U.S. patent application number 16/245816 was filed with the patent office on 2019-05-09 for binding molecules to the human ox40 receptor.
This patent application is currently assigned to Pfizer Inc.. The applicant listed for this patent is Bristol-Myers Squibb Company, Pfizer Inc.. Invention is credited to Peter BRAMS, Brigitte DEVAUX, Rory F. FINN, Ronald P. GLADUE, Haichun HUANG, Heidi N. LEBLANC, Wei LIAO, Jing MIN, Timothy J. PARADIS, Arvind RAJPAL, Barrett R. THIELE, Kristopher TOY, Yanli WU, Yi WU.
Application Number | 20190135932 16/245816 |
Document ID | / |
Family ID | 40552119 |
Filed Date | 2019-05-09 |
United States Patent
Application |
20190135932 |
Kind Code |
A1 |
MIN; Jing ; et al. |
May 9, 2019 |
BINDING MOLECULES TO THE HUMAN OX40 RECEPTOR
Abstract
The present disclosure provides isolated binding molecules that
bind to the human OX40R, nucleic acid molecules encoding an amino
acid sequence of the binding molecules, vectors comprising the
nucleic acid molecules, host cells containing the vectors, methods
of making the binding molecules, pharmaceutical compositions
containing the binding molecules, and methods of using the binding
molecules or compositions.
Inventors: |
MIN; Jing; (Chesterfield,
MO) ; WU; Yanli; (Ballwin, MO) ; FINN; Rory
F.; (Manchester, MO) ; THIELE; Barrett R.;
(St. Louis, MO) ; LIAO; Wei; (Chesterfield,
MO) ; GLADUE; Ronald P.; (Stonington, CT) ;
RAJPAL; Arvind; (San Francisco, CA) ; PARADIS;
Timothy J.; (Richmond, RI) ; BRAMS; Peter;
(Sacramento, CA) ; DEVAUX; Brigitte; (Palo Alto,
CA) ; WU; Yi; (Milpitas, CA) ; TOY;
Kristopher; (San Jose, CA) ; LEBLANC; Heidi N.;
(Mountain View, CA) ; HUANG; Haichun; (Fremont,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pfizer Inc.
Bristol-Myers Squibb Company |
New York
Princeton |
NY
NJ |
US
US |
|
|
Assignee: |
Pfizer Inc.
New York
NY
Bristol-Myers Squibb Company
Princeton
NJ
|
Family ID: |
40552119 |
Appl. No.: |
16/245816 |
Filed: |
January 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15807351 |
Nov 8, 2017 |
10196452 |
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16245816 |
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14692948 |
Apr 22, 2015 |
9840562 |
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15807351 |
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13474466 |
May 17, 2012 |
9028824 |
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14692948 |
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13099196 |
May 2, 2011 |
8236930 |
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13474466 |
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12332944 |
Dec 11, 2008 |
7960515 |
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13099196 |
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61013947 |
Dec 14, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2317/92 20130101; A61K 2039/505 20130101; C07K 16/2878
20130101; A61P 37/04 20180101; C07K 2317/565 20130101; A61P 35/04
20180101; A61P 35/02 20180101; A61P 31/00 20180101; C07K 2317/14
20130101; C07K 2317/21 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1.-28. (canceled)
29. An isolated monoclonal antibody that binds to human OX40R,
comprising: (a) a heavy chain CDR1 comprising an amino acid
sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising an amino
acid sequence of SEQ ID NO: 2; and a heavy chain CDR3 comprising an
amino acid sequence of SEQ ID NO: 3; or (b) a heavy chain CDR1
comprising an amino acid sequence of SEQ ID NO: 13; a heavy chain
CDR2 comprising an amino acid sequence of SEQ ID NO: 14; and a
heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:
15, wherein said isolated monoclonal antibody comprises 1, 2, 3 or
4 conservative amino acid substitutions in one or more heavy chain
CDR sequences; and wherein said isolated monoclonal antibody: (a)
binds to human OX40R with a K.sub.D of 5.times.10.sup.-9 M or less;
and (b) has agonist activity on human OX40R.
30. The antibody according to claim 29, further comprising: (a) a
light chain comprising a light chain CDR1 comprising an amino acid
sequence of SEQ ID NO: 4; a light chain CDR2 comprising an amino
acid sequence of SEQ ID NO: 5; and a light chain CDR3 comprising an
amino acid sequence of SEQ ID NO: 6; or (b) a light chain
comprising a light chain CDR1 comprising an amino acid sequence of
SEQ ID NO: 16; a light chain CDR2 comprising an amino acid sequence
of SEQ ID NO: 17; and a light chain CDR3 comprising an amino acid
sequence of SEQ ID NO: 18.
31. The antibody according to claim 29, which is a human antibody,
or a chimeric or humanized antibody.
32. A composition comprising an antibody according to claim 29 and
optionally a pharmaceutically acceptable carrier.
33. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes an antibody according to claim 29.
34. A vector comprising the nucleic acid molecule of claim 33.
35. The vector according to claim 34, further comprising an
expression control sequence operably linked to the nucleic acid
molecule.
36. A host cell comprising the vector according to claim 34.
37. An in vitro method of inhibiting growth of tumor cells,
comprising contacting the tumor cells with an antibody according to
claim 29, wherein the antibody is in an amount effective to inhibit
the growth of the tumor cells.
38. A method of treating cancer in a mammal in need thereof,
comprising administering to the mammal a therapeutically effective
amount of the composition of claim 32.
39. The method according to claim 38, wherein the cancer is
selected from the group consisting of hepatocellular carcinoma,
squamous cell carcinoma, head and neck cancer, renal cell
carcinoma, melanoma, breast cancer, prostate cancer, colorectal
cancer, lung cancer, and hematological cancer.
40. The antibody according to claim 30, which is a human antibody,
or a chimeric or humanized antibody.
41. A composition comprising an antibody according to claim 30 and
optionally a pharmaceutically acceptable carrier.
42. A method of enhancing an immune response in a mammal in need
thereof, comprising administering to the mammal a therapeutically
effective amount of the composition of claim 32.
43. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes an antibody according to claim 30.
44. A vector comprising the nucleic acid molecule of claim 43.
45. The vector according to claim 34, further comprising an
expression control sequence operably linked to the nucleic acid
molecule.
46. A host cell comprising the vector according to claim 45.
47. An in vitro method of inhibiting growth of tumor cells,
comprising contacting the tumor cells with an antibody according to
claim 30, wherein the antibody is in an amount effective to inhibit
the growth of the tumor cells.
48. A method of enhancing an immune response in a mammal in need
thereof, comprising administering to the mammal a therapeutically
effective amount of the composition of claim 41.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/013,947 filed on 14 Dec. 2007, which is
incorporated herein by reference in its entity.
JOINT RESEARCH AGREEMENT
[0002] The disclosure and claims herein were made as a result of
activities undertaken within the scope of a joint research
agreement in effect on or before the date the claimed invention was
made between Pfizer Inc. and Medarex, Inc.
BACKGROUND
[0003] The present disclosure relates to antibodies, and
particularly to antibodies that bind to the OX40 receptor.
[0004] Enhancing anti-tumor T cell function represents a powerful
and novel approach for cancer treatment. Crucial components
involved with generating an effective anti-tumor T cell response
include enhancing CD4+ helper T cell activity to promote the
generation of anti-tumor cytolytic T cells, and providing survival
signals for memory and effector T cells. A key receptor that has
been shown to mediate these responses is the OX40 receptor.
Sugamura, K., Ishii, N., Weinberg, A. Therapeutic targeting of the
effector T-cell co-stimulatory molecule OX40. Nature Rev. Imm. 4:
420-431 (2004); Hori, T. Roles of OX40 in the pathogenesis and
control of diseases. Intn. J. Hematology. 83: 17-22 (2006).
[0005] The OX40 receptor (OX40R) (also known as CD1134, TNFRSF4,
ACT-4, ACT35, and TXGP1L) is a member of the TNF receptor
superfamily. The OX40R is found to be expressed on activated CD4+
T-cells. High numbers of OX40R+ T cells have been demonstrated
within tumors (tumor infiltrating lymphocytes) and in the draining
lymph nodes of cancer patients (Vetto, J. T. et al. 1997. Presence
of the T-cell activation marker OX-40 on tumor infiltrating
lymphocytes and draining lymph nodes cells from patients with
melanoma and head and neck cancers. Am. J. Surg. 174: 258-265;
Weinberg, A. D. et al. Engagement of the OX-40 receptor in vivo
enhances antitumor immunity. J. Immunol. 164: 2160-69 (2000);
Petty, J. K., et al. Survival in human colorectal cancer correlates
with expression of the T-cell costimulatory molecule OX-40 (CD134).
Am. J. Surg. 183: 512-518 (2002)). It was shown in tumor models in
mice that engagement of the OX40R in vivo during tumor priming
significantly delayed and prevented the appearance of tumors as
compared to control treated mice (Weinberg et al., 2000).
Therefore, it has been contemplated to enhance the immune response
of a mammal to an antigen by engaging the OX40R through the use of
an OX40R binding agent (WO 99/42585; Weinberg et al., 2000).
SUMMARY
[0006] The present disclosure provides isolated binding molecules
that bind to the human OX40R, including OX40R antibodies,
antigen-binding fragments of the OX40R antibodies, and derivatives
of the OX40R antibodies. In some embodiments the binding molecule
binds to the human OX40R with a K.sub.D of 1.times.10.sup.-7 M or
less and has agonist activity on the human OX40R. In some further
embodiments, the binding molecule is a human monoclonal antibody
that specifically binds to the human OX40R with a K.sub.D of 100 nM
or less.
[0007] The present disclosure also provides a composition that
comprises one or more of the binding molecules and a
pharmaceutically acceptable carrier. In some embodiments, the
binding molecule is a human monoclonal OX40R antibody or an
antigen-binding fragment thereof. The composition may further
comprise additional pharmaceutical agents, such as chemotherapeutic
agents, immunotherapeutic agents, and homornal therepeutic
agents.
[0008] The present disclosure further provides therapeutic and
diagnostic methods using the binding molecules. In some
embodiments, the disclosure provides a method of treating or
preventing cancer in a mammal, comprising administering to the
mammal a therapeutically effective amount of a binding molecule or
a composition comprising a binding molecule. In some other
embodiments, the disclosure provides a method of enhancing an
immune response in a mammal, comprising administering to the mammal
a therapeutically effective amount of a binding molecule or a
composition comprising a binding molecule. In some particular
embodiments the binding molecule used in the methods is a human
monoclonal OX40R antibody or an antigen-binding fragment
thereof.
[0009] The present disclosure further provides nucleic acid
molecules that encode an amino acid sequence of a binding molecule,
vectors comprising such nucleic acids, host cells comprising the
vectors, and methods of preparing the binding molecules.
[0010] The disclosure also provides other aspects, which will be
apparent from the entire disclosure, including the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1a and 1b are graphs showing that antibody 11D4
specifically binds to the OX40R;
[0012] FIG. 2 is a graph showing the effect of cross-linked
antibody 11D4 on OX40R-stimulated luciferase activity;
[0013] FIG. 3 is a graph showing the effect of antibody 11D4 on
IL-2 production by alloantigen primed T cells;
[0014] FIG. 4 is a graph showing the effect of antibody 11D4 on
anti-CD3 induced IL-2 production by primary T cells;
[0015] FIG. 5 is a graph showing the effect of antibody 11D4 on
anti-CD3 induced IL-2 production by cynomolgus primary T cells;
[0016] FIG. 6 shows the saturation binding curves with antibody
11D4 using cynomolgus PBMC's from 14 donors stimulated with
anti-CD3 and anti-CD28;
[0017] FIG. 7 shows the saturation binding curves with antibody
11D4 using human PBMC's from 17 donors stimulated with anti-CD3 and
anti-CD28;
[0018] FIG. 8 is a graph showing the effect of antibody 11D4 on the
growth of B cell lymphoma Raji in SCID mice;
[0019] FIG. 9 is a graph showing the effect of antibody 11D4 on the
growth of B cell lymphoma Raji 21 days after tumor injection;
[0020] FIG. 10 is a graph showing the effects of a single injection
of antibody 11D4 on the growth of the prostate tumor PC-3 in SCID
mice;
[0021] FIG. 11 is a graph showing the effect of antibody 11D4 on
the growth of the prostate tumor PC-3 in SCID mice 27 days after
tumor injection;
[0022] FIG. 12 is a graph showing the effect of antibody 11D4 on
the growth of the colon carcinoma LOVO in SCID mice;
[0023] FIG. 13 is a graph showing the effect of antibody 11D4 on
the growth of the colon carcinoma LOVO in SCID mice 25 days after
tumor injection;
[0024] FIG. 14 is a graph showing the effect of antibody 11D4 on
the growth of the breast tumor BT474 in SCID mice; and
[0025] FIG. 15 is a graph showing the effect of antibody 11D4 on
the growth of the breast tumor BT474 in SCID mice.
DETAILED DESCRIPTION
Definitions
[0026] The term "agonist" refers to a binding molecule, as defined
herein, which upon binding to the OX40R, (1) stimulates or
activates the OX40R, (2) enhances, increases, promotes, induces, or
prolongs an activity, function, or presence of the OX40R, or (3)
enhances, increases, promotes, or induces the expression of the
OX40R.
[0027] The term "antibody" refers to an immunoglobulin molecule
that is typically composed of two identical pairs of polypeptide
chains, each pair having one "light" (L) chain and one "heavy" (H)
chain. Human light chains are classified as kappa and lambda light
chains. Heavy chains are classified as mu, delta, gamma, alpha, or
epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA,
and IgE, respectively. Within light and heavy chains, the variable
and constant regions are joined by a "J" region of about 12 or more
amino acids, with the heavy chain also including a "D" region of
about 3 or more amino acids. Each heavy chain is comprised of a
heavy chain variable region (abbreviated herein as HCVR or V.sub.H)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, C.sub.H1, C.sub.H2 and C.sub.H3.
Each light chain is comprised of a light chain variable region
(abbreviated herein as LCVR or V.sub.L) and a light chain constant
region. The light chain constant region is comprised of one domain,
CL. The constant regions of the antibodies may mediate the binding
of the immunoglobulin to host tissues or factors, including various
cells of the immune system (e.g., effector cells) and the first
component (C1q) of the classical complement system. The V.sub.H and
V.sub.L regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
variable regions of each heavy/light chain pair (V.sub.H and
V.sub.L), respectively, form the antibody binding site. The
assignment of amino acids to each region or domain is in accordance
with the definitions of Kabat Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol.
196:901-917; Chothia et al. (1989) Nature 342:878-883. The term
"antibody" encompasses an antibody that is part of an antibody
multimer (a multimeric form of antibodies), such as dimers,
trimers, or higher-order multimers of monomeric antibodies. It also
encompasses an antibody that is linked or attached to, or otherwise
physically or functionally associated with, a non-antibody moiety.
Further, the term "antibody" is not limited by any particular
method of producing the antibody. For example, it includes, inter
alia, recombinant antibodies, monoclonal antibodies, and polyclonal
antibodies.
[0028] The term "antibody derivative" or "derivative" of an
antibody refers to a molecule that is capable of binding to the
same antigen (e.g., OX40R) that the antibody binds to and comprises
an amino acid sequence of the antibody linked to an additional
molecular entity. The amino acid sequence of the antibody that is
contained in the antibody derivative may be the full-length
antibody, or may be any portion or portions of a full-length
antibody. The additional molecular entity may be a chemical or
biological molecule. Examples of additional molecular entities
include chemical groups, amino acids, peptides, proteins (such as
enzymes, antibodies), and chemical compounds. The additional
molecular entity may have any utility, such as for use as a
detection agent, label, marker, pharmaceutical or therapeutic
agent. The amino acid sequence of an antibody may be attached or
linked to the additional entity by chemical coupling, genetic
fusion, noncovalent association or otherwise. The term "antibody
derivative" also encompasses chimeric antibodies, humanized
antibodies, and molecules that are derived from modifications of
the amino acid sequences of an OX40R antibody, such as conservation
amino acid substitutions, additions, and insertions.
[0029] The term "antigen-binding fragment" of an antibody refers to
one or more portions of a full-length antibody that retain the
ability to bind to the same antigen (e.g., OX40R) that the antibody
binds to. The term "antigen-binding fragment" also encompasses the
portion of an antibody that is part of a larger molecule formed by
covalent or noncovalent association of the antibody portion with
one or more additional molecular entities. Examples of additional
molecular entities include amino acids, peptides, or proteins, such
as the streptavidin core region, which may be used to make a
tetrameric scFv molecule (Kipriyanov et al., (1995) Human
Antibodies and Hybridomas 6:93-101), a cysteine residue, a marker
peptide, or a C-terminal polyhistidine tag, which may be used to
make bivalent and biotinylated scFv molecules (Kipriyanov et al.,
(1994) Mol. Immunol. 31:1047-1058).
[0030] The term "binding molecule" encompasses (1) antibody, (2)
antigen-binding fragment of an antibody, and (3) derivative of an
antibody, each as defined herein.
[0031] The term "binds to OX40R" or "binding to OX40R" refers to
the binding of a binding molecule, as defined herein, to the OX40R
in an in vitro assay, such as a BIAcore assay. Binding means a
binding affinity (K.sub.D) of 1.times.10.sup.-6 M or less.
[0032] The term "chimeric antibody" refers to an antibody that
comprises amino acid sequences derived from two or more different
antibodies. The two or more different antibodies may be from the
same species or from two or more different species.
[0033] The term "conservative amino acid substitution" refers to
substitution of an amino acid residue by another amino acid
residue, wherein the side chain R groups of the two amino acid
residues have similar chemical properties (e.g., charge or
hydrophobicity). Examples of groups of amino acids that have side
chains with similar chemical properties include 1) aliphatic side
chains: glycine, alanine, valine, leucine, and isoleuncine; 2)
aliphatic-hydroxyl side chains: serine and threonine; 3)
amide-containing side chains: asparagine and glutamine; 4) aromatic
side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side
chains: lysine, arginine, and histidine; 6) acidic side chains:
aspartic acid and glutamic acid; and 7) sulfur-containing side
chains: cysteine and methionine. Conservative amino acid
substitution groups can be, for example, valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine,
glutamate-aspartate, and asparagine-glutamine.
