U.S. patent application number 17/672346 was filed with the patent office on 2022-09-01 for fcrn antagonists and methods of use.
The applicant listed for this patent is ARGENX BV, THE BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Christophe Blanchetot, Johannes de Haard, Torsten Dreier, Nicolas G. H. Ongenae, Peter Ulrichts, E. Sally Ward Ober.
Application Number | 20220275035 17/672346 |
Document ID | / |
Family ID | 1000006337116 |
Filed Date | 2022-09-01 |
United States Patent
Application |
20220275035 |
Kind Code |
A1 |
Ulrichts; Peter ; et
al. |
September 1, 2022 |
FCRN ANTAGONISTS AND METHODS OF USE
Abstract
Provided are novel FcRn antagonist compositions comprising a
variant Fc region that binds specifically to FcRn with increased
affinity and reduced pH dependence relative to the native Fc
region. Also provided are FcRn antagonists with enhanced CD16
binding affinity. Also provided are methods of treating
antibody-mediated disorders (e.g. autoimmune diseases) using the
these FcRn antagonist compositions, nucleic acids encoding the FcRn
antagonist compositions, recombinant expression vectors and host
cells for making the FcRn antagonist compositions, and
pharmaceutical compositions comprising the FcRn antagonist
compositions.
Inventors: |
Ulrichts; Peter;
(Destelbergen, BE) ; Blanchetot; Christophe;
(Destelbergen, BE) ; Dreier; Torsten; (Sint
Martens Latem, BE) ; de Haard; Johannes; (NA
Oudelande, NL) ; Ward Ober; E. Sally; (Dallas,
TX) ; Ongenae; Nicolas G. H.; (Gent, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARGENX BV
THE BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM |
Zwijnaarde
Austin |
TX |
BE
US |
|
|
Family ID: |
1000006337116 |
Appl. No.: |
17/672346 |
Filed: |
February 15, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15821104 |
Nov 22, 2017 |
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17672346 |
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14580771 |
Dec 23, 2014 |
10316073 |
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15821104 |
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61920547 |
Dec 24, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/4703 20130101;
C07K 2317/52 20130101; C07K 2317/94 20130101; C07K 2317/524
20130101; C07K 2317/526 20130101; A61K 38/1709 20130101; A61K 38/00
20130101; C07K 2317/41 20130101; A61K 45/06 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61K 45/06 20060101 A61K045/06; A61K 38/17 20060101
A61K038/17 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with government support under Grant
No. R01 AR 56478 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1-44. (canceled)
45. A method of treating immune thrombocytopenia purpura (ITP) in a
subject, the method comprising administering to the subject an
effective amount of an isolated FcRn antagonist consisting of a
variant Fc region, wherein said variant Fc region consists of two
Fc domains which form a homodimer, wherein the amino acid sequence
of each of the Fc domains consists of SEQ ID NO: 2.
46. The method of claim 45, wherein the Fc domains of the variant
Fc region comprise an N-linked glycan having a bisecting GlcNac at
EU position 297 of the Fc domains.
47. The method of claim 45, wherein the FcRn antagonist is
administered to the subject simultaneously or sequentially with an
additional therapeutic agent.
48. The method of claim 47, wherein the additional therapeutic
agent is an anti-inflammatory agent.
49. The method of claim 47, wherein the additional therapeutic
agent is a leukocyte depleting agent.
50. The method of claim 49, wherein the leukocyte depleting agent
is a B-cell depleting agent.
51. The method of claim 50, wherein the B-cell depleting agent is
an antibody.
52. The method of claim 51, wherein the antibody specifically binds
to a cell surface marker selected from the group consisting of
CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD70, CD72,
CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85,
and CD86.
53. The method of claim 47, wherein the additional therapeutic
agent is an antibody selected from the group consisting of
rituximab, daclizumab, basiliximab, muronomab-CD3, infliximab,
adalimumab, omalizumab, efalizumab, natalizumab, tocilizumab,
eculizumab, golimumab, canakinumab, ustekinumab, belimumab, and any
combination thereof.
54. A method of treating immune thrombocytopenia purpura (ITP) in a
subject, the method comprising administering to the subject an
effective amount of an isolated FcRn antagonist consisting of a
variant Fc region, wherein said variant Fc region consists of two
Fc domains which form a homodimer, wherein the amino acid sequence
of each of the Fc domains consists of SEQ ID NO: 3.
55. The method of claim 54, wherein the Fc domains of the variant
Fc region comprise an N-linked glycan having a bisecting GlcNac at
EU position 297 of the Fc domains.
56. The method of claim 54, wherein the FcRn antagonist is
administered to the subject simultaneously or sequentially with an
additional therapeutic agent.
57. The method of claim 56, wherein the additional therapeutic
agent is an anti-inflammatory agent.
58. The method of claim 56, wherein the additional therapeutic
agent is a leukocyte depleting agent.
59. The method of claim 58, wherein the leukocyte depleting agent
is a B-cell depleting agent.
60. The method of claim 59, wherein the B-cell depleting agent is
an antibody.
61. The method of claim 60, wherein the antibody specifically binds
to a cell surface marker selected from the group consisting of
CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD70, CD72,
CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85,
and CD86.
62. The method of claim 56, wherein the additional therapeutic
agent is an antibody selected from the group consisting of
rituximab, daclizumab, basiliximab, muronomab-CD3, infliximab,
adalimumab, omalizumab, efalizumab, natalizumab, tocilizumab,
eculizumab, golimumab, canakinumab, ustekinumab, belimumab, and any
combination thereof.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 15/821,104, filed Nov. 22, 2017, which is a division of
U.S. patent application Ser. No. 14/580,771, filed Dec. 23, 2014,
now U.S. Pat. No. 10,316,073, which claims priority to U.S.
Provisional Patent Application Ser. No. 61/920,547, filed Dec. 24,
2013, the entire disclosures of which are hereby incorporated
herein by reference.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 9, 2022, is named 727006_AGX5-015DV2_ST25 and is 6,509
bytes in size.
BACKGROUND
[0004] Immunoglobulin gamma (IgG) antibodies play a key role in the
pathology of many disorders, such as autoimmune diseases,
inflammatory diseases, and disorders in which the pathology is
characterized by over-expression of IgG antibodies (e.g.,
hypergammaglobulinemia) (see e.g. Junghans, Immunologic Research 16
(1):29 (1997)).
[0005] The half-life of IgG in the serum is prolonged relative to
the serum half-life of other plasma proteins (Roopenian et al., J.
Immunology 170:3528 (2003); Junghans and Anderson, Proc. Natl.
Acad. Sci. USA 93:5512 (1996)). This long half-life is due, in
part, to the binding of the Fc region of IgG to the Fc receptor,
FcRn. Although FcRn was originally characterized as a neonatal
transport receptor for maternal IgG, it also functions in adults to
protect IgG from degradation. FcRn binds to pinocytosed IgG and
protects the IgG from transport to degradative lysosomes by
recycling it back to the extracellular compartment. This recycling
is facilitated by the pH dependent binding of IgG to FcRn, where
the IgG/FcRn interaction is stronger at acidic endosomal pH than at
extracellular physiological pH.
[0006] When the serum concentration of IgG reaches a level that
exceeds available FcRn molecules, unbound IgG is not protected from
degradative mechanisms and will consequently have a reduced serum
half-life. Thus, inhibition of IgG binding to FcRn reduces the
serum half-life of IgG by preventing IgG endosomal recycling of
IgG. Accordingly, agents that antagonize the binding of IgG to FcRn
may be useful for regulating, treating or preventing
antibody-mediated disorders, such as autoimmune diseases,
inflammatory diseases, etc. One example of a method of antagonizing
IgG Fc binding to FcRn involves the generation of blocking
antibodies to FcRn (see e.g WO2002/43658). Peptides have also been
identified that bind to and antagonize FcRn function (see e.g. U.S.
Pat. Nos. 6,212,022 and 8,101,186). In addition, full-length IgG
antibodies comprising variant Fc receptors with enhanced FcRn
binding and decreased pH dependence have also been identified that
antagonize FcRn binding to IgG (see e.g. 8,163,881). However, there
is a need in the art for improved agents that antagonize FcRn
binding to IgG for use in the treatment of antibody-mediated
disorders.
SUMMARY
[0007] The present disclosure provides novel FcRn antagonist
compositions. These compositions generally comprise a variant Fc
region, or FcRn-binding fragment thereof, that binds specifically
to FcRn with increased affinity and reduced pH dependence relative
to the native Fc region. The invention is based, in part, on the
surprising finding that an isolated variant Fc region (e.g., a
variant Fc region comprising the amino acids Y, T, E, K, F, and Y
at EU positions (EU numbering) 252, 254, 256, 433, 434, and 436
respectively) is a more efficacious FcRn antagonist in vivo than a
full-length antibody comprising that variant Fc region. The FcRn
antagonist compositions of the present disclosure are particularly
useful for reducing the serum levels of Fc-containing agents (e.g.,
antibodies and immunoadhesins). Accordingly, the instant disclosure
also provides methods of treating antibody-mediated disorders (e.g.
autoimmune diseases) using the FcRn antagonist compositions
disclosed herein. Also provided are nucleic acids encoding the FcRn
antagonist compositions, recombinant expression vectors and host
cells for making the FcRn antagonist compositions, and
pharmaceutical compositions comprising the FcRn antagonist
compositions.
