2039/505 20130101; C07K 2317/41
20130101; C07K 16/00 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. A pharmaceutical preparation formulated for subcutaneous
administration, said preparation comprising polypeptides comprising
an Fc region, wherein at least 50% of branched glycans on the Fc
region are di-sialylated by way of NeuAc-.alpha. 2,6-Gal terminal
linkages.
2. The pharmaceutical preparation of claim 1, wherein said
polypeptides are present in said pharmaceutical preparation at a
concentration of 50-250 mg/mL.
3. The pharmaceutical preparation of claim 1, wherein less than 50%
of branched glycans on the Fc region are mono-sialylated on the
.alpha. 1,3 arm by way of a NeuAc-.alpha. 2,6-Gal terminal
linkage.
4. The pharmaceutical preparation of claim 1, wherein less than 50%
of branched glycans on the Fc region are mono-sialylated on the
.alpha. 1,6 arm by way of a NeuAc-.alpha. 2,6-Gal terminal
linkage.
5. A pharmaceutical preparation comprising polypeptides having an
Fc region, wherein at least 50% of branched glycans on the Fc
region are di-sialylated by way of NeuAc-.alpha. 2,6-Gal terminal
linkages and less than 50% of branched glycans on the Fc region are
mono-sialylated on the .alpha. 1,3 arm by way of a NeuAc-.alpha.
2,6-Gal terminal linkage.
6. A pharmaceutical preparation comprising polypeptides having an
Fc region, wherein at least 50% of branched glycans on the Fc
region are di-sialylated by way of NeuAc-.alpha. 2,6-Gal terminal
linkages and less than 50% of branched glycans on the Fc region are
mono-sialylated on the .alpha. 1,6 arm by way of a NeuAc-.alpha.
2,6-Gal terminal linkage.
7. A pharmaceutical preparation comprising polypeptides having an
Fc region, wherein at least 85% of branched glycans on the Fc
region are di-sialylated by way of NeuAc-.alpha. 2,6-Gal terminal
linkages.
8. The pharmaceutical preparation of claim 1, wherein said
polypeptides consist essentially of an Fc region.
9. The pharmaceutical preparation of claim 1, wherein said
polypeptides further comprise a Fab region, a heterologous
polypeptide sequence, or a non-polypeptide moiety.
10. The pharmaceutical preparation of claim 9, wherein at least 10%
of branched glycans on the Fab region or heterologous polypeptide
sequence of said polypeptides are mono-sialylated or
di-sialylated.
11. The pharmaceutical preparation of claim 9, wherein less than
80% of branched glycans on the Fab region or heterologous
polypeptide sequence of said polypeptides are mono-sialylated or
di-sialylated.
12. The pharmaceutical preparation of claim 1, wherein said
polypeptides are recombinant polypeptides.
13. The pharmaceutical preparation of claim 1, wherein said
polypeptides are derived from plasma.
14. The pharmaceutical preparation of claim 12, wherein said
polypeptides are IgG polypeptides or said polypeptides consist
essentially of an Fc region derived from IgG polypeptides.
15. A method of increasing reticulated platelets in a subject in
need thereof, comprising administering to the subject a
pharmaceutical preparation of claim 1.
16. A method of producing new platelets in a subject in need
thereof, comprising administering to the subject a pharmaceutical
preparation of claim 1.
17. A method of increasing reticulated platelets or producing new
platelets in a subject in need thereof, comprising administering to
the subject a pharmaceutical preparation comprising polypeptides
comprising an Fc region, wherein at least 85% of branched glycans
on the Fc region are di-sialylated by way of NeuAc-.alpha. 2,6-Gal
terminal linkages.
18. The method of claim 15, wherein the subject is not being
treated with thrombopoietin or a thrombopoietin receptor agonist or
the subject did not respond to treatment with thrombopoietin or a
thrombopoietin receptor agonist.
19. The method of claim 15, wherein the subject has immune-related
thrombocytopenia.
20. The method of claim 15, further comprising, before and/or after
the administering step, the step of determining the total platelet
count and/or the reticulated platelet count in the subject.
21. The method of claim 20, further comprising, after the
determining step, the step of adjusting the dose of the
administered pharmaceutical preparation.
22. The pharmaceutical preparation of claim 13, wherein said
polypeptides are IgG polypeptides or said polypeptides consist
essentially of an Fc region derived from IgG polypeptides.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/891,778, filed Oct. 16, 2013, which is hereby
incorporated by reference in its entirety.
BACKGROUND
[0002] Therapeutic glycoproteins are an important class of
therapeutic biotechnology products, and therapeutic Fc containing
glycoproteins, such as IVIg, Fc-receptor fusions, and antibodies
(including murine, chimeric, humanized, and human antibodies and
fragments thereof) account for the majority of therapeutic biologic
products.
SUMMARY OF THE INVENTION
[0003] The invention encompasses, in part, the discovery that
Fc-containing polypeptides that include branched glycans and that
are di-sialylated on the branched glycan (e.g., on an .alpha. 1,3
and/or .alpha. 1,6 arm in the Fc region's N-linked glycosylation
site), with, e.g., a NeuAc-.alpha. 2,6-Gal terminal linkage,
exhibit improved biological activity, e.g., relative to a reference
glycoprotein, e.g., in the treatment of hematological disease,
e.g., immune-related thrombocytopenia (ITP). The present disclosure
provides, in part, methods for treating hematological disease,
e.g., immune-related thrombocytopenia and related diseases by
administering compositions containing such Fc-containing
polypeptides as well as methods for evaluating, identifying, and/or
producing (e.g., manufacturing) such polypeptides.
[0004] In one aspect, the invention features a pharmaceutical
preparation formulated for subcutaneous administration (e.g., at a
concentration of 50-250 mg/mL, e.g., 50-100 mg/mL, 75-125 mg/mL,
100-150 mg/mL, 125-175 mg/mL, 150-200 mg/mL, 175-225 mg/mL, 200-250
mg/mL). This preparation includes polypeptides having an Fc region,
wherein at least 50% (e.g., 60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%,
94%, 95%, 97%, 98% up to and including 100%) of branched glycans on
the Fc region are di-sialylated by way of NeuAc-.alpha. 2,6-Gal
terminal linkages. In some embodiments, less than 50% (e.g., less
than 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%) of branched
glycans on the Fc region are mono-sialylated (e.g., on the .alpha.
1,3 arm or the .alpha. 1,6 arm) by way of a NeuAc-.alpha. 2,6-Gal
terminal linkage.
[0005] In another aspect, the invention features a pharmaceutical
preparation including polypeptides having an Fc region, wherein at
least 50% (e.g., 60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%,
97%, 98% up to and including 100%) of branched glycans on the Fc
region are di-sialylated by way of NeuAc-.alpha. 2,6-Gal terminal
linkages and less than 50% (e.g., less than 40%, 30%, 20%, 10%,
15%, 5%, 4%, 3%, 2%, 1%) of branched glycans on the Fc region are
mono-sialylated on the .alpha. 1,3 arm by way of a NeuAc-.alpha.
2,6-Gal terminal linkage.
[0006] In another aspect, the invention features a pharmaceutical
preparation comprising polypeptides having an Fc region, wherein at
least 50% (e.g., 60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%,
97%, 98% up to and including 100%) of branched glycans on the Fc
region are di-sialylated by way of NeuAc-.alpha. 2,6-Gal terminal
linkages and less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%,
4%, 3%, 2%, 1%) of branched glycans on the Fc region are
mono-sialylated on the .alpha. 1,6 arm by way of a NeuAc-.alpha.
2,6-Gal terminal linkage.
[0007] In another aspect, the invention features a pharmaceutical
preparation comprising polypeptides having an Fc region, wherein at
least 85% of branched glycans on the Fc region are di-sialylated by
way of NeuAc-.alpha. 2,6-Gal terminal linkages.
[0008] In some embodiments of any of the foregoing preparations,
the polypeptides consist essentially of an Fc region. In other
embodiments of any of the foregoing preparations, the polypeptides
further include a Fab region, a heterologous polypeptide sequence
such as a biological receptor sequence (e.g., the polypeptides are
Fc-receptor fusion proteins), or a heterologous non-polypeptide
moiety.
[0009] In certain embodiments, at least 10% (e.g., 20%, 30%, 40%,
50%, 60% 70% or more) of branched glycans on the Fab region or
heterologous polypeptide sequence of the polypeptides are
mono-sialylated or di-sialylated. In other embodiments, less than
80% (e.g., 70%, 60, 50%, 40%, 30%, 20%, 10%, 5% or less) of
branched glycans on the Fab region or heterologous polypeptide
sequence of the polypeptides are mono-sialylated or
di-sialylated.
[0010] In some embodiments of any of the foregoing preparations,
the polypeptides are recombinant polypeptides. In other embodiments
of any of the foregoing preparations, the polypeptides are derived
from plasma, e.g., human plasma. In certain embodiments, the
polypeptides are IgG polypeptides (e.g., IgG1, IgG2, IgG3 or IgG4)
or the polypeptides consist essentially of an Fc region derived
from IgG polypeptides.
[0011] In another aspect, the invention features a method of
increasing reticulated platelets in a subject in need thereof,
comprising administering to the subject any one of the foregoing
preparations.
[0012] In another aspect, the invention features a method of
producing new platelets in a subject in need thereof, comprising
administering to the subject any one of the foregoing
preparations.
[0013] In another aspect, the invention features a method of
increasing reticulated platelets or producing new platelets in a
subject in need thereof, comprising administering to the subject a
pharmaceutical preparation comprising polypeptides comprising an Fc
region, wherein at least 85% of branched glycans on the Fc region
are di-sialylated by way of NeuAc-.alpha. 2,6-Gal terminal
linkages.
[0014] In some embodiments of any of the foregoing methods, the
subject is not being treated with thrombopoietin or a
thrombopoietin receptor agonist (e.g., romiplostim, eltrombopag).
In some embodiments of any of the foregoing methods, the subject
has failed treatment with thrombopoietin or a thrombopoietin
receptor agonist (e.g., romiplostim, eltrombopag). In other
embodiments of any of the foregoing methods, the subject has a
hematological disease such as immune-related thrombocytopenia. In
certain embodiments of any of the foregoing methods, the method
further includes, after the administering step, the step of
determining the total platelet count and/or the reticulated
platelet count in the subject, e.g., wherein the total platelet
count and/or the reticulated platelet count increases as a result
of the administering step. In some embodiments, the method further
includes after the determining step, the step of adjusting the dose
of the administered pharmaceutical preparation.
DESCRIPTION OF THE FIGURES
[0015] FIG. 1 is a schematic illustration of a common core
pentasaccharide (Man).sub.3(GlcNAc)(GlcNAc) of N-glycans.
[0016] FIG. 2 is a schematic illustration of an IgG antibody
molecule.
[0017] FIG. 3A depicts an exemplary ST6 sialyltransferase amino
acid sequence (SEQ ID NO:1). FIG. 3B depicts an exemplary ST6
sialyltransferase amino acid sequence (SEQ ID NO:2). FIG. 3C
depicts an exemplary ST6 sialyltransferase amino acid sequence (SEQ
ID NO:3).
[0018] FIG. 4 is a schematic illustration of a reaction scheme for
ST6 sialyltransferase (fucose: triangles, N-acetylglucosamine:
squares, mannose: dark circles, galactose: light circles, sialic
acid: diamonds).
[0019] FIG. 5 is a graphic representation of relative abundance of
glycans at various times during a sialylation reaction with ST6
sialyltransferase.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Antibodies are glycosylated at conserved positions in the
constant regions of their heavy chain. For example, IgG antibodies
have a single N-linked glycosylation site at Asn297 of the CH2
domain. Each antibody isotype has a distinct variety of N-linked
carbohydrate structures in the constant regions. For human IgG, the
core oligosaccharide normally consists of
GlcNAc.sub.2Man.sub.3GlcNAc, with differing numbers of outer
residues. Variation among individual IgG's can occur via attachment
of galactose and/or galactose-sialic acid at one or both terminal
GlcNAc or via attachment of a third GlcNAc arm (bisecting
GlcNAc).
[0021] The present disclosure encompasses, in part, pharmaceutical
preparations including polypeptides having an Fc region having
particular levels of branched glycans that are sialylated on both
of the branched glycans in the Fc region (e.g., with a
NeuAc-.alpha. 2,6-Gal terminal linkage). The levels can be measured
on an individual Fc region (e.g., the number of branched glycans
that are sialylated on an .alpha.1,3 arm, an .alpha.1,6 arm, or
both, of the branched glycans in the Fc region), or on the overall
composition of a preparation of polypeptides (e.g., the number or
percentage of branched glycans that are sialylated on an .alpha.1,3
arm, an .alpha.1,6 arm, or both, of the branched glycans in the Fc
region in a preparation of polypeptides).
[0022] The inventors have discovered that Fc-region containing
polypeptides having branched glycans that are preferentially
di-sialylated (e.g., with NeuAc-.alpha. 2,6-Gal terminal linkages)
exhibit improved biological activity, e.g., relative to a reference
glycoprotein, and are useful in the treatment of immune-related
thrombocytopenia and related diseases.
[0023] Preparations useful herein can be obtained from any source.
In some instances, providing or obtaining a preparation (e.g., such
as a biologic drug substance or a precursor thereof), e.g., that is
or includes a polypeptide, can include providing a host cell, e.g.,
a mammalian host cell (e.g., a CHO cell) that is genetically
engineered to express a polypeptide (e.g., a genetically engineered
cell); culturing the host cell under conditions suitable to express
the polypeptide (e.g., mRNA and/or protein); and, optionally,
purifying the expressed polypeptide, e.g., in the form of a
recombinant fusion protein) from the cultured cell, thereby
producing a preparation.
DEFINITIONS
[0024] As used herein, "acquire or acquiring (e.g., acquiring
information)" means obtaining possession of a physical entity, or a
value, e.g., a numerical value, by "directly acquiring" or
"indirectly acquiring" the physical entity or value. "Directly
acquiring" means performing a process (e.g., performing an assay or
test on a sample or "analyzing a sample" as that term is defined
herein) to obtain the physical entity or value. "Indirectly
acquiring" refers to receiving the physical entity or value from
another party or source (e.g., a third party laboratory that
directly acquired the physical entity or value). "Directly
acquiring" a physical entity includes performing a process, e.g.,
analyzing a sample, that includes a physical change in a physical
substance, e.g., a starting material. Exemplary changes include
making a physical entity from two or more starting materials,
shearing or fragmenting a substance, separating or purifying a
substance, combining two or more separate entities into a mixture,
performing a chemical reaction that includes breaking or forming a
covalent or non-covalent bond. "Directly acquiring" a value
includes performing a process that includes a physical change in a
sample or another substance, e.g., performing an analytical process
which includes a physical change in a substance, e.g., a sample,
analyte, or reagent (sometimes referred to herein as "physical
analysis"), performing an analytical method, e.g., a method which
includes one or more of the following: separating or purifying a
substance, e.g., an analyte, or a fragment or other derivative
thereof, from another substance; combining an analyte, or fragment
or other derivative thereof, with another substance, e.g., a
buffer, solvent, or reactant; or changing the structure of an
analyte, or a fragment or other derivative thereof, e.g., by
breaking or forming a covalent or non-covalent bond, between a
first and a second atom of the analyte; or by changing the
structure of a reagent, or a fragment or other derivative thereof,
e.g., by breaking or forming a covalent or non-covalent bond,
between a first and a second atom of the reagent. Exemplary
analytical methods are shown in Table 1.