[0034] The term "epitope" refers to the part of an antigen that is
capable of specific binding to an antibody, or T-cell receptor, or
otherwise interacting with a molecule. "Epitope" is also known in
the art as "antigenic determinant." An epitope generally consists
of chemically active surface groupings of molecules such as amino
acids or carbohydrate or sugar side chains and generally have
specific three dimensional structural characteristics, as well as
specific charge characteristics. An epitope may be "linear" or
"conformational." Once a desired epitope on an antigen is
determined, antibodies to that epitope can be generated, e.g.,
using the techniques described herein. The generation and
characterization of antibodies may also elucidate information about
desirable epitopes. From this information, it is then possible to
competitively screen antibodies for binding to the same epitope. An
approach to achieve this is to conduct cross-competition studies to
find antibodies that competitively bind with one another, i.e., the
antibodies compete for binding to the antigen. A high throughput
process for "binning" antibodies based upon their cross-competition
is described in PCT Publication No. WO 03/48731.
[0035] The term "germline" refers to the nucleotide sequences of
the antibody genes and gene segments as they are passed from
parents to offspring via the germ cells. The germline sequence is
distinguished from the nucleotide sequences encoding antibodies in
mature B cells which have been altered by recombination and
hypermutation events during the course of B cell maturation.
[0036] The term "host cell" refers to a cell into which an
expression vector has been introduced. The term encompasses not
only the particular subject cell but also the progeny of such a
cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not be identical to the parent cell, but are still
included within the scope of the term "host cell." The term "human
antibody" refers to an antibody consisting of amino acid sequences
of human immunoglobulin sequences only. A human antibody may
contain murine carbohydrate chains if produced in a mouse, in a
mouse cell or in a hybridoma derived from a mouse cell. Human
antibodies may be prepared in a variety of ways known in the
art.
[0037] The term "humanized antibody" refers to a chimeric antibody
that contains amino acid residues derived from human antibody
sequences. A humanized antibody may contain some or all of the CDRs
from a non-human animal antibody while the framework and constant
regions of the antibody contain amino acid residues derived from
human antibody sequences.
[0038] The term "mammal" refers to any animal species of the
Mammalia class. Examples of mammals include: humans; laboratory
animals such as rats, mice, simians and guinea pigs; domestic
animals such as cats, dogs, rabbits, cattle, sheep, goats, horses,
and pigs; and captive wild animals such as lions, tigers,
elephants, and the like.
[0039] The term "isolated nucleic acid" refers to a nucleic acid
molecule of genomic, cDNA, or synthetic origin, or a combination
thereof, which is separated from other nucleic acid molecules
present in the natural source of the nucleic acid. For example,
with regard to genomic DNA, the term "isolated" includes nucleic
acid molecules which are separated from the chromosome with which
the genomic DNA is naturally associated. Preferably, an "isolated"
nucleic acid is free of sequences which naturally flank the nucleic
acid (i.e., sequences located at the 5' and 3' ends of the nucleic
acid of interest) in the genomic DNA of the organism from which the
nucleic acid is derived.
[0040] The term "isolated antibody" or "isolated binding molecule"
refers to an antibody or a binding molecule that: (1) is not
associated with naturally associated components that accompany it
in its native state; (2) is free of other proteins from the same
species; (3) is expressed by a cell from a different species; or
(4) does not occur in nature. Examples of isolated antibodies
include an OX40R antibody that has been affinity purified using
OX40R, an OX40R antibody that has been generated by hybridomas or
other cell line in vitro, and a human OX40R antibody derived from a
transgenic animal.
[0041] The term "K.sub.D" refers to the equilibrium dissociation
constant of a particular antibody-antigen interaction and is used
to describe the binding affinity between a ligand (such as an
antibody) and a protein (such as the OX40R). The smaller the
equilibrium dissociation constant, the more tightly bound the
ligand is, or the higher the affinity between ligand and protein. A
Kr can be measured by surface plasmon resonance, for example using
the BIACORE.TM. system. An assay procedure using the BIACORE.TM.
system (BIAcore assay) is described in the Examples section of this
disclosure.
[0042] The term "off rate" or "kd" refers to the dissociation rate
constant of a particular antibody-antigen interaction. A
dissociation rate constant can be measured by surface plasmon
resonance, for example using the BIACORE.TM..
[0043] The term "OX40R antibody" refers to an antibody, as defined
herein, capable of binding to the human OX40R.
[0044] The terms "OX40 receptor" and "OX40R" are used
interchangeably in the present application, and include the human
OX40R, as well as variants, isoforms, and species homologs thereof.
Accordingly, human binding molecules disclosed herein may, in
certain cases, also bind to the OX40R from species other than
human. In other cases, the binding molecules may be completely
specific for the human OX40R and may not exhibit species or other
types of cross-reactivity.
[0045] The term "specifically bind to the human OX40R" in reference
to the interaction of a binding molecule, e.g., an antibody, with
its binding partner, e.g., an antigen, means that the K.sub.D of a
binding molecule for binding to CD40, CD137, or CD271 is more than
100 fold the K.sub.D for its binding to the human OX40R, as
determined in an in vitro assay.
[0046] The term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid molecule in a host cell.
Examples of vectors include plasmids, viral vectors, naked DNA or
RNA expression vectors, cosmid or phage vectors. Some vectors are
capable of autonomous replication in a host cell into which they
are introduced (e.g., bacterial vectors having a bacterial origin
of replication and episomal mammalian vectors). Some vectors can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome (e.g., non-episomal mammalian vectors). Certain vectors are
capable of directing the expression of genes to which they are
operatively linked, and therefore may be referred to as "expression
vectors."
[0047] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2.sup.nd Edition, E. S. Golub and D. R. Gren, Eds.,
Sinauer Associates, Sunderland, Mass. (1991)).
[0048] Binding Molecules that Bind to the Human OX40R
[0049] The present disclosure provides isolated binding molecules
that bind to the human OX40R, including OX40R antibodies,
antigen-binding fragments of the OX40R antibodies, and derivatives
of the OX40R antibodies. The binding molecules are characterized by
at least one of the following functional properties: (a) bind to
the human OX40R with a K.sub.D of 1.times.10.sup.-6 M or less; (b)
have agonist activity on the human OX40R; (c) do not bind to CD40
receptor at concentration up to 500 nM; (d) do not bind to CD137
receptor at concentrations up to 500 nM; (e) do not bind to CD271
receptor at concentrations up to 500 nM; (f) are capable of
enhancing IL-2 production by isolated human T cells; (g) are
capable of enhancing immune response; (h) are capable of inhibiting
tumor cell growth; and (i) have therapeutic effect on a cancer. In
some embodiments the binding molecule binds to the human OX40R with
a K.sub.D of 1.times.10.sup.-7 M or less, or 1.times.10.sup.-8 M or
less, or 5.times.1.times.10.sup.-9 M or less.
[0050] Human OX40R Antibodies
[0051] In some first aspects, the present disclosure provides a
human antibody that binds to the human OX40R. In some embodiments,
the human antibody is a monoclonal antibody that specifically binds
to the human OX40R with a K.sub.D of 100 nM or less, preferably 10
nM or less, and has agonist activity on the human OX40R. One
example of such human antibodies is the human monoclonal antibody
11D4. The amino acid sequence of the heavy chain and amino acid
sequence of the variable region of the heavy chain (V.sub.H) of
antiboduy 11D4 are shown in SEQ ID NOs: 9 and 7, respectively. The
amino acid sequence of the light chain and the amino acid sequence
of the variable region of the light chain (V.sub.L) of antibody
11D4 are shown in SEQ ID NOS: 10 and 8, respectively. The isotypes
of antibody 11D4 are IgG2 for the heavy chain and Kappa for the
light chain. The allotypes of antibody 11D4 are G2(n-) for the
heavy chain and Km3 for the light chain. The mature heavy and light
chain amino acid sequences are derived from conceptual translation
of DNA sequences in the expression constructs. Antibody 11D4
contains no framework mutations in the heavy chain or light chain,
but contains one mutation in the heavy chain CDR2.
[0052] Another illustrative antibody of the disclosure is the human
monoclonal antibody 18D8. The amino acid sequence of the V.sub.H
region and V.sub.L region of antibody 18D8 is shown in SEQ ID NOs:
19 and 20, respectively. The amino acid sequence of the heavy chain
and light chain is shown in SEQ ID NOS: 21 and 22,
respectively.
[0053] Given that 11D4 and 18D8 bind to the OX40R, the V.sub.H and
V.sub.L sequences of each of them can be "mixed and matched" with
other OX40R antibodies to create additional antibodies. The binding
of such "mixed and matched" antibodies to the OX40R can be tested
using the binding assays known in the art, including an assay
described in the Examples. In one case, when V.sub.H and V.sub.L
regions are mixed and matched, a V.sub.H sequence from a particular
V.sub.H/V.sub.L pairing is replaced with a structurally similar
V.sub.H sequence. Likewise, in another case a V.sub.L sequence from
a particular V.sub.H/V.sub.L pairing is replaced with a
structurally similar V.sub.L sequence.
[0054] Accordingly, in some embodiments, the disclosure provides an
isolated OX40R antibody that comprises: (1) a heavy chain variable
region of antibody 11D4 or 18D8, (2) a heavy chain variable region
comprising an amino acid sequence of SEQ ID NOs: 7 or 19, or (3) a
heavy chain variable region comprising an amino acid sequence
encoded by a nucleic acid sequence of SEQ ID NOs: 11 or 23. In some
other embodiments, the disclosure provides an isolated OX40R
antibody that comprises: (1) a light chain variable region of
antibody 11D4 or 18D8, (2) a light chain variable region comprising
an amino acid sequence of SEQ ID NOs: 8 or 20, or (3) light chain
variable region comprising an amino acid sequence encoded by a
nucleic acid sequence of SEQ ID NOs: 12 or 24.
[0055] In another aspect, the disclosure provides antibodies that
comprise the CDR1, CDR2, and CDR3 of the heavy chain variable
region (V.sub.H) and CDR1, CDR2, and CDR3 of the light chain of
11D4 or 11D8. The amino acid sequence of the V.sub.H CDR1, V.sub.H
CDR2, and V.sub.H CDR3 of 11D4 is shown in SEQ ID NOs: 1, 2, and 3,
respectivelly. The amino acid sequence of the V.sub.L CDR1, V.sub.L
CDR2, and V.sub.L CDR3 of antibody 11D4 is shown in SEQ ID NOs: 4,
5, and 6, respectivelly. The amino acid sequence of the V.sub.H
CDR1, V.sub.H CDR2, and V.sub.H CDR3 of antibody 18D8 is shown in
SEQ ID NOs: 13, 14, and 15, respectively. The amino acid sequence
of the V.sub.L CDR1, V.sub.L CDR2, and V.sub.L CDR3 of antibody
18D8 is shown in SEQ ID NOs: 16, 17, and 18, respectivelly. The CDR
regions are delineated using the Kabat system (Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242).
[0056] Given that 11D4 and 18D8 bind to the human OX40R and that
antigen-binding specificity is provided primarily by the CDR1,
CDR2, and CDR3 regions, the V.sub.H CDR1, CDR2, and CDR3 sequences
and V.sub.L CDR1, CDR2, and CDR3 sequences can be "mixed and
matched" to create additional OX40R antibodies. For example, CDRs
from different OX40R antibodies can be mixed and matched, although
each antibody will typically contain a V.sub.H CDR1, CDR2, and CDR3
and a V.sub.L CDR1, CDR2, and CDR3. The binding of such "mixed and
matched" antibodies to the OX40R can be tested using the binding
assays described above and in the Examples (e.g., ELISAs, Biacore
analysis). In one case, when V.sub.H CDR sequences are mixed and
matched, the CDR1, CDR2 and/or CDR3 sequence from a particular
V.sub.H sequence is replaced with a structurally similar CDR
sequence(s). Likewise, when V.sub.L CDR sequences are mixed and
matched, the CDR1, CDR2 and/or CDR3 sequence from a particular
V.sub.L sequence typically is replaced with a structurally similar
CDR sequence(s). It will be readily apparent to an ordinarily
skilled artisan that novel V.sub.H and V.sub.L sequences can be
created by substituting one or more V.sub.H and/or V.sub.L CDR
region sequences with structurally similar sequences from the CDR
sequences disclosed herein.
[0057] Accordingly, in some embodiments, the disclosure provides
(1) an isolated monoclonal antibody that comprises at least one CDR
selected from V.sub.H CDR1, V.sub.H CDR2, or V.sub.H CDR3 of
antibody 11D4 or 18D8. In some other embodiments, the disclosure
provides an isolated monoclonal antibody that comprises at least
one CDR selected from V.sub.L CDR1, V.sub.L CDR2 or V.sub.L CDR3 of
antibody 11D4 or 18D8. In some further embodiments, the disclosure
provides an isolated monoclonal antibody that comprises at least
one CDR selected from: a V.sub.H CDR1 comprising the amino acid
sequence of SEQ ID NOs: 1 or 13, or a sequence that differs from
SEQ ID NOs: 1 or 3 by 1, 2, 3, or 4 conservative amino acid
substitutions; a V.sub.H CDR2 comprising the amino acid sequence of
SEQ ID NOs: 2 or 14 or a sequence that differs from SEQ ID NOs: 2
or 14 by 1, 2, 3, or 4 conservative amino acid substitutions; and a
V.sub.H CDR3 comprising the amino acid sequence of SEQ ID NOs: 3 or
15 or a sequence that differs from SEQ ID NOs: 3 or 15 by 1, 2, 3,
or 4 conservative amino acid substitutions.
[0058] In still some further embodiments, the disclosure provides
an isolated monoclonal antibody that comprises at least one CDR
selected from: a V.sub.L CDR1 comprising the amino acid sequence of
SEQ ID NOs: 4 or 16 or a sequence that differs from SEQ ID NOs: 4
or 16 by 1, 2, 3, or 4 conservative amino acid substitutions; a
V.sub.L CDR2 comprising the amino acid sequence of SEQ ID NOs: 5 or
17 or a sequence that differs from SEQ ID NOs: 5 or 17 by 1, 2, 3,
or 4 conservative amino acid substitutions; and a V.sub.L CDR3
comprising the amino acid sequence of SEQ ID NOs: 6 or 18 or a
sequence that differs from SEQ ID NOs: 6 or 18 by 1, 2, 3, or 4
conservative amino acid substitutions.
[0059] In some cases, the C-terminal lysine of the heavy chain of
an OX40R antibody is cleaved (Harris R. J., J. of Chromotography,
705: 129-134 (1995)). The heavy and/or light chain(s) of the OX40R
antibodies may optionally include a signal sequence.
[0060] The class (e.g., IgG, IgM, IgE, IgA, or IgD) and subclass
(e.g., IgG1, IgG2, IgG3, or IgG4) of the OX40R antibodies may be
determined by any suitable method. In general, the class and
subclass of an antibody may be determined using antibodies that are
specific for a particular class and subclass of antibody. Such
antibodies are commercially available. The class and subclass can
be determined by ELISA, or Western Blot as well as other
techniques. Alternatively, the class and subclass may be determined
by sequencing all or a portion of the constant domains of the heavy
and/or light chains of the antibodies, comparing their amino acid
sequences to the known amino acid sequences of various class and
subclasses of immunoglobulins, and determining the class and
subclass of the antibodies. The OX40R antibodies can be an IgG, an
IgM, an IgE, an IgA, or an IgD molecule. For example, the OX40R
antibodies can be an IgG that is an IgG1, IgG2, IgG3, or an IgG4
subclass. Thus, another aspect of the disclosure provides a method
for converting the class or subclass of an OX40R antibody to
another class or subclass. In some cases, a nucleic acid molecule
encoding a V.sub.L or V.sub.H that does not include sequences
encoding C.sub.L or C.sub.H is isolated using methods well-known in
the art. The nucleic acid molecule then is operatively linked to a
nucleic acid sequence encoding a C.sub.L or C.sub.H from a desired
immunoglobulin class or subclass. This can be achieved using a
vector or nucleic acid molecule that comprises a C.sub.L or C.sub.H
chain, as described above. For example, an OX40R antibody that was
originally IgM can be class switched to an IgG. Further, the class
switching may be used to convert one IgG subclass to another, e.g.,
from IgG1 to IgG2. Another method for producing an antibody
comprising a desired isotype comprises the steps of isolating a
nucleic acid encoding a heavy chain of an OX40R antibody and a
nucleic acid encoding a light chain of an OX40R antibody, isolating
the sequence encoding the V.sub.H region, ligating the V.sub.H
sequence to a sequence encoding a heavy chain constant domain of
the desired isotype, expressing the light chain gene and the heavy
chain construct in a cell, and collecting the OX40R antibody with
the desired isotype.
[0061] Antigen-Binding Fragments
[0062] In another aspect, the present disclosure provides
antigen-binding fragments of any of the human OX40R antibodies as
described herein above. In some embodiments, the antigen-binding
fragment is selected from: (1) a light chain of an OX40R antibody;
(2) a heavy chain of an OX40R antibody; (3) a variable region from
the light chain of an OX40R antibody; (4) a variable region from
the heavy chain of an OX40R antibody; (5) one or more CDRs (two,
three, four, five, or six CDRs) of an OX40R antibody; or (6) three
CDRs from the light chain and three CDRs from the heavy chain of an
OX40R antibody. In some particular embodiments, the disclosure
provides an antigen-binding fragment of antibody 11D4 or 18D8. In
some other particular embodiments, the antigen-binding fragments of
an OX40R antibody include: (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the V.sub.H and
C.sub.H1 domains; (iv) a Fv fragment consisting of the V.sub.L and
V.sub.H domains of a single arm of an antibody; (v) a dAb fragment
(Ward et al., (1989) Nature 341:544-546), which consists of a
V.sub.H domain; (vi) an isolated CDR, and (vii) single chain
antibody (scFv), which is a polypeptide comprising a VL region of
an antibody linked to a VH region of an antibody. Bird et al.,
(1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883. An antigen-binding fragment may also
comprise two or more shorter fragments, either from the same heavy
chain or same light chain, or from different chains.
Antigen-binding fragments, such as Fab and F(ab').sub.2 fragments,
can be prepared from whole antibodies using conventional
techniques, such as papain or pepsin digestion, respectively, of
whole antibodies. They can also be obtained using recombinant DNA
techniques, as described herein.
[0063] Antibody Derivatives
[0064] In some further aspects, the present disclosure provides
derivatives of any of the OX40R antibodies as described herein
above.
[0065] In one particular aspect, the antibody derivative is derived
from modifications of the amino acid sequences of 11D4 or 18D8.