[0008] The FcRn antagonists disclosed herein are particularly
advantageous over previously described FcRn antagonist compositions
and known treatments for antibody-mediated disorders. For example,
the FcRn antagonists disclosed herein are smaller and more potent
than intravenous gamma globulin (IVIG), the current treatment for
many antibody-mediated disorders. Accordingly, the effective dose
of the disclosed FcRn antagonists can be far less than that of
IVIG. Moreover, IVIG is isolated and purified from human donors
and, as a consequence, suffers from considerable batch-batch
variation. The FcRn antagonists compositions disclosed herein can
be recombinantly produced or chemically synthesized and, therefore,
are far more homogeneous. As demonstrated herein, the FcRn
antagonists disclosed herein are also surprisingly more efficacious
than full-length IgG antibodies comprising variant Fc receptors,
such as set forth in Vaccaro et al., Nature Biotech 23(9) 1283-1288
(1997).
[0009] Accordingly, in one aspect, the instant disclosure provides
an isolated FcRn antagonist comprising a variant Fc region or
FcRn-binding fragment thereof, wherein the Fc region or fragment
comprises the amino acids Y, T, E, K, F, and Y at EU positions 252,
254, 256, 433, 434, and 436 respectively, and wherein the FcRn
antagonist is not a full-length antibody.
[0010] In certain embodiments, the FcRn antagonist does not
comprise an antibody variable region or a CH1 domain. In certain
embodiments, the FcRn antagonist does not comprise a free cysteine
residue. In certain embodiments, the Fc region is an IgG Fc region
(e.g., a human IgG Fc region). In certain embodiments, the Fc
region is an IgG1 Fc region (e.g., a human IgG1 Fc region). In
certain embodiments, the Fc region is a chimeric Fc region.
[0011] In certain embodiments, the FcRn antagonist comprises the
variant Fc region amino acid sequence set forth in SEQ ID NO:1. In
certain embodiments, the FcRn antagonist comprises a variant Fc
region wherein the amino acid sequence of the Fc domains of the
variant Fc region consists of the amino acid sequence set forth in
SEQ ID NO: 1, 2, or 3. In certain embodiments, the FcRn antagonist
consists of a variant Fc region wherein the amino acid sequence of
the Fc domains of the variant Fc region consists of the amino acid
sequence set forth in SEQ ID NO: 2.
[0012] In certain embodiments, the FcRn antagonist comprises a
variant Fc region that has altered (increased or decreased)
affinity for an Fc receptor relative to the affinity of a wild-type
IgG1 Fc region for the Fc gamma receptor. In certain embodiments,
the variant Fc has increased affinity for CD16a.
[0013] In certain embodiments, the FcRn antagonist comprises a
variant Fc region that does not comprise an N-linked glycan at EU
position 297. In certain embodiments, the FcRn antagonist comprises
a variant Fc region that comprises an afucosylated N-linked glycan
at EU position 297. In certain embodiments, the FcRn antagonist
comprises a variant Fc region that comprises an N-linked glycan
having a bisecting GlcNac at EU position 297.
[0014] In certain embodiments, the FcRn antagonist comprises a
variant Fc region linked to a half-life extender. In certain
embodiments, the half-life extender is polyethylene glycol or human
serum albumin. In certain embodiments, the instant disclosure
provides an FcRn antagonist composition comprising a plurality of
FcRn antagonist molecules disclosed herein, wherein at least 50%
(optionally, at least 60, 70, 80, 90, 95, or 99%) of the molecules
comprise a variant Fc region, or FcRn-binding fragment thereof,
having an afucosylated N-linked glycan. In certain embodiments, the
instant disclosure provides an FcRn antagonist composition
comprising a plurality of FcRn antagonist molecules disclosed
herein, wherein at least 50% (optionally, at least 60, 70, 80, 90,
95, or 99%) of the molecules comprise a variant Fc region, or
FcRn-binding fragment thereof, comprising an N-linked glycan having
a bisecting GlcNac.
[0015] In certain embodiments, the instant disclosure provides an
FcRn antagonist composition comprising a plurality of FcRn
antagonist molecules as disclosed herein, wherein greater than 95%
the of the FcRn antagonist molecules in the composition are
monomers (e.g., greater than 95, 96, 97, 98, 99, 99.1, 99.2, 99.3,
99.4, 99.5, 99.6, 99.7, 99.8, 99.9%).
[0016] In certain embodiments, the instant disclosure provides an
FcRn antagonist composition comprising a plurality of FcRn
antagonist molecules disclosed herein, wherein less than 5% the of
the FcRn antagonist molecules in the composition are present in
aggregates, (e.g., less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,
0.5, 0.4, 0.3, 0.2, 0.1%).
[0017] In certain embodiments, the instant disclosure provides an
FcRn antagonist composition comprising a plurality of FcRn
antagonist molecules disclosed herein, wherein the composition is
substantially free of FcRn antagonist molecule degradation
products.
[0018] In another aspect, the instant disclosure provides
pharmaceutical compositions comprising an FcRn antagonist or FcRn
antagonist composition disclosed herein and a pharmaceutically
acceptable carrier or excipient.
[0019] In another aspect, the instant disclosure provides a method
of inhibiting FcRn function in a subject, the method comprising
administering to the subject an effective amount of an
FcRn-antagonist composition disclosed herein.
[0020] In another aspect, the instant disclosure provides a method
of reducing the serum levels of an Fc-containing agent in subject
that has been administered the Fc-containing agent, the method
comprising administering to subject an effective amount of an
FcRn-antagonist composition disclosed herein. In certain
embodiments, the Fc-containing agent is an antibody or
immunoadhesin. In certain embodiments, the Fc-containing agent is a
therapeutic or diagnostic agent. In certain embodiments the
Fc-containing agent is an imaging agent. In certain embodiments,
the Fc-containing agent is an antibody drug conjugate.
[0021] In another aspect, the instant disclosure provides a method
of treating an antibody-mediated disorder in a subject, the method
comprising administering to the subject an effective amount of an
FcRn-antagonist composition disclosed herein. In certain
embodiments, the antibody-mediated disorder is hyperglobulinemia.
In certain embodiments, the antibody-mediated disorder is a disease
or disorder that is treatable using intravenous immunoglobulin
(IVIG). In certain embodiments, the antibody-mediated disorder is a
disease or disorder that is treatable using plasmapheresis and/or
immunoadsorption.
[0022] In certain embodiments, the antibody-mediated disorder is an
autoimmune disease. In certain embodiments, the autoimmune disease
is selected from the group consisting of allogenic islet graft
rejection, alopecia areata, ankylosing spondylitis,
antiphospholipid syndrome, autoimmune Addison's disease,
Alzheimer's disease, antineutrophil cytoplasmic autoantibodies
(ANCA), autoimmune diseases of the adrenal gland, autoimmune
hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis,
autoimmune neutropenia, autoimmune oophoritis and orchitis,
autoimmune thrombocytopenia, autoimmune urticaria, Behcet's
disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome,
celiac sprue-dermatitis, chronic fatigue immune disfunction
syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP),
Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome,
cold agglutinin disease, Crohn's disease, dermatomyositis, dilated
cardiomyopathy, discoid lupus, epidermolysis bullosa acquisita,
essential mixed cryoglobulinemia, factor VIII deficiency,
fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease,
Guillain-Barre, Goodpasture's syndrome, graft-versus-host disease
(GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic
membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic
thrombocytopenic purpura (ITP), IgA neuropathy, IgM
polyneuropathies, immune mediated thrombocytopenia, juvenile
arthritis, Kawasaki's disease, lichen planus, lichen sclerosus,
lupus erythematosus, Meniere's disease, mixed connective tissue
disease, mucous membrane pemphigoid, multiple sclerosis, type 1
diabetes mellitus, Multifocal motor neuropathy (MMN), myasthenia
gravis, paraneoplastic bullous pemphigoid, pemphigoid gestationis,
pemphigus vulgaris, pemphigus foliaceus, pernicious anemia,
polyarteritis nodosa, polychrondritis, polyglandular syndromes,
polymyalgia rheumatica, polymyositis and dermatomyositis, primary
agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic
arthritis, relapsing polychondritis, Reynaud's phenomenon, Reiter's
syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's
syndrome, solid organ transplant rejection, stiff-man syndrome,
systemic lupus erythematosus, Takayasu arteritis, toxic epidermal
necrolysis (TEN), Stevens Johnson syndrome (SJS), temporal
arteritis/giant cell arteritis, thrombotic thrombocytopenia
purpura, ulcerative colitis, uveitis, dermatitis herpetiformis
vasculitis, anti-neutrophil cytoplasmic antibody-associated
vasculitides, vitiligo, and Wegner's granulomatosis.
[0023] In certain embodiments, the autoimmune disease is an
autoimmune channelopathy. In certain embodiments, the channelopathy
is selected from the group consisting of autoimmune limbic
encephalitis, epilepsy, neuromyelitis optica, Lambert-Eaton
myasthenic syndrome, myasthenia gravis, anti-N-Methyl-D-aspartate
(NMDA) receptor encephalitis,
anti-.alpha.-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) receptor encephalitis, Morvan syndrome, neuromyotonia,
pediatric autoimmune neuropsychiatric disorders associated with
streptococcal infection (PANDAS), and Glycine receptor
antibody-associated disorder.
[0024] In certain embodiments, the FcRn antagonist is administered
to the subject simultaneously or sequentially with an additional
therapeutic agent. In certain embodiments, the additional
therapeutic agent is an anti-inflammatory agent. In certain
embodiments, the additional therapeutic agent is rituximab,
daclizumab, basiliximab, muronomab-cd3, infliximab, adalimumab,
omalizumab, efalizumab, natalizumab, tocilizumab, eculizumab,
golimumab, canakinumab, ustekinumab, or belimumab. In certain
embodiments, the additional therapeutic agent is a leucocyte
depleting agent. In certain embodiments, the additional therapeutic
agent is a B-cell depleting agent. In certain embodiments, the
B-cell depleting agent is an antibody, e.g., an antibody that
specifically binds to CD10, CD19, CD20, CD21, CD22, CD23, CD24,
CD37, CD53, CD70, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81,
CD82, CD83, CD84, CD85, or CD86.