[0025] As used herein, the term "antibody" refers to a polypeptide
that includes at least one immunoglobulin variable region, e.g., an
amino acid sequence that provides an immunoglobulin variable domain
or immunoglobulin variable domain sequence. For example, an
antibody can include a heavy (H) chain variable region (abbreviated
herein as V.sub.H), and a light (L) chain variable region
(abbreviated herein as V.sub.L). In another example, an antibody
includes two heavy (H) chain variable regions and two light (L)
chain variable regions. The term "antibody" encompasses
antigen-binding fragments of antibodies (e.g., single chain
antibodies, Fab, F(ab').sub.2, Fd, Fv, and dAb fragments) as well
as complete antibodies, e.g., intact immunoglobulins of types IgA,
IgG, IgE, IgD, IgM (as well as subtypes thereof). The light chains
of the immunoglobulin can be of types kappa or lambda.
[0026] As used herein, a "batch" of a preparation refers to a
single production run. Evaluation of different batches thus means
evaluation of different production runs or batches. As used herein
"sample(s)" refer to separately procured samples. For example,
evaluation of separate samples could mean evaluation of different
containers or vials of the same batch or from different batches. A
batch can include a drug substance batch or a drug product
batch.
[0027] As used herein, the term "constant region" refers to a
polypeptide that corresponds to, or is derived from, one or more
constant region immunoglobulin domains of an antibody. A constant
region can include any or all of the following immunoglobulin
domains: a C.sub.H1 domain, a hinge region, a C.sub.H2 domain, a
C.sub.H3 domain (derived from an IgA, IgD, IgG, IgE, or IgM), and a
C.sub.H4 domain (derived from an IgE or IgM).
[0028] As used herein, "evaluating," e.g., in the
evaluation/evaluating, identifying, and/or producing aspects
disclosed herein, means reviewing, considering, determining,
assessing, analyzing, measuring, and/or detecting the presence,
absence, level, and/or ratio of one or more parameters in a
preparation to provide information pertaining to the one or more
parameters. In some instances, evaluating can include performing a
process that involves a physical change in a sample or another
substance, e.g., a starting material. Exemplary changes include
making a physical entity from two or more starting materials,
shearing or fragmenting a substance, separating or purifying a
substance, combining two or more separate entities into a mixture,
performing a chemical reaction that includes breaking or forming a
covalent or non-covalent bond. "Evaluating" can include performing
an analytical process which includes a physical change in a
substance, e.g., a sample, analyte, or reagent (sometimes referred
to herein as "physical analysis"), performing an analytical method,
e.g., a method which includes one or more of the following:
separating or purifying a substance, e.g., an analyte, or a
fragment or other derivative thereof, from another substance;
combining an analyte, or fragment or other derivative thereof, with
another substance, e.g., a buffer, solvent, or reactant; or
changing the structure of an analyte, or a fragment or other
derivative thereof, e.g., by breaking or forming a covalent or
non-covalent bond, between a first and a second atom of the
analyte; or by changing the structure of a reagent, or a fragment
or other derivative thereof, e.g., by breaking or forming a
covalent or non-covalent bond, between a first and a second atom of
the reagent.
[0029] As used herein, the term "Fc region" refers to a dimer of
two "Fc polypeptides," each "Fc polypeptide" including the constant
region of an antibody excluding the first constant region
immunoglobulin domain. In some embodiments, an "Fc region" includes
two Fc polypeptides linked by one or more disulfide bonds, chemical
linkers, or peptide linkers. "Fc polypeptide" refers to the last
two constant region immunoglobulin domains of IgA, IgD, and IgG,
and the last three constant region immunoglobulin domains of IgE
and IgM, and may also include part or the entire flexible hinge
N-terminal to these domains. For IgG, "Fc polypeptide" comprises
immunoglobulin domains Cgamma2 (C.gamma.2) and Cgamma3 (C.gamma.3)
and the lower part of the hinge between Cgamma1 (C.gamma.1) and
C.gamma.2. Although the boundaries of the Fc polypeptide may vary,
the human IgG heavy chain Fc polypeptide is usually defined to
comprise residues starting at T223 or C226 or P230, to its
carboxyl-terminus, wherein the numbering is according to the EU
index as in Kabat et al. (1991, NIH Publication 91-3242, National
Technical Information Services, Springfield, Va.). For IgA, Fc
polypeptide comprises immunoglobulin domains Calpha2 (C.alpha.2)
and Calpha3 (C.alpha.3) and the lower part of the hinge between
Calpha1 (C.alpha.1) and C.alpha.2. An Fc region can be synthetic,
recombinant, or generated from natural sources such as IVIg.
[0030] An "Fc region-containing polypeptide" is a polypeptide that
includes all or a substantial portion of an Fc region. Examples of
an Fc region-containing polypeptide preparation include, e.g., a
preparation of Fc fragments, a preparation of antibody molecules, a
preparation of Fc-fusion proteins (e.g., an Fc-receptor fusion
protein), and a preparation of pooled, polyvalent immunoglobulin
molecules (e.g., IVIg). Such an Fc region-containing polypeptide
may be recombinant (e.g., a recombinant Fc fragment preparation or
a recombinant antibody preparation) or naturally derived (such as
IVIg).
[0031] As used herein, "glycan" is a sugar, which can be monomers
or polymers of sugar residues, such as at least three sugars, and
can be linear or branched. A "glycan" can include natural sugar
residues (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic
acid, galactose, mannose, fucose, hexose, arabinose, ribose,
xylose, etc.) and/or modified sugars (e.g., 2'-fluororibose,
2'-deoxyribose, phosphomannose, 6'sulfo N-acetylglucosamine, etc.).
The term "glycan" includes homo and heteropolymers of sugar
residues. The term "glycan" also encompasses a glycan component of
a glycoconjugate (e.g., of a polypeptide, glycolipid, proteoglycan,
etc.). The term also encompasses free glycans, including glycans
that have been cleaved or otherwise released from a
glycoconjugate.
[0032] As used herein, the term "glycoprotein" refers to a protein
that contains a peptide backbone covalently linked to one or more
sugar moieties (i.e., glycans). The sugar moiety(ies) may be in the
form of monosaccharides, disaccharides, oligosaccharides, and/or
polysaccharides. The sugar moiety(ies) may comprise a single
unbranched chain of sugar residues or may comprise one or more
branched chains. Glycoproteins can contain O-linked sugar moieties
and/or N-linked sugar moieties.
[0033] As used herein, "immune-related thrombocytopenia" refers to
disorders in which there is a relative decrease of platelets in the
blood caused by increased destruction of platelets by the immune
system. Non-limiting examples of immune-related thrombocytopenia
disorders include idiopathic thrombocytopenic purapura, neonatal
alloimmune thrombocytopenia, post-transfusion purapura, and
systemic lupus erythematosus related thrombocytopenia.
[0034] As used herein, "IVIg" is a preparation of pooled,
polyvalent IgG, including all four IgG subgroups, extracted from
plasma of at least 1,000 human donors. IVIg is approved as a plasma
protein replacement therapy for immune deficient patients. The
level of IVIg Fc glycan sialylation varies between about 10-20%
among IVIg preparations. As used herein, the term "derived from
IVIg" refers to polypeptides which result from manipulation of
IVIg. For example, polypeptides purified from IVIg (e.g., enriched
for sialylated IgGs, modified IVIg (e.g., IVIg IgGs enzymatically
sialylated), or Fc regions of IVIg (e.g., papain digested and
sialylated) are derived from IVIg.
[0035] As used herein, an "N-glycosylation site of an Fc
polypeptide" refers to an amino acid residue within an Fc
polypeptide to which a glycan is N-linked. In some embodiments, an
Fc region contains a dimer of Fc polypeptides, and the Fc region
comprises two N-glycosylation sites, one on each Fc
polypeptide.
[0036] As used herein "percent (%) of branched glycans" refers to
the number of moles of glycan X relative to total moles of glycans
present, wherein X represents the glycan of interest.
[0037] As used herein "percent (%) sequence identity" with respect
to a sequence is defined as the percentage of amino acid residues
or nucleotides in a candidate sequence that are identical with the
amino acid residues or nucleotides in the reference sequence, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes. Alignment for
purposes of determining percent sequence identity can be achieved
in various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, ALIGN or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. In one embodiment, the length of a
reference sequence aligned for comparison purposes is at least 30%,
e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100%
of the length of the reference sequence. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position. In some instances a product will
include amino acid variants, e.g., species that differ at terminal
residues, e.g., at one, two, three, or four N-terminal residues
and/or one C-terminal residue. In instances of such cases the
sequence identity which is compared is the identity between the
primary amino acid sequences of the most abundant active species in
each of the products being compared. In some instances sequence
identity refers to the amino acid sequence encoded by a nucleic
acid that can be used to make the product.
[0038] The term "pharmaceutically effective amount" or
"therapeutically effective amount" refers to an amount (e.g., dose)
effective in treating a patient, having a disorder or condition
described herein. It is also to be understood herein that a
"pharmaceutically effective amount" may be interpreted as an amount
giving a desired therapeutic effect, either taken in one dose or in
any dosage or route, taken alone or in combination with other
therapeutic agents.
[0039] "Pharmaceutical preparations" and "pharmaceutical products"
can be included in kits containing the preparation or product and
instructions for use.
[0040] "Pharmaceutical preparations" and "pharmaceutical products"
generally refer to compositions in which the final predetermined
level of sialylation has been achieved, and which are free of
process impurities. To that end, "pharmaceutical preparations" and
"pharmaceutical products" are substantially free of ST6Gal
sialyltransferase and/or sialic acid donor (e.g., cytidine
5'-monophospho-N-acetyl neuraminic acid) or the byproducts thereof
(e.g., cytidine 5'-monophosphate).
[0041] "Pharmaceutical preparations" and "pharmaceutical products"
are generally substantially free of other components of a cell in
which the glycoproteins were produced (e.g., the endoplasmic
reticulum or cytoplasmic proteins and RNA), if recombinant.
[0042] As used herein, "polynucleotide" (or "nucleotide sequence"
or "nucleic acid molecule") refers to an oligonucleotide,
nucleotide, or polynucleotide, and fragments or portions thereof,
and to DNA or RNA of genomic or synthetic origin, which may be
single- or double-stranded, and represent the sense or anti-sense
strand.
[0043] As used herein, "polypeptide" (or "amino acid sequence" or
"protein") refers to a glycoprotein, oligopeptide, peptide,
polypeptide, or protein sequence, and fragments or portions
thereof, and to naturally occurring or synthetic molecules. "Amino
acid sequence" and like terms, such as "polypeptide" or "protein,"
are not meant to limit the indicated amino acid sequence to the
complete, native amino acid sequence associated with the recited
protein molecule.
[0044] "Predetermined level" as used herein, refers to a
pre-specified particular level of one or more particular glycans,
e.g., branched glycans having a sialic acid on an .alpha.1,3 arm,
and/or branched glycans having a sialic acid on an .alpha.1,6 arm,
and/or branched glycans having a sialic acid on an .alpha.1,3 arm
and on an .alpha.1,6 arm. In some embodiments, a predetermined
level is an absolute value or range. In some embodiments, a
predetermined level is a relative value. In some embodiments, a
predetermined level is the same as or different (e.g., higher or
lower than) a level of one or more particular glycans (e.g.,
branched glycans having a sialic acid on an .alpha.1,3 arm, and/or
branched glycans having a sialic acid on an .alpha.1,6 arm, and/or
branched glycans having a sialic acid on an .alpha.1,3 arm and on
an .alpha.1,6 arm) in a reference, e.g., a reference polypeptide
product, or a level specified in a reference document such as a
pharmaceutical specification, a monograph, alert limit, or master
batch record for a pharmaceutical product.
[0045] In some embodiments, a predetermined level is an absolute
level or range of (e.g., number of moles of) one or more glycans
(e.g., branched glycans having a sialic acid on an .alpha.1,3 arm,
and/or branched glycans having a sialic acid on an .alpha.1,6 arm,
and/or branched glycans having a sialic acid on an .alpha.1,3 arm
and on an .alpha.1,6 arm) in a polypeptide preparation. In some
embodiments, a predetermined level is a level or range of one or
more glycans (e.g., branched glycans having a sialic acid on an
.alpha.1,3 arm, and/or branched glycans having a sialic acid on an
.alpha.1,6 arm, and/or branched glycans having a sialic acid on an
.alpha.1,3 arm and on an .alpha.1,6 arm) in a polypeptide
preparation relative to total level of glycans in the polypeptide
preparation. In some embodiments, a predetermined level is a level
or range of one or more glycans (e.g., branched glycans having a
sialic acid on an .alpha.1,3 arm, and/or branched glycans having a
sialic acid on an .alpha.1,6 arm, and/or branched glycans having a
sialic acid on an .alpha.1,3 arm and on an .alpha.1,6 arm) in a
polypeptide preparation relative to total level of sialylated
glycans in the polypeptide preparation. In some embodiments, a
predetermined level is expressed as a percent.
[0046] By "purified" (or "isolated") refers to a polynucleotide or
a polypeptide that is removed or separated from other components
present in its natural environment. For example, an isolated
polypeptide is one that is separated from other components of a
cell in which it was produced (e.g., the endoplasmic reticulum or
cytoplasmic proteins and RNA). An isolated polynucleotide is one
that is separated from other nuclear components (e.g., histones)
and/or from upstream or downstream nucleic acids. An isolated
polynucleotide or polypeptide can be at least 60% free, or at least
75% free, or at least 90% free, or at least 95% free from other
components present in natural environment of the indicated
polynucleotide or polypeptide.
[0047] "Reference polypeptide" refers to a polypeptide having
substantially the same amino acid sequence as (e.g., having about
95-100% identical amino acids of) a polypeptide described herein,
e.g., a polypeptide to which it is compared. In some embodiments, a
reference polypeptide is a therapeutic polypeptide described
herein, e.g., an FDA approved therapeutic polypeptide.
