AU2016202118A1 - Carrier immunoglobulins and uses thereof - Google Patents

Abstract

Disclosed is an isolated antigen binding protein, such as but not limited to, an antibody or antibody fragment. Also disclosed are pharmaceutical compositions and medicaments comprising the antigen binding protein, isolated nucleic acid encoding it, vectors, host cells, and hybridomas useful in methods of making it. In some embodiments the antigen binding protein comprises one to twenty-four pharmacologically active chemical moieties conjugated thereto, such as a pharmacologically active polypeptide.

Description

- 1 CARRIER IMMUNOGLOBULINS AND USES THEREOF The present application is a divisional application of Australian Application No. 2014200459, which is incorporated in its entirety herein by reference. [0001] This application claims the benefit of U.S. Provisional Application No. 61/210,594, filed March 20, 2009, which is hereby incorporated by reference in its entirety. [0002] The instant application contains an ASCII "txt" compliant sequence listing submitted via EFS-WEB on March 19, 2010, which serves as both the computer readable form (CRF) and the paper copy required by 37 CF. R. Section 1.821(c) and 1.821(e), and is hereby incorporated by reference in its entirety. The name of the "txt" file created on March 18, 2010, is: A-1537 WO-PCTSeqListO3l8lO-368_ST25.txt, and is 545 kb in size. [0003] Throughout this application various publications are referenced within parentheses or brackets. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. BACKGROUND OF THE INVENTION [0004] 1. Field of the Invention. [0005] This invention relates to carrier antibodies to which one or more pharmacologically active chemical moieties can be conjugated for improved pharmacokinetic characteristics. [0006] 2. Discussion of the Related Art. [0007] A "carrier" moiety refers to a pharmacologically inactive molecule to which a pharmacologically active chemical moiety, such as a non-peptide organic moiety (i.e., "small molecule") or a polypeptide agent, can be covalently conjugated or fused. Effective carriers have been sought to prevent or mitigate in vivo degradation of pharmacologically active moieties by proteolysis or other in vivo activitydimiishin cMhen modtications of the phArmacoiogicaiiy active chemical moiety, or o0 reuce renal clearance, to enhance in vivo half if or ofher pharmacokinetic properties of a therapeutic, such as increasing he rate of absorption rducing toxicity OPrimmun~ogen ieity, Uiproving solubiliy and/I'1or increingli man uacturabiy or sra abity, compared to an uncougated form of the pharmacoiogiealliy active moiety. [00 ] Exmles of uch carnes moieties that haine been employed m the pharmaccuticai i dustrv incude poyhyblene gatycol (see. C 11amg et al, Srvthiopoietin conuattes utWlh polyethylene ly col, W 0 01 2 0 21 immunoglobulim F' oua se e g Fez er aL -Modihed perndh is th apcutie emts, US Patent Nom u60>i 3 P uman serum albumin yee, o Row'n ta Albumin fusion protems US Patent N9 2S6,,S and U'S 2005 104051, Rtidon et aL Protecton of endogeous therapeute peptdes from vetidase acmnntv thr ough conjugauton vo bHood components US (8817, tramsthyetin ocCe .Wakit at. U se ot'transth reto tpqpdcproten fusions to mntease th serut halt-ide of pharmacologicailasin. e pfrdes/Proteins US 00301A 0 i At 160/00100 \A I or ths roxineatibuig globulim, or a cormboauon such as irmmmoglobul it cham, beaxv cain) ad e domain (the heterotrimene combuntion a socalled "hemibod A for eumple as decried in Sulvan or al. TOxo Pept 'bmpune Agent\, P( I US2007702831, published as WI) 200:8i0:842 Phannacologicallv actve moieties hee also been conjugaed to a peptide or small molecule that has an affinilf a long h scram protein. (See, 0.0. Mlancy cl al., Method and compositins for imereasing the erum halflife of pharnmacologicaly adiv agens by binding to transth\ ieli-n.selectA gandy US Patent No 5,714 142 Suto et al , serumn albummhinding moieties US ~'00 3 '00&3 Al1: Jones ct al , Pharmacentea acive e ionjuagates, I \ Patent % .. 