AU2015264940B2 - 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

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 [002a] 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.

[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 _

2015264940 07 Dec 2015 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 yftamsn D alter the responsiveness of bone to parathyroid hormone and calcitonin» p304] Osteoprotegedn ligand (OPGL), which is a member of the

TNF family of cytokines, promotes formation of osteoclasts through binding to the receptor activator of Nr-gB (RANK, also called osteoclast differentiation and activation receptor, or ODAR), Osteoprotegerin (OPG), on the other hand,

Inhibits the formation of osteoclasts by sequestering ORGL and preventing OPGL association with ODAR. Thus, the amount of OPGL associated with ODAR correlates with the equilibrium between bone deposition and resorption.

[DOS] After skeletal maturity, the amount of bone In the skeleton reflects the balance tor 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 mess 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 tire rats of bone synthesis or an increese in the rate of bone destruction or both, A common form of osteopenia is primary osteoporosis, also

2015264940 07 Dec 2015 referred to as posfmenopausai 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 done resorption with a normal rate of hone 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 inteMrochanterfc fracture in women 45 years and older. Elderly males may develop symptomatic osteoporosis between the apes of SO 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 osteociastogenesis, i(XPj 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 bisphosphonr selective estrogen receptor modifiers (SERMs; have emerged tor the and treatment of reduced bone mass. Thus, Il may be useful to combine those treatments with molecules that can regulate the activity of OPGL In certain osteopenia disorders,

2015264940 15 Dec 2017 [007a] 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 [007b] According to a first aspect, the present invention provides an antibody or binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR1, CDR2 and CDR3 ofSEQ ID NO: 13 and wherein the light chain comprises an amino acid sequence of SEQ ID NO: 14 wherein the antibody interacts with an osteoprotegerin ligand (OPGL).

[007c] According to a second aspect, the present invention provides a pharmaceutical composition comprising an antibody or binding fragment thereof of the invention and a pharmaceutically acceptable excipient, diluent or carrier.

[007d] According to a third aspect, the present invention provides an isolated composition comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a heavy chain and the second polynucleotide encodes a light chain, and wherein the first and second polynucleotides together encode an antibody or binding fragment thereof of the invention.

[007e] According to a fourth aspect, the present invention provides a method of treating bone loss in a subject comprising administering an antibody or binding fragment thereof of the invention or the pharmaceutical composition of the invention or the composition of the invention.

[007f] According to a fifth aspect, the present invention provides a host cell comprising the composition of the invention.

[007g] According to a sixth aspect, the present invention provides a method of producing an antibody or binding fragment thereof that interacts with osteoprotegerin ligand (OPGL) comprising culturing a host cell of the invention.

2015264940 15 Dec 2017 [007h] According to a seventh aspect, the present invention provides an antibody or binding fragment thereof when produced by the method of the invention.

[007i] According to an eighth aspect, the present invention provides use of an antibody or binding fragment thereof of the invention or a composition of the invention in the preparation of a medicament for treating bone loss.

[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.

[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

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2015264940 07 Dec comprises an amino acid sequence as set forth in SEQ ID NO; 4 or a fra •hereof [0131 in certain embodiments, the invention provides for an antibody comprising a -heavy chain and alight chain, wherein thelight chain, comprises a variable region comprising an amirioaoid 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, end wherein the first variable region comprises a sequence that has at least &0% 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 IO NQ: >4, and (ci wherein foe antibody interacts with sn osteoprotegorin ligand (QPGL).

[015] In certain embodiments, the first variable region comprises a sequence thaf.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 35% identity to the amino acid sequence set forth in SEQ ID NO: 14, [046] in certain embodiments, the first variable region comprises a sequence that has at least 9S% identity to the amino acid sequence set forth in SEQ ID NO; 13, and the second variable reg-son comprises a sequence that has at least S9% Identity to the amino acid sequence set forth In SEQ ID NO; 14.

2015264940 07 Dec 2015 r

in certain embodiments, the invention provides for a heavy chain, comprising an amino acid sequence as set forth in SEQ ID MO: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 -n 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 sot forth in SEQ ID NO;4 or a fragment thereof, in certain embodiments, the Invention provides for a light chain comprising an ammo acid sequence as set forth in SEQ ID ME): 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')z antibodies s f020j - In certain embodiments, a pharmaceutical composition comprising an antibody of the invention is provided. In certain embodiments, a pharmaceutical composition compr.smg a ihorapeuticsily effective amount of an antibody to OPGL Is provided.

[XI] in certain embodiments, a pharmaceutical composition comprises an antibody to GPGL and at least one therapeutic agent selected from

2015264940 07 Dec 2015 a bone morphogenic factor, transfarming growth facter-β (TGr 'β), an inssrleukin1 01-1} inhibitor, iL-Ira, Kirteret™, a TNFa inhibitor, a soluble TNrn receptor, Enbrei™, an anti-TNFo antibody, Esroicade™, 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, fiuonde, calcium, a non-sterolddi sntimfiammatory drug (NSAID), a COX-2 Inhibitor, Celebrex™, Vioxx™; an immunosuppressant, methotrexate, teflunomide, a serine protease Inhibitor, a secretory leukocyte protease Inhibitor (SLPI), an IL-6 Inhibitor,, an antibody to IL6, an IL-8 inhibitor, an antibody to IL-8, an IL-IS inhibitor, an IL-18 binding protein, an IL-18 antibody, an lnterieukirt-1 converting enzyme-(ICS) modulator, a fibroblast growth factor (FGF), an FGF modulator, a PAF antagonist, a keratlnocyte 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 lipapofysaccharide (LPS) levels, a noradrenaline, a noradrenaline mimetic, and a noradrenaline modulator.

[022]· In certain embodiments of the invention. a method cf treating an osteopenia disorder Is provided, comprising sdmlntsfering a pharmaceutically effective amount of an antibody, In certain embodiments, a method of treating an osteopento disorder comprising administering a pharmaceutical composition Is provided,

2015264940 07 Dec [Θ23] ip certain: embodiments, a method of treating .an inflammatory condffion with attendant bone loss in a patient comprising administering a pharmaceutical composition is provided.

[024] in certains embodiments, a method of treating an acfotmmuR© condlWn with attendant bone loss in a patient comprising administering a pharmaceutical composition is provided, [025] in csnain embodiments: a method of treating rheumatoid arthritis in a patient, comprising administering a pharmaceutical composition or the Invention is provided, (02©J 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 OR THE FIGURES [027] Figure 1 shows a cDNA sequence encoding the oORGL-rt antibody heavy chain. (SEQ ID NO: 1), [023] Figure 2 shows the amino acid sequence of the aOPGL-1 antibody heavy chain (SEQ ID RO; 2).

[029] Figure 3 shows a cDNA sequence encoding the gOPGL-1 antibody light chain (SEQ ID NO; 3), [030] Figure 4 shows the amino acid sequence of the «QPGl-1 antibody light chain (SEQ ID NO; 4),

2015264940 07 Dec 2015 (0311 Figure 5 shows a schematic diagram of the oOPGL»1 kappa light chain expression plasmid i/pBSRalS, [G32] Figure 6 shews a schematic diagram of the aGPGL-l IqG heavy· chain expression plasmid, aOPGL-l-k [033] Figure 7 shows dose-dependent binding of oOPGl-l to

OPGL-oosted HA plates.

: (034} Figure 8 shows specific binding of qOPGl-1 to memhrant hound OPGL.

[055] Figure 9 shews inhibition of aOPGL-1 binding to OPGLcoated ElA plates by soluble OP-SI..

]036] Figure 10 shows specific: binding of oOPGL-1 to OPGLcoalsd EiA pleres, [033] Figure 11 shows dose-dependent inhibition of osteoclast formation by oOPGU-t.

[038] Figure 12 shows dose-dependent inhibition of OPGL binding to ODARby oOPGL-1.

[039] Figure 13 shows the mean serum concentration finis profiles after administering s single doss of aQPGL-1 to Cynomolgus Monkeys.

[040] Figure 14 snows the mean percent change in serum N-Tx concentration after administering a single dose of oOPGLG to Oynomolgus Monkeys.

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2015264940 07 Dec

Figure 5 shows the n:san percent change he urine N-Tx joncentraticn after administering a single dose of aOPGL·! to Cynomolgus

Monkeys.

[042] Figure 16 shows antibody positive and negative serumconcentration time profiles after administering a Gynometgus Monkeys.

:ng!e dose of gOPGL-1 to [043] Figure 17 shows the amino add sequence of the dOPGL-d antibody heavy chain variable region (SEQ ID NO: 13),

1944) Figure 18 shows the a ml no acid sequence of the aOPGL-1 antibody light chain variable region (SEQ ID NO; 14), (04SJ Figure 19 shows, a eeli culture precess for production of gOPGL-1 _K [046} Figure 20 shows the serum calcium percent change after administering a single dose of oOPGL-1 to Cynoreoigus monkeys.

[047) Figure 21 shows the mean serum Alkaline Phosphatase percent change after administering a single dose of uOPGL-1 to Cvnomoigus monkeys,

DETAILED DESCRIPTION OF CERTAIN PREFERRS jODiMENTS pMSj The seqtton headings used: herein are for organizational purposes only and are not to be construed as ism-ting the subject matter described. Ail references cited in this application are expressly incorporated by reference herein for any purpose.

2015264940 07 Dec 2015

Definitions

1049} Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture end transformation (e.g., electroporation, lipofectson). Enzymatic reactions and purification techniques nW be performed according to manufacturer’s specifications or as commonly sccompfished in the art. or as described herein. The foregoing techniques and procedures rosy 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)5, which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized tn connection with, and the laboratory procedures end techniques of, analytical chemistry, synthetic organic chemistry, and medicinal snd pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthases, 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 fbf tewing meanings:

(051| The term isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some

2015264940 07 Dec 2015 combination thereof, which by virtue of its origin the Isolated polynucleotide (1) is not associated with all or a portion of a polynucleotide ip which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide which it is not linked to in nature, or {3) doss not occur in nature as part of a larger sequence, [052] The term “isolated orofeln referred to herein means a protein encoded by cDNA, recombinant RNA, or synthetic origin or some combination thereof, winch 1) is free of at least some proteins with which it would normally be found, (2) is essentially free of other proteins from the seme source, e,g„ from the same species, (3) is expressed by a ceil from a dtteent 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 ammo acids of the native sequence. The term ‘'polypeptide also encompasses aOPGL-1 (as described below, SEQ iD NO: 2 and SEQ ID MO: 4), or sequences that have deletions, additions, and/or substitutions of one or more amino acid of οΟΕ'ΘΕ-Ι, According to certain embodiments, the invention comprises the human heavy chain immunoglobulin moieoule represented by Figure 2 (SEQ ID MO: 2) arid tire human light chain immunoglobulin molecule represented by Figure 4 (SEQ ID NO: 4), or fragments of .analogs thereat [054] The term naturaiiy-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 •’S J4L

2015264940 07 Dec 2015 viruses) that can be isolated from a source in nsture and which has not been intentionally modified by man in the laboratory or otherwise is natoraily-occurrtng, >055) The term operably linked as used herein refers to components that arc in s 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 will's the control sequences.

[DS6J The tend ’toontrdi sequence as used herein: refers to polynucleotide sequences which may effect the expression and processing of coding sequences to which they are ligated, The names of such control sequences may differ depending upon the host organism. According to certain embodiments, control sequences for prokaryotes may include promoter, ribosomai binding she. and transcription termination sequence. Accc-fdlng 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.

[QS7] The term ’’polynucleotide'' as referred to herein means a polymeho form of nucleotides of at least 10 bases in length. In certain embodiments, the bases may be ribonucleotides or deoxydhormcieotides or a modified form cf either tope of nucleotide. The term includes single and double stranded forms cf DNA,

F053I The term oligonucleotide referred to herein includes naturally occurring, and modified nucleotides linked together by naturally

2015264940 07 Dec 2015 occurring, and/or non-naturaily occurring oligonucleotide linkages. Oligonucleotides are a polynucisotlde subset generally comprising a length of 200 buses 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. Olloonuoteofides 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 deoxyribcnucieotides and ribonucleotides. The term modified nucleotides'¹ includes nucleotides with modified or substituted sugar groups and the like. The term ’‘oiigd.nucieotide linkages Includes otigonucfeotides linkages such as p tios phcrothioste, p h os pfi orc d it h lost e. pho sp h oroseleneats.

phosphorodlselenoate, phosphoroar.iloihtoats, phoshoranliadate, phesphoroamidatss and the like. SaOtB.g., LaPianche etai. Nuci. Acids Res. 14:9081 (1986): Stec etal. J. Am, Chem. See. 106:6077 (1934): Stein et at Nuci Adds Res. 16:3209 (1988); Zen et al Anti-Cancer Drug Design 6:539 (1991);

Zon st al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (r. Eckstein, Ed., Oxford University Press, Oxford England (1931)): Stec et al U.S. Pat, No, 5,151,510; Uhimann and Peyman Chemical Reviews 90:643 (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 net Irrufad

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2015264940 07 Dec 2015 to, those described In Computations! Molecular Biology, desk, A.M„ sd.. Oxford University Press, New York (1S88); Biocomputing? informatics and Genome Projects. Smith, D.W,, ed„ Academic Press, New York (198-3); Computer Analysts of Sequence Data,. Part 1, Griffin, AM. and Griffin, ecs,, Humana

Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G„ Academic Press (1937); Sequence Analysis Primer, Gribskov, M. and Deveraux, J., eds„ M. Stockton Press, New York (1991); and Carilio era/., SIAM J, A.pp/fed Mato,, 48:1073 (1988).

[081J Preferred methods to determine identity: a® designed to give

Iks 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 ere not limited to, the GCG program package, including GAP (Devereux ei a/., Nuci, Acid. Res., 12:387 (1984): Genet,cs Computer Group, University of Wisconsin, Madison, Wl, SLASTP, BtABTM and PASTA (AfeGhul to a/.,../, Mo/. SM 215:403-41 Q (1990)), The BLASTX program is publicly available from the National Center tor Biotechnology Information (NCBl) and other sources (SLAST fVfeni/s/, Altschul tost. NCB/NLM/NIH Bethesda, MD 20894: Altschui ei at, supra (1SSO)), The well-known Smith Waterman algorithm may also be used to determine identity, [062) Certain alignment schemes tor aligning two amino acid sequences may result to the matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity

2015264940 07 Dec 2015 even though there is no significant relationship oetween the two fuil-iengtn 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,

For example, using the Computer algorithm GAR (Genetics

Computer Group, University of Wisconsin,. Madison, WI), two polypeptides for which the percent sequence Identity Is to be determined ere 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 ‘‘diagonal¹¹ 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 s comparison matrix such as PAM 250 or BUOSUM 62 are used in conjunction with the algorithm, In certain embodiments, a standard comparison matrix (see Dayhoff et. a/., Arias of Protein,Sequence and Structure, 6(3)(1 STB) for the PAM 250 comparison matrix; Henikoff et si, Proc, Nad. Acad, Sc/ USA, 89:10915» 10919 (1992) for the 8LOSUM 62 comparison matrix) Is also used by vhe algorithm.

[064) In certain embodiments, the parameters for a polypeptide sequence comparison include the following:

Algorithm: Needleman ef«/,, J. Mof S;o/., 48:443-453 (1970); Comparison matrix: BLOSUM 62 from Henikoft era/,, supra (1992); Gap Penalty: 12

Gap Length Penalty: 4

2015264940 07 Dec 2015

Threshold of Similarity; 0 [0S5J The GAP program may be asWi with, tte above· paraffletem.

in certain embodimente, the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm, [QS@] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Irnmunoiogy-A Synthesis (2nd Edition, E, S, Golub end 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 conventions! amino acids,, unnatural amino acids such as α-, α-d’substituted arnlno acids, N~slkyl amino acids, lactic acid, and other unconventional amino acids may afed be suitable •components for polypeptides of the present Invention,. Examples of unconventional amine acids include; 4-hydraxyproiine, y-carfcoxygiutamale. εΝ,Ν,Ν-tnmsthyHysine. e-N-sc&tyiiysine, O-phosphoserine, N-acetylserine. Nformylmetftionine, 3-mathylhistidina, 5-hydfcxyiysine, σ-Ν-rnethyiarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyprofine). in the pciypsptlds notation used herein, the left-hand direction Is the amino terminal direction end the right-hand direction Is the cerboxy-termlnai direction, in accordance with standard usage and convention, [Q67] Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of double errs vied polynucleotide sequences ss referred to as the 5' direction. The

2015264940 07 Dec dwefen of 5' to 3 addiUsn ofmdscent RNA franscnpfe Is raferred;tb as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which sre 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 end which are 3’ to the 3’ end of the RNA transcript are referred to as downstream sequences, [06SJ Conservative amine acid substitutions may encompass nonnaturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis ip biological systems. These include peptidornimetics and other reversed or inverted forms of amino acid moieties.

[069} Naturally occurring residues may be divided into c4asses based on common side chain properties:

1) hydrophobic: norieucine, Met, Ala, Val, Leu, tie:

2) neutral hydrophilic: Cys, Ser, Thr, Asm 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]' Per example, nonroonservabve substitutions may involve tris exchange of a member qf 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.

1.8

2015264940 07 Dec 2015 [0711 in making such changes, according to certain embodiments, the hydropathic index ci amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of Its bycirophobicity and charge characteristics. They are: isofeucine (+4.5); valine (+4,2): leucine (+3,8): phenytefertine (+2,8); cysteine/cystlne (+2,5); methionine (+1.9); alanine (+1.8): glycine (-0,4); threonine 00.7): serine (-0.5); 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 ef a/., J. Me/. S?oi<, 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, to making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included, to certain embodiments, those which ar© within £f are included, and in: certain embodiments, those within :±0 J: are included, [073) ^; If is also unddrsfood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophlHcity, 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 hydrophlllcsty of a protein, as governed

IS

2015264940 07 Dec 2015 by the bydroghlHclty cf its adjacent amino acids, correlates with its immunogeniafty and antigenicity, ie,, with a hfetegfcaf property of the protein, '[0743 The following hydrophsiic'rtv values have been assigned to these amino acid residues: arginine- (+3.G); lysine .(+3,0); aspartate (+3.0 ± 1); .glutamate (+3.0 ± 1); senna (+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 hycrophificny values, In certain embodiments, the substitution of amino acids whose hydropMieity values are within ±2 is Included, in oenain embodiments, those which are within ±1 are Included, and in certain embodiments, those within ±0,5 ere included. One may also identify epitopee from primary' amino acid sequences on the basis cf hydrcphiliolty. These regions are also referred to as “epitopic core regions.” [0751 Exemplary amino acid substitutions are set forth in Table 1,

Table 1: Amino Acid Substitutions

Original Residues	Exemplary Substitutions	Preferred Substitutions
Ala	Val, Leu, lie	Val
..... Arc...	Lye, Gin, Asn	Lys
Asn	Gln	Gin
Asp'	Glu	Git;
Cys	Ssr, Ala	Ser
Girt	Asn	Asn
Glu	Asp	Asp
Giy	Pro, Ala	Ala
His	Asn. Gln, Lys, Arg	Arg
lie	Leu, Vsi, Met, Ala, Phe, Norieucine	Leu

2015264940 07 Dec 2015

j Leu	Norleucine, ile, Val Met, Ala, Phe	lie
f	Lys	Arg, 1,4 Dlaminobutyrtc Acid, Gin, Asn	.....
	Met	Leu, Phe, lie	Leu
	Phe	Leu. Val, lie, Ala, Tyr	Leu
Pro	.Ala	Gly
	Ser	Thr, Ala, Cys	Thr
	Thr	Ser	Ser
	Tm	Tyr. phe	... Tyr
	‘M —	Trp, Phe, Thr, Ser	Phe
	Val	lie, Met, Leu, Phe. .Ala, Nodeucine	Leu

(076J ,A skilled artisan wiii be able io determine suitable variants of the polypeptide as set forth herein using well-known techniques, In certain emDocuments, one skilled ;n 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 mofecufes that are conserved among simitar polypeptides, in certain embodiments, even areas that may be important for biotogicai activity or or structure may ba subject to conservative amine acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure,

1077] Additionally, one skilled in the art can review structurefunction 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 id simifer proteins, One

2015264940 07 Dec 2015 skilled in the art may opt for chemically similar amino add substitutions for such predicted important amino acid residues, [076] 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 amine 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 io be on the sumacs of the protein, sines such residues may be involved In important interactions with other molecules. Moreover, one skilled In the art may generate •test variants containing a singie amino acid substitution at each desired amino acid residue. The variants can then bo 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, or 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 a one or in combination with other mutations, [075] A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in β/οήοοή., 7(4):422427 0996), Chou efa< Biochemistry, 13(2):222-245 (1374): Chou efe/., Biochemistry, 113(2):211-222 (1874): Chou et&l... Adz. Enzymei. Retet Areas ο

CM

2015264940 07 Dec

Mc-L 47:45-148 (1978): Chou ei s/.„ Ann. Rev. S/ocften?., 47:251-278 and Chou &t s'., S/opbys. A, 28:267-584 (1970), Moreover, computer programs are currently available to assist with predicting secondary structure. One meihna Ά piWer.ne secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 3Q%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural database (PD8) has provided enhanced predictability of secondary structure, including the potential cumber of folds within a polypeptide’s or protein’s structure. See Holm ere/., MM Acid. Res., 27(12244-24? (1909). it has been suggested (Brenner etai, Cum Op, Struct, Biol., 7(3):389-376 (1937)) that there are a limited number of folds in a given polypeptide oh protein and that once a critical number of sfmetures have been resolved, structural prediction will become dramatically more accurate.