[0048] As used herein, the term "sialylated" refers to a glycan
having a terminal sialic acid. The term "mono-sialylated" refers to
branched glycans having one terminal sialic acid, e.g., on an
.alpha.1,3 arm or an .alpha.1,6 arm. The term "di-sialylated"
refers to a branched glycan having a terminal sialic acid on two
arms, e.g., both an .alpha.1,3 arm and an .alpha.1,6 arm.
[0049] As used herein, the term "ST6 sialyltransferase" refers to a
polypeptide whose amino acid sequence includes at least one
characteristic sequence of and/or shows at least 100%, 99%, 98%,
97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,
84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%,
71% or 70% identity with a protein involved in transfer of a sialic
acid to a terminal galactose of a glycan through an .alpha.2,6
linkage (e.g., ST6 Gal-I). A wide variety of ST6 sialyltransferase
sequences are known in the art, such as those described herein; in
some embodiments, an ST6 sialyltransferase shares at least one
characteristic sequence of and/or shows the specified degree of
overall sequence identity with one of the ST6 sialyltransferases
set forth herein (each of which may be considered a "reference" ST6
sialyltransferase). In some embodiments, an ST6 sialyltransferase
as described herein shares at least one biological activity with a
reference ST6 sialyltransferase as set forth herein. In some such
embodiment, the shared biological activity relates to transfer of a
sialic acid to a glycan.
[0050] The term "subject," as used herein, means any subject for
whom diagnosis, prognosis, or therapy is desired. In one
embodiment, the subject is a human.
[0051] The term "thrombopoietin receptor agonist," as used herein,
refers to pharmaceutical agents that stimulate platelet production
in the bone marrow through interaction with the thrombopoietin
receptor.
[0052] The term "treatment" or "treating," as used herein, refers
to administering a therapy in an amount, manner, and/or mode
effective to improve a condition, symptom, or parameter associated
with a disorder or condition or to prevent or reduce progression of
a disorder or condition to a degree detectable to one skilled in
the art. An effective amount, manner, or mode can vary depending on
the subject and may be tailored to the subject. The term "not being
treated," as used herein, means a subject is not currently being
administered a therapy.
[0053] As used herein, the terms "coupled," "linked," "joined,"
"fused," and "fusion" are used interchangeably. These terms refer
to the joining together of two more elements or components by
whatever means, including chemical conjugation or recombinant
means.
[0054] While the present disclosure provides exemplary units and
methods for the evaluation, identification, and production methods
disclosed herein, a person of ordinary skill in the art will
appreciate that performance of the evaluation, identification, and
production methods herein is not limited to use of those units
and/or methods. For example, "percent of branched glycans" provided
herein are generally described, as a value for a glycan or
structure relative to total glycan or structure on a mol/mol basis.
A person of skill in the art understands that although the use of
other metrics or units (e.g., mass/mass, mole percent vs. weight
percent) to measure a described parameter might give rise to
different absolute values than those described herein, a test
preparation meets a disclosed target value even if other units or
metrics are used, as long as the test preparation meets the herein
disclosed value when the herein disclosed units and metrics are
used, e.g., allowing for the sensitivity (e.g., analytical
variability) of the method being used to measure the value.
I. Polypeptides
[0055] Examples of an Fc region-containing polypeptide preparation
include, e.g., a preparation of Fc fragments, a preparation of
antibody molecules, a preparation of Fc-fusion proteins (e.g., an
Fc-receptor fusion protein), and a preparation of pooled,
polyvalent immunoglobulin molecules (e.g., IVIg). Fc
region-containing polypeptides may be recombinant or naturally
derived.
[0056] Naturally derived polypeptides that can be used in the
methods of the invention include, for example, intravenous
immunoglobulin (IVIg) and polypeptides derived from IVIg (e.g.,
polypeptides purified from IVIg (e.g., enriched for sialylated
IgGs), modified IVIg (e.g., IVIg IgGs enzymatically sialylated), or
Fc regions of IVIg (e.g., papain digested and sialylated)).
[0057] Recombinant Fc region-containing polypeptides that can be
used in the methods of the invention can be, for example, expressed
in and purified from CHO cells and sialylated using human ST6-Gal
sialtransferase enzyme (expressed in and purified from E. coli
cells) or expressed in and purified from CHO cells and sialylated
using human ST6-Gal sialtransferase enzyme (expressed in and
purified from CHO cells).
A. N-Linked Glycosylation
[0058] N-linked oligosaccharide chains are added to a protein in
the lumen of the endoplasmic reticulum. Specifically, an initial
oligosaccharide (typically 14-sugar) is added to the amino group on
the side chain of an asparagine residue contained within the target
consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid
except proline. The structure of this initial oligosaccharide is
common to most eukaryotes, and contains three glucose, nine
mannose, and two N-acetylglucosamine residues. This initial
oligosaccharide chain can be trimmed by specific glycosidase
enzymes in the endoplasmic reticulum, resulting in a short,
branched core oligosaccharide composed of two N-acetylglucosamine
and three mannose residues. One of the branches is referred to in
the art as the ".alpha. 1,3 arm," and the second branch is referred
to as the ".alpha. 1,6 arm," as denoted in FIG. 1.
[0059] N-glycans can be subdivided into three distinct groups
called "high mannose type," "hybrid type," and "complex type," with
a common pentasaccharide core (Man (.alpha. 1,6)-(Man(.alpha.
1,3))-Man(.beta. 1,4)-GlcpNAc(.beta. 1,4)-GlcpNAc(.beta. 1,N)-Asn)
occurring in all three groups.
[0060] After initial processing in the endoplasmic reticulum, the
polypeptide is transported to the Golgi where further processing
may take place. If the glycan is transferred to the Golgi before it
is completely trimmed to the core pentasaccharide structure, it
results in a "high-mannose glycan."
[0061] Additionally or alternatively, one or more monosaccharides
units of N-acetylglucosamine may be added to the core mannose
subunits to form a "complex glycan." Galactose may be added to the
N-acetylglucosamine subunits, and sialic acid subunits may be added
to the galactose subunits, resulting in chains that terminate with
any of a sialic acid, a galactose or an N-acetylglucosamine
residue. Additionally, a fucose residue may be added to an
N-acetylglucosamine residue of the core oligosaccharide. Each of
these additions is catalyzed by specific glycosyl transferases.
[0062] "Hybrid glycans" comprise characteristics of both
high-mannose and complex glycans. For example, one branch of a
hybrid glycan may comprise primarily or exclusively mannose
residues, while another branch may comprise N-acetylglucosamine,
sialic acid, galactose, and/or fucose sugars.
[0063] Sialic acids are a family of 9-carbon monosaccharides with
heterocyclic ring structures. They bear a negative charge via a
carboxylic acid group attached to the ring as well as other
chemical decorations including N-acetyl and N-glycolyl groups. The
two main types of sialyl residues found in polypeptides produced in
mammalian expression systems are N-acetyl-neuraminic acid (NeuAc)
and N-glycolylneuraminic acid (NeuGc). These usually occur as
terminal structures attached to galactose (Gal) residues at the
non-reducing termini of both N- and O-linked glycans. The
glycosidic linkage configurations for these sialyl groups can be
either .alpha. 2,3 or .alpha. 2,6.
[0064] Fc regions are glycosylated at conserved, N-linked
glycosylation sites. For example, each heavy chain of an IgG
antibody has a single N-linked glycosylation site at Asn297 of the
C.sub.H2 domain. IgA antibodies have N-linked glycosylation sites
within the C.sub.H2 and C.sub.H3 domains, IgE antibodies have
N-linked glycosylation sites within the C.sub.H3 domain, and IgM
antibodies have N-linked glycosylation sites within the C.sub.H1,
C.sub.H2, C.sub.H3, and C.sub.H4 domains.
[0065] Each antibody isotype has a distinct variety of N-linked
carbohydrate structures in the constant regions. For example, IgG
has a single N-linked biantennary carbohydrate at Asn297 of the
C.sub.H2 domain in each Fc polypeptide of the Fc region, which also
contains the binding sites for C1q and Fc.gamma.R. For human IgG,
the core oligosaccharide normally consists of
GlcNAc.sub.2Man.sub.3GlcNAc, with differing numbers of outer
residues. Variation among individual IgG can occur via attachment
of galactose and/or galactose-sialic acid at one or both terminal
GlcNAc or via attachment of a third GlcNAc arm (bisecting
GlcNAc).
B. Antibodies
[0066] The basic structure of an IgG antibody is illustrated in
FIG. 2. As shown in FIG. 2, an IgG antibody consists of two
identical light polypeptide chains and two identical heavy
polypeptide chains linked together by disulphide bonds. The first
domain located at the amino terminus of each chain is variable in
amino acid sequence, providing the antibody binding specificities
found in each individual antibody. These are known as variable
heavy (V.sub.H) and variable light (V.sub.L) regions. The other
domains of each chain are relatively invariant in amino acid
sequence and are known as constant heavy (C.sub.H) and constant
light (C.sub.L) regions. As shown in FIG. 2, for an IgG antibody,
the light chain includes one variable region (V.sub.L) and one
constant region (C.sub.L). An IgG heavy chain includes a variable
region (V.sub.H), a first constant region (C.sub.H1), a hinge
region, a second constant region (C.sub.H2), and a third constant
region (C.sub.H3). In IgE and IgM antibodies, the heavy chain
includes an additional constant region (C.sub.H4).
[0067] Antibodies described herein can include, for example,
monoclonal antibodies, polyclonal antibodies, multispecific
antibodies, human antibodies, humanized antibodies, camelized
antibodies, chimeric antibodies, single-chain Fvs (scFv),
disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id)
antibodies, and antigen-binding fragments of any of the above.
Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or
IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2) or
subclass.
[0068] The term "Fc fragment," as used herein, refers to one or
more fragments of an Fc region that retains an Fc function and/or
activity described herein, such as binding to an Fc receptor.
Examples of such fragments include fragments that include an
N-linked glycosylation site of an Fc region (e.g., an Asn297 of an
IgG heavy chain or homologous sites of other antibody isotypes),
such as a CH2 domain. The term "antigen binding fragment" of an
antibody, as used herein, refers to one or more fragments of an
antibody that retain the ability to specifically bind to an
antigen. Examples of binding fragments encompassed within the term
"antigen binding fragment" of an antibody include a Fab fragment, a
F(ab').sub.2 fragment, a Fd fragment, a Fv fragment, a scFv
fragment, a dAb fragment (Ward et al., (1989) Nature 341:544-546),
and an isolated complementarily determining region (CDR). These
antibody fragments can be obtained using conventional techniques
known to those with skill in the art, and the fragments can be
screened for utility in the same manner as are intact
antibodies.
[0069] Reference Fc region-containing polypeptides described herein
can be produced by any method known in the art for the synthesis of
antibodies (see, e.g., Harlow et al., Antibodies: A Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO 92/22324;
WO 98/46645).
[0070] Additional reference Fc region-containing polypeptides
described herein are bispecific antibodies and multivalent
antibodies, as described in, e.g., Segal et al., J. Immunol.
Methods 248:1-6 (2001); and Tutt et al., J. Immunol. 147: 60
(1991).
C. Polypeptide Conjugates
[0071] The disclosure includes polypeptides (or Fc regions or Fc
fragments thereof containing one or more N-glycosylation sites)
that are conjugated or fused to one or more heterologous moieties
and that have different levels of sialylated glycans relative to a
corresponding reference polypeptide. Heterologous moieties include,
but are not limited to, peptides, polypeptides, proteins, fusion
proteins, nucleic acid molecules, small molecules, mimetic agents,
synthetic drugs, inorganic molecules, and organic molecules. In
some instances, a reference polypeptide is a fusion protein that
comprises a peptide, polypeptide, protein scaffold, scFv, dsFv,
diabody, Tandab, or an antibody mimetic fused to an Fc region, such
as a glycosylated Fc region. The fusion protein can include a
linker region connecting the Fc region to the heterologous moiety
(see, e.g., Hallewell et al. (1989), J. Biol. Chem. 264, 5260-5268;
Alfthan et al. (1995), Protein Eng. 8, 725-731; Robinson &
Sauer (1996)).
[0072] In some instances, a reference fusion protein includes an Fc
region (or an Fc fragment containing one or more N-glycosylation
sites thereof) conjugated to a heterologous polypeptide of at least
10, at least 20, at least 30, at least 40, at least 50, at least
60, at least 70, at least 80, at least 90, or at least 100 amino
acids.
[0073] In some instances, a reference fusion protein can include an
Fc region (or Fc fragment containing one or more N-glycosylation
sites thereof) conjugated to marker sequences, such as a peptide to
facilitate purification. A particular marker amino acid sequence is
a hexa-histidine peptide, such as the tag provided in a pQE vector
(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). Other
peptide tags useful for purification include, but are not limited
to, the hemagglutinin "HA" tag, which corresponds to an epitope
derived from the influenza hemagglutinin protein (Wilson et al.,
1984, Cell 37:767) and the "Flag" tag.
[0074] In other instances, a reference polypeptide (or an Fc region
or Fc fragment containing one or more N-glycosylation sites
thereof) is conjugated to a diagnostic or detectable agent. Such
fusion proteins can be useful for monitoring or prognosing the
development or progression of disease or disorder as part of a
clinical testing procedure, such as determining the efficacy of a
particular therapy. Such diagnosis and detection can be
accomplished by coupling the polypeptide to detectable substances
including, but not limited to, various enzymes, such as but not
limited to horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; prosthetic groups,
such as, but not limited to, streptavidin/biotin and avidin/biotin;
fluorescent materials, such as, but not limited to, umbelliferone,
fluorescein, fluorescein isothiocynate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; luminescent materials, such as, but not limited to,
luminol; bioluminescent materials, such as but not limited to,
luciferase, luciferin, and aequorin; radioactive materials, such as
but not limited to iodine (.sup.131I, .sup.125I, .sup.123I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.115In, .sup.113In, .sup.112In, .sup.111In), technetium
(.sup.99Tc), thallium (.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga),
palladium (.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe),
fluorine (.sup.18F), .sup.153Sm, .sup.177Lu, .sup.153Gd,
.sup.159Gd, .sup.149Pm, .sup.140La, .sup.169Yb, .sup.175Yb,
.sup.166Ho, .sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re,
.sup.142Pr, .sup.105Rh, .sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn,
.sup.85Sr, .sup.32P, .sup.51Cr, .sup.54Mn, .sup.75Se, .sup.113Sn,
and .sup.117Sn; positron emitting metals using various positron
emission tomographies, non-radioactive paramagnetic metal ions, and
molecules that are radiolabelled or conjugated to specific
radioisotopes.