4.25). [0009] F'ischer a A decrihed a pt deimnogr otdi t iige n whic nmnoglouh consisted of two heanv chains o oe ciand two iht ehatus in ie the imunoglobuainas notanionabiitolobid in (Fschr ct aL, A peptid" imuogiobulin cornat WO 2(00O4$463 A I.V Qili xet nvno rw~ are nnn~o3 f. i1dn exeiw n bmivadefcecyo xpeso. nvtoSdit SnM1MA~y N 'RENN N [0011] The invention relates to antigen ending protein. The inventive antigen binding proteins, including antibodies and antibody fragmnents, have reliable exp ression and purnfcation character iNtc Re suting. in products that are stabko and relatively uniform, and have outstanding pharmakokineic (P>K) properties in rats and cynomnogous monkeys. The inventive tige bin'ding proteins are toundi to speciftically hind to dinitrophenol (DN?) or keyhole limrpet hemocynanin (1KL-I). but have not been detected to bind to human proteins. cells or isue. Those antigen binding prioteins can be used for many purposes, including, but not ited to, quality control or analytical standards for amibody-base d drogs and as controls for biologically relvn t o~t-atched an tibodies [0012] in some emibodimients. the antigen hinding rotein of the present iention uwed as a camer tor phammeologicaiiy ae chcrtmal aoies, ae y small molecules peptides and/or proteins to enhance their la pK perties The phamacologicaly actie moeties ca be conmgated, se ,oralently bound, to the inmeum an iumaunoglobuin by a chenical comiuuton trae un, ro ffuw vh reconmbmant genetic expression, they can be fbsed to the antigen binding protein. [0013] The inventon also provide raterias amd method foi producing such imventiv e imnunoglobul ins mneludng iohted nuclei idsO' that eedoel 'hn ector e and I:olated host cWIls, and hybridonas Also prded are ioated nucleic acids encodmg' any of the imm umoglobul in heavy and/o W ght chan sequences anidtot YR and of N INequences addor COR sequences disclosed, here C, ah reiated embodimemn an ex presstoa vector .. mpism any ot'the aitementioned nucicic acids is provided. In stil another embodlment a host cell s provided convprising ny of the atoremioncd nuclei, acids or ex resin vectors [0 ]4 The inventive immung~lobui cdan be ue d in the nianufaculre of'a pharmnacetica cmpostiln ornmedicumnt.71he inventie pharmaceutica corriposition or nmedicanient comprises the nmwnnotjliti~ cninagatod wih i hateacologinaungent, and a pharinacticidl acgaledvtari phaineniaooea idly a4ctive acnpdb dauet earmner or exei piert. [0015 Numetou methods comemplaned in the preat mwennun. Tor exanp. a method is provided imolving culenn the aforemennoned host el comparing the expressin vctor of'the inntion such dh it the encoded antigen 1 lo exps i eed. A nthoC is alai provided invokn ng ultut ng the aforementioned hbribdona im a culture medium under conditions permitting expressmn of the antincn binding protein by the Inodomlau Satch methods can. also comprise the step of recovern te antigen tmdmg protein tom the host cell cuure, hI a related embodintm. an isoatead angu bmdig potemin produced by the dremeuoned method presided 100161 The oregong sum is not ended to defne evry aspect of the mntvion, and addiionaspets are described other sections much as the Detailed Dwncption of Embodhment. The entre dociunent is ended to be related as a unified disclosure, Ad it should be understood that ab combinions o features desnihed herein are contemplated, evon if the eombinaion of actures are not t'orud together in the sa sentence or paraguph. or sectin of this docment. [00 1 in addition to the foregoing, the invention includes, as an additional aspect al embodimentcs of te i nventimmonanoer in scope in any way than the variations dened by species paragraphs above. For example, certain assets of the invention that are described as a genus, and it should be understood that every member of a genus is, hidividuall, an aspect of the invention, AI.o, aspects described as a genus or select inn a membere &f a genus, should be understood to embrace conmnations ot two or more members of the genus, Although the appi icant(s) invented the full scope of the invention described herein, the applicants do not imend to claim subject matter described in the prior art work of others. Therefore, in the event that statutory prior art witin the scope of a claim is brought to the attention of the appliants b a Patent Office or other entity or individual, the applicants) reserve the right to exercise amendment rhits under applicable patent laws to redefine the subject AM. ot' such ~ cTh% to spccificAy cxc kdc. such st~tutor~ prior wt or ob'uous vurta0ons ot Statutory prior U4 trorn tixe ~?covc of such a C~ JVfl. \taririons