U.S. patent application number 14/416869 was filed with the patent office on 2015-07-30 for glycoproteins with anti-inflammatory properties.
This patent application is currently assigned to Momenta Pharmaceuticals, Inc.. The applicant listed for this patent is Momenta Pharmaceuticals, Inc.. Invention is credited to Enrique Arevalo, Carlos J. Bosques, Hetal Sarvaiya, Nathaniel Washburn.
Application Number | 20150210753 14/416869 |
Document ID | / |
Family ID | 49997976 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150210753 |
Kind Code |
A1 |
Sarvaiya; Hetal ; et
al. |
July 30, 2015 |
GLYCOPROTEINS WITH ANTI-INFLAMMATORY PROPERTIES
Abstract
Glycoproteins having particular sialylation patterns, and
methods of making and using such glycoproteins, are described.
Inventors: |
Sarvaiya; Hetal; (Quincy,
MA) ; Washburn; Nathaniel; (Littleton, MA) ;
Arevalo; Enrique; (Dorchester, MA) ; Bosques; Carlos
J.; (Arlington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Momenta Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Momenta Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
49997976 |
Appl. No.: |
14/416869 |
Filed: |
July 25, 2013 |
PCT Filed: |
July 25, 2013 |
PCT NO: |
PCT/US13/52040 |
371 Date: |
January 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61676253 |
Jul 26, 2012 |
|
|
|
61768027 |
Feb 22, 2013 |
|
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Current U.S.
Class: |
424/133.1 ;
435/68.1; 436/512; 530/387.3 |
Current CPC
Class: |
C07K 2317/52 20130101;
C07K 2317/55 20130101; C07K 1/1077 20130101; C07K 2317/41 20130101;
A61P 37/00 20180101; C12P 21/005 20130101; C07K 14/435 20130101;
A61K 38/00 20130101; C07K 16/00 20130101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C07K 1/107 20060101 C07K001/107; C07K 14/435 20060101
C07K014/435 |
Claims
1. A method of producing a pharmaceutical preparation comprising
glycoproteins comprising an Fc region, wherein the branched glycans
on the Fc region are selectively sialylated on the .alpha.1-3 arm
at a predetermined level comprising: contacting a sialyltransferase
enzyme with a preparation comprising glycoproteins comprising an
IgG Fc region under conditions suitable for sialylation of a
plurality of said branched glycans by the enzyme; measuring the
level of branched glycans having a sialic acid on said .alpha.1-3
arm and/or on the .alpha.1-6 arm; processing said preparation into
a pharmaceutical preparation if said level is equivalent to said
predetermined level; thereby producing a pharmaceutical preparation
comprising glycoproteins comprising an Fc region, wherein the
branched glycans on the Fc region are selectively sialylated on the
.alpha.1-3 arm at a predetermined level.
2. The method of claim 1, wherein said predetermined level is at
least 95% of branched glycans having a sialic acid on said
.alpha.1-3 arm.
3. The method of claim 1, wherein said predetermined level is
20-90% of branched glycans having a sialic acid on said .alpha.1-3
arm.
4. The method of claim 1, wherein said sialyltransferase enzyme is
a ST6Gal-I enzyme.
5. The method of claim 1, wherein said .alpha.1,3 arm of the
branched glycans are sialylated with a NeuAc-.alpha.2,6-Gal
terminal linkage.
6. A method of increasing anti-inflammatory activity of a reference
glycoprotein preparation, comprising: providing a reference
glycoprotein preparation comprising glycoproteins comprising an IgG
Fc region; and sialylating the branched glycans on the Fc region on
the .alpha.1-3 arm of a plurality of said branched glycans to
produce a sialylated glycoprotein preparation; wherein said
glycoproteins in said reference glycoprotein preparation are not
IgG glycoproteins or do not consist essentially of an Fc region
derived from IgG glycoproteins; and wherein said sialylated
glycoprotein preparation has an increased level of
anti-inflammatory activity relative to the level of
anti-inflammatory activity of said reference glycoprotein
preparation.
7. The method of claim 6, further comprising measuring in said
sialylated glycoprotein preparation the level of said branched
glycans having a sialic acid on the .alpha.1-3 arm and/or measuring
the level of said branched glycans having a sialic acid on the
.alpha.1-6 arm.
8. The method of claim 6, further comprising processing said
sialylated glycoprotein preparation into a pharmaceutical
preparation if the level of branched glycans having a sialic acid
on the .alpha.1-3 arm and/or the level of branched glycans having a
sialic acid on the .alpha.1-6 arm meets a predetermined level.
9. The method of claim 8, wherein said the predetermined level is
at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 100% of the branched glycans
having a sialic acid on the .alpha.1,3 arm.
10. The method of claim 8, wherein said predetermined level is less
than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less of the
branched glycans having a sialic acid on the .alpha.1,6 arm.
11. A method of increasing anti-inflammatory activity of a
reference glycoprotein preparation, comprising: providing a
reference glycoprotein preparation comprising glycoproteins
comprising an IgG Fc region; and sialylating the branched glycans
on the Fc region on the .alpha.1-3 arm of a plurality of said
branched glycans to produce a sialylated glycoprotein preparation;
measuring in said sialylated glycoprotein preparation the level of
said branched glycans having a sialic acid on the .alpha.1-3 arm
and/or measuring the level of said branched glycans having a sialic
acid on the .alpha.1-6 arm; and processing said sialylated
glycoprotein preparation into a pharmaceutical preparation if the
level of branched glycans having a sialic acid on the .alpha.1-3
arm and/or the level of branched glycans having a sialic acid on
the .alpha.1-6 arm meets a predetermined level; wherein said
sialylated glycoprotein preparation has an increased level of
anti-inflammatory activity relative to the level of
anti-inflammatory activity of said reference glycoprotein
preparation.
12. The method of claim 11, wherein said predetermined level of
branched glycans having a sialic acid on the .alpha.1-3 arm is at
least 95% and said predetermined level of branched glycans having a
sialic acid on the .alpha.1-6 arm is less than 5%.
13. The method of claim 11, wherein said predetermined level of
branched glycans having a sialic acid on the .alpha.1-3 arm is
between 20-90%.
14. The method of claim 6, wherein said sialylated glycoprotein
preparation has a level of anti-inflammatory activity that is at
least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
125%, 150%, 175%, 200%, 300%, 400%, 500%, or more, higher than the
level of anti-inflammatory activity of said reference glycoprotein
preparation.
15. A method of manufacturing a pharmaceutical product comprising
glycoproteins comprising an IgG Fc region comprising: providing a
preparation comprising glycoproteins comprising an IgG Fc region;
measuring the level of branched glycans on the Fc region in said
preparation having a sialic acid on the .alpha.1-3 arm and/or on
the .alpha.1-6 arm; and processing the preparation into a
pharmaceutical product if the level of said branched glycans having
a sialic acid on the .alpha.1-3 arm and/or on the .alpha.1-6 arm is
equivalent to a predetermined level, thereby manufacturing a
pharmaceutical product comprising glycoproteins comprising an IgG
Fc region.
16. The method of claim 15, wherein the predetermined level is a
pharmaceutical specification of greater than 25% branched glycans
having a sialic acid on the .alpha.1-3 arm and/or less than 40%
branched glycans having a sialic acid on the .alpha.1-6 arm.
17. The method of claim 15, further comprising measuring an
anti-inflammatory activity of the preparation.
18. The method of claim 17, wherein said anti-inflammatory activity
is measured in vivo or in vitro.
19. The method of claim 1, wherein said preparation is a
preparation of antibodies.
20. The method of claim 1, wherein said preparation is formulated
for subcutaneous administration.
21. The method of claim 1, wherein said glycoproteins are present
in said preparation at a concentration of 50-250 mg/mL.
22. The method of claim 1, wherein said glycoproteins consist
essentially of an Fc region.
23. The method of claim 1, wherein said glycoproteins further have
a Fab region.
