U.S. patent application number 16/215263 was filed with the patent office on 2019-06-06 for humanized anti-ox40 antibodies and uses thereof.
The applicant listed for this patent is MedImmune, LLC. Invention is credited to Melissa Damschroder, Qun Du, Scott A. Hammond, Michael Oberst.
Application Number | 20190169303 16/215263 |
Document ID | / |
Family ID | 55130798 |
Filed Date | 2019-06-06 |
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United States Patent
Application |
20190169303 |
Kind Code |
A1 |
Hammond; Scott A. ; et
al. |
June 6, 2019 |
Humanized Anti-OX40 Antibodies and Uses Thereof
Abstract
The disclosure provides humanized anti-OX40 antibodies. Also
provided are methods of making such antibodies, and methods of use,
e.g., treatment of cancer.
Inventors: |
Hammond; Scott A.;
(Gaithersburg, MD) ; Oberst; Michael;
(Gaithersburg, MD) ; Du; Qun; (Gaithersburg,
MD) ; Damschroder; Melissa; (Gaithersburg,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MedImmune, LLC |
Gaithersburg |
MD |
US |
|
|
Family ID: |
55130798 |
Appl. No.: |
16/215263 |
Filed: |
December 10, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15635847 |
Jun 28, 2017 |
10150815 |
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16215263 |
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14877547 |
Oct 7, 2015 |
9738723 |
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15635847 |
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62062431 |
Oct 10, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2317/732 20130101; C07K 2317/73 20130101; C07K 2317/92
20130101; C07K 16/2878 20130101; C07K 2317/33 20130101; C07K
2317/24 20130101; C07K 2317/515 20130101; C07K 2317/34 20130101;
C07K 2317/51 20130101; C07K 2317/734 20130101; A61K 2039/505
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1-70. (cancelled)
71. An antibody or antigen-binding fragment thereof, comprising: a
humanized heavy chain variable region (VH) and a humanized light
chain variable region (VL); wherein the VH comprises an amino acid
sequence with the formula: HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4,
wherein HFW 1 is SEQ ID NO: 6 or SEQ ID NO: 7, HCDR1 is SEQ ID NO:
8, HFW2 is SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
12, or SEQ ID NO: 13, HCDR2 is SEQ ID NO: 14, SEQ ID NO: 15 or SEQ
ID NO: 16, HFW3 is SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ
ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID
NO: 24, HCDR3 is SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27,
and HFW4 is SEQ ID NO: 28; wherein the VL comprises the amino acid
sequence SEQ ID NO: 29 or SEQ ID NO: 32; and wherein the antibody
or antigen-binding fragment thereof can specifically bind to human
OX40.
72. The antibody or antigen-binding fragment thereof of claim 71,
wherein the VH comprises the amino acid sequence of SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID
NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,
SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID
NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO: 67.
73. The antibody or antigen-binding fragment thereof of claim 71
further comprising a light chain constant region or fragment
thereof fused to the C-terminus of the VL, wherein the light chain
constant region is a human kappa constant region.
74. The antibody or antigen-binding fragment thereof of claim 71,
further comprising a heavy chain constant region or fragment
thereof fused to the C-terminus of the VH, wherein the heavy chain
constant region is a human IgG1 constant region, a human IgG4P
constant region, a human IgG1TM constant region, or a murine IgG1
constant region.
75. The antibody or antigen-binding fragment thereof of claim 71
further comprising the heavy chain amino acid sequence SEQ ID NO:
71 and the light chain amino acid sequence SEQ ID NO: 30.
76. The antibody or antigen-binding fragment of claim 71, wherein
the antigen-binding fragment is an Fv fragment, an Fab fragment, an
F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv
fragment, or an sc(Fv)2 fragment, or any combination thereof.
77. The antibody or antigen-binding fragment thereof of claim 71,
which can specifically bind to OX40 as expressed on Jurkat cells,
primary activated CD4+ or CDS+ T cells from human, cynomolgus
monkey, rhesus monkey, or any combination thereof and which does
not bind to murine or rat OX40.
78. The antibody or antigen-binding fragment thereof of claim 71,
which does not cross react with related Tumor Necrosis Factor
Receptor Superfamily (TNFRSF) proteins.
79. The antibody or antigen-binding fragment thereof of claim 71,
which can induce dose-dependent proliferation of activated CD4+ T
cells and dose-dependent cytokine release in primary activated
human CD4+ T cells in a plate-based assay.
80. The antibody or antigen-binding fragment thereof of claim 79,
wherein the cytokine is IFN.gamma., TNF.alpha., IL-5, IL-10, IL-2,
IL-4, IL-13, IL-8, IL-12 p70, IL-1, or any combination thereof.
81. The antibody or antigen-binding fragment thereof of claim 71,
which can activate the NFKB pathway in OX40 expressing T cells in
the presence of Fc.gamma.R-expressing cells.
82. The antibody or antigen-binding fragment thereof of claim 71,
which can trigger complement-dependent or antibody-dependent
cellular cytotoxicity against OX40-expressing cells.
83. The antibody or antigen-binding fragment thereof of claim 71,
wherein administration of an effective dose to a subject in need of
cancer treatment can inhibit tumor growth in the subject.
84. The antibody or antigen-binding fragment thereof of claim 83,
wherein the tumor growth inhibition is achieved in the presence of
T cells.
85. The antibody or antigen-binding fragment thereof of claim 84,
wherein tumor growth is inhibited by at least 10%, at least 20%, at
least 30%, at least 40%, and least 50%, at least 60%, or at least
70% compared to administration of an isotype-matched control
antibody or antigen-binding fragment thereof.
86. A composition comprising the antibody or antigen-binding
fragment thereof of claim 71 and a carrier.
87. A polynucleotide comprising a nucleic acid that encodes the
antibody or antigen-binding fragment thereof of claim 71.
88. The polynucleotide of claim 87, wherein the polynucleotide
comprises the nucleic acid of SEQ ID NO: 60, the nucleic acid of
SEQ ID NO: 31, the nucleic acid of SEQ ID NO: 72, or any
combination thereof.
89. A vector comprising the polynucleotide of claim 87.
90. A host cell comprising the polynucleotide of claim 87.
91. A method of producing an antibody or antigen-binding fragment
thereof, comprising culturing the host cell of claim 90 under
conditions in which the antibody or antigen-binding fragment
thereof encoded by the polynucleotide is expressed; and recovering
the antibody or antigen-binding fragment thereof.
92. A method to promote survival or proliferation of activated T
cells, comprising contacting activated T cells with the antibody or
antigen-binding fragment thereof of claim 71, wherein the antibody
or antigen-binding fragment thereof can specifically bind to OX40
on the surface of the T cells, wherein the activated T cells are
activated CD4+ T cells, activated CDS+T cells, or a combination
thereof.
93. A method of inducing cytokine release from activated T cells,
comprising contacting activated T cells with the antibody or
antigen-binding fragment thereof of claim 71, wherein the antibody
or antigen-binding fragment thereof can specifically bind to OX40
on the surface of the T cells; and releasing a cytokine from the
activated T cells.
94. The method of claim 93, wherein the cytokine is IFN.gamma.,
TNF.alpha., IL-5, IL-10, IL-2, IL-4, IL-13, IL-8, IL-12 p70, IL-1,
or any combination thereof.
95. A method of promoting T cell activation, comprising contacting
T cells with the antibody or antigen-binding fragment thereof of
claim 71, wherein the antibody or antigen-binding fragment thereof
can specifically bind to OX40 on the surface of the T cells.
96. The method of claim 95, wherein T cell activation can be
measured through stimulation of the NFKB signal transduction
pathway.
97. The method of claim 95, wherein the T cells are activated CD4+
T cells, activated CDS+ T cells, or a combination thereof.
98. The method of claim 97, wherein the activated CD4+ T cells are
human CD4+ T cells, cynomolgus monkey CD4+ T cells, rhesus monkey
CD4+ T cells, or a combination thereof.
99. The method of claim 95, wherein the contacting comprises
administering an effective amount of the antibody or
antigen-binding fragment thereof to a subject.
100. A method of treating cancer in a subject, comprising
administering to a subject in need of treatment an effective amount
of the composition of claim 86.
101. The method of claim 100, wherein the cancer is a solid
tumor.
102. The method of claim 101, wherein administration of the
composition can inhibit tumor growth, can promote tumor reduction,
or both.
103. The method of claim 102, wherein tumor growth inhibition is
achieved in the presence of T cells.
104. A method of enhancing an immune response in a subject
comprising administering to a subject in need thereof a
therapeutically effective amount of the antibody or antigen-binding
fragment thereof of claim 71.
105. The antibody or antigen-binding fragment thereof of claim 71,
which binds to an epitope of human OX40 that falls within amino
acids 108 to 146 of SEQ ID NO: 91.
106. The antibody or antigen-binding fragment thereof of claim 105,
wherein the epitope comprises at least amino acids leucine 116 and
alanine 126 of SEQ ID NO: 91.
107. The antibody or antigen-binding fragment thereof of claim 71,
which binds to a mouse OX40 variant comprising SEQ ID NO: 92,
except for a Q113L mutation and a V124A mutation.
108. An isolated peptide consisting of 100 or fewer amino acids and
comprising amino acids 108 to 146 of SEQ ID NO: 91 except for one,
two, three, four, five, or six single amino acid substitutions,
deletions, or insertions at any position except L116 and A126,
wherein the peptide can be specifically bound by the antibody or
antigen-binding fragment thereof of claim 71.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation application of U.S.
patent application Ser. No. 15/635,847, filed Jun. 28, 2017 (now
U.S. Pat. No. 10,150,815), which is a Divisional application of
U.S. application Ser. No. 14/877,547, filed Oct. 7, 2015 (now U.S.
Pat. No. 9,738,723); and claims benefit of U.S. Provisional
Application No. 62/062,431, filed Oct. 10, 2014. The above listed
applications are incorporated by reference herein in their entirety
for all purposes.
REFERENCE TO SEQUENCE LISTING
[0002] This application incorporates by reference a Sequence
Listing submitted with this application as a text file entitled
"18-1672-USDIVCON SEQUENCE LISTING
(2017-06-28-OX40H-100US1)_jec.TXT" and having a size of 111
kilobytes.
BACKGROUND
[0003] OX40 (CD134; TNFRSF4) is a tumor necrosis factor receptor
found primarily on activated CD4.sup.+ and CD8.sup.+ T cells,
regulatory T (Treg) cells and natural killer (NK) cells (Croft et
al., 2009, Immunol Rev. 229:173-91). OX40 has one known endogenous
ligand, OX40 ligand (OX40L; CD152; TNFSF4), which exists in a
trimeric form and can cluster OX40, resulting in potent cell
signaling events within T cells. Id. Signaling through OX40 on
activated CD4.sup.+ and CD8.sup.+ T cells leads to enhanced
cytokine production, granzyme and perforin release, and expansion
of effector and memory T cell pools (Jensen et al., 2010, Semin
Oncol. 37:524-32). In addition, OX40 signaling on Treg cells
inhibits expansion of Tregs, shuts down the induction of Tregs and
blocks Treg-suppressive function (Voo et al., 2013, J Immunol.
191:3641-50; Vu et al., 2007, Blood. 110:2501-10).
[0004] Immunohistochemistry studies and early flow cytometry
analyses showed that OX40 is expressed on T cells infiltrating a
broad range of human cancers (Baruah et al., 2011, Immunobiology
217:668-675; Curti et al, 2013, Cancer Res. 73:7189-98; Ladanyi et
al, 2004, Clin Cancer Res. 10:521-30; Petty et al, 2002, Am J Surg.
183:512-8; Ramstad et al, 2000, Am J Surg. 179:400-6; Sarff et al,
2008, Am J Surg. 195:621-5; discussion 625; Vetto et al, 1997, Am J
Surg. 174:258-65). While not wishing to be bound by theory, OX40
expression on tumor-infiltrating lymphocytes correlates with longer
survival in several human cancers, suggesting that OX40 signals can
play a role in establishing an antitumor immune response (Ladanyi
et al., 2004, Clin Cancer Res. 10:521-30; Petty et al., 2002, Am J
Surg. 183:512-8).
[0005] In a variety of nonclinical mouse tumor models, agonists of
OX40, including antibodies and OX40 ligand fusion proteins, have
been used successfully with promising results (Kjaergaard et al.,
2000, Cancer Res. 60:5514-21; Ndhlovu et al., 2001, J Immunol.
167:2991-9; Weinberg et al., 2000, J Immunol. 164:2160-9).
Co-stimulating T cells through OX40 promoted anti-tumor activity
that in some cases was durable, providing long-lasting protection
against subsequent tumor challenge (Weinberg et al., 2000, J
Immunol. 164:2160-9). Treg-cell inhibition and co-stimulation of
effector T cells were shown to be necessary for tumor growth
inhibition of OX40 agonists (Piconese et al., 2008, J Exp Med.
205:825-39). Many strategies and technologies have been explored to
enhance the anti-tumor effect of OX40 agonist therapy through
combinations with vaccines, chemotherapy, radiotherapy, and
immunotherapy (Jensen et al., 2010, Semin Oncol. 37:524-32; Melero
et al., 2013, Clin Cancer Res. 19:997-1008).
SUMMARY
[0006] This disclosure provides antibodies that bind to OX40, e.g.,
human OX40. In certain aspects the antibodies provided are
humanized antibodies. For example, this disclosure provides an
antibody or antigen-binding fragment thereof that includes a
humanized heavy chain variable region (VH) and a humanized light
chain variable region (VL), where the VH includes an amino acid
sequence with the formula:
HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4,
[0007] where HFW1 is SEQ ID NO: 6 or SEQ ID NO: 7, HCDR1 is SEQ ID
NO: 8, HFW2 is SEQ ID NO: 9 (WIRX39HPGKGLEX47X48G; where X39 is Q
or K, X47 is W or Y, and X48 is I or M), HCDR2 is SEQ ID NO: 14,
SEQ ID NO: 15 or SEQ ID NO: 16, HFW3 is SEQ ID NO: 17
(RITINX71DTSKNQX78SLQLNSVTPEDTAVYX91CAR; where X71 is P or R, X78
is F or Y, and X91 is Y or F , HCDR3 is SEQ ID NO: 25, SEQ ID NO:
26, or SEQ ID NO: 27), and HFW4 is SEQ ID NO: 28, where the VL
includes the amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 32;
and where the antibody or fragment thereof can specifically bind to
human OX40. In certain aspects, the amino acid sequence of HFW2 is
SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 1, and
in certain aspects the amino acid sequence of HFW3 is SEQ ID NO:
18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ
ID NO: 23, or SEQ ID NO: 24.
[0008] In certain aspects, the VH of the provided antibody or
fragment thereof includes the amino acid sequence SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID
NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,
SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID
NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO: 67. In certain
aspects, the VL of the provided antibody or fragment thereof
includes the amino acid sequence SEQ ID NO: 29 and the VH includes
the amino acid sequence SEQ ID NO: 59.
[0009] In certain aspects, the provided antibody or fragment
thereof further includes a light chain constant region or fragment
thereof fused to the C-terminus of the VL, e.g., a human kappa
constant region or a human lambda constant region. In certain
aspects, the provided antibody or fragment thereof further includes
a heavy chain constant region or fragment thereof fused to the
C-terminus of the VH, e.g., a human IgG1 constant region, a human
IgG4P constant region, a human IgG1TM constant region or a murine
IgG1 constant region. In certain aspects the heavy chain constant
region is a human IgG1 constant region. In certain aspects, the
provided antibody or fragment thereof includes the heavy chain
amino acid sequence SEQ ID NO: 71 and the light chain amino acid
sequence SEQ ID NO: 30. An antigen-binding fragment of an antibody
as provided by this disclosure can be, e.g., an Fv fragment, an Fab
fragment, an F(ab')2 fragment, an Fab' fragment, a dsFv fragment,
an scFv fragment, or an sc(Fv)2 fragment, or any combination
thereof
[0010] In certain aspects, the provided antibody or fragment
thereof can specifically bind to human, cynomolgus monkey, or
rhesus monkey OX40, e.g., can specifically bind to OX40 as
expressed on Jurkat cells, primary activated CD4+ or CD8+ T cells
from human, cynomolgus monkey, rhesus monkey, or any combination
thereof. In certain aspects, the provided antibody or fragment
thereof does not bind to murine or rat OX40. In certain aspects,
the provided antibody or fragment thereof does not cross react with
related TNFRSF proteins.
[0011] In certain aspects, the provided antibody or fragment
thereof can have a binding affinity for human OX40 expressed on
primary activated human CD4+ T cells of about 250 pM to about 370
pM as measured by flow cytometry, e.g., about 312 pM. In certain
aspects, the provided antibody or fragment thereof can achieve 20%
receptor occupancy on primary activated human CD4+ T cells (EC20)
at about 63 to about 93 pM, 50% receptor occupancy on primary
activated human CD4+ T cells (EC50) at about 250 to about 370 pM,
and 90% receptor occupancy on primary activated human CD4+ T cells
(EC90) at about 2290 to about 3330 pM as measured by flow
cytometry. For example, in certain aspects, EC20 is about 78 pM,
EC50 is about 312 pM, and EC90 is about 2810 pM.
[0012] In certain aspects, the provided antibody or fragment
thereof can have a binding affinity for human OX40 expressed on
OX40-overexpressing Jurkat cells of about 250 pM to about 600 pM as
measured by flow cytometry, e.g., about 424 pM. In certain aspects,
the provided antibody or fragment thereof can achieve EC20 on
OX40-overexpressing Jurkat cells at about 60 to about 150 pM, EC50
on OX40-overexpressing Jurkat cells at about 250 to about 600 pM,
and EC90 on OX40-overexpressing Jurkat cells at about 2260 to about
4380 pM as measured by flow cytometry. For example, in certain
aspects, EC20 is about 106 pM, EC50 is about 424 pM, and EC90 is
about 3820 pM.
[0013] In certain aspects, the provided antibody or fragment
thereof can have a binding affinity for cynomolgus monkey OX40
expressed on primary activated cynomolgus monkey CD4+ T cells of
about 340 pM to about 820 pM as measured by flow cytometry, e.g.,
about 580 pM. In certain aspects, the provided antibody or fragment
thereof can have a binding affinity for rhesus monkey OX40
expressed on primary activated rhesus monkey CD4+ T cells of about
130 pM to about 600 pM as measured by flow cytometry, e.g., about
370 pM.
[0014] In certain aspects, the provided antibody or fragment
thereof can induce dose-dependent proliferation of activated CD4+ T
cells and dose-dependent cytokine release in primary activated CD4+
T cells in a plate-based assay. For example, in certain aspects, a
20% maximal proliferation response (EC20) can be achieved in
primary activated human CD4+ T cells at an antibody concentration
of about 14 pM to about 28 pM, a 50% maximal proliferation response
(EC50) can be achieved in primary activated human CD4+ T cells at
an antibody concentration of about 0.3 pM to about 130 pM, and a
90% maximal proliferation response (EC90) can be achieved in
primary activated human CD4+ T cells at an antibody concentration
of about 50 pM to about 90 pM, all as measured by flow cytometry.
In certain aspects, EC20 is about 21 pM, EC50 is about 28 pM, and
EC90 is about 72 pM. The released cytokine in primary activated
human CD4+ T cells can be one, two, three or more of, without
limitation, IFN.gamma., TNF.alpha., IL-5, IL-10, IL-2, IL-4, IL-13,
IL-8, IL-12 p'70, IL-1.beta., or any combination thereof, for
example, IFN.gamma., TNF.alpha., IL-5, IL-10, IL-13, or any
combination thereof. In certain aspects, the provided antibody or
fragment thereof can achieve CD4+ T cell proliferation and cytokine
release in primary activated cynomolgus monkey CD4+ T cells and in
primary activated rhesus monkey CD4+ T cells.
[0015] In certain aspects, the provided antibody or fragment
thereof can activate the NF.kappa.B pathway in OX40 expressing T
cells in the presence of Fc.gamma.R-expressing cells. For example,
the OX40-expressing T cells can be OX40-overexpressing Jurkat
NF.kappa.B-luciferase reporter cells that produce luciferase in
response to stimulation of the NF.kappa.B signaling pathway. In
certain aspects, the provided antibody or fragment thereof can
trigger complement-dependent or antibody-dependent cellular
cytotoxicity against OX40-expressing cells. In certain aspects, the
provided antibody or fragment thereof can bind to human C1 q and
trigger NK-mediated antibody-dependent cellular cytotoxicity
against the OX40-expressing cells.
[0016] In certain aspects, administration of an effective dose of
the provided antibody or fragment thereof to a subject in need of
cancer treatment can inhibit tumor growth in the subject. For
example, the tumor growth inhibition can be achieved in the
presence of T cells. In certain aspects, tumor growth is inhibited
by at least 10%, at least 20%, at least 30%, at least 40%, and
least 50%, at least 60%, or at least 70% compared to administration
of an isotype-matched control antibody or fragment thereof.
[0017] This disclosure further provides a composition including the
antibody or fragment thereof as described above, and a carrier.
[0018] This disclosure further provides a polynucleotide that
includes a nucleic acid that encodes the provided antibody or
fragment thereof, or encodes a polypeptide subunit of the provided
antibody or fragment thereof. In certain aspects, the provided
polynucleotide includes the nucleic acid of SEQ ID NO: 60, the
nucleic acid of SEQ ID NO: 31, the nucleic acid of SEQ ID NO: 72,
or any combination thereof. This disclosure further provides a
vector that includes the provided polynucleotide and a host cell
that includes the provided polynucleotide or the provided vector.
In another aspect, the disclosure provides a method of producing an
antibody or fragment thereof, where the method includes culturing
the provided host cell under conditions in which the antibody or
fragment thereof encoded by the polynucleotide is expressed, and
recovering the antibody or fragment thereof.
[0019] In additional aspects, the disclosure provides a method to
promote survival or proliferation of activated T cells, where the
method includes contacting activated T cells with the provided
antibody or fragment thereof, and where the antibody or fragment
thereof can specifically bind to OX40 on the surface of the T
cells.
[0020] In additional aspects, the disclosure provides a method of
inducing cytokine release from activated T cells, where the method
includes contacting activated T cells with the provided antibody or
fragment thereof, and where the antibody or fragment thereof can
specifically bind to OX40 on the surface of the T cells. In certain
aspects the released cytokine can be one, two, three or more of,
without limitation, IFN.gamma., TNF.alpha., IL-5, IL-10, IL-2,
IL-4, IL-13, IL-8, IL-12 p'70, IL-1.beta., or any combination
thereof, e.g., IFN.gamma., TNF.alpha., IL-5, IL-10, IL-13, or any
combination thereof. In certain aspects, the activated T cells are
activated CD4+ T cells, activated CD8+ T cells, or a combination
thereof. In certain aspects, the activated CD4+ T cells are human
CD4+ T cells, cynomolgus monkey CD4+ T cells, rhesus monkey CD4+ T
cells, or a combination thereof
[0021] In additional aspects, the disclosure provides a method of
promoting T cell activation, where the method includes contacting T
cells with the provided antibody or fragment thereof, where the
antibody or fragment thereof can specifically bind to OX40 on the
surface of the T cells. In certain aspects, T cell activation can
be measured through stimulation of the NF.THETA.B signal
transduction pathway. In certain aspects, the T cells are activated
CD4+ T cells, activated CD8+ T cells, or a combination thereof. In
certain aspects, the activated CD4+ T cells are human CD4+ T cells,
cynomolgus monkey CD4+ T cells, rhesus monkey CD4+ T cells, or a
combination thereof. In certain aspects, the contacting includes
administering an effective amount of the antibody or fragment
thereof to a subject.
[0022] In additional aspects, the disclosure provides a method of
treating cancer in a subject, where the method includes
administering to a subject in need of treatment an effective amount
of the provided antibody or fragment thereof, or the provided
composition. In certain aspects, the cancer is a solid tumor. In
certain aspects, administration of the antibody or fragment thereof
or composition can inhibit tumor growth, can promote tumor
reduction, or both. In certain aspects, tumor growth inhibition is
achieved in the presence of T cells.
[0023] In additional aspects, the disclosure provides a method of
enhancing an immune response in a subject, where the method
includes administering to a subject in need thereof a
therapeutically effective amount of the provided antibody or
fragment thereof, or the provided composition.
[0024] In the therapeutic methods provided by this disclosure, the
subject to be treated can be a human subject.
[0025] In certain aspects, the provided antibody or fragment
thereof can bind to an epitope of human OX40 that falls within
amino acids 108 to 146 of SEQ ID NO: 91. In certain aspects, the
epitope includes at least amino acids leucine 116 (L116) and
alanine 126 (A126) of SEQ ID NO: 91. In certain aspects, the
provided antibody or fragment thereof can bind to a mouse OX40
variant that has the amino acid sequence of SEQ ID NO: 92, except
for a Q113L mutation and a V124A mutation.
[0026] This disclosure further provides an isolated peptide
consisting of 100 or fewer amino acids, where the peptide can be
specifically bound by the provided antibody or fragment thereof. In
certain aspects, the peptide includes amino acids 116 to 126 of SEQ
ID NO: 91. In certain aspects, the peptide includes amino acids 108
to 146 of SEQ ID NO: 91 except for one, two, three, four, five, or
six single amino acid substitutions, deletions, or insertions at
any position except L116 and A126.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0027] FIG. 1. Mutations in humanized 9B12 VH regions: Numbers in
the clonal nick name represent the position of the amino acid in
VH, according to Kabat numbering. Mabs 1, 2, 5 and 8 are VH
chimeric variants paired with humanized VL. Mabs 10-17 are
humanized VH variants with mouse back mutations. Mabs 18-27 are
variants engineered to remove potential sequence liabilities.
Variable regions of Mab24 and mAb27 were grafted onto different
heavy constant regions for the resulting isotype variants named
mAb28-30 and mAb31-32, mAb37, respectively.
[0028] FIGS. 2A-D. Binding of OX40mAb24 and 9B12 to OX40 Expressed
on the Surface of Primary Activated Human CD4.sup.+ T cells.
MFI=mean fluorescence intensity of AlexaFluor.RTM. 647 labeled
secondary anti-human antibody binding to OX40mAb24 (FIGS. 2A-B) or
AlexaFluor.RTM. 488 labeled anti-mouse secondary antibody binding
to 9B12 (FIGS. 2C-D) on primary human CD4.sup.+ T cells.
[0029] FIGS. 3A-F. Binding of OX40mAb24 and 9B12 to Human OX40
Expressed on Jurkat T cells. MFI=mean fluorescence intensity of
AlexaFluor.RTM. 647 labeled secondary anti-human antibody binding
to OX40mAb24 (FIGS. 3A-C) or AlexaFluor.RTM. 488 labeled anti-mouse
secondary antibody binding to 9B12 (FIGS. 3D-F) on Jurkat T
cells.
[0030] FIGS. 4A-B. Binding of OX40mAb24 to TNFRSF-expressing HEK293
cells and OX40-expressing Jurkat T cells. (FIG. 4A) Transient
expression of TNFRSF members, as indicated to left of histograms,
in HEK293 cells, and binding to TNFRSF-specific mAbs or to
OX40mAb24, as indicated above histograms. Gray histogram,
fluorochrome-conjugated isotype control antibody binding for
TNFRSF-specific mAb, or goat anti-human AlexaFluor.RTM. 647
secondary antibody binding control for OX40mAb24; Open histogram,
TNFRSF-specific mAb or OX40mAb24 binding. (FIG. 4B): Binding of
OX40mAb24 to OX40-expressing Jurkat as a positive control. Gray
histogram, goat anti-human AlexaFluor.RTM. 647 secondary antibody
binding control; Open histogram, OX40mAb24 binding.
[0031] FIGS. 5A-B. Binding of OX40mAb24 (OX40mAb29)
(complementarity-determining regions) CDRs and 9B12 to recombinant
human TNFRSF members by ELISA. Results of ELISA assays
demonstrating specific binding of OX40 by OX40mAb29 (FIG. 5A) and
9B12 (FIG. 5B). OX40mAb29 contains the CDRs of OX40mAb24. Antibody
binding of OX40 was compared to binding of other human TNFRSF
proteins.
[0032] FIG. 6. Schematic diagram of the OX40mAb24 plate-bound
bioactivity assay. Q=OX40mAb24; Y=anti-human CD3 antibody clone
OKT3.
[0033] FIGS. 7A-C. Human CD4.sup.+ T cell Proliferation in response
to OX40mAb24 and 9B12. (FIG. 7A) CD4 T cell proliferation of four
independent donors mediated by plate-immobilized OX40mAb24 in
combination with sub-mitogenic TCR stimulation (anti-CD3). Data
points were normalized to the lower asymptotic value of raw data
curves prior to graphing to enhance visualization of the dynamic
range of each response. (FIG. 7B) Representative raw data from
Donor 651 demonstrating proliferation-driven by OX40mAb24 plus
sub-mitogenic TCR stimulation (anti-CD3) and the relative lack of
proliferation mediated by the R347 human IgG1 control mAb, soluble
OX40mAb24 in the presence or absence of concomitant TCR signaling,
anti-CD3 mAb alone without OX40mAb24, and by plate-immobilized
OX40mAb24 in the absence of anti-CD3 mAb. (FIG. 7C) CD4 T cell
proliferation of four independent donors mediated by
plate-immobilized 9B12 in combination with sub-mitogenic TCR
stimulation. Symbols, mean values; Error bars, standard deviation
of the mean; n=3 technical replicates for OX40mAb24, R347 human
IgG1 control mAb, and 9B12 all in combination with anti-CD3; n=2
technical replicates for soluble OX40mAb24, and CD3 in the absence
of OX40mAb24, plate bound OX40mAb24 with no anti-CD3, and soluble
OX40mAb24 with no anti-CD3.
[0034] FIGS. 8A-E. Human CD4.sup.+ T cell Cytokine Release in
Response to OX40mAb24. Representative OX40mAb24 induced human CD4 T
cell cytokine release for Donor 651, including (FIG. 8A)
IFN.gamma., (FIG. 8B) TNF.alpha. (FIG. 8C) IL-10 (FIG. 8D) IL-13,
and (FIG. 8E) IL-5. Symbols, mean values; Error bars, standard
deviation of the mean; n=3 technical replicates for OX40mAb24 and
R347 human IgG1 control mAb, both in combination with anti-CD3; n=2
technical replicates for soluble OX40mAb24, soluble OX40mAb24 with
no anti-CD3, and anti-CD3 in the absence of OX40mAb24.
[0035] FIGS. 9A-E. Human CD4.sup.+ T cell Cytokine Release in
Response to 9B12. Representative 9B12 induced human CD4 T cell
cytokine release for Donor 651, including (FIG. 9A) IFN.gamma.,
(FIG. 9B) TNF.alpha. (FIG. 9C) IL-10 (FIG. 9D) IL-13, and (FIG. 9E)
IL-5. Symbols, mean values; Error bars, standard deviation of the
mean; n=3 technical replicates for 9B12 and mouse IgG1 control mAb,
both in combination with anti-CD3; n=2 technical replicates for
soluble 9B12, soluble 9B12 with no anti-CD3, and anti-CD3 in the
absence of 9B12.
[0036] FIG. 10 is a schematic illustrating cell systems used for
measuring OX40mAb24 and 9B12 bioactivity. OX40mAb24 cross linking
by Fc.gamma.R-expressing cells mediates the clustering and
activation of OX40 on the cell surface of an OX40-expressing Jurkat
NF.kappa.B-luciferase reporter cell line, resulting in the
NF.kappa.B-mediated production of luciferase that can be measured
as a surrogate for OX40 activation. Fc.gamma.R=fragment
crystallizable gamma receptor; NF.kappa.B=nuclear factor kappa
B.
[0037] FIGS. 11A-D. Bioactivity of OX40mAb24 in OX40-expressing
Jurkat NF.kappa.B Reporter Cells With and Without Cross-linking by
Fc.gamma.R. Concentration-dependent activity (in RLU) of
OX40mAb24-induced signaling through human OX40 expressed on the
cell surface of a Jurkat NF.kappa.B-luciferase reporter cell line
in a 2-cell bioassay. Reporter activity after cross-linking of
OX40mAb24 by (FIG. 11A) CD32A-expressing HEK293 cells, and HEK293
parental cells (HEK), or in the presence of (FIG. 11B)
CD32B-expressing HEK293 cells, (FIG. 11C) Raji B cells, or (FIG.
11D) CD45.sup.+ cells isolated from a primary lung tumor. Data is
representative of other results found in Table 5-2. mAb=monoclonal
antibody, RLU=relative light units.
[0038] FIGS. 12A-D. Bioactivity of 9B12 in OX40-expressing Jurkat
NF.kappa.B Reporter Cells using Fc.gamma.R Cross-linking by
Different Cell Types in 2-Cell Bioactivity Assays.
Concentration-dependent activity (in RLU) of 9B12 induced signaling
through human OX40 expressed on the cell surface of a Jurkat
NF.kappa.B-luciferase reporter cell lines in a two-cell bioassay.
Reporter activity after cross-linking of 9B12 by (FIG. 12A)
CD32A-expressing HEK293 cells, (FIG. 12B) CD32B-expressing HEK293
cells, (FIG. 12C) Raji B cells, or (FIG. 12D) CD45.sup.+ cells
isolated from a primary lung tumor. Data is representative of other
results found in Table 5-3. mAb=monoclonal antibody; RLU=relative
light units.
[0039] FIGS. 13A-B. Natural Killer Cell-mediated Antibody-Dependent
Cellular Cytotoxicity of OX40mAb24, Experiment 1. (FIG. 13A)
Specific killing of OX40-expressing activated CD4.sup.+ T cells by
human NK cells from an allogeneic, left, or autologous, right, NK
and CD4.sup.+ T cell donor pairs using 10 .mu./mL of 9B12,
OX40mAb24, or the IgG1 triple mutant (mAb29) or human IgG4P (mAb28)
versions of OX40mAb24. (FIG. 13B) Lysis of Toledo B cells by NK
cells from donors 350 and 351 in the presence of rituximab, but not
R347 human IgG1 isotype control antibody. Technical replicates were
conducted in triplicate. Error bars represent standard error of the
mean. ADCC=antibody-drug-dependent-cytotoxicity; mAb=monoclonal
antibody; NK=natural killer.
[0040] FIGS. 14A-B. Natural Killer Cell-mediated Antibody-Dependent
Cellular Cytotoxicity of OX40mAb24, Experiment 2. (FIG. 14A)
Specific killing of OX40-expressing activated CD4 T cells by human
NK cells from an allogeneic, left, or autologous, right, NK and CD4
T cell donor pairs using 10 .mu./mL of control R347 human IgG1,
9B12, OX40mAb24, the human IgG4P (mAb28) or the IgG1 triple mutant
(mAb29) versions of OX40mAb24. (FIG. 14B) Lysis of Toledo B cells
by NK cells from donors 558 and 589 in the presence of rituximab,
but not R347 human IgG1 isotype control antibody. Technical
replicates were conducted in triplicate. Error bars represent
standard error of the mean.
ADCC=antibody-drug-dependent-cytotoxicity; mAb=monoclonal antibody;
NK=natural killer.
[0041] FIGS. 15A-D. Assessment of Natural Killer Cell-mediated
Antibody-Dependent Cellular Cytotoxicity of OX40mAb24 and 9B12,
Experiment 3. (FIGS. 15A-B) Specific killing of OX40-expressing
human CD4 T cells mediated by OX40mAb24, but not by 9B12, using
primary human NK cells from two separate donors as indicated.
(FIGS. 15C-D) Lysis of Toledo B cells by NK cells from donors 363
and 504 in the presence of rituximab, but not R347 human IgG1
isotype control antibody. Technical replicates were conducted in
duplicate. Error bars represent standard error of the mean.
ADCC=antibody-drug-dependent-cytotoxicity; mAb=monoclonal antibody;
NK=natural killer.
[0042] FIGS. 16A-D. Assessment of Natural Killer Cell-mediated
Antibody-Dependent Cellular Cytotoxicity of OX40mAb24, Experiment
4. (FIGS. 16A-B) Specific killing of OX40-expressing human CD4 T
cells mediated by OX40mAb24 using primary human NK cells from two
separate donors as indicated. (FIGS. 16C-D) Lysis of Toledo B cells
by NK cells from donors 464 and 532 in the presence of rituximab,
but not R347 human IgG1 isotype control antibody. Technical
replicates were conducted in duplicate. Error bars represent
standard error of the mean.
ADCC=antibody-drug-dependent-cytotoxicity; mAb=monoclonal antibody;
NK=natural killer.
[0043] FIGS. 17A-B. Assessment of Natural Killer Cell-mediated
Antibody-Dependent Cellular Cytotoxicity of OX40mAb24, Experiment
5. Specific killing of OX40-expressing human CD4.sup.+ T cells
mediated by OX40mAb24, using primary human NK cells from donors 601
(FIG. 17A) and 602 (FIG. 17B) as indicated. Error bars represent
standard error of the mean.
ADCC=antibody-drug-dependent-cytotoxicity; mAb=monoclonal antibody;
NK=natural killer.
[0044] FIGS. 18A-B. Assessment of OX40mAb24 and 9B12 binding to
purified human C1q protein. The indicated concentrations of
purified human C1q protein were injected onto the biosensor chip.
Blank represents injections of PBS/0.005% Tween 20 vehicle alone.
FIG. 18A: OX40mAb24 binding to purified human C1q. FIG. 18B: 9B12
binding to purified human C1q.
[0045] FIGS. 19A-B. OX40mAb24 Activity in Cyno/Rhesus
OX40-expressing Jurkat NF.kappa.B-luciferase Clone B2 and LCL8664
Rhesus B-Cell Bioactivity Assays. Concentration-dependent induction
of NF.kappa.B activity by OX40mAb24 (in RLU) in a cyno/rhesus OX40
expressing Jurkat NF.kappa.B-luciferase reporter cell line combined
with rhesus B-cell line LCL8664. Data shown for 2 independent
assays (FIG. 19A and FIG. 19B) with 4 replicates for each data
point. Error bars represent standard error of the mean are not
visible due to scale. RLU=relative light units.
[0046] FIGS. 20A-B. 9B12 Activity in Cyno/Rhesus OX40-expressing
Jurkat NF.kappa.B-luciferase Clone B2 and LCL8664 Rhesus B-Cell
Bioactivity Assays. Concentration-dependent induction of NF.kappa.B
activity by 9B12 (in RLU) in a cyno/rhesus OX40 expressing Jurkat
NF.kappa.B-luciferase reporter cell line combined with rhesus
B-cell line LCL8664. Data shown for 2 independent assays (FIG. 20A
and FIG. 20B) with 4 replicates for each data point. Error bars
represent standard error of the mean. RLU=relative light units.
[0047] FIGS. 21A-B. OX40mAb24 and 9B12 Activity in Cyno/Rhesus
OX40-expressing Jurkat NF.kappa.B-luciferase Clone B2 and Fc Gamma
Receptor-Expressing Rhesus Immune Cells Bioactivity Assay.
Concentration-dependent induction of NF.kappa.B activity by
OX40mAb24 (FIG. 21A) and 9B12 (FIG. 21B) (in RLU) in a cyno/rhesus
OX40 expressing Jurkat NF.kappa.B-luciferase reporter cell line
combined with Fc.gamma. receptor-expressing rhesus immune cells.
Two replicates per data point. RLU=relative light units.
[0048] FIGS. 22A-D. OX40mAb24 and 9B12 Cause Proliferation of
Primary Rhesus CD4 T Cells. OX40mAb24 (FIGS. 22A-B) and 9B12 (FIGS.
22C-D) induced cell division in primary activated rhesus CD4.sup.+
T cells. Data is shown for 2 independent assays with triplicate
wells. Error bars represent standard error of the mean.
[0049] FIGS. 23A-B. Effect of OX40mAb24 and 9B12 on Growth of A375
Cells in a Mouse Xenograft Model--Experiment 1. Six NOD/SCID mice
in each group were engrafted SC on Day 1 with A375 cells mixed with
alloreactive human CD4.sup.+ and CD8.sup.+ T cell lines at E:T
ratio 1:6. OX40mAb24 (FIG. 23A) and 9B12 (FIG. 23B) and isotype
control (FIGS. 23A-B) were administered IP on Days 3, 5, 7, 10 and
12. Mean values of tumor volumes are shown. A comparison between
OX40mAb24-treated (FIG. 23A) or 9B12-treated (FIG. 23B) and the
isotype control-treated animals was made on Day 25 and 18,
respectively, and intergroup differences were analyzed for
statistical significance by a Mann-Whitney rank sum test. Error
bars represent standard error of the mean. *: TGI>68%, P<0.05
as compared to the isotype-control group. E:T=effector-to-target
ratio; IP intraperitoneal; NOD/SCID=non-obese diabetic/severe
combined immunodeficient; SC=subcutaneous; TGI=tumor growth
inhibition.
[0050] FIGS. 24A-B. Effect of OX40mAb24 and 9B12 on Growth of A375
Cells in a Mouse Xenograft Model--Experiment 2. Six NOD/SCID mice
in each group were engrafted SC on Day 1 with A375 cells mixed with
alloreactive human CD4.sup.+ and CD8.sup.+ T cell lines at E:T
ratio 1:6. OX40mAb24 (FIG. 24A) and 9B12 (FIG. 24B) and isotype
control (FIGS. 24A-B) were administered IP on Days 3, 5, 7, 10 and
12. Mean values of tumor volumes are shown. A comparison between
OX40mAb24-treated (FIG. 24A) or 9B12-treated (FIG. 24B) and the
isotype control-treated animals was made on Day 18, and intergroup
differences were analyzed for statistical significance by a
Mann-Whitney rank sum test. Error bars represent standard error of
the mean. #: TGI.gtoreq.75%, P.ltoreq.0.0004 as compared to the
isotype-control group. *: TGI=53%, P.ltoreq.0.05 as compared to the
isotype-control group. E:T=effector-to-target ratio;
IP=intraperitoneal; NOD/SCID=non-obese diabetic/severe combined
immunodeficient; SC=subcutaneous; TGI=tumor growth inhibition.
[0051] FIG. 25. Effect of OX40mAb24 on Growth of A375 Cells in a
Mouse Xenograft Model--Experiment 3. Six NOD/SCID mice in each
group were engrafted SC on Day 1 with A375 cells mixed with
alloreactive human CD4.sup.+ and CD8.sup.+ T cell lines at E:T
ratio 1:6. OX40mAb24 was administered IP on Days 3, 6, 8, 10 and
13. Mean values of tumor volumes are shown. A comparison between
OX40mAb24-treated and the isotype control-treated animals was made
on Day 28, and intergroup differences were analyzed for statistical
significance by a Mann-Whitney rank sum test. Error bars represent
standard error of the mean. *: TGI=75%, P<0.05 as compared to
the isotype-control group; E:T=effector-to-target ratio;
IP=intraperitoneal; NOD/SCID=non-obese diabetic/severe combined
immunodeficient; SC=subcutaneous; TGI=tumor growth inhibition.
[0052] FIGS. 26A-B. Effect of OX86 mIgG2a on Growth of CT26 Cell
Line in a Mouse Syngeneic Model. Ten BALB/c mice in each group were
inoculated SC on Day 1 with CT26 cells. Control (negative control)
and test article (OX86 mIgG2a) were administered IP on Days 9 and
12 (arrows in (FIG. 26A)). Mean (FIG. 26A) and individual (FIG.
26B) values of tumor volumes are shown. A comparison between OX86
mIgG2a-treated and the negative control-treated animals was made,
and intergroup differences were analyzed for statistical
significance by a one-way ANOVA using GraphPad Prism 6.0 software.
Error bars represent standard error of the mean. *: TGI>50%,
P<0.0001 as compared to the isotype-control group on Day 21.
IP=intraperitoneal; SC=subcutaneous; TGI=tumor growth
inhibition.
[0053] FIGS. 27A-B. Effect of OX86 mIgG2a on Growth of CT26 Cell
Line in a Mouse Syngeneic Model. Twelve BALB/c mice in each group
were inoculated SC on Day 1 with CT26 cells. Test article (OX86
mIgG2a) was administered IP on Days 13 and 16 (arrows in
(FIG.27A)). Mean (FIG. 27A) and individual (FIG. 27B) values of
tumor volumes are shown. A comparison between OX86 mIgG2a-treated
and the untreated control animals was made, and intergroup
differences were analyzed for statistical significance by a one-way
ANOVA using GraphPad Prism 6.0 software. Error bars represent
standard error of the mean. *: TGI>50%, P<0.0001 as compared
to the untreated control group on Day 24. IP=intraperitoneal;
SC=subcutaneous; TGI=tumor growth inhibition.
[0054] FIGS. 28A-B. Effect of OX86 mIgG2a on Growth of MCA205 Cell
Line in a Mouse Syngeneic Model. Fourteen C57BL/6 mice in each
group were inoculated SC on Day 1 with MCA205 cells. Control
article (isotype control) and test article (OX86 mIgG2a) were
administered IP on Days 11 and 14. Mean (FIGS. 28A) and individual
(FIGS. 28B) values of tumor volumes are shown. A comparison between
OX86 mIgG2a-treated and the isotype control-treated animals was
made, and intergroup differences were analyzed for statistical
significance by a one-way ANOVA using GraphPad Prism 6.0 software.
Error bars represent standard error of the mean. *: TGI>50%,
P<0.0001 as compared to the isotype-control group on Day 27.
IP=intraperitoneal; SC=subcutaneous; TGI=tumor growth
inhibition.
[0055] FIGS. 29A-B. Effect of OX86 mIgG2a on Growth of 4T1 Cell
Line in a Mouse Syngeneic Model. Twelve BALB/c mice in each group
were inoculated on Day 1 with 4T1 cells. Control article (isotype
control) and test article (OX86 mIgG2a) were administered IP on
Days 13, 16, 20, and 23. Mean (FIG. 29A) and individual (FIG. 29B)
values of tumor volumes are shown. A comparison between OX86
mIgG2a-treated and the isotype control-treated animals was made,
and intergroup differences were analyzed for statistical
significance by a one-way ANOVA using GraphPad Prism 6.0 software.
Error bars represent standard error of the mean.
IP=intraperitoneal; SC=subcutaneous; TGI=tumor growth
inhibition.
[0056] FIG. 30. Amino Acid Alignment of the Extracellular Domains
of Human and Mouse OX40 molecules. Human OX40 (NCBI reference
sequence NP_003318.1) shares 60% sequence identity with mouse OX40
(NCBI reference sequence NP_035789.1). The alignment was performed
using the method of Clustal W. The extracellular domains of OX40
are detailed. Amino acids that differ between human and mouse in
the CRD3 domain are shown with arrows. The two critical epitope
residues L116 and A126 are boxed. CRD=cysteine-rich domain.
[0057] FIGS. 31A-B. Nomenclature and Schematic Representation of
Chimeric Human/Mouse OX40 Variants. Chimeric human/mouse OX40
variants were constructed by swapping in or out various domains or
residues of mouse OX40 (open) into human (solid) (KO) or of human
OX40 amino acids into mouse OX40 (KI). Mutations for individual
amino acids or combinations were shown with a red (KO) and green
(KI) arrows. CRD=cysteine-rich domain; KI=knock-in; KO=knock-out;
TM=transmembrane domain.
[0058] FIGS. 32A-C. FACS Analysis of Binding of OX40mAb24 to
Chimeric Human/Mouse OX40 Variants. All variants were transiently
expressed using 293F cells for binding characterization with FACS
analysis using OX40mAb24 and its parental mouse mAb, 9B12.
Expression levels were monitored using anti-human and mouse OX40
polyclonal antibodies. (FIG. 32A) Using domain-swapped chimeric
variants, the CRD3 domain was identified as the epitope-containing
domain. OX40mAb24 and 9B12 do not bind the KO variants encoding for
mouse CRD3 domain (KO_CRD3 and KO_CRD3+4), and recognize the KI
variant (KI_CRD3) encoding for human CRD3 domain. (FIG. 32B)
Additionally, critical epitope residues were determined as L116 and
A126 in CRD3 domain by mutating individual or combinations of amino
acids, which differ between human and mouse in the CRD3 domain
(FIG. 1). The binding of OX40mAb24 and 9B12 was abolished when
replacing human residues L116 and A126 with the mouse counterparts
(KO_L116+A126). (FIG. 32C) The KI/gain-of-function variants confirm
the importance of these two critical residues. Grafting L116, A126,
or the combination to mouse OX40 led to the binding of OX40mAb24
and 9B12.
[0059] FIGS. 33A-D. Expression Levels of Ki67 and ICOS on
Peripheral Blood and Splenic CD4.sup.+ T cells Following
Administration of OX86 mIgG2a to Naive Mice. Seven naive BALB/c
mice in each group were inoculated intraperitoneally on Day 1 with
control articles (saline and NIP228 IgG2a isotype control) and test
article (OX86 mIgG2a) at the indicated dose levels. Blood (FIG. 33A
and FIG. 33C) was collected on the indicated Days and spleens from
five groups were isolated on Day 10. Expression levels of Ki67
(FIG. 33A and FIG. 33B) and ICOS (FIG. 33C and FIG. 33D) on
CD4.sup.+ T cells were measured by flow cytometry. Mean values of
the percentage of CD4 cells in blood expressing Ki67 (FIG. 33A) and
ICOS (FIG. 33C) are shown for each group. Percentage of CD4 cells
in the spleen expressing Ki67 (FIG. 33B) and ICOS (FIG. 33D) were
plotted for each animal of individual groups. Error bars represent
the standard error of the mean. *: P.ltoreq.0.05 are marked in FIG.
33A and FIG. 33C; P values are listed for each group with
significance in FIG. 33B and FIG. 33D.
[0060] FIGS. 34A-B. Correlation of Ki67 and ICOS Expression on
Peripheral Blood and Splenic CD4.sup.+ T cells Following
Administration of OX86 mIgG2a to Naive Mice. A comparison was made
between the percentage of Ki67 and ICOS positive CD4 T cells
isolated from peripheral blood (FIG. 34A) and spleens (FIG. 34B) of
individual animals shown in FIG. 12 10 days following treatment
with control articles (saline and NIP228 IgG2a isotype control) and
test article (OX86 mIgG2a) at the indicated dose levels.
Measurements for individual mice were plotted. Linear regression
analysis was performed using GraphPad Prism 6.0 software on the
resulting group of data sets to determine a best-fit line for the
data. The coefficient of determination (r2) and significance that
the slope is non-zero (P value) are provided for each graph.
