Note: Descriptions are shown in the official language in which they were submitted.
DESCRIPTION
METHODS AND COMPOSITIONS FOR TREATING AUTOIMMUNE AND
INFLAMMATORY CONDITIONS
BACKGROUND OF THE INVENTION
[0001] This application claims the benefit of priority to U.S. Provisional
Patent
Application Serial No. 61/994,239, filed May 16, 2014, and U.S. Provisional
Patent
Application Serial No. 62/014,504, filed June 19, 2014.
[0002]
1. Field of the Invention
[0003] The present invention relates generally to the field of medicine.
More
particularly, it concerns pharmaceutical compositions for enhancing tolerance
to antigens and
for treating inflammatory and autoimmune disorders.
2. Background
[0004] Autoimmune and inflammatory diseases arise from an abnormal immune
response of the body against substances and tissues normally present in the
body. This may be
restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a
particular tissue in
different places (e.g., Goodpasture's disease which may affect the basem*nt
membrane in both
the lung and the kidney). Autoimmune and auto-inflammatory diseases affect up
to 50 million
people in America alone, and the cause of autoimmunity remains unknown.
[0005] The treatment of these diseases is typically with immunosuppression¨
medication that decreases the immune response. Conventional immunotherapies
using
immunosuppressants, such as cyclosporine, tacroliums, methotrexate or anti-
TNFa/IL-6 non-
specifically suppress the function of T cell including non-pathogenic T cells
in the host.
Therefore, treatment with these immunesuppressants often results in the
development of
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severe infections and sometimes leads to the lethal consequences. There is a
need in the art
for therapeutics that treat autoimmune responses without global
immunosuppression.
SUMMARY OF THE INVENTION
[0006] This disclosure fulfills a need in the art by providing methods and
compositions for delivering the anti-inflammatory cytokine, IL-10, to antigen
presenting cells
(APCs) to suppress and alter the pathophysiologic functions of APCs in the
subjects using
APC-targeted antibody operatively linked to IL-10 or a fragment thereof.
Targeted delivery
of anti-inflammatory cytokines to the APCs in the patients is expected to
result in more
effective and pro-longed immune tolerance in the patients. Accordingly,
aspects of the
disclosure relate to a method for inhibiting an inflammatory or autoimmune
response in a
subject in need thereof comprising administering to the subject a
therapeutically effective
amount of an antigen presenting cell (APC)-targeted antibody operatively
linked to IL-10 or a
fragment thereof.
[0007] In some embodiments, the disclosure relates to a method for
preventing or
treating graft versus host disease in a subject in need thereof comprising
administering to the
subject a therapeutically effective amount of DC-ASGPR operatively linked to
IL-10 or a
fragment thereof.
[0008] Further aspects relate to a method of inducing immune tolerance in a
subject
in need thereof comprising administering to the subject a therapeutically
effective amount of
an APC-targeted antibody operatively linked to IL-10 or a fragment thereof.
Other aspects
relate to a method of suppressing a T cell response in a subject in a subject
having or at risk
of developing an inflammatory response by administering to the subject a
therapeutically
effective amount of an APC-targeted antibody operatively linked to IL -10 or a
fragment
thereof.
[0009] The term "operatively linked" refers to a situation where two
components are
combined to form the active complex prior to binding at the target site. For
example, an
antibody conjugated to one-half of a cohesin-dockerin complex and a cytokine
(e.g. IL-10) or
other molecule (e.g. antigen) complexed to the other one-half of the cohesin-
dockerin
complex are operatively linked through complexation of the cohesin and
dockerin molecules.
The term operatively linked is also intended to refer to covalent or chemical
linkages that
conjugate two molecules together.
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[0010] Yet
further aspects relate to methods and compositions for treating undesired
and/or abnormal immune responses without non-specific suppression of the host
immune
system. In particular, an anti-DC-ASGPR antibody or antigen binding fragment
thereof can
be used in compositions and methods described herein for generating anti-
pathogenic
antigen-specific T regulatory cells and/or for decreasing pathogenic T cell
responses.
[0011] The term
"anti-pathogenic antigen-specific T regulatory cells" refers to T cells
with beneficial and therapeutic properties. In one embodiment, the anti-
pathogenic antigen-
specific T regulatory cells are alloantigen-specific T regulatory cells. In
another
embodiment, the anti-pathogenic antigen-specific T regulatory cells is one
that produces IL-
10. The anti-pathogenic antigen-specific T regulatory cells may also be a CD4+
T cell.
[0012] The term
pathogenic T cell responses refers to abnormal or undesired T cell
responses that contribute to the pathology of autoimmune disease or to the
pathology of graft
versus host disease (GVHD) or graft rejection. In one embodiment, the
pathogenic T cell
response is an allogeneic T cell response. In a further embodiment, the
pathogenic T cell
response comprises allogeneic CD4+ and CD8+ T cells. In one embodiment, the
pathogenic
T cell response is one that comprises immune cells of the tissue graft.
[0013] A
further aspect of the disclosure relates to a method for preventing or
treating
GVHD in a subject in need thereof comprising administering to the subject an
anti-DC-
ASGPR antibody or antigen binding fragment thereof.
[0014] Graft-
versus-host disease (GVHD) is a common complication following an
allogeneic tissue transplant. It is commonly associated with stem cell or bone
marrow
transplant but the term also applies to other forms of tissue graft. Immune
cells (white blood
cells) in the tissue (the graft) recognize the recipient (the host) as
"foreign". The transplanted
immune cells then attack the host's body cells. GVHD may also occur after a
blood
transfusion if the blood products used have not been irradiated.
[0015] In
another instance, the disclosure describes a method for preventing or
treating graft rejection in a subject in need thereof comprising administering
to the subject an
anti-DC-ASGPR antibody or antigen binding fragment thereof.
[0016] Graft
rejection occurs when transplanted tissue is rejected by the recipient's
immune system, which destroys the transplanted tissue. Graft rejection may
also be referred
to as transplant rejection or host versus graft disease.
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[0017] In certain embodiments, the antibody or antigen binding fragment
specifically
binds to DC-ASGPR and activates DC-ASGPR. DC-asialoglycoprotein receptor (DC-
ASGPR) is a scavenger receptor carrying an immunoreceptor tyrosine-based
activation
motiflike motif. ASGPR may also be known as ASGR1, ASGPR1, CLEC4H1, and HL-1.
In
one embodiment, the antibody or antigen binding fragment thereof binds to
human DC-
ASGPR.
[0018] In some embodiments, the APC-targeted antibody targets one or more
APCs
of the group Langerhans cells, macrophages, dendritic cells, B cells, and
peripheral blood
mononuclear cells. In further embodiments, the APC-targeted antibody is
selected from an
antibody that specifically binds to MHC class I, MHC class II, CD1d, CD2, CD3,
CD4, CD8,
CD11b, CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45,
CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASGPR,
CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-
1, B7-2, IFN-y receptor, IL-2 receptor, ICAM-1, Fc y receptor, LOX-1, and
ASPGR.
[0019] In other embodiments, the APC-targeted antibody targets Langerhans
cells.
One example of an APC-targeted antibody to Langerhans cells is anti-Langerin.
In further
embodiments, the APC-targeted antibody targets macrophages. For example, the
APC-
targeted antibody may be anti-MARCO.
[0020] In yet further embodiments, the APC-targeted antibody targets one or
more
APCs of the group dendritic cells, B cells, and macrophages. In specific
embodiments, the
APC-targeted antibody targets dendritic cells. In some embodiments, the APC-
targeted
antibody comprises anti-CD40. In further embodiments, the anti-CD40 antibody
comprises
anti-CD40 clone 12E12 or fragments thereof. As shown in Example 1, anti-CD40
(12E12)-
IL-10 suppressed the expression of CD86. In some embodiments, the anti-CD40
antibody
comprises one or more CDRs having a sequence of SEQ ID NOS:31-33 and 37-39. In
other
embodiments, the anti-CD40 antibody comprises a heavy chain comprising one or
more
CDRs of SEQ ID NOS:31-33. In further embodiments, the anti-CD40 antibody
comprises a
light chain comprising one or more CDRs of SEQ ID NOS:37-39.
[0021] In specific embodiments, the anti-CD40 antibody is a humanized
antibody
comprising a heavy chain comprising three CDRs, wherein CDR1 comprises SEQ ID
NO:31,
CDR2 comprises SEQ ID NO:32, and CDR3 comprises SEQ ID NO:33. In further
embodiments, the anti-CD40 antibody is a humanized antibody comprising a light
chain
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comprising three CDRs, wherein CDR1 comprises SEQ ID NO:37, CDR2 comprises SEQ
ID
NO:38. and CDR3 comprises SEQ ID NO:39.
[0022] In some embodiments, the APC-targeted antibody comprises anti-DC-
ASGPR
or anti-Dectin-1. The anti-DC-ASGRP or anti-Dectin-1 may be one known in the
art or
described herein. In some embodiments the antibody comprises a variable region
comprising
an amino acid sequence selected from the sequences of SEQ ID NOs: 3, 8, 62,
64. 66, or 68.
In some embodiments, the antibody comprises a heavy or light chain with an
amino acid
sequence selected from the sequences of SEQ ID NOs: 1, 7, 61, 63, 65, 67, or
69-72. In some
embodiments, the antibody comprises one or more CDRs from the variable
reagion, heavy
chain, or light chain of SEQ ID NOs: 1, 3, 7, 8, 61, 62, 63, 64, 65, 66, 67,
or 68-72.
[0023] In some embodiments, the APC-targeted antibody comprises anti-DCIR.
In
specific embodiments, anti-DCIR antibody comprises anti-DCIR clone 9E8 or
fragments
thereof. In further embodiments, the anti-DCIR antibody comprises one or more
CDRs
having a sequence of SEQ ID NOS:18-20 or 24-26. In other embodiments, the anti-
DCIR
antibody comprises a heavy chain comprising one or more CDRs of SEQ ID NOS:18-
20. In
yet further embodiments, the anti-CD40 antibody comprises a light chain
comprising one or
more CDRs of SEQ ID NOS:24-26.
[0024] Certain aspects of the disclosure relate to a method for inhibiting
an
inflammatory or autoimmune response or for inducing tolerance in a subject in
need thereof
comprising administering to the subject a therapeutically effective amount of
an anti-CD40
antibody operatively linked to IL-10 or a fragment thereof, wherein the anti-
CD40 is a
humanized antibody having three heavy chain CDRs comprising an amino acid
sequence of
SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2), and SEQ ID NO:33 (CDR3) and three
light chain CDRs comprising an amino acid sequence of SEQ ID NO:37 (CDR1), SEQ
ID
NO:38 (CDR2), and SEQ ID NO:39 (CDR3).
[0025] Further aspects relate to a method for inhibiting an inflammatory or
autoimmune response or for inducing tolerance in a subject in need thereof
comprising
administering to the subject a therapeutically effective amount of an anti-DOR
antibody
operatively linked to IL-10 or a fragment thereof, wherein the anti-DCIR is a
humanized
antibody having three heavy chain CDRs from the variable region of anti-DCIR
9E8 heavy
chain (SEQ ID NO:17) and three light chain CDRs from the variable region of
anti-DCIR
9E8 light chain (SEQ ID NO:23).
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[0026] Further aspects relate to a method for inhibiting an inflammatory or
autoimmune response in a subject in need thereof comprising administering to
the subject a
therapeutically effective amount of an anti-DC-ASGPR antibody operatively
linked to IL-10
or a fragment thereof, wherein the anti-DC-ASGPR antibody is a humanized
antibody having
three heavy chain CDRs and three light chain CDRs from the variable regions of
an anti-DC-
ASGPR heavy chain and light chain pair selected from SEQ ID NO:3 and 8; SEQ ID
NO:58
and 60; SEQ ID NO:62 and 64; or SEQ ID NO:66 and 68; or is a humanized
antibody having
three heavy chain CDRs and three light chain CDRs from the heavy and light
chains of an
anti-DC-ASGPR heavy chain and light chain pair selected from SEQ ID NO:69 and
70 and
SEQ ID NO:71 and 72.
[0027] In some embodiments, the APC-targeted antibody or antibody conjugate
or
antigen binding fragment thereof comprises an amino acid sequence that is at
least or at most
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar (or
any
derivable range therein) to an APC-targeted antibody or antigen binding
fragment of any of
SEQ ID NOS:1, 2, 3. 7, 8, 10, 11, 13, 15-20, 22-26, 28-33, 35-39, or 45-114
(or any range
derivable therein). In further embodiments, the APC-targeted antibody
conjugate or antigen
binding fragment thereof comprises a variable region comprising an amino acid
sequence that
is at least or at most 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%. 98%, or 99% (or
any
range derivable therein) identical or similar to the APC-targeted antibody
variable region
described herein as SEQ ID NOS: 3, 8, 17, 23, 30, 36, 46, 48, 50, 52, 54, 56,
58, 60, 62, 64,
66, 68, 79, 81, 83, 85, 87, 89, 108, 110, 112, and 114. In further
embodiments, the antibody
comprises a CDR having an amino acid sequence corresponding to a CDR in any
one of SEQ
ID NOS: 2, 3,7, 8, 11, 13, 16, 17-20, 22-26, 29-33, 35-39, or 45-114 (or any
derivable range
therein). In some embodiments, the antibody comprises the CDRs of SEQ ID
NOS:18-20,
24-26, 31-33, or 37-39. In further embodiments, the APC-targeted antibody or
antigen
binding fragment thereof comprises a heavy or light chain amino acid sequence
that is at least
or at most 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any range
derivable therein) identical or similar to the APC-targeted antibody or
antigen binding
fragment of any of SEQ ID NOs:1, 2,7, 10, 11, 13, 15, 16, 22, 28, 29, 35, 45,
47, 49, 51, 53,
55, 57, 59, 61, 63, 65, 67, 69-78, 80, 82, 84, 86, 88, 90-107, 109, 111, or
113. In certain
embodiments, the antibody conjugate or antigen binding fragment thereof
comprises CDR1,
CDR2, and/or CDR3 from the heavy and/or light chain variable region of a APC-
targeted
antibody described herein. In certain embodiments, the antibody conjugate or
antigen binding
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fragment thereof comprises all three CDRs from the light chain variable region
and/or all
three CDRs from the heavy chain variable region of a APC-targeted antibody
described
herein.
[0028] In
certain embodiments, the antibody or antigen binding fragment specifically
binds to DC-ASGPR and activates DC-ASGPR. DC-asialoglycoprotein receptor (DC-
ASGPR) is a scavenger receptor carrying an immunoreceptor tyrosine-based
activation
motiflike motif. ASGPR may also me known as ASGR1, ASGPR1, CLEC4H1, and HL-1.
In one embodiment, the antibody or antigen binding fragment thereof binds to
human DC-
ASGPR.
[0029] In some
embodiments, the antibody or antigen binding fragment of the
methods and compositions described herein is an anti-DC-ASGPR antibody and
comprises an
amino acid sequence that is at least or at most 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%,
98%, or 99% (or any derivable range therein) identical or similar to the DC-
ASGPR antibody
or antigen binding fragment of any of SEQ ID NO: 2, 3, 7, 8, and 61-72 (or any
range
derivable therein). In a further embodiment. the DC-ASGPR antibody or antigen
binding
fragment thereof may include a polypeptide, peptide, or protein that is, is at
least, or is at
most 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any range
derivable
therein) identical or similar to an ASGPR binding polypeptide, such as Anti-
ASGPR_49C11_7H (heavy chain), SEQ ID NO:2; Anti-ASGPR_49C11_7K (light chain),
SEQ ID NO:7; anti-hASGPR_6.3H9.1D11H (heavy chain), SEQ ID NO:69; anti-
hASGPR_6.3H9.1D11K (light chain), SEQ ID NO:70; anti-hASGPR_5H8.1D4H (heavy
chain), SEQ ID NO:71; anti-hASGPR_5H8.1D4K (light chain), SEQ ID NO: 72; Anti-
ASGPR_4G2.2_ (heavy chain), SEQ ID NO: 57; Anti-ASGPR_4G2.2_ (light chain),
SEQ ID
NO: 59; Anti-ASGPR __ 5F1OH (heavy chain), SEQ ID NO:61; Anti ASGPR __ 5F1OH
(light
chain), SEQ ID NO: 63; Anti-ASGPR1H11 (heavy chain), SEQ ID NO: 65; or Anti-
ASGPRIH11 (light chain). SEQ ID NO: 67. In further embodiments, the DC-ASGPR
antibody or antigen binding fragment thereof comprises a variable region
comprising an
amino acid sequence that is at least or at most 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%,
98%, or 99% identical or similar to the DC-ASGPR antibody or antigen binding
fragment of
any of SEQ ID NOs: 3, 8, 62, 64, 66, and 68. In some embodiments, the antibody
comprises
at least or exactly one, two, or all three CDRs of a variable region from a
heavy or light chain
amino acid sequence selected from SEQ ID NO:2, 7, 57, 59, 61, 63, 65, 67, and
69-72. In
some embodiments, the antibody comprises at least or exactly one, two, or all
three CDRs of
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a variable region from a heavy or light chain variable region amino acid
sequence selected
from SEQ ID NO:3, 8, 58, 60, 62, 64, 66, and 68. In further embodiments, the
antibody
comprises at least or exactly 1, 2, 3, 4, 5, or 6 (or any derivable range
therein) CDRs from a
heavy and light chain antibody fragment selected from SEQ ID NOS: 2 and 7, SEQ
ID NOS:
57 and 59; SEQ ID NOS: 61 and 63; SEQ ID NOS: 65 and 67; SEQ ID NOS: 69 and
70; or
SEQ ID NOS: 71 and 72. In some embodiments, the antibody comprises at least or
exactly 1,
2, 3, 4, 5, or 6 (or any derivable range therein) CDRs from a heavy and light
chain variable
region antibody fragment selected from SEQ ID NOS: 3 and 8, SEQ ID NOS: 58 and
60;
SEQ ID NOS: 62 and 64; or SEQ ID NOS: 66 and 68..
[00301 The ASGPR antibody or antigen binding fragments described herein may
include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20,
21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95.
96, 97, 98, 99, 100 or
more variant amino acids within at least, or at most 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,
127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,
145, 146, 147,
148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,
163, 164, 165,
166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,
181, 182, 183,
184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,
199, 200, 201,
202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,
217, 218, 219,
220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,
235, 236, 237,
238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400,
500, 550, 1000 or
more contiguous amino acids, or any range derivable therein, of SEQ ID NO: 2,
3, 7, 8, and
61-72.
[0031] The APC-targeted antibody conjugate or antigen binding fragments
described
herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72,
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73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97,
98, 99, 100 or more variant amino acids (or any range derivable therein)
within at least, or at
most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73. 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84. 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120,
121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,
154, 155, 156,
157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,
172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,
190, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,
208, 209, 210,
211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228,
229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,
244, 245, 246,
247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids,
or any range
derivable therein, of any of SEQ ID NOs:1-5, 7-8, 10-11, 13, 15-20, 22-26. 28-
33, 35-39, or
45-114..
[0032] Embodiments are provided in which the APC-targeted antibody or
antigen
binding fragments comprises one or more CDR domains from an antibody that
specifically
binds to an antigen presenting cell surface protein. In particular
embodiments, the APC-
targeted antibody or antigen binding fragment thereof comprises one, two,
three, four, five,
six, or more CDR domains from among the VH or VL domain of the monoclonal
antibodies
listed herein in SEQ ID NOS: 3. 8, 17, 23, 30, 36, 46, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66,
68, 79, 81, 83, 85, 87, 89, 108, 110, 112, and 114. In certain aspects, the
APC-targeted
antibody or antigen binding fragment thereof comprises six CDR domains from
among the
VH or VL domains of the monoclonal antibodies: anti-Dectin-1 clone 11B6.4,
15E2.5, or
2D8.2D4; ASGPR clone 49C11, 4G2.2, 5F10, 1H11, 6.3H9.1D11, or 5H8.1D4; anti-
CD40
clone 12E12, 12B4.2C10, 24A3, or 11B6.1C3; anti-Lox-1 clone 11C8, 10F9, or
15C4; anti-
DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2, 29G10.3D9, 31A6.IF5, 3C2.2D9,
6C8.1G9,
9E8, or 2C9; or anti-Langrin clone 15B10 or 2G3. In some embodiments, the APC-
targeted
antibody or antigen binding fragment thereof comprises a sequence at least or
at most 70%,
75%, 80%, 85%, 90%, 95%, or 99% (or any range derivable therein) identical to
the VH or
VL domain of the monoclonal antibodies: anti-Dectin-1 clone 11B6.4, 15E2.5, or
2D8.2D4;
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ASGPR clone 49C11, 4G2.2, 5F10, 1H11, 6.3H9.1D11, or 5H8.1D4; anti-CD40 clone
12E12, 12B4.2C10, 24A3, or 11B6.1C3; anti-Lox-1 clone 11C8, 10F9, or 15C4;
anti-DCIR
clone 24A5.4A5, 24E7.3H9, 29E9.2E2, 29G10.3D9, 31A6.IF5, 3C2.2D9, 6C8.1G9,
9E8, or
2C9; or anti-Lanuin clone 15B10 or 2G3. Embodiments are provided in which the
APC-
targeted antibody or antigen binding fragment thereof comprises the VH domain
from the
monoclonal antibodies listed herein and/or the VL domain from the monoclonal
antibodies
listed herein. In further embodiments, the monoclonal antibody is selected
from: anti-Dectin-
1 clone 11B6.4, 15E2.5, or 2D8.2D4; ASGPR clone 49C11, 4G2.2, 5F10, 1H1l ,
6.3H9.1D11, or 5H8.1D4; anti-CD40 clone 12E12, 12B4.2C10, 24A3, or 11B6.1C3;
anti-
Lox-1 clone 11C8, 10F9, or 15C4; anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2,
29G10.3D9, 31A6.IF5, 3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrin clone
15B10 or
2G3.