Amino acid sequences of any regions of the antibody chains may be
modified, such as framework regions, CDR regions, or constant
regions. The modifications can be introduced by standard techniques
known in the art, such as site-directed mutagenesis and random
PCR-mediated mutagenesis, and may comprise natural as well as
non-natural amino acids.
[0066] Types of modifications include substitutions, insertions,
deletions, or combinations thereof, of one or more amino acids of
an OX40R antibody. In some embodiments, the antibody derivative
comprises 1, 2, 3, or 4 amino acid substitutions in the heavy chain
CDRs and/or one amino acid substitution in the light chain CDRs. In
some embodiments, a derivative of an OX40R antibody comprises one
or more amino acid substitutions relative to the germline amino
acid sequence of the human gene. In a particular embodiment, one or
more of those substitutions from germline is in the CDR2 region of
the heavy chain. In another particular embodiment, the amino acid
substitutions relative to the germline are at one or more of the
same positions as the substitutions relative to germline in
antibodies 11D4 or 18D8. In another embodiment, the amino acid
substitution is to change one or more cysteines in an antibody to
another residue, such as, without limitation, alanine or serine.
The cysteine may be a canonical or non-canonical cysteine. The
substitution can be made in a CDR or framework region of a variable
domain or in the constant domain of an antibody. Another type of
amino acid substitution is to eliminate asparagine-glycine pairs,
which form potential deamidation sites, by altering one or both of
the residues. In still other embodiments, the amino acid
substitution is a conservative amino acid substitution. In one
embodiment, the antibody derivative has 1, 2, 3, or 4 conservative
amino acid substitutions in the heavy chain CDR regions relative to
the amino acid sequences of 11D4 or 18D8.
[0067] Another type of modification of an OX40R antibody is the
alteration of the original glycosylation pattern of the antibody.
The term "alteration" refers to deletion of one or more
carbohydrate moieties found in the antibody, and/or adding one or
more glycosylation sites that are not present in the antibody.
Glycosylation of antibodies is typically N-linked. N-linked refers
to the attachment of the carbohydrate moiety to the side chain of
an asparagine residue. Addition of glycosylation sites to the
antibody is conveniently accomplished by altering the amino acid
sequence such that it contains one or more of the above-described
tripeptide sequences (for N-linked glycosylation sites).
[0068] Still another type of modification involves removal of any
carbohydrate moieties present on the antibody which may be
accomplished chemically or enzymatically. Chemical deglycosylation
requires exposure of the antibody to a compound, such as
trifluoromethanesulfonic acid, or an equivalent compound. This
treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the antibody intact. Chemical deglycosylation is described
by Sojahr, H. T., and Bahl, O. P., Arch. Biochem. Biophys. 259
(1987) 52-57 and by Edge, A. S., et al. Anal. Biochem. 118 (1981)
131-137. Enzymatic cleavage of carbohydrate moieties on antibodies
can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura, N. R., and Bahl, O. P.,
Meth. Enzymol. 138 (1987) 350-359.
[0069] Examples of other modifications include acetylation,
acylation, amidation, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links,
formation of cystine, formylation, hydroxylation, iodination,
methylation, myristoylation, oxidation, pegylation, proteolytic
processing, phosphorylation, prenylation, and sulfation.
[0070] In a further aspect, there is provided an antibody
derivative that comprises an OX40R antibody, or antigen-binding
fragment thereof, as described herein, linked to an additional
molecular entity. Examples of additional molecular entities include
pharmaceutical agents, peptides or proteins, and detection agent or
labels. Specific examples of pharmaceutical agents that may be
linked to an OX40R antibody include cytotoxic agents or other
cancer therapeutic agents, and radioactive isotopes. Specific
examples of peptides or proteins that may be linked to an OX40R
antibody include antibodies, which may be the same OX40R antibody
or a different antibody. Specific examples of detection agents or
labels that may be linked to an OX40R antibody include (1)
fluorescent compounds, such as fluorescein, fluorescein
isothiocyanate, rhodamine, 5-dimethylamine-1-naphtthalenesulfonyl
chloride, phycoerythrin, and lanthanide phosphors; (2) enzymes,
such as horseradish peroxidase, .beta.-galactosidase, luciferase,
alkaline phosphatase, and glucose oxidase; (3) biotin; (4) a
predetermined polypeptide epitope recognized by a secondary
reporter, such as leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, and epitope tags. In a
particular embodiment, the antibody derivative is an OX40R antibody
multimer, which is a multimeric form of an OX40R antibody, such as
antibody dimers, trimers, or higher-order multimers of monomeric
antibodies. Individual monomers within an antibody multimer may be
identical or different, i.e., they may be heteromeric or homomeric
antibody multimers. Individual antibodies within a multimer may
have the same or different binding specificities. Multimerization
of antibodies may be accomplished through natural aggregation of
antibodies. For example, some percentage of purified antibody
preparations (e.g., purified IgG1 molecules) spontaneously form
protein aggregates containing antibody homodimers, and other
higher-order antibody multimers. Alternatively, antibody homodimers
may be formed through chemical linkage techniques known in the art,
such as through using heterobifunctional crosslinking agents.
Suitable crosslinkers include those that are heterobifunctional,
having two distinctly reactive groups separated by an appropriate
spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester,
succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate, and
N-succinimidyl S-acethylthio-acetate) or homobifunctional (such as
disuccinimidyl suberate). Such linkers are commercially available
from Pierce Chemical Company, Rockford, Ill. Antibodies can also be
made to multimerize through recombinant DNA techniques known in the
art.
[0071] In still another aspect, the antibody derivative is a
chimeric antibody, which comprises an amino acid sequence of a
human OX40R antibody described herein above. In one example, one or
more CDRs from a human OX40R antibody is combined with CDRs from an
antibody from a non-human animal, such as mouse or rat. In another
example, all of the CDRs of the chimeric antibody are derived from
human OX40R antibodies. In another example, the CDRs from more than
one human OX40R antibody are combined in a chimeric antibody.
Further, a chimeric antibody may comprise the framework regions
derived from one human OX40R antibody and one or more CDRs from one
or more different human antibodies. Chimeric antibodies can be
generated using conventional methods known in the art. In some
particular embodiments, the chimeric antibody comprises one, two,
or three CDRs from the heavy chain variable region or from the
light chain variable region of an antibody selected from antibody
11D4 or 18D8.
[0072] Examples of other antibody derivatives provided by the
present disclosure include single chain antibodies, diabodies,
domain antibodies, nanobodies, and unibodies. A "single-chain
antibody" (scFv) consists of a single polypeptide chain comprising
a V.sub.L domain linked to a V.sub.H domain wherein V.sub.L domain
and V.sub.H domain are paired to form a monovalent molecule. Single
chain antibody can be prepared according to method known in the art
(see, for example, Bird et al., (1988) Science 242:423-426 and
Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). A
"diabody" consists of two chains, each chain comprising a heavy
chain variable region connected to a light chain variable region on
the same polypeptide chain connected by a short peptide linker,
wherein the two regions on the same chain do not pair with each
other but with complementary domains on the other chain to form a
bispecific molecule. Methods of preparing diabodies are known in
the art (See, e.g., Holliger P. et al., (1993) Proc. Natl. Acad.
Sci. USA 90:6444-6448, and Poljak R. J. et al., (1994) Structure
2:1121-1123). Domain antibodies (dAbs) are small functional binding
units of antibodies, corresponding to the variable regions of
either the heavy or light chains of antibodies. Domain antibodies
are well expressed in bacterial, yeast, and mammalian cell systems.
Further details of domain antibodies and methods of production
thereof are known in the art (see, for example, U.S. Pat. Nos.
6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European
Patents 0368684 & 0616640; WO005/035572, WO04/101790,
WO04/081026, WO04/058821, WO04/003019 and WO03/002609. Nanobodies
are derived from the heavy chains of an antibody. A nanobody
typically comprises a single variable domain and two constant
domains (CH2 and CH3) and retains antigen-binding capacity of the
original antibody. Nanobodies can be prepared by methods known in
the art (See e.g., U.S. Pat. Nos. 6,765,087, 6,838,254, WO
06/079372). Unibodies consist of one light chain and one heavy
chain of a IgG4 antibody. Unibodies may be made by the removal of
the hinge region of IgG4 antibodies. Further details of unibodies
and methods of preparing them may be found in WO2007/059782.
[0073] Methods of Producing the Binding Molecules
[0074] Binding molecules as disclosed herein can be produced by
techniques known in the art, including conventional monoclonal
antibody methodology, e.g., the standard somatic cell hybridization
technique of Kohler and Milstein (Nature 256: 495, (1975)), as well
as other techniques such as viral or oncogenic transformation of B
lymphocytes.
[0075] Immunization of Non-Human Animals
[0076] The disclosure also provides a method for making OX40R
antibodies or antigen-binding fragments thereof, which comprises
immunizing a non-human animal that comprises human immunoglobulin
loci with an OX40R antigen, and isolating the antibody from the
immunized animal or from cells derived from the immunized
animal.
[0077] Examples of suitable non-human animals include a transgenic
or transchromosomic animal, such as HuMAb Mouse.RTM., KM
Mouse.RTM., "TC mice," and Xenomouse.TM.. The HuMAb Mouse.RTM.
(Medarex, Inc.) contains human immunoglobulin gene miniloci that
encode unrearranged human heavy (.mu. and .gamma.) and .kappa.
light chain immunoglobulin sequences, together with targeted
mutations that inactivate the endogenous .mu. and .kappa. chain
loci (see e.g., Lonberg, et al. (1994) Nature 368: 856-859).
Accordingly, the mice exhibit reduced expression of mouse IgM or
.kappa., and in response to immunization, the introduced human
heavy and light chain transgenes undergo class switching and
somatic mutation to generate high affinity human IgG.kappa.
monoclonal antibodies (See, e.g., Harding, F. and Lonberg, N.
(1995) Ann. N.Y. Acad. Sci. 764:536-546). Preparation and use of
the HuMAb Mouse.RTM., and the genomic modifications carried by such
mice, is well know in the art (See, e.g., Fishwild, D. et al.
(1996) Nature Biotechnology 14: 845-851). The KM Mice.TM. carry a
human heavy chain transgene and a human light chain transchromosome
and are described in detail in WO 02/43478. The Xenomouse.TM.
(Abgenix, Inc.) contains large fragments of the human
immunoglobulin loci and is deficient in mouse antibody production.
This animal model is well known in the art (See, e.g., U.S. Pat.
Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584; and 6,162,963).
"TC mice" are also engineered mice carrying both a human heavy
chain transchromosome and a human light chain transchromosome. Such
mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci.
USA 97:722-727.
[0078] The OX40R antigen for use to immunize the animal may be
isolated and/or purified OX40R and is preferably a human OX40R. In
one embodiment, the OX40R antigen is a fragment of the human OX40R,
preferably the extracellular domain of the OX40R. In another
embodiment, the OX40R antigen is a fragment that comprises at least
one epitope of the human OX40R. In another embodiment, the OX40R
antigen is a cell that expresses OX40R on its cell surface, more
particularly a cell that overexpresses the OX40R on its cell
surface. Immunization of the animals may be done by any suitable
method known in the art. (See, e.g., Harlow and Lane, Antibodies: A
Laboratory Manual, New York: Cold Spring Harbor Press, 1990).
Particular methods for immunizing non-human animals such as mice,
rats, sheep, goats, pigs, cattle and horses are well known in the
art (See, e.g., Harlow and Lane (1990); U.S. Pat. No. 5,994,619).
Example 1 provides a method for immunizing HuMab mice.
[0079] After immunization of the animal with an OX40R antigen,
antibodies and/or antibody-producing cells can be obtained from the
animal. In one embodiment, serum is obtained from the animal and an
immunoglobulin fraction may be obtained from the serum, or the
OX40R antibodies may be purified from the serum.
[0080] The OX40R antibodies may also be produced using
antibody-producing immortalized cells prepared from cells isolated
from the immunized animal. After immunization, the lymph node
and/or splenic B cells are collected from the animal and
immortalized by suitable means. Methods of immortalizing cells
include, but are not limited to, transfecting them with oncogenes,
infecting them with an oncogenic virus and cultivating them under
conditions that select for immortalized cells, subjecting them to
carcinogenic or mutating compounds, fusing them with an
immortalized cell, e.g., a myeloma cell, and inactivating a tumor
suppressor gene (See, e.g., Harlow and Lane, supra). In a
particular embodiment, the splenic B cells collected from the
immunized animal are fused to immortalized myeloma cells to form
antibody-producing immortalized hybridomas. The myeloma cells
preferably do not secrete immunoglobulin polypeptides (a
non-secretory cell line). Immortalized hybridomas are screened
using the OX40 antigen (e.g., the OX40R, a portion thereof, or a
cell expressing the OX40R). The initial screening may be performed,
for example, using an enzyme-linked immunoassay (ELISA) or a
radioimmunoassay. An example of ELISA screening is described in WO
00/37504.
[0081] The OX40R antibody-producing cells, e.g., hybridomas, are
selected, cloned, and further screened for desirable
characteristics, including robust growth, high antibody production,
and desirable antibody characteristics, as discussed further below.
Hybridomas can be expanded in vivo in syngeneic animals, in animals
that lack an immune system, e.g., nude mice, or in cell culture in
vitro.
[0082] Thus, methods are provided for producing a cell that
produces a human monoclonal OX40R antibody or an antigen-binding
fragment thereof, comprising: (a) immunizing a non-human transgenic
animal with an OX40R antigen; (b) allowing the animal to mount an
immune response to the OX40R antigen; (c) isolating
antibody-producing cells from the animal; and (d) immortalizing the
antibody-producing cells. In one embodiment, the method further
comprises (e) creating individual monoclonal populations of the
immortalized antibody-producing cells; and (f) screening the
immortalized antibody-producing cells that produce a desired OX40R
antibody.
[0083] Nucleic Acids, Vectors, Host Cells, and Recombinant Methods
of Producing OX40R Antibodies
[0084] Another aspect of the disclosure provides an isolated
nucleic acid molecule encoding an amino acid sequence of a binding
molecule that binds the human OX40R. The amino acid sequence
encoded by the nucleic acid molecule may be any portion of an
intact antibody, such as a CDR, a sequence comprising one, two, or
three CDRs, or a variable region of a heavy chain or light chain,
or may be a full-length heavy chain or light chain. In some
embodiments, the nucleic acid molecule encodes an amino acid
sequence that comprises (1) a CDR3 region, particularly a heavy
chain CDR3 region, of antibodies 11D4 or 18D8; (2) a variable
region of a heavy chain or variable region of a light chain of
antibodies 11D4 or 18D8; or (3) a heavy chain or a light chain of
antibodies 11D4 or 18D8. In other embodiments, the nucleic acid
molecule encodes a polypeptide that comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22.
Instill other embodiments, the nucleic acid molecule is selected
from the group consisting of SEQ ID NOs: 11, 12, 23, and 24.
[0085] The nucleic acid molecules provided by the disclosure may be
obtained from any source that produces an OX40R antibody. mRNA from
OX40R antibody-producing cells may be isolated by standard
techniques, cloned and/or amplified using PCR and library
construction techniques, and screened using standard protocols to
obtain nucleic acid molecules encoding an amino acid sequence of an
OX40R antibody. The mRNA may be used to produce cDNA for use in the
polymerase chain reaction (PCR) or cDNA cloning of antibody genes.
In one embodiment, the nucleic acid molecule is obtained from a
hybridoma that expresses an OX40R antibody, as described above,
preferably a hybridoma that has as one of its fusion partners a
non-human transgenic animal cell that expresses human
immunoglobulin genes. In another embodiment, the hybridoma is
derived from a non-human, non-transgenic animal.
[0086] A nucleic acid molecule encoding the heavy chain of an OX40R
antibody may be constructed by fusing a nucleic acid molecule
encoding the heavy variable region with a nucleic acid molecule
encoding a constant region of a heavy chain. Similarly, a nucleic
acid molecule encoding the light chain of an OX40R antibody may be
constructed by fusing a nucleic acid molecule encoding the light
chain variable region with a nucleic acid molecule encoding a
constant region of a light chain. The nucleic acid molecules
encoding the VH and VL chain may be converted to full-length
antibody genes by inserting them into expression vectors already
encoding heavy chain constant and light chain constant regions,
respectively, such that the VH segment is operatively linked to the
heavy chain constant region (CH) segment(s) within the vector and
the VL segment is operatively linked to the light chain constant
region (CL) segment within the vector. Alternatively, the nucleic
acid molecules encoding the VH or VL chains are converted into
full-length antibody genes by linking, e.g., ligating, the nucleic
acid molecule encoding a VH chain to a nucleic acid molecule
encoding a CH chain using standard molecular biological techniques.
The same may be achieved using nucleic acid molecules encoding VL
and CL chains. The sequences of human heavy and light chain
constant region genes are known in the art. See, e.g., Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH
Publ. No. 91-3242, 1991. Nucleic acid molecules encoding the
full-length heavy and/or light chains may then be expressed from a
cell into which they have been introduced and the OX40R antibody
isolated.
[0087] The nucleic acid molecules may be used to recombinantly
express large quantities of OX40R antibodies, as described below.
The nucleic acid molecules may also be used to produce other
binding molecules provided by the disclosure, such as chimeric
antibodies, single chain antibodies, immunoadhesins, diabodies,
mutated antibodies, and antibody derivatives, as described
elsewhere herein. In one embodiment, a nucleic acid molecule is
used as probe or PCR primer for specific antibody sequences. For
instance, a nucleic acid molecule probe may be used in diagnostic
methods or a nucleic acid molecule PCR primer may be used to
amplify regions of DNA that could be used, inter alia, to isolate
nucleic acid sequences for use in producing variable regions of the
OX40R antibodies.
[0088] Once DNA molecules encoding the V.sub.H and V.sub.L segments
of an OX40R antibody are obtained, these DNA molecules can be
further manipulated by recombinant DNA techniques, for example to
convert the variable region genes to full-length antibody chain
genes, to Fab fragment genes, or to a scFv gene. In these
manipulations, a V.sub.L- or V.sub.H-encoding DNA molecule is
operatively linked to another DNA molecule encoding another
polypeptide, such as an antibody constant region or a flexible
linker. The term "operatively linked," as used in this context,
means that the two DNA molecules are joined such that the amino
acid sequences encoded by the two DNA molecules remain
in-frame.
