Title of Invention

COMPOSITIONS AND METHODS RELATING TO ANTI IGF-1 RECEPTOR ANTIBODIES

Abstract The present invention provides compositions and methods relating to or derived from anti-IGF-1R antibodies. In particular embodiments, the invention provides fully human, humanized, or chimeric anti­IGF-1R antibodies that bind human IGF-1R, IGF-1R-binding fragments and derivatives of such antibodies, and IGF-1R-binding polypeptides comprising such fragments. Other embodiments provide nucleic acids encoding such antibodies, antibody fragments and derivatives and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating or diagnosing subjects having IGF-1R-related disorders or conditions.
Full Text

COMPQSITIONS AND METHODS RELATIN G TO ANTI-IGF-1RECEPTOR ANTIBODIES
REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application Ser. No. 60/638,961, filed OB Decenfeex 22.2004: and is incorporated by reference herehx
FIELD OF THE INVENTION
Ibis application provides coanpositions and methods xelatzng to anti-IGF-1 receptor antibodies.
BACKG3ROUND OF THE INVENTION
InsuHn-iike growth factors 1 and 2 (IGF-1 and IGF-2, respectrvely) promote the differentiation and proÜfexaticm of a wide variety of marnrnalian cell types.
IGF-1 and IGF-2 botb circulate widely throughout the body in plasma, Tbey exert their efFects on ceDs by binding to and activating the IGF-1 receptor (IGF-1R). IGF-1R is a meniber of the family of tyrosine kinase growth factor receptors. Its amino acid sequence is about 70% identical to that of the insutm receptor.
Abnormal IGF-1. IGF-2, andVor IGF-1R activities are associated with a number of medical conditions. including vaxious types of cancer, growth defects (e.g., acromegaly. gigantism, and small statnre). Psoriasis, atherosclerosis, post angioplasty smooth rnuscle restonsis of blood vessels, diabetes. microvasular prolifexation, neuropathy, loss of muscle mass. and osteoporosis.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides nucleotide sequences encoding light chain variable dornains LI througb L52 and heavy chain variable domains Hl through H52.
Figure 2 provides amino acid sequences of light chain variable domains LI through L52. CDR and FR regions are indic2ted.
Figure 3 provides amino acid sequences of heavy chain variable domains Hl through H52. CDR and FR regions are indicated.
Figure 4 provides amino acid sequences of the light chain CDR1 regions of light chain variable domains LI through L52. Consensus sequences for groups of related CDR sequences are also provided.
Figure 5 provides arnino acid sequences of the light chain CDR2 regions of light chain variable domains LI through L52. Consensus sequences for groups of related CDR sequences are also provided.
Figure 6 provides arnino acid sequences of the light chain CDR3 regions of light chain variable domains LI through L52. Consensus sequences for groups of related CDR sequences are also provided.
Figure 7 provides arnino acid sequences of the heavy chain CDR1 regions of heavy chain variable domains Hl through H52. Consensus sequences for groups of related CDR sequences are also provided.
Figure 8 provides amino acid sequences of the heavy chain CDR2 regions of heavy chain variable domains Hl through H52. Consensus sequences for groups of related CDR sequences are also provided.

Fignre 9 provides amino acid sequences of the heavy chain CDR3 regions of heavy chain variable iomains Hl rhrough H52. Consensus sequences fbr gioups of related CDR sequences are also provided.
Figure 10 provides the amino acid sequence of a rmman IGF-1R extracellulax domain fused to a bnman IgGl Fe region (uiiderhned) with an interraiing caspace-3 cleavage site (bold).
Figure 11 provides the amino acid seqnmrr of a Imman insulin reeeptor extracellrilar domain fosed to a hnmaii IgGl Fe region (underlmed).
Figure 12 provides the protein sequence of a human IGF-1R extocdlulfrr domain (inchioing signal peptide)fcsedattheC-tennmns withcMckenavidm. TbeHHäaängmetintbelGF-lRECD isdesignaied position 1 in frris fignre.
Fignre 13 provides the Polypeptide seqaence of a hmnan kappa rtg^g chain antibody constant region and a human IgGl heavy chain antibody conssant regkax
Fignre 14 provides a graph Ühistrarjng mal fbar pfease-displayed antibodies bind significantly better to an IGF-IR-Fc molecule than they bind to an insnrhvrcceptDr-Fc or a Trnrrhy- Fe.
Figure 15 provides graphs ilhistrarrng the aiähry af certain antibodies to compete for binding to IGF-lRwithlGF-1 andIGF-2.
Figure 16 provides graphs iBustrating the abüiry of certain antibodies to inhibit the growth of 32D huIGF-lR-H&S-l cells.
Figure 17 provides graphs illustrating the ability of certam antibodies to inhibit the growth of BaJb/C 3T3 hu 1GF-1R cells.
SUMMARY OF THE INYENTION
In one aspect, the present invention provides an isolated antigen binding protein comprising either: a. a light chain CDR3 comprising a sequence selected from the group consisting of: i. a light chain CDR3 sequence that differs by no more than a total of rwo amino acid additions, sxtbstitntions, and/or deletions from a CDR3 sequence selected fromtbe group cos^isting of the light chain CDR3 sequences of L1-L52 as showninFigure6;ii. M X1X2X3X4X5PX£X,: in. Q Q XsX^X^XnP X]2T; and iv. QSYX13X14Xi5 N Xi6Xi7XiS; b. a heavy chain CDR3 comprismg a sequence selected from the group consisting of: i. a heavy chain CDR3 sequence that differs by no more than a total of three arrrmo acid additions. substitutions, and'or deletions fjotn a CDR3 sequence selected from the group consisting of the heavy chain CDR3 sequences of H1-H52 as shown in Figure 9: iL X^X^X^X^X^X^Xs^X^X-^FD L iii. X^X^ X3oX31X32X33X54X35X^X37X35MD V; iv. D S S X3o;orc. the light chain CDR3 sequence of (a) and the heavy chain CDR3 sequence of (b); wherein X-t is a glutarnine residue or a glutamate residue, X2 is an alarxme residue. a glycine residue. a uireoriine residue, or a serine residue, X3 is a leucine residue, a Phenylalanine residue, or a threonine residue, XL* is grurarnine residue. a glutamate residue, or a histidine residue, X5 is a threonine residue, a methionine residue. a tryptophan residue, or a valine residue, X5 is a glycine residue, an alanine residue. a valine residue, a leucine residue, an isoleucine residue, a proline residue, a Phenylalanine residue, a methionine residue, a tryptophan residue, or a cysteine residue, X7 is threonine residue, an alanine residue, or a serine residue, XE is an arginine residue, a serine residue. a leucine residue, or an alanine residue, X9 is an asparagine residue, a serine residue. or a histidine residue. X-o is an asparagine residue or a serine residue, X; 1 is a tryptophan residue, a valine residue, a tyrosine

residue, a proline residne. OT a phenylalanrne residne, X-^ is a leucine residue, a tyrosine residue, or an isoleucine residne, Xu is an aspartate residue or a glmamine residne, Xu is a serine residue or a proline residue, X» is a serine residue, a tyrosine residue, an aspartate residne, or an ala-ninr residue, X16 is a glutaxoine residue, an arginine residue, a valine resädae, or a tryptophan residue, Xn is an arginine residue. a valine residue, an isoleucine residue, or no resiöbc, Xu is a valine residue or no residue, X]9 is a glutamate residue OT no residue, Xzois a tyrosine residue. a grycine residue, a serine residue, or no residue, X2i is a serine residue. an asparagme residne, a tryptophan residue, a yftHaimtr xesädDe, as aspartate residue, or no residne. X^ is a serine residne, an aspartate resutae, a Tryptophan residne, an abmne residue, an argrnme residue, a rnreonine residne, a glntamiite residne, a ieoäne resädoe, a ghHamate residne, or no residne. X23 is a serine residue, a glycine residne, an asparagme reskfae, a tteomne resäöae. a tryptophan residue. a valine residue, an alanine residue, or an isolencine reskte. X34IS an aigiimy. reskfce, a grntaTrrme residue, a tyrosine residne. a valine residne, an alaTrmg residne. a grycine resHHje, a serine residue, a Phenylalanine residue, or a tryptophan residue, X25 is an asparagine residue. a teocine residne, an aspartate residue, a threonine residue, a tryptophan residue, a ryrosine residne, a valine residne, an alanine residue, or a histidine residue, X26 is an aspartate residue, a serine residne, an asparagine residne, or a grrrrarmrie residne, X2- is an alanine residue or a proline residue, X^ is an afc-nrne residue or no residue, Xs is a glutamate residue, a tyrosine residue, a glycine residue, or no residne, Xx>is an argrnme residne, a serine residue, or no residue, X^ is a glycirje residue, an aspartate residne, a valine residne, a serine residue, or no residue, X32 is a serine residue, an aspartate residue, a glycine residue, or no residue. X33 is a Phenylalanine residue, an aspartate residue. a tyrosine residue, a glycine residne, a serine residue, a histidine residue, a tryptophan residue, or no residue. X54 is a tryptophan residue, an aspartate residne, a tyrosine residue. a serine residue, or no residue, X35 is an aspartate residue, a glutamate residue, an arginine residue, a serine residue, a glycine residue, a tyrosine residue, or a tryptophan residne, X^is a tyrosine residue, a lysine residue, an isoleucine residue, a leucine residue or a Phenylalanine residiie, X37 is a tyrosine residue, a serine residne, a Phenylalanine residue, an aspartate residue. or a glycine residne, X3g is a glycine residue, an asparagine residue, or a tyrosine residue, X39 is a valine residne, a glycine residue, or a serine residue, and said antigen binding protern binds specificalry to human 1GF-IR In one ernbodiinent the isolated antigen binding protein comprises an amino acid sequence selected frorn the group consisting of: a. a light chain CDR1 sequence that difTers by no more than a total of six amino. acid additions, substitutions, and'or deletions frorn a CDR1 sequence of L1-L52 as shown in Figure 4; b. a light chain CDR2 sequence that difJers by no more than a total of two arnino acid additions, substitutions. and/or deletions &om a CDR2 sequence of Ll-L52 as shown in Figure 5: c. a light chain CDR3 sequence that differs by no more than a total of three amino acid additions. substitutions. and/or deletions frorn a CDR3 sequence of L1-L52 as shown in Figure 6; d. a heavy chain CDR1 sequence that difTers by no more than a total of two amino acid additions. substitutions, and/or deletions frorn a CDR1 sequence of H1-H52 as shown in Figure 7; e. a heavy chain CDR2 sequence that difTers by no more than a total of five arnino acid additions, substitutions, and/or deletions from a CDR2 sequence of H1-H52 as shown rnFigure 8; and f. a heavy chain CDR3 sequence that difTers by no more than a total of fovrr arnino acid additions, substitutions, and/or deletions from a CDR3 sequence of H1-H52 as shown in Figure 9. In another ernbodiment, the isolated antigen binding protein comprises an amino acid sequence selected from the group consisting of: a. a light chain CDR1

sequence that differs by no more than a total of five amino acid additionss substitutions, and/or deletions from a CDR1 sequence of L3-L52 as shown in Figure 4; b. a Hght chain QDR2 sequence that differs by no more than a total of one amino acid addition, Substitution, or deletion from a CDR2 sequence of L1-L52 as shown in Figure 5; c. a light chain CDK3 sequence that differs by no more than a total of two arrrno acid additions, substitutions, and/or deletions from a CDR3 sequence of L1-L52 as shown in Figure 6; & a heavy chain CDR1 sequence that differs by no more than a total of one amino acid addition, Substitution, or deletion from a CDR1 sequence of H1-H52 as sbown in Figure 7; e. a heavy chain CDR2 sequence that dilüas by no more tftan a total of four amino acid additions, siibsttatioBS, and/or deletions from a CDR2 sequence of H1-H52 as shown in Figure 8: and f. a heavy chain CDR3 seqnence that differs by no more ^trp a total of three amino acid additions, substrtutions, and/or deletions from a CDR3 sequence of Hl -H52 as shown in Figare 9. In another embodiment, the isolated antigen binding protein comprises an amino acid seqnence selected from the group consisting of: a. a tight chain CDR1 sequence that differs by no more ärep a total of four amino acid additions. substitutions. and/or deletions from a CDR1 sequence of L1-L52 as shown in Figure 4: b. a light chain CDR2 sequence of L1-L52 as shown in Figure 5; c. a light chain CDR3 sequence that differs by no more than a total of one amino acid addition, Substitution, or deletion from a CDR3 sequence of L1-L52 as shown in Figure 6; d. a heavy chain CDR1 sequence of H1-H52 as shown in Figure 7; e. a heavy chain CDR2 sequence that diflers by no more than a total of three amino acid additions, substiturionss and/or deletions from a CDR2 sequence of H1-H52 as shown in Figure 8; and f. a heavy chain CDR3 sequence that differs by no more than a total of two amino acid additions, substitutions, and/or deletions from a CDR3 sequence of H1-H52 as shown in Figure 9. In another embodiment, the isolated antigen binding protein comprises an amino acid sequence selected from the group consisting of: a. a light chain CDR1 sequence that differs by no more than a total of three amino acid additions. substitutions. and/or deletions from a CDR1 sequence of L1-L52 as shown in Figure 4; b. a light chain CDR3 sequence of L1-L52 as shown in Figure 6: c. a heavy chain CDR2 sequence that differs by no more ihan a total of two amino acid additions, substitutions, and/or deletions from a CDR2 sequence of H1-H52 as shown in Figure 8; and & a heavy chain CDR3 sequence that differs by no more than a total of one amino acid addröon, Substitution, or deletion from a CDR3 sequence of H1-H52 as shown in Figure 9. In another embodiment, the isolated antigen binding protein comprises an amino acid sequence selected from the group consisting of: a. a light chain CDR1 sequence that differs by no more than a total of two amino acid additions, substitutions, and/or deletions from a CDR1 sequence of L1-L52 as shown in Figure 4; b. a heavy chain CDR2 sequence that differs by no more man a total of one amino acid addition, Substitution, or deletion from a CDR2 sequence of H1-H52 as shown in Figure 8; and c. a heavy chain CDR3 sequence of H2-H52 as shown in Figure 9. In another embodiment, the isolated antigen binding protein comprises an amino acid sequence selected from the group consisting of: a. a light chain CDR1 sequence that differs by no more than a total of one amino acid addition, Substitution, or deletion from a CDR1 sequence of L1-L52 as shown in Figure 4; and b. a heavy chain CDR2 sequence of H1-H52 as shown in Figure 8. In another embodiment the isolated antigen binding protein comprises a CDR1 sequence of L1-L52 as shown in Figure 4. In another embodiment the isolated antigen binding protein comprises a sequence selected from the group consisting of: a. a light chain CDR1 sequence selected from the group consisting of: i. RSSQSLLHSNGYNYLD; ii. RASQ(G/S)(VV)(G/S)X(Y/F)L(A/N); and iii. RSSQS(UI)XXXXX; b. a

light chain CDR2 sequence selected from the group consisting o£ i. LGSNRAS; ii. AASTLQS: and iii. EDNXRPS; c. a heavy chain CDR1 sequence selected from the group consisting of: i. SSNWWS; iL XYYWS; and ÜL SYAM(S/H): and d a heavy chain CDR2 sequence selected from the group consisting of: i (E/I)(I/V)(Y/^(H,^SGST(>J/Y)\l\TSIiCS; and ü XIS(G'S)SG(G,S)STyYADSVKG; wherein amino acid residue Symbols enclosed in parentheses iderrtüy aheruative residues for the same position in a sequence. each X is mdependenily any aminn acid residue, and each Z is mdependenüy a glycine residue, an alanine residue, a vamae residne, a leocine residne, an isokncine rcskfae, a proÜne residue, a Phenylalanine residue, a memiofniiie residne, a tryptophan residae, or a cyssczne resiöae. m anomer embodirnent, the isolated antigen binding protein comprises a heavy chain CDR3 sequence tbat difFers by no more man a total of two amino acid additions, snbsttetions» and/or drtefirre from a CDR3 sequence of H1-H52 as sbown in Figure 9. In anofher embodirnent, the isolffffd ajrftpm bmdrng protein comprises a heavy chain CDR3 sequence that differs by no more fhart a total of one amino acid afVidwin substüxrtion. OT deletion from a CDR3 sequence of H1-H52 as sbown in Figure 9. m anodier rrr#x>dTPTerit me isolated antigen binding protein comprises a heavy chain CDR3 sequence of H1-H52 as shown in Figure 9. In another embodirnent, the isolated antigen binding protein comprises rwo amino acid sequences selected from the group consisting of: a. a light chain CDR1 sequence that difiers by no more man a total of six amino acid additions, substitutions, and/or deletions from a CDR1 sequence of L1-L52 as shown in Figure 4; b. a light chain CDR2 sequence that differs by no more than a total of two arnino acid additions, substitutions, and/or deletions from a CDR2 sequence of L1-L52 as sbown in Figure 5; c. a light chain CDR3 sequence that difiers by no more than a total of three arnino acid additions, substitutions, and/or deletions from a CDR3 sequence of L1-L52 as shown in Figure 6; d a heavy chain CDR1 sequence that differs by no more than a total of two amino acid additions. substitutions, and'or deletions from a CDR1 sequence of H1-H52 as shown in Figure 7; e. a heavy chain CDR2 sequence that diäers by no more than a total of frve arnino acid additions, siibstrtutions, and/or deletions from a CDR2 sequence of H1-H52 as shown in Figure 8; and f. a heavy chain CDR3 sequence'rhat differs by no more than a total of four amino acid additions. substitutions, and/or deletions from a CDR3 sequence of H1-H52 as shown in Figure 9. In another embodirnent, the isolated antigen binding protein comprises three arnino acid sequences selected from the group consisting of a. a light chain CDR1 sequence that differs by no more than a total of six arrrino acid additions, substitutions, and/or deletions from a CDR1 sequence of L1-L52 as shown in Figure 4; b. a light chain CDR2 sequence that differs by no more man a total of two amino acid additions, substitutions. and/or deletions from a CDR2 sequence of L1-L52 as shown in Figure 5; c. a light chain CDR3 sequence that difiers by no more than a total of three amino acid additions, siibstrtutions, and/or deletions from a CDR3 sequence of L1-L52 as shown in Figure 6; d a heavy chain CDR1 sequence that differs byno more than a total of two amino acid additions, substitutions, and/or deletions froma CDR1 t sequence of H1-H52 as shown in Figure 7; e. a heavy chain CDR2 sequence that difiers by no more than a total of five amino acid additions, substitutions, and/or deletions from a CDR2 sequence of H1-H52 as shown in Figure 8; and f. a heavy chain CDR3 sequence that difiers by no more tha-n a total of four arnino acid additions. substitutions. and/or deletions from a CDR3 sequence of H1-H52 as shown in Figure 9. In another embodiment the isolated antigen binding protein comprises four amino acid sequences selected from the group consisting of: a. a light chain CDR1 sequence that differs by no more than a total of six

ixmno acid additions, substitutions, and/or deletions from a CDR1 sequence of L1-L52 as shown in Figure 4; b. a light chain CDR2 sequence that difiers by no morc than a total of two amino acid additions. äubstitutions, and/or deletions from a CDR2 sequence of L1-L52 as shown in Figure 5; c. a light chain CDR3 sequence that differs by no more than a total of three amino acid additions, substitutions, and/or deletions from a CDR3 sequence of L1-L52 as shown in Figure 6; d. a heavy chain CDR1 sequence that differs by no morc tfrsn a total of two amino acid additions. substitutions, and/or deletions from a CDR1 sequence of H1-H52 as shown in Figure 7; e. a heavy chain CDR2 sequence tfaat differs by no more than a total of frve a-rrnnn acid additions, sur^stitations, and/or deletions from a CDR2 sequenee of H1-H52 as shown in Figure 8; and f. a heavy chain CDR3 sequence that difiers by no more than a total of four amino acid additions, substitutions. and/or deletions from a CDR3 sequence of H1-H52 as shown in Figure 9. In anotber ernbodnrjent the isolated antigen binding protein compErises frve amino acid sequences selected fromfhe group consistmg of a. a light chain CDR1 sequence that difers by no more than a total of six amino acid additions. substitutions, and/or deletions froma CDR1 sequence of L1-L52 as shown in Figure 4; b. a light chain CDR2 sequence that differs by no more than a total of two amino acid additions, substitutions, and/or deletions from a CDR2 sequence of L1-L52 as shown in Figure 5: c. a light chain CDR3 sequence that difiers by no more than a total of three amino acid additions, substitutions, and/or deletions from a CDR3 sequence of L1-L52 as shown in Figure 6; d. a heavy chain CDR1 sequence that differs by no more than a total of two amino acid additions, substitutions, and/or deletions from a CDR1 sequence of H1-H52 as shown in Figure 7; e. a heavy chain CDR2 sequence that differs by no more than a total of frve amino acid additions, substitutions, and/or deletions from a CDR2 sequence of H1-H52 as shown in Figure 8: and f. a heavy chain CDR3 sequence that differs by no more than a total of four amino acid additions. substitutions, and/or deletions from a CDR3 sequence of H1-H52 as shown in Figure 9. In another embodiment, the isolated antigen binding protein comprises: a. a light chain CDR1 sequence that differs by no more than a total of six amino acid additions, substitutions, and/or deletions from a CDR1 sequence of L1-L52 as shown in Figure 4; b. a light chain CDR2 sequence that difiers by no more than a total of two amino acid additions, substitutions, and/or deletions from a CDR2 sequence of L1-L52 as shown in Figure 5; c. a light chain CDR3 sequence that differs by no more tha-n a total of three amino acid additions. substitutions, and/or deletions from a CDR3 sequence of L1-L52 as shown in Figure 6; d. a heavy chain CDR1 sequence that differs by no more than a total of two ainino acid additions. substitutions, and/or deletions from a CDR1 sequence of H1-H52 as shown in Figure 7; e. a heavy chain CDR2 sequence that differs by no more than a total of frve amino acid additions. substitutions, and/or deletions from a CDR2 sequence of H1-H52 as shown in Figure 8; and f. a heavy chain CDR3 sequence that differs by no more than a total of four amino acid additions, substitutions, and/or deletions from a CDR3 sequence of Hl-H52 as shown in Figure 9. In another embodiment the isolated antigen binding protein comprises eithen a. a light chain variable domain cornprising: i. a light chain CDR1 sequence shown in Figure 4; iL a Hebt chain CDR2 sequence shown in Figure 5: and ÜL a light chain CDR3 sequence shown in Figure 6; b. a heavy chain variable domain cornprising: i. a heavy chain CDR1 sequence shown in Figure 7; iL a heavy chain CDR2 sequence shown in Figure 8; and iii. a heavy chain CDR3 sequence shown in Figure 9; or c. the light chain variable domain of (a) and the heavy chain variable domain of (b). In another embodiment, the-isolated antigen binding protein comprises eithen a. light chain CDR1, CDR2, and CDR3 sequences

•hgt eacb is identical to the CDR1. CDR2. and CDR3 sequences. respcctively, of the same Hght chain variable domain sequence selected fron: the group consisting of L1-L52; b. hcavy chain CDR1. CDR2, and CDR3 sequences that eacb is identical to the CDR1, CDR2. and CDR3 sequences, respectively. of tue same hcavy chain variable domain sequence selected from the group consistmg of H1-H52; or c. the light chain CDR1, CDR2, and CDR3 sequences of (a) and the heavy chain CDR1, CDR2, and CDR3 sequences of (b).
In another aspect, the present invention provides an isolated antigen binding protein comprising eitben a. a hght chain variable domain sequence selected from the group consistTng o£ i a sequence of amino acids at least 80% identical to a light chain variable domain sequence of L1-L52 as shown in Figure 2; iL. a sequence of amino acids comprising at least 15 contiguous amino acid residues of a hght chain variable domain sequence of L1-L52 as shown in Figure 2; in. a sequence of arnino acids encodedby a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding a light chain variable domain sequence of L1-L52 as shown in Figure 1; andiv. a sequence of arnino acids encodedby a polynucleotide sequence that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of a light chain variable domain sequence of LI -L52 as shown in Figure 1; b. a heavy chain variable domain sequence selected from the group consisting of i a sequence of arnino acids at least 80% identical to a heavy chain variable domain sequence of H1-H52 as shown in Figure 2; ii. a sequence of amino acids comprising at least 15 contiguous arnino acid residues of a heavy chain variable domain sequence of H1-H52 as shown in Figure 2;üi a sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding a heavy chain variable domain sequence of H1-H52 as shown in Figure 1; and iv. a sequence of arnino acids encoded by a polynucleotide sequence that hybridizes under moderately stringent conditions to the complement of a polynucleotide consisting of a heavy chain variable domain sequence of H1-H52 as shown in Figure 1; or c. the hght chain variable domain of (a) and the heavy chain variable domain of (b); wherein said antigen binding protein binds to human IGF4R. In one embodirnent the isolated antigen binding protein comprises either a, a hght chain variable domain sequence selected from the group consisting of: i. a sequence of amino acids at least 85% identical to a Hght chain variable domain sequence of L1-L52 as shown in Figure 2; ü a sequence of arnino acids comprising at least 25 contiguous arrrino acid residues of a Hght chain variable domain sequence of L1-L52 as shown in Figure 2; iü a sequence of arnino acids encoded by a polynucleotide sequence that is at least 85% identical to a polynucleotide sequence encoding a light chain variable domain sequence of L1-L52 as shown in Figure 1: and iv. a sequence of amino acids encoded by a polynucleotide sequence that hybridizes under highly stringent conditions to the complement of a polynucleotide consistmg of a hght chain variable domain sequence of L1-L52 as shown in Figure l;b. a heavy chain variable domain sequence selected from the group consisting of i. a sequence of amino acids at least 85% identical to a heavy chain variable domain sequence of H1-H52 as shown in Figure 2; ii. a sequence of amino acids comprising at least 25 contiguous amino acid residues of a heavy chain variable domain sequence of Hl -H52 as shown in Figure 2; iii. a sequence of amino acids encoded by a polynucleotide sequence that is at least 85% identical to a polynucleotide sequence encoding a heavy chain variable domain sequence of H1-H52 as shown in Figure 1; and iv. a sequence of arnino acids encoded by a polynucleotide sequence that hybridizes under highly stringent conditions to the complement of a polynucleotide consistmg of a heavy chain variable domain sequence of H1-H52 as shown in Figure 1; or c)

le light cham variable domain of (a) and the beavy chain variable domain of (b). In another embodiment, le isolated antigen binding protein comprises either: a. a light chaia variable domain sequence selected "om the group consisting of: i. a sequence of amino acids at least 90% identical to a light chain variable -omain sequence of L1-L52 as shown in Figure 2; iL a sequence of amino acids comprising at least 35 onüguous amino acid residues of a light chain variable domain sequence of LI -L52 as shown in Figure 2; xid iü a sequence of amino acids encoded by a polynucleotide sequence that is at least 90% identical to a ►olynucleotide sequence encoding a light chain variable domain sequence of L1-L52 as shown in Figure 1: tud b. a heavy chain variable domain sequence selected from the group consisting of: i. a sequence of urnno acids at least 90% identical to a heavy chain variable domain sequence of H1-H52 as shown in rigtrre 2; iL a sequence of arirmn acids comprising at least 35 conti gnous aamno acid residues of a heavy :hain variable domain sequence of H1-H52 as shown in Figure 2; and iü. a sequence of amino acids encoded by a polynucleotide sequence that is at least 90% identical to a polynucleotide sequence encoding a heavy chain variable domain sequence of H1-H52 as shown in Figure 1; or c) the light chain variable domain of (a) and the heavy chain variable domain of (b). In another embodiment the isolated antigen binding protein comprises eithen a. a light chain variable domain sequence selected from the group consisting of. L a sequence of amino acids at least 95% identical to a light chain variable domain sequence of L1-L52 as shown in Figure 2; iL a sequence of amino acids comprising at least 50 contiguous amino acid residues of a light chain variable domain sequence of L1-L52 as shown in Figure 2; and iii. a sequence of amino acids encoded by a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence encoding a light chain variable domain sequence of L1-L52 as shown in Figure 3; andb. a heavy chain variable domain sequence selected from the group consisting of: L a sequence of amino acids at least 95% identical to a heavy chain variable domain sequence of H1-H52 as shown in Figure 2; ü. a sequence of amino acids comprising at least 50 contiguous amino acid residues of a heavy chain variable domain sequence of H1-H52 as shown in Figure 2; and iü a sequence of amino acids encoded by a polynucleotide sequence that is at least 95% identical to a polynucleotide sequence encoding a heavy chain variable domain sequence of H1-H52 as shown in Figure 1; or c) the light chain variable domain of (a) and the heavy chain variable domain of (b). In another embociinent, the isolated antigen binding protein comprises either: a. a light chain variable domain sequence selected from the group consisting of: i. a sequence of amino acids at least 97% identical to a light chain variable domain sequence of L1-L52 as shown in Figure 2; ii. a sequence of amino acids comprising at least 75 contiguous ami.no acid residues of a light chain variable domain sequence of L1-L52 as shown in Figure 2: and iii. a sequence of amino acids encoded by a polynucleotide sequence that is at least 97% identical to a polynucleotide sequence encoding a light chain variable domain sequence of L1-L52 as shown in Figure 1; and b. a heavy chain variable domain sequence selected from the group consisting of: i. a sequence of amino acids at least 97% identical to a heavy chain variable domain sequence of H1-H52 as shown in Figure 2: iL a sequence of amino acids comprising at least 75 contiguous amino acid residues of a heavy chain variable domain sequence of H1-H52 as shown. in Figure 2; and iü. a sequence of amino acids encoded by a polynucleotide sequence that is at least 97% identical to a polynucleotide sequence encoding a heavy chain variable domain sequence of H1-H52 as shown in Figure I; or c) the light chain variable domain of (a) and the heavy chain variable domain of (b). In another embodiment the isolated antigen binding proteüi comprises either: a. a light chain variable

lomain sequence selected from the group consisting of: i. a sequence of amino acids at least 99% identical o a light chain variable domain sequence of L1-L52 as shown in Figure 2; ii. a sequence of amino acids ^omprising at least 90 contiguous amino acid residues of a light chain variable domain sequence of L1-L52 is shown in Figure 2: and iü a sequence of amino acids encoded by a polynucleotide sequence rhat is at east 99% identical to a polynucleotide sequence encoding a light chain variable domain sequence of Ll-L52 as shown in Figure 1; and b. a heavy chain variable domain sequence selected from the group consisting of: i. a sequence of amino acids at least 99% identical to a heavy chain variable domain sequence of H1-H52 as shown in Figure 2; ü a sequence of amino acids comprising at least 90 contiguous gm »im acid residues of a heavy chain variable domain sequence of H1-H52 as shown in Figure 2: and iü. a sequence of amino acids encoded by a polynucleotide sequence mal is at least 99% identical to a porynucleotide sequence encoding a "heavy chain variable domain sequence of H1-H52 as shown in Figure 1; OT c. the light chain variable domain of (a) and the heavy chain variable domain of (b). In another eTnho^TTTv-riT the isolated anlägen binding protein comprises eithen a. a light chain variable domain sequence selected from the group consisting of L1-L52 as shown in Figure 2; b. a heavy chain variable domain sequence selected from the group consisting of H1-H52 as shown in Figure 3; or c. the light chain variable domain of (a) and the heavy chain variable domain of (b). In another embodiment, the isolated antigen binding protein comprises a combination of a light chain variable domain and a heavy chain variable domain selected from the group of combihations consisting of: L1H1, L2H2. L3H3. L4H4, L5H5. LeH^LTHV.LgHS.LPro.LlOHlO.LllHlULllHl^LlBHlS^MHHLlSHlS^ieHl^LlTHn, L1SH18: L19H19, L20.H20, L21H21, L22H22, L23H23: L24H24. L25H25;L26H26: L27H27: L2SH2S: L29H29, L30H30; L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L3SH38, L39H39, L40H40, L41H4L L42H4Z L43H43, L44H44, L45H45.. L46H46, L47H47, L48H48, L49H49, L50H50, L51H51. and L52H52. In another embodiment the isolated antigen binding protein further comprises: a. the kappa light chain constant sequence of Figure 13. b. the IgGl heavy chain constant sequence of Figure 13, or c. the kappa light chain constant sequence of Figure 13 and the IgGl heavy chain constant sequence of Figure 13. In another ernbodirnent, the isolated antigen binding protein wbenbound to IGF-1R: a. inbibits IGF-1R; b. acüvates IGF-1R; c. cross-competes with a reference antibody for binding to IGF-1R; d. binds to ihe same epitope of IGF-1R as ssid reference antibody; e. binds to IGF-1R with substantially the same Kd as said reference antibody; or f. binds to 1GF-1R with substantially the sarne offrate as said reference antibody; wherein said reference antibody comprises a combination of light chain and heavy chain variable domain sequences selected from the group of combinations consisting of LI Hl. L2H2, L3H3,. L4H4S L5H5, L6H6, L7H7, LSHS, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15; L16H16: L17H17S LI8H18, L19H19, L20, H20: L21H2L L22H22, L23H23, L24H24, L25H25, L26H26: L27H27, L28H28: L29H29, L30H30, L31H31, L32H32; L33H33, L34H34, L35H35: L36H36, L37H37, L3SH3S, L39H39, L40H40, L41H4I, L42H42: L43H43: L44H44, L45H45, L46H46, L47H47, L48H48: L49H49: L50H5Ö, L51H51, and L52H52. In another embodiment the isolated antigen binding protehx when bound to a human IGF-1R, inhibits binding of IGF-1 and/or IGF-2 to said human IGF-1R. -In another embodiment, the isolated antigen binding protein inhlbits the growth of a cancer cell by greater thaxi about 80% in ^he presence of a growth stimulant selected from ih^ group consisting of serum, IGF-1, and IGF-2. In another embodiment, said cancer cell is an MCF-7 human breast Cancer cell. In another embodiment the