24. The method of claim 1, wherein said glycoproteins are derived
from plasma.
25. The method of claim 1, wherein said glycoproteins are
recombinant glycoproteins.
26. The method of claim 1, wherein said glycoproteins are IgG
glycoproteins or said glycoproteins consist essentially of an Fc
region derived from IgG glycoproteins.
27. A pharmaceutical preparation comprising sialylated
glycoproteins produced by the method of claim 1.
28. A pharmaceutical preparation comprising glycoproteins
comprising an Fc region, wherein at least 95% of branched glycans
on the Fc region have a sialic acid on the .alpha.1-3 arm and do
not have a sialic acid on the .alpha.1-6 arm, and wherein said
preparation has anti-inflammatory activity.
29. A pharmaceutical preparation comprising glycoproteins
comprising an Fc region, wherein 20-90% of branched glycans on the
Fc region have a sialic acid on the .alpha.1-3 arm and do not have
a sialic acid on the .alpha.1-6 arm, and wherein said preparation
has anti-inflammatory activity.
30. A pharmaceutical preparation comprising a plurality of
glycoproteins comprising an IgG Fc region, wherein the IgG Fc
region of each of the plurality of glycoproteins comprises a first
branched glycan sialylated on the .alpha.1-3 arm, and wherein said
pharmaceutical preparation has anti-inflammatory activity.
31. The pharmaceutical preparation of claim 30, wherein said IgG Fc
region of the plurality of glycoproteins further comprises a second
branched glycan.
32. The pharmaceutical preparation of claim 30, said IgG Fc region
of the plurality of glycoproteins further comprises a high mannose
glycan.
33. The pharmaceutical preparation of claim 30, said IgG Fc region
of the plurality of glycoproteins further comprises a second
branched glycan sialylated on the .alpha.1-3 arm.
34. The pharmaceutical preparation of claim 30, wherein said IgG Fc
region of the plurality of glycoproteins further comprises a second
branched glycan sialylated on the .alpha.1-6 arm.
35. The pharmaceutical preparation of claim 30, wherein the
plurality of glycoproteins comprising an IgG Fc region comprises at
least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 100% of the glycoproteins in said
preparation.
36. The pharmaceutical preparation of claim 27, wherein said
pharmaceutical preparation has a level of anti-inflammatory
activity that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%, or more,
higher than a level of anti-inflammatory activity of a reference
glycoprotein preparation.
37. The pharmaceutical preparation of claim 27, wherein said
pharmaceutical preparation is a preparation of antibodies.
38. The pharmaceutical preparation of claim 27, wherein said
pharmaceutical preparation is formulated for subcutaneous
administration.
39. The pharmaceutical preparation of claim 27, wherein said
glycoproteins are present in said preparation at a concentration of
50-250 mg/mL.
40. The pharmaceutical preparation of claim 27, wherein said
glycoproteins consist essentially of an Fc region.
41. The pharmaceutical preparation of claim 27, wherein said
glycoproteins further have a Fab region.
42. The pharmaceutical preparation of claim 27, wherein said
glycoproteins are derived from plasma.
43. The pharmaceutical preparation of claim 27, wherein said
glycoproteins are recombinant glycoproteins.
44. The pharmaceutical preparation of claim 27, wherein said
glycoproteins are IgG glycoproteins or said glycoproteins consist
essentially of an Fc region derived from IgG glycoproteins.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application Nos. 61/676,253, filed Jul. 26, 2012 and 61/768,027,
filed Feb. 22, 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
[0003] The invention encompasses the discovery that Fc-containing
glycoproteins comprising branched glycans that are sialylated on an
.alpha.1-3 arm of the branched glycan in the Fc region, e.g., with
a NeuAc-.alpha.2,6-Gal terminal linkage, exhibit improved
anti-inflammatory properties, e.g., relative to a reference
glycoprotein. Accordingly, the present disclosure encompasses such
glycoproteins, as well as methods of making and methods of using
such glycoproteins.
[0004] In one aspect, the invention features a method of producing
a pharmaceutical preparation including glycoproteins having an Fc
region, wherein the branched glycans on the Fc region are
selectively sialylated on the .alpha.1-3 arm at a predetermined
level. This method includes: contacting a sialyltransferase enzyme
with a preparation including glycoproteins having an IgG Fc region
under conditions suitable for sialylation of a plurality of the
branched glycans by the enzyme; measuring the level of branched
glycans having a sialic acid on the .alpha.1-3 arm and/or on the
.alpha.1-6 arm; processing the preparation into a pharmaceutical
preparation if the level is equivalent to the predetermined level;
thereby producing a pharmaceutical preparation including
glycoproteins having an Fc region, wherein the branched glycans on
the Fc region are selectively sialylated on the .alpha.1-3 arm at a
predetermined level.
[0005] In some embodiments, the predetermined level is at least 95%
(e.g., at least 96%, 97%, 98%, 99%, up to and including 100%) of
branched glycans having a sialic acid on the .alpha.1-3 arm. In
other embodiments, the predetermined level is 20-90% (e.g., 20-30%,
25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%,
65-75%, 70-80%, 75-85%, 80-90%) of branched glycans having a sialic
acid on the .alpha.1-3 arm.
[0006] In certain embodiments, the sialyltransferase enzyme is a
ST6Gal-I enzyme.
[0007] In further embodiments, the .alpha.1,3 arm of the branched
glycans are sialylated with a NeuAc-.alpha.2,6-Gal terminal
linkage.
[0008] In another aspect, the invention features a method of
increasing anti-inflammatory activity of a reference glycoprotein
preparation. This method includes: providing a reference
glycoprotein preparation including glycoproteins having an IgG Fc
region; and sialylating the branched glycans on the Fc region on
the .alpha.1-3 arm of a plurality of the branched glycans to
produce a sialylated glycoprotein preparation; wherein the
glycoproteins in the reference glycoprotein preparation are not IgG
glycoproteins or do not consist essentially of an Fc region derived
from IgG glycoproteins; and wherein the sialylated glycoprotein
preparation has an increased level of anti-inflammatory activity
relative to the level of anti-inflammatory activity of the
reference glycoprotein preparation.
[0009] In some embodiments, the method further includes measuring
in the sialylated glycoprotein preparation the level of the
branched glycans having a sialic acid on the .alpha.1-3 arm and/or
measuring the level of the branched glycans having a sialic acid on
the .alpha.1-6 arm. In other embodiments, the method further
includes processing the sialylated glycoprotein preparation into a
pharmaceutical preparation if the level of branched glycans having
a sialic acid on the .alpha.1-3 arm and/or the level of branched
glycans having a sialic acid on the .alpha.1-6 arm meets a
predetermined level (e.g., at least about 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of
the branched glycans having a sialic acid on the .alpha.1,3 arm
and/or less than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or
less of the branched glycans having a sialic acid on the .alpha.1,6
arm).
[0010] In another aspect, the invention features a method of
increasing anti-inflammatory activity of a reference glycoprotein
preparation. This method includes: providing a reference
glycoprotein preparation including glycoproteins having an IgG Fc
region; and sialylating the branched glycans on the Fc region on
the .alpha.1-3 arm of a plurality of the branched glycans to
produce a sialylated glycoprotein preparation; measuring in the
sialylated glycoprotein preparation the level of the branched
glycans having a sialic acid on the .alpha.1-3 arm and/or measuring
the level of the branched glycans having a sialic acid on the
.alpha.1-6 arm; and processing the sialylated glycoprotein
preparation into a pharmaceutical preparation if the level of
branched glycans having a sialic acid on the .alpha.1-3 arm and/or
the level of branched glycans having a sialic acid on the
.alpha.1-6 arm meets a predetermined level; wherein the sialylated
glycoprotein preparation has an increased level of
anti-inflammatory activity relative to the level of
anti-inflammatory activity of the reference glycoprotein
preparation.