[0075] Techniques for conjugating therapeutic moieties to
antibodies are well known (see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd
Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc.
1987)).
D. Sialyltransferase Polypeptides
[0076] Methods and compositions described herein include the use of
a sialyltransferase enzyme, e.g., an .alpha. 2,6 sialyltransferase
(e.g., ST6 Gal-I). A number of ST6 sialyltransferases are known in
the art and are commercially available (see, e.g., Takashima,
Biosci. Biotechnol. Biochem. 72:1155-1167 (2008); Weinstein et al.,
J. Biol. Chem. 262:17735-17743 (1987)). ST6 Gal-I catalyzes the
transfer of sialic acid from a sialic acid donor (e.g., cytidine
5'-monophospho-N-acetyl neuraminic acid) to a terminal galactose
residue of glycans through an .alpha. 2,6 linkage. The sialic acid
donor reaction product is cytidine 5'-monophosphate. FIGS. 3A-3C
depict three exemplary ST6 sialyltransferase amino acid sequences
(SEQ ID NOs:1-3). In some embodiments, an ST6 sialyltransferase has
or includes an amino acid sequence set forth in SEQ ID NO:1, SEQ ID
NO:2, or in amino acid residues 95-416 of SEQ ID NO:3, or a
characteristic sequence element thereof or therein. In some
embodiments, an ST6 sialyltransferase has at least 100%, 99%, 98%,
97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,
84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%,
71%, or 70% overall sequence identity with one or more of SEQ ID
NO:1, SEQ ID NO:2, or amino acid residues 95-416 of SEQ ID NO:3.
Alternatively or additionally, in some embodiments, an ST6
sialyltransferase includes at least about 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 75, 100, or 150 or more contiguous amino acid
residues found in SEQ ID NO:1, SEQ ID NO:2, or amino acid residues
95-416 of SEQ ID NO:3.
[0077] In some embodiments, an ST6 sialyltransferase differs from
an amino acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, or
in amino acid residues 95-416 of SEQ ID NO:3, or characteristic
sequence elements thereof or therein, by one or more amino acid
residues. For example, in some embodiments, the difference is a
conservative or nonconservative substitution of one or more amino
acid residues. Conservative substitutions are those that substitute
a given amino acid in a polypeptide by another amino acid of
similar characteristics. Typical conservative substitutions are the
following replacements: replacement of an aliphatic amino acid,
such as alanine, valine, leucine, and isoleucine, with another
aliphatic amino acid; replacement of a serine with a threonine or
vice versa; replacement of an acidic residue, such as aspartic acid
and glutamic acid, with another acidic residue; replacement of a
residue bearing an amide group, such as asparagine and glutamine,
with another residue bearing an amide group; exchange of a basic
residue, such as lysine and arginine, with another basic residue;
and replacement of an aromatic residue, such as phenylalanine and
tyrosine, with another aromatic residue.
[0078] In some embodiments, an ST6 sialyltransferase polypeptide
includes a substituent group on one or more amino acid residues.
Still other useful polypeptides are associated with (e.g., fused,
linked, or coupled to) another moiety (e.g., a peptide or
molecule). For example, an ST6 sialyltransferase polypeptides can
be fused, linked, or coupled to an amino acid sequence (e.g., a
leader sequence, a secretory sequence, a proprotein sequence, a
second polypeptide, or a sequence that facilitates purification,
enrichment, or stabilization of the polypeptide).
II. Methods for Producing Sialylated Polypeptides
[0079] The present disclosure relates to Fc region-containing
polypeptide preparations (e.g., IVIg, Fc, or IgG antibodies) having
higher levels of branched glycans that are sialylated on an .alpha.
1,3 and 1,6 arm of the branched glycans in the Fc region (e.g.,
with a NeuAc-.alpha. 2,6-Gal or NeuAc-.alpha. 2,3-Gal terminal
linkage), relative to a corresponding reference polypeptide
preparation. The higher levels can be measured on an individual Fc
region (e.g., an increase in the number of branched glycans that
are sialylated on an .alpha. 1,3 arm of the branched glycans in the
Fc region), or the overall composition of a preparation of
polypeptides can be different (e.g., a preparation of polypeptides
can have a higher number or a higher percentage of branched glycans
that are sialylated on an .alpha. 1,3 arm and an .alpha. 1,6 arm of
the branched glycans in the Fc region) relative to a corresponding
preparation of reference polypeptides).
[0080] In exemplary methods, Fc molecules were obtained or produced
from various sources, glycan compositions were characterized, and
activities were determined. The Fc molecules were tested for their
ability to increase reticulated platelets in immune-related
thrombocytopenia models.
[0081] ST6 Gal-I sialyltransferase catalyzes the transfer of sialic
acid from a sialic acid donor (e.g., cytidine
5'-monophospho-N-acetyl neuraminic acid) to a terminal galactose
residue of glycans through an .alpha. 2,6 linkage. The present
disclosure exploits the discovery that ST6 sialyltransferase
catalyzes the transfer of sialic acid to branched glycans (e.g., Fc
branched glycans) comprising an .alpha. 1,3 arm and an .alpha. 1,6
arm in an ordered fashion. As shown in FIG. 4, ST6
sialyltransferase transfers a sialic acid to an .alpha. 1,3 arm of
a branched glycan, which can be followed by transfer of a second
sialic acid to an .alpha. 1,6 arm (yielding a disialylated branched
glycan), and can further be followed by removal of sialic acid from
an .alpha. 1,3 arm (yielding a branched glycan having a sialic acid
on an .alpha. 1,6 arm). Accordingly, by controlling and/or
modulating activity (e.g., kinetics) of ST6 sialyltransferase,
polypeptides having particular sialylation patterns can be
produced.
[0082] Any parameter generally known to affect enzyme kinetics can
be controlled and/or modulated to produce a polypeptide preparation
having a predetermined level of sialic acid on an .alpha. 1,3 arm
of a branched glycan, on an .alpha. 1,6 arm of a branched glycan,
and/or on an .alpha. 1,3 arm and an .alpha. 1,6 arm of a branched
glycan. For example, reaction time, ST6 sialyltransferase
concentration and/or specific activity, branched glycan
concentration, sialic acid donor concentration, sialic acid donor
reaction product concentration, pH, buffer composition, and/or
temperature can be controlled and/or modulated to produce a
polypeptide preparation having a desired level of sialylation
(e.g., .alpha. 1,3 arm and/or .alpha. 1,6 arm sialylation).
[0083] In some embodiments, to preferentially sialylate an
.alpha.1,3 arm of branched glycans (e.g., having an .alpha. 1,3 arm
and an .alpha. 1,6 arm), branched glycans are contacted in vitro
with an ST6 sialyltransferase under limited reaction conditions.
Such limited reaction conditions are selected such that addition of
a sialic acid to an .alpha. 1,3 arm is enhanced relative to
addition of a sialic acid to an .alpha. 1,6 arm (e.g., rate of
transfer of a sialic acid to an .alpha. 1,3 arm ("R.sub.a.sup.1,3")
exceeds rate of transfer of a sialic acid to an .alpha. 1,6 arm
("R.sub.a.sup.1,6"). In some embodiments, limited reaction
conditions are further selected such that removal of a sialic acid
from an .alpha.1,6 arm is enhanced relative to addition of a sialic
acid to an .alpha. 1,6 arm (e.g., rate of removal of a sialic acid
from an .alpha. 1,6 arm ("R.sub.r.sup.1,6") exceeds rate of
transfer of a sialic acid to an .alpha. 1,6 arm
("R.sub.a.sup.1,6"). Limited reaction conditions can include, for
example, reduced reaction time, reduced enzyme concentration and/or
activity, reduced amount of branched glycans, reduced level of
sialic acid donor, and/or reduced temperature.
[0084] In some embodiments, to preferentially sialylate an
.alpha.1,6 arm of branched glycans (e.g., having an .alpha. 1,3 arm
and an .alpha. 1,6 arm), branched glycans can be contacted in vitro
with an ST6 sialyltransferase under extended reaction conditions.
Such extended reaction conditions are selected such that addition
of a sialic acid to an .alpha. 1,6 arm is enhanced relative to
removal of a sialic acid from an .alpha. 1,6 arm (e.g., rate of
transfer of a sialic acid to an .alpha. 1,6 arm ("R.sub.a.sup.1,6")
exceeds rate of removal of a sialic acid from an .alpha. 1,6 arm
("R.sub.r.sup.1,6")). In some embodiments, extended reaction
conditions are further selected such that, after initial conditions
that enhance addition of sialic acid to an .alpha. 1,3 arm,
conditions are extended such that removal of a sialic acid from an
.alpha. 1,3 arm is eventually enhanced relative to addition of a
sialic acid to an .alpha. 1,3 arm (e.g., rate of removal of a
sialic acid from an .alpha. 1,3 arm ("R.sub.r.sup.1,3") exceeds
rate of transfer of a sialic acid to an .alpha. 1,3 arm
("R.sub.a.sup.1,3")). Extended reaction conditions can include, for
example, increased reaction time, increased enzyme concentration
and/or activity, increased amount of branched glycans, increased
level of sialic acid donor, and/or increased temperature.
[0085] In some embodiments, to preferentially sialylate both an
.alpha. 1,3 arm and an .alpha. 1,6 arm of branched glycans (e.g.,
having an .alpha. 1,3 arm and an .alpha. 1,6 arm), branched glycans
are contacted in vitro with an ST6 sialyltransferase under
intermediate reaction conditions. Such intermediate reaction
conditions are selected such that addition of a sialic acid to an
.alpha. 1,3 arm is enhanced relative to removal of a sialic acid
from an .alpha. 1,3 arm (e.g., rate of transfer of a sialic acid to
an .alpha. 1,3 arm ("R.sub.a.sup.1,3") exceeds rate of removal of a
sialic acid from an .alpha. 1,3 arm ("R.sub.r.sup.1,3"). In some
embodiments, intermediate reaction conditions are further selected
such that addition of a sialic acid to an .alpha. 1,6 arm is
enhanced relative to removal of a sialic acid from an .alpha. 1,6
arm (e.g., rate of addition of a sialic acid to an .alpha. 1,6 arm
("R.sub.a.sup.1,6") exceeds rate of removal of a sialic acid from
an .alpha. 1,6 arm ("R.sub.r.sup.1,6"). Intermediate reaction
conditions can include, for example, intermediate reaction time,
intermediate enzyme concentration and/or activity, intermediate
amount of branched glycans, intermediate level of sialic acid
donor, and/or intermediate temperature. In some embodiments,
intermediate reaction conditions further include supplementing the
sialic acid donor at least once during the reaction. In some
embodiments, intermediate reaction conditions further include
removing a sialic acid donor reaction product at least once during
the reaction. In some embodiments, intermediate reaction conditions
further include supplementing the sialic acid donor reaction
product at least once during the reaction.
[0086] In some embodiments, a polypeptide, e.g., a glycosylated
antibody, is sialylated after the polypeptide is produced. For
example, a polypeptide can be recombinantly expressed in a host
cell (as described herein) and purified using standard methods. The
purified polypeptide is then contacted with an ST6
sialyltransferase (e.g., a recombinantly expressed and purified ST6
sialyltransferase) in the presence of reaction conditions as
described herein. In certain embodiments, the conditions include
contacting the purified polypeptide with an ST6 sialyltransferase
in the presence of a sialic acid donor, e.g., cytidine
5'-monophospho-N-acetyl neuraminic acid, manganese, and/or other
divalent metal ions. In some embodiments, IVIg is used in a
sialylation method described herein.
[0087] In some embodiments, chemoenzymatic sialylation is used to
sialylate polypeptides. Briefly, this method involves sialylation
of a purified branched glycan, followed by incorporation of the
sialylated branched glycan en bloc onto a polypeptide to produce a
sialylated polypeptide.
[0088] A branched glycan can be synthesized de novo using standard
techniques or can be obtained from a polypeptide preparation (e.g.,
a recombinant polypeptide, Fc, or IVIg) using an appropriate
enzyme, such as an endoglycosidase (e.g., EndoH or EndoF). After
sialylation of the branched glycan, the sialylated branched glycan
can be conjugated to a polypeptide using an appropriate enzyme,
such as a transglycosidase, to produce a sialylated
polypeptide.
[0089] In one exemplary method, a purified branched N-glycan is
obtained from a polypeptide (e.g., a polypeptide preparation, e.g.,
IVIg) using an endoglycosidase. The purified branched N-glycan is
then chemically activated on the reducing end to form a chemically
active intermediate. The branched N-glycan is then further
processed, trimmed, and/or glycosylated using appropriate known
glycosidases. The branched glycan is then sialylated using an ST6
sialylation as described herein. After engineering, the desired
branched N-glycan is transferred onto a polypeptide using a
transglycosidase (such as a transglycosidase in which glycosidic
activity has been attenuated using genetically engineering).
[0090] In some embodiments, a branched glycan used in methods
described herein is a galactosylated branched glycan (e.g.,
includes a terminal galactose residue). In some embodiments, a
branched glycan is galactosylated before being sialylated using a
method described herein. In some embodiments, a branched glycan is
first contacted with a galactosyltransferase (e.g., a
beta-1,3-galactosyltransferase) and subsequently contacted with an
ST6 sialyltransferase as described herein. In some embodiments, a
galactosylated glycan is purified before being contacted with an
ST6 sialyltransferase. In some embodiments, a galactosylated glycan
is not purified before being contacted with an ST6
sialyltransferase. In some embodiments, a branched glycan is
contacted with a galactosyltransferase and an ST6 sialyltransferase
in a single step.
[0091] In some embodiments, a host cell is genetically engineered
to express a polypeptide described herein and one or more
sialyltransferase enzymes, e.g., an ST6 sialyltransferase. In some
embodiments, the host cell is genetically engineered to further
express a galactosyltransferase. The genetically engineered host
cell can be cultured under conditions sufficient to produce a
particular sialylated polypeptide. For example, to produce
polypeptides preferentially sialylated on .alpha.1,3 arms of
branched glycans, a host cell can be genetically engineered to
express a relatively low level of ST6 sialyltransferase, whereas to
produce polypeptides preferentially sialylated on .alpha.1,6 arms
of branched glycans, a host cell can be genetically engineered to
express a relatively high level of ST6 sialyltransferase. In some
embodiments, to produce polypeptides preferentially sialylated on
.alpha.1,3 arms of branched glycans, a genetically engineered host
cell can be cultured in a relatively low level of sialic acid
donor, whereas to produce polypeptides preferentially sialylated on
.alpha.1,6 arms of branched glycans, a genetically engineered host
cell can be cultured in a relatively high level of sialic acid
donor.