[080] Additional methods of predicting secondary structure include ’’threading (Jones, D,, Cum. Opin, Struct. Bici. 7(3):377-87 (1897); Sippi of at, Structure, 4(1):13-19 (1998)), profile analysis (Bowie eta/,, Science, 253:164:70 (1991); Gribskov et a/., Mete, Enzym., 183:148-159 (1990): Grlbskov ets/., Proc. Nat, Acsd. 8tte₍ 84(13):4355-4358 (1987)), and evolutionary linkage (See Rote, supra (1999), and Brenner, supra (1997)).

[081} In certain embodiments, antibody variants include giycpsytation variants wherein the number and/or type of glycosyiaticn 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

2015264940 07 Dec 2015 of N-linked glycosylation sites thsn the native prorien. An fcMinkeri giycosyl&tion sits is characterized by the sequence: Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated -as X may be any amino acid residue except proline. The substitution of amino acid residues io create this sequence provides a potential new site for the addition often HMinked carbohydrate chain. Alternatively, substitutions which eiiminate this sequence will remove an existing N-linked carbohydrate chain, Also provided is a rearrangement of N-iinked carbohydrate chains wherein cue or more Ν-ilnked glycosyiafion sites (typically those that am naturaliy occurring) are eliminated and ora or more new N-linked sites ere created. Additional preferred antibody variants Include cysteine variants wherein one or more cysteine residues are deleted from or substituted for soother amino acid (e,g._; serine) as compared to the parent amino acid •sequence. Cysteine variants may be useful when sntsbcdles 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, [Q82J According to certain embodiments:» amino acid substitutions are those which: ( !) 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 physlocochernlcai or functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino

Ο

CN

2015264940 07 Dec acid substitutions) may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain's) forming intermoiecuiar contacts), in certain embodiments, a conservative amino acid substitution typically may not substantially chance 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. rl. Freeman and Company, New York (1384)); Introduction to Protein Structure (C, Branded and j. Tooze, eds,, Garland Publishing, New York, N.Y. (1891)); and Thornton et at Nature 354:105 (1381). which are each Incorporated herein by reference.

(Q83] The term ’’polypeptide fragment” .as used herein refers to a polypeptide that has an aminc-terrnina! andfor carboxy-terminal deletion, in certain embodiments, fragments are at least 5 to 48? amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, δ, 8, 10. 14, 20, 50, 70, 100, 150. 200, 250, 300, 350, 400, or 450 arninc acids long.

[084] .< Peptide analogs are commonly used ^!n the pharmaceutics! indristry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound sire termed peptide mi.msiics¹¹ or peptidcmimetics”. Fauchere, J, Adv, Drug Res, 15:28 (1986): Veber end FreidingerTINS p.332 (1985): and Evans et al, J. Med. Chem. 30:12.29 (1987), ;h are incorporated herein by reference for any purpose. Such compounds

2015264940 07 Dec 2015 are often developed with the aid of computerized molecular modeling'. Peptide mirnetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect Qaneraiiy, peptidomimeiics ere structurally similar to a paradigm polypeptide (I.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, out have one or mors peptide linkages optionally replaced by a linkage selected from: -CHg ΝΗ--, ~CK₂ S~~ -CH₂ -CH_g ~CW“CH-{cis and trans), -C0CK₂ -CH(OH)GHs -, and -CHg SO-, by methods wail known in the art. Systematic substitution of one or mere amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-iysine in piece of Llysins) 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 bymethods known in the art (Rfeo and Gierasch Ann. Rev, Biochem. SI :337 (1S92), incorporated herein by reference for any purpose); for example, by adding internal cysteine residues capable of forming Intramolecular disulfide bridges which cycliza the peptltfc.

((385] · Antibody or antibody pepiide(s) refer to an Intact antibody, or $ binding fragment thereof that competes with fho 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

2015264940 07 Dec 2015 dagmento include, but are not limited to, Fab, Fab¹, F(eb'}2, rv, and single-chain (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· seouence to confer specificity for an OPGL. A fufi-length heavy chain Includes a variable region domain, v’k, and three constant region domains, Cwt, Cm2, and C_h3. The Vh domain is at the amino-terminus of the polypeptide, and the Ch3 domain is at the carbody-terminus, The term “heavy chain, as used herein, encompasses a full-length heavy chain and vagmento thereof, A full-length light chain includes a variable region domain, vT, and a constant region domain, C_L, Like fee heavy chain, the variable region oomain of the light chain is at the amino-terminus of the polypeptide. The term light chain, as used herein, encompasses a fulllength light chain and fragments thereof, A Fab fragment is comprised of one light chain and the C_H1 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 feat contains more· of the constant region/between fee Cfel i=hd C_H2 domains, such that an interchain disulfide bend can ba formed between two heavy chains to form a Ffab')2 mclecule, The Fv region comprises the variable regions from both the heavy and light chains, but lacks the constant regions,. Singfe-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

2015264940 07 Dec forms an antigen-binding region. Single chain antibodies are discussed in detail In WO 801649 and U.S, Patent Nos. 4,848,778 and 8,280.203..

[087] A. bivalent antibody other than a multispecific or multlfancttonal antibody, in certain embodiments, typfcaliy is hnderstocd to have each cf Its binding sites identical, |©88J 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%, 80%, 30%, 85%. or more (as measured In an in v/too competitive binding assay), [OSSj The term epitope” Includes any polypeptide determinant capable of specific binding to an immunogiobutin or T-ceii receptor, in certain embodiments, epitope determinants intoude 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, snd/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 specificaily bind an antigen when it preferentially recognizes Ils target antigen in a complex mixture cf proteins and/or macromoiecules. In certain embodiments, on antibody is said io specifically bind an antigen when the dissociation constant is si μΜ, In certain embodiments, when the dissociation constant is art00 nM, and in certain embodiments, when the dissociation constant ss <10 nM

2015264940 07 Dec 2015 [030] The term agent is used herein to denote a chemical compound, a mixture of chemical compounds, a bloiooical macromoiecuie, or an extract’ made from biological materials.

(WI] 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 ano 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 To, 111 In, 125 i, 131 I), fluorescent labels (e.g,, HTC, rhodamine, lanthanide phosphors}, enzymatic labels (e.g., horseradish peroxidase» 3-geiactosidase, luciferase, alkaline phosphatase), chemiluminescent, bioiinyi groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.a„ 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 sterie hindrance.

jO9S] The term “biological sample”, as used herein, includes, but is not limited to, any quantity of s substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, monkeys, refs, rabbits, and other animals. Such substances include, but ere not limited to,

2015264940 07 Dec 2015 blood, serum, urine, cells, omens, tissues, bone, bone marrow, lymph nodes, and skin.

[0t3f The term osteopenia disorder* includes, but is not limited to osteoporosis, osteopenia, Paget's disease, lytic bone metestases, periodontitis, rheumatoid arthritis, and bone less due to immobilization, in addition to these bene 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 io 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 ooservsd in the absence of the modulator. In certain embodiments, a modulator is sn inhibitor, which decreases the magnitude of at least one activity or function of a moiecuie. Certain exemplary activities and functions of a molecule include, taut are not limited to, binding affinity, enzymatic activity, and signal transduction. Certain exemplary'· inhibitors Include, but are not limited to, proteins, peptides, antibodies, peptlbodles, carbohydrates or small organic rpoieouies. Pootibodies are described; e.g., In WOOi,’83525_s

2015264940 07 Dec 2015 pSS: As used herein. toubstantlally pure means an object species is the predominant species present (i.e., on a molar basis It is mors 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) cf all macromolecular species present. In certain embodiments, a substantially pure composition will comprise more than about 80%, 86%, 30%, 35%, or 99% of all macfomolsr species present in the composition. In certain embodiments, the object species is puufed to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromoleculsr species, [037] The term patient includes human and animal subjects.

[0931 In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of tor means and/or unless stated otherwise. Furthermore, the use of the term including”, as well as other forms, such as includss” 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 then one subunit unless specifically -stated. btbenvfesy [099] Osteoprotegerln Ligand (OPGL), a member of the tumor necrosis factor (TNr) family of cytokines, is involved in the formation of osteoclasts. increased osteoclast activity' correlates with a number of osteopensc

2015264940 07 Dec 2015 disorders, including post-menopausal osteoporosis, Paget's disease, lytic bone mafsstases, and rheumatoid arthritis. Therefore, a reduction in OPGL activity may result in a decrease in osteoclast activity and may reduce the severity of osteopenia disorders. According to certain embodiments of the Invention, antibodies directed to OPGL may be used treat osteopenia disorders, Including by not limited to, those mentioned above.

10100] In certain embodiments of the present invention, there·is provided a fully human monoclonal antibody against human osteopmtegerin ligand (OPGL), in certain embodiments, nucleotide sequences encoding, and amino acid sequences comprising, heavy end light chain immunoglobulin molecules, particuiany sequences corresponding to the variable regions are provided. In certain embodiments, sequences corresponding to complementarity deterrrilning regions (CBiTs), specifically from CORI through CDR3, are provided. According to certain embodiments, a hybridoma coll line expressing such an Immunoglobulin molecule and monoclonal antibody is also provided, tri certain embodiments, purified human monoclonal antibody against human OPGL is provided.

[01011 The ability to clone and reconstruct megabase-sized human loci in yeast artificial chnomosomes (Y.ACs) and to introduce them into the mouse germh'ne provides an approach to elucidating the functional components of very large or crudely mapped led as well as generating useful models of human disesse.· Furthermore, the utilization of such technology for substitution of mouse loci with their human equivalents court· p cvide unique insights inte the

2015264940 07 Dec 2015 expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression,

10i OS] An important practical application of such a strategy is the ‘'humanization of the mouse humorai immune system, introduction of’ human immunoglobulin (!g) 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 me r ole in S-ceii development. Furthermore, such a strategy could provide a source for production of fully human monoclonal antibodies (MAbs). In cartam embodiments, fully tinman 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 ortho administered antibodies, in certain embodiments, fully human antibodies may be used in tine treatment of chronic and recurring human diseases, such es osteoporosis, inflammation, sutoimmunify, and cancer, which may Involve repeated antibody administrations, (0103] · One can engineer mouse strains deficient In mouse antibody praduction with iarga 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* 3y exploiting mouse machinery for antibody diversification and selection and the lack of

2015264940 07 Dec 2015 immunological tolerance to- human proteins, the reproduced human antibody repertoire in these mouse «trams may yield high affinity antibodies against any antigen of interest, including human antigens. Using the bybridoma tschnciogy, antigen-specific human MAbs with the desired specificity may ba produced and selected.

[01041 in certain embodiments, one may use constant regions from species other than human along with the human variable reglon(s).

Naturally Occurring Antibody Structure

10105] Nafurally occurring antibody structural units typically comprise a tetramar. 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 kOe), The amino-terminal portion of each chain typically Inciudss a variable region of about 100 to 110 or more amino acids that typically ts 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, end define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG nas several subclasses, including, but not limited to, IgGT lgG2. igG3, sod lgG4, ioM has subclasses including, but not limited to, lgM1 and igM£. IgA Is similarly subdivided into subclasses including, but not limited to,

2015264940 07 Dec 2015 igA1 and jgA2. Within full-length light and heavy chains, typically, foe variable and constant regions are joined by aΛΓ region of about 12 dr more amino adds,.

with ‘he heavy chain sisc includina e D reqlcn of about 10 more amino

ΛΓ’ϊγ»’»

See, e.g., Fundamental immunology Ch. 7 (Paul, W., ed., 2nd &L 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 (PR) 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 rosy enable binding to a specific epitope. From N-terminas to Gterminal, both Sight and heavy chain variable regions typically comprise the domains FR1, CDR1, PR2, CDR2, PR'S, CDRS and FR4. The assignment, of amino acids to each domain Is typlcaliy in accordance with the definitions of Rabat Sequences of Proteins of immunological interest (National Institutes of Health, Bethesda, Md, (1987 and 1991)), or Chothla & Leah J. Mol. Biol, 106:901-917 (1987); Chcthia et al. Nature 342:878-883 (T

Bispecific or Bifunctional Antibodies

Γ0107Ι A bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy/Iight chain pairs and two different binding sites. Bispecific antibodies may ba produced by a variety of methods

2015264940 07 Dec 2015 including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, s.g., Songsivliei & Lachmann Clin. Exp. Immunol 79:315-321 KosteJny et al. -J. Immunol 143:.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 immunisation with full-length OPGL, solubie 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, tor example, bv immunization ot transgenic animals capable of producing human antibodies (see, tor example, PCT Published Application No. W0 33/12227).

10109] In certain embodiments, the complementarity determining regions (CDRs) of the light and heavy chain variable regions of oGPGL-1 may be grafted to framework regions (bps) from the same, or another, species, in certain embodiments, the OCRs of the light and heavy chain variable regions of uOPGL-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 ere aligned to identify a consensus amine add sequence, in certain embodiments, the FRs of the eOFGL~1 heavy chain or light chain are replaced with the PRs from a different heavy chain or light chain. In

2015264940 07 Dec 2015 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 he used with a constant region that is different from the constant region of oORGh-rt. 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. 8,130,370, 5,683,782, 5,693,701, 5,588,039, and 5,530,101, which are hereby Incorporated by reference for any purpose, [01 101 According to certain embodiments, antibodies of the invention are prepared through the utilization of s transgenic mouse that has a substantial portion of the human antibody producing genome Inserted but that is rendered deficient m the production of endogenous, murine, antibodies. Such mica, then, are capable of producing human immunoglobulin molecules and antibodies and are deficient In the production of murine immunoglobulin mdleeutes and antibodies,. Technologies utilized for achieving this resdtf bfe 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 98/74893, which is hereby incorporated by reference for any purpose. See also Mendez et al. Nature Genetics 15:148-156 (1997), which is hereby incorporated by reference for any purpose.

f

2015264940 07 Dec 2015 [01 iii According to certain embodiments, fully human monoctensl 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 8-celisi from the mica that express antibodies are obtained. Such recovered ceils are fused with a myeloid-type cell line to prepare immortal hybridoma cel; 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 ceil line that produces antibodies specific to OPGL is provided, [0412) in certain embodiments, antibodies of the invention are produced by hybridoma fines AMG 3,L AMG 6 u·, AfdG 6,6, 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 err;Pediments, the antibodies of the invention bind to OPGL with a dissociation constant (Kb) of between approximately 0.23 end Q.29 rM in certain embodiments of the invention, the antibodies bind to OPGL with a Kb of less than 0,23 nM.

(0113] In certain embodiments, the antibodies of the invention are of the lg©2 isofype, In certain embodiments of the invention, the antibodies comprise a human kappa fight 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 thsn:to:eoonsfant:regtort;toF the lgG2 isotype,

2015264940 07 Dec 2015 ίθ 11-] in certain embodiments, conservative modifications to the heavy and light chains cf aGPGL4 (and comespohding; mcdifioafidns to the encoding nucieot oefo will produce antibodies to OPGL having functional and chemical characteristics similar to those of oOPGL-1, in contrast, substantial modifications in the functional and/or chemical characteristics of oOPGL-1 rosy be accomplished by selecting substitutions in the amino acid sequence of the heavy end light chains that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the suhstlfuticn, for example, as a sheet or helical conformation, (b) the charge or hydropbobichy of the moiecuie at the target site, or fo) the hulk of the side chain.

[0115] For example, a conservative amino acid substitution may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge cf the amino acid residue at that position. Furthermoie, any native residue in fhe polypeptide may also be substituted with alanine, as has Peen previously described for “alanine scanning muiagenes is, fQl toj Dessred amino acid substitutions (whether conservative or non-consen/ative) can be determined by those skilled in the art at the time such substitutions are desired, in certain embodiments, amino acid substitutions can he used to identify Important residues cf oOPGL.-1, or to increase or decreass the affinity of the antibodies to OPGL described herein, [01171 In certain embodiments, antibodies of fhe present invention cae be expressed in ceil lines other than hybridoma cell lines. In certain

2015264940 07 Dec 2015 embodiments, sequencss ..encoding partfcular antibedsssfosn be used for transformation of a suitable mammalian host ceil. According io 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 veal vector) and transducing a host ceil with the virus for 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 4,968,456 (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 infrcdbctfon of beferoiogou® polynucleotides into mammalian ceils are well known in the art and include, but. are not limited to.

dextran-mediated transfection^. csfoiurh^pbcsphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microtnjection of the DNA into nuclei.

(01183 Mammalian ceil lines available as hosts for expression are wel! known in the art and include, out are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC). Including but not limited to Chinese hamster ovary (C-HO) calls, HeLa cells, baby hamster kidney (SblM) cells, monkey kidney colls (COS), human hepateealiulaf carcinoma cells (e.g., Hop G2), and a number of other celt 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,.

2015264940 07 Dec 2015

According to certain embodiments, antibodies of the invention are useful for detecting OPGL In bloiogtcai 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 end bone resorption,. in certain embodiments, antibodies to OPGL may block OPGL binding to O.OAE, which may result in a block In the signal transduction cascade and loss of NF-kB mediated transcription activation, .Assays for measuring hir-RB-medlated transcription activation using, s.g., a luciferase reporter assay, are known to those skilled in the art..

