AU2019213305B2 - Antibodies to OPGL - Google Patents

Abstract

Antibodies that interact with osteoprotegerin ligand (OPGL) are described. Methods of treating osteopenic disorders by administering a pharmaceutically effective amount of antibodies to OPGL are described. Methods of detecting the amount of OPGL in a sample using antibodies to OPGL are described.

Description

ANTIBODIES TO OPGL

[001] The present application is a divisional application of Australian Application No. 2018202668, which is incorporated in its entirety herein by reference.

[001a] This application claims priority to U.S. Provisional Application Serial No. 60/301, 172, filed June 26, 2001 , which is incorporated herein by reference for any purpose.

FIELD OF THE INVENTION

[002] The present invention relates to antibodies that bind osteoprotegerin ligand (OPGL). Compositions and methods for the treatment of bone diseases, such as osteoporosis, bone loss from arthritis, Paget's disease, and osteopenia, are also described.

BACKGROUND OF THE INVENTION

[003] Bone tissue provides support for the body and includes mineral (including calcium and phosphorous), a matrix of collagenous and noncollagenous proteins, and cells. Living bone tissue exhibits a dynamic equilibrium between formation of bone, which is called deposition, and breakdown of bone, which is called resorption. Three types of cells found in bone, osteocytes, osteoblasts and osteoclasts, are involved in this equilibrium. Osteoblasts promote formation of bone tissue whereas osteoclasts are associated with resorption. Resorption, or the dissolution of bone matrix and mineral, is a fast and efficient process compared to bone formation and can release large amounts of mineral from bone. Osteoclasts are involved in the regulation of the normal remodeling of skeletal tissue and in resorption induced by hormones. For instance, resorption is stimulated by the secretion of parathyroid hormone in response to decreasing concentrations of calcium ion in extracellular fluids. In contrast, inhibition of resorption is a function of calcitonin.

In addition, metabolites of vitamin D alter the responsiveness of bone to

parathyroid hormone and calcitonin.

[004] Osteoprotegerin ligand (OPGL), which is a member of the

TNF family of cytokines, promotes formation of osteoclasts through binding to the

receptor activator of NF-KB (RANK, also called osteoclast differentiation and

activation receptor, or ODAR). Osteoprotegerin (OPG), on the other hand,

inhibits the formation of osteoclasts by sequestering OPGL and preventing OPGL

association with ODAR. Thus, the amount of OPGL associated with ODAR

correlates with the equilibrium between bone deposition and resorption.

[005] After skeletal maturity, the amount of bone in the skeleton

reflects the balance (or imbalance) of bone formation and bone resorption. Peak

bone mass occurs after skeletal maturity prior to the fourth decade. Between the

fourth and fifth decades, the equilibrium shifts and bone resorption dominates.

The inevitable decrease in bone mass with advancing years starts earlier in

females than males and is distinctly accelerated after menopause in some

females (principally those of Caucasian and Asian descent).

[006] Osteopenia is a condition relating generally to any decrease

in bone mass to below normal levels. Such a condition may arise from a

decrease in the rate of bone synthesis or an increase in the rate of bone

destruction or both. A common form of osteopenia is primary osteoporosis, also referred to as postmenopausal and senile osteoporosis. This form of osteoporosis is a consequence of the universal loss of bone with age and is often a result of increase in bone resorption with a normal rate of bone formation. Many white females in the United States develop symptomatic osteoporosis. A direct relationship exists between osteoporosis and the incidence of hip, femoral, neck and inter trochanteric fracture in women 45 years and older. Elderly males may develop symptomatic osteoporosis between the ages of 50 and 70. Osteoporosis may, in certain instances, result from increased levels or activity of OPGL. Thus, it would be useful to have molecules that can regulate the activity of OPGL in osteoclastogenesis.

[007] Several factors have been identified which may contribute to

postmenopausal and senile osteoporosis. They include alteration in hormone levels

accompanying aging and inadequate calcium consumption attributed to decreased

intestinal absorption of calcium and other minerals. Certain treatments have included

hormone therapy or dietary supplements in an attempt to retard the process. More

recently, anti-resorptive agents such as bisphosphonates and selective estrogen

receptor modifiers (SERMs) have emerged for the prevention and treatment of

reduced bone mass. Thus, it may be useful to combine those treatments with

molecules that can regulate the activity of OPGL in treating certain osteopenic

disorders.

[007a] Any discussion of the prior art throughout the specification

should in no way be considered as an admission that such prior art is widely known

or forms part of common general knowledge in the field.

[007b] Unless the context clearly requires otherwise, throughout the

description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

SUMMARY OF THE INVENTION

[007c] According to a first aspect, the present invention provides a

recombinant antibody that comprises a heavy chain and a light chain, wherein:

(a) said antibody is a monoclonal antibody comprising: (i) a human IgG2

heavy chain comprising SEQ ID NO:13, and (ii) a human kappa light chain

comprising SEQ ID NO:14; and

(b) the antibody is expressed from a mammalian host cell; and

(c) said antibody binds to an osteoprotegerin ligand (OPGL) and inhibits

binding of OPGL to an osteoclast differentiation and activation receptor (ODAR).

[007d] According to a second aspect, the present invention provides a

recombinant antibody that comprises a heavy chain and a light chain, wherein:

(a) said heavy chain comprises (i) a heavy chain variable region

comprising SEQ ID NO:13, and (ii) a heavy chain constant region comprising the

constant region of SEQ ID NO: 2, or having one carboxy-terminal amino acid

deletion of the constant region of SEQ ID NO: 2; and

(b) said light chain comprises (i) a light chain variable region comprising

SEQ ID NO:14; and (ii) a light chain constant region comprising the constant region

of SEQ ID NO: 4; and

(c) said antibody binds to an osteoprotegerin ligand (OPGL) and inhibits

binding of OPGL to an osteoclast differentiation and activation receptor (ODAR).

[007e] According to a third aspect, the present invention provides a

recombinant antibody that comprises a heavy chain and a light chain, wherein:

(a) said heavy chain comprises an amino acid sequence of SEQ ID NO:2

from residue 20 to residue 467, or having one carboxy-terminal amino acid deletion

of residue 20 to residue 467 of SEQ ID NO:2; and the light chain comprises an

amino acid sequence of SEQ ID NO:4 from residue 21 to residue 235;

(b) the heavy chain consists of an amino acid sequence of SEQ ID NO:2

from residue 20 to residue 467, or having one carboxy-terminal amino acid deletion

of residue 20 to residue 467 of SEQ ID NO:2; and the light chain consists of an

amino acid sequence of SEQ ID NO:4 from residue 21 to residue 235.

[007f] According to a fourth aspect, the present invention provides a

pharmaceutical composition comprising a therapeutically effective amount of the

antibody of the invention and a pharmaceutically acceptable diluent, carrier,

solubilizer, emulsifier, preservative and/or adjuvant.

[007g] According to a fifth aspect, the present invention provides a

method of treating a bone disorder characterized by a net bone loss, comprising

administering an antibody of the invention, or a pharmaceutical composition of the

invention.

[007h] According to a sixth aspect, the present invention provides a

method of preventing or mitigating the onset of bone loss caused by metastatic

cancer, comprising administering prophylactically an antibody of the invention, or a

pharmaceutical composition of the invention.

[007i] According to a seventh aspect, the present invention provides a

method of treating an existing condition of bone loss due to metastasis, comprising

4a administering an antibody of the invention, or a pharmaceutical composition of the invention.

[007j] According to an eighth aspect, the present invention provides a

use of an antibody of the invention, or a pharmaceutical composition of the invention

for the manufacture of a medicament for treating a bone disorder characterized by a

net bone loss.

[007k] According to a ninth aspect, the present invention provides a

use of an antibody of the invention, or a pharmaceutical composition of the invention

for the manufacture of a medicament for preventing or mitigating the onset of bone

loss caused by metastatic cancer.

[0071] According to a tenth aspect, the present invention provides a

use of an antibody of the invention, or a pharmaceutical composition of the invention

for the manufacture of a medicament for treating an existing condition of bone loss

due to metastasis.

[008] In certain embodiments, the invention provides for an antibody,

comprising a heavy chain and a light chain, wherein the heavy chain comprises an

amino acid sequence as set forth in SEQ ID NO: 2 or a fragment thereof, and the

light chain comprises an amino acid sequence as set forth in SEQ ID NO: 4 or a

fragment thereof.

[009] In certain embodiments, the invention provides for an antibody,

comprising a heavy chain and a light chain, wherein the heavy chain comprises a

variable region comprising an amino acid sequence as set forth in SEQ ID NO: 13 or

a fragment thereof, and wherein the light chain comprises a variable region

comprising an amino acid sequence as set forth in SEQ ID NO: 14 or a fragment

thereof.

4b

[010] In certain embodiments, the invention provides for an antibody

comprising a heavy chain and a light chain, wherein the heavy chain comprises an

amino acid sequence as set forth in SEQ ID NO: 2 or a fragment thereof.

[011] In certain embodiments, the invention provides for an antibody

comprising a heavy chain and a light chain, wherein the heavy chain comprises a

variable region comprising an amino acid sequence as set forth in SEQ ID NO: 13 or

a fragment thereof.

[012] In certain embodiments, the invention provides for an antibody

comprising a heavy chain and a light chain, wherein the light chain

4c comprises an amino acid sequence as set forth in SEQ ID NO: 4 or a fragment thereof.

[013] In certain embodiments, the invention provides for an

antibody comprising a heavy chain and a light chain, wherein the light chain

comprises a variable region comprising an amino acid sequence as set forth in

SEQ ID NO: 14 or a fragment thereof.

[014] In certain embodiments, the invention provides for an

antibody, comprising a heavy chain and a light chain, (a) wherein the heavy

chain comprises a first variable region, and wherein the first variable region

comprises a sequence that has at least 90% identity to the amino acid sequence

set forth in SEQ ID NO: 13, and (b) wherein the light chain comprises a second

variable region, and wherein the second variable region comprises a sequence

that has at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

14, and (c) wherein the antibody interacts with an osteoprotegerin ligand (OPGL).

[015] In certain embodiments, the first variable region comprises a

sequence that has at least 95% identity to the amino acid sequence set forth in

SEQ ID NO: 13, and the second variable region comprises a sequence that has

at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 14.

[016] In certain embodiments, the first variable region comprises a

sequence that has at least 99% identity to the amino acid sequence set forth in

SEQ ID NO: 13, and the second variable region comprises a sequence that has

at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 14.

[017] In certain embodiments, the invention provides for a heavy

chain, comprising an amino acid sequence as set forth in SEQ ID NO:2 or a

fragment thereof. In certain embodiments, the invention provides for a heavy

chain comprising a variable region and a constant region, wherein the variable

region comprises an amino acid sequence as set forth in SEQ ID NO: 13 or a

fragment thereof.

[018] In certain embodiments, the invention provides for a light

chain, comprising an amino acid sequence as set forth in SEQ ID NO:4 or a

fragment thereof. In certain embodiments, the invention provides for a light chain

comprising an amino acid sequence as set forth in SEQ ID NO: 14 or a fragment

thereof.

[019] In certain embodiments of the invention, single chain

antibodies are provided. In certain embodiments of the invention, single chain Fv

antibodies are provided. In certain embodiments of the invention, Fab antibodies

are provided. In certain embodiments of the invention, Fab' antibodies are

provided. In certain embodiments of the invention, (Fab')2 antibodies are

provided.

[020] In certain embodiments, a pharmaceutical composition

comprising an antibody of the invention is provided. In certain embodiments, a

pharmaceutical composition comprising a therapeutically effective amount of an

antibody to OPGL is provided.

[021] In certain embodiments, a pharmaceutical composition

comprises an antibody to OPGL and at least one therapeutic agent selected from a bone morphogenic factor, transforming growth factor-P (TGF-p), an interleukin

1 (IL-1) inhibitor, IL-Ira, KineretTM, a TNFa inhibitor, a soluble TNFa receptor,

EnbreI TM, an anti-TNFa antibody, Remicade TM, a D2E7 antibody, a parathyroid

hormone, an analog of a parathyroid hormone, a parathyroid hormone related

protein, an analog of a parathyroid hormone related protein, a prostaglandin, a

bisphosphonate, an alendronate, fluoride, calcium, a non-steroidal anti

inflammatory drug (NSAID), a COX-2 inhibitor, Celebrex T M, Vioxx TM;an

immunosuppressant, methotrexate, leflunomide, a serine protease inhibitor, a

secretory leukocyte protease inhibitor (SLPI), an IL-6 inhibitor, an antibody to IL

6, an IL-8 inhibitor, an antibody to IL-8, an IL-i8 inhibitor, an IL-18 binding

protein, an IL-18 antibody, an Interleukin-1 converting enzyme (ICE) modulator, a

fibroblast growth factor (FGF), an FGF modulator, a PAF antagonist, a

keratinocyte growth factor (KGF), a KGF-related molecule, a KGF modulator; a

matrix metalloproteinase (MMP) modulator, a nitric oxide synthase (NOS)

modulator, a modulator of glucocorticoid receptor, a modulator of glutamate

receptor, a modulator of lipopolysaccharide (LPS) levels, a noradrenaline, a

noradrenaline mimetic, and a noradrenaline modulator.

[022]- In certain embodiments of the invention, a method of treating

an osteopenic disorder is provided, comprising administering a pharmaceutically

effective amount of an antibody. In certain embodiments, a method of treating an

osteopenic disorder comprising administering a pharmaceutical composition is

provided.

[023] In certain embodiments, a method of treating an

inflammatory condition with attendant bone loss in a patient comprising

administering a pharmaceutical composition is provided.

[024] In certain embodiments, a method of treating an

autoimmune condition with attendant bone loss in a patient comprising

administering a pharmaceutical composition is provided.

[025] In certain embodiments, a method of treating rheumatoid

arthritis in a patient, comprising administering a pharmaceutical composition of

the invention is provided.

[026] In certain embodiments of the invention, a method of

detecting the level of OPGL in a biological sample is provided, comprising

contacting the sample with an antibody.

BRIEF DESCRIPTION OF THE FIGURES

[027] Figure 1 shows a cDNA sequence encoding the aOPGL-1

antibody heavy chain (SEQ ID NO: 1).

[028] Figure 2 shows the amino acid sequence of the aOPGL-1

antibody heavy chain (SEQ ID NO: 2).

[029] Figure 3 shows a cDNA sequence encoding the aOPGL-1

antibody light chain (SEQ ID NO: 3).

[030] Figure 4 shows the amino acid sequence of the aOPGL-1

antibody light chain (SEQ ID NO: 4).

r31] Figure 5 shows a schematic diagram of the aOPGL-1 kappa

light chain expression plasmid aOPGL-1-Kappa/pDSRa19.

[032] Figure 6 shows a schematic diagram of the aOPGL-1 IgG2

heavy chain expression plasmid, aOPGL-1-IgG2/pDSRa19.

[033] Figure 7 shows dose-dependent binding of aOPGL-1 to

OPGL-coated EIA plates.

[034] Figure 8 shows specific binding of aOPGL-1 to membrane

bound OPGL.

[035] Figure 9 shows inhibition of aOPGL-1 binding to OPGL

coated EIA plates by soluble OPGL.

[036] Figure 10 shows specific binding of aOPGL-1 to OPGL

coated EIA plates.

[037] Figure 11 shows dose-dependent inhibition of osteoclast

formation by aOPGL-1.

[038] Figure 12 shows dose-dependent inhibition of OPGL binding

to ODAR by aOPGL-1.

[039] Figure 13 shows the mean serum concentration time profiles

after administering a single dose of aOPGL-1 to Cynomogus Monkeys.

[040] Figure 14 shows the mean percent change in serum N-Tx

concentration after administering a single dose of aOPGL-1 to Cynomolgus

Monkeys.

[041] Figure15 shows the mean percent change in urine N-Tx

concentration after administering a single dose of aOPGL-1 to Cynomolgus

Monkeys.

[042] Figure 16 shows antibody positive and negative serum

concentration time profiles after administering a single dose of aOPGL-1 to

Cynomolgus Monkeys.

[043] Figure 17 shows the amino acid sequence of the aOPGL-1

antibody heavy chain variable region (SEQ ID NO: 13).

[044] Figure 18 shows the amino acid sequence of the aOPGL-1

antibody light chain variable region (SEQ ID NO: 14).

[045] Figure 19 shows a cell culture process for production of

aOPGL-1.

[046] Figure 20 shows the serum calcium percent change after

administering a single dose of aOPGL-1 to Cynomolgus monkeys.

[047] Figure 21 shows the mean serum Alkaline Phosphatase

percent change after administering a single dose of aOPGL-1 to Cynomogus

monkeys.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

[048] The section headings used herein are for organizational

purposes only and are not to be construed as limiting the subject matter

described. All references cited in this application are expressly incorporated by

reference herein for any purpose.

Definitions

[049] Standard techniques may be used for recombinant DNA,

oligonucleotide synthesis, and tissue culture and transformation (e.g.,

electroporation, lipofection). Enzymatic reactions and purification techniques

may be performed according to manufacturer's specifications or as commonly

accomplished in the art or as described herein. The foregoing techniques and

procedures may be generally performed according to conventional methods well

known in the art and as described in various general and more specific

references that are cited and discussed throughout the present specification.

See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold

Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is

incorporated herein by reference for any purpose. Unless specific definitions are

provided, the nomenclatures utilized in connection with, and the laboratory

procedures and techniques of, analytical chemistry, synthetic organic chemistry,

and medicinal and pharmaceutical chemistry described herein are those well

known and commonly used in the art. Standard techniques may be used for

chemical syntheses, chemical analyses, pharmaceutical preparation, formulation,

and delivery, and treatment of patients.

[050] As utilized in accordance with the present disclosure, the

following terms, unless otherwise indicated, shall be understood to have the

following meanings:

[051] The term "isolated polynucleotide" as used herein shall

mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide" is found in nature, (2) is linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

[052] The term "isolated protein" referred to herein means a

protein encoded by cDNA, recombinant RNA, or synthetic origin or some

combination thereof, which (1) is free of at least some proteins with which it

would normally be found, (2) is essentially free of other proteins from the same

source, e.g., from the same species, (3) is expressed by a cell from a different

species, or (4) does not occur in nature.

[053] The term "polypeptide" is used herein as a generic term to

refer to native proteins, or sequences that have deletions, additions, and/or

substitutions of one or more amino acids of the native sequence. The term

"polypeptide" also encompasses aOPGL-1 (as described below, SEQ ID NO: 2

and SEQ ID NO: 4), or sequences that have deletions, additions, and/or

substitutions of one or more amino acid of aOPGL-1. According to certain

embodiments, the invention comprises the human heavy chain immunoglobulin

molecule represented by Figure 2 (SEQ ID NO: 2) and the human light chain

immunoglobulin molecule represented by Figure 4 (SEQ ID NO: 4), or fragments

or analogs thereof.