[0092] Recombinant expression of a gene, such as a nucleic acid
encoding a reference polypeptide and/or a sialtransferase described
herein, can include construction of an expression vector containing
a polynucleotide that encodes a reference polypeptide and/or a
sialtransferase. Once a polynucleotide has been obtained, a vector
for the production of the reference polypeptide can be produced by
recombinant DNA technology using techniques known in the art. Known
methods can be used to construct expression vectors containing
polypeptide coding sequences and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination.
[0093] An expression vector can be transferred to a host cell by
conventional techniques, and the transfected cells can then
cultured by conventional techniques to produce reference
polypeptides.
[0094] A variety of host expression vector systems can be used
(see, e.g., U.S. Pat. No. 5,807,715). Such host-expression systems
can be used to produce polypeptides and, where desired,
subsequently purified. Such host expression systems include
microorganisms such as bacteria (e.g., E. coli and B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing polypeptide coding
sequences; yeast (e.g., Saccharomyces and Pichia) transformed with
recombinant yeast expression vectors containing polypeptide coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing polypeptide
coding sequences; plant cell systems infected with recombinant
virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g. Ti plasmid) containing polypeptide coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293,
NS0, and 3T3 cells) harboring recombinant expression constructs
containing promoters derived from the genome of mammalian cells
(e.g., metallothionein promoter) or from mammalian viruses (e.g.,
the adenovirus late promoter; the vaccinia virus 7.5K
promoter).
[0095] For bacterial systems, a number of expression vectors can be
used, including, but not limited to, the E. coli expression vector
pUR278 (Ruther et al., 1983, EMBO 12:1791); pIN vectors (Inouye
& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke
& Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like.
pGEX vectors can also be used to express foreign polypeptides as
fusion proteins with glutathione 5-transferase (GST).
[0096] For expression in mammalian host cells, viral-based
expression systems can be utilized (see, e.g., Logan & Shenk,
1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). The efficiency of
expression can be enhanced by the inclusion of appropriate
transcription enhancer elements, transcription terminators, etc.
(see, e.g., Bittner et al., 1987, Methods in Enzymol.
153:516-544).
[0097] In addition, a host cell strain can be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Different host
cells have characteristic and specific mechanisms for the
post-translational processing and modification of proteins and gene
products. Appropriate cell lines or host systems can be chosen to
ensure the correct modification and processing of the polypeptide
expressed. Such cells include, for example, established mammalian
cell lines and insect cell lines, animal cells, fungal cells, and
yeast cells. Mammalian host cells include, but are not limited to,
CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T,
HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not
endogenously produce any immunoglobulin chains), CRL7O3O and
HsS78Bst cells.
[0098] For long-term, high-yield production of recombinant
proteins, host cells are engineered to stably express a
polypeptide. Host cells can be transformed with DNA controlled by
appropriate expression control elements known in the art, including
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, and selectable markers. Methods commonly
known in the art of recombinant DNA technology can be used to
select a desired recombinant clone.
[0099] In some embodiments, a reference Fc region-containing
polypeptide is recombinantly produced in cells as described herein,
purified, and contacted with a sialtransferase enzyme in vitro to
produce Fc region-containing polypeptides containing higher levels
of glycans having higher levels of sialic acid on the .alpha. 1,3
arms and .alpha. 1,6 arms of the branched glycans with a
NeuAc-.alpha. 2,6-Gal terminal linkage, relative to the reference
polypeptide. In some embodiments, a purified reference polypeptide
is contacted with the sialtransferase in the presence of CMP-sialic
acid, manganese, and/or other divalent metal ions.
[0100] A reference Fc region-containing polypeptide can be purified
by any method known in the art for purification, for example, by
chromatography (e.g., ion exchange, affinity, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. For
example, a reference antibody can be isolated and purified by
appropriately selecting and combining affinity columns such as
Protein A column with chromatography columns, filtration, ultra
filtration, salting-out and dialysis procedures (see Antibodies: A
Laboratory Manual, Ed Harlow, David Lane, Cold Spring Harbor
Laboratory, 1988). Further, as described herein, a reference
polypeptide can be fused to heterologous polypeptide sequences to
facilitate purification.
[0101] In some embodiments, a polypeptide can be purified using a
lectin column by methods known in the art (see, e.g., WO 02/30954).
For example, a preparation of polypeptides can be enriched for
polypeptides containing glycans having sialic acids in .alpha. 2,6
linkage as described in, e.g., WO2008/057634. Following enrichment
of polypeptides containing glycans having sialic acids in .alpha.
2,6 linkage, the glycan composition of such polypeptides can be
further characterized to identify polypeptides having sialic acids
attached to the .alpha. 1,3 arm and .alpha. 1,6 arm of a branched
glycan. Preparations of polypeptides containing a predetermined
level of glycans having sialic acids in .alpha. 2,6 linkage on the
.alpha. 1,3 arm and .alpha. 1,6 arm can be selected for use, e.g.,
for therapeutic use. Such compositions can have increased levels of
anti-inflammatory activity.
[0102] In accordance with the present disclosure, there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are described in the literature (see, e.g., Green &
Sambrook, Molecular Cloning: A Laboratory Manual, Fourth Edition
(2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.; DNA Cloning: A Practical Approach, Volumes I and II (Glover
and Hames, eds. 1995); Oligonucleotide Synthesis (M. J. Gait ed.
1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins
eds. (1985)); Transcription And Translation (B. D. Hames & S.
J. Higgins, eds. (1984)); R. I. Freshney, Culture of Animal Cells:
A Manual of Basic Technique and Specialized Application (2010);
Immobilized Cells and Enzymes (IRL Press, (1986)); J. M. Guisan,
Immobilization of Enzymes and Cells (2013); B. Perbal, A Practical
Guide To Molecular Cloning (1984); T. A. Brown, Essential Molecular
Biology: A Practical Approach Volume I (2000); T. A. Brown,
Essential Molecular Biology: A Practical Approach Volume II (2002);
F. M. Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley & Sons, Inc. (1994).
[0103] Glycan compositions can be characterized using methods
described in, e.g., Barb, Biochemistry 48:9705-9707 (2009);
Anumula, J. Immunol. Methods 382:167-176 (2012); Gilar et al.,
Analytical Biochem. 417:80-88 (2011).
Glycan Evaluation
[0104] Glycans of polypeptides can be evaluated using any methods
known in the art. For example, sialylation of glycan compositions
(e.g., level of branched glycans that are sialylated on an
.alpha.1,3 arm and/or an .alpha.1,6 arm) can be characterized using
methods described in, e.g., Barb, Biochemistry 48:9705-9707 (2009);
Anumula, J. Immunol. Methods 382:167-176 (2012); Gilar et al.,
Analytical Biochem. 417:80-88 (2011); Wuhrer et al., J. Chromatogr.
B. 849:115-128 (2007). In some embodiments, in addition to
evaluation of sialylation of glycans, one or more parameters
described in Table 1 are evaluated.
[0105] In some instances, glycan structure and composition as
described herein are analyzed, for example, by one or more,
enzymatic, chromatographic, mass spectrometry (MS), chromatographic
followed by MS, electrophoretic methods, electrophoretic methods
followed by MS, nuclear magnetic resonance (NMR) methods, and
combinations thereof. Exemplary enzymatic methods include
contacting a polypeptide preparation with one or more enzymes under
conditions and for a time sufficient to release one or more
glycan(s) (e.g., one or more exposed glycan(s)). In some instances,
the one or more enzymes include(s) PNGase F. Exemplary
chromatographic methods include, but are not limited to, Strong
Anion Exchange chromatography using Pulsed Amperometric Detection
(SAX-PAD), liquid chromatography (LC), high performance liquid
chromatography (HPLC), ultra performance liquid chromatography
(UPLC), thin layer chromatography (TLC), amide column
chromatography, and combinations thereof. Exemplary mass
spectrometry (MS) include, but are not limited to, tandem MS,
LC-MS, LC-MS/MS, matrix assisted laser desorption ionisation mass
spectrometry (MALDI-MS), Fourier transform mass spectrometry
(FTMS), ion mobility separation with mass spectrometry (IMS-MS),
electron transfer dissociation (ETD-MS), and combinations thereof.
Exemplary electrophoretic methods include, but are not limited to,
capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose
gel electrophoresis, acrylamide gel electrophoresis,
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by
Western blotting using antibodies that recognize specific glycan
structures, and combinations thereof. Exemplary nuclear magnetic
resonance (NMR) include, but are not limited to, one-dimensional
NMR (1 D-NMR), two-dimensional NMR (2D-NMR), correlation
spectroscopy magnetic-angle spinning NMR (COSY-NMR), total
correlated spectroscopy NMR (TOCSY-NMR), heteronuclear
single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple
quantum coherence (HMQC-NMR), rotational nuclear overhauser effect
spectroscopy NMR (ROESY-NMR), nuclear overhauser effect
spectroscopy (NOESY-NMR), and combinations thereof.
[0106] In some instances, techniques described herein may be
combined with one or more other technologies for the detection,
analysis, and or isolation of glycans or polypeptides. For example,
in certain instances, glycans are analyzed in accordance with the
present disclosure using one or more available methods (to give but
a few examples, see Anumula, Anal. Biochem., 350(1):1, 2006; Klein
et al., Anal. Biochem., 179:162, 1989; and/or Townsend, R. R.
Carbohydrate Analysis" High Performance Liquid Chromatography and
Capillary Electrophoresis., Ed. Z. El Rassi, pp 181-209, 1995;
WO2008/128216; WO2008/128220; WO2008/128218; WO2008/130926;
WO2008/128225; WO2008/130924; WO2008/128221; WO2008/128228;
WO2008/128227; WO2008/128230; WO2008/128219; WO2008/128222;
WO2010/071817; WO2010/071824; WO2010/085251; WO2011/069056; and
WO2011/127322, each of which is incorporated herein by reference in
its entirety). For example, in some instances, glycans are
characterized using one or more of chromatographic methods,
electrophoretic methods, nuclear magnetic resonance methods, and
combinations thereof. In some instances, methods for evaluating one
or more target protein specific parameters, e.g., in a polypeptide
preparation, e.g., one or more of the parameters disclosed herein,
can be performed by one or more of following methods.
TABLE-US-00001 TABLE 1 Exemplary methods of evaluating parameters:
Method(s) Relevant literature Parameter C18 UPLC Mass Spec.* Chen
and Flynn, Anal. Biochem., Glycan(s) 370: 147-161 (2007) (e.g.,
N-linked glycan, exposed N-linked Chen and Flynn, J. Am. Soc. Mass
glycan, glycan detection, glycan Spectrom., 20: 1821-1833 (2009)
identification, and characterization; site specific glycation;
glycoform detection (e.g., parameters 1-7); percent glycosylation;
and/or aglycosyl) Peptide LC-MS Dick et al., Biotechnol. Bioeng.,
C-terminal lysine (reducing/non-reducing) 100: 1132-1143 (2008) Yan
et al., J. Chrom. A., 1164: 153-161 (2007) Chelius et al., Anal.
Chem., 78: 2370- 2376 (2006) Miller et al., J. Pharm. Sci., 100:
2543- 2550 (2011) LC-MS (reducing/non- Dick et al., Biotechnol.
Bioeng., reducing/alkylated) 100: 1132-1143 (2008) Goetze et al.,
Glycobiol., 21: 949-959 (2011) Weak cation exchange Dick et al.,
Biotechnol. Bioeng., (WCX) chromatography 100: 1132-1143 (2008)
LC-MS (reducing/non- Dick et al., Biotechnol. Bioeng., N-terminal
pyroglu reducing/alkylated) 100: 1132-1143 (2008) Goetze et al.,
Glycobiol., 21: 949-959 (2011) PeptideLC-MS Yan et al., J. Chrom.
A., 1164: 153-161 (reducing/non-reducing) (2007) Chelius et al.,
Anal. Chem., 78: 2370- 2376 (2006) Miller et al., J. Pharm. Sci.,
100: 2543- 2550 (2011) Peptide LC-MS Yan et al., J. Chrom. A.,
1164: 153-161 Methionine oxidation (reducing/non-reducing) (2007);
Xie et al., mAbs, 2: 379-394 (2010) Peptide LC-MS Miller et al., J.
Pharm. Sci., 100: 2543- Site specific glycation
(reducing/non-reducing) 2550 (2011) Peptide LC-MS Wang et al.,
Anal. Chem., 83: 3133-3140 Free cysteine (reducing/non-reducing)
(2011); Chumsae et al., Anal. Chem., 81: 6449- 6457 (2009)
Bioanalyzer Forrer et al., Anal. Biochem., 334: 81-88 Glycan (e.g.,
N-linked glycan, exposed N- (reducing/non-reducing)* (2004) linked
glycan) (including, for example, glycan detection, identification,
and characterization; site specific glycation; glycoform detection;
percent glycosylation; and/or aglycosyl) LC-MS (reducing/non- Dick
et al., Biotechnol. Bioeng., Glycan (e.g., N-linked glycan, exposed
N- reducing/alkylated)* 100: 1132-1143 (2008) linked glycan) *
Methods include Goetze et al., Glycobiol., 21: 949-959 (including,
for example, glycan detection, removal (e.g., enzymatic, (2011)
identification, and characterization; site chemical, and physical)
Xie et al., mAbs, 2: 379-394 (2010) specific glycation; glycoform
detection; of glycans percent glycosylation; and/or aglycosyl)
Bioanalyzer Forrer et al., Anal. Biochem., 334: 81-88 Light chain:
Heavy chain (reducing/non-reducing) (2004) Peptide LC-MS Yan et
al., J. Chrom. A., 1164: 153-161 Non-glycosylation-related peptide
(reducing/non-reducing) (2007) modifications (including, for
example, Chelius et al., Anal. Chem., 78: 2370- sequence analysis
and identification of 2376 (2006) sequence variants; oxidation;
Miller et al., J. Pharm. Sci., 100: 2543- succinimide; aspartic
acid; and/or site- 2550 (2011) specific aspartic acid) Weak cation
exchange Dick et al., Biotechnol. Bioeng., Isoforms (including, for
example, charge (WCX) chromatography 100: 1132-1143 (2008) variants
(acidic variants and basic variants); and/or deamidated variants)
Anion-exchange Ahn et al., J. Chrom. B, 878: 403-408 Sialylated
glycan chromatography (2010) Anion-exchange Ahn et al., J. Chrom.