[0025] In another aspect, the instant disclosure provides a nucleic
acid molecule encoding an FcRn-antagonist disclosed herein. In
another aspect, the instant disclosure provides an expression
vector comprising a nucleic acid molecule encoding an
FcRn-antagonist disclosed herein. In another aspect, the instant
disclosure provides a host cell comprising an expression vector or
nucleic acid encoding an FcRn-antagonist disclosed herein. In
another aspect, the instant disclosure provides a method of
producing an FcRn-antagonist, the method comprising culturing a
host cell disclosed herein under conditions such that an
FcRn-antagonist is expressed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 depicts the results of experiments to determine the
effect of Fc-Abdeg and HEL-Abdeg on the serum levels of a tracer
antibody (FR70-hIgG1) in cynomolgous monkey.
[0027] FIG. 2 depicts the results of experiments to determine the
effect of Fc-Abdeg and HEL-Abdeg on total IgG serum levels in
cynomolgous monkey.
[0028] FIG. 3 depicts the results of experiments the effect of
Fc-Abdeg and HEL-Abdeg on albumin levels in cynomolgous monkey.
[0029] FIG. 4 depicts the results of experiments to determine the
effect of Fc-Abdeg and IVIG on the serum levels of a tracer
antibody (FR70-hIgG1) in cynomolgous monkey.
[0030] FIG. 5 depicts the results of ELISA assays comparing the
affinity of Fc-Abdeg, Fc-Abdeg-POT and Fc-Abdeg-S239D/I332E for
human CD16a.
[0031] FIG. 6 depicts the results of ELISA assays comparing the
affinity of Fc-Abdeg, Fc-Abdeg-POT and Fc-Abdeg-S239D/I332E for
murine CD16-2.
[0032] FIG. 7 depicts the results of experiments to determine the
effect of Fc-Abdeg, Abdeg-POT and Fc-AbdegS239D/I332E on
anti-CD20-induced ADCC-signal using the Promega's Raji-based ADCC
reporter bioassay.
[0033] FIG. 8 depicts the results of experiments to determine the
effect of Fc-Abdeg and Abdeg-POT on anti-CD70-induced lysis of
CD70+U266 cells in vitro.
[0034] FIG. 9 depicts the results of experiments to determine the
effect of Fc-Abdeg, Fc-Abdeg-POT, Fc-Abdeg-S239D/I332E and IVIG on
platelet levels in an acute murine model for immune
thrombocytopenia.
[0035] FIG. 10 depicts the result of an exemplary gelfiltration
purification of Fc-Abdeg.
DETAILED DESCRIPTION
[0036] The present disclosure provides novel FcRn antagonist
compositions. These compositions generally comprise a variant Fc
region, or FcRn-binding fragment thereof, that binds specifically
to FcRn with increased affinity and reduced pH dependence relative
to the native Fc region. The invention is based, in part, on the
surprising finding that an isolated variant Fc region (e.g., a
variant Fc region comprising the amino acids Y, T, E, K, F, and Y
at EU positions 252, 254, 256, 433, 434, and 436 respectively) is a
more efficacious FcRn antagonist in vivo than a full-length
antibody comprising that variant Fc region. The FcRn antagonist
compositions of the present disclosure are particularly useful for
reducing the serum levels of Fc-containing agents (e.g., antibodies
and immunoadhesins). Accordingly, the instant disclosure also
provides methods of treating antibody-mediated disorder (e.g.
autoimmune diseases) using the FcRn antagonist compositions
disclosed herein. Also provided are nucleic acids encoding the FcRn
antagonist compositions, recombinant expression vectors and host
cells for making the FcRn antagonist compositions, and
pharmaceutical compositions comprising the FcRn antagonist
compositions.
I. DEFINITIONS
[0037] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The meaning and scope of the terms should be clear,
however, in the event of any latent ambiguity, definitions provided
herein take precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular.
Generally, nomenclature used in connection with, and techniques of,
cell and tissue culture, molecular biology, immunology,
microbiology, genetics and protein and nucleic acid chemistry and
hybridization described herein are those well known and commonly
used in the art.
[0038] In order that the present invention may be more readily
understood, certain terms are first defined.
[0039] As used herein the term "FcRn antagonist" refers to any
agent comprising an Fc region (e.g., a variant Fc region disclosed
herein) that binds specifically to FcRn through the Fc region and
inhibits the binding of immunoglobulin to FcRn, with the proviso
that the agent is not a full length IgG antibody.
[0040] As used herein, the term "Fc region" refers to the portion
of a native immunoglobulin formed by the Fc domains of its two
heavy chains. A native Fc region is homodimeric.
[0041] As used herein, the term "variant Fc region" refers to an Fc
region with one or more alteration relative to a native Fc region.
Alteration can include amino acid substitutions, additions and/or
deletions, linkage of additional moieties, and/or alteration the
native glycans. The term encompasses heterodimeric Fc regions where
each of the constituent Fc domains is different. Examples of such
heterodimeric Fc regions include, without limitation, Fc regions
made using the "knobs and holes" technology as described in, for
example, U.S. Pat. No. 8,216,805, which is incorporated by
reference herein in its entirety. The term also encompasses single
chain Fc regions where the constituent Fc domains are linked
together by a linker moiety, as described in, for example,
US20090252729A1 and US20110081345A1, which are each incorporated by
reference herein in their entirety.
[0042] As used herein, the term "Fc domain" refers to the portion
of a single immunoglobulin heavy chain beginning in the hinge
region just upstream of the papain cleavage site and ending at the
C-terminus of the antibody. Accordingly, a complete Fc domain
comprises at least a portion of a hinge (e.g., upper, middle,
and/or lower hinge region) domain, a CH2 domain, and a CH3
domain.
[0043] As used herein the term "FcRn binding fragment" refers to a
portion of an Fc region that is sufficient to confer FcRn
binding.
[0044] As used herein, the term "EU position" refers to the amino
acid position in the EU numbering convention for the Fc region
described in Edelman, G. M. et al., Proc. Natl. Acad. USA, 63,
78-85 (1969) and Kabat et al, in "Sequences of Proteins of
Immunological Interest", U.S. Dept. Health and Human Services, 5th
edition, 1991.
[0045] As used herein, the term "CH1 domain" refers to the first
(most amino terminal) constant region domain of an immunoglobulin
heavy chain that extends from about EU positions 118-215. The CH1
domain is adjacent to the VH domain and amino terminal to the hinge
region of an immunoglobulin heavy chain molecule, and does not form
a part of the Fc region of an immunoglobulin heavy chain.
[0046] As used herein, the term "hinge region" refers to the
portion of a heavy chain molecule that joins the CH1 domain to the
CH2 domain. This hinge region comprises approximately 25 residues
and is flexible, thus allowing the two N-terminal antigen binding
regions to move independently. Hinge regions can be subdivided into
three distinct domains: upper, middle, and lower hinge domains
(Roux et al. J. Immunol. 161: 4083 (1998)). The FcRn antagonists of
the instant disclosure can include all or a portion of a hinge
region.
[0047] As used herein, the term "CH2 domain" refers to the portion
of a heavy chain immunoglobulin molecule that extends from about EU
positions 231-340.
[0048] As used herein, the term "CH3 domain" includes the portion
of a heavy chain immunoglobulin molecule that extends approximately
110 residues from N-terminus of the CH2 domain, e.g., from about
position 341-446 (EU numbering system).
[0049] As used herein, the term "FcRn" refers to a neonatal Fc
receptor. Exemplary FcRn molecules include human FcRn encoded by
the FCGRT gene as set forth in RefSeq NM_004107.
[0050] As used herein, the term "CD16" refers to Fc.gamma.RIII Fc
receptors that are required for Antibody-Dependent Cell-mediated
Cytotoxicity (ADCC). Exemplary CD16 molecules include human CD16a
as set forth in RefSeq NM_000569.
[0051] As used herein, the term "free cysteine" refers to native or
engineered cysteine amino acid residue that exists in a
substantially reduced form in a mature FcRn antagonist.
[0052] As used herein, the term "antibody" refers to immunoglobulin
molecules comprising four polypeptide chains, two heavy (H) chains
and two light (L) chains interconnected by disulfide bonds, as well
as multimers thereof (e.g., IgM). Each heavy chain comprises a
heavy chain variable region (abbreviated VH) and a heavy chain
constant region. The heavy chain constant region comprises three
domains, CH1, CH2 and CH3. Each light chain comprises a light chain
variable region (abbreviated VL) and a light chain constant region.
The light chain constant region comprises one domain (CL). The VH
and VL regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions
(CDRs), interspersed with regions that are more conserved, termed
framework regions (FR).
[0053] As used herein the term "N-linked glycan" refers to the
N-linked glycan attached to the nitrogen (N) in the side chain of
asparagine in the sequon (i.e., Asn-X-Ser or Asn-X-Thr sequence,
where X is any amino acid except proline) present in the CH2 domain
of an Fc region. Such N-Glycans are fully described in, for
example, Drickamer K, Taylor M E (2006). Introduction to
Glycobiology, 2nd ed., which is incorporated herein by reference in
its entirety.