[054] The term "naturally-occurring" as used herein as applied to

an object refers to the fact that an object can be found in nature. For example, a

polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.

[055] The term "operably linked" as used herein refers to

components that are in a relationship permitting them to function in their intended

manner. For example, a control sequence "operably linked" to a coding

sequence is ligated in such a way that expression of the coding sequence is

achieved under conditions compatible with the control sequences.

[056] The term "control sequence" as used herein refers to

polynucleotide sequences which may effect the expression and processing of

coding sequences to which they are ligated. The nature of such control

sequences may differ depending upon the host organism. According to certain

embodiments, control sequences for prokaryotes may include promoter,

ribosomal binding site, and transcription termination sequence. According to

certain embodiments, control sequences for eukaryotes may include promoters

and transcription termination sequence. In certain embodiments, "control

sequences" can include leader sequences and/or fusion partner sequences.

[057] The term "polynucleotide" as referred to herein means a

polymeric form of nucleotides of at least 10 bases in length. In certain

embodiments, the bases may be ribonucleotides or deoxyribonucleotides or a

modified form of either type of nucleotide. The term includes single and double

stranded forms of DNA.

[058] The term "oligonucleotide" referred to herein includes

naturally occurring, and modified nucleotides linked together by naturally occurring, and/or non-naturally occurring oligonucleotide linkages.

Oligonucleotides are a polynucleotide subset generally comprising a length of

200 bases or fewer. In certain embodiments, oligonucleotides are 10 to 60

bases in length. In certain embodiments, oligonucleotides are 12, 13, 14, 15, 16,

17, 18, 19, or 20 to 40 bases in length. Oligonucleotides may be single stranded

or double stranded, e.g. for use in the construction of a gene mutant.

Oligonucleotides of the invention may be sense or antisense oligonucleotides.

[059] The term "naturally occurring nucleotides" includes

deoxyribonucleotides and ribonucleotides. The term "modified nucleotides"

includes nucleotides with modified or substituted sugar groups and the like. The

term "oligonucleotide linkages" includes oligonucleotides linkages such as

phosphorothioate, phosphorodithioate, phosphoroselenoate,

phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,

phosphoroamidate, and the like. See, e.g., LaPlanche et al. Nucl. Acids Res.

14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl.

Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design 6:539 (1991);

Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F.

Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S.

Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990), the

disclosures of which are hereby incorporated by reference for any purpose. An

oligonucleotide can include a label for detection.

[060] Identity and similarity of related and polypeptides can be

readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford

University Press, New York (1988); Biocomputing: Informatics and Genome

Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer

Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana

Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje,

G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and

Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo et al., SIAM

J. Applied Math., 48:1073 (1988).

[061] Preferred methods to determine identity are designed to give

the largest match between the sequences tested. Methods to determine identity

are described in publicly available computer programs. Preferred computer

program methods to determine identity between two sequences include, but are

not limited to, the GCG program package, including GAP (Devereux et al., Nuc.

Acid. Res., 12:387 (1984); Genetics Computer Group, University of Wisconsin,

Madison, WI, BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol.,

215:403-410 (1990)). The BLASTX program is publicly available from the

National Center for Biotechnology Information (NCBI) and other sources (BLAST

Manual, Altschul et al. NCB/NLM/NIH Bethesda, MD 20894; Altschul et al., supra

(1990)). The well-known Smith Waterman algorithm may also be used to

determine identity.

[062] Certain alignment schemes for aligning two amino acid

sequences may result in the matching of only a short region of the two

sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, in certain embodiments, the selected alignment method

(GAP program) will result in an alignment that spans at least 50 contiguous

amino acids of the target polypeptide.

[063] For example, using the computer algorithm GAP (Genetics

Computer Group, University of Wisconsin, Madison, WI), two polypeptides for

which the percent sequence identity is to be determined are aligned for optimal

matching of their respective amino acids (the "matched span", as determined by

the algorithm). In certain embodiments, a gap opening penalty (which is

calculated as 3X the average diagonal; the "average diagonal" is the average of

the diagonal of the comparison matrix being used; the "diagonal" is the score or

number assigned to each perfect amino acid match by the particular comparison

matrix) and a gap extension penalty (which is usually 1/10 times the gap opening

penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are

used in conjunction with the algorithm. In certain embodiments, a standard

comparison matrix (see Dayhoff et al., Atlas of Protein Sequence and Structure,

(3)(1978) for the PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad.

Sci USA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also

used by the algorithm.

[064] In certain embodiments, the parameters for a polypeptide

sequence comparison include the following:

Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970); Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992); Gap Penalty: 12 Gap Length Penalty: 4

Threshold of Similarity: 0

[0651 The GAP program may be useful with the above parameters. In certain embodiments, the aforementioned parameters are the default

parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

[066] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology--A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids,

unnatural amino acids such as x-, a-disubstituted amino acids, N-alkyl amino

acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of

unconventional amino acids include: 4-hydroxyproline, y-carboxyglutamate, 6

N,N,N-trimethyllysine, c-N-acetyllysine, O-phosphoserine, N-acetylserine, N

formylmethionine, 3-methylhistidine, 5-hydroxylysine, a-N-methylarginine, and

other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[067] Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of

double-stranded polynucleotide sequences is referred to as the 5'direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5'to the 5'end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3'to the 3'end of the RNA transcript are referred to as "downstream sequences".

[0681 Conservative amino acid substitutions may encompass non

naturally occurring amino acid residues, which are typically incorporated by

chemical peptide synthesis rather than by synthesis in biological systems. These

include peptidomimetics and other reversed or inverted forms of amino acid

moieties.

[069] Naturally occurring residues may be divided into classes

based on common side chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, lle; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.

[070] For example, non-conservative substitutions may involve the

exchange of a member of one of these classes for a member from another class.

Such substituted residues may be introduced into regions of the human antibody

that are homologous with non-human antibodies, or into the non-homologous

regions of the molecule.

[071] in making such changes, according to certain embodiments,

the hydropathic index of amino acids may be considered. Each amino acid has

been assigned a hydropathic index on the basis of its hydrophobicity and charge

characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);

phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);

glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3);

proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);

asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[072] The importance of the hydropathic amino acid index in

conferring interactive biological function on a protein is understood in the art.

Kyte et al., J. Mol. BioL, 157:105-131 (1982). It is known that certain amino acids

may be substituted for other amino acids having a similar hydropathic index or

score and still retain a similar biological activity. In making changes based upon

the hydropathic index, in certain embodiments, the substitution of amino acids

whose hydropathic indices are within ±2 is included. In certain embodiments,

those which are within ±1 are included, and in certain embodiments, those within

±0.5 are included.

[073] It is also understood in the art that the substitution of like

amino acids can be made effectively on the basis of hydrophilicity, particularly

where the biologically functional protein or peptide thereby created is intended for

use in immunological embodiments, as in the present case. In certain

embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

[074] The following hydrophilicity values have been assigned to

these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ±I 1);

glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine

(0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine(

1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine(

2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based upon

similar hydrophilicity values, in certain embodiments, the substitution of amino

acids whose hydrophilicity values are within ±2 is included, in certain

embodiments, those which are within ±1 are included, and in certain

embodiments, those within ±0.5 are included. One may also identify epitopes

from primary amino acid sequences on the basis of hydrophilicity. These regions

are also referred to as "epitopic core regions."

[075] Exemplary amino acid substitutions are set forth in Table 1.

Table 1: Amino Acid Substitutions Original Exemplary Preferred Residues Substitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, GIn, Asn Lys Asn Gin Gin Asp Glu Glu Cys Ser, Ala Ser Gin Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, GIn, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe, Norleucine

Leu Norleucine, lie, lie Val, Met, Ala, Phe Lys Arg, 1,4 Diamino- Arg butyric Acid, Gin, Asn Met Leu, Phe, lie Leu Phe Leu, Val, lie, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val lie, Met, Leu, Phe, Leu Ala, Norleucine

[076] A skilled artisan will be able to determine suitable variants of

the polypeptide as set forth herein using well-known techniques. In certain

embodiments, one skilled in the art may identify suitable areas of the molecule

that may be changed without destroying activity by targeting regions not believed

to be important for activity. In certain embodiments, one can identify residues

and portions of the molecules that are conserved among similar polypeptides. In

certain embodiments, even areas that may be important for biological activity or

for structure may be subject to conservative amino acid substitutions without

destroying the biological activity or without adversely affecting the polypeptide

structure.

[077] Additionally, one skilled in the art can review structure

function studies identifying residues in similar polypeptides that are important for

activity or structure. In view of such a comparison, one can predict the

importance of amino acid residues in a protein that correspond to amino acid

residues which are important for activity or structure in similar proteins. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted important amino acid residues.

[078] One skilled in the art can also analyze the three-dimensional

structure and amino acid sequence in relation to that structure in similar

polypeptides. In view of such information, one skilled in the art may predict the

alignment of amino acid residues of an antibody with respect to its three

dimensional structure. In certain embodiments, one skilled in the art may choose

not to make radical changes to amino acid residues predicted to be on the

surface of the protein, since such residues may be involved in important

interactions with other molecules. Moreover, one skilled in the art may generate

-testvariants containing a single amino acid substitution at each desired amino

acid residue. The variants can then be screened using activity assays known to

those skilled in the art. Such variants could be used to gather information about

suitable variants. For example, if one discovered that a change to a particular

amino acid residue resulted in destroyed, undesirably reduced, OF unsuitable

activity, variants with such a change may be avoided. In other words, based on

information gathered from such routine experiments, one skilled in the art can

readily determine the amino acids where further substitutions should be avoided

either alone or in combination with other mutations.

[079] A number of scientific publications have been devoted to the

prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422

427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et aL.,

Biochemistry, 113(2):211-222 (1974); Chou et aL., Adv. EnzymoL. Reat. Areas

Mo. Biol, 47:45-148 (1978); Chou et aL., Ann. Rev. Biochem., 47:251-276 and

Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer programs are

currently available to assist with predicting secondary structure. One method of

predicting secondary structure is based upon homology modeling. For example,

two polypeptides or proteins which have a sequence identity of greater than 30%,

or similarity greater than 40% often have similar structural topologies. The recent

growth of the protein structural database (PDB) has provided enhanced

predictability of secondary structure, including the potential number of folds within

a polypeptide's or protein's structure. See Holm et al., Nucl. Acid. Res.,

27(1):244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct.

Biol, 7(3):369-376 (1997)) that there are a limited number of folds in a given

polypeptide or protein and that once a critical number of structures have been

resolved, structural prediction will become dramatically more accurate.

[080] Additional methods of predicting secondary structure include

"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); SippI et al.,

Structure, 4(1):15-19 (1996)), "profile analysis" (Bowie et aL., Science, 253:164

170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al.,

Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage" (See

Holm, supra (1999), and Brenner, supra (1997)).

[081] In certain embodiments, antibody variants include

glycosylation variants wherein the number and/or type of glycosylation site-has

been altered compared to the amino acid sequences of the parent polypeptide.

In certain embodiments, protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protien. An N-linkedglycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of anN-linked carbohydrate chain.

Alternatively, substitutions which eliminate this sequence will remove an existing

N-linked carbohydrate chain. Also provided is a rearrangement ofN-linked

carbohydrate chains wherein one or moreN-linked glycosylation sites (typically

those that are naturally occurring) are eliminated and one or more newN-linked

sites are created. Additional preferred antibody variants include cysteine variants

wherein one or more cysteine residues are deleted from or substituted for

another amino acid (e.g., serine) as compared to the parent amino acid

sequence. Cysteine variants may be useful when antibodies must be refolded

into a biologically active conformation such as after the isolation of insoluble

inclusion bodies. Cysteine variants generally have fewer cysteine residues than

the native protein, and typically have an even number to minimize interactions

resulting from unpaired cysteines.

[082] According to certain embodiments, amino acid substitutions

are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility

to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter

binding affinities, and/or (4) confer or modify other physiocochemical or functional

properties on such polypeptides. According to certain embodiments, single or

multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). In certain embodiments, a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins,

Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and

Company, New York (1984)); Introduction to Protein Structure (C. Branden and

J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at.

Nature 354:105 (1991), which are each incorporated herein by reference.

[083] The term "polypeptide fragment" as used herein refers to a

polypeptide that has an amino-terminal and/or carboxy-terminal deletion. In

certain embodiments, fragments are at least 5 to 467 amino acids long. It will be

appreciated that in certain embodiments, fragments are at least 5, 6, 8, 10, 14,

, 50, 70, 100, 150, 200, 250, 300, 350, 400, or 450 amino acids long.

[084] Peptide analogs are commonly used in the pharmaceutical

industry as non-peptide drugs with properties analogous to those of the template

peptide. These types of non-peptide compound are termed "peptide mimetics" or

"peptidomimetics". Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and

Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987),

which are incorporated herein by reference for any purpose. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from: --CH 2 NH--, --CH 2 S--, --CH 2 -CH 2 -- , --CH=CH-(cis and trans), -- COCH 2 -- , --CH(OH)CH 2 --, and --CH 2 SO--, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L lysine) may be used inc ertain embodiments to generate more stable peptides.

In addition, constrained peptides comprising a consensus sequence or a

substantially identical consensus sequence variation may be generated by

methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992),

incorporated herein by reference for any purpose); for example, by adding

internal cysteine residues capable of forming intramolecular disulfide bridges

which cyclize the peptide.

[085] "Antibody" or "antibody peptide(s)" refer to an intact

antibody, or a binding fragment thereof that competes with the intact antibody for

specific binding. In certain embodiments, binding fragments are produced by

recombinant DNA techniques. In certain embodiments, binding fragments are

produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single-chain antibodies.

[086] The term "heavy chain" includes any polypeptide having

sufficient variable region sequence to confer specificity for an OPGL. The term

"light chain" includes any polypeptide having sufficient variable region-sequence

to confer specificity for an OPGL. A full-length heavy chain includes a variable

region domain, VH, and three constant region domains, CHI, CH2, and OH 3 . The

VH domain is at the amino-terminus of the polypeptide, and the CH 3 domain is at

the carbody-terminus. The term "heavy chain", as used herein, encompasses a

full-length heavy chain and fragments thereof. A full-length light chain includes a

variable region domain, VL, and a constant region domain, CL. Like the heavy

chain, the variable region domain of the light chain is at the amino-terminus of

the polypeptide. The term "light chain", as used herein, encompasses a full

length light chain and fragments thereof. A Fab fragment is comprised of one

light chain and the CHi and variable regions of one heavy chain. The heavy

chain of a Fab molecule cannot form a disulfide bond with another heavy chain

molecule. A Fab'fragment contains one light chain and one heavy chain that

contains more of the constant region;between the COH and CH 2 domains, such

that an interchain disulfide bond can be formed between two heavy chains to

form a F(ab')2 molecule. The Fv region comprises the variable regions from both

the heavy and light chains, but lacks the constant regions. Single-chain

antibodies are Fv molecules in which the heavy and light chain variable regions

have been connected by a flexible linker to form a single polypeptide chain which forms aantigen-binding region. Single chain antibodies are discussed indetail in WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.

[087] A bivalent antibody other than a "multispecific" or "multifunctional" antibody, in certain embodiments, typically is understood to

have each of its binding sites identical.

[088] An antibody substantially inhibits adhesion of a ligand to a receptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60%, 80%, 85%, or more (as measured in an in vitro competitive binding assay).

[089] The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In certain embodiments, an antibody is said to specifically bind an antigen when the

dissociation constant is 51 M, in certain embodiments, when the dissociation

constant is 100 nM, and in certain embodiments, when the dissociation constant is 510 nM.

[090] The term "agent" is used herein to denote a chemical

compound, a mixture of chemical compounds, a biological macromolecule, or an

extract made from biological materials.

[091] As used herein, the terms "label" or "labeled" refers to

incorporation of a detectable marker, e.g., by incorporation of a radiolabeled

amino acid or attachment to a polypeptide of biotin moieties that can be detected

by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic

activity that can be detected by optical or colorimetric methods). In certain

embodiments, the label or marker can also be therapeutic. Various methods of

labeling polypeptides and glycoproteins are known in the art and may be used.

Examples of labels for polypeptides include, but are not limited to, the following:

radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In,

125 1, 131 1), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),

enzymatic labels (e.g., horseradish peroxidase, p-galactosidase, luciferase,

alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined

polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper

pair sequences, binding sites for secondary antibodies, metal binding domains,

epitope tags). In certain embodiments, labels are attached by spacer arms of

various lengths to reduce potential steric hindrance.

[092] The term "biological sample", as used herein, includes, but is

not limited to, any quantity of a substance from a living thing or formerly living

thing. Such living things include, but are not limited to, humans, mice, monkeys,

rats, rabbits, and other animals. Such substances include, but are not limited to, blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph nodes, and skin.

[093] The term "osteopenic disorder" includes, but is not limited to, osteoporosis, osteopenia, Paget's disease, lytic bone metastases, periodontitis,

rheumatoid arthritis, and bone loss due to immobilization. In addition to these

bone disorders, certain cancers are known to increase osteoclast activity and

induce bone resorption, such as breast, prostate, and multiple myeloma. These

cancers are now known to produce factors that result in the over-expression of

OPGL in the bone, and lead to increased osteoclast numbers and activity.

[094] The term "pharmaceutical agent or drug" as used herein

refers to a chemical compound or-composition capable of inducing a desired

therapeutic effect when properly administered to a patient.

[095] The term "modulator," as used herein, is a compound that

changes or alters the activity or function of a molecule. For example, a

modulator may cause an increase or decrease in the magnitude of a certain

activity or function of a molecule compared to the magnitude of the activity or

function observed in the absence of the modulator. In certain embodiments, a

modulator is an inhibitor, which decreases the magnitude of at least one activity

or function of a molecule. Certain exemplary activities and functions of a

molecule include, but are not limited to, binding affinity, enzymatic activity, and

signal transduction. Certain exemplary inhibitors include, but are not limited to,

proteins, peptides, antibodies, peptibodies, carbohydrates or small organic

molecules. Peptibodies are described, e.g., in WO01/83525.

[096] As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more

abundant than any other individual species in the composition). In certain

embodiments, a substantially purified fraction is a composition wherein the object

species comprises at least about 50 percent (on a molar basis) of all

macromolecular species present. In certain embodiments, a substantially pure

composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all

macromolar species present in the composition. In certain embodiments, the

object species is purified to essential homogeneity (contaminant species cannot

be detected in the composition by conventional detection methods) wherein the

composition consists essentially of a single macromolecular species.

[097] The term patient includes human and animal subjects.

[098] In this application, the use of the singular includes the plural

unless specifically stated otherwise. In this application, the use of "or" means

"and/or" unless stated otherwise. Furthermore, the use of the term "including",

as well as other forms, such as "includes" and "included", is not limiting. Also,

terms such as "element" or "component" encompass both elements and

components comprising one unit and elements and components that comprise

more than one subunit unless specifically stated otherwise.