[0033] In certain embodiments, the APC-targeted antibody or antigen binding
fragment thereof is recombinant. In certain aspects, the recombinant
polypeptide comprises
at least 90%, 95%, or 99% of one or more CDR domains from the VH or VL domain
of the
anti-Dectin-1 clone 11B6.4, 15E2.5. or 2D8.2D4; ASGPR clone 49C11, 4G2.2,
5F10. 1H11,
6.3H9.1D11, or 5H8.1D4; anti-CD40 clone 12E12, 12B4.2C10, 24A3, or 11B6.1C3;
anti-
Lox-1 clone 11C8, 10F9, or 15C4; anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2,
29G10.3D9, 31A6.1F5, 3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrin clone
15B10 or
2G3 monoclonal antibodies. In some embodiments, the recombinant polypeptide
comprises
two, three, four, five, six, or more CDR domains from the VH or VL domain of
the anti-
Dectin-1 clone 11B6.4, 15E2.5, or 2D8.2D4; ASGPR clone 49C11, 4G2.2, 5F10,
1H11,
6.3H9.1D11, or 5H8.1D4; anti-CD40 clone 12E12, 12B4.2C10, 24A3, or 11B6.1C3;
anti-
Lox-1 clone 11C8. 10F9, or 15C4; anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2,
29G10.3D9, 31A6.IF5, 3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrin clone
15B10 or
2G3 monoclonal antibodies.
[0034] In some embodiments, a recombinant polypeptide comprises i) CDR1
(SEQ
ID NO:37), CDR2 (SEQ ID NO:38), and/or CDR3 (SEQ ID NO:39) from the variable
light
chain of anti-CD40 12E12; and/or ii) CDR1 (SEQ ID NO:31), CDR2 (SEQ ID NO:32),
and/or CDR3 (SEQ ID NO:33) from the variable heavy chain of 12E12. In some
embodiments, a recombinant polypeptide comprises i) CDR1, CDR2, and/or CDR3
from the
variable light chain of anti-DCIR 9E8; and/or ii) CDRL CDR2, and/or CDR3 from
the
variable heavy chain of 9E8.
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[0035] Certain aspects are directed to methods of inhibiting an
inflammatory response
or inducing tolerance in a subject in need thereof comprising administering to
the subject an
effective amount of one or more APC-targeted antibody or antigen binding
fragment thereof
operatively linked to IL-10. The antibody can be a purified polyclonal
antibody, a purified
monoclonal antibody, a recombinant polypeptide, or a fragment thereof. In
certain aspects
the antibody is humanized or human. In still further aspects the antibody is a
recombinant
antibody segment. In certain aspects a monoclonal antibody includes one or
more of anti-
Dectin-1 clone 11136.4, 15E2.5, or 2D8.2D4; ASGPR clone 49C11, 4G2.2, 5F10,
1H11,
6.3H9.1D11, or 5H8.1D4; anti-CD40 clone 12E12, 12B4.2C10, 24A3, or 11B6.1C3;
anti -
Lox-1 clone 11C8, 10F9, or 15C4; anti-DCIR clone 24A5.4A5, 24E7.3H9, 29E9.2E2,
29G10.3D9, 31A6.IF5, 3C2.2D9, 6C8.1G9, 9E8, or 2C9; or anti-Langrin clone
15B10 or
2G3. An antibody can be administered at a dose of 0.1, 0.5, 1, 5, 10, 50, 100
mg or 1.1g/kg to
5, 10, 50, 100, 500 mg or ..tg/kg, or any range derivable therein.
[0036] The methods described herein provide a dose sparing effect such that
the
targeted delivery of IL-10 requires a smaller amount or dose to achieve the
same effect as a
non-targeted IL-10. In certain embodiments, the therapeutically effective
amount of the
APC-targeted antibodies operatively linked to IL-10 is at least 5, 10, 20, 50,
100, 500, or
1000 fold less than the dose of non-targeted IL-10. In further embodiments,
the
therapeutically effective amount of the APC-targeted antibodies operatively
linked to IL-10 is
greater than 50%, greater than 75%, greater than 80%, greater than 90% or
greater than 99%
less than the effective amount of the dose of non-targeted IL-10. The
therapeutically
effective amount of non-targeted IL-10 is known in the art, and may vary
depending on the
disease to be treated. In certain embodiments, the effective amount of non-
targeted IL-10 is
1, 5, 10, or 20 vg/kg. In one embodiment, the effective amount of non-targeted
IL-10 is 5
[Ig/kg. In other embodiments, the therapeutically effective amount of the APC-
targeted
antibodies operatively linked to IL-10 is at least 5 fold less than the dose
of non-targeted IL-
10.
[0037] In certain embodiments, the antibody is a human antibody, humanized
antibody, recombinant antibody, hi-specific antibody, chimeric antibody, a
nanobody, a
DARPin, an antibody derivative, a veneered antibody, a diabody, a monoclonal
antibody, or a
polyclonal antibody. In a specific embodiment, the antibody is a humanized
antibody.
[0038] In certain embodiments, the antibody is a non-naturally occurring
antibody. In some
embodiments, the antibody is non-naturally occurring since it comprises at
least two
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polypeptide segments from different sources. The different sources may be
different
mammals, such as human and mouse, for example.
[0039] In some
embodiments of the methods described herein, the subject is a human
subject. The term "subject," "individual" or "patient" is used interchangeably
herein and
refers to a vertebrate, for example a primate, a mammal or preferably a human.
Mammals
include, but are not limited to equines, canines, bovines, ovines, murines,
rats, simians,
humans, farm animals, sport animals and pets.
[0040] In some
embodiments, the subject is one that has an autoimmune disease or an
inflammatory disorder. The autoimmune disease or inflammatory disorder may be
one
known in the art and/or described herein. In some embodiments, the autoimmune
disease or
inflammatory disorder is selected from rheumatoid arthritis, allergy, asthma,
systemic onset
juvenile arthritis, inflammatory bowel disease, systemic lupus erythematosus,
multiple
sclerosis, type 1 diabetes, graft rejection, graft versus host disease,
colitis, and Crohn's
disease.
[0041] In some
embodiments, the subject is at risk for the development of a disease
mediated by a pathogenic T cell response. In further embodiments, the subject
is one that is
suffering from or at risk of suffering from an autoimmune disease or an auto-
inflammatory
disease. In a specific embodiment, the autoimmune disease or auto-inflammatory
disease is
selected from rheumatoid arthritis, allergy, asthma, systemic onset juvenile
arthritis,
inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis,
type 1
diabetes, graft rejection, graft versus host disease, colitis, and Crohn's
disease.
[0042] In some
embodiments, the subject is one that will receive or has received
transplanted tissues. In a related embodiment, the transplanted tissue is an
allograft. An
allograft (also known as allotransplantation, allogeneic transplant, or
hom*ograft) is the
transplantation of cells, tissues, or organs, to a recipient from a
genetically non-identical
donor of the same species. In a related embodiment, the subject is one that
has a
complication from the transplanted tissue, wherein the complication is graft
rejection or
GVHD.
[0043] In some
embodiments, the APC-targeted antibody is administered prior to
tissue transplantation. When the
antibody or antigen binding fragment thereof is
administered prior to tissue transplantation, the method may further comprise
the prevention
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of a complication relating to the transplanted tissue, wherein the
complication comprises
GVHD or graft rejection.
[0044] In some embodiments, the APC-targeted antibody is administered after
tissue
transplantation. When the antibody or antigen binding fragment thereof is
administered after
tissue transplantation, the method may further comprise treating a
complication from the
transplanted tissue, wherein the complication comprises GVHD or graft
rejection.
[0045] The tissue used in transplantation may be any tissue known in the
art to be
therapeutically useful for transplantation. Non-limiting examples of tissue
transplantations
include anterior cruciate ligament (ACL); joint reconstruction in the knee and
ankle; meniscal
replacement; reconstruction due to cancer or trauma; ridge augmentation in
dental
procedures; shoulder repair; spinal fusion; urological tissues; skin
transplants; corneal
transplants; heart transplants; heart valves; lung transplantation; intestinal
transplantation
such as isolated small bowel, intestine, or multivisceral; liver transplants;
kidney transplants;
bone marrow transplants; bone allograft; and ligament or tendon allograft.
[0046] In one embodiment, the transplanted tissue comprises immune cells.
The term
immune cells includes cells of the immune system that are involved in
defending the body
against both infectious disease and foreign materials. Immune cells may
include, for
example, neutorphils, eosinophils, basophils, lymphocytes such as b cells and
t cells, and
monocytes. T cells may include, for example, CD4+, CD8+, T helper cells,
cytotoxic T cells,
T cells, regulatory T cells, suppressor T cells, and natural killer cells.
[0047] In another embodiment, the transplanted tissue comprises stem cells.
Stem
cell types are known in the art. Non-limiting examples of stem cells include
hematopoietic
stem cells, neural stem cells, and embryonic stem cells. In one embodiment,
the stem cells
are hematopoietic stem cells. In a further embodiment, the transplanted tissue
comprises
bone marrow. In a yet further embodiment, the transplanted tissue comprises
blood. In
another embodiment, the transplanted tissue comprises skin cells.
[0048] In some embodiments, the APC-targeted antibody operatively linked to
IL-10
or a fragment thereof is administered in an amount effect for the maintenance
of pathogen-
specific immunity in the subject.
[0049] The IL-10 polypeptide may be a polypeptide or fragment of an IL-10
protein
known in the art or described herein by accession number NP_000563.1. In some
embodiments, the IL-10 polypeptide comprises SEQ ID NO:5. In some embodiments,
IL-10
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is covalently linked to the antibody. In some embodiments, the covalent
linkage is through a
peptide bond. The IL-10 polypeptide may also be linked to the antibody through
binding
polypeptides. In one embodiment, the binding polypeptides are dockerin and
cohesin.
[0050] In some embodiments, the method further comprises administration of
an
antigen or allergen. The antigen or allergen may be operatively linked to the
APC-targeted
antibody or to IL-10. In some embodiments, the antigen or allergen is
covalently linked (i.e.
by a peptide bond) to the APC-targeted antibody, antigen binding fragment
thereof, or IL-10.
When the antigen or allergen is operatively linked to the APC-targeted
antibody, antigen
binding fragment thereof, or IL-10, it may be linked through binding
polypeptides. Binding
peptides include, for example, dockerin and cohesin.
[0051] In further embodiments, the compositions or methods do not comprise
an
antigen or allergen or the administration of an allergy or antigen. For
example, an antigen or
allergen is not operatively (either directly or indirectly) linked to the APC-
targeted antibody.
In some embodiments, the compositions consists essentially of an antigen
presenting cell
(APC)-targeted antibody operatively linked to IL-10 or a fragment thereof.
[0052] In further embodiments, the compositions or methods do not comprise
a TLR
molecule or the administration of a TLR molecule.
[0053] In some embodiments, the antibody may comprise a y4 constant region.
In a
related embodiment, the y4 constant region comprises a substitution of
glutamic acid for
leucine at residue 235. In another embodiment, y4 constant region comprises a
substitution
of proline for serine at residue 228 in the hinge region.
[0054] In certain embodiments, the methods comprises multiple
administrations of
the composition. The administrations may be days, weeks, months, years, or
decades apart.
The compositions comprising the conjugate described herein may be administered
orally,
intravenously, subcutaneously, intradermally, intramuscularly, intranasally,
by injection, by
inhalation, mucosally, and/or by using a nebulizer.
[0055] In certain embodiments of the methods described herein, the anti-DC-
ASGPR
antibody or antigen binding fragment is administered in a therapeutically
effective amount.
In certain embodiments, the antibody or antigen binding fragment is
administered in an
amount that increases production of IL-10 in the subject. In a further
embodiment, the
antibody or antigen binding fragment is administered in an amount whereby the
subject
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maintains pathogen-specific immunity after administration of the antibody or
antigen binding
fragment.
[0056] In some embodiments, the anti-DC-ASGPR antibody or antigen binding
fragment thereof may be administered in a pharmaceutical composition. In some
embodiments, the pharmaceutical composition does not contain an antigen or
does not
contain detectable amounts of an antigen. In a further embodiment, the
pharmaceutical
composition consists essentially of an anti-DC-ASGPR antibody. In further
embodiments,
the antibody or antigen binding fragment thereof is not conjugated to an
antigen or is not is
not conjugated to a dockerin or cohesion molecule. In yet further embodiments,
the antibody
is not covalently or operatively linked to an antigen.
[0057] The term "operatively linked" refers to a situation where two
components are
combined to form the active complex prior to binding at the target site. For
example, an
antibody conjugated to one-half of a cohesion-docerin complex and an antigen
complexed to
the other one-half of the cohesion-docerin complex are operatively linked
through
complexation of the cohesion and docerin molecules.
[0058] Also disclosed herein are compositions comprising the antibodies and
antibody conjugates as described herein.
[0059] Aspects of the disclosure relate to APC-targeted antibodies and APC-
targeted
antibodies conjugated to IL-10 in pharmaceutical compositions and for use in
the preparation
of medicaments for treating an autoimmune and/or inflammatory condition
described herein.
[0060] Aspects also relate to an APC-targeted antibody or an APC-targeted
antibody
conjugated to IL-10 in pharmaceutical compositions and for use in the
preparation of
medicaments for inducing immune tolerance or suppressing a T cell response in
a subject
having or at risk of developing an autoimmune or inflammatory response,
wherein the
autoimmune or inflammatory response is caused by an autoimmune or inflammatory
disease
described herein.
[0061] As used herein the specification, "a" or "an" may mean one or more.
As used
herein in the claim(s), when used in conjunction with the word "comprising",
the words "a"
or "an" may mean one or more than one. A composition with the words
"consisting
essentially of' is intended to exclude any active ingredients not specifically
recited in the
composition. Examples of active ingredients include cytokines, TLRs, antigens,
adjuvants,
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etc... In any of the embodiments described herein, embodiments consisting
essentially of the
recited elements is also contemplated.
[0062] The use of the term "or" in the claims is used to mean "and/or"
unless
explicitly indicated to refer to alternatives only or the alternatives are
mutually exclusive,
although the disclosure supports a definition that refers to only alternatives
and "and/or." As
used herein "another" may mean at least a second or more.
[0063] Throughout this application, the term "about" is used to indicate
that a value
includes the inherent variation of error for the device, the method being
employed to
determine the value, or the variation that exists among the study subjects.
[0064] Other objects, features and advantages of the present invention will
become
apparent from the following detailed description. It should be understood,
however, that the
detailed description and the specific examples, while indicating preferred
embodiments of the
invention, are given by way of illustration only, since various changes and
modifications
within the spirit and scope of the invention will become apparent to those
skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] The following drawings form part of the present specification and
are included
to further demonstrate certain aspects of the present invention. The invention
may be better
understood by reference to one or more of these drawings in combination with
the detailed
description of specific embodiments presented herein.
[0066] FIG. 1 shows that recombinant fusion proteins of antibody and IL-10
target
human APCs.
[0067] FIG. 2 shows that antibody-IL-10 fusion proteins inhibit DC
maturation
induced by E. coli lipopolysaccharide.
[0068] FIG. 3 demonstrates the dose sparing effects of the targeted IL-10
fustion
proteins.
[0069] FIG. 4 shows that treatment of PBMCs from healthy donors with anti-
DC-
ASGPR mAb reduces the proliferation of CD4+ and CD8+ T cells from MHC-
mismatched
donors. Summary of data generated with PBMCs from 6 pairs of MHC-mismatched
healthy
donors (Mean SD).
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[0070] FIG. 5
shows that blocking IL-10 partially recovers allogeneic CD4+ T cells
proliferation induced by anti-DC-ASGPR-activated PBMCs.
[0071] FIG. 6
shows that allogeneic CD4+ T cells cultured with anti-DC-ASGPR-
activated PBMCs secrete decreased IFNy but increased IL-10 during
restimulation.
[0072] FIG. 7
demonstrates the effect of anti-DC-ASGPR mAb on delay of
xenogenic GVHD in NOG mice.
[0073] FIG. 8
shows the hypothetical pathways of DC-ASGPR-induced suppression
of allogeneic T cell responses.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0074] Methods
and compositions described herein can be used to treat or prevent
inflammatory and/or autoimmune disorders or for inducing immune tolerance. It
was
discovered that delivering the anti-inflammatory cytokine, IL-10, to human
antigen
presenting cells (APCs) can suppress and alter the pathophysiologic functions
of APCs in the
patients. It is contemplated that targeted delivery of anti-inflammatory
cytokines to the APCs
in the patients is expected to result in more effective and pro-longed immune
tolerance in the
patients. Delivering IL-10 to APCs can directly suppress ongoing inflammatory
reaction in a
short term period and can also induce regulatory T cells which can prolong the
effectiveness
of the treatment. Furthermore, the methods described herein provide a dose
sparing effect
such that the targeted delivery of IL-10 requires a smaller amount or dose to
achieve the same
effect as a non-targeted IL-10.
I. Antibodies
[0075] Methods
and compositions of the disclosure relate to APC-targeted antibodies
and antibody binding fragments thereof. In some embodiments, the antibodies
are
operatively linked to IL-10. As used herein, an "antibody" includes whole
antibodies and any
antigen binding fragment or a single chain thereof. Thus the term "antibody"
includes any
protein or peptide-containing molecule that comprises at least a portion of an
immunoglobulin molecule. Examples
of such include, but are not limited to a
complementarity determining region (CDR) of a heavy or light chain or a ligand
binding
portion thereof, a heavy chain or light chain variable region, a heavy chain
or light chain
constant region, a framework (FR) region or any portion thereof or at least
one portion of a
binding protein.
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[0076] The antibody can be any of the various antibodies described herein,
non-
limiting, examples of such include a polyclonal antibody, a monoclonal
antibody, a chimeric
antibody, a recombinant antibody, a human antibody, a veneered antibody, a
diabody, a
humanized antibody, an antibody derivative, a recombinant humanized antibody,
or a
derivative or fragment of each thereof.
[0077] Antibodies can be generated using conventional techniques known in
the art
and are well-described in the literature. Several methodologies exist for
production of
polyclonal antibodies. For example, polyclonal antibodies are typically
produced by
immunization of a suitable mammal such as, but not limited to, chickens,
goats, guinea pigs,
hamsters, horses, mice, rats, and rabbits. An antigen is injected into the
mammal, induces the
B-lymphocytes to produce immunoglobulins specific for the antigen.
Immunoglobulins may
be purified from the mammal's serum. Common variations of this methodology
include
modification of adjuvants, routes and site of administration, injection
volumes per site and
the number of sites per animal for optimal production and humane treatment of
the animal.
For example, adjuvants typically are used to improve or enhance an immune
response to
antigens. Most adjuvants provide for an injection site antigen depot, which
allows for a stow
release of antigen into draining lymph nodes. Other adjuvants include
surfactants which
promote concentration of protein antigen molecules over a large surface area
and
immunostimulatory molecules. Non-limiting examples of adjuvants for polyclonal
antibody
generation include Freund's adjuvants, Ribi adjuvant system. and Titermax.
Polyclonal
antibodies can be generated using methods known in the art some of which are
described in
U.S. Pat. Nos. 7.279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746;
6,322,788; 5,686,073;
and 5,670,153.
[0078] Unless specified otherwise, the antibodies can be polyclonal or
monoclonal
and can be isolated from any suitable biological source, e.g., murine, rat,
sheep or canine.
[0079] In a specific embodiment, the antibody is a monoclonal antibody. As
used
herein, "monoclonal antibody" refers to an antibody obtained from a
substantially
hom*ogeneous antibody population. Monoclonal antibodies are highly specific, as
each
monoclonal antibody is directed against a single determinant on the antigen.
The antibodies
may be detectably labeled, e.g., with a radioisotope, an enzyme which
generates a detectable
product, a fluorescent protein, and the like. The antibodies may be further
conjugated to
other moieties, such as members of specific binding pairs, e.g., biotin
(member of biotin-
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avidin specific binding pair), and the like. The antibodies may also be bound
to a solid
support, including, but not limited to. polystyrene plates or beads, and the
like.
[0080] Monoclonal antibodies can be generated using conventional hybridoma
techniques known in the art and well-described in the literature. For example,
a hybridoma is
produced by fusing a suitable immortal cell line (e.g., a myeloma cell line
such as, but not
limited to, Sp2/0. Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8,653, Sp2
SA3, Sp2
MAI, Sp2 SS1, Sp2 SA5, U397, MIA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-
562, COS, RAJI, NIH 313, HL-60, MLA 144, NAMAIWA. NEURO 2A, CHO, PerC.6,
YB2/0) or the like, or heteromyelomas, fusion products thereof, or any cell or
fusion cell
derived there from, or any other suitable cell line as known in the art, with
antibody
producing cells, such as, but not limited to, isolated or cloned spleen,
peripheral blood,
lymph, tonsil, or other immune or B cell containing cells, or any other cells
expressing heavy
or light chain constant or variable or framework or CDR sequences, either as
endogenous or
heterologous nucleic acid, as recombinant or endogenous, viral, bacterial,
algal, prokaryotic,
amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat,
sheep, primate,
eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA
or
RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the
like or
any combination thereof. Antibody producing cells can also be obtained from
the peripheral
blood or, preferably the spleen or lymph nodes, of humans or other suitable
animals that have
been immunized with the antigen of interest. Any other suitable host cell can
also be used for
expressing-heterologous or endogenous nucleic acid encoding an antibody,
specified
fragment or variant thereof. The fused cells (hybiidomas) or recombinant cells
can be isolated
using selective culture conditions or other suitable known methods, and cloned
by limiting
dilution or cell sorting, or other known methods.
[0081] Other suitable methods of producing or isolating antibodies of the
requisite
specificity can be used, including, but not limited to, methods that select
recombinant
antibody from a peptide or protein library (e,g., but not limited to, a
bacteriophage, ribosome,
oligonucleotide, cDNA, or the like, display library; e.g., as available from
various
commercial vendors such as MorphoSys (Martinsreid/Planegg, Del.), BioInvent
(Lund,
Sweden), AfTitech (Oslo, Norway) using methods known in the art. Art known
methods are
described in the patent literature some of which include U.S. Pat. Nos.
4,704,692; 5,723.323;
5,763,192; 5,814,476; 5.817,483; 5,824,514; 5,976,862. Alternative methods
rely upon
immunization of transgenic animals (e.g., SCID mice, Nguyen et al. (1977)
Microbiol.
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Immunol. 41:901-907 (1997); Sandhu et al. (1996) Crit, Rev. Biotechnol. 16:95-
118; Eren et
al. (1998) Mumma 93:154-161 that are capable of producing a repertoire of
human
antibodies, as known in the art and/or as described herein. Such techniques,
include, but are
not limited to, ribosome display Wanes et al. (1997) Proc. Natl. Acad. Sci.