[0089] The isolated DNA molecule encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA molecule to another DNA molecule encoding
heavy chain constant regions (CH1, CH2 and CH3). The sequences of
human heavy chain constant region genes are known in the art (see,
e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most
preferably is an IgG1 or IgG2 constant region. The IgG1 constant
region sequence can be any of the various alleles or allotypes
known to occur among different individuals, such as Gm(1), Gm(2),
Gm(3), and Gm(17). These allotypes represent naturally occurring
amino acid substitutions in the IgG1 constant regions. For a Fab
fragment heavy chain gene, the V.sub.H-encoding DNA can be
operatively linked to another DNA molecule encoding only the heavy
chain CH1 constant region. The CH1 heavy chain constant region may
be derived from any of the heavy chain genes.
[0090] The isolated DNA molecule encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA
molecule to another DNA molecule encoding the light chain constant
region, C.sub.L. The sequences of human light chain constant region
genes are known in the art (see e.g., Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242) and DNA fragments encompassing these regions can be
obtained by standard PCR amplification. The light chain constant
region can be a kappa or lambda constant region. The kappa constant
region may be any of the various alleles known to occur among
different individuals, such as Inv(1), Inv(2), and Inv(3). The
lambda constant region may be derived from any of the three lambda
genes.
[0091] To create a scFv gene, the V.sub.H- and V.sub.L-encoding DNA
fragments are operatively linked to another fragment encoding a
flexible linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the V.sub.H and V.sub.L sequences
can be expressed as a contiguous single-chain protein, with the
V.sub.L and V.sub.H regions joined by the flexible linker (See
e.g., Bird et al., (1988) Science 242:423-426; Huston et al.,
(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al.,
(1990) Nature 348:552-554). The single chain antibody may be
monovalent, if only a single V.sub.H and V.sub.L are used,
bivalent, if two V.sub.H and V.sub.L are used, or polyvalent, if
more than two V.sub.H and V.sub.L are used. Bispecific or
polyvalent antibodies may be generated that bind specifically to
OX40R and to another molecule.
[0092] In another aspect, the present disclosure provides a vector,
which comprises a nucleic acid molecule described herein above. The
nucleic acid molecule may encode a portion of a light chain or
heavy chain (such as a CDR or a variable region), a full-length
light or heavy chain, polypeptide that comprises a portion or
full-length of a heavy or light chain, or an amino acid sequence of
an antibody derivative or antigen-binding fragment. To express a
binding molecule, a DNA molecule encoding partial or full-length
binding molecule is inserted into an expression vector such that
the DNA molecule is operatively linked to transcriptional and
translational control sequences. In this context, the term
"operatively linked" is intended to mean that the DNA molecule is
ligated into a vector such that transcriptional and translational
control sequences within the vector serve their intended function
of regulating the transcription and translation of the DNA
molecule. The expression vector and expression control sequences
are chosen to be compatible with the expression host cell used.
Expression vectors include, for example, plasmids, retroviruses,
adenoviruses, adeno-associated viruses (AAV), plant viruses such as
cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, and
EBV derived episomes. The DNA molecule encoding an amino acid
sequence of the light chain and DNA molecule encoding an amino acid
sequence of the heavy chain can be inserted into separate vectors
or in the same vector. The DNA molecule is inserted into the
expression vector by any suitable methods (e.g., ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are
present).
[0093] An example of a suitable expression vector is one that
encodes a functionally complete human C.sub.H or C.sub.L
immunoglobulin sequence, with appropriate restriction sites
engineered so that any V.sub.H or V.sub.L sequence can be inserted
and expressed. The expression vector also can encode a signal
peptide that facilitates secretion of the amino acid sequence of
the antibody chain from a host cell. The DNA encoding the amino
acid sequence of an antibody chain may be cloned into the vector
such that the signal peptide is linked in-frame to the amino
terminus of the amino acid sequence of the antibody chain. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0094] In addition to the nucleic acid sequence encoding an amino
acid sequence of an OX40R antibody (antibody chain genes), the
expression vectors carry regulatory sequences that control the
expression of the antibody chain genes in a host cell. The design
of the expression vector, including the selection of regulatory
sequences, may depend on such factors as the choice of the host
cell to be transformed, the level of expression of protein desired,
and so forth. Regulatory sequences for mammalian host cell
expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such
as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the
SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major
late promoter (AdMLP)), polyoma and strong mammalian promoters such
as native immunoglobulin and actin promoters. For further
description of viral regulatory elements, and sequences thereof,
see e.g., U.S. Pat. Nos. 5,168,062, 4,510,245, and 4,968,615.
[0095] In addition to the antibody chain nucleic acid sequences and
regulatory sequences, the recombinant expression vectors may carry
additional sequences, such as sequences that regulate replication
of the vector in host cells and selectable marker genes. The
selectable marker gene facilitates selection of host cells into
which the vector has been introduced (see e.g., U.S. Pat. Nos.
4,399,216, 4,634,665 and 5,179,017). Selectable marker genes
include the dihydrofolate reductase (DHFR) gene (for use in
dhfr-host cells with methotrexate selection/amplification), the
neomycin phosphotransferase gene (for G418 selection), and the
glutamate synthetase gene. The design of the expression vector,
including the selection of regulatory sequences, may depend on a
number of factors, such as the choice of the host cell to be
transformed, the level of expression of protein desired, and so
forth. Nucleic acid molecules encoding binding molecules and
vectors comprising these nucleic acid molecules can be used for
transformation of a suitable host cell for recombinant production
of a binding molecule. A suitable host cell is transformed with one
or more expression vectors carrying nucleic acid molecules encoding
an amino acid sequence of a binding molecule such that the amino
acid sequence is expressed in the host cell and, typically,
secreted into the medium in which the host cell is cultured and
from which medium the amino acid sequence can be recovered.
Transformation of host cells can be by carried out by any suitable
method know in the art, such as those disclosed in U.S. Pat. Nos.
4,399,216, 4,912,040, 4,740,461, and 4,959,455.
[0096] The host cell may be a mammalian, insect, plant, bacterial,
or yeast cell. Examples of mammalian cell lines suitable as host
cells include Chinese hamster ovary (CHO) cells, NSO cells, SP2
cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster
kidney (BHK) cells, African green monkey kidney cells (COS), human
hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a
number of other cell lines. Examples of insect cell lines include
Sf9 or Sf21 cells. Examples of plant host cells include Nicotiana,
Arabidopsis, duckweed, corn, wheat, potato, and so forth. Bacterial
host cells include E. coli and Streptomyces species. Examples of
yeast host cells include Schizosaccharomyces pombe, Saccharomyces
cerevisiae, and Pichia pastoris.
[0097] Amino acid sequences of a binding molecule expressed by
different cell lines or in transgenic animals may have different
glycosylation. However, all binding molecules encoded by the
nucleic acid molecules provided herein, or comprising the amino
acid sequences provided herein are part of the present invention,
regardless of the glycosylation of the binding molecules.
[0098] In another aspect, the present disclosure provides a method
for producing an OX40R antibody or antigen-binding fragment thereof
using phage display. The method comprises (a) synthesizing a
library of human antibodies on phage, (b) screening the library
with the OX40R or a portion thereof, (c) isolating phage that binds
the OX40R or a portion thereof, and (d) obtaining the antibody from
the phage. One exemplary method for preparing the library of
antibodies comprises the step of: (a) immunizing a non-human animal
comprising human immunoglobulin loci with OX40R or an antigenic
portion thereof to create an immune response; (b) extracting
antibody-producing cells from the immunized animal; (c) isolating
RNA encoding heavy and light chains of the OX40R antibodies from
the extracted cells; (d) reverse transcribing the RNA to produce
cDNA; (e), amplifying the cDNA; and (f) inserting the cDNA into a
phage display vector such that antibodies are expressed on the
phage. Recombinant human OX40R antibodies or antigen binding
fragments thereof can be isolated by screening a recombinant
combinatorial antibody library. The library may be a scFv phage
display library, generated using human V.sub.L and V.sub.H cDNAs
prepared from mRNA isolated from B cells. Methods for preparing and
screening such libraries are known in the art. Kits for generating
phage display libraries are commercially available (e.g., the
Pharmacia Recombinant Phage Antibody System, catalog no.
27-9400-01; and the Stratagene SurfZAP.TM. phage display kit,
catalog no. 240612).
[0099] In one case, to isolate and produce human OX40R antibodies
with the desired characteristics, a human OX40R antibody as
described herein is first used to select human heavy and light
chain sequences having similar binding activity toward OX40R using
methods known in the art, such as the the epitope imprinting
methods described in WO 93/06213. The antibody libraries used in
this method may be scFv libraries prepared and screened as
described in WO 92/01047, McCafferty et al., Nature 348:552-554
(1990); and Griffiths et al., EMBO J. 12:725-734 (1993). The scFv
antibody libraries may be screened using human CCR2 as the
antigen.
[0100] Once initial human V.sub.L and V.sub.H regions are selected,
"mix and match" experiments are performed, in which different pairs
of the initially selected V.sub.L and V.sub.H segments are screened
for OX40R binding to select V.sub.L/V.sub.H pair combinations.
Additionally, to further improve the quality of the antibody, the
V.sub.L and V.sub.H segments of the V.sub.L/V.sub.H pair(s) can be
randomly mutated, within the CDR3 region of V.sub.H and/or V.sub.L,
in a process analogous to the in vive somatic mutation process
responsible for affinity maturation of antibodies during a natural
immune response. This in vitro affinity maturation can be
accomplished by amplifying V.sub.H and V.sub.L domains using PCR
primers complimentary to the V.sub.H CDR3 or V.sub.L CDR3,
respectively, which primers have been "spiked" with a random
mixture of the four nucleotide bases at certain positions such that
the resultant PCR products encode V.sub.H and V.sub.L segments into
which random mutations have been introduced into the V.sub.H and/or
V.sub.L CDR3 regions. These randomly mutated V.sub.H and V.sub.L
segments can be re-screened for binding to OX40R.
[0101] Following screening and isolation of an OX40R antibody or
antigen binding portion from a recombinant immunoglobulin display
library, nucleic acids encoding the selected binding molecule can
be recovered from the display package (e.g., from the phage genome)
and subcloned into other expression vectors by recombinant DNA
techniques. If desired, the nucleic acid can further be manipulated
to create other antibody forms, as described below. To express a
recombinant human antibody isolated by screening of a combinatorial
library, the DNA encoding the antibody is cloned into a recombinant
expression vector and introduced into mammalian host cells, as
described above.
[0102] Pharmaceutical Compositions
[0103] In another aspect, the present disclosure provides a
composition, e.g., a pharmaceutical composition, containing one or
a combination of binding molecules provided by the disclosure, and
optionally a pharmaceutically acceptable carrier. The compositions
can be prepared by conventional methods known in the art.
[0104] In some embodiments, the composition comprises an OX40R
antibody or an antigen-binding fragment thereof. In a particular
embodiment, the composition comprises antibody 11D4 or antibody
18D8, or a antigen-binding fragment of either antibody. In still
other embodiments, the composition comprises a derivative of
antibody 11D4 or antibody 18D8.
[0105] The term "pharmaceutically acceptable carrier" refers to any
inactive substance that is suitable for use in a formulation for
the delivery of a binding molecule. A carrier may be an
antiadherent, binder, coating, disintegrant, filler or diluent,
preservative (such as antioxidant, antibacterial, or antifungal
agent), sweetener, absorption delaying agent, wetting agent,
emulsifying agent, buffer, and the like. Examples of suitable
pharmaceutically acceptable carriers include water, ethanol,
polyols (such as glycerol, propylene glycol, polyethylene glycol,
and the like) dextrose, vegetable oils (such as olive oil), saline,
buffer, buffered saline, and isotonic agents such as sugars,
polyalcohols, sorbitol, and sodium chloride.
[0106] The compositions may be in any suitable forms, such as
liquid, semi-solid, and solid dosage forms. Examples of liquid
dosage forms include solution (e.g., injectable and infusible
solutions), microemulsion, liposome, dispersion, or suspension.
Examples of solid dosage forms include tablet, pill, capsule,
microcapsule, and powder. A particular form of the composition
suitable for delivering a binding molecule is a sterile liquid,
such as a solution, suspension, or dispersion, for injection or
infusion. Sterile solutions can be prepared by incorporating the
antibody in the required amount in an appropriate carrier, followed
by sterilization microfiltration. Generally, dispersions are
prepared by incorporating the antibody into a sterile vehicle that
contains a basic dispersion medium and other carriers. In the case
of sterile powders for the preparation of sterile liquid, methods
of preparation include vacuum drying and freeze-drying
(lyophilization) to yield a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. The various dosage forms of the
compositions can be prepared by conventional techniques known in
the art.
[0107] The relative amount of a binding molecule included in the
composition will vary depending upon a number of factors, such as
the specific binding molecule and carriers used, dosage form, and
desired release and pharmacodynamic characteristics. The amount of
a binding molecucle in a single dosage form will generally be that
amount which produces a therapeutic effect, but may also be a
lesser amount. Generally, this amount will range from about 0.01
percent to about 99 percent, from about 0.1 percent to about 70
percent, or from about 1 percent to about 30 percent relative to
the total weight of the dosage form.
[0108] In addition to the binding molecule, one or more additional
therapeutic agents may be included in the composition. Examples of
the additional therapeutic agents are described herein below. The
suitable amount of the additional therapeutic agent to be included
in the composition can be readily selected by a person skilled in
the art, and will vary depending on a number of factors, such as
the particular agent and carriers used, dosage form, and desired
release and pharmacodynamic characteristics. The amount of the
additional therapeutic agent included in a single dosage form will
generally be that amount of the agent which produces a therapeutic
effect, but may be a lesser amount as well.
[0109] Use of the Binding Molecules and Pharmaceutical
Compositions
[0110] Binding molecules and pharmaceutical compositions comprising
a binding molecule provided by the present disclosure are useful
for therapeutic, dignostic, or other purposes, such as enhancing an
immune response, treating cancer, enhancing efficacy of other
cancer therapy, or enhancing vaccine efficacy, and have a number of
utilities, such as for use as medicaments or diagnostic agents.
Thus, in another aspect, the present disclosure provides methods of
using the binding molecules or pharmaceutical compositions.
[0111] In one particular aspect, methods are provided for enhancing
immune response in a mammal, comprising administering to the mammal
a therapeutically effective amount of a binding molecule provided
by the disclosure. In some embodiments, the binding molecule is an
OX40R antibody or antigen-binding fragment thereof and the mammal
is a human. In a further embodiment, the binding molecule is
antibody 11D4 or antibody 18D8, or an antigen-binding fragment of
either antibody. The term "enhancing immune response" or its
grammatical variations, means stimulating, evoking, increasing,
improving, or augmenting any response of a mammal's immune system.
The immune response may be a cellular response (i.e. cell-mediated,
such as cytotoxic T lymphocyte mediated) or a humoral response
(i.e. antibody mediated response), and may be a primary or
secondary immune response. Examples of enhancement of immune
response include increased CD4+ helper T cell activity and
generation of cytolytic T cells. The enhancement of immune response
can be assessed using a number of in vitro or in vivo measurements
known to those skilled in the art, including, but not limited to,
cytotoxic T lymphocyte assays, release of cytokines (for example
IL-2 production), regression of tumors, survival of tumor bearing
animals, antibody production, immune cell proliferation, expression
of cell surface markers, and cytotoxicity. Typically, methods of
the disclosure enhance the immune response by a mammal when
compared to the immune response by an untreated mammal or an animal
not treated using the claimed methods. In one embodiment, the
method enhances a cellular immune response, particularly a
cytotoxic T cell response. In another embodiment, the cellular
immune response is a T helper cell response. In still another
embodiment, the immune response is a cytokine production,
particularly IL-2 production.
[0112] In another particular aspect, the present disclosure
provides a method of treating cancer in a mammal, comprising
administering to the mammal a therapeutically effective amount of a
binding molecule provided by the disclosure. The term "treating
cancer" or "treatment of cancer" refers to causing a desirable or
beneficial effect in a mammal diagnosed with a cancer. The
desirable or beneficial effect may include inhibition of further
growth or spread of cancer cells, death of cancer cells, inhibition
of reoccurrence of cancer, reduction of pain associated with the
cancer, or improved survival of the animal. Inhibition of
reoccurrence of cancer contemplates cancer sites and surrounding
tissue which have previously been treated by radiation,
chemotherapy, surgery, or other techniques. The effect can be
either subjective or objective. For example, if the animal is
human, the human may note improved vigor or vitality or decreased
pain as subjective symptoms of improvement or response to therapy.
Alternatively, the clinician may notice a decrease in tumor size or
tumor burden based on physical exam, laboratory parameters, tumor
markers or radiographic findings. Some laboratory signs that the
clinician may observe for response to treatment include
normalization of tests, such as white blood cell count, red blood
cell count, platelet count, erythrocyte sedimentation rate, and
various enzyme levels. Additionally, the clinician may observe a
decrease in a detectable tumor marker. Alternatively, other tests
can be used to evaluate objective improvement, such as sonograms,
nuclear magnetic resonance testing and positron emissions testing.
In some embodiments, the binding molecule is an OX40R antibody or
an antigen-binding fragment thereof provided by the disclosure. In
a further embodiment the binding molecule is antibody 11D4 or 18D8,
or an antigen-binding fragment of either antibody. In a further
embodiment, the mammal is a human.
[0113] In another particular aspect, the present disclosure
provides a method of preventing cancer in a mammal, comprising
administering to the mammal a therapeutically effective amount of a
binding molecule provided by the disclosure. The term "preventing
cancer" or "prevention of cancer" refers to delaying, inhibiting,
or preventing the onset of a cancer in a mammal in which the onset
of oncogenesis or tumorigenesis is not evidenced but a
predisposition for cancer is identified whether determined by
genetic screening, for example, or otherwise. The term also
encompasses treating a mammal having premalignant conditions to
stop the progression of, or cause regression of, the premalignant
conditions towards malignancy. Examples of premalignant conditions
include hyperplasia, dysplasia, and metaplasia. In some
embodiments, the binding molecule is an OX40R antibody or a
fragment thereof provided by the disclosure. In a further
embodiment the binding molecule is antibody 11D4 or 18D8, or an
antigen-binding fragment of either antibody. In a further
embodiment, the mammal is a human.