of the invention defined by such au~cnded claims aTh arc PreMed as aspects of the in BRIEF DESCRTI) NN ONTH PRAiNGS [0018). Fiure A-N shows shernatic structur' ofi some embodiments of'a compOsitin of th euitionthat inclde on or nore nits of a phamancohigieay actixe toxmi peptide analog (squipgle) ttsed. ia itn optional Peptidy inker moiety sueh as bIn not hNuted to 1- or L 1 described herein with coe or more domams of an imimunoglobuin These schematics show a more ty plel I1, tdthough they are ended to apply as w el to igCes, whbich wil hve 4 disniide bonds in thiehmgnc and a di ffercnt arran'tent ot' the disultidk hoad bnldou the beav y and light chain gmd ,mdpZ~ C' andle an lg~ figure I A. represents a miono a lemt heterodimenic Fe-toxin peptide analog fusio wt the toxin peptide anuu iuse'd to the In denieal mnd of one ofibe nInuloohbUn R domain mfonomerh lgure 1 14 reresets a i alem hormouneticFc-toxsn peptide analog tson, wOn h~ Ootude an'higs uedto the( C-te'rin"l cods of both of the nmmunlothttln -e domain monomers hggre 1( r eresents a mnono alcnt beterodimnene toxin pepude adog-e fion ith the toxin pepthde analog f'sed to the NfetrinAl cnd of one of the immunelobulin Fe domain monomes Ftgurc I r presents a bi\alot homodie toxin peptide anaog 'e fls~ln, with to peptd xtaag s iused to the N -ermmral ends of both of the nornunoglobul in Fe. domain meonmers Fngur\ I F represents a monov alent herotumv'ne Fe tov in peptide anslos A bT compnsuw an umrnunovjooulin heavw chain MC) - munouuin hght chain C ) an immnoolobulin Fe monomer w ith a toxm ppnde analog fesed to us '-temunu in Ficnre IF represents a mOnoalenTm heterotetradrcie HI 1 anybody i-A\xi pepide analog fusion, wih a toxin peptde analog fused to Ohe { errunal end of oln of the [C monomners. Fanmre' IG represents a hiMalent M a.nbody Ab IC-toin peptde analog fuson having toxmn peptide analog on he I'rmmad ends of' both Hf monomers. Figure i represent% a mnuoaent H IVT toxin pept analw4 Ab with tou xn peptide analog fused to the N-termma' end of one of ie 1t r oOmei ta gtire 1 1 t'epreSent a mon0xsl i 1l toxin peptide snaloy)C AN. vo id tL toxin peptide analog. fIsed to the Nerminl end of'one u he' HU monomer [,ine U represent a monovalent lT Ab L(toxin pepide analog tusion 6k., Ilt4txn peptide ad fusion 1 PC 2its C)) with The toxi peptde anlog fused tohe f-term d of one of the I ( monomers. Figure If represents a bivalent UT A \b2 t oxi m petide antilog fuion . (toim pepnde anaog tim 201hi uh mio p e analogs Red to the C-ermial end of both of the LC monomers Figure 1 L renresntsi a valnt i-T At L C-axin pertude ajnalotT UCAoim Peptide analog (ue., 2(L(-toxm peptide analog fusin) 1 Ut'-tuin peptide anao fusson 4 1lt, with the toxin pentakanalogs Imsed to the (-termnai ends of both ofthe K monomers arid one of the FR' monomers. Fiue represents a hi talent atihody' with a toxin peptide analog tmely inerted into an internal loop of'the immunoglobulin Fe domain of each li monomer. Figure IN representa a monovaet aiibody with a toxin peptide anulo moiety inserted ito an itr0a loop of the immunogobuiin kF doamn of one of the ti1 nmers Dn or Wi mers wll fo sotmaneouy im certain host cells upon expression of a dcosvn bonuclete acid 'D\) consovet encodinga smga e ehain. other ho-c e lO the cells cn be placed in conditions taong, r funnaon of dimeisctners or the dihoes/im ers cain be formed m utm if more than one U monomer, Ft moommer, or im tinolulin Fe domai monomer is part of a sngi embodiment the individual monomers can be. if desired. identical or dtiffurnt fom each other [009 FiguZ2 demonsates by Pathpress eetrophvsig that the -InJA4 HGMC-ShKO :-3 Q 6K) Ab:T) M 9 3 de d in Eampn 4 and 5,is more potent in h oking human 3uent figuree 2 than human K .i current (F iure 2. [0020 Figure 3A shows a Coomassic brilliant blue stained Tris-glycine 4-20% SDS-PAGE of the final monovalent -35, Q 6K]/ant-KtL H Ab product. described in Example 4 herein. Lants $12 waere loaded as follows: lane I: Novex Mark 12 wide range protein standards (10 p lane 2 5 pg product, non-reduced: lane 3: blank; lane 4: 20 pg product, non-remed- ho 5bank; lane 6: 10 pg product, non-reduced; lane 7: Novex Mark 12 ide ange protein standards (10 l): lane 8: 0.5 pg product, reduced; lane 9: blank lane 10: 2,0 pg product, reduced; lane 1i: blank; lane !I: 10 pg product, reduced.