B, 878: 403-408 Sulfated glycan chromatography (2010)
1,2-diamino-4,5- Hokke et al., FEBS Lett., 275: 9-14 Sialic acid
methylenedioxybenzene (1990) (DMB) labeling method LC-MS Johnson et
al., Anal. Biochem., 360: 75- C-terminal amidation 83 (2007) LC-MS
Johnson et al., Anal. Biochem., 360: 75- N-terminal fragmentation
83 (2007) Circular dichroism Harn et al., Current Trends in
Secondary structure (including, for spectroscopy Monoclonal
Antibody Development and example, alpha helix content and/or beta
Manufacturing, S. J. Shire et al., eds, sheet content) 229-246
(2010) Intrinsic and/or ANS dye Harn et al., Current Trends in
Tertiary structure (including, for example, fluorescence Monoclonal
Antibody Development and extent of protein folding) Manufacturing,
S. J. Shire et al., eds, 229-246 (2010) Hydrogen-deuterium Houde et
al., Anal. Chem., 81: 2644- Tertiary structure and dynamics
(including, exchange-MS 2651 (2009) for example, accessibility of
amide protons to solvent water) Size-exclusion Carpenter et al., J.
Pharm. Sci., Extent of aggregation chromatography 99: 2200-2208
(2010) Analytical Pekar and Sukumar, Anal. Biochem.,
ultracentrifugation 367: 225-237 (2007)
[0107] References listed in Table 1 are hereby incorporated by
reference in their entirety or, in the alternative, to the extent
that they pertain to one or more of the methods disclosed in Table
1. Other methods for evaluating one or more parameters are
disclosed in the examples.
III. Treatment of Immune-Related Thrombocytopenia
[0108] The inventors have discovered that biological activity of
Fc-containing molecules is enhanced by sialylation of two branches
of branched glycans. Accordingly, Fc region-containing polypeptides
described herein (e.g., Fc region-containing polypeptides
containing glycans containing sialic acid on an .alpha. 1,3 arm and
an .alpha. 1,6 arm of branched glycans with a NeuAc-.alpha. 2,6-Gal
terminal linkage) have increased activity relative to a reference
polypeptide. Current treatments for immune-related thrombocytopenia
include IVIg infusions, platelets transfusions, and treatment with
thrombopoietin or thrombopoietin receptor agonist, e.g.,
romiplostim (NPLATE.RTM., Amgen) and eltrombopag (PROMACTA.RTM.,
GlaxoSmithKline).
IV. Pharmaceutical Compositions and Administration
[0109] A polypeptide of the present disclosure, e.g., an Fc
region-containing polypeptide comprising branched glycans that are
sialylated on both an .alpha. 1,3 arm and an .alpha. 1,6 arm of the
branched glycan in the Fc region, e.g., with a NeuAc-.alpha.
2,6-Gal terminal linkage, can be incorporated into a pharmaceutical
composition and can be useful in the treatment of immune-related
thrombocytopenia. Such a pharmaceutical composition is useful as an
improved composition for the prevention and/or treatment of
diseases relative to the corresponding reference polypeptide.
Pharmaceutical compositions comprising a polypeptide can be
formulated by methods known to those skilled in the art. The
pharmaceutical composition can be administered parenterally in the
form of an injectable formulation comprising a sterile solution or
suspension in water or another pharmaceutically acceptable liquid.
For example, the pharmaceutical composition can be formulated by
suitably combining the sulfated polypeptide with pharmaceutically
acceptable vehicles or media, such as sterile water and
physiological saline, vegetable oil, emulsifier, suspension agent,
surfactant, stabilizer, flavoring excipient, diluent, vehicle,
preservative, binder, followed by mixing in a unit dose form
required for generally accepted pharmaceutical practices. The
amount of active ingredient included in the pharmaceutical
preparations is such that a suitable dose within the designated
range is provided.
[0110] The sterile composition for injection can be formulated in
accordance with conventional pharmaceutical practices using
distilled water for injection as a vehicle. For example,
physiological saline or an isotonic solution containing glucose and
other supplements such as D-sorbitol, D-mannose, D-mannitol, and
sodium chloride may be used as an aqueous solution for injection,
optionally in combination with a suitable solubilizing agent, for
example, alcohol such as ethanol and polyalcohol such as propylene
glycol or polyethylene glycol, and a nonionic surfactant such as
polysorbate 80.TM., HCO-50 and the like.
[0111] Non-limiting examples of oily liquid include sesame oil and
soybean oil, and it may be combined with benzyl benzoate or benzyl
alcohol as a solubilizing agent. Other items that may be included
are a buffer such as a phosphate buffer, or sodium acetate buffer,
a soothing agent such as procaine hydrochloride, a stabilizer such
as benzyl alcohol or phenol, and an antioxidant. The formulated
injection can be packaged in a suitable ampoule.
[0112] Route of administration can be parenteral, for example,
administration by injection, transnasal administration,
transpulmonary administration, or transcutaneous administration.
Administration can be systemic or local by intravenous injection,
intramuscular injection, intraperitoneal injection, subcutaneous
injection.
[0113] The term "subcutaneous administration" refers to
introduction of a drug under the skin of an animal or human patient
(e.g., by subcutaneous infusion or subcutaneous bolus), preferably
within a pocket between the skin and underlying tissue, by
relatively slow, sustained delivery from a drug receptacle. The
pocket may be created by pinching or drawing the skin up and away
from underlying tissue. In particular embodiments, an extracellular
matrix degrading enzyme (e.g., a hyaluronidase or any extracellular
matrix degrading enzyme described herein) is administered at each
of the sites (e.g., prior to administration of the composition
and/or during the non-delivery period). In particular embodiments,
the extracellular matrix degrading enzyme is co-infused with the
composition.
[0114] Convenient sites for subcutaneous administration include the
shoulder, upper arm, thigh, and abdomen. In particular embodiments
of the above methods, the composition is administered into subcutis
or fat at a depth between 2 mm and 10 mm below the dermis of the
subject.
[0115] The term "subcutaneous infusion" refers to introduction of a
drug under the skin of an animal or human patient, preferably
within a pocket between the skin and underlying tissue, by
relatively slow, sustained delivery from a drug receptacle for a
period of time including, but not limited to, 30 minutes or less,
or 90 minutes or less. Optionally, the infusion may be made by
subcutaneous implantation of a drug delivery pump implanted under
the skin of the animal or human patient, wherein the pump delivers
a predetermined amount of drug for a predetermined period of time,
such as 30 minutes, 90 minutes, or a time period spanning the
length of the treatment regimen.
[0116] The term "subcutaneous bolus" refers to drug administration
beneath the skin of an animal or human patient, where bolus drug
delivery is preferably less than approximately 15 minutes, more
preferably less than 5 minutes, and most preferably less than 60
seconds. Administration is preferably within a pocket between the
skin and underlying tissue, where the pocket is created, for
example, by pinching or drawing the skin up and away from
underlying tissue.
[0117] The term "extracellular matrix degrading enzyme" means an
enzyme that can break down extracellular matrix at the site of
infusion, resulting in improved tissue permeability for an
composition infused at the site. Extracellular matrix degrading
enzymes include enzymes catalyzing the hydrolysis of hyaluronic
acid (hyaluronan), a glycosaminoglycan, chondroitin, or collagen,
such as a hyaluronidase, glycosaminoglycanase, collagenase (e.g.
cathepsin), serine proteases, thiol proteases, and matrix
metalloproteases, of which the human enzymes are preferred and the
recombinant human enzymes are most preferred. Examples of such
enzymes which can be used in the methods and compositions of the
invention are described in U.S. Pat. Nos. 4,258,134; 4,820,516;
7,871, 607; 7,767,429; 7,829,081; 7,846,431; 7,871,607; 8,187,855;
and 8,105,586, and U.S. Patent Publication Nos. 20090304665;
20110053247; 20120101325; and 20110008309, each of which is
incorporated by reference. Human hyaluronidases which can be used
in the methods and compositions of the invention are also
described, for example, in U.S. Pat. Nos. 3,945,889; 6,057,110;
5,958,750; 5,854,046; 5,827,721; and 5,747,027, each of which is
incorporated herein by reference. Commercially available
hyaluronidases which can be used in the methods and compositions of
the invention include HYDASE.TM. (PrimaPharm Inc.), VITRASSE.RTM.
(ISTA Pharmaceuticals), AMPHADASE.RTM. (Amphastar Pharmaceuticals),
and HYLENEX.RTM. (sold by Halozyme Therapeutics).
[0118] A suitable means of administration can be selected based on
the age and condition of the patient. A single dose of the
pharmaceutical composition containing a modified polypeptide can be
selected from a range of 0.001 to 1000 mg/kg of body weight. On the
other hand, a dose can be selected in the range of 0.001 to 100000
mg/body weight, but the present disclosure is not limited to such
ranges. The dose and method of administration varies depending on
the weight, age, condition, and the like of the patient, and can be
suitably selected as needed by those skilled in the art.
EXAMPLES
Example 1
Preparation of Sialylated Glycoproteins
[0119] The sialylation of IVIg by the sialyltransferase ST6 was
analyzed. IVIg was first galactosylated and then sialylated. The
reactions were performed sequentially. There was no purification
between galactosylation and sialylation reactions. The relative
abundance of glycoforms was analyzed following the sialylation
reactions.
Galactosylation
[0120] A reaction was set up that contained the following
components at the concentrations indicated in Table 2:
TABLE-US-00002 TABLE 2 Galactosylation conditions (Target s2IVIG)
Final Constituent concentration MOPS (pH 7.4) 50 mM MnCl.sub.2 8 mM
IVIg 125 mg/ml B4GalT1 (100 u/ml) 1.04 mg/g-IVIG UDP-Galactose 5
mM
[0121] The reaction was incubated for 24-72 hours at 37.degree.
C.
B. Sialylation
[0122] To an aliquot of the galactosylation reaction were added
CMP-NANA, MOPS buffer and ST6Gal1. The final volume was adjusted so
that the final concentration of components in the reaction was as
indicated in Table 3.
TABLE-US-00003 TABLE 3 Sialylation conditions Constituent Final
concentration MOPS (pH 7.4) 50 mM IVIg 115 mg/ml CMP-NANA (6
.times. 8 mM) 48 mM ST6Gal1 (SEQ ID NO: 1) 3.5 mg/g-IVIG
[0123] The reaction was incubated at 37.degree. C. Aliquots were
extracted at the times indicated in FIG. 5 and frozen at
-20.degree. C. for later analyses.
C. Results
[0124] As shown in FIG. 5, the predominant glycoform changed over
time from G2F to A1F (1,3) to A2F to A1F (1,6). The results are
summarized in the reaction scheme depicted in FIG. 4. As shown in
FIG. 4, the product glycoform can change between G2F, A1F (1,3),
A2F, and A1F (1,6) during the course of a reaction due to competing
addition (forward reaction) and removal (back reaction) steps.
[0125] The sialyltransferase ST6 can add sialic acid to either
branch of a substrate's biantennary N-glycan. However, these
results demonstrate that addition to each branch happens at
different rates, resulting in different end products depending on
the reaction conditions. Addition of sialic acid to the .alpha.1,3
branch is faster than addition to the .alpha.1,6 branch.
[0126] These data also demonstrate that sialyltransferase ST6 can
also catalyze the removal of sialic acids from N-glycans. The
removal of sialic acid from the .alpha.1,3 branch is faster than
removal from the .alpha.1,6 branch. This can surprisingly lead to
the production of Fc glycans substantially or primarily
monosialylated on the .alpha.1,6 branch by modulating reaction
conditions.
[0127] This Example demonstrates that reaction conditions can be
controlled to produce a glycoprotein product having a predetermined
or target sialylation levels. Such conditions can include time, ST6
sialyltransferase concentration, substrate concentration, donor
sugar nucleotide concentration, product nucleotide concentration,
pH, buffer composition, and/or temperature.
Example 2
Dose Response of IVIg, S1-IVIg, S2-IVIg, and Des-IVIg in a Chronic
ITP Mouse Model
[0128] The effect of IVIg, S1-IVIg, S2-IVIg, and Des-IVIg at
varying doses in an anti-CD41 antibody mediated ITP mouse model was
analyzed.
A. Study Design
[0129] Sixty-six to seventy two mice were given 1.5 .mu.g/mouse of
rat anti-CD41 antibody (Ab) clone MWReg30 (BioLegend cat#133910)
once daily for 4 days (on Days 1, 2, 3 and 4), intraperitoneally.
Six to twelve mice were dosed in the same manner with a rat IgG1, k
isotype control (Bio Legend cat#400414). All mice were dosed once
intravenously with saline control, IVIg, S1-IVIg, S2-IVIg, or
desialylated-IVIg (Des-IVIg) at different doses 1 to 2 hours after
the third anti-CD41 Ab injection (Table 4). Mice were bled on Day 4
(4 h after the forth anti-CD41 injection) and on Day 5 (24 h after
the forth anti-CD41 Ab injection) to quantitate total platelet and
reticulated platelet levels. To confirm that platelet depletion was
successful, a subgroup of mice was bled on Day 3, prior to
treatment.
TABLE-US-00004 TABLE 4 IVIg, S1-IVIg, S2-IVIg, and Des-IVIg dose
response study details Induction (1.5 .mu.g IP) Treatment Agent
Group # n 4 daily doses (200 uL IV) Dose Timing of Dosing Blood
Sampling 1 6 anti-CD41 Saline 200 .mu.L 1-2 h post 3. anti-CD41
dose Day 3, 4 and Day 5 2 6 Rat IgG1 Saline 200 .mu.L 1-2 h post 3.
anti-CD41 dose Day 4 and Day 5 3 8 anti-CD41 IVIg Gammagard 0.5
g/kg 1-2 h post 3. anti-CD41 dose Day 4 and Day 5 4 8 anti-CD41
IVIg Gammagard 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4 and Day 5
5 8 anti-CD41 S1-IVIg 0.5 g/kg 1-2 h post 3. anti-CD41 dose Day 4
and Day 5 6 8 anti-CD41 S1-IVIg 1 g/kg 1-2 h post 3. anti-CD41 dose
Day 4 and Day 5 7 8 anti-CD41 S2-IVIg 0.5 g/kg 1-2 h post 3.
anti-CD41 dose Day 4 and Day 5 8 8 anti-CD41 S2-IVIg 1 g/kg 1-2 h
post 3. anti-CD41 dose Day 4 and Day 5 9 8 anti-CD41 Des-IVIg 0.5
g/kg 1-2 h post 3. anti-CD41 dose Day 4 and Day 5 10 8 anti-CD41
Des-IVIg 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4 and Day 5
B. Methods
ITP Induction in Mice:
[0130] In vivo studies were conducted using female C57BL/6 mice
(18-22 g, Charles Rivers Labs, MA). All procedures were performed
in compliance with the Animal Welfare Act and with the Guide for
the Care and Use of Laboratory Animals.