[0054] As used herein the term "afucosylated" refers to an N-linked
glycan which lacks a core fucose molecule as described in U.S. Pat.
No. 8,067,232, the contents of which is incorporated by reference
herein in its entirety.
[0055] As used herein the term "bisecting GlcNac" refers to an
N-linked glycan having an N-acetylglucosamine (GlcNAc) molecule
linked to a core mannose molecule, as described in U.S. Pat. No.
8,021,856, the contents of which is incorporated by reference
herein in its entirety.
[0056] As used herein, the term "antibody-mediated disorder" refers
to any disease or disorder caused or exacerbated by the presence of
an antibody in a subject.
[0057] As used herein, the term "Fc-containing agent" is any
molecule that comprises an Fc region.
[0058] As used herein, the term "leucocyte depleting agent" refers
to an agent that reduces the number of leucocytes in a subject upon
administration.
[0059] As used herein, the term "B-cell depleting agent" refers to
an agent that reduces the number of B-cells in a subject upon
administration.
[0060] As used herein, the term "T-cell depleting agent" refers to
an agent that reduces the number of T-cells in a subject upon
administration.
[0061] As used herein, the term "autoimmune channelopathy" refers
to a disease caused by autoantibodies against an ion channel
subunit or a molecule that regulates the channel.
[0062] As used herein, the term "treat," "treating," and
"treatment" refer to therapeutic or preventative measures described
herein. The methods of "treatment" employ administration to a
subject, an antibody or antigen binding fragment thereof of the
present invention, for example, a subject having an IL-6-associated
disease or disorder (e.g. inflammation and cancer) or predisposed
to having such a disease or disorder, in order to prevent, cure,
delay, reduce the severity of, or ameliorate one or more symptoms
of the disease or disorder or recurring disease or disorder, or in
order to prolong the survival of a subject beyond that expected in
the absence of such treatment. As used herein, the term "subject"
includes any human or non-human animal.
[0063] As used herein, the term "immunoadhesin" refers to an
antibody-like molecule, which comprises a functional domain of a
binding protein (e.g., a receptor, ligand, or cell-adhesion
molecule) with an Fc region.
II. FCRN ANTAGONISTS
[0064] In one aspect, the invention provides novel FcRn antagonist
compositions. In general, these compositions comprise a variant Fc
region, or FcRn-binding fragment thereof, that binds specifically
to FcRn with increased affinity and reduced pH dependence relative
to a native Fc region. These FcRn antagonists inhibit the binding
of Fc-containing agents (e.g., antibodies and immunoadhesins) to
FcRn in vivo, which results in an increased rate of degradation of
the Fc-containing agents and, concomitantly, a reduced serum level
of these agents.
[0065] The instant specification discloses, for the first time,
that an isolated variant Fc region (e.g., a variant Fc region
comprising the amino acids Y, T, E, K, F, and Y at EU positions
252, 254, 256, 433, 434, and 436 respectively) is a more
efficacious FcRn antagonist in vivo than a full-length antibody
comprising the same variant Fc region. Accordingly, in certain
embodiments, the FcRn antagonist compositions are not full-length
antibodies. In certain embodiments, the FcRn antagonist
compositions do not comprise an antibody variable domain. In
certain embodiments, the FcRn antagonist compositions do not
comprise an antibody variable domain or a CH1 domain. However, in
certain embodiments, the FcRn antagonist compositions may comprise
a variant Fc region linked to one or more additional binding
domains or moieties, including antibody variable domains.
[0066] Any Fc region can be altered to produce a variant Fc region
for use in the FcRn antagonist compositions disclosed herein. In
general, an Fc region, or FcRn-binding fragment thereof, is from a
human immunoglobulin. It is understood, however, that the Fc region
may be derived from an immunoglobulin of any other mammalian
species, including for example, a Camelid species, a rodent (e.g. a
mouse, rat, rabbit, guinea pig) or non-human primate (e.g.
chimpanzee, macaque) species. Moreover, the Fc region or portion
thereof may be derived from any immunoglobulin class, including
IgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype,
including IgG1, IgG2, IgG3 and IgG4. In certain embodiments, the Fc
region is an IgG Fc region (e.g., a human IgG region). In certain
embodiments, the Fc region is an IgG1 Fc region (e.g., a human IgG1
region). In certain embodiments, the Fc region is a chimeric Fc
region comprising portions of several different Fc regions.
Suitable examples of chimeric Fc regions are set forth in
US20110243966A1, which is herein incorporated by reference in its
entirety. A variety of Fc region gene sequences (e.g. human
constant region gene sequences) are available in the form of
publicly accessible deposits. It will be appreciated that the scope
of this invention encompasses alleles, variants and mutations of Fc
regions.
[0067] An Fc region can be further truncated or internally deleted
to produce a minimal FcRn-binding fragment thereof. The ability of
an Fc-region fragment to bind to FcRn can be determined using any
art recognized binding assay e.g., ELISA.
[0068] To enhance the manufacturability of the FcRn antagonists
disclosed herein, it is preferable that the constituent Fc regions
do not do comprise any non-disulphide bonded cysteine residues.
Accordingly, in certain embodiments the Fc regions do not comprise
a free cysteine residue.
[0069] Any Fc variant, or FcRn-binding fragment thereof, that binds
specifically to FcRn with increased affinity and reduced pH
dependence relative to the native Fc region can be used in the FcRn
antagonist compositions disclosed herein. In certain embodiments,
the variant Fc region comprises amino acid alterations,
substitutions, insertions and/or deletions that confer the desired
characteristics. In certain embodiments, the variant Fc region or
fragment comprises the amino acids Y, T, E, K, F, and Y at EU
positions 252, 254, 256, 433, 434, and 436 respectively.
Non-limiting examples of amino acid sequences that can be used in
variant Fc regions are set forth in Table 1, herein. In certain
embodiments, the amino acid sequence of the Fc domains of the
variant Fc region comprises the amino acid sequence set forth in
SEQ ID NO:1. In certain embodiments, the amino acid sequence of the
Fc domains of the variant Fc region consists of the amino acid
sequence set forth in SEQ ID NO:1, 2, or 3. In certain embodiments
an FcRn-antagonist consists of a variant Fc region, wherein the
amino acid sequence of the Fc domains of the variant Fc region
consists of the amino acid sequence set forth in SEQ ID NO:1, 2, or
3.
TABLE-US-00001 TABLE 1 Amino acid sequences of non-limiting
examples of variant Fc regions SEQ ID NO Amino Acid Sequence 1
CPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALKFHYTQKSLSLSP G 2
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALKFHYTQKS LSLSPGK 3
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALKFHYTQKS LSLSPG
Amino acids at EU positions 252, 254, 256, 433, and 434 are
underlined
[0070] In certain embodiments, the variant Fc region has altered
(e.g., increased or decreased) binding affinity for an additional
Fc receptor. The variant Fc region can have altered (e.g.,
increased or decreased) binding affinity for one or more of
Fc.gamma. receptors e.g., Fc.gamma.RI (CD64), Fc.gamma.RIIA (CD32),
Fc.gamma.RIIB (CD32), Fc.gamma.RIIIA (CD16a), and Fc.gamma.RIBB
(CD16b). Any art recognized means of altering the affinity for an
additional Fc receptor can be employed. In certain embodiments, the
amino acid sequence of the variant Fc region is altered.
[0071] In certain embodiments, the variant Fc region comprises a
non-naturally occurring amino acid residue at one or more positions
selected from the group consisting of 234, 235, 236, 239, 240, 241,
243, 244, 245, 247, 252, 254, 256, 262, 263, 264, 265, 266, 267,
269, 296, 297, 298, 299, 313, 325, 326, 327, 328, 329, 330, 332,
333, and 334 as numbered by the EU index as set forth in Kabat.
Optionally, the Fc region may comprise a non-naturally occurring
amino acid residue at additional and/or alternative positions known
to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821;
6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO
02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO
05/040217, the contents of which are incorporated by reference
herein in their entirety).
[0072] In certain embodiments, the variant Fc region comprises at
least one non-naturally occurring amino acid residue selected from
the group consisting of 234D, 234E; 234N, 234Q, 234T, 234H, 234Y,
234I, 234V, 234F, 235A, 235I), 235R, 235I, 235P, 235S, 235N, 235Q,
235I, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E; 239N, 239Q,
239F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W, 241 L, 241Y,
241E, 241R, 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247V, 247G,
252Y, 254T, 256E, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M,
264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N,
265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T,
266M, 267Q, 267L, 269I, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N,
296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298I, 298I,
298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 313F,
325Q, 325L, 325I; 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W,
327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V,
328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330I, 330C, 330L,
330Y, 330V, 330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E,
332N, 332Q, 332T, 332H, 332Y, and 332A as numbered by the EU index
as set forth in Kabat. Optionally, the Fc region may comprise
additional and/or alternative non-naturally occurring amino acid
residues known to one skilled in the art (see, e.g., U.S. Pat. Nos.
5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO
01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and
WO 05/040217, the contents of which are incorporated by reference
herein in their entirety).
[0073] Other known Fc variants that may be used in the FcRn
antagonists disclosed herein include without limitations those
disclosed in Ghetie et al., 1997, Nat. Biotech, 15:637-40; Duncan
et al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol.,
147:2657-2662; Lund et al, 1992, Mol. Immunol., 29:53-59; Alegre et
al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc
Natl. Acad Sci USA, 92:11980-11984; Jefferis et al; 1995, Immunol
Lett., 44:111417; Lund et al., 1995, Faseb J.; 9:115-119; Jefferis
et al, 1996, Immunol Lett., 54:101-104; Lund et al, 1996, J.