R(002i Figure 3B shows size exeiusio chromatography on 50 g of the final mOovaent Ec-L0-ShK[1 -35, Q16K nt-IK Ab product, described in Example 4- injected onto a P'henomenx Bi1Sep SEC-3000 column (R8 x 300 mm) in 50 mM Na I PO 4 250 mM, NC and p1H 69 at i mmn observing the absorance a 28O no [0022] Figure 3 ows an LC-MNS alysis of the final sample ofmnoovaent F LO1-ShK[l35, Qi6K]nti-KLi Ab described in Example 4 The product was chromatographed through a Waters MassIPREP micro desalting column using a Waters ACQUITY UPLC system, The coluhmn was set at 80* and the protein euted using a lineag'ret of increavmg acetonirtrie concentration in 0. % formic acid. Part of the colnun affluent was diced into a Wats LCT Premir E SI-TOF mass spectroeter tor mass analss T iistrument was r n the postive V moe, The capillary voltage was set at 3,200 V and the cone.voltage at 80 V, Th mas\ spectrum was acquired fro mx00 to 3000tMtn/z and deconvoluted using the Max Entu software provided by the instrument manufacturer. [00231 Fhgure 4A shows a Coomassie brilliant blue stained Tris-lyome 4-20% SOS-PAGE of the final monovalent anti-K IT H- M0-ShK I 35, QJ6K] Ab product deserved in Example 4, Lans 1- 12 wene loaded as follows: ln 1:- Novex Mark1i2 wide range protein standard (1 pIt):~1 lane 2: 0,5ipg product, non-reued: lane 1 blank, lane 4: .20 pg produce , non-reduced; lane 5:blank ian -0 pg product, non-reduced; lane '7: Novcx M irk wide range protein statndiard (10 pi) tane t: 0.5 pg product reduced: lane 9: blank; lane 10: 2.0 pg produt, reduced; lane. 1: bant; lane 12: 10 pg product, reduced, 0024] Figure 4B shows size exclusion chromatography on 25 pg of the final monovaemn anti-KLH 120.6 HC-L 10-Sh K f 5, Q16K] tiody product deerbed in Exampi 4, injected onto a PMenormenex BioSep SEC-3000 colum n( T x 300 mno) in 50 tmiM NaH.POA 250 tM NaUL and pH 6,9 at I nL/nin deteting the absorne a 280 nm. The deflection observed at aboutI i ii m in injection related artetaet [00:51 i igure 4C shomu a MA LLD) nvrs spectral analysis of the final sample of MOnovalent ati KI 1 IM'4 10-bKf~305. Q 161] A, dscribeNd i ESample 4, analyzed usimp a ,\bejromss \MALDI micro NM \ nmas \peetronmeter equipped with a f rogoen haer. Th e storpre w as tun at post et linea de T he mnstrunment's vottage w as set at 1 \. mnd the high mass detector w as set at. \ enseun was produced by accumulating dta ninm abu 200 laser shots internall mass eanbratin uvas achie ed usmg puut proteins o f known molecular masses [00i] Figur e S \ shows a Counmsie bnltant blue stained Tis-utiymc -4-20% SSP,\G of th~e final Niva lenat aKLi h& l LI (P-ShK [1 -3 Q 16K At\ product, desrbed in Example 4U Lane 1-12 were loaded as 'oilows lane 1 Nov Matk 12 waie range protein standards 0 I; lne 2: 0( [t product. non-edueed: kae 3 Klnk 1a : pg product non-reduced lma e 5:hmr, hoe : 10 pg product. non