U.S. patent application number 16/590164 was filed with the patent office on 2020-01-30 for methods to manipulate alpha-fetoprotein (afp).
This patent application is currently assigned to THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. The applicant listed for this patent is THE BRIGHAM AND WOMEN'S HOSPITAL, INC.. Invention is credited to Kristi BAKER, Richard S. BLUMBERG, Amit GANDHI, Michal PYZIK.
Application Number | 20200031928 16/590164 |
Document ID | / |
Family ID | 54333408 |
Filed Date | 2020-01-30 |
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United States Patent
Application |
20200031928 |
Kind Code |
A1 |
BLUMBERG; Richard S. ; et
al. |
January 30, 2020 |
METHODS TO MANIPULATE ALPHA-FETOPROTEIN (AFP)
Abstract
As demonstrated herein, soluble human FcRn binds to AFP with
affinities greater than observed with albumin, and is able to
interfere with FcRn-mediated protection of and functional
associations with IgG. Accordingly, provided herein, in some
aspects, are compositions and methods to inhibit FcRn and AFP
interactions in diseases or disorders where elevated AFP levels are
associated with immunosuppression. Also provided herein, in some
aspects, are compositions and methods to enhance or potentiate FcRn
and AFP interactions in diseases or disorders with decreased AFP
levels or diseases or disorders where increasing AFP levels
increasing with immunosuppression.
Inventors: |
BLUMBERG; Richard S.;
(Waltham, MA) ; BAKER; Kristi; (Edmonton, CA)
; PYZIK; Michal; (Cambridge, MA) ; GANDHI;
Amit; (Billerica, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BRIGHAM AND WOMEN'S HOSPITAL, INC. |
Boston |
MA |
US |
|
|
Assignee: |
THE BRIGHAM AND WOMEN'S HOSPITAL,
INC.
Boston
MA
|
Family ID: |
54333408 |
Appl. No.: |
16/590164 |
Filed: |
October 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16284005 |
Feb 25, 2019 |
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16590164 |
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15306665 |
Oct 25, 2016 |
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PCT/US2015/026860 |
Apr 21, 2015 |
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16284005 |
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62101539 |
Jan 9, 2015 |
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61984252 |
Apr 25, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/3955 20130101;
A61K 45/06 20130101; C07K 16/44 20130101; A61P 43/00 20180101; C07K
2317/76 20130101; C07K 2317/77 20130101; A61P 35/00 20180101; C07K
16/283 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
DK-53056 awarded by the National Institutes of Health (NIH). The
government has certain rights in the invention
Claims
1-61. (canceled)
62. A method to inhibit or reduce FcRn and alpha-fetoprotein (AFP)
interactions in a disease or disorder associated with elevated AFP
levels comprising administering a therapeutically effective amount
of a pharmaceutical composition comprising an inhibitor of AFP-FcRn
and a pharmaceutically acceptable carrier, wherein said inhibitor
of AFP-FcRn inhibits binding between alpha-fetoprotein (AFP) and
FcRn.
63. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits or blocks the AFP binding site on FcRn.
64. The method of claim 62, wherein the inhibitor of AFP-FcRn is an
antibody or antigen-binding fragment thereof.
65. The method of claim 64, wherein the antibody or antigen-binding
fragment thereof is a chimeric, humanized, or completely human
antibody or antigen-binding fragment thereof.
66. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between Y521 and/or V522 of AFP and R42 of
FcRn.
67. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between P492 of AFP and R69 of FcRn.
68. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between Q441 and/or V493 of AFP and E44 of
FcRn.
69. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between H534 and/or E589 of AFP and N173 of
FcRn.
70. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between the hydrophobic core of AFP and FcRn.
71. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between L484, V493, V497, and/or F512 of AFP and
V57, W59, and/or W61 of FcRn.
72. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between T443 of AFP and E62 and/or W59 of
FcRn.
73. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between D529 of AFP and 5230 of FcRn.
74. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between S527 and/or D528 of AFP and E50 and/or 67Y
of .beta.2m complexed with FcRn.
75. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between R604 of AFP and the carbonyl oxygen at E50
of .beta.2m complexed with FcRn.
76. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between Q597 of AFP and E69 of .beta.2m complexed
with FcRn.
77. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between E106 of AFP and H161 of FcRn.
78. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between S135 of AFP and H161 of FcRn.
79. The method of claim 62, wherein the inhibitor of AFP-FcRn
inhibits binding between F531, F533, F552, and/or F575 of AFP and
W53 of FcRn.
80. The method of claim 62, wherein the subject has or has been
diagnosed with cancer.
81. The method of claim 80, further comprising administering an
anticancer therapy or agent to the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Continuation Application of U.S. Ser.
No. 16/284,005 filed on Feb. 25, 2019, which is a Continuation
Application of U.S. Ser. No. 15/306,665 filed on Oct. 25, 2016,
which is a 35 U.S.C. .sctn. 371 National Phase Entry Application of
International Application No. PCT/US15/26860 filed Apr. 21, 2015,
and which claims benefit under 35 U.S.C. .sctn. 119(e) of U.S.
Provisional Application No. 61/984,252 filed Apr. 25, 2014, and
U.S. Provisional Application No. 62/101,539 filed Jan. 9, 2015, the
contents of each of which are incorporated herein by reference in
their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 26, 2019, is named 043214-081613USC2-16284005_SL.txt and is
22,577 bytes in size.
TECHNICAL FIELD
[0004] The technical field relates to compositions and methods for
modulating alpha-fetoprotein levels and activities.
BACKGROUND
[0005] Alpha-fetoprotein (AFP) is a major plasma protein in the
fetus, where it is produced by the yolk sac and liver (Ingram et
al., 1981). In an adult, its concentration is very low, except when
a tumor, such as a hepatoma or teratoma, is present. The
alpha-fetoprotein and albumin genes are syntenic, and mammalian AFP
and serum albumin genes are believed to have arisen through
duplication of an ancestral gene 300 to 500 million years ago.
SUMMARY
[0006] The compositions and methods described herein are based, in
part, on the discovery that alpha-fetoprotein (AFP) is a third
ligand for the neonatal Fc receptor. As demonstrated herein,
soluble human FcRn binds to AFP with affinities greater than
observed with albumin, and is able to interfere with FcRn-mediated
protection of and functional associations with IgG. As further
shown herein, the AFP binding site on FcRn overlaps with the
albumin binding sites on FcRn, and antibodies that are specific for
the albumin site on hFcRn can decrease FcRn-mediated AFP transport.
As also demonstrated herein, the binding of FcRn to AFP occurs over
a much wider pH range than that observed for IgG and albumin, which
typically bind under acidic pH conditions. In addition, provided
herein are single nucleotide polymorphisms in AFP that can impact
binding of AFP with human FcRn, such as, for example, G109R, R487S,
and S445L that increase AFP-FcRn binding, and T451I and D536V, that
decrease AFP-FcRn binding.
[0007] Accordingly, provided herein, in some aspects, are
compositions and methods to inhibit FcRn and AFP interactions in
diseases or disorders where elevated AFP levels are associated with
immunosuppression. Also provided herein, in some aspects, are
compositions and methods to enhance or potentiate FcRn and AFP
interactions in diseases or disorders with decreased AFP levels or
diseases or disorders where AFP levels increase with
immunosuppression.
[0008] In some aspects, provided herein are pharmaceutical
compositions comprising an inhibitor of AFP-FcRn and a
pharmaceutically acceptable carrier, wherein said inhibitor of
AFP-FcRn inhibits binding between AFP and FcRn.
[0009] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn comprises a T451I
and/or D536V polymorphism of wild-type AFP.
[0010] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between Y521 and/or V522 of AFP and R42 of FcRn.
[0011] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between P492 of AFP and R69 of FcRn.
[0012] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between Q441 and/or V493 of AFP and E44 of FcRn.
[0013] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between H534 and/or E589 of AFP and N173 of FcRn.
[0014] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between the hydrophobic core of AFP and FcRn.
[0015] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between L484, V493, V497, and/or F512 of AFP and V57, W59, and/or
W61 of FcRn.
[0016] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between T443 of AFP and E62 and/or W59 of FcRn.
[0017] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between D529 of AFP and S230 of FcRn.
[0018] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between S527 and/or D528 of AFP and E50 and/or 67Y of .beta.2m
complexed with FcRn.
[0019] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between R604 of AFP and the carbonyl oxygen at E50 of 02m complexed
with FcRn.
[0020] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between Q597 of AFP and E69 of .beta.2m complexed with FcRn.
[0021] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between E106 of AFP and H161 of FcRn.
[0022] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between S135 of AFP and H161 of FcRn.
[0023] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits binding
between F531, F533, F552, and/or F575 of AFP and W53 of FcRn.
[0024] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn is an antibody or
antigen-binding fragment thereof, a small molecule compound, or an
RNA or DNA aptamer.
[0025] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment thereof
is a chimeric, humanized, or completely human antibody or
antigen-binding fragment thereof.
[0026] In some embodiments of these aspects and all such aspects
described herein, the inhibitor of AFP-FcRn inhibits or blocks the
AFP binding site on FcRn.
[0027] Also provided herein, in some aspects, are pharmaceutical
compositions comprising an AFP-FcRn potentiator and a
pharmaceutically acceptable carrier.
[0028] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator comprises a G109R,
R487S, and/or S445L polymorphism of wild-type AFP that increases
AFP-FcRn binding.
[0029] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
Y521 and/or V522 of AFP and R42 of FcRn.
[0030] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
P492 of AFP and R69 of FcRn.
[0031] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
Q441 and/or V493 of AFP and E44 of FcRn.
[0032] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
H534 and/or E589 of AFP and N173 of FcRn.
[0033] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
the hydrophobic core of AFP and FcRn.
[0034] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
L484, V493, V497, and/or F512 of AFP and V57, W59, and/or W61 of
FcRn.
[0035] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
T443 of AFP and E62 and/or W59 of FcRn.
[0036] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
D529 of AFP and S230 of FcRn.
[0037] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
S527 and/or D528 of AFP and E50 and/or 67Y of .beta.2m complexed
with FcRn.
[0038] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
R604 of AFP and the carbonyl oxygen at E50 .beta.2m complexed with
FcRn.
[0039] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
Q597 of AFP and E69 of .beta.2m complexed with FcRn.
[0040] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
E106 of AFP and H161 of FcRn.
[0041] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
S135 of AFP and H161 of FcRn.
[0042] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator enhances binding between
531, F533, F552, and/or F575 of AFP and W53 of FcRn.
[0043] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator is an antibody or
antigen-binding fragment thereof, a small molecule compound, an RNA
or DNA aptamer, or an AFP functional fragment.
[0044] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment thereof
is a chimeric, humanized, or completely human antibody or
antigen-binding fragment thereof.
[0045] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator binds FcRn and mimics
AFP binding.
[0046] In some embodiments of these aspects and all such aspects
described herein, the AFP-FcRn potentiator binds or physically
interacts with AFP or FcRn, and enhances or promotes interactions
between AFP and FcRn.
[0047] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises Y521 and/or
V522 of AFP and can interact with R42 of FcRn.
[0048] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises P492 of AFP
and can interact with R69 of FcRn.
[0049] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises Q441 and/or
V493 of AFP and can interact with E44 of FcRn.
[0050] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises H534 and/or
E589 of AFP and can interact with N173 of FcRn.
[0051] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises L484, V493,
V497, and/or F512 of AFP and can interact with V57, W59, and/or W61
of FcRn.
[0052] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises T443 of AFP
and can interact with E62 and/or W59 of FcRn.
[0053] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises D529 of AFP
and can interact with S230 of FcRn.
[0054] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises S527 and/or
D528 of AFP and can interact with E50 and/or 67Y of .beta.2m
complexed with FcRn.
[0055] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises R604 of AFP
and can interact with the carbonyl oxygen at E50 of .beta.2m
complexed with FcRn.
[0056] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises Q597 of AFP
and can interact with E69 of .beta.2m complexed with FcRn.
[0057] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises E106 of AFP
and can interact with H161 of FcRn.
[0058] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises S135 of AFP
and can interact with H161 of FcRn.
[0059] In some embodiments of these aspects and all such aspects
described herein, the AFP-functional fragment comprises F531, F533,
F552, and/or F575 of AFP and can interact with W53 of FcRn.
[0060] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises SEQ ID NO: 4
or AFP (1-575).
[0061] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises SEQ ID NO: 5
or AFP (484-575).
[0062] In some aspects, provided herein are methods to inhibit or
reduce FcRn and AFP interactions in a disease or disorder
associated with elevated AFP levels comprising administering a
therapeutically effective amount of any of the pharmaceutical
compositions comprising an AFP-FcRn inhibitor described herein to a
subject in need thereof.
[0063] In some embodiments of these aspects and all such aspects
described herein, the subject has or has been diagnosed with
cancer.
[0064] In some embodiments of these aspects and all such aspects
described herein, the subject has or has been diagnosed with a
cancer or tumor of primitive origin, a tumor of liver origin, such
as a hepatoma, a tumor of biliary origin, such as
cholangiocarcinoma, stomach cancer, pancreatic cancer, or a
teratocarcinoma.
[0065] In some embodiments of these aspects and all such aspects
described herein, the method further comprises administering an
anti-cancer therapy or agent to the subject.
[0066] In some embodiments of these aspects and all such aspects
described herein, the method further comprises administering
administering a tumor or cancer antigen.
[0067] In some aspects, provided herein are methods to increase or
potentiate FcRn and AFP interactions in diseases or disorders
associated with decreased AFP levels, or where increasing AFP
levels is beneficial, comprising administering a therapeutically
effective amount of any of the pharmaceutical compositions
comprising an AFP-FcRn potentiator described herein to a subject in
need thereof.
[0068] In some embodiments of these aspects and all such aspects
described herein, the subject in need is pregnant or is at risk for
having a problem with establishing and/or maintaining a
pregnancy.
[0069] In some embodiments of these aspects and all such aspects
described herein, the subject has or has been diagnosed with an
autoimmune disease or disorder.
[0070] In some embodiments of these aspects and all such aspects
described herein, the subject has or has been diagnosed with host
versus graft disease (HVGD), is an organ or tissue transplant
recipient, or a recipient of an allogenic transplant.
Definitions
[0071] Unless otherwise defined herein, scientific and technical
terms used in connection with the present application shall have
the meanings that are commonly understood by those of ordinary
skill in the art to which this disclosure belongs. It should be
understood that this invention is not limited to the particular
methodology, protocols, and reagents, etc., described herein and as
such can vary. The terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention, which is defined solely
by the claims. Definitions of common terms in immunology, and
molecular biology can be found in The Merck Manual of Diagnosis and
Therapy, 19th Edition, published by Merck Sharp & Dohme Corp.,
2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The
Encyclopedia of Molecular Cell Biology and Molecular Medicine,
published by Blackwell Science Ltd., 1999-2012 (ISBN
9783527600908); and Robert A. Meyers (ed.), Molecular Biology and
Biotechnology: a Comprehensive Desk Reference, published by VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner
Luttmann, published by Elsevier, 2006; Janeway's Immunobiology,
Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor &
Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's
Genes XI, published by Jones & Bartlett Publishers, 2014
(ISBN-1449659055); Michael Richard Green and Joseph Sambrook,
Molecular Cloning: A Laboratory Manual, 4.sup.th ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN
1936113414); Davis et al., Basic Methods in Molecular Biology,
Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN
044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch
(ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in
Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley
and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols
in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and
Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John
E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach,
Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN
0471142735, 9780471142737), the contents of which are all
incorporated by reference herein in their entireties.
[0072] As used herein, the terms "AFP-FcRn inhibitor" and "alpha
fetoprotein and FcRn inhibitor," "inhibitor of AFP-FcRn," or
"inhibitor of AFP and FcRn interactions" refer to a molecule or
agent that significantly blocks, inhibits, reduces, or interferes
with the interaction between AFP and FcRn and their resultant
biological or functional activity in vitro, in situ, and/or in
vivo, including activity of downstream pathways mediated by AFP
binding to FcRn and signaling, such as, for example, transcytosis
of AFP, inhibition of T cell stimulation by IgG comprising immune
complex-primed dendritic cells, AFP-mediated inhibition of immune
responses, and/or increased serum half-life of AFP. Exemplary
AFP-FcRn inhibitors contemplated for use in the various aspects and
embodiments described herein include, but are not limited to,
antibodies or antigen-binding fragments thereof that specifically
bind to one or more amino acid residues or epitopes on AFP and/or
FcRn involved in the binding and/or interactions of AFP and FcRn,
and inhibit/reduce/block AFP and FcRn interactions and/or binding;
small molecule agents that target or specifically bind one or more
amino acid residues on AFP and/or FcRn involved in the binding
and/or interactions of AFP and FcRn, and inhibit/reduce/block AFP
and FcRn interactions and/or binding; RNA or DNA aptamers that bind
to AFP and/or FcRn and and inhibit/reduce/block AFP and FcRn
interactions and/or binding; and/or AFP fragments or fusion
polypeptides thereof that block endogenous AFP interactions with
FcRn.
[0073] "Decreased/decreasing interaction between AFP and FcRn,"
"reduced/reducing interaction between AFP and FcRn," "inhibits
binding," or "inhibited/inhibiting interaction between AFP and
FcRn" as used interchangeably herein, generally means either
reducing or inhibiting the interaction between or binding of AFP
and FcRn by at least 5%, at least 10%, at least 25%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98%, or more, compared to the interaction between AFP
and FcRn under the same conditions but without the presence of
AFP-FcRn inhibitors described herein. Assays for measuring such
inhibition or reduced interactions are known in the art and are
described herein in the Examples.
[0074] As used herein, the terms "AFP-FcRn potentiator,"
"potentiator of AFP-FcRn interaction," "AFP-FcRn activator agent,"
and "AFP-FcRn agonist agent" refer to a molecule or agent that
mimics or up-regulates (e.g., increases, potentiates or
supplements) the biological activity of AFP binding to FcRn in
vitro, in situ, and/or in vivo, including downstream pathways
mediated by AFP binding to FcRn and signaling, such as, for
example, transcytosis of AFP, inhibition of T cell stimulation by
immune complex-primed dendritic cells, AFP-mediated inhibition of
immune responses, and/or increased serum half-life of AFP. An
AFP-FcRn potentiator or agonist can be, in some embodiments, an AFP
protein fragment or derivative thereof having at least one
bioactivity of the wild-type AFP. An AFP-FcRn potentiator can also
be a compound which increases the interaction of AFP with FcRn, for
example. Exemplary AFP-FcRn potentiators or agonists contemplated
for use in the various aspects and embodiments described herein
include, but are not limited to, antibodies or antigen-binding
fragments thereof that specifically bind to AFP bound to FcRn and
enhance the interaction and/or block FcRn binding to albumin and/or
IgG but allow binding of AFP to FcRn; RNA or DNA aptamers that bind
to FcRn and mimic AFP binding to FcRn; AFP structural analogs or
AFP fragment, derivatives, or fusion polypeptides thereof; and
small molecule agents that target or bind to FcRn and act as
functional mimics of AFP binding to FcRn.
[0075] As used herein, "antibodies" or "antigen-binding fragments"
thereof include monoclonal, human, humanized or chimeric
antibodies, single chain antibodies, Fab fragments, F(ab')
fragments, fragments produced by a Fab expression library, and/or
binding fragments of any of the above. Antibodies also refer to
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain antigen or
target binding sites or "antigen-binding fragments." The
immunoglobulin molecules described herein 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 of immunoglobulin molecule, as is
understood by one of skill in the art.
[0076] The terms "antibody fragment" or "antigen-binding fragment"
include: (i) the Fab fragment, having V.sub.L, C.sub.L, V.sub.H and
C.sub.H1 domains; (ii) the Fab' fragment, which is a Fab fragment
having one or more cysteine residues at the C-terminus of the
C.sub.H1 domain; (iii) the Fd fragment having V.sub.H and C.sub.H1
domains; (iv) the Fd' fragment having V.sub.H and C.sub.H1 domains
and one or more cysteine residues at the C-terminus of the CH1
domain; (v) the Fv fragment having the V.sub.L and V.sub.H domains
of a single arm of an antibody; (vi) a dAb fragment (Ward et al.,
Nature 341, 544-546 (1989)) which consists of a V.sub.H domain or a
V.sub.L domain; (vii) isolated CDR regions; (viii) F(ab').sub.2
fragments, a bivalent fragment including two Fab' fragments linked
by a disulphide bridge at the hinge region; (ix) single chain
antibody molecules (e.g. single chain Fv; scFv) (Bird et al.,
Science 242:423-426 (1988); and Huston et al., PNAS (USA)
85:5879-5883 (1988)); (x) "diabodies" with two antigen binding
sites, comprising a heavy chain variable domain (V.sub.H) connected
to a light chain variable domain (V.sub.L) in the same polypeptide
chain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al.,
Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) "linear
antibodies" comprising a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995);
and U.S. Pat. No. 5,641,870); and modified versions of any of the
foregoing (e.g., modified by the covalent attachment of
polyalkylene glycol (e.g., polyethylene glycol, polypropylene
glycol, polybutylene glycol) or other suitable polymer).
[0077] As used herein, an "epitope" can be formed both from
contiguous amino acids, or noncontiguous amino acids juxtaposed by
tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents, whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5, about 9, or
about 8-10 amino acids in a unique spatial conformation. An
"epitope" includes the unit of structure conventionally bound by an
immunoglobulin V.sub.H/V.sub.L pair. Epitopes define the minimum
binding site for an antibody, and thus represent the target of
specificity of an antibody. In the case of a single domain
antibody, an epitope represents the unit of structure bound by a
variable domain in isolation. The terms "antigenic determinant" and
"epitope" can also be used interchangeably herein.