Immunol., 157:4963-4969; Armour et al., 1999, Eur J Immunol
29:2613-2624; Idusogie et al, 2000, J. Immunol., 164:4178-4184;
Reddy et al, 2000, J. Immunol., 164:1925-1933; Xu et al., 2000,
Cell Immunol., 200:16-26; Idusogie et al, 2001, J. Immunol.,
166:2571-2575; Shields et al., 2001; J Biol. Chem., 276:6591-6604;
Jefferis et al, 2002, Immunol 82:57-6:5; Presta et al., 2002,
Biochem Soc Trans., 30:487-490); U.S. Pat. Nos. 5,624,821;
5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022;
5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 6,821,505;
6,277,375; U.S. Patent Publication Nos. 2004/0002587 and PCT
Publications WO 94/29351; WO 99/58572; WO 00/42072; WO 02/060919;
WO 04/029207; WO 04/099249; WO 04/063351, the contents of which are
incorporated by reference herein in their entirety.
[0074] In certain embodiments, the variant Fc region is a
heterodimer, where the constituent Fc domains are different from
each other. Methods of producing Fc heterodimers are known in the
art (see e.g., U.S. Pat. No. 8,216,805, which is incorporated by
reference herein in its entirety). In certain embodiments, the
variant Fc region is a single chain Fc region, where the
constituent Fc domains are linked together by a linker moiety.
Methods of producing single chain Fc regions are known in the art
(see e.g., US20090252729A1 and US20110081345A1, which are each
incorporated by reference herein in their entirety).
[0075] It is believed that pathogenic IgG antibodies observed in
autoimmune diseases are either the pathogenic triggers for these
diseases or contribute to disease progression and mediate disease
through the inappropriate activation of cellular Fc receptors.
Aggregated autoantibodies and/or autoantibodies complexed with self
antigens (immune complexes) bind to activating Fc receptors,
causing numerous autoimmune diseases (which occur in part because
of immunologically mediated inflammation against self tissues) (see
e.g., Clarkson et al., NUM 314(9), 1236-1239 (2013));
US20040010124A1; US20040047862A1; and US2004/0265321A1, which are
each incorporated by reference herein in their entirety).
Accordingly, to treat antibody-mediated disorders (e.g. autoimmune
diseases), it would be advantageous to both remove the deleterious
autoantibodies and to block the interaction of the immune complexes
of these antibodies with activating Fc receptors (e.g., Fc.gamma.
receptors, such as CD16a).
[0076] Accordingly, in certain embodiments, the variant Fc region
of the FcRn antagonist exhibits increased binding to CD16a (e.g.,
human CD16a). This is particularly advantageous in that it allows
the FcRn antagonist to additionally antagonize the immune
complex-induced inflammatory response of autoantibodies being
targeted for removal by FcRn inhibition. Any art recognized means
of increasing affinity for CD16a (e.g., human CD16a) can be
employed. In certain embodiments, the FcRn-antagonist comprises a
variant Fc-region comprising an N-linked glycan (e.g., at EU
position 297). In this case it is possible to increase the binding
affinity of the FcRn-antagonist for CD16a by altering the glycan
structure. Alterations of the N-linked glycan of Fc regions are
well known in the art. For example, afucosylated N-linked glycans
or N-glycans having a bisecting GlcNac structure have been shown to
exhibit increased affinity for CD16a. Accordingly, in certain
embodiments, the N-linked glycan is afucosylated. Afucosylation can
be achieved using any art recognized means. For example, an
FcRn-antagonist can be expressed in cells lacking fucosyl
transferase, such that fucose is not added to the N-linked glycan
at EU position 297 of the variant Fc region (see e.g., U.S. Pat.
No. 8,067,232, the contents of which is incorporated by reference
herein in its entirety). In certain embodiments, the N-linked
glycan has a bisecting GlcNac structure. The bisecting GlcNac
structure can be achieved using any art recognized means. For
example, an FcRn-antagonist can be expressed in cells expressing
beta1-4-N-acetylglucosaminyltransferase III (GnTIII), such that
bisecting GlcNac is added to the N-linked glycan at EU position 297
of the variant Fc region (see e.g., U.S. Pat. No. 8,021,856, the
contents of which is incorporated by reference herein in its
entirety). Additionally or alternatively, alterations of the
N-linked glycan structure can also be achieved by enzymatic means
in vitro.
[0077] In certain embodiments, the instant disclosure provides
FcRn-antagonist compositions wherein a portion of the
FcRn-antagonist molecules contained therein comprise altered glycan
structures. In certain embodiments, the FcRn-antagonist composition
comprises a plurality of FcRn-antagonist molecules disclosed
herein, wherein at least 50% (optionally, at least 60, 70, 80, 90,
95, or 99%) of the molecules comprise an Fc region or FcRn-binding
fragment thereof having an afucosylated N-linked glycan. In certain
embodiments, the FcRn-antagonist composition comprising a plurality
of FcRn-antagonist molecules disclosed herein, wherein at least 50%
(optionally, at least 60, 70, 80, 90, 95, or 99%) of the molecules
comprise an Fc region or FcRn-binding fragment thereof comprising
an N-linked glycan having a bisecting GlcNac.
[0078] In certain embodiments, the variant Fc region does not
comprise an N-linked glycan. This can be achieved using any art
recognized methods. For example, the Fc variant can be expressed in
a cell that is incapable of N-linked glycosylation. Additionally or
alternatively, the amino acid sequence of the Fc variant can be
altered to prevent or inhibit N-linked glycosylation (e.g., by
mutation of the NXT sequon). Alternatively, the Fc variant can be
synthesized in an acellular system (e.g., chemically
synthesized).
[0079] In certain embodiments, FcRn-antagonist molecules may be
modified, e.g., by the covalent attachment of a molecule (e.g., a
binding or imaging moiety) to the FcRn-antagonist such that
covalent attachment does not prevent the FcRn-antagonist from
specifically binding to FcRn. For example, but not by way of
limitation, the FcRn-antagonist may be modified by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization
by known protecting blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc.
[0080] In certain embodiments, the FcRn antagonist comprises a
variant Fc region linked to a half-life extender. As used herein,
the term "half-life extender" refers to any molecule that, when
linked to an FcRn antagonist disclosed herein, increases the
half-life of an FcRn antagonist. Any half-life extender may be
linked (either covalently or non-covalently) to the FcRn
antagonist. In certain embodiments, the half-life extender is
polyethylene glycol or human serum albumin. In certain embodiments,
the FcRn antagonist is linked to a binding molecule that
specifically binds to a half-life extender present in a subject,
such as a blood-carried molecule or cell, such as serum albumin
(e.g., human serum albumin), IgG, erythrocytes, etc.
[0081] The FcRn antagonists disclosed herein have excellent
manufacturability. For example, as shown in Example 5 herein, they
can be expressed at high levels in mammalian cells (e.g., at 6 g/L
in CHO cells in a 10 L stirred tank bioreactor). Moreover, after
Protein A purification, the resultant purified FcRn antagonist
composition has a very high percentage of FcRn antagonist monomers,
and contains an extremely low level of FcRn antagonist protein
aggregates and degradation products. Accordingly, in certain
embodiments, the instant disclosure provides an FcRn antagonist
composition comprising a plurality of FcRn antagonist molecules as
disclosed herein, wherein greater than 95% the of the FcRn
antagonist molecules in the composition are monomers (e.g., greater
than 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7,
99.8, 99.9%). In certain embodiments, the instant disclosure
provides an FcRn antagonist composition comprising a plurality of
FcRn antagonist molecules disclosed herein, wherein less than 5%
the of the FcRn antagonist molecules in the composition are present
in aggregates, (e.g., less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,
0.5, 0.4, 0.3, 0.2, 0.1%). In certain embodiments, the instant
disclosure provides an FcRn antagonist composition comprising a
plurality of FcRn antagonist molecules disclosed herein, wherein
the composition is substantially free of FcRn antagonist molecule
degradation products.
III. USES OF FCRN ANTAGONISTS
[0082] The FcRn antagonist compositions of the present disclosure
are particularly useful for reducing the serum levels of
Fc-containing agents (e.g., antibodies and immunoadhesins).
Accordingly, in one aspect the instant disclosure provides a method
of inhibiting FcRn function in a subject, the method generally
comprising administering to the subject an effective amount of an
FcRn antagonist composition (e.g., a pharmaceutical composition)
disclosed herein.
[0083] The reduction of serum levels of Fc-containing agents (e.g.,
antibodies and immunoadhesins) is particularly applicable to the
treatment of antibody-mediated disorders (e.g. autoimmune
diseases). Accordingly, in one aspect the instant disclosure
provides methods of treating antibody-mediated disorders (e.g.
autoimmune diseases) using the FcRn antagonist compositions
disclosed herein.