[0061] FIGS. 35A-D. Expression Levels of Ki67 and ICOS on
Peripheral Blood and Splenic CD8.sup.+ T cells Following
Administration of OX86 mIgG2a to Naive Mice. Seven naive BALB/c
mice in each group were inoculated intraperitoneally on Day 1 with
control articles (saline and NIP228 IgG2a isotype control) and test
article (OX86 mIgG2a) at the indicated dose levels. Blood (panels A
and C) was collected on the indicated Days and spleens from five
groups were isolated on Day 10. Expression levels of Ki67 (FIG. 35A
and FIG. 35B) and ICOS (FIG. 35C and FIG. 35D) on CD8.sup.+ T cells
were measured by flow cytometry. Mean values of the percentage of
CD8 cells in blood expressing Ki67 (FIG. 35A) and ICOS (FIG. 35C)
are shown for each group. Percentage of CD8 cells in the spleen
expressing Ki67 (FIG. 35B) and ICOS (FIG. 35D) were plotted for
each animal of individual groups. Error bars represent the standard
error of the mean. *: P.ltoreq.0.05 are marked in FIG. 35A and FIG.
35C; P values are listed for each group with significance in FIG.
35B and FIG. 35D.
[0062] FIGS. 36A-B. Correlation of Ki67 and ICOS Expression on
Peripheral Blood and Splenic CD8.sup.+ T cells Following
Administration of OX86 mIgG2a to Naive Mice. A comparison was made
between the percentage of Ki67 and ICOS positive CD8.sup.+ T cells
isolated from peripheral blood (FIG. 36A) and spleens (FIG. 36B) of
individual animals shown in FIG. 12 10 days following treatment
with control articles (saline and NIP228 IgG2a isotype control) and
test article (OX86 mIgG2a) at the indicated dose levels.
Measurements for individual mice were plotted. Linear regression
analysis was performed using GraphPad Prism 6.0 software on the
resulting group of data sets to determine a best-fit line for the
data. The coefficient of determination (r2) and significance that
the slope is non-zero (P value) are provided for each graph.
[0063] FIGS. 37A-D. Effect of Mouse OX86 IgG2a on Growth of the
CT26 Cell Line in Syngeneic Mouse Model Lacking Inhibitory
(Fcgr2b-/-) or Activating (Fcer1g-/-) Fc Gamma Receptors. Groups of
eight Balb/c mice genetically engineered to lack the inhibitory
Fc.gamma. receptor IIb (Fcgr2b-/-; FIG. 37A and FIG. 37B) or the
activating Fc.gamma. receptors (Fcer1g-/-; FIG. C and FIG. D) were
inoculated SC on Day 1 with CT26 cells. Control articles
(saline/untreated and OX86 mIgG1 D265A mutant/isotype control) and
test article (OX86 mIgG2a) were administered IP on Days 4 and 7.
Mean (FIG. 37A and FIG. 37C) and individual (FIG. 37B and FIG. 37D)
tumor volumes are shown. A comparison between OX86 mIgG2a-treated
and the untreated and isotype control-treated animals was made, and
intergroup differences were analyzed for statistical significance
by a one-way ANOVA using GraphPad Prism 6.0 software. Error bars
represent standard error of the mean. SC=subcutaneous;
IP=intraperitoneal; TGI=tumor growth inhibition.
[0064] FIGS. 38A-D. Effect of Mouse OX86 IgG2a on Growth of the
MCA205 Cell Line in a Syngeneic Mouse Model Lacking Inhibitory
(Fcgr2b-/-) or Activating (Fcer1g-/-) Fc Gamma Receptors. Groups of
eight C57BL/6 mice genetically mutated to lack the inhibitory
Fc.gamma. receptor IIb (Fcgr2b-/-; FIG. 38A and FIG. 38B) or the
activating Fc.gamma. receptors (Fcer1g-/-; C and D were inoculated
SC on Day 1 with MCA205 cells. Control articles (saline/untreated
and OX86 mIgG1 D265A mutant/isotype control) and test article
(mOX40L FP) were administered IP on Days 4 and 7. Mean (FIG. 38A
and FIG. 38C) and individual (FIG. 38B and FIG. 38D) tumor volumes
are shown. A comparison between mOX40L FP-treated and the untreated
and isotype control-treated animals was made, and intergroup
differences were analyzed for statistical significance by a one-way
ANOVA using GraphPad Prism 6.0 software. Error bars represent
standard error of the mean. *: TGI.gtoreq.95%, P.ltoreq.0.023 as
compared to the untreated and isotype-control groups on study day
20. SC=subcutaneous; IP=intraperitoneal; TGI=tumor growth
inhibition.
[0065] FIGS. 39A-B. Expression Levels of Ki67 in CD4.sup.+ T cells
Isolated from Peripheral Blood, Spleen and CT26 Tumor Following
Administration of OX86 Mouse IgG2a to Mice Lacking Inhibitory
(Fcgr2b-/-) or Activating (Fcer1g-/-) Fc Gamma Receptor. Groups of
four Balb/c mice genetically engineered to lack the inhibitory
Fc.gamma. receptor IIb (Fcgr2b-/-; FIG. 39A) or the activating
Fc.gamma. receptors (Fcer1g-/-; FIG. 39B) were inoculated SC on Day
1 with CT26 cells; this study is independent of, but was conducted
similarly to, the study presented in FIG. 12. Control articles
(saline/untreated and OX86 mIgG1 D265A mutant/isotype control) and
test article (OX86 mIgG2a) were administered IP on Days 4 and 7.
Blood, spleens and tumors were isolated on Day 14 for FIG. 39A and
Day 13 for FIG. 39B. Expression levels of Ki67 in CD4.sup.+ T cells
were measured by flow cytometry. Symbols represent the percentage
of Ki67 positive CD4.sup.+ T cells from each tissue of individual
mice; horizontal bar represents the mean values for each group.
Intergroup differences were analyzed for statistical significance
by a one-way ANOVA using GraphPad Prism 6.0 software, and indicated
with a horizontal bar with the calculated P values.
SC=subcutaneous; IP=intraperitoneal.
[0066] FIGS. 40A-B. Expression Levels of Ki67 in CD8.sup.+ T cells
Isolated from Peripheral Blood, Spleen and CT26 Tumor Following
Administration of OX86 Mouse IgG2a to Mice Lacking Inhibitory
(Fcgr2b-/-) or Activating (Fcer1g-/-) Fc Gamma Receptors. Groups of
four Balb/c mice, genetically engineered to lack the inhibitory
Fc.gamma. receptor IIb (Fcgr2b-/-; FIG. 40A) or the activating
Fc.gamma. receptors (Fcer1g-/-; FIG. 40B), were inoculated SC on
Day 1 with CT26 cells; this study is independent of but was
conducted similarly to the study presented in FIG. 12. Control
articles (saline/untreated and OX86 mIgG1 D265A mutant/isotype
control) and test article (OX86 mIgG2a) were administered IP on
Days 4 and 7. Blood, spleens and tumor were isolated on Day 14 for
FIG. 40A and Day 13 for FIG. 40B. Expression levels of Ki67 on
CD8.sup.+ T cells were measured by flow cytometry. Symbols
represent the percentage of Ki67 positive CD8.sup.+ T cells from
each tissue of individual mice; horizontal bar represents the mean
values. Intergroup differences were analyzed for statistical
significance by a one-way ANOVA using GraphPad Prism 6.0 software,
and indicated with a horizontal bar with the calculated P values.
SC=subcutaneous; IP=intraperitoneal.
[0067] FIGS. 41A-B. Expression Levels of Ki67 in CD4 T cells
Isolated from Draining Lymph Node, Spleen and MCA205 Tumor
Following Administration of OX86 Mouse IgG2a to Mice Lacking
Inhibitory (Fcgr2b.sup.-/-) or Activating (Fcer1g.sup.-/-) Fc Gamma
Receptors. Groups of four C57BL/6 mice, genetically engineered to
lack the inhibitory Fc.gamma. receptor IIb (Fcgr2b.sup.-/-; FIG.
41A) or the activating Fc.gamma. receptors (Fcer1g.sup.-/-; FIG.
41B), were inoculated SC on Day 1 with MCA205 cells; study is
independent but conducted similarly to the study presented in FIG.
12. Control articles (saline/untreated and OX86 mIgG1 D265A
mutant/isotype control) and test article (OX86 mIgG2a) were
administered IP on Days 4 and 7. Draining lymph nodes, spleens and
tumor were isolated on Day 20 for FIG. 41B. Expression levels of
Ki67 in CD4.sup.+ T cells were measured by flow cytometry. Symbols
represent the percentage of Ki67 positive CD4.sup.+ T cells from
each tissue of individual mice; horizontal bar represents the mean
values. Intergroup differences were analyzed for statistical
significance by a one-way ANOVA using GraphPad Prism 6.0 software,
and indicated with a horizontal bar with the calculated P values.
SC=subcutaneous; IP=intraperitoneal.
[0068] FIGS. 42A-B. Expression Levels of Ki67 in CD8 T cells
Isolated from Draining Lymph Node, Spleen and MCA205 Tumor
Following Administration of OX86 Mouse IgG2a to Mice Lacking
Inhibitory (Fcgr2b-/-) or Activating (Fcer1g-/-) Fc Gamma
Receptors. Groups of four C57BL/6 mice, genetically engineered to
lack the inhibitory Fc.gamma. receptor IIb (Fcgr2b-/-; FIG. 42A) or
the activating Fc.gamma. receptors (Fcer1g-/-; FIG. 42B), were
inoculated SC on Day 1 with MCA205 cells; this study is independent
of but was conducted similarly to the study presented in FIG. 12.
Control articles (saline and mOX40L FP D265A mutant control) and
test article (OX86 mIgG2a) were administered IP on Days 4 and 7.
Draining lymph nodes, spleens and tumor were isolated on Day 20.
Expression levels of Ki67 on CD8.sup.+ T cells were measured by
flow cytometry. Symbols represent the percentage of Ki67 positive
CD8.sup.+ T cells from each tissue of individual mice; horizontal
bar represents the mean values. Intergroup differences were
analyzed for statistical significance by a one-way ANOVA using
GraphPad Prism 6.0 software, and indicated with a horizontal bar
with the calculated P values. SC=subcutaneous;
IP=intraperitoneal.
[0069] FIGS. 43A-B. Reduction of Regulatory T Cells by OX40mAb24.
FIG. 43A: Percentages of human CD4.sup.+Foxp3.sup.+ Treg cells
measured in the peripheral blood of mice engrafted with human
immune cells just prior to KLH immunization and treatment with
NIP228 huIgG1 control (huIgG1) or OX40mAb24. No mAb indicates no
immunization and no mAb treatment. FIG. 43B: Percentages of human
CD4.sup.+FoxP3.sup.+ Treg cells in the peripheral blood of mice in
the same groups after immunization and treatment with mAbs.
Statistical p values are from one-way ANOVA. Hu=human;
mAb=monoclonal antibody; Treg=regulatory T cells.
[0070] FIGS. 44A-D. Expansion of Effector and Memory CD4 T Cells
Relative to Regulatory T Cells after OX40mAb24 Treatment. Ratios of
human CD4.sup.+ T cells to human Treg cells in the peripheral blood
of mice engrafted with human immune cells either pre-treatment
(left) or post-treatment (right) with KLH immunization followed by
NIP228 huIgG1 mAb (huIgG1) or OX40mAb24, as indicated, for Total
CD4.sup.+ (FIG.44A); CD4.sup.+ effector (Teff) (FIG.44B); CD4.sup.+
effector memory (Tem) (FIG. 44C); and CD4.sup.+ central memory T
cells (Tcm) (FIG. 44D). No mAb indicates no immunization as well as
no mAb treatment. Statistical p values are from one-way ANOVA.
Hu=human; mAb=monoclonal antibody; Tcm=central memory Tcell;
Teff=effector T cell; Tem=effector memory T cell.
[0071] FIGS. 45A-B. Increased CD8.sup.+ Effector to Regulatory T
Cell Ratio in Mice Treated with OX40mAb24. Ratios of human
CD8.sup.+ effector T cells to human Treg cells in the peripheral
blood of mice engrafted with human immune cells either
pre-treatment (FIG. 45A) or post-treatment (FIG. 45B) with KLH
immunization followed by NIP228 huIgG1 mAb (huIgG1) or OX40mAb24,
as indicated. No mAb indicates no immunization as well as no mAb
treatment. Statistical p value from one-way ANOVA comparing all
groups is indicated. Hu=human; mAb=monoclonal antibody;
Teff=effector T cell; Treg=regulatory T cell.
[0072] FIGS. 46A-C. Increased CD25 (IL-2 Receptor) Levels on
CD8.sup.+ T Cells in Mice Treated with OX40mAb24. Percentage of
human CD25 (IL-2 receptor) positive cells among human CD8.sup.+ T
cells in the peripheral blood of mice engrafted with human immune
cells either post-treatment (left) or pre-treatment (right) with no
treatment (no mAb), or KLH immunization followed by NIP228 huIgG1
mAb (huIgG1) or OX40MAB24, as indicated, for (FIG. 46A) Total
CD8.sup.+, (FIG. 46B) CD8.sup.+ effector (Teff), (FIG. 46C)
CD8.sup.+ effector memory (Tem) T cells. No mAb indicates no
immunization as well as no mAb treatment. Statistical p values are
from one-way ANOVA. Hu=human; mAb=monoclonal antibody;
Teff=effector T cell; Tem=effector memory T cell.
DETAILED DESCRIPTION
[0073] Engagement of the OX40 receptor on T cells, e.g., CD4.sup.+
T cells during, or shortly after, priming by an antigen results in
an increased response of the T cells, e.g., CD4.sup.+ T cells to
the antigen. In the context of the present disclosure, the term
"engagement" refers to binding to and stimulation of at least one
activity mediated by the OX40 receptor. For example, engagement of
the OX40 receptor on antigen specific T cells, e.g., CD4.sup.+ T
cells can result in increased T cell proliferation as compared to
the response to antigen alone, and increased cytokine production.
The elevated response to the antigen can be maintained for a period
of time substantially longer than in the absence of OX40 receptor
engagement. Thus, stimulation via the OX40 receptor enhances the
antigen specific immune response by boosting T cell recognition of
antigens, e.g., tumor antigens.
[0074] OX40 agonists can enhance antigen specific immune responses
in a subject, such as a human subject, when administered to the
subject during or shortly after priming of T cells by an antigen.
OX40 agonists include OX40 ligand ("OX40L"), such as soluble OX40L
fusion proteins and anti-OX40 antibodies or fragments thereof. A
specific example is a humanized antibody that specifically binds to
OX40, thereby triggering signaling. A collection of humanized
anti-OX40 monoclonal antibodies are provided by this disclosure.
Also described are nucleic acids including polynucleotide sequences
that encode such antibodies. This disclosure also provides methods
for enhancing an antigen specific immune response in a subject
using humanized anti-OX40 monoclonal antibodies.
DEFINITIONS
[0075] It is to be noted that the term "a" or "an" entity refers to
one or more of that entity; for example, "a binding molecule," is
understood to represent one or more binding molecules. As such, the
terms "a" (or "an"), "one or more," and "at least one" can be used
interchangeably herein.
[0076] Furthermore, "and/or" where used herein is to be taken as
specific disclosure of each of the two specified features or
components with or without the other. Thus, the term and/or" as
used in a phrase such as "A and/or B" herein is intended to include
"A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the
term "and/or" as used in a phrase such as "A, B, and/or C" is
intended to encompass each of the following embodiments: A, B, and
C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A
(alone); B (alone); and C (alone).
[0077] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of
Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the
Oxford Dictionary Of Biochemistry And Molecular Biology, Revised,
2000, Oxford University Press, provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[0078] Units, prefixes, and symbols are denoted in their Systeme
International de Unites (SI) accepted form. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, amino acid sequences are written left to right in amino
to carboxy orientation. The headings provided herein are not
limitations of the various aspects or aspects of the disclosure,
which can be had by reference to the specification as a whole.
Accordingly, the terms defined immediately below are more fully
defined by reference to the specification in its entirety.
[0079] As used herein, the term "non-naturally occurring"
substance, composition, entity, and/or any combination of
substances, compositions, or entities, or any grammatical variants
thereof, is a conditional term that explicitly excludes, but only
excludes, those forms of the substance, composition, entity, and/or
any combination of substances, compositions, or entities that are,
or that might be at any time, determined or interpreted by a judge
or an administrative or judicial body to be,
"naturally-occurring."
[0080] As used herein, the term "polypeptide" is intended to
encompass a singular "polypeptide" as well as plural
"polypeptides," and refers to a molecule composed of monomers
(amino acids) linearly linked by amide bonds (also known as peptide
bonds). The term "polypeptide" refers to any chain or chains of two
or more amino acids, and does not refer to a specific length of the
product. Thus, peptides, dipeptides, tripeptides, oligopeptides,
"protein," "amino acid chain," or any other term used to refer to a
chain or chains of two or more amino acids are included within the
definition of "polypeptide," and the term "polypeptide" can be used
instead of, or interchangeably with any of these terms. The term
"polypeptide" is also intended to refer to the products of
post-expression modifications of the polypeptide, including without
limitation glycosylation, acetylation, phosphorylation, amidation,
and derivatization by known protecting/blocking groups, proteolytic
cleavage, or modification by non-naturally occurring amino acids. A
polypeptide can be derived from a biological source or produced by
recombinant technology, but is not necessarily translated from a
designated nucleic acid sequence. It can be generated in any
manner, including by chemical synthesis.
[0081] A polypeptide as disclosed herein can be of a size of about
3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or
more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or
more, or 2,000 or more amino acids. Polypeptides can have a defined
three-dimensional structure, although they do not necessarily have
such structure. Polypeptides with a defined three-dimensional
structure are referred to as folded, and polypeptides which do not
possess a defined three-dimensional structure, but rather can adopt
a large number of different conformations, and are referred to as
unfolded. As used herein, the term glycoprotein refers to a protein
coupled to at least one carbohydrate moiety that is attached to the
protein via an oxygen-containing or a nitrogen-containing side
chain of an amino acid, e.g., a serine or an asparagine.
[0082] By an "isolated" polypeptide or a fragment, variant, or
derivative thereof is intended a polypeptide that is not in its
natural milieu. No particular level of purification is required.
For example, an isolated polypeptide can be removed from its native
or natural environment. Recombinantly produced polypeptides and
proteins expressed in host cells are considered isolated as
disclosed herein, as are native or recombinant polypeptides which
have been separated, fractionated, or partially or substantially
purified by any suitable technique.
[0083] As used herein, the term "non-naturally occurring"
polypeptide, or any grammatical variants thereof, is a conditional
term that explicitly excludes, but only excludes, those forms of
the polypeptide that are, or that might be at any time, determined
or interpreted by a judge or an administrative or judicial body to
be, "naturally-occurring."
[0084] Other polypeptides disclosed herein are fragments,
derivatives, analogs, or variants of the foregoing polypeptides,
and any combination thereof. The terms "fragment," "variant,"
"derivative" and "analog" as disclosed herein include any
polypeptides which retain at least some of the properties of the
corresponding native antibody or polypeptide, for example,
specifically binding to an antigen. Fragments of polypeptides
include, for example, proteolytic fragments, as well as deletion
fragments, in addition to specific antibody fragments discussed
elsewhere herein. Variants of, e.g., a polypeptide include
fragments as described above, and also polypeptides with altered
amino acid sequences due to amino acid substitutions, deletions, or
insertions. In certain aspects, variants can be non-naturally
occurring. Non-naturally occurring variants can be produced using
art-known mutagenesis techniques. Variant polypeptides can comprise
conservative or non-conservative amino acid substitutions,
deletions or additions. Derivatives are polypeptides that have been
altered so as to exhibit additional features not found on the
original polypeptide. Examples include fusion proteins. Variant
polypeptides can also be referred to herein as "polypeptide
analogs." As used herein a "derivative" of a polypeptide can also
refer to a subject polypeptide having one or more amino acids
chemically derivatized by reaction of a functional side group. Also
included as "derivatives" are those peptides that contain one or
more derivatives of the twenty standard amino acids. For example,
4-hydroxyproline can be substituted for proline; 5-hydroxylysine
can be substituted for lysine; 3-methylhistidine can be substituted
for histidine; homoserine can be substituted for serine; and
ornithine can be substituted for lysine.
[0085] A "conservative amino acid substitution" is one in which one
amino acid is replaced with another amino acid having a similar
side chain. Families of amino acids having similar side chains have
been defined in the art, including basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., glycine, alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For
example, substitution of a phenylalanine for a tyrosine is a
conservative substitution. In certain embodiments, conservative
substitutions in the sequences of the polypeptides and antibodies
of the present disclosure do not abrogate the binding of the
polypeptide or antibody containing the amino acid sequence, to the
antigen to which the binding molecule binds. Methods of identifying
nucleotide and amino acid conservative substitutions which do not
eliminate antigen-binding are well-known in the art (see, e.g.,
Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al.,
Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl.
Acad. Sci. USA 94:.412-417 (1997)).
[0086] The term "polynucleotide" is intended to encompass a
singular nucleic acid as well as plural nucleic acids, and refers
to an isolated nucleic acid molecule or construct, e.g., messenger
RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can
comprise a conventional phosphodiester bond or a non-conventional
bond (e.g., an amide bond, such as found in peptide nucleic acids
(PNA)). The terms "nucleic acid" or "nucleic acid sequence" refer
to any one or more nucleic acid segments, e.g., DNA or RNA
fragments, present in a polynucleotide.
[0087] By an "isolated" nucleic acid or polynucleotide is intended
any form of the nucleic acid or polynucleotide that is separated
from its native environment. For example, gel-purified
polynucleotide, or a recombinant polynucleotide encoding a
polypeptide contained in a vector would be considered to be
"isolated." Also, a polynucleotide segment, e.g., a PCR product,
which has been engineered to have restriction sites for cloning is
considered to be "isolated." Further examples of an isolated
polynucleotide include recombinant polynucleotides maintained in
heterologous host cells or purified (partially or substantially)
polynucleotides in a non-native solution such as a buffer or
saline. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of polynucleotides, where the transcript is not one
that would be found in nature. Isolated polynucleotides or nucleic
acids further include such molecules produced synthetically. In
addition, polynucleotide or a nucleic acid can be or can include a
regulatory element such as a promoter, ribosome binding site, or a
transcription terminator.
[0088] As used herein, a "non-naturally occurring" polynucleotide,
or any grammatical variants thereof, is a conditional definition
that explicitly excludes, but only excludes, those forms of the
polynucleotide that are, or that might be at any time, determined
or interpreted by a judge or an administrative or judicial body to
be, "naturally-occurring."
[0089] As used herein, a "coding region" is a portion of nucleic
acid which consists of codons translated into amino acids. Although
a "stop codon" (TAG, TGA, or TAA) is not translated into an amino
acid, it can be considered to be part of a coding region, but any
flanking sequences, for example promoters, ribosome binding sites,
transcriptional terminators, introns, and the like, are not part of
a coding region. Two or more coding regions can be present in a
single polynucleotide construct, e.g., on a single vector, or in
separate polynucleotide constructs, e.g., on separate (different)
vectors. Furthermore, any vector can contain a single coding
region, or can comprise two or more coding regions, e.g., a single
vector can separately encode an immunoglobulin heavy chain variable
region and an immunoglobulin light chain variable region. In
addition, a vector, polynucleotide, or nucleic acid can include
heterologous coding regions, either fused or unfused to another
coding region. Heterologous coding regions include without
limitation, those encoding specialized elements or motifs, such as
a secretory signal peptide or a heterologous functional domain.
[0090] In certain embodiments, the polynucleotide or nucleic acid
is DNA. In the case of DNA, a polynucleotide comprising a nucleic
acid which encodes a polypeptide normally can include a promoter
and/or other transcription or translation control elements operably
associated with one or more coding regions. An operable association
is when a coding region for a gene product, e.g., a polypeptide, is
associated with one or more regulatory sequences in such a way as
to place expression of the gene product under the influence or
control of the regulatory sequence(s). Two DNA fragments (such as a
polypeptide coding region and a promoter associated therewith) are
"operably associated" if induction of promoter function results in
the transcription of mRNA encoding the desired gene product and if
the nature of the linkage between the two DNA fragments does not
interfere with the ability of the expression regulatory sequences
to direct the expression of the gene product or interfere with the
ability of the DNA template to be transcribed. Thus, a promoter
region would be operably associated with a nucleic acid encoding a
polypeptide if the promoter was capable of effecting transcription
of that nucleic acid. The promoter can be a cell-specific promoter
that directs substantial transcription of the DNA in predetermined
cells. Other transcription control elements, besides a promoter,
for example enhancers, operators, repressors, and transcription
termination signals, can be operably associated with the
polynucleotide to direct cell-specific transcription.
[0091] A variety of transcription control regions are known to
those skilled in the art. These include, without limitation,
transcription control regions which function in vertebrate cells,
such as, but not limited to, promoter and enhancer segments from
cytomegaloviruses (the immediate early promoter, in conjunction
with intron-A), simian virus 40 (the early promoter), and
retroviruses (such as Rous sarcoma virus). Other transcription
control regions include those derived from vertebrate genes such as
actin, heat shock protein, bovine growth hormone and rabbit
.beta.-globin, as well as other sequences capable of controlling
gene expression in eukaryotic cells. Additional suitable
transcription control regions include tissue-specific promoters and
enhancers as well as lymphokine-inducible promoters (e.g.,
promoters inducible by interferons or interleukins).
[0092] Similarly, a variety of translation control elements are
known to those of ordinary skill in the art. These include, but are
not limited to ribosome binding sites, translation initiation and
termination codons, and elements derived from picornaviruses
(particularly an internal ribosome entry site, or IRES, also
referred to as a CITE sequence).
[0093] In other embodiments, a polynucleotide can be RNA, for
example, in the form of messenger RNA (mRNA), transfer RNA, or
ribosomal RNA.
[0094] Polynucleotide and nucleic acid coding regions can be
associated with additional coding regions which encode secretory or
signal peptides, which direct the secretion of a polypeptide
encoded by a polynucleotide as disclosed herein. According to the
signal hypothesis, proteins secreted by mammalian cells have a
signal peptide or secretory leader sequence which is cleaved from
the mature protein once export of the growing protein chain across
the rough endoplasmic reticulum has been initiated. Those of
ordinary skill in the art are aware that polypeptides secreted by
vertebrate cells can have a signal peptide fused to the N-terminus
of the polypeptide, which is cleaved from the complete or "full
length" polypeptide to produce a secreted or "mature" form of the
polypeptide. In certain embodiments, the native signal peptide,
e.g., an immunoglobulin heavy chain or light chain signal peptide
is used, or a functional derivative of that sequence that retains
the ability to direct the secretion of the polypeptide that is
operably associated with it. Alternatively, a heterologous
mammalian signal peptide, or a functional derivative thereof, can
be used. For example, the wild-type leader sequence can be
substituted with the leader sequence of human tissue plasminogen
activator (TPA) or mouse .beta.-glucuronidase.
[0095] Disclosed herein are certain binding molecules, or
antigen-binding fragments, variants, or derivatives thereof. Unless
specifically referring to full-sized antibodies, the term "binding
molecule" encompasses full-sized antibodies as well as
antigen-binding subunits, fragments, variants, analogs, or
derivatives of such antibodies, e.g., engineered antibody molecules
or fragments that bind antigen in a manner similar to antibody
molecules, but which use a different scaffold.
[0096] As used herein, the term "binding molecule" refers in its
broadest sense to a molecule that specifically binds to a receptor,
e.g., an epitope or an antigenic determinant. As described further
herein, a binding molecule can comprise one of more "antigen
binding domains" described herein. A non-limiting example of a
binding molecule is an antibody or fragment thereof that retains
antigen-specific binding.
[0097] As used herein, the terms "binding domain," "receptor
binding domain," or "antigen binding domain" refer to a region of a
binding molecule that is necessary and sufficient to specifically
bind to an epitope. For example, an "Fv," e.g., a variable heavy
chain and variable light chain of an antibody, either as two
separate polypeptide subunits or as a single chain, is considered
to be a "binding domain." Other binding domains include, without
limitation, the variable heavy chain (VHH) of an antibody derived
from a camelid species, or six immunoglobulin complementarity
determining regions (CDRs) expressed in a heterologous framework,
e.g., a different germline or species, or in a different scaffold,
e.g., a fibronectin scaffold. A "binding molecule" as described
herein can include one, two, three, four, five, six, seven, eight,
nine, ten, eleven, twelve or more "antigen binding domains."
[0098] The terms "antibody" and "immunoglobulin" can be used
interchangeably herein. An antibody (or a fragment, variant, or
derivative thereof as disclosed herein) includes at least the
variable domain of a heavy chain (for camelid species) or at least
the variable domains of a heavy chain and a light chain. Basic
immunoglobulin structures in vertebrate systems are relatively well
understood. See, e.g., Harlow et al., Antibodies: A Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless
otherwise stated, the term "antibody" encompasses anything ranging
from a small antigen-binding fragment of an antibody to a full
sized antibody, e.g., an IgG antibody that includes two complete
heavy chains and two complete light chains, an IgA antibody that
includes four complete heavy chains and four complete light chains
and optionally includes a J chain and/or a secretory component, or
an IgM antibody that includes ten or twelve complete heavy chains
and ten or twelve complete light chains and optionally includes a J
chain.
[0099] As will be discussed in more detail below, the term
"immunoglobulin" comprises various broad classes of polypeptides
that can be distinguished biochemically. Those skilled in the art
will appreciate that heavy chains are classified as gamma, mu,
alpha, delta, or epsilon, (.gamma., .mu., .alpha., .delta.,
.epsilon.) with some subclasses among them (e.g., .gamma.1-y4 or
.alpha.1-.alpha.2)). It is the nature of this chain that determines
the "class" of the antibody as IgG, IgM, IgA IgG, or IgE,
respectively. The immunoglobulin subclasses (isotypes) e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, IgA.sub.2,
etc. are well characterized and are known to confer functional
specialization. Modified versions of each of these classes and
isotypes are readily discernible to the skilled artisan in view of
the instant disclosure and, accordingly, are within the scope of
this disclosure.
[0100] Light chains are classified as either kappa or lambda
(.kappa., .lamda.). Each heavy chain class can be associated with
either a kappa or lambda light chain. In general, the light and
heavy chains are covalently bonded to each other, and the "tail"
portions of the two heavy chains are bonded to each other by
covalent disulfide linkages or non-covalent linkages when the
immunoglobulins are generated either by hybridomas, B cells or
genetically engineered host cells. In the heavy chain, the amino
acid sequences run from an N-terminus at the forked ends of the Y
configuration to the C-terminus at the bottom of each chain. The
basic structure of certain antibodies, e.g., IgG antibodies,
includes two heavy chain subunits and two light chain subunits
covalently connected via disulfide bonds to form a "Y" structure,
also referred to herein as an "H2L2" structure.
[0101] Both the light and heavy chains are divided into regions of
structural and functional homology. The terms "constant" and
"variable" are used functionally. In this regard, it will be
appreciated that the variable domains of both the variable light
(VL) and variable heavy (VH) chain portions determine antigen
recognition and specificity. Conversely, the constant domains of
the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer
biological properties such as secretion, transplacental mobility,
Fc receptor binding, complement binding, and the like. By
convention the numbering of the constant region domains increases
as they become more distal from the antigen binding site or
amino-terminus of the antibody. The N-terminal portion is a
variable region and at the C-terminal portion is a constant region;
the CH3 (or CH4 in the case of IgM) and CL domains actually
comprise the carboxy-terminus of the heavy and light chain,
respectively.
[0102] As indicated above, a binding domain, e.g., an antibody
variable region allows the binding molecule to selectively
recognize and specifically bind a receptor, or an epitope on an
antigen. That is, the VL domain and VH domain, or subset of the
complementarity determining regions (CDRs), of a binding molecule,
e.g., an antibody combine to form the variable region that defines
a three dimensional antigen binding site. More specifically, the
antigen binding site is defined by three CDRs on each of the VH and
VL chains. Certain antibodies form larger structures. For example,
IgA can form a molecule that includes two H2L2 units, a J chain,
and a secretory component, all covalently connected via disulfide
bonds, and IgM can form a pentameric or hexameric molecule that
includes five or six H2L2 units and optionally a J chain covalently
connected via disulfide bonds.
[0103] The six "complementarity determining regions" or "CDRs"
present in an antibody antigen-binding domain are short,
non-contiguous sequences of amino acids that are specifically
positioned to form the binding domain as the antibody assumes its
three dimensional configuration in an aqueous environment. The
remainder of the amino acids in the binding domain, referred to as
"framework" or "FW" regions, show less inter-molecular variability.
The framework regions largely adopt a .beta.-sheet conformation and
the CDRs form loops which connect, and in some cases form part of,
the .beta.-sheet structure. Thus, framework regions act to form a
scaffold that provides for positioning the CDRs in correct
orientation by inter-chain, non-covalent interactions. The binding
domain formed by the positioned CDRs defines a surface
complementary to the epitope on the immunoreactive antigen. This
complementary surface promotes the non-covalent binding of the
antibody to its cognate epitope. The amino acids that make up the
CDRs and the framework regions, respectively, can be readily
identified for any given heavy or light chain variable region by
one of ordinary skill in the art, since they have been defined in
various different ways (see, "Sequences of Proteins of
Immunological Interest," Kabat, E., et al., U.S. Department of
Health and Human Services, (1983); and Chothia and Lesk, J. Mol.
Biol., 196:901-917 (1987), which are incorporated herein by
reference in their entireties).
[0104] In the case where there are two or more definitions of a
term which is used and/or accepted within the art, the definition
of the term as used herein is intended to include all such meanings
unless explicitly stated to the contrary. A specific example is the
use of the term "complementarity determining region" ("CDR") to
describe the non-contiguous antigen combining sites found within
the variable region of both heavy and light chain polypeptides.
These particular regions have been described, for example, by Kabat
et al., U.S. Dept. of Health and Human Services, "Sequences of
Proteins of Immunological Interest" (1983) and by Chothia et al.,
J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by
reference. The Kabat and Chothia definitions include overlapping or
subsets of amino acids when compared against each other.
Nevertheless, application of either definition (or other
definitions known to those of ordinary skill in the art) to refer
to a CDR of an antibody or variant thereof is intended to be within
the scope of the term as defined and used herein, unless otherwise
indicated. The appropriate amino acids which encompass the CDRs as
defined by each of the above cited references are set forth below
in Table 1 as a comparison. The exact amino acid numbers which
encompass a particular CDR will vary depending on the sequence and
size of the CDR. Those skilled in the art can routinely determine
which amino acids comprise a particular CDR given the variable
region amino acid sequence of the antibody.
TABLE-US-00001 TABLE 1 CDR Definitions.sup.1 Kabat Chothia VH CDR1
31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34
26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96 .sup.1Numbering of
all CDR definitions in Table 1 is according to the numbering
conventions set forth by Kabat et al. (see below).
[0105] Kabat et al. also defined a numbering system for antibody
heavy and light chains, e.g., antibody variable domain sequences
that is applicable to any antibody. One of ordinary skill in the
art can unambiguously assign this system of "Kabat numbering" to
any variable domain sequence, without reliance on any experimental
data beyond the sequence itself. As used herein, "Kabat numbering"
refers to the numbering system set forth by Kabat et al., U.S.
Dept. of Health and Human Services, "Sequence of Proteins of
Immunological Interest" (1983). Unless use of the Kabat numbering
system is explicitly noted, however, consecutive numbering is used
for all amino acid sequences in this disclosure.
[0106] Binding molecules, e.g., antibodies or antigen-binding
fragments, variants, or derivatives thereof include, but are not
limited to, polyclonal, monoclonal, human, humanized, or chimeric
antibodies, single chain antibodies, epitope-binding fragments,
e.g., Fab, Fab' and F(ab').sub.2, Fd, Fvs, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv), fragments
comprising either a VL or VH domain, fragments produced by a Fab
expression library. ScFv molecules are known in the art and are
described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin or
antibody molecules encompassed by this disclosure can be of any
type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin
molecule.
[0107] By "specifically binds," it is generally meant that a
binding molecule, e.g., an antibody or fragment, variant, or
derivative thereof binds to an epitope via its antigen binding
domain, and that the binding entails some complementarity between
the antigen binding domain and the epitope. According to this
definition, a binding molecule is said to "specifically bind" to an
epitope when it binds to that epitope via its antigen binding
domain more readily than it would bind to a random, unrelated
epitope. The term "specificity" is used herein to qualify the
relative affinity by which a certain binding molecule binds to a
certain epitope. For example, binding molecule "A" can be deemed to
have a higher specificity for a given epitope than binding molecule
"B," or binding molecule "A" can be said to bind to epitope "C"
with a higher specificity than it has for related epitope "D."
[0108] A binding molecule, e.g., an antibody or fragment, variant,
or derivative thereof disclosed herein can be said to bind a target
antigen with an off rate (k(off)) of less than or equal to, e.g.,
5.times.10.sup.-2 sec.sup.-1, 10.sup.-2 sec.sup.-1,
5.times.10.sup.-3 sec.sup.-1, 10.sup.-3 sec.sup.-1,
5.times.10.sup.-4 sec.sup.-1, 10.sup.-4 sec.sup.-1,
5.times.10.sup.-5 sec.sup.-1, or 10.sup.-5 sec.sup.-1
5.times.10.sup.-6 sec.sup.-1, 10.sup.-6 sec.sup.-1,
5.times.10.sup.-7 sec.sup.-1 or 10.sup.-7 sec.sup.-1.
[0109] A binding molecule, e.g., an antibody or antigen-binding
fragment, variant, or derivative disclosed herein can be said to
bind a target antigen with an on rate (k(on)) of greater than or
equal to, e.g., 10.sup.3 M.sup.-1 sec.sup.-1,
5.times.10.sup.3M.sup.-1 sec.sup.-1, 10.sup.4 M.sup.-1 sec.sup.-1,
5.times.10.sup.4 M.sup.-1 sec.sup.-1, 10.sup.5 M.sup.-1 sec.sup.-1,
5.times.10.sup.5 M.sup.-1 sec.sup.-1, 10.sup.6 M.sup.-1 sec.sup.-1,
or 5.times.10.sup.6 M.sup.-1 sec.sup.-1 or 10.sup.7 M.sup.-1
sec.sup.-1.
[0110] A binding molecule, e.g., an antibody or fragment, variant,
or derivative thereof is said to competitively inhibit binding of a
reference antibody or antigen binding fragment to a given epitope
if it preferentially binds to that epitope to the extent that it
blocks, to some degree, binding of the reference antibody or
antigen binding fragment to the epitope. Competitive inhibition can
be determined by any method known in the art, for example,
competition ELISA assays. A binding molecule can be said to
competitively inhibit binding of the reference antibody or antigen
binding fragment to a given epitope by at least 90%, at least 80%,
at least 70%, at least 60%, or at least 50%.
[0111] As used herein, the term "affinity" refers to a measure of
the strength of the binding of an individual epitope with one or
more binding domains, e.g., of an immunoglobulin molecule. See,
e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring
Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. As used
herein, the term "avidity" refers to the overall stability of the
complex between a population of binding domains and an antigen.
See, e.g., Harlow at pages 29-34. Avidity is related to both the
affinity of individual binding domains in the population with
specific epitopes, and also the valencies of the immunoglobulins
and the antigen. For example, the interaction between a bivalent
monoclonal antibody and an antigen with a highly repeating epitope
structure, such as a polymer, would be one of high avidity. An
interaction between a between a bivalent monoclonal antibody with a
receptor present at a high density on a cell surface would also be
of high avidity.
[0112] Binding molecules or antigen-binding fragments, variants or
derivatives thereof as disclosed herein can also be described or
specified in terms of their cross-reactivity. As used herein, the
term "cross-reactivity" refers to the ability of a binding
molecule, e.g., an antibody or fragment, variant, or derivative
thereof, specific for one antigen, to react with a second antigen;
a measure of relatedness between two different antigenic
substances. Thus, a binding molecule is cross reactive if it binds
to an epitope other than the one that induced its formation. The
cross reactive epitope generally contains many of the same
complementary structural features as the inducing epitope, and in
some cases, can actually fit better than the original.
[0113] A binding molecule, e.g., an antibody or fragment, variant,
or derivative thereof can also be described or specified in terms
of their binding affinity to an antigen. For example, a binding
molecule can bind to an antigen with a dissociation constant or
K.sub.D no greater than, e.g., 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M,
5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-M, 10.sup.-10 M,
5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
[0114] Antibody fragments including single-chain antibodies or
other binding domains can exist alone or in combination with one or
more of the following: hinge region, CH1, CH2, CH3, or CH4 domains,
J chain, or secretory component. Also included are antigen-binding
fragments that can include any combination of variable region(s)
with one or more of a hinge region, CH1, CH2, CH3, or CH4 domains,
a J chain, or a secretory component. Binding molecules, e.g.,
antibodies, or antigen-binding fragments thereof can be from any
animal origin including birds and mammals. The antibodies can be
human, murine, donkey, rabbit, goat, guinea pig, camel, llama,
horse, or chicken antibodies. In another embodiment, the variable
region can be condricthoid in origin (e.g., from sharks). As used
herein, "human" antibodies include antibodies having the amino acid
sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries or from animals transgenic for
one or more human immunoglobulins and can in some instances express
endogenous immunoglobulins and some not, as described infra and,
for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0115] As used herein, the term "heavy chain subunit" includes
amino acid sequences derived from an immunoglobulin heavy chain, a
binding molecule, e.g., an antibody comprising a heavy chain
subunit includes at least one of: a VH domain, a CH1 domain, a
hinge (e.g., upper, middle, and/or lower hinge region) domain, a
CH2 domain, a CH3 domain, a CH4 domain, or a variant or fragment
thereof. For example, a binding molecule, e.g., an antibody or
fragment, variant, or derivative thereof can include, in addition
to a VH domain, a CH1 domain; CH1 domain, a hinge, and a CH2
domain; a CH1 domain and a CH3 domain; a CH1 domain, a hinge, and a
CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain, and a
CH3 domain. In certain aspects a binding molecule, e.g., an
antibody or fragment, variant, or derivative thereof can include,
in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3
domain, a CH4 domain, and a J chain. Further, a binding molecule
for use in the disclosure can lack certain constant region
portions, e.g., all or part of a CH2 domain. It will be understood
by one of ordinary skill in the art that these domains (e.g., the
heavy chain subunit) can be modified such that they vary in amino
acid sequence from the original immunoglobulin molecule.
[0116] The heavy chain subunits of a binding molecule, e.g., an
antibody or fragment thereof, can include domains derived from
different immunoglobulin molecules. For example, a heavy chain
subunit of a polypeptide can include a CH1 domain derived from an
IgG1 molecule and a hinge region derived from an IgG3 molecule. In
another example, a heavy chain subunit can include a hinge region
derived, in part, from an IgG1 molecule and, in part, from an IgG3
molecule. In another example, a heavy chain subunit can comprise a
chimeric hinge derived, in part, from an IgG1 molecule and, in
part, from an IgG4 molecule.
[0117] As used herein, the term "light chain subunit" includes
amino acid sequences derived from an immunoglobulin light chain.
The light chain subunit includes at least one of a VL or CL (e.g.,
C.kappa. or C.lamda.) domain.
[0118] Binding molecules, e.g., antibodies or antigen-binding
fragments, variants, or derivatives thereof can be described or
specified in terms of the epitope(s) or portion(s) of an antigen
that they recognize or specifically bind. The portion of a target
antigen that specifically interacts with the antigen binding domain
of an antibody is an "epitope," or an "antigenic determinant." A
target antigen can comprise a single epitope or at least two
epitopes, and can include any number of epitopes, depending on the
size, conformation, and type of antigen.
[0119] As previously indicated, the subunit structures and three
dimensional configuration of the constant regions of the various
immunoglobulin classes are well known. As used herein, the term "VH
domain" includes the amino terminal variable domain of an
immunoglobulin heavy chain and the term "CH1 domain" includes the
first (most amino terminal) constant region domain of an
immunoglobulin heavy chain. The CH1 domain is adjacent to the VH
domain and is amino terminal to the hinge region of a typical
immunoglobulin heavy chain molecule.
[0120] As used herein the term "CH2 domain" includes the portion of
a heavy chain molecule that extends, e.g., from about amino acid
244 to amino acid 360 of an IgG antibody using conventional
numbering schemes (amino acids 244 to 360, Kabat numbering system;
and amino acids 231-340, EU numbering system; see Kabat EA et al.
op. cit. The CH3 domain extends from the CH2 domain to the
C-terminal of the IgG molecule and comprises approximately 108
amino acids. Certain immunoglobulin classes, e.g., IgM, further
include a CH4 region.
[0121] As used herein, the term "hinge region" includes the portion
of a heavy chain molecule that joins the CH1 domain to the CH2
domain. This hinge region comprises approximately 25 amino acids
and is flexible, thus allowing the two N-terminal antigen binding
regions to move independently.
[0122] As used herein the term "disulfide bond" includes the
covalent bond formed between two sulfur atoms. The amino acid
cysteine comprises a thiol group that can form a disulfide bond or
bridge with a second thiol group. In certain IgG molecules, the CH1
and CL regions are linked by a disulfide bond and the two heavy
chains are linked by two disulfide bonds at positions corresponding
to 239 and 242 using the Kabat numbering system (position 226 or
229, EU numbering system). In certain aspects provided herein, a
human IgG4 Fc domain can be mutated in the hinge region to insure
disulfide bond formation between two hinge regions, specifically, a
serine to proline mutation at position 228 (according to EU
numbering). Human IgG4 Fc domains comprising the S228P mutation are
referred to herein as "IgG4P Fc domains."
[0123] As used herein, an "Fc-TM region" is a human IgG Fc region
that comprises amino acid substitutions of L234F, L235E and P331S
as numbered by the EU index as set forth in Kabat and exhibits
reduced or ablated effector (ADCC and/or CDC) function, reduced or
ablated binding to Fc receptors, and/or reduced or ablated
toxicities. See, e.g., U.S. Patent Application Publication No.
2011/0059078, which is incorporated herein by reference in its
entirety.
[0124] As used herein, the term "chimeric antibody" refers to an
antibody in which the immunoreactive region or site is obtained or
derived from a first species and the constant region (which can be
intact, partial or modified) is obtained from a second species. In
some embodiments the target binding region or site will be from a
non-human source (e.g. mouse or primate) and the constant region is
human.
[0125] The terms "multispecific antibody, or "bispecific antibody"
refer to an antibody that has binding domains for two or more
different epitopes within a single antibody molecule. Other binding
molecules in addition to the canonical antibody structure can be
constructed with two binding specificities. Epitope binding by
bispecific or multispecific antibodies can be simultaneous or
sequential. Triomas and hybrid hybridomas are two examples of cell
lines that can secrete bispecific antibodies. Bispecific antibodies
can also be constructed by recombinant means. (Strohlein and Heiss,
Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9
(2010)). A bispecific antibody can also be a diabody.
[0126] As used herein, the term "engineered antibody" refers to an
antibody in which the variable domain in either the heavy and light
chain or both is altered by at least partial replacement of one or
more amino acids in either the CDR or framework regions. In certain
aspects entire CDRs from an antibody of known specificity can be
grafted into the framework regions of a heterologous antibody.
Although alternate CDRs can be derived from an antibody of the same
class or even subclass as the antibody from which the framework
regions are derived, CDRs can also be derived from an antibody of
different class, e.g., from an antibody from a different species.
An engineered antibody in which one or more "donor" CDRs from a
non-human antibody of known specificity are grafted into a human
heavy or light chain framework region is referred to herein as a
"humanized antibody." In certain aspects not all of the CDRs are
replaced with the complete CDRs from the donor variable region and
yet the antigen binding capacity of the donor can still be
transferred to the recipient variable domains. Given the
explanations set forth in, e.g., U. S. Pat. Nos. 5,585,089,
5,693,761, 5,693,762, and 6,180,370, it will be well within the
competence of those skilled in the art, either by carrying out
routine experimentation or by trial and error testing to obtain a
functional engineered or humanized antibody.
[0127] As used herein the term "engineered" includes manipulation
of nucleic acid or polypeptide molecules by synthetic means (e.g.
by recombinant techniques, in vitro peptide synthesis, by enzymatic
or chemical coupling of peptides or some combination of these
techniques).
[0128] As used herein, the terms "linked," "fused" or "fusion" or
other grammatical equivalents can be used interchangeably. These
terms refer to the joining together of two more elements or
components, by whatever means including chemical conjugation or
recombinant means. An "in-frame fusion" refers to the joining of
two or more polynucleotide open reading frames (ORFs) to form a
continuous longer ORF, in a manner that maintains the translational
reading frame of the original ORFs. Thus, a recombinant fusion
protein is a single protein containing two or more segments that
correspond to polypeptides encoded by the original ORFs (which
segments are not normally so joined in nature.) Although the
reading frame is thus made continuous throughout the fused
segments, the segments can be physically or spatially separated by,
for example, in-frame linker sequence. For example, polynucleotides
encoding the CDRs of an immunoglobulin variable region can be
fused, in-frame, but be separated by a polynucleotide encoding at
least one immunoglobulin framework region or additional CDR
regions, as long as the "fused" CDRs are co-translated as part of a
continuous polypeptide.
[0129] In the context of polypeptides, a "linear sequence" or a
"sequence" is an order of amino acids in a polypeptide in an amino
to carboxyl terminal direction in which amino acids that neighbor
each other in the sequence are contiguous in the primary structure
of the polypeptide. A portion of a polypeptide that is
"amino-terminal" or "N-terminal" to another portion of a
polypeptide is that portion that comes earlier in the sequential
polypeptide chain. Similarly a portion of a polypeptide that is
"carboxy-terminal" or "C-terminal" to another portion of a
polypeptide is that portion that comes later in the sequential
polypeptide chain. For example in a typical antibody, the variable
domain is "N-terminal" to the constant region, and the constant
region is "C-terminal" to the variable domain.
[0130] The term "expression" as used herein refers to a process by
which a gene produces a biochemical, for example, a polypeptide.
The process includes any manifestation of the functional presence
of the gene within the cell including, without limitation, gene
knockdown as well as both transient expression and stable
expression. It includes without limitation transcription of the
gene into messenger RNA (mRNA), and the translation of such mRNA
into polypeptide(s). If the final desired product is a biochemical,
expression includes the creation of that biochemical and any
precursors. Expression of a gene produces a "gene product." As used
herein, a gene product can be either a nucleic acid, e.g., a
messenger RNA produced by transcription of a gene, or a polypeptide
which is translated from a transcript. Gene products described
herein further include nucleic acids with post transcriptional
modifications, e.g., polyadenylation, or polypeptides with post
translational modifications, e.g., methylation, glycosylation, the
addition of lipids, association with other protein subunits,
proteolytic cleavage, and the like.