jolated antigen binding protern binds to human IGF-] R with a selecüvity that is at least fifty times greater bau its selectivity for human insulin receptor. In another embodiment, tbe isolated antigen binding protein ohibits tumor growth in vivo. In another ernbodiment, the isolated antigen binding protein inhlbits IGF-1R oedizted tyrosine phosphorya^vn In another einbornrnetil. tbe isolated antigen binding protein pecifically binds to the IGF-IR of a non-human prirnate, a cynomologous monkey, a cbimpanzee, a non-arimate ma-rm-nal a rodent, a nxxise, a rat, a hamstex, a guinea pig, a cat or a dog. In another embodiment, ne isolated antigen binding piutein comprises: a. a human antibody; b. a hrrmanized antibody; c. a ±nneric antibody, et a mcnoctaal antibody, e. a pofydonal antibody. £ a recombinant antibody, g. an mdgen-binding antibody frzznxxst h_ a singk cten antibody: i. adiabodyj. atriabody;k. a tetrabody 1. a Fab fragment; m. a 'F(zb'}2 few'^-'tf n. a domani antibody, o. an IgD antibody; p. an IgE antibody, q. an IgM antibody, r. an IgGl antibody; s. an IeG2 antibody; t an IgG3 antibody, u. an IgG4 antibody; or v. an IgG4 antibody having at käst one iDntatkm in a hinge region fhat aDeviates a tendency to form intra-H cham disulnde bond.
In another aspect tbe preseni hrv^ntioc nrovides an isolated poiynucleotide compxising a sequence lhat encodes the light chain, tbe beavy chartL or bom of said antigen binding protein. In one embodiment, said poiynucleotide comprises s light cbain variable domain nucleic acid sequence of Figure 1 and/or a heavy chain variable domain nucleic acid sequence of Figure 1. In another embodirnent, a plasmid comprises said isolated poiynucleotide. In another embodiment, said plasmid is an expression vector. In another embodiment. an isolated cell comprises said poiynucleotide. In another embodiment, a chromosome of said cell comprises said poiynucleotide. In another embodiment said cell is a hybridoma. In another embodiment, an expression vector comprises said poiynucleotide. In another embodiment said cell is a CHO cell. In another embodiment the present invention provides a method of makmg an antigen binding protein that binds human IGF-IR. comprising incubating said isolated cell under conditions that aliow it to express said antigen binding protein.
In another aspect. the present irrvention provides a pharmaceutical composition comprising the antigen binding protein. In one embodiment, the present invention provides a method of treating a condition in a subject comprising acmnnistering to said subject said pharmaceutical composition, wherein said condition is, treatable by redacing the actrvity of IGF-1R in said subject In another embodiment said subject is a human being. In another emrx>diment said condition is multiple myeloma, a hquid rumor, liver Cancer, a rhymus disorder, a T-cell mediated antoimmune disease, an endocronological disorder, ischemia, or a neurodegenerative disorder. In another embodichnent, said liquid tumor is selected from the group consisting of acute lymphocyric leukemia (ALL) and chronic myelogenous leukemia (CML): wherein said liver cancer is selected from the group coiisistmg of hepatoma, hepatoceDular Carcinoma, cholangiocarcinoma. angiosarcomas. hernangjosarcomas, hepatoblastoma; wherein said thymus disorder is selected from the group consisting of thymoma and thyroiditis. wherein said T-cell mediated autoimmune disease is selected from the group consistinp. of Multiple Sclerosis, Rheumatoid Arthritis. Systemic Lupus Erythematosus (SLE). Grave's Disease, Hashimoto's Thyroiditis, Myasthenia Gravis. Auto-Immune Thyroiditis. Bechet's Disease, wherein said endocrinological disorder is selected from the group consisting of Type II Diabetes. hyperrhyroidisTn. hypothyroidism, thyroiditis, hyperadrenocorticism, and hypoadrenocorticisrD; wherein said ischerrria is post cardiac infarct ischemia. or wherein said


benign prostatic hyperplasia, breast Cancer, prostate Cancer, bone Cancer, hing Cancer, colorectal cancer, cervical Cancer. synovial sarcoma. diarrnea associated with merastanc carcmoki. vasoactive intestinal peptide secreting tumors, gigantism, Psoriasis, atherosclerosis, smooth nmsck restenosis of blood vessels, inappropriate microvascular proliferation. ghoblastotaa. meduHoblastoma, bcac and neck squamous cell cancer. oral Cancer, oral leukoplakia, prostate intraepithelial neoplasia. anal cancer, esopbaeeal cancer. gastric Cancer, bone cancer. metastatic cancer, rx>rycydiernia rubra vera. a brmgn COTKÜÖOE rekied to oxidatrve stress. retLnopatby of prerrmturity. Acute Respiratory Distress Syixfromr. an ovenktse of acesamrnophen, broncbopulmonary dysplasia, cystic fibrosis, rang fibrosis, aod dsbeöc renDOpsrby- In anotber embodiment the method fnrtber cornprising adiiMMStoing to said snbject a secood treaimerri- In anotber embodinieni, said second treatrnent is admirdstered to said subject befbre and/cr säxaahasscoask? wirb and/or alter said pharmaceurical composition is adanrnsrered to said sabject In anoäiex exnrxxnrnenx. said second treatrnent comprises radiaüon treatrnent, surgexy. or a second pharnsaceatkal conx>osrtiotL In anotber embodirnent, said second pharmaceutical composition comprises an agem selected from the group consisring of a corticosteroid, an anti-emetic, ondansetron hydrochloride. granisetron rrydrochloride. metroclopramide, domperidone, haloperidol, cyclizine, lorazepam, procnlorperazine, dexamefhasonc. levomepromazine, tropisetron, a cancer Vaccine, a GM-CSF mhibiting agent, a GM-CSF DNA Vaccine, a cell-based Vaccine, a dendritic cell Vaccine, a recombinant viral Vaccine., a beat sbock protein (HSP) Vaccine, an allooeneic rumor Vaccine, an autoloeous tumor Vaccine, an analeesic. ibaprofen. nanroxen. cboline magnesium trisalicylate, an oxycodone hydrochloride, an anti-angiogenic agent an anti-vascular agent bevacizumab. an anti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGF receptor fragment an anti-TWEAK antibody, an anti-TWEAK receptor antibody, a soluble TWEAK receptor fragment, AMG 706, AMG 386, an anti-proliferative agent a famesyl protein transferase inbibitor. an avß3 Inhibitor, an avß5 inbibitor. a p53 inbibitor, a Kit receptor inhibitor, a ret receptor inhibitor. a PDGFR inbibitor, a growth hormone secretion inhibitor, an angiopoietin inhibitor, a tumor infiltrating macrophage-inhibiting agent a c-fms inbibiting agent, an anti-c-fms antibody, an CSF-1 rr±ibitTng agent an ann-CSF-1 antibody, a soluble c-fms fragment, pegvisomant gemcitabine, panitinnumab. irinotbecan, and SN-38. In anotber errbodiment said metbod comprises adrninistering to said subject a thrrd trearment In anotber embodiment said condition is a cancer, said second treatrnent corrrprises adxrxiriisteTing paniturnrxmab. and said third treatrnent comprises admxinstering gemcitabine. In anotber emb
overdcse of acetammophen, broncbopnlnionar>T dysplasia. cystic fibrosis, lung flbrosis, and diabetic reimopathy.
In another aspect the present invention provides a method of increasing the longevity of a subject comprising adinmistering to said subject said phannaceutical compositiorL
In aDother aspect, the present invention provides a metbod of decreasiog IGF-1R activity in a subject in need thereof comprising adininistering to said subject said pharmacemical composition.
In another aspect, the present invention provides a metbod of decreasing IGF-IR signaimg in a subject in need tbereof comprising aduikiistering to said subject said pbarmaocutical composition.
In another aspect tbe present invention provides a method of inhibirmg the binding of IGF-1 and/or IGF-2 to IGF-IR in a subject in need thereof comprising adrnmisteriiig to said subject Said pharmaceutical composition-
DETAELED DESCRIPTION OF THE INVENTION
The present invention provides compositions, kits, and methods relatmg to molecnles rhst bind to the Insulin-Like Growrh Factor Receptor ("IGF-IR"), including molecnles that agonize or antagonize IGF-IR, such as anti-IGF-lR antibodies, antibody fragments, and antibody derivatives, e.g., antagonistic anti-IGF-1R antibodies» antibody fragments, or antibody derivatives. Also provided are nucleic acids, and derivatives and fragments thereof, comprising a sequence of nucleotides that encodes all or a portion of a Polypeptide that binds to IGF-IR e.g., a nucleic acid encoding all or part of an anti-IGF-lR antibody, antibody fragment, or antibody derivative, plasmids and vectors comprising such nucleic acids, and cells or cell lines comprising such nucleic acids and'or vectors and plasmids. The provided methods include, for example, methods of makmg, identüying, or isolating molecules that bind to IGF-IR. such as anti-IGF-lR antibodies, methods of deteimining whether a molecule binds to IGF-IR, methods of deterniining whetber a molecule agonizes or antagonizes IGF-IR, methods of making compositions, such as pharmaceutical compositions. comprising a molecule that binds to IGF-IR, and methods for administering a molecule that binds IGF-IR to a subject for example. methods for treating a condition mediated by IGF-IR, and for agonizing or antagonizrag a biological activity of IGF-IR, IGF-1, and'or IGF-2 in vivo or in vitro.
Polynucleoride and Polypeptide sequences are indicated using Standard one- or three-letter abbreviations. Unless otherwise indicated Polypeptide sequences have their amino terrrrrni at the left and their carboxy terarmi at the right and single-stranded nucleic acid sequences. and the top Strand of double-stranded nucleic acid sequences. have their 5' termini at the left and their 3' ternrini ax the right A particular Polypeptide or polynucleoride sequence also can be described by explaining how it differs from a reference seauence.
Polynucleoride and polypeptide sequences of particular light and heavy chain variable domains are shown in Figures 1, 2 and 3„ where they are labeled. for example. LI ("light chain variable domain 1"), Hl ("heavy chain variable domain 1"), etc. Antibodies comprising a light chain and heavy chain from Figures 2 and 3 are indicated by corobming the name of the light chain and the name of the heavy chain variable domains. For example. (tL4H7." indicates an antibody comprising the light chain variable domain of LA and the heavy chain variable domain of H7.

Unless otherwise defined herein, scientific and tecbirlcal terms used in connection with the present invention shall have the meanings that are commonly uiiderstood by those of ordinary skill in the art Further, unless otherwise required by context singulai terms shall include pluralities and plural terms shall include the Singular. Generally. nomenclatures used in connection with, and techniques o£ cell and tissue culrure, molecular biology. immunology, microbiology, genetics and protein and micleic acid chemistry and hybridizarion described herein are those well known and commonly used in the art The rnethods and techniques of the present invention are generally perfonned accordmg to conventional methods well known in the art and as described in various general and more specific references that are cited and discossed throughout the present specincation unless otherwise indicated. See. e.g.. Sambrook et al. Molecular aoning: A Laboratory Manual, 2d ed.. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. N.Y. (1989) and Ausubel et al» Currem Protocols in Molecular Biology. Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor laboratory Press. Cold Spring Harbor. N.Y. (1990). which are incorporated herein by reference. Enzymaric reactions and purification techniques are performed according to manufacturer's specüäcations, as commonly accomplished in the art or as described herein. The terminology used in connection with, and the laboratory procednres and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art Standard techniques can be used for chenrical syntheses, chemica] analyses. phanriaceutical preparaticox, forrnulation, and delivery, and treatment of patients.
The following terms, unless otherwise indicated, shall be undexstood to have the following meanings:
The term "isolated molecule" (where the molecule is, for example, a Polypeptide, a polynucleotide. or an antibody) is a molecule thatby virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native State, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species. or (4) does not occur in nature. Thus; a molecule that is chemically synthesizecl or synthesized in a cellular System different from the cell from which it naturally originales, will be "isolated" from its naturally associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art Molecule purity or homogeneity may be assayed by a nurober of means well known in the art. For example, tbe purity ofa Polypeptide sample maybe assayed using Polyacrylamide gel electrophoresis and staining of the gel to visualize the polypepüde using techniques well known in the art For certain purposes. higher resolution may be provided by using HPLC or other means well known in the art for purification.
The terms "IGF-1R inhibitor" and trTGF-lR antagonisf' are used rnterchangeably. Each is a molecule that detectably inhibits at least one function of IGF-1R. Conversely, an "IGF-IR agonist" is a molecule that detectably increases at least one function of IGF-IR. The Inhibition caused by an IGF-1R inhibitor need not be complete so long as it is detectable using an assay. Any assay of a function of IGF-1R can be used, examples of which are provided herein. Examples of functions of IGF-1R that can be inhibited by an IGF-1R inhibitor, or increased by an IGF-1R agonist inchzde binding to IGF-1. IGF-12. and/or another IGF-lR-activating molecule. kmase activity, downstream signaüng, and so on. Focamples of types

f IGF-1R inhibitors and IGF-1R agonists include, but are not limited to, IGF-1R binding Polypeptides such ; antigen binding proteins (e.g., IGF-1R iahibiting antiben binding proteins), antibodies, antibody Zements, s"nd antibody derivatives.
The terms "peptide," tlpohpepride" and "protein" eaeh refers to a molecule comprising two or sore amino acid residues joined to each other by peptide bonds. These terms encornpass, e.g., native and nröcial proteins. protein fxagments and porypepti.de analogs (such as ranteins. varianis, and fusion Toffisos) of a protein seqnence as well as post-translationaily, or omerwise covalentiy or non-covalently, oodifiec nmtmncL A peptide, Polypeptide, or protein may be monomeric or polymeric.
The tercn "polypeptide firagment1' as used herein refers to a Polypeptide that has an ammo-tenrrmal md/oer carboxy-terrrjinal deletioB as compared to a correspontüns full-length protein. Fragments can be, for ;xairok,atl£ast5, 6,7, 8,9,10, 11,12,13, 14,15,20,50,70,80,90, 100, 150 or200 amino acids in £2gÜL Fragmems can also be, for example, at most 1,000, 750, 500, 250, 200, 175, 150,125, 100, 90, 80: 70, 60, 50, 40. 30, 20, 15, 14. 13, 12, 11, or 10 amino acids in iength. A fragment canttrröier comprise, at eirher ox both of rs ends, one or rnore additional amino acids, for example, a sequence of amino acids from a öifferent naüirally-occurring protein (e.g.y an Fe or leucine zipper domain) or an artificial amino acid seqaence (e.g., an artificial linker sequence).
Polypeptides of the ürvention include Polypeptides that have been modiüed in any way and for any reason, for example, to: (1) reduce suseeptibility to proteolysis, (2) reduce suseeptibility to Oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding af5niti.es, and (4) confer or modify other physicochemical or functional properties. Analogs include muteins of a Polypeptide. For example. Single or multiple armno acid substitutions (e.g., conservative amino acid substitutions) may be made in the naturally oecurring sequence (e.g.. in the portion of the polypeptide outside the domain(s) forrning mtermolecular contacts. A "conservative amino acid Substitution" is one that does not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a hehx that oecurs in the parent sequence, or disrupt other types of secondary strueture that characterize the parent sequence or are necessary for its functionality). Examples of art-recognized polypeptide secondary and tertiär)^ struetures are described in Proteins, Structures and Molecular Prrnciples (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Strueture (C. Branden and J. Tooze. eds.5 Gariand Publishing, New York, N.Y. (1991)): and Thomton et at. Nature 354:105 (1991), which are eacb incorporated herein by Teference.
The present invenüon also provides non-peptide analogs of IGF-1R binding Polypeptides. Non-peptide analogs are commonly used in the pharmaceutical industry as drugs with properties analogous to those of the template peptide. These types of non-peptide Compound are termed "peptide rnimetics1' or "peptidomimetics". Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger UNS p.392 (1985); and Evans et dl. J. Med. Chem. 30:1229 (1987), which are incorporated herein byreference. Peptide rnimetics that are structurally similar to rherapeuticaily useful peptides may be used to produce an equivalent therapeutic or prophyrlactic effect Generali)', peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a Polypeptide that has a desired biochemical properry or pharmacological activity). such as a human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisring of: —CH2NH—, —CH2S—. «CH2~CH2~, —CH—CH-(c£y and trans). —

COCHi-; -CH(OH)CH2-, and -CH>SO-? by merhods weil known in the ait Systematic Substitution of one ormore amino acids of a conscnsus sequence with a D-?rnrno acid of die same type (e.g., D-lysine in place of L-lysine) may also be used to generate more stable peptides. In addition, constrained peptides comprisirig a consensus sequence ar z subsrsnnally idenrical coiisensus sequence Variation may be generaledby methods known in die zr, (Rizo and Gierascb Ann. Rcv. Biochem, 61:387 (1992), incorporated herein by reference). for example, by admrtg internal cysteine residnes capable of forrrring intramolecular disulfide bridges wiacfa c^hze the pepbde.
A 4*varianf of a Polypeptide (e.g„ an atäibody) coraprises an amino acid sequence wberein one or more amino acid residnes are insex&d imn. deteted üum sodror snbsüüiied into die amino acid sequence relative to another Polypeptide ssqaeocc WitattK of the invenaon include fdsion proteins.
A "derivative*' of a Polypeptide is a porypeptide (e-g-5 an antibody) that has been cherndcaUy modified, e.g., via conjugaücm to anolfaex rherrmr>~\ wcästj soch as, for example, polyeüiylene glycoL albutnin (eg.. immsn serum aTDumin), päospöcffyiznoiL and giycosylatioiL Uniess otherwise indicated, the tenn "antibody^* incrudes- in addition to antibodies comprising two fhll-lengrh heavy chains and two füll-lengrh light chains, derivatives. ^^^riaI^s. nagments. and rrnnnns Tbereo£ examples of which are described below.
An "antigen binding protein" is a protein comprising a portion diat binds to an antigen and, optionally, a scaffold or framework portion that allows die antigen binding portion to adopt a conformadon that promotes binding of the antigen binding protein to die antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g.. an antigen binding portion of an antibody), antibody derivatives, and antibody analogs. The antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold wirb grafted CDRs or CDR derivatives. Such scaffoids include, but are not limited to. antibody-öerived scaffolds comprising mntations introduced to. for example, stabilize tbe tineendirnensional structure of übe antigen binding protein as well as wholly synthetic scaffolds comprising, for example. a biocompatible poryiner. See. for example. Korndorfer et al.s 2003, Proteins: Structure, Function, and Bioinfonnarics. Votene 53. Issue 1:121-129: Roque et al.. 2004. Biotechnol. Prog. 20:639-654. In addition, peptide antibody rnimetics ("PAMs") can be used. as well as scaffoids based on antibody rnimetics utiliring flbronection corziponents as a scaffold.
An antigen binding protein can have. for example. the structure of a naturally occurring immunoglobulin. An "mimunogiobniin"' is a tetrameric molecule. In a naturally occurring Immunoglobulin, each tetramer is composed of two identical pairs of Polypeptide chains. each pair having one ,Tlight" (about 25 kDa) and one "heavy1' chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more arrrino acids primarily responsible for antigen recognition. The carboxy-tepm'nal portion of each chain defines a constant region primarily responsible for effector function. Human ligbt chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody1 s isotype as IgM, IgD, IgG, IgA, and IgE, respecdvely. Within light and heavy chains. übe variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a MD" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (PauL W.. ed., 2nd ed. Raven Press, N.Y.

(1989)) (incoiporated by reference in its entirety for all purposes). The variable regions ofeach light/beavy chain pair form the antibody binding site such that an iniact iinrnnnoglobiilin has two binding sites.
Naturally occurring inirnimoglobulin chains exhibit the same general structirre of relatively conserved. framework regions (FR) joined by three hypervaxiable regions. also called canpicziieiitaiüy detexmining regions or CDRs, From N-tcnrnpus to C-terminns, both light and heavy cheins comprise the domains FRL CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assigmnent of amino aciös so each domain is in accordance with the definitions of Kabat et al. in Sequences of Proteins qflmrmmologicad htteresL 5 Ed., US Dept of Health and Human Services, PHS, NIE NIH Pubbeation no. 91-3242, 1991.
An "ariribodyn refers to an intact immunoglobolin or to an antigen binding ponxxi mereof -OBS. competes with the intact antibody for specific binding, unless ötherwise speeified. Antigen binrimg portions may be prodneed by recombinant DNA teckniques or by enzymatic or chankal c lesvage of imact arüibodies. Antigen binding portions include, tnter alia. Fab. Fab\ Ffab^ Fv, domain antibodies (dAbsl and complementarity determining region (CDR) fragmenrs. single-chain antibodies (scFv). chnneric antibodies, diabodies. triabodies, tetrabodies, and Polypeptides that contain at least a porrion of an irnrnunogiobulin that is suf&cient to confer specific antigen binding to the Polypeptide.
A Fab fragment is a monovalent fragment having the V^. VH. CL and QJ1 domains: a F(abT)-fragment is a bivalent fragment having two Fab fragments linked by a disulflde bridge ai the hinge region: a Fd fragment has the VH and CH1 domains; an Fv fragment has the VL and VH domains of a Single arm of an antibody; and a dAb fragment has a VH domain, a VL domain. or an antigen-binding fragment of a VH or VL domain (US Pat No. 6,846:634; 6:696;245, US App. Pub. No. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958, Ward etal, Nature 341:544-546, 1989).
A single-chain antibody (scFv) is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino aeid residues) to form a contmuous protein chain whexein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et dl.., 1988, Science 242:423-26 and Huston et al., 1988, Proc. NatL Acad Sei USA 85:5S79-83). Diabodies are bivalent antibodies comprising two Polypeptide chains, ^herein each Polypeptide chain comprises VH and VL domains joined by a linker that is too shon to a3ow for parring berween two domains on the same chain. fcns allowiag each domain to pair with £ cornplementary domain on another Polypeptide chain (see, e.g., Holliger a al„ 1993, Proc. Natl. Acad. Sei USA 90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). If the two Polypeptide chains of a diabody are idenücal, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to rnake a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies cornprising three and four Polypeptide chains, respectively, and forrning three and fonr antigen binding sites. respectively, which can be the same or different.
Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody may be identified using the System described by Kabat ei al. in Sequences of Proteins of Irnmtraological Interest, 5th Ed., US Dept of Health and Hnman Services, PHS, NIH, NIH Publication no: 91-3242, 1991. One or more CDRs may be incorporated into a molecnle either covalently or noncovalentiy to make it an antigen binding protein. An antigen bindins protein may incorporate the CDR(s) as part of a larger

Polypeptide chain, may covalently link the CDR(s) to another Polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs penrdt rhe antigen binding protein to speciacaUy bind to a particular antigen of interest
An antigen binding protein may bave one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be difFerent For example, a nanirally occurrmg HrcmaTi jnimunoglobulin typically has two identical binding sites, white a "bispecific" or '"bifunctional" antibody has two düferent binding sites.
The tenn "human antibody" includes all antibodies that have one or more variable and constant regions derived from human iinnnmoglobtiim sequences. In one embodirnent all of the variable and consraHt domains are derived from human Immunoglobulin sequences (a fully human antibody). These antibodies may be prepared in a vaiiety of ways, examples of which are described below. inchiding through the immrrnizarion with an antigen of interest of a mouse that is geneticalry modified to express aiiribodies derived from human heavy and/or light chain-encoding genes.
A humanized antibody has a sequence that diners frorn the sequence of an antibody derived from a non-human species by one or more amino acid substitutions. deletions. and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject. In one embodirnent certahi amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody. In another embodirnent, the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species. In another embodirnent, one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for imnnmospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes. such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies may be found in U.S. PaL Nos. 6,054.297, 5.886.152 and 5,877,293.
The tenn "chimeric antibody" refers to an antibody th3t contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodirnent, one or more of the CDRs are derived from a human anti-IGF-lR antibody. In another embodirnent, all of the CDRs sie derived from a human antiTGF-lR antibody. In another embodirnent, the CDRs frommore than one human anti-IGF-lR antibodies are mixed and matched in a chimeric antfoody. For instance. a chimeric antibody may comprise a CDR1 from the light chain of a fest human anti-IGF-lR antibody, a CDR2 and a CDR3 from the light chain of a second human anfi-IGF-lR antibody, and the CDRs from the heavy chain from a third anti-IGF-lR antibody. Further, the framework regions may be derived from one of the same anti-IGF-lR antibodies, from one or more difrerent antibodies, such as a human antibody, or from a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and/or light chain is identical with, homologous to. or derived from an antibody from a particular species or belonging to a particular antibody dass or subclass, while the remainder of the chain(s) is/are identical with, homologous 1 to, or derived from an antibody (-ies) from another species or belonging to another antibody dass or

subclass. Also included are fragments of such antibodies that cxhfoit the desired biological activity (i.e., tbe ability to specifically bind IGF-1R). See, e.g.t U.S. Patent No. 4.816,567 and Morrison, 1985, Science 229:1202-07.
A "rieutralizing antibody" or "an inhibitory antibody" is an antibody that innfoits Ihs binding of IGF- 1R to IGF-I and/or IGF-2 when an excess of tbe anti-IGF-lR antibody reduces the amount of IGF-1 and'ox IGF-2 bound to IGF-1R by at least about 20% using tbe assay described in Exaiuple 9. In various enixK&nenrs, die antibody reduces tbe amount of IGF-I and/or IGF-2 bound to IGF-1R by at least 30%., 40%, 50%, 60%, 70%, 75%, 80%: 85%, 90%, 95%, 97%: 99%, and 99.9%.
An "actrvating antibody" is an antibody that activates IGF-1R by at least about 20% when added to a cell tissue or organism expressing IGF-1R. where "100% activation" is tbe level of activation achieved under physiological conditions by tbe same molar amount of IGF-1 and/or IGF-2. In various emboäunents, tbe antibody activates IGF-1R activity by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, or 1000%.
Fragments or analogs of antibodies can be readily prepared by tbose of ordinary skill in tbe art following tbe teachings of this specificatiorj and using tecbniques well-known in tbe art. Prefexred amino-and caxboxy-tennini of fraginents or analogs occux near boundaries of functional domains. Stnicüiral and functional domains can be identified by comparison of tbe nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Compirterized comparison methods can be used to ideirtify sequence motifs or predicted protein conformation domains tbat occur in otber proteirts of kncwn structure and/or function. Methods to identify' protein sequences tbat fold into a known three-dimensional structure are known. See, e.g., Bowie et al., 1991. Science 253:164.
A "CDR grafted antibody" is au antibody cbmprising one or more CDRs derived from an antibody of a particular species or isotype and the fxamework of anotber antibod}' of the same or tiifferent species or isotype.
A "multi-specific antibody" is an antibody that recognizes more than one epitope on one or more antigens. A subclass of this type of antibody is a "bi-specific antibody" which recognizes wo distinct epitopes on the same or different antigens.
An antigen binding protein "specmcally binds" to an antigen {e.g.. human IGF-1R) if iibinds to the antigen with a dissociation constant of 1 nanomolar or less.
An "antigen binding domain," "antigen binding region," or "antigen binding siten is a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's speciScity and afSnity for the antigen. For an antibody that specifically binds to its antigen, this v>iU include at least part of at least one of its GOR domains.
An "epitope" is the portion of a molecule tbat is bound by an antigen binding protein (e.g., by an antibody). An epitope can comprise non-contiguous portions of the molecule (eg\, in a Polypeptide, amino acid residues that are not contiguous in the polypeptide's prrrnary sequence but that, in tbe context of tbe Polypeptide ;s tertiary and quaternary structure. are near enough to each other to be bound by an antigen binding protein).