[099] Osteoprotegerin Ligand (OPGL), a member of the tumor

necrosis factor (TNF) family of cytokines, is involved in the formation of

osteoclasts. Increased osteoclast activity correlates with a number of osteopenic disorders, including post-menopausal osteoporosis, Paget's disease, lytic bone metastases, and rheumatoid arthritis. Therefore, a reduction in OPGL activity may result in a decrease in osteoclast activity and may reduce the severity of osteopenic disorders. According to certain embodiments of the invention, antibodies directed to OPGL may be used treat osteopenic disorders, including by not limited to, those mentioned above.

[0100] In certain embodiments of the present invention, there-is provided a fully human monoclonal antibody against human osteoprotegerin ligand (OPGL). In certain embodiments, nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to the variable regions are provided. In certain embodiments, sequences corresponding to complementarity determining regions (CDR's), specifically from CDR1 through CDR3, are provided. According to certain embodiments, a hybridoma cell line expressing such an immunoglobulin molecule and monoclonal antibody is also provided. In certain embodiments, purified human monoclonal antibody against human OPGL is provided.

[0101] The ability to clone and reconstruct megabase-sized human loci in yeast artificial chromosomes (YACs) and to introduce them into the mouse germline provides an approach to elucidating the functional components of very large or crudely mapped loci as well as generating useful models of human disease. Furthermore, the utilization of such technology for substitution of mouse loci with their human equivalents could provide unique insights into the expression and regulation of human gene products duringdevelopment, their communication with other systems, and their involvement in disease induction and progression.

[0102] An important practical application of such a strategy is the

"humanization" of the mouse humoral immune system. Introduction of human

immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been

inactivated offers the opportunity to study the mechanisms underlying

programmed expression and assembly of antibodies as well as their role in B-cell

development. Furthermore, such a strategy could provide a source for

production of fully human monoclonal antibodies (MAbs). In certain

embodiments, fully human antibodies are expected to minimize the immunogenic

and allergic responses intrinsic to mouse or mouse-derivatized Mabs, and thus,

in certain embodiments, increase the efficacy and safety of the administered

antibodies. In certain embodiments, fully human antibodies may be used in the

treatment of chronic and recurring human diseases, such as osteoporosis,

inflammation, autoimmunity, and cancer, which may involve repeated antibody

administrations.

[0103] One can engineer mouse strains deficient in mouse antibody

production with large fragments of the human Ig loci in anticipation that such

mice would produce human antibodies in the absence of mouse antibodies.

Large human Ig fragments may preserve the large variable gene diversity as well

as the proper regulation of antibody production and expression. By exploiting the

mouse machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, thereproduced human antibody repertoire in these mouse strains may yield high affinity antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen-specific human MAbs with the desired specificity may be produced and selected.

[0104] In certain embodiments, one may use constant regions from

species other than human along with the human variable region(s).

Naturally Occurring Antibody Structure

[0105] Naturally occurring antibody structural units typically

comprise a tetramer. Each such tetramer typically is composed of two identical

pairs of polypeptide chains, each pair having one full-length "light" (in certain

embodiments, about 25 kDa) and one full-length "heavy" chain (in certain

embodiments, about 50-70 kDa). The amino-terminal portion of each chain

typically includes a variable region of about 100 to 110 or more amino acids that

typically is responsible for antigen recognition. The carboxy-terminal portion of

each chain typically defines a constant region that may be responsible for

effector function. Human light chains are typically classified as kappa and

lambda light chains. Heavy chains are typically classified as mu, delta, gamma,

alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and

IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1,

IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1

and IgM2. IgA is similarly subdivided into subclasses including, but not limited to,

IgAl andl gA2. Within full-length light and heavy chains, typically, 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 10 more amino acids.

See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,

N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The

variable regions of each light/heavy chain pair typically form the antigen binding

site.

[0106] The variable- regions typically exhibit the same general

structure of relatively conserved framework regions (FR) joined by three hyper

variable regions, also called complementarity determining regions or CDRs. The

CDRs from the two chains of each pair typically are aligned by the framework

regions, which may enable binding to a specific epitope. From N-terminal to C

terminal, both light and heavy chain variable regions typically comprise the

domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of

amino acids to each domain is typically in accordance with the definitions of

Kabat Sequences of Proteins ofImmunological Interest (National Institutes of.

Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.

196:901-917 (1987); Chothia et aL Nature 342:878-883 (1989).

Bispecific or Bifunctional Antibodies

[0107] A bispecific or bifunctional antibody typically is an artificial

hybrid antibody having two different heavy/light chain pairs and two different

binding sites. Bispecific antibodies may be produced by a variety of methods including, but notlimited to, fusion of hybridomas orlinking of Fab' fragments.

See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990),

Kostelny et al. J. Immunol. 148:1547-1553 (1992).

Preparation of Antibodies

[0108] According to certain embodiments, certain antibodies

specifically binding to OPGL are encompassed by the invention. In certain

embodiments, the antibodies may be produced by immunization with full-length

OPGL, soluble forms of OPGL, or a fragment thereof. In certain embodiments,

the antibodies of the invention may be polyclonal or monoclonal, and/or may be

recombinant antibodies. In certain embodiments, antibodies of the invention are

human antibodies prepared, for example, by immunization of transgenic animals

capable of producing human antibodies (see, for example, PCT Published

Application No. WO 93/12227).

[0109] In certain embodiments, the complementarity determining

regions (CDRs) of the light and heavy chain variable regions of aOPGL-1 may be

grafted to framework regions (FRs) from the same, or another, species. In

certain embodiments, the CDRs of the light and heavy chain variable regions of

aOPGL-1 may be grafted to consensus human FRs. To create consensus

human FRs, in certain embodiments, FRs from several human heavy chain or

light chain amino acid sequences are aligned to identify a consensus amino acid

sequence. In certain embodiments, the FRs of the aOPGL-1 heavy chain or light

chain are replaced with the FRs from a different heavy chain or light chain. In certain embodiments, rare amino acids in the FRs of the heavy and light chains of aOPGL-1 are not replaced, while the rest of the FR amino acids are replaced.

Rare amino acids are specific amino acids that are in positions in which they are

not usually found in FRs. In certain embodiments, the grafted variable regions

from aOPGL-1 may be used with a constant region that is different from the

constant region of aOPGL-1. In certain embodiments, the grafted variable

regions are part of a single chain Fv antibody. CDR grafting is described, e.g., in

U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101,

which are hereby incorporated by reference for any purpose.

[0110] According to certain embodiments, antibodies of the

invention are prepared through the utilization of a transgenic mouse that has a

substantial portion of the human antibody producing genome inserted but that is

rendered deficient in the production of endogenous, murine, antibodies. Such

mice, then, are capable of producing human immunoglobulin molecules and

antibodies and are deficient in the production of murine immunoglobulin

molecules and antibodies. Technologies utilized for achieving this result are

disclosed in the patents, applications, and references disclosed in the

specification, herein. In certain embodiments, one may employ methods such as

those disclosed in PCT Published Application No. WO 98/24893, which is hereby

incorporated by reference for any purpose. See also Mendez et al. Nature

Genetics 15:146-156 (1997), which is hereby incorporated by reference for any

purpose.

[0111] According to certain embodiments, fully human monoclonal

antibodies specific for OPGL are produced as follows. Transgenic mice

containing human immunoglobulin genes are immunized with the antigen of

interest. Lymphatic cells (such as B-cells) from the mice that express antibodies

are obtained. Such recovered cells are fused with a myeloid-type cell line to

prepare immortal hybridoma cell lines, and such hybridoma cell lines are

screened and selected to identify hybridoma cell lines that produce antibodies

specific to the antigen of interest. In certain embodiments, the production of a

hybridoma cell line that produces antibodies specific to OPGL is provided.

[0112] In certain embodiments, antibodies of the invention are

produced by hybridoma lines AMG 6.1, AMG 6.4, AMG 6.5, AMG 7.1, and AMG

7.2. In certain embodiments, antibodies of the invention are produced by

hybridoma lines AMG 6.1, AMG 6.4, and AMG 6.5. In certain embodiments, the

antibodies of the invention bind to OPGL with a dissociation constant (Kd) of

between approximately 0.23 and 0.29 nM. In certain embodiments of the

invention, the antibodies bind to OPGL with a Kd of less than 0.23 nM.

[0113] In certain embodiments, the antibodies of the invention are

of the IgG2 isotype. In certain embodiments of the invention, the antibodies

comprise a human kappa light chain and a human IgG2 heavy chain. In certain

embodiments, the antibodies of the invention have been cloned for expression in

mammalian cells. In certain embodiments, the variable regions of the antibodies

are ligated to a constant region other than the constant region for the IgG2

isotype.

[0114] In certain embodiments, conservative modifications to the

heavy and light chains of aOPGL-1 (and corresponding modifications to the

encoding nucleotides) will produce antibodies to OPGL having functional and

chemical characteristics similar to those of aOPGL-1. In contrast, substantial

modifications in the functional and/or chemical characteristics of aOPGL-1 may

be accomplished by selecting substitutions in the amino acid sequence of the

heavy and light chains that differ significantly in their effect on maintaining (a) the

structure of the molecular backbone in the area of the substitution, for example,

as a sheet or helical conformation, (b) the charge or hydrophobicity of the

molecule at the target site, or (c) the bulk of the side chain.

[0115] For example, a "conservative amino acid substitution" may

involve a substitution of a native amino acid residue with a nonnative residue

such that there is little or no effect on the polarity or charge of the amino acid

residue at that position. Furthermore, any native residue in the polypeptide may

also be substituted with alanine, as has been previously described for "alanine

scanning mutagenesis."

[0116] Desired amino acid substitutions (whether conservative or

non-conservative) can be determined by those skilled in the art at the time such

substitutions are desired. In certain embodiments, amino acid substitutions can

be used to identify important residues of aOPGL-1, or to increase or decrease

the affinity of the antibodies to OPGL described herein.

[0117] In certain embodiments, antibodies of the present invention

can be expressed in cell lines other than hybridoma cell lines. In certain embodiments, sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell. According to certain embodiments, transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455

(which patents are hereby incorporated herein by reference for any purpose). In

certain embodiments, the transformation procedure used may depend upon the

host to be transformed. Methods for introduction of heterologous polynucleotides

into mammalian cells are well known in the art and include, but are not limited to,

dextran-mediated transfection, calcium phosphate precipitation, polybrene

mediated transfection, protoplast fusion, electroporation, encapsulation of the

polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

[0118] Mammalian cell lines available as hosts for expression are

well known in the art and include, but are not limited to, many immortalized cell

lines available from the American Type Culture Collection (ATCC), including but

not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster

kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma

cells (e.g., Hep G2), and a number of other cell lines. In certain embodiments,

cell lines may be selected through determining which cell lines have high

expression levels and produce antibodies with constitutive OPGL binding

properties.

rll aocran nibde

[L019 According to certain embodiments, antibodies of the

invention are useful for detecting OPGL in biological samples. In certain

embodiments, this allows the identification of cells or tissues which produce the

protein. In certain embodiments, antibodies which bind to OPGL and block

interaction with other binding compounds may have therapeutic use in

modulating osteoclast differentiation and bone resorption. In certain

embodiments, antibodies to OPGL may block OPGL binding to ODAR, which

may result in a block in the signal transduction cascade and loss of NF-kB

mediated transcription activation. Assays for measuring NF-kB-mediated

transcription activation using, e.g., a luciferase reporter assay, are known to

those skilled in the art.

[0120] In certain embodiments, methods are provided of treating a

bone disorder comprising administering a therapeutically effective amount of an

antibody to OPGL. In certain embodiments, methods ar provided of treating a

bone disorder comprising administering a therapeutically effective amount of an

antibody to OPGL and another therapeutic agent. In certain such embodiments,

the additional therapeutic agent is administered in a therapeutically effective

amount. In certain embodiments, the bone disorder is a disorder characterized

by a net bone loss, including but not limited to, osteopenia and osteolysis. In

certain embodiments, treatment with an antibody to OPGL is used to suppress

the rate of bone resorption. Therefore, in certain embodiments, treatment may

be used to reduce the rate of bone resorption where the resorption rate is above

normal, or to reduce bone resorption to below normal levels in order to compensate for below normal levels of bone formation. In certain embodiments, antibodies can be tested for binding to OPGL in the absence or presence of OPG and examined for their ability to inhibit OPGL-mediated osteclastogenesis and/or bone resorption.

[0121] Conditions which may be treated according to certain

embodiments include, but are not limited to, the following:

Osteoporosis, including, but not limited to, primary osteoporosis,

endocrine osteoporosis (including, but not limited to, hyperthyroidism,

hyperparathyroidism, Cushing's syndrome, and acromegaly), hereditary

and congenital forms of osteoporosis (including, but not limited to,

osteogenesis imperfecta, homocystinuria, Menkes' syndrome, Riley-Day

syndrome), and osteoporosis due to immobilization of extremities;

Paget's disease'of bone (osteitis deformans) in adults and

juveniles;

Osteomyelitis, i.e., an infectious lesion in bone, leading to bone

loss;

Hypercalcemia, including, but not limited to, hypercalcemia

resulting from solid tumors (including, but not limited to, breast, lung and

kidney) and hematologic malignacies (including, but not limited to, multiple

myeloma, lymphoma and leukemia), idiopathic hypercalcemia, and

hypercalcemia associated with hyperthyroidism and renal function

disorders;

Osteopenia, including but not limited to, osteopenia following

surgery, osteopenia induced by steroid administration, osteopenia

associated with disorders of the small and large intestine, and osteopenia

associated with chronic hepatic and renal diseases;

Osteonecrosis, i.e., bone cell death, including, but not limited to,

osteonecrosis associated with traumatic injury, osteonecrosis associated

with Gaucher's disease, osteonecrosis associated with sickle cell anemia,

osteonecrosis associated with systemic lupus erythematosus,

osteonecrosis associated with rheumatoid arthritis, osteonecrosis

associated with periodontal disease, osteonecrosis associated with

osteolytic metastasis, and osteonecrosis associated with other condition;

and

Loss of cartilage and joint erosion associated with rheumatoid

arthritis.

[0122] In certain embodiments, an antibody to OPGL may be used

alone or with at least one additional therapeutic agents for the treatment of bone

disorders. In certain embodiments, an antibody to OPGL is used in conjunction

with a therapeutically effective amount of an additional therapeutic agent.

Exemplary therapeutic agents that may be administered with an antibody to

OPGL include, but are not limited to, the bone morphogenic factors designated

BMP-1 through BMP-12; transforming growth factor-P (TGF-p) and TGF-p family

members; interleukin-1 (IL-1) inhibitors, including, but not limited to, IL-1ra and

derivatives thereof and KineretTM; TNFa inhibitors, including, but not limited to, solubleTNFreceptors, EnbrelTM, anti-TNFa antibodies, RemicadeTM, and D2E7 antibodies; parathyroid hormone and analogs thereof; parathyroid related protein and analogs thereof; E series prostaglandins; bisphosphonates (such as alendronate and others); bone-enhancing minerals such as fluoride and calcium; non-steroidal anti-inflammatory drugs (NSAIDs), including, but not limited to,

COX-2 inhibitors, such as CelebrexTM and Vioxx TM; immunosuppressants, such as

methotrexate or leflunomide; serine protease inhibitors, including, but not limited

to, secretory leukocyte protease inhibitor (SLPI); IL-6 inhibitors (including, but not

limited to, antibodies to IL-6), IL-8 inhibitors (including, but not limited to,

antibodies to IL-8); IL-18 inhibitors (including, but not limited to, IL-18 binding

protein and IL-18 antibodies); Interleukin-1 converting enzyme (ICE) modulators;

fibroblast growth factors FGF-1 to FGF-10 and FGF modulators; PAF

antagonists; keratinocyte growth factor (KGF), KGF-related molecules, and KGF

modulators; matrix metalloproteinase (MMP) modulators; Nitric oxide synthase

(NOS) modulators, including, but not limited to, modulators of inducible NOS;

modulators of glucocorticoid receptor; modulators of glutamate receptor;

modulators of lipopolysaccharide (LPS) levels; and noradrenaline and

modulators and mimetics thereof.

[0123] In certain embodiments, an antibody to OPGL is used with

particular therapeutic agents to treat various inflammatory conditions,

autoimmune conditions, or other conditions with attendant bone loss. In certain

embodiments, in view of the condition and the desired level of treatment, two,

three, or more agents may be administered. In certain embodiments, such agents may be provided together byinclusion in the same formulation. In certain embodiments, such agents and an antibody to OPGL may be provided together by inclusion in the same formulation. In certain embodiments, such agents may be provided together by inclusion in a treatment kit. In certain embodiments, such agents and an antibody to OPGL may be provided together by inclusion in a treatment kit. In certain embodiments, such agents may be provided separately. In certain embodiments, when administered by gene therapy, the genes encoding protein agents and/or an antibody to OPGL may be included in the same vector. In certain embodiments, the genes encoding protein agents and/or an antibody to OPGL may be under the control of the same promoter region. In certain embodiments, the genes encoding protein agents and/or an antibody to OPGL may be in separate vectors.

[0124] In certain embodiments, the present invention is directed to therapies comprising an antibody to OPGL and at least one interleukin-1 (IL-1) inhibitor, and methods of treatment using such therapies. In certain embodiments, a therapy comprises an antibody to OPGL and an IL-1 inhibitor and at least one additional molecule described herein. In certain embodiments, methods of treatment use IL-1 inhibitors and/or TNF-q inhibitors in conjunction with an antibody to OPGL. In certain embodiments, an antibody to OPGL in combination with IL-1 inhibitors and/or TNF-q inhibitors may be used for treatment of conditions such as asthma, rheumatoid arthritis, and multiple sclerosis.

[0125] Interleukin-1 (IL-1) is an anti-inflammatory cytokine. In

certain instances, IL-1 is a mediator in many diseases andmedical conditions. In

certain instances, IL-1 is manufactured by cells of the macrophage/monocyte

lineage. In certain instances, IL-1 is produced in two forms: IL-1 alpha (IL-1a)

and IL-1 beta (IL-1p).

[0126] A disease or medical condition is considered to be an

"interleukin-1 mediated disease" if the spontaneous or experimental disease or

medical condition is associated with elevated levels of IL-1 in bodily fluids or

tissue and/or if cells or tissues taken from the body produce elevated levels of IL

1 in culture. In certain embodiments, such interleukin-1 mediated diseases are

also recognized by the following additional two conditions: (1) pathological

findings associated with the disease or medical condition can be mimicked

experimentally in animals by administration of IL-1 or upregulation of expression

of IL-1; and (2) a pathology induced in experimental animal models of the

disease or medical condition can be inhibited or abolished by treatment with

agents that inhibit the action of IL-1. In certain embodiments, one or more of the

above conditions are met in an IL-1-mediated disease. In certain embodiments,

all three of the conditions are met in an IL-1-mediated disease.