[0011] In some embodiments, the predetermined level of branched
glycans having a sialic acid on the .alpha.1-3 arm is at least 95%
(e.g., at least 96%, 97%, 98%, 99%, up to and including 100%) and
said predetermined level of branched glycans having a sialic acid
on the .alpha.1-6 arm is less than 5%. In other embodiments, the
predetermined level of branched glycans having a sialic acid on the
.alpha.1-3 arm is between 20-90% (e.g., 20-30%, 25-35%, 30-40%,
35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%,
75-85%, 80-90%).
[0012] In some embodiments, the sialylated glycoprotein preparation
has a level of anti-inflammatory activity that is at least about
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%,
175%, 200%, 300%, 400%, 500%, or more, higher than the level of
anti-inflammatory activity of the reference glycoprotein
preparation.
[0013] In another aspect, the invention features a method of
manufacturing a pharmaceutical product including glycoproteins
having an IgG Fc region. This method includes: providing a
preparation including glycoproteins having an IgG Fc region;
measuring the level of branched glycans on the Fc region in the
preparation having a sialic acid on the .alpha.1-3 arm and/or on
the .alpha.1-6 arm; and processing the preparation into a
pharmaceutical product if the level of the branched glycans having
a sialic acid on the .alpha.1-3 arm and/or on the .alpha.1-6 arm is
equivalent to a predetermined level, thereby manufacturing a
pharmaceutical product including glycoproteins having an IgG Fc
region.
[0014] In some embodiments, the predetermined level is a
pharmaceutical specification of greater than 25% (e.g., greater
than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, up to and including 100%)
branched glycans having a sialic acid on the .alpha.1-3 arm and/or
less than 40% (e.g., less than 40%, 35%, 30%, 25%, 20%, 15%, 10%,
5%, 4%, 3%, 2%, 1%) branched glycans having a sialic acid on the
.alpha.1-6 arm.
[0015] In other embodiments, the method further includes measuring
(e.g., in vivo or in vitro) an anti-inflammatory activity of the
preparation.
[0016] In some embodiments of any of the foregoing methods, the
preparation is a preparation of antibodies.
[0017] In other embodiments of any of the foregoing methods, the
preparation is formulated for intravenous or subcutaneous
administration.
[0018] In certain embodiments of any of the foregoing methods, the
glycoproteins are present in the preparation at a concentration of
50-250 mg/mL.
[0019] In further embodiments, the glycoproteins consist
essentially of an Fc region.
[0020] In other embodiments of any of the foregoing methods, the
glycoproteins further have a Fab region.
[0021] In some embodiments of any of the foregoing methods, the
glycoproteins are derived from plasma.
[0022] In certain embodiments of any of the foregoing methods, the
glycoproteins are recombinant glycoproteins.
[0023] In further embodiments, the glycoproteins are IgG
glycoproteins or said glycoproteins consist essentially of an Fc
region derived from IgG glycoproteins.
[0024] In another aspect, the invention features a pharmaceutical
preparation including sialylated glycoproteins produced by any of
the foregoing methods.
[0025] In another aspect, the invention features a pharmaceutical
preparation including glycoproteins having an Fc region, wherein at
least 95% (e.g., at least 96%, 97%, 98%, 99%, up to and including
100%) of branched glycans on the Fc region have a sialic acid on
the .alpha.1-3 arm and do not have a sialic acid on the .alpha.1-6
arm, and wherein the pharmaceutical preparation has
anti-inflammatory activity.
[0026] In another aspect, the invention features a pharmaceutical
preparation including glycoproteins having an Fc region, wherein
20-90% (e.g., 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%,
50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%) of branched
glycans on the Fc region have a sialic acid on the .alpha.1-3 arm
and do not have a sialic acid on the .alpha.1-6 arm, and wherein
the pharmaceutical preparation has anti-inflammatory activity.
[0027] In another aspect, the invention features a pharmaceutical
preparation including a plurality of glycoproteins having an IgG Fc
region, wherein the IgG Fc region of each of the plurality of
glycoproteins includes a first branched glycan sialylated on the
.alpha.1-3 arm, and wherein the pharmaceutical preparation has
anti-inflammatory activity.
[0028] In some embodiments, the IgG Fc region of the plurality of
glycoproteins further comprises a second branched glycan. In other
embodiments, the IgG Fc region of the plurality of glycoproteins
further comprises a high mannose glycan. In certain embodiments,
the IgG Fc region of the plurality of glycoproteins further
comprises a second branched glycan sialylated on the .alpha.1-3
arm. In further embodiments, the IgG Fc region of the plurality of
glycoproteins further comprises a second branched glycan sialylated
on the .alpha.1-6 arm.
[0029] In some embodiments, the plurality of glycoproteins having
an IgG Fc region includes at least about 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of
the glycoproteins in the pharmaceutical preparation.
[0030] In certain embodiments of any of the foregoing
pharmaceutical preparations, the pharmaceutical preparation has a
level of anti-inflammatory activity that is at least about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%,
200%, 300%, 400%, 500%, or more, higher than a level of
anti-inflammatory activity of a reference glycoprotein
preparation.
[0031] In other embodiments of any of the foregoing pharmaceutical
preparations, the pharmaceutical preparation is a preparation of
antibodies.
[0032] In some embodiments of any of the foregoing pharmaceutical
preparations, the pharmaceutical preparation is formulated for
subcutaneous administration.
[0033] In certain embodiments of any of the foregoing
pharmaceutical preparations, the glycoproteins are present in said
preparation at a concentration of 50-250 mg/mL.
[0034] In further embodiments of any of the foregoing
pharmaceutical preparations, the glycoproteins consist essentially
of an Fc region.
[0035] In other embodiments of any of the foregoing pharmaceutical
preparations, the glycoproteins further have a Fab region.
[0036] In some embodiments of any of the foregoing pharmaceutical
preparations, the glycoproteins are derived from plasma.
[0037] In certain embodiments of any of the foregoing
pharmaceutical preparations, the glycoproteins are recombinant
glycoproteins.
[0038] In further embodiments of any of the foregoing
pharmaceutical preparations, the glycoproteins are IgG
glycoproteins or said glycoproteins consist essentially of an Fc
region derived from IgG glycoproteins.
[0039] In some embodiments, the pharmaceutical preparations of the
invention have increased efficacy in the treatment of rheumatoid
arthritis, X-linked agammagloulinemia, hypogammaglobulinemia, an
acquired compromised immunity condition, immune thrombocytopenia,
Kawasaki disease, allogeniec bone marrow transplant, chronic
lymphocytic leukemia, common variable immunodeficiency, pediatric
HIV, a primary immunodeficiency, chronic inflammatory demyelinating
polyneuropathy, adult HIV, Alzhemier's disease, autism, Behcet's
disease, capillary leak syndrome, chronic fatigue syndrome,
clostridium difficile colitis, dermatomyositis and polymyositis,
Grave's ophthalmopathy, muscular dystrophy, inclusion body
myositis, infertility, Lambert-Eaton syndrome, Lennox-Gastaut,
Lupus erythematosus, multifocal motor neuropathy, multiple
sclerosis, myasthenia gravis, neonatal alloimmune thrombocytopenia,
parvovirus B19, pemphigus, post-transfusion purpura, renal
transplant rejection, spontaneous abortion/miscarriage, Sjogren's
syndrome, stiff person syndrome, opsoclonus myoclonus, severe
sepsis and septic shock, toxic epidermal necrolysis, multiple
myeloma, Wegener's granulomatosis, Churg-Strauss syndrome, and
acute infections relative to IgG (e.g., IVIG).