[0078] As used herein, "small molecule inhibitors" include, but are
not limited to, small peptides or peptide-like molecules, soluble
peptides, and synthetic non-peptidyl organic or inorganic
compounds. A small molecule inhibitor or antagonist can have a
molecular weight of any of about 100 to about 20,000 daltons (Da),
about 500 to about 15,000 Da, about 1000 to about 10,000 Da.
[0079] The term "therapeutically effective amount" therefore refers
to an amount of the inhibitors or potentiators described herein,
using the methods as disclosed herein, that is sufficient to
provide a particular effect when administered to a typical subject.
An effective amount as used herein would also include an amount
sufficient to delay the development of a symptom of the disease,
alter the course of a symptom disease (for example but not limited
to, slow the progression of a symptom of the disease), or reverse a
symptom of the disease. Thus, it is not possible to specify the
exact "effective amount". However, for any given case, an
appropriate "effective amount" can be determined by one of ordinary
skill in the art using only routine experimentation.
[0080] A "cancer" or "tumor" as used herein refers to an
uncontrolled growth of cells which interferes with the normal
functioning of the bodily organs and systems. A subject that has a
cancer or a tumor is a subject having objectively measurable cancer
cells present in the subject's body. Included in this definition
are benign tumors and malignant cancers, as well as dormant tumors
or micrometastases. Cancers which migrate from their original
location and seed vital organs can eventually lead to the death of
the subject through the functional deterioration of the affected
organs. Hemopoietic cancers, such as leukemia, are able to
out-compete the normal hemopoietic compartments in a subject,
thereby leading to hemopoietic failure (in the form of anemia,
thrombocytopenia and neutropenia) ultimately causing death.
[0081] The term "anti-cancer therapy" refers to a therapy useful in
treating cancer. Examples of anti-cancer therapeutic agents
include, but are not limited to, e.g., surgery, chemotherapeutic
agents, growth inhibitory agents, cytotoxic agents, agents used in
radiation therapy, anti-angiogenesis agents, apoptotic agents,
anti-tubulin agents, and other agents to treat cancer, such as
anti-HER-2 antibodies (e.g., HERCEPTIN.RTM.), anti-CD20 antibodies,
an epidermal growth factor receptor (EGFR) antagonist (e.g., a
tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib
(TARCEVA.RTM.)), platelet derived growth factor inhibitors (e.g.,
GLEEVEC.TM. (Imatinib Mesylate)), a COX-2 inhibitor (e.g.,
celecoxib), interferons, cytokines, antagonists (e.g., neutralizing
antibodies) that bind to one or more of the following targets
ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF
receptor(s), TRAIL/Apo2, and other bioactive and organic chemical
agents, etc. Combinations thereof are also specifically
contemplated for the methods described herein.
[0082] As used herein, the terms "tumor antigen" and "cancer
antigen" are used interchangeably to refer to antigens which are
differentially expressed by cancer cells and can thereby be
exploited in order to target cancer cells. Cancer antigens are
antigens which can potentially stimulate apparently tumor-specific
immune responses. Some of these antigens are encoded, although not
necessarily expressed, by normal cells. These antigens can be
characterized as those which are normally silent (i.e., not
expressed) in normal cells, those that are expressed only at
certain stages of differentiation and those that are temporally
expressed such as embryonic and fetal antigens. Other cancer
antigens are encoded by mutant cellular genes, such as oncogenes
(e.g., activated ras oncogene), suppressor genes (e.g., mutant
p53), and fusion proteins resulting from internal deletions or
chromosomal translocations. Still other cancer antigens can be
encoded by viral genes such as those carried on RNA and DNA tumor
viruses. Many tumor antigens have been defined in terms of multiple
solid tumors: MAGE 1, 2, & 3, defined by immunity;
MART-1/Melan-A, gp100, carcinoembryonic antigen (CEA), HER-2,
mucins (i.e., MUC-1), prostate-specific antigen (PSA), and
prostatic acid phosphatase (PAP). In addition, viral proteins such
as hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV)
have been shown to be important in the development of
hepatocellular carcinoma, lymphoma, and cervical cancer,
respectively. However, due to the immunosuppression of patients
diagnosed with cancer, the immune systems of these patients often
fail to respond to the tumor antigens.
[0083] As used herein, the phrase "at risk for having a problem
with establishing and/or maintaining a pregnancy" refers to a
subject (e.g., a human) that is predisposed to experiencing a
problem with establishing and/or maintaining a pregnancy. This
predisposition may be genetic (e.g., a particular genetic tendency
to experience a problem with establishing and/or maintaining a
pregnancy, such as heritable disorders), or due to other factors
(e.g., age, prior experience of a problem with establishing and/or
maintaining a pregnancy, drug or alcohol use, environmental
conditions, exposures to detrimental compounds present in the
environment, etc.). Thus, it is not intended that the present
invention be limited to any particular risk, nor is it intended
that the present invention be limited to any particular problem
with establishing and/or maintaining a pregnancy.
[0084] As used herein, an "autoimmune disease" refers to a class of
diseases in which a subject's own antibodies react with host tissue
or in which immune effector T cells are autoreactive to endogenous
self-peptides and cause inflammation and/or destruction of tissue.
Thus an immune response is mounted against a subject's own
antigens, referred to as self-antigens. A "self-antigen" as used
herein refers to an antigen of a normal host tissue. Normal host
tissue does not include cancer cells.
BRIEF DESCRIPTION OF THE FIGURES
[0085] FIG. 1 demonstrates that hAFP is transcytosed by hFcRn at
acidic and neutral pH. Transcytosis of human AFP in MDCK II cells
co-expressing human FcRn and .beta.2microglobulin (hFcRn/.beta.2m)
or vector control at pH 6 and 7.4. B.fwdarw.A Basolateral to apical
direction, A.fwdarw.B Apical to Basolateral direction.
[0086] FIG. 2 demonstrates that hAFP is transcytosed by mouse FcRn.
Transcytosis of human AFP in MDCK II cells co-expressing mouse FcRn
and 132microglobulin (mFcRn/m.beta.2m) or vector control at pH 7.4.
B.fwdarw.A Basolateral to apical direction, A.fwdarw.B Apical to
Basolateral direction.
[0087] FIG. 3 demonstrates that AFP transcytosis by hFcRn is
blocked by ADM31 antibody, which specifically binds to an epitope
on FcRn that contains the albumin binding site. Transcytosis of
human AFP in MDCK II cells co-expressing human FcRn and
.beta.2microglobulin (mFcRn/m.beta.2m) or vector control at pH 7.4
in presence of anti-human FcRn antibody (ADM31) or isotype control.
B.fwdarw.A Basolateral to apical direction.
[0088] FIG. 4 demonstrates that AFP hinders FcRn-mediated
transcytosis of IgG. Transcytosis of human IgG in MDCK II cells
co-expressing human FcRn and .beta.2microglobulin (hFcRn/h.beta.2m)
or vector control at pH 6 which have been pre-incubated with hAFP
or Human Serum Albumin (HSA) as control at pH 7.4. Basolateral to
apical direction is shown.
[0089] FIG. 5 demonstrates that AFP binds to human and mouse FcRn
at neutral pH. SPR analyses of hAFP binding to hFcRn (left panel)
or mFcRn (right panel) at neutral pH.
[0090] FIG. 6 demonstrates that AFP binds to hFcRn at acidic pH.
SPR analyses of hAFP binding to hFcRn at pH 6.
[0091] FIG. 7 demonstrates that AFP inhibits T cell stimulation by
IgG-immune complex (IC) primed dendritic cells (DC). hAFP blocks
proliferation (IL-2 secretion) of CD8+(OT-I, left panel) or
CD4+(OT-II, right panel) T cells in response to antigen in IgG-IC
from bone marrow (BM). DC from hFCGRT/hB2M/mFcgrt-/- human FcRn and
.beta.2-microglobulin transgenic and mouse FcRn knockout)mice were
treated with 100 .mu.g/ml of IgG or IHH-IgG (FcRn-defective IgG) in
association with 0, 0.5, 1, or 5 .mu.g/ml of OVA in presence of 100
.mu.g/ml of hAFP and then co-cultured with either OVA-specific CD8+
or CD4+ T cells. 24 after the stimulation IL-2 secretion in the
supernatants were measured by ELISA.
[0092] FIG. 8 demonstrates that ADM31 blocks AFP-FcRn-mediated
inhibitory functions. ADM31 .alpha.-hFcRn monoclonal antibody
blocks hAFP inhibition of CD8+ T cell IL-2 secretion in response to
antigen in IgG-IC. BMDC from hFCGRT/hB2M/mFcgrt-/- mice were
treated with 100 .mu.g/ml of IgG or IHH-IgG in association with 0.5
.mu.g/ml OVA in presence of 50 .mu.g/ml of hAFP or HSA and 50
.mu.g/ml of ADM31 or isotype control, and then co-cultured with
OVA-specific CD8+ T cells. 24 after the stimulation IL-2 secretion
in the supernatants were measured by ELISA.
[0093] FIG. 9 demonstrates that ADM31 blocks AFP-FcRn-mediated
inhibitory functions. ADM31 .alpha.-hFcRn monoclonal antibody
blocks hAFP inhibition of CD8+ T cell proliferation in response to
antigen in IgG-IC. BMDC from hFCGRT/hB2M/mFcgrt-/- mice were
treated with 100 .mu.g/ml of IgG or IHH-IgG in association with 0.5
.mu.g/ml OVA in presence of 50 .mu.g/ml of hAFP or HSA and 50
.mu.g/ml of ADM31 or isotype control, and then co-cultured with
CD8+ T cells labelled with eFluor670 Proliferation Dye. 72 hrs
later the cells were acquired. Percent of proliferated cells is
displayed.
[0094] FIG. 10 demonstrates that administration of hAFP results in
increased clearance of hIgG antibodies from systemic circulation.
hFCGRT/hB2M/mFcgrt-/- mice were injected with hIgG and the
following day with hAFP. 24, 48 and 72 hrs later blood samples were
collected and the amount of hIgG was quantified by ELISA and
compared to Day 0. The results illustrate that AFP injection
resulted in faster clearance of hIgG from circulation.
[0095] FIG. 11 shows an AFP homology model derived from human serum
albumin (HSA) Crystal Structure (PDB ID: 4NOF). Based on high
homology between HSA and AFP, a structural model of AFP was built
and superimposed on FcRn:HSA:Fc-YTE structure (PDB ID 4N0U) with
RMSD of 0.072. All the figures were drawn using PyMOL (DELANO
SCIENTIFIC) and labels were added using ADOBE.RTM. Photoshop.
[0096] FIG. 12 depicts superimposition of AFP model on HSA (left
panel) or FcRn-HSA-IgG ternary complex crystal structure (PDB ID:
4N0U) (right panel). FIG. 13 depicts HSA Y497/V498 residues are
conserved in AFP (Y521/V522) and interact with FcRn R42. HSA/AFP
have conserved residues in Domain III that establish binding to
FcRn.
[0097] FIG. 14 demonstrates that HSA P468 residue is conserved in
AFP (P492) and interacts with FcRn R69. HSA/AFP conserved residues
in Domain III that establish binding to FcRn
[0098] FIG. 15 demonstrates that HSA Q417/V469 residues are
conserved in AFP (Q441/V493) and interact with FcRn E44. HSA/AFP
conserved residues in Domain III establish binding to FcRn AFP. HSA
V469/AFPV493 make backbone contacts with conserved HSA H464/AFP
H488.
[0099] FIG. 16 demonstrates that HSA H510/E565 residues are
conserved in AFP (H534/E589) and interact with FcRn N173. HSA/AFP
conserved residues in Domain III establish binding to FcRn.
[0100] FIG. 17 demonstrates that hydrophobic core centered on HSA
L460/V469/V473/F488 is conserved in AFP (L484/V493/V497/F512) and
interacts with FcRn V57/W59/W61. HSA/AFP conserved residues in
Domain III establish binding to FcRn.
[0101] FIG. 18 demonstrates that HSA S419 residue is not conserved
in AFP (T443) yet is able to interact with FcRn E62/W59. HSA/AFP
non-conserved residues in Domain III preserve AFP binding to
FcRn.
[0102] FIG. 19 demonstrates that HSA E505 non-conserved residue in
AFP (D529) preserves binding to FcRn S230. HSA/AFP non-conserved
residues in Domain III preserve AFP binding to FcRn.
[0103] FIG. 20 demonstrates that AFP 5527/D528 residues make
contacts with .beta.2m E50 and 67Y that are not present in HSA
(N503, A504) providing new interactions. HSA/AFP non-conserved
residues that increase AFP binding to FcRn through new contacts
with .beta.2m and is not pH dependent.
[0104] FIG. 21 demonstrates that AFP R604 makes additional contacts
with .beta.2m E50, providing new interactions. HSA/AFP
non-conserved residues increase AFP binding to .beta.2m. HSA Q580
lacks these interactions.
[0105] FIG. 22 demonstrates that AFP Q597 residue is better
positioned to make contacts with .beta.2m E69 providing stronger
interaction. HSA/AFP non-conserved residues establish new and
increased AFP-.beta.2m interactions. HSA K573 lacks these
interactions.
[0106] FIG. 23 demonstrates that AFP (E106) conserved residue (with
HSA E82) makes long range interaction with FcRn H161. Conserved
HSA/AFP residues in Domain that interact with FcRn.
[0107] FIG. 24 demonstrates that AFP S135 allows AFP interface to
come closer to FcRn and makes .about.3 .ANG. interactions with FcRn
H161, which is absent in HSA. AFP Domain I-FcRn interaction
indicates neutral pH binding. Nearby conserved proline in HSA/AFP
occupy same space in interface.
[0108] FIG. 25 demonstrates that a substantially conserved
hydrophobic core in AFP (F531/F533/F552/F575) centered on FcRn W53:
AFP F552 results in stronger AFP-FcRn interactions than HSA A528.
AFP-FcRn interactions are consistent with neutral pH binding. AFP
(F531/F533/F552/F575) vs HSA (F507/F509/A528/F551)
DETAILED DESCRIPTION
[0109] Compositions and methods are provided herein that relate to
the discoveries described herein that alpha fetal protein (AFP) is
a third ligand for the neonatal Fc receptor or FcRn.
FcRn and Alpha Fetoprotein
[0110] FcRn, also known as the neonatal Fc receptor, is encoded by
the Fcgrt gene. It is a MHC class I-like transmembrane protein
consisting of a heavy chain containing three extracellular domains
(.alpha.1, .alpha.2 and .alpha.3), a single pass transmembrane
domain and a short cytoplasmatic tail (Burmeister et al., 1994a,b;
Martin et al., 2001). For proper function, the FcRn heavy chain
non-covalently associates with the common .beta.2-microglobulin
subunit as a light chain, which interacts with FcRn via residues on
the underside of the .alpha.1-.alpha.2 platform and the side of the
.alpha.3 domain (West & Bjorkman, 2000). Although the tertiary
structure resembles MHC class I molecules with which it shares
22-29% sequence homology (Simister & Mostov, 1989), the mouse
and human FcRn genes are located outside the MHC locus, on
chromosomes 7 and 19, respectively (Ahouse et al., 1993; Kandil et
al., 1996). In further divergence from classical MHC molecules, the
sites where peptide residues bind to MHC class I molecules are
occluded in FcRn by an arginine side chain and a proline residue,
so that FcRn does not present peptide antigens to T-cells
(Burmeister et al., 1994a,b).
[0111] Most serum proteins have a short serum half-life (about 1-2
days). However, two types of serum proteins, namely albumin and
antibodies of the IgG class, have greatly extended serum
half-lives. For example, most subclasses of IgG have a half-life of
about 10-20 days in humans. The Fc region of IgG is required for
this extension of half-life. Thus, truncated IgG polypeptides
carrying only the Fc region, and potentially also proteins carrying
a short FcRn binding peptide sequence (FcBP) (Sockolosky et al.
Proc Natl Acad Sci USA 2012, 109, 16095-100), also show such
extended serum half-life. Moreover, when the Fc region is fused
with a fusion partner (e.g., a biologically active protein), this
Fc fusion protein shows an extended serum half-life due to its
interaction with FcRn.
[0112] The mechanism by which FcRn extends the serum half-life of
IgG and IgG Fc fusion proteins is well established (Ghetie and
Ward, 2000, 2002; Roopenian and Akilesh, 2007). FcRn is localized
in the endosomal compartments of many cell types, including
vascular endothelium. Serum proteins are constantly being
endocytosed and directed to the early endosomal vesicles. FcRn is
harbored primarily in this acidified vesicle. In this acidified
environment, the Fc region binds FcRn, and the IgG/FcRn complex is
then recycled either apically or basolaterally back to the plasma
membrane, whereupon exposure to the neutral pH 7.2 extracellular
environment results in its release into the circulation. In
contrast, other endocytosed proteins that do not bind FcRn are not
rescued, and thus continue through the endosomal route to catabolic
elimination, resulting in their short half-life. The biochemical
mechanism by which the Fc region of IgG binds FcRn in an acidic
environment is understood. The CH2-CH3-hinge region of the Fc
region contains solvent exposed histidine residues, which when
protonated, engage residues on FcRn with sufficient affinity to
permit IgG to exploit the FcRn recycling pathway to escape
catabolic elimination.
[0113] Between different species, FcRn exhibits considerable
structural variations, which most likely account for the molecule's
different ligand binding specificity and slight variations in its
functions. The peptide sequences of rat and mouse FcRn, for
example, are 91% homologous (Ahouse et al., 1993), whereas the
extracellular region of human FcRn shares only 65% amino acid
sequence identity with rat FcRn (Story et al., 1994). Bovine FcRn,
on the other hand, displays 77% homology to its human counterpart,
but exhibits further divergence from rodent FcRn (Kacskovics et
al., 2000). Similarly, although mouse and rat FcRn exhibit
promiscuous binding to multiple different species of IgG such as
horse, rabbit and human, human FcRn binding is significantly more
restricted and limited to itself and rabbit (Ober et al.,
2001).
[0114] Elucidation of the crystal structure revealed that two FcRn
molecules bind to a single IgG in a 2:1 stoichiometry (Huber et
al., 1993; Sanchez et al., 1999; Schuck et al., 1999). Each IgG
heavy chain contains three constant regions (Huber et al., 1976)
with one of the FcRn molecules binding to the CH2-CH3 interface of
the IgG Fc region (Huber et al., 1993; Sanchez et al., 1999; Schuck
et al., 1999; West & Bjorkman, 2000). Such binding between IgG
and FcRn occurs in a strictly pH-dependent manner with low micro-
to nanomolar affinity at pH<6.5 but no binding at pH 7.5
(Raghavan et al., 1995). Several amino acids on both molecules have
been identified to be critical for this interaction. Site-directed
mutagenesis approaches have revealed that the residues Ile253,
His310 and His435 of IgG play a central role in the interaction
with FcRn, as shown within different species (mouse, human and rat)
as well as for interspecies binding (Firan et al., 2001; Kim et
al., 1994, 1999; Martin et al., 2001; Medesan et al., 1997;
Raghavan et al., 1995; Shields et al., 2001). The pKa of His is
6.0-6.5 such that several histidine residues of IgG become
protonated below physiologic pH, allowing for the formation of salt
bridges with acidic residues on FcRn which in doing so provides the
structural basis for the strict pH dependency of IgG-FcRn
interactions.
[0115] As initially identified in the interaction between rat FcRn
and rat IgG2a, residues on FcRn involved in binding IgG include
Glu117, Glu118, Glu132, Trp133, Glu135 and Asp137 on the .alpha.2
helix (Martin et al., 2001). Although these residues are generally
conserved between different species and the main tertiary structure
of FcRn with three extracellular ligand-binding domains is
preserved, differences between rodent and human FcRn have been
described at specific residues and contribute to IgG binding
(Vaughn et al., 1997). While human FcRn contains only a single
N-glycan moiety in its .alpha.2 domain, rat FcRn possesses three
additional N-glycan moieties in the .alpha.1, .alpha.2 and .alpha.3
domains (Ahouse et al., 1993; Kuo et al., 2009; Martin et al.,
2001; West & Bjorkman, 2000). The Asn128 residue in the
.alpha.2 domain of rat FcRn, which is lacking in human FcRn, binds
to IgG forming a functional "carbohydrate handshake" (Martin et
al., 2001; Vaughn & Bjorkman, 1998). In another example, human
FcRn displays very limited interspecies IgG binding, extending only
to rabbit IgG (Ober et al., 2001), whereas human IgG can bind to
cynomolgus FcRn (Bitonti et al., 2004; Dall'Acqua et al., 2006;
Zalevsky et al., 2010). Cynomolgus and human IgG have been
demonstrated to bind equally well to cynomolgus monkey FcRn
(Dall'Acqua et al., 2006), thereby further strengthening the
evolutionary significance of the interaction between the Fc region
and FcRn. Rodent FcRn, however, is known to be promiscuous by
binding to IgG molecules from a variety of species including human,
rabbit and bovine IgG as discussed above (Ober et al., 2001).
Murinization of human FcRn by mutating the poorly conserved Leu137
residue within the .alpha.2 domain of human FcRn to the murine
counterpart (glutamic acid) confers binding of human FcRn to mouse
IgG1 and IgG2a while reducing binding to human IgG1 twofold (Zhou
et al., 2003). The L137E mutation demonstrates that single docking
topologies are vitally important in the binding of FcRn to IgG.