USA, 94:4937-
4942; Hanes et al, (1998) Proc. Natl. Acad. Sci. USA 95:14130-14135); single
cell antibody
producing technologies (e,g., selected lymphocyte antibody method ("SLAM")
(U.S. Pat. No.
5,627,052, Wen et al, (1987) J. Immunol 17:887-892; Babcook et al. (1996)
Proc. Natl. Acad.
Sci. USA 93:7843-7848); gel microdroplet and flow cytometry (Powell et al.
(1990)
Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass).; Gray et al.
(1995) J. Imm.
Meth. 182:155-163; and Kenny et al, (1995) Bio. Technol. 13:787-790); B-cell
selection
(Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134).
[0082] The
terms "polyclonal antibody" or "polyclonal antibody composition" as
used herein refer to a preparation of antibodies that are derived from
different B-cell lines.
They are a mixture of immunoglobulin molecules secreted against a specific
antigen, each
recognizing a different epitope.
[0083] The term
"mouse antibody" as used herein, is intended to include antibodies
having variable and constant regions derived from mouse germline
immunoglobulin
sequences.
[0084] As used
herein, chimeric antibodies are antibodies whose light and heavy
chain genes have been constructed, typically by genetic engineering, from
antibody variable
and constant region genes belonging to different species. In one embodiment,
the antibody is
a mouse/human chimeric antibody.
[0085] In
further embodiments, the antibody comprises a modification and is an
"antibody derivative." The term
"antibody derivative" includes post-translational
modification to linear polypeptide sequence of the antibody or fragment. For
example, U.S.
Pat. No. 6,602,684 B1 describes a method for the generation of modified glycol-
forms of
antibodies, including whole antibody molecules, antibody fragments, or fusion
proteins that
include a region equivalent to the Fc region of an immunoglobulin, having
enhanced Fe-
mediated cellular toxicity, and glycoproteins so generated.
[0086] The
antibodies provided herein also include derivatives that are modified by
the covalent attachment of any type of molecule to the antibody such that
covalent attachment
does not prevent the antibody from generating an anti-idiotypic response.
Antibody
- 20 -
derivatives include, but are not limited to, antibodies that have been
modified by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand
or other protein,
etc. Additionally, the derivatives may contain one or more non-classical amino
acids.
[0087] Antibody derivatives can also be prepared by delivering a
polynucleotide
encoding an antibody to a suitable host such as to provide transgenic animals
or mammals,
such as goats, cows, horses, sheep, and the like, that produce such antibodies
in their milk.
These methods are known in the art and are described for example in U.S. Pat.
Nos. 5,827,690;
5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.
[0088] Antibody derivatives also can be prepared by delivering a
polynucleotide to
provide transgenic plants and cultured plant cells (e.g., but not limited to
tobacco, maize, and
duckweed) that produce such antibodies, specified portions or variants in the
plant parts or in
cells cultured therefrom. Antibody derivatives have also been produced in
large amounts from
transgenic plant seeds including antibody fragments, such as single chain
antibodies (scFv's),
including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998)
Plant Mol. Biol.
38:101-109. Thus, antibodies can also be produced using transgenic plants,
according to know
methods.
[0089] Antibody derivatives also can be produced, for example, by adding
exogenous
sequences to modify immunogenicity or reduce, enhance or modify binding,
affinity, on-rate,
off-rate, avidity, specificity, half-life, or any other suitable
characteristic. Generally part or all
of the non-human or human CDR sequences are maintained while the non-human
sequences
of the variable and constant regions are replaced with human or other amino
acids.
[0090] The term "variable region" refers to a portion of the antibody that
gives the
antibody its specificity for binding antigen. The variable region is typically
located at the ends
of the heavy and light chains. Variable loops of 13-strands, three each on the
light (VL) and
heavy (VH) chains are responsible for binding to the antigen. These loops are
referred to as the
"complementarity determining regions" (CDRs).
[0091] In general, the CDR residues are directly and most substantially
involved in
influencing antigen binding. Humanization or engineering of antibodies can be
performed
using any known method such as, but not limited to, those described in U.S.
Pat. Nos.
5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323;
5,766,886;
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5,714,352; 6,204,023; 6,180,370: 5,693,762; 5,530.101; 5,585,089; 5,225,539;
and
4,816,567.
[0092] The term "constant region" refers to a portion of the antibody that
is identical
in all antibodies of the same isotype. The constant region differs in
antibodies of different
isotypes.
[0093] In one embodiment, the antibody is a humanized antibody. As used
herein,
the term "humanized antibody" or "humanized immunoglobulin" refers to a
human/non-
human chimeric antibody that contains a minimal sequence derived from non-
human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins
(recipient antibody) in which residues from a variable region of the recipient
are replaced by
residues from a variable region of a non-human species (donor antibody) such
as mouse, rat,
rabbit, or non-human primate having the desired specificity, affinity and
capacity.
Humanized antibodies may comprise residues that are not found in the recipient
antibody or
in the donor antibody. The humanized antibody can optionally also comprise at
least a
portion of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin, a non-human antibody containing one or more amino acids in a
framework
region, a constant region or a CDR, that have been substituted with a
correspondingly
positioned amino acid from a human antibody. In general, humanized antibodies
are
expected to produce a reduced immune response in a human host, as compared to
a non-
humanized version of the same antibody. The humanized antibodies may have
conservative
amino acid substitutions which have substantially no effect on antigen binding
or other
antibody functions. Conservative substitutions groupings include:glycine-
alanine, valine-
leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine,
serine-threonine
and asparagine-glutamine.
[0094] Chimeric, humanized or primatized antibodies can be prepared based
on the
sequence of a reference monoclonal antibody prepared using standard molecular
biology
techniques. DNA encoding the heavy and light chain immunoglobulins can be
obtained from
the hybridoma of interest and engineered to contain non-reference (e.g.,
human)
immunoglobulin sequences using standard molecular biology techniques. For
example, to
create a chimeric antibody, the murine variable regions can be linked to human
constant
regions using methods known in the art (U.S. Pat. No. 4,816,567). To create a
humanized
antibody, the murine CDR regions can be inserted into a human framework using
methods
known in the art (U.S. Pat. No. 5,225,539 and U.S. Pat. Nos. 5,530,101;
5,585,089; 5,693,762
- 22 -
and 6,180,370). Similarly, to create a primatized antibody the murine CDR
regions can be
inserted into a primate framework using methods known in the art (WO 93/02108
and WO
99/55369). Methods of determining CDRs from the sequence of a variable region
are known
in the art (see, for example, Zhao and Lu, "A germline knowledge based
computational
approach for determining antibody complementarity determining regions." Mol.
Immunol.,
(2010) 47(4):694-700).
[0095] Techniques for making partially to fully human antibodies are known
in the art
and any such techniques can be used. According to one embodiment, fully human
antibody
sequences are made in a transgenic mouse which has been engineered to express
human heavy
and light chain antibody genes. Multiple strains of such transgenic mice have
been made which
can produce different classes of antibodies. B cells from transgenic mice
which are producing
a desirable antibody can be fused to make hybridoma cell lines for continuous
production of
the desired antibody. (See for example, Russel et al. (2000) Infection and
Immunity April
2000:1820-1826; Gallo et al. (2000) European J. of Immun. 30:534-540; Green
(1999) J. of
Immun. Methods 231:11-23; Yang et al. (1999A) J. of Leukocyte Biology 66:401-
410; Yang
(1999B) Cancer Research 59(6):1236-1243; Jakobovits (1998) Advanced Drug
Reviews
31:33-42; Green and Jakobovits (1998) J. Exp. Med. 188(3):483-495; Jakobovits
(1998) Exp.
Opin. Invest. Drugs 7(4):607-614; Tsuda et al. (1997) Genomics 42:413-421;
Sherman-Gold
(1997) Genetic Engineering News 17(14); Mendez et al. (1997) Nature Genetics
15:146-156;
Jakobovits (1996) Weir's Handbook of Experimental Immunology, The Integrated
Immune
System Vol. IV, 194.1-194.7; Jakobovits (1995) Current Opinion in
Biotechnology 6:561-566;
Mendez et al, (1995) Genomics 26:294-307; Jakobovits (1994) Current Biology
4(8):761-763;
Arbones et al. (1994) :Immunity 1(4):247-260; Jakobovits (1993) Nature
362(6417):255-258;
Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555; and U.S.
Pat. No.
6,075,181.)
[0096] Antibodies also can be modified to create chimeric antibodies.
Chimeric
antibodies are those in which the various domains of the antibodies' heavy and
light chains are
coded for by DNA from more than one species. See, e.g., U.S. Pat. No.
4,816,567.
[0097] Alternatively, antibodies can also be modified to create veneered
antibodies.
Veneered antibodies are those in which the exterior amino acid residues of the
antibody of one
species are judiciously replaced or "veneered" with those of a second species
so that the
antibodies of the first species will not be immunogenic in the second species
thereby reducing
the immunogenicity of the antibody. Since the antigenicity of a protein is
primarily
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Date Recue/Date Received 2021-10-08
dependent on the nature of its surface, the immunogenicity of an antibody
could be reduced by
replacing the exposed residues which differ from those usually found in
another mammalian
species antibodies. This judicious replacement of exterior residues should
have little, or no,
effect on the interior domains, or on the interdomain contacts. Thus, ligand
binding properties
should be unaffected as a consequence of alterations which are limited to the
variable region
framework residues. The process is referred to as "veneering" since only the
outer surface or
skin of the antibody is altered, the supporting residues remain undisturbed.
[0098] The procedure for "veneering" makes use of the available sequence
data for
human antibody variable domains compiled by Kabat et al. (1987) Sequences of
Proteins of
Immunological interest, 4th ed., Bethesda, Md., National Institutes of
Health,. Non-limiting
examples of the methods used to generate veneered antibodies include EP
519596; U.S. Pat.
No. 6,797,492; and described in Padlan et al. (1991) Mol. Immunol. 28(4-5):489-
498.
[0099] The term "antibody derivative" also includes "diabodies" which are
small
antibody fragments with two antigen-binding sites, wherein fragments comprise
a heavy chain
variable domain (VH) connected to a light chain variable domain (VL) in the
same polypeptide
chain. (See for example, EP 404,097; WO 93/11161; and Hollinger et al. (1993)
Proc. Natl.
Acad. Sci. USA 90:6444-6448.) 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. (See also, U.S.
Pat. No.
6,632,926 to Chen et al, which discloses antibody variants that have one or
more amino acids
inserted into a hypervariable region of the parent antibody and a binding
affinity for a target
antigen which is at least about two fold stronger than the binding affinity of
the parent antibody
for the antigen).
[00100] The term "antibody derivative" further includes engineered antibody
molecules,
fragments and single domains such as scFv, dAbs, nanobodies, minibodies,
Unibodies, and
Affibodies & Hudson (2005) Nature Biotech 23(9):1126-36; U.S. Patent
Publication US
2006/0211088; PCT Publication W02007/059782; U.S. Pat. No. 5,831,012).
[00101] The term "antibody derivative" further includes "linear
antibodies". The
procedure for making linear antibodies is known in the art and described in
Zapata et al.
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(1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pair
of tandem Ed
segments (VH-CH 1-VH-CH1) which form a pair of antigen binding
regions.
Linear antibodies can be bispecific or monospecific.
[00102] Antibodies can be recovered and purified from recombinant cell
cultures by
known methods including, but not limited to, protein A purification, ammonium
sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. High
performance liquid chromatography ("HPLC") can also be used for purification.
[00103] It also is possible to determine without undue experimentation,
whether an
antibody has the same specificity as antibodies contemplated herein by
determining whether
the antibody being tested prevents an antibody from binding the protein or
polypeptide with
which the antibody is normally reactive. If the antibody being tested competes
with an
antibody used in embodiments described herein as shown by a decrease in
binding by the
monoclonal antibody, then it is likely that the two antibodies bind to the
same or a closely
related epitope. Alternatively, one can pre-incubate an antibody for use in
embodiments with
a protein with which it is normally reactive, and determine if the antibody
being tested is
inhibited in its ability to bind the antigen. If the antibody being tested is
inhibited then, in all
likelihood, it has the same, or a closely related, epitopic specificity as the
antibody for use in
embodiments described herein.
[00104] The term "antibody" also is intended to include antibodies of all
immunoglobulin isotypes and subclasses unless specified otherwise. An isotype
refers to the
genetic variations or differences in the constant regions of the heavy and
light chains of an
antibody. In humans, there are five heavy chain isotypes: IgA, IgD, IgG. IgE.
and IgM and
two light chain isotypes: kappa and lambda. The IgG class is divided into four
isotypes:
IgGl, IgG2, IgG3 and IgG4 in humans, and IgG 1, IgG2a, IgG2b and IgG3 in mice.
They
share more than 95% hom*ology in the amino acid sequences of the Fc regions but
show
major differences in the amino acid composition and structure of the hinge
region. Particular
isotypes of a monoclonal antibody can be prepared either directly by selecting
from an initial
fusion, or prepared secondarily, from a parental hybridoma secreting a
monoclonal antibody
of different isotype by using the sib selection technique to isolate class
switch variants using
the procedure described in Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA
82:8653 or
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Spira et al. (1984) J. Immunol. Methods 74:307. Alternatively, recombinant DNA
techniques
may be used.
[00105] The isolation of other monoclonal antibodies with the specificity
of the
monoclonal antibodies described herein can also be accomplished by one of
ordinary skill in
the art by producing anti-idiotypic antibodies. Herlyn et al. (1986) Science
232:100. An anti-
idiotypic antibody is an antibody which recognizes unique determinants present
on the
monoclonal antibody of interest.
[00106] In some aspects, it will be useful to detectably or therapeutically
label the
antibody. Methods for conjugating antibodies to these agents are known in the
art. For the
purpose of illustration only, antibodies can be labeled with a detectable
moiety such as a
radioactive atom, a chromophore, a fluorophore, or the like. Such labeled
antibodies can be
used for diagnostic techniques, either in vivo, or in an isolated test sample.
[00107] In certain embodiments, the antibody or antigen binding fragment
further
comprises a modification. The modification may be a conservative amino acid
mutation
within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions, of conservative
amino acid
mutations in the Fc hinge region, pegylation, conjugation to a serum protein,
conjugation to
human serum albumin, conjugation to a detectable label, conjugation to a
diagnostic agent,
conjugation to an enzyme, conjugation to a fluorescent, luminescent, or
bioluminescent
material, conjugation to a radioactive material, or conjugation to a
therapeutic agent.
[00108] As used herein, the term "label" intends a directly or indirectly
detectable
compound or composition that is conjugated directly or indirectly to the
composition to be
detected, e.g., polynucleotide or protein such as an antibody so as to
generate a "labeled"
composition. The term also includes sequences conjugated to the polynucleotide
that will
provide a signal upon expression of the inserted sequences, such as green
fluorescent protein
(GFP) and the like. The label may be detectable by itself (e.g. radioisotope
labels or
fluorescent labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a
substrate compound or composition which is detectable. The labels can be
suitable for small
scale detection or more suitable for high-throughput screening. As such,
suitable labels
include, but are not limited to radioisotopes, fluorochromes, chemiluminescent
compounds,
dyes, and proteins, including enzymes. The label may be simply detected or it
may be
quantified . A response that is simply detected generally comprises a response
whose
existence merely is confirmed, whereas a response that is quantified generally
comprises a
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response having a quantifiable (e.g., numerically reportable) value such as an
intensity,
polarization, and/or other property. In luminescence or fluorescence assays,
the detectable
response may be generated directly using a luminophore or fluorophore
associated with an
assay component actually involved in binding, or indirectly using a
luminophore or
fluorophore associated with another (e.g., reporter or indicator) component.
[00109] Examples
of luminescent labels that produce signals include, but are not
limited to bioluminescence and chemiluminescence. Detectable luminescence
response
generally comprises a change in, or an occurrence of, a luminescence signal.
Suitable
methods and luminophores for luminescently labeling assay components are known
in the art
and described for example in Haugland, Richard P. (1996) Handbook of
Fluorescent Probes
and Research Chemicals (6th ed.). Examples of luminescent probes include,
but are not
limited to, aequorin and luciferases.
[00110] Examples
of suitable fluorescent labels include, but are not limited to,
fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,
methyl-
coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM.,
and Texas
Red. Other suitable optical dyes are described in the Haugland, Richard P.
(1996) Handbook
of Fluorescent Probes and Research Chemicals (6th ed.).
[00111] In
another aspect, the fluorescent label is functionalized to facilitate covalent
attachment to a cellular component present in or on the surface of the cell or
tissue such as a
cell surface marker. Suitable
functional groups, including, but not are limited to,
isothiocyanate groups, amino groups, hal oacetyl groups, maleimides,
succinimidyl esters, and
sulfonyl halides, all of which may be used to attach the fluorescent label to
a second
molecule. The choice of the functional group of the fluorescent label will
depend on the site
of attachment to either a linker, the agent, the marker, or the second
labeling agent.
[00112]
Attachment of the fluorescent label may be either directly to the cellular
component or compound or alternatively, can by via a linker. Suitable binding
pairs for use
in indirectly linking the fluorescent label to the intermediate include, but
are not limited to,
antigens/antibodies, e.g., rhodamine/anti-rhodamine, biotin/avidin and
biotin/strepavidin.
[00113] The
coupling of antibodies to low molecular weight haptens can increase the
sensitivity of the antibody in an assay. The haptens can then be specifically
detected by
means of a second reaction. For example, it is common to use haptens such as
biotin, which
- 27 -
reacts avidin; or dinitrophenol, pyridoxal, and fluorescein, which can react
with specific anti-
hapten antibodies.
[00114] The variable region of an antibody can be modified by mutating
amino acid
residues within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions to improve
one or
more binding properties (e.g., affinity) of the antibody. Mutations may be
introduced by site-
directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody
binding, or
other functional property of interest, can be evaluated in appropriate in
vitro or in vivo assays.
Preferably conservative modifications are introduced and typically no more
than one, two,
three, four or five residues within a CDR region are altered. The mutations
may be amino acid
substitutions, additions or deletions.
[00115] Framework modifications can be made to the antibodies to decrease
immunogenicity, for example, by "backmutating" one or more framework residues
to the
corresponding germline sequence.
[00116] In addition, an antibody may be engineered to include modifications
within the
Fe region to alter one or more functional properties of the antibody, such as
serum half-fife,
complement fixation, Fe receptor binding, and/or antigen-dependent cellular
cytotoxicity.
Such modifications include, but are not limited to, alterations of the number
of cysteine residues
in the hinge region to facilitate assembly of the light and heavy chains or to
increase or decrease
the stability of the antibody (U.S. Pat. No. 5,677,425) and amino acid
mutations in the Fc hinge
region to decrease die biological half life of the antibody (U.S. Pat. No.
6,165,745).
[00117] Additionally, one or more antibodies may be chemically modified.
Glycosylation of an antibody can be altered, for example, by modifying one or
more sites of
glycosylation within the antibody sequence to increase the affinity of the
antibody for antigen
(U.S. Pat. Nos. 5,714,350 and 6,350,861). Alternatively, to increase antibody-
dependent cell-
mediated cytotoxicity, a hypofucosylated antibody having reduced amounts of
fucosyl residues
or an antibody having increased bisecting GleNac structures can be obtained by
expressing the
antibody in a host cell-- with altered glycosylation mechanism (Shields,
R. L. et al., 2002
J. Biol. Chem. 277:26733-26740; Umana et al., 1999 Nat. Biotech. 17:176-180).
[00118] Antibodies can be pegylated to increase biological half-life by
reacting the
antibody or fragment thereof with polyethylene glycol (PEG) or a reactive
ester or aldehyde
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Date Recue/Date Received 2021-10-08
derivative of PEG, under conditions in which one or more PEG groups become
attached to the
antibody or antibody fragment. Antibody pegylation may be carried out by an
acylation
reaction or an alkylation reaction with a reactive PEG molecule (or an
analogous reactive
watersoluble polymer). As used herein, the term "polyethylene glycol" is
intended to
encompass any of the forms of PEG that have been used to derivatize other
proteins, such as
mono (CI-CIO) alkoxy- or aryl oxy-polyethyl ene glycol or polyethylene glycol-
maleimide. The
antibody to be pegylated can be an aglycosylated antibody. Methods for
pegylating proteins
are known in the art and can be applied to one or more antibodies(EP 0 154 316
and EP 0 401
384).
[00119] Additionally, antibodies may be chemically modified by conjugating
or fusing
the antigen-binding region of the antibody to serum protein, such as human
serum albumin, to
increase half-life of the resulting molecule. Such approach is for example
described in EP
0322094 and EP 0 486 525.
[00120] The antibodies or fragments thereof may be conjugated to a
diagnostic agent
and used diagnostically, for example, to monitor the development or
progression of a disease
and determine the efficacy of a given treatment regimen. Examples of
diagnostic agents
include enzymes, prosthetic groups, fluorescent materials, luminescent
materials,
bioluminescent materials, radioactive materials, positron emitting metals
using various
positron emission tomographies, and nonradioactive paramagnetic metal ions.
The detectable
substance may be coupled or conjugated either directly to the antibody or
fragment thereof, or
indirectly, through al inker using techniques known in the art. Examples of
suitable enzymes
include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase. Examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent
materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin. An example of a luminescent
material includes
luminol. Examples of bioluminescent materials include luciferase, luciferin,
and aequorin.
Examples of suitable radioactive material include Indium-111, Lutetium-171,
Bismuth-212,
Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-
125,
Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-111, Gallium-67,
Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166, Holmium-166,
Rhenium-186, Ithenium-188, Rhenium-189, Lead-212, Radium-223, Actinium-225,
Iron-59,
Selenium-75, Arsenic-77, Strontium-89, Molybdenum-99, Rhodium-
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1105, Palladium-109, Praseodymium-143, Promethium-149, Erbium-169, Iridium-
194, Gold-
198, Gold-199, and Lead-211. Monoclonal antibodies may be indirectly
conjugated with
radiometal ions through the use of bifunctional chelating agents that are
covalently linked to
the antibodies. Chelatin2 agents may be attached through amities (Meares et
al., 1984 Anal.
Biochem. 142: 68-78); sulfhydral groups (Koyama 1994 Chem. Abstr. 120:
217262t) of
amino acid residues and carbohydrate groups (Rodwell et al. 1986 PNAS USA 83:
2632-
2636; Quadri et al. 1993 Nucl. Med. Biol. 20: 559-570).