[.0120] in certain embodiments, methods are provided of irsating a bone disorder comprising administering a therapeutically effective amount of an antibody to OPGL, in certain embodiments, methods ar ©provided of treating a bene disorder comprising administering a therapeutically effective amount of an antibody to OPGL arid another therapeutic agent. In certain such embodiments. I.ha additional therapeutic agent is administered in a therapeutically effective amount, in certain embodiments, the boos 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 toreduce hone resorption to below no rmal levels in order to

2015264940 07 Dec 2015 compensate ter below normal isvsls of bone formation.. In certain embodiments, antibodies can be tested for binding to OPQL in the absence or presence of OPG and examined for their ability to inhibit QPGL-mediated ostedastogenesis and/or bone resorption, i'Ql 2 i} Conditions which may be treated according to certain embodiments include, but are not limited io, the foiiowing:

Osteoporosis, including, but not limited to, primary osteoporosis, endocrine osteoporosis Onaluding, but not tatted to.,; hyperthyroidism^ hyperparathyroidism, Cushing's syndrome, ?and acromegaly), hereditary and congenital forms of osteoporosis (including, but not limited to, osteogenesis imperfecta, hornocystinuria, Menkes' syndrome, Riley-Day syndrome), and osteoporosis doe to immobilization of extremities;

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

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 hemstoiogic mah'gnacles (including, but not limited to, multiple mye<otna, lymphoma and leukemia), idiopathic hypercalcemia, and hypercalcemia associated with hyperthyroidism and renal function disorders;

2015264940 07 Dec 2015

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 arid renal diseases;

Osteonecrosis, i.e,, bone ceil death, Including, but not limited to, osteonecrosis associated with traumatic irslury, osteonecrosis associated with Gaucher's disease, osteonecrosis associated with sickle call 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 [01221 in certain embodiments, an antibody to OPGL may be used alone or with et isast one additional therapeutic agents for the treatment of bone disorders, in certain embodiments, an antibody to OPGL is used in conjunction with a therapeufieatly effective amount of an additional therapeutic agent. Exemplary therapeutic agents that may be administered with an antibody to OPC3L include, but are not limited to, the bone morphogenic factors designated BMP-1 through BMP-12: transforming growth factor# (TGF-β) and TGF-β family members; interleukln-1 (IL-1) inhibitors, Including, but not limited to, ite-lra and derivatives thereof and Kinerei™; TNFu inhibitors, including, but not limited to,

2015264940 07 Dec 2015 soluble TNFo teceptorsp Enbrel™, antETHFo antibodies, Remfcade¹**· and D2E7 antibodtes; 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 limbed to, COX-2 Inhibitors, such as Celebrex™ and Vioxx™; immunosuppressants, such as methotrexate or ieilunomide; serine protease inhibitors, including, but not limited to, secretory leukocyte protease inhibitor (SLPt); IL-β Inhibitors (including, but not limited to, antibodies to 11.-6), IL-S inhibitors (including, but not limited to, antibodies to IL~8); IL~18 inhibitors (including, but not limited to, IL-18 binding nrotein and IL-'l8 antibodies); lnterleukin-1 converting enzyme (ICE) modulators;

•fibroblast growth factors FGF-t to FSF-10 and f GF modulators; FAF antagonists; keratinooyts growth factor (KEF), KGF-related molecules, end KGF modulators; matrix metalloproteinase (MM?) modulators; Nitric oxide synthase (NOS) modulators, including, but not limited to, modulators of inducible NOS; modulators of glucocorticoid receptor; modulators of glutamate reccoto’” modulators of iipepolysacuharlde (LPS) levels; and noradrenaline and mod u Iators and mimetics thereot {01233 Id -certain embodiments , an antibody to OFGL 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 admintetersd, fn certain: embodiments, such

2015264940 07 Dec 2015 agents may be provided together by inciusion sn the same formulation. In certain embodiments, such agents sod 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 gens therapy, the genes >enooding 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 toe 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 interteukto-l (IL-1) inhibitor, and methods of treatment using such therapies, to, certain embodiments, a therapy comprises an antibody to OPGL and an IL-1 inhibitor and at least one additions? molecule described herein. In certain embodiments, methods of treatment use IL-1 Inhibitors and/or TNr-g inhibitors in coniuneiion with sn antibody to OPGL, in certain embodiments, an antibody to OPGL in combination with IL-1 inhibitors and/or TNF-ς inhibitors may be usee for treatment of conditions such as asthma, rheumatoid arthritis, and multiple sclerosis.

4o

2015264940 07 Dec 2015

p.;l2S] interleukin-1 (H..-1) is so anil-inflammatory cytokine, to certain instances, IL-l is a mediator in many diseases and medical conditions. In certain Instances, IL-1 is manufactured by ceils of the macrophage/monocyto lineage, in certain instances, iL-1 Is produced in two forms: iL-1 alpha (IL·lx) and IL-I beta (IL· 1β>, [0126] A disease or medical condition is considered to be an dnierleukin-l 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 ILΊ in culture. In certain embodiments, such interleukln-1 mediated diseases are also recognised by the following additional two conditions; (1) pathological findings associated wtoh the disease or medical condition can be mimicked experimentally in animals by administration of IL-l or upregulation of expression of IL-1; end (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 11.-1, In certain embodiments, one or more of the above conditions are met in an IL-1-mediated disease, to certain embodiments, all three or the conditions are met in an SL-1-medMted disease.

[012¾ Acute and chronic Interiaukln-i (IL-1) -mediated diseases include, but are not limited to, ibs following: acute pancreatitis; amyotrophic lateral sclerosis (ALS, cr Lou Gehrig’s disease); Atehelrners disease; cachexia/anoroxis. including, but not limited to, AIDS-induced cachexia: asthma arid other pulmonary diseases; atherosclerosis: autoimmune vasculitis: chronic·;.

2015264940 07 Dec 2015

Clostridium-associated diarrhea; coronary conditions and indications, including, hut not limited to. congestive bean failure, coronary restenosis, myocardial infarction, rnyocarciia! dysfunction (e.g.. related to sepsis), and coronary artery bypass graft; cancer, Including, but not limited to, leukemias, including, but not limited to, multiple myelorns 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; hemcborragic 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.cp, 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, canc-ar-r-siated 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 ο

ΓΝ

2015264940 07 Dec inflammatory condition resulting from, e.g., strain, sprain, cartilage damage, trauma, orthopedic surgery, infection, or other disease processes, (0128] in certain embodiments, an iL-d inhibitor may be any protein or molecule capable of specifically preventing activation of cellular receptors to 11.-1, which may result from any number of mechanisms, Exemplary mechanisms include, but. are not limited to, downregulating 11-1 production, binding free IL-1, intertenng with ib-1 binding to its receptor, interfering with formation of the it-1 receptor complex (i.e., association of H..-1 receptor with IL-1 receptor accessory protein), and interfering with modulation toll 1 signaling, after binding to its receptor.

(0129] Certain lnterteukin-1 inhibitors include, but are not limited to, 1L--1 receptor antagonists, Including, but not limited to, Kineret™, lL-1ra, IL- ira variants, and it-lra derivatives, which ere collectively termed IL-Ira 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 nor limited to, soluble 11.-1 receptors (see, e.g,, U, S. Pet. No. 5,492,888, U. S. P< No, 5,488,032, and U- & Pat No. 5,464,937, U, S. Pat No, 5,319,071, and U.S, Pat. Να. 5,180,812, which are hereby incorporated by reference for any purpose); snti4L.-1 monoclonal antibodies (see, e.g., WO $1501997, WO 9402627., WO 9006371, U.S.Pat No. 4,935,343, EP 304/78 Rp 267611 and £P 220063, which are hereby incorporated by reference for any purpose); IL-I eceptor accessory proteins and antibodies thereto (see, e.g., WO 98/23067 and WO 98/37773, which are hereby incorporated by reference for any purpose))

2015264940 07 Dec 2015

Inhibitors of lnterteukin-1 bate converting enzyme (ICE) or caspase I (see, e.g., WO 99/46248, WO 99/47545, end WO 99/47154, which are hereby incorporated by reference ter any purpose), which may he used to inhibit IL-1 beta production and secretion; Interteukln-lbeta protease inhibitors; and other compounds and proteins that block in viva synthesis or extracellular release of it-1.

[0130] Exemplary IL-1 inhibitors are disclosed, e.g,. in US Pat Nos, δ 747,444: 5,353.032; 5,603,035; 5,343,905: 5,359,032; 5,866,576; 5,869,660; 5,869,315; 5,872.095; 5,955,480; 5,965,564; international (WO) patent applications 98/21957, 96/09323, 91/17184. 98/40907, 98/32733, 98/42325, 98/44940, 98/47892, 38/56377, 99/03837, 99/06428. 99/06042, 91/17249, 98/32733, 98/17861, 97/08174, 95/34326, 98/38428, 99/36415;. European (EP) patent applications 534978 and 894785; and French patent application FE 2782514. The disclosures of all of the aforementioned references are hereby incorporated by reference for any purpose.

[0131] inferieukin~1 receptor antagonist (IL-1 ra) 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-1aand IL-Ιβ. Certain receptor antagonists, including IL-1 re and variants and derivatives thereof, as well as methods of making and using them, are described in U,S, Patent No. 5,975,222; WO 91/06285; WO 91/17184; AU 9173636; WO 82/15221: WO 93/21946; WO 94/06457; WO 94/21275; PR 2706772; WO 94/21235; DE 4219626, WO 94/20517; WO 8S/22793;WO 57/28828; and WO 39/36341, which are incorporated herein by reference for any purpose. In certain embodiments, an IL-1 receptor antagonist may be

IS

2015264940 07 Dec 2015 glycosylated, in_: certain embodiments,. an IL-1 receptor antagonist maybe nongiycosylated.

(Si 32) Tnres forms of i L-1 ra_:and variants thereof are described in

U.S. Pat. No. 5.075,222 (the ‘222 patent). The first form, called “IL-II in the ’222 patent, is characterized as a 22-23 kD molecule on SDS-rAGE with an approximate isoelectric point of 4.8, which elutes from a Mono Q FPLC column at around 52 mM NaCI in Tris buffer, pH 7,6. Tee second form, IL-Imp, is characterized as a 22-23 kO protein, which elutes from a Mono Q column at 48 mM NaCI, Both IL-lraa and IL-1 rap are glycosylated. The third form, IL-1 rax, Is characterized as a 20 kD protein, which elutes from a Mono Q column at 48 mM NaCI and ia 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 canes into certain ceil types, and expressing those genes to produce the inhibitors.

10133) In certain embodiments, deletions, insertions, and/or substitutions (individually or collectively referred to as '‘vanant(s)) are made within the amino acid sequences of IL-1 ra. in certain embodiments, an IL-lra variant is bfoiooicaily 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 TNFo Inhibitor, and methods of treatment using such therapies, in certain embodiments, a therapy comprises an antibody to OPGL and a TNFo Inhibitor and at least one additional molecule described hereto.

SO

2015264940 07 Dec 2015 [01351 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 medteai condition that is associated with elevated levels of TNF in bodily fluids or tissue and/or in which cehs 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 sotibn of TNF, (01361 Certain acute and chronic TNF-meds'aisd diseases Include, out 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 io sepsis), and coronary anew bypass graft; depression; diabetes, Including, but not limited to, juvenile onset Type 1 diabetes, diabetes meliltus, and insulin resistance (including, but not limited to, insulin resistance associated with obesity)*, endometriosis, endometritis, and related conditions; fibromyalgia and analgesia; craft versus host rejection; hyperalgesia; Infiammatory bowel diseases, inciuding, but not limited to, Crohn's disease and Clostridium difflcile-assoclated diarrhea: ischemia. Including, but not limited to, ;W

2015264940 07 Dec 2015 cerebral ischemia, which includes, but is not limrled to, brain injury as a result οι trauma, epilepsy, hemorrhage, and/or stroke: lung disease, including, hut nut limited to, adult respiratory distress syndrome, asthma, and pulmonary fibrosis; multiple sclerosis; nsuromf ammatory diseases; ocular diseases and conditions, including, but not limited: to, corneal transplant, ocular degeneration and uveitis; pam, including, but not limited to, cancer-related pain; pancreatitis; periodontal diseases; Pityriasis rubra pilaris (PRP); prostatitis, including bacterial and nonbacterial 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, butrtot limited to, juvenile rheumatoid arthritis), seronegative polyarthritis, ankylosing spondylitis, Reiter’s syndrome and reactive arthritis, Still's disease, psoriatic arthritis, enteropathic arthritis, polymyositis, darmatomyositis, scleroderma, system Ic Seferosls» Vssoulitis (a< g., Kawasaki ’ s d isea se), cerebral: vasculitis, Lyme disease, staphyioccccaMnduced (“septic) arthritis, Sjogren’s syndrome, rheumatic fever, polychondritis end polymyalgia rheumatica and giant ceil artoritis): septic shock; side effects from radiation therapy; systemic lupus erythematosus (SLE); temporal mandibular joint dis-aass; thyroiditis; and tissue transplantation and/or an inflammatory condftion, e#, resetting from strain, sprain, cartilage damage, trsume, orthopedic surgery, infection (s.g„ HIV,

Clostridium dlfftoife and related species) or other disease process.

[0137) in certain embodiments, TNF inhibitors may act by at least one of downregulatinc or Inhibiting TNF production, binding free TNF, interfering

2015264940 07 Dec 2015 with TNF binding io its receptor, and interfering with .modulation of TNF signaling after binding to its receptor. The term TNF inhibitor' includes, but is not limited io. solubilized TNF receptors, including, but not limited to, soluble tumor necrosis factor receptor type * (sTNF-RI; also ceiled the p5S receptor), Soluble turner necrosis factor receptor type ii (also celled the p75 receptor), end Enbrei™: antibodies to TNF, Including, but not limited to, Remicade™ and D2E7 (see, e.g., U.S. Patent. Nos, 6,090.382 end 6,258,562); antibodies to TNF receptor; sTNF-RI (see, e.g., WO S8/24463), etanercept (Enbreto), AvakioeInhibitors of TNF-a converting enzyme (TACE); and other molecules that affect TNF activity.

[Q138] Exemplary TNF-α inhibitors are described, e,g_;, in European patent applications EP 208 378; EP 422 339; EP 393 438; EP 398 327; EP 412 486: EP 413 014, EP 417 563 EP 433 900; EP 464 533; EP 512 528; EP 526 905; EP 588 928; BP 607 77S_:, which describes the use of jefiunbmide· tor inhibition of TNF-o: EP 683 210; EP 542 795; EP 818 439; BP 664 128; EP 542 795; EP 741 707; EP 874 819 ; EP 882 714; EP 880 970; EP 648 783; EP 731 791; EP 896 988; EP 550 376; EP 882 714; EP 853 063; EP 550 376; EP 943 818; EP 933 121; EP 614 984 ; EP 853 083; U.S. Paient Nos, 5,138.021;

5,>329.117; 5,948,833; 8,807,862; 5,695,953: 5,834,438; 5,817,822; 5330742; 5,834,438; 5,881,558; 5.853,977; 5,359,037; 5,512.544; 5,695,953; 5,811,261;

5.833,145; 5,863,923; 5.	,866,616; 5.641,673; 5,860,677;	5,869,511; 5,872.	,146;
5,854,003: 5.856.1 SI; 8.	.677,222; 5,877,200; 5,877,151;	8,888,010; 5,869	,860;
5,859,207; 5,891,883; $,	,677,180; 6,955,480; 5,955,476;	5,955,435; 5,094	,35¾
5,090,119; 5,952,820; 5,	.962,481: International patent sp	’plications WO 90)	Ί357

2015264940 07 Dec 2015

WO 91/03553, WO 92/01002, WO 92/73095, WO 92/16221, WO 93/07863, WO 93/21946. WO 93/19777, WO @5/34326. WO 96/28546, WO 98/27298, WO @8/30641, WG 96/360. WO 9e/38iWWO @7/18207, WO 97/15SB1, WO 97/12S02, WO 96/26861, WO 96/12735, WO @8/11209, WO 38/39328, WO 98/33316, WO 88/38859, WO 93/39313, WO 98/42659, WO 98/39329, WO 93/43959, WO 98/46288, WO 98/47863, WO 96/33172, WO 98/2Q925, WO 97/37974, WO 97/37973, WO 97/47599, WO 96/35711, WO 98/51665, WO @8/43946, WO 95/04046, WO 98/56377. WO 97/12244,-WO 99/08364, WO 39/00363, WO 98/57936, WO 99/01449, WO 99/01139. WO 88/56788, WO @8/56756, WO 98/53842, WO 98/52948, WO @8/52937, WO 99/02510, WO 97/43250, WO 99/85410, WO 99/08042, WO S9/09Q22, WO 99/08638. WO 99/07679, WO 99/09965, WO 39/07704, WO 99/08041, WO 99/37818, WO 99/37625. WO G7/11S8S, WO 39/60238, WO 93/47872, WO 99/48491;

Japanese patent applications 10147531, 10231286, 10259140, and 10130149, 10316570, 0801431, and 127,800/1391; German application no. 19731521; and British application nos, 2 218 101,2 326 881,2 246 589. The disclosures of all of the aforementioned references are hereby inccrporared fey reference for any purpose, [0139} EP 393 438 and ER 422 339 describe the amine acid and nucleic acid sequences of a soluble TNr receptor type I (also known as sTNr R-l or 30kDa TIME inhlhifer) and a soluble TNr receptor typ© 11 (also known as s'FNFR-ls or 40kDs TNF inhibitor), which are collectively termed “sTNFRs. EP 393 438 and EP 422 339 also describe modified forms of sTNFR-l end sTNFFMI,

2015264940 07 Dec 2015 ingfoding, but net Med t© fragments, funotfonat denvatives,_: and variants. Furthermore, EP 393 438 and EP 422 339 describe methods forisoiatsng genes that code for the inhibitors, cloning the genes into suitable vectors, transforming or transfecting the genes Into certain ceil types, and expressing the genes to produce the inhibitors.

(O140J sTNFR-l and sTNFR-li are members of the nerve growth factor/TNF receptor supsrfamiiy of receptors, which includes the nerve growth factor receptor (NGFJ, the B cell antigen CD40, 4-lBB,tho rat T-cell antigen MRG 0X40, the fas antigen, and the CD27 and CD30 antigens (Smith et at (1990) Science, 248:1019-1023), A conserved feature of that group of ceil 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 43 cysteine residues st positions that are well conserved (Smith et si. (1990), aa).

(0141 ] EP 393 438 teaches a 40kDa TNF inhibitor .451 and a 40kOa

TNF inhibitor ASS, which are truncated versions of the foil-length recombinant

40kDa TNF inhibitor protein, Δ51 and Δ53 have 51 or 53 amine acids, raspeetiyafe deieted from the carboxyl terminus of the mature protein.

[0142) Published PCT Application No, WO 98/01556 describes truncated forms of sTNFR-l and sTNFR-ll that do not contain the fourth domain (amino acid residues Thr'ⁱ²⁷-Asn'^!Sl of sTNFR-l and amino acid residues Pm¹⁴¹Thmra of ssYNFR-il}; a portion of the third domain (amino acid residues Asn¹¹¹Cys¹²⁶ of sTNFR-l and amine acid residues Proⁱ²³-Lys^U0 of sTNFR-ll); and,

2015264940 07 Dec 2015 optionally, do not contain a portion of the first domain (amino aoidT Cys¹⁸of sTNFR-ί and amino add residues Leu-Cys³² of sTNr'FNi). in certain embodiments, the truncated s 1 NFRs include the proteins represented by the formula R_riCys^-Cys^3]..R_{2 anc}j R^Cys^Cys'^J-Rs. These proteins are truncated forms of sTfoFR-l and sTNFR-!i, respectively.

. [01431 As used herein, R<-(Cys'^f9-Cys¹⁰³]-Rg represents one or more proteins wherein iCys^-Cys·⁰³} is residues 13 through 103 of sTNFR~i, the sequence of which is provided in Figure 1 of WO 98/01355; wherein F>.₅ represents a methicnylaled or nonmethienyiated amine «group of Cys¹⁸ or one or more amino-term inpi amino acid residuesfoeiecied from Cys¹⁸ to Asp\· and wherein Fh represents a carboxy group of Cys¹⁰³ or one or more carboxyterminal amino acid residues selected from Phe¹^to Leu¹¹⁰.

[Q144] Exempiary truncated s'TNFRTs of the present invention include, but are not limited to. sTNFFW 2.6D/C105, sTNFR-l 2.6D/G106, sTNFR-l :2.6D/NW5, sTNFR-i 2.3D/dS, sTNFR-l 2.3D/d1S, sTNFR-i 2.3D/d15, either methionylated or nonmethionylafeci, and variants and derivatives thereof.

Certain exemplary truncated sTNFR-Ts are described, e,g., in published PCT Application No.. WO 98/01555.