Apart from the residues discussed above, Ile1 on .beta.2m
contributes to IgG binding, most likely by interacting with
hydrophobic residues at position 309 of the IgG-Fc domain.
[0116] Accordingly, the term "FcRn" as used herein, refers to the
molecule comprising the 365 amino acid FcRn large subunit p51
precursor having the amino acid sequence of:
MGVPRPQPWALGLLLFLLPGSLGAESHLSLLYHLTAVSSPAPGTPAFWVSGWLGPQQYLS
YNSLRGEAEPCGAWVWENQVSWYWEKETTDLRIKEKLFLEAFKALGGKGPYTLQGLLGCE
LGPDNTSVPTAKFALNGEEFMNFDLKQGTWGGDWPEALAISQRWQQQDKAANKELTFLLF
SCPHRLREHLERGRGNLEWKEPPSMRLKARPSSPGFSVLTCSAFSFYPPELQLRFLRNGL
AAGTGQGDFGPNSDGSFHASSSLTVKSGDEHHYCCIVQHAGLAQPLRVELESPAKSSVLV
VGIVIGVLLLTAAAVGGALLWRRMRSGLPAPWISLRGDDTGVLLPTPGEAQDADLKDVNV IPATA,
(SEQ ID NO: 1), as described by, e.g., NP_001129491.1 or
NP_004098.1, which non-covalently associates with the .beta.2
microgobulin (".beta.2m") chain having the amino acid sequence of:
MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLL
KNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID
NO: 2), as described by, e.g., NP_004039.1, together with any
naturally occurring allelic, splice variants, and processed forms
thereof. Typically, FcRn refers to human FcRn. The term "FcRn" is
also used to refer to truncated forms or fragments of the FcRn
polypeptide that retains an FcRn function or activity of interest
as described herein, such as, for example, binding to AFP.
Reference to any such forms of FcRn can be identified in the
application, e.g., by "FcRn (24-110)." Specific residues of FcRn
can be referred to as, for example, "FcRn(53) or "W53 of FcRn," or
"E69 of .beta.2m of FcRn."
[0117] As described herein, the inventors have discovered that a
third ligand for FcRn is alpha fetoprotein (AFP). As demonstrated
herein, human AFP is transcytosed by FcRn at both acidic and
neutral pHs, and increasing amounts of human AFP can inhibit
FcRn-mediated transcytosis of IgG, and results in increased IgG
clearance from systemic circulation, as well as decreased T cell
stimulation by IgG immune complexes.
[0118] Alpha-fetoprotein (AFP) is a major plasma protein in the
fetus, where it is produced by the yolk sac and liver (Ingram et
al., 1981). In an adult, its concentration is very low, except when
a tumor, such as a hepatoma or teratoma is present. The
alpha-fetoprotein and albumin genes are syntenic, and mammalian AFP
and serum albumin genes are believed to have arisen through
duplication of an ancestral gene 300 to 500 million years ago.
After birth, AFP is down-regulated thousands of fold, such that it
is not expressed at high levels in a host under homeostatic
conditions. It can become subsequently elevated and expressed at
high levels during processes associated with particular types of
pathology, such as cancers, particularly in tumors of liver origin
(e.g., hepatoma), tumors of the biliary system (e.g.,
cholangiocarcinoma), and in tumors of primitive origin and that are
poorly differentiated, such as teratocarcinomas. In addition,
elevated AFP levels can occur during chronic liver inflammatory
processes, liver regeneration, and during immune activation, such
as allogeneic responses.
[0119] Accordingly, the term "AFP" as used herein, refers to the
609 amino acid polypeptide having the amino acid sequence of:
MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATY
KEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEKEILEKYGHSDCCSQSEEG
RHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILL
WAARYDKIIPSCCKAENAVECFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITV
TKLSQKFTKVNFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKIT
ECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSR
RHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEELQKYIQESQALAKRSCGLFQ
KLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADII
IGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQA
QGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLI
SKTRAALGV (SEQ ID NO: 3), as described by, e.g., NP_001125.1,
together with any naturally occurring allelic, splice variants, and
processed forms thereof. Typically, AFP refers to human AFP. The
term "AFP" can also, in some embodiments, be used to refer to
truncated forms or fragments of the AFP polypeptide that retain an
AFP function or activity of interest as described herein, such as,
for example, binding to FcRn. Reference to any such forms of AFP
can be identified in the application, e.g., by "AFP (211-402)."
Specific residues of AFP can be referred to as, for example,
"AFP(531) or "F531 of AFP."
[0120] The discovery as described herein that AFP is a third ligand
for FcRn provides novel compositions and methods for the treatment
of conditions in which modulating the level of AFP is
therapeutic.
Inhibitors and Potentiators of AFP-FcRn Interactions
[0121] Provided herein are compositions and methods thereof based,
in part, on the discovery that alpha-fetoprotein (AFP) is a third
ligand for the neonatal Fc receptor. As demonstrated herein,
soluble human FcRn binds to AFP with affinities greater than
observed with albumin, and less than that of IgG. As further shown
herein, the AFP binding site on FcRn overlaps directly with both
the albumin binding sites on FcRn binding sites on FcRn and
indirectly with the IgG binding sites mainly through interactions
with .beta.2-microglobulin. IgG interactions with FcRn include
amino acid contact sites within .beta.2-microglobulin. Antibodies
that are specific for the albumin site on hFcRn can decrease
FcRn-mediated AFP transport. As also demonstrated herein, the
binding of FcRn to AFP occurs over a much wider pH range than that
observed for IgG and albumin, which typically bind under acidic pH
conditions. In addition, provided herein are single nucleotide
polymorphisms in AFP that can impact binding of AFP with human
FcRn, such as, for example, G109R, R487S, and S445L that increase
AFP-FcRn binding, and T451I and D536V, that decrease AFP-FcRn
binding.
[0122] Accordingly, provided herein, in some aspects, are
compositions and methods to inhibit or reduce FcRn and AFP
interactions in diseases or disorders where elevated AFP levels are
associated with immunosuppression. Also provided herein, in some
aspects, are compositions and methods thereof to enhance or
potentiate FcRn and AFP interactions in diseases or disorders with
decreased AFP levels or diseases or disorders where increasing AFP
levels is therapeutic, such as subjects in need of increasing
immunosuppression.
[0123] In some aspects, provided herein are compositions, such as
pharmaceutical compositions, comprising inhibitors of AFP-FcRn.
Such inhibitors are used to inhibit/block the interaction between
AFP and FcRn and/or reduce transcytosis of human AFP, and/or reduce
serum half-life of AFP. In particular, in some embodiments of the
aspects described herein, such AFP-FcRn inhibitors can be used to
inhibit or block the AFP binding site on FcRn, which overlaps with
the albumin binding sites on FcRn.
[0124] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn comprises a T451I and/or
D536V polymorphism of wild-type AFP that decreases AFP-FcRn
binding.
[0125] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between Y521 and/or V522 of AFP and R42 of FcRn.
[0126] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between P492 of AFP and R69 of FcRn.
[0127] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between Q441 and/or V493 of AFP and E44 of FcRn.
[0128] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between H534 and/or E589 of AFP and N173 of FcRn.
[0129] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding between
the hydrophobic core of AFP and FcRn. In some such embodiments, an
inhibitor of AFP-FcRn inhibits binding and/or interactions between
L484, V493, V497, and/or F512 of AFP and V57, W59, and/or W61 of
FcRn.
[0130] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between T443 of AFP and E62 and/or W59 of FcRn.
[0131] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between D529 of AFP and S230 of FcRn.
[0132] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding between
S527 and/or D528 of AFP and E50 and/or 67Y of 32m complexed with
FcRn.
[0133] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding between
R604 of AFP and the carbonyl oxygen at E50 of .beta.2m complexed
with FcRn.
[0134] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between Q597 of AFP and E69 of .beta.2m complexed with
FcRn.
[0135] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between E106 of AFP and H161 of FcRn.
[0136] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between S135 of AFP and H161 of FcRn.
[0137] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between F531, F533, F552, and/or F575 of AFP and W53
of FcRn.
[0138] As used herein, the terms "AFP-FcRn inhibitor" and "alpha
fetoprotein and FcRn inhibitor," "inhibitor of AFP-FcRn," or
"inhibitor of AFP and FcRn interactions" refer to a molecule or
agent that significantly blocks, inhibits, reduces, or interferes
with the interaction between AFP and FcRn and their resultant
biological or functional activity in vitro, in situ, and/or in
vivo, including activity of downstream pathways mediated by AFP
binding to FcRn and signaling, such as, for example, transcytosis
of AFP, inhibition of T cell stimulation by immune complex-primed
dendritic cells, AFP-mediated inhibition of immune responses,
and/or increased serum half-life of AFP. Exemplary AFP-FcRn
inhibitors contemplated for use in the various aspects and
embodiments described herein include, but are not limited to,
antibodies or antigen-binding fragments thereof that specifically
bind to one or more amino acid residues or epitopes on AFP and/or
FcRn involved in the binding and/or interactions of AFP and FcRn,
and inhibit/reduce/block AFP and FcRn interactions and/or binding;
small molecule agents that target or specifically bind one or more
amino acid residues on AFP and/or FcRn involved in the binding
and/or interactions of AFP and FcRn, and inhibit/reduce/block AFP
and FcRn interactions and/or binding; RNA or DNA aptamers that bind
to AFP and/or FcRn and inhibit/reduce/block AFP and FcRn
interactions and/or binding; and/or AFP fragments or fusion
polypeptides thereof that block endogenous AFP interactions with
FcRn.
[0139] As used herein, an AFP-FcRn inhibitor has the ability to
reduce or decrease the interaction between AFP and FcRn and/or
their resultant biological or functional activity in vitro, in
situ, and/or in vivo by at least 5%, 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 95%, at least 98%, at least
99%, or more, relative to the interaction and/or activity in the
absence of the AFP-FcRn inhibitor.
[0140] "Decreased/decreasing interaction between AFP and FcRn,"
"reduced/reducing interaction between AFP and FcRn," "inhibits
binding," or "inhibited/inhibiting interaction between AFP and
FcRn" as used interchangeably herein, generally means either
reducing or inhibiting the interaction between or binding of AFP
and FcRn by at least 5%, at least 10%, at least 25%, at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, at least 98%, or more, compared to the interaction between AFP
and FcRn under the same conditions but without the presence of
AFP-FcRn inhibitors described herein.
[0141] In some embodiments of the compositions, methods, and uses
described herein, the AFP-FcRn inhibitor is an antibody or
antigen-binding fragment thereof. In some embodiments of the
aspects described herein, such AFP-FcRn inhibitors can be used to
inhibit or block the AFP binding site on FcRn, which overlaps with
the albumin binding sites on FcRn, as described herein. In some
embodiments, an antibody or antigen-binding fragment inhibitor of
AFP-FcRn binds to an epitope that comprises the AFP binding site on
FcRn.
[0142] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
Y521 and/or V522 of AFP and R42 of FcRn.
[0143] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
P492 of AFP and R69 of FcRn.
[0144] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
Q441 and/or V493 of AFP and E44 of FcRn.
[0145] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
H534 and/or E589 of AFP and N173 of FcRn.
[0146] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding between the hydrophobic core
of AFP and FcRn. In some such embodiments, the antibody or
antigen-binding fragment inhibitor of AFP-FcRn inhibits binding
and/or interactions between L484, V493, V497, and/or F512 of AFP
and V57, W59, and/or W61 of FcRn.
[0147] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
T443 of AFP and E62 and/or W59 of FcRn.
[0148] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
D529 of AFP and S230 of FcRn.
[0149] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding between S527 and/or D528 of
AFP and E50 and/or 67Y of .beta.2m complexed with FcRn.
[0150] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding between R604 of AFP and the
carbonyl oxygen at E50 of .beta.2m complexed with FcRn.
[0151] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
Q597 of AFP and E69 of .beta.2m complexed with FcRn.
[0152] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
E106 of AFP and H161 of FcRn.
[0153] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
S135 of AFP and H161 of FcRn.
[0154] In some embodiments of these aspects and all such aspects
described herein, the antibody or antigen-binding fragment
inhibitor of AFP-FcRn inhibits binding and/or interactions between
F531, F533, F552, and/or F575 of AFP and W53 of FcRn.
[0155] Antibodies or antigen-binding fragments thereof that are
specific for or that selectively bind AFP, FcRn, and/or AFP bound
to FcRn, suitable for use in the compositions and for practicing
the methods described herein are preferably monoclonal, and can
include, but are not limited to, human, humanized or chimeric
antibodies, comprising single chain antibodies, Fab fragments,
F(ab') fragments, fragments produced by a Fab expression library,
and/or binding fragments of any of the above. Antibodies also refer
to immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain antigen or
target binding sites or "antigen-binding fragments." The
immunoglobulin molecules described herein 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 of immunoglobulin molecule, as is
understood by one of skill in the art.
[0156] Examples of antibody fragments encompassed by the terms
antibody fragment or antigen-binding fragment as described herein
include: (i) the Fab fragment, having V.sub.L, C.sub.L, V.sub.H and
C.sub.H1 domains; (ii) the Fab' fragment, which is a Fab fragment
having one or more cysteine residues at the C-terminus of the
C.sub.H1 domain; (iii) the Fd fragment having V.sub.H and C.sub.H1
domains; (iv) the Fd' fragment having V.sub.H and C.sub.H1 domains
and one or more cysteine residues at the C-terminus of the CH1
domain; (v) the Fv fragment having the V.sub.L and V.sub.H domains
of a single arm of an antibody; (vi) a dAb fragment (Ward et al.,
Nature 341, 544-546 (1989)) which consists of a V.sub.H domain or a
V.sub.L domain; (vii) isolated CDR regions; (viii) F(ab').sub.2
fragments, a bivalent fragment including two Fab' fragments linked
by a disulphide bridge at the hinge region; (ix) single chain
antibody molecules (e.g. single chain Fv; scFv) (Bird et al.,
Science 242:423-426 (1988); and Huston et al., PNAS (USA)
85:5879-5883 (1988)); (x) "diabodies" with two antigen binding
sites, comprising a heavy chain variable domain (V.sub.H) connected
to a light chain variable domain (V.sub.L) in the same polypeptide
chain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al.,
Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) "linear
antibodies" comprising a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995);
and U.S. Pat. No. 5,641,870); and modified versions of any of the
foregoing (e.g., modified by the covalent attachment of
polyalkylene glycol (e.g., polyethylene glycol, polypropylene
glycol, polybutylene glycol) or other suitable polymer).
[0157] With respect to a target or antigen, the term "ligand
interaction site" on the target or antigen means a site, epitope,
antigenic determinant, part, domain or stretch of amino acid
residues on the target or antigen that is a site for binding to a
ligand, receptor or other binding partner, a catalytic site, a
cleavage site, a site for allosteric interaction, a site involved
in multimerisation (such as homomerization or heterodimerization)
of the target or antigen; or any other site, epitope, antigenic
determinant, part, domain or stretch of amino acid residues on the
target or antigen that is involved in a biological action or
mechanism of the target or antigen, i.e., AFP, FcRn, or AFP bound
to FcRn. For example, in some embodiments, a ligand interaction
site on FcRn can be any site to which IgG binds or interacts, or
any site to which albumin binds or interacts, or any site to which
AFP binds or interacts or which when interacting with AFP affects
the conformation of the binding sites for albumin and/or IgG within
the FcRn/.beta.2-microglobulin heterodimeric complex. More
generally, a "ligand interaction site" can be any site, epitope,
antigenic determinant, part, domain or stretch of amino acid
residues on a target or antigen to which a binding site of an
AFP-FcRn inhibitor described herein can bind such that the
interaction or binding between AFP and FcRn (and/or any pathway,
interaction, signalling, biological mechanism or biological effect
mediated by AFP binding to FcRn is involved) is modulated.
[0158] In the context of an antibody or antigen-binding fragment
thereof, the term "specificity" or "specific for" refers to the
number of different types of antigens or antigenic determinants to
which a particular antibody or antigen-binding fragment thereof can
bind. The specificity of an antibody or antigen-binding fragment or
portion thereof can be determined based on affinity and/or avidity.
The affinity, represented by the equilibrium constant for the
dissociation (K.sub.D) of an antigen with an antigen-binding
protein, is a measure for the binding strength between an antigenic
determinant and an antigen-binding site on the antigen-binding
protein: the lesser the value of the K.sub.D, the stronger the
binding strength between an antigenic determinant and the
antigen-binding molecule. Alternatively, the affinity can also be
expressed as the affinity constant (K.sub.A), which is 1/K.sub.D).
As will be clear to the skilled person, affinity can be determined
in a manner known per se, depending on the specific antigen of
interest. Accordingly, an antibody or antigen-binding fragment
thereof as defined herein is said to be "specific for" a first
target or antigen compared to a second target or antigen when it
binds to the first antigen with an affinity (as described above,
and suitably expressed, for example as a K.sub.D value) that is at
least 10 times, such as at least 100 times, and preferably at least
1000 times, and up to 10.000 times or more better than the affinity
with which said amino acid sequence or polypeptide binds to another
target or polypeptide. Preferably, when an antibody or
antigen-binding fragment thereof is "specific for" a target or
antigen, compared to another target or antigen, it is directed
against said target or antigen, but not directed against another
target or antigen.
[0159] However, as understood by one of ordinary skill in the art,
in some embodiments, where a binding site on a target is shared or
partially shared by multiple, different ligands, an antibody or
antigen binding fragment thereof can specifically bind to a target,
such as FcRn, and have the functional effect of
inhibiting/preventing binding of multiple, different ligands, such
as AFP, albumin, and/or IgG. For example, as demonstrated herein,
the ADM31 antibody inhibits AFP binding to FcRn, as well as binding
of albumin to FcRn (Sand, K. M., et B. Dalhus, G. J. Christianson,
M. Bern, S. Foss, J. Cameron, D. Sleep, M. Bjoras, D. C. Roopenian,
I. Sandlie and J. T. Andersen (2014). "Dissection of the neonatal
Fc receptor (FcRn)-albumin interface using mutagenesis and
anti-FcRn albumin-blocking antibodies." J Biol Chem 289(24):
17228-17239).
[0160] Avidity is the measure of the strength of binding between an
antigen-binding molecule and the pertinent antigen. Avidity is
related to both the affinity between an antigenic determinant and
its antigen binding site on the antigen-binding molecule, and the
number of pertinent binding sites present on the antigen-binding
molecule. Typically, antigen-binding proteins will bind to their
cognate or specific antigen with a dissociation constant (K.sub.D
of 10.sup.-5 to 10.sup.-12moles/liter or less, and preferably
10.sup.-7 to 10.sup.-12 moles/liter or less and more preferably
10.sup.-8 to 10.sup.-12 moles/liter (i.e. with an association
constant (K.sub.A) of 10.sup.5 to 10.sup.12 liter/moles or more,
and preferably 10.sup.7 to 10.sup.12 liter/moles or more and more
preferably 10.sup.8 to 10.sup.12 liter/moles). Any K.sub.D value
greater than 10.sup.-4 mol/liter (or any K.sub.A value lower than
10.sup.4 M.sup.-1) is generally considered to indicate non-specific
binding. The K.sub.D for biological interactions which are
considered meaningful (e.g., specific) are typically in the range
of 10.sup.-10 M (0.1 nM) to 10.sup.-5 M (10000 nM). The stronger an
interaction is, the lower is its K.sub.D. Preferably, a binding
site on a AFP-FcRn inhibitor antibody or antigen-binding fragment
thereof described herein will bind to AFP and/or FcRn with an
affinity less than 500 nM, preferably less than 200 nM, more
preferably less than 10 nM, such as less than 500 pM. Specific
binding of an antigen-binding protein to an antigen or antigenic
determinant can be determined in any suitable manner known per se,
including, for example, Scatchard analysis and/or competitive
binding assays, such as radioimmunoassays (RIA), enzyme
immunoassays (EIA) and sandwich competition assays, and the
different variants thereof known per se in the art; as well as
other techniques as mentioned herein.
[0161] In some embodiments of the compositions, methods, and uses
described herein, the AFP-FcRn inhibitor is a monoclonal
antibody.
[0162] The term "monoclonal antibody," as used herein, refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each antibody in a
monoclonal preparation is directed against the same, single
determinant on the antigen. It is to be understood that the term
"monoclonal antibody" refers to an antibody that is derived from a
single clone, including any eukaryotic, prokaryotic, or phage
clone, and not the method by which it is produced. The term
"monoclonal antibody" as used herein is not limited to antibodies
produced through hybridoma technology, and the modifier
"monoclonal" is not to be construed as requiring production of the
antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the invention can be made
by the hybridoma method first described by Kohler et al., Nature
256:495 (1975), or later adaptations thereof, or can be made by
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The
"monoclonal antibodies" can also be isolated from phage antibody
libraries using the techniques described in Clackson et al., Nature
352:624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597
(1991), for example.
[0163] In some embodiments of the compositions, methods, and uses
described herein, the AFP-FcRn inhibitor is a chimeric antibody
derivative of an antibody or antigen-binding fragment thereof that
binds AFP, FcRn, and/or AFP bound to FcRn.
[0164] As used herein, the term"chimeric antibody" refers to an
antibody molecule in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA 81:6851-6855 (1984)). Chimeric antibody molecules can
include, for example, one or more antigen binding domains from an
antibody of a mouse, rat, or other species, with human constant
regions. A variety of approaches for making chimeric antibodies
have been described and can be used to make chimeric antibodies
containing the immunoglobulin variable region which recognizes the
desired antigen, e.g., AFP and/or FcRn. See, for example, Takeda et
al., 1985, Nature 314:452; Cabilly et al., U.S. Pat. No. 4,816,567;
Boss et al.; Tanaguchi et al., European Patent Publication
EP171496; European Patent Publication 0173494, United Kingdom
patent GB 2177096B).