[0131] Terms such as "treating" or "treatment" or "to treat" or
"alleviating" or "to alleviate" refer to therapeutic measures that
cure, slow down, lessen symptoms of, and/or halt or slow the
progression of an existing diagnosed pathologic condition or
disorder. Terms such as "prevent," "prevention," "avoid,"
"deterrence" and the like refer to prophylactic or preventative
measures that prevent the development of an undiagnosed targeted
pathologic condition or disorder. Thus, "those in need of
treatment" can include those already with the disorder; those prone
to have the disorder; and those in whom the disorder is to be
prevented.
[0132] OX40, or "OX40 receptor" is a protein (also variously termed
CD134, tumor necrosis factor receptor superfamily member 4, and
ACT-35) expressed on the surface of activated T cells, e.g.,
CD4.sup.+ and CD8.sup.+ T cells, as well as on Foxp3.sup.+CD4.sup.+
regulatory T cells (Tregs). Naive CD4.sup.+ and CD8.sup.+ T cells
do not express OX40 (Croft, M., (2010) Ann Rev Immunol
28:57-78).
[0133] "OX40 ligand" ("OX40L") (also variously termed tumor
necrosis factor ligand superfamily member 4, gp34, TAX
transcriptionally-activated glycoprotein-1, and CD252) is found
largely on antigen presenting cells (APCs), and can be induced on
activated B cells, dendritic cells (DCs), Langerhans cells,
plamacytoid DCs, and macrophages (Id.). Other cells, including
activated T cells, NK cells, mast cells, endothelial cells, and
smooth muscle cells can express OX40L in response to inflammatory
cytokines (Id.). OX40L specifically binds to the OX40 receptor. The
human protein is described in PCT Publication No. WO 95/21915. The
mouse OX40L is described in U.S. Pat. No. 5,457,035. OX40L is
expressed on the surface of cells and includes an intracellular, a
transmembrane and an extracellular receptor-binding domain. A
functionally active soluble form of OX40L can be produced by
deleting the intracellular and transmembrane domains as described,
e.g., in U.S. Pat. Nos. 5,457,035 and 6,312,700, and WO 95/21915,
the disclosures of which are incorporated herein for all purposes.
A functionally active form of OX40L is a form that retains the
capacity to bind specifically to OX40, that is, that possesses an
OX40 "receptor binding domain." Methods of determining the ability
of an OX40L molecule or derivative to bind specifically to OX40 are
discussed below. Methods of making and using OX40L and its
derivatives (such as derivatives that include an OX40 binding
domain) are described in WO 95/21915, which also describes proteins
comprising the soluble form of OX40L linked to other peptides, such
as human immunoglobulin ("Ig") Fc regions, that can be produced to
facilitate purification of OX40 ligand from cultured cells, or to
enhance the stability of the molecule after in vivo administration
to a mammal (see also, U.S. Pat. No. 5,457,035 and PCT Publication
No. WP 2006/121810, both of which are incorporated by reference
herein in their entireties).
[0134] By "subject" or "individual" or "animal" or "patient" or
"mammal," is meant any subject, particularly a mammalian subject,
for whom diagnosis, prognosis, or therapy is desired. Mammalian
subjects include humans, domestic animals, farm animals, and zoo,
sports, or pet animals such as dogs, cats, guinea pigs, rabbits,
rats, mice, horses, swine, cows, bears, and so on. As used herein,
phrases such as "a subject that would benefit from therapy" and "an
animal in need of treatment" includes subjects, such as mammalian
subjects, that would benefit from administration of a humanized
anti-OX40 antibody. Such antibodies, can be used, e.g., for a
diagnostic procedures and/or for treatment or prevention of a
disease, e.g., cancer.
Humanized Anti-OX40 Antibodies and Antigen-Binding Fragments
Thereof
[0135] The present disclosure relates to antibodies, e.g.,
humanized antibodies, which specifically bind to OX40. In certain
aspects, an antibody or fragment thereof as provided herein can be
isolated. In certain aspects, an antibody or fragment thereof as
provided herein can be substantially pure. In certain aspects an
antibody or fragment thereof as provided herein can be
non-naturally occurring.
[0136] In certain aspects, this disclosure provides a humanized
anti-OX40 antibody or an antigen-binding fragment thereof
comprising the VH and VL of OX40mAb5, OX40mAb8, OX40mAb10,
OX40mAb11, OX40mAb12, OX40mAb13, OX40mAb14, OX40mAb15, OX40mAb16,
OX40mAb17, OX40mAb18, OX40mAb19, OX40mAb20, OX40mAb21, OX40mAb22,
OX40mAb23, OX40mAb24, OX40mAb25, OX40mAb25a, OX40mAb26, OX40mAb27,
OX40mAb28, OX40mAb29, OX40mAb30, OX40mAb31, OX40mAb32, or
OX40mAb37. In certain aspects, this disclosure provides a humanized
anti-OX40 antibody or an antigen-binding fragment thereof
comprising the heavy chain and light chain of OX40mAb5, OX40mAb8,
OX40mAb10, OX40mAb11, OX40mAb12, OX40mAb13, OX40mAb14, OX40mAb15,
OX40mAb16, OX40mAb17, OX40mAb18, OX40mAb19, OX40mAb20, OX40mAb21,
OX40mAb22, OX40mAb23, OX40mAb24, OX40mAb25, OX40mAb25a, OX40mAb26,
OX40mAb27, OX40mAb28, OX40mAb29, OX40mAb30, OX40mAb31, or
OX40mAb32.
[0137] In certain aspects this disclosure provides a humanized
anti-OX40 antibody or an antigen-binding fragment thereof
comprising an antibody VH and an antibody VL, wherein the VL
comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%,
95%, or 100% identical to the reference amino acid sequence SEQ ID
NO: 29 or SEQ ID NO: 32.
[0138] In certain aspects this disclosure provides a humanized
anti-OX40 antibody or an antigen-binding fragment thereof
comprising an antibody VH and an antibody VL, where the VL
comprises SEQ ID NO: 29 or SEQ ID NO: 32.
[0139] The disclosure further provides a humanized anti-OX40
antibody or an antigen-binding fragment thereof comprising an
antibody VH and an antibody VL, wherein the VH comprises VH-CDR1,
VH-CDR2, and VH-CDR3 amino acid sequences identical to, or
identical except for eight, seven, six, five, four, three, two, or
one single amino acid substitutions, deletions, or insertions in
one or more of the VH-CDRS to: the VHCDR1 amino acid sequence SEQ
ID NO: 8, the VHCDR2 amino acid sequence SEQ ID NO: 14, SEQ ID NO:
15, or SEQ ID NO: 16, and the VHCDR3 amino acid sequence SEQ ID NO:
25, SEQ ID NO: 26, or SEQ ID NO: 27.
[0140] The disclosure further provides a humanized anti-OX40
antibody or an antigen-binding fragment thereof comprising an
antibody VH and an antibody VL, wherein the VH comprises an amino
acid sequence with the formula:
HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4,
wherein HFW1 is SEQ ID NO: 6 or SEQ ID NO: 7, HCDR1 is SEQ ID NO:
8, HFW2 is SEQ ID NO: 9, HCDR2 is SEQ ID NO: 14, SEQ ID NO: 15 or
SEQ ID NO: 16, HFW3 is SEQ ID NO: 17, HCDR3 is SEQ ID NO: 25, SEQ
ID NO: 26, or SEQ ID NO: 27, and HFW4 is SEQ ID NO: 28. In certain
aspects the amino acid sequence of HFW2 is SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In certain aspects the
amino acid sequence of HFW3 is SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID
NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO:
24.
[0141] Moreover, the disclosure provides a humanized anti-OX40
antibody or an antigen-binding fragment thereof comprising an
antibody VH and an antibody VL, wherein the VH comprises an amino
acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%
identical to the reference amino acid sequence SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ
ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:
61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO: 67.
[0142] In one aspect, the disclosure provides a humanized anti-OX40
antibody or an antigen-binding fragment thereof comprising an
antibody VH and an antibody VL, where the VL comprises the amino
acid sequence SEQ ID NO: 29 and the VH comprises the amino acid
sequence SEQ ID NO: 59.
[0143] A humanized anti-OX40 antibody or an antigen-binding
fragment thereof as provided herein can include, in addition to a
VH and a VL, a heavy chain constant region or fragment thereof. In
certain aspects the heavy chain constant region is a human heavy
chain constant region, e.g., a human IgG constant region, e.g., a
human IgG1 constant region or a human IgG4 constant region. As
described elsewhere herein, in certain aspects a heavy chain
constant region or fragment thereof, e.g., a human IgG constant
region or fragment thereof, can include one or more amino acid
substitutions relative to a wild-type IgG constant region, where
the modified IgG has one or more desirable properties relative to a
wild-type IgG constant region. For example, the human IgG constant
region can be an IgG4P constant region, or an IgG1-TM constant
region, as described elsewhere herein.
[0144] A humanized anti-OX40 antibody or an antigen-binding
fragment thereof as provided herein can include, in addition to a
VH and a VL, and optionally a heavy chain constant region or
fragment thereof, a light chain constant region or fragment
thereof. In certain aspects the light chain constant region is a
kappa or lambda light chain constant region, e.g., a human kappa
constant region or a human lambda constant region. In a specific
aspect, the light chain constant region is a human kappa constant
region.
[0145] In certain aspects the disclosure provides a humanized
anti-OX40 antibody or an antigen-binding fragment thereof
comprising an antibody heavy chain or fragment thereof and an
antibody light chain or fragment thereof, where the heavy chain
comprises the amino acid sequence SEQ ID NO: 71, and the light
chain comprises the amino acid sequence SEQ ID NO: 30.
[0146] A humanized anti-OX40 antibody or an antigen-binding
fragment thereof as provided herein can be, e.g., monoclonal,
polyclonal, recombinant, multispecific, or any combination thereof.
A humanized anti-OX40 antibody or antigen-binding fragment can be,
e.g., an Fv fragment, an Fab fragment, an F(ab')2 fragment, an Fab'
fragment, a dsFv fragment, an scFv fragment, an sc(Fv)2 fragment,
an Fd fragment, a disulfide linked Fv fragment, a V-NAR domain, an
IgNar, an intrabody, an IgG.DELTA.CH2, a minibody, a F(ab')3
fragment, a tetrabody, a triabody, a diabody, a single-domain
antibody, a DVD-Ig, a Fcab fragment, an mAb2 fragment, an (scFv)2
fragment, or a scFv-Fc fragment.
[0147] In certain aspects, a humanized anti-OX40 antibody or an
antigen-binding fragment thereof as provided herein can
specifically bind to human, cynomolgus monkey, or rhesus monkey
OX40. In certain aspects, a humanized anti-OX40 antibody or an
antigen-binding fragment thereof as provided herein can
specifically bind to the surface of primary human, cyno, or rhesus
CD4 T cells or Jurkat cells. In certain aspects, a humanized
anti-OX40 antibody or an antigen-binding fragment thereof as
provided herein can specifically bind to OX40 as expressed on
activated CD4.sup.+ or CD8.sup.+ T cells from human, cynomolgus
monkey, rhesus monkey, or any combination thereof. In certain
aspects, a humanized anti-OX40 antibody or an antigen-binding
fragment thereof as provided herein can specifically bind to OX40
as expressed on primary activated CD4.sup.+ T cells from human,
cynomolgus monkey, rhesus monkey, or any combination thereof. In
certain aspects, a humanized anti-OX40 antibody or an
antigen-binding fragment thereof as provided herein does not bind
to murine or rat OX40. In certain aspects, a humanized anti-OX40
antibody or an antigen-binding fragment thereof as provided herein
does not cross react with related TNFRSF proteins, e.g., the TNFRSF
proteins listed in Table 3-1 in Example 3 below.
[0148] A humanized anti-OX40 antibody or antigen-binding fragment
thereof as provided herein can contain one or more conservative
amino acid changes, e.g., up to ten conservative changes (e.g., two
substituted amino acids, three substituted amino acids, four
substituted amino acids, or five substituted amino acids, etc.),
provided that the changes can be made in the polypeptide without
changing a biochemical function of the humanized anti-OX40 antibody
or antigen-binding fragment thereof, e.g., specifically binding to
OX40, thereby triggering signaling. For example, one or more
conservative changes can be made in a receptor binding domain of an
antibody as provided herein without blocking its ability to bind to
OX40.
[0149] Additionally, part of a polypeptide domain can be deleted
without impairing or eliminating all of its functions. Similarly,
insertions or additions can be made in the polypeptide chain, for
example, adding epitope tags, without impairing or eliminating its
functions, as described below. Other modifications that can be made
without materially impairing one or more functions of a polypeptide
include, for example, in vivo or in vitro chemical and biochemical
modifications that incorporate unusual amino acids. Such
modifications include, for example, acetylation, carboxylation,
phosphorylation, glycosylation, labeling, e.g., with radionuclides,
and various enzymatic modifications, as will be readily appreciated
by those of ordinary skill in the art. A variety of methods for
labeling polypeptides, and labels useful for such purposes, are
well known in the art, and include radioactive isotopes such as
.sup.32P, fluorophores, chemiluminescent agents, enzymes, and
antiligands.
[0150] A humanized anti-OX40 antibody or antigen-binding fragment
thereof as provided herein can further include a heterologous
agent, e.g., a stabilizing agent, an immune response modifier, or a
detectable agent. In certain aspects the heterologous agent
comprises one or more additional polypeptide sequences fused to the
polypeptide subunit via a peptide bond, such as a signal sequence
(e.g., a secretory signal sequence), a linker sequence, an amino
acid tag or label, or a peptide or polypeptide sequence that
facilitates purification. In certain aspects, the heterologous
polypeptide can be fused to the N-terminus or the C-terminus of
either a heavy chain or light chain antibody subunit, or fragment
thereof, as long as the functional characteristics of the domains
are maintained.
[0151] In certain aspects, the heterologous agent can be chemically
conjugated to a humanized anti-OX40 antibody or antigen-binding
fragment thereof as provided herein. Exemplary heterologous agents
that can be chemically conjugated to the polypeptide subunit
include, without limitation, linkers, drugs, toxins, imaging
agents, radioactive compounds, organic and inorganic polymers, and
any other compositions which can provide a desired activity that is
not provided by the polypeptide subunit itself. Specific agents
include, without limitation, polyethylene glycol (PEG), a cytotoxic
agent, a radionuclide, an imaging agent, biotin.
[0152] In certain aspects, the disclosure provides certain
anti-OX40 antibodies to be used as controls or research tools. For
example, the disclosure provides a humanized anti-OX40 antibody or
antigen-binding fragment thereof as described above, in which the
VH and VL are fused to a murine IgG1 heavy chain and a murine kappa
light chain, respectively. This "reverse chimera" is useful for in
vivo characterization in rhesus monkeys. In another example, the VH
region on a humanized anti-OX40 antibody as provided herein can be
attached to various heavy chain constant regions with altered
effector functions, e.g., human IgG4P or human IgG1TM, described
elsewhere herein.
[0153] A humanized anti-OX40 antibody or antigen-binding fragment
thereof as provided herein can specifically bind to OX40 as
expressed on primary activated T cells, e.g., primary activated
CD4.sup.+ T cells, primary activated CD8.sup.+ T cells and/or
regulatory T cells, from human, cynomolgus monkey, rhesus monkey,
or any combination thereof. In certain aspects, a humanized
anti-OX40 antibody or antigen-binding fragment thereof as provided
herein, e.g., OX40mAb24, can bind to human OX40 expressed on
primary activated human CD4.sup.+ T cells with a binding affinity
of about 0.01 pM to about 1 nM, e.g., about 1 pM to about 500 pM,
e.g., about 100 pM to about 400 pM, e.g., about 250 pM to about 370
pM, all as measured by flow cytometry. For example, a humanized
anti-OX40 antibody or an antigen-binding fragment thereof can bind
to human OX40 expressed on primary activated human CD4.sup.+ T
cells with a binding affinity of about 0.1 pM, about 0.5 pM, about
1 pM, about 10 pM, about 50 pM, about 100 pM, about 150 pM, about
200 pM, about 250 pM, about 275 pM, about 300 pM, about 325 pM,
about 350 pM, about 370 pM, about 400 pM, about 425 pM, about 450
pM, about 475 pM, about 500 pM, about 550 pM, about 600 pM, about
650 pM, about 700 pM, about 750 pM, or about 1 nM, all as measured
by flow cytometry. In certain aspects, a humanized anti-OX40
antibody or antigen-binding fragment thereof as provided herein can
bind to human OX40 expressed on primary activated human CD4.sup.+ T
cells with a binding affinity of about 312 pM. Binding affinity can
be measured by a number of different methods and/or instruments,
and the relative binding affinities can vary depending on the
method or instrument, as is well understood by persons or ordinary
skill in the art.
[0154] A humanized anti-OX40 antibody or antigen-binding fragment
thereof as provided herein can occupy and cross-link some or all of
the OX40 molecules on the surface of a cell, e.g., a primary
activated human CD4.sup.+ T cell. In certain aspects, a humanized
anti-OX40 antibody or antigen-binding fragment thereof as provided
herein can bind to human OX40 expressed on primary activated human
CD4.sup.+ T cells, and can achieve 20% receptor occupancy on
primary activated human CD4.sup.+ T cells (EC.sub.20) at a
concentration of about 0.01 pM to about 1 nM, e.g., about 0.1 pM to
about 500 pM, e.g., about 1 pM to about 200 pM, e.g., about 10 pM
to about 100 pM, e.g., about 50 pM to about 100 pM, e.g., about 63
pM to about 93 pM, e.g., about 1 pM, about 10 pM, about 20 pM,
about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM,
about 78 pM, about 80 pM, about 90 pM, about 100 pM, or about 150
pM, as measured by flow cytometry. In certain aspects, a humanized
anti-OX40 antibody or antigen-binding fragment thereof as provided
herein can achieve 20% receptor occupancy on primary activated
human CD4.sup.+ T cells (EC.sub.20) at a concentration of about 78
pM., as measured by flow cytometry. In certain aspects, a humanized
anti-OX40 antibody or antigen-binding fragment thereof as provided
herein can bind to human OX40 expressed on primary activated human
CD4.sup.+ T cells, and can achieve 50% receptor occupancy on
primary activated human CD4.sup.+ T cells (EC.sub.50) at a
concentration of about 0.01 pM to about 1 nM, e.g., about 1 pM to
about 500 pM, e.g., about 100 pM to about 400 pM, e.g., about 250
pM to about 370 pM, e.g., about 100 pM, about 150 pM, about 200 pM,
about 250 pM, about 275 pM, about 300 pM, about 325 pM, about 350
pM, about 370 pM, about 400 pM, about 425 pM, about 450 pM, about
475 pM, or about 500 pM, all as measured by flow cytometry. In
certain aspects, a humanized anti-OX40 antibody or antigen-binding
fragment thereof as provided herein can achieve 50% receptor
occupancy on primary activated human CD4.sup.+ T cells (EC.sub.50)
at a concentration of about 312 pM., as measured by flow cytometry.
In certain aspects, a humanized anti-OX40 antibody or an
antigen-binding fragment thereof can bind to human OX40 expressed
on primary activated human CD4.sup.+ T cells, and can achieve 90%
receptor occupancy on primary activated human CD4.sup.+ T cells
(EC.sub.90) at a concentration of about 100 pM to about 100 nM,
e.g., about 500 pM to about 10 nM, e.g., about 1 nM to about 500
nM, e.g., about 2 nM to about 4 nM, e.g., about 1 nM, about 2 nM,
about 3 nM, about 4 nM, or about 5 nM, as measured by flow
cytometry. In certain aspects, a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can achieve 90%
receptor occupancy on primary activated human CD4.sup.+ T cells
(EC.sub.90) at a concentration of about 2290 pM to about 3330 pM,
e.g., about 2810 pM., as measured by flow cytometry.
[0155] In certain aspects, a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can bind to
human OX40 expressed on OX40-overexpressing Jurkat cells with a
binding affinity of about 250 pM to about 600 pM, e.g., about 424
pM as measured by flow cytometry.
[0156] In certain aspects, a humanized anti-OX40 antibody or an
antigen-binding fragment thereof can bind to human OX40 expressed
on OX40-overexpressing Jurkat cells, and can achieve EC.sub.20 at a
concentration of about 60 to about 150 pM, EC.sub.50 at a
concentration of about 250 to about 600 pM, and EC.sub.90 at a
concentration about 2260 to about 4390 pM as measured by flow
cytometry. In certain aspects, a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can bind to
human OX40 expressed on OX40-overexpressing Jurkat cells and can
achieve EC.sub.20 at a concentration of about 106 pM, EC.sub.50 at
a concentration of about 424 pM, and EC.sub.90 at a concentration
of about 3820 pM, as measured by flow cytometry.
[0157] In another example, a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can bind to
cynomolgus monkey OX40 expressed on primary activated cynomolgus
monkey CD4.sup.+ T cells with a binding affinity of about 340 pM to
about 820 pM, e.g., about 580 pM, as measured by flow cytometry. In
another example, a humanized anti-OX40 antibody or antigen-binding
fragment thereof as provided herein can bind to rhesus monkey OX40
expressed on primary activated rhesus monkey CD4.sup.+ T cells with
a binding affinity of about 130 pM to about 600 pM, e.g., about 370
pM, as measured by flow cytometry.
[0158] In certain aspects, a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can induce
dose-dependent proliferation of activated CD4.sup.+ T cells in a
plate-based assay. For example, in an in vitro assay a humanized
anti-OX40 antibody or antigen-binding fragment thereof as provided
herein, e.g., OX40mAb24, a 20% maximal proliferation response
(EC.sub.20) can be achieved in primary activated human CD4.sup.+ T
cells at an antibody concentration of about 14 pM to about 28 pM,
e.g., about 21 pM, a 50% maximal proliferation response (EC.sub.50)
can be achieved in primary activated human CD4.sup.+ T cells at an
antibody concentration of about 0.3 pM to about 130 pM, e.g., about
28 pM, and a 90% maximal proliferation response (EC.sub.90) can be
achieved in primary activated human CD4.sup.+ T cells at an
antibody concentration of about 50 pM to about 90 pM, e.g., about
72 pM, all as measured by flow cytometry.
[0159] In certain aspects, a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can induce
dose-dependent cytokine release from activated CD4.sup.+ T cells,
e.g., human primary activated CD4.sup.- T cells. In certain
aspects, the released cytokine is IFN.gamma., TNF.alpha., IL-5,
IL-10, IL-2, IL-4, IL-13, IL-8, IL-12 p70, IL-1.beta., or any
combination thereof. In certain aspects, the cytokine is
IFN.gamma., TNF.alpha., IL-5, IL-10, IL-13 or any combination
thereof. Similarly, a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can achieve
CD4.sup.+ T cell proliferation and cytokine release in primary
activated cynomolgus monkey CD4.sup.+ T cells and in primary
activated rhesus monkey CD4.sup.+ T cells.
[0160] In additional aspects, a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can activate
the NF.kappa.B pathway in OX40 expressing T cells in the presence
of Fc.gamma.R-expressing cells. For example, a humanized anti-OX40
antibody or antigen-binding fragment thereof as provided herein can
activate the NF.kappa.B pathway in OX40-expressing Jurkat
NF.kappa.B-luciferase reporter cells that produce luciferase in
response to stimulation of the NF.kappa.B signaling pathway.
Alternatively, a humanized anti-OX40 antibody or antigen-binding
fragment thereof as provided herein can activate the NF.kappa.B
pathway in cells expressing human OX40, cynomolgus monkey OX40 or
rhesus monkey OX40.
[0161] In yet another aspect a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can facilitate
cancer treatment, e.g., by slowing tumor growth, stalling tumor
growth, or reducing the size of existing tumors, when administrated
as an effective dose to a subject in need of cancer treatment. In
certain aspects the facilitation of cancer treatment can be
achieved in the presence of T cells. In certain aspects, a
humanized anti-OX40 antibody or antigen-binding fragment thereof as
provided herein, when administered as an effective dose to a
subject in need of treatment, can reduce tumor growth by at least
10%, at least 20%, at least 30%, at least 40%, and least 50%, at
least 60%, or at least 70% compared to administration of an
isotype-matched control antibody.
[0162] In yet further aspects, a humanized anti-OX40 antibody or
antigen-binding fragment thereof as provided herein can induce
proliferation of activated, OX40-expressing T cells through binding
to OX40, and at the same time trigger complement-dependent or
antibody-dependent cellular cytotoxicity against the
OX40-expressing T cells, e.g., activated CD4.sup.+ T cells,
activated CD8.sup.+ T cells and/or regulatory T cells. Moreover in
certain aspects, a humanized anti-OX40 antibody or antigen-binding
fragment thereof as provided herein can induce proliferation of
OX40-expressing T cells, e.g., activated, OX40-expressing
CD4.sup.+, CD8.sup.+ T cells and/or regulatory T cells through
binding to OX40, and at the same time bind to C1q or trigger NK
cell-mediated antibody-dependent cellular cytotoxicity of
OX40-expressing T cells, e.g., activated CD4.sup.+ T cells,
activated CD8+ T cells and/or regulatory T cells.
[0163] In certain aspects the disclosure provides an anti-OX40
antibody or fragment thereof comprising a humanized VH attached to
a murine heavy chain constant region and a humanized VL attached to
a murine light chain constant region, wherein the heavy chain
comprises SEQ ID NO: 81 and the light chain comprises SEQ ID NO:
83. In certain aspects the heavy chain constant region can be,
e.g., a murine IgG1 constant region. In certain aspects the light
chain constant region can be, e.g., a murine kappa constant
region.
[0164] In certain aspects the disclosure provides a rat-anti-mouse
OX40 antibody or antigen-binding fragment thereof comprising a rat
VH and a rat VL, where the VH comprises the amino acid sequence SEQ
ID NO: 85 and the VL comprises the amino acid sequence SEQ ID NO:
88. In certain aspects, this antibody or fragment thereof further
comprises a light chain constant region or fragment thereof fused
to the C-terminus of the VL. The light chain constant region can
be, e.g., a murine kappa constant region. In certain aspects, this
antibody or fragment thereof further comprises a heavy chain
constant region or fragment thereof fused to the C-terminus of the
VH. The heavy chain constant region can be, e.g., a murine IgG2a
constant region. In certain aspects this rat-anti-mouse OX40
antibody comprises the heavy chain amino acid sequence SEQ ID NO:
86 and the light chain amino acid sequence SEQ ID NO: 89. In
certain aspects, a rat-anti-mouse OX40 antibody or fragment thereof
as provided herein can specifically bind to mouse OX40. In certain
aspects, administration of an effective dose of a rat-anti-mouse
OX40 antibody or fragment thereof as provided herein to mouse can
inhibit mouse cancer cell line growth in the mouse.
[0165] In certain aspects a rat-anti-mouse OX40 antibody fragment
as provided herein can be, e.g., an Fv fragment, an Fab fragment,
an F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv
fragment, or an sc(Fv)2 fragment, or any combination thereof.
Polynucleotides Encoding Humanized Anti-OX40 Antibodies, Fragments,
or Subunits
[0166] This disclosure provides polynucleotides comprising nucleic
acid sequences that encode a humanized anti-OX40 antibody or an
antigen-binding fragment thereof. For example, the disclosure
provides a polynucleotide, or two or more polynucleotides,
comprising a nucleic acid sequence that encodes a humanized
anti-OX40 antibody or a subunit of a humanized anti-OX40 antibody,
or encodes an antigen-binding fragment of such an antibody. The
polynucleotides of the disclosure can be in the form of RNA or in
the form of DNA. DNA includes cDNA, genomic DNA, e.g., modified
genomic DNA, and synthetic DNA; and can be double-stranded or
single-stranded, and if single stranded can be the coding strand or
non-coding (anti-sense) strand.
[0167] In certain aspects, a polynucleotide can be isolated. In
certain aspects, a polynucleotide can be substantially pure. In
certain aspects a polynucleotide can be non-naturally occurring. In
certain aspects a polynucleotide can be cDNA or derived from cDNA.
In certain aspects a polynucleotide can be recombinantly produced.
In certain aspects a polynucleotide can comprise the coding
sequence for the mature polypeptide fused in the same reading frame
to a polynucleotide which aids, for example, in expression and
secretion of a polypeptide from a host cell (e.g., a leader
sequence which functions as a secretory sequence for controlling
transport of a polypeptide from the cell). The polypeptide having a
leader sequence is a preprotein and can have the leader sequence
cleaved by the host cell to form the mature form of the
polypeptide.
[0168] In certain aspects the disclosure provides a polynucleotide
comprising a nucleic acid that encodes a humanized anti-OX40
antibody or an antigen-binding fragment thereof as provided herein,
or a polypeptide subunit of a humanized anti-OX40 antibody or an
antigen-binding fragment thereof as provided herein.
[0169] Also provided are polynucleotides comprising nucleic acid
sequences comprising one or a small number of deletions, additions
and/or substitutions. Such changes can be contiguous or can be
distributed at different positions in the nucleic acid. A
substantially identical nucleic acid sequence can, for example,
have 1, or 2, or 3, or 4, or even more nucleotide deletions,
additions and/or substitutions. In certain aspects, the one or more
deletions, additions and/or substitutions do not alter the reading
frame encoded by the polynucleotide sequence, such that a modified
("mutant") but substantially identical polypeptide is produced upon
expression of the nucleic acid.
[0170] In certain aspects this disclosure provides an isolated
polynucleotide comprising a nucleic acid encoding an antibody VL,
wherein the VL comprises an amino acid sequence at least 70%, 75%,
80%, 85%, 90%, 95%, or 100% identical to the reference amino acid
sequence SEQ ID NO: 29 or SEQ ID NO: 32. In certain aspects, the
polynucleotide encodes an antibody light chain and comprises a
nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%
identical to the reference nucleic acid sequence SEQ ID NO: 31.
[0171] In certain aspects this disclosure provides an isolated
polynucleotide comprising a nucleic acid encoding an antibody VL,
wherein the VL comprises SEQ ID NO: 29 or SEQ ID NO: 32.
[0172] The disclosure further provides an isolated polynucleotide
comprising a nucleic acid encoding an antibody VH, wherein the VH
comprises VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences
identical to, or identical except for eight, seven, six, five,
four, three, two, or one single amino acid substitutions,
deletions, or insertions in one or more of the VH-CDRS to: the
VHCDR1 amino acid sequence SEQ ID NO: 8, the VHCDR2 amino acid
sequence SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, and the
VHCDR3 amino acid sequence SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID
NO: 27.
[0173] The disclosure further provides an isolated polynucleotide
comprising a nucleic acid encoding an antibody VH, wherein the VH
comprises an amino acid sequence with the formula:
HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4,
wherein HFW1 is SEQ ID NO: 6 or SEQ ID NO: 7, HCDR1 is SEQ ID NO:
8, HFW2 is SEQ ID NO: 9, HCDR2 is SEQ ID NO: 14, SEQ ID NO: 15 or
SEQ ID NO: 16, HFW3 is SEQ ID NO: 17, HCDR3 is SEQ ID NO: 25, SEQ
ID NO: 26, or SEQ ID NO: 27, and HFW4 is SEQ ID NO: 28. In certain
aspects the amino acid sequence of HFW2 is SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In certain aspects the
amino acid sequence of HFW3 is SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID
NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO:
24.
[0174] Moreover, the disclosure provides an isolated polynucleotide
comprising a nucleic acid encoding an antibody VH, wherein the VH
comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%,
95%, or 100% identical to the reference amino acid sequence SEQ ID
NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,
SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO: 67. In
certain aspects, the polynucleotide comprises a nucleotide sequence
at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the
reference nucleic acid sequence SEQ ID NO: 34, SEQ ID NO: 36, SEQ
ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:
46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ
ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:
64, SEQ ID NO: 66, or SEQ ID NO: 68.
[0175] In certain aspects the disclosure provides a polynucleotide
comprising the nucleic acid of SEQ ID NO: 60, the nucleic acid of
SEQ ID NO: 31, the nucleic acid of SEQ ID NO: 72, or any
combination thereof.
[0176] Further provided is a vector comprising a polynucleotide as
described above. Suitable vectors are described elsewhere herein,
and are known to those of ordinary skill in the art.
[0177] In certain aspects, the disclosure provides a composition,
e.g., a pharmaceutical composition, comprising a polynucleotide or
vector as described above, optionally further comprising one or
more carriers, diluents, excipients, or other additives.
[0178] In certain aspects, the disclosure provides a polynucleotide
composition comprising: a polynucleotide that comprises a nucleic
acid encoding a VH, and polynucleotide that comprises a nucleic
acid encoding a VL. According to this aspect, the VL and VH
together can comprise a VL amino acid sequence at least 70%, 75%,
80%, 85%, 90%, 95%, or 100% identical to the reference amino acid
sequence SEQ ID NO: 29 or SEQ ID NO: 32, and a VH comprising: (a)
VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences identical to, or
identical except for eight, seven, six, five, four, three, two, or
one single amino acid substitutions, deletions, or insertions in
one or more of the VH-CDRS to: the VHCDR1 amino acid sequence SEQ
ID NO: 8, the VHCDR2 amino acid sequence SEQ ID NO: 14, SEQ ID NO:
15, or SEQ ID NO: 16, and the VHCDR3 amino acid sequence SEQ ID NO:
25, SEQ ID NO: 26, or SEQ ID NO: 27; (b) an amino acid sequence
with the formula:
HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4,
wherein HFW1 is SEQ ID NO: 6 or SEQ ID NO: 7, HCDR1 is SEQ ID NO:
8, HFW2 is SEQ ID NO: 9, HCDR2 is SEQ ID NO: 14, SEQ ID NO: 15 or
SEQ ID NO: 16, HFW3 is SEQ ID NO: 17, HCDR3 is SEQ ID NO: 25, SEQ
ID NO: 26, or SEQ ID NO: 27, and HFW4 is SEQ ID NO: 28; or (c) an
amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%
identical to the reference amino acid sequence SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ
ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:
61, SEQ ID NO: 63, SEQ ID NO: 65, or SEQ ID NO: 67. For VH (b), the
amino acid sequence of HFW2 can be SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, or SEQ ID NO: 13; and/or the amino acid sequence of
HFW3 can be SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:
21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
[0179] In a polynucleotide composition as described above, the
polynucleotide comprising a nucleic acid encoding a VH and the
polynucleotide comprising a nucleic acid encoding a VL can reside
in a single vector, or can be on separate, non-identical vectors.
Accordingly the disclosure provides one or more vectors comprising
the polynucleotide composition described above.
[0180] In some cases, a polynucleotide composition encoding a VH
and VL as described above can encode a humanized antibody or
antigen-binding fragment thereof that can specifically bind to
OX40, e.g., human OX40, cynomolgus monkey OX40, and/or rhesus
monkey OX40.
[0181] In certain aspects the disclosure provides an isolated
polynucleotide comprising a nucleic acid that encodes an anti-OX40
antibody or fragment thereof comprising a humanized VH attached to
a murine heavy chain constant region and a humanized VL attached to
a murine light chain constant region, wherein the heavy chain
comprises SEQ ID NO: 81 and the light chain comprises SEQ ID NO:
83. In certain aspects the heavy chain constant region can be,
e.g., a murine IgG1 constant region. In certain aspects the light
chain constant region can be, e.g., a murine kappa constant
region.
[0182] In certain aspects the disclosure provides an isolated
polynucleotide comprising a nucleic acid that encodes a
rat-anti-mouse OX40 antibody or antigen-binding fragment thereof
comprising a rat VH and a rat VL, where the VH comprises the amino
acid sequence SEQ ID NO: 85 and the VL comprises the amino acid
sequence SEQ ID NO: 88. In certain aspects, this antibody or
fragment thereof further comprises a light chain constant region or
fragment thereof fused to the C-terminus of the VL. The light chain
constant region can be, e.g., a murine kappa constant region. In
certain aspects, this antibody or fragment thereof further
comprises a heavy chain constant region or fragment thereof fused
to the C-terminus of the VH. The heavy chain constant region can
be, e.g., a murine IgG2a constant region. In certain aspects this
rat-anti-mouse OX40 antibody comprises the heavy chain amino acid
sequence SEQ ID NO: 86 and the light chain amino acid sequence SEQ
ID NO: 89. In certain aspects, a rat-anti-mouse OX40 antibody or
fragment thereof as provided herein can specifically bind to mouse
OX40. In certain aspects, administration of an effective dose of a
rat-anti-mouse OX40 antibody or fragment thereof as provided herein
to mouse can inhibit mouse cancer cell line growth in the
mouse.
[0183] This disclosure further provides a host cell comprising a
polynucleotide, polynucleotide composition, or vector as provided
above, where the host cell can, in some instances, express an
antibody or antigen-binding fragment thereof that specifically
binds to OX40, e.g., human OX40, cynomolgus monkey OX40, or rhesus
monkey OX40. Such a host cell can be utilized in a method of making
an antibody or antigen-binding fragment thereof as provided herein,
which method includes (a) culturing the host cell and (b) isolating
the antibody or antigen-binding fragment thereof expressed from the
host cell.
[0184] Polynucleotide variants are also provided. Polynucleotide
variants can contain alterations in the coding regions, non-coding
regions, or both. In some aspects polynucleotide variants contain
alterations that produce silent substitutions, additions, or
deletions, but do not alter the properties or activities of the
encoded polypeptide. In some aspects, polynucleotide variants are
produced by silent substitutions due to the degeneracy of the
genetic code. Polynucleotide variants can be produced for a variety
of reasons, e.g., to optimize codon expression for a particular
host (change codons in the human mRNA to those by a bacterial host
such as E. coli). Vectors and cells comprising the polynucleotides
described herein are also provided.
[0185] In some aspects, a DNA sequence encoding a humanized
anti-OX40 antibody or an antigen-binding fragment thereof can be
constructed by chemical synthesis using an oligonucleotide
synthesizer. Such oligonucleotides can be designed based on the
amino acid sequence of the desired polypeptide and selecting those
codons that are favored in the host cell in which the recombinant
polypeptide of interest will be produced. Standard methods can be
applied to synthesize an isolated polynucleotide sequence encoding
an isolated polypeptide of interest. For example, a complete amino
acid sequence can be used to construct a back-translated gene.
Further, a DNA oligomer containing a nucleotide sequence coding for
the particular isolated polypeptide can be synthesized. For
example, several small oligonucleotides coding for portions of the
desired polypeptide can be synthesized and then ligated. The
individual oligonucleotides can contain 5' or 3' overhangs for
complementary assembly.
[0186] Once assembled (by synthesis, site-directed mutagenesis or
another method), the polynucleotide sequences encoding a particular
isolated polypeptide of interest can be inserted into an expression
vector and operatively linked to an expression control sequence
appropriate for expression of the protein in a desired host. Proper
assembly can be confirmed, e.g., by nucleotide sequencing,
restriction mapping, and/or expression of a biologically active
polypeptide in a suitable host. In order to obtain high expression
levels of a transfected gene in a host, the gene can be operatively
linked to or associated with transcriptional and translational
expression control sequences that are functional in the chosen
expression host.
[0187] In certain aspects, recombinant expression vectors are used
to amplify and express DNA encoding a humanized anti-OX40 antibody
or an antigen-binding fragment thereof. Recombinant expression
vectors are replicable DNA constructs which have synthetic or
cDNA-derived DNA fragments encoding a polypeptide chain of a
humanized anti-OX40 antibody or an antigen-binding fragment
thereof, operatively linked to suitable transcriptional or
translational regulatory elements derived from mammalian,
microbial, viral or insect genes. In one example, a transcriptional
unit can comprise an assembly of (1) a genetic element or elements
having a regulatory role in gene expression, for example,
transcriptional promoters or enhancers, (2) a structural or coding
sequence which is transcribed into mRNA and translated into
protein, and (3) appropriate transcription and translation
initiation and termination sequences, as described in detail below.
Such regulatory elements can include an operator sequence to
control transcription. The ability to replicate in a host,
conferred, e.g., by an origin of replication, and a selection gene
to facilitate recognition of transformants can additionally be
incorporated. DNA regions are operatively linked when they are
functionally related to each other. For example, DNA for a signal
peptide (secretory leader) is operatively linked to DNA for a
polypeptide if it is expressed as a precursor which participates in
the secretion of the polypeptide; a promoter is operatively linked
to a coding sequence if it controls the transcription of the
sequence; or a ribosome binding site is operatively linked to a
coding sequence if it is positioned so as to permit translation.
Structural elements intended for use in yeast expression systems
include a leader sequence enabling extracellular secretion of
translated protein by a host cell. Alternatively, where a
recombinant protein is expressed without a leader or transport
sequence, the protein can include an N-terminal methionine. This
methionine can optionally be subsequently cleaved from the
expressed recombinant protein to provide a final product.
[0188] The choice of expression control sequence and expression
vector will depend upon the choice of host. A wide variety of
expression host/vector combinations can be employed. Useful
expression vectors for eukaryotic hosts, include, for example,
vectors comprising expression control sequences from SV40, bovine
papilloma virus, adenovirus and cytomegalovirus. Useful expression
vectors for bacterial hosts include known bacterial plasmids, such
as plasmids from E. coli, including pCR 1, pBR322, pMB9 and their
derivatives, wider host range plasmids, such as M13 and filamentous
single-stranded DNA phages.
[0189] Suitable host cells for expression of a humanized anti-OX40
antibody or an antigen-binding fragment thereof include
prokaryotes, yeast, insect or higher eukaryotic cells under the
control of appropriate promoters. Prokaryotes include gram negative
or gram positive organisms, for example E. coli or bacilli. Higher
eukaryotic cells include established cell lines of mammalian origin
as described below. Cell-free translation systems could also be
employed. Additional information regarding methods of protein
production, including antibody production, can be found, e.g., in
U.S. Patent Publication No. 2008/0187954, U.S. Pat. Nos. 6,413,746
and 6,660,501, and International Patent Publication No. WO
04009823, each of which is hereby incorporated by reference herein
in its entirety.
[0190] Various mammalian or insect cell culture systems can also be
employed to express a humanized anti-OX40 antibody or an
antigen-binding fragment thereof. Expression of recombinant
proteins in mammalian cells can be performed because such proteins
are generally correctly folded, appropriately modified and
completely functional. Examples of suitable mammalian host cell
lines include HEK-293 and HEK-293T, the COS-7 lines of monkey
kidney cells, described by Gluzman (Cell 23:175, 1981), and other
cell lines including, for example, L cells, C127, 3T3, Chinese
hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression
vectors can comprise nontranscribed elements such as an origin of
replication, a suitable promoter and enhancer linked to the gene to
be expressed, and other 5' or 3' flanking nontranscribed sequences,
and 5' or 3' nontranslated sequences, such as ribosome binding
sites, a polyadenylation site, splice donor and acceptor sites, and
transcriptional termination sequences. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, BioTechnology 6:47 (1988).
[0191] A humanized anti-OX40 antibody or an antigen-binding
fragment thereof produced by a transformed host, can be purified
according to any suitable method. Such standard methods include
chromatography (e.g., ion exchange, affinity and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for protein purification. Affinity tags
such as hexahistidine, maltose binding domain, influenza coat
sequence and glutathione-S-transferase can be attached to the
protein to allow easy purification by passage over an appropriate
affinity column. Isolated proteins can also be physically
characterized using such techniques as proteolysis, nuclear
magnetic resonance and x-ray crystallography.
[0192] For example, supernatants from systems that secrete
recombinant protein into culture media can be first concentrated
using a commercially available protein concentration filter, for
example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate can be applied to
a suitable purification matrix. Alternatively, an anion exchange
resin can be employed, for example, a matrix or substrate having
pendant diethylaminoethyl (DEAE) groups. The matrices can be
acrylamide, agarose, dextran, cellulose or other types commonly
employed in protein purification. Alternatively, a cation exchange
step can be employed. Suitable cation exchangers include various
insoluble matrices comprising sulfopropyl or carboxymethyl groups.
Finally, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify a humanized anti-OX40 antibody or
an antigen-binding fragment thereof. Some or all of the foregoing
purification steps, in various combinations, can also be employed
to provide a homogeneous recombinant protein.
[0193] A humanized anti-OX40 antibody or an antigen-binding
fragment thereof produced in bacterial culture, can be isolated,
for example, by initial extraction from cell pellets, followed by
one or more concentration, salting-out, aqueous ion exchange or
size exclusion chromatography steps. High performance liquid
chromatography (HPLC) can be employed for final purification steps.
Microbial cells employed in expression of a recombinant protein can
be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell lysing
agents.
[0194] Methods known in the art for purifying antibodies and other
proteins also include, for example, those described in U.S. Patent
Publication Nos. 2008/0312425, 2008/0177048, and 2009/0187005, each
of which is hereby incorporated by reference herein in its
entirety.
Pharmaceutical Compositions and Administration Methods
[0195] Methods of preparing and administering a humanized anti-OX40
antibody or an antigen-binding fragment thereof as provided herein,
to a subject in need thereof, e.g., to enhance an immune response
in a cancer patient, e.g., to inhibit or reduce tumor growth, are
well known to or can be readily determined by those skilled in the
art. The route of administration of a humanized anti-OX40 antibody
or an antigen-binding fragment thereof can be, for example, oral,
parenteral, by inhalation or topical. The term parenteral as used
herein includes, e.g., intravenous, intraarterial, intraperitoneal,
intramuscular, subcutaneous, rectal, or vaginal administration.
While all these forms of administration are clearly contemplated as
suitable forms, another example of a form for administration would
be a solution for injection, in particular for intravenous or
intraarterial injection or drip. Usually, a suitable pharmaceutical
composition can comprise, without limitation, a buffer (e.g.
acetate, phosphate or citrate buffer), a surfactant (e.g.
polysorbate), a stabilizer agent (e.g. human albumin), etc. In
other methods compatible with the teachings herein, a humanized
anti-OX40 antibody or an antigen-binding fragment thereof as
provided herein can be delivered directly to the site of the
adverse cellular population thereby increasing the exposure of the
diseased tissue to the therapeutic agent.
[0196] Certain pharmaceutical compositions provided herein can be
orally administered in an acceptable dosage form including, e.g.,
capsules, tablets, aqueous suspensions or solutions. Certain
pharmaceutical compositions also can be administered by nasal
aerosol or inhalation. Such compositions can be prepared as
solutions in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
and/or other conventional solubilizing or dispersing agents.
[0197] The amount of a humanized anti-OX40 antibody or an
antigen-binding fragment thereof that can be combined with carrier
materials to produce a single dosage form will vary depending upon
the subject treated and the particular mode of administration. The
composition can be administered as a single dose, multiple doses or
over an established period of time in an infusion. Dosage regimens
also can be adjusted to provide the optimum desired response (e.g.,
a therapeutic or prophylactic response).
[0198] By "therapeutically effective dose or amount" or "effective
amount" is intended an amount of a humanized anti-OX40 antibody or
an antigen-binding fragment thereof, that when administered brings
about a positive therapeutic response with respect to treatment of
a patient with a disease or condition to be treated.
Kits
[0199] This disclosure further provides kits that comprise a
humanized anti-OX40 antibody or an antigen-binding fragment thereof
as described herein and that can be used to perform the methods
described herein. In certain embodiments, a kit comprises at least
one purified humanized anti-OX40 antibody or an antigen-binding
fragment thereof, in one or more containers. One skilled in the art
will readily recognize that the disclosed humanized anti-OX40
antibody can be readily incorporated into one of the established
kit formats that are well known in the art.
Immunoassays
[0200] A humanized anti-OX40 antibody or an antigen-binding
fragment thereof can be assayed for specific and/or selective
binding by any method known in the art. The immunoassays that can
be used include but are not limited to competitive and
non-competitive assay systems using techniques such as Western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), fluorescent focus assay (FFA), "sandwich" immunoassays,
immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al., eds, (1994) Current
Protocols in Molecular Biology (John Wiley & Sons, Inc., NY)
Vol. 1, which is incorporated by reference herein in its
entirety).
[0201] Methods and reagents suitable for determination of binding
characteristics of a humanized anti-OX40 antibody as provided
herein are known in the art and/or are commercially available.
Equipment and software designed for such kinetic analyses are
commercially available (e.g., BIAcore.RTM., BIAevaluation.RTM.
software, GE Healthcare; KINEXA.RTM. Software, Sapidyne
Instruments).
Methods of Immune Enhancement and Treatment
[0202] The enhancement of an antigen-specific immune response in a
subject (e.g., a mammalian subject, such as a human subject) by
engaging OX40 on activated T cells, e.g., activated CD4.sup.+ T
cells and/or activated CD8.sup.+ T cells, during or after antigen
activation can be accomplished using a wide variety of methods. The
method of choice will primarily depend upon the type of antigen
against which it is desired to enhance the immune response, and
various methods available are discussed below. Whatever method is
selected, a humanized anti-OX40 antibody or an antigen-binding
fragment thereof can be administered to a subject, e.g., a human
patient such that it is presented to T cells of the subject during
or shortly after priming of the T cells by antigen.
[0203] In certain aspects, the disclosure provides a method to
promote survival or proliferation of activated T cells, e.g.,
activated CD4.sup.+ T cells and/or activated CD8.sup.+ T cells,
comprising contacting the activated T cells, e.g., activated
CD4.sup.+ T cells and/or activated CD8.sup.+ T cells, with a
humanized anti-OX40 antibody or an antigen-binding fragment
thereof, under conditions where the humanized anti-OX40 antibody
can specifically bind to OX40 on the surface of the T cells, e.g.,
activated CD4.sup.+ T cells and/or activated CD8.sup.+ T cells. In
certain aspects the contacting is in vitro. In certain aspects the
contacting is in vivo, e.g. via administration of an effective dose
of the humanized anti-OX40 antibody to a subject in need of
treatment. In certain aspects the contacting can occur at the same
time as T cell activation, e.g., antigen activation, in certain
aspects the contacting can occur after T cell activation.