reduced; lane ~, Nosex Mark 12 wide ange potein standards ( 0 pO;lane 0.5 pg product, reduced; lane 9: blak, lane 10: 21 pg product, redured; la ne 11: blmk; ane 12: 10 gpmduct reduced. [0027] :g 513 Shos size vision crmatog on2 go the final bivalenA fnPL F=ll I 0ShKfl V35. QI ]Atromtdi idinLaipe4 eced onto Phnomenex B yoSe SEC000 column ( x rMn) 50 M NaliPOM00 mM NaCI. and n6.9 ad n g ch bsotbance at 240 rdeflection observed at about 1 n iaetd a 8] Figue $C sh a MA ) mas speetr a sif de final s peo nsn a Micromass MALD iero MX mass specometer equipped with a. nitogen lasr. The samnipe was rn at posive nearmode The sintum s getsw at 12 A and tha ib mass deo aseso ata a aciee pn A d prOtes of known molcu mes. -AGE of the final monovalentKdl iIC-3 $ 5. Q16 Ab produce.

described in Examuple 4, Lanes 1-12 were loaded as follows: lane 1: Novex Mark 12 wide rang protein standards 10 I lan, 2 0,5 pgproduct, non-reduced; lane .3 blank; lane 4: 2.0 pgu product, non-reduced; ane 5:blank: ane 6: 10 pprduct, non reduced; lane 7: Novex Mark! 2 wide range protein standards: 10 pl; lane 8; 05 pg product, reduced; lane 9:; lan 1: 2.0 g product, reduced; lane I: blank; lane 12; 10 p product, reduced, [003f] Figare 1B h size exclusion chromatography on 20 pg of tHe inal monovakent antol- H HC'- 10-SbKf2'~35 QW16 A b product descAbed in Exwarpk -L injected onto a Phenomen e i1:0e Ski 3000 coumn 8 0 1 m in 50 rM aNiHPos 250 moM N ( and pH 6 9 at imL min deteenng the aberhance at 280 nm. he deflection obred at about i t minm ection-reluted artefact [0031] Finure 6C show an LCM-S massspectral analysis of the final sample of monovaient antKLH HC-L 0-ShK[2-35 Q16K] Adb described in Example 4. The product was chromatographed through a Waters MvsaPR micro desalting colunmn using a Waters ACQUITY UPLC system. The colum was set at 0*C and the protin etd using a linear gradient of increasing actonitrile concentration in 0. 1 formic acid. Part of the column effluent was diverted into a Waters LCT Premier ES -TOE mass spectronetet for mass analysis. The instrument was run in the positiveVmode, The capillary voltage was set at 3.200 V and the cone voltage at 80 V The mass spectrum was acquired fomN 800 to 3000 rn/ and deconvoduted using the MIaxEnti so ftware provided by the iti.trumeont amuf actuirer. i[X032] Fhire. 7 shows results of phamckiet stuies(sne-ubuaes dose 6 ng/kg) performed in Sprague-Dawley rats, Open squares represent data for monovalent F/Fc-LjO-ShK( 1-35, Q69K) heerodimoerof SEQ D NO: ' and SEQ IDNO:26) closed cir present data for monovalent anti-KLH antibody-Shk(i 3, Q16K) (teramner of SEQ 1D N. 28, SEQ iDNO:29, SEQ 10 NO:28, and SEQ 1D NO:32); and closed triangles represent data tor monovalent anti-KEH antibody (ioop)~ShK(1-35, Q16K) (tetramnerof SEQ ID NO; 28; SEQ ID NO:35: SEQ ID NO:28; and SEQ ID N:34). described in Exanle 5 and Table 7H.