[0165] In some embodiments of the compositions, methods, and uses
described herein, the AFP-FcRn inhibitor is a humanized antibody
derivative of an antagonist antibody or antigen-binding fragment
thereof that binds AFP, FcRn, and/or AFP bound to FcRn.
[0166] Humanized forms of non-human (e.g., murine) antibodies are
chimeric antibodies which contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies can comprise residues which are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0167] In some embodiments of the compositions, methods, and uses
comprising any of the AFP-FcRn inhibitor antibodies or
antigen-binding fragments thereof described herein, the AFP-FcRn
inhibitor antibody or antigen-binding fragment is an antibody
derivative. For example, but not by way of limitation, antibody
derivatives include antibodies that have been modified, e.g., by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. Any
of numerous chemical modifications can be carried out by known
techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, etc. Additionally, the
derivative can contain one or more non-classical amino acids, or
alternative scaffolds such as centyrins, DARPINS, or fynomers
engineered to bind FcRn and inhibit AFP.
[0168] The AFP-FcRn inhibitor antibodies and antigen-binding
fragments thereof described herein can be generated by any suitable
method known in the art. Monoclonal and polyclonal antibodies
against, for example, FcRn, are known in the art. To the extent
necessary, e.g., to generate antibodies with particular
characteristics or epitope specificity, the skilled artisan can
generate new monoclonal or polyclonal AFP-FcRn inhibitor antibodies
as briefly discussed herein or as known in the art.
[0169] Polyclonal antibodies can be produced by various procedures
well known in the art. For example, AFP, FcRn, or fragments thereof
comprising one or more of the AFP and/or FcRn interaction sites,
can be administered to various host animals including, but not
limited to, rabbits, mice, rats, etc. to induce the production of
sera containing polyclonal antibodies specific for the protein.
Polyclonal antibodies are preferably raised in animals by multiple
subcutaneous (sc) or intraperitoneal (ip) injections of the
relevant antigen and an adjuvant. It can be useful to conjugate the
antigen to a protein that is immunogenic in the species to be
immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, or soy-bean trypsin inhibitor using a bifunctional
or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxy-succinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups. Various other adjuvants can
be used to increase the immunological response, depending on the
host species, and include but are not limited to, Freund's
(complete and incomplete), mineral gels such as aluminum hydroxide,
surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum. Suitable
adjuvants are also well known to one of skill in the art.
[0170] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. Various methods for making monoclonal antibodies described
herein are available in the art. For example, the monoclonal
antibodies can be made using the hybridoma method first described
by Kohler et al., Nature, 256:495 (1975), or any later developments
thereof, or by recombinant DNA methods (U.S. Pat. No. 4,816,567).
For example, monoclonal antibodies can be produced using hybridoma
techniques including those known in the art and taught, for
example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold
Spring Harbor Laboratory Press, 2nd ed., 1988); Hammer-ling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). Methods for producing and screening for
specific antibodies using hybridoma technology are routine and well
known in the art. In another example, antibodies useful in the
methods and compositions described herein can also be generated
using various phage display methods known in the art, such as
isolation from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et
al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks et al.,
Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0171] In some embodiments of the compositions, methods, and uses
described herein, completely human antibodies are used as AFP-FcRn
inhibitors, which are particularly desirable for the therapeutic
treatment of human patients.
[0172] Human antibodies can be made by a variety of methods known
in the art, including phage display methods described above using
antibody libraries derived from human immunoglobulin sequences. See
also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications
WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO
96/33735, and WO 91/10741, the contents of which are herein
incorporated by reference in their entireties.
[0173] Human antibodies can also be produced using transgenic mice
which express human immunoglobulin genes, and upon immunization are
capable of producing a full repertoire of human antibodies in the
absence of endogenous immunoglobulin production. For an overview of
this technology for producing human antibodies, see, Lonberg and
Huszar, 1995, Int. Rev. Immunol. 13:65-93. For a detailed
discussion of this technology for producing human antibodies and
human monoclonal antibodies and protocols for producing such
antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047;
WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat.
Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;
5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, the
contents of which are herein incorporated by reference in their
entireties. In addition, companies such as Abgenix, Inc. (Freemont,
Calif.) and Medarex (Princeton, N.J.) can be engaged to provide
human antibodies directed against a selected antigen using
technology similar to that described above. See also, e.g.,
Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);
Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al.,
Year in Immuno., 7:33 (1993); and Duchosal et al. Nature 355:258
(1992), the contents of which are herein incorporated by reference
in their entireties. Alternatively, phage display technology
(McCafferty et al., Nature 348:552-553 (1990)) can be used to
produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. Human antibodies can also be generated by in
vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275, the contents of which are herein incorporated by
reference in their entireties). Completely human antibodies which
recognize a selected epitope can be generated using a technique
referred to as "guided selection." In this approach a selected
non-human monoclonal antibody, e.g., a mouse antibody, is used to
guide the selection of a completely human antibody recognizing the
same epitope (Jespers et al., 1994, Bio/technology 12:899-903).
[0174] "An "Fv" fragment is an antibody fragment which contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy and one light chain variable domain in
tight association, which can be covalent in nature, for example in
scFv. It is in this configuration that the three CDRs of each
variable domain interact to define an antigen binding site on the
surface of the V.sub.H-V.sub.L dimer. Collectively, the six CDRs or
a subset thereof confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific for an antigen) has the ability
to recognize and bind antigen, although usually at a lower affinity
than the entire binding site.
[0175] As used herein, "antibody variable domain" refers to the
portions of the light and heavy chains of antibody molecules that
include amino acid sequences of Complementarity Determining Regions
(CDRs; ie., CDR1, CDR2, and CDR3), and Framework Regions (FRs).
V.sub.H refers to the variable domain of the heavy chain. V.sub.L
refers to the variable domain of the light chain. According to the
methods used in this invention, the amino acid positions assigned
to CDRs and FRs may be defined according to Kabat (Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md., 1987 and 1991)). Amino acid numbering of antibodies
or antigen binding fragments is also according to that of
Kabat.
[0176] As used herein, the term "Complementarity Determining
Regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino
acid residues of an antibody variable domain the presence of which
are necessary for antigen binding. Each variable domain typically
has three CDR regions identified as CDR1, CDR2 and CDR3. Each
complementarity determining region may comprise amino acid residues
from a "complementarity determining region" as defined by Kabat
(i.e. about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the
light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (i.e. about residues 26-32
(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain
and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). In some instances, a complementarity determining region
can include amino acids from both a CDR region defined according to
Kabat and a hypervariable loop. For example, the CDRH1 of the human
heavy chain of antibody 4D5 includes amino acids 26 to 35.
[0177] "Framework regions" (hereinafter FR) are those variable
domain residues other than the CDR residues. Each variable domain
typically has four FRs identified as FR1, FR2, FR3 and FR4. If the
CDRs are defined according to Kabat, the light chain FR residues
are positioned at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88
(LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are
positioned about at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94
(HCFR3), and 103-113 (HCFR4) in the heavy chain residues. If the
CDRs comprise amino acid residues from hypervariable loops, the
light chain FR residues are positioned about at residues 1-25
(LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the
light chain and the heavy chain FR residues are positioned about at
residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113
(HCFR4) in the heavy chain residues. In some instances, when the
CDR comprises amino acids from both a CDR as defined by Kabat and
those of a hypervariable loop, the FR residues will be adjusted
accordingly. For example, when CDRH1 includes amino acids H26-H35,
the heavy chain FR1 residues are at positions 1-25 and the FR2
residues are at positions 36-49.
[0178] As used herein, a "chimeric antibody" refers to a molecule
in which different portions of the antibody are derived from
different animal species, such as antibodies having a variable
region derived from a murine monoclonal antibody and a human
immunoglobulin constant region. Methods for producing chimeric
antibodies are known in the art. See e.g., Morrison, Science, 1985,
229:1202; Oi et al, 1986, Bio-Techniques 4:214; Gillies et al.,
1989, J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715;
4,816,567; and 4,816,397, the contents of which are herein
incorporated by reference in their entireties.
[0179] "Humanized antibodies," as the term is used herein, refer to
antibody molecules from a non-human species, where the antibodies
that bind the desired antigen, i.e., AFP, FcRn, and/or AFP bound to
FcRn, have one or more CDRs from the non-human species, and
framework and constant regions from a human immunoglobulin
molecule. Often, framework residues in the human framework regions
will be substituted with the corresponding residue from the CDR
donor antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., 1988, Nature 332:323.
Antibodies can be humanized using a variety of techniques known in
the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and
5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology, 1991, 28(4/5):489-498; Studnicka et
al., 1994, Protein Engineering 7(6):805-814; Roguska. et al, 1994,
PNAS 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332),
the contents of which are herein incorporated by reference in their
entireties. Accordingly, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), the
contents of which are herein incorporated by reference in their
entireties, by substituting rodent CDRs or CDR sequences for the
corresponding sequences of a human antibody. Accordingly, such
"humanized" antibodies are chimeric antibodies (U.S. Pat. No.
4,816,567, the contents of which are herein incorporated by
reference in its entirety) wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0180] The "Fab" fragment contains a variable and constant domain
of the light chain and a variable domain and the first constant
domain (C.sub.H1) of the heavy chain. F(ab').sub.2 antibody
fragments comprise a pair of Fab fragments which are generally
covalently linked near their carboxy termini by hinge cysteines
between them. Other chemical couplings of antibody fragments are
also known in the art.
[0181] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally the Fv polypeptide
further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains, which enables the scFv to form the desired
structure for antigen binding For a review of scFv, see Pluckthun
in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg
and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
[0182] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H and
V.sub.L). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in,
for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0183] The expression "linear antibodies" refers to the antibodies
described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995).
Briefly, these antibodies comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0184] Various techniques have been developed for the production of
antibody or antigen-binding fragments. The antibodies described
herein can be fragmented using conventional techniques and the
fragments screened for utility in the same manner as described
above for the whole antibodies. Traditionally, these fragments were
derived via proteolytic digestion of intact antibodies (see, e.g.,
Morimoto et al., Journal of Biochemical and Biophysical Methods
24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). For
example, Fab and F(ab')2 fragments of the bispecific and
multispecific antibodies described herein can be produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes
such as papain (to produce Fab fragments) or pepsin (to produce
F(ab') 2 fragments). F(ab') fragments contain the variable region,
the light chain constant region and the C.sub.H1 domain of the
heavy chain. However, these fragments can now be produced directly
by recombinant host cells. For example, the antibody fragments can
be isolated from the antibody phage libraries discussed above.
Alternatively, Fab'-SH fragments can be directly recovered from E.
coli and chemically coupled to form F(ab')2 fragments (Carter et
al., Bio/Technology 10:163-167 (1992)). According to another
approach, F(ab')2 fragments can be isolated directly from
recombinant host cell culture. Other techniques for the production
of antibody fragments will be apparent to the skilled practitioner.
In other embodiments, the antibody of choice is a single chain Fv
fragment (scFv). See WO 93/16185.
[0185] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in
Enzymology 203:46-88; Shu et al., 1993, PNAS 90:7995-7999; and
Skerra et al., 1988, Science 240:1038-1040. For some uses,
including the in vivo use of antibodies in humans as described
herein and in vitro proliferation or cytotoxicity assays, it is
preferable to use chimeric, humanized, or human antibodies.
[0186] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result an improvement
in the affinity of the antibody for antigen, compared to a parent
antibody which does not possess those alteration(s). Preferred
affinity matured antibodies will have nanomolar or even picomolar
affinities for the target antigen. Affinity matured antibodies are
produced by procedures known in the art. Marks et al.
Bio/Technology 10:779-783 (1992) describes affinity maturation by
V.sub.H and V.sub.L domain shuffling. Random mutagenesis of CDR
and/or framework residues is described by: Barbas et al. Proc Nat.
Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol.
Biol. 226:889-896 (1992).
[0187] As used herein "complementary" refers to when two
immunoglobulin domains belong to families of structures which form
cognate pairs or groups or are derived from such families and
retain this feature. For example, a V.sub.H domain and a V.sub.L
domain of a natural antibody are complementary; two V.sub.H domains
are not complementary, and two V.sub.L domains are not
complementary. Complementary domains can be found in other members
of the immunoglobulin superfamily, such as the V.sub..alpha. and
V.sub..beta. (or .gamma. and .delta.) domains of the T-cell
receptor. Domains which are artificial, such as domains based on
protein scaffolds which do not bind epitopes unless engineered to
do so, are non-complementary. Likewise, two domains based on, for
example, an immunoglobulin domain and a fibronectin domain are not
complementary.
[0188] In some embodiments of the compositions, methods, and uses
described herein, the AFP-FcRn inhibitor is a small molecule
inhibitor, agent, or compound. In some embodiments of the aspects
described herein, such AFP-FcRn small molecule inhibitors or small
molecule inhibitors of AFP-FcRn can be used to inhibit or block the
AFP binding site on FcRn, which overlaps with the albumin and IgG
binding activities on FcRn, as described herein.
[0189] Such small molecule inhibitors include, but are not limited
to, small peptides or peptide-like molecules, soluble peptides, and
synthetic non-peptidyl organic or inorganic compounds. A small
molecule inhibitor or antagonist can have a molecular weight of any
of about 100 to about 20,000 daltons (Da), about 500 to about
15,000 Da, about 1000 to about 10,000 Da.
[0190] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between Y521 and/or V522 of AFP and R42
of FcRn.
[0191] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between P492 of AFP and R69 of
FcRn.
[0192] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between Q441 and/or V493 of AFP and E44
of FcRn.
[0193] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between H534 and/or E589 of AFP and
N173 of FcRn.
[0194] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding between the hydrophobic core of AFP and FcRn. In some such
embodiments, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between L484, V493, V497, and/or F512
of AFP and V57, W59, and/or W61 of FcRn.
[0195] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between T443 of AFP and E62 and/or W59
of FcRn.
[0196] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between D529 of AFP and S230 of
FcRn.
[0197] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding between S527 and/or D528 of AFP and E50 and/or 67Y of
.beta.2m complexed with FcRn.
[0198] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding between R604 of AFP and the carbonyl oxygen at E50 of
.beta.2m complexed with FcRn.
[0199] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between Q597 of AFP and E69 of .beta.2m
complexed with FcRn.
[0200] In some embodiments of these aspects and all such aspects
described herein the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between E106 of AFP and H161 of
FcRn.
[0201] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between S135 of AFP and H161 of
FcRn.
[0202] In some embodiments of these aspects and all such aspects
described herein, the small molecule inhibitor of AFP-FcRn inhibits
binding and/or interactions between F531, F533, F552, and/or F575
of AFP and W53 of FcRn.
[0203] In some embodiments of the compositions, methods, and uses
described herein, an AFP-FcRn inhibitor is an RNA or DNA aptamer
that binds or physically interacts with AFP, and blocks
interactions between AFP and FcRn. In some embodiments of the
compositions, methods, and uses described herein, an AFP-FcRn
inhibitor is an RNA or DNA aptamer that binds or physically
interacts with FcRn, and blocks interactions between AFP and FcRn.
In some embodiments of the cocompositions, methods, and uses
described herein, the aptamer comprises at least one RNA or DNA
aptamer that binds to the FcRn large subunit p51 heavy chain
precursor of FcRn. In some embodiments of the compositions,
methods, and uses described herein, the aptamer comprises at least
one RNA or DNA aptamer that binds to the .beta.2m subunit of
FcRn.
[0204] In some embodiments of these aspects and all such aspects
described herein, the the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between Y521 and/or V522 of
AFP and R42 of FcRn.
[0205] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between P492 of AFP and R69 of
FcRn.
[0206] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between Q441 and/or V493 of
AFP and E44 of FcRn.
[0207] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between H534 and/or E589 of
AFP and N173 of FcRn.
[0208] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding between the hydrophobic core of AFP and FcRn. In
some such embodiments, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between L484, V493, V497,
and/or F512 of AFP and V57, W59, and/or W61 of FcRn.
[0209] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between T443 of AFP and E62
and/or W59 of FcRn.
[0210] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between D529 of AFP and S230
of FcRn.
[0211] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding between S527 and/or D528 of AFP and E50 and/or 67Y
of .beta.2m complexed with FcRn.
[0212] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding between R604 of AFP and the carbonyl oxygen at E50
of .beta.2m complexed with FcRn.
[0213] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between Q597 of AFP and E69 of
.beta.2m complexed with FcRn.
[0214] In some embodiments of these aspects and all such aspects
described herein the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between E106 of AFP and H161
of FcRn.
[0215] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between S135 of AFP and H161
of FcRn.
[0216] In some embodiments of these aspects and all such aspects
described herein, the RNA or DNA aptamer inhibitor of AFP-FcRn
inhibits binding and/or interactions between F531, F533, F552,
and/or F575 of AFP and W53 of FcRn.
[0217] AFP-FcRn inhibitors for use in the compositions, methods,
and uses described herein can be identified or characterized using
methods known in the art, such as protein-protein binding assays,
biochemical screening assays, immunoassays, and cell-based assays,
which are well known in the art, including, but not limited to,
those described herein in the Examples and Figures.
[0218] For example, to identify a molecule that inhibits
interaction between AFP and FcRn, transcytosis assays can be used,
as described herein. For example, cells, such as MDCK II cells,
that co-express human FcRn and .beta.2m, can be tested for
basolateral to apical transcytosis of AFP in the presence of a
putative AFP-FcRn inhibitor (i.e., a test agent) and a control
agent. If AFP transcytosis is inhibited by the presence of the test
agent, relative to the transcytosis in the presence of the control
agent, the test agent can be deemed a candidate inhibitor that
inhibits binding between AFP and FcRn. Additional assays, such as,
for example, Biacore assays, can be used to further determine
whether and how a candidate agent inhibits binding between FcRn and
AFP.
[0219] Also provided herein, in some aspects, are compositions,
such as pharmaceutical compositions, comprising potentiators of
AFP-FcRn interactions. Such potentiators are used to
enhance/increase/potentiate the interaction between AFP and FcRn
and/or increase transcytosis of human AFP, and/or increase serum
half-life of AFP, thereby increasing immunosuppressive activities
of AFP in the treatment of disorders and conditions in need of
enhanced AFP levels, including autoimmune disorders, transplant
patients, and high-risk pregnancies, for example.
[0220] As used herein, the terms "AFP-FcRn potentiator,"
"potentiator of AFP-FcRn interaction," AFP-FcRn activator agent,"
and "AFP-FcRn agonist agent" refer to a molecule or agent that
mimics or up-regulates (e.g., increases, potentiates or
supplements) the biological activity of AFP binding to FcRn in
vitro, in situ, and/or in vivo, including downstream pathways
mediated by AFP binding to FcRn and signaling, such as, for
example, transcytosis of AFP, inhibition of T cell stimulation by
immune complex-primed dendritic cells, AFP-mediated inhibition of
immune responses, and/or increased serum half-life of AFP. An
AFP-FcRn potentiator or agonist can be, in some embodiments, an AFP
protein fragment or derivative thereof having at least one
bioactivity of the wild-type AFP. An AFP-FcRn potentiator can also
be a compound which increases the interaction of AFP with FcRn, for
example. Exemplary AFP-FcRn potentiators or agonists contemplated
for use in the various aspects and embodiments described herein
include, but are not limited to, antibodies or antigen-binding
fragments thereof that specifically bind to AFP bound to FcRn and
enhance the interaction and/or block FcRn binding to albumin and/or
IgG but allow binding of AFP to FcRn; RNA or DNA aptamers that bind
to FcRn and mimic AFP binding to FcRn; AFP structural analogs or
AFP functional fragments, derivatives, or fusion polypeptides
thereof; and small molecule agents that target or bind to FcRn and
act as functional mimics of AFP binding to FcRn.
[0221] As used herein, an AFP-FcRn potentiator has the ability to
increase or enhance the activity of AFP binding to FcRn or
mimic/replicate the downstream functional consequences mediated by
AFP binding to FcRn by at least 5%, 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 95%, at least 98%, at least
99%, at least 100%, at least 1.5-fold, at least 2-fold, at least
5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at
least 100-fold, at least 1000-fold, or more relative to the
activity or expression level in the absence of the AFP-FcRn
potentiator.
[0222] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator comprises a G109R, R487S,
and/or S445L polymorphism of wild-type AFP that increases AFP-FcRn
binding.
[0223] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between Y521 and/or V522 of AFP and R42 of FcRn.
[0224] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between P492 of AFP and R69 of FcRn.
[0225] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between Q441 and/or V493 of AFP and E44 of FcRn.
[0226] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between H534 and/or E589 of AFP and N173 of FcRn.
[0227] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding between
the hydrophobic core of AFP and FcRn. In some such embodiments, an
AFP-FcRn potentiator enhances binding and/or interactions between
L484, V493, V497, and/or F512 of AFP and V57, W59, and/or W61 of
FcRn.
[0228] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between T443 of AFP and E62 and/or W59 of FcRn.
[0229] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between D529 of AFP and S230 of FcRn.
[0230] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding between
S527 and/or D528 of AFP and E50 and/or 67Y of .beta.2m complexed
with FcRn.
[0231] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding between
R604 of AFP and the carbonyl oxygen at E50 of .beta.2m complexed
with FcRn.