[0084] Any antibody-mediated disorder can be treated using the FcRn
antagonist compositions disclosed herein. In certain embodiments,
the antibody-mediated disorder is one that is amenable to treatment
by IVIG. In certain embodiments, the antibody-mediated disorder is
an autoimmune disease. Non-limiting autoimmune diseases include
allogenic islet graft rejection, alopecia areata, ankylosing
spondylitis, antiphospholipid syndrome, autoimmune Addison's
disease, Alzheimer's disease, antineutrophil cytoplasmic
autoantibodies (ANCA), autoimmune diseases of the adrenal gland,
autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune
myocarditis, autoimmune neutropenia, autoimmune oophoritis and
orchitis, autoimmune thrombocytopenia, autoimmune urticaria,
Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's
syndrome, celiac sprue-dermatitis, chronic fatigue immune
disfunction syndrome, chronic inflammatory demyelinating
polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial
pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's
disease, dermatomyositis, discoid lupus, essential mixed
cryoglobulinemia, factor VIII deficiency,
fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease,
Guillain-Barre, Goodpasture's syndrome, graft-versus-host disease
(GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonary
fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA
neuropathy, IgM polyneuropathies, immune mediated thrombocytopenia,
juvenile arthritis, Kawasaki's disease, lichen planus, lupus
erythematosus, Meniere's disease, mixed connective tissue disease,
multiple sclerosis, type 1 diabetes mellitus, Multifocal motor
neuropathy (MMN), myasthenia gravis, paraneoplastic bullous
pemphigoid, pemphigus vulgaris, pemphigus foliaceus, pernicious
anemia, polyarteritis nodosa, polychrondritis, polyglandular
syndromes, polymyalgia rheumatica, polymyositis and
dermatomyositis, primary agammaglobulinemia, primary biliary
cirrhosis, psoriasis, psoriatic arthritis, Reynaud's phenomenon,
Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma,
Sjogren's syndrome, solid organ transplant rejection, stiff-man
syndrome, systemic lupus erythematosus, Takayasu arteritis, toxic
epidermal necrolysis (TEN), Stevens Johnson syndrome (SJS),
temporal arteritis/giant cell arteritis, thrombotic
thrombocytopenia purpura, ulcerative colitis, uveitis, dermatitis
herpetiformis vasculitis, anti-neutrophil cytoplasmic
antibody-associated vasculitides, vitiligo, and Wegner's
granulomatosis.
[0085] In certain embodiments, the autoimmune disease is an
autoimmune channelopathy. Non-limiting channelopathies include
neuromyelitis optica, Lambert-Eaton myasthenic syndrome, myasthenia
gravis, anti-N-Methyl-D-aspartate (NMDA) receptor encephalitis,
anti-.alpha.-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) receptor encephalitis, Morvan syndrome, and Glycine receptor
antibody-associated disorder.
[0086] The FcRn antagonist compositions of the instant disclosure
are particularly suited to treating antibody-mediated disorders
characterized by an over production of serum immunoglobulin.
Accordingly, in certain embodiments, the FcRn antagonist
compositions are used to treat hypergammaglobulinemia.
[0087] The FcRn antagonist compositions can also be used in
combination with one or more additional therapeutic agents. In
certain embodiments, the additional therapeutic agent is an
anti-inflammatory agent. Any inflammatory agent can be used in
combination with the compositions disclosed herein. In certain
embodiments, the therapeutic agent is rituximab, daclizumab,
basiliximab, muronomab-CD3, infliximab, adalimumab, omalizumab,
efalizumab, natalizumab, tocilizumab, eculizumab, golimumab,
canakinumab, ustekinumab, or belimumab. In certain embodiments, the
additional therapeutic agent is leucocyte depleting agent (e.g.,
B-cell or T-cell depleting agent). Any leucocyte depleting agent
can be used in combination with the FcRn antagonist compositions
disclosed herein. In certain embodiments, the leucocyte depleting
agent is a B-cell depleting agent. In certain embodiments, the
leucocyte depleting agent is an antibody against a cell surface
marker. Suitable cell surface markers include, without limitation,
CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD70, CD72,
CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85,
or CD86 The FcRn antagonist and the additional therapeutic agent(s)
can be administered to the subject simultaneously or sequentially,
via the same or different route(s) of administration.
[0088] The FcRn antagonist compositions of the instant disclosure
are also well suited to rapidly reducing the serum levels of an
Fc-containing agent in subject. Such rapid clearance is
advantageous in cases where the Fc-containing agent is toxic (e.g.,
an antibody-drug conjugate or an agent that is immunogenic) because
it reduces the exposure of the subject to the drug. Rapid clearance
is also advantageous in cases where the Fc-containing agent is an
imaging agent that requires a low serum level of the agent to
facilitate imaging. Accordingly, in certain embodiments, the FcRn
antagonist compositions are used to reduce the serum levels of an
Fc-containing agent in subject that has been administered the
Fc-containing agent. The serum levels of any Fc-containing agent
(e.g., therapeutic or diagnostic agent) can be reduced using the
FcRn antagonist compositions disclosed herein. Non limiting
examples of Fc-containing agents include imaging agents (e.g.,
labeled antibodies), antibody drug conjugates, or immunogenic
agents (e.g., non-human antibodies or immunoadhesins). The FcRn
antagonist can be administered simultaneously with the
Fc-containing agent or sequentially (e.g., before or after the
Fc-containing agent).
[0089] Furthermore, in diseases or conditions requiring
administration of a therapeutic agent, the subject will often
develop antibodies (e.g., anti-drug antibodies) against the
therapeutic agent, which, in turn, prevent the therapeutic agent
from being available for its intended therapeutic purpose or cause
an adverse reaction in the subject. Accordingly, the FcRn
antagonist compositions disclosed herein can also be used to remove
antibodies (e.g., anti-drug antibodies) against the therapeutic
agent that develop in a subject.
[0090] The FcRn antagonist compositions disclosed herein can also
be used in combination with the therapeutic protein to enhance the
benefit of the therapeutic protein by reducing the levels of IgG;
wherein, IgG antibodies are responsible for the decreased
bioavailability of a therapeutic protein. In certain embodiments
the instant disclosure provides a method of treating a disorder
resulting from an immune response to a clotting factor comprising
administering to a subject a therapeutically effective amount of an
FcRn antagonist compositions disclosed herein. Suitable clotting
factors include, without limitation, fibrinogen, prothrombin,
factor V, factor VII, factor VIII, factor IX, factor X, factor XI,
factor XII, factor XIII, or von Willebrand's factor. This method
may be used to regulate or treat, or prevent an immune response to
a clotting factor in a patient suffering, e.g., from hemophilia A
or hemophilia B. In certain embodiments, the method may be used to
regulate or treat an immune response to, e.g., therapeutic
erythropoietin in a patient suffering from pure red cell aplasia
(PRCA).
[0091] FcRn is responsible for transporting maternal antibodies
across the placenta to the fetus in a pregnant woman. Accordingly,
if a pregnant female is administered an Fc-containing agent (e.g.,
a therapeutic antibody), the agent may come in contact with the
fetus as a result of the FcRn-mediated transport across the
placenta. To avoid any potential deleterious effect of the
Fc-containing agent on fetal development, it would be advantageous
to block FcRn function. Accordingly, the instant disclosure
provides a method of preventing placental transfer of an
Fc-containing agent (e.g., a therapeutic antibody) to the fetus in
a pregnant woman, the method comprising administering to the woman
an FcRn antagonist compositions disclosed herein, either
simultaneously or sequentially (prior or post) with the
Fc-containing agent.
[0092] The FcRn antagonist compositions disclosed herein can also
be used to treat inflammatory disorders including, but not limited
to, asthma, ulcerative colitis and inflammatory bowel syndrome
allergy, including allergic rhinitis/sinusitis, skin allergies
(urticaria/hives, angioedema, atopic dermatitis), food allergies,
drug allergies, insect allergies, mastocytosis, arthritis,
including osteoarthritis, rheumatoid arthritis, and
spondyloarthropathies.
[0093] Successful implementation of gene therapy for the treatment
of a disease or condition may be hampered by the development of
antibodies specific to the therapeutic protein encoded by the
transgene as well as possibly to the vector used to deliver the
transgene. Accordingly, the FcRn antagonist compositions disclosed
herein can be administered in combination with gene therapy to
enhance the benefit of the encoded therapeutic protein by reducing
the levels of IgG. These methods are particularly useful in
situations where IgG antibodies are responsible for the decreased
bioavailability of a gene therapy vector or the encoded therapeutic
protein. The gene therapy vector may be, e.g., a viral vector such
as adenovirus and adeno-associated virus. Diseases that can be
treated using gene therapy include, but are not limited to, cystic
fibrosis, hemophilia, PRCA, muscular dystrophy, or lysosomal
storage diseases, such as, e.g., Gaucher's disease and Fabry's
disease.
[0094] One skilled in the art would be able, by routine
experimentation, to determine what an effective, non-toxic amount
of FcRn antagonist composition would be for the purpose of treating
an antibody-mediated disorder. For example, a therapeutically
active amount of a polypeptide may vary according to factors such
as the disease stage (e.g., stage I versus stage IV), age, sex,
medical complications (e.g., immunosuppressed conditions or
diseases) and weight of the subject, and the ability of the
antibody to elicit a desired response in the subject. The dosage
regimen may be adjusted to provide the optimum therapeutic
response. For example, several divided doses may be administered
daily, or the dose may be proportionally reduced as indicated by
the exigencies of the therapeutic situation. Generally, however, an
effective dosage is expected to be in the range of about 0.1 to
10,000 mg/kg body weight per day e.g., about 1 to 1000, about
10-500, or about 50-250 or mg/kg body weight per day (e.g., about
70 mg/kg body weight per day.
IV. PHARMACEUTICAL COMPOSITIONS
[0095] In another aspect, the instant disclosure provides
pharmaceutical compositions comprising an FcRn antagonist or FcRn
antagonist composition disclosed herein and a pharmaceutically
acceptable carrier or excipient. Examples of suitable
pharmaceutical carriers are described in Remington's Pharmaceutical
Sciences by E. W. Martin. Examples of excipients can include
starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,
chalk, silica gel, sodium stearate, glycerol monostearate, talc,
sodium chloride, dried skim milk, glycerol, propylene, glycol,
water, ethanol, and the like. The composition can also contain pH
buffering reagents, and wetting or emulsifying agents.