The ^percent identity" of two polynucleotide or two Polypeptide sequences is determined by c-nrnparing ihr sequences using ins GAP Computer program (a part of the GCG Wisconsin Package, Version 10.3 (Accetys. San Diego, CA)) using its default parameters.
The "^ ■ N ^poiytnicleotide," "oligonucleoIide', and "nucleic acid^ are used interchangeably
throughoul and include DNA molecules (e.g-, cDNA or genomic DNA), RNA molecules (e.g.s mRNA),
* analogs of tae DNA or RNA generated nsrog nucleotide analogs (e.g., pcptide nucleic acids and non-
naioralry ocoaiing nucleotide analogsX and hybrids thereo£ The nucleic acid molecule can be single-
stranded ox äorok-siraDried- In ooe snixKiküercL the micleic acid molecules of the invenfioB coüipiise a
contignous open readmg ferne encoding an antibody, or a fragrnerjt derivative, mutein, or variant rhereo£
of die nrvennon-
Two EHEie'SiiSDded polynadeotides are "the complerDent" of each otiier if their sequences can be ahgned in an ami-paiHÖe] orieifiianoc soch that every nucleotide in one polynucleotide is opposite its cornplemensry nuckoäde in the other polynucleotide, without the introduction of gaps, and without unpaired rrncleotiäes ai the 5' or rhe 3! end of either sequence. A polynucleotide is "a^rnplementary', to another porynncleotide if the two polynucleotides can hybridize to one another under moderately stringent conditions. Thns, 3 polynncleotide can be complementary to another polynucleotide without being its complement.
A 'Vector"' is a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid Segments canbe ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenovinises and adeno-associated viruses), wherein additional DNA Segments can be introdnced into the viral genome. Certain vectors are capable of autonomous replication in a host cell imo which they are irrrroduced (e.g.s bacterial vectors comprising a bacterial origin of replication and episomal rnarnmalian vectors). Orher vectors (e.g., non-episomal mammalian vectors) are integrated into rhe gcnome of z host cell lipon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector* is a type of vector that can direct the expression of a chosen polynucleoode.
A rmcleoride sequence is "operabfy linked" to a regulatory sequence if the regulatory sequence afTects the expression {e.g., the leveL tiraing, or locarion of expression) of the nucleotide sequence. A "regulatory secne-nce'5 is a nucleic acid that afTects the expression {e.g., the level, Urning, or locauon of expression) of a nucleic acid to which it is operably linked. The regulatory sequence caru for example, exeri its effects directly on the regulated nucleic acid, or throngh the action of one or more oiher molecules (eg., Polypeptides that bind to the regulatory sequence an&'or the nucleic acid). Examples of regulatory sequences inchide promoters, enhancers and other expression control elements {e.g.. polyadenylation Signals). Frrrthex examples of regulatory sequences are descnbed in, for example. GoeddeL 1990. Gene Expression Technology: Methoös in Enzymology 185, Academic Press, San Diego, CA and Baron et al.. 1995, Nucleic Acids Res. 23:3605-06.
A uhost cell" is a cell that can be used to express a nucleic acicL e.g., a nucleic acid of the invention. A host cell can be a prokaryote. for example. E. coli, or it can be a eukaryote. for example. a single-ceHed eukaryote (e.g., a yeast or other fungus). a plant cell (e.g., a tobacco or tomato plant cell), an

aniroa] cell {e.g.. a human cell, a monkey celL a hamster cell 2 rat cell, a monse cell or an insect cell) or a hybridoma. Examples of host celis include tbe COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al, 1981, Cell 23:175): L cells, C127 celis, 3T5 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and reiaied cell lines which grow in sertzm-free media (see Rasmussen et al, 1998, Cytotechnology 28:31) or CHO scainDX-BlL which is deficient in DHFR (see Urlaub et al, 1980, Proc. NatL Acad Sei USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA ceD hne öerrved &om tbe Afiiean graa mookey kidi^y cell hue CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), h\mm eaiayuük. kidney ccDs soch as 293, 293 EBNA or MSR 293. human epidexmal A431 cells. human Colo205 cefis. oter trans&nrjed JMHHU* cell hnes. normal diploid cells, cell strains derived &om in vitro cnhnre of prinery tissoe, lyiiigfv e^plants, EL-60. U93 7. HaK or hirkat cells. Typically, a host cell is a cuhnred ceE mar canbe trarisiaarjed or tr^nsiected with a polypeptsde-encodhig rmeieie aeid, wirich can then be expressed in ifae host ceSL The phrase Recombinant host cell" can be used to denote a host ceQ fhst rss been transfonned or rransfected with a nucleic aeid to be expressed. A host cell also can be a cell thai compnses the rroclek aeid but does not express it at a desired level unless a regulatory sequence is rnrrodnced into the hos* cell such that it becomes operably linked with the nucleic arid, It is understood that me tenn host ceD refers not only to the particular subjeet cell bin to the progeny or potential progeny of such a ceÜ. Becacse certain modincations may oeeur in sueeeeding generations due to. e.g.. mutation ar environmental infhsence, snch progeny may not in fact be identical to the parent cell but are still inchided witrriri the scope of the term as used herein.
IGF-1R
IGF-lRis a transmembrane reeeptor tyrosine kinase (Bhime-Jensen et al. 2001,Naüire 411:355-65). The human IGF-1R is synthesized as a 1367 amino aeid precursor Polypeptide that includes a 30 amino aeid Signal pepride removed dnring translocation iso the endoplasmic retieuhrrn (Swiss-Prot: P08069). Tne 1GF-1R proreeeptor is glycosylated and ckaved by 2 protease at positions 708-711 (counting firom the first amino aeid following the signal peptide sequence) dnring maturation in the ER-golgi resultmg in the fonnation of an a-chain (1-707) and a ß-chain (712-1337) thai remain linked by disuinde bonds (Bhaumick ei al, 1981, Proc Natl Acad Sei USA 78:4279-53, Chemausek et al., 1981, Biochemistry 20:7345-50, Jacobs et al, 1983, Proc Natl Acad Sei USA 80:1228-31, LeBon et al, 1986, J Biol Chem 261:7635-89, EUeman, er al, 2000, Biochem J 347:771-79). The predorninant form of the IGF-1R (and INSR) that exists on the cell-surface is a proteolytically processed and glycosylated (aß)2 dimer joined covalently by ODe or more disulSde bonds.
The extracellular portion of the IGF-1R consists of ihe a-chain and 191 amino aeids of the ß-chain (712-905). The reeeptor contains a single transmembrane spanning seqnence (906-929) and a 408-residue cytoplasmic domain that includes a functional tyrosine kinase (Rubin et al. 1983, Natnre 305:438-440). Comparative sequence analysis has revealed that the IGF-1R is composed of 11 distinet structural motifs (reviewed by Adams etal, 2000, Cell Mol Life Sei 57:1050-93, Marino-Buslje et al, 1998, FEBS Ltrs 441:331-36, Ward et al, 2001, BMC Bioinformatics 2:4). The N-terminal half of the extracellular domain contains two homologous comains referred to as LI (1-151) and L2 (299-461) (Ward et al, 2001, supra) separated by a cysteine-rich (CR) region (152-298) consisting of several structural modules with disulfide

iinkages that abgn with repeating units present in tbe TNF receptor and lamimn (Waid et at.. 1995, Proteins 22:141-53). The crystal structnre of the LI—CR-L2 domain has been solved (Garrett et aL, 1998. Nature 394:395-99). Tbe L2 domain is followed by tbree fibronectin type m domains (Marino-Buslje et o/..,1998. suprcL Mulbern et al., 1998, Trends Biocbem Sei 23:465-66, Ward et al, 1999, Growrb Factors 16*315-22). The first Fnin domain (FnEH-l, 461-579) is 118 amino aeids in length. The second Fnlü domain (FnIÜ-2L 580-798) is dismpted by a major insert sequence (TD) of about 120 amino aeids in length. Tbe ID domain incrades a furin protease cleavage site that separates tbe a and ß cbams of tbe mamre receptor. The trdrd FnHI domain (FnIH-3) is locaied entirely in tbe ß-chain (799-901) tenniiiating several residnes before tbe tiansmembraiie sequence. Tbe catalytic domain of tbe 1GF-1R tyrosme kinase is located between ^mmfi aeids positions 973-1229. and its stxucorre bas been solved (Faverynkis et al.. 2001, Natnre Structnral Bk>l 8:1058-63. Pantsch et aL, 2001, Structnre 9:955-65). Tbe kinase is üanked by two regntetory regions, tbe jirr^rrrf-mhr?™> region (930-972) and a 108 amino aeid C-tennina] tau (1220-1337) (Surrnacz et al, 1995, Experimenlal Cell Res 21S:370-80; Hongo etal., 1996. Oncogene 12:1231-38). The two regülatory regions contain ryrosine residnes that serve as docking sites for signal transducing proteins wben pbospborylated by tbe activatedIGF-lRtyrosine kinase (reviewed by Baserga (ed.), 1998 The IGF-1 Recepior in Normal and Abnormal Growth, Honnones and Growth Factors in Development and Neoplasia. WDey-Liss. Inc., Adams et al, 2000, CeU Mol Life Sei 57:1050-93).
Tbe IGF-1R amino arid sequence is about 70% identical to tbe insulin receptor (INSR; Swiss-Prot: P06213). The bighest homology between tbe reeeptors is located in the tyrosine kinase domain (84%); tbe lowest idenüry is in the CR region and the C-terminus. The IGF-IR is also highly related (- 55% identical) to tbe insulin related receptor (IRR; Swiss-Prot: P14616).
Human IGF-IR can be activated by the insulin-like growth factors, IGF-1 and IGF-2 and insulin" (INS) (Hill et aL, 1985: Pediatric Research 19:879-86). IGF-1 and IGF-2 are encoded nonallelic genes (Brissenden et aL, 1984, Nature 310: 781-8, Bell et aL, 1985, Proceedings of tbe National Academy of Sciences of the United States of America 82: 6450-54), and both genes express alternative proteins retaisd by differential RNA splicing and protein processing. Tbe most common and well-studied matuxe forms of IGF-1 and IGF-2 are respectively 70 and 67 amino aeids in length (Jansen et al.. 1983. Nature 306:609-: l, Dull et aL, 1984. Nature 310: 777-81). These proteins (and their isoforms) are identical at 11/21 posrnons to the insulin A-peptide, and identical at 12/30 positions wirb tbe insulin B-peptide.
IGF-IR is expressed in all cells rypes in tbe normal adult anirnal except for liver hepatoeyres znd
rnarure B-cells. Human bloodplasma contains high concenrrarjons of IGF-1 and IGF-2. and IGF-1 cznbe
detected in most tissues. The receptor is an integral component of tbe pbysiological meebanism Controlling
organ size and bomeostasis. Without being bound to a particular theory. the "Somatomedin Hypothesis^
states that Growth Hormone (GH) mediated somatic growth that oecurs during chüdbood and adolescence . . -.
is dependent on the endocrine form of IGF-1 that is mainry produced and secretedby the bver (Daugbaday. 2000, Pediatric Nephrology 14: 537^0). Tbe synthesis of hepatic IGF-1 is stimulated by GH release in tbe pituitary in response to hypothalamic GHRH (GH releasing bormone). The serum concentration of IGF-1 increases over 100 fold between ages 5-15 in bumans. Tbe hioavailabüity of IGF-1 is regulated by IGF binding protein 3 (IGFBP3) wirb approximately 99% of the growth factor compartmentalized in tbe bound state. Primary IGF-1 deficiency arising formpartial gene deletions, and secondary IGF-1 deficiency

:sulting from defects in GH producrion or signaling are not lethal (Woods, 1999, IGF Deficiency in ontemporary Endocrinology: The IGF System. R a. R Rosenfeld, C. Jr. Totowa, ed_£, Humana Press, NJ: 51-74). The affecteä individuals exhibh growm retardation at birth. grow slowly and can face certain CNS DBonnaüties.
IGF-1R signaling promotes cell growth and survival through the IRS adaptex protern-dependent Ovation of the PBKinase/Ail pathway. IGF-1R transuiLs a signal to its major Substrates. IRS-1 through 3S4 and the Shc protcins (Blakesley er al.. 1999, IGF-1 receptorfimction: transducing the IGF-1 signal ito intracellular events in The IGF System, R G. a_ R Rosenfeld. Jr. CT. Totowa. ed_s, Humana Press, U: 143-63). This results in activation of the Ras/RaSMAP kmase and PI3 Kinase/Akt signaling pathways. lowever. induction of Akt-mediated ceü survival via IRS is the dominant parlrway response upon IGF tiniulation of most cells. See Figure 10.
mtigen bindrng proteins
In one aspect, the present invention provides antigen binding proteins {e.g. antibodies, antibody ragments, antibody derivatives, antibody nmteins, and antibody variants), thatbind to IGF-1R, e.g.. human GF-1R.
Antigen binding proteins in accordance with the present invention include antigen binding proteins hat inhibit a biological activity of IGF-1R Examples of such biological activrües include binding a ;ignaling molecule {e.g., IGF-1 and/or IGF-2), and transducing a signal in response to binding a signaling nolecule.
Different antigen binding proteins may bind to diffeTent domains or epitopes of IGF-1R or act by lifferent mechanisms of action. Examples include but are not limited to antigen binding proteins that nterfere with binding of IGF-1 and/or IGF-2 to IGF-1R or that inhibit signal transduction. The site of iction may be, for example, intracellular (e.g., by interfering with an intracellular signaling cascade) or sxtraceUuIar. An antigen binding protein need not completely inhibit an IGF-1 and/oi IGF-2 induced activity to find use in the present invention; rather, antigen binding proteins that reduce a particulax activity of IGF-1 an&'or IGF-2 are contemplatec for use as well. (Discussioris herein of paracular roechanisins of action for IGF-lR-binding antigen binding proteins in treating particular diseases are illustrative only, and the methods presented herein are not bound thereby.)
It has been observed that IGF-1 and IGF-2 each exhfbrts biphasic binding to IGF-1R. High affmity binding has been reported to have a KD in me ränge of 0.2 nM; high affmity binding, about ten fold higher. Thus. in one embodiment, the present invention provides an IGF-1R inhibitor that inhibits both the high and low afanity binding of IGF-1 and/or IGF-2 to IGF-R It has been suggested that the high aifinity binding, rather than the low afnnity binding, of IGF-1 and;or IGF-2" to IGF-1R is required for the confonnation change that activates the tyrosine kinase activity of IGF-1R. Thus, in another embodiment the IGF-1R inhibitor preferentially inhibits the high afünity binding of IGF-1 and/or IGF-2 to IGF-1R as compared to the low affmity binding.
In another aspect, the present invention provides antigen binding proteins that comprise a light chain variable region selected from the group consisting of LI through L52 and/or a heavy chain variable region selected from the group consisting of Hl through R52, and fxagrnents, derivatives, muteins. and

variants thereof (see Figures 2 and 3). Such an antigen binding protein can be denoted using tbe nomenclature "LxHy", wberein "x" corresponds to the number of the light chain variable region and "y" corresponds to the number of the heavy CD? in variable region as fhey arc labeled in Figures 2 and 3. For example. L2H1 refers to an antigen binding protein ?öth a ligbt chain variable region comprising tbe amino aeid sequence of L2 and a heavy chain variable region comprising the amino aeid sequence of Hl, as shown in Figures 2 and 3. Figures 2 and 3 also indicate the locatkra of tbe CDR and framework regions of eacb of tbese variable domain sequences. The CDR regions of each ligbt and heavy chain also are gronped by type and by sequence sirnüarity in Figures 4 through 9. Antigen binding proteins of the inverrtion include, for example, antigen binding proteins having a combination of light chain and heavy chain variable domains selected from the group of combinations consisting of L1H1,L2KL L3H3, L4H4: L5H5, L6H6, L7H7, LSHS, L9H9, L10E10, LI 1H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31, L32H32, L33H33, I34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L4ÖH40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49: L50H50, L51H51, and L52H52.
In one embodiment, the present invention provides an antigen binding protein comprising a light
chain variable domain comprising a sequence of amino aeids that diffeTS firom the seqnence of a light chain
variable domain selected frorn the group consisting of LI through L52 only at 15. 14. 13, 12, 11, 10,9,8,7,
6, 5. 4, 3, 2. or 1 residues, wherein each such sequence difference is independently either a deletion.
insertion, or Substitution of one amino aeid residue. In another embodiment the bght-chain variable
domain comprises a sequence of amino aeids that is at least 70%, 75%: S0%: 85%, 90%, 95%, 97%, or 99%
identical to the sequence of a light chain variable domain selected from the group consisting of LI through
L52. In another embodiment the light chain variable domain comprises a sequence of amino aeids that is
encoded by a nucleotidc sequence that is at least 70%, 75%; 80%, 85%, 90%, 95%, 97%, or 99% identical
to a nucleotide sequence that encodes a light chain variable domain selected from the group consisting of
LI through L52. In another embodiment the light chain variable domain comprises a sequence of amino
aeids that is encoded by a polynucleotide that hybridizes under rnoderately stringent conditions to the
complement of a polynucleotide that encodes a light chain variable domain selected from the group
consisting of LI through L52. In another embodiment tbe light chain variable domain comprises a
sequence of amino aeids that is encoded by a polynucleotiGe that hybridizes under rnoderately stringent
conditions to the complement of a polynucleoride that encodes a ligbt chain variable domain selected from
the group consisting of LI through L52. In another embodiment, the light chain variable domain comprises
a sequence of amino aeids that is encoded by a polynucleotide that hybridizes under rnoderately stringent
conditions to a complement of a light chain polymicleotide selected from Figure 1. -■ -
In another embodiment, tbe present invention provides an antigen binding protein- comprising a heavy chain variable domain comprising a sequence of amino aeids that differs from the sequence of a heavy chain variable domain selected from the group consisting of Hl through H52 only at 15, 14, 13, 12, 31, 10, 9, 8, 7. 6, 5, 4. 3. 2, or 1 residue(s), wherein each such sequence difference is independently either a deletion. insertion, or Substitution of one amino aeid residue. In another embodiment the heavy chain variable domain comprises a sequence of amino aeids that is at least 70%. 75%, 80%, 85%, 90%, 95%.

97%. or 99% identicai to tbe sequence of a heavy chain variable domain selected from the group consisting of Hl through H52. In another emrx>diment, the heavy chain variable domain comprises a sequence of airrinn acids that is er>coded by a nucleotide sequence that is at least 70%. 75%, 80%, 85%. 90%, 95%, 97%. or 99% iderucal zo a nncleotide sequence that encodes a heavy charn variable domain selected from the group consisting of Hl through H52. In another embodiment, the heavy cbain variable domain comprises a sequecce of amino acids that is encoded by a polynucleotide that hybridizes linder moderaiely strtngent corjdirions ta the carcpieanent of a porynacleoüde tbat encodes a heavy cham variable domain selected fjom the groop consiscing of Hl through H52. ha another exnbodiment tbe heavy chain variable domain comprises a seqneuce of amiüo acids that is encoded by a polynucleotide that hybridizes under moderaiery strmgear ccmdrrkKs to the compisineiri of a polynncleoride that encodes a heavy chain variable domain selected üuui the groap cousisöng of Hl thron gh H52. In another ernbodiment. the heavy chain variable dornain coxaprises a sequence of aznino acids that is encoded by a polynucleotide that hybridizes under rnoderaiely srringe-ii coaditiocs to a canTplement of a heavy chain polynucleotide selected from Figure 1.
Parücular embodrmerrrs of amigen binding proteins of the present invention compnse one or moxe amino acid sequences that are identicai to the amino acid sequences of one or more of the CDRs and/or FRs iliustrated in Figures 2 through 9. In one emborörnent the antigen binding protein comprises a light chain CDR1 sequence ühistraied in Figure 4. In another embodiment, the antigen binding protein comprises a light chain CDR2 sequence iliustrated in Figure 5. In another embodirnent the antigen binding protein comprises a light chain CDR3 sequence iliustrated in Figure 6. In another embodiment the antigen binding protein comprises a heavy chain CDR1 sequence iliustrated in Figure 7. In another embodiment. the. antigen binding protein comprises a heavy chain CDR2 sequence iliustrated in Figure 8. In another embodiment, the antigen binding protein comprises a heavy cbain CDR3 sequence iliustrated in Figure 9. In another embodrmenr the antigen binding protein comprises a light chain FR1 sequence iliustrated in Figure 2. In another embodiment the andgen binding protein comprises a light chain FR2 sequence iliustrated in Figure 2. In another embodiment the antigen binding protein comprises a light chain FRS sequence iliustrated in Figure 2. In another embodiment the antigen binding protein comprises a light chain FR4 sequence üiustraied in Figure 2. In another embodiment the antigen binding protein comprises a heavy chain FR1 sequence iliustrated in Figure 3. In another embodiment the antigen binding protein comprises a heavy chain FR2 sequence iliustrated in Figure 3. In another embodiment the antigen binding protein comprises a heavy chain FR3 sequence iliustrated in Figure 3. In another embodiment the antigen binding protein comprises a heavy chain FR4 sequence iliustrated in Figure 3.
In one embodiment the present invention provides an antigen binding protein that comprises one or more CDR sequences that dirTer from a CDR sequence sho"wn in Figures 2 through 9 by no more than 5, 4, 3. 2, or 1 amino acid residues.
In one embodiment, the present invention provides an antigen binding protein that comprises at least one CDR from L1-L52 and/or H1-H52. as shown in Figures 2 through 9. and at least one CDR sequence from an anü-IGF-lR antibody described in US Pat App. Pub. Nos. 03/0235582, 04/0228859, 04/0265307,. 04/0886503, 05/0008642, 05/0084906, 05/0186203, 05/0244408, PCT Pub. Nos. WO 03/059951, WO 03/100008, WO 04/071529A2, WO 04/083248, WO 04/087756, WO 05/016967, WO

05/016970, or WO 05/058967 (each of which is incorporated herein by reference in its entirety for all purposes) wherein the antigen binding protein binds to IGF-1 receptor. In another embodiment. the antigen binding protein cornprises 2, 3. 4, or 5 CDR sequences froniLl-L52 and/or H1-K52, as sbown in Figures 2 through 9. In another erribodimeiit, the antigen brn±ng protein cornprises 2. 3. 4, or 5 CDR sequences from an anti-IGF-lR antibody described in US Pat App. Pub. Nos. 03/0235582, 04/0228859, 04/0265307, 04/0886503, 05/0008641 05/0084906, 05/0186203, 05/0244408, PCTPub. Nos. WO 03/059951, WO 03/1OOOO8, WO 04/071529 A2, WO 04/083248, WO 044)87?56, WO 054)16967, WO 05/016970, or WO 05/058967. In another exobodimeiiL ai least one of the antigen bnariing prosem's CDR3 sequences is a CDR3 seqnence &om LI-L52 and/or H1-K52, as shown in Figures 2, 3, 6. and 9. hi another embodimenl nie antigen binnrnp. proteia's light chain CDR3 seqnence is a Tignt cfcgm CDR3 seqnence fromLl-L52 as sho"wn in Figures 2 and 6 and the antigen binding protein5 s heavy chain CDR3 seqoence is a heavy chain sequence from Hl -H52 as sbown in Figures 3 and 9. In ano&er eabodiireia, the amigec binding protein cornprises 1, 2, 3, 4. or 5 CDR sequences that each mdepeiiöeiiny düreasby 6, 5, 4. 3. 2. I. or 0 single amino acid addirions, substitnrions, and'or deletions fei a CDR seqnence of L1-L52 and'or H1-H52, and rhe antigen binding protein further cornprises 1. 2, 3, 4, or 5 CDR sequences that each independently difFers by 6, 5. 4. 3, 2, 1. or 0 Single ainino acid aädifions, siibstmrrions, and/or dekdons from a CDR seqnence of US Pat App. Pub. Nos. 03/0235582, 04/0228859, 04/0265307, O4/08S65O3, 05/0008642, 05/0084906, 05/0186203, 05/0244408, PCT Pub. Nos. WO 03/059951.. WO 03/100008, WO 04/071529A2, WO 04/083248. WO 04/087756, WO 05/016967.. WO 05/016970. or WO 05/058967. In another embodrment the CDR sequence(s) from US Pat App. Pub. Nos. 03/0235582, 04/0228859, 04/0265307, 04/0886503, 05/0008642, 05/0084906, 05/0186203, 05/0244408, PCT Pub. Nos. WO 03/059951, WO 03/100008, WO 04/071529A2: WO 04/083248, WO 04/087756, WO 05/016967. WO 05/016970, or WO 05/058967. In another ernbodiment, the CDR sequence(s) are from (an) antibody-ies) that bind(s) to the L2 portion of the extracellular domain of IGF-1 receptor. In another erc6odirnent, the antigen binding protein does not comprise a light chain CDR3 sequence and'or z heavy chain CDR3 sequence from an anü-IGF-lR antibody from US Pat App. Pub. Nos. 03/0235582, 04-0228859, 04/0265307, 04/0886503, 05/0008642, 05/0084906, 05/0186203, 05/0244408, PCT Pub. Nos. WO 03/059951: WO 03T0OOO8, WO 04/071529.A2, WO 04/083248, WO 04/087756, WO 05/016967, WO 05/016970, er WO 05/058967.
In one embodiment the present invention provides an anhgen binding protein that cornprises a light Cham CDR1 comprising the sequence RSSQSLLEDCjX2GYNX5LX4 (SEQ ID NO:236), wherein X. is a serine or a threonine residue, X2 is an asparagine, serine, or histidine residue, X3 is a tyrosine or a Phenylalanine residue. and X* is an aspartate er an aspaxagine residue. In another embodiment, the light chain CDR1 cornprises the sequence TRSSGX]DC2X;NY\7Q (SEQ ID NO:237), wherein Xi is a serine or an aspartate residue, X2 is an alanine or an aspartate residue, and X: is a serine or an asparagine residue. In another ernbodiment, the light chain CDR1 cornprises the seqnence RASQX^XjXjXsLX^ (SEQ ID NO:238). wherein Xj is a glycine or a serine residue. X2 is an isoleucine, valine, or proline residue, and X5 is a serine, glycine, or tyrosine residue, Xi is any arnino acid residue, X5 is z Phenylalanine, tyrosine, asparagine. or Tryptophan residue. and X* is an alanine or an asparagine residue. In another embodiment X2 is an isoleucine or valine residue, X3 is a glycine or serine residue, X* is an arginine, serine, asparagine, serine, tyrosine, or isoleucine residue, and X

valine residue, X2 is a serine or a Phenylalanine residue, X3 is an asparagine, tyrosine, or threonine residue, and XA is an alanine or an aspartate residue.- In another embodirnent tbe CDR2 comprises the seqüence AXiSX2LX3S (SEQ ED NO:240). wherein Xi is an alanine or a threonine residue, X2 is a threonine or a glycine residue. and X3 is a glutainine or a glutamate residue. In another embodiment. the CDH2 comprises the sequence XÄNXsRPS (SEQ ID NO:241), wherein Xj is a gbtamate, glnlHminr, or glycine resüae, X2 is an aspartate or rysine residue. and X3 is any arnino aeid residae.
In one embodiinent the present invention provides an antigen binding protein mal comprises a hghi riigTTi CDR3 comprising the sequence MXÄXtXÄPXÄ (SEQ ED NCh242); wherein Xl is a gfatamme or glutamate residue, X2 is an alanine. glycine, serine, or rrnxonine residoe, X3 is a leucine or threonine residue. X* is a glmamine, glutamate, or histHÜne residue, X5 is a mreoirine, Tryptophan, merhiomne. or vaüne residue, X* is a nonpolar side chain residue, and X? is a rhieonine, serine, or glarrin^ residue. In another embodiment the CDR3 comprises the seqoence QQX5X2X5X4PX5T (SEQ ID NCh243), wherein Xi is an arginine, serine, leucine. or alanine residue, X2 is an asparagine. serine. orhisridirje residue. X3 is a serine or an asparagine residue, X4 is a nonpolar side chain residue, and X5 is a leucine. isoleucine, tyrosine. or tryptophan residue. In another ernbodiment the CDR3 comprises the sequence QSYX1SX2NX3X4V (SEQ ID NO:244). wherein X, is an aspartate or a ghiramine residue, X2 is a serine or an aspartate residue. X3 is a glutamine, valine. or tryptophan residue, and X4 is an arginine residue or no residue.
In one embodiment the present invention provides an antigen binding pxotein that comprises a heavy chain CDR1 comprising the sequence XjX^XsWWS (SEQ ID NO:245), wherein X) is a serine residue or no residue. X2 is a serine or asparagine residue, and X3 is an asparagine residue and an isolencine residue. In another embodiment, the heavy chain CDR1 comprises the sequence XiX2YWS (SEQ ID KO:2-6), wherein X] is a glycine. asparagine. or aspartate residue, and X2 is a tyrosine or phenvlabnoe residue. In another embodiment the heavy chain CDR1 comprises the sequence SYX1X2X3 (SEQ ED NO:247). wherein X] is an alanine or glycine residue, X2 is a rnethionine or isoleucine residue. and X; is a serine or histidine residue.
In one embodiment the present invention provides an antigen binding protein that comprises 2 heavy chain CDR2 comprising the sequence XiX2X3X4X5GXaX-YN?SLX8S (SEQ ID NO:24S), wberm X-, is a glutamate. tyrosine. or serine residue. X2 is a isoleucine or valine residue. X3 is a tyrosine, asparagine. or serine residue, X4 is a histidine, tyrosine, aspartate. or proline residue. X5 is a serine or arginine residue, X$ is a serine or asparagine residue, X7 is an asparagine or tyrosine residue, and Xg is a lysine or glutamate residue. In another embodiment, the heavy chain CDR2 comprises the sequence XJISX^XIXSXÖXTYYADSVKG (SEQ ID NO:249), wherein X, is a threonine, alanine, valine, or tyrosine residue, X2 is a glycine, serine, or tyrosine residue, X3 is a serine, asparagine, or aspartate residue. X4 is a glycine or serine residue, X5 is a glycine, serine, or aspartate residue, X$ is a serine, threonine, or asparagine residue, and X7 is a threonine, lysine, or isoleucine residue.
In one embodiment, the present invention provides an antigen binding protein that comprises a heavy chain CDR3 comprising the sequence X1X2X3X4X5X€XTX8XQFDI (SEQ ED N"O:250), wherein X-, is a


arginine, threonine, glutarnine, leucine, or glutamate residue, or no residue. X5 is a serine. glycine, asparagine, threonine. tryptophan. alanine, valine, or isoleucine residue. X$ is an arginine, gltrtarnine, tyrosine, valine, alanine. glycine, serine, Phenylalanine, or Tryptophan residue, X-, is a leucine. asparagine, aspartate. threonine, tryptophan. tyrosine, valine, alanine. or histidine residue. Xg is an aspartate. serine. asparagine, or glutaxmne residue, and X9 is an alamme- or a proline residue. In anothex en&odhrjenL the heavy chain CDR3 comprises the sequence X,X2X3XiX5X6X,XsXoX1oXnMDV (SEQ K) NO:251), wherein Xj is an pla-nfnp. residue, or no residue. X2 is a glutamate, tyrosine, or glycine residue, or no residue, X2 is a serine or arginine residue, or DO residue. X In one embodiment, the present invenüon provides an isolated antigen binding protein. comprising either: a. a light chain CDR3 comprising a sequence selected from the groiro consisting on i. a iight chain CDR3 sequence selected from the group consisting of the light chain CDR5 sequences of L1-L52 as shown

inFigure 6: ii. MQALQTPZT; iü. QQ(R/S)(X7S)(S/N)ZPLT: and iv. QSYDSSNXJV: b. a heavy chain CDR3 comprising a sequence selected from the group consisring of: L a heavy chaf-n CDR3 sequence that differs by no more than a total of three ainino acid addinons. substitutions. 01 deletions from a CDR3 sequence selected fron the group consistiiLg of the heavy chain CDR3 sequences of H1-H52 as shown in Figure9;iL SRLDAFDI; iü. SXYDYYGMDV; iv. HRXDXAWYFDL; and v. DSSG;orc. rhelighi chain CDR3 sequence of (a) and the heavy chain CDR3 sequence of (b); wberein amino acid residue Symbols enclosed in parentheses ideniüy alternative residues for the same position in a sequence. each X is independently any amino acid residue. each Z is independently a glycme residue, an alanine residne, a valine residue, a leucine residue, an isoleucine residue, a proline residue. a Phenylalanine residue, a memionine residue. a tryptophan residue. or z cysteine residue. each J is independentiy a gfetarnme residue. an arginine residue. a valine residue. or a tryptophan residue. and the antigen binding protein binds to human IGF-1R.
The nucieotide sequences of Figure 1. or the arnino acid sequences of Figures 2 through 9. can be aitered, for example, by random mutagenesis or by site-directed mutagenesis (e.g.. ohgonucleotide-directed site-specific mutagenesis) to create an altered polynucleotide comprising one or more particular nucieotide substitutions. deletions, or inserrions as compared to the non-mutated polynucleotide. Examples of techniques for rnaVrng such alterations are described in Walder et ah, 1986.Gene 42:133; Bauer et aI1985. Gene 37:73; Craik. BioTechniques, January 1985, 12-19; Smith etat, 1981, GeneticEngineering: Principles and 7./etf?ods. Plenum Press: andU.S. Patent Nos. 4,518,584 and 4,737.462. These and other methods can be used to make. for example, derivatives of anti-IGF-lR antibodies that have a desired property, for example. increased af&nity, avidity, or specificity for IGF-1R. increased activity or stability in vivo or in vitro, or reduced in vivo side-effects as compared to the underivatized antibody.
Other derivatives of anti- IGF-1R antibodies mithin the scope of rhis invention include covalent or aggregatdve conjugates of anti-IGF-lR antibodies, or fragments thereof, with other proteins or Polypeptides, such as by expression of recombinant fusion proteins comprising heterologous Polypeptides fused to the N-ierminus or C-terminus of an anti- IGF-1R antibody Polypeptide. For example, the conjugated pepride may be a heterologous signal (or leader) Polypeptide. eg\. the yeast alpha-factor leader. or a pepride such as an epitope tag. Antigen binding protem-contairdng fusion proteins can comprise peptides added to faciiitate puriScation or identiScation of antigen binding protein (e.g., poly-His). An antigen binding protein also can be linked to the FLAG peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:255) as described in Hopp et ah: Bio/Technology 6:1204, 1988, and U.S. Patent 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody (mAb), enabiing rapid assay and facile purificarion of expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given Polypeptide are commercially available (Sigma, St Louis. MO).
Oligomers that contain one or more antigen binding proteins may be employed as IGF-1R antagonists. Oligomers may be in the form of covalentlv-linked or non-covalentry-linked dimers. trimers. or higher oligomers. Ohgomers comprising two or more antigen binding protein are contemplated for use.


One erobodiment is directed to oligomers cornprising multiple antigen binding proteins joined via covalent or non-covalsnt interactians between peptide moieties fused to theantigen binding proteins. Such peptides may be peptide linkers (spacers). or peptides that have the property of promoting oligomerizaüorL Leurine zippers and certain Polypeptides derived from antibodies are among the peptides that can promote oligomerizatioD of antigen binding proteins attached thereto. as described in more detail below.
In parti^alar cinbodiments, the oligomers comprise from two to four antigen binding proteins. The antigezi binding pLOtcios of the oügomer may be in any form, such as any of the fbrms described above. e-g.r v^ttMiiT^ or fegmeans. Preferabry, the oligomers comprise antigen binding proteins that have IGF-1R binding acüviiy.
In one enrodinient an oiigomex is prepared using Polypeptides derived from iinmunoglobnlms. PreparHikHi of fesion pfoteins cornprising certain beterologous Polypeptides fused to varions portions of antibody^DfTrveGrx>rypeptiäes (inclnding the Fe domain) has been described, e.g., by Ashkenazi et al.y 199i? PNAS USA 85:10535; Byrn er al9 1990, Nature 344:677: and Hollenbaugh er ci., 1992 "Construction of T,i:nriK>globgth Fusion Proteins", in Current Protocob in Immun•ology, Suppl. 4, pages 10.19.1 -10.19.11.
One embodimect of the present invenüon is directed to a dimer cornprising two fusion proteins createdby rusing an IGF-1R binding fragment of au anti- IGF-1R antibody to the Fe region of an antibody. The dimer can be made by, for example, inserting a gene fusion encoding the fusion protein into an appropriate expression vector. expressing the gene fusion in host cells transformed with the recombinant expression vector. and aUowing the expressed fusion protein to assemble much Hks antibody molecules, whereupon interchain disulfide bonds form between the Fe moieties to yield the dimer.
The term "Fe Polypeptide" as used herein includes native and mutein forms of Polypeptides derived from the Fe region of an antibody. Truncated forms of such Polypeptides containiag the hinge region that premotes dimerization also are included. Fusion proteins comprising Fe moieties (and oligomers formed tbereSrom) offer the advantage of facile purificatioü by afnnity chromatography over Protein A or Protein G columns.
One suitable Fe Polypeptide, described in PCT applicauon WO 93/10151 (hereby incorporated by reference), is a singie chain Polypeptide extending from the N-tenmnal hinge region to the native C-terminns of the Fe region of a human IgGl antibody. Another useful Fe Polypeptide is the Fe mutein described in U.S. Patent 5:457;035 and in Baum et al, 1994. EMBO J. 13:3992-4001. The arnino aeid sequence of this mrr.ein is identical to that of the native Fe sequence presented in WO 93/10151. except that arnino arid 19 has been changed from Leu to Ala. amino aeid 20 has been changed from Leu to Glu, and amino aeid 22 has been changed from Gly to .Ala. Tne mutein exhibits reduced afnnity for Fe reeeptors.
In other embodiments. the variable poraon of the heavy and Altematively, the oligomer is a fusion protein cornprising multiple antigen binding proteins, with er wrihoul peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233.
Another method for preparing oligomeric antigen binding proteins involves use of a leucine zipper. Leucine ziuper domains are peptides that promote oligomerization of the proteins in which they are found.