[00121] Additional suitable conjugated molecules include ribonuclease
(RNase),
DNase I, an antisense nucleic acid. an inhibitory RNA molecule such as a siRNA
molecule,
an immunostimulatory nucleic acid, aptamers, ribozymes, triplex forming
molecules, and
external guide sequences. Aptamers are small nucleic acids ranging from 15-50
bases in
length that fold into defined secondary and tertiary structures, such as stem-
loops or G-
quartets, and can bind small molecules, such as ATP (U.S. Pat. No. 5,631,146)
and
theophiline (U.S. Pat. No. 5,580,737), as well as large molecules, such as
reverse
transcriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No.
5,543,293). Ribozymes
are nucleic acid molecules that are capable of catalyzing a chemical reaction,
either
intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid
substrates
through recognition and binding of the target substrate with subsequent
cleavage. Triplex
forming function nucleic acid molecules can interact with double-stranded or
single-stranded
nucleic acid by forming a triplex, in which three strands of DNA form a
complex dependant
on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules can bind
target regions
with high affinity and specificity.
[00122] The functional nucleic acid molecules may act as effectors,
inhibitors,
modulators, and stimulators of a specific activity possessed by a target
molecule, or the
functional nucleic acid molecules may possess a de novo activity independent
of any other
molecules. In one embodiment, the antibody is a stimulator of dendritic cells
[00123] The conjugated agents can be linked to the antibody directly or
indirectly,
using any of a large number of available methods. For example, an agent can be
attached at
the hinge region of the reduced antibody component via disulfide bond
formation, using
cross-linkers such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP), or via
a carbohydrate
moiety in the Fc region of the antibody (Yu et al. 1994 Int. J. Cancer 56:
244; Upeslacis et al.,
"Modification of Antibodies by Chemical Methods," in Monoclonal antibodies:
principles
and applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995);
Price,
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"Production and Characterization of Synthetic Peptide-Derived Antibodies," in
Monoclonal
antibodies: Production, engineering and clinical application, Ritter et al.
(eds.), pages 60-84
(Cambridge University Press 1995)).
[00124]
Techniques for conjugating agents to antibodies are well known (Amon et al.,
"Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal
Antibodies And Cancer Therapy, Reisfeld et al. (eds.). pp. 243-56 (Alan R.
Liss, Inc. 1985);
Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery
(2nd Ed.),
Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84:
Biological
And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And
Future Prospective Of The Therapeutic Use Of Radiolabeted Antibody in Cancer
Therapy",
in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.
(eds.), pp. 303-
16 (Academic Press 1985), and Thorpe et al., The Preparation And Cytotoxic
Properties Of
Antibody-Toxin Conjugates" 1982 Immunol. Rev. 62:119-58),
[00125]
Antibodies or antigen-binding regions thereof can be linked to another
functional molecule such as another antibody or ligand for a receptor to
generate a bi-specific
or multi-specific molecule that binds to at least two or more different
binding sites or target
molecules. Linking of the antibody to one or more other binding molecules,
such as another
antibody, antibody fragment, peptide or binding mimetic, can be done, for
example, by
chemical coupling, genetic fusion, or noncovalent association. Multi-specific
molecules can
further include a third binding specificity, in addition to the first and
second target epitope.
[00126] Bi-
specific and multi-specific molecules can be prepared using methods
known in the art. For example, each binding unit of the hi-specific molecule
can be
generated separately and then conjugated to one another. When the binding
molecules are
proteins or peptides, a variety of coupling or cross-linking agents can be
used for covalent
conjugation. Examples of cross-linking agents include protein A, carbodiimide,
N-
succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitroberizoic acid)
(DTNB), o-
phenylenedimaleimide (oRDM), N-succinimidy1-3-(2-pyridyldithio)propionate
(SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-I-carboxylate (sulfo-
SMCC)
(Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu et al., 1985 Proc. Natl.
Acad. Sci. USA
82:8648). When the binding molecules are antibodies, they can be conjugated by
sulfhydryl
bonding of the C-terminus hinge regions of the two heavy chains.
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[00127] The antibodies or fragments thereof may be linked to a moiety that
is toxic to
a cell to which the antibody is bound to form "depleting" antibodies. These
antibodies are
particularly useful in applications where it is desired to deplete an NK cell.
[00128] The antibodies may also be attached to solid supports, which are
particularly
useful for immunoassays or purification of the target antigen. Such solid
supports include, but
are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,
polyvinyl chloride or
polypropylene.
[00129] The antibodies also can be bound to many different carriers. Thus,
compositions are also provided containing the antibodies and another
substance, active or
inert. Examples of well-known carriers include glass, polystyrene,
polypropylene,
polyethylene, dextran, nylon, amylase, natural and modified cellulose,
polyacrylamide,
agarose, and magnetite. The nature of the carrier can be either soluble or
insoluble for
purposes of embodiments described herein. Those skilled in the art will know
of other
suitable carriers for binding monoclonal antibodies, or will be able to
ascertain such, using
routine experimentation.
II. Constructs
[00130] All examples of H chain constructs are typically used in co-
transfection of
CHO cells with matching L chain vectors. Also, in some embodiments
immunotherapeutics
will have humanized variable regions.
[00131] The following depicts APC-targeted antibodies and antibody-IL10
fusion
proteins useful in the methods and compositions described herein.
Anti-ASGPR-49C11-hIL-10
[00132] SEQ ID NO:1 shows a fusion protein of the heavy chain of the anti-
ASGPR
49C11 antibody fused through a linker to human IL-10. The linker is underlined
and the IL-
amino acid sequence is in bold italics.
[00133] mAnti-AS GPR_49C 11_7H-LV-hIgG4H-C-Flex-v 1 -hIL-10] antibody, SEQ
ID
NO:1:
VQLQESGPDLVKPS Q SLSLTCTVTGYS ITS GYSWHWIRQFPGNKLEWMGYILFS GS T
NYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARSNYGSFASWGQGTLVTVS
AAKTTGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFE
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GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP
REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV
YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSRLTVDKSRVVQEGN V FS CS V MHEALHNHYTQKSLSLS LGKA S QTPTN TIS VTPTN
N STPTNNSNPKPNPASYGQGTQSENSCIRFPGNLYNMLRDLRDAFSK VKTFFQMKD
QLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGEN
IXTLRLRLRRCHRELPCENKSKAVEQVKNAINKLQEKGIYKAMSEFDIFINYIEAYM
TMKIRN (SEQ ID NO:1).
[00134] The heavy chain of the anti-ASGPR 49C11 antibody from above is SEQ
ID
NO:2:
VQLQESGPDLVKPS Q SLSLTCTVTGYS ITS GYSWHWIRQFPGNKLEWMGYILFS GS T
NYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARSNYGSFASWGQGTLVTVS
AAKTTGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFE
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP
REEQFN STYR V VS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV
YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFF
LYSRLTVDKSRVVQEGN VFSCS VMHEALHNHYTQKSLSLSLGKAS (SEQ ID NO:2).
[00135] The H chain variable region of anti-ASGPR 49C11 is shown in SEQ ID
NO.:3:
QLQESGPDLVKPS QSLSLTCTVTGYSITS GYSWHWIRQFPGNKLEWMGYILFS GS TN
YNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARSNYGSFASWGQGTLVTVSA
AKTT (SEQ ID NO:3).
[00136] The linker shown above is SEQ ID NO:4:
QTPTNTISVTPTNNSTPTNNSNPKPNP (SEQ ID NO:4).
[00137] The hIL-10 amino acid sequence from the Anti-ASGPR-49C11-hIL-10 is
shown in SEQ ID NO:5:
ASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDF
KGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLP
CENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO:5).
[00138] The DNA sequence of the mAnti-ASGPR_49C11_7H-LV-hIgG4H-C-Flex-
vl-hIL-10 antibody is shown in SEQ ID NO:6:
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ATGAGAGCGCTGATTCTTTTGTGCCTGTTCACAGCCTTTCCTGGTATCCTGTCTGA
TGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCA
CTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAGCTGGCACTGGA
TCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACTCTTCAGTGG
TAGCACTAACTACAACCCATCTCTGAAAAGTCGAATCTCTATCACTCGAGACACA
TCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCA
CATATTTCTGTGCAAGATCTAACTATGGTTCCTTTGCTTCCTGGGGCCAAGGGACT
CTGGTCACTGTCTCTGCAGCCAAAACAACGGGCCCATCCGTCTTCCCCCTGGCGC
CCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGG
ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG
GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGT
AGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGG
TCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTC
CTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCA
CGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGT
ACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACA
CCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC
TGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCT'TCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATG
TC U CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAG
CCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTG
ACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCC
GCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGC
AACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTT
TCTTTCAAATGAAGGATCAGCTGGACAACTTG ____________________________________ 1'1
GTTAAAGGAGTCCTTGCTGGA
GGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTAC
CTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCAT
GTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGT
CATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAAT
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GCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGAC
ATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA (SEQ
ID NO:6).
[00139] The corresponding light chain amino acid sequence, mAnti-
ASGPR_49C11_7K-LV-hIgGK-C, is shown in SEQ ID NO:7:
QIVLTQSPAIMS AS PGEKVTMTC SAS S SVSHMHWYQQKS GTSPKRWIYDTSRLAS GV
PARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSHPWSFGGGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QES VTEQD S KD S TY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:7).
[00140] The L chain variable region of anti-ASGPR 49C11 is shown in SEQ ID
NO.:8:
QIVLTQSPAIMSASPGEKVTMTCSASS SVSHMHWYQQKSGTSPKRWIYDTSRLASGV
PARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSHPWSFGGGTKLE (SEQ ID NO:8)
[00141] The DNA sequence of mAnti-ASGPR_49C11_7K-LV-hIgGK-C is shown in
SEQ ID NO:9:
ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAAT
ATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCA
GGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTCACATGCAC
TGGTACCAGCAGAAGTCAGGCACTTCCCCCAAAAGATGGA ___________________________ 1 1
TATGACACATCC
AGACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTT
ACTCTCTCACAATCAGCAGCATGGAGGCTGA AGATGCTGCCACTTATTACTGCCA
GC AGTGGA GT AGTC ACCC A TGGTCGTTCGGTGG AGGC ACC A A ACTCGAGATC A A
ACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG
AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG
CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGA
GTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGA
CGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCC
ATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
(SEQ ID NO:9).
Anti-CD40-24A3-hIL-10
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[00142] SEQ ID NO:10 shows a fusion protein of the heavy chain of the anti-
CD40
24A3 antibody fused through a linker to human IL-10. The linker is underlined
and the IL-10
amino acid sequence is in bold italics.
[00143] manti-hCD40_24A3.3F1_H-LV-hIgG4H-C -Flex- vl-hIL-10 antibody, SEQ
ID NO:10:
VQLQESGPDLVKPS Q SLSLTCTVTGYS ITSD YSWHWIRQFPGNKLEWMGYIYYS GS T
NYNPSLKSRISITRDTSKNQFFLQLNSVTTEDSATYFCARFYYGYSFFDYWGQGTTLT
VS S AKTKGPS VFPLAPC SRS TSE S TAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPA
VLQS S GLYS LS S VVTVPS S SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE
FEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP
QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFS CS VMHEALHNHYTQKSLSLSLGKAS QTPTNTISVTP
TNNSTPTNNSNPKPNPASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQM
KDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLG
ENLKILRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKWYKAMSEFDIFINYIEAY
MTMKIRN (SEQ ID NO:10).
[00144] The heavy chain of the anti-CD40 24A3 antibody from above is SEQ ID
NO:11:
VQLQESGPDLVKPS Q SLSLTCTVTGYS ITSD YSWHWIRQFPGNKLEWMGYIYYS GS T
NYNPSLKSRISITRDTSKNQFFLQLNSVTTEDSATYFCARFYYGYSFFDYWGQGTTLT
VS S AKTKGPS VFPLAPC SRS TSES TAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPA
VLQS S GLYS LS S VVTVPS S SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE
FEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP
QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO:11).
[00145] The linker shown above is SEQ ID NO:4 and the IL-10 amino acid
sequence
is shown as SEQ ID NO:5.
[00146] The DNA sequence of the manti-hCD40_24A3.3F1_H-LV-hIgG4H-C-Flex-
v -h/L-10 antibody is shown in SEQ ID NO: l2,:
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ATGAGAGTGCTGATTCTTTTGTGCCTGTTCACAGCCTTTCCTGGTATCCTGTCTGA
TGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCA
CTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGATTATAGCTGGCACTGGA
TCCGGCAGTTCCCAGGAAACAAACTGGAATGGATGGGCTACATATATTACAGTG
GTAGCACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACAC
ATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACTCAGCC
ACATATTTCTGTGCA AGATTTTACTACGGTTATAGCTTCT ___________________________ 1'1
GACTACTGGGGCCA
AGGCACCACTCTCACAGTCTCCTCAGCCAAAACAAAGGGCCCATCCGTCTTCCCC
CTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTG
GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC
TCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCT
GCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCA
AATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATC
AGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCT
GAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTC
AACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG
GAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG
ACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGT
CCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGG
TGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGG
GGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACA
GAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATC
AGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCC
AACCCCGCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTC
CCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGA
AGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTT
GCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAG
TTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAG
GCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGG
CGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTG
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AAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAG
TTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACT
GA (SEQ ID NO:12).
[00147] The corresponding light chain amino acid sequence, manti-
hCD40_24A3.3F1_K-LV-hIgGK-C, is shown in SEQ ID NO:13:
QIVLTQSPAFMS AS PGEKVTMTC S AS S S VS YMHWYQQKSGTS PKRWIYDTS KLA S G
VPARFS GS GSGTS YS LTIS SMEAEDAATYYC QQWS S NPLTFGAGTKLEIKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAS (SEQ ID NO:13).
[00148] The DNA sequence of manti-hCD40_24A3.3F1_K-LV-hIgGK-C is shown in
SEQ ID NO:14:
ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAGT
ATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCATTCATGTCTGCATCTCCA
GGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTCAGTTACATGCAC
TGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCC
AAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTT
ACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCA
GCAGTGGAGTAGTAACCCACTCACGTTCGGTGCTGGGACCAAGCTCGAGATCAA
ACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG
AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG
CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGA
GTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGA
CGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCC
ATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGCTA
GCTAG (SEQ ID NO:14).
Anti-DCIR-9E8-hIL-10
[00149] SEQ ID NO:15 shows a fusion protein of the heavy chain of the anti-
DCIR
9E8 antibody fused through a linker to human IL-10. The linker is underlined
and the IL-10
amino acid sequence is in bold italics.
[00150] mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v1-hIL-10 antibody, SEQ ID
NO:15:
QVTLKESGPGILQPS QTLS LTC S FS GFS LS TS GMGLSWIRQPSGKGLEWLAHIYWDD D
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KRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVW
GAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQS S GLY S LS SVVTVPS S S LGTKTYTCNVDHKPS NTKVDKRVES KYGPP
CPPCPAPEFEGGPS V FLFPPKPKDTLMIS RTPEVTC V V VD V S QEDPE V QFN WY VD GV E
VHN AKTKPREEQFN S TY RV VS V LTVLHQDWLN GKEY KCKVS N KGLPS SIEKTISKAK
GQPREPQV YTLPPSQEEMTKN QVS LTCLVKGFYPS D IA VEWES N GQPENN YKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKS LS LS LG KA SQTPTN
TISVTPTNNSTPTNNSNPKPNPASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKT
FFQMKIVLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHV
NSLGENLKTLRLRLRRCHRELPCENKSKAVEQVKNAFNKNEKGIYKAMSEFDIFIN
YIEAYMTMKIRN (SEQ ID NO:15).
[00151] The heavy chain of the anti-DCIR 9E8 antibody from above is SEQ ID
NO:16:
QVTLKES GPGILQPS QTLS LTC S FS GFS LS TS GMGLSWIRQPSGKGLEWLAHIYWDD D
KRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVW
GAGTT VTV S S AKTKGPS V FPLAPCS RSTS ESTAALGCLV KD YFPEP V TV S WN SGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP
CPPCPAPEFEGGPS VFLFPPKPKDTLMIS RTPEVTCVVVD V S QEDPEV QFN VVYV D G VE
VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
G QPREPQVYTLPPS QEEMTKNQVS LTCLVKG FYPS D IA VEWES NG QPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLCK AS (SEQ
ID NO:16).
[00152] The H chain variable region of anti-DCIR 9E8 is shown in SEQ ID
NO.:17:
QVTLKES GPGILQPS QTLS LTC S FS GFS LS TS GMGLSWIRQPSGKGLEWLAHIYWDD D
KRYNPSLKSRLTISKDTSSNQVFLKITIVDTADAATYYCARSSHYYGYGYGGYFDVW
GAGTTVTVS.
[00153] The linker shown above is SEQ ID NO:18:
QTPTNTISVTPTNNSTPTNNSNPKPNP (SEQ ID NO:18).
[00154] The IL-10 amino acid sequence is shown as SEQ ID NO:19:
AS PGQGTQS EN SCTHFPGN LPN MLRD LRDAFS R V KTFFQMKD QLD NLLLKES LLED F
KGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVN SLGENLKTLRLRLRRCHRFLP
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CENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID
NO:19).
[00155] The corresponding DNA sequence for the IL-10 gene is shown as SEQ
ID
NO:20:
CGCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGG
CAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACT
TTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGG
AGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTA
CCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCA
TGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTG
TCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAA
TGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGA
CATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA
(SEQ ID NO:20).
[00156] The DNA sequence of mAnti-DCIR_9E8_H-LV-hIgG4H-C-Flex-v 1-hIL-10
antibody is shown in SEQ ID NO:21:
ATGAACAGGCTTACTTCCTCATTGCTGCTGCTGATTGTCCCTGCATATGTCCTGTC
CCAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTC
AGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTCTGAG
CTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGG
GATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAG
GATACCTCCAGCAACCAGGTTTTCCTCAAGATCACCATTGTGGACACTGCAGATG
CTGCCACATACTACTGTGCTCGAAGCTCCCATTACTACGGTTATGGCTACGGGGG
ATACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACG
AAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCA
CAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTC
GTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG
TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG
GCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGG
ACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGA
GTTCGAAGGGGGACCATCAGTCTTCCTGFICCCCCCAAAACCCAAGGACACTCTC
A TG A TCTCCCGG ACCCCTGAGGTCACGTGCGTGGTGGTGG ACGTG AGCC AGG A A
G ACC CCG AGGTCCA GTTC A ACTGGTACGTGGATGGCGTGGAGGTGCATA ATGCC
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AAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTC
CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTC
TCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGG
CAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACC
AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCC
TCCCGTGCTGG ACTCCGACGGCTCCTTCTTCCTCTACAGC AGGCT AACCGTGG AC
A AGAGCAGGTGGCAGGAGGGGA ATGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACA ACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTA A AGCTAGTC
AGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCA
ACAACAGCAACCCCAAGCCCAACCCCGCTAGCCCAGGCCAGGGCACCCAGTCTG
AGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCG
AGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAA
CTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAA
GCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGA
ACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGA
CCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAG
CAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCAT
CTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATG
ACAATGAAGATACGAAACTGA (SEQ ID NO:21).
[00157] The corresponding light chain amino acid sequence, mAnti-DCIR_9E8_K-
LV-hIgGK-C, is shown in SEQ ID NO:22:
NIVLTQSPASLAVSLGQRATISCRASESIHSYGNSFLHWYQQKPGQPPKLLIYLASNLE
SGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTFUGGTKLEIKRTVAA
PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:22).
[00158] The L chain variable region of anti-DCIR 9E8 is shown in SEQ ID
NO.:23:
NIVLTQSPASLAVSLGQRATISCRASESIHS YGNSFLHWYQQKPGQPPKLLIYLASNLE
SGVPARFSGSGSRTDFTLTIDPVEADDAATY .... YCQQNNEDPW TEGGGTKLEIK.
[00159] The DNA sequence of the L chain variable region of the anti-DCIR
9E8 is
shown in SEQ ID NO:24:
AACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGG
CCACCATATCCTGCAGAGCCAGTGAAAGTATTCATAGTTATGGCAATAGTTTTCT
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GCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCA
TCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGCGGCAGTGGGTCTAGGACA
GACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACT
GTCAGCAAAATAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTCGAGA
TCAAA (SEQ ID NO:24).
[00160] The leader sequence prior to the light chain amino acid sequence
comprises:
METDTLLLWVLLLWVPGSTG (SEQ ID NO:25).
[00161] The corresponding DNA sequence of the leader sequence comprises:
ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCA
CAGGT (SEQ ID NO:26).
[00162] The DNA sequence of mAnti-DCIR_9E8_K-LV-hIgGK-C is shown in SEQ
ID NO:27:
ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCA
CAGGTAACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCA
GAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTATTCATAGTTATGGCAATAG
TTTTCTGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTAT
CTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGCGGCAGTGGGTCTA
GGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTA
TTACTGTCAGCAAAATAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCT
CGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATC
CCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACT
CCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCA
GCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCG
AAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG
AGTGTTAG (SEQ ID NO:27).
Anti-CD40-12E12-hIL-10
[00163] SEQ ID NO:28 shows a fusion protein of the heavy chain of the anti-
DCIR
9E8 antibody fused through a linker to human IL-10. The linker is underlined
and the IL-10
amino acid sequence is in bold italics.
[00164] mAnti- CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-v1-hIL-10 antibody, SEQ
ID NO:28:
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EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGG
STYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQ
GTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP
CPAPEFEGGPS VFLFPPKPKDTLMISRTPE VTC V VVDVSQEDPEVQFNWY VDGVEVH
NAKTKPREEQFN STYR V VS VLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK A S QTPTNTI
SVTPTNNSTPTNNSNPKPNPASPGQGTQSENSCHIFPGNLPNMLRDLRDAFSRVKTF
FQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVN
SLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYI
EAYMTMKIRN (SEQ ID NO:28).
[00165] The heavy chain of the anti-CD40 12E12 antibody from above is SEQ
ID
NO:29:
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGG
STYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQ
GTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSS V VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP
CPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NA KTKPR EEQFNS TYR VVSVLTVLHQDWLNG KEYKC KVSNKGLP SSIEKTIS KA KG Q
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO:29).
[00166] The H chain variable region of anti-CD40 12E12 is shown in SEQ ID
NO. :30:
EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGG
STYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWGQ
GTSVTVS.