[0096] The pharmaceutical composition can be formulated for
parenteral administration (e.g., intravenous or intramuscular) by
bolus injection. Formulations for injection can be presented in
unit dosage form, e.g., in ampoules or in multidose containers with
an added preservative. The compositions can take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
pyrogen free water.
[0097] FcRn antagonists may be linked to chelators such as those
described in U.S. Pat. No. 5,326,856. The peptide-chelator complex
may then be radiolabeled to provide an imaging agent for diagnosis
or treatment of diseases or conditions involving the regulation of
IgG levels.
V. PRODUCTION OF FCRN ANTAGONISTS
[0098] In one aspect, the invention provides polynucleotides,
vectors and host cells encoding the FcRn antagonists disclosed
herein. Methods of making an FcRn antagonists comprising expressing
these polynucleotides are also provided.
[0099] Polynucleotides encoding the FcRn antagonists disclosed
herein are typically inserted in an expression vector for
introduction into host cells that may be used to produce the
desired quantity of the claimed FcRn antagonists. Accordingly, in
certain aspects, the invention provides expression vectors
comprising polynucleotides disclosed herein and host cells
comprising these vectors and polynucleotides.
[0100] The term "vector" or "expression vector" is used herein for
the purposes of the specification and claims, to mean vectors used
in accordance with the present invention as a vehicle for
introducing into and expressing a desired gene in a cell. As known
to those skilled in the art, such vectors may easily be selected
from the group consisting of plasmids, phages, viruses and
retroviruses. In general, vectors compatible with the instant
invention will comprise a selection marker, appropriate restriction
sites to facilitate cloning of the desired gene and the ability to
enter and/or replicate in eukaryotic or prokaryotic cells.
[0101] Numerous expression vector systems may be employed for the
purposes of this invention. For example, one class of vector
utilizes DNA elements, which are derived from animal viruses such
as bovine papilloma virus, polyoma virus, adenovirus, vaccinia
virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40
virus. Others involve the use of polycistronic systems with
internal ribosome binding sites. Additionally, cells that have
integrated the DNA into their chromosomes may be selected by
introducing one or more markers that allow selection of transfected
host cells. The marker may provide for prototrophy to an
auxotrophic host, biocide resistance (e.g., antibiotics) or
resistance to heavy metals such as copper. The selectable marker
gene can either be directly linked to the DNA sequences to be
expressed, or introduced into the same cell by cotransformation.
Additional elements may also be needed for optimal synthesis of
mRNA. These elements may include signal sequences, splice signals,
as well as transcriptional promoters, enhancers, and termination
signals.
[0102] More generally, once a vector or DNA sequence encoding an
FcRn antagonist has been prepared, the expression vector may be
introduced into an appropriate host cell. That is, the host cells
may be transformed. Introduction of the plasmid into the host cell
can be accomplished by various techniques well known to those of
skill in the art. These include, but are not limited to,
transfection (including electrophoresis and electroporation),
protoplast fusion, calcium phosphate precipitation, cell fusion
with enveloped DNA, microinjection, and infection with intact
virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors"
Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds.
(Butterworths, Boston, Mass. 1988). Most preferably, plasmid
introduction into the host is via electroporation. The transformed
cells are grown under conditions appropriate to the production of
the FcRn antagonist, and assayed for FcRn antagonist expression.
Exemplary assay techniques include enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (MA), or fluorescence-activated
cell sorter analysis (FACS), immunohistochemistry and the like.
[0103] As used herein, the term "transformation" shall be used in a
broad sense to refer to the introduction of DNA into a recipient
host cell that changes the genotype and consequently results in a
change in the recipient cell.
[0104] Along those same lines, "host cells" refers to cells that
have been transformed with vectors constructed using recombinant
DNA techniques and encoding at least one heterologous gene. In
descriptions of processes for isolation of polypeptides from
recombinant hosts, the terms "cell" and "cell culture" are used
interchangeably to denote the source of FcRn antagonist unless it
is clearly specified otherwise. In other words, recovery of FcRn
antagonist from the "cells" may mean either from spun down whole
cells, or from the cell culture containing both the medium and the
suspended cells.
[0105] In one embodiment, the host cell line used for FcRn
antagonist expression is of mammalian origin; those skilled in the
art can determine particular host cell lines, which are best suited
for the desired gene product to be expressed therein. Exemplary
host cell lines include, but are not limited to, DG44 and DUXB11
(Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical
carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with
SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3
(mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse
myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human
lymphocyte), 293 (human kidney). In one embodiment, the cell line
provides for altered glycosylation, e.g., afucosylation, of the
FcRn antagonist expressed therefrom (e.g., PER.C6.RTM. (Crucell) or
FUT8-knock-out CHO cell lines (Potelligent.TM. Cells) (Biowa,
Princeton, N.J.)). In one embodiment NSO cells may be used. CHO
cells are particularly preferred. Host cell lines are typically
available from commercial services, the American Tissue Culture
Collection or from published literature.
[0106] In vitro production allows scale-up to give large amounts of
the desired FcRn antagonist. Techniques for mammalian cell
cultivation under tissue culture conditions are known in the art
and include homogeneous suspension culture, e.g. in an airlift
reactor or in a continuous stirrer reactor, or immobilized or
entrapped cell culture, e.g. in hollow fibers, microcapsules, on
agarose microbeads or ceramic cartridges. If necessary and/or
desired, the solutions of polypeptides can be purified by the
customary chromatography methods, for example gel filtration,
ion-exchange chromatography, chromatography over DEAE-cellulose
and/or (immuno-) affinity chromatography.
[0107] Genes encoding the FcRn antagonists of the invention can
also be expressed in non-mammalian cells such as bacteria or yeast
or plant cells. In this regard it will be appreciated that various
unicellular non-mammalian microorganisms such as bacteria can also
be transformed; i.e. those capable of being grown in cultures or
fermentation. Bacteria, which are susceptible to transformation,
include members of the enterobacteriaceae, such as strains of
Escherichia coli or Salmonella; Bacillaceae, such as Bacillus
subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae.
It will further be appreciated that, when expressed in bacteria,
the FcRn antagonists can become part of inclusion bodies. The FcRn
antagonists must be isolated, purified and then assembled into
functional molecules. In addition to prokaryotes, eukaryotic
microbes may also be used. Saccharomyces cerevisiae, or common
baker's yeast, is the most commonly used among eukaryotic
microorganisms although a number of other strains are commonly
available.
[0108] In addition to cell-based expression systems, the FcRn
antagonists can also be produced using acellular or chemically
synthetic methods. In certain embodiments, the FcRn antagonists are
produced by in vitro chemical synthesis.
VI. EXEMPLIFICATION
[0109] The present invention is further illustrated by the
following examples, which should not be construed as further
limiting. The contents of Sequence Listing, figures and all
references, patents, and published patent applications cited
throughout this application are expressly incorporated herein by
reference.
Example 1: Effect of Fc-Abdeg on Serum IgG Levels in Cynomolgus
Monkeys
[0110] The effect of a human anti-lysozyme IgG (HEL-Abdeg) and a
human IgG Fc region (Fc-Abdeg), comprising the amino acids Y, T, E,
K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436,
respectively (Fc-Abdeg; SEQ ID NO:2), on serum IgG levels of a
tracer antibody was determined in cynomolgus monkeys. Specifically,
cynomolgus monkeys were administered 1 mg/kg of an anti-murine CD70
hIgG1 tracer antibody (FR70-hIgG1; Oshima et al., Int Immunol
10(4): 517-26 (1998)) by i.v. bolus injection. Animals were infused
5 minutes later with either 7 mg/kg Fc-Abdeg, 20 mg/kg HEL-Abdeg,
or PBS (2 monkeys per group). Infusion was performed within 1 hour
and animals were administered a volume of 10 ml/kg. Blood samples
(3.times.150 .mu.l) were taken at 5 min prior to dosing
("pre-dose") and 5 min, 2 h, 6 h, 24 h, 48 h, 72 h, 96 h and 120 h
after completion of the infusion. Tracer levels were determined by
performing a mCD70-binding ELISA and data were plotted relative to
tracer levels at end of dosing (FIG. 1). Total cynomolgus IgG
levels were also determined (FIG. 2). The results of these
experiments show that Fc-Abdeg reduced tracer antibody more
efficiently than equimolar amounts of HEL-Abdeg.
[0111] In addition to its key role in the IgG salvage pathway, FcRn
is also involved in albumin homeostasis (Chaudhury et al., J Exp
Med. 197(3):315-22 (2003). FcRn interacts with IgG-Fc and albumin
at distinct sites and binding can happen concurrently (Andersen et
al., Nat Commun. 3:610 (2012)). Conceptually, blockage of IgG
recycling using Abdeg-modified molecules should not interfere with
albumin-FcRn interaction. This hypothesis was confirmed in a mouse
in vivo study, where the authors showed no influence of an
Abdeg-equipped hIgG1 molecule on albumin levels (Patel et al., J
Immunol 187(2): 1015-22 (2011)). In the experiment described above,
albumin levels were also determined at day -3, day 3 and day 17
after the completion of the infusion. Analogous to the mouse study,
no significant changes in albumin levels were observed after
Fc-Abdeg or HEL-Abdeg treatment (see FIG. 3).