[01451 As used herein, ^:!R3“[Cys³²-Cysⁿ⁵]“R<s represents one or more proteins wherein fCysWoysiisj Is residues Cys³'²through Cysl® of sTNFR-B. the sequence of which Is provided in Figure 8 of WO 98/01555; wherein R₃ represents a malhienylatad er nonrnethionyjafsd amine group of

Cys³² or one or more a.mloo-termlnel amino aclci residues selected from Gys^

2015264940 07 Dec 2015

Leu¹; and wherein R.4 represents a carboxy group of Cys'¹³or one or more carbpxyderminal amino acid residues selected from Als^11&#o Arg¹²², [0146] in certain embodiments, the present invention is directed to therapies comprising an entibcdy 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 teas! 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 mguiate both endogenous and Wading 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 tote 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 bean isolates 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 1 PI), alpha

2015264940 07 Dec 2015 l-antiehymctrypsin (alpha lAchy), alpha Aanticoilagensse (alpha 1AC), and :nler-aipha~trypsin Inhibitor (halpha-l), (04 50j In certain instances, a disturbance of the prbtease/prote innshkor balance can load to proteose-mediated tissue destruction: including, but not limited to, emphysema, arthritis, glomerulonephritis, periodontitis, muscuiar dystrophy, tumor Invasion, and various other pathological conditions, in certain situations, e.g, severe pathological processes such as sepsis or acute leukemia, tine amount of free proteolytic enzymes present may Increase due to the release of enzyme from secretory cells·.

[0131] Furthermore, in certain instances, a diminished regulating inhibitor capacity of the organism may also cause alterations In the pretease/prelease inhibitor balance. A hbrtfertihg exarapfe-of such a diminished: reguieting inhibitor capacity Is an alpha 1~protes.se 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 die 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 eiastase are nonlimiting examples of a class of proteases known as serine proteases, [0154] in certain instances, leukocyte eiastase. when released extraceSiuiariy, ctegtados co nnsctive tissue and other vaiuabte proteins,. White a

2015264940 07 Dec 2015 normally functioning organism degrades a carta» amount of connective tissue and ether proteins, fhe presence of an excessive amount of leukocyte elastase may be associated with various pathological slates, 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 norms!, a protease inhibitor has bean sought which is specific tor leukocyte elastase. Such a. protease Inhialtor may be useful if it were capable of being isolated or prepared in a purified form and in sufficient quantities fo be pharmaceutically useful.

10155] Certain leukocyte elastase inhibitors are described, e.g., in Sehiessler at an, Acid-Stable inhibitors of Granulocyte Neutral Proteases In Human Mucous Secretions: Biochemistry and Possible Biological Function, in feMSgteaSMWainan Havemann et al.

(ads), Urban arte Schwarzeoberg, inc. (1978), and in Travis and Sslvesen. Ann. MBte· 52: 655-709 {1983).

[01531 in certain instances, trypsin initiates degradation of certain soft organ tissue, such as pancreatic tissue, during a variety cf acute conditions, Inciuding, but not limited to, pancreafltfs. A trypsin inhibitor may be useful If it could be isolated and prepared in a purified form and in sufficient quantities to be pharmacedfioailyiisefuL [0157j Cafhepsin G Is another protease present -n leukocytes.. In certain embodiments, cathspsln G is capable of degrading a variety of proteins in vitro, including those of the complement pathway. Pancreatic elastase is another

2015264940 07 Dec 2015 protease that may have a rale in psncseatihs. Thus, Inhibitor for these proteases may also be of pharmaceutical vaiue.

[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 sold residues. By analogy, if 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 professes, in certain embodiments, a member of this class inhibits every serine protease.

f0159] An exemplary serine- protease fphibftor is secretory leukocyte protease inhibitor (SLP1) and fragments and analogs thereof. Exemplary serins protease inhibitors also include, but are not limited to, anu-teukoprotease (ALP), mucous protease inhibitor (MPi), human seminal plasma inhibitor-i (HUSI-I), bronchial mucus inhibitor (BMI), and cervical mucus Inhibitor (CUSI). In certain embodiments, a serine protease inhibitors also may be IPS modulator. See, e.g., Jin et al (1997), Cell 38(3): 417-26. in certain embodiments, these molecules are wel ed for use In conditions leading to bone loss because they are prefetorifially directed todhe cartilage,.

[0160] ' Exemplary serine orotease inhibitors are described, e.g., In U.S. Pat. No. 4,789,130: U.S, Pat. No. 5.300,400; 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 sdiieofjyely termed serine protease Inhibitors/’

2015264940 07 Dec 2015 [0161! ;l~1 3 is a pro-inflammatory cytokine teat was found to indues interferon-γ snd was previously named interferon gamma inducing lector (IGIF). in certain instances, IL-l has peen shown to upraguiate IL-18 production, and IL· 18 Induces production of a number of proinflammatory cytokines. Including 1L-S and MMP-1. See, e.g., Dim-relic et al. (1998), J. Leukocyte Biol. S3: 658-64. In certain instances, caspase I is also Important tor IL-13 production. Experiments also suggest that TNr-π regulates IL-13 production, and that simultaneous inhibition of TNF-α and IL-1 S u o -mis against liver toxicity. See, e.g,, Faogioni et al. (2000). PNAS 97: 2367-72.

[01623 1L-1S acts to vivo through a receptor system reminiscent of the IL-1 system. IL-18 interacts wkn a cell surface receptor (1L--13R), which interacts with.an accessory protein (IL-ISRAcP). IL-'IS-meclated signaling proceeds upon formation of the complex of IL-18, IL-ISR, and IL-ISRAcP, A natural inhibitor for IL-18 Is IL-13bp. In certain embodiments,. iL-1 Sbp acts as a decoy receptotoby binding to IL-13 molecules and preventing interaction with IL~ [0163) In certain embodiments, the present invention Is directed to therapies comprising an antibody to OPGL and at least one IL-13 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 additions) 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

SI

2015264940 07 Dec 2015 conditions that may be treated with an antibody to OPGL and at least one IL-13 inhibitor according to certain embodiments include, but are not limited to, arthritis, including, but not limited to rheumatoid arthritis: systemic iupus eiythsmaiosus (SL£); graft versus host disease (GvHD); hepatitis; sepsis; and the less of bon® and os [016-4] Exemplary' IL~18 inhibitors include, but are not limited to, antibodies that bind to iL-18; antibodies that bind to 1L-18R; antibodies that bind to IL-ISRAeP; iL-18bp; IL-18R fragments (e.g.. a smuUitoed extracellular domain of ins iL-18 receptor); peptides that bind to IL-18 and reduce or prevent its interaction with 11..-18R; peptides that bind to IL-1 SR and reduce or prevent its Interaction with iL-18 or with IL-18RAcP; peptides that bind to IL-13RAcP 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, fLMSR, and ILiSRA_c?

[01601 Certain 11.-18 Inhibitors are described, e.g., in US Pat No. 6,912,324, issued July 14, 1994; EP 0 962 531, published Dec. 8, 1999; EP 712 931, published Now 16, 1994: US Pet. No. 5,914,253, issued duly 14.. 4994; WO 97/24441, published July 10. 1997; US Pat No, 8,880,283. Issued May 9,2000; EP 860 902, published Deo. 20, 1996; EP 304 585, published Sep. 16, 199S; WO 98/41232, published Sep, 24,109:3: US Pat No. 5,054,487, issued April 25,

2000; WO 99/03063, published Aug 14, 1997; WO 9-9/22780, published NOV. 3, 1997; WO 99/37772, published Jan. 23, 1998; WO 99/37773, published March 20, 1398; EP 0 974 800, published Jan.. 25, 2000: WO 00/12555, published Mar.

2015264940 07 Dec 2015

0,2000; Japanese patent application JP 111,399/94, published Oct, 31, 1097; 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 tc OPGL may be used with et least one therapeutic agent for Inflammation. In certain embodiments, an antibody to £3PSLmayfee used with at least one fHerapeutic agentfbr an immune disorder. Exemplary therapeutic agents for inflammation and immune disorders Include, but are not limited to, corticosteroids, including, but not limited to, predniselcne; nonsteroidal anti-inflammatory drugs (NSAlDs), including, but not limited to, cyclooxygenase type 1 (CGX-1) and cyclooxygenase type 2. (COX£ ) inhibitors; disease modifying antirheumatic drugs (DMARDs), Including, but not limited to, methotrexate, hydroxychloroquine, cnioraquine, cyclosporine, gold compounds (such as auranofin, aurothiomalate and aurotnioglucosa), and ieikmomtde; type IV phosophodiesterase inhbitcrs. including, but not limited to, Rolipram and Pentexlfylilris; Tacrolimus (FK-506); Sirollmue (rapamycin); mycophenolic acid; 5-ilpoxygenase Inhibitors, including, but not limited to, Ziieuion; moduiators of interleu&n-S (IL~S): small molecule modulators of 38 kDa mitogen-activated protein kinase (p3S-MAPK); smell molecule modulators of intracellular molecules Involved In inflammation pathways, wherein such intracellular molecuies include, but are not limited to, ink, IKK» NP~xS, ZAP7Q, and lex. Certain exemplary therapeutic agents for Inflammation are described, e.g.. in C.A. Dsnarello and L.L, Motdawer Proinflarnroatory and Anil-inflammatory. Cytokines In Rheumatoid Arthritis: A Primer for Clinicians Third Edition (2001)

2015264940 07 Dec 2015

Amgen inc. Thousand Oaks., CA. Certain exemplary therapeutic agents for inftammatian and autoimmune diseases include, but are not limited to, interferon gamma (iFN~y) modulators; modulators of OX4Q/OX4CL (including soluble forms of 0X40); modulators of 4-183/4ΊΒΒ ligand (including soluble forms of 4-1 BB); and modulators of S ceil-T ceil ccsilmulatory pathways, including, but not limited to, modulators of the receptor ligand pairs C028/S7, C04Q/CQ40L,

ICOS/B7RP1, and AGP-3/TACi/BA.FFR (AGP-3 binds to both TAG! and BAFF'R receptors). Certain exemplary modulators of 3 cell-T call oostlmulstory pathways include, but are not limited to. Inhibitors of CD2S, 37.1, and 37.2 (including soluble forms of 37.1 or B7.2 and soluble forms of CTLA4, both of which ra/re fused to a heterologous peptide or protein which reduces or prevents degradation and/or increases hslfdife, 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 CC40 which may be fused to a heterologous peptide or protein); inhibitors of ICOS and B7RP1 (including soluble forms of ICOS which may he fused to a heterologous peptide or protein) and inhibitors of AGP-3, TAGI and BARER {including soluble forms of TAG I and SAFFRj. iCOS, B7RR1 and Inhibitors thereof are described, e.g., in WOOO/4824Q, AGR-3, TAGI and SAPPR end inhibitors thereof are described, e.g., in WOOO/4774C, WO01/35872, W002/15273, WO98/3S381, and von Bulow and Bram (1897) Selene® 278H 33140,.

2015264940 07 Dec [0 Wj in certain embodiments, an antibody to OPGL is used to treat bone toss, including, but not limited to, bone loss resulting from osteoiytic destruction of bone caused by malignant or metastatic tumors, 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, esophageal· rectal, bladder. cervical, ovarian, and liver cancers, as well as cancer of the gastraintestlona? tract, in certain embodiments, an anybody m OPGL may be used to treat bone ioss associated with, e.g., cortsin hematoioglcai 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 feast one other therapeutic agent, including, but not limited to, et 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 on® or more of the following; artthracyclines, taxol, tamoxifene, doxorubicin, 5-ftuorouracll, and other drugs known In the art. In certain embodiments, a cancer therapy agent Is a .luteinizing hormone-releasing hormone (LHRH) antagonist. in certain errtbodimente, a LWEH antagonist te a peptide antagonist.

(0169] in certain embodiments, an LHRH antagonist comprises the peptide: Ac-D-NaM-Ci-Phe-D'-PaPSef-N-Me-TyrtD’Asn-Leu-LysfiPrJ-PrcO-Aia'· NH2 (SEQ ID NO; 20), where Mai is 3rt2mapthyS)elanteyS: 4-Ci-Phe is (4^!6@

Ο <N

2015264940 07 Dec chlorophenyl)aianinyf; Pal Is 3-(3^!-pyndyl}slanlnyi; and Lys(iPr) is N~epsiion-2propyi-lysinyf.

[0170] in certain embodiments, sn LHRH antaaonist 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 [0171] Exemplary therapeutic antiboaies according to certain embodiments include, but are not limited to, mouse, mouse-human chimeric, CDR-grafted, humanised and fully human antibodies, and synthetic antibodies, including, but not limited to, these selected by screening antibody libraries. Exemplary antibodies include, but ere not limited to, those which bind to ceil surface proteins Her2, CDC20, CDC33, mucin-like glycoprotein, and epidermal growth rector receptor (EGrR) present on tumor cells, and optionally Induce a cytostatic and/or cytotoxic effect on tumor cells displaying these proteins. Exemplary antibodies also Include HERCEFTiN™ ftrasteumsb}, which may be used to treat breast cancer and other forms of cancer,, and RITUXAN™ (rituximab), ZEVALIN™ (ibritumomab tiuxetan), and LYMPHOCtOE™ iepratuzurnab), which may be used to treat non-Hodgkin’s lymphoma and other forms of cancer. Certain examplary antibodies also include ERBITUX™ (IMCC225], SEXxAR™ (iodine 131. tdsitumomab), and Campath.

[Cl72] in certain embodiments, cancer therapy agents are polypeptides which selectively induce apoptosis in turner colls, including, but not limited to, the- TNF-relafed polypeptide TRAIL, in certain embodiments, an

2015264940 07 Dec 2015 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 administered prophylactisiiy to prevent or mitigate the onset of bone loss by metastatic cancer. In certain embodiments, an antibody to OPGL may he aarmnistesed for the treatment of an existing condition of bone loss due to metastasis, [0 ’ 7'0 :_n certain embodiments, an antibody to OPGL may he 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 bene lesions yisibf©: by radiotogioal 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 re injury, in certain Instances, the skeletal lesions that occur during myeloma not only lead to bone fractures, taut also deformity and occasionally nerve compression, particularly In the vertebral spine. In soma 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 nsk of fractures and spinel deformity.

S7

2015264940 07 Dec 2015 :0174) to certain instances, myeloma cells do not directly participate in boas destruction, but instead produce exiraceltoiar ssgnais that toad to osteoclast differentiation. and activation, in certain instances, ©sfeoofeste produce high levels of the cytokine IL-6, particularly when they become activated, IL-S is a 8-ceil growth factor, and contributes to the growth of both murine and human myeloma cells to wire. Myeloma ceils may also either directly or indirectly produce OPGL, which may result in local bone lysis surrounding the myeloma : : t ceils embedded in bone marrow spaces, to certain instances, tbs normal osteoclasts adjacent to the myeloma cell In turn produce IL-6, which may load to local expansion of the tumor cells. Myeloma cells expand In a clonal fashion and may occupy bone spaces that are crested by inappropriate bone resorption.

.0 ‘75] it has been observed that OpG administration in rodents Induces rapid death of the osteoclast population (see, e.g,. Lacey etai. (2000) Am, J. Pathol 167:436-446). A reduction In the number of osteoclasis may counteract the effect of increased IL-6 production by those ceils 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] 6-ceIls express the receptor for OPGL, ODAR. Myeloma calls also express ODAR, and In addition may produce OPGL. In certain instances, the expression of both OPGL and ODAR in the same ceil population may create an autocrine stimulus that affects survival of the myeloma ceil. Thus,

2015264940 07 Dec 2015 in certain embodiments, administration of an antibody to OPGL may reduce tumor cell survival, thereby decreasing or eliminating the tumor burden seen in myotoma patfents, (0177] in certain embodiments, the invention provides for pharmaceutics! -cornposifions 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 pharmaceutics! 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 onsraddiftonal therapeurtc agenf is selected from bene morphogenic factors designated 3MP-1 through BMP-12; transforming growth factor-6 lTGF-β) and TGP-β family members: interleukin-'! (IL-’i) inhibitors, including, but not limited to. IL»1 ra and derivatives thereof and Klnerst™; TNFa Inhibitors, including, but not limited to, a soluble TNFa receptor, Enbrei™, antlTNFo antibodies. Remicade™. and D2E7 antibody; parathyroid hormone and analogs thereof, parathyroid related protein and analogs thereof; E series prostaglandins; bisphosphonates (such as alendronate and others); baneenhancing minerals such os fluoride and .calcium; non-steroidai anti-inflammatory drugs (NSAIDs), induoing COX'2 inhibitors, such as Celebrex* and VIgxx™; immunbWprewnis, such as methmrexsts or teflunomide; serine protease ο

ΓΝ

2015264940 07 Dec inhibitors such ss secretory leukocyte protease inhibitor (SLPi); 11-6 inhibitors (e.g,, antibodies to iL-8), IL-8 inhibitors (e,g.,_f antibodies to IL-8); IL-18 inhibitors (e,g<, IL-18 binding protein or IL-18 antibodies); Interleukin-I converting enzyme (ICE) modulators: fibroblast growth factors f-GF-1 to PGr-10 and F

GF modulators; PAF antagonists; fcerafinocyis growth factor (KGF), KGF-reiated molecules, or KGF modulators; matrix metalloproteinase (MMF) modulators; Nitric oxide synthase (NOS) modulators, including modulators of inducible NOS; modulators of Glucocorticoid receptor; modulators of glutamate receptor; modulators of lipopolysaeehanda (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 . condehtrations 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 tp, 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-KCi, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ©thyienediamlne ieiraacetie acid (EDTA)); complexing

2015264940 07 Dec 2015 agents (such as caffeine, polyvinylpyrrolidone, beta-eydo dextrin or hydroxypropWbeta-cyclodextrirh: fillers; monosaccharides: dfsaocharides; and other carbohydrates (such as glucose, mannose or dextrins);.prcieins (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 chicrlds, benzoic acid, salicylic acid, ihlmerossi, phenethyl alcohol, msthylparabsn, propylparaben, chlorffexidine, sorbic acid or hydrogen peroxide); solvents (such as giycerirp propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol Or sorbitol); suspending· agents; surfactants or wetting agents (such as pluronlcs, PEG. sorbitan esters, poiysorbates such as poiysorhate 20, poiysorbate 80, triton, tromethamine, lecithin, cholesterol, fyloxapsh; stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferabiy sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remzngton's Prienmeceuffoa/ Sciences, IS’⁴¹ Edition, A.R, Gennaro, ed,, Mack Publishing Company (1990).

[0181] - In certain embodiments, an antibody to OPGL and/or a therepeuffc molecule is linked to a half-life extending vehicle known in the art. Such vehlcies include, but are not limited to, the rc domain, polyethylene glycol, and dextran. Such vehicles are described, e,g., In U.S. .Application Serial Mo. 09/428,082 snd published PCT Application Mo, WO 99/25044, which are he env incorporated by reference tor any purpose,

2015264940 07 Dec 2015 (01821 in certain, embodiments, the optimal phaimaceWsi 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,.forSample, Pemfrtgtortfepharthacedifcsf Sctences, wpM In certain embodiments, such compositions may influence the physical state, stability, rate of in VfVo release and rate of in vivo clearance of the antibodies of the Invention.

101831 Jn-certain embodiments, the primary vehicle dreamier in a pharmaceutical composition may be either aqueous or nan-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 sLipptemented with other materials common ihteQmposiiions for perenterai 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 O'PGL. with or without at least one additional therapeutic agents, may be prepared for storage by mixing hie selected composition having the desired degree of purity with optional formulation agents (Pem/ngten's Pharmaceuf/ca/ Sc/encee. supra) in the term of a lyophilized cake or an aqueous solution.

Further, In certain embodiments, a composition comprising an antibody to C-PGL,

2015264940 07 Dec 2015 with or without at feast one additional therapeutic agents, may be formulated as a lyophilisate using appropriate excipients such as sucrose.