Leucine zippers were originally identüied in several DNA-binding proteins (Laaaschuls et al. 1988. Science 240:1759). and have since been found in s variety of difFsrem proteins. Among the kaown leucine zippers are naturally o-ccuiring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine ziDper rionsHs suitable foi producirg sc'^ble oHgoroeric proteins are described in PCT application WO 94/10308, and the leucine zipper derivec rom hing Surfactant protein D (SPD) described in Hoppe et al.. 1994. FEBS Leiters 344:191. hereby incoeporsted by reference. The usc of a modified leucine zipper that aliows for stähle tnincnzation of a heterwoeous piotein fhsed fbexetu is described in Fanslow et al, 1994, Semin. IrmmmoL 6:267-78. In one ^miA-4i recmiirM.'iynTfhskmproteins comprising an anti- IGF-1R antibody' iragment or derivative fused to 2 ViM.inri zipper peptide are expressed in suitable host cells, and the sorable oligomeric anti- 1GF-IR antibody ragmenB or derivatives ifeat form are recovexed frorn the culture supemaianT.
In one aspect The present invention ^xrhäes antigen bmdmg piotsiiis that interfere wrfh rhe binding of IGF-1 and^or IGF-2 to an IGF-1 R Such antigen brrvfag proerrns can be made against IGF-IR, or a fxagmeirt variant OT derivative thexeofL and screeued in convemiösa] assays for the abiliiy to interfere witb binding of IGF-1 and/or IGF-2 to IGF-IR Exarnples of STtirabk assays are assays that test the antigen binding proteins for the abiliry to inhibit binding of IGF-1 and/or IGF-2 to cells expressing IGF-IR. or that test antigen binding uroteias for the ability to reducs a biological or ceHnlar response that results &ora the binding of IGF-1 and'oi IGF-2 to cell surface IGF-1"R reeeptors.
In another aspect the present invention provides an antigen binding protein that blocks the binding of IGF-1 and'br IGF-2 to IGF-IR but does not significantly block the binding of insulin to insulin reeeptor (INS-R). In one embodiment, the antigen binding protein does not bind to INS-R In another embodiment, the antigen binding protein binds to the INS-R with such a low affrmty that it does not effectively block the binding of insulin to ENS-R. In another embodiment, the antigen binding protein binds to INS-R, but antigen binding protein-bound INS-R can stiH bind to insulin. In another embodiment, the antigen binding protein*s selectivity for IGF-1R is at least 50 rmes grearsr than its selectivity for insulin reeeptor. In another embodiment the selectivity- of the zrhgen binding protein is more than 100 times greater than its selectivity for insulin reeeptor.
In another aspect the present invention prevides an antigen binding protein that demonstrates speoies selectivity. In one embodiment the airigen binding protein binds to one or more rnammalian IGF-IR for example, to human IGF-IR and one or more of mouse. rat guinea pig, hamster. gerbil, cat. rabbit dog. goat sheep, cow, horse. cameL and non-hnman prirnate IGF-IR. In another embodiment the antigen binding protein binds to one or more prirnate IGF-IR, for example. to human IGF-1R and one or more of cynomologous. rnarrnoset rhesus, and chimpanzee IGF-IR. In another embodiment the antigen binding protein binds specincally to human, cynomologous. marmoset rhesus, or chimpanzee IGF-1R. In another embodiment the antigen binding protein does not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, cat rabbit dog, goat sheep, cow, horse, camel, and non-human primate IGF-IR. In another embodiment the antigen binding protein does not bind to a New World monkey species such as a rnarrnoset. In another embodiment the antigen binding protein does not exhibit specific binding to anv naturally orairring protein other than IGF-1R In another embeniirnent, the antigen binding protein does not exhibit specific binding to any naturally oecurring protein othex than mammalian IGF-1R. In another

embodiment, the antigen binding protein does not exhibit specific binding to any naturally occurnng protein other thg-n primäre IGF- 1R. In another embodiment the antigen binding protein does not e>±ibit specific binding to any naturally occxtrring protein othcr than human IGF-IR. In another embodiment the Antigen binding protein speciücaily binds to mouse. rat cynomolgus monkey, and human IGF-IR. In another embodiment. the antigen binding protein specincally binds to mouse. rat, cynomolgus monkey, and 'i » -^ IGF-1R with a srmiLax binding af&nity. In another embodiment the antigen binding protein blocks bbdmg of human IGF-1 and IGF-2 with mouse, rat, cynomolgus monkey. and hnman IGF-IR. In another embodnnent the antigen binding protein blocks binding of human IGF-1 and IGF-2 with mouse. rat cynomolgus monkey. and human IGF-IR with srmilar K,;. In another emfoodiincnt the antigen binding protein blocks binding of human IGF-1 and IGF-2 with monse, rat. cynomolgus monkey. and human IGF-1R with a K; of between about 0.57 and about 0.61 n.M.
One rnay östermme the selectivity of an antigen binding protein rot an IGF-1 R using methods we£ known in the an and following the teachings of me specirlcatioTL For exampie. ose rnay oete^ntine :ne selectivity using Western blot FACS. ELISA or RIA.
In another aspect the present invention provides an IGF-1R binding antigen binding protem (for exampie. an anti-IGF-lR antibody), that has one or more of the following characteristics: binds 10 hoch human and murine IGF-IR, inhibits the binding of both IGF-1 and IGF-2 to human IGF-IR, rnhibits the binding of both IGF-1 and IGF-2 to murine IGF-IR, preferentially rnhibits the high affinity binding of IGF-1 and/or of IGF-2 to IGF-IR. binds to the L2 domain of IGF-IR, causes relatively little down-regulation of cell-surface expressed IGF-1 R after 17 bours of exposure (as compared to MAB391 (R&D Systems. Minneapolis. MK); e.g.. amount of IGF-IR is reduced by less than 20%). causes a level of down-regulation of cell-surface expressed IGF-IR on Colo-205 or MiaPaCa-2 xenografc tumor cells in mice as MAB391 after four weeks of once weekly doses of 200 micrograms.
Antigen-bmding fragments of antigen binding proteins of the invention may be prodnced by couventional Techniques. Examples of such fragments incfude, but are not limited to, F2b and F(ab*)_
fragments. Antibody fragments and derivatives procuced by genetic engineering techniques also are contemp lated.
Additional embodiments include chimeric antibodies, e.g., humanized versions of non-buman (e.g.. rrjurinej monoclonal antibodies. Such humanized antibodies may be prepared by known techmques. and offer the advantage of reduced immun ogeniciry vvhen the antibodies are admindstered to humans. In one embodiment a humanized monoclonal antibody comprises the variable domain of a murine antibody (or all or part of the antigen binding site thereof) and a constant domain derived fjom a human antibody. Afternatively, a humanized antibody fragment may comprise the antigen binding site of a rnurine monoclonal antibody and a variable domain fragment (tedring the antigen-binding site) derived from a human antibody. Procedures for the procucrion of chimeric and farther engineered monoclonal antibodies include those described in Riechmann et al. 1988, Nature 332:323. Liu et al, 1987, Proc. Nat Acad. Sei. USA 84:3439. Lamck et al, 1989, Bio/Technology 7:934; and Winter et al., 1993, 7TPS 14:139. In one embodiment the chimeric antibody is a CDR grafted antibody. Techniques for humanizieg antibodies are discussed in, e.g., U.S. Pat App. No. 10/194,975 (pubhshed February 27: 2Ö03), U.S. Pat. No.s 5,S69:619;

5,225.539, 5.821,337, 5,859,205, Fadian et al, 1995, FASEB J. 9:133-39, and Taniura ei al, 2000,1 Immunol. 164:1432-41.
Procedura havc been developed for generating human or partially human antibodies in non-mrman animals. For example, mice in which cme or more endogenous rmnöimoglobuhx genes have been inactivated by various means have been preparedL Human immunoglobulin genes have been introdnced ruto the raice to replace the inactivated mouse genes. Antibodies produced in the arirmal incorporate human imnnmoglobuhn Polypeptide cbains encoded by the human genetic Tn?trri?1 introdnced into the gTmral In one embodimeiiL a non-hnman aninial such as a transgenic mouse, is inimimi/sd wim an IGF-1R Polypeptide, such tfaat antibodies directed agahist the IGF-1R Polypeptide are generated in the animaL One example of a suitable mmnmogen is & sohible hirnren IGF-1R. such as a Polypeptide comprising me extracelhüar domain of the protem of Figure 10. or ofher mmnmogerdc fragment of the protein of Figure 10. Examples of techm'qnes for proänction and use of transgenic arrirnals for the procacnon of bnman or partially human antibodies are descnhed in U.S. Patents 5,814,318, 5,569.825, and 5.545.806. Davis ez al, 2003, Producüon of human antibodies from transgenic mice in Lo, ed. .antibody Erigineering: Metbods and Protocols, Humana Press, NJ:191-2Q0, Kellermann ei al, 2002, Curr Opin Biotechnol. 13:593-97, Rüssel et al, 2000,. Infect Immun, 68:1820-26, Gallo er al, 2000: Eur J Immun. 30:534-40, Davis ex al, 1999, Cancer Metastasis Rev. 18:421-25, Green, 1999, J Immunol Methods. 231:11-23, Jakobovits, 1998, Advanced Drug Delivery Reviews 31:33-42, Green et al, 1998, J Exp Med. 188:483-95, Jakobovits A, 1998, Exp. Opin. luvest Drugs. 7:607-14, Tsuda et al, 1997, Genomics. 42:413-21, Mendez et al, 1997, Nat Genet 15:146-56, Jakobovits, 1994, Curr Biol. 4:761-63, Arbones ei al, 1994, Immunity. 1:247-60, Green et al,
1994, Nat Genet. 7:13-21, Jakobovits et al, 1993, Nature. 362:255-58, Jakobovits et al, 1993, Proc Natl
Acad Sei USA. 90:2551-55. Chen, J., M. Trounstine, F. \V. Alt, F. Young, C. Kurafcara, J. Lormg, D.
Huszar. "Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deietion of the
JH locus." International Immunology 5 (1993): 647-656, Choi ei al, :993, Nature Genetics 4: 117-23.
Fisbwild ei al, 1996, Nature Biotechnology 14: 845-51, Harding ei al, 1995. Annais of the New York
Academy of Sciences, Lonberg ei al. 1994. Nature 368: 856-59, Lonberg. 1994, Transgenic Approaches io
Human Monoclonal Annbodies in Handbook of Experimental Phannacclogy 113: 49-101. Lonberg et al,
1995. Internal Review- of Immunology 13: 65-93, Neuberger, 1996. Nature Biotechnology 14: £26. Taylor
ei al, 1992. Nucleic Acids Research 20: 6287-95. Tavlor et al.. 3994. International Immunolosv 6: 579-91.
Tomizuka et al, 1997, Nature Genetics 16: 133-43, Tomizuka et al. 2000, Proceedmgs of me National
Academy of Sciences USA 97: 722-27, Tuaillon ei al, 1993, Proceedinp of the National Academy of
Sciences USA 90; 3720-24, and Tuaillon et al, 1994, Journal of Immunology 152: 2912-20.
In anomer aspect the present invention provides monoclonal aritibodies mat bind to IGF-1R. Monoclonal antibodies may be produced using any technique known in the art e.g., by irnmortalizing spieen cells harvestsd from the transgenic anirnal after completion of the immtmizarion schedule. The spieen cells can be irrrnortalized using any technique known in the art eg., by fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in hybridoma-producing fusion proceduies preferably are non-antrbody-producing, have high fusion efSciency. and enzyme deficiencies that render them incapabie of grovy-ing in certain selective media which support the growm of only the desrred fused cells (hybridemas). Examples of suitable cell lines for use inmouse fusions include Sp-20, P3-X63/Ag8, P3-

X63-AgS.653s NSl/l.Ag4 L Sp210-Agl4, F0: NSO/U MPC-II.. MPC11-X45-GTG 3.7 and S394/5XX0 Bui; examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IRSS3F and 4B210. Öfter cell lines useful for cell fusions are U-266, GM1500-GRG2, LICR-LOK-HMy2 and UC729-6.
In one embodiment a hybridoma cell line is produced by innrniniziEg an p^imal (e.g.. a transgenic anirr.al having human innmmoglobuliD sequences) wirb an IGF-1R inrmuaogcii; harvesnng spieen cells from the immunized animal; fusing the harvested spieen cells to a myeloma cell line, therefcy generating hybridoina cells: establishing hybridoiua cell lines from the bybridoma cells. and idennfying a bybridoma cell line that prodnces an antibody that binds an IGF-1R Polypeptide. Such bybridoma cell hnes. and. anti-IGF-1R monoclonal antfDodies produced by mem, are encompassed by the present invention
Monoclonal antibodies secreted by a hybridoma cell line can be pnrified nsmg any technique known in the an. Hybridomas or mAbs may be mrther screened to idenrify mAbs witnparticular propernes, such as the abiliiy to block an 1GF-1 and'or IGF-2 indnced acüviiy. Exarnples of such screens are provided in the examples below.
Molecular evohition of the complementarity determining regions (CDRs) in the center of the antibody binding site also has been used to isolate antibodies wirb increased aiSnity, for example. antibodies havirig increased affiniry for c-erbB-2, as described by Schier et cl„ 1996. J. Mol. Biol. 263:551. Accordingly. such techniques are useful in preparing antibodies to IGF-1R.
Antigen binding proteins directed against an TGF-1R can be used, for example, in assays to detect the presence ofIGF-lR Polypeptides, either in vitro or in vrvo. The antigen binding proteins also may be employed in purirying IGF-1R proteins by immunoaffinity chromatography. Those antigen binding proteins that additionally can block binding of IGF-1 and/or IGF-2 to IGF-1R may be used to inhibit a biological acrivity that results from such binding. Blocking antigen binding proteins can be used in the metheds of the present invention. Such antigen binding proteins tbat fanetion as IGF-1 and/or IGF-2 antagonists may be employed in treating any IGF-1 and'or IGF-2-induced condition, including hur not limited to cancer. In one embodiment, a human anti- 1GF-1R monoclonal antibody generated by procedures involving immtmization of transgenic rrice is employed in treating such conditions.
Antigen binding proteins may be employed in an in vitro procedura, or administered in vivo to inhibit an IGF-1 and'or IGF-2-induceo biological activiry. Disorders eaused or exaeerbated (directly or indirectly) by the interaenon of IGF-1 and/or-IGF-2 vririi cell sumee IGF-1 R. examples of which are provided above, thus may be treated. In one embodiment, the present invention provides a therapeutic method comprising in vivo adiroinistration of an IGF-1 and'or IGF-2 blocking antigen binding protein to a mammal in need thereof in an amount effective for reducing an IGF-1 and'or IGF-2-induced biological activiry.
Antigen binding proteins of the invention include partiaily human and fuüy human monoclonal antibodies that inhibit a biological activity of IGF-1 and also inhibit a biological activity of IGF-2. One embodiment is directed to a human monoclonal antibody that at least partiaily blocks binding of IGF-1 and of IGF-2 to a cell that expresses human IGF-1R. In one embodiment, the antibodies are generated bv immunizing a transgenic mouse with an IGF-1R immunogen In another embodiment, the immunogen is a human IGF-IR Polypeptide (e.g.. a soluble fragment comprising all or part of the IGF-1R extracellular

omain). Hybridoma cell lines derived from such irnmunized mke, wherein the hybridoma secretes a lonoclonal antibody that binds IGF-1R, also are provided herein
Although human, partially human, or humanized antibodies will be suitable for rnany applications, srccularly those involving administration of the antibody to a human subject other types of atirigen inding proteins will be suitable for certain applications. The non-human antibodies of the invention can c, for example. derived from any anul)ody-producing animal, such as mouse, rat rabbit. goat, donkey. or on-taman psimate (such as monkey (e.g., cynomologous or ihesus monkey) or ape (e.g.. chnnpanzee)). 4oQ-hEmaB smbodies of me invention can be used, for example, in in vitro and cell-cuhnrc based ppöcatiaas. or any oÜier appbcahon wnere an immune response to the antibody of me invention does not iccar, is rnsignrocant can be prevenied, is not a concern, or is desired. In one emboditnent a non-human saibody of "die invention is adininistered to a non-human subject. In another embodimenL the non-human mtibody does not elicit an nnmune response in the non-human subject. In anofher ernbodiment, the non-snnan antibody is from the sarne species as me non-human subject. e.g.. a mouse antibody of the invention s adinmisTcred to a mouse. An antibody from a particular species can be made by. for example. tmrnnp-izing an anirnal ofthat species with the desired immunogen (e.g., a soluble IGF-iR Polypeptide) or using an arüfrcial System for generating antibodies ofthat species (ß.g., a bacterial or phage display-based system for generating antibodies of a particular species). or by Converting an antibody from one species into an antibody from another species by replacing, e.g.s the constant region of the antibody -with a constant regioE from me other species, or by replacing one or more amino acid residues of the antibody so that it more closely resembles the sequence of an antibody from the other species. In one ernbodiment, the antibody is a chimeric antibody comprising amino acid sequences derived from antibodies from rwo or more cifferent species.
Antigen binding proteins may be prepared by any of a number of conventional techniques. For example. they may be purüied from cells that naturally express them (e.g., an antibody can be puxified from a hybridcma that produces it). or produced in recombinant expression Systems, using any technique known in the art See. for example. Monoclonal Antibodies, Hybridomas: A Are~.v Dimension in Biological Analyses. Kennet ex ah (eds.). Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.). Cold Spring Harbor Laboratory Press. Geld Spring Harbor, NY, (1988).
Any expression system known in the art can be ^astd to make the recombinant Polypeptides of the invention. In general, host cells are transformed with a recombinant expression vector that comprises DNA encoding a desired Polypeptide. Among the host cells that may be employed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms. for example E. coli or bacilli. Higher eukaryotic cells include insect cells and established cell lines of marrrrnahan origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CR! 1651) (Gluzman et aL 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan ei ah, 1991. EMBO J. 10:2821. Appropriate cloning and expression vectors for use with bacterial. fungal, yeast, and mammalian ceilrilar hosts are described by Pouwels et ah {Cloning Vectors: A Laboratory Manual, Elsevier. New York, 1985).

The rransformed cclis can be cultured under conditions that promote expression of tbe Polypeptide. and the pol>peptide recovered by Conventions! protein puritication procedures. One such puriftcation procedura indudcs the uss of afrmity chromatography, e,g., over a matrix having all or a portion (e.g., the extracellular domain) of IGF-IR banne thereto. Polypeptides contemplated for use herein include substaiirialiy homogencous recombinant -nsT^mg'igr. anti- IGF-IR antibody Polypeptides substantiaUy free of contarninating endogenocs materials.
Andgen biading i»**»iiw> may be preparcä, sad screened fox desired properties. by any of a number of known techrnques. Cerzs&L of tbe tscänäqnes involve isoiating a nncleic- acid encoding a Polypeptide chain (or portion thereoi) of an autigea binöing proem of imcresi (e.g.. an anti-IGF-lR antibody), and -nanipmlatTnp nie nuclek acki throogh recozobiisani DNA Technology. Tbe nucleic acid may be fused to artother nncleic acid of Lutaest or altered (e.g_ by rrasageBesis or other conventional techniquss) to adö. delete. or suhstiLiir one or more *u*nr» acid residnes, for exzmple.
In one aspect. the t^esent iüventiozi provides antigen-binding fragments of an anti-IGF-lR antibody of the invention. Such fragrrjerirs can consst enrirely of antibody-derived sequences or can comprise additiona! seqoences. Exaxoples of antigen-bmding fragments include Fab. F(ab')2, Single chain antibodies, diabodies, triabodies, retrabodies. and domain antibodies. Other examples are provided in Lunde et al, 2Ö02: Biocbeca. Soc. Trans. 30:500-06.
Single chain antibodies may be formed by ünking heavy and light chain variable domain (Fv region) fragments via an ammo acid bridge (shon Peptide linker), resulting in a Single Polypeptide chain. Such sing]e-chain Fvs (scFvs) have beer) prepared by fusing DNA encoding a peptide linker between DNAs encoding the rwo variable domain Polypeptides (VL and Vj.;). The resulting Polypeptides can fold back on themselves to form antigen-bmding monomers, or they can form rnultimers (e.g., dimers, trimers. or tetramers). depending cn tbe lengrh of a flexible linker between the rwo variable domains (Kortt et ah, 1997.. Prot Eng. 10:423: Korn et ed.. 2001, BiomoL Eng. 18:95-108). By combining different VL and VH-comprising Polypeptides, coe can form nroltimeric scFvs that bind to different epitopes (Kriangkum ei ah, 2001, Biomol. Eng. 18:31-40). Techniques devclopec for tbeproduetion of Single chain antibodies include those described in U.S. Patent No. 4;946,778; BirdL 19SS: Science 242:423: Huston ei a!.s 1988, Proc. Nati Acad. Sei. USA 85:5879: Ward et al. 1989, Natsre 334:544, de Graaf er a/., 2G02r Metbods Mol Biol. 178:379-87. Single chain antibodies derived from antibodies provided herein include. but are not limited to. scFvs comprising the variable domain combizations L1H1, L2H2; L3H3. L4K4; L5H5, L6K6, L7H7, L5HSS L9K9. L10R10r LI 1H11. L12H12: L13H13. L14H14, L15H15: L16H16, L17H17, L1SH1S, L19H19, L20H20, L2IH21. L22H22: L23H23: L24H24; L25H25, L26H26: L27K27, L28H28, L29H29: L30H30, L31H3L L32H31 L33H33: L34H34; L35H35: L26H36, L37H37.. L3SH38: L39H39, L40H40, L41H41, L42K42, L43H43, L44H44; L45H45, L46H46", L47H47, L4-8H48, L49H49, L50H50, L51H5L and L52H52) are encompassea by the present invention.
Antigen binding proteins (e.g.. antibodies, antibody fragments, and antibody derivatives) of the invention can comprise any constant region kno^r: in the art The light chain constact region can be, for example. a kappa- or lambda-^.pe light chain constant region. e.g., a humankappa- or lambda-type light chain constant region. Tbe heavy chain constant region can be. for example. an alpha-, delta-, epsilon-, gainrria-. or iru-type heavy chain constant regions. e.g.. a human alpha-, delta-, epsilon-, gamma-, or niu-


Techniques are Vnown for deriving an antibody of £ differeirt subclass or isotype from an antibody of interest, i.e.. subclass swrtcrring. Tbns, IgG antibodies may be dsrrved from an IgM antibody, for example, and vice versa. Such techniques aÜow the preparatice of ncw antibodies ihat possess the axiiigsn-binding properties of a given antibody (tiie paxen: antibody), b«r also exHbit bioloocal properties associaied with an antibody isotype or subclass diferent fixnn tfaet of tbe parent anbboäv. Recombioani DNA techniques may be employed, Qoned DNA encoämg pamcafex antibody poiypeytkfes may be employed in such procednres, e.g.. DNA encoding the Consta TF oomain of an anrExwy cf me desired isotype. See also Laufe et ed., 2002, Methods Mol. Bio! 178:303-16.
In ose embodnnent. an antigen biading protein of the inventian cesresises the IgGl heavy chain domain of Figure 13 or a fraginent of the IgGl heavy chain domain of Figure 13. Ir asoüier eznbo Accordingly. the antigen binding proteins of the preseut invention incrude tbose comprising. for example, the variable domain combinations L1H1, L2HZ L3H3: L4H4: L5K5, \£H6Z L7H7; LSHS.. L9H9, LI OHIO, LI 1H11, L12H12S LI3H13: L14H14; L15H15, L16HI6. L17H17.. L18H13: L19H19, L20H20, L21H2L L22H22: L23H23B L24H24, L25H25: L26H26, L27R27. L28H28: L29H29, L30H30, L31H3L L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39: L40H40: L41H41, L42H42, L43H43: L44H44, L45H45, L46H46, L47H47; L48H4S, L49H49, L5ÖH5Ö.. L51H5L and L52H52, having a desired isotype (for example. IgA. IgGl. IgG2, IgG3, IgG-1, IgM, IgE, and IgD) as well as Fab or F(abf)z fragments thereof. Moreover, if an IgG4 is desired. it may also be desired to introdnee a point mutation (CPSCP -> CPPCP) in the hinge region as described in B'.oom et aL, 1997, Protein Science 6:407, incorporated by reference herein) to alleviate a tendency :o form intra-H chain disnlriae bonds that can lead to heterogeneity in rhe IgG4 antibodies.
Moreover. techniques for deriving antigen binding proieras having different properties (i.e.. varying afoniriei for rhe antigen to which they bind) art also known. One such technique. referred to as chain shufQing. invojves displaying inxrnunoglobulin variable domain gene rspertoires on me surface of ülamentous bacteriophage. orten referred to as phage display. Cnain shufQing has been 'astä to prepare high affinity antfbodies to the hapten 2-pheny]oxazol-5-one. as described by Marks et a'L, 1992, BioTecknology. 10:779.
In particular embodiments, antigen binding proteins of the present invention have a binding affmity (KJ for IGF-1R of at least 106, measured as described in the Examples. In other embodiments. the antigen binding proteins exhibit a Ks of at least 10', at least 108, at least IQ5, or at least 1010.
In anorher embodiment the present invention pro vi des an antigen binding prötein that has a low dissociation rate from IGF-1R. In one ernbodiment the anngen binding protein has a K^of 1x10^ s_i or lower. In another embodimenu me Ko^- is 5x10": s"' er iower. In another embodiment the Ko«- is substantially the same as an antibody having a combination of light chain and heavy chain variable domain

sequences selected from the group of combinations consistmg of L1H1. L2H2. L3H3. L4H4, L5H5. L6H6, L7H7, L8H8, L9H9.. L10HI0, L11H11, L12H12, L13H13, LI4H14, L15H!5: L16H16, L17H17, L18H1S, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L2SH2S, L29H29, L30H30, L31H31, L32H32: L33H33, L34H34, L35H35, L36H36, L37H37: L3SH3S.. L39R39: L40H40, L41H4L L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H4S, L49H49, L50H50-, L51H51, and L52H52. In another embodiment, the antigen bincing protein binds to IGF-1R wirb. substHrjüally the same K^g- as an antibody that conroises one or more CDRs mno an antibody haviag a corcbixiation oflight chain and heavy cham variable domain sequences selected from the group of corninnations consisnng of L1H1, L2H2: L3E3, L4H4, L5H5, L6H6T L7H7: L8H8: L9H9r L10H1CL LI 1H1L L12H12, L13HI3, L14H14, L15H15, L16H16, L17H17, L18H18; L19H19, L2ÖH2Ö, L21H2L L22H22, L23H23r L24H24, L25H25, L26H25. L27H27, L28H28, L29H29: L30H30.. L3IH31, L32H3Z L33H33, L34H34:L35E35, L36H36, L37H37: L38H38.. L39H39.. L40H40: L4iH4!; L42H4Z L43H43: L44B44, L45H45: L46H46, L47H47, L48H48: L49H49; L50H50, L51H51. and L52H52. In another embodiment the antigen bincing protein binds tö IGF-1R wirb substantially the same K^ as an antibody thai comprises one of me arnioo acid sequences ilhistrated in Figures 2 through 9. In another embodiinent, the aniigen binding protein binds to IGF-1R with substantially the same Ko^ as an antibody that comprises one or more CDRs from an antibody that comprises one of the amino acid sequences illustrated in Figures 2 through 9.
In another aspect the present invention provides an antigen binding protein that binds to the L2 domain of human IGF-1R. Antigen binding proteins that bind to the L2 domain can be made using any technique known in the art For example. such antigen binding proteins can be isolated using the fuU-length IGF-1R Polypeptide (e.g.. in a membrane-bound preparation), a soluble extracellular domain fragment of IGF-1R (an example of v/hich is provided in Example 1), or a smaller fragment of the IGF-1R extracellular domain comprising or consisting of the L2 domain (examples of which are provided in Example 10). Antigen binding proteins so isolated can be screened to deterrnine their binding speciücity using any method known rn the art (an example of which is provided in Example 10).
In another aspect the present invention provides an antigen binding protein that binds to human IGF-1R expressed on the surface of a cell and, when so bound, inhibits IGF-1R signaiing activity in the cell without causing a signiücant recuction in the amount of TGF-1R on the surface of the cell. Any method for determining or estimaüng the amount of IGF-1R on the surface and or in the inferior of the cell can be iiseä. In one embodiment the present invention provides an antigen binding protein that binds to me L2 domain of a human 1GF-1R expressed on the surface of a cell and. when so bound. inhibits IGF-1R signaling activity in the cell without significantly increasing the rate of internalizaticn of the 1GF-1R from the surface of the cell. In orher embodiments. binding of the antigen binding protein to the IGF- IR-expressing cell causes less man ahout 75%, 50%: 40%, 30%, 20%, 15%, 10%, 5%. 1%, or 0.1% of the cell-surface 1GF-1R to be iniernaiized. In another aspect binding of the antigen binding protein to the IGF-lR-expressing cell causes a gradual reduction in the amount of IGF-1R on the cell surface such that within a few hours of contacüng the cell wirb the antigen binding protein, lirtle or no decrease in cdl surface IGF-1R is detected, but aller several days or weeks of exposure of the cell to the antigen binding protein, a marked decrease in cell surface IGF-iR is detected.