[0114] A variety of cancers, whether malignant or benign and
whether primary or secondary, may be treated or prevented with a
method provided by the disclosure. Examples of such cancers include
lung cancers such as bronchogenic carcinoma (e.g., squamous cell
carcinoma, small cell carcinoma, large cell carcinoma, and
adenocarcinoma), alveolar cell carcinoma, bronchial adenoma,
chondromatous hamartoma (noncancerous), and sarcoma (cancerous);
heart cancer such as myxoma, fibromas, and rhabdomyomas; bone
cancers such as osteochondromas, condromas, chondroblastomas,
chondromyxoid fibromas, osteoid osteomas, giant cell tumors,
chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcomas,
malignant fibrous histiocytomas, Ewing's tumor (Ewing's sarcoma),
and reticulum cell sarcoma; brain cancer such as gliomas (e.g.,
glioblastoma multiforme), anaplastic astrocytomas, astrocytomas,
oligodendrogliomas, medulloblastomas, chordoma, Schwannomas,
ependymomas, meningiomas, pituitary adenoma, pinealoma, osteomas,
hemangioblastomas, craniopharyngiomas, chordomas, germinomas,
teratomas, dermoid cysts, and angiomas; cancers in digestive system
such as leiomyoma, epidermoid carcinoma, adenocarcinoma,
leiomyosarcoma, stomach adenocarcinomas, intestinal lipomas,
intestinal neurofibromas, intestinal fibromas, polyps in large
intestine, and colorectal cancers; liver cancers such as
hepatocellular adenomas, hemangioma, hepatocellular carcinoma,
fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and
angiosarcoma; kidney cancers such as kidney adenocarcinoma, renal
cell carcinoma, hypernephroma, and transitional cell carcinoma of
the renal pelvis; bladder cancers; hematological cancers such as
acute lymphocytic (lymphoblastic) leukemia, acute myeloid
(myelocytic, myelogenous, myeloblastic, myelomonocytic) leukemia,
chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell
leukemia), chronic myelocytic (myeloid, myelogenous, granulocytic)
leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B cell
lymphoma, mycosis fungoides, and myeloproliferative disorders
(including myeloproliferative disorders such as polycythemia vera,
myelofibrosis, thrombocythemia, and chronic myelocytic leukemia);
skin cancers such as basal cell carcinoma, squamous cell carcinoma,
melanoma, Kaposi's sarcoma, and Paget's disease; head and neck
cancers; eye-related cancers such as retinoblastoma and
intraoccular melanocarcinoma; male reproductive system cancers such
as benign prostatic hyperplasia, prostate cancer, and testicular
cancers (e.g., seminoma, teratoma, embryonal carcinoma, and
choriocarcinoma); breast cancer; female reproductive system cancers
such as uterine cancer (endometrial carcinoma), cervical cancer
(cervical carcinoma), cancer of the ovaries (ovarian carcinoma),
vulvar carcinoma, vaginal carcinoma, fallopian tube cancer, and
hydatidiform mole; thyroid cancer (including papillary, follicular,
anaplastic, or medullary cancer); pheochromocytomas (adrenal
gland); noncancerous growths of the parathyroid glands; pancreatic
cancers; and hematological cancers such as leukemias, myelomas,
non-Hodgekin's lymphomas, and Hodgekin's lymphomas.
[0115] In practicing the therapeutic methods, the binding molecules
may be administered alone as monotherapy, or administered in
combination with one or more additional therapeutic agents or
therapies. Thus, in another aspect, the present disclosure provides
a combination therapy, which comprises a binding molecule provided
by the disclosure in combination with one or more additional
therapies or therapeutic agents. The term "additional therapy"
refers to a therapy which does not employ a binding molecule
provided by the disclosure as a therapeutic agent. The term
"additional therapeutic agent" refers to any therapeutic agent
other than a binding molecule provided by the discosure. In some
embodiments, the binding molecule is antibody 11D4 or 18D8, or an
antigen-binding fragment of either antibody. In one particular
aspect, the present disclosure provides a combination therapy for
treating cancer in a mammal, which comprises administering to the
mammal a therapeutically effective amount of a binding molecule
provided by the disclosure in combination with one or more
additional therapeutic agents. In a further embodiment, the mammal
is a human.
[0116] A wide variety of cancer therapeutic agents may be used in
combination with a binding molecule. One of ordinary skill in the
art will recognize the presence and development of other cancer
therapies which can be used in combination with the methods and
binding molecules of the present disclosure, and will not be
restricted to those forms of therapy set forth herein. Examples of
categories of additional therapeutic agents that may be used in the
combination therapy for treating cancer include (1)
chemotherapeutic agents, (2) immunotherapeutic agents, and (3)
hormone therapeutic agents.
[0117] The term "chemotherapeutic agent" refers to a chemical or
biological substance that can cause death of cancer cells, or
interfere with growth, division, repair, and/or function of cancer
cells. Examples of chemotherapeutic agents include those that are
disclosed in WO 2006/088639, WO 2006/129163, and US 20060153808,
the disclosures of which are incorporated herein by reference.
Examples of particular chemotherapeutic agents include: (1)
alkylating agents, such as chlorambucil (LEUKERAN),
mcyclophosphamide (CYTOXAN), ifosfamide (IFEX), mechlorethamine
hydrochloride (MUSTARGEN), thiotepa (THIOPLEX), streptozotocin
(ZANOSAR), carmustine (BICNU, GLIADEL WAFER), lomustine (CEENU),
and dacarbazine (DTIC-DOME); (2) alkaloids or plant vinca
alkaloids, including cytotoxic antibiotics, such as doxorubicin
(ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin
(CERUBIDINE, DAUNOXOME), nemorubicin, idarubicin-(IDAMYCIN PFS,
ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE). dactinomycin
(actinomycin D, COSMEGEN), plicamycin (MITHRACIN), mitomycin
(MUTAMYCIN), and bleomycin (BLENOXANE), vinorelbine tartrate
(NAVELBINE)), vinblastine (VELBAN), vincristine (ONCOVIN), and
vindesine (ELDISINE); (3) antimetabolites, such as capecitabine
(XELODA), cytarabine (CYTOSAR-U), fludarabine (FLUDARA),
gemcitabine (GEMZAR), hydroxyurea (HYDRA), methotrexate (FOLEX,
MEXATE, TREXALL), nelarabine (ARRANON), trimetrexate (NEUTREXIN),
and pemetrexed (ALIMTA); (4) Pyrimidine antagonists, such as
5-fluorouracil (5-FU); capecitabine (XELODA), raltitrexed
(TOMUDEX), tegafur-uracil (UFTORAL), and gemcitabine (GEMZAR); (5)
taxanes, such as docetaxel (TAXOTERE), paclitaxel (TAXOL); (6)
platinum drugs, such as cisplatin (PLATINOL) and carboplatin
(PARAPLATIN), and oxaliplatin (ELOXATIN); (7) topoisomerase
inhibitors, such as irinotecan (CAMPTOSAR), topotecan (HYCAMTIN),
etoposide (ETOPOPHOS, VEPESSID, TOPOSAR), and teniposide (VUMON);
(8) epipodophyllotoxins (podophyllotoxin derivatives), such as
etoposide (ETOPOPHOS, VEPESSID, TOPOSAR); (9) folic acid
derivatives, such as leucovorin (WELLCOVORIN); (10) nitrosoureas,
such as carmustine (BiCNU), lomustine (CeeNU); (11) inhibitors of
receptor tyrosine kinase, including epidermal growth factor
receptor (EGFR), vascular endothelial growth factor (VEGF), insulin
receptor, insulin-like growth factor receptor (IGFR), hepatocyte
growth factor receptor (HGFR), and platelet-derived growth factor
receptor (PDGFR), such as gefitinib (IRESSA), erlotinib (TARCEVA),
bortezomib (VELCADE), imatinib mesylate (GLEEVEC), genefitinib,
lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib,
rituximab, trastuzumab (HERCEPTIN), cetuximab (ERBITUX),
bevacizumab (AVASTIN), and ranibizumab (LUCENTIS), lym-1 (ONCOLYM),
antibodies to insulin-like growth factor-1 receptor (IGF-1R) that
are disclosed in WO2002/053596); (12) angiogenesis inhibitors, such
as bevacizumab (AVASTIN), suramin (GERMANIN), angiostatin, SU5416,
thalidomide, and matrix metalloproteinase inhibitors (such as
batimastat and marimastat), and those that are disclosed in
WO2002055106; and (13) proteasome inhibitors, such as bortezomib
(VELCADE).
[0118] The term "immunotherapeutic agents" refers to a chemical or
biological substance that can enhance an immune response of a
mammal, Examples of immunotherapeutic agents include: bacillus
Calmette-Guerin (BCG); cytokines such as interferons; vaccines such
as MyVax personalized immunotherapy; Onyvax-P, Oncophage, GRNVAC1,
FavId, Provenge, GVAX, Lovaxin C, BiovaxID, GMXX, and NeuVax; and
antibodies such as alemtuzumab (CAMPATH), bevacizumab (AVASTIN),
cetuximab (ERBITUX), gemtuzunab ozogamicin (MYLOTARG), ibritumomab
tiuxetan (ZEVALIN), panitumumab (VECTIBIX), rituximab (RITUXAN,
MABTHERA), trastuzumab (HERCEPTIN), tositunomab (BEXXAR),
tremelimumab, CAT-3888, and agonist antibodies to CD40 receptor
that are disclosed in WO2003/040170.
[0119] The term "hormone therapeutic agent" refers to a chemical or
biological substance that inhibits or eliminates the production of
a hormone, or inhibits or counteracts the effect of a hormone on
the growth and/or survival of cancerous cells. Examples of such
agents suitable for the methods herein include those that are
disclosed in US20070117809. Examples of particular hormone
therapeutic agents include tamoxifen (NOLVADEX), toremifene
(Fareston), fulvestrant (FASLODEX), anastrozole (ARIMIDEX),
exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate
(MEGACE), goserelin (ZOLADEX), and leuprolide (LUPRON). The binding
molecules of this disclosure may also be used in combination with
non-drug hormone therapies such as (1) surgical methods that remove
all or part of the organs or glands which participate in the
production of the hormone, such as the ovaries, the testicles, the
adrenal gland, and the pituitary gland, and (2) radiation
treatment, in which the organs or glands of the patient are
subjected to radiation in an amount sufficient to inhibit or
eliminate the production of the targeted hormone.
[0120] The combination therapy for treating cancer also encompasses
the combination of a binding molecule provided by the disclosure
with surgery to remove a tumor. The binding molecule may be
administered to the mammal before, during, or after the
surgery.
[0121] The combination therapy for treating cancer also encompasses
combination of a binding molecule provided by the disclosure with
radiation therapy, such as ionizing (electromagnetic) radiotherapy
(e.g., X-rays or gamma rays) and particle beam radiation therapy
(e.g., high linear energy radiation). The source of radiation can
be external or internal to the mammal. The binding molecule may be
administered to the mammal before, during, or after the radiation
therapy.
[0122] Administration of the Binding Molecules and Compositions
[0123] The binding molecules and compositions provided by the
present disclosure can be administered via any suitable enteral
route or parenteral route of administration. The term "enteral
route" of administration refers to the administration via any part
of the gastrointestinal tract. Examples of enteral routes include
oral, mucosal, buccal, and rectal route, or intragastric route.
"Parenteral route" of administration refers to a route of
administration other than enteral route. Examples of parenteral
routes of administration include intravenous, intramuscular,
intradermal, intraperitoneal, intratumor, intravesical,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, transtracheal, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal, subcutaneous,
or topical administration. The antibodies and compositions of the
disclosure can be administered using any suitable method, such as
by oral ingestion, nasogastric tube, gastrostomy tube, injection,
infusion, implantable infusion pump, and osmotic pump. The suitable
route and method of administration may vary depending on a number
of factors such as the specific antibody being used, the rate of
absorption desired, specific formulation or dosage form used, type
or severity of the disorder being treated, the specific site of
action, and conditions of the patient, and can be readily selected
by a person skilled in the art
[0124] The term "therapeutically effective amount" of a binding
molecule refers to an amount that is effective for an intended
therapeutic purpose. For example, in the context of enhancing an
immune response, a "therapeutically effective amount" is any amount
that is effective in stimulating, evoking, increasing, improving,
or augmenting any response of a mammal's immune system. In the
context of treating cancer, a "therapeutically effective amount" is
any amount that is sufficient to cause any desirable or beneficial
effect in the mammal being treated, such as inhibition of further
growth or spread of cancer cells, death of cancer cells, inhibition
of reoccurrence of cancer, reduction of pain associated with the
cancer, or improved survival of the mammal. In a method of
preventing cancer, a "therapeutically effective amount" is any
amount that is effective in delaying, inhibiting, or preventing the
onset of a cancer in the mammal to which the binding molecule is
administered. The therapeutically effective amount of a binding
molecule usually ranges from about 0.001 to about 500 mg/kg, and
more usually about 0.05 to about 100 mg/kg, of the body weight of
the mammal. For example, the amount can be about 0.3 mg/kg, 1
mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, or 100 mg/kg of body
weight of the mammal. In some embodiments, the therapeutically
effective amount of an OX40R antibody is in the range of about
0.1-30 mg/kg of body weight of the mammal. The precise dosage level
to be administered can be readily determined by a person skilled in
the art and will depend on a number of factors, such as the type,
and severity of the disorder to be treated, the particular binding
molecule employed, the route of administration, the time of
administration, the duration of the treatment, the particular
additional therapy employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and like factors well known in the medical arts.
[0125] A binding molecule or composition is usually administered on
multiple occasions. Intervals between single doses can be, for
example, weekly, monthly, every three months or yearly. An
exemplary treatment regimen entails administration once per week,
once every two weeks, once every three weeks, once every four
weeks, once a month, once every 3 months or once every three to 6
months. Typical dosage regimens for an OX40R antibody include 1
mg/kg body weight or 3 mg/kg body weight via intravenous
administration, using one of the following dosing schedules: (i)
every four weeks for six dosages, then every three months; (ii)
every three weeks; (iii) 3 mg/kg body weight once followed by 1
mg/kg body weight every three weeks.
EXAMPLES
Example 1: Preparation of OX40R Antibodies
[0126] Illustrative antibodies in accordance with the disclosure
were prepared, selected, and assayed as follows:
[0127] Immunization with the OX40R Antigen and Selection of Mice
Producing OX40R Monoclonal Antibodies:
[0128] Fully human monoclonal antibodies to human OX40R were
prepared using human Ig transgenic mouse strains HCo7, HCo112,
Hco17, and Hco27 as well as the human transchromosomal/transgenic
strain, KM (Medarex, Inc.). These strains all express fully human
antibodies that are indistinguishable from antibodies isolated from
humans.
[0129] In the transgenic strains, both the endogenous mouse kappa
light chain gene and the endogenous mouse heavy chain gene were
homozygously disrupted as described in Chen et al. (1993) EMBO J.
12:821-830 and in Example 1 of WO 01/09187, respectively. Moreover,
they carry a human kappa light chain transgene, KCo5, as described
in Fishwild et al. (1996) Nature Biotechnology 14:845-851. In
contrast, the transgenic strains are distinct with respect to their
human heavy chain genes. The HCo7 strain carries the HCo7 human
heavy chain transgene as described in U.S. Pat. Nos. 5,545,806,
5,625,825, and 5,545,807; the HCo12 strain carries the HCo12 human
heavy chain transgene as described in Example 2 of WO 01/09187; the
Hco17 strain carries the Hco17 human heavy chain transgene as
described in Example 8 of Deshpande et al., US 2005/0191293A1; the
Hco27 strain carries the Hco27 human heavy chain transgene as
described in Example 5 of PCT/US2008/072640 filed 8 Aug. 2008. The
KM strain carries a human mini-chromosome as described in Ishida et
al., (2002), Cloning and Stem Cells, 4: 91-102.
[0130] General immunization schemes for HuMab mice are described in
Lonberg et al. (1994) Nature 368(6474): 856-859; Fishwild et al.
(1996) Nature Biotechnology 14: 845-851; and PCT Publication WO
98/24884.
[0131] HuMab mice of the HCo7, HCo12, Hco17, Hco27 and KM strains
were immunized beginning at 6-16 weeks of age with 15-25 .mu.gs of
purified human recombinant OX40R-Ig protein and murine pro-B cell
line, 300-19 (Reth, M. G. et al., Nature 312 29: 418-42, 1984; Alt,
F. et al., Cell 27: 381-390, 1981), transfected to express human
OX40R in Ribi adjuvant. The purified human recombinant OX40R-Ig
protein is a construct of the extracellular domain (amino acids
1-220) of human OX40R fused to the constant region of human IgG1.
Administration was via injection intra-peritoneally, subcutaneously
or into the footpad at 3-28 day intervals, up to a total of 10
immunizations. Immune response was monitored via ELISA and FACS
screening as described below.
[0132] Selection of HuMab Mice Producing OX40R Antibodies:
[0133] To select HuMab mice producing antibodies that bind to the
OX40R, blood from the immunized mice was obtained and analyzed by
ELISA for specific binding to purified human OX40R recombinant
protein, and by FACS for binding to a cell line expressing full
length human OX40R, and not to a control cell line not expressing
OX40R.
[0134] ELISA binding assay was as described by Fishwild et al.
(1996), Nature Biotechnology 14: 845-851. Briefly, microtiter
plates were coated using 50 .mu.l/well of a purified recombinant
OX40R-Ig solution containing 1 .mu.g/ml in PBS, and incubated
overnight at 4.degree. C. The wells were then blocked using 200
.mu.l/well of 5% chicken serum in PBS/Tween (0.05%). Dilutions of
plasma from OX40R-immunized mice were added to each well and
incubated for 1 hour at ambient temperature. The plates were washed
with PBS/Tween and then incubated with a goat-anti-human IgG Fc
polyclonal antibody conjugated with horseradish peroxidase (HRP)
for 1 hour at room temperature. After washing, the plates were
developed with ABTS substrate (Moss Inc., product #: ABTS-1000
mg/ml) and analyzed by spectrophotometer at OD 405.
[0135] FACS assay was carried out according to conventional
procedures. Briefly, OX40R-expressing 300-19 cells were incubated
with serum from immunized mice diluted at 1:20. Cells were washed
and specific antibody binding was detected with FITC-labeled
anti-human IgG Ab. Flow cytometric analyses were performed on a
FACS flow cytometry instrument (Becton Dickinson, San Jose,
Calif.).