[0232] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between Q597 of AFP and E69 of .beta.2m complexed with
FcRn.
[0233] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between E106 of AFP and H161 of FcRn.
[0234] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between S135 of AFP and H161 of FcRn.
[0235] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between F531, F533, F552, and/or F575 of AFP and W53
of FcRn.
[0236] In some embodiments of the compositions, methods, and uses
described herein, the AFP-FcRn potentiator is an antibody or
antigen-binding fragment thereof that selectively binds or
physically interacts with AFP bound to FcRn and enhances the
interaction of AFP and FcRn, and/or blocks FcRn binding to albumin
and/or IgG but allows binding of AFP to FcRn, thereby resulting in
increased transcytosis of AFP, increased inhibition of T cell
stimulation by immune complex-primed dendritic cells, increased
AFP-mediated inhibition of immune responses, and/or increased serum
half-life of AFP. Exemplary assays to measure increases or
up-regulation of downstream pathway activities are known to those
of ordinary skill in the art and are provided herein in the
Examples.
[0237] In some embodiments of the compositions, methods, and uses
described herein, an AFP-FcRn potentiator is a monoclonal antibody.
In some embodiments of the compositions, methods, and uses
described herein, an AFP-FcRn potentiator is an antibody fragment
or antigen-binding fragment, as the term is described elsewhere
herein.
[0238] In some embodiments of the compositions, methods, and uses
described herein, an AFP-FcRn potentiator is a chimeric antibody
derivative of the AFP-FcRn potentiator antibodies and
antigen-binding fragments thereof, as the term is described
elsewhere herein.
[0239] In some embodiments of the compositions, methods, and uses
described herein, an AFP-FcRn potentiator is a humanized antibody
derivative, as the term is described elsewhere herein.
[0240] In some embodiments, the AFP-FcRn potentiator antibodies and
antigen-binding fragments thereof described herein include
derivatives that are modified, i.e., by the covalent attachment of
any type of molecule to the antibody, provided that covalent
attachment does not prevent the antibody from binding to the target
antigen.
[0241] In some embodiments, the AFP-FcRn potentiator antibodies and
antigen-binding fragments thereof described herein are completely
human antibodies or antigen-binding fragments thereof, which are
particularly desirable for the therapeutic treatment of human
patients. Human antibodies can be made by a variety of methods
known in the art, and as described in more detail elsewhere
herein.
[0242] The AFP-FcRn potentiator antibodies and antigen-binding
fragments thereof described herein, as well as any of the other
antibodies or antigen-binding fragments thereof described herein in
various aspects and embodiments, can be generated by any suitable
method known in the art.
[0243] In some embodiments of the compositions, methods, and uses
described herein, the AFP-FcRn potentiator is a small molecule
potentiator, activator, or agonist, including, but not limited to,
small peptides or peptide-like molecules, soluble peptides, and
synthetic non-peptidyl organic or inorganic compounds. A small
molecule activator or agonist can have a molecular weight of any of
about 100 to about 20,000 daltons (Da), about 500 to about 15,000
Da, about 1000 to about 10,000 Da. In some embodiments of the
compositions, methods, and uses described herein, an AFP-FcRn
potentiator comprises a small molecule that binds FcRn and mimics
AFP binding. Exemplary sites of small molecule binding include, but
are not limited to, the portion of FcRn that binds specifically to
AFP, or to the portions of FcRn adjacent to the AFP binding
site.
[0244] In some embodiments of the compositions, methods, and uses
described herein, an AFP-FcRn potentiator is an RNA or DNA aptamer
that binds or physically interacts with AFP or FcRn, and enhances
or promotes interactions between AFP and FcRn.
[0245] In some embodiments of the compositions, methods, and uses
described herein, an AFP-FcRn potentiator comprises an AFP
structural analog, functional fragment, or derivative, such as an
AFP variant engineered to possess increased binding to the
FcRn/.beta.2-microglobulin complex. The term "AFP structural
analog," "AFP functional fragment," or "AFP derivative" as used
herein, refer to compounds, such as peptides, that can bind to FcRn
under physiological conditions in vitro or in vivo, wherein the
binding at least partially mimics or increases an FcRn mediated
biological activity. Suitable AFP structural analogs, functional
fragments, or derivatives can be designed and synthesized through
molecular modeling of AFP binding to FcRn, for example.
[0246] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises Y521 and/or
V522 of AFP and can interact with R42 of FcRn.
[0247] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises P492 of AFP
and can interact with R69 of FcRn.
[0248] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises Q441 and/or
V493 of AFP and can interact with E44 of FcRn.
[0249] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises H534 and/or
E589 of AFP and can interact with N173 of FcRn.
[0250] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises L484, V493,
V497, and/or F512 of AFP and can interact with V57, W59, and/or W61
of FcRn.
[0251] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises T443 of AFP
and can interact with E62 and/or W59 of FcRn.
[0252] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises D529 of AFP
and can interact with S230 of FcRn.
[0253] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises S527 and/or
D528 of AFP and can interact with E50 and/or 67Y of .beta.2m
complexed with FcRn.
[0254] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises R604 of AFP
and can interact with the carbonyl oxygen at E50 of .beta.2m
complexed with FcRn.
[0255] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises Q597 of AFP
and can interact with E69 of .beta.2m complexed with FcRn.
[0256] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises E106 of AFP
and can interact with H161 of FcRn.
[0257] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises S135 of AFP
and can interact with H161 of FcRn.
[0258] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises F531, F533,
F552, and/or F575 of AFP and can interact with W53 of FcRn.
[0259] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises
TABLE-US-00001 (SEQ ID NO: 4)
MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIF
FAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCH
EKEILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEA
YEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVE
CFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITVTKLSQKFTKV
NFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKIT
ECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIF
LASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEE
LQKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAI
TRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQC
CTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQ
EFLINLVKQKPQITEEQLEAVIADF or AFP (1-575).
[0260] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment comprises
TABLE-US-00002 (SEQ ID NO: 5)
LCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIF
HKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADF or AFP (484-575).
[0261] The term "AFP functional fragment," as used herein, refers
to a fragment of AFP that specifically binds to FcRn and can be
transcytosed across a cell membrane, and can, in some embodiments,
increase serum half-life of a protein to which it is fused or
conjugated. Accordingly, the term "functional" when used in
conjunction with a fragment, "derivative" or "variant" refers to a
protein molecule that possesses a desired biological activity that
is substantially similar to a biological activity of the entity or
molecule of which it is a fragment, derivative or variant. By
"substantially similar" in this context is meant that the
biological activity, e.g., specifically bind to FcRn and be
transcytosed across a cell membrane, and can, in some embodiments,
increase serum half-life of a protein to which it is fused or
conjugated, is at least 50% as active as a reference, e.g., a
corresponding wild-type or endogenous AFP, and preferably at least
60% as active, 70% as active, 80% as active, 90% as active, 95% as
active, 100% as active or even higher (i.e., the variant or
derivative has greater activity than the wild-type), e.g., 110% as
active, 120% as active, or more. Assays to measure the biological
activity of an AFP functional fragment are known in the art, and
non-limiting examples are provided herein in the Examples.
[0262] In some embodiments of these aspects and all such aspects
described herein, an AFP-functional fragment differs from
endogenous AFP by one or more amino acid or nucleic acid deletions,
additions, substitutions or side-chain modifications, yet retains
one or more functions or biological activities of the naturally
occurring molecule. Amino acid substitutions include alterations in
which an amino acid is replaced with a different
naturally-occurring or a non-conventional amino acid residue. Such
substitutions may be classified as "conservative," in which case an
amino acid residue contained in a polypeptide is replaced with
another naturally occurring amino acid of similar character either
in relation to polarity, side chain functionality or size.
Substitutions encompassed by variants as described herein may also
be "non conservative," in which an amino acid residue which is
present in a peptide is substituted with an amino acid having
different properties (e.g., substituting a charged or hydrophobic
amino acid with alanine), or alternatively, in which a
naturally-occurring amino acid is substituted with a
non-conventional amino acid. Also encompassed within the term
"variant," when used with reference to a polynucleotide or
polypeptide, are variations in primary, secondary, or tertiary
structure, as compared to a reference polynucleotide or
polypeptide, respectively (e.g., as compared to a wild-type
polynucleotide or polypeptide).
[0263] As used herein the term "derivative" refers to a polypeptide
that is derived from wild-type AFP as described herein, e.g., an
AFP functional fragment, and includes peptides which have been
chemically modified by techniques such as adding additional side
chains, ubiquitination, labeling, pegylation (derivatization with
polyethylene glycol), and insertion, deletion or substitution of
amino acid mimetics and/or unnatural amino acids that do not
normally occur in the sequence of wild-type AFP that is basis of
the derivative. For example, in some embodiments, an AFP derivative
can comprise a label, such as, for example, an epitope, e.g., a
FLAG epitope or a V5 epitope or an HA epitope. Such a tag can be
useful for, for example, purifying a fusion protein derivative. The
term "derivative" also encompasses a derivatized polypeptide, such
as, for example, a polypeptide modified to contain one or
more-chemical moieties other than an amino acid. The chemical
moiety can be linked covalently to the peptide, e.g., via an amino
terminal amino acid residue, a carboxy terminal amino acid residue,
or at an internal amino acid residue. Such modifications include
the addition of a protective or capping group on a reactive moiety
in the polypeptide, addition of a detectable label, and other
changes that do not adversely destroy the activity of the AFP
derivative or fusion protein. In some embodiments, an AFP
derivative contains additional chemical moieties not normally a
part of the molecule. Such moieties can improve its solubility,
absorption, biological half life, etc. The moieties can
alternatively decrease the toxicity of the molecule, or eliminate
or attenuate an undesirable side effect of the molecule, etc.
Moieties capable of mediating such effects are disclosed, for
example, in Remington's Pharmaceutical Sciences, 18th edition, A.
R. Gennaro, Ed., MackPubl., Easton, Pa. (1990).
[0264] In some embodiments, an AFP functional fragment comprises an
AFP functional fragment that differs by 1 conservative
substitution, 2 conservative substitutions, 3 conservative
substitutions, 4 conservative substitutions, 5 conservative
substitutions, 6 conservative substitutions, 7 conservative
substitutions, 8 conservative substitutions, 9 conservative
substitutions, 10 or fewer conservative substitutions, 15 or fewer
conservative substitutions, 20 or fewer conservative substitutions,
25 or fewer conservative substitutions, 30 or fewer conservative
substitutions, 35 or fewer conservative substitutions, 40 or fewer
conservative substitutions, 45 or fewer conservative substitutions,
or 50 or fewer conservative substitutions, relative to the the
sequence of the naturally occurring AFP molecule or a domain or
portion thereof of AFP having the desired biological activity.
[0265] In some embodiments, an AFP functional fragment differs by 1
or fewer non-conservative substitutions, 2 or fewer
non-conservative substitutions, 3 or fewer non-conservative
substitutions, 4 or fewer non-conservative substitutions, 5 or
fewer non-conservative substitutions, 6 or fewer non-conservative
substitutions, 7 or fewer or non-conservative substitutions, 8 or
fewer non-conservative substitutions, 9 or fewer non-conservative
substitutions, 10 or fewer or non-conservative substitutions, 15 or
fewer or non-conservative substitutions, relative to the the
sequence of the naturally occurring AFP molecule or a domain or
portion thereof of AFP having the desired biological activity.
[0266] Amino acids can be grouped according to similarities in the
properties of their side chains (in A. L. Lehninger, in
Biochemistry, second ed., pp. 73-75, Worth Publishers, New York
(1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro
(P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser
(S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp
(D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively,
naturally occurring residues can be divided into groups based on
common side-chain properties: (1) hydrophobic: Norleucine, Met,
Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn,
Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues
that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr,
Phe. Non-conservative substitutions will entail exchanging a member
of one of these classes for another class.
[0267] Preferred conservative substitutions are as follows: Ala
into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp
into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or
into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu
into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into
Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser
into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into
Val, into Ile or into Leu.
[0268] AFP-FcRn potentiators for use in the compositions, methods,
and uses described herein can be identified or characterized using
methods known in the art, such as protein-protein binding assays,
biochemical screening assays, immunoassays, and cell-based assays,
which are well known in the art, such as those described herein in
the Examples.
[0269] For the clinical use of the methods and uses described
herein, administration of the compositions comprising AFP-FcRn
inhibitors or AFP-FcRn potentiators can include formulation into
pharmaceutical compositions or pharmaceutical formulations for
parenteral administration, e.g., intravenous; mucosal, e.g.,
intranasal; ocular, or other mode of administration. In some
embodiments, the AFP-FcRn inhibitors or AFP-FcRn potentiators
described herein, can be administered along with any
pharmaceutically acceptable carrier compound, material, or
composition which results in an effective treatment in the subject.
Thus, a pharmaceutical formulation for use in the methods described
herein can contain an AFP-FcRn inhibitors or AFP-FcRn potentiator
as described herein in combination with one or more
pharmaceutically acceptable ingredients.
[0270] The phrase "pharmaceutically acceptable" refers to those
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable carrier" as used herein
means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent, media, encapsulating material, manufacturing aid (e.g.,
lubricant, talc magnesium, calcium or zinc stearate, or steric
acid), or solvent encapsulating material, involved in maintaining
the stability, solubility, or activity of an AFP-FcRn inhibitor or
AFP-FcRn potentiator. Each carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not injurious to the patient. Some examples of
materials which can serve as pharmaceutically-acceptable carriers
include: (1) sugars, such as lactose, glucose and sucrose; (2)
starches, such as corn starch and potato starch; (3) cellulose, and
its derivatives, such as sodium carboxymethyl cellulose,
methylcellulose, ethyl cellulose, microcrystalline cellulose and
cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin;
(7) excipients, such as cocoa butter and suppository waxes; (8)
oils, such as peanut oil, cottonseed oil, safflower oil, sesame
oil, olive oil, corn oil and soybean oil; (9) glycols, such as
propylene glycol; (10) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol (PEG); (11) esters, such as ethyl
oleate and ethyl laurate; (12) agar; (13) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15)
pyrogen-free water; (16) isotonic saline; (17) Ringer's solution;
(19) pH buffered solutions; (20) polyesters, polycarbonates and/or
polyanhydrides; (21) bulking agents, such as polypeptides and amino
acids (22) serum components, such as serum albumin, HDL and LDL;
(23) C2-C12 alcohols, such as ethanol; and (24) other non-toxic
compatible substances employed in pharmaceutical formulations.
Release agents, coating agents, preservatives, and antioxidants can
also be present in the formulation. The terms such as "excipient",
"carrier", "pharmaceutically acceptable carrier" or the like are
used interchangeably herein.
[0271] The AFP-FcRn inhibitors or AFP-FcRn potentiators described
herein can be specially formulated for administration of the
compound to a subject in solid, liquid or gel form, including those
adapted for the following: (1) parenteral administration, for
example, by subcutaneous, intramuscular, intravenous or epidural
injection as, for example, a sterile solution or suspension, or
sustained-release formulation; (2) topical application, for
example, as a cream, ointment, or a controlled-release patch or
spray applied to the skin; (3) intravaginally or intrarectally, for
example, as a pessary, cream or foam; (4) ocularly; (5)
transdermally; (6) transmucosally; or (7) nasally. Additionally, an
AFP-FcRn inhibitor or AFP-FcRn potentiator can be implanted into a
patient or injected using a drug delivery system. See, for example,
Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984);
Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals"
(Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S.
Pat. No. 35 3,270,960.
[0272] Further embodiments of the formulations and modes of
administration of the compositions comprising AFP-FcRn inhibitors
or AFP-FcRn potentiators that can be used in the methods described
herein are described below.
[0273] Parenteral Dosage Forms.
[0274] Parenteral dosage forms of the AFP-FcRn inhibitors or
AFP-FcRn potentiators can also be administered to a subject by
various routes, including, but not limited to, subcutaneous,
intravenous (including bolus injection), intramuscular, and
intraarterial. Since administration of parenteral dosage forms
typically bypasses the patient's natural defenses against
contaminants, parenteral dosage forms are preferably sterile or
capable of being sterilized prior to administration to a patient.
Examples of parenteral dosage forms include, but are not limited
to, solutions ready for injection, dry products ready to be
dissolved or suspended in a pharmaceutically acceptable vehicle for
injection, suspensions ready for injection, controlled-release
parenteral dosage forms, and emulsions.
[0275] Suitable vehicles that can be used to provide parenteral
dosage forms of the disclosure are well known to those skilled in
the art. Examples include, without limitation: sterile water; water
for injection USP; saline solution; glucose solution; aqueous
vehicles such as but not limited to, sodium chloride injection,
Ringer's injection, dextrose Injection, dextrose and sodium
chloride injection, and lactated Ringer's injection; water-miscible
vehicles such as, but not limited to, ethyl alcohol, polyethylene
glycol, and propylene glycol; and non-aqueous vehicles such as, but
not limited to, corn oil, cottonseed oil, peanut oil, sesame oil,
ethyl oleate, isopropyl myristate, and benzyl benzoate.
[0276] Aerosol Formulations.
[0277] An AFP-FcRn inhibitor or AFP-FcRn potentiator can be
packaged in a pressurized aerosol container together with suitable
propellants, for example, hydrocarbon propellants like propane,
butane, or isobutane with conventional adjuvants. An AFP-FcRn
inhibitor or AFP-FcRn potentiator described herein, can also be
administered in a non-pressurized form such as in a nebulizer or
atomizer. An AFP-FcRn inhibitor or AFP-FcRn potentiator described
herein, can also be administered directly to the airways in the
form of a dry powder, for example, by use of an inhaler.
[0278] Suitable powder compositions include, by way of
illustration, powdered preparations of AFP-FcRn inhibitors or
AFP-FcRn potentiators described herein, thoroughly intermixed with
lactose, or other inert powders acceptable for intrabronchial
administration. The powder compositions can be administered via an
aerosol dispenser or encased in a breakable capsule which can be
inserted by the subject into a device that punctures the capsule
and blows the powder out in a steady stream suitable for
inhalation. The compositions can include propellants, surfactants,
and co-solvents and can be filled into conventional aerosol
containers that are closed by a suitable metering valve.
[0279] Aerosols for the delivery to the respiratory tract are known
in the art. See for example, Adjei, A. and Garren, J. Pharm. Res.,
1: 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm.,
114: 111-115 (1995); Gonda, I. "Aerosols for delivery of
therapeutic and diagnostic agents to the respiratory tract," in
Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313
(1990); Anderson et al., Am. Rev. Respir. Dis., 140: 1317-1324
(1989)) and have potential for the systemic delivery of peptides
and proteins as well (Patton and Platz, Advanced Drug Delivery
Reviews, 8:179-196 (1992)); Timsina et. al., Int. J. Pharm., 101:
1-13 (1995); and Tansey, I. P., Spray Technol. Market, 4:26-29
(1994); French, D. L., Edwards, D. A. and Niven, R. W., Aerosol
Sci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10
(1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22:
263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22:
837-858 (1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995);
Patton, J. and Platz, R., Adv. Drug Del. Rev., 8: 179-196 (1992);
Bryon, P., Adv. Drug. Del. Rev., 5: 107-132 (1990); Patton, J. S.,
et al., Controlled Release, 28: 15 79-85 (1994); Damms, B. and
Bains, W., Nature Biotechnology (1996); Niven, R. W., et al.,
Pharm. Res., 12(9); 1343-1349 (1995); and Kobayashi, S., et al.,
Pharm. Res., 13(1): 80-83 (1996), contents of all of which are
herein incorporated by reference in their entirety.
[0280] The formulations of the AFP-FcRn inhibitors or AFP-FcRn
potentiators described herein, further encompass anhydrous
pharmaceutical compositions and dosage forms comprising the
disclosed compounds as active ingredients, since water can
facilitate the degradation of some compounds. For example, the
addition of water (e.g., 5%) is widely accepted in the
pharmaceutical arts as a means of simulating long-term storage in
order to determine characteristics such as shelf life or the
stability of formulations over time. See, e.g., Jens T. Carstensen,
Drug Stability: Principles & Practice, 379-80 (2nd ed., Marcel
Dekker, NY, N.Y.: 1995). Anhydrous pharmaceutical compositions and
dosage forms of the disclosure can be prepared using anhydrous or
low moisture containing ingredients and low moisture or low
humidity conditions. Pharmaceutical compositions and dosage forms
that comprise lactose and at least one active ingredient that
comprise a primary or secondary amine are preferably anhydrous if
substantial contact with moisture and/or humidity during
manufacturing, packaging, and/or storage is expected. Anhydrous
compositions are preferably packaged using materials known to
prevent exposure to water such that they can be included in
suitable formulary kits. Examples of suitable packaging include,
but are not limited to, hermetically sealed foils, plastics, unit
dose containers (e.g., vials) with or without desiccants, blister
packs, and strip packs.
[0281] Controlled and Delayed Release Dosage Forms.
[0282] In some embodiments of the aspects described herein,
AFP-FcRn inhibitors or AFP-FcRn potentiators can be administered to
a subject by controlled- or delayed-release means. Ideally, the use
of an optimally designed controlled-release preparation in medical
treatment is characterized by a minimum of drug substance being
employed to cure or control the condition in a minimum amount of
time. Advantages of controlled-release formulations include: 1)
extended activity of the drug; 2) reduced dosage frequency; 3)
increased patient compliance; 4) usage of less total drug; 5)
reduction in local or systemic side effects; 6) minimization of
drug accumulation; 7) reduction in blood level fluctuations; 8)
improvement in efficacy of treatment; 9) reduction of potentiation
or loss of drug activity; and 10) improvement in speed of control
of diseases or conditions. (Kim, Cherng-ju, Controlled Release
Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.:
2000)). Controlled-release formulations can be used to control a
compound of formula (I)'s onset of action, duration of action,
plasma levels within the therapeutic window, and peak blood levels.