In another aspect the present invention provides an antigen bindmg protein having a half-life of at least one day in vitro or in vfvo (e.g-, when adrninistered to a human subiect). In one exnbodixnent the antigen binding protein has a half-life cf at leasi ihres days. in another ernbodimsnt, the antigen binding protein has a half-life of four cays or longer. In another embodiment the antigen binding protein has a half-life of eight days or longer. In another ernbodirnent the antigen binding protein is derivatized or modined such that it has a longer half-life as compared to the underivatized or unmodiiied antigen binding protein. In another embodrroerjl the antigen binding protein confratns one or more poirxr mntations to increase serum half liie. such as described in "WO 00/09560, published Feb_24. 2000. mcorporated by reference.
Tne present invention farther provides mulü-specific antigen binding proteins, for example. bispecinc antigen binding protein. e.g., Antigen binding protein mar bind to rwo dirJereni epitopes of IGF-1R. or to an epitope of IGF-1R and an epitope of another molecule. via rwo dirrerem antigen bind ins sites or regions. Moreover, bispecinc antigen binding protein as disclosed herein can comprise an IGF-1R binding site from one of the herein-described antibodies and a second IGF-1R binding region from another of the herein-described antibodies, mcluding those described herein by reference to other pubücations. Altematively. a bispecinc antigen binding protein rnay comprise an antigen binding site from one of the herein described antibodies and a second antigen binding site from another IGF-1R antibody that is known in the art or from an antibody that is prepared by known methods or the methods described herein,
Numerous methods of preparing bispecifk antibodies are known in the art and discussed in US Patent Application 09/839.632, filed April 20. 2001 (incorporated by reference herein). Such methods include the use of hybrid-hybridomas as described by Milsteia et al., 1983, Nature 305:537, and others (U.S. Patent 4,474:893. U.S. Patent 6.106.833). and chemical coupling of antibody fragments (Brennan et aL1985, Science 229:81; Glennie et a/.,19S7: J. IrnmunoL 139:2367: U.S. Patent 6,010,902). Moreover, bispeci&c antibodies can be proöuced via recombinant means. for example by nsing leucrne zipper moieties (i.e.. from the Fos and Jun proteins, which preferentially form hetexodimers; Kostelny et al., 1992; J. Irnmnol. 14S:I547) or other lock and key interactive domain structures as described in U.S. Patent 5.582,996. Acditional useful techniques include those described in Kom ex cL 1997. supra; U.S. Patent 5,959,083; and U.S. Patent 55S07:706.
In another aspect, the antigen binding protein of the present invention comprises a derivative of an antibody. The derivatized antibody can compnse any molecule or suhsrance that imparts a desired property to the antibody, such as increased half-life in z. particular use. The derivatized antibody can comprise. for example, a detectable (or labeling) moiety (e.g., a radioactive. colorimeiric. antigenic or enzymatic molecule, a detecable bead (such as a magnetic or elecirodense (e.g.. gold) bead), or a molecule that binds to another molecule (e.g.. biotin or streptavidin)). a therapeutic or oiagnostic xnciety (e.g.. a radioactive, cytotoxic. or pharmaceutically active moietyj. or a molecule that increases the suitabihty of the antibody for a particular use (e.g., administration to a subject. such as a human subj&ct or other in vivo or in vitro uses). Hxarrrples of molecule s that can be used to derivatize an antibody include aFDumin (e.g-.. human serum alburnin) and Polyethylene glycol (PEG). Albumin-Iiriked and PEGylaied derivatives of antibodies can be prepared using techniques well known in the an. In one ernbodirnent the antibody is conjugated or othenvise linked Xo nansthyretin (TTR) or a TTR variant. Tne TTR or TTR variant can be chemically

modified with. for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyurxolidone), Polyethylene glycols, propropylene glycol homopoiymers, polypropylene oxids/ethylene oxide co-polymers, polyoxyefhylated polyols and polyvinyl alcohols. US Fat- App. No. 20030195154.
In ancther aspect, the preseni invention provides methods of Screening for a molecule that binds to IGF-1R using the antigen hinding proteins of the present invention Any svhtable Screening technique can be used. hx one erhbodirnent, an IGF-1R molecule, or a fxagment thereof to which an antigen binding protein of the present invention binds, is contacted with the antigen binding protein of the invention and with aoother molecule. wherein the othex moiecule binds to IGF-1R if it reduces the binding of the antigen binding protein to IGF-1R. Binding of the antigen binding protein can be detected using any surtable rnethoG- e.g., an ELISA. Detection of binding of the antigen binding protein to IGF-1R can be sirnplified by detectabiy labeling the antigen binding protein- as discussed above. In another embodimenl the IGF-*R4-vmrrng molecule is funher analyzed to detsxxnine wherher it rnhibits IGF-1R,IGF-1, and/ox IGF-2-mediated siginling.
Nucleic acids
In one aspect the present invention provides isolated nucleic acid molecules. The nucleic acids comprise, for example, polynucleotides that encode all or part of an antigen binding protein . for example, one or both charns of an antibody of the invention, er a fxagment, derivative- mutein, or variant thereof, polynucleotides suffkient for use as hybridizarion prohes, PCR primers or sequencing primers for identifying. analyzing, mutating or ampiifying a polynucleoride encoding a polypeptide. anti-sense nucleic acids for inhibiting expression of a polynucleoride, and complementary sequences of the foregoing. The nucleic acids canbe any length. They can be. for example. 5. 10. 15, 20,25, 30,35. 40.45. 50. 75, 100. 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleondes in length, and'or can comprise one or more additional sequences, for example, regulatory sequences. and'or be pari of a '{arger nucleic acid, for example. a vector. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and'or DNA nucleotides, and artificial variants thereof (e.g.. peptide nucleic acids).
Nucleic acids encoding antibody Polypeptides (e.g,, beavy or light chain, variable dornain only. or füll lensrJi} mav be isolated from B-cells of mice that have been irnrnunized with IGF-1R. Tue nucleic acid may be isolated. by conventional procedures such as Polymerase chain reaction (PCR).
Figure 1 provides nucleic acid sequences encoding the variable regions of the heavy and light chain variable regions shown in Figures 2 anc 3. The sküled artisan will appreciate that due to the degeneraey of the genetic code, each of the Polypeptide sequences in Figures 2 through 9 also is encoded bv a large number of other nucleic acid sequences. The present invention provides each degenerate nucleoride sequence encoding each antigen binding protein of the invention.
The invention furthex provides nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids cornprising a nucleoride sequence of Figure 1) under particülai hybridizarion condirions. Methods for hybridizina nucleic acids are well-known in the art. See, e.g-.. Current Protocols in Molecular Biolosv John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. As defxned herein, a moderately stringent hybridizarion condition uses a prewashing Solution containing 5X sodiurn chloxide/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA

(pH 8.0), hybridization buffer of about 50% formamidc. 6X SSC. and a hybridization temperature of 55° C (or other srmiiar hybridization Solutions, such as one containing about 50% forma mi de. v,irh a nybridization temperature of 42r Q. and Nvashing condiuons of 60a C. in 0.5X SSC 0.1% SDS. A stringent hybridization eondksoc hybridizes m SX SSC at 45" C, foüowed by one or more washes in Ö.IX SSC. 0.2% SDS at 6SC C. Furcbsrmore, one of skül b the an car. rnanipulate the hybridization and/or washing conditicns to incrcasc or decrease the stringency of hybridization such that nucleic acids conrprising nucleotide sequeoccs thai are ai käst 65, 70: 75, 80, 85, 90, 95, 98 or 99% identical to each other typically remain bybhdized to each ofcer. The basc parsmeteis afectmg the choice of hybridization conditioiis and gihdance for devisns snnable ccsxhricsjs are set form by. for example, Sambrook. Fritsch, and Maniatis (1989, Molecular Ckjoing: A Labcffaiory MsnnaL Cold Spring Harbox Laboratoiy Press. Colc Spring Haibor. N.Y., chapsrs 9 and 11; and Cuueui Protocols in Molecular Biology, 1995. Ausubel et al- eds.. Jona Yvüey & Sans^ bx:... secäocs 2.10 and 63-6.4), and can be readUy deterrnined by rhose having oröinary skill m the anbased OSL for exampk. the lengih and/or base composition of the DKA.
Changes can be mtrococed by irmaticn into a nucleic acid. rhereby leading tc changes in the amino acid sequence of a Polypeptide (e-g-, an antigen binding protein) that it encodes. Mutations can be introduced esing axry t^hrrknie fcoowi: in the art In one embodiment, one or more parricular amino acid residues are changed using. for exarnple. 2 sxte-directjed rrmtagenesis protocoL In another ernbodirnent one or more randomly selected residues is cbsnged using. for example, a random mutagenesis protocoL However it is made. a mutant polypepride can be expressed and screened for a desired property (e.g.. binding to 1GF-IR or blockmg the binding of IGF-1 and'or IGF-2 to IGF-1R).
Mutations can be introduced rnro a nucleic acid without significantly altering the biological activitv of a Polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid suhsriturioiis at non-essenri2l amino acid residues. In one embodiment, a nucleotide sequence provided in Figuxe I, or a desired fragment, vaxiant, er derivative thereof, is mutated such that it encodes an arnino acid secuence comprising one or mors deletions or substitations of arnino acid residues that are shown in Fi gares 2 rhrough 9 to be residnes where t\vo or more sequences differ. In another embodiment. the mutagenesis mserts an arnino acid adjacent to one or more amino acid residues shown in Figures 2 through 9 to be residues where two or more sequences differ. Altcmatively, one or more mutations can be introduced mtc a nucleic acid that selectively change the biological aenvity (e.g., binding of IGF-1 R. inhibiting IGF-1 and/or IGF-2. etc.) ofa Polypeptide that it encodes. For example, the mutation can quantitative ly or quaiitarively change the biological aenvity. Examples of quantitative changes include increasing, reducing or elinnnating the acüvity. Examples of qualitative changes include changing the antigen speciücity of an antigen binding protein.
In another aspect the present ievention pro\ides nucleic acid molecules that are suitable for use as primsrs or hybridization probes for the detection of nucleic acid sequences of the invention. A nucleic acid molecule of the rnvennon can comprise orüy a portion of a nucleic acid sequence encodmg a full-lerigth Polypeptide of the invention. for example, a fragment that can be used as a probe or primer or a fragment eacoding an aenve portion (e.g., an IGF-1R binding portion) ofa polypepride of the invention.
Probes based on the sequence ofa nucleic acid of the invention can be used to detect the nucleic acid or sirnilsj nucleic acids, for example. transcripts encoding a polypepride of the invention. The probe

can cornprise a labe! groiro, e.g., a radioisotope, a fruorescenl ccapounti, an enzyme. or an enzyme co factor. Such probes canbe used to identify a cell that expresses the Polypeptide.
In another aspect the present invention provides vectors cornprismg a nucleic aeid enccKJing a Polypeptide of The invention or £ portion thereof. Examples o: V-CJDTS include. but are not limited to, Plasmids, viral vectors, nou-episomal manamalian vectors and rxpression vectors, for example, recombinant expression vectors.
The recombinant expression vectors of the invention OB. coaoprise a nuciek aeid of the invention in a form suitable fbr expression of the nucleic arid in a host cc£_ The reccEabsnam esxiesscn vectors include one OT more regnlatory sequences, selected on ihe hasis of the bosx cslk to be Gseti ihr expression-which is operably linked to the nucleic aeid sequence to be expressed- Hesoiziasy sequences inciuee those lhat direct constinitrve expression of a nucleotide sequence in roany types of best celb (&. g_, SV40 eany gene enhancer, Rons sarcoma virus prornoter and cytornegalcRTrus pranaoer). these frer direct expression of the nucleotide sequence only in certain host cells (e.g.. tissue-speeifie resnlaiory sequences, see Voss ei G!., 19S6. Trends Biochenx Sei. 11:287. Manisiis er c/.. 1937. Science 236:1237. rneorporaied by reference herein in theii entireries), and those that direct inducible expression of a rmcleotide sequence in response to parhcular treatment or condition (e.g., the metaHothionin prometex in m^i"inaVyn cells and the tet-responsive and/or streptonrycin Tesponsive promoter in both prokaryotic and eukaryode Systems (see id.). It will be appreciated by rhose skiHed in the art that the design of the expression vsetor can depend on such factors as the choiee of the host cell to be transformed, the level of expression of protein desired. etc. 'The expression vectors of the invention can be introcueed into host cells to thereby produce proteins or peptides. including fusion proteins or peptides. encoded by nucleic acics as described herein.
In another aspect, the present invention provides host cells into which a recombinant expression vector of the invention has been introcueed. A host cell can be any prokaryotic cell (for example, E. coli) or eukaryotic cell (for example. yeast, insect, or mammaliar cells (e.g., CHO cells)). Vector DNA can be introcueed into niokaryotic or eukaryotic cells via convenheaai transformation or transfection techniques. For stable transfection of mammalian cells, ii is known thaL depending upon the expression vector and transfection technique used, only a small rraction of cells may unegrate the foreign DNA into tbeir genome. In order to identüy and select these integrants. a gene that encodes z selectable rnarker (e.g.. for resistance to annbiotics) is generally introduced into the host cells a:ong wrth me gene of interest. Freferred seleciable markers include these which confer resistance to drugs, such as G413, hygromyein and methotrexate. Cells stably transfected wirb the imroduced nucleic aeid can be ideatined by drng selecrion (e.g., cells that have incorporated the selectable marker gene will survive, wnüe the other c-ells die), among other methods.
Indications
in one aspect the present invention provides methods of treatmg a subjeet The method can. for example, have a generally salübrious effect on the subjeet, e.g.. it can increase the subjeet' s expected longe\dty. Aliemarively. the method can. for example. treat prevent, eure, relie\"e? or ameliorate ("treafl a disease, disorder. condition. or illness ("a condition"). Among übe conditions tc be rreated in aecordance Aith the present invention are conditions characterized by inanpropriate expression or acrivitj,' of IGF-1. IGF-2. and-'or IGF-1R. In sorne such conditions. the expression or activity level :s too high, and the

reatraent comprises acmiiiistering an IGF-1R antagonis; as dcscribed herein. In other such conditions, the rxpression or acnvity level is too low, and the treatment comprises admunstenng an IGF-1R agonist as isscribed herein.
One example of a type of condition that can be treaied using the methods and compositiaiis of the present invention is a condition that involves cell growth, for example, a cancerous condition. Thns, in one ernbodiment, the presset inverrüon provides conx>ositions and methods for treatrog a cancerons condition. The cancerous condition can be any cancerous condition that can be treaied nsing the coinpositions comprised herem, for example. IGF-1R antagomzing anfigen binding proteins such as anti-IGF-lR antibodies, antibody fxagmems. or antibody derivatives. Examples of cancerous conditions metade, for example, Acute Lymphoblasüc Leukemia, Adrenocortical Carcinoma, AIDS-Related Cancers, AJDS-Related Lymphoma. Anal Cancer, Chüdhood Cerebellar Astrocytorna, Chüdhood Cerebral Astrocytorna, Basal Ccü C^icmoma. Extrahepafic Büe Duct Cancer, Bladder Cancer. Osteosarcoma/Maligna-nt Fibrous Histiocytoma Bone Cancer. Bram Tumors (e.g., Brain Stein Glioma. Cerebellar Astrocytoma, Cerebral Astrocyiorna/Maiignant Glioma, Ependymoma, Medulloblastoma, Supratentorial Primitive Neuroectodermal Tumors, Visual Pathway and Hypothalamic Glioma), Breast Cancer, Bronchial Adenomas/Carcinoids. Burkitf s Lymphoma, Carcinoid Tumor, Gastromtestinal Carcinoid Tumor. Carcinoma of Unkno'wn Primary, Primary Central Nervous System. Cerebellar Astrocytorna, Cerebral Astrocytoma/Malignant Glioma. Cervical Cancer, Chüdhood Cancers. Chronic Lyrnphocytic Leukemia, Chronic Myelogenous Leukemia. Chronic Myeloproliferative Disorders, Colon Cancer. Colorectal Cancer. Cutaneous T-Cell Lymphoma. Endometrial Cancer. Ependymoma, Esophageal 'Cancer. E «ing's Family of Tumors, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Intraocular Melanoms Eye Cancer. Retinoblastoma Eye Cancer, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Germ Cell Tumors (e.g., Extracranial. Extragonadal, and Ovarian), Gestational Trophoblastic Tumor, Glioma (e.g.. Adult, Chüdhood Brain Stern, Chüdhood Cerebral Astrocytorna, Childhood Visual Pathway and Hypothalamic), Hairy Cell Leukemia, Head and Neck Cancer, Hepaiocellular (Liver) Cancer. Hodgkm's LympbGma. H\popharyngeal Cancer, Hypothalamic and Visual Pathway Glioma, Intraocular Metenoma, Islet Cell Carcinoma (Endocrine Pancreas). Kaposfs Sarcoma, Kidney (Renal Cell) Cancer, Laryngeal Cancer, Leukemia (e.g., Acute Lympboblastic, Acute Myeloid, Chronic Lyrnphocytic. Cnronic Myelogenous. and Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer, Non-Small Cell Lung Cancer, Smail Cell Lung Cancer, Lymphoma (e.g., AIDS-Related, Burkitt's. Cutaneous T-Cell, Hodgkin's. Non-Hodgkin's, and Primary Central Nervous System), Waldenström'sMacroglobulinemia, Malignant Fibrous Histiocytoma ofBone/Osteosarcoma. Medulloblastoma, Melanoms. Intraocuiai (Eye) Melanoma, Merkel Cell Carcinoma. Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma'Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplasie Syndromes, Myelodysplasie/Myeloproliferative Diseases, Myelogenous Leukemia, Chronic Myeloid Leukemia, Multrple Myeloms, Chronic Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer. Nasopharyngeal Cancer. Neuroblastoma, Oral Cancer, Oropharyngeal Cancer. Ostecsarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Ovarian Epithelial Cancer. Ovaiian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor. Pancrearic Cancer, Islet Cell Pancreatic Cancer. Paranasal Sinus

anc Nasa] Caviry Cancer. Parathyroid Cancer. PenUe Cancer. Pheochrornocytoma, Pineoblastoma, Pituitary Tumor. Plasma Cell Neoplasm/Multiple Myelons. Pleuropulrnonary Blastorna, Primär}' Central Nervous System LyTirphoma, Prostate Cancer, Rectal Cancer. RJCUZI Cell (Kicney) Cancer. Renal Pelvis and Ureter Transitional Cell Cancer. RsTmoblasioma, Rnabdomycsarcoma. Salivzry Gland Cancer, Soft Tissue Sarcoma, Uterine Sarcoma, Sezary Syndrome, non-Melanoma Skin Cancer, Merkel Cell Skin Carcinoma. Smail Inrestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Cutaneous T-Cell Lymphoma, Testicular Cancer. Thymoma. Tbymic Carcinoma, Thyroid Cancer, Gestarionai Trophoblastic Tumor. Carcinoma of Unknown Priroary Site. Cancer of Unkaown Primary Site. Urethral Cancer, Endometrial Uterine Cancer, Üterine Sarcoma. Vaginal Cancer. Visual Parhway and Hy^pothalamic Glioms. Vulvar Cancer, "Waldcnström's Macroglobulinernia, and WümsT Tumor.
Four different groups have studied a total of 425 breast Cancers, rnostry ductal in origin, and 48 normal üssues or benigD speeimens by raa^oimmunoassay (TUA^ or imnmaoMstochenn^trv ("3HC7*) (Papa et al, 1993, Cancer Research 53: 3736-40, Happcrfieid et al., 1997, Journal of Patbology 183: 412-17; Eliis et al, 1998. Breas: Cancer Research Sc Treatment 52: 175-84, Lee et al., 1998. Breast Cancer Research Sc Treatment 47: 295-302, Schnarr et al.. 2000, International Journal of Cancer 89: 506-13). These studies suggest that elevated TGF-1R expression. on the Order of 5-10 fold, is associated with favorable prognosis and biomarkers (ER.+ PR+). suggesting that estrogen and IGF cooperate in the rriaintenance or progression of well difTerentdated tumor. Sirnilarly, estrogen has been shown to be essential for the growth and survival of the ER+ MCF-7 breast cancer cell Ihre, and in this context IGF-1R is ur> regulated by estrogen treatment (reviewed in Eliis et al, 1998. Breast Cancer Research Sc Treatment 52: 175-84). Thus, in one embodiment, the present invention provides a method of treating breast cancer in a subjeet in need of such treatment. cornprising adrninistering to the subjeet an effective amount of an IGF-1R antagonist as described herein. In another embodiment the method further comprises adrriinistering a horrnone rnbibitor. e.g., an estrogen inhibitor.
A retrospective IGF-1 R IHC analysis has been reported for a collecdon of 12 colonic adenomas, 36 primary colorectal adenocarcinomas and 27 correspondmg metastases, and 34 adjacent normal tissues (Hakamerc/., 1999, Human Patbology. 30: 1I2S-33). The frequency of moderate to strong IHC staining appeared to dramatically increase vvith higher stage and tumor grade (0% normal vs. 93 % metastases). The results are consistent with RNA analysis by RNAss protection assay (**"R?A") (Freier ei al. 1999, Gut 44: 704-08). Thus. in one emboeiment the present invention provides a method of treating colon cancer in a subjeet in need of such treatment cornprising adrninistenng to the subjeet an effective amount of an IGF-1R antagonist as described herein.
High plasma IGF-1 and reduced !GFbp3 h men 40-80 years old is associated with increased prostate cancer risk (Chan et al. 1998. Science 279: 563-6). High IGF-1 is associated with a risk of other Cancers including breast (Hankmson et al, 1998, Lancet 351: 1393-96), colon (Ma et al., 1999. Journal of the National Cancer Institute 91: 620-25) and hing (Yu et al.. 1999. Journal of the National Cancer Institute 91: 151-56). In transgenic mouse models. tumor ineidence is increased by IGF-1 overexpression in diverse locations (Bol et al, 1997, Oncogene 14: 1725-34; DiGiovanni et al.., 2000, Cancer Research 60: 1561-70: DiGiovanni ei al, 2000. Proceedings of the National Academy of Sciences of the United States of America 97: 3455-60, Hadsell et al. 2000, Oncogene 19: 8S9-98). These mouse studies point to a role for both

seruxri and srromal produced IGF-1. Thus. in one embodiment the present invention provides a metnod. ot treaüiig a subject in need of such treatment, comprising aömoinistering to the subject an effectrve amount of an antagonist of IGF-1 R as described herein, ^-herein the antagonist inhibits the actvaticm of IGF-1 R by IGF-1. In another embodirnent the subject has Cancer. In another ernbodiment the subject has a mmor. In another ernbodiment, the Cancer is prostate, breast colon or lung Cancer.
It has been observed that bone is the major source of IGF-1 in the body. Thus, in one aspect the present invention provides con^ositioiis and methods for inhfbitmg IGF-1R in a bone of a subject. In one enibodiment an IGF-1R inhibitor of the present invention is acmmstered to a subject that has. or is at risk for developing, a rumor in a bone. The rumor can be, for example, a primary tnmor or a metastatic rumor. The treatrnent optsonally further comprises aidministering to the subject one or more addiüoria] therapeutic andV'or palliative treatmenrs, for example. an anü-rnmor treatment (e~g„ chemotherapy. radiation therapy. or anti-hormone therapy) or a treatment mar inhibits bone tuxnover {e.g., denosumab (Amgen Inc.. Thousand Oaks: CA)}.
IGF-2 is overexpressed in a varieTy of tumors and srromal tissues. IGF-2 levels appear especiaily high (as rauch as 40 fold) in primary liver Cancers (Cariani et al, 1988. Cancer Research 48: 6S44-49) and acenocarcinoma of the colon (Freier et al, 1999. Gut 44: 704-08). Many of the overerowth disorders are associated wirb an increased incidence of childhood tumors. Five to ten percent cf individuals wirb either the prenatal growth disorder Beckwith-Weidmann Syndrome (BWS) or beniihyperplasia develop tumors such as nephroblastoma, adrenal Carcinoma, and neuroblastoma (reviewedby Morison et al., 1998, Molecular Medicine Today 4: 110-05). The turnor-predisposing factor in these children appears to be the mosaic loss of maternal IGF-2 gene imprinting, or duplicarion of the patemal chromosomal arm (1 ip) that carries IGF-2. Both eiterations would mcrease the level of IGF-2 expression. IGF-2 overexpression as a result of mosaic uniparental disomy or loss of IGF-2 ixnprinting has also been detected in Wiims tumors. Growth disorders are not observed in these children even though the IGF-2 gene alterations also occur in some normal tissues, perhaps reflectrng the tissue distribution of the affected cells. Imprinnng of the matemal IGF-2 gene also occurs in mice, and the effects of IGF-2 overexpression are consistent with the human Situation (Cariani et als 1991. Journal of Hepatology 13: 220-26, Schirmacher et al, 1992. Cancer Research 52; 2549-56; Harris er cL 1998, Oncogene 16: 203-09). The incidence of tumors and organomegaly increases in mice that transgenically express excess IGF-2 (Christofori ei al, 1994, Nature 369: 414-18. Ward et al, 1994. Proceedings of the National Academy of Sciences of the Unired States of America 91: 10365-9. Wolf et al.. 1994. Endocrrnology 135: 1877-S6, Bates ei al, 1995. British Journal of Cancer 72: 1189-93, Hassan et al, 2000, Cancer Research 60: 1070-76). Local IGF-2 overexpression increases the spontaneous appearance of prostate, rnammary. intestinal, liver and epidermal tumors. Plasma specific expression using liver promoters elevate hepatocellular carcinomas and lyrnphoma. Thus, in one ernbodiment the present invention provides a method of treating a subject in need of such trearment^ comprlsing adrrrinistering to the subject an effective amount of an antagonistof IGF-1 Ras described herein. Tvherein the antagonist inhibits the activation of IGF-1R by IGF-2. La another ernbodiment the subject has cancer. In another ernbodiment the .subject has a tumor. In another ernbodiment the subject has liver cancer. adenocarcinoma of the colon. Beckwith-WeidTpann Syndrome, hernihyperplasia, neohroblastoma. adrenal Carcinoma, neuroblastoma. mosaic loss of maternal IGF-2 gene imprmting. dupHcation of the

pateraal chromosomal arm (11p). increased 1GF-2 expression, a minor {e.g.. a prostate, mammary. intestiiial liver. epidermsl. or Vvilms tnmor). organomegaly. hepatocellular Carcinoma, or lymphorna.
In another aspect. the invennon provides methods ofpre vermag or inhibrang a Cancer from srjreadins to another r?ar; ef the bodv. or of treatine: a cancer that has snreac to another part of the bodv. In one tmbodimeni the canrrz aas spread to a regional lyrnph node. In another embodiment, the cancer is metastatic. The nrimary nzrnor can be any krnd of tumor. for example, an adenocarcinoma tumor (e.g.. a prostate adenocarcrnoictt tarnor, a besäst csrcmoma tumor, or a renal cell Carcinoma tumor). a non-smaU ceü or small cell kmg caiaccx tamor, a örynrid cancer tnmor. eic. Tbe site of the metastatic tnmor can be anywberc in the body. h can be. ibr exampk. n bone. the lymph System limg, Drain, eye. skin. pancrease, or liver. In ose paröctnax embcect bavmg a tumor disease is rreated witii an effective arnormt of an IGF- 1R inblnrniig ■-^mncmnp of tbe niesen: invennon such that the primary tumor is prevemed trom meiastasiziiig. In ancthex piarticmar enjbcKhmsnL. a subject having a primary tumor is treated with an effective sroouni of an 1GF-1R rnmrnrmg cöraDcsinon of the present rnvenuon such trist the primary tnmor is iniubited fron metastasizing. In another parncujar embodiment a subject having a metastatic tumor is treated with an effective amoum of an IGF-1R mbfDrrins cornoosition of die present invention such mar growth or spreadrng of the secondarv- tumor is irmibrted. In another particular embodiment a subject having a metastatic rumor is treated wirb an enective amotmt of an IGF-1R inhibiting composition of the present invention such mal the secandary tnmor is rednced in size. In a more particular embodiment, the primary tnmor is an adenocarcinoina tnmor. a non-small cell lung tumor. a small cell hing tumor. or a thyroid cancer. In another more particular embodiment the metastatic tumor is in a hone. In another more particular embodiment. a metastatic tnmor is prevented or inhibited from forming in a bone. In another more particulany definec embodiment the method comprises ueating the subject with an IGF-1R rnhfbiting composition offne present invention and ose or more other treatments (e.g., a treatment that kills ar inhibits the growth of cancer eells, such as radiahorL hormonal therapy. or chemotherapy, or a treatment that inhibits the turnc-ver cf bone, such as denosumab). non-limiting examples of which are provided herein. The one or mcre other rrsatments can incinde. for example the Standard of care for the subject"s particular condition and'or palliative care.
With out being boimd to any particular theory. mmor cells appear to depend on the PI3 Kinase/Alct signaling pathway to resist the apoptosis-inducing acrivity of cbemctherapetrücs. radiation. and anti-hormone therany. Thus, in one embodimem, the present invention provides methods of treating a subject in need of such treatmeni comprising admioistering to the subject an IGF-1R antagonist of the present invention and a chemotherapeutic, radiation- and/or an anti-hoimone therapy. This concept has been vaiidated experimentally in cell culture rnodels and rodent tumor models by anüsense and dominant negative mutations (reviewed by Baserga er cl.. 1997. Biochimica et Biophysica Acta 1332: Fl 05-26, Baserga. 2ÖO0> Oncogene 19: 5574-S1). Ln one embodiment the chemotherapeuric agents is selected from the group consisting of rnitotic mhibitors. alkylating agents. anti-metabolites. intercalating antibiotics, grcwth facior Inhibitors, cell cycle inhfoitors, enz'v^mes. topoisomerase inhibirors. anti-survival agents. biological response modifiers. anti-bormones. e.g. anti-androgens, and acti-angiogenesis agents.
One example of a chcmotherapeutic agent that can be sdministered in combination with an IGF-I receptor inhibitor of the invention is CPT-11. CPT-11 (lrinotecan hydorchloride trihydrate) is a serni

synthetic, water soluble derivative of carnptothechx a plant alkaloicl CPT-11 and an associated metabolite called SN38 inhibit topoisomerase 1 (TOPOl). This enzyme nrrodnces reversible single-strand breaks in DNA that allow unv,±nding and permit DNA repheation to preeeed. Inhibition of TOPOl pxevents religation of single-strand breaks aüer DNA. replication izsuhzzg in greaily increased chromosoma] fragmentation. This DNA damage premotes cell death by zpoptosis through the action of p53 and other Systems that monitor genome integrity. The cytotoxic effec: of CPT-11 is gencrally limited to cells that are replicatmg DNA (S-Phase). QuiesceTit cells are largely iwaSsctsd.
In anothex en&odrrnent the present invennosn prervidrs treating a sobjeci ic need thereoi wirb an effective amonnt of anIGF-lR antagonist of thepresem mverniotQ znd "with aneffeenve arnonmof an apoptosis-inducing agent
In anotber exrix>diiiient an anti-aiigiogerjesis agent sach as an MMP-2 frzsxrix-ineiElloproteinase
2) inhfoitoi. an MMP-9 (rnainx-noetaBoproieinase 9) infcäyrtor, and'or a COX-II (cyckwxy genäse TT)
mhibitor. is used in conjunetion wtöi a Compound of the inverdon. Ezainples of uscful COX-Ü Inhibitors
melude CELEBREX™ (alecoxib): BEXTRA™ (valdecoxfo), and VIOXX™ (ioecoxib). Examples of
useful matrix metaliOproteinase inhibhors are described in WO 96/33172 (pubhshed Oct 24, 1996). WO
96/27583 (published Mar. 7: 1996), European Patent Applicsdon No. 9730497;. 1 (filed JnL 8, 1997):
European Patent Application No. 99308617.2 (filed Oct. 29, 1999), WO 93/07697 (publisbed Feb. 26,
1998), WO 98/03516 (publisbed Jan 29, 1998): WO 98/34918 (pubbshed Ana. 13, 1998), WO 98/34915
(publisbed Aug. 13, 199S): WO 98/33768 (publisbed Aug. 6, 1998).. WO 98/30566 (publisbed Jul. 16,
1998): European Patent Puhbcanon 606:046 (published Jul 13. 1994). European Patent Pubh'cadon 931,788
(publisbed Jul. 28, 1999), WrO 90/05719 (pubbshed May 3h 1990), WO 99/52910 (published Oct. 21,
1999), WO 99/52889 (published Oct. 21, 1999), WO 99/29667 (published Jun. 17, 1999), PCT
International Application No. PCT/TB9S/01113 (Sied Jul 21,1998), European Patent Application No.
99302232.1 (filed Mar. 25, 1999), Great Britain patent appikanonriuinber 9912961.1 (Sied Jun. 3, 1999),
U.S. Pro-visional Application No. 60/148,464 (filed Aug. 12, 1999), U.S. Pat No. 5,863,949 (issued Jan. 26,
1999), U.S. Pat. No. 5,861,510 (issued Jan. 19, 1999), and European Patent Publicarion 780,386 (published
Jun. 25, 1997), all of vdnch are incorporated herein in tber entiredes by reference. In one enÄodirnent the
MM? inhibitor is one that does not demonstrate anhralgia. In another embodiment, the MMP inbibitor
selectively mhibits MMP-2 and/or MMP-9 relative to otber rnatrix-rnetailopTOteinases {i.e.. MMP-1. MMP-
3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MM?-10, MMP-11. MMP-12, and MMP-13). Some
specific examples of MMP inhfoitors useful in the presem irrvention are A.G-334Ö. RO 32-3555. RS 13-
0830, and the Compounds recited in the following list: 3-[[4-(4-fiuoro-pnenoxy)-benzene~ sulfonyl]-(l-
hydroxycaibamoylK:yclopentyI)-arrjino]-propionic aeid; 3-e>:o-3-[4-(4-ih]oro-pbenoxy)-
berizenesulfonylamino]-8-oxa-bicyclo[3.2.1]o- ctane-3-c^rbox%rHc aeid hydroxyarriide; (2R 3R) l-[4-(2-
chloro-4-fluoro-ben- :ryloxy)-beir&Kiesulfonyl]-3-hydroxy-3-iM^ aeid
hydxoxyarnide: 4-[4^4-fiuoro-phenoxy)-benzenesulfonylainino]-tetrahydro-py- ran-4-carboxylic aeid
bydxoxyainice; 3-[[4-(4-fluoro-phenox>7)-benzenestiIfon- y]}41-bycTOX^'carbcnrx3Yl^yclobutyI)-armno]-
propionic aeid; 4-[4-(4^hloro-pheno>:y)-benzenesulfon^ ic aeid
bydroxyamide; (R) 3-[4-(4~cbJoro-pbenoxy)-ben2enesnifonyianaino]-te- trahydro-pvran-3-carboxylic aeid hydroxyamide; (2P_ 3R) l-[4~(4-fiuoro-2-methyl-benzyloxy)-beii2snes^

peridine-2-carboxylic acid bydroxyainide; 3-[[4-(4-fluoro-pbenoxy)-bciizenes- uifony]]-(3-hydraxycafbaiDoyl-1 -methyl-ethylVamino]-propiordc acid: 3-[[4-(4-fiuorD-pb£BO>^0-^e-:2:e^e^^o^yn-(4-bydroxycaibamoyl-tetraiiydro— p>Taxi-4-yl)-amr>oj"propioaic acid: 3-sxo-3-[4-(4-chloTD-pbenoxy)-bsnzenesu- Ifoaylan3ino]-8^x£-icycIo[3J2.1]ociane-3-C2^>ox}?iic acidbydrcxyarnide: 3-endo-3-[4-xa-icyclor3.2.1 ]octane-3-carboxylic acid hydroxysirride; and (R) 3-[4-{4-fluoro-pbenoxy)-b- enzer-esulfoiiylEiiiiBo]-tdrabydro-fin2ii-3-caibox>'lic aridhyäroxyainiäs; and pharrnacetrrically acceptahle säte, sorvates. derivatives, and otber preparanons of the corrE>ounds.
Sporadic mutabons that inacrrvaie die PEIN gene product occux relatxvely ireqoenäy in most human Cancers (Yamada er al., 2001. J Cell Sei 114:2375-82, Hill ei al., 2002, Pbarroacol Sbeiapeat 93:243-51). Löss of PTEN causes the Akt phosphoryiated State to persist through loss ofrbeabürry to down-regulate stiirnalaiory signals orignastiag from 1GF-1R and other sotrrces. Tbe Status of tbe D53 tumor suppressor also rnüuences rbe acüvhy of tbe IGF-1R signaling System. In rbe ground Stare, the basal or consdtuiive transerrprion of IGF-1R is repressed by p53 via an indirect mechanism. Acrjvabonof Akt prornotes rbe pbospborylation cf rrxlrnl. which tben hinös ibe p53 tumor suppressor and prornotes its degradaüon (Mayo er al, 2002, TI3S 27:462-67), resuiting in increased 1GF-1R expression. A similar outcome is observed when p53 is inactivated by mutation. When transiently expressed in Saos-2 (a human osteosarcoma cell line) and RD (a rbabdomyosarcoma cell line). wild-type p53 is able to suppress ihe acüvity of a cotransfectec IGF-lRpromoter construet wbereas tumor-derived. mutant versions of p53 bave no effect. It has been proposed that the increased level of IGF-1R promotes tbe resistance to apoptosis associated wirb p53 loss in maiignant cells (Werner et al, 2000. Cell Mol Life Sei 57:932-42). Taus, in one embodiment the present invention provides a method of treating a cancerous condition in a subjeet in need of such treatment comprising administsring to the subjeet an effective amount of an 1GF-1R antagonist as described herein, wherein the cancerous condition is characterized by ceüs that bave a reduced expression or acüvity of p53.
The WT1 (Vviins kidney fomor suppressor 1 protein) also has been shown to bind and repress the IGF-1R promoter. Thus. in one embodiment, the present invention provides a method of treating a cancerous condiuon in a subjeet in need of such treatment cornprising administering to ihe subjeet an effective amount of an IGF-1R antagonist as described herein wherein the cancerous condition is characterized by a reduced expression or activity of WT1.
The proliferation cf ncrmal Sbroblasts has been shown to require. under denned culture condiriens, the combined acücn of IGF and a stromal growth factor (e.g. PDGF. EGF) to rarnp-up Ras/Raf'Map Kinase and promote cell cycle entry (tbe G0 to Gl transition). Ffbroblasts derived ürom IGF-]R (-'-) mice do not respond to growth factor alone. or most oncogenes (e.g. oncogexdc Ras) that activate the Ras/Raf/Map Kinase pathway. Thus. in one embodiment tbe present invention provides a method of treating a subjeet in need of such treatment comprising administering to the subjeet an IGF-1R antagonist as described herein and an agent tbat targets a growth factor and/or a growth factor reeeptor, such as a growth factor reeeptor tyrosine kmase, e.g., the EGFR, HER~2; ber-abh VEGFFL Kit, ral mTOR, CDKl/2: VEGFR2. PKCß. Mek, and'or KDR. Examples of mole-cules thattarget such growth facters andrer reeeptors rnclude parjjtumurnab (Abgenix. Fremont CA/Amgen, Thousand Oaks, CA). HERCEPTIN™ (GenenteccL South San Francisco.. CA), GLEEVEC™ (Novaitis, East Hanover, NJ). IRESSA™