[0136] Mice that developed the highest titers of OX40R antibodies
were used for fusions. Fusions were performed as described below
and hybridoma supernatants were tested for anti-OX40R activity by
ELISA and FACS.
[0137] Generation of Hybridomas Producing Human Monoclonal
Antibodies to OX40R:
[0138] The mice selected above were boosted intravenously with
OX40R-Ig at 3 days and then again at 2 days prior to sacrifice and
removal of the spleen and/or lymph nodes.
[0139] The splenocytes and/or lymph node lymphocytes isolated from
the immunized HuMab or KM mice were fused to SP2/0 non-secreting
mouse myeloma cells (ATCC, CRL-1581) using electrofusion (E-fusion,
Cyto Pulse.TM. technology, Cyto Pulse.TM. Sciences, Inc., Glen
Burnie, Md.), according to standard or manufacturer-recommended
protocols. Briefly, single cell suspensions of splenocytes and/or
lymph node lymphocytes from immunized mice were prepared and then
combined with an equal number of SP2/0 non-secreting mouse myeloma
cells; E-fusion was then performed.
[0140] The cells were then plated at 2.times.10.sup.4 cells/well in
flat bottom microtiter plate, and incubated for 10-14 days in
selective medium containing 10% fetal bovine serum, 10% P388D1
(ATCC, CRL-TIB-63) conditioned medium, 3-5% (IGEN) in DMEM
(Mediatech, Herndon, Va., Cat. No. CRL 10013, with high glucose,
L-glutamine and sodium pyruvate), 7 mM HEPES, 0.055 mM
2-mercaptoethanol, 0.1 IU/mL penicillin--0.1 mg/mL streptomycin,
and 1.times.HAT (Sigma, Cat. No. CRL-P-7185).
[0141] After 1-2 weeks, cells were cultured in medium in which the
HAT was replaced with HT. Approximately 10-14 days after cell
plating, supernatants from individual wells were screened for the
presence of human gamma, kappa antibodies. The supernatants which
scored positive for human gamma, kappa were then screened by ELISA
and FACS (using the protocol described above) for human OX40R
monoclonal IgG antibodies. The antibody-secreting hybridomas were
transferred to 24 well plates, screened again and, if confirmed
positive for human OX40R IgG monoclonal antibodies, were subcloned
at least twice by limiting dilution. The stable subclones were then
cultured in vitro to generate small amounts of antibody in tissue
culture medium for further characterization.
Example 2: Biological/Pharmacological Examples
[0142] A. In Vitro Study Procedures:
[0143] Binding to the Extracellular Domain of the OX40R:
[0144] A human OX40-Ig fusion protein was diluted in BupH.TM.
Carbonate buffer, pH 9.4 (Pierce, Rockford, Ill.) was coated onto
96-well Maxisorb plates (Nunc, Roskilde, Denmark) at 100 .mu.l/well
(0.25 .mu.g/ml) and incubated overnight at 4.degree. C. The Plates
were washed three times with wash buffer containing 0.05% Tween 20
(Sigma, St Louis, Mo.) diluted in PBS (Sigma, St Louis, Mo.) and
blocked with 300 .mu.l/well of 0.5% BSA (Sigma, St Louis, Mo.) in
PBS for 1 hour at RT.degree.. Next, the plates were washed and
incubated with anti-human OX40 reactive antibodies diluted in
blocking buffer at various concentrations (100 .mu.l/well) and
incubated for 1 hour at RT.degree.. The plates were then washed and
incubated for one hour at RT.degree. with a horse radish peroxidase
labeled anti-human kappa chain antibody (Bethyl Laboratories,
Montgomery, Tex.) at 25 ng/ml in blocking buffer. Finally, the
assay plates were washed and 100 .mu.l/well of 1-Step Turbo-TMB
substrate (Pierce, Rockford, Ill.) was added for 30 minutes at
RT.degree.. The reaction was stopped by adding an equal volume of
2M H.sub.2SO.sub.4 and absorbance was read at 450 nm on a Molecular
Devices Spectra Max 340 (Molecular Devices, Sunnyvale, Calif.).
[0145] FACS Based Binding to Cell Surface OX40R:
[0146] OX40R-expressing cell lines (see below) or activated primary
peripheral blood mononuclear cells (see below) were used to assess
binding on both the human and cynomolgus OX40 receptors. Cells were
harvested and washed (5.times.10.sup.5/tube) using wash buffer at
RT.degree.. The wash buffer consisted of PBS, 2% heat-inactivated
fetal bovine serum (Hyclone, Logan, Utah) and 0.02% sodium azide
(Sigma, St. Louis, Mo.). Next, 100 .mu.l of various concentrations
of antibody was added to the cells (starting at 30 ug/ml and using
a 3-fold titration) diluted in wash buffer containing 0.005 mg/ml
of cytocholasin B (Sigma, St. Louis, Mo.). The cells were gently
rocked at RT.degree. for 3 hours. Next, the cells were washed twice
and resuspended in 0.5 ml/tube with cold wash buffer and 10,000
events were collected and analyzed using a Becton Dickinson
FACSCalibur and CellQuest software (San Jose, Calif.).
[0147] Biacore Assay:
[0148] The Biosensor biospecific interaction analysis instrument
(BIAcore 2000) uses surface plasmon resonance to measure molecular
interactions on a CM5 sensor chip. Changes in the refractive
indices between two media, glass and carboxymethylated dextran,
caused by the interaction of molecules to the dextran side of the
sensor chip, is measured and reported as changes in arbitrary
reflectance units (RU) as detailed in the manufacturer's
application notes.
[0149] The carboxymethylated dextran surfaces on a CM5 sensor chip
were activated by derivatization with 0.05 M N-hydroxysuccinimide
mediated by 0.2 M N-ethyl-N'-(dimethylaminopropyl) carbodiimide for
7 min. Streptavidin (Sigma S-4762) at a concentration of 500
.mu.g/ml, in 10 mM Na acetate, pH 4.5, was injected onto three
surfaces (Flow Cell-2, 3 and 4) at a rate of 5 .mu.l/min and
covalently immobilized to the flow cell surfaces with approximately
2500RU's. 35 .mu.l of 10 mM Na acetate buffer was injected over
Flow cell-1 during immobilization in place of antigen to make an
activated blank surface to measure non-specific binding.
Deactivation of unreacted N-hydroxysuccinimide esters on all four
Flow cells was performed using 1M ethanolamine hydrochloride, pH
8.5. Following immobilization, the flow cells are cleaned of any
unreacted or poorly bound material with 5 regeneration injections
of 5 .mu.l of 50 mM NaOH until a stable baseline was achieved.
[0150] Biotinylated CD134-muIg (Ancell 513-030), at a concentration
of 10 .mu.g/ml at a flow rate of 5 .mu.l/min was manually injected
over Flow cells-2, 3 and 4 to achieve 3 surface densities:
Fc-2=150RU, Fc-3=375RU and Fc-4=580RU. The different density
surfaces were prepared to monitor the possibility of mass transport
limited binding during association phase and rebinding during
dissociation, both artifacts that are influenced by surface density
that must be avoided.
[0151] A dilution series of the OX40R antibodies were prepared over
a concentration range of 666 nM to 66 pM by half logs in running
buffer (0.01M HEPES, pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005%
polysorbate 20 (v/v)). The flow rate was set at 5 .mu.l/min and 25
.mu.l of each concentration point sample was injected over the
sensor chip with a regeneration injection of 5 .mu.l of 50 mM NaOH
between each concentration of antibody injected. Dissociation time
was 5 min. The data was analyzed using BIAevaluation 3.0 global fit
software (separate analysis of each concentration point).
[0152] Epitope Characterization:
[0153] 300-19 cells expressing a recombinant human OX40-CD40 fusion
construct corresponding to 1-235 amino acid sequence of OX40
(extracellular and transmembrane domain) and 216-278 amino acid
sequence of CD40 (intracellular domain) was used for antibody
epitope analysis. The OX40-CD40 expressing cell line was grown in
RPMI medium (Gibco, Grand Island, N.Y.) supplemented with 10% fetal
calf serum (Hyclone, Logan, Utah), 10 mM hepes, 1%
penicillin-streptomycin, 2 mM L-glutamine, 0.1 mM non-essential
amino acids and 0.05 mM 2-mercapthoethanol (Gibco, Grand Island,
N.Y.). 300-19.hCD134.2 cells (5.times.10.sup.5/tube) were washed
once in 3 mls of cold wash buffer (PBS, 2% FBS and 0.02% sodium
azide). The cell supernatant was aspirated and 100 .mu.l of wash
buffer containing 300 .mu.g/ml of primary unconjugated OX40
reactive antibody was added to the cell pellet, mixed and incubated
for 30 minutes at 4.degree. C. Next, a fluorochrome labeled
secondary antibody was added to the tube, mixed and incubated for
an additional 30 minutes at 4.degree. C. The OX40 reactive
flourochrome labeled antibodies included either 10 .mu.l of
phycoerythrin (PE) labeled Ber Act 35 (Caltag Laboratories,
Burlingame, Calif.), PE-labeled L106 (BD Pharmingen, San Jose,
Calif.) or Alexa Fluor 647 conjugated OX40R antibody. The OX40R
antibody was labeled with fluorochome using the Alex Fluor 647
protein labeling kit as described by the manufacturer (Molecular
Probes, Eugene, Oreg.). After staining, cells were then washed 3
times with wash buffer, resuspended in cold wash buffer and 10,000
events were collected and analyzed using a Becton Dickinson
FACSCalibur and CellQuest software (San Jose, Calif.). Antibodies
were demeaned as binding to the same epitope when the primary
antibody blocked the staining of the secondary fluorochrome labeled
antibody by more than 80%.
[0154] Antibody OX40 Ligand-OX40R Inhibition Assay:
[0155] Antibodies were tested for their ability to block the
binding of the 300-19 human-OX40 ligand (L) expressing cells to
OX40-human IgG1 fusion protein coated plates. The 300-19-OX40L cell
line was grown in RPMI medium (Gibco, Grand Island, N.Y.)
supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah), 10
mM HEPES, 1% penicillin-streptomycin, 2 mM L-glutamine, 0.1 mM
non-essential amino acids, 0.05 mM 2-mercapthoethanol and 0.5 mg/ml
Geneticin (Gibco, Grand Island, N.Y.). The OX40-human IgG1 fusion
protein contains the first 220 amino acids of the extracellular
OX40 protein. The fusion protein was coated onto to Nunc Maxisorb
plates (Nunc, Roskilde, Denmark) in 100 .mu./well (5 .mu.g/ml) in
coating buffer (BupH, Carbonate-Bicarbonate buffer, Pierce,
Rockford, Ill.) and incubated overnight at 4.degree. C. Next,
plates were blotted on a paper towel to remove fluid, blocked with
200 .mu.l/well with blocking buffer (5% Carnation Milk diluted in
PBS) and incubated at RT.degree. for two hours. Plates were washed
with PBS and various dilutions of antibody diluted in PBS were then
added (50 .mu.l/well) to the assay plate and incubated at
RT.degree. for 30 minutes. Next, 50 .mu.l/well of cells in PBS at
6.times.10.sup.5/well were added to the antibody containing wells
and incubated for an additional 60 minutes at 37.degree. C. in a 5%
CO.sub.2 humidified chamber. The plates were gently washed 2 times
with PBS to remove non-adherent cells and cell activity in the
wells was measured by adding 200 .mu.l of a 20 ug/ml Fluorescein
Diacetate (Sigma, St. Louis, Mo.) PBS solution to each well. The
plates were incubated at 37.degree. C. in a 5% CO.sub.2 humidified
chamber for 90 minutes and read using a spectrophotometer at 490
(Spectra Max 340, Molecular Devices, Sunnyvale, Calif.).
[0156] OX40R Antibody Selectivity Assay (ELISA):
[0157] Maxisorb 96-well plates (Nunc, Roskilde, Denmark) were
coated with 100 .mu.l of human TNF.alpha. receptor family member
fusion proteins at 0.25 .mu.g/ml diluted in BupH.TM. Carbonate
buffer, pH 9.4 (Pierce, Rockford, Ill.) and incubated overnight at
4.degree. C. The selectivity receptor fusion proteins tested
included CD40-Ig (Alexis Biochemicals, San Diego, Calif.), CD137-Ig
(R&D Systems, Minneapolis, Minn.) and CD271-Ig (Alexis
Biochemicals, San Diego, Calif.). Also included as the positive
control with each assay was the OX40-Ig fusion protein (in-house
construct, Bioexpress, 97/2117). Plates were then washed three
times with wash buffer containing 0.05% Tween 20 (Sigma, St Louis,
Mo.) diluted in PBS and blocked with 300 .mu.l of 0.5% BSA (Sigma,
St Louis, Mo.) in PBS (Sigma, St Louis, Mo.) for 1 hour at
RT.degree.. Next, the plates were washed and 100 .mu.l/well of
anti-human OX40 reactive antibodies were added to the plates at
various concentrations and incubated for 1 hour at RT.degree..
Plates were thoroughly washed three times and OX40R antibody
binding was detected with a horse radish peroxidase labeled
anti-human kappa chain antibody (Bethyl Laboratories, Montgomery,
Tex.) at 25 ng/ml for 1 hour at RD.degree.. Plates were then washed
three times which was followed by the addition of 100 .mu.l/well of
1-Step Turbo-TMB substrate (Pierce, Rockford, Ill.) for 30 minutes
at RT. The reaction was stopped by adding an equal volume of 2M
H.sub.2 SO.sub.4. Absorbance was read at 450 nm on a Molecular
Devices Spectra Max 340 (Molecular Devices, Sunnyvale, Calif.).
[0158] Species Cross-Reactivity: Cell Lines Expressing OX40R:
[0159] The 300-19 cell line expressing either a recombinant human
OX40-CD40 fusion construct corresponding to 1-235 of OX40R
(extracellular and transmembrane domain) and 216-278 of CD40
(intracellular domain) or the entire cynomolgus OX40R protein.
[0160] Preparation of Human T Lymphocytes:
[0161] Human whole blood was collected into heparinized syringes
(Baxter; Deerfield, Ill.) and then immediately transferred to Sigma
Accuspin tubes (Sigma, St. Louis, Mo.) for the isolation of
peripheral blood mononuclear cells (PBMC) as described by the
manufacturer. The PBMC were washed twice with DPBS and T
lymphocytes were isolated using a T cell purification column as
described by the manufacturer (R & D Systems, Minneapolis,
Minn.). Briefly, PBMCs were resuspended in 2 mls of column buffer
and loaded into a pre-washed T cell isolation column. PBMCs were
incubated for 10 minutes at room temperature and T cells were
eluted with column buffer, washed one time and resuspended TCM at
2.times.10.sup.6/ml consisting of RPMI 1640 (Sigma, St Louis, Mo.)
supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) and
L-glutamine (2 mM), Hepes (10 mM), penicillin (100 U/ml),
streptomycin (50 ug/ml) (Gibco, Grand Island, N.Y.). A 2 ml volume
of T cells containing an anti-human CD28 antibody at 1 ug/ml (clone
37407, R & D Systems, Minneapolis, Minn.) was added to the
wells of a 24 well plate pre-coated with an anti-human CD3 antibody
clone UCTH1 (R & D Systems, Minneapolis, Minn.) at 5 .mu.g/ml
in PBS. T cell cultures were stimulated for 3 days prior to being
tested for human OX40 cross-reactivity by flow cytometry.
[0162] Preparation of Cynomolgus PBMCs:
[0163] Cynomolgus whole blood was obtained using heparinized
vacutainer tubes (BD; Franklin Lakes, N.J.) and was diluted 1:4 in
PBS. Diluted whole blood was mixed and 15 mls was carefully layered
over an equal volume of Histopaque 1077 (Sigma, St Louis, Mo.). The
tubes were spun at 1000.times.g for 45 minutes at RT and the
mononuclear PBMC interface was harvested, washed once in PBS and
resuspended for 2 minutes at RT.degree. with ACK lysing buffer
(Biosource, Rockville, Md.) to remove any RBCs. After a PBS wash,
the PBMCs were counted and readjusted to 1.times.10.sup.6/ml in
tissue culture medium (TCM). TCM consisted of RPMI 1640 (Sigma, St
Louis, Mo.) supplemented with 10% fetal bovine serum (Hyclone,
Logan, Utah) and L-glutamine (2 mM), Hepes (10 mM), penicillin (100
U/ml), streptomycin (50 ug/ml) purchased from Gibco (Grand Island,
N.Y.). Next, 2 mls of the PBMC preparation containing an anti-human
CD28 cross-reactive antibody (clone CD28.2, BD Biosciences, San
Diego, Calif.) was added to the wells of a 24 well plate (Costar,
Corning, N.Y.) pre-coated with an anti-monkey CD3 antibody (clone
FN18, Biosource, Camarillo, Calif.) at 10 .mu.g/ml in PBS. PBMC
cultures were stimulated for 4 days prior to being tested for human
OX40 cross-reactivity by flow cytometry.
[0164] Preparation of Rabbit PBMCs:
[0165] Rabbit whole blood was drawn into heparinized vacutainer
tubes (BD; Franklin Lakes, N.J.) and immediately diluted 1:3 with
warm HBSS (Gibco, Grand Island, N.Y.). After mixing, 5 mls of the
diluted blood was carefully layered over and equal volume of
Lympholyte-Rabbit (Cedarlane Laboratories, Westbury, N.Y.) and
centrifuged for 30 minutes at 25.degree. C. The PBMC interface was
collected, washed twice with PBS and resuspended to
2.times.10.sup.6/ml in TCM containing PHA at 10 ng/ml (Remel,
Lenexa, Kans.). The cells were cultured for 24-48 hours.
[0166] Preparation of Canine PBMCs:
[0167] Canine whole blood was collected using heparinized
vacutainer tubes (BD; Franklin Lakes, N.J.). Next, the blood was
mixed with an equal volume of warm HBSS (Gibco, Grand Island,
N.Y.). Four mls of diluted blood was slowly layered over 3 mls of
Lympholyte-M (Cedarlane Laboratories, Westbury, N.Y.) in a 15 ml
conical tubes. The tubes were centrifuged for 20 minutes at
800.times.g and the PBMC interface was collected, washed twice with
HBSS and resuspended in TCM at 2.times.10.sup.6/ml. PBMCs were
added to the wells of a 24 well plate (2 ml/well) and the cells
were stimulated with 2 .mu.g/ml of ConA (Sigma, St. Louis, Mo.) for
48 hours.