[0204] In further aspects, the disclosure provides a method of
inducing cytokine release from activated T cells, e.g., activated
CD4.sup.- T cells and/or activated CD8.sup.+ T cells, comprising
contacting the activated T cells, e.g., activated CD4.sup.+ T cells
and/or activated CD8.sup.+ T cells, with a humanized anti-OX40
antibody or an antigen-binding fragment thereof as provided herein,
wherein the humanized anti-OX40 antibody can specifically bind to
OX40 on the surface of the activated T cells, e.g., activated
CD4.sup.+ T cells and/or activated CD8.sup.+ T cells. In certain
aspects the contacting is in vitro. In certain aspects the
contacting is in vivo, e.g. via administration of an effective dose
of the humanized anti-OX40 antibody to a subject in need of
treatment. In certain aspects the contacting can occur at the same
time as T cell activation, e.g., antigen activation, in certain
aspects the contacting can occur after T cell activation. In
certain aspects the cytokine can be IFN.gamma., TNF.alpha., IL-5,
IL-10, IL-2, IL-4, IL-13, IL-8, IL-12 p70, IL-1.beta., or any
combination thereof. In certain aspects the cytokine is IFN.gamma.,
TNF.alpha., IL-5, IL-10, IL-13, or any combination thereof.
[0205] In certain aspects, the activated T cells, e.g., activated
CD4.sup.+ T cells and/or activated CD8.sup.+ T cells are human T
cells, cynomolgus monkey T cells, rhesus monkey T cells, or a
combination thereof.
[0206] The disclosure further provides a method of promoting T cell
activation, comprising contacting T cells with a humanized
anti-OX40 antibody as provided herein, wherein the humanized
anti-OX40 antibody can specifically bind to OX40 on the surface of
the T cells. In certain aspects the contacting occurs in the
presence of antigen, e.g., a tumor antigen. In certain aspects the
method further comprises interaction of an Fc domain of the
humanized anti-OX40 antibody with a cell expressing Fc.gamma.R,
e.g., a B cell, monocyte, macrophage, myeloid or plasmacytoid
dendritic cell, follicular dendritic cell, Langerhans cell,
endothelial cell, NK cell, activated T cell, neutrophil,
eosinophil, platelet, mast cell, a CD45.sup.+ cell from a primary
human tumor or tumor-draining or non-draining lymph node, a
CD45.sup.+ cell from other secondary or tertiary lymphoid
structures, or a combination thereof. In certain aspects, the T
cell activation can be measured through stimulation of the
NF.kappa.B signal transduction pathway. In certain aspects the
contacting is in vitro. In certain aspects the contacting is in
vivo, e.g. via administration of an effective dose of the humanized
anti-OX40 antibody to a subject in need of treatment.
[0207] The disclosure further provides a method of treating cancer
in a subject, comprising administering to a subject in need of
treatment an effective amount of a humanized anti-OX40 antibody or
an antigen-binding fragment thereof, or a composition or
formulation comprising the humanized anti-OX40 antibody. In certain
aspects, the cancer is a solid tumor. According to this method,
administration of humanized anti-OX40 antibody or composition can
inhibit tumor growth; can promote tumor reduction, or both. In
certain aspects, the tumor growth inhibition is achieved in the
presence of T cells.
[0208] The terms "cancer", "tumor", "cancerous", and "malignant"
refer to or describe the physiological condition in mammals that is
typically characterized by unregulated cell growth. Examples of
cancers include but are not limited to, carcinoma including
adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and
leukemias. More particular examples of such cancers include
squamous cell cancer, small-cell lung cancer, non-small cell lung
cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's
lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer,
ovarian cancer, liver cancer such as hepatic carcinoma and
hepatoma, bladder cancer, breast cancer (including hormonally
mediated breast cancer, see, e.g., Innes et al. (2006) Br. J.
Cancer 94:1057-1065), colon cancer, colorectal cancer, endometrial
carcinoma, myeloma (such as multiple myeloma), salivary gland
carcinoma, kidney cancer such as renal cell carcinoma and Wilms'
tumors, basal cell carcinoma, melanoma, prostate cancer, vulval
cancer, thyroid cancer, testicular cancer, esophageal cancer,
various types of head and neck cancer including, but not limited
to, squamous cell cancers, and cancers of mucinous origins, such
as, mucinous ovarian cancer, cholangiocarcinoma (liver) and renal
papillary carcinoma.
[0209] This disclosure further provides a method of preventing or
treating a cancer in a subject in need thereof, comprising
administering to the subject an effective amount of a humanized
anti-OX40 antibody or an antigen-binding fragment thereof, a
composition or formulation comprising the humanized anti-OX40
antibody, or a polynucleotide, a vector, or a host cell as
described herein.
[0210] Effective doses of compositions for treatment of cancer vary
depending upon many different factors, including means of
administration, target site, physiological state of the patient,
whether the patient is human or an animal, other medications
administered, and whether treatment is prophylactic or therapeutic.
Usually, the patient is a human but non-human mammals including
transgenic mammals can also be treated. Treatment dosages can be
titrated using routine methods known to those of skill in the art
to optimize safety and efficacy.
[0211] The compositions of the disclosure can be administered by
any suitable method, e.g., parenterally, intraventricularly,
orally, by inhalation spray, topically, rectally, nasally,
buccally, vaginally or via an implanted reservoir. The term
"parenteral" as used herein includes subcutaneous, intravenous,
intramuscular, intra-articular, intra-synovial, intrasternal,
intrathecal, intrahepatic, intralesional and intracranial injection
or infusion techniques.
[0212] The disclosure further provides a method of enhancing an
immune response in a subject comprising administering to a subject
in need thereof a therapeutically effective amount of a humanized
anti-OX40 antibody or an antigen-binding fragment thereof, or a
composition or formulation comprising the humanized anti-OX40
antibody.
[0213] The subject to be treated can be any animal, e.g., mammal,
in need of treatment, in certain aspects, subject is a human
subject.
[0214] In its simplest form, a preparation to be administered to a
subject is a humanized anti-OX40 antibody or an antigen-binding
fragment thereof, administered in conventional dosage form, which
can be combined with a pharmaceutical excipient, carrier or diluent
as described elsewhere herein.
[0215] A humanized anti-OX40 antibody or an antigen-binding
fragment thereof can be administered by any suitable method as
described elsewhere herein, e.g., via IV infusion. In certain
aspects, a humanized anti-OX40 antibody or an antigen-binding
fragment thereof can be introduced into a tumor, or in the vicinity
of a tumor cell.
[0216] All types of tumors are potentially amenable to treatment by
this approach including, without limitation, carcinoma of the
breast, lung, pancreas, ovary, kidney, colon and bladder, as well
as melanomas, sarcomas and lymphomas.
[0217] Engagement of the OX40 receptor on activated T cells, e.g.,
activated CD4.sup.+ T cells and/or activated CD8.sup.+ T cells
during, or shortly after, priming by an antigen results in an
increased response of the activated T cells, e.g., activated
CD4.sup.+ T cells and/or activated CD8.sup.+ T cells to the
antigen. In the context of the present disclosure, the term
"engagement" refers to binding to and stimulation of at least one
activity mediated by the OX40 receptor. For example, engagement of
the OX40 receptor on antigen specific activated T cells, e.g.,
activated CD4.sup.+ T cells and/or activated CD8.sup.+ T cells,
results in increased T cell proliferation as compared to the
response to antigen alone, and increased cytokine production. The
elevated response to the antigen can be maintained for a period of
time substantially longer than in the absence of OX40 receptor
engagement. Thus, stimulation via the OX40 receptor enhances the
antigen specific immune response by boosting T cell recognition of
antigens, e.g., tumor antigens.
[0218] OX40 agonists can enhance antigen specific immune responses
in a subject, such as a human subject, when administered to the
subject during or shortly after priming of T cells by an antigen.
OX40 agonists include OX40 ligand ("OX40L"), such as soluble OX40L
fusion proteins and anti-OX40 antibodies or fragments thereof. A
specific example is a humanized antibody that specifically binds to
OX40, thereby triggering signaling. A collection of humanized
anti-OX40 monoclonal antibodies are provided by this disclosure.
Also described are nucleic acids including polynucleotide sequences
that encode such antibodies. This disclosure also provides methods
for enhancing an antigen specific immune response in a subject
using humanized anti-OX40 monoclonal antibodies.
OX40 Epitopes
[0219] The portion of a target molecule, e.g., an OX40 polypeptide,
which specifically interacts with the antigen binding domain of an
antibody is an "epitope," or an "antigenic determinant." A target
molecule, e.g., a polypeptide, can be a single epitope, but
typically includes at least two epitopes, and can include any
number of epitopes, depending on the size, conformation, and type
of target molecule.
[0220] The minimum size of an epitope that can be bound by an
antibody on a target polypeptide is thought to be about four to
five amino acids. Peptide or polypeptide epitopes can contain at
least seven, at least nine, at least ten, or at least about 15 or
more amino acids. Since an antibody can recognize a polypeptide
antigen in its tertiary form, the amino acids comprising an epitope
need not be contiguous. An epitope of OX40, e.g., human OX40 as
provided herein can include at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 15, at least
20, at least 25, or between about 10 to about 30 contiguous or
non-contiguous amino acids of OX40, e.g., human OX40. In certain
aspects, an epitope of OX40, e.g., human OX40 as provided herein
consists of a peptide of 100 or fewer amino acids, 75 or fewer
amino acids, 50 or fewer amino acids, 40 or fewer amino acids, 35
or fewer amino acids, 30 or fewer amino acids, 25 or fewer amino
acids, 20 or fewer amino acids, or 15 or fewer amino acids, and can
include at least 4, at least 5, at least 6, at least 7, at least 8,
at least 9, at least 10, at least 15, at least 20, at least 25, or
between about 10 to about 30 contiguous or non-contiguous amino
acids of OX40, e.g., human OX40. On the other hand, an epitope of
OX40, e.g., human OX40 as provided herein can include no more than
4, no more than 5, no more than 6, no more than 7, no more than 8,
no more than 9, no more than 10, no more than 15, no more than 20,
no more than 25, or can consist of between about 10 to about 30
contiguous or non-contiguous amino acids of OX40, e.g., human
OX40.
[0221] In certain aspects, an anti-OX40 antibody or fragment
thereof as provided herein binds to an epitope of OX40, e.g., human
OX40, rhesus monkey OX40, or cynomolgus monkey OX40 that falls
within the third cysteine rich domain (CRD3) of OX40, e.g., within
amino acids 108 to 146 of human OX40 (SEQ ID NO: 91), or a peptide
at least 17%, at least 75%, at least 80%, at least 85%, and least
90%, at least 95%, or at least 100% identical to amino acids 108 to
146 of SEQ ID NO: 91. By "falls within" the CRD3 of OX40, means
that the means that the epitope can include 4 or more, 5 or more, 6
or more 7 or more 8 or more 9 or more, 10 or more, 15 or more, or
30 or more contiguous or non-contiguous amino acids amino acids of
the region of OX40 consisting of the CRD3 region, e.g., amino acids
108 to 146 of SEQ ID NO: 91, or a peptide at least 70%, at least
75%, at least 80%, at least 85%, and least 90%, at least 95%, or at
least 100% identical to amino acids 108 to 146 of SEQ ID NO:
91.
[0222] In certain aspects the OX40 CRD3 peptide that binds the
antibody provided herein retains a leucine at the position
corresponding to amino acid 116 of SEQ ID NOL 91, and an alanine at
the position corresponding to amino acid 126 of SEQ ID NO: 91. For
example, certain anti-OX40 antibodies or fragments thereof as
provided herein bind to human OX40 but do not bind to mouse or rat
OX40. The CRD3 region of mouse OX40 stretches from about amino acid
104 to about amino acid 144 of SEQ ID NO: 92. Amino acid Q113 of
mouse OX40, SEQ ID NO: 92, corresponds to amino acid L116 of human
OX40, SEQ ID NO: 91, and amino acid V124 of mouse OX40, SEQ ID NO:
92, corresponds to amino acid A126 of human OX40, SEQ ID NO: 91. As
shown in Example 10, an OX40 antibody as provided herein, e.g.,
OX40mAb24, can bind to a variant of mouse OX40 comprising SEQ ID
NO: 92 except for a Q113L mutation and a V124A.
[0223] In certain aspects an isolated peptide is provided, the
peptide consisting of or comprising an epitope that specifically
binds an OX40 antibody as provided herein, e.g., OX40mAb24. In
certain aspects, the peptide consists of 100 or fewer amino acids,
75 or fewer amino acids, 50 or fewer amino acids, 40 or fewer amino
acids, 35 or fewer amino acids, 30 or fewer amino acids, 25 or
fewer amino acids, 20 or fewer amino acids, or 15 or fewer amino
acids, and includes at least 4, at least 5, at least 6, at least 7,
at least 8, at least 9, at least 10, at least 15, at least 20, at
least 25, or between about 10 to about 30 contiguous or
non-contiguous amino acids of the CRD3 of OX40, e.g., an OX40
region at least 70%, at least 75%, at least 80%, at least 85%, and
least 90%, at least 95%, or at least 100% identical to amino acids
108 to 146 of SEQ ID NO: 91. In certain aspects, the peptide
retains a leucine at the position corresponding to amino acid 116
of SEQ ID NO: 91, and an alanine at the position corresponding to
amino acid 126 of SEQ ID NO: 91.
[0224] Such an isolated peptide can be used, e.g., for screening
libraries for binding molecules that specifically bind to OX40, or
as an immunogen to raise anti-OX40 antibodies in a subject
animal.
[0225] This disclosure employs, unless otherwise indicated,
conventional techniques of cell biology, cell culture, molecular
biology, transgenic biology, microbiology, recombinant DNA, and
immunology, which are within the skill of the art. Such techniques
are explained fully in the literature. See, for example, Sambrook
et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.;
Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992)
Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor
Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I
and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al.
U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic
Acid Hybridization; Hames and Higgins, eds. (1984) Transcription
And Translation; Freshney (1987) Culture Of Animal Cells (Alan R.
Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986);
Perbal (1984) A Practical Guide To Molecular Cloning; the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and
Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold
Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology,
Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical
Methods In Cell And Molecular Biology (Academic Press, London);
Weir and Blackwell, eds., (1986) Handbook Of Experimental
Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986);
and in Ausubel et al. (1989) Current Protocols in Molecular Biology
(John Wiley and Sons, Baltimore, Md.).
[0226] General principles of antibody engineering are set forth in
Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ.
Press). General principles of protein engineering are set forth in
Rickwood et al., eds. (1995) Protein Engineering, A Practical
Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General
principles of antibodies and antibody-hapten binding are set forth
in: Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer
Associates, Sunderland, Mass.); and Steward (1984) Antibodies,
Their Structure and Function (Chapman and Hall, New York, N.Y.).
Additionally, standard methods in immunology known in the art and
not specifically described are generally followed as in Current
Protocols in Immunology, John Wiley & Sons, New York; Stites et
al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton
& Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980)
Selected Methods in Cellular Immunology (W.H. Freeman and Co.,
NY).
[0227] Standard reference works setting forth general principles of
immunology include Current Protocols in Immunology, John Wiley
& Sons, New York; Klein (1982) J., Immunology: The Science of
Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et
al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension
in Biological Analyses (Plenum Press, NY); Campbell (1984)
"Monoclonal Antibody Technology" in Laboratory Techniques in
Biochemistry and Molecular Biology, ed. Burden et al., (Elsevier,
Amsterdam); Goldsby et al., eds. (2000) Kuby Immunology (4th ed.;
H. Freemand & Co.); Roitt et al. (2001) Immunology (6th ed.;
London: Mosby); Abbas et al. (2005) Cellular and Molecular
Immunology (5th ed.; Elsevier Health Sciences Division); Kontermann
and Dubel (2001) Antibody Engineering (Springer Verlag); Sambrook
and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003);
Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring
Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold
Spring Harbor Press).
[0228] All of the references cited above, as well as all references
cited herein, are incorporated herein by reference in their
entireties.
[0229] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
[0230] Abbreviations and definitions of terms are listed in Table
2.
TABLE-US-00002 TABLE 2 List of Abbreviations and Definitions of
Terms Abbreviation or Term Definition 1A7 Isotype control mouse
IgG1 kappa antibody 9B12 Mouse anti-human OX40 IgG1.kappa.
monoclonal antibody A375 human melanoma cell line Aa amino acid
ADCC Antibody-dependent cellular cytotoxicity ADPE Antibody
Development and Protein Engineering ANOVA Analysis of variance Asp
aspartic acid BSA Bovine serum albumin BLASTP protein sequence
homology Basic Local Alignment Search Tool C Final tumor volumes
from the control group CD cluster of differentiation CDC
Complement-dependent cytotoxicity CD4.sup.+, OX40.sup.+ CD4
positive, OX40 positive CDR complementarity-determining regions CFA
Complete Freund's adjuvant CFSE Carboxyfluorescein succinimidyl
ester CI Confidence Interval Clq Complement component Clq CR
Complete response CRD Cysteine rich domain Cyno cynomolgus DM-L
density media- lymphocyte E:T effector-to-target (ratio) EC
effective concentration ECf effective concentration resulting in f
% of maximal effect EC.sub.20 effective concentration resulting in
20% of maximal effect EC.sub.50 half maximal effective
concentration EC.sub.90 effective concentration resulting in 90% of
maximal effect F fraction of maximal FACS fluorescence activated
cell sorting FBS fetal bovine serum Fc Fragment, crystallizable
Fcer1g-/- Genetically engineered mouse strain; lacks expression of
activating Fc gamma receptors (Fc gamma I, III, and IV) Fcgr2b-/-
Genetically engineered mouse strain; lacks expression of inhibitory
Fc gamma IIb receptor FCS Flow cytometry standard Fc.gamma.
fragment, crystallizable gamma FMO Fluorescence minus-one FP Fusion
protein G Force of gravity H hour H.sup.+L heavy plus light chains
HEK293 Human embryonic kidney cell line Hr hour HSC Hematopoietic
stem cell Hu Human ICOS Inducible T-cell co-stimulator IgG
Immunoglobulin IgG1 Immunoglobulin G1 IL-2 interleukin 2 IP
intraperitoneal IU International Units K.sub.d equilibrium binding
dissociation constant KI knock-in KLH Keyhole limpet hemocyanin KO
knock-out K.sub.p equilibrium dissociation constant Leu Leucine M
Mouse mAb monoclonal antibody mL milliliter OX40mAb24 Humanized
anti-human OX40 IgG1.kappa. monoclonal antibody MFI mean
fluorescence intensity mOX40L FP Mouse OX40 ligand mouse IgG1
fusion protein mOX40L FP Mouse OX40 ligand mouse IgG1 fusion
(Y182A) protein engineered to have reduced binding to OX40
NF.kappa.B Nuclear factor kappa-light-chain- enhancer of activated
B cells NIP228 Mouse IgG1 kappa monoclonal antibody against
4-hydroxy-3-iodo-5-nitrophenylacetic acid NK natural killer
NOD/SCID non-obese diabetic/severe combined immunodeficient NSG
Mice with genetic background Nod.Cg-
Prkd.sup.cscid112rg.sup.tm/Wy/SzJ OX40L OX40 ligand OX40L FP human
OX40 ligand IgG4P fusion protein IgG4P Y180A engineered to have
reduced binding to OX40 OX40mAb24 Humanized anti-human OX40 IgG1
.kappa. monoclonal antibody OX86 Rat anti-mouse OX40 IgG1.kappa.
monoclonal antibody PBMC peripheral blood mononuclear cells PBS
Phosphate buffered saline PHA-L Phytohemagglutinin-Leucoagglutinin
PI Propidium iodide PK/PD Pharmacokinetic/pharmacodynamics RBC red
blood cell RBCL Red blood cell lysis RBD receptor binding domain Rh
recombinant human RLU Relative light units RPMI Roswell Park
Memorial Institute 1640 medium ROA Route of administration SC
subcutaneous SD Standard Deviation SEM Standard Error of the Mean
Ser serine T Final tumor volumes from the test group Tcm Central
Memory T cell TCR T cell receptor Teff Effector T cell Tem Effector
memory T cell TGI tumor growth inhibition TIL tumor infiltrating
leukocyte TM transmembrane domain TNFR tumor necrosis factor
receptor TNFRSF tumor necrosis factor receptor superfamily TRAF2
tumor necrosis factor receptor- associated factor 2 Treg regulatory
T cells .mu.L microliter .mu.g Microgram V volume
Example 1
Humanization of Anti-human OX40 Murine MAb 9B12
[0231] Murine mAb 9B12 was humanized by grafting its CDRs onto
selected human germline frameworks. The sequences of the heavy
chain variable region (VH) and the light chain variable region (VL)
of murine mAb 9B12 were compared with the human antibody germline
sequences available in the public NCBI databases. Human acceptor
frameworks (FR) were identified based on the highest sequence
homology. When selecting an optimal acceptor framework several
criteria were considered such as matching residues that could
impact binding (residues in the Vernier zone, canonical class
residues, and VH/VL interface residues), and the potential for
immunogenicity (low germline frequency). An optimal hybrid human
acceptor FR sequence was designed for VH and VL independently by
selecting the most homologous human immunoglobulin germline segment
for each individual framework region. Three different germline
acceptor sequences were combined to form the humanized VH framework
backbone, while two different germline acceptor sequences were
selected for VL. The fully human germline acceptor template chosen
for the VH chain (9B12VH-hu) was a combination of IGVH4-34*09 (FR
1), VH4-39 (FR 2), VH6-1(FR 3) and JH4 (FR 4). VL (9B12VL-hu1) was
a combination of O18 (FR 1), O18 (FR 2), L23 (FR 3) and JK1 (FR 4).
The framework homology between the murine sequence and human
template acceptor sequence was approximately 72% for VH and 77% for
VL.
[0232] Murine framework residues which could influence or maintain
the functional conformation of the parent CDR and did not match the
human germline sequence were identified and selectively
reintroduced into the human acceptor FR to best preserve the 9B12
binding affinity and functionality. In this case, mouse FR residues
27D, 39K, 47Y, 48M, 71R, 78Y and 91F in the VH chain and 44V and
68R in the VL chain were mutated back in the human template. CDR
residues as defined by Kabat were fused into the designed acceptor
frameworks for both VH and VL for generating the humanized
antibody. Two humanized VH genes (9B12VH-hu and 9B12VH-hu39K71R)
and two VL genes (9B12VL-hu1 and 9B12VL-hu2) were synthesized by
GeneART (Thermo Fisher Scientific, Waltham, Mass.), then cloned
into the in-house pOE IgG1 expression vector.
[0233] A panel of humanized variants has been generated and
characterized in T cell-based binding and proliferation assays. The
humanized variants, containing several framework amino acids
reverted to mouse FR residues in the heavy chain variable region,
bound to OX40 on activated CD4.sup.+ T cells with comparable
affinities and similar potencies (T cell proliferation) to 9B12.
The variable light chain paired with all humanized VH variants
encodes for fully humanized frameworks. To reduce the risk of
immunogenicity, the number of mouse residues in the VH human FR was
further reduced to 3 or 4 by replacing non-influential mouse
residues with the corresponding human residues. To remove potential
sequence liabilities, the NG deamidation site in VH-CDR2, the RYD
integrin binding site and the DG isomerization site in VH-CDR3 were
proactively removed either independently or in combination. To
avoid ADCC mediated by human IgG1 Fc effector function, IgG4P and
IgG1TM Fc variants were made for the humanized lead mAbs. The
resultant IgG4P and IgG1TM variants exhibited the same binding
activity to OX40 as the IgG1 variants, but significantly reduced
ADCC activity which is most similar to the murine mAb 9B12. In
summary, the humanized variants exhibited cell-binding affinities
and in vitro potencies comparable to the parent mouse mAb 9B12. The
amino acid differences among the humanized VH variant, all of which
are paired with the humanized VL, are summarized in FIG. 1.
[0234] Reverse chimera was engineered for in vivo characterization
in rhesus monkeys. Briefly, the VH and VL of humanized mAb24 were
grafted onto the constant heavy and constant light chains of murine
9B12. In vitro characterization demonstrated the Fab portions of
the reverse chimera bound to human OX40 comparable to mAb24.
Example 2
Binding Affinity and Receptor Occupancy of OX40mAb24 to Native OX40
Expressed on the Surface of Activated Human, Non-human Primate, Rat
and Mouse T cells
2.1 Materials
[0235] Materials used in this example are listed in Table 2-1.
TABLE-US-00003 TABLE 2-1 Materials Item Source AlexaFluor .RTM.
A488 goat anti-human Life Technologies, Carlsbad, IgG (H + L) CA
AlexaFluor .RTM. A488 goat anti-mouse Life Technologies, Carlsbad,
IgG (H + L) CA AlexaFluor .RTM. A488 goat anti-rat Life
Technologies, Carlsbad, IgG (H + L) CA Antibiotic/antimycotic
solution, Life Technologies, Carlsbad, 100X CA Anti-rat CD134 (OX40
clone) antibody Biolegend, San Jose, CA Anti-rat CD3 antibody BD
Biosciences, San Jose, CA Anti-mouse CD134 (OX86 clone) MedImmune,
Gaithersburg, MD antibody Anti-rat IgG1, .kappa. isotype Biolegend,
San Jose, CA control mAb clone RTK2071 Balb/C mouse Harlan,
Indianapolis, IN Beta mercaptoethanol (BME) Life Technologies,
Carlsbad, CA Concanavalin A Sigma, St. Louis, MO
Ethylenediaminetetraacetic acid Life Technologies, Carlsbad, (EDTA)
CA Heat inactivated newborn calf serum Life Technologies, Carlsbad,
(FBS) CA Hamster anti-mouse CD3 antibody BD Biosciences, San Jose,
CA Hamster anti-mouse CD28 antibody BD Biosciences, San Jose, CA
IL-2, recombinant human Preprotech, Rocky Hill, NJ Lymphocyte
separation medium (LSM) MP Biomedicals, Santa Ana, CA Magcellect
rat CD4 T cell isolation kit R&D Systems, Minneapolis, MN
Miltenyi MACS buffer Miltenyi San, Diego, CA Mouse CD4 T cell
isolation kit Miltenyi San, Diego, CA Mouse IgG1, .kappa. isotype
control Biolegend, San Jose, CA mAb clone MOPC-21 Non-human primate
CD4 T cell Miltenyi San, Diego, CA isolation kit Non-TC treated
round-bottom VWR, Radnor, PA 96 well plates Percoll Sigma, St.
Louis, MO Phytohemagglutinin- Roche Applied Science,
Leucoagglutinin (PHA-L) Indianapolis, IN Phosphate buffered saline,
pH 7.2 Life Technologies, (PBS) Carlsbad, CA RosetteSep CD4 T cell
Stem Cell Technologies, enrichment kit Vancouver, BC RPMI-1640 Life
Technologies, Carlsbad, CA Sprague Dawley rat Harlan, Indianapolis,
IN Whole blood, sodium heparin MedImmune Blood Donor
anti-coagulated Program, Gaithersburg, MD
[0236] In this example, cell-based equilibrium binding assays were
performed to measure the apparent affinity of OX40mAb24 binding to
OX40 expressed on the cell surface of human, non-human primate, rat
and mouse T cells. Additionally, the equilibrium binding assays
were utilized to determine the concentrations of OX40mAb24 that
achieve 20%, 50%, or 90% human OX40 receptor occupancy on activated
human CD4.sup.+ T cells.
[0237] OX40mAb24 concentrations required to achieve 20%, 50% or 90%
receptor occupancy were also determined for binding of 9B12, the
murine anti-human OX40 monoclonal antibody from which OX40mAb24 was
derived, to human and non-human primate OX40 expressed on the
surface of CD4.sup.+ T cells for a comparison of OX40mAb24 and 9B12
binding.
2.2 Assays
[0238] 2.2.1 Binding of OX40mAb24 to Primary Human CD4.sup.+ T
Cells and OX40-Expressing Jurkat T cells
[0239] The apparent equilibrium binding consent (K.sub.d) for
OX40mAb24 binding to human OX40 and the concentrations required to
bind 20%, 50%, or 90% of cell surface human OX40 receptor at
equilibrium were calculated from binding curves of OX40mAb24 to
OX40-expressing activated primary human CD4.sup.+ T cells or human
OX40-overexpressing Jurkat T cells. Similar experiments were
performed at the same time to assess binding of 9B12 to these cells
for comparison with OX40mAb24 values.
[0240] Primary human CD4.sup.+ T cells were first isolated from
sodium heparin anti-coagulated whole blood obtained from healthy
donors through the MedImmune Blood Donor Program using a RosetteSep
CD4.sup.+ T cell enrichment kit (Stem Cell Technologies, Vancouver,
BC) and a modified manufacturer's protocol.
[0241] Primary human CD4.sup.+ T cells were cultured for 48 h with
2 .mu.g/mL PHA-L and 20 IU/mL rhIL-2 to activate T cells and
up-regulate OX40. Activated T cells, which were >95% viable,
were subsequently used in OX40mAb24 binding experiments. All donors
represent unique individuals; that is, repeat binding experiments
were not performed with CD4.sup.+ T cells from the same donor.
[0242] Human OX40-overexpressing Jurkat NF.kappa.B-luciferase clone
64 cells were cultured in complete RPMI +10% FBS prior to binding
experiments, without the need for activation.
[0243] OX40mAb24 (10 .mu.g/mL) or 9B12 (10 .mu.g/mL) was diluted
over a 17 point 2-fold dilution series. OX40mAb24 was added to
100,000 cells (activated primary CD4.sup.+ T cells or human
OX40-overexpressing Jurkat NFkB-luciferase clone 64 cells) per well
and incubated for one hour at 4.degree. C. For background binding
subtraction, cells were incubated in the presence of secondary
antibody alone. The control R347 human IgG1 monoclonal antibody and
mouse IgG1 isotype clone MOPC-21 were used in experiments with OX40
over-expressing Jurkat T cells to demonstrate the specificity for
OX40 binding of OX40mAb24 or 9B12, respectively. Following
incubation, cells were washed three times with 200 .mu.L of cold
(4.degree. C.) FACS buffer and incubated with 100 .mu.L of FACS
buffer (PBS+2% heat-inactivated newborn calf serum) containing 10
.mu.g/mL AlexaFluor.RTM. 647 labeled goat anti-human IgG secondary
antibody (for binding to OX40mAb24) or 10 .mu.g/mL AlexaFluor.RTM.
488 labeled goat anti-mouse IgG secondary antibody (for binding to
9B12) and 5 .mu.g/mL propidium iodide (PI). Following secondary
antibody incubation, cells were washed and suspended in 100 .mu.L
FACS buffer for flow cytometry analysis on a BD LSRII flow
cytometer as described below.
2.2.2 OX40mAb24 Binding to Mouse CD4.sup.+ T Cells
[0244] OX40mAb24 was investigated for binding to mouse OX40
expressed on activated primary CD4.sup.+ T cells. Similar
experiments were performed at the same time to assess binding of
9B12 to these cells in comparison with OX40mAb24. Mouse CD4.sup.+ T
cells were isolated from harvested normal Balb/C mouse spleens
according to the following protocol:
[0245] Spleens were mashed against a 70 .mu.M nylon filter to
release splenocytes and the filter rinsed with 1 mL complete medium
(RPMI-1640 plus 10% FBS, 1% antibiotic/antimycotic solution and 55
.mu.M beta mercaptoethanol [BME]). Splenocytes were pelleted and
the supernatant discarded. The pellet was treated with 5 mL of IX
red blood cell (RBC) lysis buffer and incubated to lyse RBCs.
Osmolarity was restored by addition of complete medium at the end
of incubation time.
[0246] Cells were pelleted, washed in Miltenyi MACS buffer (PBS pH
7.2+0.5% bovine serum albumin (BSA)+2 mM ethylenediaminetetraacetic
acid [EDTA]) and the supernatant was discarded. The pellet was
suspended in cold MACS buffer and counted with a ViCell counter to
determine cell number and viability.
[0247] Mouse CD4.sup.+ T cell isolation was performed with a
Miltenyi process kit (San Diego, Calif.) according to
manufacturer's instructions, and isolated cells were suspended in
complete medium
[0248] Mouse CD4.sup.+ T cells (150,000 per well in 100 .mu.L
complete medium) were cultured overnight in 96-well plates coated
with 2 .mu.g/mL each hamster anti-mouse CD3 and hamster anti-mouse
CD28 antibodies to activate T cells and induce OX40 expression.
Activated CD4.sup.+ T cells were removed from the incubation plate
and 100,000 cells were transferred to each well of a non-tissue
culture-treated 96-well round-bottom plate for binding assays and
washed once with FACS buffer. Binding was performed with 10
.mu.g/mL of OX40mAb24, 9B12 and rat anti-mouse OX40, clone OX86
(positive control) antibodies each serially diluted 3-fold in FACS
buffer for a 10-point data curve. For negative controls, 10
.mu.g/mL R347 human IgG1, MOPC-21 mouse IgG1 or RTK2071 rat IgG1
were diluted 6-fold for a 3 point data curve. FACS buffer (50
.mu.L) containing OX40mAb24, or antibodies, was added to CD4.sup.+
T cells in duplicate and incubated. Following primary incubation,
cells were washed with 200 .mu.L of 4.degree. C. FACS buffer and
incubated with 50 .mu.L of FACS buffer containing 10 .mu.g/mL
AlexaFluor.RTM. 488 labeled goat anti-human secondary, 10 .mu.g/mL
AlexaFluor.RTM. 488 labeled goat anti-mouse secondary or
AlexaFluor.RTM. 488 labeled goat anti-rat secondary antibody and 5
.mu.g/mL PI. Following secondary antibody incubation, cells were
washed with 4.degree. C. FACS buffer (200 .mu.L per wash) and
suspended in 100 .mu.L FACS buffer for flow cytometry analysis on a
BD LSRII flow cytometer as described in Section 2.3.
2.2.3 OX40mAb24 Binding to Rat CD4.sup.+ T Cells
[0249] OX40mAb24 was investigated for binding to rat OX40 expressed
on activated primary CD4.sup.+ T cells. Similar experiments were
performed at the same time with 9B12 to allow comparison with
OX40mAb24. Rat CD4.sup.+ cells were isolated from freshly harvested
normal Sprague-Dawley rat spleens according to the protocol
described above for the isolation of mouse splenocytes, except that
rat CD4.sup.+ T cell isolation was performed with an R&D
Systems Magellect kit (Minneapolis, Minn.) according to
manufacturer's instructions.
[0250] Rat CD4.sup.+ T cells (1.times.10.sup.6 per mL complete
medium) were cultured overnight in a T75 cell culture flask with 1
.mu.g/mL concanavalin A (Con A) and 500 IU/mL IL-2, to activate T
cells and induce OX40 expression, and incubated overnight.
Activated CD4.sup.+ T cells were removed from the flask and 100,000
cells were transferred to each well of a non-tissue culture treated
96-well round-bottom plate for binding assays and washed with FACS
buffer. Binding was performed with 10 .mu.g/mL OX40mAb24, 9B12 or
mouse anti-rat CD134, clone OX40 (positive control) antibody
serially diluted 3-fold for a 10 point data curve in FACS buffer.
For negative controls, 10 .mu.g/mL R347 human IgG1 or mouse IgG1
clone MOPC-21 were serially diluted 6-fold for a 3 point data
curve. 100 .mu.L of OX40mAb24 control protein, 9B12 or clone OX40
antibody was added to CD4.sup.+ T cells in duplicate and incubated.
Following primary incubation, cells were washed with 200 .mu.L of
4.degree. C. FACS buffer per wash and incubated with 100 .mu.L of
FACS buffer containing 10 .mu.g/mL AlexaFluor.RTM. 488 labeled goat
and human, or AlexaFluor.RTM. 488 labeled goat anti-mouse secondary
antibody and 5 .mu.g/mL PI. Following secondary antibody
incubation, cells were processed for flow cytometry as described in
Section 2.3.
2.2.4 OX40mAb24 Binding to Cynomolgus Monkey CD4.sup.+ T Cells
[0251] OX40mAb24 was investigated for binding to cynomolgus monkey
(cyno) OX40 expressed on activated primary CD4.sup.+ T cells.
Similar experiments were performed at the same time with 9B12 to
allow comparison with OX40mAb24 values. Cyno CD4.sup.+ T cells were
isolated from sodium heparin anti-coagulated whole blood obtained
from healthy cyno donors (N=2) from World Wide Primates (Miami,
Fla.) according to the following protocol:
[0252] Whole blood was layered onto 30 mL of 60% Percoll in a 50 mL
conical centrifuge tube. Blood was centrifuged and peripheral blood
mononuclear cells (PBMC) were collected at the interface and washed
with cold (4.degree. C.) Miltenyi MACS buffer at 1200 RPM for 10
minutes. Supernatant was discarded and the pellet was treated with
5 mL of 1.times. RBC lysis buffer and incubated. Complete medium
(RPMI with 10% FBS and 1% antibiotics/antimycotics) was added to
the pellet to stop the lysis process at the end of incubation
time.
[0253] Cells were pelleted and washed with 20 mL of cold (4.degree.
C.) Miltenyi MACS buffer. The supernatant was discarded and the
cell pellet was suspended in cold (4.degree. C.) MACS buffer and
counted with a ViCell counter to determine cell number and
viability.
[0254] Cyno CD4.sup.+ T cell isolation was performed with a
Miltenyi non-human primate kit according to manufacturer's
instructions, then CD4.sup.+ T cells counted on a ViCell counter
and suspended at 1.times.10.sup.6 per mL in complete medium as
described above.
[0255] Cyno CD4.sup.- T cells (1.times.10.sup.6 per mL in complete
medium) were incubated for 48 hours in a T75 cell culture flask
with 2 .mu.g/mL PHA-L and 20 IU/mL IL-2, to activate T cells and
induce OX40 expression. Activated CD4.sup.+ T cells were removed
from the flask and 100,000 cells were transferred to each well of a
non-tissue culture treated 96-well round-bottom plate for binding
assays and washed with 200 .mu.L FACS buffer. Binding was performed
with 10 .mu.g/mL OX40mAb24 or 9B12 serially diluted 4-fold in FACS
buffer for an 11-point data curve, or both R347 human IgG1 or mouse
IgG1 clone MOPC-21 (negative controls) serially diluted 6-fold for
a 3 point data curve. OX40mAb24, 9B12 or control protein was added
to CD4.sup.+ T cells and incubated. Following primary incubation,
cells were washed with 200 .mu.L of cold (4.degree. C.) FACS buffer
per wash and incubated with 100 .mu.L of FACS buffer containing 10
.mu.g/mL AlexaFluor.RTM. 488 labeled goat anti-human secondary
antibody or 10 .mu.g/mL AlexaFluor.RTM. 488 labeled goat anti-mouse
secondary and 5 .mu.g/mL PI. Following secondary antibody
incubation, cells were processed for flow cytometry as described in
Section 2.3.
2.2.5 OX40mAb24 Binding to Rhesus Monkey CD4.sup.+ T Cells
[0256] OX40mAb24 was investigated for binding to rhesus macaque
OX40 expressed on activated primary CD4.sup.+ T cells. Similar
experiments were performed at the same time with 9B12 to allow
comparison with OX40mAb24 values. Rhesus CD4.sup.+ T cells were
isolated from sodium heparin anti-coagulated whole blood obtained
from healthy rhesus donors (N=2) from World Wide Primates (Miami,
Fla.) according to the following protocol:
[0257] Heparinized rhesus blood (20 mL) was diluted 1:1 with PBS
and layered onto 15 ml of 95% LSM in a 50 ml conical centrifuge
tube. Blood was centrifuged, PBMC were collected at the interface
and washed twice with cold Miltenyi MACS buffer at 400.times.g for
30 minutes. Supernatant was discarded and the pellet was treated
with 5 mL of IX RBC lysis buffer and incubated. Complete medium
(RPMI with 10% FBS and 1% antibiotics/antimycotics) was added to
the pellet to stop the lysis process at the end of incubation time.
Cells were pelleted and washed with 20 mL of cold Miltenyi MACS
buffer. Supernatant was discarded and the pellet was suspended in
cold MACS buffer and counted with a ViCell counter to determine
cell number and viability. Rhesus CD4.sup.+ T cell isolation was
performed with a Miltenyi non-human primate kit (San Diego, Calif.)
according to manufacturer's instructions.
[0258] Rhesus CD4.sup.+ T cells (1.times.10.sup.6 per mL in
complete medium) were cultured for 48 hours in a T75 cell culture
flask with 5 .mu.g/mL Con-A and 1000 IU/mL IL-2 to activate T cells
and induce OX40 expression. Binding to 100,000 activated rhesus
CD4.sup.+ T cells was performed with 10 .mu.g/mL OX40mAb24 and 9B12
serially diluted 3-fold in FACS buffer for a 10 point (experiment
1) or 12 point data curve (experiment 2) and 10 .mu.g/mL human and
mouse isotype controls diluted 6-fold for 2 data points (experiment
1) or 4 data points (experiment 2). AlexaFluor.RTM. 488-labeled
goat anti-human secondary antibody and AlexaFluor.RTM. 488 labeled
goat anti-mouse secondary binding were as described above for cyno
CD4.sup.+ T cells, and flow cytometry as described in Section
2.3.
2.3 Flow Cytometry
[0259] Flow cytometry in the assays described in Section 2.1 was
performed using an LSRII flow cytometer (Becton-Dickinson, San
Jose, Calif.). Flow Jo cytometry analysis software (TreeStar,
Ashland, Oreg.) was used to determine OX40mAb24, 9B12 and control
protein binding to cells. Wells containing OX40-expressing cells
(unstained, no PI or secondary antibody), cells bound to
AlexaFluor.RTM. 488- or AlexaFluor.RTM. 647-labeled secondary
antibody reagent only or cells permeabilized with 0.1% saponin and
treated with 10 .mu.g/mL PI were prepared for single-stain
compensation controls. After fluorescence compensation, live (PI
negative) cells were gated and the mean fluorescence intensity
(MFI) of secondary antibody was determined.
2.4 Calculations
2.4.1 Determination of Apparent Equilibrium Dissociation Constant
(K.sub.d)
[0260] GraphPad Prism version 5.01 for Windows, GraphPad Software,
San Diego Calif. USA, www.graphpad.com, was used to plot MFI of
OX40mAb24 binding versus protein concentration (M) to create
binding curves from which apparent K.sub.d was determined. To
determine the apparent K.sub.d for OX40mAb24 and 9B12 binding to
human, mouse, rat, cyno, or rhesus monkey OX40, a non-linear
regression (curve fit) equation for one site (specific) binding was
employed.
2.4.2 Determination of 20%, 50%, and 90% Receptor Occupancy
Values
[0261] The amount of a monoclonal antibody (mAb) bound to its
receptor can be estimated from the following binding
relationship:
Receptor (A)+mAb (B)receptor-mAb complex (AB) Equation 1
[0262] The binding dissociation constant (K.sub.d) of the
respective antibody is represented by:
Kd = [ A ] [ B ] [ AB ] Equation 2 ##EQU00001##
Where [A] and [B] represent the concentrations of free receptor and
antibody, respectively. Finally, the fractional occupancy, fraction
(F) of all receptor molecules that are bound to the antibody, can
be calculated by:
F = [ AB ] [ A ] + [ B ] Equation 3 ##EQU00002##
Formation of equation 2 and substitution into equation 3 results
in:
F = [ A ] [ B ] / Kd [ A ] + ( [ A ] [ B ] / Kd ) Equation 4
##EQU00003##
Simplification of equation 4 results in:
F = [ B ] [ B ] + Kd Equation 5 ##EQU00004##
Therefore, at equilibrium condition, the fraction of all receptor
molecules that are bound to the antibody can be calculated if the
concentration of free mAb and the dissociation constant K.sub.d of
the respective antibody are known.
[0263] Derivation of this formula for the calculation of the
concentration of antibody required for a fractional receptor
occupancy expressed as F, leads to the following formula:
[B]=F*Kd/(1-F) Equation 6
Where [B] equals the concentration of mAb, in this case OX40mAb24.
This formula (Equation 6) was used for the calculation of the
concentrations of OX40mAb24 required for 20, 50, and 90% receptor
occupancy (F=0.20, 0.50 or 0.90) from cell binding experiments from
which the K.sub.d value was calculated using the non-linear
regression (curve fit) equation for one site (specific) binding
described above.
2.5 Statistical Methods
[0264] A 2-sided unpaired Student's t test with 95% confidence
level and Welch's correction to account for data sets with
different standard deviations was utilized in Graphpad Prism
software to determine statistically significant differences between
apparent K.sub.d values or apparent receptor occupancy values
determined for OX40mAb24 binding to OX40 on activated primary human
CD4 T cells versus OX40-expressing Jurkat T cells. Descriptive
statistics (i.e., mean and standard error of the mean) are
presented in the summary figures and tables.
2.6 Results
2.6.1 Binding of OX40mAb24 to Primary Human CD4.sup.+ T Cells
[0265] OX40mAb24 bound to activated human CD4.sup.+ T cells with a
mean apparent K.sub.d of 312 pM and 20%, 50% and 90% receptor
occupancy values of 78.1, 312, and 2810 pM, respectively; n=6
binding assays with six independent T cell donors (FIGS. 2A and 2B,
and Table 2-2).
[0266] In comparison, 9B12 bound to activated human CD4.sup.+ T
cells with a mean K.sub.d of 669 pM, and 20%, 50% and 90% receptor
occupancy values of 167, 669, and 6020 pM, respectively (FIGS. 2C
and 2D and Table 2-2). The ratio between 9B12 and OX40mAb24
apparent K.sub.d values was therefore 2.1 to 1, and reflects a
similar binding affinity to human OX40 for the murine and humanized
monoclonal antibodies
TABLE-US-00004 TABLE 2-2 Apparent Affinity (K.sub.d) and Receptor
Occupancy Values for Binding of OX40mAb24 or 9B12 to
OX40-expressing Activated Primary Human CD4.sup.+ T cells Binding N
K.sub.d EC.sub.20 EC.sub.50 EC.sub.90 Protein donors (StdErr), pM
(StdErr), pM (StdErr), pM (StdErr), pM OX40mAb24 6 312 (57.9) 78.1
(14.5) 312 (57.9) 2810 (521) 9B12 6 669 (137) 167 (34.3) 669 (137)
6020 (1230) EC.sub.20 = effective concentration resulting in 20% of
maximal effect; EC.sub.50 = half maximal effective concentration;
EC.sub.90 = effective concentration resulting in 90% of maximal
effect; K.sub.d = equilibrium binding dissociation constant; StdErr
= standard error of the mean.
2.6.2 Binding of OX40mAb24 to a Jurkat T Cell Line Engineered to
Over-Express Human OX40
[0267] OX40mAb24 bound to OX40-expressing Jurkat T cells with a
mean K.sub.d of 424 pM and 20%, 50% and 90% receptor occupancy
values of 106, 424, and 3820 pM, respectively. FIG. 3A-C and Table
2-3. There were no statistically significant differences in
apparent binding affinity or receptor occupancy of OX40mAb24 to
OX40 expressed by activated primary human CD4.sup.+ T cells and
OX40 over-expressing Jurkat T cells (p=0.59).
[0268] In comparison, 9B12 bound these cells with a mean K.sub.d of
726 pM and 20%, 50% and 90% receptor occupancy values of 182, 726,
and 6540 pM, respectively (FIGS. 3D-F and Table 2-3). The ratio
between the apparent K.sub.d values for 9B12 and OX40mAb24 was
therefore 1.7 to 1, similar to the ratio calculated for binding to
OX40 on activated human CD4.sup.+ T cells.
TABLE-US-00005 TABLE 2-3 Apparent Affinity (K.sub.d) and Receptor
Occupancy Values for Binding of OX40mAb24 or 9B12 to Human
OX40-overexpressing Jurkat NFkB-luciferase Clone 64 cells Binding N
K.sub.d EC.sub.20 EC.sub.50 EC.sub.90 Protein experiments (StdErr),
pM (StdErr), pM (StdErr), pM (StdErr), pM OX40mAb24 3 424 (173) 106
(43.3) 424 (173) 3820 (1560) 9B12 3 726 (308) 182 (76.9) 726 (308)
6540 (2770) EC.sub.20 = effective concentration resulting in 20% of
maximal effect; EC.sub.50 = half maximal effective concentration;
EC.sub.90 = effective concentration resulting in 90% of maximal
effect; K.sub.d = equilibrium binding dissociation constant; StdErr
= standard error of the mean.
2.6.3 Binding of OX40mAb24 to Mouse or Rat CD4.sup.+ T Cells
[0269] Neither OX40mAb24 nor 9B12 bound to activated mouse or rat
CD4.sup.+ T cells (data not shown). Positive staining of activated
mouse and rat CD4.sup.+ T cells was observed with commercial
anti-mouse and anti-rat OX40 antibodies, clones OX86 and OX40,
respectively. 9B12 did not bind activated mouse nor rat CD4.sup.+ T
cells (data not shown).
2.6.4 Binding of OX40mAb24 to Cynomolgus and Rhesus Monkey
CD4.sup.+ T Cells
[0270] OX40mAb24 bound to activated cynomolgus cells with a mean
K.sub.d of 581 pM. The cyno K.sub.d was 1.9-fold higher than the
human K.sub.d (Table 2-4).
[0271] 9B12 bound the activated cyno CD4.sup.+ T cells with a mean
K.sub.d of 1088 pM (Table 2-4), which resulted in a ratio between
9B12 and OX40mAb24 apparent K.sub.d values of 1.9 to 1.
[0272] OX40mAb24 bound to activated rhesus monkey CD4.sup.+ T cells
with a mean K.sub.d of 369 pM (Table 2-4). The rhesus K.sub.d was
1.2-fold higher than the human K.sub.d.
[0273] 9B12 bound the activated rhesus monkey CD4.sup.+ T cells
with a mean K.sub.d of 713 pM (Table 2-4), which results in a ratio
between 9B12 and OX40mAb24 apparent K.sub.d values not shown of 2.8
to 1.
TABLE-US-00006 TABLE 2-4 Apparent Affinity (K.sub.d) for Binding of
OX40mAb24 or 9B12 to Cynomolgus and Rhesus Monkey OX40-expressing
Activated Primary CD4.sup.+ T cells Cynomolgus Rhesus Binding N
K.sub.d N K.sub.d Protein experiments (StdErr), pM experiments
(StdErr), pM OX40mAb24 2 581 (238) 2 369 (236) 9B12 2 1088 (37) 2
713 (559) K.sub.d = equilibrium binding dissociation constant;
StdErr = standard error of the mean.