[0112] In a subsequent experiment, the antibody-depleting potency
of Fc-Abdeg was compared to IVIG. Specifically, cynomolgus monkeys
were administered with 1 mg/kg tracer antibody (FR70-hIgG1) 2 days
prior to dosing with 70 mg/kg Fc-Abdeg or 2 g/kg IVIG (2 monkeys
per group). Infusion of Fc-Abdeg and IVIG was performed within 4
hours and animals were administered a volume of 20 ml/kg. Blood
(3.times.150 .mu.l) samples were taken 5 min prior to dosing
("pre-dose"), and 5 min, 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, 120 h,
and 168 h after completion of the infusion. Tracer levels were
determined by mCD70-binding ELISA and plotted relative to pre-dose
levels (FIG. 4). In comparison with IVIG treatment at clinical dose
(2 g/kg), 70 mg/kg Fc-Abdeg showed significantly enhanced kinetics
of tracer clearance and was also able to clear more efficiently
(>95% tracer clearance in 4 days for Abdeg versus .about.75% in
7 days for IVIG).
Example 2: Effect of Afucoslyation on Fc-Abdeg Affinity for Human
CD16a and Murine CD16-2
[0113] The binding affinity of Fc-Abdeg for hCD16a was determined
and compared to the afucosylated form (Fc-Abdeg-POT). In the same
experiment an Fc-Abdeg variant showing improved affinity for all
Fc.gamma.Rs was included ("Fc-Abdeg-S239D/I332E). Specifically, a
Maxisorp plate was coated with 100 ng/well of Neutravidin
Biotin-binding Protein (ThermoScientific, 31000) and incubated
overnight at 4.degree. C. The following day, the plate was blocked
with PBS+1% casein for 2 hours at room temperature. Subsequently,
100 .mu.l/well of a 250 ng/ml solution (dilution in PBS+0.1%
casein) of biotinylated hCD16a (Sino Biological Inc.,
10389-H27H1-B) was added to the plate and incubated for 1 hour at
room temperature prior to applying a concentration gradient of
Fc-Abdeg or Fc-Abdeg-POT molecules (1 .mu.M-0.005 nM) for a further
hour. Binding to hCD16a was detected using an HRP-conjugated
polyclonal goat anti-human Fc antibody (Jackson ImmunoResearch,
109-035-008) (incubation 1 hour at RT, dilution 1/50,000 in
PBS+0.1% casein), followed by addition of 100 .mu.l room
temperature-equilibrated TMB (SDT-reagents #s TMB). Plates were
incubated for 10 minutes prior to addition of 100 .mu.l 0.5N
H.sub.2SO.sub.4 and OD450 nm measurement. EC50 values were
determined using GraphPad Prism software. The results of these
experiments, set forth in FIG. 5, show that defucosylation of the
Fc-Abdeg molecule results in a >30-fold increase in affinity for
hCD16a (EC.sub.50=13 nM for the Fc-Abdeg-POT vs. EC.sub.50>0.4
.mu.M for the fucosylated Fc-Abdeg). As expected, binding affinity
of the Fc-Abdeg-S239D/I332E variant for hCD16a was increased
compared to wild-type Fc-Abdeg (EC.sub.50=6 nM).
[0114] Using a similar experimental procedure as described above,
the binding affinity for murine CD16-2 (Sino Biological Inc.,
50036-M27H-B) was determined. The results of these experiments, set
forth in FIG. 6, again show an increased affinity of the
afucosylated variant compared to the wild-type Fc-Abdeg
(EC.sub.50=11 nM vs. EC.sub.50>100 nM). The fold increase in
affinity for mCD16-2 of the Fc-Abdeg-POT variant over the wild-type
Fc-Abdeg is lower compared to that observed for binding to human
CD16a. This effect was not observed for the Fc-Abdeg-S239D/I332E
variant (EC.sub.50=2 nM), which has a similar fold increase in
affinity over wild type Fc-Abdeg for both the human and murine CD16
(EC.sub.50=2 nM).
[0115] Autoantibodies complexed with self antigens bind to
activating Fc.gamma.Rs and thereby trigger the autoimmune diseases,
which occur in part because of immunologically mediated
inflammation against self tissue. The ability of Fc-Abdeg to
antagonize the interaction of autoimmune antibodies and
Fc.gamma.RIII receptors on NK cells was evaluated in two ADCC-based
assays.
[0116] Initially, an ADCC reporter bioassay (Promega, G7016) was
used to analyze the competitive hCD16a binding potency of Fc-Abdeg,
Fc-Abdeg-POT and Fc-Abdeg-S239D/I332E. Specifically, 10,000
CD20-expressing Raji cells (target cells) were incubated with
60,000 Jurkat cells expressing hCD16a (effector cells) in presence
of 100 ng/ml anti-CD20 antibody and increasing concentration of
competitor. Cells were incubated for 6 hours at 37.degree. C. prior
to measuring the bioluminescence signal, which is a measure of
ADCC-activity. The luciferase signal was plotted relative to the
signal obtained by 100 ng/ml anti-CD20 in the absence of competitor
(see FIG. 7). These experiments demonstrate that both Fc-Abdeg-POT
and Fc-Abdeg-S239D/I332E efficiently block the anti-CD20-induced
ADCC signal, whilst incubation with wild-type Fc-Abdeg does not
lead to competitive binding for hCD16a expressed on Jurkat
cells.
[0117] In a next ADCC assay, inhibition of the lytic activity of an
anti-hCD70 antibody (27B3-hIgG1) by Fc-Abdeg and Fc-Abdeg-POT was
tested as a measure of competitive hCD16 binding. Specifically,
about 50,000 hCD70-expressing U266 cells were spiked into about
300,000 freshly purified PBMCs from a healthy donor in the presence
of 50 ng/ml of the anti-hCD70 antibody and a concentration-gradient
of Fc-Abdeg, Fc-Abdeg-POT and IVIG. The U266 cells were incubated
for two days, and subsequent cell lysis was analyzed by FACS using
a marker specific for the U266 cells (CD28). The results of these
experiments, set forth in FIG. 8, show that the anti-CD70 antibody
efficiently lyses U266 cells and that this depletion could be
attenuated in a dose-dependent fashion by addition of Fc-Abdeg-POT
but not by wild-type Fc-Abdeg nor IVIG. These data demonstrate that
Fc-Abdeg POT has enhanced competitive CD16a binding properties
relative to wild-type Fc-Abdeg and IVIG.
Example 3: Murine Acute ITP Model
[0118] The therapeutic potency of Fc-Abdeg, Fc-Abdeg-POT,
Fc-Abdeg-S239D/I332E molecules was tested in a mouse model of acute
immune thrombocytopenia. Specifically, C57BL/6 mice were treated
with IVIG (20 mg/animal), Fc-Abdeg (1 mg/animal), Fc-Abdeg-POT (1
mg/animal), Fc-Abdeg-S239D/I332E (1 mg/animal) or saline via the
intraperitoneal infusion (5 animals/group). Prior to treatment, a
blood sample was withdrawn for a baseline measurement of platelet
counts. One hour later, mice were treated with 5 .mu.g/animal of
the anti-mouse platelet antibody MWReg30 (Nieswandt et al., Blood
94:684-93 (1999)). Platelet counts were monitored over 24 hours.
Platelet counts were normalized relative to the initial counts for
each mouse and platelets numbers were determined using flow
cytometry via anti-CD61 staining. The results of these experiments,
set forth in FIG. 9, demonstrate that pretreatment with Fc-Abdeg
reduces MWReg30-induced thrombocytopenia with a similar potency
compared to a 7-fold higher molar dose of IVIG, and further, that
blockade of Fc.gamma.Rs by Fc-Abdeg POT and Fc-Abdeg-S239D/I332E
had a synergistic beneficial effect in this model, as seen by the
improved platelet counts at the 180 and 1440 minutes time
points.
Example 4: Manufacturability Fc-Abdeg
[0119] Fc-Abdeg (comprising Fc domains having SEQ ID NO:2) was
produced in CHO cells (Evitria, Switzerland) by transient
transfection. Following transfection, high titers of Fc-Abdeg were
detected in the supernatants (between 200 and 400 mg/ml). A similar
favorable production profile was seen when Fc-Abdeg was expressed
from an expression construct stably integrated into the CHO
GS-XCEED cell line (Lonza, Great-Britain). On average, stable
transfectants yielded 3 g/L and several clones were identified
which produced up to 6 g/L Fc-Abdeg in a 10 L stirred tank
bioreactor.
[0120] The manufacturability of the Fc-Abdeg was further
investigated by analysis of aggregates and degradation products
following protein A-purification of the aforementioned Fc-Abdeg
production runs. Specifically, 137 .mu.g of Fc-Abdeg was loaded on
a Superdex 200 10/300 GL gelfiltration column (GE Healthcare)
coupled to an AktaPurifier chromatography system. Results of this
experiment, set forth in FIG. 10, showed that only a very small
percentage of Fc-Abdeg aggregates was observed (.about.0.5%),
whilst no Fc-Abdeg degradation products were detected.
Additionally, applying various stress conditions (freeze-thaw,
rotational or temperature stress) to the protein A-purified
Fc-Abdeg did not lead to any apparent change in physicochemical and
functional properties. Taken together, these data demonstrate the
excellent manufacturability of the Fc-Abdeg.
Sequence CWU 1
1
31221PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe1 5 10 15Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Tyr Ile Thr Arg Glu Pro 20 25 30Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val 35 40 45Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr 50 55 60Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val65 70 75 80Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 85 90 95Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 100 105 110Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 115 120
125Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
130 135 140Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly145 150 155 160Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp 165 170 175Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp 180 185 190Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu Lys 195 200 205Phe His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 210 215 2202227PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5
10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Tyr 20 25 30Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205His Glu Ala Leu Lys Phe His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys2253226PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5
10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Tyr 20 25 30Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205His Glu Ala Leu Lys Phe His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly225
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