[0127] Acute and chronic interleukin-1 (IL-1) -mediated diseases

include, but are not limited to, the following: acute pancreatitis; amyotrophic

lateral sclerosis (ALS, or Lou Gehrig's disease); Alzheimer's disease;

cachexia/anorexia, including, but not limited to, AIDS-induced cachexia; asthma

and other pulmonary diseases; atherosclerosis; autoimmune vasculitis; chronic fatigue syndrome; Clostridium associated illnesses, including, but not limited to,

Clostridium-associated diarrhea; coronary conditions and indications, including,

but not limited to, congestive heart failure, coronary restenosis, myocardial

infarction, myocardial dysfunction (e.g., related to sepsis), and coronary artery

bypass graft; cancer, including, but not limited to, leukemias, including, but not

limited to, multiple myeloma leukemia and myelogenous (e.g., AML and CML),

and tumor metastasis; diabetes (including, but not limited to, insulin-dependent

diabetes); endometriosis; fever; fibromyalgia; glomerulonephritis; graft versus

host disease and/or transplant rejection; hemohorragic shock; hyperalgesia;

inflammatory bowel disease; inflammatory conditions of a joint, including, but not

limited to, osteoarthritis, psoriatic arthritis, and rheumatoid arthritis; inflammatory

eye disease, including, but not limited to, those associated with, for example,

corneal transplant; ischemia, including, but not limited to, cerebral ischemia

(including, but not limited to, brain injury as a result of, e.g., trauma, epilepsy,

hemorrhage or stroke, each of which may lead to neurodegeneration);

Kawasaki's disease; learning impairment; lung diseases (including, but not

limited to, acute respiratory distress syndrome, or ARDS); multiple sclerosis;

myopathies (e.g., muscle protein metabolism, including, but not limited to, muscle

protein metabolism in sepsis); neurotoxicity (including, but not limited to, such

condition induced by HIV); osteoporosis; pain, including, but not limited to,

cancer-related pain; Parkinson's disease; periodontal disease; pre-term labor;

psoriasis; reperfusion injury; septic shock; side effects from radiation therapy;

temporal mandibular joint disease; sleep disturbance; uveitis; and an inflammatory condition resulting from, e.g., strain, sprain, cartilage damage, trauma, orthopedic surgery, infection, or other disease processes.

[0128] In certain embodiments, an IL-1 inhibitor may be any protein

or molecule capable of specifically preventing activation of cellular receptors to

IL-1, which may result from any number of mechanisms. Exemplary

mechanisms include, but are not limited to, downregulating IL-1 production,

binding free IL-1, interfering with IL-1 binding to its receptor, interfering with

formation of the IL-1 receptor complex (i.e., association of IL-1 receptor with IL-1

receptor accessory protein), and interfering with modulation of IL-1 signaling after

binding to its receptor.

[0129] Certain interleukin-1 inhibitors include, but are not limited to,

IL-1 receptor antagonists, including, but not limited to, Kineret TM, IL-1ra, IL-1ra

variants, and IL-1ra derivatives, which are collectively termed "IL-1ra proteins;"

anti-IL-1 receptor monoclonal antibodies (see, e.g., EP 623674, which is hereby

incorporated by reference for any purpose); IL-1 binding proteins, including, but

not limited to, soluble IL-1 receptors (see, e.g., U. S. Pat. No. 5,492,888, U. S.

Pat. No. 5,488,032, and U. S. Pat. No. 5,464,937, U. S. Pat. No. 5,319,071, and

U.S. Pat. No. 5,180,812, which are hereby incorporated by reference for any

purpose); anti-IL-1 monoclonal antibodies (see, e.g., WO 9501997, WO

9402627, WO 9006371, U.S.Pat. No. 4,935,343, EP 364778, EP 267611 and EP

220063, which are hereby incorporated by reference for any purpose); IL-1

receptor accessory proteins and antibodies thereto (see, e.g., WO 96/23067 and

WO 99/37773, which are hereby incorporated by reference for any purpose); inhibitors of interleukin-1 beta converting enzyme (ICE) or caspase I (see, e.g.,

WO 99/46248, WO 99/47545, and WO 99/47154, which are hereby incorporated

by reference for any purpose), which may be used to inhibit IL-1 beta production

and secretion; interleukin-1beta protease inhibitors; and other compounds and

proteins that block in vivo synthesis or extracellular release of IL-.

[0130] Exemplary IL-1 inhibitors are disclosed, e.g., in US Pat. Nos.

,747,444; 5,359,032; 5,608,035; 5,843,905; 5,359,032; 5,866,576; 5,869,660;

,869,315; 5,872,095; 5,955,480; 5,965,564; International (WO) patent

applications 98/21957, 96/09323, 91/17184, 96/40907, 98/32733, 98/42325,

98/44940, 98/47892, 98/56377, 99/03837, 99/06426, 99/06042, 91/17249,

98/32733, 98/17661, 97/08174, 95/34326, 99/36426, 99/36415;. European (EP)

patent applications 534978 and 894795; and French patent application FR

2762514. The disclosures of all of the aforementioned references are hereby

incorporated by reference for any purpose.

[0131] Interleukin-1 receptor antagonist (IL-1ra) is a human protein

that acts as a natural inhibitor of interleukin-1 and is a member of the IL-1 family,

which includes IL-1a and IL-1p. Certain receptor antagonists, including IL-1ra

and variants and derivatives thereof, as well as methods of making and using

them, are described in U.S. Patent No. 5,075,222; WO 91/08285; WO 91/17184;

AU 9173636; WO 92/16221; WO 93/21946; WO 94/06457; WO 94/21275; FR

2706772; WO 94/21235; DE 4219626, WO 94/20517; WO 96/22793;WO

97/28828; and WO 99/36541, which are incorporated herein by reference for any

purpose. In certain embodiments, an IL-1 receptor antagonist may be glycosylated. In certain embodiments, an !L-1 receptor antagonist may be non glycosylated.

[0132] Three forms of IL-1ra and variants thereof are described in

U.S. Pat. No. 5,075,222 (the'222 patent). The first form, called "L-1i" in the'222

patent, is characterized as a 22-23 kD molecule on SDS-PAGE with an

approximate isoelectric point of 4.8, which elutes from a Mono Q FPLC column at

around 52 mM NaCl in Tris buffer, pH 7.6. The second form, IL-1rap, is

characterized as a 22-23 kD protein, which elutes from a Mono Q column at 48

mM NaCl. Both IL-1raa and IL-1rap are glycosylated. The third form, IL-1rax, is

characterized as a 20 kD protein, which elutes from a Mono Q column at 48 mM

NaCl and is non-glycosylated. The'222 patent also describes certain methods

for isolating genes that code for the inhibitors, cloning those genes in suitable

vectors, transforming and transfecting those genes into certain cell types, and

expressing those genes to produce the inhibitors.

[0133] In certain embodiments, deletions, insertions, and/or

substitutions (individually or collectively referred to as "variant(s)") are made

within the amino acid sequences of IL-1ra. In certain embodiments, an IL-1ra

variant is biologically active (e.g., possesses the ability to inhibit IL-1).

[0134] In certain embodiments, the present invention is directed to

therapies comprising an antibody to OPGL and at least one TNFa inhibitor, and

methods of treatment using such therapies. In certain embodiments, a therapy

comprises an antibody to OPGL and a TNFa inhibitor and at least one additional

molecule described herein.

[0135] Certain diseases and medical conditions are mediated by

TNF and may be categorized as inflammatory conditions. As used herein, a

"TNF-mediated disease" includes, but is not limited to, a disease or medical

condition that is associated with elevated levels of TNF in bodily fluids or tissue

and/or in which cells or tissues taken from the body produce elevated levels of

TNF in culture. In certain embodiments, a disease is a TNF-mediated disease if

(1) pathological findings associated with the disease or medical condition can be

mimicked experimentally in animals by the administration or upregulation of

expression of TNF and/or (2) a pathology induced in experimental animal models

of the disease or medical condition can be inhibited or abolished by treatment

with agents that inhibit the action of TNF.

[0136] Certain acute and chronic TNF-mediated diseases include,

but are not limited to: cachexia and anorexia; cancer, including, but not limited to,

leukemia; chronic fatigue syndrome; coronary conditions and/or indications,

including, but not limited to, congestive heart failure, coronary restenosis,

myocardial infarction, myocardial dysfunction (including but not limited to, such

condition related to sepsis), and coronary artery bypass graft; depression;

diabetes, including, but not limited to, juvenile onset Type 1 diabetes, diabetes

mellitus, and insulin resistance (including, but not limited to, insulin resistance

associated with obesity); endometriosis, endometritis, and related conditions;

fibromyalgia and analgesia; graft versus host rejection; hyperalgesia;

inflammatory bowel diseases, including, but not limited to, Crohn's disease and

Clostridium difficile-associated diarrhea; ischemia, including, but not limited to, cerebral ischemia, which includes, but is not limited to, brain injury as a result of trauma, epilepsy, hemorrhage, and/or stroke; lung disease, including, but not limited to, adult respiratory distress syndrome, asthma, and pulmonary fibrosis; multiple sclerosis; neuroinflammatory diseases; ocular diseases and conditions, including, but not limited to, corneal transplant, ocular degeneration and uveitis; pain, including, but not limited to, cancer-related pain; pancreatitis; periodontal diseases; Pityriasis rubra pilaris (PRP); prostatitis, including bacterial and non bacterial prostatitis, and related conditions; psoriasis and related conditions; pulmonary fibrosis; reperfusion injury; rheumatic diseases, including, but not limited to, rheumatoid arthritis, osteoarthritis, juvenile arthritis (including, but not limited to, juvenile rheumatoid arthritis), seronegative polyarthritis, ankylosing spondylitis, Reiter's syndrome and reactive arthritis, Still's disease, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic sclerosis, vasculitis (e.g., Kawasaki's disease), cerebral vasculitis,

Lyme disease, staphylococcal-induced ("septic") arthritis, Sjagren's syndrome,

rheumatic fever, polychondritis and polymyalgia rheumatica and giant cell

arteritis); septic shock; side effects from radiation therapy; systemic lupus

erythematosus (SLE); temporal mandibular joint disease; thyroiditis; and tissue

transplantation and/or an inflammatory condition, e.g., resulting from strain,

sprain, cartilage damage, trauma, orthopedic surgery, infection (e.g., HIV,

Clostridium difficile and related species) or other disease process.

[0137] In certain embodiments, TNF inhibitors may act by at least

one of downregulating or inhibiting TNF production, binding free TNF, interfering with TNF binding to its receptor, and interfering with modulation of TNF signaling after binding to its receptor. The term "TNF inhibitor" includes, but is not limited to, solubilized TNF receptors, including, but not limited to, soluble tumor necrosis factor receptor type I (sTNF-RI; also called the p55 receptor), soluble tumor necrosis factor receptor type II (also called the p75 receptor), and Enbrel TM ; antibodies to TNF, including, but not limited to, Remicade TM and D2E7 (see, e.g.,

U.S. Patent Nos. 6,090,382 and 6,258,562); antibodies to TNF receptor; sTNF-RI

(see, e.g., WO 98/24463), etanercept (Enbre"), Avakineh; inhibitors of TNF-a

converting enzyme (TACE); and other molecules that affect TNF activity.

[0138] Exemplary TNF-a inhibitors are described, e.g., in European

patent applications EP 308 378; EP 422 339; EP 393 438;EP 398 327; EP 412

486; EP 418 014, EP 417 563, EP 433 900; EP 464 533; EP 512 528; EP 526

905; EP 568 928; EP 607 776, which describes the use of leflunomide for

inhibition of TNF-a; EP 663 210; EP 542 795; EP 818 439; EP 664 128; EP 542

795; EP 741 707; EP 874 819 ; EP 882 714; EP 880 970; EP 648 783; EP 731

791; EP 895 988; EP 550 376; EP 882 714; EP 853 083; EP 550 376; EP 943

616; EP 939 121; EP 614 984; EP 853 083; U.S. Patent Nos. 5,136,021;

,929,117; 5,948,638; 5,807,862; 5,695,953; 5,834,435; 5,817,822; 5830742;

,834,435; 5,851,556; 5,853,977; 5,359,037; 5,512,544; 5,695,953; 5,811,261;

,633,145; 5,863,926; 5,866,616; 5,641,673; 5,869,677; 5,869,511; 5,872,146;

,854,003; 5,856,161; 5,877,222; 5,877,200; 5,877,151; 5,886,010; 5,869,660;

,859,207; 5,891,883; 5,877,180; 5,955,480; 5,955,476; 5,955,435; 5,994,351;

,990,119; 5,952,320; 5,962,481; International patent applications WO 90/13575,

WO 91/03553, WO 92/01002, WO 92/13095, WO 92/16221, WO 93/07863, WO

93/21946, WO 93/19777, WO 95/34326, WO 96/28546, WO 98/27298, WO

98/30541, WO 96/38150, WO 96/38150, WO 97/18207, WO 97/15561, WO

97/12902, WO 96/25861, WO 96/12735, WO 96/11209, WO 98/39326, WO

98/39316, WO 98/38859, WO 98/39315, WO 98/42659, WO 98/39329, WO

98/43959, WO 98/45268, WO 98/47863, WO 96/33172, WO 96/20926, WO

97/37974, WO 97/37973, WO 97/47599, WO 96/35711, WO 98/51665, WO

98/43946, WO 95/04045, WO 98/56377, WO 97/12244, WO 99/00364, WO

99/00363, WO 98/57936, WO 99/01449, WO 99/01139, WO 98/56788, WO

98/56756, WO 98/53842, WO 98/52948, WO 98/52937, WO 99/02510, WO

97/43250, WO 99/06410, WO 99/06042, WO 99/09022, WO 99/08688, WO

99/07679, WO 99/09965, WO 99/07704, WO 99/06041, WO 99/37818, WO

99/37625, WO 97/11668, WO 99/50238, WO 99/47672, WO 99/48491;

Japanese patent applications 10147531, 10231285, 10259140, and 10130149,

10316570, 11001481, and 127,800/1991; German application no. 19731521; and

British application nos. 2 218 101, 2 326 881, 2 246 569. The disclosures of all

of the aforementioned references are hereby incorporated by reference for any

purpose.

{0139] EP 393 438 and EP 422 339 describe the amino acid and

nucleic acid sequences of a soluble TNF receptor type I (also known as sTNFR

or 30kDa TNF inhibitor) and a soluble TNF receptor type lI (also known as

sTNFR-II or 40kDa TNF inhibitor), which are collectively termed "sTNFRs". EP

393 438 and EP 422 339 also describe modified forms of sTNFR-l and sTNFR-I, including, but not limited to fragments, functional derivatives, and variants.

Furthermore, EP 393 438 and EP 422 339 describe methods for isolating genes

that code for the inhibitors, cloning the genes into suitable vectors, transforming

or transfecting the genes into certain cell types, and expressing the genes to

produce the inhibitors.

[0140] sTNFR- and sTNFR-II are members of the nerve growth

factor/TNF receptor superfamily of receptors, which includes the nerve growth

factor receptor (NGF), the B cell antigen CD40, 4-1BB, the rat T-cell antigen

MRC OX40, the fas antigen, and the CD27 and CD30 antigens (Smith et al.

(1990) Science, 248:1019-1023). A conserved feature of that group of cell

surface receptors is a cysteine-rich extracellular ligand binding domain, which

can be divided into four repeated motifs of about forty amino acids that contain 4

6 cysteine residues at positions that are well conserved (Smith et al. (1990),

supra).

[0141] EP 393 438 teaches a 40kDa TNF inhibitor A51 and a 40kDa

TNF inhibitor A53, which are truncated versions of the full-length recombinant

kDa TNF inhibitor protein. A51 and A53 have 51 or 53 amino acids,

respectively, deleted from the carboxyl terminus of the mature protein.

[0142] Published PCT Application No. WO 98/01555 describes

truncated forms of sTNFR-I and sTNFR-Il that do not contain the fourth domain 41 (amino acid residues Thr 127-Asn 16 1of sTNFR-l and amino acid residues Pro

Thr 17 9 of sTNFR-I); a portion of the third domain (amino acid residues Asn 111

Cys1 2 6 of sTNFR-1 and amino acid residues Pro 123-Lys 140 of sTNFR-II); and, optionally, do not contain a portion of the first domain (amino acid residues Asp1

Cys19 of sTNFR-I and amino acid residues Leu-Cys 32 of sTNFR-ll). In certain

embodiments, the truncated sTNFRs include the proteins represented by the

formula Rl-[Cys 19-Cys 103]-R2 and R4-[Cys32 -Cys115-R .5 These proteins are

truncated forms of sTNFR-I and sTNFR-l, respectively.

. [0143] As used herein, "R-[Cys1 9-Cys1o 3]-R 2" represents one or

more proteins wherein [Cys1 9 -Cys10 3] is residues 19 through 103 of sTNFR-l, the

sequence of which is provided in Figure 1 of WO 98/01555; wherein R1

represents a methionylated or nonmethionylated amine group of Cys 19 or one or

more amino-terminal amino acid residues selected from Cys18 to Asp1; and

wherein R 2 represents a carboxy group of Cys103 or one or more carboxy

terminal amino acid residues selected from Pheo4 to Leu 110 .

[0144] Exemplary truncated sTNFR-'s of the present invention include, but are not limited to, sTNFR- 2.6D/C105, sTNFR- 2.6D/C106, sTNFR

2.6D/N105, sTNFR-1 2.3D/d8, sTNFR-1 2.3D/d18, sTNFR-1 2.3D/d15, either

methionylated or nonmethionylated, and variants and derivatives thereof.

Certain exemplary truncated sTNFR-1's are described, e.g., in published PCT

Application No. WO 98/01555.

[0145] As used herein, "R3-[Cys 3 2 -Cys11 5]-R4" represents one or

more proteins wherein [Cys 3 2 -Cys11 5] is residues Cys 3 2 through Cys 115 of

sTNFR-II, the sequence of which is provided in Figure 8 of WO 98/01555;

wherein R 3 represents a methionylated or nonmethionylated amine group of

Cys3 2 or one or more amino-terminal amino acid residues selected from Cys 31 to

Leu 1 ; and wherein R4 represents a carboxy group of Cys115 or one or more

carboxy-terminal amino acid residues selected from Ala1 16 to Arg 122

.

[0146] In certain embodiments, the present invention is directed to

therapies comprising an antibody to OPGL and at least one serine protease

inhibitor, and methods of treatment using such therapies. In certain

embodiments, a therapy comprises an antibody to OPGL and a serine protease

inhibitor and at least one additional molecule described herein.