Quantitation of Total Platelets:
[0131] Blood samples were collected by submandibular bleed into
EDTA coated tubes, and then run on a VetScan Instrument for
platelet level determination. Total platelet levels were analyzed
using One-Way ANOVA with Dunnett's or Bonferroni's post-test.
Quantitation of Reticulated Platelets:
[0132] To evaluate and quantitate for the presence of reticulated
(young) platelets which contain residual RNA, whole blood was
sequentially stained for total platelets (anti-CD61) followed by
staining for the RNA with thiazole orange (RNA-binding dye,
commercially available as ReticCount Reagent from BD Biosciences).
This analysis was performed for blood samples collected on Day
5.
[0133] Ten microliters of whole blood was transferred into the
bottom of a 5 mL FACS tube. Five microliters of anti-mouse CD61-PE
antibody (BD Biosciences) was added directly to the whole blood and
samples were mixed thoroughly by pipetting. Samples were incubated
at room temperature for 5 minutes in the dark. Two milliliters of
ReticCount reagent (BD Biosciences) was added to each sample and
samples incubated for a minimum of 30 minutes at room
temperature.
[0134] Samples were acquired on a FACS Canto flow cytometer (BD
Biosciences). Total platelets were identified by forward and side
scatter characteristics of the cells and distinguished from
erythrocytes by gating on CD61-PE positive events. A total 10,000
platelet events were recorded for each sample. Using FlowJo
software, a gate was set on the reticulated platelets (CD61
positive and thiazole orange positive) using samples from isotype
control treated mice to achieve a rate of 6-10% reticulated
platelets (normal rate). The same gate was then applied to all
subsequent samples and treatment groups to calculate percentages of
reticulated and non-reticulated platelets. Total counts of
reticulated and non-reticulated platelets for each sample were
calculated by multiplying the total number of platelets measured in
the VetScan Instrument by the percentage of the platelet fraction.
Total reticulated and non-reticulated platelet levels were analyzed
using One-Way ANOVA with Dunnett's or Bonferroni's post-test.
[0135] In addition to the overall platelet quantification using the
VetScan Instrument, numbers of reticulated platelets were also
determined using ReticCount, anti-CD61-PE labeled Ab and flow
cytometry.
C. Results
[0136] The results of the IVIg, S1-IVIg, S2-IVIg, and Des-IVIg dose
response study are shown in Table 5.
TABLE-US-00005 TABLE 5 Total, reticulated, and non-reticulated
platelet counts (10.sup.9/L) on Day 5 Anti-CD41 Ab (1.5 .mu.g)
Treatment Isotype desialylated Control IVIg S1-IVIg S2-IVIg IVIg
Disease (1.5 .mu.g) Dose Induction Saline Saline 0.5 g/kg 1 g/kg
0.5 g/kg 1 g/kg 0.5 g/kg 1 g/kg 0.5 g/kg 1 g/kg n per group 6 6 8 7
6 8 7 8 8 7 Total 733 .+-. 73 240 .+-. 200 282 .+-. 79 429 .+-. 116
355 .+-. 137 356 .+-. 89 362 .+-. 94 501 .+-. 102 224 .+-. 51 182
.+-. 78 Platelets .times. 10.sup.9/L[mean .+-. SD] Reticulated 70
.+-. 18 97 .+-. 24 109 .+-. 76 268 .+-. 84 196 .+-. 64 234 .+-. 81
304 .+-. 73 391 .+-. 86 175 .+-. 42 134 .+-. 56 Platelets .times.
10.sup.9/L [mean .+-. SD] Non-Reticulated 663 .+-. 64 143 .+-. 182
173 .+-. 49 161 .+-. 77 159 .+-. 85 122 .+-. 63 58 .+-. 41 111 .+-.
69 49 .+-. 32 48 .+-. 26 Platelets .times. 10.sup.9/L [mean .+-.
SD]
Example 3
Comparison of IVIg, S1-IVIg, S2-IVIg, and Des-IVIg in a Chronic ITP
Mouse Model
[0137] The effect of IVIg, S1-IVIg, S2-IVIg, and Des-IVIg in an
anti-CD41 antibody mediated ITP mouse model was analyzed.
A. Study Design
[0138] Sixty-six to seventy two mice were given 1.5 .mu.g/mouse of
rat anti-CD41 antibody (Ab) clone MWReg30 (BioLegend cat#133910)
once daily for 4 days (on Days 1, 2, 3 and 4), intraperitoneally.
Six to twelve mice were dosed in the same manner with a rat IgG1, k
isotype control (Bio Legend cat#400414). All mice were dosed once
intravenously with saline control, IVIg, S1-IVIg, S2-IVIg, or
desialylated-IVIg (Des-IVIg at different doses 1 to 2 hours after
the third anti-CD41 Ab injection (Table 6). Mice were bled on Day 4
(4 h after the forth anti-CD41 injection) and on Day 5 (24 h after
the forth anti-CD41 Ab injection) to quantitate total platelet and
reticulated platelet levels. To confirm that platelet depletion was
successful, a subgroup of mice was bled on Day 3, prior to
treatment. On Day 4 bone marrow cells were isolated to quantitate
megakaryocytes.
TABLE-US-00006 TABLE 6 IVIg, S1-IVIg, S2-IVIg, and Des-IVIg
comparison study details Induction (1.5 .mu.g IP) Treatment Agent
Group # n 4 daily doses (200 uL IV) Dose Timing of Dosing Blood
Sampling 1 12 anti-CD41 Saline 200 .mu.L 1-2 h post 3. anti-CD41
dose Day 3, Day 4, and Day 5 2 12 Rat IgG1 Saline 200 .mu.L 1-2 h
post 3. anti-CD41 dose Day 4 and Day 5 3 12 anti-CD41 IVIg
Gammagard 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4 and Day 5 4 12
anti-CD41 S1-IVIg 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4 and Day
5 5 12 anti-CD41 S2-IVIg 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4
and Day 5 6 12 anti-CD41 Des-IVIg 1 g/kg 1-2 h post 3. anti-CD41
dose Day 4 and Day 5
B. Methods
ITP Induction in Mice:
[0139] In vivo studies were conducted using female C57BL/6 mice
(18-22 g, Charles Rivers Labs, MA). All procedures were performed
in compliance with the Animal Welfare Act and with the Guide for
the Care and Use of Laboratory Animals.
Quantitation of Total Platelets:
[0140] Blood samples were collected by submandibular bleed into
EDTA coated tubes, and then run on a VetScan Instrument for
platelet level determination. Total platelet levels were analyzed
using One-Way ANOVA with Dunnett's or Bonferroni's post-test.
Quantitation of Reticulated Platelets:
[0141] To evaluate and quantitate for the presence of reticulated
(young) platelets which contain residual RNA, whole blood was
sequentially stained for total platelets (anti-CD61) followed by
staining for the RNA with thiazole orange (RNA-binding dye,
commercially available as ReticCount Reagent from BD Biosciences).
This analysis was performed for blood samples collected on Day
5.
[0142] Ten microliters of whole blood was transferred into the
bottom of a 5 mL FACS tube. Five microliters of anti-mouse CD61-PE
antibody (BD Biosciences) was added directly to the whole blood and
samples were mixed thoroughly by pipetting. Samples were incubated
at room temperature for 5 minutes in the dark. Two milliliters of
ReticCount reagent (BD Biosciences) was added to each sample and
samples incubated for a minimum of 30 minutes at room
temperature.
[0143] Samples were acquired on a FACS Canto flow cytometer (BD
Biosciences). Total platelets were identified by forward and side
scatter characteristics of the cells and distinguished from
erythrocytes by gating on CD61-PE positive events. A total 10,000
platelet events were recorded for each sample. Using FlowJo
software, a gate was set on the reticulated platelets (CD61
positive and thiazole orange positive) using samples from isotype
control treated mice to achieve a rate of 6-10% reticulated
platelets (normal rate). The same gate was then applied to all
subsequent samples and treatment groups to calculate percentages of
reticulated and non-reticulated platelets. Total counts of
reticulated and non-reticulated platelets for each sample were
calculated by multiplying the total number of platelets measured in
the VetScan Instrument by the percentage of the platelet fraction.
Total reticulated and non-reticulated platelet levels were analyzed
using One-Way ANOVA with Dunnett's or Bonferroni's post-test. In
addition to the overall platelet quantification using the VetScan
Instrument, numbers of reticulated platelets were also determined
using ReticCount, anti-CD61-PE labeled Ab and flow cytometry.
Quantitation of Megakaryocytes in the Bone Marrow:
[0144] On Day 4 (1 day after IVIg treatment and 4 h after the
4.sup.th anti-CD41 antibody injection) of the study, bone marrow
was extracted from one femur per mouse by using a syringe with a 25
gauge needle, flushing the bone shaft repeatedly with 0.5 mL of
media. Cell suspensions were filtered through a nylon mesh and
fixed in 4% paraformaldehyde for 15 minutes on ice. Cells were
washed twice with PBS buffer containing 10% culture grade normal
bovine serum, resuspended and counted using a ViCell cell counter.
Cells were resuspended at 1.times.10.sup.6 cells/m L. Cytospin
slides were prepared with 0.5 mL per slide. The slides were
air-dried and stored at 80.degree. C. until use.
[0145] After blocking, cells were stained with anti-CD41 (rat
anti-mouse CD41; clone: MWReg30, cat#133910, Biolegend; diluted
1:150 in PBS contains 10% normal donkey serum) by
immunohistochemistry using a BondMax instrument (Leica) and the Rat
Polink-2 open kit protocol. Slides were counter stained with
hematoxylin, mounted, and cover slipped.
[0146] Stained slides were imaged using a Vectra microscope system
under 4.times. and 20.times. magnification. Images were spectrally
unmixed, segmented, then quantified for megakaryocyte count as well
as CD41 signal intensity using Inform software. Total, mean and
maximum signals as well as signal area was calculated for each
category. Data were normalized to total cell numbers and reported
as total OD signal or per cell ratio. Data were transferred into
Excel and Graph Pad Prism, graphed and analyzed for statistically
significant differences.
C. Results
[0147] The results of the IVIg, S1-IVIg, S2-IVIg, and Des-IVIg
comparison study are shown in Table 7.
TABLE-US-00007 TABLE 7 Total, reticulated, and non-reticulated
platelet counts (10.sup.9/L) on Day 5 and Megakaryocyte count in
bone marrow cells (MK/10.sup.6 BM cells) on Day 4 Anti-CD41 Ab (1.5
.mu.g) Treatment Isotype Desialylated Control IVIg S1-IVIg S2-IVIg
IVIg (1.5 .mu.g) Dose Disease Induction Saline Saline 1 g/kg 1 g/kg
1 g/kg 1 g/kg n per group 6 6 6 6 6 6 Total Platelets 739 .+-. 84
277 .+-. 203 290 .+-. 113 563 .+-. 138 578 .+-. 209 220 .+-. 97
[mean .+-. SD] Reticulated 61 .+-. 11 98 .+-. 57 131 .+-. 60 184
.+-. 34 261 .+-. 49 130 .+-. 46 Platelets [mean .+-. SD]
Non-Reticulated 679 .+-. 75 179 .+-. 168 159 .+-. 60 379 .+-. 133
318 .+-. 177 90 .+-. 56 Platelets [mean .+-. SD] Megakaryocytes 328
.+-. 132 323 .+-. 51 341 .+-. 162 360 .+-. 31 496 .+-. 115 372 .+-.
87 in Bone Marrow [mean .+-. SD]
Example 4
Comparison of IVIg, rFc, S1-rFc, S2-rFc, and Des-IVIg in a Chronic
ITP Mouse Model
[0148] The effect of IVIg, rFc, S1-rFc, S2-rFc, and Des-IVIg in an
anti-CD41 antibody mediated ITP mouse model was analyzed.
A. Study Design
[0149] Sixty-six to seventy two mice were given 1.5 .mu.g/mouse of
rat anti-CD41 antibody (Ab) clone MWReg30 (BioLegend cat#133910)
once daily for 4 days (on Days 1, 2, 3 and 4), intraperitoneally.
Six to twelve mice were dosed in the same manner with a rat IgG1, k
isotype control (Bio Legend cat#400414). All mice were dosed once
intravenously with saline control, IVIg, recombinant Fc (rFc),
S1-rFc, S2-rFc, or Des-IVIg at different doses 1 to 2 hours after
the third anti-CD41 Ab injection (Table 8). Mice were bled on Day 4
(4 h after the forth anti-CD41 injection) and on Day 5 (24 h after
the forth anti-CD41 Ab injection) to quantitate total platelet and
reticulated platelet levels. To confirm that platelet depletion was
successful, a subgroup of mice was bled on Day 3, prior to
treatment. On Day 5 bone marrow cells were isolated to quantitate
megakaryocytes.
TABLE-US-00008 TABLE 8 IVIg, rFc, S1-rFc, S2-rFc, and Des-IVIg
comparison study details Induction (1.5 .mu.g IP) Treatment Agent
Blood Group # n 4 daily doses (200 uL IV) Dose Timing of Dosing
Sampling 1 12 anti-CD41 Saline 200 .mu.L 1-2 h post 3. anti-CD41
dose Day 3, Day 4, and Day 5 2 12 Rat IgG1 Saline 200 .mu.L 1-2 h
post 3. anti-CD41 dose Day 4 and Day 5 3 12 anti-CD41 IVIg
Gammagard 1 g/kg 1-2 h post 3. anti-CD41 dose Day 4 and Day 5 4 12
anti-CD41 rFc 0.3 g/kg 1-2 h post 3. anti-CD41 dose Day 4 and Day 5
5 12 anti-CD41 S1-rFc 0.3 g/kg 1-2 h post 3. anti-CD41 dose Day 4
and Day 5 6 12 anti-CD41 52-rFc 0.3 g/kg 1-2 h post 3. anti-CD41
dose Day 4 and Day 5 7 12 anti-CD41 Des-IVIg 1 g/kg 1-2 h post 3.
anti-CD41 dose Day 4 and Day 5
B. Methods
ITP Induction in Mice:
[0150] In vivo studies were conducted using female C57BL/6 mice
(18-22 g, Charles Rivers Labs, MA). All procedures were performed
in compliance with the Animal Welfare Act and with the Guide for
the Care and Use of Laboratory Animals.
Quantitation of Total Platelets:
[0151] Blood samples were collected by submandibular bleed into
EDTA coated tubes, and then run on a VetScan Instrument for
platelet level determination. Total platelet levels were analyzed
using One-Way ANOVA with Dunnett's or Bonferroni's post-test.
Quantitation of Reticulated Platelets:
[0152] To evaluate and quantitate for the presence of reticulated
(young) platelets which contain residual RNA, whole blood was
sequentially stained for total platelets (anti-CD61) followed by
staining for the RNA with thiazole orange (RNA-binding dye,
commercially available as ReticCount Reagent from BD Biosciences).