(AsiraZeneca, Wilmingion DE)5 ERBITUX™, (ImClonc, New York, NY)r AVASTIN"™, (Genentech), PTK787 (Novartis), SUl 1248 (Pfizer.. New York, NY), TARCEVA™ (OSI Pharmaccuncals, Melvüle, KY)S 43-9006 (Bayer, West Haven CT), CCI-779 (WyetL Madison, NJ): RAD001 (Novartis), BMS-387052 (Bristol-Myers Squibb. New York., NY), IMC-1C! 1 (ImClone), LY333531 (Eli Lilly, Indianapolis, IK), PD 184352 (Pfizer), 2C4 (Genentech), and GW2016 (GiaxoSimthKLne, Research Triangle Park, NC).
The role of IGF-1R in hematological malignancies has been reviewed by (No\*ak et ah, 2003. insuiin-Like Growth Factors and Hematological Malignxmcies in Insulin-Like Growth Factors, LeRoitn et al. ed.s. Landes Bioscience). A functiona] role for die IGF-1R in herotopoietic maügnancies is demorostraied by. for example. the abüity of IGF-1R monoclonal antibodies to block transformed cell growth in cultnre. IGF-I has been found to enhance growth of freshly isolated human acute myelogenous leukemia and acute lymphoblastic leukerrha blasts. Wirb respect to T cell malignancies. IGF-I bas been shown to inihience the growth of murine lympboma cells bearing a pre-T cell pheootype and. immaüire and rnatizre primary human T lineage acute lymphoblastic leukemia ceils were found to express high numbers of IGF-1R. Tlius. in one embodirnent the present invention provides methods of trear'ng E hematological rnalignancy in 2 subject in need thereof comprising administering to the subject an anragonist of 1GF-1R as described herein. In anothex embodirnent, the rnalignancy is an acute myelogenous leukemia. an acute lymphoblastic leukemia. or a T cell rnalignancy.
In another aspect the presem invention provides methods of identirying subjects who are more likely to benent from treatrnent using the compositions and/or methods of treatment of the present invention. Such methods can enable a earegiver to better tailor a therapeutic regimen to a parücukr subiect's needs and reduce the likelihood of an ineffective or counterproducüve course of treatrnent. In one embodirnent, the present invention provides a method of determining whether a subject is a candidate for treatment using a corrxposition or method as described herein comprising detennining whether a target cell type in the subject expresses IGF-1R. wherein if the target cell type expresses IGF-1R, thenthe subject is a candidate for treatment In another embodirnent. the method cornprises deteronning the approximate average number of IGF-IRmolecules per target cell, wherein 10". 10"". 10". 10\ or 106 IGF-1R per cell indicates that the subject is a candidate for treatment. The approximate average number of IGF-1R moiecules per target cell can be deteimined using any technique known in the art, for example, by staining a sample comprising cells of the target cell type wiih an IGF-1R bindmg molecule, and detecting the amount of IGF-lRbinding molecule bound to the sample, where the amount of IGF-1R binding molecule detected is proportional to the average number of IGF-1R moiecules in the sample. In another embodirnent the method cornprises cornparing the approximate average number of IGF-1R moiecules per target cell to a reference Standard- wherein if the aporoximate average number of IGF-1R moiecules per target cell is greater than the reference Standard, then the subject is more ükeiy to beneüt from treatment using the compositions and/or methods of treatment of the present invention- In another embodirnent the target cell type is a cancerous cell type, In another embodirnent, the target cell type is a colon Cancer cell type, a breast cancer cell type, anNSCLC cell type, or a lenkende cell type.
In another embodirnent a subject who is a candidate for treatment is identified by detecting IGF-1 anoVor IGF-2 in the target cell type, or in the Stratum of the target cell type. In another embodirnent, the

target cell type is a cancerous cell type. In another embodiment the target cell type is a coion Cancer cell type, a breast cancer cell type, an KSCLC cell type, or a leiikemic cell type.
In another embodiment a subject who is H candidate for treatment is identfied by detecting activiiy of IGF-lR-mediated signaling in the target cell type, wherein IGF-lR-mediated signaling in the target cell type intiicstes that the subject is a candidate for treatment. Examples of molecules that can be mcnrrtored for lGF-lP.-dependent changes are shown in Figure 10, such as molecules in the PB/Akt patirway, e.g., IGF-1IL IRS adapters, Akt exe. Such molecules can be irxraitored for, for example, a change in ohosohoTylaikjn ^nr; eg., an increase in phosphorylation. Phosphospecific antibodies that recognize rhe activasd fanrs of diese proteia markers are highly developed, and these reagenrs have nroven to be reüabie for rrnmnnobks detection in expeiimental Systems.
The compcsiikjas and/or metbods of the present invenrion also cari be used. for example, in cosmenc rearmesrrs. m Terexmaxy treafments. to increase longevity. to treat reprodnetive defects. and to rear a vanery of giuv..! related disorders.
Taerapeciic methods and adnTmistration of antigen binding proteins
Certain rnethods provided herein comprise adminisrering an IGF-1R binding antigen binding protein ;o a subject thereby reducing an IGF-1-induced biologicai response thai plays a role in a particular condition. In partic^iaT embodiraents, methods of the invenrion involve contacting endogenous IGF-1R tvith an IGF-1R binding antigen binding protein. e.g.. via administration to a subject or in an ex vivo procedura.
The term "Treatrnenf' encompasses alleviation orprevention of at least one Symptom or other aspect of i disorder. er reduetion of disease severity, and the like. An antigen binding protein need not effeci a complete eure, or eradicate every symptom or manifestation of a disease. to constitute a viable rherat?eutic aeent As is recognized in the pertinent field. drugs employed as thexapeuric agents rnay reduce the severity of a given disease State, but need not abolish every manifestation of the disease to be regarded as usefoi merapeuik agents. Similarly. a prophylacrically atiministered treatment need not be completely efiective in preventmg the onset of a condition in order to constitute a viable prophylactic agent Simply reduemg the impact of a disease (for example. by reducing the number or severity of its Symptoms, or by increasing the erTectiveness of another treatment or by producing another beneficial eneci). or reducing the Likelihooc that the disease will oeeur or morsen in a subject is sufScient One embodiment of the invention is directed to a method comprising aohninistermg to s patient an IGF-1R antagonist in an amount and for a time suffkient to ineuce a sustained improvement over baseline of an indicator that refjects the severity of the particular disorder.
As is understood in the pertinent neld, pharmaceuticai coinpositions comprising the molecules of . the mvention are atiministered to a subject in a manner appropriate to the indication. Pharmaceuticai composinons may be admiriistered by any suitable technique, including but not limited to parenterally. topicaily, or by inhalation. If injeeted, the pharmaceuticai composition can be adrninistered, for example. ■via inrra-arncular. intravenous. intramuscular, intralesional. intraperitonea] or subeutareous routes. by bolus injeetion. or continuous infusion. Localized administration. e.g. at a site of disease or injury is coniemplated, as are transdermal delivery and sustained reje-ase from implants. Delivery by inhalation

includes, for example. nasal or oral inhalarien, use of a nebulizer. inhalanon of rhe antagonist in aerosol form, and the like. Other alternatives inchirie eyedrops; oral preparations inciuding puls. syrups. lozenges or chewing gmzi; and topical prepararioiis such as iotions. gels. sprays, and ointments.
Use of antigen binding proteins in ex vrvc procedm-es also is contempiaiecL For example. a patienrs blood or other hodüy fluid may be contacted wirb an antigen binding protein thar binds IGF-1R ex vfvo. The antigen binding protein may be botnd to a suitabie insomble marrix or solid support material.
A-dvantageousry. antigen binding pröicins are admirdssered in the form of a composition coaipfising one or more additioiiai cömpooaas such as a physioipgdry Hy acceptable cairier, excipdent or düuent OptionaHy. tbe composition addinasalry cxErpises ose or more pbysioiosäcalh' active agents. for example, a second IGF-1 receptOT-mbibning sufastance. an aim-angkrtEnric substance. a cbenaotherapeutic substance, an analgesic substance. sc., noD-exciasrve examples of ~irich are provided herein. In various particular eriirxxiiirients.. the cccEposrrion cxerssises esse, two. mree. fbur. Sve, or six physiologically active agents in additdon to an IGF- 1R binding antigen bmdmg protem
In one embociinenL the phaxinaceEtical axzposzxkm comprise an antigen binding protein of tbe invention together wmi one or mcre suhstances selected from "die group consisting of a bufFer, an anüoxidant such as ascorbic acid a low rxiDlecular weigbi poKpsptide (such, as those having fewer tban 10 amino acids). a proiein. an arnino acid, a caibohydrzte such as glncose. sucrose or dextrms. a chelating agent such as EDTA, glutathione. a stabüizer, and an. excrpienr Neutral bufFered saline or saline mixed wirb conspecific serum albumin are examples of appropriate düuents. In accordance vnxh appropriate indastry Standards, preservatives such as benzyl alcobol may also be added. Tbe composition may be formulated as a lyophilizate using appropriate excipient Solutions (e.g., sucrose) as düuents. Suitable cornponents are nontoxic to recipients at tbe dosages and concentrations employed. Further examples of components that may be employed in pbarmaceutica] formuladons are presented in Remington's Pharmaceutical Sciences, 16" Ed. (19S0) and 20te E& (2000), Mack Publishing Company. Easton PA.
Kits for use by medical Dracnrjoners include an IGF-1 receptor-inmlritmg substance of tbe invention and a label or other Instructions for use in treating any of tbe conditions ciscussed berein. In one embodirnent, the kit includes a sterile prrpararion of cne or more IGF-IR binding antigen binding proteins. ■Ä'hich may be in the form of a composition as disclosed above. and may be in one er more vials.
Dosages zr<. tbe frequency of adzninistration may van aecording to such factors as the route administration narticular antigen binding proteins employed. nature and severiry disease be treated whether condirion is acute or chronic size general condition subjeet. appropriate dosages can detennined by procedures knovn ir. pertinent art e.g. in clinical trials that involve dose escalation studies.> An IGF-1 reeeptor inhibiring substance of the invention may be adrmnistered. for example. once or more fhan once. e.g-., at regulär intervais over a period of ttme. In parnetdar ernbodiments, an-antigen binding protem is adnrinistered over a period of at least a monm or more. e.g.. for one, two, or three months or even mdefmitely. For treating chronic condinons. long-tenn treatment is generallV most effecrive. However, for treating acute condinons. administration for shorter periods. e.g, from one to six weeks. may be surncient. In general, the antigen binding protein is adrriinistered until the paüent manifests a medicaliy relevant cegree ot rmprovement over baseline for rhe chosen indicator or indicators.

Pardcular embodiments of tbe presenl invention involve achnniisteTing an antigen binding protein et a dosage of from about 1 ng of antigen bitiding protein per kg of subjeefs weightper öay ("lng,Tcz'dayTr) to aboirt 10 rng/kg/day. more preferably from about 500 ng/kg/day to about 5 mg/kg/day. and most preferably from about 5 ug/kg/day to about 2 mg/kg/day, to a subjeet In additional ernbodiineu-s, ar antigen binding protein is administered to adults one time per weck, two trrnes per week, or tbiee or n»re times per week, to treat an IGF-1 and/or IGF-2 mediated disease, conditior or disorder. e.g.. a medacal disorder disclosed herein. If injeeted. the efective amouni of antigen binding protein per adnlt öosc nsy ränge from 1-20 mg/m", and preferably is about 5-12 mg/m". Altemgriveiy. a Sät dose may be aerninistered: me amounr may ränge from 5-100 mg/'dose. One ränge for a flat dose is abom 20-30 mg Der dose. In one ernbodiment of the invention. a üat dose of 25 mg/'dose is repeaiedry adixäzostered by imecüon. If a rorrte of a^hinnistration other than irhecrion is used. rhe dose is appropriately adjussd in aecordance wilh Standard medica] practices. One example of a rherapeatic regimen invoives rmexxmg a dose of about 20-30 mg of antigen bmding protein to one to three ümes per week over a period o: ax i^asx three weeks. mough treatment for longer periods may be necesszry to induce the desired degree of irrrprovemenL For pediatric subjeets (age 4-ITj. one exemplary suitable regimen involves the subeutaneous iniection of 0.4 mg/kg, up to a rnaximum dose of 25 mg of antigen binding protein adrniinstered two or three times per week.
Particalar embodiments of the methods provided herein involve subeutaneous injeerjon of from 0-5 mg to 10 mg. preferably from 3 to 5 mg. of an antigen binding protein, onoe or twice per week. Another embodiment is directed to pulmonary administrarion {e.g., by nebulizer) of 3 or more mg of antigen binding protein once a week.
Examples of therapeuü'c regimens provided herein comprise subeutaneous injecüoa of an antigen binding protein once a week. at a dose of 1.5 to 3 mg, io treat a condirion in which IGF-1R signaiing plays a role. Examples of such conditions are provided herein and inchide. for example. Cancer, acrornegzly and orher overgrovvth disorders, diabetes. obesity, macular degeneration, and aging. Weekly adminisrratien cf tätigen binding protein is continued until a desired result is achievecL e.g.. the subjeet *s Symptoms subside. Treatment may resume as needed. or, alternativ ei y, rnarntenance doses may be administered.
Other examples of therapeutic regimens provided herein comprise subeutaneous or intravenous sdnainistration of a dose of 1. 3. 5. 6, 7. S. 9. 10. 11. 12., 15, or 20 rni'iligrams of an IGF-1R inhlbrtor of the present invention per kilogram body mass of the subjeet (mgkgj. The dose canbe admrmstereä once io the subiect, o: more than once at a certain interval, for example. once a day. three times a week, rwice £ week. once a week. three times a month, twice a month. once a month, osee every two months. ciice every three months, once every six months. or once a year. The cirration of the treatment. and any changes to the dose and/'or frequericy of treatment can be altered or varied during the course of treatment in order to meet the particular needs of the subjeet
In another embodiment an antigen binding protein is aöhirmistered to the subjeet in an arnount and for s time sufneient to induce an irrrprovemenL preferably a sustained improvernent. in at jeast one ir.aicator that refiects the severity of the disorder that is being treated. Various indicators that refect the extern of the subject's iliness. disease er condirion may be assessed for detenriining whether the amount and time of the treatment is suföcient. Such indicators includc, for example, clirocally recognized indicators of disease

severiry, syrnptorns. or manifestario-is of the disorder in cuesdon. In one embodiment, an improvement is considered to be sustained if the subject exhibits the izrrprovement on at least two occasicns separaten by two to four weeks. The degxee of iinprovernent generally is deiermined by a physician, who may -maVe this detennination based cn siens. r.inptoins. hiousies- or otber test resuits, and who mav also emnlov questionnaires that are adrninistered to the subject, such as qualiry-of-Iife quesnoniiaires developed for a given disease.
Elevated levels of IGF-1 and/or IGF-2 are associated with a nrrmber of disorders. mchiding. for example, Cancer (e.g... hing? prostate, breast and colon Cancers), and acromegaly and otber overgrowfh disorders (cg., constmmonally tall chüdren). Subjecrs with a grven disorder rnay be screenecl to iaeririry those individnals who have elevated IGF-1 and/or IGF-2 levels. thereby idenüfving the subjects who inay beneöt most äom treatment with an IGF-1 R hm&mg antigen binding protein. Thns. treatroent methods orovided herein optionally comprise a nrst step of rneasruing a subjecrs IGF-1 and/or IGF-2 levels. An anlägen binding protein rnay be admmistered to a subject in "wbom IGF-1 and'or IGF-2 levels are elevated above normal In one ernbodirnent the present invention prcrides a method of treating an overgrcwth disorder (e.g., acromegaly) comprising admirhstering to a subject in need thereof an antigen binding protein of the present invention and pegviscmant
A subject*s levels of IGF-1 and'or IGF-2 may be monitoted hefore. during and/or after treatment with an antigen binding protein, to detect changes, if any, in their levels. For some disorders. the incidence . of elevated IGF-1 and/or IGF-2 levels may vary according to such factors as the stage of the disease or the particiliar form of the disease. Known techniques may be employed foi measuring IGF-1 and/or IGF-2 levels, e.g.. in a subjecfs serum. IGF-1 and/or IGF-2 levels in blood samples may be measuxed using any suitable technique, for example. ELISA.
Particnlar ernbodiments of methods and compositions of the invention involve the use of an antigen binding protein and one or more additional IGF-1R antagonists. for example, two or more antigen binding proteins of the invention, or an antigen binding protein of the invention and one or more otber IGF-1R antagonists. In furtber ernbodiments, antigen binding protein are adrninistered alone or in combinarion with other agents useful for treating the concinon with which the patient is afHicted. Examples of such agents incltice both proteinaceous and non-proteinaceous drags. When multiple thera.peutics are co-administered dcsages may be adjusted accordingly, as is recognized in the pertinent art "Co-adrninistration" and combination therapy are not limited to simultan eous administration, but also ine lüde treatment regimens in which an antigen bindirg protein is adrninistered 2t least once during a course of treatment that involves administering at least one other therapeutic agent to the padeni.
ExamDles of other agents that may be co-administered with an antigen binding protein are other antigen binding proteins or therapeunc Polypeptides that aie chosen according to the parricular condirion to be txeated AJtematively. non-proteinaeeous drugs that are useful in treating one of the paricular conditions discussed above rnay be co-administered with an IGF-1R antagonist.
Combination theranv
In another aspect the present invention provides a method of treating a subject with an IGF-1P. inhibiting antigen binding protein and one or more other treatments. In one ernbodirnent such a

combinarJcn therapy achieves synergy or an additive effect by. for example, attacking muitbie siies or molecular targets in a tumor. Types of combination therapies that can be used in connection -cito rhs present invention include inrfbiting or acüvaühg (as appropriate) mump3s nodes in a Single disease-related pathway, multiple pathways in a target cell and multiple cell types mithin a targe: rissue (e.g., Vsimin a rumor). For example, an IGF-1R inhibixor of tbe present invention can be combined with a ireatmeui tost inhibits IGF-1, promotes apoptcsis, inhibits angiogenesis, or inhibits macrophage. In anotber embodiment a taxgeted agent that. when used by itselr fafls to eiicit a therapeufically desired effect could be used to, for example. sensitize Cancer cells or augmeni treatment effect of other agents. In anotber emhodroent an IGF-IR inhibitor according to ihe mventdon is used in combination wirb, a cytotoxic drug or other targetec agent thai induces apoptcsis. In anotber ernbodiment, an IGF-1R inhibitor is used in combrnation ^im one or more aaents that inhfoii cüfferent tarsets that are invoived in cell survival (e.g.. PKIB. mTORl differeni receptor tyrosine kbiases (e-g.t ErbBl. ErbB2, c-Met, c-kit), or cUfferenT cell types (cg., KDR inMbitors. c-fms). In anotber embodiment, an IGF-1 R inhibitor of ihe mveniioi: is added to the existing Standard of care for a particular eondmon Examples of therapeutic agents include. but are not limited to. gemcitabine. taxol, taxotere. and CPT-11.
In another embodiment a combination therapy method comprises arrrrrim'stering to the subject wc. three, four, five. six, or more of the IGF-IR agonists or antagonists described herein. In another embodiment, tbe method comprises administering to the subject two or more treatments that togetber inhibit or activate (directly or indirectly) IGF-lR-mediated signal transduction. Examples of such methods include using combinations of two or more IGF-IR inhibiting anrigen binding progeins. of an IGF-IR inhibiting antigen binding protein and one ormore other IGF-1, IGF-2, and'or IGF-IR agonists or antagonists {e.g., IGF-1 and'or IGF-2 binding Polypeptides, IGF-IR binding Polypeptides, IGF-1 and'or IGF-2 derivatives. . anti-IGF-1 and'or IGF-2 antibodies, anti-sense nucleic acids against IGF-1, IGF-2, and/or IGF-IR, or other molecules that bind to IGF-1, IGF-2, and/or IGF-IR Polypeptides or nucleic acids). or of an IGF-IR inhibiting antigen binding protein and one or more other treatments (e.g., surgery. ultrasound radiotherapy. chemotherapy, or treatment with another anti-cancer agen:). as described, for example, in US Fat. No. 5,473,054 (issued Dec. 5: 1995), 6,051,593 (issued April 18s 2000), 6..0S4..0S5 (issued july A 2000), 6,506..763 (issued Jan. 14, 2003), US Pat. App. Pub. No.s 03/0092631 (published May 15, 2003): 03/0165502 (published Sept. 4r 2003), 03/0235582 (published Dec. 25. 2003), 04/OSS6503 (published May 6, 2034), 05/0272637 (published Dec. 8, 2005), PCT Pub. Ser. No.s WO 99/60C23 (pubiisbed Nov. 25, * 1999V WO 02053596 (published July 11, 2002), WO O2/O727S0 (publishsd Sept. 19, 2002); WO 03/0272^5 (published March 3, 2003): WO 03/020698 (published March 13, 2003), WO 03/059551 (pubiisbed July 24, 2003), WO 03/100003 (published Dec. 4, 2003): WO 03/106621 (published Dec. 24, 2002), WO 04/071529 (published August 26, 2004), WO 04/033248 (published SeuL 30, 2004), WO 04/087756 (published Oct. 14, 2004), WO 05/112969 (published Dec. 1, 2005), Kuli eioL, 1983, J Bio! Chem 255:6561-66, Flier ei alt 1986, Proc Natl Acad Sei USA 83:664~66S, Conover et alz 1987: J Cell Physiol 133:560-66, Rohlik ei alt 19S7,BiochemBiophys Res Comm 149:276-81, Arteaga et aL: 19S9, J Clinical Investigation 84:1418-23. .Arteaga etat, 1989, Cancer Res 49:6237-^1, Gansler er c/., 1989, American J Pathol 135:961-66, Gustafson et ai, 1990, J Biol Chem 265:18663-67, Steele-Perlrins ei ah, 1990, BiochemBiophys Res Comm 171:1244-51. Cullen et al. 1992. Mol Endocrinol 6:91-300, Soos et

aL 1992, J Biol Chem 267:12955-63, Xiong et al, 1992, Proc Nail Acac Sei USA 89:5356-60. Broker er al, 1993, Euro J Cancer 29A:562-69, Furlanetto et aL, 1993, Cancer Res 53:2522^26, Li er al., 1993, 3iocbem Biopbys Res Comm 196:92-98, Kalebic et ed.. 1994, Cancer Res 54:5531-34, Lahm et al, 1994,
Ird J Cancer 58:452-59, Zia er sL 1996, J Cell Biochern Supp 24:269-75: Jansson er al. 1997, J Biol Cnem 272:S189-97, Scotlandi et al, 199S, Cancer Res 58:4127-31, Logie et al, 1999, Li et a!.: 2000, Cancer Inarrranol Imrrsmoiherapy 49:243-52, J Mol Endocrinol 23:23-32, De Meyts et al., 2002, Natsre Reviews 1:769-83, Haüey ez al. 2002, Mol Cancer Tnerapeutics 1:1349-53, Maloney et al, 2003, Cancer Research 633073^S3, Bnnnrms- d, 2003. Cancer Research 63:8912-21, and KaiavitaJri et al., 2004, Hormones 3:27-36.. (each mccKTDarssd herein by reference in res entiretyj may be employed in methods and cornposräons of the presem invenrioiL Furüiermore. one or more anti-IGF-lR arrrihodies or antibody dai * dives can be rtsed in exmfornarjon with one or more molecules or orher trearmenis. ^herein the oiher raoleccies's) aud/ar TreaüXüeii'/s") do not erhectry bind to or affect IGF-IR. IGF-1. or TGF-2, bnt ?rhich zxnnbrnaiion is cfecrive fbr treatinä or nreventin£ a conditioiL such as Cancer or an oversrowüi disorder (£.£.. acrome^aiyj. T^ Q^ embocrrneriL one or more of the mojecule(s) anci/or treatmentfs} treats or prrvems a conetrnon that is caused by cne or more of the other molecule(s) or treatment(s) in rhe course of tberapv. e.g., nausea- fztigue, alopecia, cachexia, insomxda, etc. In every case wbere a combinatioD of raolecales and/or orhsT treatments is used. the individual molecule(s) anö/or treanoent(s) can be adnÖGisrered in any ordex, over any Iength of time, which is effective, e.g.. siniultaneously. consecutively, or aitemately. In one embodimert, the method of treatment comprises completing a first course of trearment vrith one molecuie or other treatment before begiririing a second course of treatment. The Iength of time between the end of the first course of treatment and beginning of the second course of treatment can be any Iength of Time thai ailows the total course of therapy to be effective, e.g., seconds, minutes. hours, davs. wseks, months. or even years.
In anofber embodimenu the method comprises administenng one or more of the IGF-1 R anragonists described herein arid one or more other treatments (e.g., a therapeutic er palliative treatment). for example. anti-cancer treatmenTS (such as surgery, ultrasour.d, raöiotherapy. chernotberapy. or treatment niib another anti-cancer agent). Where a method comprises admiiiisTering more than one treatment to a Fuhjeci. it is TO be understood that the order. tirning. number. coBcemration, and voiuroe of the a-dmini=tranons is limited onlv by the medical reqnixements and hmitations of the treatment i.e.. rwo rreatments can be administered to the subjeet, e.g.. simultaneously. consecutively. aherr.alety. er aecording to any ether regimen. Examples of agents that can be adrninisiered m combination wiih the IG7-1R antagonists deschbea herein include. but are not limited to. neutrophil-boosting agents. irinothecan. SN-3S. gerncitabine. herstatin. or an IGF-1 R-binding herstatin derivative (as described, for example. in US Fat App. No. 05/0272637): AVASTIN® (Genentech, South San Francisco, CA): HERCEPTIN® (Genentech), RTTUXAN^ (Genentech), ARIMIDEX® (AsrraZeneca, Wümingtoru DE), IRESSA® (AstraZeneca), BEXX.AR® (Corixa, Seattle, WA), ZEVALIN® (Biogen Idee, Cambridge, MA), ERBITÜX® (Imclone S>-sierns In:.: New York, NY): GEMZAR® (Eh Lilly and Co.. Indianapolis, IN), CAMP70SAR® (Pfizer, New Yori-i NY)/GLEE\^EC® (Novartis), SU-11248 (Pfjzer), BMS-354825 (Bristol-Myers Squibb), panirumumab (Abgenix. Fremont CA/Amgen Inc.. Thousand Oaks, CA), and denosumab (Amgen Inc.. Thonsand Oaks. CA).

The fcDo^ving examples. both actual and propheric. are provided fcr übe purpose of ülustrating specific einbodiinents or fearares of the instant invenrion and do not limit its scope.
EXAMPLE I: Preparatiori o: Antibodies
This example demonstraies a method of prrparing antibodies recogniziiig the IGF-i receptor. IGF-1 receptorPolypeptides may be employed as i;..,.*i itogens in generatmg monoclonal antibodies by convenüonai tecbniques. It is recognized tnat pohypepodcs in varioas ICBZDS may be employed as innmmogens, e.g., feil length proteins. fjagmenis therrol fusion uioteiia. mereof such as Fe fnsions. ceils -expressirig the recombinanx protein on die cell surfacc, ezc~
To snxninaiize an exanxple 01 sueb a procednre, an IGF-1R nnmEzaogsn emnisiSed m complete Freund's adjuvant is injeetsd subcntaneously into Lewis ras, in zmamäs ranging irem 3 0-100 ul. Three weeks laier. the immizrüzed -nfrnak areboesred wräi addJDOcal "i*turrK>ggn errmisifieä in incomrsjere Freund's adjuvant and hoosred every three weeks theresfer. Sernm semples are- periocically raken by retro-orbital bieeding or tail-tip excision fcr tesnng by dor-bkrt assay. ELISA (errzynx-Iirked irxErrunosorbent assay), or inhibition of binding of li-\G¥-'i or '^-IGt-l 10 exrracts of IGrF-lR-expressing ceils. FoIIowmg detection of an appronriaie antibody titer. positive ^^KIS are given a ^HT^I inrravenous injeetion of antigen in saline. Three to four days later, übe grrirngl.g are sacriöced, splenocytes barvested and fused to the murine myeloma cell line AG8653. The xesuliing hybridoina cell iroes are piated in rxruitiple rniexotiter plates in a HAT selective medium (bypoxanthine. arninopterin. and tbymidine) to mbibit proliferation of non-fused ceils, myeloma hybrids, and spieen cell hybrid«.
Hybridoma clones thus generated are screened fcr reaeiivity wifh IGF-IR. Initial Screening of bybridoma supernatanls utilizes an antibody capture and binding of parrialiy purified '"I-IGF-I receptor. Hybridomas mar are positive in this Screening meibod are tested by a modined antibody capture 10 detect bybridoma ceils lines tbat are producing blocking antibody. Hybridomas ihat secrete a monoclonal antibody capable of inhibitrng 125I-IGF-1 binding in eelis expressing 1GF-1R are thus detected. Such hydridomas üben axe injeeted into the peritoneal ca^iries of nude mice to produce aseites coritaining high concantrafiens (>1 mg'ml) of anti-IGF-lR monoclonal antibody. The resiihing monoclonal antibodies may be purified by ammordum sulfate preeipitation foüowed by gel exclusion chromatography, and'or afrinity chiomatography based on binding of antibody to Protein G.
Suriilar methods can be used to generate human antibodies in transgenic mice. See. e.g., Chen et aL, 1993. niernaL Iirimunol. 5: 647-56: Chen ei aL. 1993: EMBO J. 12: 521-30: CtioiercL, i 993.. Nature Genetics4; 117-23; Fishvvild ei cl, 1996,-Natuxe Bicreca 14: S45-51; Harding et al.: I995: Annais New York Acad. Sei: Lonberg ei aL, 1994, Nature 36S: S56-59: Lcnberg, 1994: Kandbook Exper.l Pharmacol. 113: 49-101; Lonberg" er ai.s 1995, Internal Rev. Immunol. 13: 65-93: MorrisoiL 1994: Nature 368: 812-13; Neuberger, 1996, Nature Biotech. 14: 826: Taylor et aL, 1992: yt-ac. Acids Res. 20: 62S7-95: Taylor eta!.,-1994, totemat. Immunol 6: 579-91; Toinizuka er aL, 1997. Nature Genetdcs 16: 133-43; Tornizuka et aL, 2000; Proc. Nai. Acad Sei. USA 97: T22-27; Tuaülon ei aLr 1993, Proc. Nat Acad. Sei. USA.90: 3720-24; Tuaillon er aL. 1994, J. Immunol. 152: 2912-20; P.ussel ei iL, 2000, Infection and Immuniry April 2000: 1S20-26; Gallo ez aL, 2000, Eur. J. ImmuncL 30: 534-^0; Davis er al.. 1999r Cancer Meiasssis Rev. 15:421-25: Green, 1999. J. Immunol. Methods 231:11-23: Jakoboviis. 1998, Advanced Drug Delivery Rev.