In particular, controlled- or extended-release dosage forms or
formulations can be used to ensure that the maximum effectiveness
of an AFP-FcRn inhibitor or AFP-FcRn potentiator is achieved while
minimizing potential adverse effects and safety concerns, which can
occur both from under-dosing a drug (i.e., going below the minimum
therapeutic levels) as well as exceeding the toxicity level for the
drug.
[0283] A variety of known controlled- or extended-release dosage
forms, formulations, and devices can be adapted for use with the
AFP-FcRn inhibitors or AFP-FcRn potentiators described herein.
Examples include, but are not limited to, those described in U.S.
Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719;
5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;
5,354,556; 5,733,566; and 6,365,185 B1, each of which is
incorporated herein by reference in their entireties. These dosage
forms can be used to provide slow or controlled-release of one or
more active ingredients using, for example, hydroxypropylmethyl
cellulose, other polymer matrices, gels, permeable membranes,
osmotic systems (such as OROS.RTM. (Alza Corporation, Mountain
View, Calif. USA)), multilayer coatings, microparticles, liposomes,
or microspheres or a combination thereof to provide the desired
release profile in varying proportions. Additionally, ion exchange
materials can be used to prepare immobilized, adsorbed salt forms
of the disclosed compounds and thus effect controlled delivery of
the drug. Examples of specific anion exchangers include, but are
not limited to, DUOLITE.RTM. A568 and DUOLITE.RTM. AP143
(Rohm&Haas, Spring House, Pa. USA).
[0284] In some embodiments of the methods described herein, the
AFP-FcRn inhibitors or AFP-FcRn potentiators for use in the methods
described herein are administered to a subject by sustained release
or in pulses. Pulse therapy is not a form of discontinuous
administration of the same amount of a composition over time, but
comprises administration of the same dose of the composition at a
reduced frequency or administration of reduced doses. Sustained
release or pulse administrations are particularly preferred when
the disorder occurs continuously in the subject, for example where
the subject has continuous or chronic symptoms of a viral
infection. Each pulse dose can be reduced and the total amount of
the AFP-FcRn inhibitors or AFP-FcRn potentiators described herein
administered over the course of treatment to the subject or patient
is minimized.
[0285] The interval between pulses, when necessary, can be
determined by one of ordinary skill in the art. Often, the interval
between pulses can be calculated by administering another dose of
the composition when the composition or the active component of the
composition is no longer detectable in the subject prior to
delivery of the next pulse. Intervals can also be calculated from
the in vivo half-life of the composition. Intervals can be
calculated as greater than the in vivo half-life, or 2, 3, 4, 5 and
even 10 times greater the composition half-life. Various methods
and apparatus for pulsing compositions by infusion or other forms
of delivery to the patient are disclosed in U.S. Pat. Nos.
4,747,825; 4,723,958; 4,948,592; 4,965,251 and 5,403,590.
Methods of Treatment and Uses of AFP-FcRn Inhibitors and
Potentiators
[0286] As demonstrated herein, alpha-fetoprotein (AFP) is a third
ligand for the neonatal Fc receptor and soluble human FcRn binds to
AFP with affinities greater than observed with albumin, and less
than that of IgG. As further shown herein, the AFP binding site on
FcRn interferes with the albumin and IgG binding activities of
FcRn, and antibodies that are specific for the albumin site on
hFcRn can decrease FcRn-mediated AFP transport. As also
demonstrated herein, the binding of FcRn to AFP occurs over a much
wider pH range than that observed for IgG and albumin, which
typically bind under acidic pH conditions. In addition, provided
herein are single nucleotide polymorphisms in AFP that can impact
binding of AFP with human FcRn, such as, for example, G109R, R487S,
and S445L that increase AFP-FcRn binding, and T451I and D536V, that
decrease AFP-FcRn binding.
[0287] Accordingly, provided herein, in some aspects, are methods
to inhibit or reduce FcRn and AFP interactions in diseases or
disorders where elevated AFP levels are associated with
immunosuppression comprising administering a therapeutically
effective amount of a pharmaceutical composition comprising an
AFP-FcRn inhibitor to a subject in need thereof.
[0288] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor comprises a T451I and/or
D536V polymorphism of wild-type AFP that decreases AFP-FcRn
binding.
[0289] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between Y521 and/or V522 of AFP and R42 of FcRn.
[0290] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between P492 of AFP and R69 of FcRn.
[0291] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between Q441 and/or V493 of AFP and E44 of FcRn.
[0292] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between H534 and/or E589 of AFP and N173 of FcRn.
[0293] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding between
the hydrophobic core of AFP and FcRn. In some such embodiments, an
AFP-FcRn inhibitor inhibits binding and/or interactions between
L484, V493, V497, and/or F512 of AFP and V57, W59, and/or W61 of
FcRn.
[0294] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between T443 of AFP and E62 and/or W59 of FcRn.
[0295] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between D529 of AFP and S230 of FcRn.
[0296] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding between
S527 and/or D528 of AFP and E50 and/or 67Y of .beta.2m complexed
with FcRn.
[0297] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding between
R604 of AFP and the carbonyl oxygen at E50 of .beta.2m complexed
with FcRn.
[0298] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between Q597 of AFP and E69 of .beta.2m complexed with
FcRn.
[0299] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between E106 of AFP and H161 of FcRn.
[0300] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between S135 of AFP and H161 of FcRn.
[0301] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn inhibitor inhibits binding and/or
interactions between F531, F533, F552, and/or F575 of AFP and W53
of FcRn.
[0302] In some embodiments of these aspects and all such aspects
described herein, a subject having a disease or disorder associated
with elevated AFP levels has or has been diagnosed with cancer.
[0303] By "metastasis" is meant the spread of cancer from its
primary site to other places in the body. Cancer cells can break
away from a primary tumor, penetrate into lymphatic and blood
vessels, circulate through the bloodstream, and grow in a distant
focus (metastasize) in normal tissues elsewhere in the body.
Metastasis can be local or distant. Metastasis is a sequential
process, contingent on tumor cells breaking off from the primary
tumor, traveling through the bloodstream, and stopping at a distant
site. At the new site, the cells establish a blood supply and can
grow to form a life-threatening mass. Both stimulatory and
inhibitory molecular pathways within the tumor cell regulate this
behavior, and interactions between the tumor cell and host cells in
the distant site are also significant.
[0304] Metastases are most often detected through the sole or
combined use of magnetic resonance imaging (MRI) scans, computed
tomography (CT) scans, blood and platelet counts, liver function
studies, chest X-rays and bone scans in addition to the monitoring
of specific symptoms.
[0305] Examples of cancer include but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular examples of such cancers include, but are not limited
to, basal cell carcinoma, biliary tract cancer; bladder cancer;
bone cancer; brain and CNS cancer; breast cancer; cancer of the
peritoneum; cervical cancer; cholangiocarcinoma; choriocarcinoma;
colon and rectum cancer; connective tissue cancer; cancer of the
digestive system; endometrial cancer; esophageal cancer; eye
cancer; cancer of the head and neck; gastric cancer (including
gastrointestinal cancer); glioblastoma; hepatic carcinoma;
hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx
cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung, and
squamous carcinoma of the lung); lymphoma including Hodgkin's and
non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral
cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian
cancer; pancreatic cancer; prostate cancer; retinoblastoma;
rhabdomyosarcoma; rectal cancer; cancer of the respiratory system;
salivary gland carcinoma; sarcoma; skin cancer; squamous cell
cancer; stomach cancer; teratocarcinoma; testicular cancer; thyroid
cancer; uterine or endometrial cancer; cancer of the urinary
system; vulval cancer; as well as other carcinomas and sarcomas; as
well as B-cell lymphoma (including low grade/follicular
non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL;
intermediate grade/follicular NHL; intermediate grade diffuse NHL;
high grade immunoblastic NHL; high grade lymphoblastic NHL; high
grade small non-cleaved cell NHL; bulky disease NHL; mantle cell
lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), tumors of primitive origins and Meigs' syndrome.
[0306] In some embodiments of these methods and all such methods
described herein, a subject having a disease or disorder associated
with elevated AFP levels has or has been diagnosed with a cancer or
tumor of primitive origin, a tumor of liver origin, such as a
hepatoma, a tumor of biliary origin, such as cholangiocarcinoma,
stomach cancer, pancreatic cancer, or a teratocarcinoma.
[0307] In some embodiments of these methods and all such methods
described herein, the methods further comprise administering an
anti-cancer therapy or agent to a subject in addition to the
AFP-FcRn inhibitors described herein.
[0308] The term "anti-cancer therapy" refers to a therapy useful in
treating cancer. Examples of anti-cancer therapeutic agents
include, but are not limited to, e.g., surgery, chemotherapeutic
agents, growth inhibitory agents, cytotoxic agents, agents used in
radiation therapy, anti-angiogenesis agents, apoptotic agents,
anti-tubulin agents, and other agents to treat cancer, such as
anti-HER-2 antibodies (e.g., HERCEPTIN.RTM.), anti-CD20 antibodies,
an epidermal growth factor receptor (EGFR) antagonist (e.g., a
tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib
(TARCEVA.RTM.)), platelet derived growth factor inhibitors (e.g.,
GLEEVEC.TM. (Imatinib Mesylate)), a COX-2 inhibitor (e.g.,
celecoxib), interferons, cytokines, antagonists (e.g., neutralizing
antibodies) that bind to one or more of the following targets PD1,
PDL1, PDL2, TIM3 or any TIM family member, CEACAM1 or any CEACAM
family member, ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA
or VEGF receptor(s), TRAIL/Apo2, and other bioactive and organic
chemical agents, etc. Combinations thereof are also specifically
contemplated for the methods described herein.
[0309] In some embodiments, an anti-cancer therapy comprises an
immunotherapy such as adoptive cell transfer. "Adoptive cell
transfer," as used herein, includes immunotherapies involving
genetically engineering a subject or patient's own T cells to
produce special receptors on their surface called chimeric antigen
receptors (CARs). CARs are proteins that allow the T cells to
recognize a specific protein (antigen) on tumor cells. These
engineered CAR T cells are then grown in the laboratory until they
number in the billions. The expanded population of CART cells is
then infused into the patient. After the infusion, the T cells
multiply in the subject's body and, with guidance from their
engineered receptor, recognize and kill cancer cells that harbor
the antigen on their surfaces.
[0310] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Rel.sup.86, Rel.sup.88, Sm.sup.153, Bi.sup.212, P32 and
radioactive isotopes of Lu), chemotherapeutic agents, and toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including active
fragments and/or variants thereof.
[0311] In some embodiments of these methods and all such methods
described herein, the methods further comprise administering a
chemotherapeutic agent to the subject being administered the
AFP-FcRn inhibitors described herein.
[0312] Non-limiting examples of chemotherapeutic agents can include
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.RTM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE, vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11) (including the
treatment regimen of irinotecan with 5-FU and leucovorin);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment
regimen (FOLFOX); lapatinib (TYKERB.); inhibitors of PKC-alpha,
Raf, H-Ras, EGFR (e.g., erlotinib (TARCEVA.RTM.)) and VEGF-A that
reduce cell proliferation and pharmaceutically acceptable salts,
acids or derivatives of any of the above. In addition, the methods
of treatment can further include the use of radiation or radiation
therapy.
[0313] As used herein, the terms "chemotherapy" or
"chemotherapeutic agent" refer to any chemical agent with
therapeutic usefulness in the treatment of diseases characterized
by abnormal cell growth. Such diseases include tumors, neoplasms
and cancer as well as diseases characterized by hyperplastic
growth. Chemotherapeutic agents as used herein encompass both
chemical and biological agents. These agents function to inhibit a
cellular activity upon which the cancer cell depends for continued
survival. Categories of chemotherapeutic agents include
alkylating/alkaloid agents, antimetabolites, hormones or hormone
analogs, and miscellaneous antineoplastic drugs. Most if not all of
these agents are directly toxic to cancer cells and do not require
immune stimulation. In one embodiment, a chemotherapeutic agent is
an agent of use in treating neoplasms such as solid tumors. In one
embodiment, a chemotherapeutic agent is a radioactive molecule. One
of skill in the art can readily identify a chemotherapeutic agent
of use (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014,
Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning;
Principles of Cancer Therapy, Chapter 85 in Harrison's Principles
of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer
Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology,
Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; Baltzer
L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed.
St. Louis, Mosby-Year Book, 1995; Fischer D S (ed): The Cancer
Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book,
2003)).
[0314] By "radiation therapy" is meant the use of directed gamma
rays or beta rays to induce sufficient damage to a cell so as to
limit its ability to function normally or to destroy the cell
altogether. It will be appreciated that there will be many ways
known in the art to determine the dosage and duration of treatment.
Typical treatments are given as a one time administration and
typical dosages range from 10 to 200 units (Grays) per day.
[0315] In some embodiments of these methods and all such methods
described herein, the methods further comprise administering a
tumor or cancer antigen to a subject being administered the
AFP-FcRn inhibitors described herein.
[0316] A number of tumor antigens have been identified that are
associated with specific cancers. As used herein, the terms "tumor
antigen" and "cancer antigen" are used interchangeably to refer to
antigens which are differentially expressed by cancer cells and can
thereby be exploited in order to target cancer cells. Cancer
antigens are antigens which can potentially stimulate apparently
tumor-specific immune responses. Some of these antigens are
encoded, although not necessarily expressed, by normal cells. These
antigens can be characterized as those which are normally silent
(i.e., not expressed) in normal cells, those that are expressed
only at certain stages of differentiation and those that are
temporally expressed such as embryonic and fetal antigens. Other
cancer antigens are encoded by mutant cellular genes, such as
oncogenes (e.g., activated ras oncogene), suppressor genes (e.g.,
mutant p53), and fusion proteins resulting from internal deletions
or chromosomal translocations. Still other cancer antigens can be
encoded by viral genes such as those carried on RNA and DNA tumor
viruses. Many tumor antigens have been defined in terms of multiple
solid tumors: MAGE 1, 2, & 3, defined by immunity;
MART-1/Melan-A, gp100, carcinoembryonic antigen (CEA), HER-2,
mucins (i.e., MUC-1), prostate-specific antigen (PSA), and
prostatic acid phosphatase (PAP). In addition, viral proteins such
as hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV)
have been shown to be important in the development of
hepatocellular carcinoma, lymphoma, and cervical cancer,
respectively. However, due to the immunosuppression of patients
diagnosed with cancer, the immune systems of these patients often
fail to respond to the tumor antigens.
[0317] By "reduce" or "inhibit" in terms of the cancer treatment
methods described herein is meant the ability to cause an overall
decrease preferably of 20% or greater, 30% or greater, 40% or
greater, 45% or greater, more preferably of 50% or greater, of 55%
or greater, of 60% or greater, of 65% or greater, of 70% or
greater, and most preferably of 75% or greater, 80% or greater, 85%
or greater, 90% or greater, or 95% or greater, for a given
parameter or symptom. Reduce or inhibit can refer to, for example,
the symptoms of the disorder being treated, the presence or size of
metastases or micrometastases, the size of the primary tumor, the
presence or the size of the dormant tumor, etc.
[0318] As used herein, "alleviating a symptom of a cancer or tumor"
is ameliorating any condition or symptom associated with the cancer
such as the symptoms of the cancer being treated, the presence or
size of metastases or micrometastases, the size of the primary
tumor, the presence or the size of the dormant tumor, etc. As
compared with an equivalent untreated control, such as a subject
prior to the administration of the AFP-FcRn inhibitors, such
reduction or degree of prevention is at least 5%, 10%, 20%, 40%,
50%, 60%, 80%, 90%, 95%, or more as measured by any standard
technique known to one of ordinary skill in the art. A patient or
subject who is being treated for a cancer or tumor is one who a
medical practitioner has diagnosed as having such a condition.
Diagnosis can be by any suitable means.
[0319] Also provided herein, in some aspects, are methods to
increase or potentiate FcRn and AFP interactions in diseases or
disorders associated with decreased AFP levels or where increasing
AFP levels is beneficial comprising administering a therapeutically
effective amount of a pharmaceutical composition comprising an
AFP-FcRn potentiator to a subject in need thereof.
[0320] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator comprises a G109R, R487S,
and/or S445L polymorphism of wild-type AFP that increases AFP-FcRn
binding.
[0321] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between Y521 and/or V522 of AFP and R42 of FcRn.
[0322] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between P492 of AFP and R69 of FcRn.
[0323] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between Q441 and/or V493 of AFP and E44 of FcRn.
[0324] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between H534 and/or E589 of AFP and N173 of FcRn.
[0325] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding between
the hydrophobic core of AFP and FcRn. In some such embodiments, an
AFP-FcRn potentiator enhances binding and/or interactions between
L484, V493, V497, and/or F512 of AFP and V57, W59, and/or W61 of
FcRn.
[0326] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between T443 of AFP and E62 and/or W59 of FcRn.
[0327] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between D529 of AFP and S230 of FcRn.
[0328] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding between
S527 and/or D528 of AFP and E50 and/or 67Y of .beta.2m complexed
with FcRn.
[0329] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding between
R604 of AFP and the carbonyl oxygen at E50 of .beta.2m complexed
with FcRn.
[0330] In some embodiments of these aspects and all such aspects
described herein, an AFP-FcRn potentiator enhances binding and/or
interactions between Q597 of AFP and E69 of .beta.2m complexed with
FcRn.
[0331] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between E106 of AFP and H161 of FcRn.
[0332] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between S135 of AFP and H161 of FcRn.
[0333] In some embodiments of these aspects and all such aspects
described herein, an inhibitor of AFP-FcRn inhibits binding and/or
interactions between F531, F533, F552, and/or F575 of AFP and W53
of FcRn.
[0334] In some embodiments of the compositions, methods, and uses
described herein, the AFP-FcRn potentiator is an antibody or
antigen-binding fragment thereof that selectively binds or
physically interacts with AFP bound to FcRn and enhances the
interaction of AFP and FcRn, and/or blocks FcRn binding to albumin
and/or IgG but allows binding of AFP to FcRn, thereby resulting in
increased transcytosis of AFP, increased inhibition of T cell
stimulation by immune complex-primed dendritic cells, increased
AFP-mediated inhibition of immune responses, and/or increased serum
half-life of AFP.
[0335] In some embodiments of these methods and all such methods
described herein, a subject in need of increased AFP levels or
increased AFP and FcRn interaction or binding is a pregnant
subject.
[0336] In some embodiments of these methods and all such methods
described herein, a subject in need of increased AFP levels or
increased AFP and FcRn interaction or binding is a subject at risk
for having a problem with establishing and/or maintaining a
pregnancy.
[0337] In some embodiments, prior to administrating the
pharmaceutical compositions comprising AFP-FcRn potentiators
described herein to a subject, a subject is first identified as one
who has one or more of the following: placental insufficiency (See,
e.g., Lepercq and Mahieu-Caputo, 1998, Horm. Res. 49(suppl 2):
14-19); a specific maternal weight and/or height prior to pregnancy
(e.g., that is identified as being at risk for non full-term
pregnancy); low weight gain during pregnancy; maternal history of
non-full term pregnancies (e.g., spontaneous abortion, stillbirth,
neonatal death, and/or premature parturition); previous offspring
with low birth weight; specific maternal activities placing the
pregnancy at risk (e.g., smoking, alcohol and/or drug use, and/or
poor nutrition); early intrauterine infections; maternal medical
diseases; multiparous pregnancies; a history of or newly
experienced complications arising during pregnancy; and/or a
general desire for establishing pregnancy and/or maintenance of a
healthy pregnancy.
[0338] In some embodiments of these methods and all such methods
described herein, a subject in need of increased AFP levels or
increased AFP and FcRn interaction or binding has or has been
diagnosed with an autoimmune disease or disorder.
[0339] The methods described herein can, in some aspects, be
employed in the context of treatment for an autoimmune disease.
[0340] Accordingly, in some embodiments of these methods and all
such methods described herein, the autoimmune diseases to be
treated or prevented using the methods described herein, include,
but are not limited to: rheumatoid arthritis, Crohn's disease or
colitis, multiple sclerosis, systemic lupus erythematosus (SLE),
autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's
thyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigus
vulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, scleroderma with anti-collagen
antibodies, mixed connective tissue disease, polymyositis,
pernicious anemia, idiopathic Addison's disease,
autoimmune-associated infertility, glomerulonephritis (e.g.,
crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus (type 1 diabetes mellitus;
insulin-dependent diabetes mellitus). Autoimmune disease has been
recognized also to encompass atherosclerosis and Alzheimer's
disease. In some embodiments of the aspects described herein, the
autoimmune disease is selected from the group consisting of
multiple sclerosis, type-I diabetes, Hashimoto's thyroiditis,
Crohn's disease or colitis, rheumatoid arthritis, systemic lupus
erythematosus, gastritis, autoimmune hepatitis, hemolytic anemia,
autoimmune hemophilia, autoimmune lymphoproliferative syndrome
(ALPS), autoimmune uveoretinitis, glomerulonephritis,
Guillain-Barre syndrome, psoriasis and myasthenia gravis.