This analysis was performed for blood samples collected on Day
5.
[0153] Ten microliters of whole blood was transferred into the
bottom of a 5 mL FACS tube. Five microliters of anti-mouse CD61-PE
antibody (BD Biosciences) was added directly to the whole blood and
samples were mixed thoroughly by pipetting. Samples were incubated
at room temperature for 5 minutes in the dark. Two milliliters of
ReticCount reagent (BD Biosciences) was added to each sample and
samples incubated for a minimum of 30 minutes at room
temperature.
[0154] Samples were acquired on a FACS Canto flow cytometer (BD
Biosciences). Total platelets were identified by forward and side
scatter characteristics of the cells and distinguished from
erythrocytes by gating on CD61-PE positive events. A total 10,000
platelet events were recorded for each sample. Using FlowJo
software, a gate was set on the reticulated platelets (CD61
positive and thiazole orange positive) using samples from isotype
control treated mice to achieve a rate of 6-10% reticulated
platelets (normal rate). The same gate was then applied to all
subsequent samples and treatment groups to calculate percentages of
reticulated and non-reticulated platelets. Total counts of
reticulated and non-reticulated platelets for each sample were
calculated by multiplying the total number of platelets measured in
the VetScan Instrument by the percentage of the platelet fraction.
Total reticulated and non-reticulated platelet levels were analyzed
using One-Way ANOVA with Dunnett's or Bonferroni's post-test.
[0155] In addition to the overall platelet quantification using the
VetScan Instrument, numbers of reticulated platelets were also
determined using ReticCount, anti-CD61-PE labeled Ab and flow
cytometry.
Quantitation of Megakaryocytes in the Bone Marrow:
[0156] On Day 5 of the study (ITP-010; 2 days after IVIg treatment
and 24 h after the 4.sup.th anti-CD41 antibody injection), bone
marrow was extracted from one femur per mouse by using a syringe
with a 25 gauge needle, flushing the bone shaft repeatedly with 0.5
mL of media. Cell suspensions were filtered through a nylon mesh
and fixed in 4% paraformaldehyde for 15 minutes on ice. Cells were
washed twice with PBS buffer containing 10% culture grade normal
bovine serum, resuspended and counted using a ViCell cell counter.
Cells were resuspended at 1.times.10.sup.6 cells/m L. Cytospin
slides were prepared with 0.5 mL per slide. The slides were
air-dried and stored at 80.degree. C. until use.
[0157] After blocking, cells were stained with anti-CD41 (rat
anti-mouse CD41; clone: MWReg30, cat#133910, Biolegend; diluted
1:150 in PBS contains 10% normal donkey serum) by
immunohistochemistry using a BondMax instrument (Leica) and the Rat
Polink-2 open kit protocol. Slides were counter stained with
hematoxylin, mounted, and cover slipped.
[0158] Stained slides were imaged using a Vectra microscope system
under 4.times. and 20.times. magnification. Images were spectrally
unmixed, segmented, then quantified for megakaryocyte count as well
as CD41 signal intensity using Inform software. Total, mean and
maximum signals as well as signal area was calculated for each
category. Data were normalized to total cell numbers and reported
as total OD signal or per cell ratio. Data were transferred into
Excel and Graph Pad Prism, graphed and analyzed for statistically
significant differences.
C. Results
[0159] The results of IVIg, rFc, S1-rFc, S2-rFc, and Des-IVIg
comparison study are shown in Table 9.
TABLE-US-00009 TABLE 9 Total, reticulated, and non-reticulated
platelet counts (10.sup.9/L) and Megakaryocyte count in bone marrow
cells (MK/10.sup.6 BM cells) on Day 5 Anti-CD41 Ab (1.5 .mu.g)
Isotype Treatment Control IVIg rFc S1-rFc S2-rFc Des IVIg (1.5
.mu.g) Dose Disease Induction Saline Saline 1 g/kg 0.3 g/kg 0.3
g/kg 0.3 g/kg 1 g/kg n per group 6 6 6 6 6 6 6 Total Platelets 734
.+-. 124 189 .+-. 83 401 .+-. 103 277 .+-. 226 379 .+-. 198 369
.+-. 89 160 .+-. 67 [mean .+-. SD] Reticulated 64 .+-. 10 73 .+-.
39 125 .+-. 42 116 .+-. 55 178 .+-. 60 234 .+-. 95 122 .+-. 56
platelets [mean .+-. SD] Non-Reticulated 670 .+-. 117 116 .+-. 70
277 .+-. 106 161 .+-. 191 201 .+-. 157 135 .+-. 46 38 .+-. 25
platelets [mean .+-. SD] Megakaryocytes in 126 .+-. 64 1.0 .+-. 0.7
275 .+-. 87 237 .+-. 57 218 .+-. 68 345 .+-. 44 81 .+-. 53 Bone
Marrow [mean .+-. SD]
[0160] While the methods have been described in conjunction with
various instances and examples, it is not intended that the methods
be limited to such instances or examples. On the contrary, the
methods encompass various alternatives, modifications, and
equivalents, as will be appreciated by those of skill in the art.
Sequence CWU 1
1
31418PRTHomo sapiens 1Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala
Gly Leu Leu Ala Ser 1 5 10 15 Ser Arg Ala Gly Ser Ser Pro Leu Leu
Ala Met Glu Trp Ser His Pro 20 25 30 Gln Phe Glu Lys Leu Glu Gly
Gly Gly Ser Gly Gly Gly Ser Gly Gly 35 40 45 Ser Trp Ser His Pro
Gln Phe Glu Lys His Ala His Ala His Ser Arg 50 55 60 Lys Asp His
Leu Ile His Asn Val His Lys Glu Glu His Ala His Ala 65 70 75 80 His
Asn Lys Glu Leu Gly Thr Ala Val Phe Gln Gly Pro Met Arg Arg 85 90
95 Ala Ile Arg Gly Arg Ser Phe Gln Val Trp Asn Lys Asp Ser Ser Ser
100 105 110 Lys Asn Leu Ile Pro Arg Leu Gln Lys Ile Trp Lys Asn Tyr
Leu Ser 115 120 125 Met Asn Lys Tyr Lys Val Ser Tyr Lys Gly Pro Gly
Pro Gly Ile Lys 130 135 140 Phe Ser Ala Glu Ala Leu Arg Cys His Leu
Arg Asp His Val Asn Val 145 150 155 160 Ser Met Val Glu Val Thr Asp
Phe Pro Phe Asn Thr Ser Glu Trp Glu 165 170 175 Gly Tyr Leu Pro Lys
Glu Ser Ile Arg Thr Lys Ala Gly Pro Trp Gly 180 185 190 Arg Cys Ala
Val Val Ser Ser Ala Gly Ser Leu Lys Ser Ser Gln Leu 195 200 205 Gly
Arg Glu Ile Asp Asp His Asp Ala Val Leu Arg Phe Asn Gly Ala 210 215
220 Pro Thr Ala Asn Phe Gln Gln Asp Val Gly Thr Lys Thr Thr Ile Arg
225 230 235 240 Leu Met Asn Ser Gln Leu Val Thr Thr Glu Lys Arg Phe
Leu Lys Asp 245 250 255 Ser Leu Tyr Asn Glu Gly Ile Leu Ile Val Trp
Asp Pro Ser Val Tyr 260 265 270 His Ser Asp Ile Pro Lys Trp Tyr Gln
Asn Pro Asp Tyr Asn Phe Phe 275 280 285 Asn Asn Tyr Lys Thr Tyr Arg
Lys Leu His Pro Asn Gln Pro Phe Tyr 290 295 300 Ile Leu Lys Pro Gln
Met Pro Trp Glu Leu Trp Asp Ile Leu Gln Glu 305 310 315 320 Ile Ser
Pro Glu Glu Ile Gln Pro Asn Pro Pro Ser Ser Gly Met Leu 325 330 335
Gly Ile Ile Ile Met Met Thr Leu Cys Asp Gln Val Asp Ile Tyr Glu 340
345 350 Phe Leu Pro Ser Lys Arg Lys Thr Asp Val Cys Tyr Tyr Tyr Gln
Lys 355 360 365 Phe Phe Asp Ser Ala Cys Thr Met Gly Ala Tyr His Pro
Leu Leu Tyr 370 375 380 Glu Lys Asn Leu Val Lys His Leu Asn Gln Gly
Thr Asp Glu Asp Ile 385 390 395 400 Tyr Leu Leu Gly Lys Ala Thr Leu
Pro Gly Phe Arg Thr Ile His Cys 405 410 415 Pro Gly 2375PRTHomo
sapiens 2Gly Ser Tyr Tyr Asp Ser Phe Lys Leu Gln Thr Lys Glu Phe
Gln Val 1 5 10 15 Leu Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp
Ser Gln Ser Val 20 25 30 Ser Ser Ser Ser Thr Gln Asp Pro His Arg
Gly Arg Gln Thr Leu Gly 35 40 45 Ser Leu Arg Gly Leu Ala Lys Ala
Lys Pro Glu Ala Ser Phe Gln Val 50 55 60 Trp Asn Lys Asp Ser Ser
Ser Lys Asn Leu Ile Pro Arg Leu Gln Lys 65 70 75 80 Ile Trp Lys Asn
Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser Tyr Lys 85 90 95 Gly Pro
Gly Pro Gly Ile Lys Phe Ser Ala Glu Ala Leu Arg Cys His 100 105 110
Leu Arg Asp His Val Asn Val Ser Met Val Glu Val Thr Asp Phe Pro 115
120 125 Phe Asn Thr Ser Glu Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile
Arg 130 135 140 Thr Lys Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser
Ser Ala Gly 145 150 155 160 Ser Leu Lys Ser Ser Gln Leu Gly Arg Glu
Ile Asp Asp His Asp Ala 165 170 175 Val Leu Arg Phe Asn Gly Ala Pro
Thr Ala Asn Phe Gln Gln Asp Val 180 185 190 Gly Thr Lys Thr Thr Ile
Arg Leu Met Asn Ser Gln Leu Val Thr Thr 195 200 205 Glu Lys Arg Phe
Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile Leu Ile 210 215 220 Val Trp
Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys Trp Tyr Gln 225 230 235
240 Asn Pro Asp Tyr Asn Phe Phe Asn Asn Tyr Lys Thr Tyr Arg Lys Leu
245 250 255 His Pro Asn Gln Pro Phe Tyr Ile Leu Lys Pro Gln Met Pro
Trp Glu 260 265 270 Leu Trp Asp Ile Leu Gln Glu Ile Ser Pro Glu Glu
Ile Gln Pro Asn 275 280 285 Pro Pro Ser Ser Gly Met Leu Gly Ile Ile
Ile Met Met Thr Leu Cys 290 295 300 Asp Gln Val Asp Ile Tyr Glu Phe
Leu Pro Ser Lys Arg Lys Thr Asp 305 310 315 320 Val Cys Tyr Tyr Tyr
Gln Lys Phe Phe Asp Ser Ala Cys Thr Met Gly 325 330 335 Ala Tyr His
Pro Leu Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn 340 345 350 Gln
Gly Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala Thr Leu Pro 355 360
365 Gly Phe Arg Thr Ile His Cys 370 375 3402PRTHomo sapiens 3Met
Ile His Thr Asn Leu Lys Lys Lys Phe Ser Tyr Phe Ile Leu Ala 1 5 10
15 Phe Leu Leu Phe Ala Leu Ile Cys Val Trp Lys Lys Gly Ser Tyr Glu
20 25 30 Ala Leu Lys Leu Gln Ala Lys Glu Phe Gln Val Thr Lys Ser
Leu Glu 35 40 45 Lys Leu Ala Ile Gly Ser Gly Ser Gln Ser Thr Ser
Ala Ser Ile Lys 50 55 60 Gln Asp Ser Lys Pro Gly Ser Gln Val Leu
Ser His Leu Arg Val Thr 65 70 75 80 Ala Lys Val Lys Pro Gln Ser Pro
Tyr Gln Val Trp Asp Lys Asn Ser 85 90 95 Ser Ser Lys Asn Leu Asn
Pro Arg Leu Gln Lys Ile Leu Lys Asn Tyr 100 105 110 Leu Ser Met Asn
Lys Tyr Lys Val Ser Tyr Lys Gly Pro Gly Pro Gly 115 120 125 Val Lys
Phe Ser Val Glu Ala Leu Arg Cys His Leu Arg Asp Arg Val 130 135 140
Asn Val Ser Met Ile Glu Ala Thr Asp Phe Pro Phe Asn Thr Thr Glu 145
150 155 160 Trp Glu Gly Tyr Leu Pro Lys Glu Asn Phe Arg Thr Lys Ala
Gly Pro 165 170 175 Trp His Arg Cys Ala Val Val Ser Ser Ala Gly Ser
Leu Lys Ser Ser 180 185 190 His Leu Gly Lys Glu Ile Asp Ser His Asp
Ala Val Leu Arg Phe Asn 195 200 205 Gly Ala Pro Val Ala Asp Phe Gln
Gln Asp Val Gly Met Lys Thr Thr 210 215 220 Ile Arg Leu Met Asn Ser
Gln Leu Ile Thr Thr Glu Lys Gln Phe Leu 225 230 235 240 Lys Asp Ser
Leu Tyr Asn Glu Gly Ile Leu Ile Val Trp Asp Pro Ser 245 250 255 Leu
Tyr His Ala Asp Ile Pro Asn Trp Tyr Lys Lys Pro Asp Tyr Asn 260 265
270 Phe Phe Glu Thr Tyr Lys Ser Tyr Arg Lys Leu Tyr Pro Ser Gln Pro
275 280 285 Phe Tyr Ile Leu Arg Pro Gln Met Pro Trp Glu Leu Trp Asp
Ile Ile 290 295 300 Gln Glu Ile Ala Pro Asp Arg Ile Gln Pro Asn Pro
Pro Ser Ser Gly 305 310 315 320 Met Leu Gly Ile Ile Ile Met Met Thr
Leu Cys Asp Gln Val Asp Val 325 330 335 Tyr Glu Phe Leu Pro Ser Lys
Arg Lys Thr Asp Val Cys Tyr Tyr His 340 345 350 Gln Lys Phe Phe Asp
Ser Ala Cys Thr Met Gly Ala Tyr His Pro Leu 355 360 365 Leu Phe Glu
Lys Asn Met Val Lys Gln Leu Asn Glu Gly Thr Asp Glu 370 375 380 Asp
Ile Tyr Ile Phe Gly Lys Ala Thr Leu Ser Gly Phe Arg Thr Ile 385 390
395 400 His Cys
* * * * *