[0147] Endogenous proteolytic enzymes may degrade invading

organisms, antigen-antibody complexes, and certain tissue proteins that are no

longer necessary or useful. Infective agents may introduce additional proteolytic

enzymes into the organism. Protease inhibitors may regulate both endogenous

and invading proteolytic enzymes.

[0148] In certain embodiments, naturally occurring protease

inhibitors serve to control endogenous proteases by limiting their reactions locally

and temporally. In certain embodiments, protease inhibitors may inhibit

proteases introduced into the body by infective agents. In certain instances,

tissues that are particularly prone to proteolytic attack and infection, including,

but not limited to, those of the respiratory tract, are rich in protease inhibitors.

[0149] Protease inhibitors comprise approximately 10% of the

human plasma proteins. At least eight inhibitors have been isolated from this

source and characterized in the literature. These include, but are not limited to,

alpha 2-macroglobulin (alpha 2M), alpha 1-protease inhibitor (alpha 1PI), alpha

1-antichymotrypsin (alpha lAchy), alpha 1-anticollagenase (alpha 1AC), and

inter-alpha-trypsin inhibitor (1-alpha-1).

[0150] In certain instances, a disturbance of the protease/protease

inhibitor balance can lead to protease-mediated tissue destruction, including, but

not limited to, emphysema, arthritis, glomerulonephritis, periodontitis, muscular

dystrophy, tumor invasion, and various other pathological conditions. In certain

situations, e.g. severe pathological processes such as sepsis or acute leukemia,

the amount of free proteolytic enzymes present may increase due to the release

of enzyme from secretory cells.

[0151] Furthermore, in certain instances, a diminished regulating

inhibitor capacity of the organism may also cause alterations in the

protease/protease inhibitor balance. A nonlimiting example of such a diminished

regulating inhibitor capacity is an alpha 1-protease inhibitor deficiency, which is

correlated with the development of pulmonary emphysema.

[0152] In certain instances, serious damage to the organism can

occur when such aberrant conditions are present unless measures can be taken

to control the proteolytic enzymes. Therefore, protease inhibitors have been

sought that can be administered to an organism to control proteolytic enzymes.

[0153] Leukocyte elastase, trypsin, cathepsin G, and pancreatic

elastase are nonlimiting examples of a class of proteases known as serine

proteases.

[0154] In certain instances, leukocyte elastase, when released

extracellularly, degrades connective tissue and other valuable proteins. While a normally functioning organism degrades a certain amount of connective tissue and other proteins, the presence of an excessive amount of leukocyte elastase may be associated with various pathological states, including, but not limited to, emphysema and rheumatoid arthritis. In certain embodiments, to counteract the effects of leukocyte elastase when it is present in amounts greater than normal, a protease inhibitor has been sought which is specific for leukocyte elastase. Such a protease inhibitor may be useful if it were capable of being isolated or prepared in a purified form and in sufficient quantities to be pharmaceutically useful.

[0155] Certain leukocyte elastase inhibitors are described, e.g., in Schiessler et al., "Acid-Stable Inhibitors of Granulocyte Neutral Proteases in

Human Mucous Secretions: Biochemistry and Possible Biological Function", in

Neutral Proteases of Human Polymorphoneuclear Leucocytes, Havemann et al.

(eds), Urban and Schwarzenberg, Inc. (1978), and in Travis and Salvesen, Ann

Rev. Biochem. 52: 655-709 (1983).

[0156] In certain instances, trypsin initiates degradation of certain

soft organ tissue, such as pancreatic tissue, during a variety of acute conditions,

including, but not limited to, pancreatitis. A trypsin inhibitor may be useful if it

could be isolated and prepared in a purified form and in sufficient quantities to be

pharmaceutically useful.

[0157] Cathepsin G is another protease present in leukocytes. In

certain embodiments, cathepsin G is capable of degrading a variety of proteins in

vitro, including those of the complement pathway. Pancreatic elastase is another protease that may have a role in pancreatitis. Thus, inhibitors for these proteases may also be of pharmaceutical value.

[0158] In certain embodiments, the substrate specificity and

sensitivity to different inhibitors of serine proteases are believed to result from

changes in only a few amino acid residues. By analogy, it may be possible to

conceive of a class of serine protease inhibitors in which changes in a relatively

few amino acids will result in inhibition of different proteases. In certain

embodiments, a member of this class inhibits every serine protease.

[0159] An exemplary serine protease inhibitor is secretory leukocyte

protease inhibitor (SLPI) and fragments and analogs thereof. Exemplary serine

protease inhibitors also include, but are not limited to, anti-leukoprotease (ALP),

mucous protease inhibitor (MPI), human seminal plasma inhibitor-I (HUSI-l),

bronchial mucus inhibitor (BMI), and cervical mucus inhibitor (CUSI). In certain

embodiments, a serine protease inhibitors also may be LPS modulator. See,

e.g., Jin et al. (1997), Cell 88(3): 417-26. In certain embodiments, these

molecules are well-suited for use in conditions leading to bone loss because they.

are preferentially directed to the cartilage.

[0160] Exemplary serine protease inhibitors are described, e.g., in

U.S. Pat. No. 4,760,130; U.S. Pat. No. 5,900,400; and U.S. Pat. No. 5,633,227;

which are herein incorporated by reference for any purpose. The molecules

disclosed in the foregoing references as well as any variants or analogues

thereof are collectively termed "serine protease inhibitors."

[01611 IL-18 is a pro-inflammatory cytokine that was found to induce

interferon-y and was previously named interferon gamma inducing factor (GIF).

In certain instances, IL-1 has been shown to upregulate IL-18 production, and IL

18 induces production of a number of proinflammatory cytokines, including IL-6

and MMP-1. See, e.g., Dinarello et al. (1998), J. Leukocyte Biol. 63: 658-64. In

certain instances, caspase I is also important for IL-18 production. Experiments

also suggest that TNF-a regulates IL-18 production, and that simultaneous

inhibition of TNF-a and IL-18 protects against liver toxicity. See, e.g., Faggioni et

al. (2000), PNAS 97: 2367-72.

[0162] IL-18 acts in vivo through a receptor system reminiscent of

the IL-1 system. IL-18 interacts with a cell surface receptor (IL-18R), which

interacts with an accessory protein (IL-18RAcP). IL-18-mediated signaling

proceeds upon formation of the complex of IL-18, IL-18R, and IL-18RAcP. A

natural inhibitor for IL-18 is IL-18bp. In certain embodiments, IL-18bp acts as a

"decoy receptor" by binding to IL-18 molecules and preventing interaction with IL

18R.

[0163] In certain embodiments, the present invention is directed to

therapies comprising an antibody to OPGL and at least one IL-18 inhibitor, and

methods of treatment using such therapies. In certain embodiments, a therapy

comprises an antibody to OPGL and an IL-18 inhibitor and at least one additional

molecule described herein. Exemplary conditions that may be treated according

to certain embodiments include, but are not limited to, inflammation, autoimmune

diseases, IL-1 mediated diseases, and TNF-mediated diseases. Exemplary conditions that may be treated with an antibody to OPGL and at least one IL-18 inhibitor according to certain embodiments include, but are not limited to, arthritis, including, but not limited to rheumatoid arthritis; systemic lupus erythematosus

(SLE); graft versus host disease (GvHD); hepatitis; sepsis; and the loss of bone

and cartilage accompanying these diseases.

[0164] Exemplary IL-18 inhibitors include, but are not limited to,

antibodies that bind to IL-18; antibodies that bind to IL-18R; antibodies that bind

to IL-18RAcP; IL-18bp; IL-18R fragments (e.g., a solubilized extracellular domain

of the IL-18 receptor); peptides that bind to IL-18 and reduce or prevent its

interaction with IL-I8R; peptides that bind to IL-18R and reduce or prevent its

interaction with IL-18 or with IL-18RAcP; peptides that bind to IL-I8RAcP and

reduce or prevent its interaction with IL-18R; and small molecules that reduce or

prevent IL-18 production or the interaction between any of IL-18, IL-18R, and IL

18RAcP.

[0165] Certain IL-18 inhibitors are described, e.g., in US Pat. No.

,912,324, issued July 14, 1994; EP 0 962 531, published Dec. 8, 1999; EP 712

931, published Nov. 15, 1994; US Pat. No. 5,914,253, issued July 14,1994; WO

97/24441, published July 10, 1997; US Pat. No. 6,060,283, issued May 9, 2000;

EP 850 952, published Dec. 26, 1996; EP 864 585, published Sep. 16,1998; WO

98/41232, published Sep. 24, 1998; US Pat. No. 6,054,487, issued April 25,

2000; WO 99/09063, published Aug 14,1997; WO 99/22760, published Nov. 3,

1997; WO 99/37772, published Jan. 23,1998; WO 99/37773, published March

, 1998; EP 0 974 600, published Jan. 26, 2000; WO 00/12555, published Mar.

62.

9, 2000; Japanese patent application JP 111,399/94, published Oct. 31, 1997;

Israel patent application IL 121554 AO, published Feb. 8, 1998; which are

incorporated herein by reference for any purpose.

[0166] In certain embodiments, an antibody to OPGL may be used

with at least one therapeutic agent for inflammation. In certain embodiments, an

antibody to OPGL may be used with at least one therapeutic agent for an

immune disorder. Exemplary therapeutic agents for inflammation and immune

disorders include, but are not limited to, corticosteroids, including, but not limited

to, prednisolone; nonsteroidal anti-inflammatory drugs (NSAIDs), including, but

not limited to, cyclooxygenase type 1 (COX-1) and cyclooxygenase type 2 (COX

2 ) inhibitors; disease modifying antirheumatic drugs (DMARDs), including, but

not limited to, methotrexate, hydroxychloroquine, chloroquine, cyclosporine, gold

compounds (such as auranofin, aurothiomalate and aurothioglucose), and

leflunomide; type IV phosophodiesterase inhbitors, including, but not limited to,

Rolipram and Pentoxifylline; Tacrolimus (FK-506); Sirolimus (rapamycin);

mycophenolic acid; 5-lipoxygenase inhibitors, including, but not limited to,

Zileuton; modulators of interleukin-6 (IL-6); small molecule modulators of 38 kDa

mitogen-activated protein kinase (p38-MAPK); small molecule modulators of

intracellular molecules involved in inflammation pathways, wherein such

intracellular molecules include, but are not limited to, jnk, IKK, NF-KB, ZAP70,

and Ick. Certain exemplary therapeutic agents for inflammation are described,

e.g., in C.A. Dinarello and L.L. Moldawer Proinflammatory and Anti-Inflammatory

Cytokines in Rheumatoid Arthritis: A Primer for Clinicians Third Edition (2001)

Amgen Inc. Thousand Oaks, CA. Certain exemplary therapeutic agents for

inflammation and autoimmune diseases include, but are not limited to, interferon

gamma (IFN-y) modulators; modulators of OX40/OX40L (including soluble forms

of OX40); modulators of 4-1BB/4-1BB ligand (including soluble forms of 4-1BB);

and modulators of B cell-T cell costimulatory pathways, including, but not limited

to, modulators of the receptor ligand pairs CD28/7, CD40/CD40L,

ICOS/B7RP1, and AGP-3/TACI/BAFFR (AGP-3 binds to both TACI and BAFFR

receptors). Certain exemplary modulators of B cell-T cell costimulatory pathways

include, but are not limited to, inhibitors of CD28, B7.1, and B7.2 (including

soluble forms of B7.1 or B7.2 and soluble forms of CTLA4, both of which may be

fused to a heterologous peptide or protein which reduces or prevents

* degradation and/or increases half-life, reduces toxicity, reduces immunogenicity,

or increases biological activity of a therapeutic protein by increasing solubility or

circulating half-life); inhibitors of CD40 and CD40L (including soluble forms of

CD40 which may be fused to a heterologous peptide or protein); inhibitors of

ICOS and B7RPI (including soluble forms of ICOS which may be fused to a

heterologous peptide or protein) and inhibitors of AGP-3, TACI and BAFFR

(including soluble forms of TACI and BAFFR). ICOS, B7RP1 and inhibitors

thereof are described, e.g., in WOOO/46240. AGP-3, TACI and BAFFR and

inhibitors thereof are described, e.g., in WOOO/47740, WO01/85872,

W002/15273, W098/39361, and von Bulow and Bram (1997) Science 278:138

140.

[0167] In certain embodiments, an antibody to OPGL is used to

treat bone loss, including, but not limited to, bone loss resulting from osteolytic

destruction of bone caused by malignant or metastatictumors. In certain

embodiments, an antibody to OPGL may be used to treat bone loss associated

with cancer. Exemplary cancers include, but are not limited to, breast, prostate,

thyroid, kidney, lung, esophogeal, rectal, bladder, cervical, ovarian, and liver

cancers, as well as cancer of the gastrointestional tract. In certain embodiments,

an antibody to OPGL may be used to treat bone loss associated with, e.g.,

certain hematological malignancies, including, but not limited to, multiple

myeloma and lymphoma, including Hodgkin's Disease.

[0168] In certain embodiments, an antibody to OPGL is

administered alone. In certain embodiments, an antibody to OPGL is

administered with at least one other therapeutic agent, including, but not limited

to, at least one other cancer therapy agent. Exemplary cancer therapy agents

include, but are not limited to, radiation therapy and chemotherapy. In certain

embodiments, chemotherapy may involve treatment with one or more of the

following: anthracyclines, taxol, tamoxifene, doxorubicin, 5-fluorouracil, and other

drugs known in the art. In certain embodiments, a cancer therapy agent is a

luteinizing hormone-releasing hormone (LHRH) antagonist. In certain

embodiments, a LHRH antagonist is a peptide antagonist.

[0169] In certain embodiments, an LHRH antagonist comprises the

peptide: Ac-D-Nal-4-Cl-Phe-D-Pal-Ser-N-Me-Tyr-D-Asn-Leu-Lys(iPr)-Pro-D-Ala

NH2 (SEQ ID NO: 20), where Nal is 3-(2-napthyl)alaninyl; 4-CI-Phe is (4' chlorophenyl)alaninyl; Pal is 3-(3'-pyridyl)alaninyl; and Lys(iPr) is N-epsilon-2 propyl-lysinyl.

[0170] In certain embodiments, an LHRH antagonist is an LHRH

antagonist decapeptide. Certain exemplary decapeptides are described, e.g., in

U.S. Patent No. 5,843,901, which is herein incorporated by reference for any

purpose.

[0171] Exemplary therapeutic antibodies according to certain

embodiments include, but are not limited to, mouse, mouse-human chimeric,

CDR-grafted, humanized and fully human antibodies, and synthetic antibodies,

including, but not limited to, those selected by screening antibody libraries.

Exemplary antibodies include, but are not limited to, those which bind to cell

surface proteins Her2, CDC20, CDC33, mucin-like glycoprotein, and epidermal

growth factor receptor (EGFR) present on tumor cells, and optionally induce a

cytostatic and/or cytotoxic effect on tumor cells displaying these proteins.

Exemplary antibodies also include HERCEPTIN TM (trastuzumab), which may be

used to treat breast cancer and other forms of cancer, and RITUXANTM

(rituximab), ZEVALIN T M (ibritumomab tiuxetan), and LYMPHOCIDE T M

(epratuzumab), which may be used to treat non-Hodgkin's lymphoma and other

forms of cancer. Certain examplary antibodies also include ERBITUXTM (IMC

C225), BEXXAR TM(iodine 131 tositumomab), and Campath.

[0172] In certain embodiments, cancer therapy agents are

polypeptides which selectively induce apoptosis in tumor cells, including, but not

limited to, the TNF-related polypeptide TRAIL. In certain embodiments, an antibody to OPGL may be administered at least one of prior to, concurrent with, and subsequent to treatment with a cancer therapy agent. In certain embodiments, an antibody to OPGL may be administeredprophylactially to prevent or mitigate the onset of bone loss by metastatic cancer. In certain embodiments, an antibody to OPGL may be administered for the treatment of an existing condition of bone loss due to metastasis.

[0173] In certain embodiments, an antibody to OPGL may be used

to prevent and/or treat bone loss associated with multiple myeloma and/or to

prevent and/or treat the disease itself. Multiple myeloma is a B cell derived

tumor that may result in significant morbidity and/or mortality. In certain

instances, a clinical manifestation of multiple myeloma is focal bone loss, which

may be due to increased osteoclast activation in localized regions. Many

myeloma patients present with bone lesions visible by radiological analysis and

suffer from skeletal pain. In certain instances, patients with myeloma are

susceptible to pathological fractures of involved bone, which may occur either

spontaneously or due to injury. In certain instances, the skeletal lesions that

occur during myeloma not only lead to bone fractures, but also deformity and

occasionally nerve compression, particularly in the vertebral spine. In some

patients, a pathological increase in serum calcium (hypercalcemia) occurs, and

may cause significant problems during disease treatment. In certain

embodiments, an antibody to OPGL may be administered to patients to reduce or

block bone resorption and release of calcium, which may reduce the risk of

fractures and spinal deformity.

[0174] In certain instances, myeloma cells do not directly participate

in bone destruction, but instead produce extracellular signals that lead to

osteoclast differentiation and activation. In certain instances, osteoclasts

produce high levels of the cytokine IL-6, particularly when they become activated.

IL-6 is a B-cell growth factor, and contributes to the growth of both murine and

human myeloma cells in vitro. Myeloma cells may also either directly or indirectly

produce OPGL, which may result in local bone lysis surrounding the myeloma

cells embedded in bone marrow spaces. In certain instances, the normal

osteoclastsadjacent to the myeloma cell in turn produce IL-6, which may lead to

local expansion of the tumor cells. Myeloma cells expand in a clonal fashion and

may occupy bone spaces that are created by inappropriate bone resorption.

[0175] It has been observed that OPG administration in rodents

induces rapid death of the osteoclast population (see, e.g., Lacey et al. (2000)

Am. J. Pathol. 157:435-448). A reduction in the number of osteoclasts may

counteract the effect of increased IL-6 production by those cells and may

therefore affect the growth and survival of myeloma cells within trabecular bone.

Thus, in certain embodiments, administration of an antibody to OPGL to a

myeloma patient may not only block the hyper resorption of bone, but may also

affect the expansion and survival of the tumor itself.

[0176] B-cells express the receptor for OPGL, ODAR. Myeloma

cells also express ODAR, and in addition may produce OPGL. In certain

instances, the expression of both OPGL and ODAR in the same cell population

may create an autocrine stimulus that affects survival of the myeloma cell. Thus, in certain embodiments, administration of an antibody to OPGL may reduce tumor cell survival, thereby decreasing or eliminating the tumor burden seen in myeloma patients.

[0177] In certain embodiments, the invention provides for

pharmaceutical compositions comprising a therapeutically effective amount of an

antibody to OPGL together with a pharmaceutically acceptable diluent, carrier,

solubilizer, emulsifier, preservative and/or adjuvant.