31:33-42; Green ei al, I99S: J. Exp. Med. 18S: 483-95; Jakobovhs, 1998, Ex?. Opin. luvest Drugs 7: 607-14; Tsud2 et aL 1997; Genomics 42: 413-21; Mendez e: al, 1991 s Nature Genetics 15: 146-56; Jakobovits, 1996, Weir's Handbook of Experimental Immunology, The Iniegrated Tmrrmne System VcL IV. 194.1-194.7; Mendez ei z!.A995: Genomics 26: 294-307; Jskobovits, 1994, Currem: Bio! 4: 761-63; Aibonss. 1994, Immuniry 1: 247-60; Green et aL 1994; Nature Geneties 7: 13-21; Jakobovits et cl.9 1993.Narure 362: 255-5S: Jakobo\its et ed., 1993s. Proc. Nat Acad Sri. USA 90: 2551-55.
EXAMPLE 2: Isolation of Human IGF-lR(ECD)-C3-rauIgGl
This exampie provides a metbod of makmg a sohible fegmem of IGF-1R useful for raising antibodies. Cloning of pDSRa:buIGF-IR(ECD)^-:naIgGlFc
PrkDsrs 2S30-36:
5' AGC>^-GCT-TCCÄCCATasjiGTC7N3GCT-CCGGAGGA.GG 3' SEQ ID KG:256} and 2S30-3S:
5' ATTTG7xCGACTT-CGTCCAGA'?GGArGA.\Grr~-TTCAT 3', BSQ TD NO: 257) wexe used to arnplify the human IGF-1R extraeellular doinain (1-906) cDNA sequence. The primers included a Kozak translation initiation sequence (underlined above) preceding the starr codon, restrictLon sites for subsequeni subcloning, and a caspace-3 site, which is inserted next to the extraeellular domain C-terminus. PCR was performed on a PerldnEimer 2400 (PerkinElmer, Torrance. CA) under the following conditions: ] cycle al 95c C for 2 nun, 23 eycies at 95°C for 20 sec; 58.5° C for 30 sec. and 72° C for 3 min, and 1 cycle at 72° C for 10 rnin. Final reacüon conditions were IXpfii TURBO® buffer (Stratagene: La Jolla: CA): 200 uM dNTTs. 2 uM each primen 5 13 pfu TURBO® (Stratagene) and 1 ng remplate DNA. The PCPv. produet was purüüed using a Clontech Nucleospin Cohimn (Clontech, Palo Alto, CA) aecording to the manufacrurers instruetions. digested with Hind HI and Sal I (Roche. Indianapolis, IN) and gel purined. The human IGF-1R insert was ligated into Hind JK pDSRo:hiiIGFlR(ECD)-C3-muIgGlFc Plasmid Base ?aix Number:




Medium (Invitrogen) supplemented with 30% dialyzed fetal bovine serum. Ix pgnicilHp-streptomycin (Invitrogen)) and expression levels evaluated by wettern blot To psrfonn this assay, 0.5 rnl of serum free medium was added to a siegle well confiuent cells cultured in a 24 well plate (Falcon). The conditionec medium was rscovcrcd after 48hr. Samples for westem blotting were nm in 10% Tris-glycme gel (Kovex). and blotted on 0.45 um Nitrocellulose membrane (Invitrogen), using the Mini Trans-Blot cell (Biorad). The blotted membranes were incubated wirb rabbit ann-mouse TgG Fe antibody, conjugated with Horsexadisb Peroxidase (Pierce). The clone expressing tbe kighest level of IGF-1 R(ECD)~C3-TnuIgG IFc was expanded in DHFR selection medium and 2x10' cells were inoculated into 50 roller botöes each (Coming) in 250 ml ofnigb-glucose DMEM (Invitrogen). 10% dialyzed FBS (Invitrogen). Ix glutamine (Invitrogen). Ix Noc essetrdai amino aeids (Invitrogen). Ix sodiuni pymvate (Invnrogen). Medium was gassed with 10% CO^/balance air for 5 seconds before capping the roller botde. Roller bottles were kept at 37c C on roDer racks spinn in g at 0.75 rpm.
\Vhen cells reached approximately S5-9Q% confluency (after approxiinaiely 5-6 days in culture), growrh medium was discarded. cells washed with 100 ml PBS and 200 ml producrion medium was added (50 % DMEM (Invitrogen)/ 50 % F12 (Invitrogen), lx glutamine (Invitrogen), :x non-essential amino acids (Invitrogen). Ix sodium pymvate (Invitrogen), 1.5% DMSO (Sigma)). The conditionec medium was harvested and replaced at one week intervals. The resulting 30 liters of conditioned medium were fältered through a 0.45 um cellulose acetate fUter (Coming. Acton, MA).
Pmükation of hu IGF-3R(ECDK3-muIgGlFc
Tne resulting fjltrate rrom the conditioned medium was concentrated 20-fold using a spiral-wcund cartridse (molecular weieht cut-off = 10 kDa). then diluted 1:1 with 3 MKQ. 1 M alvcine. nH 9.0 to bring the final sali concentration to 1.5 M KCl, 0.5 M glyeme, pH 9.0. This sample was applied to. a rProtein A-Sepharose column (Amersham Pharmacia Biotech, Uppsala. Sweden) which had been equilibrated in 1.5 M KCL 0.5 M glycine. pH 9.0. The column was washed with 40 colurrm volumes of the same buffer. then eiuied with 20 column volumes of 0.1 M glycine-HCl, pH 2.S. Five-mL fractions were collecred and immediately neutralized with 1 mL of 1 M Tris-HCL pH 7.5. Fractions containing hu!GFiR(ECD)-C3-muIgGFc were identiüed by SDS-PAGE. pooled, and dialyzed against phosphate-bnffered saline. The yield was 2.4 mg/L of conditiened medium. Tne major protein species detected were the mature a and B chains and murine Fe. each of which appeared to be properly glycosylated based on theu elevated and beterogeneous molecular weights. Unprocessed IGF-1R(ECD), as well as glycosylated but nox proteolytically cleaved IGF-1R(CED); was also presert in ihe preparation. The shift in bands to higher moiecular weights under non-icducing condiiions indicates that disulfide linkages joined the q and ß chains. Ajnino-terrninal sequencing of the final produet mdicated that 60% of the protein "was correctiy pfocessed betvveen the a- and ß-ebains of IGF-1R(ECD), while 40% remained unprocessed.
EXAMPLE 3: Isolation of Human INSR(ECD)-rauIgGl
This example presents a method of cloning and expressing a solubls fragment of the human insu]in reeeptor. Cloning of pDSRa:huTNSR(ECD)-rnuIgGlFc

Pracrs 2830-40: 5' AGC^AGCi?TCCACCA'TGGGC.\CCGGGQGCCGG 3' SEQ XD WO: 259
(Bind HI Site ^mderlined) and 2830-41:
z' ATTTGTC^^T'TC^^CAAX^ 3' SSQ XD NC: 2 60
(5a/ I site underlmed) wer- used to grnplüy the hnman INSR extraceDiüar domain (i-929) from and INSR parmtEJ plamid gpeonfpg tbe B form of the INSR splice variant (Ullrich et al.t 1985, Nature 313:756-61; Ebss er aL. 1985. Cell 40:747-58). The priniers included a Kozak translation mitiaticro sequence preceding tbe scart codon and resciciion süss for snbsequeni sub-cloning. PCR was perfonned cm a PeikmEkner 2400 -JDÖCZ Ute fbBowiag caodiüons: 1 cycle at 95D C for 2 nun, 32 cycles at 95° C for 30 sec, 58.5° C for 30 sec and 72c C for 3 sinn, anc 1 cycle at T2V C for 10 min.' Final reaction conditions were IXpfu TURBO® bafer, 200 uM dNTPs, 2 iM eaefe primer, 5 Ü pfii TURBO® (Stratagene) and 10 ng template DNA. The PCR prodoc: was purxsee rsing a NUCLEOSPIN® Colirmn (BD Biosciences ClontecüL Palo Alta CA) accui;7";^ tc the r^znirtacmrer"s insiractkms, digested wirb //ind HE and 5a/1 (Roche), and gel puriSea mior to ligaüon into Hinä JK-SaJ I digested pDSRa-miilgG 1. Tbe integrity of the inserr was conSrmed by DNA secpffnring. The protem sequence of ibe INSR-muFc is sbown in Figure 11. Tbe final expression vector is described in Table 2. Table 2



Expression of hu INSRf£CDVC3-nmIgGlFc
AM-i/D CHOd- cells were transfected "v-itn 15 urn of üuearizsd expression vector pDSRa:huINSR(ECD) -mulgGIFc usrag FUGENE™ 6 üpofection reagent (Roche Diaenostics Corp., Indianapolis. IN), then cultured under condnions to allovv expression and secretion of protein into the cell raedimiL Colonies vvere selected and acahxed as descnbed above.
Pmificatira of ha INTSR(hCDVC3-jnnIgGlr:
Tne üliered condiüoned medium containiiLg biINSR(ECDVmuIgGFc was ccncentrated 17-fold using a spiral-wound cartridge (molecular weight cut-off = 10 kDa). then diluted 1:1 väth 3 M KG, 1 M glycme. pH 9.0 io bring the final salt ccncentraticn to 1.5 M KCL 0.5 M glycine, pK 9.0. This sample was applied to 2 rProtein A-Sepharose column (Pharmacia) which had been eqmlibrated in 1.5 M KCL 0.5 M glycine, pH 9.0. Tne column was washed with 40 colunm volumes of the same buffer. then eiuted with 20 column volumes of 0.1 M glycine-HCL pH 2.8. Five-mL fracrions were collected and irnmediately neurralized with 1-mL of 1 M Tris-HCL pH 7.5. FrsctioEs containing huINTSR(£CD)-muIgGFc were identifieä by SDS-PAGE. pooled. and dialyzed agarns: phosphate-buffered saline. Tne yield '^vas 0.9 mg/L of condjtioijed medium. Tne major protein species were the mature a and ß cbains and rnurine Fe. Fach of these species appeared to be propeily glycosylated based on its elevated and heterogeneous molecular

weight. Unprocessed IKSR (ECD) as weil as glycosylated but aot proteolytically cleavsd IXSR (CED) also was present in the preparatioiL The shift in bandi io higher molecnlar weights under non-rechicin2 conditions indicated that disnlfide linkages joined the a and 5 chams. Arrrntv-tw-mi-nal sec*jencing of the final product indicated that 37% of the protein was correctly processed between the a- and ;-±ains of INSRfECD). whüe 23% remained unprocessed-
EXAMPLE 3: Initial Screen for Anti-IGF-IR phage Fab
This example provides a metbod of identifying a^ti-IGF-lR antibodies.
A Target Quest Q Fab library ("the TQ library": Target QUSSL Maastricht the NecedsndsX -Ätdch was conSTTUCted using peripheral blood iymphocytes from four heairhy donors and splerne rymphocytss from one patient wirb gastric Carcinoma, w-as obtained. The library drvexsny was 3.7 x 20"" ZKSDSS, containing 3x20" heavy chains. The source. Screening methods. and charactexizanon of ±e irsary nsve been pnblisbed (de Haard er cd. !999: J Bio! Chem 274:1821S-30). Dynabeads (200 ul) M-50 Lbcoaad (catalog # 140.02. Dynal Lake Success. NY) were washed 3 rimes with PBS. resusoended m 200 LÜ of IGFlR(ECD)-C3-mFc tc a coBcerjrratiorj of 0.5 uM in PBS, and incubated at 4° C on z rotato: cvemdgirL The IGF~lR(ECD)-C3-mFc coatedbeads were washed 3x wiih 1 ml of 2% non-fät dry unjk : wim 1ml PBS/0.1% Tween 20. 2x with PBS with a change of tubes between different wash solunens. Bound phage was eluted with 1 ml of 0.1M TEA (pHll) for 10 minutes. then immediately separated frcm die beads and neutralized with 0.5 ml of 1 M Tris.HG. The eluted phage pool was mixen with 4 ml 2x TT brcrh (10 g yeast extract 16 g bacto-tryptone. 5 g NaCl per liter of watcr) and 5 ml of TG1 bacrerial c^dr^re (O.D. ;90 about 0.5) in a 50-rcl conical tube. The infection rnixture was incubate at 37° C in an inoibaicr for 30 min,, then centrifugec ar 3500 rpm for 20 min, The cell pellet was resusperjded in 1500 u! 2>:Y7-CG broth and 300 ul were spread on each of five 2xYT-CG (2>: YT broih containing 100 ug.'ml carberJ-rllir. and 2% gjucose) plates. After 20 hours of bcubation at 30° C. 4 ml of 2x YT-AG were addec tc zr_zh ciate and the ce'.Is were r&covered with. cell scraper from the plates. This step was repeated tiree rimes. A small oortion of the recovered celis was used for phage rescue (see below). The rernairiing cell suspensicr. was centrifuged at 3500 rpm for 20 min. Tne cell pellet was si'.srended ir.to an amour: of 50% glyceroi roughiy half the volume of the pellet size and stored at -S0° C.
In order to resene phage, the plated-amplified cell Suspension was used to inoculate 40 ml of 2x ^T-CG io an OD--^ of about 0.05. The culture was incubated at 37s C on a shaker to OD

carbenicillin and 40 ug/ml kanarnycir) and transferred 10 nvo 250-ml flasks and incubated at 30= C wrth sbaläng at 270 rpm for 20 hours. The over-night culture was centrifuged at 4000 rpm. for 20 min to removal cell debris. The centrifaganon was repeated to ensure the removal of cell dehns. About 3/5 volurne of PEG Solution (20% PEG 8000. 2.5 M NsCI) was added to übe supsrnatani to precipitate the phage rsanicles. The rnixture was incubated on ice for at least 1 hour; followed by centrifugation at 4O00 rpm for 20 n-Hn to collect the precipitated phage particles. The phage pellet was re-suspended into 1 ml of PBS and transferred to a microfuge tube. Tne phage Suspension was len on ice for 3 hour to allow compkte Suspension of phage particles. and clarüüeä by cenrrifiigation at 14.000 rpm for 2 mrn to remove übe residual cell debris. Phage precipiranon step was repeated- The frnal phage pellet was suspended into PBS after clarificatiorL The rescued phage Suspension was used in the next round of selection.
Four rounds of selection were performed that incinded alteraüoris of various Standard binding parameters. The second round ofselection was identica] to thefirst round ofselection. Variadons in rnpui phage number and elution reagent w-ere introduced in rounds three and four. For the round rhree selecricn. 5xl0!1 pfu ofphages were selected and bound phages were eluted erther with 1 uM IGF-I t'catalog = 13769, Sigma. St. Louis, MO) or with a 1 uM concentrarion of a chimeric aTR3-hurc antibody to yield rwo round-three pools, TQ4-3IS and TQ4-3CA. Round four selection was carried out on rescued phage pools from both round three pools. Two rounds of negative selection with mouse IgG Fc-coated DYNABRADS® (Dynal Biotech. Oslo, Norway) were includec to remove mouse Fe binders prior to actual IGF-1R sclccriorL Tne fficubation trme for negative selection was 30 minutes each. 3.78xlOn pfu of TQ4-3IS pool and 3.75xl0'2 pfu of TQ4-3CA pool were selected separately. Bound phage were eluted with \ uM IGF-2 (catalog # I2526: Sigma, St Louis; MO) to yield two rcund-4 pools, TQ4-4ISI2 and TQ4-4CA12. The sequence of about 96-192 phage DNA inserts was determined at each elution step.
In some cases, a secondary screen was done. Phagemid DNA mixtures of the total TQ library, and the selected phage amplifred afler several rounds ofselection against IGF-1R. were prepared using a DNA. Maxiprep kit aecording to the inanufacturers instruetions (Qiagen. Valencia. CA). All four DNA preparations were digested with Asc I and EcoR I (New England Biolab. Beverly. MA). The resultrng two Asc VEcoR I fragments were separated on preparative 0.5% agarose gels. Tue 2.1 kb n-agmenis c^ntaining heavv chains were gel puriäed from the IGF-1R selected phage. Tne 3.9 kb fragments coniainieg the light chams and pCESl vector poruon were gel puriüed from the total TQ library DNA. The 2." '^ fragments were ligated to the 3.9 kb fragments frum the DNA sample of TQ library in 3:1 ratio. The hgaiec DNA was precipitated and used to transform TG1 cells by electroporation. Tne library size of the resx:ec light chain shuftled secondary library was S.SxIO5. -After sequencing 96 randomly picked clones. 76 unique light chain sequence: were obtained. indicating that the artempt to shuffle light chains. was successfui.
The bindine;. washins anc elution condition for screenins the liaht chain shufLe librarv were essentially ±e same as decribed for the intial screen. However. several variations were included to merease selection pressure for amplincahon of IGF-1R binders with higher afnnities. especially those with significantly slower off-rates. Tnese parameters were: higher number of input phage {2-3.7 x 101" pfa), srnallerbead volume {,100 ul fer round one. 50 ul for round two. and 25 ül for round ihreei. and sxtended specific elution nme up to 20 hours. Elution buffers were 0.1 M TEA for round one (RD1), i uM IGF-1 in 0.4% MPBS for RD2 and 1 uM IGF-1 or IGF-2 in 0.4% MPBS for RD3. In RD2 and RD3; binders that

were eluted in 15 min or 2 hours were discaxded Eiun'on was continned and eluted phage-s were ccliected after 8-10 hours and agarn after 20 hcrars.
Phage Fab ELISA Screen
In 96-weIl 2-m3 deep-well blocks. 480 ui/well 2xYT-CG broth was inoculated wirb 20 ul of oveniight cultares of tbe incividual clones. then incubated at 37° C. 300 rpm for 3 hours. To each welL 50 ul of 1:3 diluted M13K07 hsiper pbage were addec to infect the cells. The block was incubated at 373 C without shaking for 30 iminrrtes- and then shaken gently for anotner 30 minutes at 150 rpnx The block was centrifuged at 3600 rpm for 20 mimnes ro pellet the infected cells. Tbe cell pellet in each ^=ell was suspended into 480 ul of 2xYT-CK (2xYT broth c^nlainiiig 100 ug/ml carbenicillin and -^0 ug/ml kanaraycin). and mcubared at 30= C ovemigbi fcr ahout 20 hours. Tne cell debris was separaied by centrifugation at 3600 rpm for 20 mimnes. The rescued pbage sroematant was used in the r-hage ELISA to check for IGF- 1R-specific. INSR-cross reactive. or mouse Fe binding cf mdividual clones.
Three seis of Nunc MaxiSorb Irnrnunoplates were coated with 100 ul/well of IGF-lR-C3-mrc ai 5 ug/mL rNSR-mFc at 5 ug-ml, or mouse IgGl (catalog #010-0103, Rockland. Gilberts ville, PA ) at 2 ug/ml in PBS. respectively. at 4= C oveniight. Tne coated plates were washed 3x with 300 ui/well of PBS. Tne wasbed plates were blocked with 300 ul/well 2% MPBS at room temperatore for one houx. Meanwhile. rescued phages of incividual clones were pre-blocked by mixing 170 ul of rescued phage with 170 ul of4% MPBS. The blocked plates were washed 5>: with 300 ul/well TBST (TBS: 10 mM Tris-HCL pK 7.5, 1 mM EDTA, 150 mM NaCl; Tween-20. 0.1%). 100 ul/well of pre-blocked phage dilutions were distribuled to each set of coated plaie. which were incubated at room temperamre on a rocker for 90 minutes. The plates were wasbed 5x with 300 uTwelI TBST. 100 ul/well of anti-M33-HRP in 2% MPBS (1:3000 dilutiorL catalog Dirmber 27-9421-03. Amersham Pharmacia Biotech) were distributed, and plates were incubated at room temperatore cn rocker for orje hour. The plates were washed 5x with 300 ul/well TBST. 100 ul/well of tbe Substrate 1-Step™ ABTS (Pierce Biotecbnology. Rockford- IL. catalog number 37615) were added. Plates were incubated for cne hour. OD-^ was measured for signal ceiection.
The phage displayed antibodies exhibited essenrially no crossrsactivity with the insulin reeeptor and murine Fe domaiu. Tse signal observed in the IGF-1R ELISA is therefore specific for the IGF-IR exTraceliular domain. Results trom sirnilar assays for four of the phage-displayed antibodies are shown in figure 14.
The DNA inserts of IGF-1R oositive, INSR and mu JgGI negative, clones were sequenced. Fifry-two umque Fab sequences were identiüed. having the following combinatjons of hght chain and heavy cham variable demain sequences: L1H1, L2H2, L3Hi; L4Fx4, L5H5: L6H6, L7H7, LSHS: L9H9, LI OHIO, L]1H11:L12H12SL]3K13:L14H14,L15H155L^
L22H22, L23H23, L24H24, L25H25, L26H26, L27H27, L2SH2Sr L29H29, L30H30, L31H3L L32H32, L33H33: L34H54.. L35H35, L36H36, L37H37, L3SH38, L39H39, L40H40, L41H4L L42K42: L43H43, L44H44: L45H45, L46H46, L47H47; L4SH48; L49H49: L50H50, L51H51, and L52H52. wherein "Lx" indicates light chain variable domain number "x" and "Hx" indicates heavy chain variable dornain number

"x." Figure 1 presents the polyrmcleotide seqnences of eacb of these light and heavy variable dornains. Figures 2 and 3 present me corresponding amino acid sequences.
EXAMPLE 4: Subclcdng cf VH and \\ into IgG 1 expression vectcrs
Tkis example nresenis a method of subcloniag the previously idenuüed variable domain sequences into an IgG 1 expression vector.
CoasCTctkm of i>D5Rc20 aad-pDSRg20:bIgGlCH
Tbe pDSRo20:hIgGICH expression vector (WO 90/14363) ^zs a derivahve of pDSR19:bIgGlQ-: isee ^S. Prcvisiana: Patent Application No. 60/370,407, filed April 5: 2ÖG2; "Human Anti-OPGL Xantrelrrrnc Antibodies A_s Selective OPGL Pathway Inhibitors." iacorporated herein by reference in its eriirrety). The pDSxexl9:hIgGlCH plasmid encoded a rat variable region-lmniEn constsnt region IgGl {rVhhChl}. Tbe plasmid v-,-as constmcisd by the three-piece ligation ofXba I and 3smB I terrnioated ra: antibody variable region PCR produet. the human IgGl consian: region (C:~:1, hinge. C>-: and CH3 doroains) derjved bv Sei I cleavage and gel Isolation of the BsniB I and Sai I fragment from the linear plasmid pDSRal9:higGi CH [Hinä TR and BSTKB I ends) and a linearized pDSRc!9 withA&a I and Sai I ends. pDSRoi20 was prodneed by changing nucleotide 2563 in pDSRal 9 fxom a guanosine to an adenosine by site dixected mntagenesis. Tne heavy chain expression vector. pDSRa20:hIgGlCH rat variable region-hmnan consiani region IgGl (rVh/hChl), is 6163 base pairs and contains the 7 functional regions describe-d in Table 3. Table 3
Plasmid Base ?aii Nrnnber



The linear plasmid pDSRo20:hIgG! CH ^*as prepared by digesting die pDSR20: rat variable region'nurnan constant regicn IgGl plasmid widi die restriction enzyrnes Xha I and BsmB I to remove ihe rat variable regicn and purified using a QIAquick Gel Extraction kit Tue linear plasmid pDSRa20:higGl Gr; ccntaining die 1.0 kb? human leG i constant regicn domsin \vas used ;o accept ann-JGF-1R variable heavy chain coding sc-querices.
ConsirjctioE of die and-IGF-lR IgGl Bea">" Chain Expression Clones
The sequence coding for tbe anri-IGF-lR variable region of tbe heavy cbains was arnpliSed dom phagemid DNA -A-ith cornpleirientary oligcnucleodde prirners. Primers for.ppI>Tnera.se chain reacdcn (PCR) were designed to incorporste a Hind TU site, Xha I site. Kozak sequence (CCACC) and signal sequence (translated pepüde is MDMRVPAQLLCTLLLLWLRGARC; SEQ ID NO:263) onto ihe 5r end of tbe variable region. while a BsiriB I site was added onto die 3' end cf die PCR proauct. The PCR products vvere digested widi Xha I and BsmB I. and üben c-cned into the Xha l-Bsmü I linear pDSRa20:higGlCH expression vector ccntaining ihe human IgGl consent regicn (Figure 13). The final expression veciors contained die seven runctional regions described in Table 4.


Constraction of ibe anti-IGF-iR IgGl Variable Cham E>ipressicn Clones.
The "iight cbains encoded in anti-IGF-iR ubüge were ejtber kappa or lambcla ciass. They were cloned using one of nvo approaches. CompleirieDtary primers wers desigced to add a Hinä TU site. au Xba I site. Kozak secuence (CCACC) and signal sequence {translated peptide is
MDMRVPAQLLGLLLLV^LRGARC, SEQ JD NO:264) were added to the 5S end of the ccKÜng regier Tbose chains that bad enror-free coding regions were clcaed as fuJ3-]engtb prodncts. Tbe full-lengtb light cbains were cloned as Xba I and Sal I fragments into tbe expression vector pDSRa20. T^t fr-a: expression vectors coniaiaec the seven funcnonal regions descnbed in Table 5.



Some kappa clones had errors in their constant regions wben compared to natural hrman constan: region sequence. To eliminate these diserepancies. tbe kappa variable region was amplified with a prirner that would introduce an Xba I site into tbe 5" end and a BsrnB I site ialo tbe 3' end. This fragmerri was tben ligated along wirb a buman kappa constant region (Figure 13) wim a compafible BsrnB I on the 5' end and a 3%Sal I ends into pDSRa20 with Xba 1 and Sa! I ends.
EXAMPLE5: Trarisient Expression of.-antibodies
Tois example provides a merhod cf transienrly expressing anti-IGF-lR antibodies.
The antibodies were expressed transiently in senirn-free Suspension acapted 293T cells. Al: transfections were performed as 250 mL cultures. Brieüy, 1.25 x 30' cells (5.0 x 10' cells'niL x 250 mL) were cenrrifuged EI 2.500 RPM for 10 mmutes at 4C C to remove the conditioned roediim. The cells were resuspended in semm-free DMEM and cenünfuged again at 2.500 RPM for 10 rmnutes at 4C C. After aspiratmg the wasb Solution, tbe cells were resuspended in growtb medium [DMEM/F12 (3:1) ~ Ix lnsulin-Transfernn-Seieniuin Supplement - IX Pen Strep Glut + 2mM L-Glutarnine + 20 mM HEPES - 0.01% Pluronic F681 in a 500 mL Spinner flask cultare. The Spinner flask culture was mainiained on inagnetic stir plate at 125 RPM which was placed in a humidified incubalor maintained at 37c C and 5% C02. The plasmid DNA was incubated with tbe transfection reagent in a 50 mL conical tube. Tbe DNA-transfecrion reagent compjex was prepared in 5% of rbe nnai culture volume in seium-free DMEM. One microgram cf plasmid DNA per müliliter of culture was irrst added to serorn-free DMEM, followeä by 1 ui X-TremeGene RO-1539/mL culture. The compIex.es were incubated at room temperature for approxirnately 30 minutes and tben added to tbe cells in tbe Spinner flask. Tbe transfection expression was perfonned for 7 Gays, afrer wbicb tbe conditioned medium was- harvested by centrifugation at 4.000 RPM for 60 minutes at 4" C.
If the initial transfection faüed to yield the required 100 ug purined antibody, those clones were re-expressed in roller bottles. These transfections used 293T 2dberent cells grov%n and maintained in DMEM supplementea with 5% FBS •*■ Ix Non-Esseutial Amino Acids -f Ix Pen Strep Glut -*- Ix Sodium Pyruvate. Anproximatelv. 4-5 x 10' 293T cells were seeded in a S50 cm* roller bottles ovemight Tne p-e'oously sesded cells were then transfectec the foiiowing dav nsing EUGENE™ 6 rrsT;sfection reagen:. Tne DN'A — transfection realen: mixtuxe was nrecared in annrcximatelv in 6.75 mL serurn-free DMEM. f 75 Li*; FUGENE™ 6 transfection reagent v,as fhrst added foilowed by 112.5 ug plasmid DNA. Tne compjex was incubated at room temperature for 30 minores. The entire mixture was tben added to a rolle- bettle. Tbe roller bortle was infused with. ^ 5D4 CO: gas mixture, capped tightly and placed in a 37° C incubalor on a roller rack rotating at 0.35 RPM 77ns transfection was performed for 24 bours afrer wbicb tbe medium was replaced with 100 mL DMEM - IXInsulm-Trsnsfemn-Selenium Supplement - IX Pen Strep Glu -r IX Non-Essemial Amino Acids - IX Sodium Pyruvate. Typicaüy. 2-3 harvests (100ml) were obtained from