[0184) in dertaiR 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, [04851 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 physiotegfcatpH or at a slightly lower pH, typically within a pH range of from about S to about 8.

[0188] 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 distiiiad’water in which the antibody to OPGL, with nr 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 mlcrospheres, bioerodibie particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide for the ccntroiled or sustained

2015264940 07 Dec 2015 release of the product which may then be deilversd 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 deliver/ devices may be used to introduce the desired molecule [0187] .'n certain embodiments, a phamteoeuftcaf: 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/USS4/Q0187S, 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 hioavaiiabillty is matomfead and pfe-eyalerrifc degradation is minimized. In certain embodiment.

at least one additional agent can be included to facilitate absorption of the ο

CM

2015264940 07 Dec antibody to OPGL and/or any additions! 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 pharmaceuffesi composition may involve an effective quantity of antibodies to OPGL, with or without at least one additions! 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 pushas magrtosiurh: stearate, stearic acid, ortalo., [01WJ Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving antibodies to OPGL, with or without at least one addiOonal therapeutic agents, in sustained- or controiieddelivery formulations, in certain embodiments, techniques for formutetlng a variety of other sustained” or confrafteri-rieiivery means, such as liposome carriers, bta-erodibie microparticles or porous beads and depot injections, are also known to those skiited in the eft. Gee for example, PCT Application No. PCT/US93/00S29 which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions, in certam

2015264940 07 Dec 2015

- anodiments, sustained-release preparations may include semipermeal polymer matrices in the form of shaped articles, e.g, terns, or micro-capsules. ' Sustained release matrices may include polyesters, hydrogels, polyiasfidee (U.S, 3,773,319 and EP 058,481), copolymers of L-giutamrc add and gamma ethyl-Lgiutamate (Sidman, at $/., 8/opo/ymers, 22:547-558 (1383)), poly (2mydnoxyefhylmethacrylate) (Langer ei a/., J, β/omocf. Water, Res., 15:167-277 (1981) and Langer, Cftem, Tech., 12:98-105 {1982)), ethylene vinyl acetate (Longer of a/., supra) or pdy-Q(-)~3~bydro?xyhutyrlc 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,, Eppsteln ef a/., Proc. Nsff. Acad. Sot USA. 82:3688-3692 (1985): EP 038,876; EP 088,048

143,943, [0131] The pharmaceutical composition to be used for rr? wVo administration typically is sterile. In certain embodiments, ibis may be accomplished by filtration through steniefiitetteBmambranes» te certain embodiments, where the composition Is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution'. In certain embodiments, the composition for parenteral administration may be stored in lyophilized form or in a solution. Is certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vlat having a stopper plerceabi

7B

2015264940 07 Dec 2015 [0102] , in ceiteir embodiments, once the pharmaceutical composition has been formulated, it may be stored in sterile yfofe as a solution, suspension, gel, emulsion, solid, oras a dehydrated or lyophilized powder. In certain embodiments, such formulations may he stored either in a ready-to-use form or. in a form (e.g., lyophilized) that is reconstituted prior fo administration..

(0183) In certain embodiments, the present invention is directed to kits for producing a single-dose administration unit, fo certain embodiments, the kits may each contain both a first container having a dried protein and a second container having an aqueous formulation, fo certain encodsmenls of this Invention, kite containing single and multi-chambered pre-filled syringes (e.g,, liquid syringes and lyosyringes) are included;

(0134] in certain embodiments, the effective amount cd a pharmaceutical composition comprising an antibody to OPGL, with cr without at least one additional therapeutic agent, fo bo employed therapeutically wdl depend, for example, upon the therapeutic context and objectives. One skilled in me art will appreciate that fhe 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 ranee from about 0.1 pg/kg ?r

2015264940 07 Dec 2015 to up to shout 100 mg/kg or more, depending on the factors mentioned above, in certs in embodiments, the dosage rosy range from 0,1 pg/kg up to about 100 mg/kg; or 1 ug/kg up to about 100 mg/kg: or 5 ug/kg up to about 100 mg/kg, [0195] b 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 moiecuie) overtime, ores a continuous infusion via an implantation devise or oatheten Furfhdf refinement of the appropriate dosage is routinely made by those of ordinary skill tn 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 doseresponse data, (D18®J In certain embodiments, the route of administration of the pharmaceutical composition is In accord with known methods, e.g, orally, through injection by intravenous, intrapentoneal, intracerebral (intra-parenchymal), intracerebroventncuiar, intramuscular, intra-ocular, intraarterial, intraportai, or intralesional routes; by sustained release systems or by impiantetlon devices, tn certain embodiments, the compositions may be administered by bolus injection dr continuously by Infusion, or by Implantation device.

ο

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2015264940 07 Dec

In certain, ambodlmems, the oomposition may be liy via implantation of a membrane, sponge or another appropriate material onto which the desired moiecule has been absorbed or encapsulated, in certain embodiments, where an impiantation device is used, the device may he implanted Into any suitable tissue o: organ, and delivery of the desired molebuiB may be via diffusion, feecNreteas© bolus, oreonfinuous administration,

101981 Irt certain obodirhents, If may fee desirable to use a pharmaceutical composition comprising an antibody to OPGL, with or without at least one additional therapeutic agent, to an ex wo. manner, tri such instances, ceils, tissues anfe/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 wh*cb the ceils, tissues and/or organs are subsequently Implanted back info the patient.

[01 SSI frt certain embodiments, aft antibody to^: GPBL and/or any additional therapeutic agents can be delivered by Implanting certain cells that have been genettesily 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, ft-ie ceils may fee Immortalized, in certain embodiments, in order to decrease the chance of an Immunological response, the oelis may be encapsulated to avoid infiltration of surrounding tissues, in certain embodiments, the encapsulation materials are typically biocompatlfeie,

2015264940 07 Dec 2015 semPpermeebte polymeric enclosures ©r membranes that allow the release o the protein product's) but prevent the destruction of the colls by the patient’s Immune system or by other detrimental factors from the surrounding tissues.

EXAMPLES [02001 The following examples, including the experiments conducted and results achieved are provided for illustrative purposes on are not to be construed as limiting the present invention.

Example 1

Cloning the oQPGL· Heavy and Light Chains [02011 CKO cells expressing the full-length human OPGL cDNA are used to immunize transgenic mice containing human irnrnuoogiobulin genes. Lymph nodes from the immunized mice are fused to murine myeloma ceiis to generate hybridomas. Supernatants from the hybridoma lines are tested In an ELISA assay for anWedias that react with human CPBL. Ant.l-GPGL expressing hybridcma 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, 0.28 nM, and 0.23 hM< 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 foe cOPGL-1 fight Chain cDNA, white AMG 6.4 is used to clone the aOPGL-1 heavy chain cDNA

2015264940 07 Dec 2015

Cloning of the oQRGL-1 ilqbf cAalr?

F0202] The aOPGL-l kappa light chain vanable region is obtained using PCR amplification methods from first strand cDNA prepared from A.MG 6.5 total RNA. First strand cDNA is prepared from AMG 6,-5 total RNA using a random primer with an Wended 5-adapter (S’GGCCGGATAGGCCTCACNNNNNNT-3' (SEQ ID NO: 15)) and the materials and methods provided by the Gthco Superscript II ™ Preamplificanon System to First Strand cDNA Synthesis kit (cat. no. 18089-011). The oligonucleotides below are used for the PCR:

5' kappa RACE p rimer:

5’ - GAT GAC CCA GTC TCC AGO CAC CCT G - 3’ (SEQ ID NO: 5) SFXappaRACEprtmef:

5' - AA6 GGT CAG AGG GCA AAG GAT GG - 3·' (SEQ ID NO: 6) (0203] The amplified DNAs. ere cloned Into pCRlNTOPO (Invitrogen) and the resulting plasmids am sequenced. The fcapsa chain consensus sequence is used to design primers for PCR amplification of the full-length oOPGL-1 kappa chain. The 5' aORGL-1 kappa primer Incorporates a Xbel site (TCYAGA) for cloning end a “CCACC” Kozak sequence before the initiator Met codon. The 3' eOPGL-1 kappa primer incorporates a Sa/i site (GTCGAG) following the stop codon for cloning,

S’ oQPGL-i kappa primer:

5'~CAA CTC TAG A CC ACC ATG GAA ACC CCA GCG-3^f (SEQ ID NO: “) Aoal Site Kodak Μ E T R A (SEQ ID NO: 18)

2015264940 07 Dec 2015 kappa primer,

GAG GTC GAO TTA TGA AC.A CTC TCC CCT GTT GAA <3 -3^! (SEQ IQ MO: S)

Sgf Sits ) ID NO:

[0204] The fif^tength eOPGL-4 kappaohain oDNA clone is obtained using the AMO 6.5 first strand cDMA, described above, by FOR ampirficadon with the 5' and 3’ oOPGL-1 kappa pnmers, The PCR reaction generates a 738 bp fragment encoding the 235 amine acid residues (including the 20 amino acid kappa chain signal sequence) of the aOPGL-1 kappa chain (Figure 4, SEQ ID RO; 4). Following purification using a QIAquick PCR Purification kit (Qiagen cat. na.2SW4), this fragment is used to construct tire kappa light chain expression Vector, [0205] The 738 bp full-length agment generated above Is cut with Xdqi and Sato is purified using the Rrpmega Wizard DMA Gieari-Up System {Promegs cat, no. A7100), and is cloned into pDSRcdS to generate plasmid aOPGL-1-kappa/pDSRctlS (Figure 5). pDSRortS 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 pDSRc.19, pDSRo2 Is mr in tbs following ways; the FSH poiyA is shortened by approximately 1400 palm, to SSc base pairs, and now ends at the Ndci site; the dihydrofolate reductase {PHrR} promoter now contains 209 base pairs, having been shortened from the S' end by approximately 1 kiio'oase; and an approximately 550 base pair Belli fragment from the DHFR poiyA sequence is deleted.

2015264940 07 Dec 2015 [0206]

The ctOPGL-l kappa light chain expression cion® is sequenced to confirm that it coded fob the same peptide That is identified in the

AMG 5,5 hybridoma. The final expression vector, aOPGL-1-kappa,'pDSRsxIS, Is 5476 bp and contains the 7 functional regions shown in Table 2,·

Table 2; Features οΙ'αΟΡΟΕ-Ι-Ιτ^ρρ^/ρΟδΡΙίχΙδ

Pfesmfo Sase

Fair Number:

to 381 A transcription fermlnatlon/polyadenylation signal from the cc-subunit of the bovine pituitary glycoprotein hormone (a-FSH) (Goodwin, et al. Noc.fe/o Acids Res. 1983 11: 6573-82: Genbank Accession Number X0Q004)

882 to 2027 A mouse dihydrofolate reductase (DHFR) minigene containing the endogenous mouse DHFR promoter, the cDNA coding sequences, end the DHFR transcription terminatfon/ooiyadenylation signals (Gasser et al. Free Ate# Acad Sc/ U S A. 1982 78:8522-6.: Nunbsrg et al, Ce//1980 19:355-84: Setoer et al, J Bigs Chem. 1982 2S7: 5143-7: McGropan et al, d Biel Chew. 1935 260; 2307-14)

2031 to pSR322 sequences containing the ampicillin resistance marker 3947 gene and the origin for replication of the plasmid In E. coli (Genbank Accession Number 101749)

394S to An SV40 early promoter, enhancer and origin of replication

4292 (Takebe et al, Mo! Ceil Blok 1988 8; 466-72., Genbank Accession Number 302400}

4293 to A translational enhancer element from the HTLV-1 LTR domain

4585 ' (Seikl et al, Rrac Acad Sci U S A. 1983 30: 3618-22,

Genbank Accession Number 102029)

4574 fo An intron from the SV40 18S, 19S splice donor/acceptor signals 4730 (Okayama and Berg, Mo/ Cer/Sfo/. 1983 3: Ξ80-9, Genbank

Accession Number J02400)

4750 to The aOPGL-1 Kappa light chain cDNA between the Xbal and Sa/I 5476 sites

Ό2οη

A circular plasmid map of the vector is shown in Figure 5,

2015264940 07 Dec 2015 the aOPGL-1 heavy chain [0206] The eOPGL-1 lgG2 heavy chain Is cloned from AMG 6.4 hybridoma double-stranded cDNA produced with the Clontech Marathon™ cDNA Amplification Kit (cat. no. K1302-1), Amplification of AMG 6.4 heavy chain cDNA Is accomplished by 5·' end 3' rapid amplification of cDNA ends (RACE) techniques performed with human germline lgG2 heavy chain constant region specific primers (shown below) and RACE primers and ether materials and methods provided in the Marathon™ cDNA amplifioation kit.

¢0 igG2 RACE .pother

5·'- GGC ACG GTC ACC ACC CTG CTG AG -3' (SCO ID NO: 9}

3' IgGS. RACE primer

S'- CCT CCA CCA AGG GCC CAT CGG TOT -3' (SEQ ID NO: 10) [0200( The 600 bp 5' RACE product and 1200 bp 3' RACE product are cloned into pCR.2<1 (invitrogen) end are sequenced. This sequence information Is used to design aOPGU-1 heavy chain specific, primers for the cloning of the foil-length sequence. The heavy chain 5' pnrnar (5^f oOPGL-1 igG2 Primer) is directed against the sense strand and has a Hindi ll site and consensus Kozak sequence before the natural start site,. The heavy chain 3' primer (T oORGL-l lgG2 Primer) is an antisense primer that contains a Ga/Ί site arid stop codon after trie last amino acid of the heavy chain igG2 sequr

2015264940 07 Dec 2015

S' oQPGt-T lgG2 Primer:

S^!- CAGAAGCTtGACC^Cg ATG GAG TTT GGG CTG AGC TGG CTT ΤΪ7 GTT GTG GC ~ 3' (SEQ id MO;: 1)

Hsftdli! Kazak Μ E aQPGL-1 lg<32 Primer;

Q. L S W L F t, V A (SEQ © HQ: 1

S’- GOA rCTCGAC TTA TCA TTT AGC GGG AGA GAG GGA GAG - 3' (SEQ |O MO' 12)

Sail (S£&toW is) [0210] The double-stranded cDNA described above Is used to generate the idll-length heavy chain cDNA by PCR amplification with the S'- and 3'~ aOPGL-1 lgG2 primers, The PCR generates a 1433 bp fragment encoding the 467 amino acid residues. (includingThe 19 amino acid IgG signal sequence): of the aOPGL-1 lgG2 heavy chain protein (Figure 2, SEQ ID NO; 2). Following purification using a QlAguick PCR Purification kit (Qiagen cat. no,23104), this fragment is used to construct the heavy chain expression vector as fellows.

[0211] DNA encoding the full-length lgG2 heavy fragment generated above Is cot with F&idlli and Call, purified using a QIAqasck Gel Extraction kit (Qiagen cat no.2S704), and this fragment Is cloned into pDSRalS. The resulting expression plasmid is named aOPGL-1 -JgGz/pDSRal9 (Figure 6). All vector components tire identical to aOPGL-l’kappa/pDSRal9 vector, described above, except the aQPGL-1 lgG2 heavy chain cDNA replaces the ’01.4 kappa lidht chain cDNA. between theXbal andSa/l steto The aOPQL2015264940 07 Dec 2015 igG2 heavy chain expression done is sequenced to confirm that it coded for } the same polypeptide that is Identified in the AMG 6.4 hybridoma.

aGPGL-1 expression in GHO cells (0212} Stable expression of aOPGL-1 antibody is achieved by cotransfection of aOPGL-1-kappa/pDSRal9 and aOPGL-1 -lgG2/pDSR<x19 plasmids into dihydrofoiafe 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 1.5x10θ AM-1/D cells grown in CKO d“ medium (DMEM-high glucose, 10% fetal bovine serum,

1% penicillin/ streptomycin/giutamine, 1X NaPyruvate, 1% nonessentiai amino acids (NEAA))(Gibco®) and 1% hf supplement (Gibco®)) the day before transfections (Day 0). On day one, 400 μί of serum-free RPMi 1840 medium (Gibco®) is aliquoted into a 12x75 mm polypropylene tuba. Twenty four microliters of 7rans!T®-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.S pg of aOPGL1-kappa/pDSRa19 and 7.5 pg of aOPGL-1 ~lgG2/pDSRa19, digested with Pvu1) is then added dropwise to the mixture and is incubated at room temperature for minutes.

[0214] The CHO d- medium is removed from the ceils, which are washed with 10 ml of Duibeccc's phosphate buffered saline (Gibco®). Six

2015264940 07 Dec 2015 rnftRifters of serum-fres MEM medium supplemented with KT, L-glu . NEAA,

No pyruvate (Gibco©) is added to the ceils. The DNA/LT1 complex Is added dropwlse to the plates, which are gently rocked hack and forth to distribute the □ KA evenly over the ceils, .After 8 hours In a tissue culture Incubator, the medium is replaced with fresh CKO d- medium. Forty eight hours later the cells are spilt to teh 100 mm culture dishes In CKO select medium (DMEM high glucose, 10% dialyzed fetal bovine serum (FBS), 1% penicillin /streptomycin /glutamine, 1% nonessential amino acids arid IX Ka pyruvate) (Giboo©),

Medium Is changed twice weekly until colonies appeared, [0215] After 1C~14 days, colonies ere picked using c mm cloning discs (Labours®) soaked In 1 x trypsin-EDTA (Glbcc®) and ere cultured in 24 well tissue culture plates with CKO select medium. When the cells become confluent, serum free media (CKO 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 aGPGL··! heavy chain, and goat antKbumsn Lapps chain antibody (Pierce, Rockford, It) followed by KRP-oomugated rabbit anti-goaf IgG(H-i-L) antibody (Pierce, Rockford, IL) to detect the aOPGL-1 light chain. The highest expressing clones era expanded and stored In liquid nitrogen.

2015264940 07 Dec 2015 id Creation of Gel! ..me I25Q

CHQ cells producing oOPGL-1 are cloned by two rounds of limiting dilution in 96 well plates under serum-ires ccndh.cns. The clones are selected based on production end growth characteristics In various suspension vessels, EIAs are performed to select the clone that produces me Highest level of cOPGLd, Growth characteristics, including doubling times and densities then measured by growing the clones in 100 ml, 250 ml.. 500 mL 1 I, and 3 i spinner flasks, as well as In 31 Aplikon bioreactors. The clone with the f dsuhOrig time that reaches the highest density in culture fe sefeeted, end fe designated Ceil Line 125Q. When the cions has expanded to yield sufficient celts to freeze 360 ampules at approximately 1x10⁷ ceils/mL, ceils are resuspended in a cr/opresarvaflvs, serum-free medium (90% VM-Soy Eaten Medium (see Table 3 for details} supplemented with 10 ml/L nonessential amino acids and 10 ml/L L-Giutamine (Glbco/LTI/lnvitrogen), 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.

10217} Based on growth and production In small-scale spinners and rger scale bioreactors, Cell Line 125Q is chosen as the cell line for manufacturing of o'OPGL-1.

2015264940 07 Dec 2015

Cell Cbitbre [0218] aOPGL-1 is produced by expression in Ceil Line 125Q, a clonal line of CKO cells that expresses oOPGL-1 from plasmids oOPGL1~koppe/pDSRa19 end oOPGL-1 ~lgG2/pOSRa19, 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 Saleh Medium (see Table 3 for composition) supplemented with 10 mi/L nonessential amino acids and 10 ml/L L-giutamihe (Gibco/LTI/invitrogen) (VM-Soy Sapp) in 125 mi erlenrneyer shakers at 100 rpm for 5 days. The entire culture is then used to inoculate VM-Soy Supp in a 500 mi spinner flask to 3x10^y viable ceils/ml (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 3£S vc/mi, and is grown with 70 rpm spinning for <M days.