[0341] In some embodiments of these methods and all such methods
described herein, a subject in need of increased AFP levels or
increased AFP and FcRn interaction or binding has or has been
diagnosed with host versus graft disease (HVGD). In a further such
embodiment, the subject being treated with the methods described
herein is an organ or tissue transplant recipient. In some
embodiments, the methods are used for increasing transplantation
tolerance in a subject. In some such embodiments, the subject is a
recipient of an allogenic transplant.
[0342] The transplant can be any organ or tissue transplant,
including but not limited to heart, kidney, liver, skin, pancreas,
bone marrow, skin or cartilage. "Transplantation tolerance," as
used herein, refers to a lack of rejection of the donor organ by
the recipient's immune system.
[0343] As used herein, in regard to any of the compositions,
methods, and uses comprising AFP-FcRn inhibitors or potentiators
described herein, the terms "treat," "treatment," "treating," or
"amelioration" refer to therapeutic treatments, wherein the object
is to reverse, alleviate, ameliorate, inhibit, slow down or stop
the progression or severity of a condition associated with, a
disease or disorder. The term "treating" includes reducing or
alleviating at least one adverse effect or symptom of a disease or
disorder. Treatment is generally "effective" if one or more
symptoms or clinical markers are reduced. Alternatively, treatment
is "effective" if the progression of a disease is reduced or
halted. That is, "treatment" includes not just the improvement of
symptoms or markers, but also a cessation of at least slowing of
progress or worsening of symptoms that would be expected in absence
of treatment. Beneficial or desired clinical results include, but
are not limited to, alleviation of one or more symptom(s),
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable. The
term "treatment" of a disease also includes providing relief from
the symptoms or side-effects of the disease (including palliative
treatment).
[0344] The terms "subject," "patient," and "individual" as used in
regard to any of the methods described herein are used
interchangeably herein, and refer to an animal, for example a
human, recipient of the inhibitors described herein. For treatment
of disease states which are specific for a specific animal such as
a human subject, the term "subject" refers to that specific animal.
The terms "non-human animals" and "non-human mammals" are used
interchangeably herein, and include mammals such as rats, mice,
rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The
term "subject" also encompasses any vertebrate including but not
limited to mammals, reptiles, amphibians and fish. However,
advantageously, the subject is a mammal such as a human, or other
mammals such as a domesticated mammal, e.g. dog, cat, horse, and
the like. Production mammal, e.g. cow, sheep, pig, and the like are
also encompassed in the term subject.
[0345] The term "effective amount" as used herein refers to the
amount of a AFP-FcRn inhibitor or potentiator described herein,
needed to alleviate at least one or more symptom of the disease or
disorder being treated, and relates to a sufficient amount of
pharmacological composition to provide the desired effect, e.g.,
increase or decrease serum AFP levels. The term "therapeutically
effective amount" therefore refers to an amount of the inhibitors
or potentiators described herein, using the methods as disclosed
herein, that is sufficient to provide a particular effect when
administered to a typical subject. An effective amount as used
herein would also include an amount sufficient to delay the
development of a symptom of the disease, alter the course of a
symptom disease (for example but not limited to, slow the
progression of a symptom of the disease), or reverse a symptom of
the disease. Thus, it is not possible to specify the exact
"effective amount". However, for any given case, an appropriate
"effective amount" can be determined by one of ordinary skill in
the art using only routine experimentation.
[0346] Effective amounts, toxicity, and therapeutic efficacy can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dosage can
vary depending upon the dosage form employed and the route of
administration utilized. The dose ratio between toxic and
therapeutic effects is the therapeutic index and can be expressed
as the ratio LD50/ED50. Compositions, methods, and uses that
exhibit large therapeutic indices are preferred. A therapeutically
effective dose can be estimated initially from cell culture assays.
Also, a dose can be formulated in animal models to achieve a
circulating plasma concentration range that includes the IC50,
which achieves a half-maximal inhibition of measured function or
activity) as determined in cell culture, or in an appropriate
animal model. Levels in plasma can be measured, for example, by
high performance liquid chromatography. The effects of any
particular dosage can be monitored by a suitable bioassay. The
dosage can be determined by a physician and adjusted, as necessary,
to suit observed effects of the treatment.
[0347] The AFP-FcRn inhibitors or potentiators described herein can
be administered to a subject in need thereof by any appropriate
route which results in an effective treatment in the subject. As
used herein, the terms "administering," and "introducing" are used
interchangeably and refer to the placement of an AFP-FcRn
inhibitors or potentiators into a subject by a method or route
which results in at least partial localization of such agents at a
desired site, such as a tumor site or site of inflammation, such
that a desired effect(s) is produced.
[0348] In some embodiments, the AFP-FcRn inhibitors or potentiators
described herein can be administered to a subject by any mode of
administration that delivers the agent systemically or to a desired
surface or target, and can include, but is not limited to,
injection, infusion, instillation, and inhalation administration.
To the extent that polypeptide agents can be protected from
inactivation in the gut, oral administration forms are also
contemplated. "Injection" includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intraventricular, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intraarticular, sub capsular, subarachnoid,
intraspinal, intracerebro spinal, and intrasternal injection and
infusion.
[0349] The phrases "parenteral administration" and "administered
parenterally" as used herein, refer to modes of administration
other than enteral and topical administration, usually by
injection. The phrases "systemic administration," "administered
systemically", "peripheral administration" and "administered
peripherally" as used herein refer to the administration of the
AFP-FcRn inhibitors or potentiators, other than directly into a
target site, tissue, or organ, such that it enters the subject's
circulatory system and, thus, is subject to metabolism and other
like processes.
[0350] It is understood that the foregoing description and the
following examples are illustrative only and are not to be taken as
limitations upon the scope of the invention. Various changes and
modifications to the disclosed embodiments, which will be apparent
to those of skill in the art, may be made without departing from
the spirit and scope of the present invention. Further, all
patents, patent applications, and publications identified are
expressly incorporated herein by reference for the purpose of
describing and disclosing, for example, the methodologies described
in such publications that might be used in connection with the
present invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents are
based on the information available to the applicants and do not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0351] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that could be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0352] Exemplary embodiments of the various aspects disclosed
herein can be described by one or more of the following numbered
paragraphs: [0353] A. A pharmaceutical composition comprising an
inhibitor of AFP-FcRn and a pharmaceutically acceptable carrier,
wherein said inhibitor of AFP-FcRn inhibits binding between
alpha-fetoprotein (AFP) and FcRn. [0354] B. The pharmaceutical
composition of paragraph A, wherein the inhibitor of AFP-FcRn
comprises a T451I and/or D536V polymorphism of wild-type AFP.
[0355] C. The pharmaceutical composition of any one of paragraphs
A-B, wherein the inhibitor of AFP-FcRn inhibits binding between
Y521 and/or V522 of AFP and R42 of FcRn. [0356] D. The
pharmaceutical composition of any one of paragraphs A-C, wherein
the inhibitor of AFP-FcRn inhibits binding between P492 of AFP and
R69 of FcRn. [0357] E. The pharmaceutical composition of any one of
paragraphs A-D, wherein the inhibitor of AFP-FcRn inhibits binding
between Q441 and/or V493 of AFP and E44 of FcRn. [0358] F. The
pharmaceutical composition of any one of paragraphs A-E, wherein
the inhibitor of AFP-FcRn inhibits binding between H534 and/or E589
of AFP and N173 of FcRn. [0359] G. The pharmaceutical composition
of any one of paragraphs A-F, wherein the inhibitor of AFP-FcRn
inhibits binding between the hydrophobic core of AFP and FcRn.
[0360] H. The pharmaceutical composition of any one of paragraphs
A-G, wherein the inhibitor of AFP-FcRn inhibits binding between
L484, V493, V497, and/or F512 of AFP and V57, W59, and/or W61 of
FcRn. [0361] I. The pharmaceutical composition of any one of
paragraphs A-H, wherein the inhibitor of AFP-FcRn inhibits binding
between T443 of AFP and E62 and/or W59 of FcRn. [0362] J. The
pharmaceutical composition of any one of paragraphs A-I, wherein
the inhibitor of AFP-FcRn inhibits binding between D529 of AFP and
S230 of FcRn. [0363] K. The pharmaceutical composition of any one
of paragraphs A-J, wherein the inhibitor of AFP-FcRn inhibits
binding between S527 and/or D528 of AFP and E50 and/or 67Y of
.beta.2m complexed with FcRn. [0364] L. The pharmaceutical
composition of any one of paragraphs A-K, wherein the inhibitor of
AFP-FcRn inhibits binding between R604 of AFP and the carbonyl
oxygen at E50 of 02m complexed with FcRn. [0365] M. The
pharmaceutical composition of any one of paragraphs A-L, wherein
the inhibitor of AFP-FcRn inhibits binding between Q597 of AFP and
E69 of .beta.2m complexed with FcRn. [0366] N. The pharmaceutical
composition of any one of paragraphs A-M, wherein the inhibitor of
AFP-FcRn inhibits binding between E106 of AFP and H161 of FcRn.
[0367] O. The pharmaceutical composition of any one of paragraphs
A-N, wherein the inhibitor of AFP-FcRn inhibits binding between
S135 of AFP and H161 of FcRn. [0368] P. The pharmaceutical
composition of any one of paragraphs A-O, wherein the inhibitor of
AFP-FcRn inhibits binding between F531, F533, F552, and/or F575 of
AFP and W53 of FcRn. [0369] Q. The pharmaceutical composition of
any one of paragraphs A-P, wherein the inhibitor of AFP-FcRn is an
antibody or antigen-binding fragment thereof, a small molecule
compound, or an RNA or DNA aptamer. [0370] R. The pharmaceutical
composition of paragraph Q, wherein the antibody or antigen-binding
fragment thereof is a chimeric, humanized, or completely human
antibody or antigen-binding fragment thereof. [0371] S. The
pharmaceutical composition of any one of paragraphs A-R, wherein
the inhibitor of AFP-FcRn inhibits or blocks the AFP binding site
on FcRn. [0372] T. A pharmaceutical composition comprising an
AFP-FcRn potentiator and a pharmaceutically acceptable carrier.
[0373] U. The pharmaceutical composition of paragraph T, wherein
the AFP-FcRn potentiator comprises a G109R, R487S, and/or S445L
polymorphism of wild-type alpha-fetoprotein (AFP) that increases
AFP-FcRn binding. [0374] V. The pharmaceutical composition of any
one of paragraphs T-U, wherein the AFP-FcRn potentiator enhances
binding between Y521 and/or V522 of AFP and R42 of FcRn. [0375] W.
The pharmaceutical composition of any one of paragraphs T-V,
wherein the AFP-FcRn potentiator enhances binding between P492 of
AFP and R69 of FcRn. [0376] X. The pharmaceutical composition of
any one of paragraphs T-W, wherein the AFP-FcRn potentiator
enhances binding between Q441 and/or V493 of AFP and E44 of FcRn.
[0377] Y. The pharmaceutical composition of any one of paragraphs
T-X, wherein the AFP-FcRn potentiator enhances binding between H534
and/or E589 of AFP and N173 of FcRn. [0378] Z. The pharmaceutical
composition of any one of paragraphs T-Y, wherein the AFP-FcRn
potentiator enhances binding between the hydrophobic core of AFP
and FcRn. [0379] AA. The pharmaceutical composition of any one of
paragraphs T-Z, wherein the AFP-FcRn potentiator enhances binding
between L484, V493, V497, and/or F512 of AFP and V57, W59, and/or
W61 of FcRn. [0380] BB. The pharmaceutical composition of any one
of paragraphs T-AA, wherein the AFP-FcRn potentiator enhances
binding between T443 of AFP and E62 and/or W59 of FcRn. [0381] CC.
The pharmaceutical composition of any one of paragraphs T-BB,
wherein the AFP-FcRn potentiator enhances binding between D529 of
AFP and S230 of FcRn. [0382] DD. The pharmaceutical composition of
any one of paragraphs T-CC, wherein the AFP-FcRn potentiator
enhances binding between S527 and/or D528 of AFP and E50 and/or 67Y
of (32m complexed with FcRn. [0383] EE. The pharmaceutical
composition of any one of paragraphs T-DD, wherein the AFP-FcRn
potentiator enhances binding between R604 of AFP and the carbonyl
oxygen at E50 .beta.2m complexed with FcRn. [0384] FF. The
pharmaceutical composition of any one of paragraphs T-EE, wherein
the AFP-FcRn potentiator enhances binding between Q597 of AFP and
E69 of .beta.2m complexed with FcRn. [0385] GG. The pharmaceutical
composition of any one of paragraphs T-FF, wherein the AFP-FcRn
potentiator enhances binding between E106 of AFP and H161 of FcRn.
[0386] HH. The pharmaceutical composition of any one of paragraphs
T-GG, wherein the AFP-FcRn potentiator enhances binding between
S135 of AFP and H161 of FcRn. [0387] II. The pharmaceutical
composition of any one of paragraphs T-HH, wherein the AFP-FcRn
potentiator enhances binding between 531, F533, F552, and/or F575
of AFP and W53 of FcRn. [0388] JJ. The pharmaceutical composition
of any one of paragraphs T-II, wherein the AFP-FcRn potentiator is
an antibody or antigen-binding fragment thereof, a small molecule
compound, an RNA or DNA aptamer, or an AFP functional fragment.
[0389] KK. The pharmaceutical composition of paragraph JJ, wherein
the antibody or antigen-binding fragment thereof is a chimeric,
humanized, or completely human antibody or antigen-binding fragment
thereof. [0390] LL. The pharmaceutical composition of any one of
paragraphs T-KK, wherein the AFP-FcRn potentiator binds FcRn and
mimics AFP binding [0391] MM. The pharmaceutical composition of any
one of paragraphs T-LL, wherein the AFP-FcRn potentiator binds or
physically interacts with AFP or FcRn, and enhances or promotes
interactions between AFP and FcRn. [0392] NN. The pharmaceutical
composition of paragraph MM, wherein the AFP-functional fragment
comprises Y521 and/or V522 of AFP and can interact with R42 of
FcRn. [0393] OO. The pharmaceutical composition of any one of
paragraphs JJ or NN, wherein the AFP-functional fragment comprises
P492 of AFP and can interact with R69 of FcRn. [0394] PP. The
pharmaceutical composition of any one of paragraphs JJ or NN-OO,
wherein the AFP-functional fragment comprises Q441 and/or V493 of
AFP and can interact with E44 of FcRn. [0395] QQ. The
pharmaceutical composition of any one of paragraphs JJ or NN-PP,
wherein the AFP-functional fragment comprises H534 and/or E589 of
AFP and can interact with N173 of FcRn. [0396] RR. The
pharmaceutical composition of any one of paragraphs JJ or NN-QQ,
wherein the AFP-functional fragment comprises L484, V493, V497,
and/or F512 of AFP and can interact with V57, W59, and/or W61 of
FcRn. [0397] SS. The pharmaceutical composition of any one of
paragraphs JJ or NN-RR, wherein the AFP-functional fragment
comprises T443 of AFP and can interact with E62 and/or W59 of FcRn.
[0398] TT. The pharmaceutical composition of any one of paragraphs
JJ or NN-SS, wherein the AFP-functional fragment comprises D529 of
AFP and can interact with S230 of FcRn. [0399] UU. The
pharmaceutical composition of any one of paragraphs JJ or NN-TT,
wherein the AFP-functional fragment comprises S527 and/or D528 of
AFP and can interact with E50 and/or 67Y of .beta.2m complexed with
FcRn. [0400] VV. The pharmaceutical composition of any one of
paragraphs JJ or NN-UU, wherein the AFP-functional fragment
comprises R604 of AFP and can interact with the carbonyl oxygen at
E50 of 132m complexed with FcRn. [0401] WW. The pharmaceutical
composition of any one of paragraphs JJ or NN-VV, wherein the
AFP-functional fragment comprises Q597 of AFP and can interact with
E69 of .beta.2m complexed with FcRn. [0402] XX. The pharmaceutical
composition of any one of paragraphs JJ or NN-WW, wherein the
AFP-functional fragment comprises E106 of AFP and can interact with
H161 of FcRn. [0403] YY. The pharmaceutical composition of any one
of paragraphs JJ or NN-XX, wherein the AFP-functional fragment
comprises S135 of AFP and can interact with H161 of FcRn. [0404]
ZZ. The pharmaceutical composition of any one of paragraphs JJ or
NN-YY, wherein the AFP-functional fragment comprises F531, F533,
F552, and/or F575 of AFP and can interact with W53 of FcRn. [0405]
AAA. A method to inhibit or reduce FcRn and alpha-fetoprotein (AFP)
interactions in a disease or disorder associated with elevated AFP
levels comprising administering a therapeutically effective amount
of a pharmaceutical composition comprising an AFP-FcRn inhibitor of
any one of paragraphs A-S to a subject in need thereof. [0406] BBB.
The method of paragraph AAA, wherein the subject has or has been
diagnosed with cancer. [0407] CCC. The method of any one of
paragraphs AAA or BBB, wherein the subject has or has been
diagnosed with a cancer or tumor of primitive origin, a tumor of
liver origin, such as a hepatoma, a tumor of biliary origin, such
as cholangiocarcinoma, stomach cancer, pancreatic cancer, or a
teratocarcinoma. [0408] DDD. The method of any one of paragraphs
AAA-CCC, further comprising administering an anti-cancer therapy or
agent to the subject. [0409] EEE. The method of any one of
paragraphs AAA-DDD, further comprising administering a tumor or
cancer antigen. [0410] FFF. A method to increase or potentiate FcRn
and alpha-fetoprotein (AFP) interactions in diseases or disorders
associated with decreased AFP levels or where increasing AFP levels
is beneficial comprising administering a therapeutically effective
amount of a pharmaceutical composition comprising an AFP-FcRn
potentiator of any one of paragraphs T-ZZ to a subject in need
thereof. [0411] GGG. The method of paragraph FFF, wherein the
subject in need is pregnant or is at risk for having a problem with
establishing and/or maintaining a pregnancy. [0412] HHH. The method
of paragraph FFF, wherein the subject has or has been diagnosed
with an autoimmune disease or disorder. [0413] III. The method of
paragraph FFF, wherein the subject has or has been diagnosed with
host versus graft disease (HVGD), is an organ or tissue transplant
recipient, or a recipient of an allogenic transplant.
Examples
[0414] hAFP transcytosis assays were performed as previously
described for IgG. In brief, MDCK II cells expressing
h.beta..sub.2m and hFcRn or m.beta..sub.2m and mFcRn or vector
controls (expressing only 1102m) were grown to confluence on
transwells (Costar) and allowed to polarize over 4 days. 12 hours
before the transcytosis experiment, the medium was changed to
serum-free media without antibiotics. On the day of experiment, the
transwells were incubated with HBSS pH 7.4 for 20 minutes before
placing on a new 12 well plate (Costar) where the input chamber
contained AFP HBSS pH 6.0 or 7.4 (with) and the exit chamber
contained HBSS pH 7.4. For blocking AFP transcytosis with the mouse
anti-human FcRn antibody (ADM31) or isotype control (IgG2b),
transwells were pre-incubated for 20 min with the respective
antibodies in HBSS pH 7.4 prior to the addition of AFP to the same
side of the chamber. After 2 hours incubation in 36.degree. C. and
5% CO.sub.2, the medium at the opposite chamber was harvested and
the hAFP concentration was measured using ELISA method. For the AFP
inhibition of IgG transcytosis, AFP and IgG were both added in HBSS
pH 6 to the input chamber and 2 hours later the medium at the
opposite chamber was harvested and IgG was quantified using ELISA
method.
[0415] In vitro cross-presentation assays were carried out by
pulsing 1.times.10.sup.5 isolated DC with preformed immune
complexes (0.5 .mu.g/ml NIP-conjugated OVA+100 .mu.g/ml anti-NIP
IgG or anti-NIP IHH-IgG) for 2-3 h followed by extensive washing
and the addition of 2.times.10.sup.5 purified OT-I CD8.sup.+ T
cells. The presentation assays were carried out accordingly with
the distinction of utilizing 2.times.10.sup.5 purified OT-II
CD4.sup.+ T cells. Depending on condition, DCs were pre-incubated
with hAFP (50 or 100 .mu.g/ml) or HSA (50 or 100 .mu.g/ml) in
presence or absence of ADM31 or IgG2b isotype control (75 or 100
.mu.g/ml). Immune complexes were formed using ovalbumin conjugated
to the hapten NIP (4-hydroxy-3-iodo-5-nitrophenylacetic acid) and
NIP-specific chimeric IgG (IgG) or IHH-IgG. IHH-IgG is a mutational
variant of the chimeric IgG protein which contains a NIP-specific
mouse Fab fragment and a human IgG1 Fc fragment and which has been
rendered incapable of FcRn binding due to the introduction of
mutations in three critical amino acids in the Fc region which are
required for FcRn ligation. Cytokine secretion was measured after
24 h or 48 h by ELISA. For the measurement of proliferation, OT-I
CD8.sup.+ T cells were stained with eFluor670 Proliferation Dye
according to the manufacturer's instructions (eBioscience) and
stimulated as described above.
[0416] For in vivo enhanced clearance of hIgG upon hAFP
administration effect, hFCGRT/hB2M/mFcgrt-/- mice were injected
i.p. with hIgG (100 .mu.g/mouse) and the following day with hAFP
(100 .mu.g/mouse). 24, 48 and 72 hrs later blood samples were
collected and the amount of hIgG was quantified by ELISA and
compared to Day 0.
[0417] Surface plasmon resonance was conducted using a Biacore 3000
instrument (GE Healthcare) with CMS sensor chips coupled with
recombinant human FcRn & h.beta..sub.2m heterodimer protein or
mouse FcRn & m.beta..sub.2m (1000 RU). The coupling was
performed by injecting 25 .mu.g/mL of the protein diluted in 10 mM
sodium acetate pH 5.0 using the amine coupling kit (GE Healthcare).
Phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween
20) at pH 6.0 or pH 7.4 was used as running buffer. Glycine pH 2.5
was used for regeneration of the flow cells. hAFP (SinoBiological)
was injected at 25.degree. C. with a flow rate of 25 .mu.l/min, and
data were analyzed using the BIAevaluation 4.1 software where the
sensorgrams were zero adjusted and reference cell values
subtracted.
[0418] FIG. 1 demonstrates that human AFP (hAFP) is transcytosed by
human FcRn (hFcRn) at acidic and neutral pH. Transcytosis of AFP by
MDCK II cells co-expressing FcRn and .beta.2microglobulin
(hFcRn/.beta.2m) or vector control at pH 6 and 7.4. B.fwdarw.A
Basolateral to apical direction, A.fwdarw.B Apical to basolateral
direction.
[0419] FIG. 2 demonstrates that hAFP is transcytosed by mouse FcRn.
Transcytosis of AFP by MDCK II cells co-expressing mouse FcRn and
.beta.2microglobulin (mFcRn/m.beta.2m) or vector control at pH 7.4.
B.fwdarw.A Basolateral to apical direction, A.fwdarw.B Apical to
basolateral direction.
[0420] FIG. 3 demonstrates that hAFP transcytosis by hFcRn is
blocked by ADM31 antibody. Transcytosis of AFP by MDCK II cells
co-expressing hFcRn, h.beta.2m (mFcRn/m.beta.2m) or vector control
at pH 7.4 in presence of anti-human FcRn antibody (ADM31) or
isotype control (IgG2b). B.fwdarw.A Basolateral to apical
direction.
[0421] FIG. 4 demonstrates that AFP hinders FcRn-mediated
transcytosis of IgG. Transcytosis of human IgG by MDCK II cells
co-expressing hFcRn, h.beta.2m, or vector control at pH 6 which
have been pre-incubated with hAFP or Human Serum Albumin (HSA) as
control at pH 7.4. Basolateral to apical direction is shown.
[0422] FIG. 5 demonstrates that AFP binds to human and mouse FcRn
at neutral pH. SPR analyses of hAFP binding to hFcRn (left panel)
or mFcRn (right panel) at neutral pH.
[0423] FIG. 6 demonstrates that AFP binds to hFcRn at acidic pH.
SPR analyses of hAFP binding to hFcRn at pH 6.
[0424] FIG. 7 demonstrates that AFP inhibits T cell stimulation by
IgG-IC primed DC. hAFP blocks proliferation (IL-2 secretion) of
CD8.sup.+ (OT-I, left panel) or CD4.sup.+ (OT-II, right panel) T
cells in response to antigen in IgG-IC. BMDC from
hFCGRT/hB2M/mFcgrt.sup.-/- mice were treated with 100 .mu.g/ml of
IgG or IHH-IgG in association with 0, 0.5, 1, or 5 .mu.g/ml of OVA
in presence of 100 .mu.g/ml of hAFP and then co-cultured with
either OVA-specific CD8.sup.+ or CD4.sup.+ T cells. 24 after the
stimulation IL-2 secretion in the supernatants were measured by
ELISA.
[0425] FIG. 8 demonstrates that ADM31 blocks AFP-FcRn-mediated
inhibitory functions. ADM31, a monoclonal anti-hFcRn antibody
blocks hAFP inhibition of CD8.sup.+ T cell IL-2 secretion in
response to antigen in IgG-IC. BMDC from hFCGRT/hB2M/mFcgrt.sup.-/-
mice were treated with 100 .mu.g/ml of IgG or IHH-IgG in
association with 0.5 .mu.g/ml OVA in presence of 50 .mu.g/ml of
hAFP or HSA and 50 .mu.g/ml of ADM31 or isotype control, and then
co-cultured with OVA-specific CD8.sup.+ T cells. 24 after the
stimulation IL-2 secretion in the supernatants were measured by
ELISA.
[0426] FIG. 9 demonstrates that ADM31 blocks AFP-FcRn-mediated
inhibitory functions. ADM31 blocks hAFP inhibition of CD8.sup.+ T
cell proliferation in response to antigen in IgG-IC. BMDC from
hFCGRT/hB2M/mFcgrt.sup.-/- mice were treated with 100 .mu.g/ml of
IgG or IHH-IgG in association with 0.5 .mu.g/ml OVA in presence of
50 .mu.g/ml of hAFP or HSA and 50 .mu.g/ml of ADM31 or IgG2b
isotype control, and then co-cultured with CD8.sup.+ T cells
labelled with eFluor670 Proliferation Dye. 72 hrs later the cells
were acquired. Percent of proliferated cells is displayed.
[0427] FIG. 10 demonstrates that administration of hAFP results in
increased clearance of hIgG antibodies from systemic circulation.
hFCGRT/hB2M/mFcgrt.sup.-/- mice were injected with hIgG and the
following day with hAFP. 24, 48 and 72 hrs later blood samples were
collected and the amount of hIgG was quantified by ELISA and
compared to Day 0. The results illustrate that AFP injection
resulted in faster clearance of hIgG from circulation.
[0428] FIG. 11 shows an AFP homology model derived from HSA Crystal
Structure (PDB ID: 4N0F).
[0429] FIG. 12 depicts superimposition of AFP model on FcRn-HSA-IgG
ternary complex crystal structure (PDB ID: 4N0U).
[0430] FIG. 13 depicts HSA Y497/V498 residues are conserved in AFP
(Y521/V522) and interact with FcRn R42. HSA/AFP conserved residues
in Domain III that establish binding to FcRn.
[0431] FIG. 14 demonstrates that HSA P468 residue is conserved in
AFP (P492) and interacts with FcRn R69. HSA/AFP conserved residues
in Domain III that establish binding to FcRn
[0432] FIG. 15 demonstrates that HSA Q417/V469 residues are
conserved in AFP (Q441/V493) and interact with FcRn E44. HSA/AFP
conserved residues in Domain III that establish binding to FcRn
AFP. HSA V469/AFPV493 make backbone contacts with conserved HSA
H464/AFP H488.
[0433] FIG. 16 demonstrates that HSA H510/E565 residues are
conserved in AFP (H534/E589) and interact with FcRn N173. HSA/AFP
conserved residues in Domain III establish binding to FcRn.
[0434] FIG. 17 demonstrates that hydrophobic core centered on HSA
L460/V469/V473/F488 is conserved in AFP (L484/V493/V497/F512) and
interacts with FcRn V57/W59/W61. HSA/AFP conserved residues in
Domain III establish binding to FcRn.
[0435] FIG. 18 demonstrates that HSA S419 residue is not conserved
in AFP (T443) yet is able to interact with FcRn E62/W59. HSA/AFP
non-conserved residues in Domain III preserve AFP binding to
FcRn.
[0436] FIG. 19 demonstrates that HSA E505 non-conserved residue in
AFP (D529) preserves binding to FcRn S230. HSA/AFP non-conserved
residues in Domain III preserve AFP binding to FcRn.
[0437] FIG. 20 demonstrates that AFP S527/D528 residues make
contacts with .beta.2m E50 and 67Y that are not present in HSA
(N503, A504) providing new interactions. HSA/AFP non-conserved
residues that increase AFP binding to FcRn through new contacts
with .beta.2m and is not pH dependent.
[0438] FIG. 21 demonstrates that AFP R604 makes additional contacts
with .beta.2m E50, providing new interactions. HSA/AFP
non-conserved residues increase AFP binding to .beta.2m. HSA Q580
lacks these interactions.
[0439] FIG. 22 demonstrates that AFP Q597 residue is better
positioned to make contacts with .beta.2m E69 providing stronger
interaction. HSA/AFP non-conserved residues establish new and
increased AFP-132m interactions. HSA K573 lacks these
interactions.
[0440] FIG. 23 demonstrates that AFP (E106) conserved residue (with
HSA E82) makes long range interaction with FcRn H161. Conserved
HSA/AFP residues in Domain I interact with FcRn.
[0441] FIG. 24 demonstrates that AFP S135 allows AFP interface to
come closer to FcRn and makes .about.3 .ANG. interactions with FcRn
H161, which is absent in HSA. AFP Domain I-FcRn interaction
suggests neutral pH binding. Nearby conserved proline in HSA/AFP
occupy same space in interface.
[0442] FIG. 25 demonstrates a larger hydrophobic core in AFP
(F531/F533/F552/F575) centered on FcRn W53: AFP F552 results in
stronger AFP-FcRn interactions than HSA A528. AFP-FcRn interactions
indicates neutral pH binding. AFP (F531/F533/F552/F575) versus HSA
(F507/F509/A528/F551).
TABLE-US-00003 TABLE 1 SUMMARY OF INTERACTIONS OF AFP WITH FCRN HSA
AFP FcRn Contact Interaction Residues Residues Residues change
HSA/AFP Conserved Interactions with FcRn Y497 Y521 R42 = V498 V522
P468 P492 R69 = Q417 Q441 E44 = V469 V493 H464 H488 H510 H534 N173
= E565 E589 L460 L484 V57 = V469 V493 W59 V473 V497 W61 F488 F512
D89 D112 N149 = E82 E106 H161 = S419 T443 E62, W59 = E505 D529 S230
= Novel AFP Interactions with FcRn -- S135 H161 .uparw. Binding
F507 F531 W53 .uparw. Binding F509 F533 F551 F575 A528 F552 P421
S445 Q56 .uparw. Binding HSA AFP .beta..sub.2-Microglobulin
Residues Residues Residues Novel AFP Interactions with Beta-2
-Microglobulin N503 S527 E50 .uparw. Binding A504 D528 Y67 Q580
R604 K573 Q597 E69 .uparw. Binding
Sequence CWU 1
1
311365PRTHomo sapiens 1Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu
Gly Leu Leu Leu Phe1 5 10 15Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser
His Leu Ser Leu Leu Tyr 20 25 30His Leu Thr Ala Val Ser Ser Pro Ala
Pro Gly Thr Pro Ala Phe Trp 35 40 45Val Ser Gly Trp Leu Gly Pro Gln
Gln Tyr Leu Ser Tyr Asn Ser Leu 50 55 60Arg Gly Glu Ala Glu Pro Cys
Gly Ala Trp Val Trp Glu Asn Gln Val65 70 75 80Ser Trp Tyr Trp Glu
Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys 85 90 95Leu Phe Leu Glu
Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110Leu Gln
Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120
125Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp
130 135 140Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu
Ala Ile145 150 155 160Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala
Asn Lys Glu Leu Thr 165 170 175Phe Leu Leu Phe Ser Cys Pro His Arg
Leu Arg Glu His Leu Glu Arg 180 185 190Gly Arg Gly Asn Leu Glu Trp
Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205Ala Arg Pro Ser Ser
Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe 210 215 220Ser Phe Tyr
Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu225 230 235
240Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser
245 250 255Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu
His His 260 265 270Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln
Pro Leu Arg Val 275 280 285Glu Leu Glu Ser Pro Ala Lys Ser Ser Val
Leu Val Val Gly Ile Val 290 295 300Ile Gly Val Leu Leu Leu Thr Ala
Ala Ala Val Gly Gly Ala Leu Leu305 310 315 320Trp Arg Arg Met Arg
Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg 325 330 335Gly Asp Asp
Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp 340 345 350Ala
Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala 355 360
3652119PRTHomo sapiens 2Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala
Leu Leu Ser Leu Ser1 5 10 15Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys
Ile Gln Val Tyr Ser Arg 20 25 30His Pro Ala Glu Asn Gly Lys Ser Asn
Phe Leu Asn Cys Tyr Val Ser 35 40 45Gly Phe His Pro Ser Asp Ile Glu
Val Asp Leu Leu Lys Asn Gly Glu 50 55 60Arg Ile Glu Lys Val Glu His
Ser Asp Leu Ser Phe Ser Lys Asp Trp65 70 75 80Ser Phe Tyr Leu Leu
Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp 85 90 95Glu Tyr Ala Cys
Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile 100 105 110Val Lys
Trp Asp Arg Asp Met 1153609PRTHomo sapiens 3Met Lys Trp Val Glu Ser
Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr1 5 10 15Glu Ser Arg Thr Leu
His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu 20 25 30Asp Ser Tyr Gln
Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr 35 40 45Ile Phe Phe
Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser 50 55 60Lys Met
Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp65 70 75
80Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu
85 90 95Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser
Asp 100 105 110Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe
Leu Ala His 115 120 125Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe
Gln Val Pro Glu Pro 130 135 140Val Thr Ser Cys Glu Ala Tyr Glu Glu
Asp Arg Glu Thr Phe Met Asn145 150 155 160Lys Phe Ile Tyr Glu Ile
Ala Arg Arg His Pro Phe Leu Tyr Ala Pro 165 170 175Thr Ile Leu Leu
Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys 180 185 190Cys Lys
Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr 195 200
205Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys
210 215 220Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile
Thr Val225 230 235 240Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn
Phe Thr Glu Ile Gln 245 250 255Lys Leu Val Leu Asp Val Ala His Val
His Glu His Cys Cys Arg Gly 260 265 270Asp Val Leu Asp Cys Leu Gln
Asp Gly Glu Lys Ile Met Ser Tyr Ile 275 280 285Cys Ser Gln Gln Asp
Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys 290 295 300Leu Thr Thr
Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp305 310 315
320Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp
325 330 335Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe
Leu Ala 340 345 350Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln
Leu Ala Val Ser 355 360 365Val Ile Leu Arg Val Ala Lys Gly Tyr Gln
Glu Leu Leu Glu Lys Cys 370 375 380Phe Gln Thr Glu Asn Pro Leu Glu
Cys Gln Asp Lys Gly Glu Glu Glu385 390 395 400Leu Gln Lys Tyr Ile
Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys 405 410 415Gly Leu Phe
Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu 420 425 430Val
Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met 435 440
445Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu
450 455 460Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp
Ile Ile465 470 475 480Ile Gly His Leu Cys Ile Arg His Glu Met Thr
Pro Val Asn Pro Gly 485 490 495Val Gly Gln Cys Cys Thr Ser Ser Tyr
Ala Asn Arg Arg Pro Cys Phe 500 505 510Ser Ser Leu Val Val Asp Glu
Thr Tyr Val Pro Pro Ala Phe Ser Asp 515 520 525Asp Lys Phe Ile Phe
His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala 530 535 540Leu Gln Thr
Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys545 550 555
560Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser
565 570 575Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val
Cys Phe 580 585 590Ala Glu Glu Gly Gln Lys Leu Ile Ser Lys Thr Arg
Ala Ala Leu Gly 595 600 605Val4575PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 4Met Lys Trp Val Glu
Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr1 5 10 15Glu Ser Arg Thr
Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu 20 25 30Asp Ser Tyr
Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr 35 40 45Ile Phe
Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser 50 55 60Lys
Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp65 70 75
80Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu
85 90 95Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser
Asp 100 105 110Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe
Leu Ala His 115 120 125Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe
Gln Val Pro Glu Pro 130 135 140Val Thr Ser Cys Glu Ala Tyr Glu Glu
Asp Arg Glu Thr Phe Met Asn145 150 155 160Lys Phe Ile Tyr Glu Ile
Ala Arg Arg His Pro Phe Leu Tyr Ala Pro 165 170 175Thr Ile Leu Leu
Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys 180 185 190Cys Lys
Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr 195 200
205Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys
210 215 220Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile
Thr Val225 230 235 240Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn
Phe Thr Glu Ile Gln 245 250 255Lys Leu Val Leu Asp Val Ala His Val
His Glu His Cys Cys Arg Gly 260 265 270Asp Val Leu Asp Cys Leu Gln
Asp Gly Glu Lys Ile Met Ser Tyr Ile 275 280 285Cys Ser Gln Gln Asp
Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys 290 295 300Leu Thr Thr
Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp305 310 315
320Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp
325 330 335Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe
Leu Ala 340 345 350Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln
Leu Ala Val Ser 355 360 365Val Ile Leu Arg Val Ala Lys Gly Tyr Gln
Glu Leu Leu Glu Lys Cys 370 375 380Phe Gln Thr Glu Asn Pro Leu Glu
Cys Gln Asp Lys Gly Glu Glu Glu385 390 395 400Leu Gln Lys Tyr Ile
Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys 405 410 415Gly Leu Phe
Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu 420 425 430Val
Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met 435 440
445Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu
450 455 460Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp
Ile Ile465 470 475 480Ile Gly His Leu Cys Ile Arg His Glu Met Thr
Pro Val Asn Pro Gly 485 490 495Val Gly Gln Cys Cys Thr Ser Ser Tyr
Ala Asn Arg Arg Pro Cys Phe 500 505 510Ser Ser Leu Val Val Asp Glu
Thr Tyr Val Pro Pro Ala Phe Ser Asp 515 520 525Asp Lys Phe Ile Phe
His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala 530 535 540Leu Gln Thr
Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys545 550 555
560Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe 565
570 575592PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Leu Cys Ile Arg His Glu Met Thr Pro Val Asn
Pro Gly Val Gly Gln1 5 10 15Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg
Pro Cys Phe Ser Ser Leu 20 25 30Val Val Asp Glu Thr Tyr Val Pro Pro
Ala Phe Ser Asp Asp Lys Phe 35 40 45Ile Phe His Lys Asp Leu Cys Gln
Ala Gln Gly Val Ala Leu Gln Thr 50 55 60Met Lys Gln Glu Phe Leu Ile
Asn Leu Val Lys Gln Lys Pro Gln Ile65 70 75 80Thr Glu Glu Gln Leu
Glu Ala Val Ile Ala Asp Phe 85 90615PRTHomo sapiens 6Leu Glu Val
Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu1 5 10 15715PRTHomo
sapiens 7Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp
Asp1 5 10 1587PRTHomo sapiens 8Glu Lys Thr Pro Val Ser Asp1
597PRTHomo sapiens 9Glu Met Thr Pro Val Asn Pro1 51054PRTHomo
sapiens 10Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn
Leu Gly1 5 10 15Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro 20 25 30Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
Leu Cys Val Leu 35 40 45His Glu Lys Thr Pro Val 501154PRTHomo
sapiens 11Pro Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr Arg Lys
Met Ala1 5 10 15Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp Lys
Leu Leu Ala 20 25 30Cys Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His
Leu Cys Ile Arg 35 40 45His Glu Met Thr Pro Val 501258PRTHomo
sapiens 12Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln
Ile Lys1 5 10 15Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro
Lys Ala Thr 20 25 30Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala
Ala Phe Val Glu 35 40 45Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr 50
551358PRTHomo sapiens 13Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val
Ala Leu Gln Thr Met1 5 10 15Lys Gln Glu Phe Leu Ile Asn Leu Val Lys
Gln Lys Pro Gln Ile Thr 20 25 30Glu Glu Gln Leu Glu Ala Val Ile Ala
Asp Phe Ser Gly Leu Leu Glu 35 40 45Lys Cys Cys Gln Gly Gln Glu Gln
Glu Val 50 551431PRTHomo sapiens 14Gln Leu Cys Val Leu His Glu Lys
Thr Pro Val Ser Asp Arg Val Thr1 5 10 15Lys Cys Cys Thr Glu Ser Leu
Val Asn Arg Arg Pro Cys Phe Ser 20 25 301531PRTHomo sapiens 15His
Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly Val Gly1 5 10
15Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe Ser 20 25
301611PRTHomo sapiens 16Lys Lys Val Pro Gln Val Ser Thr Pro Thr
Leu1 5 101711PRTHomo sapiens 17Lys Lys Ala Pro Gln Leu Thr Ser Ser
Glu Leu1 5 101811PRTHomo sapiens 18Glu Phe Asn Ala Glu Thr Phe Thr
Phe His Ala1 5 101911PRTHomo sapiens 19Ala Phe Ser Asp Asp Lys Phe
Ile Phe His Lys1 5 102011PRTHomo sapiens 20Pro Lys Glu Phe Asn Ala
Glu Thr Phe Thr Phe1 5 102111PRTHomo sapiens 21Pro Pro Ala Phe Ser
Asp Asp Lys Phe Ile Phe1 5 102211PRTHomo sapiens 22Lys Leu Val Ala
Ala Ser Gln Ala Ala Leu Gly1 5 102311PRTHomo sapiens 23Lys Leu Ile
Ser Lys Thr Arg Ala Ala Leu Gly1 5 10249PRTHomo sapiens 24Cys Phe
Ala Glu Glu Gly Lys Lys Leu1 5259PRTHomo sapiens 25Cys Phe Ala Glu
Glu Gly Gln Lys Leu1 52611PRTHomo sapiens 26Leu Arg Glu Thr Tyr Gly
Glu Met Ala Asp Cys1 5 102711PRTHomo sapiens 27Ile Leu Glu Lys Tyr
Gly His Ser Asp Cys Cys1 5 102815PRTHomo sapiens 28His Lys Asp Asp
Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu1 5 10 152915PRTHomo
sapiens 29Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro
Glu1 5 10 153049PRTHomo sapiens 30Thr Phe Thr Phe His Ala Asp Ile
Cys Thr Leu Ser Glu Lys Glu Arg1 5 10 15Gln Ile Lys Lys Gln Thr Ala
Leu Val Glu Leu Val Lys His Lys Pro 20 25 30Lys Ala Thr Lys Glu Gln
Leu Lys Ala Val Met Asp Asp Phe Ala Ala 35 40 45Phe3149PRTHomo
sapiens 31Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val
Ala Leu1 5 10 15Gln Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys
Gln Lys Pro 20 25 30Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala
Asp Phe Ser Gly 35 40 45Leu
* * * * *