[0168] Preparation of Murine and Rat PBMCs:
[0169] Rat whole blood collected in heparinized syringes was
diluted 1:3 in warm HBSS. Next, 5 mls was carefully layered over an
equal volume of Lympholyte-Rat (Cedarlane Laboratories, Westbury,
N.Y.). The tubes were centrifuged for 20 minutes at 1500 RPM. The
PBMCs interface was collected, washed twice and the cell pellet was
re-adjusted to 2.times.10.sup.6/ml in TCM. Two mls of cells were
added to each well of a 24 well plate and stimulated for 24-48
hours with PHA (Remel, Lenexa, Kans.) at 10 ng/ml prior to flow
cytometry staining.
[0170] Flow Cytometry Staining for Species Cross-Reactivity:
[0171] Stimulated mouse, rat, rabbit, dog and cynomolgus PBMCs and
the 300-19 cell line expressing the cynomolgus OX40 receptor were
used to test for human OX40 antibody species cross-reactivity.
Human OX40 expressing activated T lymphocytes and OX40 transduced
300-19 cells were used as positive controls. Cells
(5.0.times.10.sup.5/tube) were washed once in cold wash buffer
(PBS, 2% FBS and 0.02% sodium azide) and 100 .mu.l/tube of Alexa
Fluor 647 conjugated control or OX40 reactive antibodies at 5 ug/ml
was added to each tube. The antibodies were labeled using an Alex
Fluor 647 protein labeling kit as described by the manufacturer
(Molecular Probes, Eugene, Oreg.). The cells were incubated in the
dark with fluorochrome antibodies on ice for 30 minutes, washed
three times and resuspended in 0.5 ml wash buffer for analysis.
Antibody staining was measured and analyzed using a Becton
Dickinson FACSCalibur and CellQuest software (San Jose,
Calif.).
[0172] Luciferase Activity Assay:
[0173] 293T cells containing the extracellular domain of OX40 and
the intracellular domain of CD40 fused to a NfkB reporter
containing luciferase were prepared. Cells were harvested, washed
and resuspended into phenol red free complete medium (DMEM
containing 10% fetal bovine serum, HEPES buffer, nonessential amino
acids and L-glutamine) at density of 0.5.times.10.sup.6 cell/ml. 80
ul of cells were plated into each assay well of a 96 well plate
(PerkinElmer, parts number 6005680). Test antibodies were added to
each well alone or in the presence of a cross linking antibody Fab'
goat anti-human IgG (Jackson ImmunoResearch, West Grove, Pa.). The
plate was incubated overnight at 37 C. 100 ul of luciferase
(Promega, Bright-glo luciferassay system, Cat. # E2620) was added
the next day and the amount of luciferase activity was measured
using a syntillation counter (TopCount, Packard-NXT).
[0174] Human .alpha.CD3 IL-2 Assay:
[0175] Human whole blood was collected in heparinized (Baxter;
Deerfield, Ill.) syringes, layered over Accuspin tubes (Sigma; St.
Louis, Mo.) and centrifuged for 15 minutes at 2000 rpm's. The buffy
coat was collected, washed with PBS (Sigma, St. Louis, Mo.), and
red blood cells lysed with water. T cells were separated out by
human CD3.sup.+ enrichment columns (R&D; Minneapolis, Minn.),
counted and adjusted to 1.times.10.sup.6/ml in RPMI media (Gibco;
Grand Island, N.Y.) containing: 10% fetal calf serum (Hyclone;
Logan, Utah), 10 mM hepes, 1% penicillin-streptomycin, 2 mM
L-glutamine and 0.1 mM non-essential amino acids (all Gibco).
Concurrently, human anti-CD3.epsilon. clone #UCHT1 (R&D
systems, Minneapolis, Minn.) was placed at 2.5 .mu.gs/ml in PBS
into 24 well plates (Costar; Corning, N.Y.) and incubated for 2
hours at 37.degree. C. The plates were washed 3.times. with PBS and
the following added to the wells: T cells at 1.times.10.sup.6/well,
serial dilutions of OX40 antibodies (or IgG.sub.2 KLH control) and
F(ab').sub.2 goat anti-human IgG Fc.gamma. to cross link (added at
2.5 ug/mL). Supernatants were pulled at 48 and 72 hours and IL-2
levels were assessed by ELISA (R&D).
[0176] Cynomolgus .alpha.CD3 IL-2 Assay:
[0177] Cynomolgus monkey whole blood was collected in heparinized
tubes (BD; Franklin Lakes, N.J.), diluted 1:4 in PBS, layered over
Histopaque 1077 (Sigma, St Louis, Mo.) and centrifuged for 45
minutes at 2200 rpm's. The buffy coat was collected, washed with
PBS, and red blood cells lysed with water. Cells were adjusted to
1.times.10.sup.6/ml and added to 24 well plates that had been
pre-coated for 2 hours with varying concentrations of monkey
anti-CD3, clone FN-18 (Biosource; Camarillo, Calif.) at 37.degree.
C. Serial dilutions of OX40 antibody (or IgG.sub.2 KLH control), as
well as F(ab').sub.2 goat anti-human IgG Fc.gamma. at 2.5 ug/mL
were added to the wells. Supernatants were collected at 24 and 48
hours and IL-2 levels were assessed by ELISA (Biosource, Camarillo,
Calif.).
[0178] Alloantigen Primed T Cells Assay:
[0179] Freshly isolated human T cells (see above) were incubated
with mitomycin c treated allogeneic tumor cells (Raji) for 3-4
days. T cells were then harvested, washed, and rested for 1 day in
fresh media prior to stimulating with 11D4. The level of IL-2 was
assessed 24 hours latter by ELISA (R&D systems, Minneapolis,
Minn.).
[0180] B. In Vivo Study Procedures
[0181] SCID-Beige Human Tumor Models Using Mice Engrafted with
Human T Cells and Dendritic Cells:
[0182] SCID-beige mice (Taconic #CBSBG-MM) were acclimated for 5-7
days after arrival prior to use. The following tumor cell lines
were used: RAJI, ATCC #CCL-86; BT-474, ATCC #HTB-20; PC-3,
ATCC#-1435; and LoVo, ATCC# CCL-229.
[0183] Purified T lymphocytes (T cells) and monocyte derived
dendritic cells were prepared from human blood as follows: Human
mononuclear cells were collected from heparinized blood using Sigma
Accuspin Tubes #A7054. Cells were collected, placed in a T75 flask,
and incubated for 3 hrs at 37.degree. C. in a humidified incubator
under 5% CO.sub.2. The non-adherent cells were collected and saved
(see below). The flask containing the adherent cells was incubated
with 20 ml RPMI complete medium (containing 10% fetal calf serum)
supplemented with IL-4 (R&D) at 10 ng/ml and GM-CSF (R&D)
at 100 ng/ml. The culture was then incubated for 6-7 days at
37.degree. C. in a humidified incubator under 5% CO.sub.2. The
non-adherent monocyte derived dendritic cells were then collected
by decanting and rinsing flask several times with RPMI complete
medium.
[0184] The initial non-adherent mononuclear cells were used to
purify T cells via high affinity negative selection using T cell
enrichment columns (R&D) as per manufacturer's instructions.
Purified T cells are cryo-preserved in Recovery-Cell Culture Medium
at 10.sup.7/ml and stored in liquid Nitrogen until use. Tumor cells
(1.times.10.sup.7) were injected subcutaneously (SC) with T cells
(1.times.10.sup.6) and monocyte-derived dendritic cells
(5.times.10.sup.5) from the same donor, at 0.2 mL/mouse. Tumor
growth was monitored over time with calipers.
[0185] C. Results for Antibody 11D4
[0186] (1) In Vitro Studies:
[0187] Certain properties of antibody 11D4 from in vitro studies
are summarized in Table 3.
[0188] Antibody 11D4 Binds to the OX40R with High Affinity.
[0189] This was demonstrated by using an IgG1 fusion protein
containing the extracellular domain of the OX40R and on whole cells
(OX40R+ transfected cells and activated primary T cells). In
examples using the IgG1 fusion protein, 11D4 bound to the
extracellular domain of the OX40R with an EC.sub.50 of 0.5+/-0.18
.mu.g/mL (3.5 nM). This binding was confirmed on 300-19 pre-B cells
expressing the full length extracellular domain of the OX40R (no
binding was observed on parental 300-19 cells). The EC.sub.50 for
binding to OX40R transfected cells was 0.2+/-0.16 .mu.g/mL (1.7
nM). In order to confirm that binding was observed on primary T
cells, peripheral blood T cells were isolated from multiple human
donors and stimulated with anti-CD3 and anti-CD28 for 2 days to
upregulate the expression of the OX40R. Saturation binding data on
these T cells indicated that 11D4 binds with an EC.sub.50 of
0.6+/-1.0 .mu.g/mL (4.0 nM, N=17 donors). These data demonstrate
that 11D4 avidly binds to the OX40R.
[0190] In order to further characterize this binding, data was
collected to assess the region on the extracellular domain of the
OX40R where 11D4 interacts and to also determine whether the
receptor was internalized following binding. Competition binding
data to the OX40R IgG1 fusion protein indicated that 11D4 competes
for binding with OX40 ligand expressing cells providing evidence
that 11D4 interacts at the ligand binding region of the receptor.
In addition, 11D4 does not cross-compete with two commercially
available OX40R antibodies, BerAct35 and L106, for binding to T
cells as assessed by FACS analysis. FACS analysis using
non-competing detection antibodies indicated that the OX40R was not
internalized following the pre-incubation of primary, activated T
cells with 11D4 for 30 minutes. Its binding affinity, determined by
Biacore analysis using the OX40R extracellular domain fusion
protein as the immobilized ligand, indicated that the equilibrium
dissociation constant (KD) of 11D4 for binding was 0.48 nM. These
analyses also estimated the off rate constant (kd) of 11D4 to be
5.72 E-05 l/s. Therefore, 11D4 binds with high affinity to the
ligand binding region of the OX40R, has a slow off-rate constant,
and does not internalize the receptor following binding.
[0191] Antibody 11D4 Selectively Binds to the OX40R.
[0192] The selectivity of 11D4 for the OX40R was assessed against
other members of the TNFR superfamily using data related to IgG1
fusion protein constructs containing the respective extracellular
domain of the related receptor. These receptors included the CD40
receptor, 4-1BB receptor (CD137) and the nerve growth factor
receptor (CD271). In all cases, no significant binding was observed
at concentrations up to 100 .mu.g/mL (700 nM) on these receptors.
When compared to binding observed to the OX40R fusion protein
(EC.sub.50=0.5 ug/ml), these data demonstrate that 11D4 is
>100-fold selective for the OX40R vs other related family
members tested. (See FIGS. 1a and 1b).
[0193] Functional Activity of Antibody 11D4:
[0194] The functional activity of 11D4 was demonstrated on both
OX40R+ transfected cells and on primary T cells. In these assays,
11D4 demonstrated agonist activity when added to cells with or
without a seondary antibody, F(ab').sub.2 goat anti-human IgG
Fc.gamma..
[0195] In the first set of experiments, 11D4 was assessed for
agonist activity using 293 cells transfected with the extracellular
and transmembrane domain of the OX40R fused to the intracellular
domain of CD40 with an NFkB luciferase reporter. In this assay,
11D4 enhanced signaling through the OX40R with a mean EC.sub.50 of
0.33 .mu.g/mL (2.2 nM, N=4). A representative
concentration-response curve for the induction of luciferase by
11D4 is shown in FIG. 2. In the absence of the F(ab').sub.2
secondary antibody, the magnitude of luciferase activity was
reduced 4-fold along with the EC.sub.50.
[0196] As further evidence for the agonist activity of 11D4,
antigen-specific T cells were generated. Freshly isolated human T
cells were incubated with mitomycin c treated allogeneic tumor
cells (Raji) for 3-4 days. T cells were then harvested, washed, and
rested for 1 day in fresh media prior to stimulating with 11D4.
FACS analysis indicated a high level of OX40R expression on these
cells even after resting. 11D4 induced high levels of IL-2 by these
cells, in some cases exceeding 100 ng/mL (FIG. 3). The average
EC.sub.50 for this response from 2 separate examples was
0.008+/-0.006 .mu.g/mL. In the absence of 11D4, only minimal levels
of IL-2 were secreted by these cells.
[0197] 11D4 also enhances the IL-2 production by the primary human
T cells stimulated by anti-CD3. Although the signal to noise ratio
in this assay was low in some assays due to the induction of IL-2
by anti-CD3 alone, 11D4 enhanced IL-2 production when added with
F(ab').sub.2 goat anti-human IgG Fc.gamma.. No activity was
observed for 11D4 on freshly isolated T cells in the absence of
anti-CD3. The magnitude of IL-2 augmentation by 11D4 ranged from
2.3 to 57-fold vs. anti-CD3 alone depending on the donor and the
amount of IL-2 generated by anti-CD3. The effect of 11D4 on IL-2
production by primary human T cells stimulated with 2.5 .mu.g/mL
anti-CD3 is represented in FIG. 4 (using an 8 point concentration
curve with 1:3 dilutions). The average EC.sub.50 calculated from
those data which used 8-point concentration response curves was
0.042+/-0.01 .mu.g/mL (see Table 4).
[0198] Functional activity of 11D4 on IL-2 production was also
assayed using monkey cells stimulated with anti-CD3 and 11D4 (along
with F(ab').sub.2 secondary antibody). Results are represented in
FIG. 5 and Table 5. These data indicated that the EC.sub.50 for
11D4 was similar between monkey and human cells (0.022 vs 0.042
.mu.g/mL for human cells), but the magnitude of IL-2 induced above
that of anti-CD3 alone was significantly less using Cynomolgus T
cells (approx. 35-fold, 5762+/-4748 pg/mL IL-2 for human cells
(N=21) vs 261+/-294 pg/mL IL-2 for monkey cells (N=9).
[0199] Species Cross-Reactivity:
[0200] 11D4 was assessed for its ability to bind to T cells from
multiple species. T cells were isolated from mouse, rat, rabbit,
dog, and monkey and activated with either anti-CD3 plus anti-CD28
or mitogen. No binding was observed to mouse, rat, rabbit or dog
cells as indicated by FACS analysis. The lack of binding to mouse
OX40R was also confirmed by ELISA using a commercially available
fusion protein containing the extracellular domain of the murine
OX40R. In contrast, 11D4 binds to Cynomolgus monkey T cells as
determined in a saturation binding assay by FACS. The range of
EC.sub.50 values obtained using different monkeys is shown in FIG.
6. For comparison, the range of EC.sub.50 values obtained using
human cells is shown in FIG. 7. Although variable, the range of
EC.sub.50 values was similar between monkey and human cells (mean
values are 0.354 .mu.g/mL for monkey vs 0.566 .mu.g/mL for human
cells).
[0201] (2) In Vivo Studies:
[0202] The lack of 11D4 cross-reactivity with the murine OX40R
required the development of a xenogenic tumor model using Severe
Combined Immunodeficient (SCID) beige mice. SCID-beige mice lack
murine T and B lymphocytes and NK cells making them ideal
recipients for the engraftment of human immune cells and the growth
of human tumors. Four tumor cell lines representing diverse tumor
types were tested in this in vivo model. None of the tumor lines
expressed OX40R. In all cases, tumor cells (1.times.10.sup.7) were
injected subcutenaously (SC) with T cells (1.times.10.sup.6) and
monocyte derived dendritic cells (5.times.10.sup.5) from the same
donor. 11D4 administered by intraperitoneal (IP) injection
inhibited tumor growth up to 98% in these models as summarized in
Table 6. The IP route of administration was chosen for 11D4 due to
its ease of administration and rapid dissemination into the
peripheral blood.
[0203] Efficacy of 11D4 Against a B Cell Lymphoma in SCID-Beige
Mice:
[0204] SCID-beige mice were injected SC with the Burkitt's B cell
lymphoma, Raji, together with human T cells and monocyte-derived
dendritic cells. Mice received a single IP injection of either 11D4
or an isotype control antibody (IgG2 anti-KLH) at the time of tumor
injection. As shown in FIG. 8, 11D4 decreased the rate of tumor
growth in treated animals. The tumor size in each individual animal
(N=10) on day 21 after challenge is shown in FIG. 9, illustrating
64% inhibition in tumor growth by a dose level of 10 mg/kg. No
activity was observed in the absence of T cells and dendritic
cells.
[0205] Efficacy of 11D4 in a Prostate Tumor Model:
[0206] SCID-beige mice were injected SC with the prostate
adenocarcinoma PC-3 together with human T cells and
monocyte-derived dendritic cells. Mice received a single IP
injection of either 11D4 or an isotype control antibody (IgG2
anti-KLH) at the time of tumor injection. The results, which are
represented in FIG. 10, show that 11D4 treatment resulted in a
dose-dependent inhibition of tumor growth. The tumor size in each
individual animal (N=10) from this study on day 27 after challenge
is shown in FIG. 11, illustrating a 70% inhibition in tumor growth
when animals were administered a single injection of 1.0 mg/kg
11D4, and 90% inhibition at a dose of 10 mg/kg. The plasma levels
of 11D4 determined on day 27 in these animals were 6.2 .mu.g/mL at
the 1.0 mg/kg dose level.
[0207] Efficacy of 11D4 in a Colon Carcinoma Tumor Model:
[0208] SCID-beige mice were injected SC with the colorectal
adenocarcinoma LoVo together with human T lymphocytes and
autologous monocyte-derived dendritic cells. Mice received a single
IP injection of either 11D4 or a control antibody (IgG2 anti-KLH)
at the time of tumor injection. The results, which are represented
in FIG. 12, show that 11D4 dose dependently decreased tumor growth
in these animals. The tumor size in each individual animal (N=10)
from this study on day 27 after challenge is shown in FIG. 13,
illustrating a 64% inhibition in tumor growth using a single dose
of 1.0 mg/kg and a 87% inhibition of tumor growth at a dose level
of 10.0 mg/kg.
[0209] Efficacy of 11D4 in a Mammary Carcinoma Tumor Model
[0210] SCID-beige mice were injected SC with the mammary carcinoma
BT474 together with human T lymphocytes and autologous
monocyte-derived dendritic cells. Mice received two injections (IP)
of either 11D4 or a control antibody (IgG2 anti-KLH) at the time of
tumor injection and again 30 days latter. The results, which are
represented in FIG. 14, show that 11D4 decreased tumor growth in
these animals. The tumor size in each individual animal (N=10) from
this study on day 85 after challenge is shown in FIG. 15
illustrating a 98% inhibition in tumor growth at a dose level of
10.0 mg/kg and 85% inhibition at a dose of 1 mg/kg.