(0219j The culture from the 3L. spinner flask is then split to two 3 L spinner flasks si 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, end grown under the same conditions. Four lifers of culture from the four SL spinner flasks is used to inoculate-10 Lof W-Soy Supp without phenol red In a 2Q L bioreactor, end tile bioreactor is run in fed-batch mods for 7-10 days, In fed-bateh mode, a nutrient feed containing concentrated media components (“Feed”, as sat ‘forth below in Table 3) is added to maintain cell growth and culture viability.

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2015264940 07 Dec (0220} The entire culture from the 20L bicreactor is then used to inoculate 70 L of VM-Soy Supp without phenol red in a 150L foloreactor, and the Goreacter is run in tod-batch mode for ,9-10 days. Finally, the entire culture from the 1S0L bioreactor Is used to inoculate approximately 880 L of VM-Scy (without supplement or phenol red) in a 2000L bioreactor, and the bforeactor is run in fedbatch mode, The rate of feed during fed-batch mode is determined such that the glucose level in the culture is maintained at O.S g/L for each bioreactcr. The cell density and glucose concentration are measured dally and the rate of toed adjusted accordingly, (0224} Production In the 2000L bioreactor lasts for approximately two weeks during which time «OPGL-1 is canstitutively produced by the cells and secreted into the cel i culture medium [0222} The production reactor is controlled at set pH, temperature, end dissolved oxygen lever, pH is 7.0 and ss 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 nt 37*C throughout the process, All gases are passed through membrane filters of pore size 0.22 pm nr less, [0.223] At the end of production, the cell broth is ted Into a disk stack centrifuge and the culture supernatant is separated from the calls. The centrals is further clarified through, a Cuno 90SP depth niter followed by a 0,2 prn Posldvne filter (Pail Co,}. The clarified conditioned media Is then concentrated by tangential flow ultrafiltration (UP) using 50kD NMWL membranes (Millipore

2015264940 07 Dec 2015

Biomax 50), The conditioned media is concentrated 15-to 3Q- fold. The resulting eoncsnfrefed oondlilofted medium (COM) is then either processed through purification or frozen for purification at a later date. The production process Is summarized in Figurel 9.

SllLOuifeeliedium [02241 The ceil culture medium for use throughout the entire coil culture process is based on Dulbecco’s Modified Ecgfe's Medium /Ham’s Nutrient r12 (DMEM/F12, rl)_f and contains supplemental levels of amino acids, additional nutrients and salts, a soy hydiolysate and recombinant human insulin fNucsliin^eZrs, 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 pre pore size prior to use.

COMPONENTS FOR BASAL Media and FEEDS

Table 3, Celt Culture Media Components

COMPONENT	VMSoy Batch Medium (cig/J..·)	FEED fmgZL)
ΠΜΕΜΖΡΠ COMPONENTS
fnorga&ic salts CaCis feshysl)	116.60	233.2
CuSO-uSHoO	0,0026	0.0052
Fs(NO?)3.9H?.O	0,1000	0,2
FeSO4.7H20	0,8340	r 1,668
κα	311,80	f 623,6
MgCls (aahyd.)	57,280	114.56
MgSO (anhyd.)	97.680	195.36
NaCl	905,9,90	1811.95

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2015264940 07 Dec

Nalfc?<XH20

NsSKPO*

ZnSO<f.7H20

Other Components D-Glucose Ma Hypoxanthine Lmolsie acid Ltpoic add Phenol Red Puiresciae.2HCi Sodiums pyruvate

Amino adds

L-Alarune J

L-Arginine ECI

L* A sparagine.H’O

I.-Aspardc acid |

L«Cysteine.ECiJisO Ϊ

L-Cysiffi3,2HCI I

L-Glutamie acid

L-Glutamine

Cdycms j

L-Histidioe.HCl.HaO :>

L-fsoIeycine i

L-Lcucine

L-Lysme· HGI

L-MeOtiooinc

L“Phs»yMasise> >

L-Proliae

L-Serins

L-Threonine j

L-Trypiophan . |

L*Tyrosine.2Na.2EfeO 1

E-Valine „ Vitamins:

Biotin [

125.00	250
142.040	284.08
0,8640	1 1.728

3151.00	| I23Q2
5.40	1 10.8
0.C90	0.18
0 2060	1 0-412
Ξ.ϊΟ	| 16.2
0,1620	0.524
; 10.00	220

26,70	;. 53.4
295.00		590
45,00		90
39.90		79,8
35,120		70.24
62,580		125,16
44.10		88.2
657.00		: 1324
52.50		105
62.950		125,9
108.940		217,88
118,10		236.2
182.50		365
34,480		58,96
70.960		141,92
5750		115
73.50	i	147
106.90	. j	213,8
18.040	I	36.08
111,580	s	223.16
105,70	•t	211.4

0.0073 I 0.0146 ο

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2015264940 07 Dec

D-Ca PaatoftmW	4.^80	8.06
Choiiae Chloride	17.960	35.92
Folic Acid	5.30	10.6
i-Inofitel	25.20	50,4
Niacinamide	4,040	S.OS
Pyridoxal HC1	4.00	S
Pyridoxine HQ	0.0620	0,124
RIbofiavm.	0.4380	0.S76
Thiarnine HCi	4340	8.68
Thymidine	0,3635	0.727
Vleimin BI2	1.360	2.72
,U)DfflONAL CO WOPENIS		1. ..
Necellin Zn, (rhu ihsvlm)	5.00	15
Seieuous Acid	: 0.0050	0.015
Sdiaaolaaiine	0.0012 .	0.0037
Triiodothyronine	0.000040	0.00012
Hydro eorti sons	0.020	0.06
Ferric Citrate	122.450	122,450
Plurorac F-68	1000.00	500
Soy Hydrolysate	6000.00	1 6000.00
NaHCOs	3000.00	3000.00

3500.00

Purification Process [0225] uOPGL~1 expressed in CHO ceils is secreted into the extracellular medium, A series of steps may be used to generate pure material· Trie 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.

2015264940 07 Dec

A, Hydrophobic Charge. Induction Chromatography (HCIC) (0228) This chromatography step removes the majority of host cell proteins and DNA, The concentrated conditioned media {COM) is filtered through a Cuno 3DSP filter and then through a Cunc VR07 charged celiatese-.feased 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 s low pH buffer (20 mM Sodium Acetate, pH 5,0), As ills elated from the column, the product Is collected based on the absorbance at £80 nm of the column effluent.

8, Viral Inactivation

1022/1 The-MEP pool is titrated to pH 3.7 and is held fcr approximately 80 minutes to inactivate potentially contaminating tetrovlrua, Following the held step, the pH is adjusted to approximately 6,0,

C. Viral Filtration {0£28j The pH-adjusted pool is filtered through a MilUpore Viresoive NFR filter or equivalent. The antibody flows through the filter white potentially contaminating viruses > 50 nm are retained.

D. Cation Exchange Chromatography (CEX) (0228] The antibody Is further purified by cation exchange chromatography using SP Sepharcse HP (/\rr.ersham Pharmacia) or equivalent. The cahon exchange chromatography step removes additional CHO cell proteins, DNA, lower molecular weight proteins, and aggregated forms of cOPCI..-t, The viral filtered pool is loaded onto the cation exchange resin. After ο

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2015264940 07 Dec loading, the column is washed with equilibration buffer (20 mM NaMES pH 8.2). The antibody is then eluted with a linear gradient' of increasing salt (20 mM NaMES pH 6.2, 0 M NaCl toZO mM NaMES pH 6,¾ 0,3 M NaCl). As it so eluted from the column, the product is collected based on the absorbance at 230 am of the column effluent.

E. Hydrophobic interaction Chromatography (NIC) [02301 The antibody is further purified by hydrophobic interaction chromatography using Phenyl Teyopesd 850S (Tosoh Blosep) or equivalent The hydrophobic Interaction chromatography step is used as a polishing step and removes additional CKO cell proteins, DNA, lower molecular weight proteins, and aggregated forms of aOPGE-1. The cation exchange pool Is conditioned before loading onto the column by addition of ammonium sulfate to a conductivity of >1 OS mS/cm at 1f5-25^eC, After loading, the column Is wearied 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, QrnM Tris pH 8 to 0M Potassium Phosphate. 20 rnM Tris pH S). As if is eluted from the column, the product is collected based on the absorbance at 2S0 nm of the column effluent,

P. Concentration and Dianiiration (0231} The HiC column pool is concentrated and dlaflite-red into formulation buffer by tangential flow ultrafiltration using 50RD NMWL. membranes (MIHIpore Biomax 50). The formulation buffer includes 10 rnM Acetate, 5% Sorbitop pH 5.2 and dOPGl-i Is at 30 mg/mL

2015264940 07 Dec 2015

Filtration and Storage [Q232] The purified bulk is passed through a 0.22 um PVDF ftfter iporeX is sarrtpted, and stored at approximately -30*0 in a secured freezer.

Example 4

Binding Specificity of aQPGL-i [0233] Antibodies that ere produced in GKO ceils 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 8.

(0234] Human OFG binds and neutralizes OPGL in rats, mice and oynomolgus monkeys, as weii as in humans, aOPGL-1 binds human OPGL with high affinity out does not. md significantly to murine OPGL (Table 4).

Table 4: Affinity of «OPGL-Ι to Ceil Membrane Expressed OPGL of Human, Cynomelgus Monkey, or Mouse Sequence.

OPGL Species	aOPGL-ϊ .Efis-i (ag/rnl)
H-;msn	to
Cynomoigus	ip
Mouse	No Specific Binding

OPGL of these species is expressed in CKO sells as the ?uil4engih_; membrane-hound piotesn. Binding of oOPGL-1 to the call surface expressed OPGL is assessed by FACS analysis of cells Incubated With aOPGL-1 and s FITC-lsbeied secondary antibody to human lgG2. aOPGL~1 binds burner, and cynomolgus OPGL but there is no specific binding to mouse OPGL.

[0235] In addition, human OPS has been reported c *hcw msk binding to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (Truneh of al. 2COO), a felatsd msmber ©f the TNF family, which shows DNA and ο

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2015264940 07 Dec •amino sold sequence homology to OPGL (Lacey et ah, does not delectably bind to other INF-related proteins- such as TNFa, ΤΝΡβ, or OSAO ligand, [0230-1 uOPGL-1 binds specifically to. OPGL on El A plates (Figure 7). Recombinant soluble OPGL (2 pg/mQ is coated onto 96-well SA plates at room temperature for is to 24 hours, After blocking with 1% BSA in PBS, varying concentrations of aOPGL-1 (approximately 2 ng/mi to 1OQO ng/mi) diluted in 1% BSA/PRS are added to the wells and the plates are incubated for about 2 hours at room temperature, Bound antibody is detected with goat snti-Human IgG (FaP'i-'KEP using TMB-H2O2 (tefrarnethyibenzidine and hydrogen peroxide) substrate cocktaiL The absorbance is read at 450nm and bSCntn.

[02371 aOPGL-1 binds specifically OPGL expressed on the surface of transfected ceils (Figure 8}„ oOFGL.~1 (100 ng/mi) diluted in FACS Buffer (PBS, 01’%BSA, O,01%Sodium Azide) is presncubated with varying concentrations of OPGL, TNFa, TNFb. TRAIL, or CD40 ligand (approximately 0.1 ng/ml to WOO ng/mi), and is then added to approximately 200,000 CHO REN 218-9 cells, which are CHO ceils sfabiy expressing membrane-bound OPGL on the ceil surface. After 1 hour at2-8°C, unbound antibody is removed by caotrifogation and washing. Ceils are then incubated for 30 minutes at 2*8*0 with FITC-labeled P(eb') 2 Goat anfi-Human IgG (Fey fragment specific). After centrifugation and washing, cell surface fluorescence is measured using flow cytometry. Figure 8 shows that binding of uORGL-1 to CHO REN 218-9 coils Is ο

Cd

2015264940 07 Dec rifle, and is cchipeflfiveiy reduced by addition of soluble GPGL, but not by iFa, TNFb, TRAiL, or CD40 ligand, in competition experiments, gOPGL-1 binding to OPGL on ΞΙΑ plates is inhibited by addition of exogenous OPGL (Figure 9), but not by the addition of TNFa, ΤΝΡβ, TRAIL, or CD40 ligand (Figure 10), This proesdora is performed in substantially the same manner as above, for binding of oOPGL-1 to OPGL on EIA plates, except a constant concentration of oOPGL-1 (IdOng/mL) is praincubated with varying concentrations of soluble OPGL or other iigands (approximately 1 ng/mi to 1900 ng/mi ter each) before it Is added to the OPGL·· nOPGL-1 Neutrafeins Acfvrtv inhibition of Qstesciasf FermdfiPn (G2393 RAW 264,7 (ATCC No. TIB-71, Manassas, VA) Is a murine ' macrophage cel) line that was derived from an .Abeison murine leukemia virusinduced turner, RAW 264,7 cells will differentiate to osteoclast-like celis 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) Ce/7 39 p, 309. and Lacey et al (1988) Ceil 93 p, 165, which are herein incorporated by reference for any purpose.

[Q240I RAW ceils are stimulated by ligand to differentiate into osteoclast-like cells, and the differentiation can be measured by TRAP activity.. a

2015264940 07 Dec 2015 property or osteoclasts. Thus, the effect of aOPGL~1 on osteoclasiogenesis can be measured.

(0241 j RAW ceils ere incubated for 4 days in the presence of a constant amount of OPGL (40 no/mi) and varying amounts of aOPGL-1 (6,3 ng/mt to 200 ng/ml) in ceil culture medium (DMEM, 10% PBS, 0.292 mg/ml L~

Glut, 100 units/ml Penicillin G, 100 pg/rni Streptomycin sulfate). At the end of 4 ' days, the ceils are stained for tartrate-resistant acid phosphatase (TRAP) activity by permsabifeation and ac-d.ficaiion, followed Py treatment with para-nitrophenyiphosphate for 5 minutes. Briefly, the media Is aspirated off of the ceils, and 100 ul of citrate buffer (410 ml 0.1M citric acid, 590 ml 0,TM citrate, trlsodlum 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 bundreif microliters of PNPP solution is then added (157,8 mg acid phosphatase reagent (Sigma 104100), 7,2 mi tartrate solution (Sigma cat, no. 387-3), and 22,8 ml citrate buffer), and plates are incubated for 3 to 5 minutes at room temperarure, The reaction is terminated by addition of 60 ul 0.5 M NaOH solution, (0242) TRAP will convert paramitrophenylphosphats to paranitrophenol, 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 dOPGL-l concentration is shown in Figure 11, and demonstrates that «OPGL-ί inhibits osteoclast formation in this assay.

2015264940 07 Dec 2015 fhhMdRofOPGL Bsr potency of aOPGL-1 is 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 (K7F.F) to detect binding of aOPGL-1 to Europium-conjugated osteoprofegerin ligand (Eu-QFGL), If oOPGL-t inhibits Eu-OPGL binding to ODAR. fluorescent output will decrease, and the amount of gOPGL-I present will be inversely related to the amount of (024-4] OPGL is febeied with europium, which emits tight at 620 am when excited with 337 nm light ODAR is fused to FLAG and to Pc, end the FcODAR-FLAG fusion protein is labeled with an anti-FLAG antibody linked to allophycocyanin (ARC), a fiuorophore which emits 665 nm light when excited by light at 620 nm. Therefore, when Eu-iabsied GP<3 ligand binds to the Fc-ODARFLAG/anti-FLAG-AFC complex, the tertiary complex will emit 565 nm light when excited with light at 337 nm,, [0245] Eu-OPGL at 0,05 pg/ml is prsincubated with various doncenfeattens. (0,1 fd 150 ng/mi) of aGPGL-1 i n assaybuffer (50 mM Tris pH 3,, 100 mM NeCI. 0,05% NaN₃, 0,1% BSA, and 0.05% Tween 20) at room temperature for approximately one hour (Prelncuhation mix), A mixture of FcODAR-FLAG (1 pg/ml) and anfi-FLAG-APC (2.5 pg/ml) is also prepared in assay buffer and Incubated st room temperature for one hour (Fluorochrome mix), Equal volumes of Preincubation mix and Fiuorochrome mix ere then combined

100

Ο

CU

2015264940 07 Dec and incubated at mem temperature for 3 hours. The fluorescence is. measured by reading plates on the Packard Discovery HTRF rnlcrppiate analyzer using sn excitation wavelength of 337 rjm and an omission wavelength of 665 nm.

[02461 When qOPGL-1 is preincubated with Eu-OPG ligand and is then mixed with Fc-QOAR-FLAG/antl-FLAG-APC, the fluorescent ihfensfrv at bbo nro decreases m a cosemanner, -as shown in Figure 1] demonstrating that aOPGLI6.2 can effectively inhibit OPGL binding to ODAR.

Pharmacokinetics in Cynomoigus Monkeys:

Six male and six female cynomclgus 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, end 4 each consists of 1 male end 1 female. Animals in Group 1 are administered a single SC dose of 1 .mg/kg oOPGL-t while animals in Groups 2, 3 and 4 are administered single IV doses of 0,1, 1.0, or -J 0,0 mg/kg of aOPGL-l, respectively, [0248J Animals are dosed with oOPGL-1 expressed from transacted Chinese hamster ovary/ (CHO) cel’s, Serum samples are taken for determination of oOPGL-1 levels, antibody analysis, and analysis of the bone turnover marker serum N-teiopeptlde (serum N-Tx), alkaline phosphatase (ALP), and serum calcium (serum Co). Urine is also collected for analysis of Ntelopeptide (urine N-'fx) and creatinine, (02491 The serum concentration-time profiles following IV administration are characterized ov a id-phasic distribution (Figure 13). initially,

2015264940 07 Dec 2015 there is a rapid distribution phase, followed by a significantly slower plateau phase, which appears to be concentration-dependent. The third is a rapid elimination phase.

jrapartmental analysis'of complete serum concent ration-time p rofltes analysis of the data up to 1 using WinNonKn Professional (vl.S), and exponential 4 days after test article administration and above

10,000 ng/mL using SAAM II (vl.1.2) are utilized to investigate the pharmacokinetics of oOPGL-1 In monkeys. The initial volume of distribution from all IV doses averages 28.0 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~l has an average distribution half-life (f^) of 6.02 hours, an extended secondary phase with a half-life (t^) that increases with dose from SS.9 hours at a dose of 0,1 mg/ko to a maximum of 444 hours at a dose of 10,0 mg/kg, Terminal elimination half-lria (tv-ΰ estimated non-compsrtmentaiiy averages 31 hours across all IV dose groups. Clearance (CL, Cb'P) of oOPG.L-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 mgi x₀₁ :(62511 After administering subcutaneously, absorption Is slow, with: average peak concentrations (C_max) of 11,800 ng/ml at 132 hr, There is high variability in the range of exposure after SC administration, resulting in an average clearance of 0,38? x 0,281 ml/br/kg and mean residence time of 202 ± 80.1 hours,. Average bioavailabillty is 89%.

102

2015264940 07 Dec 2015 (UZSzJ ; h& preceding cate are summarized in Tsoie 5,

Table· 5; Mean -;± SD) Mon-Compartmental Pharmacokinetic Parameters³¹ In Cynornofgus Monkeys After Administering a Single Dose of aQPGt~t |V and SC p Notj-Cpinparf jnestal Paraineisr Estimates

	1 US msS&r i...........................' ........		10 mg/kg
on ms-ug	Magfcg I
Parameier	Ute	j SC in	=,.5)	IV (n»2)	IV (nte) i	IV i-r-XS}
		Mean 1	SD ./	Msan	Msas I	Ms 5Ώ
	hr	j 132 I	S'3,2	0	\ j V 5	0
	mgtel	I I 1.600 i	3410	43,30	38200 |	3767)00
	hr	j 34,$ J	ι m	30.?	31.4 ί	ND
	ugteteil	| 3520 |	,7;0	233	39S0 :	99900
CL, CL/?	ml 4tf4ig	I 0.3S7	0,781	0.40?	Q.25S i	0,120
MKT	<te	i 202	30. i	84,3	124 i	519
V$s		tw	N/A	33.?	31·7 1	55,9

'’Vsiues'sTe refsoiieTfe 3 sigmficaat Inures·..................................................