[0033] Figure 8 shows results of pharnmacokinetic studies (sigle- sutbcutaneous dose =::: 6 mg/kg dose) performed in Sprag-Dawley rat for bivaent dopen squareS) and monovalent (cosed ci reles1 anti-KLH antibody-ShK(-35. QU6Krspectively, tetramcrs of [SEQ ) NO: 28, SEQ ID No32 SEQ L) NO:28 SQ lD N:321 and (SEQ ID NO: 28, SEQ ID) NO:29, SEQ U) NO:28, SEQ IID NO:32], asfurther dncribed in Example 55 and Table 73. [00341 Figure 9 shows r suits of pharmacokinetic studies (single- subcutaneous dose = 6 ) prfornmd in Sprague-Dawley rats for bivalent 4open squares) and monovalent (cosed circles} antiKLHR antibody (loopShK(1- 35, Q1 6K) (repectijvy tetrmers of [SEQ iD NO; 28, E D 35. SEQ ID NO:28, SEQ ID NO:35] and [SEQ L)D NO: 28, SEQ U) NO:34. SEQ ID) NO:28, S EQ iD) NO:35b. as further described in Example 5, and Table 7L [0035] Fugure 10 sho the result of pharmacokinetic tdies (simgle, mg suit utaneus doe in SI)N rats of nmonovalent F&ShK/'ec beteroudimer ( open squarest monovalemt Fe- hK KLIIAU (beterotra ner o~r hermbodylepen triamgle) and the hi valent ShK-Fe c headier i closed citeles), The monovlent heterodirer and beterotrimer provided much greater exposure than the bivalemt honmodimer. furtherr details on thi sudyarc prouded m Example 5 [0036] Figure I I shows analysis of antibodies on a 1.0 mm Tri5.glycine 4-20% SDS-A E (Novex) developed at 220V using reducing oadina buffer and stain with Quick~lue (Boston iologicals). Lanes were ioade d as follows (etto right): lane. 1, Novex Mark 12 standards; larw 2. 2 pg~ aDNP 3B Ab frorn transient ccil culture; lane 3, 2 pg aDNP-3B I Ab from stable cell culture; lane 4. 2 pg aDNP 31H4 Ab fromt transient cell culture; lane 5. 2 ge aDNP 31H4 Ab frm stable cLl culture; lane 6, t gg aNP 3A l AU from transient cell culture; lane 7, 2 gi IaN 3C72 Ab fromn transWicu cel cuitture; and lane 8, 2 pge aDNP 3 A4 At fmm t nsienlt cell [00371 Ft r' 2A -Ushows anaysi of atbodies on a 0 mInn- iveyne & 20% SDS-PAGE (Nose e developed at 20 t nue on-redcig lvadhuivutk-r and~ staining with Quickfilue (Bloston Biologicaht} Lanes were loaded as follows {heft to rgto: igure I2A): lane 1, Novex Mark 2andard lane 2. 0 5 pg aDNP 3A1 Ab; lane 3, 0.5 pg aDNP 3A4 A; lane 4, 0.5 p aD)NP 3C Ab !ne 0. pg aKLH 120.6 Ab; lana 6, Novex Mark i 2 standards; han 7, 5 pg a\P 3 N A b; lane 8, 5 pg aDNP 3A4 Ab lane 9. 5 pg aDNP 32 Ab; lane 0 5 pg aKLH K 120,6 Ab; (Figure 121 Lan e I Novex Mark 12 standards; iane2, 05 pg aDN P 3B1 Ab; lane 3, blank; Lane 4 Novex Mark 12 standards; lane 5, pg aDNP 3B1 Ah, [)038] Figure I3A shows analysis of antibodies on a 1.0 mm Tris-giycine 4-201% SDS-PAGEF (Noycx) developed at 220V using non-reducing loading bufr and vig with Quickilue (Boston Bfiologicas).Lune awee loaded as folows (lt to righ); nc 1, Novex Mark 12 standards; ane 2bank ne 3, 0,2 pg 0 DNP 3dI Ab; Lana 4, ,2 pg aDNP 3A1 Ab, Lane 5, blank; lane 6,0 6 pg r DNP 3BI Ai lane 7, 06 pg aDNP 3A1 Ab; lane 8 bla n; Lana 9, 1. pag DNPB I Ab; lana 10, lA pg aDNP SAl At. [0039 Figure 13 shou analysis of antibodies on a 1 0 mm BisTas 4-129 NuPA\GF (Novex) devoted at 20\ using non-rdl ny loading buffer and stain ing ah d Qul&k Bue (Boston Biologiead) Lanes ware loaded as U0llows (le to nghlila m 1, Nos Mark 12 mamards; lane2, blank Laue , 0.2 pg aD\P 31 AH lane 4. 