[0040] In some aspects, the present disclosure encompasses a
preparation, e.g., a therapeutic preparation, that includes
Fc-containing sialylated glycoproteins. In some aspects, a
preparation, e.g., a therapeutic preparation, includes a mixture of
asialylated glycoproteins, monosialylated glycoproteins (e.g.,
monosialylated on an .alpha.1-3 arm of a branched glycan (e.g.,
with a NeuAc-.alpha.2,6-Gal terminal linkage), and/or disialylated
glycoproteins (e.g., sialylated on both an .alpha.1-3 arm and an
.alpha.1-6 arm of a branched glycan). In some embodiments, a
preparation of glycoproteins includes at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, or about 100% glycoproteins that are monosialylated on an
.alpha.3 arm of a branched glycan (e.g., with a
NeuAc-.alpha.2,6-Gal terminal linkage). In some embodiments, a
preparation of glycoproteins includes less than about 25%, less
than about 20%, less than about 15%, less than about 10%, less than
about 5%, or less, asialylated and/or disialylated glycoproteins.
In some embodiments, an Fc-containing glycoprotein preparation is
selected from a preparation of Fc fragments, a preparation of
antibody molecules, a preparation of Fc-fusion proteins (e.g.,
Fc-receptor fusion proteins), and a preparation of IVIG.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The present teachings described herein will be more fully
understood from the following description of various illustrative
embodiments, when read together with the accompanying drawings. It
should be understood that the drawings described below are for
illustration purposes only and are not intended to limit the scope
of the present teachings in any way.
[0042] FIGS. 1A-1C show exemplary ST6Gal sialyltransferase
sequences. FIG. 1A depicts an exemplary ST6Gal sialyltransferase
amino acid sequence (SEQ ID NO:1). FIG. 1B depicts an exemplary
ST6Gal sialyltransferase amino acid sequence (SEQ ID NO:2). FIG. 1C
depicts an exemplary ST6 Gal sialyltransferase amino acid sequence
(SEQ ID NO:3).
[0043] FIG. 2 is a schematic illustration of a common core
pentasaccharide (Man).sub.3(GlcNAc)(GlcNAc) of N-glycans.
[0044] FIG. 3 is a schematic illustration of an IgG antibody
molecule.
[0045] FIG. 4 a panel of representations of HILIC-LC extracted ion
chromatogram of Fc glycopeptides expressed in CHO cells,
glycopeptides derived from a sialylated Fc that was sialylated
using a rhST6Gal expressed in E. coli cells, or glycopeptides
derived from a sialylated Fc that was sialylated using a rhST6Gal
expressed in CHO cells.
DETAILED DESCRIPTION
[0046] 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 IgGs 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).
[0047] The inventors have discovered that glycoproteins having
branched glycans that are preferentially sialylated on an
.alpha.1,3 arm of the branched glycan in the Fc region (e.g., with
a NeuAc-.alpha.2,6-Gal terminal linkage), have increased
anti-inflammatory properties. Described herein are glycoproteins
(e.g., antibodies or fusion proteins, such as Fc fusion proteins)
having branched glycans sialylated on an .alpha.1,3 arm of the
branched glycan in the Fc region (e.g., with a NeuAc-.alpha.2,6-Gal
terminal linkage) and have increased anti-inflammatory activity
relative to glycoproteins not having such sialylated glycans.
Methods of making and using such compositions are also
described.
DEFINITIONS
[0048] As used herein, the terms "approximately" or "about" as
applied to one or more values of interest, refer to a value that is
similar to a stated reference value. In some embodiments, the terms
"approximately" or "about" refer to a range of values that fall
within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference
value.
[0049] As used herein, the term "equivalent" refers to a
difference, for example, the percent of a particular glycoform in a
glycoprotein preparation, that does not result in a difference in
biological properties (e.g., potency, binding characteristics,
stability, or susceptibility to degradation) as compared to a
target glycoprotein preparation. In some instances, "equivalent"
refers to the allowed difference of the percent of a particular
glycoform in a glycoprotein preparation in a specification for
commercial release of a drug product under Section 351(k) of the
PHS Act.
[0050] 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 (e.g., have an .alpha. 1,3 arm and an
.alpha. 1,6 arm). 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 glycoprotein, glycolipid,
proteoglycan, etc.). The term also encompasses free glycans,
including glycans that have been cleaved or otherwise released from
a glycoconjugate.
[0051] 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.
[0052] As used herein, the term "glycoprotein preparation" refers
to a set of individual glycoprotein molecules, each of which
comprises a polypeptide having a particular amino acid sequence
(which amino acid sequence includes at least one glycosylation
site) and at least one glycan covalently attached to the at least
one glycosylation site. Individual molecules of a particular
glycoprotein within a glycoprotein preparation typically have
identical amino acid sequences but may differ in the occupancy of
the at least one glycosylation sites and/or in the identity of the
glycans linked to the at least one glycosylation sites. That is, a
glycoprotein preparation may contain only a single glycoform of a
particular glycoprotein, but more typically contains a plurality of
glycoforms. Different preparations of the same glycoprotein may
differ in the identity of glycoforms present (e.g., a glycoform
that is present in one preparation may be absent from another)
and/or in the relative amounts of different glycoforms.
[0053] As used herein, the term "pharmaceutical preparation" refers
to a preparation that comprises an active pharmaceutical ingredient
or "API" in a dosage form suitable for human or veterinary use
(e.g., a preparation in which glycoproteins are present at a
concentration of at least 20 mg/mL).
[0054] As used herein, the term "pharmaceutical product" refers to
a sterile preparation intended for human or veterinary use,
formulated for use in a subject and presented in its finished
dosage form (e.g., packaged for administration).
[0055] "Pharmaceutical preparations" and "pharmaceutical products"
can be included in kits containing the preparation or product and
instructions for use.
[0056] "Pharmaceutical preparations" and "pharmaceutical products"
generally refer to compositions in which the final predetermined
level of sialylation has been achieved. 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).
[0057] "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.
[0058] The term "glycoform" is used herein to refer to a particular
form of a glycoprotein. That is, when a glycoprotein includes a
particular polypeptide that has the potential to be linked to
different glycans or sets of glycans, then each different version
of the glycoprotein (i.e., where the polypeptide is linked to a
particular glycan or set of glycans) is referred to as a
"glycoform".
[0059] "Reference glycoprotein", as used herein, refers to a
glycoprotein having substantially the same amino acid sequence as
(e.g., having about 90-100% identical amino acids of) a
glycoprotein described herein, e.g., a glycoprotein to which it is
compared. In some embodiments, a reference glycoprotein is a
therapeutic glycoprotein described herein, e.g., an FDA approved
therapeutic glycoprotein.
[0060] "Predetermined level," as used herein, refers to a
pre-specified particular level (e.g., an absolute value or range)
of one or more particular glycans. In some embodiments, a
predetermined level is 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)
in a preparation of a reference glycoprotein. In some embodiments,
a predetermined level is expressed as a percent.
[0061] For any given parameter, in some embodiments, "percent"
refers to the number of moles of a particular glycan (glycan X)
relative to total moles of glycans of a preparation. In some
embodiments, "percent" refers to the number of moles of PNGase
F-released Fc glycan X relative to total moles of PNGase F-released
Fc glycans detected.
[0062] By "purified" (or "isolated") refers to a nucleic acid
sequence (e.g., a polynucleotide) or an amino acid sequence (e.g.,
a glycoprotein) that is removed or separated from other components
present in its natural environment or substantially free of
reactants or byproducts thereof used in its production. For
example, a purified or isolated glycoprotein 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).
A further example of a purified or isolated glycoprotein are
sialylated glycoproteins which 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) used in their production. An
isolated polynucleotide is one that is separated from other nuclear
components (e.g., histones) and/or from upstream or downstream
nucleic acid sequences. An isolated nucleic acid sequence or amino
acid sequence 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 nucleic acid sequence or
amino acid sequence.
[0063] 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.
[0064] As used herein, "polypeptide" (or "amino acid sequence" or
"protein") refers to an 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.
[0065] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0066] 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.
[0067] 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.
[0068] The term "subject", as used herein, means any subject for
whom diagnosis, prognosis, or therapy is desired. For example, a
subject can be a mammal, e.g., a human or non-human primate (such
as an ape, monkey, orangutan, or chimpanzee), a dog, cat, guinea
pig, rabbit, rat, mouse, horse, cattle, or cow.