“Hot feteimirsSif, PK swipes end tterif® pistes) $ j phase hafics ierminal phase & sol efeseryte ^'NstAppHca&fe:

[0253] dOPGL-1 causes a rapid decrease in semm fOx tevefe 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 3 SC dose of 1.0 mg/kg. Maximum effect increases with dose from approximately 80 to 91% ever the dose range of 0,1 to 1 rng/kg. However, at higher doses no further suppression is observed with maximum inhiuitlon of 91%, Mean levels of serum N-Tx return to baseline by day 28 after administering O.l.mg/kg IV and by day 70 after administering 1 mg/kg SO. Urine N~Tx shows similar trends to those of serum N-Tx, except that ail groups return to baseline values by study day 105

103^:

2015264940 07 Dec [02541 Suppression ef serdmCa jneredses with dose to a mean nadir of 31.6% below the baseline average seven days after IV administration of 10,0 mg/kg. All ether dose groups have mean decreases in serum Ca of less than 26.4% from their baseline averages. By day 17 all serum Ga tevefe in treated animals return to within 10% of them baseline averages (Figure 201.

[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 end a more prolonged suppression then 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-Ί is a bone anti-resorptive agent, (02661 The majority of animals (9 of 12) develop antibodies to «OPGL- L The incidence of antibodies to oGPGL-1 is not dose or route dependent. It is not possible to assess the effect of antibodies to aOPGL-Ί on oOPGL-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 cOPGL 1 Is cieared prior to antibody development and therefore, effects on aOPGL-1 disposition are not observed (Figure 16).

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2015264940 15 Dec 2017

Claims (30)

1. Claims

   1. An antibody or binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises CDR1, CDR2 and CDR3 of SEQ ID NO: 13 and wherein the light chain comprises an amino acid sequence of SEQ ID NO: 14 wherein the antibody interacts with an osteoprotegerin ligand (OPGL).
2. 2. The antibody or binding fragment thereof of claim 1, wherein the heavy chain comprises a first variable region comprising the amino acid sequence of SEQ ID NO:

   13 and wherein the light chain comprises a second variable region comprising the amino acid sequence of SEQ ID NO: 14.
3. 3. The antibody or binding fragment thereof of claim 1 or 2 wherein the heavy chain comprises the variable region and the constant region of SEQ ID NO: 2; and the light chain comprises the variable region and the constant region of SEQ ID NO: 4.
4. 4. The antibody or binding fragment thereof of any one of claims 1 to 3 wherein the antibody comprises (i) a human lgG2 heavy chain comprising the amino acid sequence of SEQ ID NO:13, and (ii) a human kappa light chain comprising the amino acid sequence of SEQ ID NO:14.
5. 5. The antibody or binding fragment thereof of any one of claims 1 to 4, wherein the antibody is fully human antibody.
6. 6. The antibody or binding fragment thereof of any of one of claims 1 to 5, wherein the antibody inhibits binding of osteoprotegerin ligand (OPGL) to an osteoclast differentiation activation receptor (ODAR).
7. 7. The antibody or binding fragment thereof of any one of claims 1 to 6, wherein the heavy chain and the light chain form a single-chain antibody.

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   2015264940 15 Dec 2017
8. 8. The antibody or binding fragment thereof of any one claims 1 to 7, wherein the heavy chain and the light chain form a single-chain Fv antibody.
9. 9. The antibody or binding fragment thereof of any one of claims 1 to 6, which is a Fab antibody, a Fab’ antibody or a (Fab’h antibody.
10. 10. A pharmaceutical composition comprising an antibody or binding fragment thereof of any one of claims 1 to 9 and a pharmaceutically acceptable excipient, diluent or carrier.
11. 11. An isolated composition comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a heavy chain and the second polynucleotide encodes a light chain, and wherein the first and second polynucleotides together encode an antibody or binding fragment thereof of any one of claims 1 to 9.
12. 12. The composition of claim 11, wherein the first and second polynucleotides are part of separate nucleic acid molecules.
13. 13. The composition of claim 11 or 12, wherein the first polynucleotide is part of a first vector and the second polynucleotide is part of a second vector.
14. 14. The composition of claim 13, wherein the vector is a viral vector.
15. 15. The composition of claim 13, wherein at least one of the first vector and the second vector is a viral vector.
16. 16. A method of treating bone loss in a subject comprising administering an antibody or binding fragment thereof of any one of claims 1 to 9 or the pharmaceutical composition of claim 10 or the composition of any one of claims 11 to 15.
17. 17. A method of claim 16 wherein the bone loss is associated with at least one condition selected from osteoporosis, Paget’s disease, osteomyelitis, hypercalcemia, osteopenia, osteonecrosis, an inflammatory condition, an autoimmune condition, rheumatoid arthritis, and cancer.

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    2015264940 15 Dec 2017
18. 18. The method of claim 16 or 17 wherein the bone loss is associated with an inflammatory condition, and wherein the method further comprises administering at least one additional therapeutic agent selected from an interleukin-1 (IL-1) inhibitor, IL1ra, anakinra, a TNFa inhibitor, a soluble TNFa receptor, etanercept, an anti-TNFa antibody, infliximab, a D2E7 antibody, a non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, celecoxib, rofecoxib, and leflunomide.
19. 19. The method of claim 16 or 17, wherein the bone loss is associated with an autoimmune condition, and wherein the method further comprises administering at least one additional therapeutic agent selected from an interleukin-1 (IL-1) inhibitor, IL1 ra, anakinra, a TNFa inhibitor, a soluble TNFa receptor, etanercept, an anti-TNFa antibody, infliximab, a D2E7 antibody, methotrexate, a soluble form of CTLA4, and a modulator of glucocorticoid receptor.
20. 20. The method of claim 16 or 17, wherein the bone loss is associated with rheumatoid arthritis, and wherein the method further comprises administering at least one additional therapeutic agent selected from an interleukin-1 (IL-1) inhibitor, IL-1 ra, anakinra, a TNFa inhibitor, a soluble TNFa receptor, etanercept, an anti-TNFa antibody, infliximab, a D2E7 antibody, a non-steroidal anti-inflammatory drug (NSAID), a COX-2 inhibitor, celecoxib, rofecoxib, leflunomide, methotrexate, a soluble form of CTLA4, and a modulator of glucocorticoid receptor.
21. 21. The method of claim 16 or 17, wherein the bone loss is associated with cancer, and wherein the method further comprises administering at least one additional therapeutic agent selected from a keratinocyte growth factor (KGF), a KGFrelated molecule, a KGF modulator, an anti-Her2 antibody, an anti-CDC20 antibody, and an anti-EGFR antibody, and a PAF antagonist.
22. 22. The method of claim 16 or 17, wherein the bone loss is associated with cancer, and wherein the method further comprises administering at least one of radiation therapy and chemotherapy.

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    2015264940 15 Dec 2017
23. 23. The method of claim 22, wherein the cancer is selected from breast, prostate, thyroid, kidney, lung, esophageal, rectal, bladder, cervical, ovarian, liver, gastrointestinal, multiple myeloma, lymphoma and Hodgkin’s Disease.
24. 24. A host cell comprising the composition of any one of claims 11 to 15.
25. 25. The host cell of claim 24, which is a prokaryotic host cell or a eukaryotic host cell.
26. 26. The host cell of claim 24, which is a mammalian host cell.
27. 27. The host cell of claim 26, wherein the host cell is selected from a Chinese hamster ovary cell, a HeLa cell, a baby hamster kidney cell, a monkey kidney cell, and a human hepatocellular carcinoma cell.
28. 28. A method of producing an antibody or binding fragment thereof that interacts with osteoprotegerin ligand (OPGL) comprising culturing a host cell of any one of claims 24 to 27.
29. 29. An antibody or binding fragment thereof when produced by the method of claim 28.
30. 30. Use of an antibody or binding fragment thereof of any one of claims 1 to 9 or a composition of any one of claims 11 to 15 in the preparation of a medicament for treating bone loss.

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    FIGURE 1 aOFGL-1 Heavy Chain cDNA

    DNA sequence of heavy chain expression piasmid beginning at HlndlH site, through Sail site. Start codon begins at nt 14, stop codon begins at nt 1415.

    I AAGCTTSACC ACCATGGAGT TTGGGCTGAG CTGGCTTTTT CTTGTGGCTA TTTTAAAAGG 61 TGTCCAGTGT GAGGTGCAGC TCTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC 121 CCTGAGACTC TCCTOTGCAG CCTCTGGATT CACCTTTAGC AGCTATGCCA TGAGCTGGGT 181 GCGGCAGGCT CCAGGGAAGG GGCTGGAGTG GGTCTCAGGT ATTACTGGGA GTGGTGGTAG 243 TACATACTAC gcagactccg tgaagggccg cttcaccatc tccagagaca attccaagaa 301 CA0GCTGTAT CTGCAAATGA ACAGCCTGA6 AGCCGAGGAC ACGGCCGTAT ATTACTGTGC 361 GAAAGATCCA GGGACTACGG TGATTATGAG TTGGTTCGAC CCCTGGGGCC AGGGAACCCT 421 GGTCACCGTC TCCTCAGCCT CCACCAAGGG CCCATCGGTC TTCCCCCTGG CGCCCTGCTC 481 CACCAGCACC TCCGAGAGCA CA6CGGCCCT GGGCTCCCTC GTCAAGGACT ACTTCCCCGA. 541 ACCGGTGACG GTGTCGTGGA ACTCAGGCGC TCTGACCAGC GGCGTGCACA CCTTCCCAGC 601 TGTCCTACAG TCCTCAGGAC TCTACTCCCT CAGCAGCGTG GTGACCGTGC CCTCCAGCAA 661 CTTCGGCACC CAGACCTACA CCTGCAACGT AGATCACAAG CCCAGCAACA. CCAAGGTGGA 721 CAAGACAGTT GAGCGCAAAT GTTGTGTCGA GTGCCCACCG TGCCCAGCAC CACCTCTGGC 781 AGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC 841 CCCTGAGGTC ACGTGCGTGG TGGTGGACGT GAGCCACGAA GACCCCGAGG TCCAGTTCAA 901 CFGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCACGGG AGGAGCAGTT §61 CAACAGCACG TTCCCTGTGG TCAGCCTCCT CACCGTTGTG CACCAGGACT GGCTGAACGG 1021 CAAGGAGTAC AACTGCAAGG TCTCCAACAA AGGCCTCCCA GCCCCCATCG AGAAAACCAT 1081 CTCCAAAACC AAAGGGCAGC CCCGAGAACC ACAGCTCTAC ACCCTGCCCC CATGCCGGGA 1141 GGAGATGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC A4AGGCTTCT ACCCCAGCGA 1201 CATCGCCOTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACACCTCC 1261 CATGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG CTCACCGTGG ACAAGAGCAG 1321 GTGGCAGCAG GGGAACCTCT TCTCATGCTC CGTGATGCAT GAGGCTCT'GC ACAACCACTA

    1381 CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAATGATAA GTCGAC (SEQ ID no: 1)

    1/21

    FIGURE 2

    2015264940 07 Dec 2015 aOPGL-1 Heavy Chain Amino Acid Sequence

    The IgG2 signal peptide is underlined, the variable region is in capital letters and is not underlined, and the constant region is In lower case.

    1 MEFGL5WLFL VA1LKGVQCE VQLLESGGGL SI GKGLEWVSGI TGSGGSTYYA DSVKGRFTIS 121 ttvimswfdp WGQGTivrvs sastkgpsvf 131 swnsgaltsg vbtfpavlqs sglyslssvv 241 rkccvecppc pappvagpsv flfppkpkdt 301 gvevbnaktk preeqfnstf rvvsvltvvh 361 gqprepqvyt Ippsreemtk nqvsltclvk 421 dgsfflyskl tvdksrwqqg nvfscsvmhe

    VQPGGSLRLS CAASGFTFSS YAMSWVRQAP RDNSKNTLYL QMNSLRAEDT AVYYCAKDFG plapcsrsts estaalgclv kdyfpepvtv tvpssnfgtq tytcnvdhkp sntkvdktve lirnsrtpevt cvvvdvshed pevqfnwyvd qdwlngkeyk ckvsnkglpa piektisktk gfypsdiave wesngqpenn ykttppnilds alhnbytqks Islspgk (SEQ IP no: 2)

    2/21

    FIGURE 3 oOPGL-1 kappa Light Chain cDNA

    2015264940 07 Dec 2015

    DNA sequence of kappa chain expression piasmsd sequence from Xba! sfe through Sail site. Start codon begins at nt 12; stop codon begins at nt 717

    1 TCTASACCAC CATSGAAACC CCAGCGCAGC TTCTCTTCCr CCTGCTACTC TGGCTCCCAG 61 ATACCACCGG AGAAATT6TG TTGACGCAGT CTCCAGGCAC CCTGTCTTTG TCTCCAGGGG 121 AAAGAGCCAC CCTCTCCTGT AGGGCCAGTC AGAGTG7TCG CGGCASGTAC TTAGCCTGCT 181 ACCAGCAGAA ACCTGGCCAG 6CTCCCAGSC TCCTCATCTA TGGTGCATCC AGCAGGGCCA 241 CTGGCATCCC AGACAGSTTC AGTGGCAGTG GGTCTGGGAC AGACTTCACT CTCACCATCA 301 GCAGACTGGA GCCTGAAGAT T7TGCACTGT TTTACTGTCA GCACTATGGT AGTTCACCTC 361 GGACGTTCGG CCAAGGGACC AAGGTGGAAA TCAAACGAAC TCTGGCTGCA CCATCTGTCT 421 TCATCTTCCC GCCATCTGAT GAGCAGTTGA AATCTG6AAC TGCCTCTGTT GTGTGCCTGC 481 TGAATAACTT CTATCCCAGA GAGGCCAAAG TACAGTGGAA GGTGGATAAC GCCCTCCAAT 541 CGGGTAACTC CCAGGAGAGT GTCACAGAGC AGGACAGCAA GGACA.GCACC TACAGCCTCA 601 GCA6CACCCT GACGCTGAGC AAAGGAGACT ACGAGAAACA CAAAGTCTAC GCCT-SCGAAG 661 TCACCCATCA GGGCCTGAGC TCGCCCGTCA CAAAGAGCTT CAACA6GGGA GACTGTTGAT

    721 AACTCGAC (SEQ ID NO: 3)

    3/21

    FIGURE 4 aOPGL-1 kappa Light Chain Amin© Acid Sequence

    2015264940 07 Dec 2015

    The kappa signal peptide is underlined, the variable region is in capital leters and not underlined, and the constant region is in lower case,

    1 METPAQLLFL LLLWLPDTTG EIVLTOSPGT LSLSPGERAT LSCRASQSVR 51 GRYLAUfYQQK PGQAPRLLIY GASSRATGIF DRFSGSGSGT DFTLTXSRLE 101 PEDFAVFYCQ QYGSSPRTFG QGTKVEikrt vaapsvfifp psdeqlksgt 151 aswcTInnf ypr.eakvqwk vdnalqsgns qesvteqdsk dstyslsstl

    201 tlskadyekh kvyacevthq glsspvtksf nrgec (SEQ XD no: 4}

    4/21

    2015264940 07 Dec 2015 figures

    Circular Plasmid Map of aOPGL«1»kopps/pDSR«19 Sot {5472) cOFSL- J Kappa,

    AM ¢4750) · ‘ tfftrilf J{4745),

    SV40 16s Accepter SV40JVS Sffirf-R ¢4648)

    SV40 18s, 19s Conor

    R

    SV40 pro/ori £«R1 :(39485 „&iR3(3SS) aFSH PoiyA ΛΜ(883)

    DHm

    Promoter seitnss)

    DHFR &d{.W fwi ¢3335)

    Amp

    P&R322

    DHFR PoiyA AM ¢2028)

    5/21

    FIGURE δ ο

    (Μ

    2015264940 07 Dec

    Circular Plasmid Map of oOPGL~14gG2/pDSRa1S

    Hfodl (68675

    Sai (686SS aOPGL-1 SgG2

    Wndili i4745}

    SV40 16s Accepter SV40 iVS ' 0emHJ(4WI.)

    SV40 16s, 19s ‘ Doner .....

    ΛΜΙ {4595»

    R

    SV4Q profort £cd« (3948)

    Hindi <34%)' $ed 0435}

    Avrf 0325) Amp ._Eet&i {358} aFSH PoiyA

    DHFR Promoter &si (! 595} OHFR

    Bgil<1739:

    OHFR PoiyA

    PBR322

    2015264940 07 Dec 2015

    RGURE7 aGPGW Binds io Soluble OPG Ligand Coated on the EIA Plate in a Dosedependent Manner

    96-weii EIA plates are coated with recombinant soluble OPGL. Varying concentrations of aGPGL-1 are added to the wells and incubated for about 2hrs at room temperature.. Bound antibody is detected with goat anii-Humsn fgG {Fafa^!)~Hcrse Radish Peroxidase. The absorbance is read et 450 nm end 650 nm.

    7/21

    2015264940 07 Dec 2015

    FIGURE 8 oOPGL-1 Binds Specifically to Membrane-bound OPGL aOPGL-1 binds to OPGL expressed on a ceil surface of transfected CHO REN 218-9 ceils in a dose-dependent manner. This binding is competed by exogenously added human OPGL but not by TNF-a, TMF-β, TRAIL or CD40 ligand. gOPGL-1 (100 ng/ml) is pre-incubated with varying concentrations of soluble OPGL or other ligands and is then incubated with CHO REN 218-9 cells expressing OPGL on the surface. Cells are then incubated for 30 minutes at 2-B°C with FlTC-labeied Fiab’JjGoatanti-Human IgG, Fey Fragment Specific. After centrifugation and washing cell surface fluorescence is measured using flow cytometry.

    8/21

    IGURE 9 ο

    (Ν

    2015264940 07 Dec □OPGL-1 binding to OPGL on an ElA plate is reduced competitively by exogenously added soluble OPGL.

    9/21

    2015264940 07 Dec 2015

    Figure 10 aOPGL-1 does not bind to TNF family members TNFa, TNFp, TRAIL, or CD40· ligand

    Φ TNFafaOPGL· I s TNFS,' aOPGL-l A TRAIL·' aOPGL-1 O CD40U aOPGL-1 aOPGL-1 binding to OPGL on an ElA piste is not reduced by exogenously added TNF-a, TNF-β TRAIL or CD40 Ligand.

    10/21

    FIGURE 11

    2015264940 07 Dec 2015

    Dose-dependent Inhibition by aOPGL-1 of OPG Ligand-induced TRAP Activity in RAW 264.7 Cells

    11/21

    FIGURE 12

    2015264940 07 Dec 2015

    Dose-dependent Inhibition by etOPGL-1 of Europium-labeled OPG Ligand Binding to ODAR-FLAG/anti-FLAG-APC aOPGL-1, ng/mL

    12/21 £>i FnC'Ti’f'i r?M irr*r ii λ^ϊ

    FIGURE 13

    2015264940 07 Dec 2015

    Mean (± SD) Serum-Concentration Time Profiles Following Single Dose Administration of aOPGL-1 to Cynomolgus Monkeys at Doses of 0,1,1 and 10.0 mg/kg IV (π-2/dose) and 1.0tng/kg SC (n-S/dose)

    13/21

    FIGURE 14

    2015264940 07 Dec 2015

    Mean (± SD) Percent Chang® in Seram N-Tx Concentration After Administering a Single Dose of oOPGL-1 IV (n~2/dose) or SC (n-6) to Cynomolgus Monkeys at Doses of 0.1,1 and 10.0 mg/kg.