[0211] D. Results for Antibody 18D8
[0212] (1) In Vitro Studies:
[0213] Results from in vitro studies for antibody 18D8 are
summarized in Table 7.
[0214] Effect of antibody 18D8 on anti-CD3 induced IL-2 production
by primary human T cells from different donors are also shown in
Table 8.
[0215] (2) In Vivo Studies:
[0216] Efficacy of 18D8 Against B Cell Lymphoma in a SCID-Beige
Mice Model
[0217] SCID-beige mice were injected SC with the Burkitt's B cell
lymphoma, Raji, together with human T lymphocytes and autologous
monocyte-derived dendritic cells. Mice received a single IP
injection of either 18D8 or an isotype control antibody (IgG2
anti-KLH) at the time of tumor injection. Ten animals per group
were used in each study. The results from two studies are presented
in Table 9. The results show that 18D8 produced significant
anti-tumor efficacy at the doses of 1.0 mg/kg and 10 mg/kg. No
activity was observed in the absence of T cells and dendritic
cells, suggesting that this anti-tumor effect may be immune
mediated.
[0218] Efficacy of 18D8 Against Prostate Tumor in a SCID-Beige Mice
Model
[0219] SCID-beige mice were injected SC with the prostate
adenocarcinoma PC-3 together with human T cells and autologous
monocyte-derived dendritic cells. Mice received a single IP
injection of either 18D8 or an isotype control antibody (IgG2
anti-KLH) at the time of tumor injection. Ten animals per group
were used in the study. The results are presented in Table 9. The
results show that 18D8 treatment resulted in a 42%, 90%, and 88%
inhibition of tumor growth at the doses of 0.1 mg/kg, 1.0 mg/kg,
and 10 mg/kg, respectively.
DEPOSIT INFORMATION
[0220] Applicants have deposited a culture of E. coli DH.alpha.5
containing plasmid that encodes the heavy chain of antibody 11D4
and a culture of E. coli DH.alpha.5 containing plasmid that encodes
the light chain of antibody 11D4 in the American Type Culture
collections (ATCC), 10801 University Boulevard, Manassas, Va.
20110-2209, on Jul. 10, 2007, which have been assigned deposit
numbers PTA-8524 and PTA-8525, respectively. These deposits were
made under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purpose of Patent Procedure and the Regulations thereunder
(Budapest Treaty). These deposits will be maintained without
restriction in the ATCC depository for a period of 30 years, or 5
years after the most recent request, or for the effective life of
the patent, whichever is longer, and will be replaced if the
deposits become non-viable during that period. Availability of the
deposited materials is not to be construed as a license to practice
the invention in contravention of the rights granted under the
authority of any government in accordance with its patent laws.
[0221] All references cited in this specification, including,
without limitation, all papers, publications, patents, patent
applications, books, journal articles, are hereby incorporated by
reference into this specification in their entireties. The
discussion of the references herein is intended to merely summarize
the assertions made by their authors and no admission is made that
any reference constitutes prior art.
[0222] Although the foregoing invention has been described in some
detail by way of illustrations and examples for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings herein that certain
changes and modifications may be made to the invention without
departing from the spirit or scope of the appendant claims.
TABLE-US-00001 TABLE 1 Sequence Identifiers for Antibodies 11D4 and
18D8 SEQ ID NO: Antibody Sequence 1 11D4 V.sub.H CDR1 Amino Acid 2
11D4 V.sub.H CDR2 Amino Acid 3 11D4 V.sub.H CDR3 Amino Acid 4 11D4
V.sub.L CDR1 Amino Acid 5 11D4 V.sub.L CDR2 Amino Acid 6 11D4
V.sub.L CDR3 Amino Acid 7 11D4 V.sub.H Amino Acid 8 11D4 V.sub.L
Amino Acid 9 11D4 Heavy Chain Amino Acid 10 11D4 Light Chain Amino
Acid 11 11D4 V.sub.H Nucleic Acid 12 11D4 V.sub.L Nucleic Acid 13
18D8 V.sub.H CDR1 Amino Acid 14 18D8 V.sub.H CDR2 Amino Acid 15
18D8 V.sub.H CDR3 Amino Acid 16 18D8 V.sub.L CDR1 Amino Acid 17
18D8 V.sub.L CDR2 Amino Acid 18 18D8 V.sub.L CDR3 Amino Acid 19
18D8 V.sub.H Amino Acid 20 18D8 V.sub.L Amino Acid 21 18D8 Heavy
Chain Amino Acid 22 18D8 Light Chain Amino Acid 23 18D8 V.sub.H
Nucleic Acid 24 18D8 V.sub.L Nucleic Acid
TABLE-US-00002 TABLE 2A Amino Acid Sequences for Antibody 11D4
SEQUENCE DES- (Variable region in upper case, constant CRIPTION
region in lower case, CDRs underlined) Heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAP Chain
GKGLEWVSYISSSSSTIDTADSVKGRFTISRDNAKNSLYLQ
MNSLRDEDTAVYYCARESGWYLFDYWGQGTLVTVSSastkg
psvfplapcsrstsestaalgclvkdyfpepvtvswnsgal
tsgvhtlpavlgssglyslssvvtvpssnfgtqtytcnvdh
kpsntkvdktverkccvecppcpappvagpsvflfppkpkd
tlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktk
preeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpap
iektisktkgqprepqvytlppsreemtknvsltclvkgfy
psdiavewesngqpennykttppmldsggsfflyskltvdk
srwqqgnvfscsvmhealhnhytqkslslspgk Light
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPE Chain
KAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED
FATYYCQQYNSYPPTFGGGTKVEIKrtvaapsvfifppsde
qlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvt
eqdskdstyslsstltlskadyekhkvyacevthqglsspv tksfnrgec
TABLE-US-00003 TABLE 2B Amino Acid Sequences for Antibody 18D8
SEQUENCE DES- (Variable region in upper case, constant CRIPTION
region in lower case, CDRs underlined) Heavy
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAP Chain
GKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQ
MNSLRAEDTALYYCAKDQSTADYYFYYGMDVWGQGTTVTVS
Sastkgpsyfplapcsrstsestaalgclvkdyfpepvtvs
wnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqty
tcnvdbkpsntkvdktverkccvecppcpappvagpsvflf
ppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvev
hnaktkpreeqfnstrvvsvltvvhqdwlngkeykckvsnk
glpapiektisktkgqprepqvytlppsreemtknqvsltc
lvkgfypsdiavewesngqpennykttppmldsdgsfflys
kltvdksrwqqgnvfscsvmhealhnhytqkslslspgk Light
EIVVTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPG Chain
QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPED
FAVYYCQQRSNWPTFGQGTKVEIKrtvaapsvfifppsdeq
lksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvte
qdskdstyslsstltlskadyekhkvyacevthqglsspvt ksfnrgec
TABLE-US-00004 TABLE 3 Summary of Certain in vitro Properties of
Antibody 11D4 Parameter Activity .mu.g/ml (nM) Affinity for OX40R:
(Biacore) K.sub.D 0.07 0.48 Off rate (kd) 5.7E-05 1/s Binding to
OX40R: Fusion protein extracellular domain 0.5 +/- 0.18 3.50
Saturation binding (EC.sub.50): CD3/CD28 stimulated T cells 0.6 +/-
1.00 4.00 (N = 17) OX40R+ 300-19 cells 0.2 +/- 0.16 1.70 (N = 5) In
vitro stimulation of OX40R+ 0.33 +/- 0.22 2.20 transfected cells
(luciferase) (EC.sub.50; N = 4) Augmentation of T cell activity:
CD3 induced IL-2 production 0.042 +/- 0.01 0.30 (N = 12)
Stimulation of IL-2 production by 0.008 +/- 0.006 0.04
antigen-primed cells (N = 2) Selectivity (binding) >100 .mu.g/mL
>700.00 (CD40, CD137, CD271) Values represent the mean +/- one
SD
TABLE-US-00005 TABLE 4 Effect of Antibody 11D4 on Anti-CD3 Induced
IL-2 Production by Primary Human T Cells EC.sub.50 Max IL-2 ECmax
Stimulation (.mu.g/mL) (pg/mL) (.mu.g/mL) Index Donor 0.008 4831
0.05 3.8 1 0.011 5450 0.05 2.6 2 0.014 6571 0.5 2.3 3 0.014 7271
0.05 5.9 4 0.011 6313 0.05 9.1 5 ND ND ND 7.0 6 0.010 1006 0.05 4.8
7 ND ND ND 5.9 8 ND ND ND 25.4 9 ND ND ND 57.0 10 ND ND ND 8.3 11
ND ND ND 5.1 12 ND ND ND 2.7 13 ND ND ND 4.6 14 0.014 4687 0.05 6.0
15 0.014 3012 0.05 35.2 16 ND ND ND 21.4 17 0.029 2796 0.125 3.8 18
0.052 1718 0.125 5.5 19 0.020 14190 0.56 16.8 20 0.068 1611 1.67
7.9 21 Max IL-2: Amount of IL-2 produced with 11D4 at the ECmax
over anti-CD3 alone ECmax: Concentration of 11D4 producing the
maximum level of IL-2 over anti-CD3 alone
Stimulation Index: Ratio of the maximum level of IL-2 produced with
11D4 vs the amount of IL-2 produced with anti-CD3 alone ND=not
determined. Values for the last four donors (18-21) are from dose
response curve done in 8-point 1:3 or 1:4 dilutions; all other
values represent log dilution curves. The EC.sub.50 from the 1:3
and 1:4 concentration curves was 0.042+/-0.01 ug/mL, N=4.
TABLE-US-00006 TABLE 5 Effect of Antibody 11D4 on Anti-CD3 Induced
IL-2 Production by Cynomolgus T Cells. Max IL-2 EC.sub.50 Induced
ECmax Stimulation (.mu.g/mL) (pg/mL) (.mu.g/mL) index Donor 0.007
376 0.05 3.5x 32750 0.002 116 0.05 2.2x 2325 ND ND ND 2.0x 32405
0.007 167 0.05 34.4 32081 0.011 978 0.005 5.6x 32842 ND ND ND 6.3x
2325 0.008 40 0.021 2.7x 33081 0.031 168 0.062 5.0x 33080 0.028 128
0.062 3.8x 33062 Max IL-2: Amount of IL-2 produced with 11D4 at
ECmax over anti-CD3 alone ECmax: Concentration of 11D4 producing
the maximum level of IL-2 over anti-CD3 alone Stimulation Index:
Ratio of the maximum IL-2 produced with 11D4 over the amount
produced with anti-CD3 alone ND = not determined
[0223] Values for the last three donors (33081, 33080, and 33062)
in Table 6 are from dose-response curve done in 8-point 1:3
dilutions. All other values represent log dilution curves. The
EC.sub.50 derived from those curves using 1:3 dilutions was
0.022+/-0.01; N=3.
TABLE-US-00007 TABLE 6 Human Tumor Growth Inhibition by Antibody
11D4 In SCID-beige Mice Engrafted with Human T cells and Dendritic
Cells Dosing Tumor with Study 10 1.0 0.1 0.01 Type 11D4 Duration
mg/kg mg/kg mg/kg mg/kg Raji: B cell Day 1 21 days 64% 42% 27% nd
lymphoma Raji: B cell Day 1 21 days nd 75% 42% 8% lymphoma Lovo:
colon Day 1 25 days 76% 44% 20% nd carcinoma Lovo: colon Day 1 25
days 87% 64% 15% nd carcinoma PC3: Day 1 27 days 90% 77% 45% nd
prostate PC3: Day 1 27 days 90% 70% 50% nd prostate BT474: Day 1 85
days 98% 85% 46% nd breast and 30 Values = % inhibition of tumor
growth determined at study termination nd = not detected
TABLE-US-00008 TABLE 7 Results of in vitro Studies with Antibody
18D8 Activity Parameter .mu.g/ml (nM) Affinity for OX40R (Biacore):
K.sub.D 0.49 3.38 Off rate (kd) 2.9E-04 1/s Binding to OX40R
(EC.sub.50): Fusion protein extracellular domain 0.034 +/- 0.01
0.23 Saturation binding: CD3/CD28 stimulated T cells (N = 4) 1.06
+/- 0.51 7.30 OX40R+ 300-19 cells (N = 2) 0.24 +/- 0.09 1.66
Augmentation of T Cell Activity (EC50): CD3 induced IL-2 production
(N = 4) 0.049 +/- 0.06 0.33 Stimulation of IL-2 production by 0.014
+/- 0 0.10 antigen-primed cells (N = 1) Selectivity (Binding to
CD40, CD137, >100 .mu.g/mL >700.00 CD271): Values for
activity expressed in .mu.g/ml represent the mean +/- one SD.
TABLE-US-00009 TABLE 8 Effect of Antibody 18D8 on Anti-CD3 Induced
IL-2 Production by Primary Human T Cells. EC.sub.50 Max IL-2 ECmax
Stimulation (.mu.g/mL) (pg/mL) (.mu.g/mL) Index Donor 0.013 1120
0.05 13.7 L C 0.024 4334 0.5 5.1 T H 0.024 2280 0.5 5.4 K O 0.135
1356 0.5 2.4 R N Max IL-2: Amount of IL-2 produced with 18D8 at the
ECmax over anti-CD3 alone. ECmax: Concentration of 18D8 producing
the maximum level of IL-2 over anti-CD3 alone. Stimulation Index:
Ratio of the maximum IL-2 produced with 18D8 over the amount
produced with anti-CD3 alone. Values represent log dilution
curves.
TABLE-US-00010 TABLE 9 Inhibition of Human Tumor Growth by Antibody
18D8 in SCID-beige Mice Dosing Tumor with Study Dose Level of 18D8
(mg/kg) Type 18D8 Duration 10 1.0 0.1 0.01 Raji: B cell Day 1 23
days 73% 73% 11% nd lymphoma Raji: B cell Day 1 24 days 54% 59% nd
nd lymphoma PC3: prostate Day 1 24 days 88% 90% 42% nd Values = %
inhibition of tumor growth determined at study termination nd = not
determined
Sequence CWU 1
1
2415PRTHuman 1Ser Tyr Ser Met Asn1 5217PRTHuman 2Tyr Ile Ser Ser
Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val Lys1 5 10
15Gly39PRTHuman 3Glu Ser Gly Trp Tyr Leu Phe Asp Tyr1 5411PRTHuman
4Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1 5 1057PRTHuman 5Ala
Ala Ser Ser Leu Gln Ser1 569PRTHuman 6Gln Gln Tyr Asn Ser Tyr Pro
Pro Thr1 57118PRTHuman 7Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Ser Ser
Thr Ile Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ser
Gly Trp Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val
Thr Val Ser Ser 1158107PRTHuman 8Asp 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 Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 1059444PRTHuman 9Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ser Gly Trp Tyr Leu Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
Cys Val Glu Cys 210 215 220Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe 260 265 270Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295
300Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val305 310 315 320Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Thr 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg 340 345 350Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser385 390 395 400Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410
415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
44010214PRTHuman 10Asp 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
Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys
Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro 85 90 95Thr Phe Gly Gly 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
21011354DNAHuman 11gaggtgcagc tggtggagtc tgggggaggc ttggtacagc
cgggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agctatagca
tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg ggtttcatac
attagtagta gtagtagtac catagactac 180gcagactctg tgaagggccg
attcaccatc tccagagaca atgccaagaa ctcactgtat 240ctgcaaatga
acagcctgag agacgaggac acggctgtgt attattgtgc gagagaaagc
300ggctggtacc tctttgacta ctggggccag ggaaccctgg tcaccgtctc ctca
35412321DNAHuman 12gacatccaga tgacccagtc tccatcctca ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattagc agctggttag
cctggtatca gcagaaacca 120gagaaagccc ctaagtccct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttatta ctgccaacag tataatagtt accctcccac tttcggcgga
300gggaccaagg tggagatcaa a 321135PRTHuman 13Asp Tyr Ala Met His1
51417PRTHuman 14Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp
Ser Val Lys1 5 10 15Gly1515PRTHuman 15Asp Gln Ser Thr Ala Asp Tyr
Tyr Phe Tyr Tyr Gly Met Asp Val1 5 10 151611PRTHuman 16Arg Ala Ser
Gln Ser Val Ser Ser Tyr Leu Ala1 5 10177PRTHuman 17Asp Ala Ser Asn
Arg Ala Thr1 5188PRTHuman 18Gln Gln Arg Ser Asn Trp Pro Thr1
519124PRTHuman 19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser
Ile Gly Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Lys Asp Gln Ser
Thr Ala Asp Tyr Tyr Phe Tyr Tyr Gly Met Asp 100 105 110Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 12020106PRTHuman 20Glu Ile
Val Val Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10521450PRTHuman 21Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Ser Trp Asn Ser Gly Ser
Ile Gly Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Lys Asp Gln Ser
Thr Ala Asp Tyr Tyr Phe Tyr Tyr Gly Met Asp 100 105 110Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 130 135
140Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro145 150 155 160Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr 165 170 175Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val 180 185 190Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr Tyr Thr Cys Asn 195 200 205Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys Thr Val Glu Arg 210 215 220Lys Cys Cys Val
Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly225 230 235 240Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250
255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe Arg 290 295 300Val Val Ser Val Leu Thr Val Val His Gln
Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375
380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Met385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys 45022213PRTHuman
22Glu Ile Val Val Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155
160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys 21023372DNAHuman
23gaagtgcagc tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc
60tcctgtgcag cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct
120ccagggaagg gcctggaatg ggtctcaggt attagttgga atagtggtag
cataggctat 180gcggactctg tgaagggccg attcaccatc tccagagaca
acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agctgaggac
acggccttgt attactgtgc aaaagatcag 300agtacagctg attactactt
ctactacggt atggacgtct ggggccaagg gaccacggtc 360accgtctcct ca
37224318DNAHuman 24gaaattgtgg tgacacagtc tccagccacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag
cctggtacca acagaaacct 120ggccaggctc ccaggctcct catctatgat
gcatccaaca gggccactgg catcccagcc 180aggttcagtg gcagtgggtc
tgggacagac ttcactctca ccatcagcag cctagagcct 240gaagattttg
cagtttatta ctgtcagcag cgtagcaact ggccgacgtt cggccaaggg
300accaaggtgg aaatcaaa 318
* * * * *