EXAMPLE 6: Anti-IGF-JR Antibody Small-scale Purification
This' exainple provides a method of purifying anti-IGF-IR antibodies on a smaU scaie.
Conditioned medium was flitered mrough a 0.45 um cellulcse acetate Eiter and concentrated approximately 8-fold using a Vivaflow 200 50 K tangential flow membrane (Vivascience. Gc^ttimrem Germany). rProtein A SBPHAROSE™ Fast Flow resin (Amersham Biosciences. Piscataway. NTj was washed with phosphate huffered saline (2.7 mM potasshin} Chloride, 13S mM sodium Chloride, 1.5 mM potassium phosphate. and S.l mM sodium phosphate. pH 7.4) (PBS) four rimes then directly applied to the concentrated media. T~be amoimt of resin used was based on antibody concentr^tion determined by ELISA where 1 ul of resin was used per 5 ug pntTbody. The medium was incubated ovemight at 4C C with gemle agitatlon. Tne resin was cenmfuged ai 500 g ihr 10 min. at 4° C. Tne supematanr was decantsd as the unhound fraction. The resin was washed with PBS four ticnes for one miaute at roora temperainrs wifh gextle agitatiom each time coDecting the resin by centrifagation at 500 g for 10 mtn. at 4Z C. Tne smbody was eluted by mcubatmg the resin with 1.5 volumes of 0.1 M glycine pH 3.0 for 10 mm. a: roorr temperaruie. The resin was centrifuged at 500 g for 10 ~rrm- at 4~ C and the supemstant decanted as eluted antibody. Tne elution step described above was repeated for a total of three elutions; each time the ehited material was neutxaLized warn 0.04 volumes of 1.0 M tris-HCL pH 9.2. The sample was nltered through a 0.2 um cellulose acetate filter. Protein coTicentration was determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad Laboratories. Hercules. CA) as per the supplied Instructions using Human IgG (Sigma-Aldricb, St Louis. MO) as a Standard. The sample was compared ro a Human TgG 1. K Standard (Sigma-Aldrich, St. Louis, MO) using a 4-20% tris-glycine SDS Polyacrylamide gel (SDS-PAGE) gel stsined with Coomassie hriJliant blue dye. l\o contaminating protein was visible in tbese preparations.
EXAMPLE 7; Isolation of Stahle CHO Clones Expressmg Antibodies
This exarrrole provides a method for isolatjig stable CHO cell lines expressing anti-IGF-IR. antibodies.
Stahle expression of TQ1 IC. TQ25, TQ 58 and TQ59 IgGl was achkved by ec-traiisfecricn of AMI-D CHO cells (ü.S. ?at No. 6,210,924. incorporated herein by reference in its enüxcry) wim pDSRa20 heavy and light chian IgGl expression constructs. The plasmid transfeetions were performed using LF200Ö (lnvitrogen. Carlsbad. CA) according to the mamfactursrs Instructions. Briefiy. d v 106AM1-D CHO cells wereplated 24 hours prior to transfecrion. in 100 mm chameter FALCON™ plasnc petri dishes (BD Falcon. Franklin Lakes.. NT) in 10 ml of Dulbecco's Modiüed Eagles Medium tTnvitrogen) suppiemented with 5% fetal bovine seruni lx perdcillin-streptcmvcin and glutamine (lnvitrogen). ncn-esseimal amino acies (lnvitrogen). sodium pyruvate. and HT (0.1 mM so«iiumhyx'Cxanminr, 16 nM mymicine: lnvitrogen). Approximately 15 mg of each pDSRa21 - light chain and heavy chain plasmid DNA were linearized using P\nt I (Xew: England Biolabs) and dilüted in 2 ml of OPTI-MEM® (mvitrogen). The diluted plasmids were mixed with 75 ul of L3POFECTAM3NE™ 2000 (LF2000; GIBCO/BRL) düuted in 2 ml of OP7I-MEM-S1 and the mixture was incubated for 20 min at roomisrnperarüre. Tbe following day fresh growm medium was added. The cells were cultured in complete growth medium for 45 hours. ihen plated in HT- seiection medium in 1:20 and 1:50 dilutions. Approximately 2 weeks arter mmsfection. 12-24 visible colonics were picked inte 24-well plaies. using the sterile c]oning dises (RPI). The clones

expressing the highest level of TQ11C. TQ25, TQ5S and TQ59 IgGI ^:ere identified by westem - rrnrnunohlot analysis. To perform this assay. 0.5 mi of serurn free medium was addee to a Single-well corfluenr cells cultured in a 24 well plate (BD Falcon). The conditioned medium was recovered after 24 hr, arc 10 jii of CM TÄ"ES n±ced with an equai volurae of loading bufter to mn a 1 0% Tris-Glycine po'.vacn'lainics protein gel (mvitrcgeu). The gel was transferred to a 0.45 Jim pore size rdtrocellulose :scmhrane (Invitrogea). and westem biot analysis v%*as done using 1:1000 düuüon of gcat anti-human IgG ?c hnmnuoPnre antibody (Pierce Biotechnclogy, Inc.. Rockford. 3L) and ECL as detecäon agent
EXAMPLE 8: Niid-scak Expression of Antibodies
Th25 examoie rxoviäes a methoc of excressrng anti IGF-1R antibodies expressed bv stabie CHO csZ ünes.
Tbe CHG ceti hnes made accortmig ro Exarnp'ie 7 were expandec to T-175 tissue culmre Üasks '.Talcoc) far seale-tm expressioa A confluen.; T175 Üask (approximately 2 -3 x 107 ceiis) was nseö to seed I - I>0 crc2 roiler bcmies •'Corning Life Sciences. Acton, MA), and three conflueru roiler bottles •'approximately 1-2 x 1öS cells per roiler botde) vvere used to seed 30 roHers in 250 ml of high-glucose DMEM (Invitrogen j. 10% dialyzsd FBS (Invitrogen). Ix giutamine (Invitrogen). :x non-essenüal amino acies (Invitrogen), Ix sodiinn pyruvaie (Invitrogen). Medium was infused with 10% COvbaiance air for 5 secocds before capping the roiler bottle. Roller bottles were incubated at 37° C on roiler racks spinning at ü/ö rpnx
Wnen cells reacied approximately 85-90% confiuency (appioximately 5-6 days in culmre), the gjowrh medium was discardei the cells were washed with 100 ml PBS, and 200 rnl production medium wzs adde-d (50% DMEM flnvitrogen)/ 50% Fl2 (Invitrogen). lx giutamine (Invitrogen). Ix non-essential =mino acids (mvitrogerf;. Ix sodiurnpyruvate (Invitrogen), 1.5% DMSO (Sigma). Conditioned medium was harvested every seven days for a total of four harvests.
Conditioned medium was nitered through a 0.45 um cellulcse acetate filier and corcentrated anproximately lö-fold using a Sartorius Sartocon Slice Disposable 30 K Tangential flow membrane •v5srtorius A.G. GoetringerL Germany). The concentrared material was applied ro a IG ml rProtein A Sepharose column at -° C and the Howthrough was collected as the unbound fraction. The column was ■vashed with four colurnn volumes of PBS. The bound sarunie was elutea v.im approximately four column vciumes of 0.1 M glycine pH 3.0. Tne eluate peak was collected and neutralize-d with 0.04 volumes of 1.0 V ris-HCl, pH 9.2. The eluate was dialyzed against 150 volumes of PBS ovemight at4~' C. The sample was nltered through a 0.2 um cellulose acetate nlter and protein ccncentraticn was measuied by cetermining the absorbance at 28Gnrn using an extinction coefncient of 14,000 M-l. Tne sample was compared to a Huznsn IgGI, K Standard (Sigma-Aldricb. St. Louis. Missouri. USA) using a 4-20% tris-Eivcine SDS-PAGE gel stained with Coomassie brilüant blue stahx Endotoxin leveis in each antibody prepraücn was deterrnined using the P^Totell Limulus Amebocyte Lysate Assay (Associates of Cape Cod. Inc.. Falmouth, Ma) £S ner the suppHed Instructions.
EXAMPLE 9: OKI GEN" Dose Response Competition Assays

This example provides methods for testiag tbe ability of an antiDody to block ligand bindins to IGF-1R.
An ORIGEN* bindmg assay ^-as uscd to deterrnine whcther TQI IC, TQ25, TQ 58 and TQ59 IgG! antibodies could block bgand brnörng re TGF-IR using proccdures provided by tbe maniifacturer (Igen, Inc.. Gajthersburg, MD). To labe: IGF-1 and IGF-2 wirb nitbenitini lyophilized proteins were dissolved into PBS to give a 1.0 mg/n] sohaioa. Label (ORI-TAG-NHS ester from Igen, Cat # 110034) ■fc-as added to tbe proxein at a iDolar ratio of f: 1 (bbeL rrotsni} from a label stock of 5 mg/nü in DMSO. Tbe mberure was incnbaEed at room teirosxanire (20-221; Q ibr I hr in ibe darfr rben treated wirb 20 ui 2M glycine for 10 rm-n at room tenroerainre. The labekd protein was seosraied &om tbe free label by application to an .Arnersharn Bioscienees SXP-5 coinnm (Arnershari 3iosciences. Piscataway. NJ) equiiibrated in PBS and 0-33 ml Xenons coiieozed. Tbe prot^in concentration of ihe fractions was deteinibied by Micro BGA Protein Assay CPiexce 3knschnoiogy. Inc^ Rockford. IL). Fractions two and three contained signif cant protein and were corabined. Tbe araennt of incorporated ruthenium label "was assessed using tbe foUcwiag fonnuia: nithsrniroa tris-bipyridyi cornpound (Rufbpy);~~) labeling of IGF-1 and IGF-2.
Dynal M450 pararnagnetic beads ccaied wrih sbeep anü-moose IgG was used as tbe solid support phase for tbe IGF-1 R(ECD)-C3-rnuFc. Tbe M45Ö beads were prepared for reeeptor loading by washing three rirnes with assay buffer containing lx PBS, ö.05% TWfcfcJv™ 20 (ICT Aroericas. Inc., Wilmington DE) 0.1% BSA, 0.01% sodium azide. The IGF-1 RiTCD)-C3-rnuFc was bound for 1 hr at a ratio of 50 ng reeeptor per 1x10 M450 beads in a vclume of 25 ui assay buiTer. To generale dose response data, tbe antibodies or unlabeled IGF-1 and IGF-2 facters were sdded ai increasing concentrations (10"HM to lO^M) sinrultaneously with 1 n.M Ru-IGF-1 or 2 nM Ru-IGF-2. The final reacrion volume was 100 ul. After ineubation at room tenrperarare in tbe dark for 2 hr. an MS Analyzer (Igen) was used to rernove free rurbsniurn label ed b'sand and ceterrniae rbe anoount of bgand bonnd to reeeptor. The data were expressed as tbe percent cf total ligand boimc irrnrus backgroimd rernaiiiing afuer cernpention with excess unlabeled grewth IGF1 or IGF-2. Corrrpedrion curves weis generated wiüi GrapbPad Prism Software (GraphPad Software, San Diego. CA) using 2 smglc c-onrponent equilibirrum model. Essendally all (> 98%) binding was competed with excess unlabeled gro^.th factors. Tne positive control antibodies ia tbe binding analysis were tbe rnurine anti-IGF-IR andbodies olR5 (Calbiochem. San Diego, CA) or MAB591 (R&D Systems. Minneapolis. MNj. 24-57 (Biocarta, San Diego. CA) and 1E7 (Santa Graz Bioiechnology. Inc.. Santa Cruz. CAV The negative control aiitibodv was an anti-CD2ö antibody. Ligand compeution data ^re shc-v%7i in Figure 15. Tbe YÄ and maximum inbabirior. vaiues obsen'ed for IGF-1 and IGF-2 binding rsactions are iisted in Table 6.



EXAMPLE 10: SPA Dose Response Competition Assay
This example presenis a scinnilation proximny assay (SPA) for assesessing the sfea of znnbocües on nie interaction of insulin (INS) with the insulin receptor (INSR) and of IGF-1 and IGF-2 zz IGF-IR.
1GF-1R binding reactions fcr TQ1 IC. TQ25. TQ 58 and TQ59 IgGI aaifoodies osj=z^ed 1T ?BSr 0,05% TWEEN® 20 (Maiünkrodr), 0.1% ESA (EM Science, GfobstowiL Sil 50 ng IGF-IRj.ECD}-C3-muFc; 500 ug SPA PVT anti-mouse IgG üuoroirncnospberes (Amersham) and li5T-Iabeled IGF-', er IGF-2 obtained from Amersham at a final concemradon of 0.64 nM. The total reacton volume *^s 1 Oö LÜ_ The INSR binding reactions were idendcal except they coniained 50 ng DsSR(ECD}-muFc and O.f— nM *2^-INS (Amersham). Receptor was loaded onto SPA PVT microspberes for ih at room temperEtnre prior to assembly of the binding reactions. To generale dose response data, antibodies or uclabeled growth factors were added at increasing concentrations (10"JiM io 10" M) simuitaneously with "T-labeiec growth factors. Essentially all binding was competed wirb excess unlabeled growth factors. Tne reeeptor-inäependent background, caused by random y Stimulation of the SPT PVT rmerospheres. was less tban 0.5% of the iormi :"I cpm. The data were expressed as the pereem of total Hg and bound minus background rernaining efter competinon with excess unlabeled growth IGF1 or IGF-2. Compen'tion curves wert generaicd wiib GraphPad Prism Software using a siegle cornponent e-qtülibrium modeL
EXAMPLE 11: Antibody Binding to IGF-1R
This example provides a methoc of deteeting the binding of an anti-IGF-lR anrfoocy to IGF-1R.
BIACORE® 2000, sensor chip CM5: Surfactant ?20, HBS-EP (lOmM KEPES. 0.15M NaCL 3.4m_M EDTA: 0.005% P20: pH 7.4): amine couplbg b't, lOmM acetaie pH 4.5 and K'niM ^ycine vH 1.5 all were purchased frern BIACcre, Inc. (Fiscataway, NT. Phosphare-bnnered saline (?BS. IX. nr- calcium chJoride. no magnesium chloride) was fron Gibco. Bovine serum alburnin fBSA. rracucr: v. IzG free^ was from Sigtna. Recombinant Protein G ("rProtein G") was from Pierce BiotecbncJogy.
Irnrnobilizanon of rProtein G and IGF-:R-C3-nrjpc to the sensor chip surfacc was perfonned aecording to manufacturers instracticns; using a contmuous flow of lOnaM HEPES. 0.15M NaCl. S.^nxM EDTA, 0.005% P20. pH 7.4 (HBS-EP buffer). Brief]"/, carboxyl groups on the sensor chrps's surfaces were activated by iniecüng 60 vi of a mixtuie containing 0.2 M N-ethyl-N'-(ch^ethylarninoprcrcyIy2arbodiiiriide (EDC) and 0.05 M N-hyarcxysuccinirnide (NHS). Specific surfaces were obtained by irrjeering rProtein A (Pierce) or IGF-lR-CZ-mFc ciluied in i OmM acetate, pH 4.5 at concentrations benveen 20 2nd 50 iig;'rrJ. Excess reactive groups on the surfaces were deaethared by injeeting 60 iil of I M exhanoisrrüne. Final immobilized leve-s were 5.000-6,000 resonance units (RU) for the Protein G surfaces. end -7.SO0 RTJ for

the IGF-]R-mFc surfaces. A blank, mock-coupled reference surface was also prepared on the IGF-IR-mFc senscr chip.
Tne leinene analysis of the Lnteracnori between IGF-:R-mFc and antibodies was perfonned as follows. Antibodies as well as a positive conrro! antibody (ann-IRS-CDR-huTnan-nioiise ehimera) were diluted in PBS -i- 0.0ö5% P2G + 0.1 mg'ml BSA and injected over the Protein G surfaces to Captine the antibodies. IGF-lR-mFc was diluted in PBS + 0.005% P20 - 0.1 mg/ml BSA fjom 500nM to 3.9DM, and eacb concentranon was injected over the capmred antibody surfeces. as well as over a blank Protein G surface for background subtraction, After a 10 THrrture dissociatioiL eacb surra.ce was regenerated by injecting 1 OmM glycine. pH 1.5. Kinetic analysis of rhe resultmg sensorgranis was performed using BIAEvaluation, v. 3.2 (BIACore. Inc.).
A Solution affinity analysis was done by incubating rwo different concentrations (0.2nM and InM) of antibody wiih varviag concentrations (0.0InM to 50nM) oflGF-lR-mFc in PBS + 0.005% P-20 - 0.1 mg/ml BSA. Incubations were done at room lernperarure for ai least f ve hours to allow sarnples ro reacb equilibrium. Sarnples were then injected over the rmmobüized IGF-lR-mFc surface. After the sample injection. the surfaces were regenerated by injecthg 25 ul SmM glycine. pH 1.5. The binding signa.1 obtained is proportional to the free antibody in sohrdon at equilibriuxn. Tne dissociation equilibrinni constant (Kp) was obtained frorn nonlinear regression analysis of the competition curves using a dual-curve one-site homogeneous binding model (KinExA Software v. 2.3. Sapidyne Instruments Inc.. Boise ID). The data are shown in Table 7

EXAMPLE 12: Epitope Mapping A^din-Fusion proteins
This example provides a method of deteirriiniiig the epitope of IGF-1R bound by zn anti-IGF-": "R antibody.
The subdomains of IGF-IR bound by antibodies TQ1 IC TQ25: TQ58: and TQ59 were determined using avidin-IGF-lR fusioD proteins. To express each protein the coding DNA secuenc-es of nie complete IGF-1R(ECD) was cloned into the expression vector pCep4-avidin-C such that chicken avidin sequence is joined to the C-terrnixius of the expressed IGF-IR protein. The ECD coding sequence (1-932) was PCR arnpliSed from a parental IGF-IR plasmid using PCR primers 2804-25: ■ 5' GCAAGC-TTGGa^QAAATCTGCGGGCCAG 3' SDQ ID KG: 265
and 2S26-6S: 5' An-GCGGCCGCTTCATATCC-XGT-TTTGGCCTG 2' SEQ ID NG:2c6

The primers include a 5* Hinö. TU site and a 3 * Not I site for cloning in:o pCep4a\-idin-C. Th? amiBO acid sequence of the avidin-hmnanIGF-IR(ECD) fusion proiein is sho^-c in Figure 12. The IGF-IR subdomains constracts used for epitope mapping included: LI (1-151). CR (152-298). L2 (299-4611, FnTTT-1 (461-579). FnHI-lTD (580-79$), FnIII-3 (799-901). L1^CR-L2 (1-461). and LI-CR (1-298). Tae an±io acid coordinates of the IGF-IR subdomain rspresented in each expressicr plasmid are gircn in parentbcsis. The coding sequence of each domain -wzs PCR ainplinec from a parental IGF1R cDNA clone using the following primer pairs: LI:
2804-25: (SEQ ID KO:265) 2804-19:
5' A^GCGGCCGCCCCACATTCCTTTGGGGGC 3 ' SEQ ID MC: 257 CR:
2S04-3S:
5' AGCAAGCTTGGACCTGT-GTCCAGGGACC 31 SEQ ID NO: 2 68 2804-20;
5' ATTGCGGCCGCGCAAGGACCTT'CAOIAGGG 3' SEQ ID NO: 2 69 L2:
2804-39:
5' AGCAAGCTTGCCGAAGGTCTGTGAGGAAG 3' SEQ ID NO: 27 0 2S04-23:
5' ATTGCGGCCGCACTTTCACAGGAGGCTCTC 3' SEQ ID MO: 271 FnUI-1: 280S-0S:
5' AGCAAGCTTGGACGTCCTGCATTTCACCTC 3' SEQ ID NO:272 2804-52:
5' ATTGGGGCCGCGG7X3CGAATGTACAAGATCTC 3' SEQ ID NO: 273 FnIII-2+IB: 2804-41:
5' AGCAAGCI-TGAATGCT-TCAGTTCCTTCCATTC 3' SEQ ID NO:2~4 2804-51:
5' ATTICGGCCGCAGTCCTTGCAAAGACGAAGTTG 3' SEQ ID NO:275 FnlII-3: 2g04-^2:
5' AGCAAGC7-TGATGCCCGCAGA_AGGAGCAG 3' SEQ ID NO: 275 2304-50:
5' ATTGCGGCCC-CTTTAATGGCCACTCTGGT-TTC 3' SEQ ID NO: 277 LI-i-CR-L2: 2S04-25:
5' AGCAAGC7CGGGAGAAATCTGCGGGCCAG 3' SEQ ID NO: 27 8 2804-23 (SEQIDNO:272)
LKCR:
2604-25: AGC AAG OTT GGG AGA AAT CTG CGG GCC AG {SEQ ID NC:279}

2804-20 (SEQ TD KO:270)
Tne primers included Hind HL and Not I site for cloning as described for the IGF-IR (ECD1 Tne IGF-1R subdomams were clonsd into the expression vector pCep4avidin-N such that chicken a vi dir secusoce (wi± endcgerious signal sequence) is joined to the N-tenninus of The expressed IGF-IR. nrotsins. Expression of each avidin-fusion protein was achieved by transient transfection of human 293-EBNA cells (Invrtroger) in roller botties cultures. Tue cells were grown and mamtained in DMEM supplemented wirb 5% FES - Ix Nac-Essentiai Ammo Acids + lx Pen Strep Glut - Ix Sodrum Pyruvate. Approximatsry 4-5 x 1 ö 293-EBNA ceils were seeded in B50 cm' roller botties ovexoight Tne previously seeded cells were thsn transfeosd -wirb pCep4-avidin plasmid DNA the following day using FÜGEST™ 6 transfection reageaL The DNA —transfection reagent rnixture was prepared in approxnnately in 6.75 xnL sertun-free DMB.;_ 675 dlFUGENE™ 6 transfecdon reagenr was nrst added. folicwed by 112.5 ug plasmid DNA. The compiex "Ä-as hicubaied at room temperature for 30 rninutes, Tne enüre rnixture was ihen added to a roller bettle. Tne roller bonie was gassed wirb a 5% C02 gas rnixture. capped nghtly and placed in a 37c C mrubaior on i rolier rack rotating at 0.35 RPM. Th? transfection was perfonned for 24 hours after which rhe medium was replaced wiih ICK) mL DMEM 4 IX Iimlm-Transferrin-Sekniurn Supplement 4- IX Pen Strep Ghi - IX Non- Essen tial Arnino Acids - IX Sodium Pyruvale. Harvest of the condition medium and rspiaesmeiLi with fresh medium occmred 48 hr intervais (2-3 cycles). The harvesred serum-free condiöoned medium was pooled together and clarifled by centrifugarion at 10.ÜG0 x g for 30 rninutes at 4° C.
Tne coneenrraticn cf avidin-fusion in each conditioned medium was deterrnined using a quantitative FACS based method. The avidin fusion protein in 200 ul of conditioned medium was captured by mcubatJon fer 2 hx at room teraperamre with 5 ul (~ 3.5 x 10 j of biotin coated polystyrene besds iSpherotecL Inc.. Liberiyvük. IL). The conditioned medium was removedby three cycles of centrifdgation andresuspension cf the avidm-coated beads in PBS contarrung 0.5°/? BSA (BPBS). The avidin-heads were stained with 1 ug/'ml of goat FITC-labeled anti-avidin antibody (Vector Lab Burlingame. CA) in IrrJ BPBS. After 0.5 hr ineubahon antibody-beads oomolexes were collected by cenrnfugadon at i S00 mm for 5 min and the teilet was washed three times. The FITC fluorescence was detected with a FACSCAN (Beckion Dickson Bioscience. Franklin Lakes. NT). Tne signai was converted to protein mass using s Standard eurve derived with recombinant avidin. For epitope mapping the bietm-beads were loaded v-ritb 50-IOC ns avidm-fusionDrotein pei~3.5 x 10' beads of beads by ineubarien with the appropriate amount (I -20 mi'i of conditioned medium The loaded beads were washed extensively and resuspended in Im] 3FBS. rcr all experiment the biorin-beads were blocked w;th 10% BSA in PBS prior tc loa ding fusion prctein-
Method /_, One Color Assay: Biotin-coated polystyrene beads loaded with IGF-IR (ECD) and IGF-IR snbdomain fusion proteins v.-ere mixed with 1 ug of anti-IGF-lR antibody in I ml of BPBS. After ineubation for 1 hr at room temperature. 4 ml washing buffer was added and the antibody-beads complexes were collected by centrifugarion for 5 min at 750g. The pellet was washed 3 rirnes by resusnerisien in 4 ml of BPBS. The antibody bound to a\ddin-bead complexes was detected by treatment \vith 0.5 ug-'ml Phycoervthrin-(PE) labeled goat anti-human F(ab5)2 (Southem Biotech Associates. Inc., Birmingham, AL) in 1 ml BPBS. Testen antibodies were found to bind to the awidin-fusion protein contairhng the completc

IGF-IR ECD and the L2 dom?^'n Eineiig ro LI. CR or FnJH-1 was not detected in this exr^rirnent A relative!}* weak reaction was also observed "with the LI domain,
Mezhod 2. Tvso color asscr.-: To sirnultaneously raonitor the amounts cf anti-IGF-lR monoclonal antibody and a\idIn-fusion bound to btotht-öeatis. Wj C-laheled anti-a\idin antibody "was included (1 ug/ml) was included in ihe binding rcarrxH: in combination witb 0.5 ug/rru PZ-labelsa goat anti-human IgGl. The beads were prepared for FACSCAN analysis as described for the one color assay.
Method 3, Antibody Compe^nor,. To tüeuaie for bbsüng wirb fhiorescein the antibodies were diaiyzed or resuspended at a conceznisrics of ! ZDET-IÜ* in ?3S {pH ES). Label ([6-fhiorescein-5- (and-6)-caiboxairrido] hexanoie aeid, suatinnnidyl estex 5{6VSFX] mdsed isomers from Molecular Probes (Eugene. OPv Cax No. F21SI) was adtied. to the LuutsLi ai a molar ratio 9^5:1 (label: protein) from a label stock of 5mg/ml in DMSO. The Tnixnire w-as incnbaied at -~ C ovgzdght in the dankt The labeled antibody was separated from the free label by diah-ss m P3S. The FTTC annbod>* rares obtained ranged from 3 to S. For each competition experhneni a hinähig rsactics: "»"as assembiec that contained a 50 fold excess (10-50 ug/ml) of tmlabelec competiter antibody. :.5s I(r bkrtin beads coared whh avicin fusion protein in BPBS. The FITC-labeled antibody (1 ug'mJ) "*"as added afer a 30 nnn preineubation. The process followed the one coloi method from this point forwarti.
£ach of the four tested antibodies binds to the IGF-IR L2 dornaiiL as shown in Table S. Howevex. the precise amino acid'contacts of each antibody in the IGF-IR L2 domain may differ.

EXAMPLE 13: -Antibody Binding :o Celi-Snrface IGF-IR
This example provides a method for detecting the binding of an anti-IGF-IR antibody to cell-surface expressed IGF-IR
The ability of antibodies TQ1 IC. TQ25. TQ58. and TQ59 to bind to human IGF-IR displayed on ihe cell surface was evaluated using Balb/C 3T3 fibroblasts and MCF-7 human breast cance: cells engineered to overexoress the human IGF-IR reeeptor at a level of —3-4 x 10"' moiecules t>er cell. A Balb/C 3T3 cell line that stahly overexpresses the human IGF-IR (~ 3 xlO5 reeeptors per cell) was derived using ^"ith a retre viral vector essentially as described by Pietrzkowski et al. 1992. Cell Gro*-vth Differentiation 3:199-205. MCF-7 breast cancer cells that cveiproduce huiGF-1R were transfected with apcDNA3.1 exrression vector (Tnvi trogen Corp.}. Zeocin resistant cells that express a high level of hu IGF-IR (—4 x

105 receptors per cell) were expanced aner selection by FACS using anti-IG?-" R monoclonal artibodv aIR3 and an PE-labeled goat anti murine IgG antibody (Caltag Laboratories. Bvrrirrigarne, CA). The process of selection and expansion was repeated four times.
IGF-1R Receptor antibody staining and receptor expression was rrornhored by ?ACS as feile ~_s: rhe cells were released froin T175 üasks (Coming) by washing 2 times with ex^ss ?3S (Ca/Mg free.; foliowed by treatment with 5 ml of Cell Dissociation Buffer (Sigxna) for 10 rrrm zi racm tenperature. The cells were collected by centrifugaticra and washed two times by res~ospendnig thss tn PBS aüti c^ntriiagation. For primary antibody staining. 1 ug of antibody was added TO 10* celh resasDendeti in 100 ul PBS plus 0.5% BSA (BPBS) and ine cells were incubated at 4C C for 1.5 irr. Tbc cells -Ä-ST^ collected c> eentxifiigation and washeti rwice wirb BPBS to remc^e unbocnd primary annbodv. The cells -=?ere resuspended in 100 ul of BPBS and inccbated with 1 ug of FTTC-Iabeled gGat azsi-icssD F(ah*)2 (Southern Biotecbnology Associates. Inc., BirininghanL AL) ar 4fC for 30 nhiiures. Aj~er -s-ashisg to rernove unbound FITC secendary antibody, ihe c-ells were res-jsperaded in L ml of PBS* OjfVt ESA and FITC cell fluorescence was detected with H FACSCAN (Becktori Dickson Bioscience, Franklin Lakes. XI». The fluorescence levels were ecnverted to absolute reeeotor levels using Quanrnzn rrncrobead rBangs Laboratories. Inc.. Fishers. IN) wirb predeternained IgGl binding capacrry :o generale 2 Standard cirr.e. Data retiucüon was performed with QuickCal v2.1 Software fVerity Software House. Topshain. ME") provided by the manufacrnrer.
The peak fluorescent intensity of anti-IGF-lR antibody labeling of the TGF-I3. cverexpressors was increased 10-20 fold relative to parental BaFo/'C 3T3 arjd MCF-7 cells for each of the t^sted antibodies. Tkis is the result predicted for an antibody iba: speciücally binds JGF-1R. Backgrounc fluorescence of cells tre2ted wirb no antibodies or FITC-Iaheled secondary alone were insignificant.
EXAMPLE 34: Inhibition of IGF-1R
This example presents methods of detecring Inhibition of IGF-1R by anti-IGF-lR antibodies.
32D huIGF-IR-IRS-1 Cell Inhibition
Murine 32D cells that ccexpress the human IGF-1R receptor (20K per cell) and bunzan IRS-1 have nroven 10 be a affective System tc exarnine the molecular components 1GF-IR siinabng Valentinis er -JL. 1999. J Bio! Chem 274:12423-30. Normal 32D cells express relative]}- low levels of rhe murine crthoiogs of these two gene prcducts. 32D cell normally required 1L3 for growth and surv;\ al. IGF-I or IGF-2 ean replace IL3 in 32D huIGF-JR-tIRS-I cells as showr. in Figure 16, panel A. Tnt EC-- :c 'he IGF-1 d:^se response curve was abcut 0.5 n.M. wbereas the IGF-2 EC50 (2.8 nM) is about six fold higher retlecting weaker cfüniiy of IGF-2 for IGF-1 R. To assess the ability 01 rhe antibodies TQ1 IC. TQ25; TQ5E; and TQ59 to block IGF-1 or IGF-2 Stimulation. 96-well micrctitre plates were seeded with 3Q:öö0 22D hu IGF-1R-MRS-1 cells per well in a volurne of 200 ul of RPMI (Gibco/BRL) containing 5% fetal bovine semm (Gibco'BRL) and Ix penicillin. strepromvcin. glutamine (Gihoco/BRL) and increasmg concenL-aricT- of antibody (10"l2M io lO'^f) or 30 antibody. IGF-1 (2 nM). IGF-2 (S ruvl) or noih-ing was added aftt;r 1 'r,r preincubation with antibody, "li-thyrnidine (1 uCi per well) was added at 27 hr nosi-annbod}' Eddition. The cells were harvesteü 21 hr later, and incorporation of "H- rh\Tnidine into DNA was deterrnincd fcr each

sample. The assays were performed in triplicate. An anti-CD20 antibody was used as a negative control. Each of anfcbodies TQ!*C TQ25: TQ5S: and TQ59 was abie to completely block the IGF-1 and IGF-2 mediated Stimulation of the 32D cells. The reduction of background proliferation in ihe absence of added IGF-1 and IGF-2 is dae to the irthibhion of senim IGF-1 and IGF-2. The binding data were analyzed using GraphPad PRIZM™ Software. The data are shown in Figure 16.
Balb/'C 3T3 hu IGF-1 R Cell Inhibition
IGF-1 greatly stirnulates tne mccrporarjon of ""H-thymddine by senrm-starved cuteres ofxoouse embryonic fibroblasts (Balb/C 3T3 or NTH 3T3) that cversxpress IGF-1R (-1 x 10ö IGFlRper etil). Kate et al. 1993, J Biol Chem 268:2655-61; Pienzkowsjd et d... 1992, Cell Growth Difereiriaticii 3:199-205. This phenornenon is recaprtuiaied withboth IGF-1 and IGF-2 in a Balb/C 3T3 cell line hn IGF-1R overexpressor. Both growth factors srrmnlaföd ^H-thyuddine incorporadon by aboia 20-fold. The EG* rf the IGF-1 dose response curve was aboui Ö.7 nM. vsiiereas the IGF-2 ECv, (4.4 nM) is sevenfoie higher. indicating a weaker aföniry of IGF-2 fer IGF-1R. To ass&ss the ability of a given antibody io block IGF-1 or IGF-2 Stimulation, 96-well rrhcroütre plates were seeaed with 1Q.OÖ0 cells per weil in a volume of 200 ui of DMEM (Gfbco/BRL) containing 10% calf seram (GIbco/BRL) and 1 x penicillio. streptomyciiL glutarnine (Giboco/BRL). After ovemight ineubation wben the cells were about 80% confluent the growth medium was replaced with 100 UJ DMEM contaimng 0.1% BSA after wasbing once with 200 ul PBS. Antibodies at increasing concentrations (10_1"M ic 10"6 VI), orno antibody, were added at 24 hr post-semm starvation. IGF-1 (2 nM). IGF-2 (S nM) and 3H-thyrnindine Q uCi per well) were. added after a 1 hr ■ preineubation with antibody. The cells were harvested 24 hr later. and incorporatien of "TI- th\Tricine info DNA was deterrnined for each sample. "The assays were performed in triplicate. Each tested antibody was able to completely block fhe IGF-1 and IGF-2 mediated Stimulation of Balb/C 3T3 cells: as shown in Figure 17. An anti-CD20 antibody was used as a negative control (UCD20T' m Figure 17).
Each reference cited herein is incorporated by reference in its entirety for all that it icaches and for allpurposes.





























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Patent Number 258082
Indian Patent Application Number 3203/CHENP/2007
PG Journal Number 49/2013
Publication Date 06-Dec-2013
Grant Date 29-Nov-2013
Date of Filing 20-Jul-2007
Name of Patentee AMGEN INC
Applicant Address ONE AMGEN CENTER DRIVE , THOUSAND OAKS, CA 91320-1799
Inventors:
# Inventor's Name Inventor's Address
1 CALZONE , FRANK, J., 841 PINE CREST CIRCLE ,WESTLAKE VILLAGE ,CA 91361
2 DESHPANDE , RAJENDRA , V., 168 DAYBREAK CIRCLE , THOUSAND OAKS, CA 91320,USA
3 TSAI, MEI-MEI 91 EAST GAINSBOROUGH ROAD, THOUSAND OAKS, CA 91360 ,USA
PCT International Classification Number C07K 16/22
PCT International Application Number PCT/US05/46493
PCT International Filing date 2005-12-20
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/638,961 2004-12-22 U.S.A.