[0178] In certain embodiments, the invention provides for

pharmaceutical compositions comprising a therapeutically effective amount of an

antibody to OPGL and a therapeutically effective amount of at least one

additional therapeutic agents, together with a pharmaceutically acceptable

diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. In certain

embodiments, the at least one additional therapeutic agent is selected from bone

morphogenic factors designated BMP-1 through BMP-12; transforming growth

factor-p (TGF-P) and TGF-p family members; interleukin-1 (IL-1) inhibitors,

including, but not limited to, IL-1ra and derivatives thereof and Kineret TM; TNFa

inhibitors, including, but not limited to, a soluble TNFa receptor, EnbrelTM, anti

TNFa antibodies, Remicade T M, and D2E7 antibody; parathyroid hormone and

analogs thereof, parathyroid related protein and analogs thereof; E series

prostaglandins; bisphosphonates (such as alendronate and others); bone

enhancing minerals such as fluoride and calcium; non-steroidal anti-inflammatory

drugs (NSAIDs), including COX-2 inhibitors, such as Celebrex TM and Vioxx TM ;

immunosuppressants, such as methotrexate or leflunomide; serine protease inhibitors such as secretory leukocyte protease inhibitor (SLPI); IL-6 inhibitors

(e.g., antibodies to IL-6), IL-8 inhibitors (e.g., antibodies to IL-8); IL-18 inhibitors

(e.g., IL-18 binding protein or IL-18 antibodies); Interleukin-1 converting enzyme

(ICE) modulators; fibroblast growth factors FGF-1 to FGF-10 and FGF

modulators; PAF antagonists; keratinocyte growth factor (KGF), KGF-related

molecules, or KGF modulators; matrix metalloproteinase (MMP) modulators;

Nitric oxide synthase (NOS) modulators, including modulators of inducible NOS;

modulators of glucocorticoid receptor; modulators of glutamate receptor;

modulators of lipopolysaccharide (LPS) levels; and noradrenaline and

modulators and mimetics thereof.

[0179] In certain embodiments, acceptable formulation materials

preferably are nontoxic to recipients at the dosages and concentrations

employed.

[0180] In certain embodiments, the pharmaceutical composition

may contain formulation materials for modifying, maintaining or preserving, for

example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,

stability, rate of dissolution or release, adsorption or penetration of the

composition. In certain embodiments, suitable formulation materials include, but

are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine

or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or

sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates,

phosphates or other organic acids); bulking agents (such as mannitol or glycine);

chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical

Sciences, 1 8 th Edition, A.R. Gennaro, ed., Mack Publishing Company (1990).

[0181] In certain embodiments, an antibody to OPGL and/or a

therapeutic molecule is linked to a half-life extending vehicle known in the art.

Such vehicles include, but are not limited to, the Fc domain, polyethylene glycol,

and dextran. Such vehicles are described, e.g., in U.S. Application Serial No.

09/428,082 and published PCT Application No. WO 99/25044, which are hereby

incorporated by reference for any purpose.

[0182] In certain embodiments, the optimal pharmaceutical

composition will be determined by one skilled in the art depending upon, for

example, the intended route of administration, delivery format and desired

dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In

certain embodiments, such compositions may influence the physical state,

stability, rate of in vivo release and rate of in vivo clearance of the antibodies of

the invention.

[0183] .In certain embodiments, the primary vehicle or carrier in a

pharmaceutical composition may be either aqueous or non-aqueous in nature.

For example, in certain embodiments, a suitable vehicle or carrier may be water

for injection, physiological saline solution or artificial cerebrospinal fluid, possibly

supplemented with other materials common in compositions for parenteral

administration. In certain embodiments, neutral buffered saline or saline mixed

with serum albumin are further exemplary vehicles. In certain embodiments,

pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate

buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable

substitute therefor. In certain embodiments, a composition comprising an

antibody to OPGL, with or without at least one additional therapeutic agents, may

be prepared for storage by mixing the selected composition having the desired

degree of purity with optional formulation agents (Remington's Pharmaceutical

Sciences, supra) in the form of alyophilized cake or an aqueous solution.

Further, in certain embodiments, a composition comprising an antibody to OPGL, with or without at least one additional therapeutic agents, may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0184] In certain embodiments, the pharmaceutical compositions of

the invention can be selected for parenteral delivery. In certain embodiments,

the compositions may be selected for inhalation or for delivery through the

digestive tract, such as orally. The preparation of such pharmaceutically

acceptable compositions is within the skill of the art.

[0185] In certain embodiments, the formulation components are

present in concentrations that are acceptable to the site of administration. In

certain embodiments, buffers are used to maintain the composition at

physiologicalpH or at a slightly lower pH, typically within a pH range of from

about 5 to about 8.

[0186] In certain embodiments, when parenteral administration is

contemplated, a therapeutic composition may be in the form of a pyrogen-free,

parenterally acceptable aqueous solution comprising the desired antibody to

OPGL, with or without additional therapeutic agents, in a pharmaceutically

acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is

sterile distilled water in which the antibody to OPGL, with or without at least one

additional therapeutic agent, is formulated as a sterile, isotonic solution, properly

preserved. In certain embodiments, the preparation can involve the formulation

of the desired molecule with an agent, such as injectable microspheres, bio

erodible particles, polymeric compounds (such as polylactic acid or polyglycolic

acid), beads or liposomes, that may provide for the controlled or sustained release of the product which may then be delivered via a depot injection. In certain embodiments, hyaluronic acid may also be used, and may have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices may be used to introduce the desired molecule.

[0187] In certain embodiments, a pharmaceutical composition may

be formulated for inhalation. In certain embodiments, an antibody to OPGL, with

or without at least one additional therapeutic agent, may be formulated as a dry

powder for inhalation. In certain embodiments, an inhalation solution comprising

an antibody to OPGL, with or without at least one additional therapeutic agent,

may be formulated with a propellant for aerosol delivery. In certain

embodiments, solutions may be nebulized. Pulmonary administration is further

described in PCT application no. PCT/US94/001875, which describes pulmonary

delivery of chemically modified proteins.

[0188] In certain embodiments, it is contemplated that formulations

may be administered orally. In certain embodiments, an antibody to OPGL, with

or without at least one additional therapeutic agents, that is administered in this

fashion may be formulated with or without those carriers customarily used in the

compounding of solid dosage forms such as tablets and capsules. In certain

embodiments, a capsule may be designed to release the active portion of the

formulation at the point in the gastrointestinal tract when bioavailability is

maximized and pre-systemic degradation is minimized. In certain embodiments,

at least one additional agent can be included to facilitate absorption of the antibody to OPGL and/or any additional therapeutic agents. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

[0189] In certain embodiments, a pharmaceutical composition may

involve an effective quantity of antibodies to OPGL, with or without at least one

additional therapeutic agents, in a mixture with non-toxic excipients which are

suitable for the manufacture of tablets. In certain embodiments, by dissolving the

tablets in sterile water, or another appropriate vehicle, solutions may be prepared

in unit-dose form. In certain embodiments, suitable excipients include, but are

not limited to, inert diluents, such as calcium carbonate, sodium carbonate or

bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch,

gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic

acid, or talc.

[0190] Additional pharmaceutical compositions will be evident to

those skilled in the art, including formulations involving antibodies to OPGL, with

or without at least one additional therapeutic agents, in sustained- or controlled

delivery formulations. In certain embodiments, techniques for formulating a

variety of other sustained- or controlled-delivery means, such as liposome

carriers, bio-erodible microparticles or porous beads and depot injections, are

also known to those skilled in the art. See for example, PCT Application No.

PCT/US93/00829 which describes the controlled release of porous polymeric

microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.

Sustained release matrices may include polyesters, hydrogels, polylactides (U.S.

3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L

glutamate (Sidman et a, Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl

methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and

Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al.,

supra) or poly-D(-)-3-hydroxybutyric acid (EP 133,988). In certain embodiments,

sustained release compositions may also include liposomes, which can be

prepared by any of several methods known in the art. See e.g., Eppstein et al.,

Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and

EP 143,949.

[0191] The pharmaceutical composition to be used for in vivo

administration typically is sterile. In certain embodiments, this may be

accomplished by filtration through sterile filtration membranes. In certain

embodiments, where the composition islyophilized, sterilization using this

method may be conducted either prior to or following yophilization and

reconstitution. In certain embodiments, the composition for parenteral

administration may be stored in lyophilized form or in a solution. In certain

embodiments, parenteral compositions generally are placed into a container

having a sterile access port, for example, an intravenous solution bag or vial

having a stopper pierceable by a hypodermic injection needle.

[0192] In certain embodiments, once the pharmaceutical

composition has been formulated, it may be stored in sterile vials as a solution,

suspension, gel, emulsion, solid, or as a dehydrated orlyophilized powder. In

certain embodiments, such formulations may be stored either in a ready-to-use

form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0193] In certain embodiments, the present invention is directed to

kits for producing a single-dose administration unit. In certain embodiments, the

kits may each contain both a first container having a dried protein and a second

container having an aqueous formulation. In certain embodiments of this

invention, kits containing single and multi-chambered pre-filled syringes (e.g.,

liquid syringes and lyosyringes) are included.

[0194] In certain embodiments, the effective amount of a

pharmaceutical composition comprising an antibody to OPGL, with or without at

least one additional therapeutic agent, to be employed therapeutically will

depend, for example, upon the therapeutic context and objectives. One skilled in

the art will appreciate that the appropriate dosage levels for treatment, according

to certain embodiments, will thus vary depending, in part, upon the molecule

delivered, the indication for which the antibody to OPGL, with or without at least

one additional therapeutic agent, is being used, the route of administration, and

the size (body weight, body surface or organ size) and/or condition (the age and

general health) of the patient. In certain embodiments, the clinician may titer the

dosage and modify the route of administration to obtain the optimal therapeutic

effect. In certain embodiments, a typical dosage may range from about 0.1 pg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In certain embodiments, the dosage may range from 0.1 pg/kg up to about 100 mg/kg; or I pg/kg up to about 100 mg/kg; or 5 pg/kg up to about 100 mg/kg.

[0195] In certain embodiments, the frequency of dosing will take

into account the pharmacokinetic parameters of the antibody to OPGL and/or any

additional therapeutic agents in the formulation used. In certain embodiments, a

clinician will administer the composition until a dosage is reached that achieves

the desired effect. In certain embodiments, the composition may therefore be

administered as a single dose, or as two or more doses (which may or may not

contain the same amount of the desired molecule) over time, or as a continuous

infusion via an implantation device or catheter. Further refinement of the

appropriate dosage is routinely made by those of ordinary skill in the art and is

within the ambit of tasks routinely performed by them. In certain embodiments,

appropriate dosages may be ascertained through use of appropriate dose

response data.

[0196] In certain embodiments, the, route of administration of the

pharmaceutical composition is in accord with known methods, e.g. orally, through

injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal),

intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or

intralesional routes; by sustained release systems or by implantation devices. In

certain embodiments, the compositions may be administered by bolus injection

or continuously by infusion, or by implantation device.

[0197] In certain embodiments, the composition may be

administered locally via implantation of a membrane, sponge or another

appropriate material onto which the desired molecule has been absorbed or

encapsulated. In certain embodiments, where an implantation device is used,

the device may be implanted into any suitable tissue or organ, and delivery of the

desired molecule may be via diffusion, timed-release bolus, or continuous

administration.

[0198] In certain embodiments, it may be desirable to use a

pharmaceutical composition comprising an antibody to OPGL, with or without at

least one additional therapeutic agent, in an ex vivo manner. In such instances,

cells, tissues and/or organs that have been removed from the patient are

exposed to a pharmaceutical composition comprising an antibody to OPGL, with

or without at least one additional therapeutic agent, after which the cells, tissues

and/or organs are subsequently implanted back into the patient.

[0199] In certain embodiments, an antibody to OPGL and/or any

additional therapeutic agents can be delivered by implanting certain cells that

have been genetically engineered, using methods such as those described

herein, to express and secrete the polypeptides. In certain embodiments, such

cells may be animal or human cells, and may be autologous, heterologous, or

xenogeneic. In certain embodiments, the cells may be immortalized. In certain

embodiments, in order to decrease the chance of an immunological response,

the cells may be encapsulated to avoid infiltration of surrounding tissues. In

certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.

EXAMPLES

[0200] The following examples, including the experiments

conducted and results achieved are provided for illustrative purposes only and

are not to be construed as limiting the present invention.

Example I Cloning the aOPGL-1 Heavy and Light Chains

[0201] CHO cells expressing the full-length human OPGL cDNA are

used to immunize transgenic mice containing human immunoglobulin genes.

Lymph nodes from the immunized mice are fused to murine myeloma cells to

generate hybridomas. Supernatants from the hybridoma lines are tested in an

ELISA assay for antibodies that react with human OPGL. Anti-OPGL expressing

hybridoma lines AMG 6.5, AMG 6.4, and AMG 6.1 are found to express

antibodies with high affinity for OPGL (Kd's of 0.28 nM, 0.29 nM, and 0.23 nM,

respectively), and AMG 6.5 is selected for cloning. Heavy and light chain cDNA

clones from AMG 6.5 and AMG 6.4 are identical, and AMG 6.5 is used to clone

the aOPGL-1 light chain cDNA, while AMG 6.4 is used to clone the aOPGL-1

heavy chain cDNA.

Cloninq of the aOPGL-1 liqht chain

[0202] The aOPGL-1 kappa light chain variable region is obtained

using PCR amplification methods from first strand cDNA prepared from AMG 6.5

total RNA. First strand cDNA is prepared from AMG 6.5 total RNA using a

random primer with an extended 5'-adapter (5'

GGCCGGATAGGCCTCACNNNNNNT-3'(SEQ ID NO: 15)) and the materials

and methods provided by the Gibco SuperScript 11 TM Preamplification System for

First Strand cDNA Synthesis kit (cat. no. 18089-011). The oligonucleotides

below are used for the PCR:

' kappa RACE primer:

' - GAT GAC CCA GTC TCC AGC CAC CCT G - 3' (SEQ ID NO: 5)

3' kappa RACE primer:

' - AAG GGT CAG AGG CCA AAG GAT GG - 3' (SEQ ID NO: 6)

[0203] The amplified DNAs, are cloned into pCRII-TOPO

(Invitrogen) and the resulting plasmids are sequenced. The kappa chain

consensus sequence is used to design primers for PCR amplification of the

full-length aOPGL-1 kappa chain. The 5' aOPGL-1 kappa primer incorporates a

Xbal site (TCTAGA) for cloning and a "CCACC" Kozak sequence before the

initiator Met codon. The 3' aOPGL-1 kappa primer incorporates a Sall site

(GTCGAC) following the stop codon for cloning.

' aOPGL-1 kappa primer:

'-CAA CTC TAG A CC ACC ATG GAA ACC CCA GCG-3' (SEQ ID NO: 7)

Xbal Site Kozak M E T P A (SEQ ID NO: 16)

3' aOPGL-1 kappa primer:

'-TTT GAC GTC GAC TTA TCA ACA CTC TCC CCT GTT GAA G -3' (SEQ ID NO: 8)

Safl Site * * C E G R N F (SEQ ID NO: 17)

[0204] The full-length aOPGL-1 kappa chain cDNA clone is

obtained using the AMG 6.5 first strand cDNA, described above, by PCR

amplification with the 5'and 3'aOPGL-1 kappa primers. The PCR reaction

generates a 738 bp fragment encoding the 235 amino acid residues (including

the 20 amino acid kappa chain signal sequence) of the aOPGL-1 kappa chain

(Figure 4, SEQ ID NO: 4). Following purification using a QlAquick PCR

Purification kit (Qiagen cat. no.28104), this fragment is used to construct the

kappa light chain expression vector.

[0205] The 738 bp full-length kappa fragment generated above is

cut with Xbal and Sall, is purified using the Promega Wizard DNA Clean-Up

System (Promega cat. no. A7100), and is cloned into pDSRa19 to generate

plasmid aOPGL-1-kappa/pDSRa9 (Figure 5). pDSRal9 has been described

previously (see WO 90/14363, which is herein incorporated by reference for any

purpose (see, e.g., Figure 12)). Briefly, to make pDSRa19, pDSRa2 is modified

in the following ways: the FSH polyA is shortened by approximately 1400 base

pairs, to 885 base pairs, and now ends at the Ndel site; the dihydrofolate

reductase (DHFR) promoter now contains 209 base pairs, having been

shortened from the 5' end by approximately 1 kilobase; and an approximately

550 base pair Bgl fragment from the DHFR polyA sequence is deleted.

[0206] The aOPGL-1 kappa light chain expression clone is

sequenced to confirm that it coded for the same peptide that is identified in the

AMG 6.5 hybridoma. The final expression vector, aOPGL-1-kappa/pDSRa19, is

5476 bp and contains the 7 functional regions shown in Table 2.

Table 2: Features of aOPGL-1-kappalpDSRaI9

Plasmid Base Pair Number: 2 to 881 A transcription termination/polyadenylation signal from the c-subunit of the bovine pituitary glycoprotein hormone (a-FSH) (Goodwin, et al, Nucleic Acids Res. 1983 11: 6873-82; Genbank Accession Number X00004)

882 to 2027 A mouse dihydrofolate reductase (DHFR) minigene containing the endogenous mouse DHFR promoter, the cDNA coding sequences, and the DHFR transcription termination/polyadenylation signals (Gasser et al, Proc Natl Acad Sci U S A. 1982 79:6522-6.; Nunberg et al, Cell1980 19:355-64; Setzer et al, J Biol Chem. 1982 257: 5143-7; McGrogan et al, J Biol Chem. 1985 260: 2307-14)

2031 to pBR322 sequences containing the ampicillin resistance marker 3947 gene and the origin for replication of the plasmid in E. coli (Genbank Accession Number J01749)

3949 to An SV40 early promoter, enhancer and origin of replication 4292 (Takebe et al, Mol Cell Biol. 1988 8: 466-72., Genbank Accession Number J02400)

4299to A translational enhancer element from the HTLV-1 LTR domain 4565 (Seiki et al, Proc Natl Acad Sci U S A. 1983 80: 3618-22, Genbank Accession Number J02029)

4574 to An intron from the SV40 16S, 19S splice donor/acceptor signals 4730 (Okayama and Berg, Mol Cell Biol. 1983 3: 280-9, Genbank Accession Number J02400)

4750 to The aOPGL-1 Kappa light chain cDNA between the Xbal and Sall .5476 sites

[0207] A circular plasmid map of the vector is shown in Figure 5.