Example 3
Binding Specificity of OX40mAb24 for Human OX40
[0274] In this example, flow cytometry-based cell binding assays
were performed to determine the specificity of OX40mAb24 for human
OX40, relative to other human TNFRSF members with related amino
acid sequences, which included: NGFR (TNFRSF16), LT.beta.R
(TNFRSF3), TNFR2 (TNFRSF1.beta.), GITR (TNFRSF18), CD137 (TNFRSF9),
and HVEM (TNFRSF14). In addition, the binding specificity of
OX40mAb24 for recombinant human TNFRSF members was tested in an
ELISA format, which included those mentioned above as well as DR6
(TNFRSF21), osteoprotegerin (OPG; TNFRSF11B), RANK (TNFRSF11A), FAS
(TNFRSF6), and CD40 (TNFRSF 5).
3.1 Materials
[0275] Materials used in this study are listed in Table 3-1.
TABLE-US-00007 TABLE 3-1 Materials Item Source AlexaFluor .RTM. 647
goat anti-human Life Technologies, Carlsbad, CA IgG (H + L) BioTek
.RTM. plate washer BioTek .RTM., Wincoski, VT CD137 (TNFRSF9)
pCMV6-XL5 Origene Technologies, Inc., expression vector Rockville,
MD GITR (TNFRSF18) pCMV6-XL5 Origene Technologies, Inc., expression
vector Rockville, MD Goat anti-human IgG kappa light Sigma-Aldrich,
St. Louis, MO chain HRP conjugate Goat-antimouse IgG (Fab specific)
Sigma-Aldrich, St. Louis, MO HRP conjugate HVEN (TNFRSF14)
pCMV6-XL4 Origene Technologies, Inc., expression vector Rockville,
MD Lipofectamine 2000 Life Technologies, Carlsbad, CA AF488
anti-mouse IgG1 isotype Biolegend, San Diego, CA AF488 anti-human
GITR Ebioscience, San Diego, CA AF647 anti-human NGFR BD, San Jose,
CA APC anti-human CD137 BD, San Jose, CA APC anti-mouse IgG1
isotype Biolegend, San Diego, CA Clone Manager v9 software Sci-Ed
Software, Cary, NC Fetal bovine serum (FBS), Life Technologies,
Carlsbad, CA heat inactivated LT.beta.R (TNFRSF3) pCMV6-XI.4
Origene Technologies, Inc., expression vector Rockville, MD
MaxiSorp 96 well flat bottom VWR, Radnor, PA plate Newborn calf
serum, heat Life Technologies, Carlsbad, CA inactivated NGFR
(TNFRSF16) pCMV6-XL5 Origene Technologies, Inc., expression vector
Rockville, MD PE anti-human HVEM Ebioscience, San Diego, CA PE
anti-human Lt.beta.R R&D systems, Minneapolis, MN PE anti-human
TNFRSF 1B BD, San Jose, CA PE anti-mouse IgG1 isotype Biolegend,
San Diego, CA PE anti-rat isotype Ebioscience, San Diego, CA PBS,
pH 7.2 without calcium Life Technologies, Carlsbad, CA and
magnesium Recombinant human TNFRSF1.beta. R&D systems,
Minneapolis, MN (TNFRII) Recombinant human TNFRSF3 R&D systems,
Minneapolis, MN (LT.beta.R) Recombinant human TNFRSF4 R&D
systems, Minneapolis, MN (OX40) Recombinant human TNFRSF5 R&D
systems, Minneapolis, MN (CD40) Recombinant human TNFRSF6 R&D
systems, Minneapolis, MN (FAS) Recombinant human TNFRSF9 In house
protein; lot# AMPur19 (CD137) Recombinant human TNFRSF11A R&D
systems, Minneapolis, MN (RANK) Recombinant human TNFRSF11B R&D
systems, Minneapolis, MN (OPG) Recombinant human TNFRSF14 R&D
systems, Minneapolis, MN (HVEM) Recombinant human TNFRSF16 R&D
systems, Minneapolis, MN (NGFR) Recombinant human TNFRSF18 In house
protein; lot# LBPur0025 (GITR) Recombinant human TNFRSF21 R&D
systems, Minneapolis, MN (DR6) TNFRSF1.beta. pCMV6-XL5 Origene
Technologies, Inc., expression vector Rockville, MD Tween-20
Sigma-Aldrich, St. Louis, MO
3.2 Methods
[0276] 3.2.1 Search for Proteins with Close Sequence Homology to
Human OX40
[0277] In order to identify human proteins with amino acid sequence
identity to human OX40, a protein sequence homology Basic Local
Alignment Search Tool (BLASTP) search was conducted using the
protein sequence of OX40 (CCDS 11/UniProt P43489). Nineteen TNFRSF
family members/isoforms were identified. The full-length sequences
of these proteins were verified using both the CCDS and UniProt
databases (www.uniprot.org). Clone Manager version 9 software was
used to perform an assembled alignment against the human OX40
reference using a blosum62 scoring matrix to determine the
percentage of amino acid identity between human OX40 and the
proteins identified in the BLASTP search (Table 3-2).
3.2.2 Binding Specificity of OX40mAb24 for OX40 Relative to Other
TNFRSF Members Expressed in HEK293 Cells
[0278] cDNA constructs capable of directing the expression of
individual TNFRSF members, when transfected into mammalian cells,
were obtained from Origene Technologies, Rockville, Md. These cDNA
constructs were amplified and purified by the Protein Sciences
group at MedImmune in Gaithersburg, Md. for use in transient
transfections. For individual expression of each of the TNFRSF
members, HEK293 cells were transfected using Lipofectamine 2000
(Life Technologies, Carlsbad, Calif.) combined with 0.5 .mu.g DNA
of an expression vector encoding one of the TNFRSF members,
according to the manufacturer's suggested protocol for
Lipofectamine 2000. Forty-eight hours post transfection, cells were
removed from tissue culture plates by trypsinization. Trypsin was
neutralized by the addition of serum-containing complete medium
RPMI-1640 plus 10% FBS), followed by cell pelleting and washes in
complete medium. Cells were then suspended in cold FACS buffer
(PBS+2% FBS) and plated into 96 well non-tissue culture-treated
plates for binding studies with TNFRSF member-specific mAbs (Table
3-3) and OX40mAb24.
[0279] For binding of antibodies or OX40mAb24, HEK cells were
pelleted, FACS buffer removed, and cells suspended in FACS buffer
containing 2 .mu.g/mL propidium iodide (PI) and either a
fluorochrome-labeled mAb, specific for the transfected TNFRSF
member, at a concentration recommended by the manufacturer or with
OX40mAb24 at a concentration of 1 .mu.g/mL. For binding controls,
cells were separately incubated with fluorochrome-labeled isotype
control antibodies. Cells were incubated with antibodies for 1 hour
at 4.degree. C. in the dark. Thereafter, cells incubated with
fluorochrome-labeled monoclonal antibodies were washed in cold FACS
buffer and then binding events collected and analyzed by flow
cytometry using an LSRII flow cytometer (BD Biosciences, San Jose,
Calif.) and FlowJo software, as described in Section 3.2.3. Cells
incubated with OX40mAb24 were washed in ice cold FACS buffer and
then suspended 25 .mu.g/mL of Alexa Fluor.RTM. 647 goat antihuman
IgG (H+L) secondary antibody and incubated for a further 30 minutes
at 4.degree. C. in the dark. For a secondary antibody binding
control cells were incubated in the absence of OX40mAb24, but in
the presence of fluorochrome-labeled secondary antibody alone.
Thereafter, cells were washed and suspended in cold FACS buffer for
analysis on an LSRII flow cytometer.
3.2.3 Flow Cytometry Analysis
[0280] Flow cytometry standard (FCS) data was examined using FlowJo
software (Ashland, Oreg.). To analyze mAb binding, cells were first
gated for viable (PI negative) cells, and then the mean
fluorescence intensity (MFI) of events was plotted versus total
number of events to generate binding histograms. The geometric MFI
of all viable cells was determined for each sample so that the fold
MFI over background (isotype control or secondary antibody alone)
could be determined.
3.2.4 OX40mAb24 Binding Specificity ELISA
[0281] An eight point, two-fold dilution series of each recombinant
human TNFRSF protein was prepared after dilution of stock proteins
to 5 .mu.g/mL in PBS. Subsequently, 50 .mu.L of each antigen
dilution was transferred in duplicate to wells of a Nunc 96-well
MaxiSorp flat bottom plate, and incubated overnight at 4.degree. C.
to adsorb proteins to the plate. Afterwards, plates were washed
three times with PBS in a BioTek.RTM. plate washer to remove
unbound protein. Anti OX40 mAb29, 9B12, and control antibodies were
diluted in PBS to a final concentration of 10 .mu.g/mL, and 50
.mu.L of mAb were added to each well and incubated at room
temperature for one hour to bind mAb to plate-bound protein.
Thereafter, wells were washed with PBS/Tween-20 0.1%
(volume/volume) using a BioTek.RTM. plate washer. HRP conjugated
goat anti-human or goat anti-mouse secondary antibodies at 10
.mu.g/mL was added to each well and incubated at room temperature
for 1 hour. After three washes in PBS/Tween-20 0.1%, 50 .mu.L of
TMB substrate was added to each well and incubated at room
temperature for 5 minutes to develop the colorimetric product.
Reactions were stopped by adding 50 .mu.L of 0.5 molar
H.sub.2SO.sub.4 to wells, and plates read immediately at 450 nm
using an Envision C plate reader for detection of the colorimetric
product. Results were graphed in GraphPad Prism software for
Windows, version 5.01, and binding curves generated using
non-linear regression analysis for single site binding.
3.3 Results
[0282] The protein sequence homology BLASTP search on human OX40
identified 19 human TNFRSF proteins or isoforms that shared 15-27%
amino acid sequence identity with the full-length OX40 sequence.
The proteins and their percentages of sequence identity are listed
in Table 3-2.
TABLE-US-00008 TABLE 3-2 Amino Acid Sequence Identity of Twelve
TNFRSF Members with the Highest Homology to Human OX40. Alternate %
identity UniProt protein TNFRSF member names with OX40 sequence ID
TNFRSF11A RANK, CD265 27 Q9Y6Q6 iso 2 (delta 7, 8, 9) TNFRSF6B DcR3
25 O95407 TNFRSF18 GITR, AITR 25 Q9Y5U5 iso 2 TNFRSSF10C DCR1,
TRAIL-R3 24 O14798 TNFRSF5 CD40 23 P25942 iso 2 TNFRSF18 GITR, AITR
23 Q9Y5U5 iso 3 TNFRSF18 GITR, AITR 23 Q9Y5U5 TNFRSF9 CD137, 4-1BB
22 Q07011 TNFRSF5 CD40 21 P25942 TNFRSF14 TR2, HVEM-A 21 Q92956
TNFRSF16 NGF receptor 21 P08138 TNFRSF3 LT.beta.R TNFRIII 20 P36941
TNFRSF6 Fas 20 P25445 iso 6 Tmdcl (A) TNFRSF6 Fas 20 P25445 TNFRSF3
LT.beta.R TNFRIII 19 P36941 iso 2 TNFRSF6 Fas 19 P25445 iso 7
FasExo8Del TNFRSF11B Osteoprotegerin 18 O0030 TNFRSF1B TNFR1b,
TNFR2, 18 P20333 CD120b TNFRSF11A RANK, CD265 16 Q9Y6Q6 CCDS =
consensus coding sequence; ID = identifier; NA = not applicable;
TNFRSF = tumor necrosis factor receptor superfamily;
UniProt--Universal Protein Resource
[0283] Binding of OX40mAb24 to transiently transfected HEK293 cells
that expressed human NGFR, LT.beta.R, TNFR2, GITR, CD137 or HVEM
was assessed by flow cytometry as described in Section 3.1.3 above.
Cell surface expression of human NGFR, LT.beta.R, TNFR2, GITR,
CD137 and HVEM was confirmed using commercially available
antibodies specific for each human TNFRSF protein; fold increase in
MFI compared to isotype control antibodies for each TNFRSF protein
is shown in Table 3-3. Binding of OX40mAb24 to HEK293 cells that
expressed human NGFR, LT.beta.R, TNFR2, GITR, CD137 or HVEM was not
substantially above that seen for binding of the secondary antibody
alone to those same cells (Table 3-3, FIG. 4A). In contrast,
binding of OX40mAb24 to a Jurkat cell line that constitutively
overexpresses OX40 was 48-fold greater, by mean MFI, than the
binding of fluorochrome labeled secondary antibody alone (Table 3-3
and FIG. 4B).
TABLE-US-00009 TABLE 3-3 Fold Binding of Fluorochrome-labeled
TNFRSF-specific Monoclonal Antibodies and OX40mAb24 to
TNFRSF-overexpressing HEK293 Cells or OX40mAb24 to
OX40-overexpressing Jurkat Cells. Receptor-Specific OX40mAb24
binding TNFRSF member Cell Line Transferred Commercial mAb binding
(ratio to secondary expressed with TNFRSF Member (ratio to isotype
control MFI) antibody alone MFI) OX40 Jurkat ND 48 TNFRSF16 (NGFR)
HEK293 17 2.2 TNFRSF3 (LT.beta.R) HEK293 146 1.0 TNFRSF1.beta.
HEK293 5.0 1.0 TNFRSF18 (GITR) HEK293 37 1.1 TNFRSF9 (CD137) HEK293
24 1.2 TNFRSF14 (HVEM) HEK293 69 1.1
mAb=monoclonal antibody; MFI=mean fluorescence intensity; ND=not
determined; TNFRSF=tumor necrosis factor receptor superfamily
[0284] In an ELISA format, binding of an antibody containing the
Fab arms of OX40mAb24 but with IgG1 Fc domain containing three
amino acid modifications (mAb29) was specific for OX40 (FIG. 5A),
showing no specific binding above background to recombinant human
NGFR (TNFRSF16), LT.beta.R (TNFRSF3), TNFR2 (TNFRSF1.beta.), GITR
(TNFRSF18), CD137 (TNFRSF9), HVEM (TNFRSF14), DR6 (TNFRSF21),
osteoprotegerin (OPG; TNFRSF11B), RANK (TNFRSF11A), FAS (TNFRSF6),
and CD40 (TNFRSF5). 9B12, the mouse anti-human OX40 IgG1 monoclonal
antibody that was "humanized" to create OX40mAb24, demonstrated
similar lack of binding to these TNFRSF proteins (FIG. 5B).
3.4: Conclusions
[0285] Binding of OX40mAb24 and 9B12 to human OX40 is specific, and
do not cross-react with highly related TNFRSF proteins.
Example 4
Ability of OX40mAb24 to Co-Stimulate Primary Human CD4.sup.+ T
Cells through OX40 In Vitro
[0286] In this example, the ability of OX40mAb24 to enhance
activation of T cells, in combination with activation through the
CD3/T cell receptor (TCR) complex, was assessed using a plate-based
human CD4.sup.+ T cell proliferation and cytokine release assay.
Soluble OX40mAb24 activity was also examined, as were the activity
of soluble and plate-bound OX40mAb24 in the absence of CD3/TCR
signaling.
4.1 Materials
[0287] Materials used in this study are listed in Table 4-1.
TABLE-US-00010 TABLE 4-1 Materials Item Source AlexaFluor .RTM. 647
goat anti-human IgG (H + L) Life Technologies, Carlsbad, CA
Anti-human CD4 EFluor450 .RTM. eBioscience, San Diego, CA Bovine
serum albumin (BSA) Sigma, Saint Louis, MO CFSE cell labeling kit
Life Technologies, Carlsbad, CA Complete RPMI medium: RPMI-1640 +
10% FBS Materials from Life Technologies, Carlsbad, CA Deep well
plates, polypropylene, 2 mL VWR, Radnor, PA EasySep CD4.sup.+ T
cell enrichment kit Stem Cell Technologies, Vancouver, BC Canada
FlowJo software TreeStar, Ashland, OR Goat anti-human IgG,
Fc.gamma.-specific Jackson ImmunoResearch, West Grove, PA Goat
anti-mouse IgG, Fc.gamma.-specific Jackson ImmunoResearch, West
Grove, PA Heat inactivated newborn calf serum Life Technologies,
Carlsbad, CA IL-2, recombinant human Preprotech, Rocky Hill, NJ
Leuko Pak AllCells, Alameda, CA LSM MP Biomedicals, Santa Ana, CA
LSR II flow cytometer BD Biosciences, San Jose, CA Mouse anti-human
CD3 antibody clone OKT3 Biolegend, San Diego, CA Newborn calf
serum, beat inactivated (FBS) Life Technologies, Carlsbad, CA
Non-issue culture treated round-bottom 96 well plates VWR, Radnor,
PA PHA-L Roche Applied Science, Indianapolis, IN Phosphate Buffered
Saline (PBS) pH 7.2 without Life Technologies, Carlsbad, CA Calcium
and Magnesium Prism software, v 5.01 Graphpad Software, San Diego,
CA Propidium iodide (1 mg/mL solution) Sigma, Saint Louis, MO
RosetteSep CD4.sup.+ T cell enrichment kit StemCell Technologies,
Vancouver, BC Canada RPMI-1640 Life Technologies, Carlsbad, CA
Th1/Th2 multi-cytokine detection array Mesoscale Discovery (MSD),
Rockville, MD Vi-Cell counter Beckman Coulter, Indianapolis, IN
Whole blood, sodium heparin anti-coagulated MedImmune Blood Donor
Program, Gaithersburg, MD
4.2 Assays
4.2.1 Plate-Immobilized Bioactivity of OX40mAb24
[0288] The bioactivity of OX40mAb24 was determined by measurement
of human CD4.sup.+ T cell proliferation and cytokine production in
a plate-based drug capture assay (FIG. 6).
[0289] Enriched human CD4.sup.+ T cells were isolated from healthy
donor whole blood using a RosetteSep CD4.sup.+ T cell enrichment
kit, according to the manufacturer's protocol. Assays were
performed with cells from four independent donors.
[0290] CD4.sup.+ T cells were suspended in complete RPMI culture
medium and the cell concentration adjusted to 1.0.times.10.sup.6
per mL. Final concentrations of 2 .mu.g/mL
phytohemagglutinin-leucoagglutinin (PHA-L) and 20 IU/mL recombinant
human IL-2 were added, and cells were cultured at 37.degree. C. and
5% CO.sub.2 in a humidified tissue culture incubator for 2 days to
activate T cells and up-regulate OX40.
[0291] Non-tissue culture treated round-bottom 96 well assay plates
were coated with 100 .mu.L of 2 .mu.g/mL goat anti-mouse
Fc.gamma.-specific IgG and 2 .mu.g/mL goat anti-human
Fc.gamma.-specific IgG in PBS. Goat anti-human IgG capture
antibodies were not added to wells intended for assay of soluble
OX40mAb24 activity. Plates were incubated overnight at 4.degree.
C., washed with 200 .mu.L of PBS, and blocked for 90 minutes at
37.degree. C. with 1% BSA in PBS (1% BSA/PBS). The plates were
washed with PBS and 2 ng/mL of mouse anti-human CD3 clone OKT3
reconstituted in 1% BSA/PBS was added to the plates for 90 minutes
at 37.degree. C. The plates were washed with PBS to remove unbound
OKT3, OX40mAb24, R347 human IgG1 control mAb, 9B12, and mouse IgG1
control mAb clone MOPC-21 were each reconstituted in 1% BSA/PBS
starting at 0.918 .mu.g/mL (3.0 nM) and serially diluted over a
3-fold dilution series and then added to assay plates and incubated
for 90 minutes at 37.degree. C.
[0292] Activated primary human CD4.sup.+ T cells were collected,
washed in complete RPMI medium, and the concentration adjusted to
1.0.times.10.sup.6 viable cells/mL. Cells were labelled with
carboxyfluorescein succinimidyl ester (CFSE), according to the
manufacturer's instructions, with the exception of using 1.25 .mu.M
CFSE as opposed to the recommended 5 .mu.M with an incubation of 10
minutes at 37.degree. C. After labeling, cells were suspended in
complete RPMI medium and the concentration was adjusted to
0.5.times.10.sup.6 per mL. The plates were washed with PBS and 200
.mu.L of CD4.sup.+ T cells (100,000/well) were added to each well.
For wells containing soluble OX40mAb24, OX40mAb24 was diluted in
complete RPMI medium to the highest final concentrations used for
plate-bound OX40mAb24. Cells in the plate were pelleted by
centrifugation at 380.times.g and incubated at 37.degree. C. for 3
days. After 72 hours incubation time, 40 .mu.L of cell culture
supernatant was removed for cytokine release measurement. CD4.sup.+
T cells were pelleted, and washed once with PBS containing 2% FBS
(FACS buffer). Cells were suspended in binding mix containing
anti-human CD4 eFluor450.RTM. labeled antibody for identification
of CD4.sup.+ T cells, and propidium iodide (PI) for live/non-viable
cell discrimination, and incubated for 30 minutes. Following
incubation, cells were washed in FACS buffer, re-suspended in FACS
buffer and analyzed by flow cytometry using an LSRII flow cytometer
and FlowJo software for analysis of Flow Cytometry Standard (FCS)
formatted data.
[0293] To analyze T cell proliferation, live (PI negative) events
were gated using FlowJo software, and the percentage of CD4-gated
cells showing dilution of CFSE determined as a measure of the
percentage of cells undergoing proliferation.
[0294] To analyze cytokine release, cell culture supernatants
obtained after 72 hours of culture were measured for cytokine
content using a 10-plex human Th1/Th2 cytokine analysis kit from
MesoScale Discovery (Gaithersburg, Md.) according to the
manufacturer's protocol. This kit employs an electrochemical
detection method to quantitatively measure the following human
cytokines: IFN.gamma., IL-2, IL4, IL-5, IL-8, IL-10, IL-12 p70,
IL-13, and IL-1.beta..
[0295] GraphPad Prism version 5.01 for Windows, GraphPad Software,
San Diego Calif. USA, www.graphpad.com was used to plot the log of
mAb concentration versus either proliferation or cytokine release
values. The effective concentrations resulting in 20%, 50% and 90%
maximal effect (EC.sub.20, EC.sub.50, and EC.sub.90) values for
OX40mAb24 bioactivity were calculated from sigmoidal dose-response
bioactivity curves using the ECAnything function.
4.3 Results
[0296] Proliferation data from each of the four donors and cytokine
release data from one donor are shown in FIG. 7A-C and FIG. 8A-E.
EC20, EC50, and EC90 potency values for human primary CD4+ T cell
proliferation are shown in Table 4-2; potency values for human
primary CD4+ T cell cytokine release assays in Table 4-3 through
Table 4-7; Mean proliferation and cytokine release values for
OX40mAb24 and 9B12 are shown in Table 4-8 and Table 4-9,
respectively.
[0297] OX40mAb24 co-stimulated proliferation of primary human CD4+
T cells (n=4) in a concentration-dependent manner with EC20, EC50,
and EC90 values of 21, 28, and 72 pM, respectively. 9B12
co-stimulated proliferation of CD4+ T cells with EC20, EC50, and
EC90 values of 106, 218, and 622 pM. Therefore, the ratio of 9B12
to OX40mAb24 concentrations required to induce a 50% of maximum
proliferative response was 8 to 1 (Table 4-2).
[0298] OX40mAb24 and 9B12 co-stimulated primary human CD4+ T cells
to release cytokines (n=4). Mean EC20, EC50, and EC90 values were
less potent than values for proliferation, and are summarized in
Table 4-8 and Table 4-9. Non-linear regression analysis could not
be conducted for IL-2, IL-4, IL-8, IL-12 p70, and IL-1.beta. assay
results for both mAbs, due to poorly formed or non-existent
sigmoidal dose-response curves.
TABLE-US-00011 TABLE 4-2 Mean EC.sub.20, EC.sub.50 and EC.sub.90
Values for OX40mAb24 and 9B12 in a Primary Human CD4.sup.+ T cell
Proliferation Assay Activity in absence Donor Monoclonal EC.sub.20
EC.sub.50 EC.sub.90 Soluble of TCR Number Antibody (95% CI), pM
(95% CI), pM (95% CI), pM activity stimulation 367 OX40mAb24 32
(22, 46) 38 (20, 72) 71 (17, 29) Not Tested None 661 OX40mAb24 33
(28, 39) 51 (33, 77) 128 (96, 172) Not Tested None 645 OX40mAb24
7.5 (2.7, 21) 8.3 (2.8, 25) 35 (7.1, 173) None Not Tested 651
OX40mAb24 9.6 (6.3, 15) 16 (9.1, 26) 54 (30, 99) None Not Tested
Mean OX40mAb24 21 (6.9) 28 (9.8) 72 (20) (Standard Error of the
Mean), pM 367 9B12 99.8 (80.0, 125) 156 (90.2, 269) 337 (214, 530)
Not Tested Not Tested 661 9B12 128 (113, 144) 237 (164, 342) 535
(464, 617) Not Tested Not Tested 645 9B12 77.5 (30.2, 199) 169
(67.0, 425) 686 (152, 3080) Not Tested Not Tested 651 9B12 118
(80.8, 173) 309 (201, 475) 929 (511, 1690) Not Tested Not Tested
Mean 9B12 106 (11.1) 218 (35.2) 622 (125) (Standard Error of the
Mean), pM CI = confidence interval; EC.sub.20 = effective
concentration resulting in 20% of maximal effect; EC.sub.50 = half
maximal effective concentration; EC.sub.90 = effective
concentration resulting in 90% of maximal effect; TCR = T cell
receptor.
TABLE-US-00012 TABLE 4-3 Mean EC.sub.20, EC.sub.50 and EC.sub.90
Values for IFN.gamma. Induced by OX40mAb24 or 9B12 in a Primary
Human CD4.sup.+ T Cell Bioactivity Assay Activity in absence Donor
Monoclonal EC.sub.20 EC.sub.50 EC.sub.90 Soluble of TCR Number
Antibody (95% CI), pM (95% CI), pM (95% CI), pM activity
stimulation 367 OX40mAb24 38.6 (21.3, 70.0) 57.2 (31.2, 105) 106
(34.6, 328) Not Tested None 661 OX40mAb24 ND ND ND Not Tested None
645 OX40mAb24 32.1 (20.8, 49.7) 58.2 (42.0, 80.8) 150 (77.6, 289)
None Not Tested 651 OX40mAb24 47.5 (24.5, 91.8) 77.4 (51.4, 116)
168 (86.7, 324) None Not Tested Mean OX40mAb24 39.4 (4.46) 64.3
(6.57) 141 (18.4) (Standard Error of the Mean), pM 367 9B12 ND ND
ND Not Tested Not Tested 661 9B12 ND ND ND Not Tested Not Tested
645 9B12 706 (518, 963) 2380 (1420, 3970) 16300 (5480, 48300) Not
Tested Not Tested 651 9B12 344 (274, 430) 758 (639, 900) 2660 (780,
3990) Not Tested Not Tested Mean 9B12 525 (148) 1570 (662) 9480
(5569) (Standard Error of the Mean), pM CI = confidence interval;
EC.sub.20 = effective concentration resulting in 20% of maximal
effect; EC.sub.50 = half maximal effective concentration; EC.sub.90
= effective concentration resulting in 90% of maximal effect; TCR =
T cell receptor.
TABLE-US-00013 TABLE 4-4 Mean EC.sub.20, EC.sub.50 and EC.sub.90
Values for TNF.alpha. Induced by OX40mAb24 or 9B12 in a Primary
Human CD4.sup.+ T cell Bioactivity Assay Activity in absence Donor
Monoclonal EC.sub.20 EC.sub.50 EC.sub.90 Soluble of TCR Number
Antibody (95% CI), pM (95% CI), pM (95% CI), pM activity
stimulation 367 OX40mAb24 ND ND ND Not Tested None 661 OX40mAb24 ND
ND ND Not Tested None 645 OX40mAb24 37.6 (22.5, 62.7) 54.1 (29.1,
101) 96.2 (26.7, 347) None Not Tested 651 OX40mAb24 ND ND ND None
Not Tested Mean OX40mAb24 ND ND ND (Standard Error of the Mean), pM
367 9B12 ND ND ND Not Tested Not Tested 661 9B12 ND ND ND Not
Tested Not Tested 645 9B12 306 (258, 503) 670 (527, 853) 1800
(1100, 2920) Not Tested Not Tested 651 9B12 388 (300, 502) 764
(640, 919) 2260 (1500, 3410) Not Tested Not Tested Mean 9B12 347
(33.5) 717 (38.4) 2030 (188) (Standard Error of the Mean), pM CI =
confidence interval; EC.sub.20 = effective concentration resulting
in 20% of maximal effect; EC.sub.50 = half maximal effective
concentration; EC.sub.90 = effective concentration resulting in 90%
of maximal effect; ND = not determined due to poor curve fit
(EC.sub.20, EC.sub.50, EC.sub.90) or lack of sufficient in values
to determine mean and standard error of the mean; TCR = T cell
receptor.
TABLE-US-00014 TABLE 4-5 Mean EC.sub.20, EC.sub.50 and EC.sub.90
Values for IL10 Induced by OX40mAb24 or 9B12 in a Primary Human
CD4.sup.+ T Cell Bioactivity Assay Activity in absence Donor
Monoclonal EC.sub.20 EC.sub.50 EC.sub.90 Soluble of TCR Number
Antibody (95% CI), pM (95% CI), pM (95% CI), pM activity
stimulation 367 OX40mAb24 41.6 (17.8, 97.0) 59.5 (25.4, 139) 105
(25.0, 440) Not Tested None 661 OX40mAb24 63.5 (29.6, 136) 89.5
(64.2, 125) 154 (93.3, 255) Not Tested None 645 OX40mAb24 ND ND ND
None Not Tested 651 OX40mAb24 53.0 (31.0, 90.6) 86.4 (65.0, 115)
188 (110, 321) None Not Tested Mean OX40mAb24 52.7 (6.32) 78.5
(9.53) 149 (24.1) (Standard Error of the Mean), pM 367 9B12 130
(83.3, 204) 198 (139, 284) 385 (224, 662) Not Tested Not Tested 661
9B12 ND ND ND Not Tested Not Tested 645 9B12 528 (363, 767) 1220
(858, 1740) 4630 (2050, 10400) Not Tested Not Tested 651 9B12 405
(300, 547) 796 (649, 976) 2320 (1440, 3730) Not Tested Not Tested
Mean 9B12 354 (118) 738 (296) 2445 (1227) (Standard Error of the
Mean), pM CI = confidence interval; EC.sub.20 = effective
concentration resulting in 20% of maximal effect; EC.sub.50 = half
maximal effective concentration; EC.sub.90 = effective
concentration resulting in 90% of maximal effect; ND = not
determined due to poor curve fit (EC.sub.20, EC.sub.50, EC.sub.90)
or lack of sufficient in values to determine mean and standard
error of the mean; TCR = T cell receptor.
TABLE-US-00015 TABLE 4-6 Mean EC20, EC50 and EC90 Values for IL13
Induced by OX40mAb24 or 9B12 in a Primary Human CD4.sup.+ T Cell
Bioactivity Assay Activity in absence Donor Monoclonal EC.sub.20
EC.sub.50 EC.sub.90 Soluble of TCR Number Antibody (95% CI), pM
(95% CI), pM (95% CI), pM activity stimulation 367 OX40mAb24 ND ND
ND Not Tested None 661 OX40mAb24 ND ND ND Not Tested None 645
OX40mAb24 44.7 (26.5, 75.5) 73.8 (52.2, 104) 163 (93.3, 286) None
Not Tested 651 OX40mAb24 36.2 (24.9, 52.7) 65.0 (49.6, 85.3) 164
(99.2, 273) None Not Tested Mean OX40mAb24 40.5 (4.25) 69.4 (4.40)
164 (0.50 (Standard Error of the Mean), pM 367 9B12 ND ND ND Not
Tested Not Tested 661 9B12 154 (841, 283) 238 (166, 341) 472 (278,
800) Not Tested Not Tested 645 9B12 1100 (413, 2910) 5450 (1060,
2800) 6930 (4310, 1110000) Not Tested Not Tested 651 9B12 495 (324,
756) 1770 (896, 3510) 13400 (2980, 60400) Not Tested Not Tested
Mean 9B12 583 (277) 2486 (1550) 6930 (3730) (Standard Error of the
Mean), pM CI = confidence interval; EC.sub.20 = effective
concentration resulting in 20% of maximal effect; EC.sub.50 = half
maximal effective concentration; EC.sub.90 = effective
concentration resulting in 90% of maximal effect; ND = not
determined due to poor curve fit (EC.sub.20, EC.sub.50, EC.sub.90)
or lack of sufficient in values to determine mean and standard
error of the mean; TCR = T cell receptor.
TABLE-US-00016 TABLE 4-7 Mean EC.sub.20, EC.sub.50 and EC.sub.90
Values for IL5 Induced by OX40mAb24 or 9B12 in a Primary Human
CD4.sup.+ T cell Bioactivity Assay Activity in absence Donor
Monoclonal EC.sub.20 EC.sub.50 EC.sub.90 Soluble of TCR Number
Antibody (95% CI), pM (95% CI), pM (95% CI), pM activity
stimulation 367 OX40mAb24 33.2 (21.2, 52.1) 46.7 (24.7, 88.0) 79.8
(12.3, 519) Not Tested None 661 OX40mAb24 43.4 (27.6, 68.4) 69.0
(49.7, 95.7) 144 (86.8, 238) Not Tested None 645 OX40mAb24 ND ND ND
None Not Tested 651 OX40mAb24 56.2 (35.5, 88.9) 95.9 (76.1, 121)
224 (128, 391) None Not Tested Mean OX40mAb24 44.3 (6.65) 70.5
(14.2) 149 (41.7) (Standard Error of the Mean), pM 367 9B12 ND ND
ND Not Tested Not Tested 661 9B12 ND ND ND Not Tested Not Tested
645 9B12 ND ND ND Not Tested Not Tested 651 9B12 423 (285, 627)
1090 (742, 1600) 4870 (1900, 12500) Not Tested Not Tested Mean 9B12
ND ND ND (Standard Error of the Mean), pM CI = confidence interval;
EC.sub.20 = effective concentration resulting in 20% of maximal
effect; EC.sub.50 = half maximal effective concentration; EC.sub.90
= effective concentration resulting in 90% of maximal effect; ND =
not determined due to poor curve fit (EC.sub.20, EC.sub.50,
EC.sub.90) or lack of sufficient in values to determine mean and
standard error of the mean; TCR = T cell receptor.
TABLE-US-00017 TABLE 4-8 Summary of Mean EC.sub.20, EC.sub.50 and
EC.sub.90 Values for Proliferation and Cytokine Release for
OX40mAb24 Bioactivity EC.sub.20 EC.sub.50 EC.sub.90 Readout
(StdErr), pM (StdErr), pM (StdErr), pM CD4 T cell 21 (6.9) 28 (98)
72 (20) proliferation IFN.gamma. release 39.4 (4.46) 64.3 (6.57)
141 (18.4) TNF.alpha. release ND ND ND IL10 52.7 (6.32) 78.5 (9.53)
149 (24.1) IL13 40.5 (4.25) 69.4 (4.40) 164 (0.50) IL5 44.3 (6.65)
70.5 (14.2) 149 (41.7) EC.sub.20 = effective concentration
resulting in 20% of maximal effect; EC.sub.50 = half maximal
effective concentration; EC.sub.90 = effective concentration
resulting in 90% of maximal effect; ND = not determined due to
insufficient n value to calculate a mean and standard error of the
mean; StdErr = standard error of the mean.
TABLE-US-00018 TABLE 4-9 Summary of Mean EC.sub.20, EC.sub.50 and
EC.sub.90 Values for Proliferation and Cytokine Release for 9B12
Bioactivity EC.sub.20 EC.sub.50 EC.sub.90 Readout (StdErr), pM
(StdErr), pM (StdErr), pM CD4 T cell 106 (11.1) 218 (35.2) 622
(125) proliferation IFN.gamma. release 525 (148) 1570 (662) 9480
(5569) TNF.alpha. release 347 (33.5) 717 (38.4) 2030 (188) IL10 354
(118) 738 (296) 2445 (1227) IL13 583 (277) 2486 (1547) 6934 (3732)
IL5 ND ND ND EC.sub.20 = effective concentration resulting in 20%
of maximal effect; EC.sub.50 = half maximal effective
concentration; EC.sub.90 = effective concentration resulting in 90%
of maximal effect; ND = not determined due to insufficient n value
to calculate a mean and standard error of the mean; StdErr =
standard error of the mean.
[0299] The activity of non-plate bound, soluble OX40mAb24 was
determined. Soluble OX40mAb24 did not induce either primary human
CD4.sup.+ T cell proliferation (FIG. 7A-C) or cytokine release
(FIG. 8A-E) above levels observed for either anti-CD3 antibody
alone or in the presence of R347 human IgG1 control mAb. Plate
bound anti-CD3 antibody alone produced a minimal-to-moderate level
of proliferation and cytokine release. The lack of activity
demonstrated here by soluble OX40mAb24 is in agreement with the
absence of activity observed for soluble OX40mAb24 without
cell-based cross-linking in a 2-cell bioactivity assay that
measured OX40 mediated NF.kappa.B signaling (see Example 5
below).
[0300] Likewise, OX40mAb24, either immobilized on the plate surface
or added as a soluble unbound protein, in the absence of a
sub-mitogenic anti-CD3 antibody signal induced little-to-no
CD4.sup.+ T cell proliferation (FIG. 7A-C) or cytokine release
(FIG. 8A-E). These results demonstrated that, in this study,
OX40mAb24 does not have activity in primary human CD4.sup.+ T cells
in the absence of simultaneous CD3/TCR ligation.
4.4 Conclusions
[0301] OX40mAb24 induced proliferation and cytokine release of
primary human CD4.sup.+ T cells in a concentration-dependent manner
similar to that of the antibody from which it was humanized 9B12
(FIG. 8A-E and FIG. 9A-E). OX40mAb24 demonstrated activity as a
plate-bound, but not as a soluble, protein. Furthermore, OX40mAb24
activity occurred concurrent with CD3/TCR signaling.
Example 5
Determination of the In Vitro Activity of OX40mAb24 in 2-Cell Based
Bioactivity Assays Using Jurkat NF.kappa.B-luciferase Reporter T
Cells
[0302] In this example, the ability of OX40mAb24 and 9B12 to signal
through human OX40 was assessed using a set of two-cell reporter
bioactivity assays. Measurement of T cell activation through OX40
co-stimulation was accomplished by using an OX40-overexpressing
Jurkat NF.kappa.B-luciferase T cell reporter line that produces
luciferase in response to stimulation of the NF.kappa.B signaling
pathway (FIG. 10). NF.kappa.B signaling has been reported to occur
downstream of OX40 engagement, and can correlate with other
measures of T cell activation, such as proliferation and cytokine
release (Croft M, et al., Immunol Rev. 229:173-91 (2009)). The
amount of luciferase, and thus T cell activation, was measured by
adding a luciferase substrate to cell lysates and measuring light
emitted by the reaction product using a luminometer. Bioactivity of
OX40mAb24 cross-linked using cells engineered to express different
Fc.gamma. receptor complements, as well as soluble,
non-Fc.gamma.R-crosslinked OX40mAb24 was measured.
5.1 Materials
[0303] Materials used in this study are listed in Table 5-1.
TABLE-US-00019 TABLE 5-1 Materials Item Source CD45.sup.+
microbeads Miltenyi, San Diego, CA Collagenase III Worthington
Biochemical Corporation, Lakewood, NJ DNAse I, from b ovine
pancreas Sigma, Saint Louis, MO EDTA, 0 5M pH 8.0 Life
Technologies, Carlsbad, CA Envision luminescence reader Perkin
Elmer, Waltham, MA Heat inactivated newborn calf Life Technologies,
Carlsbad, CA serum (FBS) LS column Miltenyi, San Diego, CA MACS
buffer: PBS + 0.5% Materials from Life Technologies, BSA + 2 mM
EDTA Carlsbad, CA Non-TC treated round-bottom VWR, Radnor, PA 96
well plates Prism v5.01 software GraphPad, San Diego, CA Steady-Glo
Luciferase Assay Promega, Madison, WI Solution ViCell counter
Beckman Coulter, Indianapolis, IN
5.2 Two-cell Bioactivity Assay
5.2.1 Isolation of Primary Human CD45.sup.+ Cells
[0304] Primary human CD45.sup.+ cells were isolated from human
tumors. Tumor samples were removed from transport media and placed
in sterile petri dish. Hank's Buffered Salt Solution was added and
visible necrotic tissue or any normal tissue from tumor sample was
dissected. Tissue was minced into small pieces (.about.1 mm) and
placed in a 50 mL conical tube, and Collagenase III enzyme mix (250
IU/mL collagenase III, 3 mM CaCl.sub.2, 315 .mu.g/mL DNAase 1) was
added, mixed and incubated. The digested sample was filtered
through a 70 micron filter and washed with MACS buffer. Dissociated
cells were pelleted and the cell number and viability were
determined using a ViCell counter. Cells were suspended in MACS
buffer with CD45 microbeads and incubated on ice. Cells were washed
and re-suspended in MACS buffer for positive selection of
CD45.sup.+ cells using an LS column. Bead-bound CD45.sup.+ cells
were eluted by removing the column from the magnet and adding MACS
buffer to the column. Cells were pelleted and re-suspended in
complete RPMI medium and used in bioactivity assays as described
above.
5.2.2 Assay Protocol
[0305] OX40mAb24 and 9B12 were tested for bioactivity using a
2-cell bioactivity assay. Human OX40-overexpressing Jurkat
NF.kappa.B-luciferase reporter clone 64 (OX40 Jurkat reporter) was
used to measure OX40 agonism (NF.kappa.B activity). A second,
Fc.gamma.R-expressing cell line, was used to mediate OX40mAb24
cross-linking, which clusters and activates OX40 on the OX40 Jurkat
reporter cells (FIG. 10). The Fc.gamma.R-expressing cell lines used
for cross-linking included the Raji human B cell lymphoma line,
CD32A-expressing HEK293, CD32B-expressing HEK293 or CD45+ immune
cells isolated from primary human tumors.
[0306] To determine the soluble activity of OX40mAb24, bioactivity
assays were also conducted using either parental HEK293 cells,
which are Fc.gamma.R null and therefore are unable to cross-link
OX40mAb24, or in the absence of cross-linking cells altogether.
[0307] Prior to use, OX40 Jurkat reporter cells were cultured in
complete RPMI medium in a tissue culture incubator at a density of
0.5-1.5.times.106 per mL. Cells were passaged the day prior to the
bioassay at a final density of 106 cells per mL.
[0308] OX40 Jurkat reporter cells, Fc.gamma.R-expressing cell
lines, or HEK parental cells were collected, and pelleted. To
isolate CD45+ cells from primary human tumors and normal adjacent
tissues, tissues were dissociated and CD45+ cells isolated and
re-suspended in complete RPMI medium for use in bioactivity assays,
as described below.
[0309] OX40mAb24, 9B12 and various control antibodies were serially
diluted 3-fold in complete RPMI medium.
[0310] OX40 Jurkat reporter cells plus Fc.gamma.R-expressing cells,
or HEK parental cells, were added to a 96 well plate at 100,000
cells per well. OX40mAb24, 9B12 or control antibodies were added to
cells in a dilution series with a starting concentration of 10
.mu.g/mL, and incubated at 37.degree. C. in a tissue culture
incubator.
[0311] After 16-24 hours incubation time, 100 .mu.L reconstituted
Steady-Glo luciferase assay solution (Promega, Madison, Wis.) was
added to each well and mixed to lyse cells and then incubated to
equilibrate luciferase signal. Steady-Glo/sample lysate (150 .mu.L)
was transferred from each well to a 96 well, white walled assay
plate for detection and luminescence read using a Perkin Elmer
Envision luminescence reader.
[0312] GraphPad Prism version 5.01 for Windows (GraphPad Software,
San Diego, Calif.), was used to plot the concentration of
OX40mAb24, 9B12, R347 human IgG1, mouse IgG1 clone MOPC-21 (x-axis
is log 10 of protein concentration) versus luminescence RLU
(y-axis). The EC20, EC50, and EC90 values for bioactivity were
determined using ECAnything (ECf) for f=20, f=50 and f=90 from
sigmoidal dose-response (variable slope) bioactivity curves).
5.3 Results of 2-cell Bioactivity Assays
[0313] Results for the bioactivity of OX40mAb24 and control
antibodies are shown in FIG. 11A-D, FIG. 12A-D and Table 5-2.
[0314] In the presence of an Fc.gamma.R-expressing cell line (e.g.,
CD32A-expressing HEK293, CD32B-expressing HEK293, Raji B cells, or
CD45+ cells isolated from primary human tumors), OX40mAb24
demonstrated potent stimulation of OX40-overexpressing Jurkat
NF.kappa.B reporter cells, as measured by NF.kappa.B pathway
activation. In the absence of a second cell type or in the presence
of HEK293 cells lacking exogenously expressed Fc.gamma.Rs,
OX40mAb24 exhibited minimal reporter activity (FIG. 11A-D).
[0315] Potency values (EC20, EC50, and EC90) for the two-cell
bioassays are summarized in Table 5-2. The mean EC20, EC50, and
EC90 values across all assays were 228, 751, and 5630 pM,
respectively.
[0316] Results for the bioactivity of 9B12 and control antibodies
are shown in FIG. 12A-D and the potency values are summarized in
Table 5-3. The mean EC20, EC50, and EC90 values for all assays were
519, 2530, and 41100 pM, respectively. Therefore, the ratio of
2-cell bioactivity (EC50) for 9B12 relative to that of OX40mAb24
was calculated to be 3.4 to 1.
TABLE-US-00020 TABLE 5-2 Two-cell Bioactivity of OX40mAb24
Experiment Number Fc.gamma.R-expressing cell* EC.sub.20 (95% CI) pM
EC.sub.50 (95% CI) pM EC.sub.90 (95% CI) pM 1 Raji 298 (236, 378)
1140 (982, 1340) 9850 (6500, 14300) 2 CD32A-expressing HEK 104
(78.8, 138) 437 (370, 517) 4250 (2900, 6240) 3 CD32A-expressing HEK
100 (79.5, 126) 322 (281, 370) 2060 (1530, 2780) 4 Raji 350 (259,
467) 1040 (855, 1270) 5930 (3740, 9400) 5 CD32B-expressing HEK 90.6
(75.4, 109) 270 (242, 301) 1520 (1200, 1920) 6 Raji 180 (109, 296)
796 (577, 1100) 8410 (3820, 18500) 7 CD32B-expressing HEK 280 (198,
394) 899 (710, 1140) 5730 (3290, 9970) 8 CD32B-expressing HEK 237
(196, 286) 671 (592, 762) 3510 (2630, 4670) 9 Raji 676 (443, 1030)
1320 (970, 1800) 3820 (2000, 7320) 10 Raji 297 (127, 694) 1260
(666, 2390) 12500 (2570, 60500) 11 CD32B-expressing HEK 144 (113,
184) 700 (598, 818) 8540 (5770, 12600) 12 CD45.sup.+ cells, NSCLC
33.6 (24.7, 45.8) 81.6 (68.5, 97.3) 334 (236, 471) 13 CD45.sup.+
cells, RCC 92.2 (75.4, 113) 337 (296, 383) 2630 (1940, 3550) 14
CD45.sup.+ cells, NSCLC 247 (207, 295) 772 (688, 867) 4700 (3600,
6140) 15 CD45.sup.+ cells, RCC 351 (155, 798) 1410 (782, 2530)
12700 (2960, 54500) 16 CD32A-expressing HEK 166 (145, 191) 553
(507, 603) 3720 (3060, 4530) Mean (Standard Error 228 (38.9) 751
(101) 5630 (937) of the Mean) NSCLC = non-small cell lung cancer;
RCC = renal cell carcinoma *As indicated, assays used Raji,
CD32A-expressing HEK293, CD32B-expressing HEK293 cells, or
CD45.sup.+ cells isolated from primary human tumor samples with
OX40-expressing Jurkat NF.kappa.B-luciferase clone 64 reporter
cells.
TABLE-US-00021 TABLE 5-3 Two-cell Bioactivity of 9B12 Experiment
Number Fc.gamma.R-expressing cell* EC.sub.20 pM EC.sub.50 pM
EC.sub.90 pM 1 Raji 1110 (791, 1570) 9280 (4460, 19300) 267000
(57900, 1230000) 2 CD32A-expressing HEK 74.1 (53.0, 104) 338 (279,
410) 3760 (2440, 5790) 3 CD32A-expressing HEK 66.7 (43.3, 103) 225
(175, 289) 1550 (914, 2630) 4 Raji 1040 (834, 1300) 5040 (3880,
6550) 61200 (32200, 116000) 5 CD32B-expressing HEK 249 (206, 303)
726 (641, 823) 3950 (2980, 5240) 6 Raji 548 (408, 736) 2700 (2070,
3620) 33700 (17000, 66700) 7 CD32B-expressing HEK 585 (520, 658)
1770 (1620, 1930) 10200 (8280, 12500) 8 CD32B-expressing HEK 518
(449, 598) 1340 (1210, 1480) 6050 (4800, 7630) 9 Raji 872 (504,
1510) 3070 (1910, 4950) 22600 (7000, 73100) 10 Raji 571 (212, 1540)
3330 (1080, 10200) 54400 (3400, 872000) 11 CD32B-expressing HEK 650
(458, 923) 2360 (1790, 3100) 18200 (9200, 36000) 12 CD45.sup.+
cells, NSCLC 104 (76.3, 143) 223 (185, 269) 741 (501, 1100) 13
CD45.sup.+ cells, RCC ND ND ND 14 CD45.sup.+ cells, NSCLC ND ND ND
15 CD45.sup.+ cells, RCC 356 (132, 964) 2420 (938, 6250) 50500
(4140, 616000) Mean (Standard Error 519 (96.2) 2530 (687) 41100
(19700) of the Mean) ND = not determined; NSCLC = non-small cell
lung cancer; RCC = renal cell carcinoma *As indicated, assays used
Raji, CD32A-expressing HEK293, CD32B-expressing HEK293 cells, or
CD45.sup.+ cells isolated from primary human tumor samples with
OX40-expressing Jurkat NF.kappa.B-luciferase clone 64 reporter
cells.
5-4: Conclusions
[0317] OX40mAb24 and 9B12 mediate the activation of human T cells
as measured by the stimulation of the NF.kappa.B pathway in an
OX40-overexpressing Jurkat NF.kappa.B-luciferase reporter cell
line. In the absence of cells that express Fc.gamma.Rs capable of
cross linking via the Fc domains of OX40mAb24, minimal reporter
cell-line activity was measured. However, in a 2-cell system
comprising cells that express Fc.gamma.R that are capable of
cross-linking the mAb, and a Jurkat overexpressing
NF.kappa.B-luciferase reporter line for readout of T cell
activation, OX40mAb24 and 9B12 mediated potent OX40 activation with
mean EC.sub.50 values of 751 pM and 2530 pM, respectively.