0.p aDN? L Ab; lane . blank lane o. 0r DN 3B1 AK lane 7. 06 pg aDNP ;A I Nb lane 8, bla lane 9. 1 pg aDNP 331 A\lane 1I 1 8 pg aDNP [0040] Fu 14A~ shows anaLysis of antibodies on a 0 mn Trigyce 4 20%

S

iS-PAGE (Novex) developed at 220V using non-reducing loading buffer and staining witht QuicLueL tBoston Biologicaik. Lanes were loaded as allows (left to right); (Figure 14LA: w i 0.1% SDS) in rnningn buffer) 'ane I, Novex Mark 12 standards: lane 2, 0 S pga > 3B1 AL incubated at room temperature for 10 min; lane 3, 0,5 pg aDNP 3B1 Ab incubated at 85 for 5 in; lane 4. 0.5 jg aDNP 33 Ab incubated at 100 C fr 10 min; lane 5, ank; lane 6, I tpg DNP 3B1 Ab incubated at room temperature for 10 min; lane 7, 1 pg aDNP 3BI At incubated at S5PC for 5 niw; ane 8. 1 Ig aDNP 3B Ab incubated at 100C fo tO mii; (Figure 14B: 0.4% SDS in mning buffer; 84C treatment fo 5 mint hmne Novex Mark 12 standards, lane '2, blantk; lane 3, 0.25 pg aDNP 381 Ab: !anc 4, blank; lane 5, 0.5 g aDN P 31 A b:ne 6 blank lane 7, 1 p DNP 381 Aib lane 8, blank; lane 9, 2.0 ~g aDNt> 31 Ab. (0041 I H 15 hbows anvios, usmn mo se neaaujon columns (TSK-GFL 63000\\ Xl .L mm particle A ~ . 300 mm, l)osohtioscience 08541 in serie with a 100 ink! sodi phosphate, 250 m Nl Ci at phI 6 moble phase flowed at 0.5 rntmLhnn of antibodies aDNP M. I \l, darker trace with post shoulder; aNP 3f1 h '; a ll 120.6 ('K.LI1h aDNP 3(2 0'32''. and aON'P 30 [00421 Figure 16 shows analysis of antibodies aDNP 3A ("3A"Y, aDNP 3C2 (43C2") and DNP~3A4 before and afier 3 weeks of light exposure, using two exclusion columns ITSK-(GEL (G3000SWXL 5 mm particle size. 7.8 x 300 mm, TosohBiscience 0544 i) in series with a 100 mMsodium phosphate, 250 mM Na at pH 6.8 mobil phase flowed at 0.5 mjmin, [0043] Figure 17TA-3 ,show analysis, using two size exclusion cobuns (TSK-AE L 03000 WXL, 5 mm particle size, 7.8 300 tOm, Tosohioscience, 08541) in series with a 100mM sodium phosphate, 250 mM NaCi at pH 6. mobile phase flowed at 0.5 tmL/nn of antibodies aDNP 3A4, JN 3A4YY ('W101lY aDNP 3A4-F (W 10F"), sDNP 3A4 YSS ("WV10Y/CSS), and a DNP-3A4-FSS ( 0 F/CSS" before (Fiure 1 and a fte (Figur f 1) 2 days of ight xponmtre. [0044] Figure 18 shows ion exchange analysis of aDN P a.ntibo'ies (aNP-N aDNP-3A4~, aUNP3A4-F, aDl> NPA4~YSS and aDNP-3A4-FSS. Tey were analyzed fo bomogeneity using a TOsch4s 5 PW column (10-pm parties. 