[00167] The CDRs of the The H chain variable of anti-CD40 12E12 are CDR1:
SASQGISNYLN (SEQ ID NO:31), CDR2: AYINSGGGSTYYPDTVK (SEQ ID NO:32),
and CDR3: RRGLPFHAMD (SEQ ID NO:33).
[00168] The linker shown above is SEQ ID NO:4 and the IL-10 amino acid
sequence
is shown as SEQ ID NO:5.
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[00169] The DNA
sequence of mAnti- CD40_12E12.3F3_H-LV-hIgG4H-C-Flex-vl-
hIL-10 antibody is shown in SEQ ID NO:34:
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGT ______________________________ Fl
TAAAAGGTGTCCAGTG
TGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCGGAGGGTCCCT
GAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGG
TTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAATTCTGGTG
GTGGTAGCACCTATTATCCAGACACTGTA A AGGGCCGATTCACCATCTCCAGAGA
CA A TGCC A AGA ACACCCTGTACCTGCA A A TG AGCCGGCTGA AGTCTGAGGAC AC
AGCCATGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGG
GGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCT
TCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTG
CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC
CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACA
CCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGT
CCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACC
ATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACC
CCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAG
TTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCC
CGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCAC
AGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCC
TGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGA
GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG
ACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGG
AGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
ACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACC
ATCAGCGTGACCCCCACCA ACA ACAGCACCCCCACCA ACA ACAGCA ACCCCA AG
CCCAACCCCGCTAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCAC
TTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAG
TGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTC
CTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATC
CAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATC
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AAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTA
CGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAG
GTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGT
GAGTTTGACATC __ Fl CATCAACTACATAGAAGCCTACATGACAATGAAGATACGA
AACTGA (SEQ ID NO:34).
[00170] The corresponding light chain amino acid sequence, mAnti-
CD40_12E12.3F3_K-V-hIgGK-C, is shown in SEQ ID NO:35:
DIQMTQTTS S LS AS LGDRVTIS C SAS QGIS NYLNWYQQKPDGTVKLLIYYTSILHS GVP
SRFSGSGSGTDYSLTIGNLEPEDIATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFP
PS DEQLKS GTAS VVCLLNNFYPREAKVQWKVDNALQS GNS QES VTEQD S KD STYS L
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:35).
[00171] The L chain variable region of anti-CD40 12E12 is shown in SEQ ID
NO.:36:
DIQMTQTTS S LS AS LGDRVTIS C SAS QGIS NYLNWYQQKPDGTVKLLIYYTSILHS GVP
SRFSGSGSGTDYSLTIGNLEPEDIATYYCQQFNKLPPTFGGGTKLEIK (SEQ ID NO: 36).
[00172] The CDRs of the L chain variable of anti-CD40 12E12 are CDR1:
SASQGISNYLN (SEQ ID NO:37). CDR2: YTSILHS (SEQ ID NO:38), and CDR3:
QQFNKLPPT (SEQ ID NO:39).
[00173] The DNA sequence of mAnti-CD40_12E12.3F3_K-V-hIgGK-C is shown in
SEQ ID NO:40:
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAG
ATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTAGGAGAC
AGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGT
ATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCAATTTT
ACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCT
CTCACCATCGGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGT
TTAATAAGCTTCCTCCGACGTTCGGTGGAGGCACCAAACTCGAGATCAAACGAA
CTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA
GCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGG
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GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID
NO:40).
IgG-hIL10 Control
[00174] SEQ ID NO:41 shows a fusion protein of the heavy chain of the IgG
control
antibody fused through a linker to human IL-10. The linker is underlined and
the IL-10
amino acid sequence is in bold italics.
[00175] hIgG4H-Flex-v 1-hIL-10 antibody, SEQ ID NO:41:
RLQLQESGPGLLKPS VTLS LTCTVS GDS VAS SS YYWGWVRQPPGKGLEWIGTINFS G
NMYYSPSLRSRVTMSADMSENSFYLKLDSVTAADTAVYYCAAGHLVMGFGAHWG
QGKLVSVSPASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC
PPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE
VHNAKTKPREEQFNS TYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAK
GQPREPQVYTLPPS QEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTN
TISVTPTNNSTPTNNSNPKPNPASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKT
FFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHV
NSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFIN
YIEAYMTMKIRN (SEQ ID NO:41).
[00176] The linker shown above is SEQ ID NO:4 and the IL-10 amino acid
sequence
is shown as SEQ ID NO:5.
[00177] The DNA sequence of hIgG4H-Flex-v 1-hIL-10 antibody antibody is
shown in
SEQ ID NO:42:
ATGGACCTCCTGTGCAAGAACATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGG
CGGCTCCCAGATGGGTCCTGTCCCGGCTGCAGCTGCAGGAGTCGGGCCCAGGCCT
GCTGAAGCCTTCGGTGACCCTGTCCCTCACCTGCACTGTCTCGGGTGACTCCGTC
GCCAGTAGTTCrl __ ATTACTGGGGCTGGGTCCGTCAGCCCCCAGGGAAGGGACTCG
AGTGGATAGGGACTATCAATTTTAGTGGCAATATGTATTATAGTCCGTCCCTCAG
GAGTCGAGTGACCATGTCGGCAGACATGTCCGAGAACTCCTTCTATCTGAAATTG
GACTCTGTGACCGCAGCAGACACGGCCGTCTATTATTGTGCGGCAGGACACCTCG
TTATGGGATTTGGGGCCCACTGGGGACAGGGAAAACTGGTCTCCGTCTCTCCAGC
TTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCC
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GAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTG
TCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG
CAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACAC
CAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCC
AGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAG
GACACTCTC ATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGA
GCC A GGA AGACCCCGAGGTCCA GTTC A ACTGGTACGTGGATGGCGTGG AGGTGC
ATA ATGCC A AG ACA AAGCCGCGGG A GG AGCAGTTC AACAGCACGTACCGTGTGG
TC AGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAACGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAG
CCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGG
AGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG
ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAA
CCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGC
ATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAA
AGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCAC
CCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGCCCAGGCCAGGGCAC
CCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGA
GATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGC
TGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGG
TTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAA
GCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAAC
CTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAA
ACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGA
AAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGC
CTACATGACAATGAAGATACGAAACTGA (SEQ ID NO:42).
[00178] The corresponding light chain amino acid sequence hIgGK, is shown
in SEQ
ID NO:43:
DIQMTQS PS S LSAS VGDRVTITCRA S QS IS SYLNWYQQKPGKAPKLLIYAAS SLQSGV
PS RFSGS GS GTDFTLTIS S LQPEDFATYYC QQS YS TPYTFGQGTKLEIKRTVAAPS VFIF
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PP S DEQLKS GTAS VVCLLNNFYPREAKVQWKVDNALQS GNS QESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:43).
[00179] The DNA sequence of hIgGK is shown in SEQ ID NO:44:
ATGAGGGTC CCCGC TCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCC A
GATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGG
TATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTT
TGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCA
CTCTCACCATCAGCAGTCTCCAACCTGAAGATTTTGCAACTTACTACTGTCAACA
GAGTTACAGTACCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACG
AACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA
TCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA
AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG
TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC
TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ
ID NO:44).
[00180] Shown below are further examples of antibodies and antibody
fragments
useful in the methods and compositions described herein.
Anti-Dectin-1 mAbs
[00181] manti-Dectin- 1-11B 6 .4-H- V-hIgG4H-C] ; SEQ ID NO:45 :
QVQLKESGPGLVAPS QSLS ITCS VS GFS LS NYDISWIRQPPGKGLEWLGVMWTGGGA
NYNSAFMSRLS INKDNS KS QVFLKMNNLQTDDTAIYYCVRDAVRYWNFDVWGAGT
TVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQS SGLYS LS S VVTVPS S SLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCP
APEFEGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPR
EPQVYTLPPS QEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO:45).
[00182] The above sequence is a chimera between the H chain variable region
of the
mAb 11B6.4 and the C region of hIgG4.
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[00183] The H chain variable region of the mAb 11B6.4 is shown in SEQ ID
NO:46:
QVQLKESGPGLVAPS QSLS ITCS VS GFS LS NYDISWIRQPPGKGLEWLGVMWTGGGA
NYNS AFM S RLS INKDNS KS QVFLKMNNLQTDDTAIYYCVRDAVRYWNFDVWGAGT
TVTVSSAKTK (SEQ ID NO:46).
[00184] [manti-Dectin-1-11B6.4-K-LV-hIgGK-C] is the corresponding L chain
chimera; SEQ ID NO:47:
QIVLS QS PAILS AS PGEKVTMTCRAS S S VS YIHWYQQKPGS S PKPWIYATS HLASGVP
ARFSGSGSGTSYSLTISRVEAEDTATYYCQQWSSNPFTFGSGTKLEIKRTVAAPSVFIF
PPS DEQLKS GTAS VVCLLNNFYPREAKVQWKVDNALQS GNS QES VTEQD S KDS TYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:47).
[00185] The L chain variable region of the manti-Dectin-1-11B6.4-K-LV-hIgGK-
C is
shown in SEQ ID NO:48:
QIVLS QS PAILS AS PGEKVTMTCRAS S S VS YIHWYQQKPGS S PKPWIYATS HLASGVP
ARFSGSGSGTSYSLTISRVEAEDTATYYCQQWSSNPFTFGSGTK (SEQ ID NO:48).
[00186] manti-Dectin-1-15E2.5-H-V-hIgG4H-C]; SEQ ID NO:49:
QVQLQQSGAELARPGASVKMSCKASGYTFTTYTMHWVKQRPGQGLEWIGYINPSSG
YTNYNQKFKD KATLTAD KS S S TASM QLS S LTSEDS AVYYCARERAVLVPYAMDYW
GQGTS VTV SSAKTKGPS VFPLAPCSRSTSESTAALGCLV KDYFPEP V TV S WNSGALTS
GVHTFPAVLQSSGLY SLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPP
CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AK
G QPREPQVYTLPPS QEEMTKNQVS LTCLVKGFYPS D IA VEWES NG QPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ
ID NO:49).
[00187] The above sequence is a chimera between the H chain variable region
of the
mAb 15E2.5 and the C region of hIgG4.
[00188] The H chain variable region of the mAb 15E2.5 is shown in SEQ ID
NO:50:
QVQLQQSGAELARPGASVKMSCKASGYTFTTYTMHWVKQRPGQGLEWIGYINPSSG
YTNYNQKFKD KATLTAD KS S S TASM QLS S LTSEDS AVYYCARERAVLVPYAMDYW
GQGTSVTVSSAKTK (SEQ ID NO:50).
[00189] [manti-Dectin-1-15E2.5-K-V-hIgGK-C] is the corresponding L chain
chimera;
SEQ ID NO:51:
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QIVLTQSPAVMSASPGEKVTITCTASSSLSYMHWFQQKPGTSPKLWLYSTSILASGVP
TRFS GS GSGTS YSLTISRMEAEDAATYYCQQRS S SPFTFGS GTKLEIKRTVAAPS VFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:51).
[00190] The L chain variable region of the manti-Dectin-1-15E2.5-K-V-hIgGK-
C is
shown in SEQ ID NO:52:
QIVLTQSPAVMSASPGEKVTITCTASSSLSYMHWFQQKPGTSPKLWLYSTSILASGVP
TRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSSPFTFGSGTK (SEQ ID NO:52).
[00191] manti-Dectin-1-2D8.2D4-H-V-higG4H-C]; SEQ ID NO:53:
EVQLQQSGPELEKPGASVKISCK ASGYSFTGYNMNWVKQSNGKSLEWIGNIDPYYG
DTNYNQKFKGKATLTVDKS SS TAYMHLKS LTSEDS AVYYCARPYGSEAYFAYWGQ
GTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC
PPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE
VHNAKTKPREEQFNS TYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAK
GQPREPQVYTLPPS QEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ
ID NO:53).
[00192] The above sequence is a chimera between the H chain variable region
of the
mAb 2D8.2D4 and the C region of hIgG4.
[00193] The H chain variable region of the mAb 2D8.2D4 is shown in SEQ ID
NO:54:
EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIGNIDPYYG
DTNYNQKFKGKATLTVDKS SS TAYMHLKS LTSEDS AVYYCARPYGSEAYFAYWGQ
GTLVTVSAAKTK (SEQ ID NO:54).
[00194] [manti-Dectin-1-2D8.2D4-K-V-hIgGK-C] is the corresponding L chain
chimera; SEQ ID NO:55:
DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYAAQSISGIP
SRFSGSGSGSDFTLSINGVEPEDVGVYYCQNGHSFPYTFGGGTKLEIKRTVAAPSVFIF
PP SDEQLKS GTAS VVC LLNNFYPREAKVQWKVDNALQS GNS QES VTEQD S KDS TYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:55).
[00195] The L chain variable region of the manti-Dectin-1-2D8.2D4-K-V-hIgGK-
C is
shown in SEQ ID NO:56:
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DIVMTQSPATLS VTPGDRVSLS CRAS QSISDYLHWYQQ KS HESPRLLIKYAAQS ISGIP
SRFSGSGSGSDFTLSINGVEPEDVGVYYCQNGHSFPYTFGGGTK (SEQ ID NO: 56).
Anti-DC ASGPR nabs
[00196] [mAnti-ASGPR-4G2.2-Hv-V-hIgG4H-C]; SEQ ID NO.:57:
QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQVPGKGLRWMGWMDTFT
GEPTYADDFKGRFAFSLETSASTAYLQINSLKNEDTATYFCARGGILRLNYFDYWGQ
GTTLTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP
CPAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVH
NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQV .. YTLPPS QEEMTKN QV S LTCLV KGFYPSDIAVEWESN GQPENN YKTTPPVLD
SDGSFFLYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO: 57).
[00197] The above sequence is a chimera between the H chain variable of the
mAb
4G2.2 and the C region of hIgG4.
[00198] The H chain variable of the mAb 4G2.2 is shown in SEQ ID NO.:58:
QIQLVQS GPELK KPGETVKISC KA S G YTFTNYG MNWVKQVPGKGLRWMGWMDTFT
GEPTYADDFKGRFAFSLETSASTAYLQINSLKNEDTATYFCARGGILRLNYFDYWGQ
GTTLTVSSAKTK (SEQ ID NO:58).
[00199] [mAnti-ASGPR __ 4G2.2-Kv-V-hIgGK-C] is the corresponding L chain
chimera; SEQ ID NO. :59:
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLGWYQQKPGNAPRLLISGATSLETGV
PSRFSGSGSGKDYALSITSLQTEDLATYYCQQCWTSPYTEGGGTKLEIKRTVAAPSVF
IFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQS GNS QES VTEQD S KD S TY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 59).
[00200] The L chain variable region of the mAnti-ASGPR-4G2.2-Kv-V-hIgGK-C
is
shown in SEQ ID NO. :60:
DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLGWYQQKPGNAPRLLISGATSLETGV
PSRFSGSGSGKDYALSITSLQTEDLATYYCQQCWTSPYTFGGGTKLEI (SEQ ID
NO:60).
[00201] [mAnti-ASGPR-5F10H-LV-hIgG4H-C] (SEQ ID NO.:61):
EVQLQQSGPELVKPGASVKMSCKASGYTF1DYYMKWVKQSHGKSLEWIGDINPNY
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GDTFYNQKFEGKATLTVDKSSRTAYMQLNSLTSEDSAVYYCGRGDYGYFDVVVGAG
TTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
PAPEFEGGPS V FLFPPKPKDTLMISRTPEV TC V VVDVSQEDPEV QFNW Y VDGVEVHN
AKTKPREEQFN STYR V VS VLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQP
REPQV YTLPPS QEEMTKN QVS LTCLVKGFYP SDIA VEW ESN GQPENN Y KTTPPV LD S
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLLSLGKAS (SEQ ID
NO:61).
[00202] The above sequence is a chimera between the H chain variable of the
mAb
5F10 and the C region of hIgG4.
[00203] The H chain variable of the mAb 5F10 is shown in SEQ ID NO.:62:
EVQLQQSGPELVKPGASVKMSCKASGYTFIDYYMKWVKQSHGKSLEWIGDINPNY
GDTFYNQKFEGKATLTVDKSSRTAYMQLNSLTSEDSAVYYCGRGDYGYFDVWGAG
TTVTVSSAKTK (SEQ ID NO:62).
[00204] [mAnti-ASGPR-5F10K-LV-hIgGK-C] is the corresponding L chain
chimera; SEQ ID NO. :63:
DIVMTQS HKFM S TS VGDRVSITCKAS QDVGTAVAWYQQKPGQSPKLLIYWASTRHT
GVPDRFTGSGSGTDFTLT1NNVQSEDLADYFCQQYSSNPYMFGGGTKLEIKRTVAAP
SVFIFPPSDEQLKSGTAS V VCLLN NFYPREAKVQVVKVDNALQSGNSQES VTEQDSKD
STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 63).
[00205] The L chain variable region of the mAnti-ASGPR-5F10K-LV-hIgGK-C is
shown in SEQ ID NO. :64:
DIVMTQS HKFM S TS VGDRVSITCKAS QDVGTAVAWYQQKPGQSPKLLIYWASTRHT
GVPDRFTGSGSGTDFTLTINNVQSEDLADYFCQQYSSNPYMFGGGTKLEI (SEQ ID
NO:64).
[00206] [mAnti-ASGPR-1H11H-V-hIgG4H-C] (SEQ ID NO. :65):
QLQQSGPELVKPGAS VKISCKTSGYTFTEYTMHWVRQSHGKSLEWIGGINPINGGPT
YNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARWDYGSRDVMDYWGQG
TSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
PAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHN
AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
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REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO:65).
[00207] The above sequence is a chimera between the H chain variable of the
mAb
1H11 and the C region of hIgG4.
[00208] The H chain variable of the mAb 1H11 is shown in SEQ ID NO. :66:
QLQQSGPELVKPGAS VKISCKTSGYTFTEYTMHVVVRQSHGKSLEVVIGGINPINGGPT
YNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARWDYGSRDVMDYWGQG
TSVTVSSAKTK (SEQ ID NO:66).
[00209] [mAnti-ASGPR-1H11K-LV-hIgGK-C] is the corresponding L chain
chimera,
SEQ ID NO.:67:
NIVMTQSPKSMSMSVGERVTLSCKASENVGTYVSWYQQRPEQSPKLLIYGASNRYT
GVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQTYSYIFTFGSGTKLEIKRTVAAPS
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:67).
[00210] The L chain variable region of the mAnti-ASGPR-1H11K-LV-hIgGK-C is
shown in SEQ ID NO.:68:
NIVMTQSPKSMSMSVGERVTLSCKASENVGTYVSWYQQRPEQSPKLLIYGASNRYT
GVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQTYSYIFTFGSGTKLE (SEQ ID
NO:68).
[00211] manti-hASGPR-6.3H9.1D11H (heavy chain) SEQ ID NO: 69:
VQLQQSGAELVRPGTSVKMSCEAARFTFSNYVVIGWVKQRPGHGLEVVIGDIFPGGDY
TNYNKKFKDKATLTADTSSSTAYMQLSSLTSEDS AIYYCARSDYGGYYVFDYWGQG
TTLTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN
AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID
NO:69).
[00212] manti-hASGPR_6.3H9.1D11K (light chain) SEQ ID NO: 70:
DIVMSQSPSSLAVSVGEKVTMSCKSSQNLLYSSNQKNYLAVVYQQKPGQSPKLLIYW
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AS TRES GVPDRFTGS GSGTDFTLTIS SVKAEDLAVYYCQQYYSYPYTFGGGTKLEIKR
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID
NO:70).
[00213] manti-hASGPR-5H8.1D4H (heavy chain) SEQ ID NO: 71:
AQIQLVQSGPELKKPGETVKISCKASGYTFTDYSVHWVKQAPGKGLKWMGWINTET
GEPTYADDLKGRFAFSLETSASTAYLQINNLKNEDTATYFCAKPTYRFFDYWGQGTT
LTASSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
PEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK
TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:71).
[00214] manti-hASGPR-5H8.1D4K (light chain) SEQ ID NO: 72:
DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYW
AS TRES GVPDRFTGS GSGTDFTLTIS SVQAEDLAVYYCKQSYNLWTFGGGTKLEIKRT
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID
NO:72).
anti-CD40 mAbs
[00215] anti-CD40-12B4.2C10, heavy chain, (SEQ ID NO:73:
MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYVLHW
VKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKS S S TAYMELS SLTSED S AV
YYCARGYPAYS GYAMDYWGQGTSVTVS S AKTTPPSVYPLAPGSAAQTNSMVTLGC
LVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQKGEFV (SEQ ID NO:73).
[00216] anti-CD40-12B4.2C10, light chain, SEQ ID NO:74:
MM S S AQFLG LLLLCFQGTRCDIQMTQTTS SLS A SLGDRVTIS CR A S QD IS NYLNWYQ
QKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCHHGNTLP
WTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGS
ERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN
RNEC (SEQ ID NO:74).
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[00217] anti-CD40-12B4.2C10, light chain - alternative clone (17K6), SEQ ID
NO:75:
MDFQVQIFSFLLISASVIIVISRGQIVLTQSPAILSASPGEKVTMTCSASSSVSYMYRYQQ
KPGSSPKPWIYGTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPL
TFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGAS V V CFLNNFY PKD IN V KW KIDGS E
RQNGVLN SVVTDQDSKDSTYSMSSTLTLTKDEYERHNS YTCEATHKTSTSPIVKSFNR
NEC (SEQ ID NO:75).
[00218] anti-CD40_11B6.1C3, heavy chain, SEQ ID NO:76:
MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKASGYSFTGYYMHWV
KQSHVKSLEWIGRINPYNGATSYNQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVY
YCAREDYVYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEP
VTVTWNSGSLSSGVHTFPAVLQKGEFV (SEQ ID NO:76).
[00219] anti-CD40_11B6.1C3, light chain, SEQ ID NO:77:
MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYL
HWYLQKPGQS PKLLIYKVSNRFS GVPDRFS GS GSGTDFALKIS RVEAEDLGVYFCS QS
THVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWK
IDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV
KSFNRNEC (SEQ ID NO:77).
anti-LOX-1 Abs
[00220] [mAnti-LOX-1-11C8H-LV-hIgG4H-C], heavy chain, SEQ ID NO:78:
EVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGN
SDTTYNQKFKGKAKLTAVTSTSTAYMELSSLTNEDSAVYYCTPTYYFDYWGQGTSL
TVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP
QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO:78).