[0069] 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 VH), and a light (L) chain variable region (abbreviated
herein as VL). 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.
[0070] 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 CH1 domain, a hinge region, a CH2 domain, a CH3 domain
(derived from an IgA, IgD, IgG, IgE, or IgM), and a CH4 domain
(derived from an IgE or IgM).
[0071] As used herein, the term "Fc region" refers to a dimer of
two "Fc polypeptides", each "Fc polypeptide" comprising 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 all of the 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.
[0072] An "Fc region-containing glycoprotein" is a glycoprotein
that includes all or a substantial portion of an Fc region.
Examples of an Fc region-containing glycoprotein 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 glycoprotein may be recombinant (e.g., a
recombinant Fc fragment preparation or a recombinant antibody
preparation) or naturally derived (such as IVIG).
[0073] As used herein, the term "Fc region variant" refers to an
analog of an Fc region that possesses one or more Fc-mediated
activities described herein. This term includes Fc regions
comprising one or more amino acid modifications (e.g.,
substitutions, additions, or deletions) relative to a wild type or
naturally existing Fc region. For example, variant Fc regions can
possess at least about 50% homology, at least about 75% homology,
at least about 80% homology, at least about 85%, homology, at least
about 90% homology, at least about 95% homology, or more, with a
naturally existing Fc region. For example, variant Fc regions can
possess between 1 and 5 amino acid substitutions, e.g., 1, 2, 3, 4
or 5 amino acid substitutions such as phenylalanine to alanine
substitutions. Fc region variants also include Fc regions
comprising one or more amino acid residues added to or deleted from
the N- or C-terminus of a wild type Fc region.
[0074] 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.
[0075] 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.
[0076] The terms "overexpress," "overexpression," or
"overexpressed" interchangeably refer to a protein or nucleic acid
that is transcribed or translated at a detectably greater level,
such as in a cancer cell, in comparison to a control cell. The term
includes expression due to transcription, post transcriptional
processing, translation, post-translational processing, cellular
localization (e.g., organelle, cytoplasm, nucleus, cell surface),
and RNA and protein stability, as compared to a control cell.
Overexpression can be detected using conventional techniques, e.g.,
for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins
(i.e., ELISA, immunohistochemical techniques). Overexpression can
be expression in an amount greater than about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90% or more compared to a control cell. In
certain instances, overexpression is 1-fold, 2-fold, 3-fold,
4-fold, or more, higher level of transcription or translation
compared to a control cell.
[0077] As used herein, the term "ST6Gal 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 ST6Gal
sialyltransferase sequences are known in the art. In some
embodiments, the ST6Gal sialyltransferase has at least 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity, or is 100% identical, to amino acid residues 95-416 of
SEQ ID NO:1, to SEQ ID NO:2, or to SEQ ID NO:3 (FIGS. 1A-1C).
I. Glycoproteins
[0078] Glycoproteins include, for example, any of a variety of
hematologic agents (including, for instance, erythropoietin,
blood-clotting factors, etc.), interferons, colony stimulating
factors, antibodies, enzymes, and hormones. The identity of a
particular glycoprotein is not intended to limit the present
disclosure, and any glycoprotein of interest can be a reference
glycoprotein in the present methods.
[0079] A reference glycoprotein described herein can include a
target-binding domain that binds to a target of interest (e.g.,
binds to an antigen). For example, a glycoprotein, such as an
antibody, can bind to a transmembrane polypeptide (e.g., receptor)
or ligand (e.g., a growth factor). Exemplary molecular targets
(e.g., antigens) for glycoproteins described herein (e.g.,
antibodies) include CD proteins such as CD2, CD3, CD4, CD8, CD11,
CD19, CD20, CD22, CD25, CD33, CD34, CD40, CD52; members of the ErbB
receptor family such as the EGF receptor (EGFR, HER1, ErbB1), HER2
(ErbB2), HER3 (ErbB3) or HER4 (ErbB4) receptor; macrophage
receptors such as CRIg; tumor necrosis factors such as TNF.alpha.
or TRAIL/Apo-2; cell adhesion molecules such as LFA-1, Mac1,
p150,95, VLA-4, ICAM-1, VCAM and .alpha.v.beta.3 integrin including
either .alpha. or .beta. subunits thereof (e.g., anti-CD11a,
anti-CD18 or anti-CD11b antibodies); growth factors and receptors
such as EGF, FGFR (e.g., FGFR3) and VEGF; IgE; cytokines such as
IL1; cytokine receptors such as IL2 receptor; blood group antigens;
flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4;
protein C; neutropilins; ephrins and receptors; netrins and
receptors; slit and receptors; chemokines and chemookine receptors
such as CCL5, CCR4, CCR5; amyloid beta; complement factors, such as
complement factor D; lipoproteins, such as oxidized LDL (oxLDL);
lymphotoxins, such as lymphotoxin alpha (LTa). Other molecular
targets include Tweak, B7RP-1, proprotein convertase
subtilisin/kexin type 9 (PCSK9), sclerostin, c-kit, Tie-2, c-fms,
and anti-M1.
[0080] Nonlimiting, exemplary reference glycoproteins that include
an Fc region of an antibody heavy chain include abatacept
(Orencia.RTM., Bristol-Myers Squibb), abciximab (ReoPro.RTM.,
Roche), adalimumab (Humira.RTM., Bristol-Myers Squibb), alefacept
(Amevive.RTM., Astellas Pharma), alemtuzumab (Campath.RTM.,
Genzyme/Bayer), basiliximab (Simulect.RTM., Novartis), bevacizumab
(Avastin.RTM., Roche), certolizumab (CIMZIA.RTM., UCB, Brussels,
Belgium), cetuximab (Erbitux.RTM., Merck-Serono), daclizumab
(Zenapax.RTM., Hoffmann-La Roche), denileukin diftitox (Ontak.RTM.,
Eisai), eculizumab (Soliris.RTM., Alexion Pharmaceuticals),
efalizumab (Raptiva.RTM., Genentech), etanercept (Enbrel.RTM.,
Amgen-Pfizer), gemtuzumab (Mylotarg.RTM., Pfizer), ibritumomab
(Zevalin.RTM., Spectrum Pharmaceuticals), infliximab
(Remicade.RTM., Centocor), muromonab (Orthoclone OKT3.RTM.,
Janssen-Cilag), natalizumab (Tysabri.RTM., Biogen Idec, Elan),
omalizumab (Xolair.RTM., Novartis), palivizumab (Synagis.RTM.,
Medlmmune), panitumumab (Vectibix.RTM., Amgen), ranibizumab
(Lucentis.RTM., Genentech), rilonacept (Arcalyst.RTM., Regeneron
Pharmaceuticals), rituximab (MabThera.RTM., Roche), tositumomab
(Bexxar.RTM., GlaxoSmithKline), and trastuzumab (Herceptin.RTM.,
Roche).
A. N-Linked Glycosylation
[0081] N-linked oligosaccharide chains are added to a protein in
the lumen of the endoplasmic reticulum (see Molecular Biology of
the Cell, Garland Publishing, Inc. (Alberts et al., 1994)).
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
3 glucose, 9 mannose, and 2 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 (depicted in FIG. 2,
linked to an asparagine residue). 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. 2.
[0082] N-glycans can be subdivided into three distinct groups
called "high mannose type", "hybrid type", and "complex type", with
a common pentasaccharide core (Man
(alph1,6)-(Man(alpha1,3))-Man(beta1,4)-GlcpNAc(beta
1,4)-GlcpNAc(beta 1,N)-Asn) occurring in all three groups.
[0083] After initial processing in the endoplasmic reticulum, the
glycoprotein 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".
[0084] 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.
[0085] "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.
[0086] 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 glycoproteins 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.