Therefore, the bioactivity of OX40mAb24 in the 2-cell assay system
was similar to that of 9B12.
Example 6
Ability of OX40mAb24 to Trigger Effector Function
[0318] In this example OX40mAb24 was assessed with respect to its
ability to trigger Fc effector function namely, the ability of
OX40mAb24 to trigger human natural killer (NK) cell-mediated
antibody-dependent cellular cytotoxicity (ADCC) against primary
human CD4.sup.+ T cells expressing high levels of OX40, or to bind
C1q, a prerequisite for complement-dependent cellular cytotoxicity
(CDC) by the classical complement pathway. Versions of OX40mAb24
containing either a human IgG4P Fc domain (mAb28), or a triple
mutation in the IgG1 Fc domain that reduces Fc.gamma. RIIIa binding
(mAb29) were utilized to assess the contribution of the OX40mAb24
IgG1 Fc domain to mediate ADCC activity. Also, the effector
functions of OX40mAb24 were compared to that of 9B12, a mouse
anti-human OX40 IgG1 monoclonal antibody that was humanized to
create OX40mAb24. The anti-CD20 antibody rituximab binds to B cells
expressing CD20 and was used as a positive control for ADCC
experiments. Because primary activated human CD4.sup.+ T cells do
not express CD20, a separate assay using the Toledo B cell line,
which does express CD20, was conducted to validate the activity of
NK cells used in the ADCC assay system.
6.1 Materials
[0319] Materials used in this study are listed in Table 7-1.
TABLE-US-00022 TABLE 6-1: Materials Item Source Complement protein
Clq Quidel, San Diego, CA Complete RPMI medium: RPMI-1640+ 10% FBS
Materials from Life Technologies, Carlsbad, CA DM-L medium Stem
Cell Technologies, Vancouver, BC Canada GLC biosensor chip Bio-Rad,
Hercules, CA FlowJo Software FlowJo, Ashland, OR IL-2, recombinant
human Preprotech, Rocky Hill, NJ LSR II flow cytometer BD
Biosciences, San Jose, CA Prism software, v 5.01 Graphpad Software,
San Diego, CA Propidium iodide (1 mg/mL solution) Sigma, Saint
Louis, MO ProteOn Manager 2.1 software Bio-Rad, Hercules, CA
ProteOn XPR36 instrument Bio-Rad, Hercules, CA RosetteSep CD4 T
cell enrichment kit Stem Cell Technologies, Vancouver, BC Canada
Rosette Sep Human NK cell enrichment Stem Cell Technologies,
Vancouver, BC Canada ViCell counter Beckman Coulter, Indianapolis,
IN Vybrant DiO cell labeling solution Life Technologies, Carlsbad,
CA Whole blood, sodium heparin anti-coagulated MedImmune Blood
Donor Program, MedImmune, Gaithersburg, MD
6.2 Assays
6.2.1 Antibody-Dependent Cellular Cytotoxicity
[0320] The ADCC activity of OX40mAb24 relative to that of 9B12 and
monoclonal antibodies containing the Fab arms of OX40mAb24 with
either an IgG4P Fc domain or a triple mutant (TM) human IgG1 Fc
domain, was tested using enriched primary human NK cells as
effectors and OX40-expressing primary human CD4.sup.+ T cells as
targets. As a positive control, the activity of each NK cell
preparation was tested using rituximab directed killing of the
Toledo B cell line.
[0321] For the isolation of primary human CD4.sup.+ T cells,
heparin anti-coagulated, whole blood obtained from healthy donors
through the MedImmune Blood Donor Program was processed according
to the following protocol: Stem Cell Technologies RosetteSep CD4 T
cell isolation kit antibody mix (1 mL, Stem Cell Technologies,
Vancouver, BC Canada) was added per 20 mL of whole blood, mixed,
and incubated for 20 minutes (mm) at room temperature (RT). Blood
was diluted 1:1 with sterile room temperature FACS buffer (PBS, pH
7.2 plus 2% heat inactivated newborn calf serum) and layered onto
DM-L medium followed by centrifugation for 20 min. After
centrifugation, the buffy coat containing human CD4.sup.+ T cells
was removed and the cells were washed once with RT FACS buffer and
once with RT complete RPMI medium. Cells were counted on a ViCell
counter to determine cell number and viability and CD4.sup.+ T
cells were suspended in complete RPMI medium at a concentration of
1.0.times.10.sup.6 per mL.
[0322] Primary human CD4.sup.+ T cells (1.0.times.10.sup.6 per mL
in complete RPMI medium) were cultured in a humidified tissue
culture incubator at 37.degree. C. and 5% CO.sub.2 for 48 hours
with 2 .mu.g/mL PHA-L and 20 IU/mL rhIL-2 to activate T cells and
up-regulate OX40 and were subsequently used in OX40mAb24 ADCC
assays. All donors in the figures referenced below represent unique
individuals; that is, CD4.sup.+ T cells were not isolated from the
same donor for repeat ADCC experiments.
[0323] To discriminate target T cells, or cultured Toledo B cells,
from purified human NK cells in cytotoxicity assays, the
fluorescent dye, DiO, was incorporated into the cell membrane of
target cells using the Vybrant DiO cell labeling solution
(according to the manufacturer's protocol for suspension cell
labeling). After activation and DiO labeling of primary human
CD4.sup.+ T cells and Toledo B cells, effector NK cells were
isolated from sodium heparin anticoagulated blood from the
MedImmune Blood Donor Program using the same protocol as above for
human CD4.sup.+ T cells, with the exception that 1 mL of RosetteSep
NK cell isolation kit antibody mix was used in place of 1 mL of
RosetteSep CD4.sup.+ T cell isolation kit antibody mix. Isolated
primary human NK cells were washed two times with warm complete
RPMI medium (RPMI-1640 plus 10% FBS), and then suspended in
complete RPMI at a concentration of 2.67.times.10.sup.6 per mL.
Likewise, activated primary human CD4.sup.+ T cells were also
washed two times in warm complete RPMI and suspended at a
concentration of 2.67.times.10.sup.5 per mL. Thereafter, 75 .mu.L
of primary human NK cells (200,000) and 75 .mu.L of activated
primary human CD4.sup.+ T cells (20,000) were added to wells of
sterile non-tissue culture treated round bottom 96-well plates for
an effector-to-target ratio of 10:1. In some experiments, complete
RPMI medium (50 .mu.L) containing OX40mAb24 or control mAb was
added to give a final concentration of 10 .mu.g/mL. In others,
complete RPMI (50 .mu.L) containing a 3-fold dilution series of
OX40mAb24 or control mAbs, resulting in a final concentration of 67
nM, or a 27-fold dilution series of 9B12 or R347 human IgG1
starting from 67 nM, was added to the plated cells. Rituximab (10
.mu.g/mL, control antibody) was used as a positive control in wells
containing DiO labeled, CD20-expressing Toledo B cells, in place of
OX40-expressing, activated primary human CD4.sup.+ T cells, because
activated CD4.sup.+ T cells do not express CD20. Cells in the ADCC
assay were gently pelleted by centrifugation at RT and subsequently
cultured for 24 hours.
[0324] At the end of the incubation period, cells were pelleted by
centrifugation and then suspended in FACS buffer containing 10
.mu.g/mL of propidium iodide (PT) for flow cytometry analysis on a
BD LSRII flow cytometer. Non-viable (PT positive) cells among
DiO-positive target CD4.sup.+ T cells or Toledo B cells were
discriminated using FlowJo software after fluorescence
compensation.
[0325] Graphical representation of the data for NK cell ADCC
assays, including determination of mean values and standard error
of the mean, was generated using GraphPad Prism version 5.01 for
Windows.
6.2.2 OX40mAb24 Binding to C1q
[0326] A ProteOn XPR36 instrument was used to determine the binding
of OX40mAb24 or 9B12 to human complement protein C1q purified from
pooled human sera to OX40mAb24 or 9B12 by surface plasmon
resonance. Standard amine coupling was used to immobilize OX40mAb24
or 9B12 to the surface of a GLC biosensor chip. Human C1q was
suspended in PBS/0.005% Tween 20, pH 7.4 at five concentrations
ranging from 26 nM to 1.6 nM. The samples were injected at 30
.mu.L/min for 200 seconds, and the dissociation phase was followed
for 600 seconds. Sensorgram data was processed using ProteOn
Manager 2.1 software (Bio-Rad) using 1:1 fitting.
6.3 Results
[0327] The ability of OX40mAb24, 9B12, and human IgG4P (mAb28) and
IgG1-TM (mAb29) versions of OX40mAb24 to mediate ADCC was tested at
saturating levels (10 .mu.g/mL [67 nM]) for OX40 binding (see
Example 2), using both allogeneic and autologous mixtures of NK
cells and OX40-expressing target cells (FIG. 13A and FIG. 14A).
OX40mAb24 showed measurable ADCC activity against activated human
CD4.sup.+ T cells. In contrast, 9B12, mAb28 and mAb29 showed no
ADCC activity above that of the negative controls. A
positive-control antibody against human CD20 (rituximab)
demonstrated ADCC activity using the same NK cells, demonstrating
that the NK cells were all capable of robust ADCC activity (FIG.
13B and FIG. 14B).
[0328] In dose-response experiments, OX40mAb24 demonstrated
concentration-dependent ADCC activity against activated human
CD4.sup.+ T cells (FIG. 15A-D, FIG. 16A-D, FIG. 17A-B, and Table
6-2. In contrast, 9B12 did not show any detectable ADCC activity.
In addition, the R347 human IgG1 control mAb did not show any
detectable ADCC activity, which confirms that ADCC activity
mediated by OX40mAb24 was dependent on engagement with OX40 on the
target cells. A positive-control antibody against human CD20 also
demonstrated ADCC against a CD20-expressing B cell lymphoma cell
line (FIG. 13B and FIG. 14B); this supports the validity of the
assay. Collectively, the results of these experiments confirmed
that OX40mAb24 is capable of ADCC against target cells that have
been cultured under conditions previously shown to stimulate
expression of cell surface OX40.
TABLE-US-00023 TABLE 6-2 Summary of OX40mAb24-mediated NK cell ADCC
Potency Experiment Donor Number Number EC20 (pM) EC50 (pM) EC90
(pM) 3 363 134 2380 225000 3 504 309 1370 14600 4 464 37.0 3050
3330000 4 532 111 1310 66100 5 601 54.4 501 16900 5 602 61.4 527
16000 Mean (standard error 118 (41.1) 1520 (415) 611000 (545000) of
the mean), pM
[0329] The potential for OX40mAb24 to bind to the human complement
component C1q was assessed using a surface plasmon resonance assay.
In this assay, binding to C1q was used as a surrogate for activity
in a complement dependent cytotoxicity assay (Dall'Acqua et al., J.
Immunol 177:1129-1138 (2006)). 9B12 was used as a negative control
antibody. OX40mAb24 demonstrated a concentration-dependent ability
to bind to purified human C1q protein (FIG. 18A). In contrast, 9B12
did not demonstrate any detectable binding to C1q protein (FIG.
18B).
6.4 Conclusions
[0330] OX40mAb24 binds to C1q and triggers NK-mediated ADCC against
activated CD4.sup.+ T cells.
Example 7
In Vitro Comparability Studies with OX40mAb24 and Cynomolgus and
Rhesus Monkey T Cells
[0331] In this example, the ability of OX40mAb24 to enhance
activation of T cells was determined using two-cell reporter
bioactivity assays with a Jurkat NF.kappa.B-luciferase T cell
reporter line expressing cynomolgus (cyno)/rhesus monkey OX40.
Fc.gamma. receptor-mediated drug cross-linking was mediated by
either a rhesus B cell line, or by rhesus Fc.gamma.
receptor-expressing cells in a post-red blood cell lysis whole
blood sample. OX40 activation was measured as increased luciferase
activity in response to stimulation of the NF.kappa.B signaling
pathway downstream of primary human T cell activation. NF.kappa.B
signaling is a well-studied downstream event in OX40 signaling, and
can correlate with other measures of T cell activation such as
proliferation and cytokine release (Croft M, et al., Immunol Rev.
229:173-91 (2009)). In addition, the ability of OX40mAb24 to
enhance T cell receptor-mediated activation (co-stimulation) of
rhesus T cells was tested using a plate-based bioactivity assay in
which CD4+ T cell proliferation was assessed. An isotype control
(NIP228 IgG1),was used to demonstrate specific OX40 engagement.
[0332] In parallel, the bioactivity of 9B12, the murine anti-OX40
monoclonal antibody from which OX40mAb24 was derived, was measured
to provide for a comparison of OX40mAb24 and 9B12 activity on
non-human primate OX40.
7.1 Materials
[0333] Materials used in this study are listed in Table 7.1
TABLE-US-00024 TABLE 7-1 Materials Item Source Ammonium Chloride
StemCell Technologies, Vancouver, BC Anti-human CD3 antibody, clone
SP34-2 BD Biosciences, San Jose, CA Anti-human CD4-V450 antibody,
clone L200 BD Biosciences, San Jose, CA Anti-human CD28 antibody,
clone CD28.2 BD Biosciences, San Jose, CA CFSE cell labeling kit
Life Technologies, Carlsbad, CA Concanavalin A Sigma, Saint Louis,
MO Envision multilabel plate reader Perkin Elmer, Waltham, MA Goat
anti-human Fc.gamma. antibody Jackson Immunoresearch, West Grove,
PA Goat anti-mouse Fc.gamma. antibody Jackson Immunoresearch, West
Grove, PA Heat inactivated fetal bovine serum Life Technologies,
Carlsbad, CA Interleukin-2, recombinant human Preprotech, Rocky
Hill, NJ LSR II flow cytometer BD Biosciences, San Jose, CA
Lymphocyte separation medium (LSM) MP Biomedicals, Santa Ana, CA
MACS buffer Miltenyi, San Diego, CA Nonhuman primate CD4 T cell
isolation kit Miltenyi, San Diego, CA Propidium iodide (1 mg/mL
solution) Sigma, Saint Louis, MO Rhesus monkey (Indian Origin)
whole blood, sodium Worldwide Primates, Miami, FL heparin
anti-coagulated RPMI-1640 medium Life Technologies, Carlsbad, CA
SteadyGlo Luciferase Assay Solution Promega, Madison, WI ViCell
counter Beckman Coulter, Indianapolis, IN
7.2 Assays
7.2.1 Cyno/Rhesus 2-Cell Bioactivity Assays
[0334] OX40mAb24 was tested for bioactivity using a 2-cell reporter
assay. As cyno and rhesus monkeys share identical OX40 amino acid
sequences, a cyno/rhesus OX40-expressing Jurkat
NF.kappa.B-luciferase reporter cell line (clone B2) was used for
readout of OX40 agonism (NF.kappa.B activity). To mediate OX40mAb24
cross-linking and, consequently, OX40 clustering on Jurkat reporter
cells, either an Fc.gamma. receptor-expressing rhesus B-cell line,
LCL8664, or leukocytes from whole blood of a normal rhesus monkey,
which contain Fc.gamma.R expressing cells, were used. Background
bioactivity was assessed both without the addition of OX40mAb24,
and also in the absence of the Fc.gamma.R-expressing cells. To
demonstrate the role of OX40 engagement on reporter cells, an
isotype control was used in place of OX40mAb24.
[0335] The OX40mAb24 2-cell bioactivity assay with LCL8664 was
performed according to the following protocol:
[0336] The day prior to use, cyno/rhesus OX40-expressing Jurkat
NF.kappa.B-luciferase reporter clone B2 and LCL8664 were cultured
in complete RPMI medium (RPMI with 10% fetal bovine serum [FBS] and
1% antibiotics/antimycotics) to achieve cell densities of
approximately 5.times.10.sup.6 cells/mL and 4.times.10.sup.5
cells/mL, respectively. The next day, OX40 Jurkat reporter cells
and LCL8664 were pelleted, suspended in complete medium and counted
using a ViCell counter before cell concentrations for both cell
lines were adjusted to 2.5.times.10.sup.6 in complete medium.
[0337] Clone B2 and LCL8664 cells were each added to a 96 well
round bottom non tissue-culture treated plate at 100,000 cells per
well. OX40mAb24 was added to cells in complete RPMI medium, to a
final concentration starting at 30 .mu.g/mL and diluted in 3-fold
increments. Similarly, R347 IgG1 (isotype control) was used at a
final concentration of 10 .mu.g/mL and diluted in 6-fold
increments. 9B12 and MOPC-21 (isotype control) were diluted in the
same manner. Plates were transferred to a 37.degree. C. incubator
with a humidified 5% CO.sub.2 atmosphere.
[0338] After 16 hours incubation time, 100 .mu.L reconstituted
Steady-Glo luciferase assay solution was added to each well and
mixed to lyse cells and then incubated to equilibrate luciferase
signal. Steady-Glo/sample lysate (150 .mu.L) was transferred from
each well to a 96 well, white walled assay plate for detection and
luminescence read using a Perkin Elmer Envision luminescence
reader.
[0339] The following protocol was used to acquire RBC-lysed rhesus
cells from sodium heparin anti-coagulated whole blood obtained from
a healthy Indian-origin rhesus macaque:
[0340] Fresh whole blood (5 mL) was added to a 50-mL conical tube
and 45 mL of ammonium chloride solution was added. The cell mixture
was incubated for 10 minutes on ice, then pelleted, and washed with
complete RPMI medium after which the cells were ready for use in
the 2-cell bioactivity assay.
[0341] The 2-cell bioactivity assay with rhesus RBC-lysed whole
blood cells was performed as described with the LCL8664 rhesus
B-cell line, but 10 .mu.g/mL NIP228 IgG1 was used as the
negative-control antibody.
7.2.2 Rhesus CD4 T Cell Proliferation Assay
[0342] OX40mAb24 bioactivity was determined in a CD4+ T cell
proliferation assay using activated primary rhesus CD4+ T cells and
plate-captured drug according to the following protocol. Rhesus
CD4+ T cells isolated from sodium heparin anti-coagulated whole
blood obtained from healthy rhesus monkeys (N=2) from World Wide
Primates were used as the source of responding cells.
[0343] Fresh heparinized rhesus blood was diluted 1:1 with PBS at
room temperature (RT). Then 20 mL of diluted whole blood was
overlayed onto 15 mL of 95% lymphocyte separation medium (LSM) in a
50-mL conical centrifuge tube. Blood was centrifuged at 400.times.g
for 30 minutes at room temperature without the brake. Peripheral
blood mononuclear cells were collected at the interface and washed
twice with Miltenyi MACS buffer and pelleted. The cell pellet was
treated with red cell lysis buffer for 5 minutes, and lysis buffer
was deactivated with the addition of complete RPMI medium. The
cells were washed and suspended in MACS buffer and counted with a
ViCell counter to determine cell number and viability.
[0344] Rhesus CD4+ T cells were isolated with a Miltenyi nonhuman
primate kit according to manufacturer's instructions. Then, CD4+ T
cells were counted on a ViCell counter, suspended at 1.times.106
cells/mL in complete RPMI medium with 51 .mu.g/mL Concanavalin A
and 1000 IU/mL of IL-2 and cultured at 37.degree. C. and 5% CO2 in
a humidified incubator for 2 days to activate T cells and induce
OX40 expression. Non-tissue culture treated round-bottom 96 well
assay plates were coated with 100 .mu.L of 2 .mu.g/mL goat
anti-mouse Fc.gamma.-specific IgG and 2 .mu.g/mL goat anti-human
Fc.gamma.-specific IgG in PBS. Goat anti-human IgG capture
antibodies were not added to wells intended for assay of soluble
OX40mAb24 activity. Plates were incubated overnight at 4.degree.
C., washed with 200 .mu.L of PBS, and blocked for 90 minutes at
37.degree. C. with 1% BSA in PBS (1% BSA/PBS). The plates were
washed with PBS and 2 ng/mL of anti-CD3 (clone SP34-2)
reconstituted in 1% BSA/PBS was added to the plates for 90 minutes
at 37.degree. C. The plates were washed with PBS to remove unbound
OX40mAb24, R347 human IgG1 control mAb, 9B12, and mouse IgG1
control mAb (clone MOPC-21) were each reconstituted in 1% BSA/PBS
starting at 1.01 .mu.g/mL (6.67 nM; experiment 1) or 1.5 .mu.g/mL
(10.0 nM; experiment 2) and serially diluted over a 3-fold dilution
series and then added to assay plates and incubated for 90 minutes
at 37.degree. C.
[0345] Activated primary rhesus CD4+ T cells were collected, washed
in complete RPMI medium, and the concentration adjusted to
1.0.times.106 viable cells/mL. Cells were labelled with
carboxyfluorescein succinimidyl ester (CFSE), according to the
manufacturer's instructions, with the exception of using 1.25 .mu.M
CFSE instead of the recommended 5 with an incubation of 10 minutes
at 37.degree. C. After labeling, cells were suspended in complete
RPMI and the concentration was adjusted to 1.0.times.106 per mL.
The plates were washed with PBS and 200 .mu.L of CD4+ T cells
(200,000/well) was added to each well. Then, the plate was
incubated at 37.degree. C. for 3 days.
[0346] After 72 hours incubation time, CD4+ T cells were pelleted,
and washed once with PBS containing 2% FBS (FACS buffer). Cells
were suspended in binding mix containing anti-CD4 V450.RTM. labeled
antibody for identification of CD4+ T cells, and propidium iodide
(PI) for live/non-viable cell discrimination, and incubated for 30
minutes. Following incubation, cells were washed in FACS buffer,
re-suspended in FACS buffer and analyzed by flow cytometry using an
LSRII flow cytometer and FlowJo software for analysis of Flow
Cytometry Standard (FCS) formatted data.
[0347] The assay was repeated in two independent experiments using
primary rhesus CD4+ T cells.
[0348] To assess cell proliferation, live (propidium
iodide-negative) events were gated using FlowJo software and the
percentage of CD4+-gated cells showing dilution of CFSE was
determined. Specific activity of OX40mAb24 was calculated by
subtracting the percentage of CD4+-gated cells showing dilution of
CFSE in response to anti-CD3 alone from the percentage of
CD4+-gated cells showing dilution of CSFE in response to anti-CD3
antibody plus OX40mAb24.
[0349] The concentrations of OX40mAb24 that achieved half-maximal
(EC50) responses in the Jurkat NF.kappa.B reporter assay and the
primary rhesus CD4+ T cell assay were determined using non-linear
regression analysis (log dose-response, 4-parameter fit curves) in
GraphPad Prism, version 5.01 (San Diego, Calif).
7.3 Results
7.3.1 Cyno/Rhesus 2-Cell Bioactivity Assay
[0350] Results of the cyno/rhesus 2-cell bioactivity assays are
shown in Table 7-2, FIG. 19A-B, FIG. 20A-B and FIG. 21A-B.
OX40mAb24 activated the OX40 signaling pathway, as measured by
NF.kappa.B signaling, in cyno/rhesus OX40-expressing Jurkat T cells
in the presence of the rhesus B-cell line, LCL8664, with a mean
EC50 of 1450 pM (N=2 experiments). As shown in Table 7-2, the EC50
value of OX40mAb24 activity in the cyno/rhesus 2-cell assay was 1.3
fold higher than in human 2-cell assays with the Raji B-cell line,
while 9B12 had limited activity in cyno/rhesus 2-cell assays and an
EC50 value could not be determined.
[0351] In addition, as shown in Table 7-2, OX40mAb24 activated the
OX40 signaling pathway in Jurkat T cells in the presence of rhesus
RBC-lysed whole blood, which is expected to contain Fc.gamma.
receptor-expressing cells, with an EC50 of 550 pM (N=1 experiment).
As Table 7-2 also shows, the EC50 value of 9B12 in the same assay
was 6052 pM, and was 4680 pM in a human 2-cell assay with the Raji
B-cell line.
TABLE-US-00025 TABLE 7-2 Mean Half Maximal Effective Concentration
for OX40mAb24 and 9B12 in 2-cell Bioactivity Assays 2-cell assay
format Rhesus RBC-lysed Rhesus LCL8664 B cells + whole blood +
cyno/rhesus OX40- cyno/rhesus OX40- Human Raji B cells + expressing
Jurkat NF.kappa.B- expressing Jurkat NF.kappa.B- human OX40 Jurkat
luciferase clone B2 luciferase clone B2 NF.kappa.B-luciferase clone
64 Test or Control bioactivity bioactivity bioactivity.sup.a
Article [mean EC.sub.50 (Std Err); n] [mean EC.sub.50 (Std Err); n]
[mean EC.sub.50 (Std Err); n] OX40mAb24 1450 (204); n = 2 550; n =
1 1110 (92.5); n = 5 R347 human IgG1 no activity; n = 2 NT no
activity NIP228 human NT no activity; n = 1 NT IgG1 9B12 ND 6052; n
= 1 4680 (1220); n = 5 Std Err = standard error of the mean. n =
number of experiments. NT = Not tested. ND = Not Determined.
.sup.aData from Example 5.
7.3.2 Rhesus Primary C-cell Proliferation Assay
[0352] Results for the rhesus primary T cell proliferation assay
are shown in Table 7-3, Table 7-4, and FIG. 22A-D. OX40mAb24
induced proliferation of primary rhesus CD4.sup.+ T cells with a
mean EC.sub.50 of 436 pM (Table 7-3; N=2 experiments). As shown in
Table 7-5, the EC.sub.50 value for 9B12 was 110 pM (Table 7-4; N=2
experiments). In OX40mAb24 induced proliferation of activated
primary human CD4.sup.+ T cells in a similar assay format with a
mean EC.sub.50 of 28 pM (Table 7-5; data from Example 4).
TABLE-US-00026 TABLE 7-3 Mean Half Maximal Effective Concentration
for OX40mAb24 in Primary Rhesus CD4 T cell Proliferation Assays
Test or control antibody No. of Experiments Mean EC.sub.50 (Std
Err) OX40mAb24 2 436 (309) NIP228 IgG1 2 No activity Std Err =
standard error of the mean.
TABLE-US-00027 TABLE 7-4 Mean Half Maximal Effective Concentration
for 9B12 in Primary Rhesus CD4 T cell Proliferation Assays Test or
control antibody No. of Experiments Mean EC.sub.50 (Std Err) 9B12 2
110 (13) MOPC-21 mouse IgG1 2 No activity Std Err = standard error
of the mean.
TABLE-US-00028 TABLE 7-5 Mean Half Maximal Effective Concentration
for OX40mAb24 and 9B12 in Rhesus and Human CD4 T cell Proliferation
Assays Rhesus Mean Human Mean Test or control antibody (Std Err)
(Std Err) OX40mAb24 436 (309) 28 (98) 9B12 110 (13) 218 (35) Std
Err = standard error of the mean.
7.4 Conclusions
[0353] OX40mAb24 activated the OX40 signaling pathway in a
cyno/rhesus OX40 expressing Jurkat T cell NF.kappa.B reporter cell
line in the presence of rhesus B cells or Fc.gamma.
receptor-expressing cells contained within RBC-lysed rhesus whole
blood. In addition, OX40mAb24 induced proliferation of primary
rhesus CD4.sup.+ T cells.
Example 8
Activity of OX40mAb24 in Mouse Models of Human Cancers
[0354] This example was designed to determine if OX40mAb24 is
effective as a single-agent therapy for the treatment of cancers.
This study was conducted in xenograft models of human cancers mixed
with alloreactive human T cells in immunocompromised non-obese
diabetic/severe combined immunodeficient (NOD/SCID) mice.
8.1 Materials
[0355] Materials used in this study, and their source, are listed
in Table 8-1.
TABLE-US-00029 TABLE 8-1 Materials Item Source DMEM medium
Invitrogen, Carlsbad, CA FBS Invitrogen, Carlsbad, CA Lymphocyte
separation medium VWR, West Chester, PA PBS Invitrogen, Carlsbad,
CA RPMI 1640 Invitrogen, Carlsbad, CA RosetteSep CD4.sup.+ T cell
Stem Cell Technologies, enrichment kit Vancouver, BC, Canada
RosetteSep CD8.sup.+ T cell Stem Cell Technologies, enrichment kit
Vancouver, BC, Canada RosetteSep DML medium Stem Cell Technologies,
Vancouver, BC, Canada Mitomycin C Sigma-Aldrich, St. Louis, MO
8.2 Experimental Protocols
8.2.1 Test Animals
[0356] Female NOD/SCID mice aged 5 to 9 weeks, were obtained from
Harlan Laboratories, Inc. The animals were humanely treated and
housed according to Iristithtional Animal Care and Use Committee
approved protocols in the Laboratory Animal Resources facility at
MedImmune, an Association for Animal Accreditation of Laboratory
Animal Care and United States Department of Agriculture-licensed
facility. The animals were kept in sterile micro-isolator units,
provided with sterile bedding and food, and acidified drinking
water ad libitum. Environmental conditions were standardized (room
temperature: 20.degree. C.+/-1.degree. C.; relative humidity:
50%+10%; 12-hour light-dark cycle). The animals were monitored
daily for adverse clinical signs and weekly for body weight.
8.2.2 Establishment of Xenografts
[0357] Human cancerous A375 cells originating from a human melanoma
cell line were obtained from ATCC. The cells were grown in DMEM
with 10% FCS at 37.degree. C. under 5% CO.sub.2 in a humidified
incubator. They were then harvested, washed once with PBS, then
resuspended in PBS.
[0358] Human cancerous A375 cells were harvested from cell
cultures. They were resuspended in PBS. A375 cells were
subsequently mixed with CD4.sup.+ and CD8.sup.+ T cell lines
alloreactive to A375 tumor cell lines before implantation into
animals.
[0359] To generate CD4.sup.+ and CD8.sup.+ T cell lines, human
peripheral blood mononuclear cells (PBMCs) from healthy donors were
enriched for CD4.sup.+ or CD8.sup.+ T cells by the addition of 1 mL
RosetteSep T cell enrichment product per 20 mL of whole blood. This
was followed by a 20-minute incubation and subsequent isolation by
density gradient centrifugation using RosetteSep DM-L density
medium. After centrifugation, the cells were washed 3 times with
PBS supplemented with 2% fetal bovine serum (FBS) and resuspended
in RPMI1640 medium supplemented with 10% FBS. Enriched CD4.sup.+
and CD8.sup.+ T cells were cultured separately for 7 to 10 days in
medium supplemented with recombinant human interleukin 2 (rhIL-2)
and each combined with mitomycin C-treated A375 cells. T cells were
collected and separately cultured again for 7 to 10 days in medium
supplemented with rhIL-2 and combined with mitomycin C-treated A375
cells. CD4.sup.+ and CD8.sup.+ T cells were collected and combined
at a 2:1 ratio.
[0360] A375 cells and PMBCs enriched for CD4.sup.+ and CD8.sup.+ T
cells were mixed at a ratio of 6 A375 cells to 1 T cell immediately
before implantation.
[0361] Xenografts were established by subcutaneous (SC) injection
of 3.5.times.10.sup.6 cells (human T cells mixed with A375 cells at
an effector-to-target (E:T) ratio of 1:6 and suspended in 200 .mu.L
of PBS) into the right flanks of the animals.
8.2.3 Randomization, Group Designation, and Dose Levels
[0362] Six animals were randomly assigned to each experimental
group prior to SC injection of cells. There were no animal
substitutions. Group designations and dose levels for each
experiment are listed in Table 8-2, Table 8-3 and Table 8-4. Test
and control antibodies were diluted in PBS to appropriate
concentrations and administered intraperitoneally (IP) in a total
voume of 200 .mu.L. The first dose of test and control antibodies
was administered on Day 3 or 4 after implantation of
cancer/effector T cells. The animals received up to 3 additional
doses of the test and control antibodies, as indicated in the
figtures and described in the corresponding figure description. The
formation of tumors was observed in each animal 1 or 2 times a
week. The primary endpoints in this study was either a tumor volume
of 2000 mm.sup.3 or gross tumor necrosis.
8.2.4 Tumor Measurements
[0363] Tumors were measured at intervals indicated in the figures
and tables for each experiment by caliper and tumor volumes (V)
were calculated using the following formula:
V(mm.sup.3)=(length[mm].times.width[mm].times.width[mm])/2.
[0364] For each group, the results are reported as the arithmetic
mean. Antitumor effects were expressed as percent tumor growth
inhibition (% TGI), which was calculated as follows:
% TGI=(1-[mean tumor V of treatment group].+-.[mean tumor V of
control group]).times.100
8.3 Statistical Methods
[0365] A comparison between OX40mAb24-treated or 9B12-treated, and
isotype control-treated animals was made, and intergroup
differences were analyzed for statistical significance by a
Mann-Whitney rank sum test.
[0366] Significant p-values obtained from the Mann-Whitney rank sum
test are presented in the summary tables and figures adjacent to
the arithmetic mean and standard deviation of the mean.
8.4 Results
[0367] The activity of OX40mAb24 on growth of tumors in mouse
models of human cancer was investigated in this study.
Immunodeficient NOD/SCID female animals were implanted with human
cancer cell lines mixed with alloreactive human CD4+ and CD8+ T
cell lines. CD4+ and CD8+ T cells were derived from PBMCs isolated
from healthy human donors. Animals received the first dose of the
test and control antibodies there or four days after implantation
of xenographs, and were administered additional doses of the test
and control antibodies as indicated.
[0368] In three separate experiments, OX40mAb24 plus alloreactive
human T cells significantly inhibited growth of A375 cells by up to
85% as compared to the isotype-control group (Table 8-2, Table 8-3,
and Table 8-4; FIG. 23A, FIG. 24A and FIG. 25). The control 9B12
plus alloreactive human T cells also significantly inhibited growth
of A375 cells by up to 77% as compared to the isotype-control group
(Tables 8-2 to 8-4, FIG. 23B and FIG. 24B.
TABLE-US-00030 TABLE 8-2 Treatment Groups and Percent TGI in A375
Xenograft Model on Day 18 (Experiment 2) Group .sup.a Test Antibody
Dose .sup.b (mg/kg) % TGI .sup.c 1 None; no T cells NA NA 2 None NA
NA 3 Isotype control 5 NA 4 OX40mAb24 5 79 5 OX40mAb24 2.5 75 6
OX40mAb24 1.0 85 7 9B12 5 53 IP = intraperitoneal; NA = not
applicable; TGI = tumor growth inhibition; V = volume .sup.a Number
of animals per group: 6. .sup.b All animals received 200 .mu.l of
test antibody IP on Days 4, 7, 9, and 12 .sup.c % TGI = [1 - (mean
tumor V of treatment group) / (mean tumor V of isotype control
group)] .times. 100
TABLE-US-00031 TABLE 8-3 Treatment Groups and Percent TGI in A375
Xenograft Model on Day 25 (Experiment 1) Group .sup.a Test Antibody
Dose .sup.b (mg/kg) % TGI .sup.c 1 None; no T cells NA NA 2 None NA
NA 3 Isotype control 5 NA 4 OX40mAb24 5 68 5 OX40mAb24 2.5 83 6
OX40mAb24 1.0 84 7 9B12 5 80 IP = intraperitoneal; NA = not
applicable; TGI = tumor growth inhibition; V = volume .sup.a Number
of animals per group: 6. .sup.b All animals received 200 .mu.l of
test antibody IP on Days 4, 7, 9, and 12 .sup.c % TGI = [1 - (mean
tumor V of treatment group) / (mean tumor V of isotype control
group)] .times. 100
TABLE-US-00032 TABLE 8-4 Treatment Groups and Percent TGI in A375
Xenograft Model on Day 28 (Experiment 3) Group .sup.a Test Antibody
Dose .sup.b (mg/kg) % TGI .sup.c 1 None; no T cells NA NA 2 None NA
NA 3 Isotype control 3.0 NA 4 OX40mAb24 3.0 75 5 OX40mAb24 1.0 73 6
OX40mAb24 0.3 68 7 OX40mAb24 0.1 84 8 OX40mAb24 0.03 73 IP =
intraperitoneal; NA = not applicable; TGI = tumor growth
inhibition; V = volume .sup.a N = 6. .sup.b All animals received
200 .mu.l of test antibody IP on Days 3, 6, 8, and 10 .sup.c % TGI
= [1 - (mean tumor V of treatment group) / (mean tumor V of isotype
control group)] .times. 100
8.5 Conclusions
[0369] OX40mAb24 demonstrated potent antitumor activity in mouse
models of human cancer mixed with alloreactive human T cells. The
antitumor activity of OX40mAb24 was similar to the antitumor
activity of 9B12. These results provide evidence that OX40mAb24 can
be effective as a single-agent therapy for the treatment of
patients with cancer with a T cell infiltrate.
Example 9
Rat/Mouse Anti-Mouse OX40 IgG2a Chimera Antibody Clone OX86
Inhibits the Growth of Mouse Cancer Cell Lines in Syngeneic
Models
[0370] OX40mAb24 does not cross-react to mouse (m)OX40 (See Example
2); therefore, it is not possible to test its activity in
immunocompetent mouse models. OX86 is a rat anti-mOX40 IgG1
antibody that specifically binds to and triggers signaling of mOX40
(al-Shamkhani et al., Eur. J. Immunol. 26:1695-1699 (1996)), and
has anti-tumor activity in immunocompetent mouse models of cancer
(Weinberg A D, et al., J. Immunol. 164:2160-2169 (2000)). To more
fully study the effects of OX40 agonism using a mouse surrogate
antibody with functional properties similar to OX40mAb24, a
rat/mouse anti-mOX40 IgG2a chimera antibody (OX86 mIgG2a; rat
anti-OX40 light and heavy chain variable regions with mouse IgG2a
constant regions) was generated from OX86. This example evaluates
the single-agent anti-tumor activity of OX86 mIgG2a in three mouse
models of cancer.
9.1 Materials
[0371] Materials used in this study, and their source, are listed
in Table 9-1.
TABLE-US-00033 TABLE 9-1 Materials Item Source Phosphate-buffered
saline, Life Technologies, pH 7.2 Carlsbad, CA Fetal bovine serum,
heat Life Technologies, inactivated Carlsbad, CA Roswell Park
Memorial Life Technologies, Institute 1640 medium Carlsbad, CA
0.25% Trypsin-EDTA (1x) Life Technologies, Carlsbad, CA EDTA =
Ethylenediaminetetraacetic acid.
9.2 Experimental Protocols
9.2.1 Test Animals
[0372] BALB/c and C57BL/6 mice of 6-8 weeks of age were received at
MedImmune from Harlan Laboratories, Inc. (Indianapolis, Ind.) and
allowed to acclimatize for 3 days prior to study start. Thereafter,
mice were shaved at tumor implantation sites and implanted with
microchip transponders for identification.
[0373] The animals were housed according to Institutional Animal
Care and Use Committee approved protocols in the Laboratory Animal
Resources facility at MedImmune, an Association for Animal
Accreditation of Laboratory Animal Care and United States
Department of Agriculture-licensed facility. The animals were kept
in sterile micro-isolator units, provided with sterile bedding and
food, and acidified drinking water ad libitum. Environmental
conditions were standardized (room temperature: 20.degree.
C..+-.1.degree. C.; relative humidity: 50%.+-.10%; 12-hour
light-dark cycle). The animals were monitored daily for adverse
clinical signs and bi-weekly for body weight. If hind limb
paralysis, respiratory distress, 20% body weight loss, or tumor
volume greater than 2000 mm.sup.3 were noted, the animals were
immediately sacrificed humanely by asphyxiation with CO.sub.2.
9.2.2 Establishment and Implantation of Syngeneic Tumors
[0374] CT26 and 4T1 were obtained from ATCC in Manassas, Va. MCA205
cells were obtrained from Providence Cancer Center, Portland, Oreg.
All cells were cultured in RPMI 1640 cell culture medium
supplemented with 10% FBS and grown at 37.degree. C. at 5% CO2 in a
humidified tissue culture chamber, then harvested, washed once in
FBS and then resuspended in PBS.
[0375] Allografts were established by subcutaneous (SC) injection
of 5.0.times.105 CT26 cells, or 1.0.times.105 4T1 cells suspended
in 0.1 mL of PBS into the right flank of 7- to 9-week-old BALB/c
mice, while 2.5.times.105 MCA205 cells suspended in 0.1 mL of
phosphate-buffered saline were injected into the right flank of 7-
to 9-week-old C57BL/6 mice.
9.2.3 Randomization, Group Designation, and Dose Levels
[0376] BALB/c (total of 216) and C57BL/6 (total of 70) female mice
were used in this study. BALB/c mice implanted with CT26 tumor
cells were randomly assigned after tumors grew to a mean volume of
120 mm3 per cohort, 9 days after implantation or 200 mm3 per
cohort, 13 days after implantation. BALB/c mice implanted with 4T1
tumor cells were randomly assigned after tumors grew to a mean
volume of 120 mm3 per cohort, 13 days after implantation. C57BL/6
mice were randomly assigned after tumors grew to a meanvolume of 95
mm3 per cohort, 11 days after implantation. Group designations
number of animals, dose levels, and dose schedule are presented in
Table 9-2, Table 9-3, Table 9-4, and Table 9-5. All test antibodies
and control antibodies were administered by intraperitoneal (IP)
injection. There were no animal substitutions.
[0377] Animals from each group were sacrificed when tumor volumes
reached approximately 2000 mm3 or when tumors became ulcerated or
necrotic.
TABLE-US-00034 TABLE 9-2 Study Design: CT26 Syngeneic Model Number
of Dose Dose animals schedule level Group (M/F) Treatment (study
day) (mg/kg).sup.a ROA 1 10 (F) None NA NA IP 2 10 (F) Negative
control 9, 12 2.5 IP (OX86 mIgG1 D265A) 3 10 (F) OX86 mIgG2a 9, 12
2.5 IP 4 10 (F) OX86 mIgG2a 9, 12 1.0 IP 5 10 (F) OX86 mIgG2a 9, 12
0.25 IP 6 10 (F) OX86 mIgG2a 9, 12 0.1 IP F = female; IgG1 =
immunoglobulin G1; IP = intraperitoneal; mAb = monoclonal antibody;
OX86 mIgG2a = rat anti-OX40 light and heavy chain variable regions
of clone OX86 with mouse IgG2a constant regions; OX86 mIgG1 D265A =
mouse anti-mouse OX40 mIgG1 with a point mutation in the Fc domain
that reduces its ability to bind Fcy receptors; NA = not applicable
because the animals were not treated; ROA = route of
administration. .sup.aDose volume: 0.2 mL.
TABLE-US-00035 TABLE 9-3 Study Design: CT26 Syngeneic Model Number
of animals Dose schedule Dose level Group (M/F) Treatment (study
day) (mg/kg).sup.a ROA 1 12 (F) None NA NA IP 2 12 (F) OX86 mIgG2a
13, 16 10 IP 3 12 (F) OX86 mIgG2a 13, 16 3 IP 4 12 (F) OX86 mIgG2a
13, 16 1 IP 5 12 (F) OX86 mIgG2a 13, 16 0.3 IP 6 12 (F) OX86 mIgG2a
13, 16 0.1 IP F = female; IP = intraperitoneal; 0X86 mIgG2a = rat
anti-OX40 ligh and heavy chain variable regions of clone OX86 with
mouse IgG2a constant regions; NA = not applicable because the
animals were not treated; ROA = route of administration. .sup.aDose
volume: 0.2 mL.
TABLE-US-00036 TABLE 9-4 Study Design: MCA205 Syngeneic Model
Number of animals Dose schedule Dose level Group (M/F) Treatment
(study day) (mg/kg).sup.a ROA 1 14 (F) None NA NA IP 2 14 (F)
Isotype control 11, 14 20 IP (mixture).sup.b 3 14 (F) OX86 mIgG2a
11, 14 10 IP 4 14 (F) OX86 mIgG2a 11, 14 10 IP 5 14 (F) OX86 mIgG2a
11, 14 5 IP F = female; IP = intraperitoneal; 0X86 mIgG2a = rat
anti-OX40 light and heavy chain variable regions of clone OX86 with
mouse IgG2a constant regions; NA = not applicable because the
animals were not treated; ROA = route of administration. .sup.aDose
volume: 0.2 mL. .sup.bMixture contains isotype control antibodies
with Fc domains of mIgG2a (10 mg/kg) and mIgG2b (10 mg/kg).
TABLE-US-00037 TABLE 9-5 Study Design: 4T1 Syngeneic Model Number
of animals Dose schedule Dose level Group (M/F) Treatment (study
day) (mg/kg).sup.a ROA 1 12 (F) None NA NA IP 2 12 (F) Isotype
control 13, 16, 20, 23 70 IP (mixture).sup.b 3 12 (F) OX86 mIgG2a
13, 16, 20, 23 10 IP 4 12 (F) OX86 mIgG2a 13, 16, 20, 23 5 IP 5 12
(F) OX86 mIgG2a 13, 16, 20, 23 2.5 IP 6 12 (F) OX86 mIgG2a 13, 16,
20, 23 1.0 IP 7 12 (F) OX86 mIgG2a 13, 16, 20, 23 0.25 IP F =
female; IP = intraperitoneal; 0X86 mIgG2a = rat anti-OX40 light and
heavy chain variable regions of clone OX86 with mouse IgG2a
constant regions; NA = not applicable because the animals were not
treated; ROA = route of administration. .sup.aDose volume: 0.2 mL.
.sup.bMixture contains isotype control antibodies with Fc domains
of mIgG2a (10 mg/kg), mIgG2b (20 mg/kg), rat IgG2a (20 mg/kg) and
rat IgG2b (20 mg/kg).
9.2.4 Tumor Measurements
[0378] Tumors were measured by caliper twice weekly and tumor
volumes were calculated using the following formula:
tumor volume (V)=[length (mm).times.width (mm).sup.2]/2
[0379] where length was defined as the larger side and width as the
smaller side perpendicular to the length.
[0380] Antitumor effects of each group were expressed as tumor
growth inhibition (TGI), which was calculated as follows:
% TGI=(1-[mean V of treatment group].+-.[mean V of control
group].times.100
[0381] Tumor growth responses were categorized as a complete
response (CR) if there was no measureable tumor.
9.3 Statistical Methods
[0382] One-way ANOVA was used to determine mean tumor volume
differences. In the event of a significant F test a Dunnett's or
Sidak's multiple comparison test was utilized (where appropriate).
Where applicable, a log10 transformation was applied to tumor
volumes to account for heteroscedasticity. A p value <0.05 was
considered significant.
9.4 Results
[0383] Treatment of mice with the OX86 mIgG2a results in
significantly reduced growth of CT26 and MCA205 tumor cells
compared to untreated or negative, or isotype control
antibody-treated mice control, (Table 9-6, Table 9-7 and Table 9-8;
FIG. 26A, FIG. 27A, and FIG. 28A). Treatment of mice with the OX86
mIgG2a results in delayed and reduced growth of 4T1 tumor cells
compared to untreated or isotype control antibody (Table 9-9 and
FIG. 29A).
[0384] A mixed response is often shown in syngeneic models;
however, the dramatic response of the treatment with OX86 mIgG2a is
clearer from the individual animal tumor growth graphs (FIG. 26B,
FIG. 27B, FIG. 28B and FIG. 29B). A dose response was not observed
in mice treated with OX86 mIgG2a based on TGI (Table 9-6, Table
9-7, Table 9-8 and Table 9-9; FIG. 26A, FIG. 27A, FIG. 28A and FIG.
29A) or with respect to increasing the number of animals exhibiting
complete responses (Table 9-6, Table 9-7, Table 9-8 and Table
9-9).
TABLE-US-00038 TABLE 9-6 Treatment Groups, Percent Tumor Growth
Inhibition on Day 22, and number of Complete Responders in CT26
Syngeneic Model Number of Complete Test/Control Dose .sup.b %
Responders out Group .sup.a Antibody (mg/kg) TGI .sup.c of 12 mice
.sup.d 1 None NA NA 0 2 OX86 mIgG2a 10 67 8 3 OX86 mIgG2a 3 67 6 4
OX86 mIgG2a 10 75 8 5 OX86 mIgG2a 0.3 65 8 6 OX86 mIgG2a 0.1 70 8
IP = intraperitoneal; NA = not applicable; TGI = tumor growth
inhibition; V volume. .sup.a n = 12. .sup.b All animals received
200 .mu.L of test antibody IP on Days 13 and 16. .sup.c % TGI = [1
- (mean tumor V of treatment group) / (mean tumor V of control
group)] .times. 100. .sup.d Number of animals in a group with a
tumor volume measurement recorded as zero at the end of the
study.
TABLE-US-00039 TABLE 9-7 Treatment Groups, Percent Tumor Growth
Inhibition on Day 26, and number of Complete Responders in CT26
Syngeneic Model Number of Complete Test/Control Dose .sup.b %
Responders out Group .sup.a Antibody (mg/kg) TGI .sup.c of 10 mice
.sup.d 1 None NA NA 1 2 Negative control 2.5 NA 1 (OX86 mIgG1
D265A) 3 OX86 mIgG2a 2.5 94 7 4 OX86 mIgG2a 1.0 97 8 5 OX86 mIgG2a
0.25 95 6 6 OX86 mIgG2a 0.1 92 7 IP = intraperitoneal NA = not
applicable; TGI = tumor growth inhibition; V volume. .sup.a n = 10.
.sup.b All animals received 200 .mu.L of test antibody IP on Days 9
and 12. .sup.c % TGI = [1 - (mean tumor V of treatment group) /
(mean tumor V of control group)] .times. 100. .sup.d Number of
animals in a group with a tumor volume measurement recorded as zero
at the end of the study.
TABLE-US-00040 TABLE 9-8 Treatment Groups, Percent Tumor Growth
Inhibition on Day 22, and Number of Complete Responders in MCA205
Syngeneic Model Number of Complete Test/Control Dose .sup.b %
Responders out Group .sup.a Antibody (mg/kg) TGI .sup.c of 14 mice
.sup.d 1 None NA NA 0 2 Isotype control 20 NA 0 (mixture) 3 OX86
mIgG2a 20 65 0 4 OX86 mIgG2a 10 76 4 5 OX86 mIgG2a 5 70 2
TABLE-US-00041 TABLE 9-9 Treatment Groups, Percent Tumor Growth
Inhibition on Day 25, and number of Complete Responders in 4T1
Syngeneic Model Number of Complete Test/Control Dose .sup.b %
Responders out Group .sup.a Antibody (mg/kg) TGI .sup.c of 12 mice
.sup.d 1 None NA NA 0 2 Isotype control 70 NA 0 (mixture) 3 OX86
mIgG2a 10 5 0 4 OX86 mIgG2a 5 34 2 5 OX86 mIgG2a 2.5 37 0 6 OX86
mIgG2a 1.0 22 0 7 OX86 mIgG2a 0.25 ND 0 IP = intraperitoneal; NA =
not applicable; TGI = tumor growth inhibition; V volume; ND = not
determined. .sup.a n = 12. .sup.b All animals received 200 .mu.L of
test antibody IP on Days 13, 16, 20 and 23. .sup.c % TGI = [1 -
(mean tumor V of treatment group) / (mean tumor V of control
group)] .times. 100. .sup.d Number of animals in a group with a
tumor volume measurement recorded as zero at the end of the
study.