[00221] The H chain variable of the Ab I 1C8 is shown in SEQ ID NO:79:
EVQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGN
SDTTYNQKFKGKAKLTAVTSTSTAYMELSSLTNEDSAVYYCTPTYYFDYWGQGTSL
TVSSAKTK (SEQ ID NO:79).
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[00222] [mAnti-LOX-1-11C8K-LV-hIgGK-C], light chain, SEQ ID NO:80:
DVVMTQTPLTLS VTIGQPASIS C KS S QSLLD SDGKTYLNWFLQRPGQSPKRLIYLVSK
LDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKRTV
AAPSVFIFPPSDEQLKSGTAS V VCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (SEQ ID
NO:80).
[00223] The L chain variable of the Ab 11C8 is shown in SEQ ID NO:81:
DVVMTQTPLTLS VTIGQPASIS C KS S QSLLD SDGKTYLNWFLQRPGQSPKRLIYLVSK
LDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLE (SEQ
ID NO:81)
[00224] [mAnti-LOX-1-10F9H-LV-hIgG4H-C], heavy chain, SEQ ID NO:82:
QVQLQQSGAELMKPGASVKISCKATGYTFGSYWIEWVKQRPGHGLEWIGEILPGSG
NTNYNENFKGKATFTADTS SNTAYMQLTSLT SED S AVYYCARAGIYWGQGTLVTVS
AAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSS GLYS LS S VVTVPS S SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF
EGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK
PREEQFNSTYRVVS VLTVLHQDWLNGKEY KCKVSNKGLPS S IEKTIS KA KGQPREPQ
VYTLPPS QEEMTKN QVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSF
FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID NO:82).
[00225] The H chain variable of the Ab 10F9 is shown in SEQ ID NO:83:
QVQLQQSGAELMKPGASVKISCKATGYTFGSYWIEWVKQRPGHGLEWIGEILPGSG
NTNYNENFKGK ATFTADTSSNTAYMQLTSLTSEDSAVYYC ARA GIYWG QG TLVTVS
AAKTK (SEQ ID NO:83).
[00226] [mAnti-LOX_1-10F9K-LV-hIgGK-C], light chain, SEQ ID NO:84:
DIVLTQSPAFLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYVASKQ
GSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPRTFGGGTKLEIKRTVA
APS VFIFPPSDEQLKS GTAS VVCLLNNFYPREAKVQWKVDNALQS GNS QESVTEQD S
KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 84).
[00227] The L chain variable of the Ab 10F9 is shown in SEQ ID NO:85:
DIVLTQSPAFLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYVASKQ
GSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPRTFGGGTKLE (SEQ ID
NO:85).
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[00228] [mAnti-LOX-1-15C4H-LV-hIgG4H-C], heavy chain, SEQ ID NO:86:
EIQLQQTGPELVKPGASYKISCKASGYPFTDYEVIVWYKQSHGKSLEWIGNISPYYGTT
NYNLKFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARSPNWDGAWFAHWGQ
GALVTVSAAKTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC
PPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCV V VDVSQEDPEVQFNW YVDGVE
VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLYKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ
ID NO:86).
[00229] The H chain variable of the Ab 15C4 is shown in SEQ ID NO:87:
EIQLQQTGPELVKPGASYKISCKASGYPFTDYEVIVWYKQSHGKSLEWIGNISPYYGTT
NYNLKFKGKATLTYDKSSSTAYMQLNSLTSEDSAVYYCARSPNWDGAWFAHWGQ
GALVTVSAAKTK (SEQ ID NO:87).
[00230] [mAnti-LOX-1-15C4K-LY-hIgGK-C], light chain, SEQ ID NO: 88:
DIVLTQSPASLAYSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYAASN
LESGIPARFSGSGSGTDFTLNIHPYEEEDAATYYCQQSNEDPFTFGSGTKLEIKRTVAA
PSYFIFPPSDEQLKSGTASYVCLLNNFYPREAKVQWKVDNALQSGNSQESYTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEYTHQGLSSPYTKSFNRGEC (SEQ ID NO:88).
[00231] The L chain variable of the Ab 15C4 is shown in SEQ ID NO:89:
DIVLTQSPASLAVSLGQRATISCKASQSYDYDGDSYMNWFQQKPGQPPKLLIYAASN
LESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPFTFGSGTKLE (SEQ ID
NO:89).
Anti-DCIR Abs
[00232] Anti-DCIR_24A5.4A5_H-V-hIgG4H-C, heavy chain, SEQ ID NO:90:
MOWLWNLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYSFTNYGMNW
VKQAPGKGLKWMGWINTYTGESTYADDFKGRFAFSLETSASTAYLQISNLKNEDMA
TYFCARGDFRYYYFDYWGQGTTLTGSSAKTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH
KPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA
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VEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO:90).
[00233] Anti-DCIR_24A5.4A5_K-V-hIgGK-C, light chain, SEQ ID NO:91:
MSVLTQVLALLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASGNIHNYLAWYQ
QKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINTLQPEDFGSYYCQHFWDS
WTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN
ALQSGNS QES VTEQD S KD S TYS LS S TLTLS KADYEKHKVYACEVTHQGLS SPVTKS F
NRGEC (SEQ ID NO:91).
[00234] Anti-DCIR_24E7.3H9_H-V-hIgG4H-C, heavy chain, SEQ ID NO:92:
MEWTWVFLFLLSVTAGVHS QVQLQ QS GAELMKPGAS VKIS CKATGYTFS SYWIEW
VKQRPGHGLEWIGEILPGSGRTNDNEKFKGKATFTADTS S KKAYM QLS S LTSEDS AV
YYCARRGGYSFAYWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQS S GLYS LS SVVTVPS S S LGTKTYTCNVDHKPS
NTKVD KRVES KYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS Q
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
VS NKGLPS S IEKTIS KAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
TQKSLSLSLGKAS (SEQ ID NO:92).
[00235] Anti-DCIR_24E7.3H9_K-V-hIgGK-C, light chain, SEQ ID NO:93:
MTMFS LA LLLS LLLLCVS D S RAETTVTQS PAS LSM A IGEKVTIRCVTSTD IDDDVNWY
QQKPGEPPK LLIS EG NTLR PGVPS RFS S SGYG TDFVFTIENMLSEDV ADYYCLQSGNL
PYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQS GNS QES VTEQD S KD STYS LS S TLTLS KADYEKHKVYACEVTHQGLSSPVTKS
FNRGEC (SEQ ID NO:93).
[00236] Anti-DCIR_29E9.2E2_H-VhIgG4H-C, heavy chain, SEQ ID NO:94:
MAWVWTLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW
VKQAPGKGLKWVGWINTF1 ___ GEPTYVDDFKGRFAFS LETS ASTAYLQINNLKNEDTA
TYFCARGNFRYYYFDYWGQGTTLTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVK
D YFPEPV TV SWNSGALTSGVHTFPAVLQSS GLY S LS S V VTVPSSSLGTKTYTCN VDH
KPSNTKVDKRV ES KYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMIS RTPEV TC V V VD
VS QEDPEVQFNWYVDG VEVHNAKTKPREEQFNS TYR VVS VLTVLHQDWLNGKEYK
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CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO:94).
[00237] Anti-DCIR_29E9.2E2_K-V-hIgGK-C, light chain, SEQ ID NO:95:
MSVLTQVLALLLLWLTGARCDIQMTQSPASLSASVGETVTITCRTSGNIRNYLAWYQ
QKQGKSPQLLVYNAKTLADGVPSRFGGSGSGTQYSLKINSUREDFGNYYCQHFWS
SPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKS
FNRGEC (SEQ ID NO:95).
[00238] Anti-DCIR_29G10.3D9_H-V-hIgG4H-C, heavy chain, SEQ ID NO:96:
MMGWSYIILFLVATATDVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMH
WVKQRPGEGLEWIGEINPSYGRTDYNEKFKNKATLTVAKSSSTAYMQLSSLTSEDSA
VYYCARGDYYGSSSFAYWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV
DHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLGKAS (SEQ ID NO:96).
[00239] Anti-DCIR_29G10.3D9_K-Var 1 -V-hIgGK-C, light chain. SEQ ID NO:97:
MDFQVQIFSFLLMSASVIMSRGQIVLTQSPALMSASPGEKVTMTCS ASSNISYMYWY
QQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTTSSMEAEDAATYCCQQWSS
NPPTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
FNRGEC (SEQ ID NO:97).
[00240] Anti-DCIR_29G10.3D9_K-Var2-V-hIgGK-C, light chain. SEQ ID NO:98:
MDFRVQIFSFLLMSASVIIVISRGQIVLTQSPALMSASPGEKVTMTCSASSNISYMYWY
QQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSN
PPTFGAGTKLEIKRTVAAPS VFIFPPSDEQLKSGTAS V VCLLNNFYPREAKVQWKVDN
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC (SEQ ID NO:98).
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[00241] Anti-DCIR_31A6.1F5_H-var2-V-hIgG4H-C, heavy chain, SEQ ID NO: 99:
MECNWILPFILSVISGVYSEVQLQQSGTVLARPGASVNMSCKAAGYSFTSYWVYWV
KQRPGQGLEWIGAIYPKNSRTSYNQKFQDKATLTAVTSASTAYMELSSLTNEDSAVY
YCTRPHYDSFGY WGQGTLVTVSAAKTKGPS VFPLAPCSRSTSESTAALGCLVKD YFP
EPVTVSWN SGALTSG VHTFPAVLQS SGLYS LS S V VTVPS SS LGTKTYTCN VDHKPSN
TKVD KR VES KYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCV V VD V S QE
DPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVS VLTVLHQDWLNG KEY KCKVS
NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQ
KSLSLSLGKAS (SEQ ID NO:99).
[00242] Anti-DCIR_31A6.1F5_K-var2-V-hIgGK-C, light chain, SEQ ID NO:100:
METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDSYGISFMH
WYQQKPGQPPKLLIYRAS NQES GIPARFS GS G S RTDFTLTINPVEADDVATYYC QQSN
EDPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQS GNS QES VTEQD S KD STYS LS S TLTLS KADYEKHKVYACEVTHQGLS S PVTK
SFNRGEC (SEQ ID NO:100).
[00243] Anti-DCIR_3C2.2D9_H-LV-hIgG4H-C, heavy chain, SEQ ID NO:101:
NRLTS S LLLLIVPAYVLS QQVTLKES GPGILQPS QTLS LTCS FSGFS LS TS GMGVSWIR
QPSGKGLEWLAHIYWDDDKRYNPSLKSRLTIFKDPSSNQVFLRITSVDTADTATYYC
ARNS HYYGS TYGGYFDVWGAGTTVTVS S A KTKGPS VFPLAPC S RS TS ES TAALGCLV
KDYFPEPVTVSWNS GALTSGVHTFPAVLQS S GLYS LS SVVTVPSSSLGTKTYTCNVD
HKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVS VLTVLHQDWLNGKEY
KCKVS NKGLPS S IEKTIS KAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPS DI
AVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO:101).
[00244] Anti-DCIR_3C2.2D9_K-LV-hIgGK-C, light chain, SEQ ID NO:102:
METDTLLLWVLLLGVPGSTGNIVLTQSPTSFTVSLGQRATISCRASESVHSYGNSFMH
WYQQKPGQPPKLLIYLASNVESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQN
SEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQS GNS QESVTEQD S KD S TYS LS S TLTLS KADYEKHKVYACEVTHQGLS S PV
TKSFNRGEC (SEQ ID NO:102).
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[00245] Anti-
DCIR_6C8.1G9_H-V-hIgG4H-C, heavy chain, SEQ ID NO:103:
MEWTWVFLFLLSVTAGVHS QVQLQ QS GTELM KPGASVKIS C KATGYTFSTYWIEWV
KQRPGHGLEWIGEILPGSGRTNDNEKFKGKATITADTS S KKAYMQLS SLTS EDS AVY
YCARRGGYSFAFWGQGTLVS V SAAKTKGPS V FPLAPCSRS TSES TAALGCLV KD Y FP
EPVTVSWN SGALTSG VHTFPAVLQS SGLY S LS S V VTVPS SS LGTKTYTCN VDHKPSN
TKVD KR VES KYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCV V VD V S QE
DPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVS VLTVLHQDWLNG KEY KCKVS
NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQ
KSLSLSLGKAS (SEQ ID NO:103).
[00246] Anti-
DCIR_6C8.1G9_K-V-hIgGK-C, light chain, SEQ ID NO:104:
MTMFS LALLLS LLLLCVS D S RAETTVTQS PAS LSMAIGEKVTIRCVTSTDIDDDVNWY
QQKPGEPPKLLISEGNTLRAGVPSRFSSSGYGTDFVF _______________________________ 1
IENMLSEDVADYYCLQSGNL
PYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQES
V TEQDSKDS TY S LS STLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ
ID NO:104).
[00247] Anti-
DCIR2C9H-LV-hIgG4H-V-hIgG4H-C, heavy chain, SEQ ID NO:105:
MKCSWVIFFLMAVVTGVNSEVQLQQSGAELVRPGALVKLSCKASGFNINDYYIHWV
KQRPEQGLERIGWIDPDNGNTIYDPKFQGKASITADTSPNTAYLQLSSLTSEDTAVYY
CARTRSPMVTTGFVYWGQGTVVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNS GALTS GVHTFPAVLQS S GLYS LS S VVTVPS S SLGTKTYTCNVD H
KPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VS QEDPEVQFNWYVDGVEVHNAKXKPREEQFNSTYRVVS VLTVLH QDWLNGKEY
KCKV S NKGLPS S IEKTIS KA KGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPS DI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH
NHYTQKSLSLSLGK (SEQ ID NO:105).
[00248] Anti-DCIR_2C9K-V-hIgGK-C, light chain, SEQ ID NO:106:
METDTLLLWVLLLWVPGSTGDIVLIQSPASLAVSLGQRATISCRASESVDSYVNSFM
HWYQQKPGQPPKLLIYRVSNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQS
NEDPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNS QES VTEQD S KD STYS LS S TLTLS KADYEKHKVYACEVTHQGLS S PVT
KSFNRGEC (SEQ ID NO:106).
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Anti-Langerin Abs
[00249] Anti-Langerin-15B10H-LV-hIgG4H-C, heavy chain, SEQ ID NO:107:
QVQLR QSGPELVKPG A SVKMSCKASGYTFTDYVISWVKQRTG QGLEWIGDIYPGSG
YSFYNENFKGKATLTADKS STTAYMQLSSLTSEDS A VYFC ATYYNYPFAYWGQGTL
VTVSAAKTTGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA
PEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK
TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKAS (SEQ ID
NO:107).
[00250] The H chain variable of the Ab 15B10 is shown in SEQ ID NO:108:
S VKMSCKASGYTFTDYVIS WV KQRTGQGLEWIGDIYPGSGYSFYNENFKGKATLTA
DKSSTTAYMQLSSLTSEDS AVYFCA (SEQ ID NO:108).
[00251] Anti-Langerin-15B10K-LV-hIgGK-C, light chain, SEQ ID NO:109:
DVVMTQTPLSLPVRLGDQASISCRS SQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVS
NRFSGVPDRFSGSGSGTNFTLKISRVEAEDLGLYFCSQSTHVPYTFGGGTKLEIKRTV
AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID
NO:109).
[00252] The L chain variable of the Ab 15B10 is shown in SEQ ID NO:110:
ASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTNF
TLKISRVEAEDLGLYFCS (SEQ ID NO:110).
[00253] Anti-Langerin-2G3H-LV-hIgG4H-C, heavy chain, SEQ ID NO: iii:
MTLNMLLGLRW VFFVVFYQGVHCEVQLVESGGGLVQPKGSLKLSCAASGLTFNIYA
MNWVRQAPGKGLEW VARIRNKSNN Y AT YYADS VKDRFTISRDDS QS LLYLQMNNL
KTEDTAMYYCVGRDWFDYWG QGTLVTVS A A KTKGPSVFPLAPCSRSTSEST A ALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN
VDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP
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SDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYS RLTVDKSRWQEGNVF S C SVMHE
ALHNHYTQKSLSLSLGKAS (SEQ ID NO:111).
[00254] The H chain variable of the Ab 2G3is shown in SEQ ID NO:112:
SLKLSCAASGLTFNIYAMNWVRQAPGKGLEWVARIRNKSNNYATYYADSVKDRFTI
SRDDSQSLLYLQMNNLKTEDTAMYYC (SEQ ID NO:112).
[00255] Anti -Langerin-2 G3 L-LV -hIgGK-C, light chain, SEQ ID NO:113:
MAWISLILSLLALS SGAISQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQ
EKPDHLFTGLIGGTNNRVSGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNH
WVFGGGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDN
ALQ S GNS QESVTEQD SKD S TYS LS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC (SEQ ID NO:113).
[00256] The L chain variable of the Ab 2G3is shown in SEQ ID NO:114:
VTLTC RS S TGAVTTSNYANWV QEKPDHLF TGLIGGTNNRV S GVPARF S GS LIGDKAA
LTITGAQTEDEAIYFCA (SEQ ID NO:114).
II. Interleukin 10 (IL-10)
[00257] Interleukin-10 (IL-10), also known as human cytokine synthesis
inhibitory
factor (CSIF), is an anti-inflammatory cytokine. In humans, IL-10 is encoded
by the IL 10 gene.
The mRNA sequence of human IL-10 is represented by accession No.: NM 000572.2.
The
amino acid sequence of human IL-10 is represented by accession No.: NP
000563.1 and SEQ
ID NO:5.
[00258] In some embodiments, the APC-targeted antibody or fragment thereof
is
operatively linked to an IL-10 polypeptide comprising a sequence corresponding
to a protein
sequence of an NCBI accession number NP 000563.1. In some embodiments, the IL-
10
polypeptide comprises the amino acid sequence of SEQ ID NO:5 or a fragment
thereof:
III. Antigens
[00259] Certain aspects of the disclosure include methods and compositions
concerning
antigenic components including segments, fragments, or epitopes of
polypeptides, peptides,
nucleic acids, carbohydrates, lipids and other molecules that provoke or
induce an antigenic
response, generally referred to as antigens. In one embodiment, the antigen is
a peptide. In
particular, antigens, or antigenic segments or fragments of such antigens,
which
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lead to the destruction of a cell via an immune response, can be identified
and used in the
methods and compositions described herein.
[00260] Antigens associated with various diseases and disorders are known
in the art.
It is contemplated that any antigen may be used in the methods and
compositions described
herein. In certain aspects, the antigen is one that is involved in the
etiology of an
autoimmune, allergic, or inflammatory disease known in the art and/or
described herein.
[00261] In certain aspects, the antigen is one known in the art to be
involved in
rheumatoid arthritis, allergy, asthma, systemic onset juvenile arthritis,
inflammatory bowel
disease, systemic lupus erythematosus, type 1 diabetes, and Crohn's disease.
V. Peptide Components and Proteinaceous Compositions
[00262] Polypeptides and peptides may be modified by various amino acid
deletions,
insertions, and/or substitutions. In particular embodiments, modified
polypeptides and/or
peptides are capable of modulating an immune response in a subject. As used
herein, a
"protein" or "polypeptide" or "peptide" refers to a molecule comprising at
least five amino
acid residues. In some embodiments, a wild-type version of a protein or
peptide are
employed, however, in many embodiments, a modified protein or polypeptide is
employed to
generate the antibody conjugates described herein. A "modified protein" or
"modified
polypeptide" or "modified peptide" refers to a protein or polypeptide whose
chemical
structure, particularly its amino acid sequence, is altered with respect to
the wild-type protein
or polypeptide.
[00263] Peptides include peptides that are found to be specific to
cancerous or pre-
cancerous cells in the body. These peptides may be associated with the APC-
targeted
antibodies described herein. Administration of combinations of these peptides
includes
administering a population of antibody conjugates having multiple peptides
attached and/or
administering multiple conjugate populations, each having a specific peptide
attached or a
combination of such conjugates that includes nanoparticles with 1, 2, 3, 4, 5,
6, or more
peptides attached to the APC-targeted antibody, antigen binding fragment
thereof. or IL-10
protein.
[00264] Proteinaceous compositions may be made by any technique known to
those of
skill in the art, including (i) the expression of proteins, polypeptides, or
peptides through
standard molecular biological techniques, (ii) the isolation of proteinaceous
compounds from
natural sources, or (iii) the chemical synthesis of proteinaceous materials.
The nucleotide as
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well as the protein, polypeptide, and peptide sequences for various genes have
been previously
disclosed, and may be found in the recognized computerized databases. One such
database is
the National Center for Biotechnology Information's GenBank and GenPept
databases (on the
World Wide Web). The all or part of the coding regions for these genes may be
amplified
and/or expressed using the techniques disclosed herein or as would be known to
those of
ordinary skill in the art.
[00265] Amino
acid sequence variants of antigenic epitopes and other polypeptides of
these compositions can be substitutional, insertional, or deletion variants. A
modification in a
polypeptide may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. 16,
17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97,
98, 99, 100, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115,
116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,
131, 132, 133, 134,
135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,
150, 151, 152, 153,
154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,
169, 170, 171, 172,
173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,
188, 189, 190, 191,
192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,
207, 208, 209, 210,
211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 235, 236,
237, 238, 239, 240,
241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,
256, 257, 258, 259,
260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,
275, 276, 277, 278,
279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,
294, 295, 296, 297,
298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,
313, 314, 315, 316,
317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,
332, 333, 334, 335,
336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,
351, 352, 353, 354,
355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,
370, 371, 372, 373,
374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,
389, 390, 391, 392,
393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,
408, 409, 410, 411,
412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,
427, 428, 429, 430,
431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,
446, 447, 448, 449,
450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,
465, 466,
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467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,
482, 483, 484,
485, 486, 487, 488, 489, 490, 491, 492, 493, 494. 495, 496, 497, 498, 499, 500
or more non-
contiguous or contiguous amino acids of a peptide or polypeptide, as compared
to wild-type.
A peptide or polypeptide that results in an immune response is contemplated
for use in
embodiments.