[0087] N-Linked Glycosylation in Antibodies
[0088] Antibodies are glycosylated at conserved, N-linked
glycosylation sites in the Fc regions of immunoglobulin heavy
chains. For example, each heavy chain of an IgG antibody has a
single N-linked glycosylation site at Asn297 of the CH2 domain (see
Jefferis, Nature Reviews 8:226-234 (2009)). IgA antibodies have
N-linked glycosylation sites within the CH2 and CH3 domains, IgE
antibodies have N-linked glycosylation sites within the CH3 domain,
and 10/I antibodies have N-linked glycosylation sites within the
CH1, CH2, CH3, and CH4 domains (see Arnold et al., J. Biol. Chem.
280:29080-29087 (2005); Mattu et al., J. Biol. Chem. 273:2260-2272
(1998); Nettleton et al., Int. Arch. Allergy Immunol. 107:328-329
(1995)).
[0089] 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 CH2
domain in each Fc polypeptide of the Fc region, which also contains
the binding sites for C1q and Fc.gamma.R (see Jefferis et al.,
Immunol. Rev. 163:59-76 (1998); and Wright et al., Trends Biotech
15:26-32 (1997)). 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
[0090] The basic structure of an IgG antibody is illustrated in
FIG. 3. As shown in FIG. 3, 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 (VH) and variable light (VL) regions. The other domains of
each chain are relatively invariant in amino acid sequence and are
known as constant heavy (CH) and constant light (CL) regions. As
shown in FIG. 3, for an IgG antibody, the light chain includes one
variable region (VL) and one constant region (CL). An IgG heavy
chain includes a variable region (VH), a first constant region
(CH1), a hinge region, a second constant region (CH2), and a third
constant region (CH3). In IgE and IgM antibodies, the heavy chain
includes an additional constant region (CH4).
[0091] 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 and
IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or
subclass.
[0092] 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 complementarity 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.
[0093] Reference antibodies or fragments 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). Chimeric antibodies can be produced using the methods
described in, e.g., Morrison, 1985, Science 229:1202, and humanized
antibodies by methods described in, e.g., U.S. Pat. No.
6,180,370.
[0094] Additional reference antibodies 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).
[0095] Naturally derived antibodies 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)).
[0096] IVIG is a blood product containing pooled, polyvalent IgG
extracted from the plasma of over one thousand blood donors. IVIG
is used in the treatment of rheumatoid arthritis, X-linked
agammagloulinemia, hypogammaglobulinemia, an acquired compromised
immunity condition, immune thrombocytopenia, Kawasaki disease,
allogeniec bone marrow transplant, chronic lymphocytic leukemia,
common variable immunodeficiency, pediatric HIV, a primary
immunodeficiency, chronic inflammatory demyelinating
polyneuropathy, adult HIV, Alzhemier's disease, autism, Behcet's
disease, capillary leak syndrome, chronic fatigue syndrome,
clostridium difficile colitis, dermatomyositis and polymyositis,
Grave's ophthalmopathy, muscular dystrophy, inclusion body
myositis, infertility, Lambert-Eaton syndrome, Lennox-Gastaut,
Lupus erythematosus, multifocal motor neuropathy, multiple
sclerosis, myasthenia gravis, neonatal alloimmune thrombocytopenia,
parvovirus B19, pemphigus, post-transfusion purpura, renal
transplant rejection, spontaneous abortion/miscarriage, Sjogren's
syndrome, stiff person syndrome, opsoclonus myoclonus, severe
sepsis and septic shock, toxic epidermal necrolysis, multiple
myeloma, Wegener's granulomatosis, Churg-Strauss syndrome, and
acute infections.
C. Glycoprotein Conjugates
[0097] The disclosure includes glycoproteins (or Fc regions or Fc
fragments containing one or more N-glycosylation sites thereof)
that are conjugated or fused to one or more heterologous moieties
and that have different levels of sialylated glycans relative to a
corresponding reference glycoprotein. 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 glycoprotein 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)).
[0098] Exemplary, nonlimiting reference fusion proteins include
abatacept (Orencia.RTM., Bristol-Myers Squibb), alefacept
(Amevive.RTM., Astellas Pharma), denileukin diftitox (Ontak.RTM.,
Eisai), etanercept (Enbrel.RTM., Amgen-Pfizer), and rilonacept
(Arcalyst.RTM., Regeneron Pharmaceuticals).
[0099] 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.
[0100] 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.
[0101] In other instances, a reference glycoprotein (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 glycoprotein 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.121In, .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.
[0102] 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)).
II. Sialylated Glycoproteins
[0103] Glycoproteins of the present disclosure have glycan
compositions that are different from corresponding reference
glycoproteins. For example, the present disclosure encompasses Fc
region-containing glycoprotein preparations (e.g., IVIG, Fc or IgG
antibodies) having higher levels of branched glycans that are
sialylated on an .alpha.1-3 arm of the branched glycans in the Fc
region (e.g., with a NeuAc-.alpha.2,6-Gal terminal linkage),
relative to a corresponding reference IgG antibody. 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 glycoproteins can be
different (e.g., a preparation of glycoproteins can have a higher
number or a higher percentage of branched glycans that are
sialylated on an .alpha.1-3 arm of the branched glycans in the Fc
region) relative to a corresponding preparation of reference
glycoproteins).
[0104] In some embodiments, a nucleic acid encoding a reference Fc
region-containing glycoprotein described herein is co-expressed in
a host cell with one or more sialyltransferase enzymes, e.g., an
.alpha.2,6 sialyltransferase (e.g., ST6Gal-1). Sialyltransferase
enzymes are known in the art and are commercially available.
[0105] Methods and compositions described herein include the use of
a sialyltransferase enzyme, e.g., an .alpha.2,6 sialyltransferase
(e.g., ST6Gal-1). A number of ST6Gal 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)). ST6Gal-1 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.
[0106] In some embodiments, the disclosure encompasses methods of
modifying activity of a sialyltransferase enzyme, e.g., a
sialylating activity of a sialyltransferase enzyme. In some
embodiments, activity is modified by expressing a sialyltransferase
enzyme in eukaryotic cells (e.g., yeast, insect, or mammalian cells
such as CHO cells), purifying the sialyltransferase, and contacting
the sialyltransferase with an Fc region-containing glycoprotein,
thereby preferentially sialylating the .alpha.1,3 arms of branched
glycans of the Fc region-containing glycoprotein. In some
embodiments, such sialylated Fc region-containing glycoproteins
exhibit anti-inflammatory activity.
[0107] In some embodiments, activity is modified by expressing a
sialyltransferase enzyme in prokaryotic cells (e.g., bacterial
cells, e.g., E. coli), purifying the sialyltransferase, and
contacting the sialyltransferase with an Fc region-containing
glycoprotein, thereby preferentially sialylating the .alpha.1,6
arms of branched glycans of the Fc region-containing glycoprotein.
In some embodiments, such sialylated Fc region-containing
glycoproteins do not exhibit anti-inflammatory activity.
[0108] In some embodiments, an Fc region-containing glycoprotein is
co-expressed in a host cell with a sialyltransferase enzyme (e.g.,
ST6Gal sialyltransferase), and the enzyme sialylates a branched
glycan as described herein.
[0109] In some embodiments, an Fc region-containing glycoprotein is
expressed in a host cell, and the host cell endogenously expresses
or recombinantly expresses a sialyltransferase (e.g., ST6Gal
sialyltransferase). Additionally or alternatively, the host cell is
cultured under conditions that increase the activity of a
sialyltransferase (e.g., ST6Gal sialyltransferase) in the cell,
thereby producing an Fc region-containing glycoprotein having
branched glycans sialylated as described herein.
[0110] Recombinant expression of a gene, such as a nucleic acid
encoding a reference glycoprotein and/or a sialyltransferase
described herein, can include construction of an expression vector
containing a polynucleotide that encodes a reference polypeptide
and/or a sialyltransferase. 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.
[0111] An expression vector can be transferred to a host cell by
conventional techniques, and the transfected cells can then be
cultured using conventional techniques to produce reference
polypeptides.