    14/21

    FIGURE 15

    2015264940 07 Dec 2015

    Mean (± SD) Percent Change in Urine N-Tx Concentration Following Single Dose IV (n™2/dose) or SC (n6) Administration of oOPGL-1 to Cynomolgus Monkeys at Doses of 0.1,1 and 10.0 mg/kg

    0 14 2S 42 56 70 M 98 112 125

    Tims (Day)

    15/21 ο

    ΓΗ

    2015264940 07 Dec

    Antibody Positive (open black symbols) and Negative (dosed red symbols) Animal Serum-Concentration Tim® Profiles After Administering a Single Dose of aOPGL»1 IV to Cynomolgus donkeys at a Dose of 0,1 mg/kg

    16/21

    FIGURE 17

    2015264940 07 Dec 2015

    1 EVQLLESGGG LVQPGGSLRL SCAASGFTF5 5YAMSWVRQA 41 PGKGLEWVSG XTGSGGSTYY ADSVKGRFTI SRDMSKNTLY 81 LQMNSLRAED TAVYYCAKDP GTTVIMSWFD PWGQGTLVTV

    121 55 (SEQ ID NO: 13)

    17/21

    SHl&cmTi ITC cM-iCrtEfo-r /niH c:

    2015264940 07 Dec 2015

    FIGURE 18 aOPGL-1 Light Chain Variable Region Amino Acid Sequence

    1 EIVLTQSFGT LSLSPGERAT LSCRASQSVR GRYLAWYQQK 41 PGQAPRLLIY GASSRATGIP DRFSG5G5GT DFTLTISRLE SI PEDFAVFYCQ QYGSSPRTFG QGTKVEIK (SEQ ID NO; 14)

    18/21

    FIGURE 19

    2015264940 07 Dec 2015

    Vial

    Thaw via? of 125Q

    Shakers, Spinners

    20 L Bioreactor

    Ceil Expansion

    Ceii Expansion

    ISO L Sioreaeter

    Cell Expansion

    200Q L Bioreacter

    Production Reactor

    Disk Stack Centrifuge

    Harvest: Remove ceils from conditioned medium

    Filter

    Clarify centrals

    Cross Flow Filtration: Concentrate the conditioned medium

    Proceed io purification or store frozen at -30 ± 10 °C

    19/21

    2015264940 07 Dec 2015

    FIGURE 20

    Mean Serum Calcium Percent Change From Baseline After Administering a Single Dose of ctOPGL-1 to Cynomolgus Monkeys

    Time {Day}

    20/21

    FIGURE 21

    2015264940 07 Dec 2015

    Mean Serum Alkaline Phosphatase Percent Change From Baseline After Administering a Single Dose of oOPGL-1 to Cynomolgus Monkeys

    -80 J

    0 M 2£ <52

    56 70 8«

    Tims (Day)

    21/21

    2015264940 07 Dec 2015

    ANTIBODIES TO OPGL SEQ LISTING,txt SEQUENCE LISTING <1IO> 3oyle, William J Martin, Francis H Coxvalan, Jose R Davis, C. Geoffrey <12 0> Antibodies to OPGL <130> 06843.0049-00000 <1SO> 60/301,112 <1S1> 2001-06-25 <160> 20 <170> Patentln version 3.1 <210> 1 <211> 1426 <2I2> DMA <213> Mus musculus

    <400> 1 eagcttgacc accatggagt fctgggctgag ctggcfcttfct cttgtggcta tfcfefcaaaagg 60 tgtccagtgt gaggtgcagc fcgttggagta tgggggaggc tfcggfcacagc ctggggggtc 120 cctgagactc tcetgtgcag cctctggatt caccfctfcagc agctatgcca tgagctgggfc 130 Ccgccaggct ccagggaagg ggctggagtg ggtcfccaggfc attactggga gtggtggtag 240 tacafcactac gcagactccg tgaagggccg gttcac-catc t'ccagagaea attecaagaa 300 cacgctgfcat ctgcaaatga acagcctgag agccgaggac acggocgtat attacfcgigc 360

    Page 1

    2015264940 07 Dec 2015

    AMT1SODIES TO OPGL SEQ: LISTING, txt gaaagatcca gggactacgg tg-afetatgag ttggttcgac ccctggggcc agggaaccct 420 ggtcaccgtc tcctcagccfc ccaccaaggg cccatcggtc ttccccctgg egccctgctc 430 caggagcacc feccgagagca cagcggccct gggctgccfcg gtcaaggact agttccccga 540 accggtgacg gtgtcgfcgga actcaggcgc tctgaccagc ggcgfcgcaca ccfctcccagc 600 fcgtcctacag tcctcaggec tctactcc.ct cagcagegtg gtgaccgfcgc ccfeccageaa 660 cttcggcaco- cagacctaca ectgcaacgt agatcacaag cqcagcaaca ccaaggtgga 720 caagacagfct gagcgcaaat gtfcgfcgtcga gtgcceaccg tgcccagcac cacctgtggc 780 aggaccgtca gtcttcctct tccecccaaa acccaaggac acccfcqatga tctcceggae 840 ccctgaggtc acgtgcgtgg tggtggacgt gegccacgaa gaccccgagg tccagttcaa ' 900 ctggtacgtg gacggcgtgg aggtgcat.aa tgeeaagaca aagccacggg aggagcagtfc 960 caacagcacg fctccgtgtgg fcca^cgtcct caccgttgfcg cascaggact ggctgaacgg 1020 caaggagfcac aagfcgcaagg· fcctccaacaa aggcctccca geccocatcg agaaaaccafc 108-0' ctecaaaacc aaagggcagc dccgagaacc acaggtgtac acectgcsec catcecggga 1140 ggagatgac'c aagaaccagg tcagcctgac ctgccfcggfcc iaaaggcfctcfc accccagcga 1200 catcgccgtg gagtgggaga gcaatgggca gceggagaac aactacaaga ccaeacctcc 126G cafcgcfcggac tccgacggct ccttcttcct etacagcaag ctcaccgtgg acaagagcag 1320 gtggcagcag gggaacgtct tcfccatgctc cgtgatgeat gaggefccfcgc acaaccacfca 1380 cacgcagaag agccfcctccc tgtctccggg taaatgafcaa gtcgac 1426 <210> 2 <211> 467 ' <212> PRT .

    <213> Mus muscwlua^- < 4 00> 2 Met Glu She Gly Leu £ Ser Trp Leu Phe Leu 10 Val Ila Leu Lys 15 Gly Val Gin Cvs Glu ’ 20 Val Gin Leu Lsu Glu 25 Ser Gly Gly Gly Leu 30 Val Gin Pro Gly Gly Set Leu Arg Leu Ser Cys Ala Ala Ser Gly Ehe Thr Phe

    35 4Ό 45

    Rags 2

    WXBODXSS TO OPGL SSQ LISTING. txt

    2015264940 07 Dec 2015

    Ser Ser 50 Tyr ' Ala . Met Ser Trp 55 Val Arg Gin Ala Pro 60 Gly Lys Gly Leu Gin Trp 65 Val Set Gly lie Thr 70 Gly Ser Gly Gly 75 Ser Thr Tyr Tyr ala 90 Asp Ser Val Lys Giy 85 Arg Phe Thr lie Sar 90 Arg Asp Asn Ser Lys 95 Asn Thr Leu Tyr Leu 100 Gin Met Asn Ser Leu 105. Arg Ala Glu Asp Thr 11.0 Ala Val Tyr Tyr Cys 115 Ala Lys Asp Pro Gly 120 Thr Thr Val lie Met 125 Ser Trp Phe Asp Pro 130 Trp Gly Gin Glv Thr 135 Leu Val Thr Val Ssr 140 Ser Ala Ser Thr Lys Gly 14 5 Pro Ser Val Phe Pro ISO Leu Ala Pro Cvs 155 Ser Arg Ser frt jj, Ser ISO Glu Ser Thr Ala Ala 165 Leu Gly Cys Leu Val 170 Lys Asp Tyr Phe Pro 175 Glu Pro Val Thr Val 180 Ser Trp Asn Ser Gly 185 Ala Leu Thr Ser Gly 190 Val His Thr Phe Pro. 195 Ala Val Leu Gin Ser 200 Ser Gly Leu Tyr Ser 205 Lsu Ser Ser Val Val 210 Thr Val Pro Ser Ser 215 Asn Phe siy Thr Gin 220 Thr Tyr Thr cys Asn Val 225 Asp His Lys Pro Ser 230 Asa THx Lys Val 235 Asp Lys Thr Val Glu 24 0 .Arg Lys Cys Cys Val 245 Glu Cys Pro Pro Cys 250' Pro Ala Pro Pro Val 255 Ala Gly Pro Ser Val 2 SO Phe Ian Phe Pro Pro 265 Lys Pro Lys Asp Thr 270 Leu Met XX© Se.27 Arg Thr Pro Glu Val Thr cys Val Val Val -^a-sp Val Ser His

    Page 3

    2015264940 07 Dec 2015

    ANTIBODIES TO OPGL SEQ .LISTING , txt 275 280 285 Glu Asp Fro Glu Val Gin Phe Asn Trp Tys' Val Asp •SXy VsX Glu Val. 230 295 300 Sis Asn A1 a Lys Lys Pi'o Arg Glu Glu Gin Phe Asn Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro lie 340 345 350 Glu uys Thr lie Ser Lys Thr Lys Gly Gin Pro Arg Glu Pro Gin Val 355 360 • 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Mat Thr Lvs Asn Gin Val Ser 370 375 360 Leu Thr Cys Leu Val Lys Glv Phe Tyr, Pro Ser Asp lie Ala Val Glu 38 5 390 393 400 Trp Glu Ser Asn Glv Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 4 is Het Leu Asp Ser Asp Gly Ssr Phe Phe Leu Tyr Ser Lys Leu Val 420 425 4 30 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser val Met 435 4 40 4 45 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

    450 . 455 460

    Pro Gly Lys

    465 <210> 3 <2.1.1 > 728 <212> DNA <213> Mus musculus

    Page 4

    ANTIBODIES TO OPGL SSQ LISTING. txt

    2015264940 07 Dec 2015

    <400> 3 tctagae-cac catggaaacc ccagcgcagc fctctctfccct actgctactc tggctcccag 60 ataccaccgg agaaattgtg ttgacgcagfc ctccaggcac cctgtcfcttg tctccagggg 1,20 aaagagccac cctctcctgt agggccagtc agagtgttcg cggcaggtac ttagcctggt 180 accagcagsa acctggccag gctcccaggc tcctcatcta tggtgcatcc sgcagggeca 240: ctggcafcccc agacaggttc agtggcagtg ggtetgggae agacttcact ctcaecatea 300 gcagactgga gcctgaagat tttgcagtgt tttactgtca gcagtatggt agttcaccte 3 SO ggacgtfccgg ccaagggacc aaggtggaaa tcaaacgaac tgtggctgca ccatctgtct 420 tcatcttccc gccafcctgat gagcagttga aatctggaac tgcctctgtt gtgtgcctgc 480 tgaataaett efcatcccaga gaggccaaag tacagtggaa ggtggataac fjccctccaat S40 ogggtaactc ccaggagagt gtcacagagc aggacagcaa ggacagcacc tacagcctca 600 gcagcaccct gacgct.ga.gc aaagcagact scgagaaaca caaagtctac gcctgcgaag 660 tcacccatea gggcctgagc tcgcccgtca caaagagctt caacagggga gagtgttgat 720 aagtcgac 720 <210> 4 <2X1> 235 <212> PRT <2X3> Mua musculus

    <400> 4

    Met 1 Glu Thr Pro Are 5 . Gin Leu Leu Phe Leu .10 Leu Leu Len Trp Leu: IS Pro Asp Thr Thr Gly 20 Glu lie Val Leu Thr 25 Gin Set Pro Gly Thr 30 Leu Ser Leu Ser Pro 35 Gly Glu teg Ala Thr 4.0 Leu Ser Cys Arg Ala 45 Ser Gin Ser Val Arg SO Gly teg Tyr Leu Ar a 55 Trp Tyr Gin Gin Lvs SO Pro Gly Gin Ala Pro Arg ύ@Ιζ Leu Ils Tyr Gly Ala Ser Ser Arg .A... a Thr Gly Ila Pro

    Page 5

    2015264940 07 Dec 2015

    AN TlBO DISS TO ‘ H GI- SEQ LISTING. .txt 65 70 TS SO Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie 35 90 95 Ser Arg Leu Glu Pro Glu. Asp Phe Ala Val Phe Tyr Cys Gin Gin Tyr 100 105 110 Gly Ser Ser Pro Arg Thr phe Gly Gin. Gly Thr Lys Val Glu lie Lys 113 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 130 135 140 Git; Leu Lys Ser sly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145. 150 155 160 Tyr Pro Arg. Glu Are. Lys Val Girt Trp Lys Val Asp Asn Ala Leu Gin 165 170 175 Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr .Al a Cys Glu Val Thr His Gin Gly Leu Ss.r Ser 210 215 22.0 Pro Val Thr Lys Ser Phs Asn Arg Gly Glu Cys

    225 230 235 <21 O> 5 <211> 25 <212> DNA <213> Artificial Sequence <220>

    <223> S' kappa RACE primer <400> 5 gstgacccag tctccagcca ccctg 2

    Pace 6

    ANTIBODIES TO OPGL SEQ LISTING. txt

    2015264940 07 Dec 2015 <210> 6 <211» 23 <212> DNA <213> Artificial Sequence <220» <223» 3' kappa SACS priaer <400 6 aagggtcaga ggccaaagga tgg 23 <210» 7 <211> 30 <212> ONA <2i.3> Artificial Sequence <2£0>

    <223> 5' anfci-OPSL-l kappa primer <400> 7 caactctaga ccaccatgga aaccccagcg 30 <210 8 <211> 37 <212» OKA <213» Artificial Sequence <220 <223> 3' anti-OSSL'-l kappa priper <4 OO 8 tttg&cgfceg acttatcaac ac.tctcccct gttgaag 37 <210 9 <211?· 23

    Page 7

    ANTIBODIES TO O?GL SEQ LISTING.txt

    2015264940 07 Dec 2015 <212> DNA <213> Artificial Sequence <220>

    <223> 5' IgG2 RACE primer <400> 3 ggcacggtca ccacgctgct gag <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220 <223> 3’ Ic?G2 RACE primer <400> 10 cctccaccas gggcceatcg gtct

    <210 11 <211> 51 <212> DNA <213> Artificial Sequence <220 <223?- 3’ anti-OPSL-l IgG2 Brimer <400 11 cagaagctfcg accaccatgg agttfcgggefc gagctggctt <210> 12 <211> 37 <212? DNA <213? Artificial Sequence

    ttfcctfcgtgg c

    Bags 8

    2015264940 07 Dec 2015

    ANTIBODIES TO OPGL SEQ LISTING,, txt <220 <223> 3’ anfci-OPGL-l ±gG2 Priiaar <400> 12 gcatgtcgae ttatcattta cccggagaca aggagag · 37 <210 13 <211> 122 <212 > PRT <213> Mus jausculus

    <400> 13 Glu 1 Val Gln leu Lsu 5 Glu Ser Gly Giy Gly 10 Leu Val Gln. Pro Gly 15 Gly Ser Leu Arg Leu 20 Ser cys Ala Ala Ser 25 Gly Phe Thr Phe Ser 30 Ser Tyr Ala Met Ser 35 Txp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu Glu 45 Trp Val -Ser Gly 50 lie Thr Gly Ser Gly 55 Gly Ssr Thr Tyr Tvr 60 Ala'Asp Ser Val Lys 65 Gly Arg Phe Thr Ils 70 Ser Arg Asp Asn Ser 75 Lys Asn. Thr Leu Tyr 80 Leu Gln Met A-sn Ser 85 - Leu Arg Ala Glu ftSD so Thr Ala Val Tyr Tyr 35 Cys Ala Lys Asp Pro 100 Gly Thr Thr Val lie 105 Met Ser Trp Phe Asn no Pro Trp Gly Gln Gly 115 Thr Leu Val Thr Val 120 Ser Ser <210> 14 <211 > 1 .08 <212> PRT

    Page 3

    ANilBQDIBS TO OPGL SEQ LISTING,txt <213> Mus musctilus

    2015264940 07 Dec 2015 <400> 14

    Glu 1 -L Χβ Val Leu Thr 5 Sift Ser Pro Gly Thr 10 Leu Ser Leu Ser Pro 15 Gly Gia Arg Ala Thr 20 Leu Ser Cys Arg Ala 25 Ser Gin Set Val Arg 30 Giy Arg Tyr Leu Ala 35 Trp Tyr Gin Gin Lys 40 Pro Qiy Gin. Ala Pro 4 5 Arg Leu Leu Xie Tvr 50 Gly Ala Ser Ser Arc 55' Ala Thr Gly lie Pro SO Asp Arg Phe Ser oiy 65 Ser Gly Ser Gly Thr 70 Asp Phe Thr Leu Thr 75 lie Ser Arg Leu Glu ao Pro Glu Asp Phe Ai a 85 Val Ph® Tyr cys Gin 90 Gin Tyr Gly S&r Ser 95 Pro: Arg Thr Pha Gly Gin Gly Thr Lys Val Glu lie Lys

    100 10S <210> IS <211> 24 <212> DNA <213> Artificial Sequence <220>

    <223> random primer <22 0>

    <221> miscofeature <222?· (18),.(23} <223> N is A, C, G, Or T

    Page 10

    2015264940 07 Dec 2015

    ANTIBODIES TO OPGL SEQ LISTING. txt <400» IS qgccggatag gcctcacnnn mint 24

    <210 16 <211> 5 <212» PRT <213> Artificial Sequence

    <220> <223> translation of portion of.5* anti-OPGL-1 kappa primer <400> 16

    Met Glu Thr Pro Ala 1 5 <210> 17

    <211» 6 <212» PRT <213> Artificial Sequence

    <220» <223» translation of portion of 3' anti-OEGL-l kappa primer <40Q> 17

    Gys Glu Gly Arg Asn Phe

    1 3 <21Q> 18 <211> 12 <212> PRT <213» Artificial Sequence

    <220> <223> translation of portion of 5' anti-OSGL-l IgGZ Primer <400» 19

    Page 11

    2015264940 07 Dec 2015

    ANTIBODIES TO OPGL SEQ LISTING.txt Met <314 Phe Gly I>su Ser Trp Leu phe iaa Val &ia

    1 5 10 <21Q> 19 <2U> 7 <212> PRT <213> Artificial Sequence

    <220> <223> translation of portion of 3' anti-GHGL-l Ig<32 Printer <4QQ> 13

    lys Sly Pro Ser Leu Ser Lgu 1 5

    <210> 20 <2H> 10 <212> PRT <213> Artificial Sequence

    <220» <22 3> LHRH antagonist peptide <220> <221> M2SCJEATURE <222> ill..ill <223> Xaa is &C“D-Nal» where Sai is 3-(2-napthyl)alaninyl

    <220» <221> MISC_FEMURE <222» 12} . , (2) <223> Xaa is (4 ¹-chloropiienuijalaninyl

    Page 12

    2015264940 07 Dec 2015

    ANTIBODIES TO OPGL SEQ LISTING. txt <220>

    <221> MISCJEATORE <222> {3),,13) <223> Xaa is D-Pal:, where Pal is 3---(3’-pyridyl)-alaninyl <220>

    <221> MISCFEATD8J3 <222> (5).. (S’) <223> Xaa is W-mefchyl tyrosine <220>

    < 2 21 > MI SCJBATGR2 <222> ¢6),,(6) <223> Xaa is D~asparagine <220>

    <221> MISC_FEATURE <222> (8)..(3) <223» Xaa is N-epsilon-2-propyl-lysinyl <220>

    <221> MISCJWURE <222» (10),.(10) <223» Xaa is D-alanine^NH2 <400> 20

    Xaa Xaa Xaa Ser Xaa Xaa, Leu Xaa Pro Xaa

    1 5 10

    Page 13