[00266] Deletion variants typically lack one or more residues of the native
or wild-type
amino acid sequence. Individual residues can be deleted or a number of
contiguous amino
acids can be deleted. A stop codon may be introduced (by substitution or
insertion) into an
encoding nucleic acid sequence to generate a truncated protein. Insertional
mutants typically
involve the addition of material at a non-terminal point in the polypeptide.
This may include
the insertion of one or more residues. Terminal additions, called fusion
proteins, may also be
generated.
[00267] Substitutional variants typically contain the exchange of one amino
acid for
another at one or more sites within the protein, and may be designed to
modulate one or more
properties of the polypeptide, with or without the loss of other functions or
properties.
Substitutions may be conservative, that is, one amino acid is replaced with
one of similar
shape and charge. Conservative substitutions are well known in the art and
include, for
example, the changes of: alanine to serine; arginine to lysine; asparagine to
glutamine or
histidine; aspartate to glutamate; cysteine to serine; glutamine to
asparagine; glutamate to
aspartate; glycine to proline; histidine to asparagine or glutamine;
isoleucine to leucine or
valine; leucine to valine or isoleucine; lysine to arginine; methionine to
leucine or isoleucine;
phenylalanine to tyrosine, leucine or methionine; serine to threonine;
threonine to serine;
tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to
isoleucine or
leucine. Alternatively, substitutions may be non-conservative such that a
function or activity
of a polypeptide or peptide is affected, such as avidity or affinity for a
cellular receptor(s).
Non-conservative changes typically involve substituting a residue with one
that is chemically
dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged
amino acid, and
vice versa.
[00268] Proteins may be recombinant, or synthesized in vitro.
Alternatively, a
recombinant protein may be isolated from bacteria or other host cell.
- 66 -
[00269] The term "functionally equivalent codon" is used herein to refer to
codons that
encode the same amino acid, such as the six codons for arginine or serine, and
also refers to
codons that encode biologically equivalent amino acids.
[00270] It also will be understood that amino acid and nucleic acid
sequences may
include additional residues, such as additional N- or C-terminal amino acids,
or 5' or 3' nucleic
acid sequences, respectively, and yet still be essentially as set forth in one
of the sequences
disclosed herein, so long as the sequence meets the criteria set forth above,
including the
maintenance of biological protein activity (e.g., immunogenicity). The
addition of terminal
sequences particularly applies to nucleic acid sequences that may, for
example, include various
non-coding sequences flanking either of the 5' or 3' portions of the coding
region.
[00271] It is contemplated that in composition embodiments, there is
between about
0.001 mg and about 10 mg of total protein per ml. Thus, the concentration of
protein in a
composition can be about, at least about or at most about 0.001, 0.010, 0.050,
0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5,
9.0, 9.5, 10.0, 50, 100 µg/m1 or mg/ml or more (or any range derivable
therein).
[00272] Embodiments include in some cases the administration of an APC-
targeted
antibody. In some embodiments, the methods and compositions further comprise
an antigen.
U.S. Pat. No. 4,554,101 (Hopp), teaches the identification and preparation of
antigenic epitopes
from primary amino acid sequences on the basis of hydrophilicity. Through the
methods
disclosed in Hopp, one of skill in the art would be able to identify potential
antigenic epitopes
from within an amino acid sequence and confirm their immunogenicity. Numerous
scientific
publications have also been devoted to the prediction of secondary structure
and to the
identification of epitopes, from analyses of amino acid sequences (Chou &
Fasman, 1974a,b;
1978a,b; 1979). Any of these may be used, if desired, to supplement the
teachings of Hopp in
U.S. Pat. No. 4,554,101.
IV. Pharmaceutical Compositions
[00273] Embodiments include methods and compositions for increasing immune
responses in a subject in need thereof They include compositions that can be
used to induce or
modify an immune response against an antigen e.g., a polypeptide, a peptide, a
carbohydrate,
a lipid or other molecule or molecular fragment and against developing a
condition or disease
associated with such antigen.
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[00274] It is contemplated that the APC-targeted antibody or antigen
binding fragment
thereof (and optionally antigen and optionally linked to IL-10) may be
administered with
additional adjuvants known in the art such as TLR agonists. TLR agonists may
include an
agonist to TLRI (e.g. peptidoglycan or triacyl lipoproteins), TLR2 (e.g.
lipoteichoic acid;
peptidoglycan from Bacillus subtilis, E coli 0111:B4, Escherichia coli K12, or
Staphylococcus aureus; atypical lipopolysaccharide (LPS) such as Leptospirosis
LPS and
Porphyromonas gingivalis LPS; a synthetic diacylated lipoprotein such as FSL-1
or
Pam2CSK4; lipoarabinomannan or lipomannan from M. smegm*tis; triacylated
lipoproteins
such as Pam3CSK4; lipoproteins such as MALP-2 and MALP-404 from mycoplasma;
Borrelia burgdorferi OspA; Porin from Neisseria meningitidis or Haemophilus
influenza;
Propionibacterium acnes antigen mixtures; Yersinia LcrV; lipomannan from
Mycobacterium
or Mycobacterium tuberculosis; Trypanosoma crttzi GPI anchor; Schistosoma
mansoni
ly sophosphatid ylserine; Le ishmania major lip opho sphoglyc an (LPG);
Plasmodium
ftticiparum glycophosphatidylinositol (GPI); zymosan; antigen mixtures from
Aspergillus
fumigatus or Candida albicans; and measles hemagglutinin), TLR3 (e.g. double-
stramded
RNA, polyadenylic-polyuridylic acid (Poly(A:U)); polyinosine-polycytidylic
acid
(Poly(I:C)); polyinosine-polycytidylic acid high molecular weight (Poly(I:C)
HMW); and
polyinosine-polycytidylic acid low molecular weight (Poly(I:C) LMW)). TLR4
(e.g. LPS
from Escherichia coli and Salmonella species); TLR5 (e.g. Flagellin from B.
subtilis, P.
aeruginosa, or S. typhimurium), TLR8 (e.g. single stranded RNAs such as ssRNA
with
6UUAU repeats, RNA hom*opolymer (ssPolyU naked), HIV-1 LTR-derived ssRNA
(ssRNA40), or ssRNA with 2 GUCCUUCAA repeats (ssRNA-DR)), TLR7 (e.g.
imidazoquinoline compound imiquimod, Imiquimod VacciGradeTM, Gardiquimod
VacciGradeTM, or GardiquimodTM; adenine analog CL264; base analog CL307;
guanosine
analog loxoribine; TLR7/8 (e.g. thiazoquinoline compound CL075;
imidazoquinoline
compound CL097, R848, or R848 VacciGradeTm), TLR9 (e.g. CpG ODNs); and TLR11
(e.g.
Toxoplasma gondii Profilin). In certain embodiments, the TLR agonist is a
specific agonist
listed above. In further embodiments, the TLR agonist is one that agonizes
either one TLR or
two TLRs specifically.
[00275] In certain embodiments, the methods and compositions specifically
exclude
the administration of a TLR ligand and/or agonist.
[00276] Administration of the compositions will typically be via any common
route.
This includes, but is not limited to oral, parenteral, orthotopic,
intradermal, subcutaneous,
- 68 -
intramuscular, intraperitoneal, intranasal, by inhalation, by using a
nebulizer, or by intravenous
injection. In certain embodiments, a vaccine composition may be inhaled (e.g.,
U.S. Pat. No.
6,651,655). Additional formulations which are suitable for other modes of
administration
include oral formulations. Oral formulations include such normally employed
excipients as,
for example, pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium
saccharine, cellulose, magnesium carbonate and the like. These compositions
take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders and
contain about 10% to about 95% of active ingredient, preferably about 25% to
about 70%.
[00277]
Typically, compositions are administered in a manner compatible with the
dosage formulation, and in such amount as will be therapeutically effective
and immune
modifying. The quantity to be administered depends on the subject to be
treated. Precise
amounts of active ingredient required to be administered depend on the
judgment of the
practitioner.
[00278] The
manner of application may be varied widely. Any of the conventional
methods for administration of an antibody are applicable. These are believed
to include oral
application on a solid physiologically acceptable base or in a physiologically
acceptable
dispersion, parenterally, by injection and the like. The dosage of the
pharmaceutical
composition will depend on the route of administration and will vary according
to the size and
health of the subject.
[00279] In many
instances, it will be desirable to have multiple administrations of at
most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more. The
administrations may range from
2 day to twelve week intervals, more usually from one to two week intervals.
The course of
the administrations may be followed by assays for reactive immune responses
and T cell
activity.
[00280] The
phrases "pharmaceutically acceptable" or "pharmacologically acceptable"
refer to molecular entities and compositions that do not produce an adverse,
allergic, or other
untoward reaction when administered to an animal, or human. As used
herein,
"pharmaceutically acceptable carrier" includes any and all solvents,
dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the like. The
use of such media and agents for pharmaceutical active substances is well
known in
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the art. Except insofar as any conventional media or agent is incompatible
with the active
ingredients, its use in immunogenic and therapeutic compositions is
contemplated.
[00281] The antibodies or antigen binding fragments can be formulated for
parenteral
administration, e.g., formulated for injection via the intravenous,
intradermal, intramuscular,
sub-cutaneous, or even intraperitoneal routes. In a specific embodiment, the
composition is
administered by intradermal injection. In further embodiments, the composition
is
administered by intravenous injection. The preparation of an aqueous
composition that
contains a APC-targeted antibody that modifies the subject's immune condition
will be
known to those of skill in the art in light of the present disclosure.
Typically, such
compositions can be prepared as injectables, either as liquid solutions or
suspensions; solid
forms suitable for use to prepare solutions or suspensions upon the addition
of a liquid prior
to injection can also be prepared; and, the preparations can also be
emulsified.
[00282] The pharmaceutical forms suitable for injectable use include
sterile aqueous
solutions or dispersions; formulations including sesame oil, peanut oil, or
aqueous propylene
glycol; and sterile powders for the extemporaneous preparation of sterile
injectable solutions
or dispersions. In all cases the form must be sterile and must be fluid to the
extent that it may
be easily injected. It also should be stable under the conditions of
manufacture and storage
and must be preserved against the contaminating action of microorganisms, such
as bacteria
and fungi.
[00283] The compositions may be formulated into a neutral or salt form.
Pharmaceutically acceptable salts, include the acid addition salts (formed
with the free amino
groups of the protein) and which are formed with inorganic acids such as, for
example,
hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic,
and the like. Salts formed with the free carboxyl groups can also be derived
from inorganic
bases such as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides,
and such organic bases as isopropylamine, trimethylamine, histidine, procaine
and the like.
[00284] The carrier can also be a solvent or dispersion medium containing,
for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyethylene glycol, and the like), suitable mixtures thereof, and vegetable
oils. The
prevention of the action of microorganisms can be brought about by various
antibacterial and
antifungal agents, for example. parabens, chlorobutanol, phenol, sorbic acid.
thimerosal, and
the like. In many cases, it will be preferable to include isotonic agents, for
example, sugars
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or sodium chloride. Prolonged absorption of the injectable compositions can be
brought
about by the use in the compositions of agents delaying absorption, for
example, aluminum
mono stearate and gelatin.
[00285] Sterile injectable solutions are prepared by incorporating the
active ingredients
in the required amount in the appropriate solvent with various of the other
ingredients
enumerated above, as required, followed by filtered sterilization. Generally,
dispersions are
prepared by incorporating the various sterilized active ingredients into a
sterile vehicle which
contains the basic dispersion medium and the required other ingredients from
those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, the preferred methods of preparation are vacuum-drying and freeze-
drying
techniques, which yield a powder of the active ingredient, plus any additional
desired
ingredient from a previously sterile-filtered solution thereof.
[00286] An effective amount of therapeutic or prophylactic composition is
determined
based on the intended goal. The term "unit dose" or "dosage" refers to
physically discrete
units suitable for use in a subject, each unit containing a predetermined
quantity of the
composition calculated to produce the desired responses discussed above in
association with
its administration, i.e., the appropriate route and regimen. The quantity to
be administered,
both according to number of treatments and unit dose, depends on the result
and/or protection
desired. Precise amounts of the composition also depend on the judgment of the
practitioner
and are peculiar to each individual. Factors affecting dose include physical
and clinical state
of the subject, route of administration, intended goal of treatment
(alleviation of symptoms
versus cure), and potency, stability, and toxicity of the particular
composition. Upon
formulation, solutions will be administered in a manner compatible with the
dosage
formulation and in such amount as is therapeutically or prophylactically
effective. The
formulations are easily administered in a variety of dosage forms, such as the
type of
injectable solutions described above.
VII. In Vitro or Ex Vivo Administration
[00287] As used herein, the term in vitro administration refers to
manipulations
performed on cells removed from or outside of a subject, including, but not
limited to cells in
culture. The term ex vivo administration refers to cells which have been
manipulated in vitro,
and are subsequently administered to a subject. The term in vivo
administration includes all
manipulations performed within a subject, including administrations.
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[00288] In certain aspects, the compositions may be administered either in
vitro, ex
vivo, or in vivo. In certain in vitro embodiments, isolated immune cells are
incubated with
compositions described herein. For example, isolated APCs may be incubated
with the
antibody or antibody conjugates as described herein. The cells can then be
used for in vitro
analysis, or alternatively for ex vivo administration.
VIII. Therapeutic Applications
[00289] Methods include treatment of inflammatory and autoimmune disorders
Methods may be employed with respect to individuals who has tested positive
for such
disorders or who are deemed to be at risk for developing such a condition or
related
condition.
[00290] The antibody or antigen binding fragment of the disclosure (in some
embodiments, conjugated to IL-10) can be given to induce or modify an immune
response in
a person having, suspected of having, or at risk of developing an autoimmune
condition or
complication relating to an allograft. Methods may be employed with respect to
individuals
who have tested positive for autoreactivity or allo-reactivity or who are
deemed to be at risk
for developing such a condition or related condition.
[00291] The methods described herein are particularly useful in treating or
preventing
disorders for which antigenic determinants are poorly characterized. Such
disorders include,
for example, rheumatoid arthritis, allergy, asthma, systemic onset juvenile
arthritis,
inflammatory bowel disease, and Crohn' s disease. The methods described herein
are also
particularly useful for disorders such as GVHD and graft rejection since the
antigenic
determinants of such diseases may not be known or may be different depending
on the tissue
and/or individual from which the tissue was obtained from.
[00292] It is contemplated that targeting dendritic cells (e.2. with an
anti-DC-ASGPR
antibody) inhibits autoimmune diseases but does not interfere with pathogen-
specific T cell
responses.
[00293] Embodiments can be used to treat or ameliorate a number of immune-
mediated, inflammatory, or autoimmune-inflammatory diseases, e.g., allergies,
asthma,
diabetes (e.g. type 1 diabetes), graft rejection, etc. Examples of such
diseases or disorders
also include, but are not limited to arthritis (rheumatoid arthritis such as
acute arthritis,
chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis,
acute
immunological arthritis, chronic inflammatory arthritis, degenerative
arthritis, type II
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collagen-induced arthritis, infectious arthritis, Lyme arthritis,
proliferative arthritis, psoriatic
arthritis. Still's disease, vertebral arthritis, and systemic juvenile-onset
rheumatoid arthritis,
osteoarthritis, arthritis chronica progrediente, arthritis deformans,
polyarthritis chronica
primaria, reactive arthritis, and ankylosing spondylitis), inflammatory
hyperproliferative skin
diseases, psoriasis such as plaque psoriasis, gutatte psoriasis. pustular
psoriasis, and psoriasis
of the nails, atopy including atopic diseases such as hay fever and Job's
syndrome, dermatitis
including contact dermatitis, chronic contact dermatitis, exfoliative
dermatitis, allergic
dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular
dermatitis,
seborrheic dermatitis, non-specific dermatitis, primary irritant contact
dermatitis, and atopic
dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory
diseases,
urticaria such as chronic allergic urticaria and chronic idiopathic urticaria,
including chronic
autoimmune urticaria. myositis, polymyositis/dermatomyositis, juvenile
dermatomyositis,
toxic epidermal necrolysis, scleroderma (including systemic scleroderma),
sclerosis such as
systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary
progressive
MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis,
atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis,
neuromyelitis optica
(NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease,
autoimmune-
mediated gastrointestinal diseases, colitis such as ulcerative colitis,
colitis ulcerosa,
microscopic colitis, collagenous colitis, colitis polyposa. necrotizing
enterocolitis, and
transmural colitis, and autoimmune inflammatory bowel disease), bowel
inflammation,
pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis,
respiratory
distress syndrome, including adult or acute respiratory distress syndrome
(ARDS),
meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an
autoimmune
hematological disorder, rheumatoid spondylitis, rheumatoid synovitis,
hereditary
angioedema, cranial nerve damage as in meningitis, herpes gestationis,
pemphigoid
gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden
hearing loss due to
an autoimmune condition, IgE-mediated diseases such as anaphylaxis and
allergic and atopic
rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and/or
brainstem
encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis,
granulomatous uveitis,
nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or
autoimmune uveitis,
glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or
acute
glomerulonephritis such as primary GN, immune-mediated GN, membranous GN
(membranous nephropathy), idiopathic membranous GN or idiopathic membranous
nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I
and
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Type II, and rapidly progressive GN, proliferative nephritis, autoimmune
polyglandular
endocrine failure, balanitis including balanitis circ*mscripta
plasmacellularis, balanoposthitis,
erythema annulare centrifugum, erythema dyschromicum perstans, eythema
multiform,
granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen
simplex chronicus,
lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic
hyperkeratosis,
premalignant keratosis, pyoderma gangrenosum, allergic conditions and
responses, allergic
reaction, eczema including allergic or atopic eczema, asteatotic eczema,
dyshidrotic eczema,
and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial
asthma, and
auto-immune asthma, conditions involving infiltration of T cells and chronic
inflammatory
responses, immune reactions against foreign antigens such as fetal A-B-0 blood
groups
during pregnancy, chronic pulmonary inflammatory disease, autoimmune
myocarditis,
leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus
cerebritis, pediatric
lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus
erythematosus,
alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or
subacute
cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus
disseminatus,
juvenile onset (Type I) diabetes mellitus, including pediatric insulin-
dependent diabetes
mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes) and
autoimmune
diabetes. Also contemplated are immune responses associated with acute and
delayed
hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis,
granulomatosis
including lymphomatoid granulomatosis, Wegener's granulomatosis,
agranulocytosis,
vasculitides, including vasculitis, large-vessel vasculitis (including
polymyalgia rheumatica
and gianT cell (Takayasu's) arteritis). medium-vessel vasculitis (including
Kawasaki's disease
and polyarteritis nodosa/periarteritis nodosa), microscopic polyarteritis,
immunovasculitis,
CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing
vasculitis such as
systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-
Strauss
vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis,
temporal
arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive
anemia, Diamond
Blackfan anemia, hemolytic anemia or immune hemolytic anemia including
autoimmune
hemolytic anemia (ATHA), Addison's disease, autoimmune neutropenia,
pancytopenia,
leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory
disorders,
Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such
as those
secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-
mediated
diseases, anti-glomerular basem*nt membrane disease, anti-phospholipid
antibody syndrome,
allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome,
Goodpasture's syndrome,
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Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid
such as
pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus
vulgaris,
pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus
erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome,
thermal
injury, preeclampsia, an immune complex disorder such as immune complex
nephritis,
antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM
polyneuropathies or IgM-mediated neuropathy, autoimmune or immune-mediated
thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including
chronic or
acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis,
autoimmune disease of the
testis and ovary including autoimmune orchitis and oophoritis, primary
hypothyroidism,
hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such
as
autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's
thyroiditis), or
subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism,
Grave's disease,
polyglandular syndromes such as autoimmune polyglandular syndromes (or
polyglandular
endocrinopathy syndromes), paraneoplastic syndromes, including neurologic
paraneoplastic
syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome,
stiff-
man or stiff-person syndrome, encephalomyelitis such as allergic
encephalomyelitis or
encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE),
experimental
autoimmune encephalomyelitis, myasthenia gravis such as thymoma-associated
myasthenia
gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus
myoclonus
syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's
syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, gianT
cell hepatitis,
chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid
interstitial
pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-
Barre
syndrome, Ber2er's disease (IgA nephropathy), idiopathic IgA nephropathy,
linear IgA
dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular
dermatosis, transient
acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and
pneumonocirrhosis,
autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue
(gluten
en terop ath y), refractory sprue, idiopathic sprue, cryogl obuli n emi a, am
yl otrophic lateral
sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear
disease such
as autoimmune inner ear disease (MED), autoimmune hearing loss, polychondiitis
such as
refractory or relapsed or relapsing polychondritis, pulmonary alveolar
proteinosis, Cogan's
syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's
disease/syndrome, rosacea
autoimmune, zoster-associated pain, amyloidosis, a non-cancerous
lymphocytosis, a primary
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lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign
monoclonal
gammopathy and monoclonal gammopathy of undetermined significance, MGUS),
peripheral
neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy,
migraine,
arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and
channelopathies
of the CNS, autism, inflammatory myopathy, focal or segmental or focal
segmental
glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis,
chorioretinitis,
autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure.