Cloning of the aOPGL-1 heavy chain

[0208] The aOPGL-1 IgG2 heavy chain is cloned from AMG 6.4 hybridoma double-stranded cDNA produced with the Clontech Marathon TM cDNA

Amplification Kit (cat. no. K1802-1). Amplification of AMG 6.4 heavy chain cDNA

is accomplished by 5' and 3' rapid amplification of cDNA ends (RACE)

techniques performed with human germline IgG2 heavy chain constant region

specific primers (shown below) and RACE primers and other materials and

methods provided in the Marathon TM cDNA amplification kit.

' IgG2 RACE primer

'- GGC ACG GTC ACC ACG CTG CTG AG -3' (SEQ ID NO: 9)

3' IgG2 RACE primer

'-CCTCCACCAAGGGCCCATCGGTCT-3' (SEQIDNO:10)

[0209] The 600 bp 5' RACE product and 1200 bp 3' RACE product

are cloned into pCR2.1 (Invitrogen) and are sequenced. This sequence

information is used to design aOPGL-1 heavy chain specific primers for the

cloning of the full-length sequence. The heavy chain 5' primer (5' aOPGL-1 IgG2

Primer) is directed against the sense strand and has a Hindlli site and

consensus Kozak sequence before the natural start site. The heavy chain 3'

primer (3' aOPGL-1 IgG2 Primer) is an antisense primer that contains a Sai site

and stop codon after the last amino acid of the heavy chain IgG2 sequence.

' aOPGL-1 IgG2 Primer:

'-CAGAAGCTTGACCACC ATG GAG TTT GGG CTG AGC TGG CTT TTT CTT GTG GC - 3' (SEQ ID NO: 11)

Hindll Kozak M E F G L S W L F L V A

(SEQ ID NO: 18)

3' aOPGL-1 IgG2 Primer:

'- GCA TGTCGAC TTA TCA TTT ACC CGG AGA CAG GGA GAG - 3' (SEQ ID NO: 12)

Sal * * K G P S L S L (SEQ ID NO: 19)

[0210] The double-stranded cDNA described above is used to

generate the full-length heavy chain cDNA by PCR amplification with the 5'- and

3'- aOPGL-1 IgG2 primers. The PCR generates a 1433 bp fragment encoding

the 467 amino acid residues (including the 19 amino acid IgG signal sequence)

of the aOPGL-1 IgG2 heavy chain protein (Figure 2, SEQ ID NO: 2). Following

purification using a QlAquick PCR Purification kit (Qiagen cat. no.28104), this

fragment is used to construct the heavy chain expression vector as follows.

[0211] DNA encoding the full-length IgG2 heavy fragment

generated above is cut with Hindll and Sal, purified using a QlAquick Gel

Extraction kit (Qiagen cat. no.28704), and this fragment is cloned into pDSRa19.

The resulting expression plasmid is named aOPGL-1-IgG2/pDSRa19 (Figure 6).

All vector components are identical to aOPGL-1-kappa/pDSRal9 vector,

described above, except the aOPGL-1 IgG2 heavy chain cDNA replaces the

aOPGL-1 kappa light chain cDNA between the Xbal and Safl sites. The aOPGL

1 IgG2 heavy chain expression clone is sequenced to confirm that it coded for

the same polypeptide that is identified in the AMG 6.4 hybridoma.

Example 2

aOPGL-1 expression in CHO cells

[0212] Stable expression of aOPGL-1 antibody is achieved by co

transfection of aOPGL-1-kappa/pDSRcl9 and aOPGL-1 -lgG2/pDSRa19

plasmids into dihydrofolate reductase deficient (DHFR-) Chinese hamster ovary

cells (CHO AM-1/D, U.S. Patent No. 6,210,924) followed by isolation and testing

of individual clones.

[0213] A 100 mm tissue culture dish is plated with 1.5x10 6 AM-1/D

cells grown in CHO d- medium (DMEM-high glucose, 10% fetal bovine serum,

1% penicillin/ streptomycin/glutamine, 1X NaPyruvate, 1% nonessential amino

acids (NEAA))(Gibco@) and 1% ht supplement (Gibco@)) the day before

transfections (Day 0). On day one, 400 pl of serum-free RPMI 1640 medium

(Gibco@) is aliquoted into a 12x75 mm polypropylene tube. Twenty four

microliters of TranslT@-LT1 reagent (Mirus Corporation) is added dropwise to the

medium and the mixture is allowed to incubate at room temperature for 10

minutes. A total of 15 pg of linearized plasmid DNA (7.5 pg of aOPGL

1-kappa/pDSRa19 and 7.5 pg of aOPGL-1 -IgG2/pDSRal9, digested with Pvul)

is then added dropwise to the mixture and is incubated at room temperature for

10 minutes.

[0214] The CHO d- medium is removed from the cells, which are

washed with 10 ml of Dulbecco's phosphate buffered saline (Gibco). Six milliliters of serum-free MEM medium supplemented with HT, L-glu , NEAA, and

Na pyruvate (Gibco@) is added to the cells. The DNA/LT1 complex is added

dropwise to the plates, which are gently rocked back and forth to distribute the

DNA evenly over the cells. After 6 hours in a tissue culture incubator, the

medium is replaced with fresh CHO d- medium. Forty eight hours later the cells

are split to ten 100 mm culture dishes in CHO select medium (DMEM high

glucose, 10% dialyzed fetal bovine serum (FBS), 1% penicillin /streptomycin

/glutamine, 1% nonessential amino acids and IX Na pyruvate) (Gibco@).

Medium is changed twice weekly until colonies appeared.

[0215] After 10-14 days, colonies are picked using 5 mm cloning

discs (Labcore@) soaked in 1x trypsin-EDTA (Gibcoo) and are cultured in 24

well tissue culture plates with CHO select medium. When the cells become

confluent, serum free media (CHO select medium minus FBS) is added and is

then collected 48 hours later. These conditioned media are analyzed for

antibody expression by Western blot with horse radish peroxidase (HRP)

conjugated goat anti-human IgG Fc antibody (Pierce, Rockford, IL) to detect the

aOPGL-1 heavy chain, and goat anti-human kappa chain antibody (Pierce,

Rockford, IL) followed by HRP-conjugated rabbit anti-goat IgG(H+L) antibody

(Pierce, Rockford, IL) to detect the aOPGL-1 light chain. The highest expressing

clones are expanded and stored in liquid nitrogen.

Example 3

Production of aOPGL-1

Preparation and Creation of Cell Line 125Q

[0216] CHO cells producing aOPGL-1 are cloned by two rounds of

limiting dilution in 96 well plates under serum-free conditions. The clones are

selected based on production and growth characteristics in various suspension

vessels. ElAs are performed to select the clone that produces the highest level

of aOPGL-1. Growth characteristics, including doubling times and densities are

then measured by growing the clones in 100 ml, 250 ml, 500 ml, 1 L, and 3 L

spinner flasks, as well as in 3L Aplikon bioreactors. The clone with the fastest

doubling time that reaches the highest density in culture is selected, and is

designated Cell Line 125Q. When the clone has expanded to yield sufficient

cells to freeze 360 ampules at approximately 1x107 cells/mL, cells are

resuspended in a cryopreservative, serum-free medium (90% VM-Soy Batch

Medium (see Table 3 for details) supplemented with 10 ml/L nonessential amino

acids and 10 ml/L L-Glutamine (Gibco/LTI/Invitrogen), and 10% dimethyl

sulfoxide (JT Baker)) and frozen. Ampules are stored in a limited access facility

and are submerged in liquid nitrogen in liquid nitrogen dewars.

[0217] Based on growth and production in small-scale spinners and

larger scale bioreactors, Cell Line 125Q is chosen as the cell line for

manufacturing of aOPGL-1.

Cell Culture

[0218] aOPGL-1 is produced by expression in Cell Line 125Q, a clonal line of CHO cells that expresses aOPGL-1 from plasmids aOPGL

1-kappa/pDSRa19 and aOPGL-1 -IgG2/pDSRx19. The cell culture process for

aOPGL-1 is shown in Figure 19. For each production run, cells from a vial of Cell Line 125Q are initially grown in 50 mL of VM-Soy Batch Medium (see Table 3 for composition) supplemented with 10 mi/L nonessential amino acids and 10 m/L L-glutamine (Gibco/LTI/Invitrogen) (VM-Soy Supp) in 125 ml erlenmeyer shakers at 100 rpm for 5 days. The entire culture is then used to inoculate VM-Soy Supp in a 500 ml spinner flask to 3x10viable cells/mi (3E5 vc/ml), and is grown with 70 rpm spinning for 3-4 days. The entire culture from the 500 ml spinner flask is then used to inoculate VM-Soy Supp in a 3L spinner flask to 3E5 vc/ml, and is grown with 70 rpm spinning for 3-4 days.

[0219] The culture from the 3L spinner flask is then split to two 3 L spinner flasks at 3E5 vc/ml in VM-Soy Supp without phenol red and grown under the same conditions. These spinner flask cultures are then used to inoculate four additional spinner flasks at 3E5 vc/ml in VM-Soy Supp without phenol red, and grown under the same conditions. Four liters of culture from the four 3L spinner flasks is used to inoculate 10 L of VM-Soy Supp without phenol red in a 20 L bioreactor, and the bioreactor is run in fed-batch mode for 7-10 days. In fed-batch mode, a nutrient feed containing concentrated media components ("Feed", as set forth below in Table 3) is added to maintain cell growth and culture viability.

[0220] The entire culture from the 20L bioreactor is then used to

inoculate 70 L of VM-Soy Supp without phenol red in a 150L bioreactor, and the

bioreactor is run in fed-batch mode for 9-10 days. Finally, the entire culture from

the 150L bioreactor is used to inoculate approximately 880 L of VMSoy (without

supplement or phenol red) in a 2000L bioreactor, and the bioreactor is run in fed

batch mode. The rate of feed during fed-batch mode is determined such that the

glucose level in the culture is maintained at 0.6 g/L for each bioreactor. The cell

density and glucose concentration are measured daily and the rate of feed

adjusted accordingly.

[0221] Production in the 2000L bioreactor lasts for approximately

two weeks during which time aOPGL-1 is constitutively produced by the cells and

secreted into the cell culture medium.

[0222] The production reactor is controlled at set pH, temperature,

and dissolved oxygen level: pH is 7.0 and is controlled by carbon dioxide gas and

sodium carbonate addition; dissolved oxygen is 120 mmHg and is controlled by

air, nitrogen, and oxygen gas flows. Cells are maintained at 37C throughout the

process. All gases are passed through membrane filters of pore size 0.22 pm or

less.

[0223] At the end of production, the cell broth is fed into a disk stack

centrifuge and the culture supernatant is separated from the cells. The centrate

is further clarified through a Cuno 90SP depth filter followed by a 0.2 pm

Posidyne filter (Pall Co.). The clarified conditioned media is then concentrated

by tangential flow ultrafiltration (UF) using 50kD NMWL membranes (Millipore

Biomax 50). The conditioned media is concentrated 15- to 30- fold. The

resulting concentrated conditioned medium (CCM) is then either processed

through purification or frozen for purification at a later date. The production

process is summarized in Figure19.

Cell Culture Medium

[0224] The cell culture medium for use throughout the entire cell

culture process is based on Dulbecco's Modified Eagle's Medium /Ham's Nutrient

F12 (DMEM/F12, 1:1), and contains supplemental levels of amino acids,

additional nutrients and salts, a soy hydrolysate and recombinant human insulin

(Nucellin*Zn, Eli Lilly). The components are listed in Table 3. This media is

referred to as VM-Soy. Media solutions are filtered through membrane filters of

0.2 m pore size prior to use.

Table 3. Cell Culture Media Components

COMPONENTS FOR BASAL Media and FEEDS COMPONENT VMSoy Batch Medium (mg/L) FEED (mg/L)

DMEM/F12 COMPONENTS Inorganic salts CaC12 (anhyd.) 116.60 233.2 CUSO4.5H20 0.0026 0.0052 Fe(N03)3.9H20 0.1000 0.2 FeSO4.7H20 0.8340 1.668 KCL 311.80 623.6 MgCL2 (anhyd.) 57.280 114.56 MgSO4 (anhyd.) 97.680 195.36 NaC 905.990 1811.98

WO 03/002713 PCT/USO2/20181

NaI2PO4.H-20 125.00 250 Na2HPO4 142.040 284.08 ZnSO4.7H20 0.8640 1.728

Other Components D-Glucose 3151.00 12302 Na Hypoxanthine 5.40 10.8 Linoleic acid 0.090 0.18 Lipoic acid 0.2060 0.412 Phenol Red 8.10 16.2 Putrescine.2H1- 0.1620 0.324 Sodium Pyruvate 110.00 220

Amino acids L-Alanine 26.70 53.4 L-Arginine HC1 295.00 590 L-Asparagine.H20 45.00 90 L-Aspartic acid 39.90 79.8 L-Cysteine.HC1.H20 35.120 70.24 L-Cystine.2H1 62.580 125.16 L-Glutamic acid 44.10 88.2 L-Glutamine 657.00 1314 Glycine 52.50 105 L-Histidine.HCl.H20 62.950 125.9 L-Isoleucine 108.940 217.88 L-Leucine 118.10 236.2 L-Lysine HCI 182.50 365 L-Methionine 34.480 68.96 L-Phenylalanine 70.960 141.92 L-Proline 57.50 115 L-Serine 73.50 147 L-Tbreonine 106.90 213.8 L-Tryptophan 18.040 36.08 L-Tyrosine.2Na.2H20 111.580 223.16 L-Valine 105.70 211.4

Vitamins Biotin 0.0073 0.0146

D-Ca Pantothenate 4.480 8.96 Choline Chloride 17.960 35.92 Folic Acid 5.30 10.6 i-Inositol 25.20 50.4 Niacinamide 4.040 8.08 Pyridoxal HCl 4.00 8 Pyridoxine HCl 0.0620 0.124 Riboflavin 0.4380 0.876 Thiamine HCl 4.340 8.68 Thymidine 0.3635 0.727 Vitamin B12 1.360 2.72

ADDITIONAL COMPONENTS

Nucellin Zn, (rhu insulin) 5.00 15 Selenous Acid 0.0050 0.015 Ethanolamine 0.0012 0.0037 Triiodothyronine 0.000040 0.00012 Hydrocortisone 0.020 0.06 Ferric Citrate 122.450 122.450 Pluronic F-68 1000.00 500 Soy Hydrolysate 6000.00 6000.00 NaHCO3 3000.00 3000.00

NaCl 3500.00

Purification Process

[0225] aOPGL-1 expressed in CHO cells is secreted into the

extracellular medium. A series of steps may be used to generate pure material.

The process uses hydrophobic charge induction, cation exchange, and

hydrophobic interaction chromatography along with a low pH step and viral filter.

These procedures are described below.

A. Hydrophobic Charge Induction Chromatography (HCIC)

[0226] This chromatography step removes the majority of host cell

proteins and DNA. The concentrated conditioned media (CCM) is filtered

through a Cuno 30SP filter and then through a Cuno VRO7 charged

cellulose-based filter, and then loaded on to an MEP HyperCel resin. After

loading, the column is washed with equilibration buffer (20 mM Tris pH 7.2). The

antibody is eluted from the resin using a low pH buffer (20 mM Sodium Acetate,

pH 5.0). As it is eluted from the column, the product is collected based on the

absorbance at 280 nm of the column effluent.

B. Viral Inactivation

[0227] The MEP pool is titrated to pH 3.7 and is held for

approximately 60 minutes to inactivate potentially contaminating retrovirus.

Following the hold step, the pH is adjusted to approximately 6.0.

C. Viral Filtration

[0228] The pH-adjusted pool is filtered through a Millipore Viresolve

NFR filter or equivalent. The antibody flows through the filter while potentially

contaminating viruses > 50 nm are retained.

D. Cation Exchange Chromatography (CEX)

[0229] The antibody is further purified by cation exchange

chromatography using SP Sepharose HP (Amersham Pharmacia) or equivalent.

The cation exchange chromatography step removes additional CHO cell

proteins, DNA, lower molecular weight proteins, and aggregated forms of

aOPGL-1. The viral filtered pool is loaded onto the cation exchange resin. After loading, the column is washed with equilibration buffer (20 mM NaMES pH 6.2).

The antibody is then eluted with a linear gradient of increasing salt (20 mM

NaMES pH 6.2, 0 M NaCl to20 mM NaMES pH 6.2,0.3 M NaC). As it is eluted

from the column, the product is collected based on the absorbance at 280 nm of

the column effluent.

E. Hydrophobic Interaction Chromatography (HIC)

[0230] The antibody is further purified by hydrophobic interaction

chromatography using Phenyl Toyopearl 650S (Tosoh Biosep) or equivalent. The

hydrophobic interaction chromatography step is used as a polishing step and

removes additional CHO cell proteins, DNA, lower molecular weight proteins, and

aggregated forms of aOPGL-1. The cation exchange pool is conditioned before

loading onto the column by addition of ammonium sulfate to a conductivity of

>105 mS/cm at 15-25°C. After loading, the column is washed with the

equilibration buffer (1M Potassium Phosphate pH 8). The antibody is then eluted

with a linear gradient of decreasing salt concentration (1M Potassium Phosphate,

OmM Tris pH 8 to OM Potassium Phosphate, 20 mM Tris pH 8). As it is eluted

from the column, the product is collected based on the absorbance at 280 nm of

the column effluent.

F. Concentration and Diafiltration

[0231] The HIC column pool is concentrated and diafiltered into

formulation buffer by tangential flow ultrafiltration using 50kD NMWL membranes

(Millipore Biomax 50). The formulation buffer includes 10 mM Acetate, 5%

Sorbitol, pH 5.2 and aOPGL-1 is at 30 mg/mL.

Final Filtration and Storage

[0232] The purified bulk is passed through a 0.22 tm PVDF filter

(Millipore), is sampled, and stored at approximately -30°C in a secured freezer.

Example 4

Binding Specificity of aOPGL-1

[0233] Antibodies that are produced in CHO cells that are

transfected with the two expression vectors as discussed in Examples 1 and 2

may be used in the following examples 4, 5, and 6.

[0234] Human OPG binds and neutralizes OPGL in rats, mice and

cynomolgus monkeys, as well as in humans. aOPGL-1 binds human OPGL with

high affinity but does not bind significantly to murine OPGL (Table 4).

Table 4: Affinity of aOPGL-1 to Cell Membrane Expressed OPGL of Human, Cynomolgus Monkey, or Mouse Sequence.

OPGL Species aOPGL-1 EDo (ng/mI Human 16 Cynomolgus 19 Mouse No Specific Binding OPGL of these species is expressed in CHO cells as the full-length, membrane-bound protein. Binding of aOPGL-1 to the cell surface expressed OPGL is assessed by FACS analysis of cells incubated with aOPGL-1 and a FITC-labeled secondary antibody to human IgG2. aOPGL-1 binds human and cynomolgus OPGL but there is no specific binding to mouse OPGL.