9-5 Conclusions
[0385] Single-agent treatment of tumor-bearing mice with OX86
mIgG2a results in anti-tumor activity that significantly reduces
growth of multiple tumors as compared to untreated, negative
control, and/or isotype control treated groups.
Example 10
Characterization of the Epitope of OX40mAb24
[0386] This example examines the epitope of OX40mAb24 using a
series of human/mouse OX40 chimeric variants.
10.1 Materials used in this study, and their source, are listed in
Table 10-1.
TABLE-US-00042 TABLE 10-1 Materials Item Source Anti-human
IgG-Alexa480 Invitrogen (Carlsbad, CA) Anti-sheep IgG-Alexa-480
Invitrogen (Carlsbad, CA) Anti-goat IgG-Alexa480 Invitrogen
(Carlsbad, CA) FreeStyle 293F cells Invitrogen (Carlsbad, CA)
(HEK293F cells) Goat anti-mouse OX40 R&D Systems (Minneapolis,
MN) polyclonal antibody LSRII flow cytometer BD Biosciences (San
Jose, CA) Phosphate buffered saline Invitrogen (Carlsbad, CA) Sheep
anti-human OX40 R&D Systems (Minneapolis, MN) polyclonal
antibody 293fectin transfection reagent Invitrogen (Carlsbad,
CA)
10.2 Generation of Human/Mouse Chimeric Variants
[0387] OX40mAb24 binds specifically to human OX40 (SEQ ID NO: 91)
and does not recognize mouse OX40 (SEQ ID NO: 92), despite sharing
60% identity in their amino acid (aa) sequence (FIG. 30).
Human/mouse chimeric OX40 variants were engineered by swapping
portions of the extracellular domain between human and mouse OX40.
The cDNA constructs of human and mouse OX40 were used as templates
in overlapping extension PCR to construct chimeric variants with a
transmembrane domain for cell-surface expression of the recombinant
proteins. OX40 is an approximately 45 kDa protein with three
complete cysteine-rich domains (CRDs) and one truncated
cysteine-rich domain. The structure is characteristic of the TNFR
superfamily.
[0388] Thirteen chimeric knock-out (KO/loss-of-function) variants
were constructed by replacing the following residues of the
extracellular domain of human OX40 with the corresponding mouse
OX40 aa or Alanine (FIG. 31): [0389] CRD1 (human OX40 aa 29 to 65
replaced with the mouse counterparts); [0390] CRD2 (human OX40 aa
66 to 107 replaced with the mouse counterparts); [0391] CRD3 (human
OX40 aa 108 to 146 replaced with the mouse counterparts); [0392]
CRD4+linker (human OX40 aa 147 to 214 replaced with the mouse
counterparts); [0393] CRD3+4 (human OX40 aa 108 to 167 replaced
with its mouse counterparts); [0394] A.sup.111 (human OX40 aa
alanine 111 replaced with its mouse counterpart proline); [0395]
L.sup.116 (human OX40 aa leucine 116 replaced with its mouse
counterpart glutamine); [0396] P.sup.121 (human OX40 aa proline 121
replaced with its mouse counterpart leucine); [0397] A.sup.126
(human OX40 aa alanine 126 replaced with its mouse counterpart
valine); [0398] D.sup.137 (human OX40 aa aspartic acid 137 replaced
with its mouse counterpart asparagine); [0399]
A.sup.111P.sup.121D.sup.137 (human OX40 aa Ala111, Pro121 and
Asp137 replaced with the mouse counterparts); [0400]
L.sup.116A.sup.126 (human OX40 aa Leu116 and Ala126 replaced with
the mouse counterparts); and [0401] D.sup.117S.sup.118 (human OX40
aa Asp117 and Ser118 replaced with Ala mutations).
[0402] In addition, five knock-in (KI/gain-of-function) variants
were constructed by grafting the following residues of human OX40
into mouse OX40 using the same method as described above for the KO
constructs (FIG. 31): [0403] CRD3 (human OX40 aa 108 to 146 grafted
into the corresponding mouse regions); [0404] A.sup.111 (human OX40
aa alanine 111 grafted into the corresponding mouse position);
[0405] L.sup.116 (human OX40 aa leucine 116 grafted into the
corresponding mouse position); [0406] A.sup.126 (human OX40 aa
alanine 126 grafted into the corresponding mouse position); [0407]
L.sup.116A.sup.126 (human OX40 aa leucine 116 and alanine 126
grafted into the corresponding mouse positions).
[0408] The resulting chimeric DNAs were closed into a mammalian
expression vector pEBNA for transient mammalian expression. 293F
cells were seeded at a density of 0.7.times.10.sup.6 cells/mL one
day prior to transfection. Three and a half micro grams of each
expression vector were transfected into 5 mL of HEK293 F cells
using 5 .mu.L of 293fectin transfection reagent following the
manufacturer's instructions.
10.3 Characterization of Binding of OX40mAb24 to Chimeric OX40
Variants
[0409] Forty-eight hours after transfection, HEK293F cells were
incubated with 1 .mu.g/mL of OX40mAb24 for 1 hour on ice in PBS. To
detect bound OX40mAb24 by flow cytometry, the cells were washed
3-times with cold PBS, incubated with 1 .mu.g/mL of
Alexa480-conjugated anti-human IgG antibody for 1 hr on ice, and
then analyzed using the LSRII flow cytometer.
[0410] The expression levels of all chimeric OX40 variants were
also monitored by flow cytometry; the cells were incubated with a
mixture of sheep anti-human OX40 and goat anti-mouse OX40
polyclonal antibodies at 1 .mu.g/mL in PBS for 1 hour on ice. Cells
were washed 3-times with cold PBS, and then incubated with a
mixture of Alexa480-conjugated anti-goat and anti-sheep IgG
polyclonal antibodies. After washing 3 times with cold PBS, cells
were analyzed with the LSRII flow cytometer.
10.4 Results
[0411] The epitope of OX40mAb24 was characterized using chimeric
human/mouse variants. Human OX40 and mouse OX40 share 60% identity
in the aa sequence (FIG. 30); yet, OX40mAb24 binds specifically to
human OX40 and does not recognize mouse OX40. This specificity was
employed to identify the epitope of OX40mAb24. Eighteen chimeric
variants were constructed by swapping in or out various domains of
the extracellular domain of mouse OX40 into human OX40
(KO/loss-of-function variants) or of human OX40 into mouse OX40
(KI/gain-of-function variants) (FIG. 31). All variants encoded a
transmembrane domain for cell-surface expression of the chimeric
protein. The binding characteristics of OX40mAb24 to these variants
were analyzed by flow cytometry.
[0412] The epitope of OX40mAb24 was mapped to the CRD3 domain of
human OX40 by swapping individual domains between human and mouse
OX40s. The binding of OX40mAb24 to human OX40 was abolished when
replacing human CRD3 domain with the mouse counterpart (KO_CRD3 and
KO_CRD3-4) (FIG. 32A-C). Replacement of the other human CRD domains
with mouse regions did not affect the binding of OX40mAb24
(KO_CRD1, KO_CRD2, and KO_CRD4+linker). Furthermore, grafting the
human CRD3 domain into mouse OX40 led to the binding of OX40mAb24
(KI_CRD3). All variants were expressed as monitored by anti-human
and mouse OX40s polyclonal antibodies (FIG. 32A-C). The parental
mAb of OX40mAb24, 9B12, was also characterized in the binding
study. 9B12 shows the same binding profiles to these variants as
OX40mAb24, suggesting both IgGs recognize the same epitope of the
CRD3 domain.
[0413] Furthermore, critical epitope residues Leu116 and Ala126
were identified in the CRD3 domain by mutating the amino acids that
are different between human and mouse OX40s. Five amino acids,
including Ala111, Leu116, Pro121, Ala126, and Asp137 (FIG. 30) in
the human CRD3 domain, were mutated to corresponding mouse residues
or Ala as individual amino acids or different combinations (FIG.
31). The binding of OX40mAb24 was abolished when replacing human
residues Leu116 and Ala126 with the mouse counterparts
(KO_L116+A126). Furthermore, the KI/gain-of-function variants
confirmed the importance of these two critical residues. Grafting
Leu116, Ala126, or the combination into mouse OX40 led to the
binding of OX40mAb24.
10.5 Conclusions
[0414] The epitope OX40mAb24 was mapped to the CRD3 domain of human
OX40 using human/mouse chimeric variants, with critical residues
Leu.sup.116 and Ala.sup.126. The binding profiles to all variants
between OX40mAb24 and its parental 9B12 are identical, indicating
they recognize the same epitope on human OX40.
Example 11
Pharmacodynamic Activity of the Rat/Mouse Anti-Mouse OX40 IgG2a
Chimera Antibody Clone OX86 in Naive Mice and in Mice Bearing
Syngeneic Tumors
[0415] OX86 is a rat anti-mOX40 IgG1 antibody that specifically
binds to and triggers signaling of MOX40 (al-Shamkhani et al,
1996), and has anti-tumor activity in immunocompetent mouse models
of cancer (Weinberg et al, 2000). To more fully study the effects
of OX40 agonism using a mouse surrogate antibody with functional
properties similar to OX40mAb24, this example utilizes the
rat/mouse anti-mOX40 IgG2a chimera antibody OX86 described in
Example 9. However, it is possible to draw some parallels between
the species, for example mIgG2a and human IgG1 are often considered
equivalent functionally as both isotypes are able to bind multiple
Fc.gamma. receptors, are capable of high affinity Fc.gamma.
receptor binding and are able to trigger ADCC and bind to human
C1q, a prerequisite for complement-dependent cytotoxicity (CDC) by
the classical complement pathway (Stewart et al. 2014; Dall'Acqua
et al, 2006). OX86 mIGg2a binds to mouse OX40 (see Example 9) and
was used as a surrogate mouse OX40 agonist antibody for
OX40mAb24.
[0416] In this example, the ability of OX86 mIgG2a to induce T
cells in naive and tumor bearing mice, and to enter the cell cycle
and proliferate. Further, K167 and ICOS were evaluated as a
potential biomarker of OX40 agonist activity, was investigated.
T-cell proliferation and anti-tumor activity was determined in two
syngeneic mouse models of cancer. Moreover, finally, the role of
activating (Fc.gamma. receptors I, III and IV) and/or inhibitory
(Cf.gamma. receptor IIb) Fc.gamma. receptors play in vivo activity
of OX86 mIgG2a was determined.
11.1 Materials and Methods
11.1.1 Animal Receipt and Identification
[0417] Wild-type Fcgr2b.sup.-/- or Fcer1g.sup.-/- BALB/c and
Fcgr2b.sup.-/- or Fcer1g.sup.-/- C57BL/6 mice of 6-8 weeks of age
were received at MedImmune from Harlan Laboratories, Inc.
(Indianapolis, Ind. or Charles River Laboratories (UK)) and allowed
to acclimatize for .ltoreq.15 days prior to study start.
Thereafter, mice were implanted with microchip transponders to
identify individual mice.
11.1.2 Housing
[0418] The animals were humanely treated and housed according to
Institutional Animal Care and Use Committee (USA) and Home Office
(UK) approved protocols in the Laboratory Animal Resources facility
(USA) and Biological Sciences Unit (UK) at MedImmune, an
Association for Animal Accreditation of Laboratory Animal Care and
United States Department of Agriculture-licensed facility. The
animals were kept in sterile micro-isolator units, provided with
sterile bedding and food, and acidified drinking water (USA) or tap
water (UK) ad libitum. Environmental conditions were standardized
(room temperature: 20.degree. C..+-.1.degree. C. (USA) or
21.degree. C..+-.1.degree. C. (UK); relative humidity: 50%.+-.10%
(USA) or 55.+-.10% (UK); 12-hour light-dark cycle). The animals
were monitored daily for adverse clinical signs and bi-weekly for
body weight. If hind limb paralysis, respiratory distress, 20% body
weight loss, or tumor volume greater than 2000 mm.sup.3 were noted,
the animals were immediately sacrificed humanely by cervical
dislocation or asphyxiation with CO.sub.2.
11.1.3 Establishment of Syngeneic Tumors
[0419] The CT26 cell line (mouse colon carcinoma) was obtained from
ATCC, Manassas, Va., and the cell line MCA 205 (chemically-induced
mouse soft tissue sarcoma) was obtained from the Providence Cancer
Center, Portland, Oreg. Both cell lines were maintained in RPMI
1640 medium +10% FBS at 37.degree. C., 5% CO2.
[0420] Allografts were established by subcutaneous (SC) injection
of 5.0.times.105 CT26 cells, resuspended in 0.1 mL of PBS, into the
right flank of 7- to 9-week-old wild-type, Fcgr2b-/- or Fcer1g-/-
BALB/c mice. Allografts were also established by SC injection of
2.5.times.105 MCA205 cells, resuspended in 0.1 mL of PBS, into the
right flank of 7- to 9-week-old wild-type, Fcgr2b-/- or Fcer1g-/-
C57BL/6 mice.
11.1.4 Randomization, Group Designation and Dose Levels
[0421] Wild-type (n=70), Fcgr2b.sup.-/- (n=36) or Fcer1g.sup.-/-
(n=36) BALB/c and Fcgr2b.sup.-/- (n=36) or Fcer1g.sup.-/- C57BL/6
female mice (n=36) were used in this study. All mice were randomly
assigned into treatment groups. Group designations, number of
animals, dose levels and dose schedule are presented in Table 11-1,
Table 11-2 and Table 11-3. All test articles and control articles
were administered intraperitoneally (IP). There were no animal
substitutions.
[0422] Animals from each group were sacrificed when tumor volumes
reached approximately 2000 mm.sup.3 or when tumors became ulcerated
or necrotic.
TABLE-US-00043 TABLE 11-1 Group Designation and Dose Levels of
Naive Balb/c Mice Number of Dose Dose animals schedule level Group
(M/F) Treatment (study day) (mg/kg).sup.a ROA 1, 9 7 (F), 7 (F)
Saline 1 NA IP 3, 11 7 (F), 7 (F) Isotype control 1 20 IP (NIP228
IgG2a) 4, 12 7 (F) OX86 mIgG2a 1 20 IP 5, 13 7 (F), 7 (F) OX86
mIgG2a 1 2 IP 6, 14 7 (F), 7 (F) OX86 mIgG2a 1 0.2 IP F = female; M
= male; NA = not applicable; IP = intraperitoneal; ROA = route of
administration; OX86 mIgG2a = mouse anti-mouse OX40 IgG2a
monoclonal antibody variant of OX86 .sup.aDose volume: 0.2 mL for
saline or volume adjusted to body weight; 10 mL/kg for all other
groups.
TABLE-US-00044 TABLE 11-2 Group Designation and Dose Levels in the
CT26 Syngeneic Model Number of Mouse Dose schedule Dose level Group
animals (M/F) Strain Treatment (study day) (mg/kg).sup.a ROA 1 8
(F) Balb/c None NA NA IP 2 8 (F) Fcgr2b.sup.-/- Control 4, 7 2.5 IP
(OX86 mIgG1 D265A) 3 8 (F) OX86 mIgG2a 4, 7 2.5 IP 4 8 (F) Balb/c
None NA NA IP 5 8 (F) Fcer1g.sup.-/- Control 4, 7 2.5 IP (OX86
mIgG1 D265A) 6 8 (F) OX86 mIgG2a 4, 7 2.5 IP F = female; M = male;
NA = not applicable because the animals were not treated; IP =
intraperitoneal; ROA = route of administration; OX86 mIgG2a = rat
anti-OX40 light and heavy chain variable regions of clone OX86 with
mouse IgG2a constant regions. .sup.aDose volume: 0.2 mL.
TABLE-US-00045 TABLE 11-3 Group Designation and Dose Levels in the
MCA205 Syngeneic Model Number of Mouse Dose schedule Dose level
Group animals (M/F) Strain Treatment (study day) (mg/kg).sup.a ROA
1 8 (F) C57BL/6 None NA NA IP 2 8 (F) Fcgr2b.sup.-/- Control 4, 7
7.5 IP (OX86 mIgG1 D265A) 3 8 (F) OX86 mIgG2a 4, 7 7.5 IP 4 8 (F)
C57BL/6 None NA NA IP 5 8 (F) Fcer1g.sup.-/- Control 4, 7 7.5 IP
(OX86 mIgG1 D265A) 6 8 (F) OX86 mIgG2a 4, 7 7.5 IP F = female; M =
male; NA = not applicable because the animals were not treated; IP
= intraperitoneal; ROA = route of administration; OX86 mIgG2a = rat
anti-OX40 light and heavy chain variable regions of clone OX86 with
mouse IgG2a constant regions. .sup.aDose volume: 0.2 mL.
11.1.5 Tumor Measurements
[0423] Tumors were measured by caliper twice weekly and tumor
volumes were calculated using the following formula:
tumor volume=[length (mm).times.width (mm).sup.2]/2
[0424] where length was defined as the larger side, and width as
the smaller side perpendicular to the length.
[0425] Antitumor effects of each group were expressed as tumor
growth inhibition (TGI), which was calculated as follows:
% TGI=(1-[mean tumor V of treatment group].+-.[mean tumor V of
control group]).times.100
11.1.6 Tissue Collection and Single Cell Isolation
11.1.6.1 Preparation of Mouse Blood Cells for Flow Cytometry
Analysis
[0426] Red blood cell lysis (RBCL) buffer (2 mL) was added to blood
(50 .mu.L) and incubated for 5 min. Volumes obtained from in-life
bleeds ranged from 20 .mu.L to 50 .mu.L. Terminal bleeds were 50
.mu.L. RPMI+10% fetal bovine serum (FBS) (8 mL) was added to each
sample. Cells in each sample were pelleted (300.times.g, 5 min) and
then resuspended in 0.3 mL Flow Buffer. Cell suspensions (200
.mu.L) were added to each well of a 96-well round-bottomed plate
for staining with fluorescent antibodies. Pooled group samples were
used for unstained, the single antibody staining, isotype staining
and the fluorescence-minus-one (FMO) antibody staining
controls.
11.1.6.2 Mouse Draining Lymph Node and Spleen Isolation and
Generation of Single Cell Suspensions for Flow Cytometry
Analysis
[0427] Spleens were placed in 10 mL of RPMI+1.times.Pen/Strep
solution and passed through a 40 .mu.m cell strainer. Samples were
pelleted (300 .times.g, 5 min) and then resuspended in 1 mL RBCL
buffer for 3 min. RPMI+FBS 10% (9 mL) was added to each sample.
Cell suspensions were pelleted (300.times.g, 5 min) and resuspended
in 1 mL of Flow Buffer. Cell suspensions (100 .mu.L) were added to
each well of a 96-well round-bottomed plate for staining with
fluorescent antibodies. Pooled group samples were used for
unstained, the single antibody staining, isotype staining and FMO
antibody staining controls.
11.1.6.3 Preparation of Single-cell Suspension of Mouse Tumor for
Flow Cytometry Analysis
[0428] Tumors were aseptically excised from euthanized mice, being
careful to avoid collecting connective tissue or skin, and placed
in collagenase diluted with Hanks balanced salt solution in a
6-well dish. To increase the efficiency of the enzymatic digestion
process, each tumor was minced with a scalpel or a small pair of
sharp scissors into smaller pieces. Minced tumors were transferred
into 15 mL conical tubes and placed on a shaking platform to
incubate at 37.degree. C. for 20-30 minutes. Five mL of RPMI+10%
FBS was added to each sample to deactivate the collagenase and
maintain viability of the immune cells. Samples were passed through
a 70 .mu.M cell strainer and placed into 50 mL conical tubes. An
aliquot of each sample was removed for counting. The remaining
amount sample was pelleted in a centrifuge at 330.times.g and
resuspended in FACS buffer (PBS+2% FBS) at a concentration of
1.times.10.sup.7 cells per mL.
11.1.7 Flow Cytometry Analysis
[0429] 11.1.7.1 Analysis of Tissues from Non-Tumor-Bearing Mice
[0430] Single-cell suspensions of blood or spleen cells were
pelleted (300.times.g, 5 min), resuspended in 50 .mu.L of Fc Block
solution (1:50), diluted in eBiosciences Flow Buffer and incubated
for 10 min on ice. Fifty microliters of fluorescently labelled
antibodies (2.times. stock solution) were added to each sample
(final volume 100.mu.L). Single antibody staining solutions
(extracellular) were added at 1 .mu.L per well and cell/antibody
mixtures were incubated for 30 min on ice. Cells were pelleted
(300.times.g, 5 min), washed twice (200 .mu.L Flow Buffer per well,
300.times.g, 5 min), resuspended in 50 .mu.L Fix/Penn buffer (one
part concentrate to three part diluent) and then incubated
overnight at 4.degree. C. in the dark.
[0431] Treated cells were washed twice in 1.times. Permeabilization
Buffer (diluted in water). Intracellular labelling antibodies were
diluted into Permeabilization Buffer (100 .mu.L per well) and added
to the cells, which were then incubated at room temperature in the
dark for 30 minutes. Antibodies for single marker staining
(intracellular) were added to cells at 1 .mu.L per well into 100
.mu.L Permeabilization Buffer and incubated for 30 min on ice in
the dark. Cells were washed once in Permeabilization Buffer and
resuspended in 3.7% formaldehyde solution (100 .mu.L) before being
analyzed on a Canto II flow cytometer (BD Biosciences, San Jose,
Calif.).
11.1.7.2 Analysis of Tissues from Tumor-Bearing Mice
11.1.7.2.1 Cell Surface Staining
[0432] Each sample of pelleted cells was resuspended in FACS
Buffer. Fluorescent antibody mixtures in FACS Buffer were added to
appropriate wells and incubated for 20-30 minutes at 4.degree. C.
in the dark. Cells were washed twice with FACS Buffer, resuspended
in FACS Buffer and analyzed on a LSRII flow cytometer (BD
Biosciences, San Jose, Calif.).
11.1.7.2.2 Intracellular Staining (Fixed and Permeabilized
Cells)
[0433] Each sample of pelleted cells was resuspended in 50 .mu.L of
a 1:500 dilution of a fluorescence fixable blue dye for
live/non-viable cell discrimination, and incubated for 15 minutes
at 4.degree. C. in the dark. Cells were washed once with FACS
Buffer, resuspended in 30 .mu.L PBS+4% normal mouse serum
containing Fc Block, and incubated for 15 minutes at room
temperature. Fluorescent antibody mixtures in FACS Buffer were
added to appropriate wells, and incubated for 20-30 minutes at
4.degree. C. in the dark. Cells were washed twice with FACS Buffer,
resuspended in 150 .mu.L freshly prepared FoxP3 Fix/Penn working
solution and incubated at room temperature for 30 minutes in the
dark. Cells were washed twice with 1.times. Permeabilization
Buffer, resuspended in 100 .mu.L 1.times. Permeabilization Buffer
containing antibodies to stain intracellular antigens, and
incubated at room temperature for 30 minutes in the dark. Cells
were washed once with 1.times. Permeabilization Buffer, resuspended
in FACS Buffer and analyzed on a LSRII flow cytometer.
11.1.8 Statistical Methods
[0434] One-way ANOVA was used to determine mean tumor volume
differences and mean percentage of Ki67+ or ICOS+ cells. In the
event of a significant F test, a Dunnett's or Sidak's multiple
comparison test was utilized (where appropriate). Where applicable,
a log10 transformation was applied to mean values to account for
heteroscedasticity. A p value <0.05 was considered significant.
GraphPad Prism 6.0 was employed for linear regression analysis
which generated the best fit line that best predicted Y from X, the
level of statistical significance of the line and the goodness of
fit (r.sup.2) of the determined best fit line.
11.1.9 Materials
[0435] Materials used in this study, and their source, are listed
in Table 11-4 and Table 11-5.
TABLE-US-00046 TABLE 11-4 Materials Item Source 0.25% Trypsin-EDTA
(1X) Life Technologies, Carlsbad, CA 10 .times. Permeabilization
Buffer eBioscience, UK 3.7% Formaldehyde solution Sigma-Aldrich, UK
40 and 70 .mu.m cell strainers Corning Life Sciences, Tewksbury, MA
Collagenase type 3 Worthington Biochem Corp., Lakewood, NJ Fc Block
eBioscience, UK Fetal bovine serum, heat inactivated Life
Technologies, Carlsbad, CA Fixation/Permeabilization Concentrate
eBioscience, UK Fixation/Permeabilization Diluent eBioscience, UK
Flow Buffer eBioscience, UK FoxP3 Fix/Perm Kit eBioscience, UK
Hanks buffered salt solution Life Technologies USA Heat Inactivated
Gamma Irradiated SASC Biosciences, KS, USA FBS for RBCL Buffer
neutralization LSRII and Canto II Flow Cytometers BD Biosciences,
San Jose, CA Normal mouse serum Jackson Labs, Bar harbor, MA
Pen/Strep solution Life Technologies UK Permeabilization Buffer
eBiosciences, USA Phosphate buffered saline, pH 7.2 Life
Technologies, Carlsbad, CA Red Blood Cell Lysis Buffer Sigma, UK
Roswell Park Memorial Institute Life Technologies, 1640 medium
Carlsbad, CA
TABLE-US-00047 TABLE 11-5 Fluorescent Antibodies for Flow Cytometry
Studies Item Source APC conjugated Rat IgG2a anti-CD4 eBiosciences,
UK FITC conjugated mIgG2a anti-MHC2 eBiosciences USA APC-H7
conjugated Rat IgG2a anti-CD8 BD Biosciences, UK EFluor 480
conjugated Rat IgG2a anti-Ki67 eBioscience, UK APC conjugated
isotype control Rat IgG2a eBioscience, UK APC-H7 conjugated isotype
control Rat IgGa BD Biosciences, UK eFluor conjugated isotype
control Rat IgG2a eBioscience, UK PE conjugated isotype control Rat
IgG2b eBioscience, UK PECy7 conjugated rlgG2a Ki67 eBioscience, USA
BV605 conjugated rlgG2a anti CD4 Biolegend USA BV711 conjugated
rlgG2a anti CD8 Biolegend USA Fixable Blue Life Technologies, USA
PeCy7 conjugated Isotype control rlgG2a Biolegend USA
11.2 Results
[0436] Intraperitoneal treatment of naive mice with the anti-OX40
antibody OX86 mouse IgG2a (OX86 mIgG2a) resulted in a significant,
dose-dependent and transient increase in the percentage of Ki67+
CD4+ and ICOS+CD4+ T cells in the blood over time, compared to mice
treated with control articles (FIG. 33A and C). The largest
percentage of Ki67+ CD4+ and ICOS+CD4+ T cells was detected on Day
10. A significant increase in the percentage of Ki67+CD4+ and
ICOS+CD4+ T cells was measured in the spleens of mice on Day 10
following the administration of OX86 mIgG2a, compared to mice
treated with control articles (FIG. 33B and D). A statistically
significant and strong correlation between the percentage of
Ki67+CD4+ T cells and ICOS+CD4+ T cells in the blood and spleen on
Day 10 was identified (FIGS. 34A and 34B).
[0437] Treatment of naive mice with the OX86 mIgG2a also resulted
in a significant, transient increase in the percentage of Ki67+CD8+
T cells in the blood over time, when compared to mice treated with
control articles (FIG. 35A). Increases in the percentage of
ICOS+CD8+ T cells in the blood over time (FIG. 35C), and the
percentage of Ki67+CD8+ T cells in the spleen on Day 10 (FIG. 35B),
were detected following treatment with OX86 mIgG2a, but were not
statistically significant when compared to treatment with control
articles. Treatment of naive mice with OX86 mIgG2a did induce a
significant, dose-dependent increase in the percentage of ICOS+CD8+
T cells in the spleen on Day 10, compared to mice treated with
control articles (FIG. 35D). Moderate but significant correlations
between the percentage of Ki67+CD8+ T cells and ICOS+CD8+ T cells
in the blood and spleen was determined (FIGS. 36A and 36B).
[0438] Single-agent treatment of tumor-bearing wild-type mice with
OX86 mIgG2a results in an antitumor activity that reduces growth of
two histologically different tumors as compared to untreated and
isotype control treated groups (See Example 9). Treatment with OX86
mIGg2a resulted in significantly reduced growth of MCA205 tumors on
Day 20 (FIG. 38A, Table 11-7) when compared to treatment with
control articles in C57BL/6 mice that were genetically engineered
to lack expression of the inhibitory Fc.gamma. receptor IIB
(Fcgr2b-/- mice). Identical treatment of mice with CT26 tumors
resulted in a reduced growth of tumors that did not reach
statistical significance on Day 18 (FIG. 37A, Table 11-6) when
compared to treatment with control articles in Fcgr2b-/-BALB/c
mice. No inhibition of growth, by any agent, was observed in tumor
bearing Balb/c and C57BL/6 mice genetically engineered to lack
expression of the activating Fc.gamma. receptors (Fcer1g-/- mice;
FIG. 37C; FIG. 38C). Mixed response is often observed in syngeneic
models. However, the anti-tumor response following treatment with
OX86 mIgG2a in the two different strains of Fc.gamma. receptor
knock-out mice can also be discerned from the individual animal
tumor growth graphs (FIG. 37B and D; FIG. 38B and D); treatment
with OX86 mIgG2a resulted in the inhibition of CT26 and MCA205
tumor growth and in some cases induced complete responses in
Fcgr2b-/- mice (FIG. 37B; FIG. 38B).
TABLE-US-00048 TABLE 11-6 Treatment Groups and Percent TGI and
Number of Complete Responders in CT26 Syngeneic Mouse Model Number
of Complete Test/Control Dose .sup.b Mouse % Responders out Group
.sup.a Article (mg/kg) Strain TGI .sup.c of 8 mice .sup.d 1 None NA
Balb/c NA 0 2 Isotype control 2.5 mg/kg Fcgr2b.sup.-/- NA 0 3 OX86
mIgG2a 2.5 mg/kg 15 0 4 None NA Balb/c NA 0 5 Isotype control 2.5
mg/kg Fcer1g.sup.-/- NA 0 6 OX86 mIgG2a 2.5 mg/kg <0 0 TGI =
tumor growth inhibition; NA = not applicable; IP = intraperitoneal;
V = volume .sup.a Number of animals per group: 8. .sup.b All
animals received 200 .mu.L of test article IP on Days 4 and 7.
.sup.c % TGI = [1 - (mean tumor V of treatment group) / (mean tumor
V of control group)] .times. 100; calculated on Day 18 for
Fcgr2b.sup.-/- mice and Day 16 for Fcer1g.sup.-/- mice .sup.d
Number of animals in a group with a tumor volume measurement
recorded as zero at the end of the study.
TABLE-US-00049 TABLE 11-7 Treatment Groups and Percent TGI on Day
20 and Number of Complete Responders in MCA205 Syngeneic Mouse
Model Number of Complete Test/Control Dose .sup.b Mouse %
Responders out Group .sup.a Article (mg/kg) Strain TGI .sup.c of 8
mice .sup.d 1 None NA C57BL/6 NA 0 2 Isotype control 7.5 mg/kg
Fcgr2b.sup.-/- NA 0 3 OX86 mIgG2a 7.5 mg/kg 95 8 4 None NA C57BL/6
NA 0 5 Isotype control 7.5 mg/kg Fcer1g.sup.-/- NA 0 6 OX86 mIgG2a
7.5 mg/kg 35 1 TGI = tumor growth inhibition; NA = not applicable;
IP = intraperitoneal; V = volume .sup.a Number of animals per
group: 8. .sup.b All animals received 200 .mu.L of test article IP
on Days 4 and 7. .sup.c % TGI = [1 - (mean tumor V of treatment
group) / (mean tumor V of control group)] .times. 100 .sup.d Number
of animals in a group with a tumor volume measurement recorded as
zero at the end of the study.
[0439] In parallel pharmacodynamics studies, treatment with OX86
mIgG2a compared to control articles caused a significant increase
in the percentage of Ki67+CD4+ T cells in the spleen (FIG. 39A) and
Ki67+CD8+ T cells in the peripheral blood and the spleen, but not
in the tumor (FIG. 40A), of CT26 tumor bearing Fcgr2b.sup.-/-
Balb/c mice. No increase in Ki67+ CD4+ or CD8+ T cells was detected
in the blood, spleen or tumor of CT26 tumor bearing
Fcer1g.sup.-/-Balb/c mice following treatment with OX86 mIgG2a, as
compared to control articles (FIG. 39B; FIG. 40B).
[0440] Additionally, in parallel studies, treatment with OX86
mIgG2a compared to control articles resulted in a significant
increase in the percentage of Ki67+CD4+ T cells in the draining
lymph node, spleen and tumor (FIG. 41A) and Ki67+CD8+ T cells in
the draining lymph node and the spleen (FIG. 42A) of MCA205 tumor
bearing Fcgr2b-/- C57BL/6 mice. Increases in the percentage of
Ki67+CD8+ T cells in the tumor of MCA205 tumor bearing Fcgr2b-/-
C57BL/6 mice were not statistically significant (FIG. 42A).
Treatment with OX86 mIgG2a as compared to control articles also
induced a significant increase in the percentage of Ki67+CD4+ T
cells in the draining lymph node and spleen of MCA205 tumor bearing
Fcer1g-/- C57BL/6 mice (FIG. 41B). A significant increase in the
percentage of Ki67+CD8+ T cells in the spleen of these mice was
also observed (FIG. 42B). No significant increases in the
percentage of Ki67+CD4+ T cells in the tumor (FIG. 42B) or
Ki67+CD8+ T cells in the draining lymph node or tumor of MCA205
tumor bearing Fcgr2b-/- C57BL/6 mice were detected (FIG. 42B).
11.3 Conclusions
[0441] A single dose of OX86 mIgG2a in naive mice induced a
transient increase in T-cell activation and proliferation as
measured by increased expression of ICOS and Ki67, respectively. A
significant linear correlation of the percentage of ICOS and Ki67
on CD4+ and CD8+ T cells was found. Antitumor activity of OX86
mIgG2a, measured as inhibition of the growth of CT26 and MCA205
tumors, was dependent on the expression of activating Fc.gamma.
receptors I, III and IV, but not the inhibitory Fc.gamma. receptor
IIB. Increases in peripheral blood, draining lymph node and/or
spleen T-cell proliferation (Ki67) were observed in parallel
pharmacodynamic experiments in tumor-bearing mice that expressed
the activating Fc.gamma. receptors; some T-cell proliferation was
observed in mice that expressed only the inhibitory Fc.gamma.
receptor. Together with the pharmacodynamic results from naive mice
and while not wishing to be bound by theory, a potential mechanism
for OX86 mIgG2a antitumor activity is increased peripheral and
intratumoral T-cell proliferation (Ki67). Moreover, OX86 mIgG2a in
vivo antitumor activity occurs upon expression of activating
Fc.gamma. receptors.
Example 12
T cell Pharmacodynamic Changes in Response to OX40mAb24 Therapy in
Immunocompromised Mice Engrafted with Human Hematopoietic Stem
Cells
[0442] This example investigates whether OX40mAb24 can activate and
expand human CD4+ and CD8+ memory T cells or reduce human Tregs in
immunocompromised mice with a reconstituted human immune system.
OX40mAb24 was tested in an in vivo mouse model system reconstituted
with human immune cells to determine if the drug has
immunomodulatory effects on human T cells.
12.1 Materials and Methods
12.1.1 Test Animals
[0443] Nod.Cg-PrkdcscidII2rgtm1Wy/SzJ (NSG) Mice (n=14 ages 23-26
weeks) were obtained from the Jackson Laboratory. The animals were
humanely treated and housed according to Institutional Animal Care
and Use Committee approved protocols in the Laboratory Animal
Resources facility at MedImmune, an Association for Animal
Accreditation of Laboratory Animal Care and United States
Department of Agriculture-licensed facility. The animals were kept
in sterile micro-isolator units, provided with sterile bedding and
food, and acidified drinking water ad libitum. Environmental
conditions were standardized (room temperature: 20.degree.
C..+-.1.degree. C.; relative humidity: 50%.+-.10%; 12-hour
light-dark cycle). The animals were monitored daily for adverse
clinical signs during the course of the study.
12.1.2 Pharmacodynamics of OX40mAb24 in HSC Engrafted (Humanized)
Mice
12.1.2.1 Establishment of Human HSC Engrafted Mice
[0444] Human CD34+ HSC engrafted mice were purchased from the
Jackson Laboratory. Mice were generated as follows: pooled human
CD34+ cells from multiple umbilical cord blood donors were isolated
and injected intravenously into the tail vein of
Nod.Cg-Prkdcscid112rgtm1Wy/SzJ (NSG) mice. At 12 weeks after
engraftment, mouse peripheral blood was sampled and lysed with
Pharm Lyse red blood cell lysis buffer and then analyzed by flow
cytometry for human CD45, CD19 and CD3 positive cells to determine
the degree of human immune cell engraftment in mice. Mice
successfully engrafted with 25% or more CD45+ human immune cells of
total viable blood cells were subsequently shipped to
MedImmune.
12.1.2.2 Antigen Challenge and OX40mAb24 treatment of Human HSC
Engrafted Mice
[0445] The sustained engraftment of human immune cells into NSG
mice was confirmed by flow cytometry at 22 weeks post injection of
human CD34 cells, and at 23 weeks mice were placed into 3 groups
that were left untouched or received treatment (Table 12-1). There
were no statistically significant differences in the mean
percentage of human CD45+ cell engraftment between eventual
treatment groups at week 22 prior to the start of the experiment.
Group 1 consisted of 4 mice and did not receive subcutaneous (SC)
keyhole limpet hemocyanin (KLH) immunization or antibody
administration. Group 2 consisted of 5 mice and received 300
.mu.g/50 .mu.L of KLH and 50 .mu.L of complete Freund's adjuvant
(CFA) at two independent injection SC sites. This group of mice
also received 2 mg/kg of hIgG1 isotype control antibody,
administered intraperitoneally (IP), one day after KLH
immunization. Group 3 consisted of 5 mice and received KLH/CFA SC
immunization as described above. In addition, mice were also
administered one day later a single IP dose of 2 mg/kg OX40mAb24.
On the day of KLH/CFA SC immunization, prior to immunization
(pre-treatment), and seven days later (post-treatment), whole blood
was obtained through retro-orbital bleed and cells were
immunophenotyped and counted by flow cytometry.
TABLE-US-00050 TABLE 12-1 Experimental Groups, Human CD45+ Cell
Engraftment, and Treatment Human CD45+ Cells as a Percentage of
Total Viable Human Plus Mouse CD45 Cells.sup.a- Treatment.sup.b-
Mouse Week 22 post HSC Week 23 post HSC Group Number engraftment
engraftment 1 1-1 4575 39.5 None 1 1-2 4576 68.3 None 1 1-3 4577
20.9 None 1 1-4 4578 23.8 None 2 2-1 4579 29.6 KLH immunization +
NIP228 huIgG1 2 2-2 4580 21.7 KLH immunization + NIP228 huIgG1 2
2-3 4581 27.8 KLH immunization + NIP228 huIgG1 2 2-4 4582 50.3 KLH
immunization + NIP228 huIgG1 2 2-5 4583 32.5 KLH immunization +
NIP228 huIgG1 3 3-1 4584 15.4 KLH immunization + OX40mAb24 3 3-2
4585 44.3 KLH immunization + OX40mAb24 3 3-3 4586 20.3 KLH
immunization + OX40mAb24 3 3-4 4587 16.3 KLH immunization +
OX40mAb24 3 3-5 4588 25.6 KLH immunization + OX40mAb24 Hu = human;
KLH = keyhole limpet hemocyanin; HSC = hematopoietic stem cells.
.sup.aHuman CD45+ cells determined by flow cytometry; .sup.bKLH
immunization, keyhole limpet hemocyanin plus complete Freund's
adjuvant.
12.1.2.3 Immune Cell Analysis by Flow Cytometry
[0446] Cells were immunophenotyped by flow cytometry using a whole
blood antibody binding protocol. Briefly, EDTA anti-coagulated
whole blood at a constant volume was added to wells of a deep well
plate and a T cell staining master mix added to cells to bind cell
surface antigens. After washes in FACS buffer (PBS, pH 7.2 plus 2%
heat-inactivated newborn calf serum), red blood cells (RBC) were
lysed using 1.times. RBC lysis buffer, and cells fixed and
permeabilized using EBiosciences Fix and Perm kit according to the
manufacturer's protocol. Subsequently, anti-FoxP3 and anti-Ki67
mAbs were bound to cells. Cells were washed using 1.times. fix and
perm buffer, re-suspended in FACS buffer and events analyzed using
an LSRII flow cytometer. Raw flow cytometry standard (FCS) data was
analyzed using FlowJo software, and cell populations identified
after live/dead cell discrimination and removal of doublets and
cell debris. After gating for CD4+ and CD8+ T cell populations,
memory T cells were determined by CD45RA and CCR7 expression
profile, with naive T cells defined as CD45RA+/CCR7+, effector T
cells (Teff) as CD45RA+/CCR7-, effector memory T cells (Tem) as
CD4SRA-/CCR7-, and central memory T cells (Tcm) as CD45RA-/CCR7+.
Tregs were defined as CD4+/FoxP3+ cells.
12.1.2.4 Statistical Methods
[0447] GraphPad Prism software for Windows (version 6.03) was used
for graphing and statistical analysis of data.
[0448] One-way ANOVA multiple comparisons test with Turkey's
post-test analysis was used to compare differences between
experimental group means. In the event of a significant F test, a
Sidak's multiple comparisons test was utilized. A p value of less
than 0.05 was considered significant.
12.1.3 Materials
[0449] Materials used in this study, and their source, are listed
in Table 12-2.
TABLE-US-00051 TABLE 12-2 Materials Item Source Anti-human CCR7 APC
clone 150503 R&D Systems, Minneapolis, MN Anti-human CD14 APC
clone M5E2 Biolegend, San Diego, CA Anti-human CD16 PE clone 1243
Biolegend, San Diego, CA Anti-human CD25 FITC clone Biolegend, San
Diego, CA M-A251 Anti-human CD3 FITC clone UCHT1 Biolegend, San
Diego, CA Anti-human CD4 BV605 clone Becton Dickinson, San Jose, CA
RPA-T4 Anti-human CD56 FITC clone HCD56 Biolegend, San Diego, CA
Anti-human CD8 PE-CF594 clone Becton Dickinson, San Jose, CA RPA-T8
Anti-human CD19 APC clone HIB19 Biolegend, San Diego, CA Anti-human
CD19 PE-CF594 clone Becton Dickinson, San Jose, CA HIB19 Anti-human
CD45 PE clone HI30 Biolegend, San Diego, CA Anti-human CD45 PE
clone HI30 Biolegend, San Diego, CA Anti-human FoxP3 PE clone
PHC101 eBioscience, San Diego, CA Anti-human HLA-DR BV421
Biolegend, San Diego, CA clone 3G8 Anti-human Ki67 FITC clone B56
Becton Dickinson, San Jose, CA Anti-mouse CD16/32 PE-Cy7 Biolegend,
San Diego, CA Anti-mouse CD45 PE-Cy7 clone Biolegend, San Diego, CA
30F11 Complete Freund's adjuvant (CFA) Becton Dickinson, San Jose,
CA Difco .TM. EBiosciences Fix and Perm Kit EBioscience, San Diego,
CA FlowJo Software FlowJo, Ashland, OR FoxP3/Transcription Factor
eBioscience, San Diego, CA Fixation/Permeabilization Concentrate
and Diluent Graphpad Prism software, v 6.03 Graphpad Software, San
Diego, CA Heat-inactivated newborn calf serum Life Technologies,
Frederick, MD Keyhole limpet hemocyanin Thermo Fisher Scientific,
Waltham, MA Live/Dead .RTM. Fixable Blue Dye Life Technologies,
Frederick, MD LSR II flow cytometer BD Biosciences, San Jose, CA
Mouse IgG2a APC isotype control eBioscience, San Diego, CA Mouse
IgG2b BV421 isotype control Biolegend, San Diego, CA Mouse IgG1
FITC isotype control Biolegend, San Diego, CA Phosphate-buffered
saline (PBS), Life Technologies, pH 7.2 Frederick, MD Rat IgG1
PE-CF594 isotype control Becton Dickinson, San Jose, CA Rat IgG2a
PE isotype control eBioscience, San Diego, CA Rat IgG2a PE-Cy7
isotype control Biolegend, San Diego, CA
12.2 Results
[0450] OX40mAb24 or huIgG1 control antibody was administered one
day after immunization with KLH. OX40mAb24 resulted in a
statistically significant reduction in the percentage of Treg in
peripheral blood after six days relative to the group administered
a huIgG1 isotype control antibody (p=0.019, one-way ANOVA; FIGS.
43A and 43B). No differences in the percentages of Tregs between
treatment groups were observed prior to immunization and antibody
administration.
[0451] In addition to a significant decrease in the percentage of
peripheral blood Tregs, there was a statistically significant
increase in the CD4+ Tem:Treg ratio following treatment with
OX40mAb24 relative to huIgG1 isotype matched control (p=0.042), and
trends towards significant increases for total CD4+:Treg (p=0.051),
CD4+ Teff:Treg (p=0.10), and CD4+ Tem:Treg (p=0.069) ratios (FIGS.
44A-D). Likewise, a trend towards significance was observed for an
increase in the CD8+ Teff:Treg ratio (p=0.064) in peripheral blood
of mice treated with OX40mAb24 relative to that of mice treated
with huIgG1 isotype control (FIGS. 45A-B).
[0452] CD25 (IL-2 receptor) is up-regulated on T cells following
activation, and is considered a marker of recently activated T
cells. Increases in the percentage of CD25 positive CD8+ T cells
were observed for the OX40mAb24 treatment group relative to the
huIgG1 treatment group for total CD8+ (p=0.038) effector CD8+
(p=0.076), and for effector memory CD8+ (p=0.040) T cells (FIGS.
46A-C). Therefore, agonism of OX40 by OX40mAb24 activated CD8+ T
cells relative to the control antibody.
12.3 Conclusions
[0453] In mice engrafted with human immune cells, treatment with
OX40mAb24 after antigen immunization resulted in decreased
peripheral Treg cells relative to mice receiving a human IgG1
control antibody. Likewise, the ratio of total, effector, and
memory CD4+ T cells, and effector CD8+ T cells, relative to Treg
cells were also increased in the peripheral blood of mice receiving
OX40mAb24 compared with those receiving huIgG1 control antibody.
Finally, the percentage of CD8+ T cells expressing CD25 on the cell
surface was increased after OX40mAb24 treatment, indicating an OX40
induction of peripheral CD8+ T-cell activation.