Schmidt's
syndrome, adren al i ti s, gastric atrophy, presenile dementia, dem yeli n ati
ng diseases such as
autoimmune demyelinating diseases and chronic inflammatory demyelinating
polyneuropathy, Dressler's syndrome, alopecia greata, alopecia totalis, CREST
syndrome
(calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and
telangiectasia), male and female autoimmune infertility, e.g., due to anti-
spermatozoan
antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever,
recurrent
abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome,
Cushing's
syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign
lymphocytic angiitis,
Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing
alveolitis, interstitial
lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such
as leishmaniasis,
kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's
syndrome, Caplan's
syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial
pulmonary
fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary
fibrosis, cystic
fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis
fetalis,
eosinophilic faciitis. Shulman's syndrome, Felty's syndrome, flariasis,
cycl*tis such as chronic
cycl*tis, heterochronic cycl*tis, iridocycl*tis (acute or chronic), or Fuch's
cycl*tis, Henoch-
Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID,
acquired immune
deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia,
pancreatitis,
thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination
syndromes,
congenital rubella infection. Epstein-Barr virus infection, mumps, Evan's
syndrome,
autoimmune gonadal failure. Sydenham's chorea, post-streptococcal nephritis,
thromboangitis
ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis. gianT cell
polymyalgia, chronic
hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic
keratoconjunctivitis,
idiopathic nephritic syndrome, minimal change nephropathy, benign familial and
ischemia-
reperfusion injury, transplant organ reperfusion, retinal autoirnmunity, joint
inflammation,
bronchitis, chronic obstructive airway/pulmonary disease, silicosis, aphthae,
aphthous
stomatitis, arteriosclerotic disorders, asperniogenese, autoimmune hemolysis,
Boeck's
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disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia
phacoanaphylactica,
enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis,
chronic fatigue
syndrome, febris rheumatica. Hamman-Rich's disease, sensoneural hearing loss,
haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia,
mononucleosis
infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis,
ophthalmia
symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta,
pyoderma
gan greno sum , Quervain's th yreoiditis, acquired spenic atrophy, non-
malignant thym om a,
vitiligo, toxic-shock syndrome, food poisoning, conditions involving
infiltration of T cells,
leukocyte-adhesion deficiency, immune responses associated with acute and
delayed
hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving
leukocyte
diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated
diseases,
antiglomerular basem*nt membrane disease, allergic neuritis, autoimmune
polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic
gastritis,
sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease,
nephrotic
syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome
type I, adult-
onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated
cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis,
myocarditis,
nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent
sinusitis, acute
or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an
eosinophil-related
disorder such as eosinophilia, pulmonary infiltration eosinophilia,
eosinophilia-myalgia
syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical
pulmonary
eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas
containing
eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine
autoimmune
disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous
candidiasis, Bruton's
syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich
syndrome, ataxia
telangiectasia syndrome, angiectasis, autoimmune disorders associated with
collagen disease,
rheumatism, neurological disease, lymphadenitis, reduction in blood pressure
response,
vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia,
renal ischemia,
cerebral ischemia, and disease accompanying vasculari zati on , allergic
hypersensitivity
disorders, gl omerul onephri ti de s, reperfusi on injury, i schemic re-
perfusion disorder,
reperfusion injury of myocardial or other tissues, lymphomatous
tracheobronchitis,
inflammatory dermatoses, derrnatoses with acute inflammatory components,
multiple organ
failure, bullous diseases, renal cortical necrosis, acute purulent meningitis
or other central
nervous system inflammatory disorders, ocular and orbital inflammatory
disorders,
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granulocyte transfusion-associated syndromes, cytokine-induced toxicity,
narcolepsy, acute
serious inflammation, chronic intractable inflammation, pyelitis, endarterial
hyperplasia,
peptic ulcer, valvulitis, graft versus host disease, contact hypersensitivity,
asthmatic airway
hyperreaction, and endometriosis.
[00294] Embodiments can be used to prevent, treat or ameliorate a number of
allergic
disorders. Non-limiting examples include asthma, type I diabetes, chronic
obstructive
pulmonary disease, interstitial lung disease, chronic obstructive lung
disease, chronic
bronchitis, eosinophilic bronchitis, eosinophilic pneumonia, pneumonia,
inflammatory bowel
disease, atopic dermatitis, atopy, allergy, allergic rhinitis, idiopathic
pulmonary fibrosis,
scleroderma, emphysema, breast cancer, and ulcerative colitis. Non-limiting
examples of
allergic disorders include allergic atopy and dermatitis, allergic rhinitis,
allergic asthma,
allergic responses to food (e.g. milk, egg, wheat, nut, fish, shellfish,
sulfite, soy, and casein),
environmental allergens (e.g. plant and animal allergens such as dander, dust
mites, pollen,
cedar, poison ivy, poison oak, poison sumac, etc...), insect bites (e.g. bee,
wasp, yellow
jacket, hornet, or fire ant stings), hay fever, allergic conjunctivitis,
hives, mold, medication
allergies (e.g. aspirin and penicillin), and cosmetic allergies.
[00295] In some embodiments, the compositions and methods described herein
are
used to treat an inflammatory component of a disorder listed herein and/or
known in the art.
Accordingly, the methods and compositions described herein can be used to
treat a subject
suffering from inflammation. In some embodiments, the inflammation is acute.
In other
embodiments, the inflammation is chronic. In further embodiments, the
compositions and
methods described herein are used to treat or prevent a cancer by treating or
preventing an
inflammatory component associated with the cancer. In some embodiments, the
cancer is
breast cancer.
IX. Combination Therapy
[00296] The compositions and related methods disclosed herein, particularly
administration of an APC-targeted antibody or antigen binding fragment may
also be used in
combination with the administration of traditional therapies. These include,
but are not
limited to, the administration of immunosuppressive or modulating therapies or
treatments.
Non-limiting examples of existing immunosuppressive therapies include
administration of
immunosuppressive compounds such as cyclosporine A, cyclophosphamide, FK506,
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tacrolimus, corticosteroids, azathioprine, mycophenolate mofetil, sirolimus,
rapamycin,
rapamycin analogs. deoxyspergualin, and prednisone
[00297] In one aspect, it is contemplated that an APC-targeted antibody or
antigen
binding fragment is used in conjunction with a cytokine treatment.
Alternatively, antibody
administration may precede or follow the other treatment by intervals ranging
from minutes
to weeks. In embodiments where the other agents are administered separately,
one would
generally ensure that a significant period of time did not expire between the
time of each
delivery, such that the agent and antibody would still be able to exert an
advantageously
combined effect on the subject. In such instances, it is contemplated that one
may administer
both modalities within about 12-24 h of each other and, more preferably,
within about 6-12 h
of each other. In some situations, it may be desirable to extend the time
period for
administration significantly, however, where several days (2, 3, 4, 5, 6 or 7)
to several weeks
(1, 2, 3, 4, 5, 6. 7 or 8) lapse between the respective administrations.
[00298] Administration of the anti-DC-ASGPR antibody or antigen binding
fragment
compositions to a patient/subject will follow general protocols for the
administration of such
compounds, taking into account the toxicity, if any. It is expected that the
treatment cycles
would be repeated as necessary. It also is contemplated that various standard
therapies, such
as hydration, may be applied in combination with the described therapy.
X. Examples
[00299] The following examples are included to demonstrate certain
embodiments. It
should be appreciated by those of skill in the art that the techniques
disclosed in the examples
which follow represent techniques discovered by the inventor to function well
in the practice
of the invention, and thus can be considered to constitute preferred modes for
its practice.
However, those of skill in the art should, in light of the present disclosure,
appreciate that
many changes can be made in the specific embodiments which are disclosed and
still obtain a
like or similar result without departing from the spirit and scope of the
invention.
Example 1: Targeting IL-10 to Antigen Presenting Cells
[00300] With targeted in vivo delivery of IL-10 to APCs followed by
alterations of
pathogenic functions of APC as well as the enhancement of regulatory T cell
responses, this
therapeutic strategy can be more effective and durable than non-targeted anti-
inflammatory
cytokines. Compared to non-targeted methods, targeted method described herein
requires
much less amount of IL-10 to show the same or similar effects. With the dose-
sparing-effect
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along with the delivery of IL-10 to subsets of patient's APCs, this strategy
is expected to
significantly reduce side effects of anti-inflammatory cytokine treatment, but
with better
effects.
[00301] To study the effects of APC-targeted IL-10, recombinant fusion
protein of
antibody and human IL-10 were made. Monoclonal antibodies (anti-CD40 clone
12E12,
anti-CD40 clone 24A3, anti-DCIR clone 9E8, anti-DC-ASGPR clone 49C11, and
control
IgG4) and human IL-10 fusion proteins were made.
[00302] It was found that different antibodies fused to human IL-10 can
target subsets
of human DCs in distinct patterns. The ability of antibody-IL-1 0 fusion
proteins to bind to
human DCs was measured. Both myeloid DC (mDCs) and plasmacytoid DCs (pDCs)
were
purified from human blood. DCs were incubated for 20 min in ice in the
presence of
different concentrations of recombinant fusion proteins of antibody and IL-10
(FIG. 1). After
vigorous washing, DCs were further stained with anti-human IL-10 to detect
surface bound
antibody-IL-10 fusion proteins using flow cytometry. As shown in FIG. 1, all
the
recombinant fusion proteins of antibody-IL-10 (except for the control IgG4-IL-
10) could bind
to mDCs. However, binding patterns of the individual proteins to mDCs were not
the same.
For example, anti-DCIR (9E8)-IL-10 binds to mDCs more efficiently than do the
others. In
addition, anti-CD40 (12E12)-IL-10 shows better binding to mDCs than anti-CD40
(24A3)-
IL-10. Although both anti-CD40 (12E12)-IL-10 and anti-CD40 (24A3)-IL-10 bind
to pDCs,
anti-DCIR (9E8)-IL-10 shows the besting binding to pDCs.
[00303] Taken together, these data indicate that anti-inflammatory
cytokines
(including IL-10) fused to different antibodies can target different subsets
of human APCs in
different levels, which can result in different outcomes of immune responses.
[00304] It was also found that antibody-IL-10 fusion proteins can suppress
DC
maturation. DCs are the major APCs that can induce and direct host immune
responses
toward either immunity or tolerance. It is also known that matured DCs induce
immunity
whereas immatured DCs induce immune tolerance. Therefore, the effectiveness of
antibody-
IL-10 fusion proteins on the maturation of DCs induced by Escherichia coli
lipopolysaccharide (LPS: toll-like receptor 4 ligand) was tested. Purified
blood mDCs were
cultured overnight with 0, 10, and 100 ng/ml LPS in the presence or absence of
10 lug
recombinant fusion proteins indicated or the same molar concentration of
recombinant IL-10.
mDCs were then stained with anti-CD83 and anti-CD86 to measure the expression
levels of
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these two surface molecules (indicators for DC maturation) using flow
cytometry. FIG. 2
shows that untargeted human IL-10 slightly decreased CD83 and CD86 expression.
Compared to untargeted IL-10, targeted delivery of IL-10 using recombinant
fusion proteins
of antibody and IL-10 were far more efficient to suppress the LPS-induced DC
maturation.
Anti-CD40 (12E12)-IL-10 was slightly more efficient than others to suppress
the expression
of CD86.
[00305] These data demonstrate that recombinant fusion proteins of antibody
and IL-
can efficiently target human DCs and thus can effectively suppress DC
maturation. This
indicates that targeted delivery of anti-inflammatory cytokines to human APCs
can efficiently
suppress ongoing inflammatory responses by the inhibition of APC, including
DCs,
maturation.
[00306] It was next found that targeted delivery of IL-10 to DCs using
recombinant
fusion proteins of antibody and IL-10 can efficiently suppress T cell
responses. The
effectiveness of recombinant fusion proteins of antibody and IL-10 in T cell
responses (FIG.
3) was further assessed. Purified mDCs were incubated for 2h with different
concentrations
of either recombinant human IL-10 or fusion proteins of antibody and IL-10.
CFSE-labeled
allogeneic CD4+ T cells were co-cultured for 5 days and T cell proliferation
was assessed by
measuring CFSE dilution with flow cytometry. Compared to IL-10 alone,
recombinant
fusion proteins of antibody and IL-10 were far more efficient to suppress the
allogeneic
CD4+ T cell proliferation. To result in 50% inhibition of T cell
proliferation, 54nM IL-10
was required, while only less than 5.4-0.054 nM (IL-10) was required to result
in similar
effect on T cell proliferation when IL-10 was delivered to DCs in a targeted
fashion.
[00307] Taken together, the data (FIGS. 1, 2, 3) demonstrate that 1)
recombinant
fusion proteins of antibody and anti-inflammatory cytokines can effectively
target human
APCs with different patterns, depending on APC subsets (FIG. 1); 2) they can
suppress
human APCs (including DCs) activation and maturation; and 3) can effectively
suppress T
cell responses.
Example 2: Treating GVHD with anti-DC-ASGPR
[00308] Tolerance to specific antigens is the ultimate goal for the success
of
transplantation. Over the past several decades, a large array of
immunosuppressive agents
has been developed and is being used for patients. However, immunosuppression
does not
guarantee the prevention of alloreaction over time in patients who receive
organs, tissues, and
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hematopoietic stem cell (HPSC) transplantation. As a consequence, patients
succumb to
graft-versus-host disease (GVHD) as well as serious side effects due to life-
long
immunosuppression. T cell depletion also compromises the graft-versus-leukemia
(GVL)
effects in alloITIPSC transplantation. Furthermore, controlling GVHD with
nonspecific
immunosuppression neither spares pre-existing memory cells nor discriminates
between
alloreactive and non-alloreactive T cells. Thus, although G-VE1..D could be
controlled to some
degree by immunosuppression, it is at the cost of increased incidence of graft
failure,
leukemia relapse, and compromised immunity to post-transplant infections, such
as
cytomegalovirus (CMV). Therefore, a new therapeutic strategy that can prevent
GVHD
while preserving host immunity to infections will bring great benefit to
patients.
[00309] Dendritic cells (DCs), major antigen presenting cells (APCs), can
induce host
immune responses. DCs also display functional plasticity to control immune
responses. The
ability of DCs, as immune controllers, is in part by the expression of pattern-
recognition
receptors (PRRs), including lee-ans. It was discovered that a lectin expressed
on human DCs,
DC-asialoglycoprotein receptor (DC-ASGPR), shows a unique ability to generate
antigen-
specific IL-10-producing regulatory T cells (Tregs). This applies to both self
(prostate
specific antigen) and foreign antigens (influenza HA 1), as demonstrated in
human in vitro
and non-human primates in vivo. DC-.ASGPR-induced antigen-specific Tregs
efficiently
suppress effector T cell proliferation and inflammatory cytokine expression.
It was further
discovered that signals via DC-ASGPR induce DCs to express IL-10, and this IL-
10
promotes the generation of antigen-specific Tregs. Applicants sought out to
test whether
activation of DCs via DC-ASGPR can generate alloantigen-specific Tregs and
thus can
prevent GVHD and allograft transplantation. Data shows that targeting DC-ASGPR
with
anti-DC-ASGPR antibody results in decreased allogeneic T cell responses. These
T cells can
also secrete high level of IL-10 during their reactivation in response to
alloantigens. Thus,
Applicants surmise that DC-ASGPR can be a novel therapeutic target to inhibit
such
unwanted types of immune responses in patients who undergo transplantation
surgery. This
strategy is focusing on the induction of alloantigen-specific Tregs and thus
may not interfere
with host immunity to post-transplantation infections. Therefore, it was
hypothesized that
targeting DC-ASGPR with an anti-DC-ASGPR antibody not fused to an antigen
prevents
GVHD and allograft rejection but does not interfere with host immunity to
infections.
[00310] Establishment of alloantigen-specific immune tolerance is an
ultimate goal for
the success of transplantation. The novel immunotherapeutic strategy described
herein may
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eventually permit the production of alloantigen-specific Tregs in patients
without interfering
with host immunity to post-transplantation infections. Therefore, this study
has a high
significance in both medical and immunological implications.
[00311] The approach to controlling GVHD and transplant rejection by
targeting DC-
ASGPR is highly novel and innovative in the aspects of both basic immunology
and medical
implications.
[00312] DC-ASGPR has a specialized function to generate antigen-specific
Tregs. DC-
ASGPR, a scavenger receptor, is expressed on subsets of human DCs (blood
myeloid DCs:
mDCs and skin dermal DCs but not plasmacytoid DCs: pDCs or Langerhans cells:
LCs),
monocytes, macrophages, and B cells. Endothelial cells express ASGPR, but not
DC-ASGPR.
DC-ASGPR is expressed in non-human primates (NHPs), but not in mice. Mice have
two
closely linked genes called Mg1-1 and Mg1-2 which are distantly related to
human DC-ASGPR,
the former having a closer tissue distribution profile to the single human
gene (not shown).
A. Anti-DC-ASGPR antibody treatment suppresses allogeneic CD4+ and
CD8+ T cell proliferation
[00313] To study the immunological function of DC-ASGPR, mouse monoclonal
antibodies (mAbs) specific for human DC-ASGPR were first generated. To abolish
their non-
specific bindings to FcRs, recombinant mAbs carrying mouse variable region
chimeras with
human lc chain and human IgG4 carrying two site mutations were made.
Recombinant control
mAb was also made in the same way.
[00314] It is important to note that both DC-ASGPR and Dectin-1 carry an
immunoreceptor tyrosine-based activation motif (ITAM) and can induce IL-10
expression in
DCs. However, DC-ASGPR is superior to Dectin-1 to generate Tregs (data not
shown). In
addition, anti-DC-ASGPR mAb does not induce DCs to express IL-1(3, IL-23 or IL-
12, while
anti-Dectin-1 mAb does induce these cytokines, as previously described.
[00315] Anti-DC-ASGPR mAb can suppress MI-IC-mismatched allogeneic T cell
responses: The effects of anti-DC-ASGPR mAb in 1VIHC-mismatched allogeneic T
cell
responses was tested (FIG. 4). Different numbers of PKH25-labeled PBMCs from
healthy
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donors were incubated overnight in the presence of anti-DC-ASGPR or control
mAb, and
then CFSE-labeled PBMCs from MHC-mismatched donors (total 6 pairs of MHC-
mismatched donors) were co-cultured for 5 days. The percents of CFSE CD4 and
CFSE
CD8+ T cells are presented. In the presence of control mAb. both CD4+ and CD8+
T cell
proliferations were correlated with the numbers of stimulators (PBMCs from
other donors).
However, anti-DC-ASGPR mAb significantly decreased allogeneic CD4+ and CD8+ T
cell
proliferation, particularly when the number of stimulators (X-axis) was
greater than 12.5 x
103/well. Interestingly, total numbers of CD4+ and CD8+ T cells counted at the
end of
cultures were similar in both groups (control and anti-DC-ASGPR mAb-treated
groups) (not
shown).
B. IL-10 secreted from PBMC activated with anti-DC-ASGPR contributes to
the suppression of allogeneic CD4+ and CD8+ T cell responses
[00316] Applicants further found that the decreased allogeneic T cell
proliferation by
anti-DC-ASGPR mAb was recovered (-60-70%) by neutralizing IL-10 on day 1 (2 h
before
adding MHC-mismatched PBMCs to the culture) (FIG. 5). This suggests that IL-10
secreted
from anti-DC-ASGPR-activated APCs contributes to the decreased proliferation
of T cells
from MHC-mismatched donors.
C. Anti-DC-ASGPR antibody treatment results in decreased IFNg-
producing, but increased IL-10-producing regulatory T cell responses
[00317] On day 8 of the co-culture of PBMCs from MHC-mismatched healthy
donors,
CFSEI'CDr T cells were FACS-sorted, and then restimulated for 48h with T cell-
depleted
PBMCs (from stimulators). The amounts of IL-10 and IFNy in the supernatants
were
measured (FIG. 6). MHC-mismatched CD44 T cells co-cultured anti-DC-ASGPR-
treated
PBMCs secreted decreased amount of IFNy but increased amount of IL-10 compared
to
CD4' T cells co-cultured with the same PBMC treated with control mAb. This
suggests that
treatment of PBMCs with anti-DC-ASGPR mAb promote the induction of alloantigen-
specific Tregs which could play important roles in the inhibition of GVHD and
allograft
rejection in vivo.
D. Anti-DC-ASGPR antibody treatment results in the suppression of GVHD
in vivo
[00318] Applicants further assessed the in vivo effects of anti-DC-ASGPR
mAb.
NOD/SCID/yc-/- (NOG) mice (5 mice/group) were injected intravenously (i.v.) on
day 0 with
50x106 PBMCs from healthy donors. Animals also received 3 i.v. doses of
antibodies (250
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[tg/dose) or PBS on days 0, 2, and 4. FIG. 7 shows that anti-DC-ASGPR
treatment resulted
in enhanced survival of animals (p<0.001) compared to control IgG or PBS
treatment.
[00319] Taken together, this data demonstrates that targeting DC-ASGPR with
anti-
DC-ASGPR mAb promotes antigen-specific Treg responses. It is contemplated that
this
could also apply to the in vivo establishment of alloantigen-specific Tregs.
This data and
methodology described herein is useful in the research and development of a
novel
therapeutic that can efficiently inhibit GVHD and allograft rejection without
interfering with
host immune responses to infections.
[00320] Applicants focused on novel antibodies that bind the DC-ASGPR that
can
induce DCs to secrete IL-ID and to induce IL-10-producing alloantigen-specific
Tregs in the
presence of alloantigens. Therefore, the strategy to inhibit GVHD and
allograft rejection is
based on two distinct but compensatory mechanisms (FIG. 8). First (Direct
Pathway), IL-10
secreted from DC-ASGPR-activated DCs will directly inhibit allogeneic T cell
responses in
the early time point, as shown in FIG. 5. Second (Indirect Pathway), DC-ASGPR-
induced
IL-10 can contribute to the induction of IL-10-producing alloantigen-specific
Tregs, as shown
in FIG. 6. These two pathways could result in the enhanced survival of human
PBMC-
transferred NOG mice (FIG. 7). Such alloantigen-specific Tregs express IL-10
when they
are activated at the place where alloantigens are available in vivo (Sagoo, et
al., 2011).
* * *
[00321] It is specifically contemplated that embodiments of the invention
may include
one or more elements listed or exclude one or more elements listed throughout
the
specification. For example, specific embodiments may include one specific item
listed (e.g.
antibody framework) as described herein or embodiments of the invention may
encompass
multiple items from a specific list, such as 2, 3, 4, 5, 6, 7, 8, 9. 10, 20,
30, 40, or more. The
invention may also exclude one or more listed elements, for example, some
embodiments
exclude 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or more elements listed.
Furthermore, when
ranges or numerical values are provided, it is specifically contemplated that
certain ranges or
numerical values may be excluded from the invention. Last, when the inventions
is described
in terms of including a particular feature, it is specifically contemplated
that the invention
may also exclude such feature.
[00322] All of the methods disclosed and claimed herein can be made and
executed
without undue experimentation in light of the present disclosure. While the
compositions and
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methods of this invention have been described in terms of preferred
embodiments, it will be
apparent to those of skill in the art that variations may be applied to the
methods and in the
steps or in the sequence of steps of the method described herein without
departing from the
concept, spirit and scope of the invention. More specifically, it will be
apparent that certain
agents which are both chemically and physiologically related may be
substituted for the
agents described herein while the same or similar results would be achieved.
All such similar
substitutes and modifications apparent to those skilled in the art are deemed
to be within the
spirit, scope and concept of the invention as defined by the appended claims.
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