[0112] 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).
[0113] 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).
[0114] 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).
[0115] 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.
[0116] 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.
[0117] In some embodiments, a reference Fc region-containing
glycoprotein is recombinantly produced in cells as described
herein, purified, and contacted with a sialyltransferase enzyme in
vitro to produce Fc region-containing glycoproteins containing
higher levels of glycans having higher levels of sialic acid on the
.alpha.1-3 arms of the branched glycans with a NeuAc-.alpha.2,6-Gal
terminal linkage, relative to the reference glycoprotein. In some
embodiments, a purified reference glycoprotein is contacted with
the sialyltransferase in the presence of CMP-sialic acid,
manganese, and/or other divalent metal ions.
[0118] A reference Fc region-containing glycoprotein 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
glycoprotein can be fused to heterologous polypeptide sequences to
facilitate purification.
[0119] 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., Sambrook,
Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,
Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I
and II (D. N. Glover ed. 1985); 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)); Animal Cell Culture (R. I.
Freshney, ed. (1986)); Immobilized Cells and Enzymes (IRL Press,
(1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984);
F. M. Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley & Sons, Inc. (1994).
[0120] In some embodiments, a glycoprotein can be purified using a
lectin column by methods known in the art (see, e.g., WO 02/30954).
For example, a preparation of glycoproteins can be enriched for
glycoproteins containing glycans having sialic acids in .alpha.2,6
linkage as described in e.g., WO2008/057634. Following enrichment
of glycoproteins containing glycans having sialic acids in
.alpha.2,6 linkage, the glycan composition of such glycoproteins
can be further characterized to identify glycoproteins having
sialic acids attached to the .alpha.1,3 arm of a branched glycan.
Preparations of glycoproteins containing a predetermined level of
glycans having sialic acids in .alpha.2,6 linkage on the .alpha.1,3
arm can be selected for use, e.g., for therapeutic use. Such
compositions can have increased levels of anti-inflammatory
activity.
[0121] In some embodiments, a glycoprotein, e.g., a glycosylated
antibody, is sialylated after the glycoprotein is produced. For
example, a glycoprotein can be recombinantly expressed in a host
cell (as described herein) and purified using standard methods. The
purified glycoprotein is then contacted with an ST6Gal
sialyltransferase (e.g., a recombinantly expressed and purified
ST6Gal sialyltransferase) in the presence of reaction conditions as
described herein. In certain embodiments, the conditions include
contacting the purified glycoprotein with an ST6Gal
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.
[0122] In some embodiments, chemoenzymatic sialylation is used to
sialylate glycoproteins. 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 glycoprotein.
[0123] A branched glycan can be synthesized de novo using standard
techniques or can be obtained from a glycoprotein preparation
(e.g., a recombinant glycoprotein, 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 glycoprotein.
[0124] 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
ST6Gal sialyltransferase as described herein. In some embodiments,
a galactosylated glycan is purified before being contacted with an
ST6Gal sialyltransferase. In some embodiments, a galactosylated
glycan is not purified before being contacted with an ST6Gal
sialyltransferase. In some embodiments, a branched glycan is
contacted with a galactosyltransferase and an ST6Gal
sialyltransferase in a single step.
[0125] 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).
III. Glycan Evaluation
[0126] In some embodiments, glycans of glycoproteins are analyzed
by any available suitable method. 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 glycoprotein 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 (1D-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.
[0127] 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 glycoproteins. 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 glycoprotein preparation,
e.g., one or more of the parameters disclosed herein, can be
performed by one or more of following methods.
[0128] In some instances, methods for evaluating one or more target
protein specific parameters, e.g., in a glycoprotein 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- Chen and Flynn, J. Am. Soc. Mass linked
glycan, glycan detection, Spectrom., 20: 1821-1833 (2009) glycan
identification, and characterization; site specific glycation;
glycoform detection (e.g., parameters 1-7); percent glycosylation;
and/or aglycoosyl) Peptide LC-MS Dick et al., Biotechnol. Bioeng.,
100: 1132-1143 C-terminal lysine (reducing/non-reducing) (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) Peptide LC-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., Site specific glycation (reducing/non-reducing) 100:
2543-2550 (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,
(reducing/non-reducing)* (2004) exposed N-linked glycan)
(including, for example, glycan detection, identification, and
characterization; site specific glycation; glycoform detection;
percent glycosylation; and/or aglycoosyl) LC-MS (reducing/non- Dick
et al., Biotechnol. Bioeng., Glycan (e.g., N-linked glycan,
reducing/alkylated)* 100: 1132-1143 (2008) exposed N-linked glycan)
*Methods include Goetze et al., Glycobiol., 21: 949-959 (including,
for example, glycan removal (e.g., enzymatic, (2011) detection,
identification, and chemical, and physical) Xie et al., mAbs, 2:
379-394 (2010) characterization; site specific of glycans
glycation; glycoform detection; percent glycosylation; and/or
aglycoosyl) 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 Chelius et al., Anal. Chem., example,
sequence analysis and 78: 2370-2376 (2006) identification of
sequence variants: Miller et al., J. Pharm. Sci., oxidation;
succinimide; aspartic 100: 2543-2550 (2011) acid; and/or
site-specific aspartic acid) Weak cation exchange Dick et al.,
Biotechnol. Bioeng., Isoforms (including, for example, (WCX)
chromatography 100: 1132-1143 (2008) charge 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-methylenedioxybenzene Hokke et al., FEBS Lett.,
275: 9-14 Sialic acid (DMB) labeling method (1990) LC-MS Johnson et
al., Anal. Biochem., C-terminal amidation 360: 75-83 (2007) LC-MS
Johnson et al., Anal. Biochem., N-terminal fragmentation 360: 75-83
(2007) Circular dichroism Harn et al., Current Trends in Secondary
structure (including, for spectroscopy Monoclonal Antibody
Development and example, alpha helix content Manufacturing, S. J.
Shire et al., eds, and/or beta sheet content) 229-246 (2010)
Intrinsic and/or ANS dye Harn et al., Current Trends in Tertiary
structure (including, for fluorescence Monoclonal Antibody
Development and example, extent of protein folding) Manufacturing,
S. J. Shire et al., eds, 229-246 (2010) Hydrogen-deuterium Houde et
al., Anal. Chem., Tertiary structure and dynamics exchange-MS 81:
2644-2651 (2009) (including, for example, accessibility f 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)
[0129] The literature recited above 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 for determining a
parameter described herein.
IV. Anti-Inflammatory Properties
[0130] The inventors have discovered that sialic acid-mediated
anti-inflammatory properties on Fc-containing molecules are not
only due to the level of sialylation, but due to particular
branching arrangements. Accordingly, Fc region-containing
glycoproteins described herein (e.g., Fc region-containing
glycoproteins containing glycans containing sialic acid on
.alpha.1,3 arms of branched glycans with a NeuAc-.alpha.2,6-Gal
terminal linkage) have increased anti-inflammatory properties
relative to a reference glycoprotein.
[0131] In some embodiments, Fc region-containing glycoproteins
containing sialic acid on .alpha.1,3 arms of branched glycans with
a NeuAc-.alpha.2,6-Gal terminal linkages exhibit increased
anti-inflammatory activity relative to a reference glycoprotein,
e.g., a level of anti-inflammatory activity that is 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%, at least 100%, at
least 125%, at least 150%, at least 175%, at least 200%, at least
250%, at least 300%, or higher, relative to a reference
glycoprotein.
[0132] In some embodiment, Fc region-containing glycoproteins
having sialic acids in both the .alpha.1,3 and .alpha.1,6 arms of
branched glycans may inhibit anti-inflammatory activity of
Fc-containing glycoproteins.