TABLE-US-00052 TABLE OF SEQUENCES SEQ ID NO Description Sequence 1
9B12 VL
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSKLHSGVPSR
FSGSGSRTDYSLTITDLDQEDIATYFCQQGSALPWTFGQGTKVEIK 2 LCDR1 RASQDISNYLN
3 LCDR2 YTSKLHS 4 LCDR3 QQGSALPWT 5 9B12 VH
EVQLQESGPSLVKPSQTLSLICSVTGDSFTSGYWNWIRKFPGNRLEYMGYISYNGITYHNP
SLKSRISITRDTSKNHYYLQLNSVITEDTATYFCARYRYDYDGGHAMDYWGQGTLVTVSS 6 HFW1
QVQLQESGPGLVKPSQTLSLTCAVYGGSFS 7 HFW1-variant
QVQLQESGPGLVKPSQTLSLTCAVYGDSFS 8 HCDR1 SGYWN 9 HFW2-XXX
WIRX.sub.39HPGKGLEX.sub.47X.sub.48G; where X.sub.39 is Q or K,
X.sub.47 is W or Y, and X.sub.48 is I or M 10 HFW2-variant
WIRQHPGKGLEWIG 11 HFW2-variant WIRKHPGKGLEYMG 12 HFW2-variant
WIRKHPGKGLEWIG 13 HFW2-variant WIRKHPGKGLEYIG 14 HCDR2
YISYNGITYHNPSLKS 15 HCDR2-variant YISYNAITYHNPSLKS 16 HCDR2-variant
YISYSGITYHNPSLKS 17 HFW3-XXX
RITINX.sub.71DTSKNQX.sub.78SLQLNSVTPEDTAVYX.sub.91CAR;, where
X.sub.71 is P or R, X.sub.78 is F or Y, and X.sub.91 is Y or F 18
HFW3-variant RITINPDTSKNQFSLQLNSVTPEDTAVYYCAR 19 HFW3-variant
RITINRDTSKNQYSLQLNSVTPEDTAVYFCAR 20 HFW3-variant
RITINRDTSKNQFSLQLNSVTPEDTAVYYCAR 21 HFW3-variant
RITINRDTSKNQFSLQLNSVTPEDTAVYFCAR 22 HFW3-variant
RITINRDTSKNQYSLQLNSVTPEDTAVYYCAR 23 HFW3-variant
RITINPDTSKNQYSLQLNSVTPEDTAVYFCAR 24 HFW3-variant
RITINPDTSKNQYSLQLNSVTPEDTAVYYCAR 25 HCDR3 YRYDYDGGHAMDY 26
HCDR3-variant YKYDYDAGHAMDY 27 HCDR3-variant YKYDYDGGHAMDY 28 HFW4
WGQGTLVTVSS 29 OX40mAb
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSR VL
FSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIK 30 OX40mAb
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSR light
chain FSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31 OX40Mab
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCA light
chain TCACCTGTCGGGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGG
DNA CAAGGCCCCCAAGCTGCTGATCTACTACACCAGCAAGCTGCACAGCGGCGTGCCCAGCAGA
TTCAGCGGCAGCGGCTCCGGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGG
ACTTCGCCACCTACTACTGCCAGCAGGGCTCCGCCCTGCCCTGGACCTTTGGCCAGGGCAC
CAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAG
AGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGT
CACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAA
GCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGC
CCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 32 OX40mAb
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSKLHSGVPSR
VL-hu2 FSGSGSRTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIK 33 OX40mAb5
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRQHPGKGLEWIGYISYNGITYHNP VH
SLKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 34
OX40mAb5
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGCAGCACCC
CGGCAAGGGCCTGGAATGGATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCCCGACACCAGCAAGAACCAGTTCTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 35
OX40mAb8
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEWIGYISYNGITYHNP VH
SLKSRITINRDTSKNQFSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 36
OX40mAb8
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATGGATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTTCTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 37
OX40mAb13
QVQLQESGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYMGYISYNGITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 38
OX40mAb13
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATGGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 39
OX40mAb14
QVQLQESGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 40
OX40mAb14
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 41
OX40mAb15
QVQLQRSGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP VH
SLKSRITINRDTSKNQFSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 42
OX40mAb15
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTTCTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 43
OX40mAb16
QVQLQESGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 44
OX40mAb16
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 45
OX40mAb17
QVQLQESGPGLVKPSQTLSLTCAVYGDSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP VH
SLKSRITINRDTSKNQFSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 46
OX40mAb
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH17
DNA CCTGTGCCGTGTACGGCGACAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTTCTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 47
OX40mAb18
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP VH
SLKSRITINPDTSKNQYSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 48
OX40mAb18
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCCCGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 49
OX40mAb19
QVQLQESCPCLVKPSQTLSLTCAVYCCSFSSCYWNWIRKHPCKCLEYICYISYNCITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYFCARYRYDYDGGHAMDYWGQGTLVTVSS 50
OX40mAb19
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTTCTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 51
OX40mAb20
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 52
OX40mAb20
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 53
OX40mAb21
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP VH
SLKSRITINPDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 54
OX40mAb21
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCCCGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 55
OX40mAb22
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDAGHAMDYWGQGTLVTVSS 56
OX40mAb22
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGCCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 57
OX40mAb23
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 58
OX40mAb23
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 59
OX40mAb24
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGLTVTVSS 60
OX40mAb24
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA
CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 61
OX40mAb25
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYSGITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYRYDYDGGHAMDYWGQGTLVTVSS 62
OX40mAb25
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAGCGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 63
OX40mAb25a
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYSGITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSS 64
OX40mAb25a
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAGCGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 65
OX40mAb26
EVQLQESGPSLVKPSQTLSLTCSVTGDSFTSGYWNWIRKFPGNRLEYMGYISYNAITYHNP VH
SLKSRITITRDTSKNHYYLQLNSVTTEDTATYFCARYRYDYDGGHAMDYWGQGTLVTVSS 66
OX40mAb26
GAGGTGCAGCTGCAGGAAAGCGGCCCCAGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGCAGCGTGACCGGCGACAGCTTCACCAGCGGCTACTGGAACTGGATCCGGAAGTTCCC
CGGCAACCGGCTCGAGTACATGGGCTACATCAGCTACAACGCCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCAGCATCACCCGGGACACCAGCAAGAACCACTACTACCTGCAGC
TGAACAGCGTGACCACCGAGGACACCGCCACCTACTTTTGCGCCCGGTACAGATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 67
OX40mAb27
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNP VH
SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSS 68
OX40mA27
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA VH
DNA CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT 69 Human
IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
CH chain
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK 70 Human IgG1
GCgTCgACCAAGGGCCCATCcGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGG CH
chain GCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCcTG
DNA GAACTCAGGCGCtCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA
CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA
TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA
CATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA
CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG
GCAGCCCCGAGAACCACAGGTcTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC
TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCttaagCC
TGTCTCCGGGTAAA 71 OX40mAb24
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP heavy
chain SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK 72 OX40mAb24
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA heavy
chain CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
DNA CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCg
TCgACCAAGGGCCCATCcGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCcTGGAA
CTCAGGCGCtCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTG
TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA
AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTcTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA
GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC
CTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCttaagCCTGT
CTCCGGGTAAA 73 OX40mAb28
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP heavy
chain SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSA
STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS
VMHEALHNHYTQKSLSLSLGK 74 OX40mAb28
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA heavy
chain CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
DNA CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCG
TCGACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCTTGCAGCAGAAGCACCAGCGAGAGCA
CAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAA
CAGCGGCGCTCTGACCAGCGGCGTGCATACCTTCCCCGCCGTGCTCCAGAGCAGCGGACTG
TACTCCCTGAGCAGCGTGGTGACCGTGCCTTCCAGCAGCCTGGGCACCAAGACCTACACCT
GCAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGAGCAAGTACGG
CCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTC
CCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGG
TGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTTAATTGGTACGTGGACGGCGTGGAAGT
GCATAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGTCC
GTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCA
ACAAGGGCCTGCCTAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGA
GCCCCAGGTCTACACCCTGCCACCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTG
ACCTGTCTGGTGAAAGGCTTCTATCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCC
AGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCT
GTACTCCAGACTGACCGTGGACAAGTCCAGATGGCAGGAGGGCAACGTCTTCAGCTGCTCC
GTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCA AG 75
OX40mAb29
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP heavy
chain SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK 76 OX40mAb29
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA heavy
chain CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
DNA CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCG
TCGACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCcTGGAA
CTCAGGCGCtCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTG
TGACAAAACTCACACATgcCCacCGTGCCCAGCACCTGAATTCGAGGGGGGAcCGTCAGTC
TTCCTCTTCCCCCCAAAACCCaaGgACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA
AGGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTcTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA
GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC
CTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT
CTCCGGGTAAA 77 OX40mAb31
QVQLQRSGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNP heavy
chain SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSA
STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS
VMHEALHNHYTQKSLSLSLGK 78 OX40mAb31
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA heavy
chain CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
DNA CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCG
TCGACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCTTGCAGCAGAAGCACCAGCGAGAGCA
CAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAA
CAGCGGCGCTCTGACCAGCGGCGTGCATACCTTCCCCGCCGTGCTCCAGAGCAGCGGACTG
TACTCCCTGAGCAGCGTGGTGACCGTGCCTTCCAGCAGCCTGGGCACCAAGACCTACACCT
GCAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGAGCAAGTACGG
CCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCTAGCGTGTTCCTGTTC
CCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGG
TGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTTAATTGGTACGTGGACGGCGTGGAAGT
GCATAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGTCC
GTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCA
ACAAGGGCCTGCCTAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGA
GCCCCAGGTCTACACCCTGCCACCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTG
ACCTGTCTGGTGAAAGGCTTCTATCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCC
AGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCT
GTACTCCAGACTGACCGTGGACAAGTCCAGATGGCAGGAGGGCAACGTCTTCAGCTGCTCC
GTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCTGGGCA 79
OX40mAb32
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNAITYHNP heavy
chain SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK 80 OX40mAb32
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA heavy
chain CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
DNA CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGCCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCG
TCGACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCcTGGAA
CTCAGGCGCtCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCT
GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTG
TGACAAAACTCACACATgcCCacCGTGCCCAGCACCTGAATTCGAGGGGGGAcCGTCAGTC
TTCCTCTTCCCCCCAAAACCCaaGgACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGG
CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA
AGGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTcTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA
GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC
CTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT
CTCCGGGTAAA 81 OX40mAb37
QVQLQESGPGLVKPSQTLSLTCAVYGGSFSSGYWNWIRKHPGKGLEYIGYISYNGITYHNP heavy
chain SLKSRITINRDTSKNQYSLQLNSVTPEDTAVYYCARYKYDYDGGHAMDYWGQGLTVTVSSA
KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY
TLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPK
PKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELP
IMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCM
ITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLH
EGLHNHHTEKSLSHSPGK 82 OX40mAb37
CAGGTGCAGCTGCAGGAAAGCGGCCCTGGCCTGGTCAAGCCCAGCCAGACCCTGAGCCTGA heavy
chain CCTGTGCCGTGTACGGCGGCAGCTTCAGCAGCGGCTACTGGAACTGGATCCGGAAGCACCC
DNA CGGCAAGGGCCTGGAATACATCGGCTACATCAGCTACAACGGCATCACCTACCACAACCCC
AGCCTGAAGTCCCGGATCACCATCAACCGGGACACCAGCAAGAACCAGTACTCCCTGCAGC
TGAACAGCGTGACCCCCGAGGACACCGCCGTGTACTACTGCGCCCGGTACAAATACGACTA
CGACGGCGGCCACGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCG
aaGACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCA
TGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAA
CTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTAC
ACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCA
ACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGG
TTGTAAGCCTTGCATATGTACcGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAG
CCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCA
GCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGC
TCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCC
ATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTT
TCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGT
GTAtACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATG
ATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGG
AGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAA
GCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACAT
GAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA 83 OX40mAb37
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSKLHSGVPSR light
chain FSGSGSGTDYTLTISSLQPEDFATYYCQQGSALPWTFGQGTKVEIKRADAAPTVSIFPPSS
EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK
DEYERHNSYTCEATHKTSTSPIVKSFNRNEC 84 OX40mAb37
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCA light
chain TCACCTGTCGGGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGG
DNA CAAGGCCCCCAAGCTGCTGATCTACTACACCAGCAAGCTGCACAGCGGCGTGCCCAGCAGA
TTCAGCGGCAGCGGCTCCGGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGG
ACTTCGCCACCTACTACTGCCAGCAGGGCTCCGCCCTGCCCTGGACCTTTGGCCAGGGCAC
CAAGGTGGAAATCAAGCGGGCTGATGCGGCGCCAACTGTATCCATCTTCCCACCATCCAGT
GAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAG
ACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTG
GACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAG
GACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCAC
CCATTGTCAAGAGCTTCAACAGGAATGAGTGT 85 OX86 VH
QVQLKESGPGLVQPSQTLSLTCTVSGFSLTGYNLHWVRQPPGKGLEWMGRMRYDGDTYYNS
VLKSRLSISRDTSKNQVFLKMNSLQTDDTAIYYCTRDGRGDSFDYWGQGVMVTVSS 86 OX86
heavy QVQLKESGPGLVQPSQTLSLTCTVSGFSLIGYNLHWVRQPPGKGLEWMGRMRYDGDTYYNS
chain VLKSRLSISRDTSKNQVFLKMNSLQTDDTAIYYCTRDGRGDSFDYWGQGVMVTVSSASTTP
PSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSS
SVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDV
LTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQ
DWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDF
FPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLH
NHHTEKSLSHSPGK 87 OX86 heavy
CAGGTGCAGCTGAAGGAGTCAGGACCTGGTCTGGTGCAGCCCTCACAGACCCTGTCCCTCA chain
DNA CCTGCACTGTCTCTGGGTTCTCACTAACCGGTTACAATTTACACTGGGTTCGCCAGCCTCC
AGGAAAGGGTCTGGAGTGGATGGGAAGAATGAGGTATGATGGAGACACATATTATAATTCA
GTTCTCAAATCCCGACTGAGCATCAGCAGGGACACCTCCAAGAACCAAGTTTTCTTGAAAA
TGAACAGTCTGCAAACGGATGACACAGCCATTTACTATTGTACCAGAGACGGGCGTGGTGA
CTCCTTTGATTACTGGGGCCAAGGAGTCATGGTCACAGTCTCCTCCGCGTCGACGACACCC
CCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGG
GATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCT
GTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGC
TCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACC
CGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTG
CATATGTACCGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTG
CTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATC
CCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACC
CCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAG
GACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCA
TCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTATACCATTCC
ACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTC
TTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGA
ACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCA
GAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCAC
AACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA 88 OX86 VL
DIVMTQGALPNPVPSGESASITCRSSQSLVYKDGQTYLNWFLQRPGQSPQLLTYWMSTRAS
GVSDRFSGSGSGTYFTLKISRVRAEDAGVYYCQQVREYPFTFGSGTKLEIK 89 OX86 light
DIVMTQGALPNPVPSGESASITCRSSQSLVYKDGQTYLNWFLQRPGQSPQLLTYWMSTRAS chain
GVSDRFSGSGSGTYFTLKISRVRAEDAGVYYCQQVREYPFTFGSGTKLEIKRADAAPTVSI
FPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSST
LTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 90 OX86 light
GATATTGTGATGACCCAGGGTGCACTCCCCAATCCTGTCCCTTCTGGAGAGTCAGCTTCCA chain
DNA TCACCTGCAGGTCTAGTCAGAGTCTGGTATACAAAGACGGCCAGACATACTTGAATTGGTT
TCTGCAGAGGCCAGGACAGTCTCCTCAGCTTCTGACCTATTGGATGTCTACCCGTGCATCA
GGAGTCTCAGACAGGTTCAGTGGCAGTGGGTCAGGAACATATTTCACACTGAAAATCAGTA
GAGTGAGGGCTGAGGATGCGGGTGTGTATTACTGTCAGCAAGTTCGAGAGTATCCTTTCAC
TTTCGGCTCAGGGACGAAGTTGGAAATAAAACGGGCTGATGCGGCGCCAACTGTATCCATC
TTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACA
ACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGG
CGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACC
CTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACA
AGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT 91 Human OX40
MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQN
TCVRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPG
VDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPP
ARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQ
RLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 92 Mouse OX40
MYVWVQQPTALLLLGLTLGVTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDTLCH
PCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQPRQDSGYKLGVD
CVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDRSLLATLLWETQRPTFR
PTTVQSTTVWPRTSELPSPPTLVTPEGPAFAVLLGLGLGLLAPLTVLLALYLLRKAWRLPN
TPKPCWGNSFRTPIQEEHTDAHFTLAKI
Sequence CWU 1
1
981107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp
Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Lys Leu His Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Arg Thr Asp Tyr
Ser Leu Thr Ile Thr Asp Leu Asp Gln65 70 75 80Glu Asp Ile Ala Thr
Tyr Phe Cys Gln Gln Gly Ser Ala Leu Pro Trp 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Arg
Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5 1037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Tyr
Thr Ser Lys Leu His Ser1 549PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 4Gln Gln Gly Ser Ala Leu Pro
Trp Thr1 55121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 5Glu Val Gln Leu Gln Glu Ser Gly Pro
Ser Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ser Val
Thr Gly Asp Ser Phe Thr Ser Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys
Phe Pro Gly Asn Arg Leu Glu Tyr Met 35 40 45Gly Tyr Ile Ser Tyr Asn
Gly Ile Thr Tyr His Asn Pro Ser Leu Lys 50 55 60Ser Arg Ile Ser Ile
Thr Arg Asp Thr Ser Lys Asn His Tyr Tyr Leu65 70 75 80Gln Leu Asn
Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95Arg Tyr
Arg Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115 120630PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
6Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5
10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser 20 25
30730PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Asp Ser Phe Ser 20 25 3085PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Ser Gly Tyr Trp Asn1
5914PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(4)..(4)Gln or LysMOD_RES(12)..(12)Trp or
TyrMOD_RES(13)..(13)Ile or Met 9Trp Ile Arg Xaa His Pro Gly Lys Gly
Leu Glu Xaa Xaa Gly1 5 101014PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 10Trp Ile Arg Gln His Pro Gly
Lys Gly Leu Glu Trp Ile Gly1 5 101114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Trp
Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Met Gly1 5
101214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Trp
Ile Gly1 5 101314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 13Trp Ile Arg Lys His Pro Gly Lys Gly
Leu Glu Tyr Ile Gly1 5 101416PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 14Tyr Ile Ser Tyr Asn Gly Ile
Thr Tyr His Asn Pro Ser Leu Lys Ser1 5 10 151516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Tyr
Ile Ser Tyr Asn Ala Ile Thr Tyr His Asn Pro Ser Leu Lys Ser1 5 10
151616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Tyr Ile Ser Tyr Ser Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys Ser1 5 10 151732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMOD_RES(6)..(6)Pro or
ArgMOD_RES(13)..(13)Phe or TyrMOD_RES(29)..(29)Tyr or Phe 17Arg Ile
Thr Ile Asn Xaa Asp Thr Ser Lys Asn Gln Xaa Ser Leu Gln1 5 10 15Leu
Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Xaa Cys Ala Arg 20 25
301832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 18Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
Gln Phe Ser Leu Gln1 5 10 15Leu Asn Ser Val Thr Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg 20 25 301932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu Gln1
5 10 15Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Phe Cys Ala
Arg 20 25 302032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 20Arg Ile Thr Ile Asn Arg Asp Thr
Ser Lys Asn Gln Phe Ser Leu Gln1 5 10 15Leu Asn Ser Val Thr Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 302132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
21Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln1
5 10 15Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Phe Cys Ala
Arg 20 25 302232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 22Arg Ile Thr Ile Asn Arg Asp Thr
Ser Lys Asn Gln Tyr Ser Leu Gln1 5 10 15Leu Asn Ser Val Thr Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 302332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn Gln Tyr Ser Leu Gln1
5 10 15Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Phe Cys Ala
Arg 20 25 302432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 24Arg Ile Thr Ile Asn Pro Asp Thr
Ser Lys Asn Gln Tyr Ser Leu Gln1 5 10 15Leu Asn Ser Val Thr Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 302513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Tyr
Arg Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr1 5
102613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Tyr Lys Tyr Asp Tyr Asp Ala Gly His Ala Met Asp
Tyr1 5 102713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 27Tyr Lys Tyr Asp Tyr Asp Gly Gly His
Ala Met Asp Tyr1 5 102811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 28Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser1 5 1029107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 29Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr
Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10530214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Lys Leu His Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21031642DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 31gacatccaga tgacccagag
ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60atcacctgtc gggccagcca
ggacatcagc aactacctga actggtatca gcagaagccc 120ggcaaggccc
ccaagctgct gatctactac accagcaagc tgcacagcgg cgtgcccagc
180agattcagcg gcagcggctc cggcaccgac tacaccctga ccatcagcag
cctgcagccc 240gaggacttcg ccacctacta ctgccagcag ggctccgccc
tgccctggac ctttggccag 300ggcaccaagg tggaaatcaa gcgtacggtg
gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
480gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 540ctgagcaaag cagactacga gaaacacaaa gtctacgcct
gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gt 64232107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 32Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile 35 40 45Tyr Tyr Thr
Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Arg Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10533121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 33Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Gly Ser Phe Ser Ser Gly 20 25 30Tyr Trp Asn Trp Ile Arg Gln His Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile
Thr Tyr His Asn Pro Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Pro
Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Gln Leu Asn Ser Val
Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Arg Tyr
Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser 115 12034363DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
34caggtgcagc tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg
60acctgtgccg tgtacggcgg cagcttcagc agcggctact ggaactggat ccggcagcac
120cccggcaagg gcctggaatg gatcggctac atcagctaca acggcatcac
ctaccacaac 180cccagcctga agtcccggat caccatcaac cccgacacca
gcaagaacca gttctccctg 240cagctgaaca gcgtgacccc cgaggacacc
gccgtgtact actgcgcccg gtacagatac 300gactacgacg gcggccacgc
catggactac tggggccagg gcaccctggt caccgtgtcc 360tct
36335121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 35Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Gly Ser Phe Ser Ser Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile
Thr Tyr His Asn Pro Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg
Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Gln Leu Asn Ser Val
Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Arg Tyr
Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser 115 12036363DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
36caggtgcagc tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg
60acctgtgccg tgtacggcgg cagcttcagc agcggctact ggaactggat ccggaagcac
120cccggcaagg gcctggaatg gatcggctac atcagctaca acggcatcac
ctaccacaac 180cccagcctga agtcccggat caccatcaac cgggacacca
gcaagaacca gttctccctg 240cagctgaaca gcgtgacccc cgaggacacc
gccgtgtact actgcgcccg gtacagatac 300gactacgacg gcggccacgc
catggactac tggggccagg gcaccctggt caccgtgtcc 360tct
36337121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 37Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Asp Ser Phe Ser Ser Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro
Gly Lys Gly Leu Glu Tyr Met 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile
Thr Tyr His Asn Pro Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg
Asp Thr Ser Lys Asn Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val
Thr Pro Glu Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Tyr Arg Tyr
Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser 115 12038363DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
38caggtgcagc tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg
60acctgtgccg tgtacggcga cagcttcagc agcggctact ggaactggat ccggaagcac
120cccggcaagg gcctggaata catgggctac atcagctaca acggcatcac
ctaccacaac 180cccagcctga agtcccggat caccatcaac cgggacacca
gcaagaacca gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc
gccgtgtact tctgcgcccg gtacagatac 300gactacgacg gcggccacgc
catggactac tggggccagg gcaccctggt caccgtgtcc 360tct
36339121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 39Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Asp Ser Phe Ser Ser Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro
Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile
Thr Tyr His Asn Pro Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg
Asp Thr Ser Lys Asn Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val
Thr Pro Glu Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Tyr Arg Tyr
Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser 115
12040363DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 40caggtgcagc tgcaggaaag cggccctggc
ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcga cagcttcagc
agcggctact ggaactggat ccggaagcac 120cccggcaagg gcctggaata
catcggctac atcagctaca acggcatcac ctaccacaac 180cccagcctga
agtcccggat caccatcaac cgggacacca gcaagaacca gtactccctg
240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact tctgcgcccg
gtacagatac 300gactacgacg gcggccacgc catggactac tggggccagg
gcaccctggt caccgtgtcc 360tct 36341121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
41Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Asp Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Phe Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12042363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 42caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcga
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gttctccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
tctgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36343121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
43Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Asp Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12044363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 44caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcga
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36345121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
45Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Asp Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12046363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 46caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcga
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gttctccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36347121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
47Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Phe Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12048363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 48caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cccgacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
tctgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36349121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
49Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Phe Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12050363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 50caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
tctgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36351121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
51Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12052363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 52caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36353121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
53Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12054363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 54caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cccgacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36355121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
55Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Ala Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Lys Tyr Asp Tyr Asp Ala Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12056363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 56caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acgccatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacaaatac 300gactacgacg ccggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36357121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
57Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Ala Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12058363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 58caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acgccatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36359121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
59Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12060363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 60caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacaaatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36361121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
61Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12062363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 62caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca gcggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36363121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
63Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12064363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 64caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca gcggcatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacaaatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36365121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
65Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Phe Thr Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Arg Leu Glu
Tyr Met 35 40 45Gly Tyr Ile Ser Tyr Asn Ala Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn
His Tyr Tyr Leu65 70 75 80Gln Leu Asn Ser Val Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Ala 85 90 95Arg Tyr Arg Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12066363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 66gaggtgcagc tgcaggaaag
cggccccagc ctggtcaagc ccagccagac cctgagcctg 60acctgcagcg tgaccggcga
cagcttcacc agcggctact ggaactggat ccggaagttc 120cccggcaacc
ggctcgagta catgggctac atcagctaca acgccatcac ctaccacaac
180cccagcctga agtcccggat cagcatcacc cgggacacca gcaagaacca
ctactacctg 240cagctgaaca gcgtgaccac cgaggacacc gccacctact
tttgcgcccg gtacagatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36367121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
67Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Ala Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12068363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 68caggtgcagc tgcaggaaag
cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg
cagcttcagc agcggctact ggaactggat ccggaagcac 120cccggcaagg
gcctggaata catcggctac atcagctaca acgccatcac ctaccacaac
180cccagcctga agtcccggat caccatcaac cgggacacca gcaagaacca
gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact
actgcgcccg gtacaaatac 300gactacgacg gcggccacgc catggactac
tggggccagg gcaccctggt caccgtgtcc 360tct 36369330PRTHomo sapiens
69Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33070990DNAHomo sapiens 70gcgtcgacca agggcccatc cgtcttcccc
ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcc 120tggaactcag gcgctctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
240tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagag
agttgagccc 300aaatcttgtg acaaaactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 360ccgtcagtct tcctcttccc cccaaaaccc
aaggacaccc tcatgatctc ccggacccct 420gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480tacgtggacg
gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg agaaccacag
gtctacaccc tgcccccatc ccgggaggag 720atgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
840ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa
gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 960cagaagagct taagcctgtc tccgggtaaa
99071451PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 71Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly
Gly Ser Phe Ser Ser Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro
Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile
Thr Tyr His Asn Pro Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg
Asp Thr Ser Lys Asn Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val
Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Lys Tyr
Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450721353DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 72caggtgcagc tgcaggaaag cggccctggc
ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg cagcttcagc
agcggctact ggaactggat ccggaagcac 120cccggcaagg gcctggaata
catcggctac atcagctaca acggcatcac ctaccacaac 180cccagcctga
agtcccggat caccatcaac cgggacacca gcaagaacca gtactccctg
240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact actgcgcccg
gtacaaatac 300gactacgacg gcggccacgc catggactac tggggccagg
gcaccctggt caccgtgtcc 360tctgcgtcga ccaagggccc atccgtcttc
cccctggcac cctcctccaa gagcacctct 420gggggcacag cggccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcctggaact
caggcgctct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt
gggcacccag 600acctacatct gcaacgtgaa tcacaagccc agcaacacca
aggtggacaa gagagttgag 660cccaaatctt gtgacaaaac tcacacatgc
ccaccgtgcc cagcacctga actcctgggg 720ggaccgtcag tcttcctctt
ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780cctgaggtca
catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac
840tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga
ggagcagtac 900aacagcacgt accgtgtggt cagcgtcctc accgtcctgc
accaggactg gctgaatggc 960aaggagtaca agtgcaaggt ctccaacaaa
gccctcccag cccccatcga gaaaaccatc 1020tccaaagcca aagggcagcc
ccgagaacca caggtctaca ccctgccccc atcccgggag 1080gagatgacca
agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac
1140atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac
cacgcctccc 1200gtgctggact ccgacggctc cttcttcctc tatagcaagc
tcaccgtgga caagagcagg 1260tggcagcagg ggaacgtctt ctcatgctcc
gtgatgcatg aggctctgca caaccactac 1320acgcagaaga gcttaagcct
gtctccgggt aaa 135373448PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 73Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Gly 20 25 30Tyr Trp Asn Trp
Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45Gly Tyr Ile
Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys 50 55 60Ser Arg
Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu65 70 75
80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445741344DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 74caggtgcagc tgcaggaaag cggccctggc
ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg tgtacggcgg cagcttcagc
agcggctact ggaactggat ccggaagcac 120cccggcaagg gcctggaata
catcggctac atcagctaca acggcatcac ctaccacaac 180cccagcctga
agtcccggat caccatcaac cgggacacca gcaagaacca gtactccctg
240cagctgaaca gcgtgacccc cgaggacacc gccgtgtact actgcgcccg
gtacaaatac 300gactacgacg gcggccacgc catggactac tggggccagg
gcaccctggt caccgtgtcc 360tctgcgtcga ccaagggccc cagcgtgttc
cccctggccc cttgcagcag aagcaccagc 420gagagcacag ccgccctggg
ctgcctggtg aaggactact tccccgagcc cgtgaccgtg 480tcctggaaca
gcggcgctct gaccagcggc gtgcatacct tccccgccgt gctccagagc
540agcggactgt actccctgag cagcgtggtg accgtgcctt ccagcagcct
gggcaccaag 600acctacacct gcaacgtgga ccacaagccc agcaacacca
aggtggacaa gagagtggag 660agcaagtacg gccctccctg ccccccttgc
cctgcccccg agttcctggg cggacctagc 720gtgttcctgt tcccccccaa
gcccaaggac accctgatga tcagcagaac ccccgaggtg 780acctgcgtgg
tggtggacgt gtcccaggag gaccccgagg tccagtttaa ttggtacgtg
840gacggcgtgg aagtgcataa cgccaagacc
aagcccagag aggagcagtt caacagcacc 900tacagagtgg tgtccgtgct
gaccgtgctg caccaggact ggctgaacgg caaggaatac 960aagtgcaagg
tctccaacaa gggcctgcct agcagcatcg agaagaccat cagcaaggcc
1020aagggccagc cacgggagcc ccaggtctac accctgccac ctagccaaga
ggagatgacc 1080aagaaccagg tgtccctgac ctgtctggtg aaaggcttct
atcccagcga tatcgccgtg 1140gagtgggaga gcaacggcca gcccgagaac
aactacaaga ccaccccccc tgtgctggac 1200agcgacggca gcttcttcct
gtactccaga ctgaccgtgg acaagtccag atggcaggag 1260ggcaacgtct
tcagctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag
1320tccctgagcc tgagcctggg caag 134475451PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
75Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Phe Glu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Ser Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450761353DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
76caggtgcagc tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg
60acctgtgccg tgtacggcgg cagcttcagc agcggctact ggaactggat ccggaagcac
120cccggcaagg gcctggaata catcggctac atcagctaca acggcatcac
ctaccacaac 180cccagcctga agtcccggat caccatcaac cgggacacca
gcaagaacca gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc
gccgtgtact actgcgcccg gtacaaatac 300gactacgacg gcggccacgc
catggactac tggggccagg gcaccctggt caccgtgtcc 360tctgcgtcga
ccaagggccc atccgtcttc cccctggcac cctcctccaa gagcacctct
420gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc
ggtgacggtg 480tcctggaact caggcgctct gaccagcggc gtgcacacct
tcccggctgt cctacagtcc 540tcaggactct actccctcag cagcgtggtg
accgtgccct ccagcagctt gggcacccag 600acctacatct gcaacgtgaa
tcacaagccc agcaacacca aggtggacaa gagagttgag 660cccaaatctt
gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga attcgagggg
720ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc 780cctgaggtca catgcgtggt ggtggacgtg agccacgaag
accctgaggt caagttcaac 840tggtacgtgg acggcgtgga ggtgcataat
gccaagacaa agccgcggga ggagcagtac 900aacagcacgt accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc 960aaggagtaca
agtgcaaggt ctccaacaaa gccctcccag cctccatcga gaaaaccatc
1020tccaaagcca aagggcagcc ccgagaacca caggtctaca ccctgccccc
atcccgggag 1080gagatgacca agaaccaggt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac 1140atcgccgtgg agtgggagag caatgggcag
ccggagaaca actacaagac cacgcctccc 1200gtgctggact ccgacggctc
cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1260tggcagcagg
ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
1320acgcagaaga gcctctccct gtctccgggt aaa 135377448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
77Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Ala Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Ser Lys Tyr Gly 210 215 220Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys 435 440 445781344DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 78caggtgcagc
tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg 60acctgtgccg
tgtacggcgg cagcttcagc agcggctact ggaactggat ccggaagcac
120cccggcaagg gcctggaata catcggctac atcagctaca acgccatcac
ctaccacaac 180cccagcctga agtcccggat caccatcaac cgggacacca
gcaagaacca gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc
gccgtgtact actgcgcccg gtacaaatac 300gactacgacg gcggccacgc
catggactac tggggccagg gcaccctggt caccgtgtcc 360tctgcgtcga
ccaagggccc cagcgtgttc cccctggccc cttgcagcag aagcaccagc
420gagagcacag ccgccctggg ctgcctggtg aaggactact tccccgagcc
cgtgaccgtg 480tcctggaaca gcggcgctct gaccagcggc gtgcatacct
tccccgccgt gctccagagc 540agcggactgt actccctgag cagcgtggtg
accgtgcctt ccagcagcct gggcaccaag 600acctacacct gcaacgtgga
ccacaagccc agcaacacca aggtggacaa gagagtggag 660agcaagtacg
gccctccctg ccccccttgc cctgcccccg agttcctggg cggacctagc
720gtgttcctgt tcccccccaa gcccaaggac accctgatga tcagcagaac
ccccgaggtg 780acctgcgtgg tggtggacgt gtcccaggag gaccccgagg
tccagtttaa ttggtacgtg 840gacggcgtgg aagtgcataa cgccaagacc
aagcccagag aggagcagtt caacagcacc 900tacagagtgg tgtccgtgct
gaccgtgctg caccaggact ggctgaacgg caaggaatac 960aagtgcaagg
tctccaacaa gggcctgcct agcagcatcg agaagaccat cagcaaggcc
1020aagggccagc cacgggagcc ccaggtctac accctgccac ctagccaaga
ggagatgacc 1080aagaaccagg tgtccctgac ctgtctggtg aaaggcttct
atcccagcga tatcgccgtg 1140gagtgggaga gcaacggcca gcccgagaac
aactacaaga ccaccccccc tgtgctggac 1200agcgacggca gcttcttcct
gtactccaga ctgaccgtgg acaagtccag atggcaggag 1260ggcaacgtct
tcagctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag
1320tccctgagcc tgagcctggg caag 134479451PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
79Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Ala Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Phe Glu Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Ser Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450801353DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
80caggtgcagc tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg
60acctgtgccg tgtacggcgg cagcttcagc agcggctact ggaactggat ccggaagcac
120cccggcaagg gcctggaata catcggctac atcagctaca acgccatcac
ctaccacaac 180cccagcctga agtcccggat caccatcaac cgggacacca
gcaagaacca gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc
gccgtgtact actgcgcccg gtacaaatac 300gactacgacg gcggccacgc
catggactac tggggccagg gcaccctggt caccgtgtcc 360tctgcgtcga
ccaagggccc atccgtcttc cccctggcac cctcctccaa gagcacctct
420gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc
ggtgacggtg 480tcctggaact caggcgctct gaccagcggc gtgcacacct
tcccggctgt cctacagtcc 540tcaggactct actccctcag cagcgtggtg
accgtgccct ccagcagctt gggcacccag 600acctacatct gcaacgtgaa
tcacaagccc agcaacacca aggtggacaa gagagttgag 660cccaaatctt
gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga attcgagggg
720ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc 780cctgaggtca catgcgtggt ggtggacgtg agccacgaag
accctgaggt caagttcaac 840tggtacgtgg acggcgtgga ggtgcataat
gccaagacaa agccgcggga ggagcagtac 900aacagcacgt accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc 960aaggagtaca
agtgcaaggt ctccaacaaa gccctcccag cctccatcga gaaaaccatc
1020tccaaagcca aagggcagcc ccgagaacca caggtctaca ccctgccccc
atcccgggag 1080gagatgacca agaaccaggt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac 1140atcgccgtgg agtgggagag caatgggcag
ccggagaaca actacaagac cacgcctccc 1200gtgctggact ccgacggctc
cttcttcctc tatagcaagc tcaccgtgga caagagcagg 1260tggcagcagg
ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
1320acgcagaaga gcctctccct gtctccgggt aaa 135381445PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
81Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser
Gly 20 25 30Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu
Tyr Ile 35 40 45Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro
Ser Leu Lys 50 55 60Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn
Gln Tyr Ser Leu65 70 75 80Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly
His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Lys Thr Thr Pro Pro Ser 115 120 125Val Tyr Pro Leu Ala
Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val 130 135 140Thr Leu Gly
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr
Val Pro 180 185 190Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn
Val Ala His Pro 195 200 205Ala Ser Ser Thr Lys Val Asp Lys Lys Ile
Val Pro Arg Asp Cys Gly 210 215 220Cys Lys Pro Cys Ile Cys Thr Val
Pro Glu Val Ser Ser Val Phe Ile225 230 235 240Phe Pro Pro Lys Pro
Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys 245 250 255Val Thr Cys
Val Val Val Asp Ile Ser Lys Asp Asp
Pro Glu Val Gln 260 265 270Phe Ser Trp Phe Val Asp Asp Val Glu Val
His Thr Ala Gln Thr Gln 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser
Thr Phe Arg Ser Val Ser Glu Leu 290 295 300Pro Ile Met His Gln Asp
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg305 310 315 320Val Asn Ser
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Thr
Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro 340 345
350Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr
355 360 365Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn
Gly Gln 370 375 380Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met
Asp Thr Asp Gly385 390 395 400Ser Tyr Phe Val Tyr Ser Lys Leu Asn
Val Gln Lys Ser Asn Trp Glu 405 410 415Ala Gly Asn Thr Phe Thr Cys
Ser Val Leu His Glu Gly Leu His Asn 420 425 430His His Thr Glu Lys
Ser Leu Ser His Ser Pro Gly Lys 435 440 445821335DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
82caggtgcagc tgcaggaaag cggccctggc ctggtcaagc ccagccagac cctgagcctg
60acctgtgccg tgtacggcgg cagcttcagc agcggctact ggaactggat ccggaagcac
120cccggcaagg gcctggaata catcggctac atcagctaca acggcatcac
ctaccacaac 180cccagcctga agtcccggat caccatcaac cgggacacca
gcaagaacca gtactccctg 240cagctgaaca gcgtgacccc cgaggacacc
gccgtgtact actgcgcccg gtacaaatac 300gactacgacg gcggccacgc
catggactac tggggccagg gcaccctggt caccgtgtcc 360tctgcgaaga
cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact
420aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc
agtgacagtg 480acctggaact ctggatccct gtccagcggt gtgcacacct
tcccagctgt cctgcagtct 540gacctctaca ctctgagcag ctcagtgact
gtcccctcca gcacctggcc cagcgagacc 600gtcacctgca acgttgccca
cccggccagc agcaccaagg tggacaagaa aattgtgccc 660agggattgtg
gttgtaagcc ttgcatatgt accgtcccag aagtatcatc tgtcttcatc
720ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt
cacgtgtgtt 780gtggtagaca tcagcaagga tgatcccgag gtccagttca
gctggtttgt agatgatgtg 840gaggtgcaca cagctcagac gcaaccccgg
gaggagcagt tcaacagcac tttccgctca 900gtcagtgaac ttcccatcat
gcaccaggac tggctcaatg gcaaggagtt caaatgcagg 960gtcaacagtg
cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga
1020ccgaaggctc cacaggtgta taccattcca cctcccaagg agcagatggc
caaggataaa 1080gtcagtctga cctgcatgat aacagacttc ttccctgaag
acattactgt ggagtggcag 1140tggaatgggc agccagcgga gaactacaag
aacactcagc ccatcatgga cacagatggc 1200tcttacttcg tctacagcaa
gctcaatgtg cagaagagca actgggaggc aggaaatact 1260ttcacctgct
ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc
1320cactctcctg gtaaa 133583214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 83Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr
Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Asp Ala
Ala 100 105 110Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
Thr Ser Gly 115 120 125Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
Tyr Pro Lys Asp Ile 130 135 140Asn Val Lys Trp Lys Ile Asp Gly Ser
Glu Arg Gln Asn Gly Val Leu145 150 155 160Asn Ser Trp Thr Asp Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175Ser Thr Leu Thr
Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190Thr Cys
Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200
205Phe Asn Arg Asn Glu Cys 21084642DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
84gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc
60atcacctgtc gggccagcca ggacatcagc aactacctga actggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctactac accagcaagc tgcacagcgg
cgtgcccagc 180agattcagcg gcagcggctc cggcaccgac tacaccctga
ccatcagcag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
ggctccgccc tgccctggac ctttggccag 300ggcaccaagg tggaaatcaa
gcgggctgat gcggcgccaa ctgtatccat cttcccacca 360tccagtgagc
agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac
420cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa
tggcgtcctg 480aacagttgga ctgatcagga cagcaaagac agcacctaca
gcatgagcag caccctcacg 540ttgaccaagg acgagtatga acgacataac
agctatacct gtgaggccac tcacaagaca 600tcaacttcac ccattgtcaa
gagcttcaac aggaatgagt gt 64285117PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 85Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr 20 25 30Asn Leu His
Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Arg
Met Arg Tyr Asp Gly Asp Thr Tyr Tyr Asn Ser Val Leu Lys 50 55 60Ser
Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Phe Leu65 70 75
80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Thr
85 90 95Arg Asp Gly Arg Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Val
Met 100 105 110Val Thr Val Ser Ser 11586441PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
86Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly
Tyr 20 25 30Asn Leu His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Arg Met Arg Tyr Asp Gly Asp Thr Tyr Tyr Asn Ser
Val Leu Lys 50 55 60Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn
Gln Val Phe Leu65 70 75 80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr
Ala Ile Tyr Tyr Cys Thr 85 90 95Arg Asp Gly Arg Gly Asp Ser Phe Asp
Tyr Trp Gly Gln Gly Val Met 100 105 110Val Thr Val Ser Ser Ala Ser
Thr Thr Pro Pro Ser Val Tyr Pro Leu 115 120 125Ala Pro Gly Ser Ala
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys 130 135 140Leu Val Lys
Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser145 150 155
160Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser
Thr Trp 180 185 190Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro
Ala Ser Ser Thr 195 200 205Lys Val Asp Lys Lys Ile Val Pro Arg Asp
Cys Gly Cys Lys Pro Cys 210 215 220Ile Cys Thr Val Pro Glu Val Ser
Ser Val Phe Ile Phe Pro Pro Lys225 230 235 240Pro Lys Asp Val Leu
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val 245 250 255Val Val Asp
Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe 260 265 270Val
Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu 275 280
285Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His
290 295 300Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn
Ser Ala305 310 315 320Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Thr Lys Gly Arg 325 330 335Pro Lys Ala Pro Gln Val Tyr Thr Ile
Pro Pro Pro Lys Glu Gln Met 340 345 350Ala Lys Asp Lys Val Ser Leu
Thr Cys Met Ile Thr Asp Phe Phe Pro 355 360 365Glu Asp Ile Thr Val
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn 370 375 380Tyr Lys Asn
Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val385 390 395
400Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr
405 410 415Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His
Thr Glu 420 425 430Lys Ser Leu Ser His Ser Pro Gly Lys 435
440871323DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 87caggtgcagc tgaaggagtc aggacctggt
ctggtgcagc cctcacagac cctgtccctc 60acctgcactg tctctgggtt ctcactaacc
ggttacaatt tacactgggt tcgccagcct 120ccaggaaagg gtctggagtg
gatgggaaga atgaggtatg atggagacac atattataat 180tcagttctca
aatcccgact gagcatcagc agggacacct ccaagaacca agttttcttg
240aaaatgaaca gtctgcaaac ggatgacaca gccatttact attgtaccag
agacgggcgt 300ggtgactcct ttgattactg gggccaagga gtcatggtca
cagtctcctc cgcgtcgacg 360acacccccat ctgtctatcc actggcccct
ggatctgctg cccaaactaa ctccatggtg 420accctgggat gcctggtcaa
gggctatttc cctgagccag tgacagtgac ctggaactct 480ggatccctgt
ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact
540ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt
cacctgcaac 600gttgcccacc cggccagcag caccaaggtg gacaagaaaa
ttgtgcccag ggattgtggt 660tgtaagcctt gcatatgtac cgtcccagaa
gtatcatctg tcttcatctt ccccccaaag 720cccaaggatg tgctcaccat
tactctgact cctaaggtca cgtgtgttgt ggtagacatc 780agcaaggatg
atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca
840gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt
cagtgaactt 900cccatcatgc accaggactg gctcaatggc aaggagttca
aatgcagggt caacagtgca 960gctttccctg cccccatcga gaaaaccatc
tccaaaacca aaggcagacc gaaggctcca 1020caggtgtata ccattccacc
tcccaaggag cagatggcca aggataaagt cagtctgacc 1080tgcatgataa
cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag
1140ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc
ttacttcgtc 1200tacagcaagc tcaatgtgca gaagagcaac tgggaggcag
gaaatacttt cacctgctct 1260gtgttacatg agggcctgca caaccaccat
actgagaaga gcctctccca ctctcctggt 1320aaa 132388112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
88Asp Ile Val Met Thr Gln Gly Ala Leu Pro Asn Pro Val Pro Ser Gly1
5 10 15Glu Ser Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Val Tyr
Lys 20 25 30Asp Gly Gln Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Thr Tyr Trp Met Ser Thr Arg Ala Ser
Gly Val Ser 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Arg Ala Glu Asp Ala Gly Val
Tyr Tyr Cys Gln Gln Val 85 90 95Arg Glu Tyr Pro Phe Thr Phe Gly Ser
Gly Thr Lys Leu Glu Ile Lys 100 105 11089219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
89Asp Ile Val Met Thr Gln Gly Ala Leu Pro Asn Pro Val Pro Ser Gly1
5 10 15Glu Ser Ala Ser Ile Thr Cys Arg Ser Ser Gln Ser Leu Val Tyr
Lys 20 25 30Asp Gly Gln Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Thr Tyr Trp Met Ser Thr Arg Ala Ser
Gly Val Ser 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Arg Ala Glu Asp Ala Gly Val
Tyr Tyr Cys Gln Gln Val 85 90 95Arg Glu Tyr Pro Phe Thr Phe Gly Ser
Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg Ala Asp Ala Ala Pro Thr
Val Ser Ile Phe Pro Pro Ser Ser Glu 115 120 125Gln Leu Thr Ser Gly
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 130 135 140Tyr Pro Lys
Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg145 150 155
160Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
165 170 175Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu
Tyr Glu 180 185 190Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys
Thr Ser Thr Ser 195 200 205Pro Ile Val Lys Ser Phe Asn Arg Asn Glu
Cys 210 21590657DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 90gatattgtga tgacccaggg
tgcactcccc aatcctgtcc cttctggaga gtcagcttcc 60atcacctgca ggtctagtca
gagtctggta tacaaagacg gccagacata cttgaattgg 120tttctgcaga
ggccaggaca gtctcctcag cttctgacct attggatgtc tacccgtgca
180tcaggagtct cagacaggtt cagtggcagt gggtcaggaa catatttcac
actgaaaatc 240agtagagtga gggctgagga tgcgggtgtg tattactgtc
agcaagttcg agagtatcct 300ttcactttcg gctcagggac gaagttggaa
ataaaacggg ctgatgcggc gccaactgta 360tccatcttcc caccatccag
tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 420ttgaacaact
tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga
480caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac
ctacagcatg 540agcagcaccc tcacgttgac caaggacgag tatgaacgac
ataacagcta tacctgtgag 600gccactcaca agacatcaac ttcacccatt
gtcaagagct tcaacaggaa tgagtgt 65791277PRTHomo sapiens 91Met Cys Val
Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu1 5 10 15Leu Leu
Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30Gly
Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40
45Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys
50 55 60Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys
Pro65 70 75 80Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser
Glu Arg Lys 85 90 95Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg
Cys Arg Ala Gly 100 105 110Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly
Val Asp Cys Ala Pro Cys 115 120 125Pro Pro Gly His Phe Ser Pro Gly
Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140Thr Asn Cys Thr Leu Ala
Gly Lys His Thr Leu Gln Pro Ala Ser Asn145 150 155 160Ser Ser Asp
Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175Gln
Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185
190Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu
195 200 205Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly
Leu Val 210 215 220Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala
Leu Tyr Leu Leu225 230 235 240Arg Arg Asp Gln Arg Leu Pro Pro Asp
Ala His Lys Pro Pro Gly Gly 245 250 255Gly Ser Phe Arg Thr Pro Ile
Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270Thr Leu Ala Lys Ile
27592272PRTMus musculus 92Met Tyr Val Trp Val Gln Gln Pro Thr Ala
Leu Leu Leu Leu Gly Leu1 5 10 15Thr Leu Gly Val Thr Ala Arg Arg Leu
Asn Cys Val Lys His Thr Tyr 20 25 30Pro Ser Gly His Lys Cys Cys Arg
Glu Cys Gln Pro Gly His Gly Met 35 40 45Val Ser Arg Cys Asp His Thr
Arg Asp Thr Leu Cys His Pro Cys Glu 50 55 60Thr Gly Phe Tyr Asn Glu
Ala Val Asn Tyr Asp Thr Cys Lys Gln Cys65 70 75 80Thr Gln Cys Asn
His Arg Ser Gly Ser Glu Leu Lys Gln Asn Cys Thr 85 90 95Pro Thr Gln
Asp Thr Val Cys Arg Cys Arg Pro Gly Thr Gln Pro Arg 100 105 110Gln
Asp Ser Gly Tyr Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro
115 120 125Gly His Phe Ser Pro Gly Asn Asn Gln Ala Cys Lys Pro Trp
Thr Asn 130 135 140Cys Thr Leu Ser Gly Lys Gln Thr Arg His Pro Ala
Ser Asp Ser Leu145 150 155 160Asp Ala Val Cys Glu Asp Arg Ser Leu
Leu Ala Thr Leu Leu Trp Glu 165 170 175Thr Gln Arg Pro Thr Phe Arg
Pro Thr Thr Val Gln Ser Thr Thr Val 180 185 190Trp Pro Arg Thr Ser
Glu Leu Pro Ser Pro Pro Thr Leu Val Thr Pro 195 200 205Glu Gly Pro
Ala Phe Ala Val Leu Leu Gly Leu Gly Leu Gly Leu Leu 210 215 220Ala
Pro Leu Thr Val Leu Leu Ala Leu Tyr Leu Leu Arg Lys Ala Trp225 230
235 240Arg Leu Pro Asn Thr Pro Lys Pro Cys Trp Gly Asn Ser Phe Arg
Thr 245 250 255Pro Ile Gln Glu Glu His Thr Asp Ala His Phe Thr Leu
Ala Lys Ile 260 265 270936PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 93Arg Tyr Asp Tyr Asp Gly1
5946PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 94Lys Tyr Asp Tyr Asp Ala1 5956PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 95Lys
Tyr Asp Tyr Asp Gly1 596177PRTHomo sapiens 96Met Cys Val Gly Ala
Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu1 5 10 15Leu Leu Leu Gly
Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30Gly Asp Thr
Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45Gly Asn
Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys 50 55 60Arg
Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro65 70 75
80Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys
85 90 95Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala
Gly 100 105 110Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys
Ala Pro Cys 115 120 125Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln
Ala Cys Lys Pro Trp 130 135 140Thr Asn Cys Thr Leu Ala Gly Lys His
Thr Leu Gln Pro Ala Ser Asn145 150 155 160Ser Ser Asp Ala Ile Cys
Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175Gln97175PRTMus
musculus 97Met Tyr Val Trp Val Gln Gln Pro Thr Ala Leu Leu Leu Leu
Ala Leu1 5 10 15Thr Leu Gly Val Thr Ala Arg Arg Leu Asn Cys Val Lys
His Thr Tyr 20 25 30Pro Ser Gly His Lys Cys Cys Arg Glu Cys Gln Pro
Gly His Gly Met 35 40 45Val Ser Arg Cys Asp His Thr Arg Asp Thr Leu
Cys His Pro Cys Glu 50 55 60Thr Gly Phe Tyr Asn Glu Ala Val Asn Tyr
Asp Thr Cys Lys Gln Cys65 70 75 80Thr Gln Cys Asn His Arg Ser Gly
Ser Glu Leu Lys Gln Asn Cys Thr 85 90 95Pro Thr Gln Asp Thr Val Cys
Arg Cys Arg Pro Gly Thr Gln Pro Arg 100 105 110Gln Asp Ser Gly Tyr
Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro 115 120 125Gly His Phe
Ser Pro Gly Asn Asn Gln Ala Cys Lys Pro Trp Thr Asn 130 135 140Cys
Thr Leu Ser Gly Lys Gln Thr Arg His Pro Ala Ser Asp Ser Leu145 150
155 160Asp Ala Val Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp
165 170 175986PRTArtificial SequenceDescription of Artificial
Sequence Synthetic 6xHis tag 98His His His His His His1 5
* * * * *
References