[0235] In addition, human OPG has been reported to show weak

binding to tumor necrosis factor-related apoptosis-inducingligand (TRAIL)

(Truneh et al, 2000), a related member of the TNF family, which shows DNA and amino acid sequence homology to OPGL (Lacey et al., 1998). However, OPG does not detectably bind to other TNF-related proteins such as TNFa, TNFP, or

CD40 ligand.

[0236] aOPGL-1 binds specifically to OPGL on EIA plates (Figure 7). Recombinant soluble OPGL (2 pg/ml) is coated onto 96-well EIA plates at

room temperature for 16 to 24 hours. After blocking with 1% BSA in PBS,

varying concentrations of aOPGL-1 (approximately 2 ng/ml to 1000 ng/ml) diluted

in 1% BSA/PBS are added to the wells and the plates are incubated for about 2

hours at room temperature. Bound antibody is detected with goat anti-Human

IgG (Fab')-HRP using TMB-H202 (tetramethylbenzidine and hydrogen peroxide)

substrate cocktail. The absorbance is read at 450nm and 650nm.

[0237] aOPGL-1 binds specifically OPGL expressed on the surface

of transfected cells (Figure 8). aOPGL-1 (100 ng/ml) diluted in FACS Buffer

(PBS, 0.1%BSA, 0.01%Sodium Azide) is preincubated with varying

concentrations of OPGL, TNFa, TNFb, TRAIL, or CD40 ligand (approximately 0.1

ng/ml to 1000 ng/ml), and is then added to approximately 200,000 CHO REN

218-9 cells, which are CHO cells stably expressing membrane-bound OPGL on

the cell surface. After 1 hour at 2-8°C, unbound antibody is removed by

centrifugation and washing. Cells are then incubated for 30 minutes at 2-8°C

with FITC-labeled F(ab') 2 Goat anti-Human IgG (Fcy fragment specific). After

centrifugation and washing, cell surface fluorescence is measured using flow

cytometry. Figure 8 shows that binding of aOPGL-1 to CHO REN 218-9 cells is specific, and is competitively reduced by addition of soluble OPGL, but not by addition of TNFa, TNFb, TRAIL, or CD40 ligand.

[0238] In competition experiments, aOPGL-1 binding to OPGL on

EIA plates is inhibited by addition of exogenous OPGL (Figure 9), but not by the

addition of TNFa, TNFp, TRAIL, or CD40 ligand (Figure 10). This procedure is

performed in substantially the same manner as above, for binding of aOPGL-1 to

OPGL on EIA plates, except a constant concentration of aOPGL-1 (1OOng/mL) is

preincubated with varying concentrations of soluble OPGL or other ligands

(approximately 1 ng/ml to 1000 ng/ml for each) before it is added to the OPGL

coated plates.

Example 5

aOPGL-1 Neutralizing Activity

Inhibition of Osteoclast Formation

[0239] RAW 264.7 (ATCC No. TIB-71, Manassas, VA) is a murine

macrophage cell line that was derived from an Abelson murine leukemia virus

induced tumor. RAW 264.7 cells will differentiate to osteoclast-like cells in the

presence of OPGL. The basic assay for generation of osteoclasts in culture from

RAW cells in the presence of OPGL has been described in detail in Simonet et al

(1997) Cell89 p. 309, and Lacey et al (1998) Cell93 p. 165, which are herein

incorporated by reference for any purpose.

[0240] RAW cells are stimulated by ligand to differentiate into

osteoclast-like cells, and the differentiation can be measured by TRAP activity, a property of osteoclasts. Thus, the effect of aOPGL-1 on osteoclastogenesis can be measured.

[0241] RAW cells are incubated for 4 days in the presence of a

constant amount of OPGL (40 ng/ml) and varying amounts of aOPGL-1 (6.3

ng/ml to 200 ng/ml) in cell culture medium (DMEM, 10% FBS, 0.292 mg/ml L

Glut, 100 units/mI Penicillin G, 100 pg/ml Streptomycin sulfate). At the end of 4

days, the cells are stained for tartrate-resistant acid phosphatase (TRAP) activity

by permeabilization and acidification, followed by treatment with

para-nitrophenylphosphate for 5 minutes. Briefly, the media is aspirated off of

the cells, and 100 pl of citrate buffer (410 ml 0.1M citric acid, 590 ml 0.1 M

citrate, trisodium salt, 1 mL triton X-100) is added to each well and the plates are

incubated for 3 to 5 minutes at room temperature. One hundred microliters of

PNPP solution is then added (157.8 mg acid phosphatase reagent (Sigma 104

100), 7.2 ml tartrate solution (Sigma cat. no. 387-3), and 22.8 ml citrate buffer),

and plates are incubated for 3 to 5 minutes at room temperature. The reaction is

terminated by addition of 50 pl 0.5 M NaOH solution.

[0242] TRAP will convert para-nitrophenylphosphate to para

nitrophenol, which can be quantitated by optical density measurement at 405 nm.

The TRAP activity, which is a surrogate marker for osteoclast development,

therefore correlates with the optical density at 405 nm. A plot of optical density

versus aOPGL-1 concentration is shown in Figure 11, and demonstrates that

aOPGL-1 inhibits osteoclast formation in this assay.

Inhibition of OPGL Binding to its Receptor

[0243] The potency of aOPGL-1 is demonstrated by its ability to

block the binding of OPG ligand to its cognate receptor, the osteoclast

differentiation and activating receptor (ODAR, also known as RANK). This assay

uses homogeneous time resolved fluorescent resonance (HTRF) to detect

binding of aOPGL-1 to Europium-conjugated osteoprotegerin ligand (Eu-OPGL).

If aOPGL-1 inhibits Eu-OPGL binding to ODAR, fluorescent output will decrease,

and the amount of aOPGL-1 present will be inversely related to the amount of

fluorescence.

[0244] OPGL is labeled with europium, which emits light at 620 nm

when excited with 337 nm light. ODAR is fused to FLAG and to Fc, and the Fc

ODAR-FLAG fusion protein is labeled with an anti-FLAG antibody linked to

allophycocyanin (APC), a fluorophore which emits 665 nm light when excited by

light at 620 nm. Therefore, when Eu-labeled OPG ligand binds to the Fc-ODAR

FLAG/anti-FLAG-APC complex, the tertiary complex will emit 665 nm light when

excited with light at 337 nm.

[0245] Eu-OPGL at 0.05 pg/ml is preincubated with various

concentrations (0.1 to 150 ng/ml) of aOPGL-1 in assay buffer (50 mM Tris pH 8,

100 mM NaCl, 0.05% NaN 3, 0.1% BSA, and 0.05% Tween 20) at room

temperature for approximately one hour (Preincubation mix). A mixture of Fc

ODAR-FLAG (1 pg/ml) and anti-FLAG-APC (2.5 pg/ml) is also prepared in assay

buffer and incubated at room temperature for one hour (Fluorochrome mix).

Equal volumes of Preincubation mix and Fluorochrome mix are then combined and incubated at room temperature for 3 hours. The fluorescence is measured by reading plates on the Packard Discovery HTRF microplate analyzer using an excitation wavelength of 337 nm and an emission wavelength of 665 nm.

[0246] When aOPGL-1 is preincubated with Eu-OPG ligand and is

then mixed with Fc-ODAR-FLAG/anti-FLAG-APC, the fluorescence intensity at

665 nm decreases in a dose-dependent manner, as shown in Figure 12,

demonstrating that aOPGL162 can effectively inhibit OPGL binding to ODAR.

Example 6

Pharmacokinetics in Cynomolgus Monkeys

[0247] Six male and six female cynomolgus monkeys, not greater

than 4.5 years of age and weighing 2 to 4 kg are assigned to 4 dose groups.

Group 1 consists of 3 males and 3 females. Groups 2, 3, and 4 each consists of

1 male and 1 female. Animals in Group 1 are administered a single SC dose of 1

mg/kg aOPGL-1, while animals in Groups 2, 3 and 4 are administered single IV

doses of 0.1, 1.0, or 10.0 mg/kg of aOPGL-1, respectively.

[0248] Animals are dosed with aOPGL-1 expressed from

transfected chinese hamster ovary (CHO) cells. Serum samples are taken for

determination of aOPGL-1 levels, antibody analysis, and analysis of the bone

turnover marker serum N-telopeptide (serum N-Tx), alkaline phosphatase (ALP),

and serum calcium (serum Ca). Urine is also collected for analysis of N

telopeptide (urine N-Tx) and creatinine.

[0249] The serum concentration-time profiles following IV

administration are characterized by a tri-phasic distribution (Figure 13). Initially, there is a rapid distribution phase, followed by a significantly slower plateau phase, which appears to be concentration-dependent. The third observed phase is a rapid elimination phase.

[0250] Non-compartmental analysis of complete serum

concentration-time profiles using WinNonlin Professional (v.5), and exponential

analysis of the data up to 14 days after test article administration and above

,000 ng/mL using SAAM lI (v.1.2) are utilized to investigate the

pharmacokinetics of aOPGL-1 in monkeys. The initial volume of distribution from

all IV doses averages 28.9 ml/kg, similar to plasma volume. Steady state volume

(Vss) of distribution averages 39 ml/kg across all IV doses. Exponential analysis

indicates that aOPGL-1 has an average distribution half-life (ta) of 6.02 hours,

an extended secondary phase with a half-life (t)that increases with dose from

86.9 hours at a dose of 0.1 mg/kg to a maximum of 444 hours at a dose of 10.0

mg/kg. Terminal elimination half-life (tuz) estimated non-compartmentally

averages 31 hours across all IV dose groups. Clearance (CL, CL/F) of aOPGL-1

is found to be non-linear, with animals receiving IV doses of 10 mg/kg having an

average clearance (0.120 ml/hr/kg) that is 3.3-fold lower than those receiving 0.1

mg/kg (0.401 ml/hr/kg).

[0251] After administering subcutaneously, absorption is slow, with

average peak concentrations (Cm) of 11,600 ng/ml at 132 hr. There is high

variability in the range of exposure after SC administration, resulting in an

average clearance of 0.387 ± 0.281 ml/hr/kg and mean residence time of 202±

80.1 hours. Average bioavailability is 89%.

[0252] The preceding data are summarized in Table 5.

Table 5: Mean (± SD) Non-Compartmental Pharmacokinetic Parameters' in Cynomolgus Monkeys After Administering a Single Dose of aOPGL-1 IV and SC Non-Compartmental Parameter Estimates 1.0 mg/kg 0.1 mg/kg 1.0 mg/kg 10 mg/kg Parameter Unit SC (n=6) IV (n=2) IV (n=2) IV (n=2) Mean SD Mean Mean Mean

Tinax hr 132 60.2 0 0 0 Cmax ng/ml 11600 3410 4330 38200 326000 ty, hr 34.9 11.1 30.7 31.4 NDb

AUC(o.) pg*hr/ml 3520 1750 253 3950 99900 CL, CL/F ml /hr/kg 0.387 0.281 0.401 0.256 0.120 MRT hr 202 80.1 84.8 124 519 Vss mi/kg N/AC N/A 33.7 31.7 55.9 'Values are reported to 3 significant figures bNot Determined, PK samples end during plateau (P) phase hence terminal phase is not observed 'Not Applicable

[0253] aOPGL-1 causes a rapid decrease in serum N-Tx levels

within 24 hours post dose (Figure 14). The average time of maximum effect is

observed to occur between 12 hours and 7 days post dose as IV doses increase

from 0.1 to 10 mg/kg, and between 12 hours and 11 days in animals receiving a

SC dose of 1.0 mg/kg. Maximum effect increases with dose from approximately

to 91% over the dose range of 0.1 to 1 mg/kg. However, at higher doses no

further suppression is observed with maximum inhibition of 91%. Mean levels of

serum N-Tx return to baseline by day 28 after administering 0.1.mg/kg IV and by

day 70 after administering 1 mg/kg SC. Urine N-Tx shows similar trends to those

of serum N-Tx, except that all groups return to baseline values by study day 105

(Figure 15).

[0254] Suppression of serum Ca increases with dose to a mean

nadir of 31.6% below the baseline average seven days after IV administration of

10.0 mg/kg. All other dose groups have mean decreases in serum Ca of less

than 26.4% from their baseline averages. By day 17 all serum Ca levels in

treated animals return to within 10% of their baseline averages (Figure 20).

[0255] As bone resorption and formation are intimately linked,

changes in bone formation markers (ALP) are also observed with a much slower

decline in ALP levels and a more prolonged suppression than the formation

marker, N-Tx (Figure 21). Observing bone resorption markers decrease prior to

bone formation markers (ALP) following dosing with aOPGL-1 confirms that the

aOPGL-1 is a bone anti-resorptive agent.

[0256] The majority of animals (9 of 12) develop antibodies to

aOPGL-1. The incidence of antibodies to aOPGL-1 is not dose or route

dependent. It is not possible to assess the effect of antibodies to aOPGL-1 on

aOPGL-1 pharmacokinetics above 0.1 mg/kg when no dose group has both

antibody negative and positive animals. At 0.1 mg/kg IV, the majority of aOPGL

1 is cleared prior to antibody development and therefore, effects on aOPGL-1

disposition are not observed (Figure 16).

Claims (21)

We Claim:

1. A recombinant antibody that comprises a heavy chain and a light chain,

wherein:

(a) said antibody is a monoclonal antibody comprising: (i) a human IgG2

heavy chain comprising SEQ ID NO:13, and (ii) a human kappa light chain

comprising SEQ ID NO:14; and

(b) the antibody is expressed from a mammalian host cell; and

(c) said antibody binds to an osteoprotegerin ligand (OPGL) and inhibits

binding of OPGL to an osteoclast differentiation and activation receptor (ODAR).

2. The antibody of claim 1, wherein the mammalian host cell is selected from

the group consisting of: Chinese hamster ovary cell, HeLa cell, baby hamster kidney

cell, monkey kidney cell, and human hepatocellular carcinoma cell.

3. A recombinant antibody that comprises a heavy chain and a light chain,

wherein:

(a) said heavy chain comprises (i) a heavy chain variable region

comprising SEQ ID NO:13, and (ii) a heavy chain constant region comprising the

constant region of SEQ ID NO: 2, or having one carboxy-terminal amino acid

deletion of the constant region of SEQ ID NO: 2; and

(b) said light chain comprises (i) a light chain variable region comprising

SEQ ID NO:14; and (ii) a light chain constant region comprising the constant region

of SEQ ID NO: 4; and

(c) said antibody binds to an osteoprotegerin ligand (OPGL) and inhibits

binding of OPGL to an osteoclast differentiation and activation receptor (ODAR).

4. A recombinant antibody that comprises a heavy chain and a light chain,

wherein:

(a) said heavy chain comprises an amino acid sequence of SEQ ID NO:2

from residue 20 to residue 467, or having one carboxy-terminal amino acid deletion

of residue 20 to residue 467 of SEQ ID NO:2; and the light chain comprises an

amino acid sequence of SEQ ID NO:4 from residue 21 to residue 235;

(b) the heavy chain consists of an amino acid sequence of SEQ ID NO:2

from residue 20 to residue 467, or having one carboxy-terminal amino acid deletion

of residue 20 to residue 467 of SEQ ID NO:2; and the light chain consists of an

amino acid sequence of SEQ ID NO:4 from residue 21 to residue 235.

5. A pharmaceutical composition comprising a therapeutically effective

amount of the antibody defined in any one of claims 1 to 4 and a pharmaceutically

acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.

6. A method of treating a bone disorder characterized by a net bone loss,

comprising administering an antibody of any one of claims 1-4, or a pharmaceutical

composition of claim 5.

7. The method of claim 6, further comprising administering a

therapeutically effective amount of another therapeutic agent.

8. The method of claim 6 or 7, wherein said bone disorder is osteopenia

or osteolysis.

9. The method of any one of claims 6-8, wherein the bone disorder is:

(a) an osteopenic disorder;

(b) an osteopenic disorder selected from post-menopausal osteoporosis,

Paget's disease, lytic bone metastases, and rheumatoid arthritis; or

(c) an osteopenic disorder selected from osteoporosis, periodontitis, and

bone loss due to immobilization.

10. The method of claim 6 or 7, wherein:

(a) the bone loss is associated with cancer;

(b) the bone disorder is an osteopenic disorder associated with a cancer

that increases osteoclast activity and induces bone resorption; or

(c) the bone loss results from osteolytic destruction of bone caused by

malignant or metastatic tumors.

11. The method of claim 6 or 7, wherein the bone loss is associated with

cancer, and wherein the method further comprises administering the antibody with at

least one cancer therapy agent selected from radiation therapy and chemotherapy.

12. The method of claim 11, wherein the chemotherapy involves treatment

with at least one agent selected from an anthracycline, taxol, tamoxifene,

doxorubicin, and 5-fluorouracil.

13. The method of any one of claims 10-12, wherein the cancer is breast

cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, esophageal cancer, rectal cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, or cancer of the gastrointestinal tract.

14. The method of any one of claims 10-12, wherein the cancer is a

hematological malignancy selected from the group consisting of multiple myeloma,

lymphoma, and Hodgkin's Disease.

15. The method of claim 6 or 7, wherein:

(a) the bone loss is from osteolytic destruction of bone caused by a

malignant tumor or a metastatic tumor; or

(b) the bone loss is associated with multiple myeloma.

16. The method of claim 6 or 7, wherein said bone disorder is:

(a) hypercalcemia;

(b) hypercalcemia, wherein the hypercalcemia is a result of a solid tumor;

(c) hypercalcemia according to (b), wherein the solid tumor is selected

from a solid tumor of breast, lung, and kidney;

(d) hypercalcemia, wherein the hypercalcemia is a result of hematologic

malignancy;

(e) hypercalcemia according to (d), wherein the hypercalcemia is a result

of multiple myeloma;

(f) hypercalcemia according to (d), wherein the hypercalcemia is a result

of lymphoma or leukemia; or

(g) idiopathic hypercalcemia, hypercalcemia associated with

hyperthyroidism or hypercalcemia associated with a renal function disorder.

17. A method of preventing or mitigating the onset of bone loss caused by

metastatic cancer, comprising administering prophylactically an antibody of any one

of claims 1-4, or a pharmaceutical composition of claim 5.

18. A method of treating an existing condition of bone loss due to

metastasis, comprising administering an antibody of any one of claims 1-4, or a

pharmaceutical composition of claim 5.

19. Use of an antibody of any one of claims 1-4, or a pharmaceutical

composition of claim 5 for the manufacture of a medicament for treating a bone

disorder characterized by a net bone loss.

20. Use of an antibody of any one of claims 1-4, or a pharmaceutical

composition of claim 5 for the manufacture of a medicament for preventing or

mitigating the onset of bone loss caused by metastatic cancer.

21. Use of an antibody of any one of claims 1-4, or a pharmaceutical

composition of claim 5 for the manufacture of a medicament for treating an existing

condition of bone loss due to metastasis.