V. Pharmaceutical Compositions and Administration
[0133] A glycoprotein of the present disclosure (e.g., an Fc
region-containing glycoprotein comprising branched glycans that are
sialylated on an .alpha.1,3 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 exhibit
anti-inflammatory activity. Such a pharmaceutical composition is
useful as an improved composition for the prevention and/or
treatment of diseases relative to the corresponding reference
glycoprotein. Pharmaceutical compositions comprising a glycoprotein
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 sialylated glycoprotein 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.
[0134] 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.
[0135] Nonlimiting 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 ampule.
[0136] In some instances, the level of sialylated glycans (e.g.,
branched glycans that are sialylated on an .alpha.1,3 arm of the
branched glycan in the Fc region, e.g., with a NeuAc-.alpha.2,6-Gal
terminal linkage) in a preparation of antibodies or Fc-containing
polypeptides, produced using a method described herein can be
compared to a predetermined level (e.g., a corresponding level in a
reference standard), e.g., to make a decision regarding the
composition of the polypeptide preparation, e.g., a decision to
classify, select, accept or discard, release or withhold, process
into a drug product, ship, move to a different location, formulate,
label, package, release into commerce, or sell or offer for sale
the polypeptide, e.g., a recombinant antibody. In other instances,
the decision can be to accept, modify or reject a production
parameter or parameters used to make the polypeptide, e.g., an
antibody. Particular, nonlimiting examples of reference standards
include a control level (e.g., a polypeptide produced by a
different method) or a range or value in a product specification
(e.g., an FDA label or Physician's Insert) or quality criterion for
a pharmaceutical preparation containing the polypeptide
preparation.
[0137] In some instances, methods (i.e., evaluation,
identification, and production methods) include taking action
(e.g., physical action) in response to the methods disclosed
herein. For example, a polypeptide preparation is classified,
selected, accepted or discarded, released or withheld, processed
into a drug product, shipped, moved to a different location,
formulated, labeled, packaged, released into commerce, or sold or
offered for sale, depending on whether the preselected or target
value is met. In some instances, processing may include formulating
(e.g., combining with pharmaceutical excipients), packaging (e.g.,
in a syringe or vial), labeling, or shipping at least a portion of
the polypeptide preparation. In some instances, processing includes
formulating (e.g., combining with pharmaceutical excipients),
packaging (e.g., in a syringe or vial), and labeling at least a
portion of the preparation as a drug product described herein.
Processing can include directing and/or contracting another party
to process as described herein.
[0138] In some instances, a biological activity of a polypeptide
preparation (e.g., an antibody preparation) is assessed. Biological
activity of the preparation can be analyzed by any known method. In
some embodiments, a binding activity of a polypeptide is assessed
(e.g., binding to a receptor). In some embodiments, a therapeutic
activity of a polypeptide is assessed (e.g., an activity of a
polypeptide in decreasing severity or symptom of a disease or
condition, or in delaying appearance of a symptom of a disease or
condition). In some embodiments, a pharmacologic activity of a
polypeptide is assessed (e.g., bioavailability, pharmacokinetics,
pharmacodynamics). For methods of analyzing bioavailability,
pharmacokinetics, and pharmacodynamics of glycoprotein
therapeutics, see, e.g., Weiner et al., J. Pharm. Biomed. Anal.
15(5):571-9, 1997; Srinivas et al., J. Pharm. Sci. 85(1):1-4, 1996;
and Srinivas et al., Pharm. Res. 14(7):911-6, 1997.
[0139] The particular biological activity or therapeutic activity
that can be tested will vary depending on the particular
polypeptide (e.g., antibody). The potential adverse activity or
toxicity (e.g., propensity to cause hypertension, allergic
reactions, thrombotic events, seizures, or other adverse events) of
polypeptide preparations can be analyzed by any available method.
In some embodiments, immunogenicity of a polypeptide preparation is
assessed, e.g., by determining whether the preparation elicits an
antibody response in a subject.
[0140] 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.
[0141] 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 glycoprotein 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.
[0142] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting. Unless otherwise
defined, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs. Although methods and materials
similar or equivalent to those described herein can be used in the
practice or testing of the present invention, suitable methods and
materials are described herein.
EXAMPLES
Example 1
Sialylation of Fc Molecules
[0143] Fc molecules were obtained or produced from various sources,
glycan compositions were characterized, and anti-inflammatory
activities were determined. The Fc molecules were tested for their
ability to protect mice from joint inflammation in a mouse
arthritis model using a method described in Anthony, Proc. Natl.
Acad. Sci. U.S.A. 105:19571-19578 (2008).
[0144] Fc molecules were derived from IVIG as follows. Commercial
grade IVIG was buffer exchanged in to phosphate buffered saline
(PBS) from its formulation buffer. This buffer exchanged IVIG was
digested by papain at 37.degree. C. using 5 .mu.g papain/mg of
IVIG, and the digestion was quenched with iodoacetamide. The
undigested IgG and Fc/Fab monomers were separated by size exclusion
chromatography. The Fc/Fab peak was further purified on a Protein A
column to remove the Fab fragments. The purified Fc was
concentrated before performing the sialylation reaction.
[0145] Sialylation of the Fc or IVIG substrate was performed as
follows. The substrate (75 mg/mL) was incubated at 37.degree. C.
for 24-48 hours with 50 mM UDP-galactose and 20 mU of bovine milk
beta-1,4-galactosyltransferase per mg of substrate. The
galactosylated substrate was further incubated at 37.degree. C. for
48-72 hours with 80 mM CMP-sialic Acid and the specified number of
units of alpha-2,6-sialyltransferase per mg of substrate for
sialylation. Enzyme activity was determined as described in
Anumula, Glycobiol. 22:912-917 (2012).
[0146] In another method, Fc was recombinantly expressed in and
purified from CHO cells, and was subsequently sialylated using a
recombinant sialyltransferase enzyme. The glycoprotein contained
branched glycans having higher levels of sialic acid on the
.alpha.1-3 arm of the branched glycans with a NeuAc-.alpha.2,6-Gal
terminal linkage, relative to the reference glycoprotein. As
depicted in FIG. 4 (top panel), Fc recombinantly expressed in CHO
cells contained sialic acids linked to galactose in .alpha.2,3
linkage that were attached to both the .alpha.1,3 and .alpha.1,6
arms of the branched glycans.
[0147] Interestingly, Fc that was derived from IVIG and sialylated
using human ST6Gal sialyltransferase enzyme (expressed in and
purified from E. coli cells, 6.5 mU enzyme/mg of substrate)
contained sialic acids linked to galactose in .alpha.2,6 linkage
that were preferentially attached to the .alpha.1,6 arms of the
branched glycans (FIG. 4, middle panel). When assayed in the mouse
model of inflammation, these Fc molecules did not exhibit
anti-inflammatory activity.
[0148] Surprisingly, when Fc that was derived from IVIG and
sialylated using human ST6Gal sialyltransferase enzyme (expressed
in and purified from CHO cells, 0.26 mU enzyme/mg of substrate),
the Fcs contained sialic acids were linked to galactose in
.alpha.2,6 linkage that were preferentially attached to the
.alpha.1,3 arms of the branched glycans (FIG. 4, bottom panel).
When assayed in the mouse model of inflammation, these Fc molecules
exhibited anti-inflammatory activity.
[0149] In another exemplary method, a preparation of IVIG was
obtained, and glycan composition was determined. About 5% to about
20% of the total glycans in the IVIG preparation contained one
sialic acid on each glycan (i.e., were monosialylated). Further,
greater than about 90% of these monosialylated glycans contained a
sialic acid on an .alpha.1,3 arm of the branched glycans with a
NeuAc-.alpha.2,6-Gal terminal linkage.
[0150] In another method, Fc molecules from IVIG were sialylated
with human ST6Gal sialyltransferase (recombinantly expressed in and
purified from insect cells, 0.42 mU enzyme/mg of substrate). The
sialyltransferase preferentially sialylated the .alpha.1,3 arms of
the branched glycans. These Fc molecules exhibit anti-inflammatory
activity in the mouse model of inflammation.
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
* * * * *