7.5 mom D Xt 7. em long) ui Buffer A ( 10M sodinn acetate pH 5,0 and B 10 moM sodium acetate, 600 mM Na~( , pH 5.0) flowed at I mI/min with a prlgnanvedinear gradient 0 0%E 10 Wia 355 i3t 0 i 70% It .3 An 90%N ad 3 l 0%B) [0045] Figre 19 shows an ana yis of aDNP 3B F gue B9A DNP 3A iure 9 and aDN' A4455 (Figure I9 ntiodies by non eduing C&SOS idet on of absorbanCe at20 nm A bar sd sic capiary 50 pn a302 CrA Ws Used fr the epartion analysis [00H] Fgur 20 shows an anslvsix of aDN 3 (Fg A aDN A4 (gurd 20Bur aned aDNP A4 S gare 0atibodies by ed n DS ith deetion of absorb 2 m \ baeud iica apil 0 3 rm was used for te separAnon a 'ys [0047] Hyre 21 shows an o nis of o3NPR\44 (dotted curWeN a)DNP-3 4S P5 (solid curve) and bDP-8 (dahed cmve) anooubes were analyzed by DS( using a. NJucCai VP-DSC nnere the: samples were heated from 20 C to %'O at a rate of IPC per mmin ie, The PrOteim coneentranion wa 0.5 mg mi in 1 ml sodium aceetate 9% sacmrse. j 5.0 [0048] Figure 22 shows serum concentrations of sDNP 3A4-F. aDNP 3A4-FSS, and aD)NP 31 antibodies in rats receiving a single subcutaneous injection of 5 mg/kg. as determined by EL ISA. BWood samptles were colletted at 0, 0.25. , 4. 24, 48, 72, %, 168, 336, 504M 672. 840 and 1008 hour post-dose. [0049] F'igxure 2.3 shows plasma conce.ntrations of aDNP 3A or aKLH 1246 in male cynonmolgus monkeys reciving a bous intravenous Jieton sDNP 3A4 (4 mgk) or aK 120. (3mg/kg) antibodies, rc taken perodically and plasma concenurations of the amibes was determined by ELISA, The data for aDNIP 3A4 was normalized tw 3 mgkg for comparison purpos 00501 Ni re 24 shws a Coomasic brilian lue snd TdI D-gein 4 S% SDSPAGE of the finl monvalenKll 206 Sh 5,16K Ab produce described in Example 4. Lanes 1- 12 were loaded as flows: lane 1: Novex Mark12 wide rangc protein standard 10 I) lane 2: 0,5 pg product, non-educed; lane blank: lane 4:2.0 pg product non-reduced; lane 5:Iank lane 6: 10 ppoctfnon reduced; lane 7: Novex Mark I2 wide range protein standards (10p; lane 8: 0,5 pg product. reduced; lane 9: Na; Iane 10: 2.0 g product, reduced lane II: blank; lane 12: 10 pg product, reduced, [0051] Figre 25 shows size exclusion chromatography on 25 pg of the final monovalent aKtLH 120,6 LCShK'[14~5. Q i6K] Ab product, described inE m 4, in ejected onto a Pheonmenex Bioep SEC-3000E coumn (7,8 x 300 nmn in 50 mM NaH POs 250 uM NACT PH 6 9, at 1 mrnd detecting the absorbance 2a 20 nm. [0052] Figure 26A shows non-reduci g ( Fiure 26A) and reducing (Fi re 26f) MALDM S mass spectral analysis of the fin sample of mnonovalent KLY H 120.6 LC-~ShK[ I~35, Q 16K] product, descibe ixIn Examiple 4, sing a'v Micomass~ MAL..DY micro MIX mass spectrometer equipped with a nitrogen laser, The sample was nm I positive linear mode. The instrument's votage was set at u2 k ud th high mass detector was set at 5 kV, Each spectrum~ a s produced by' accumout data frm about 200 laser shots. Extrnal mass calibration was achieved using purified protons of known molecular tmase [0053] Figure 27 shows a Coormassi brilliant blue stained Tis-yeme 4 20% SDS-PAE of the final bivale aiK H 120. LC-ShK[i35 Q16K] At product described in Example 4.Lanes 12 were loaded as follows: lane 1: Novex Marki2 wide range protein standards (10 p ) lane 2: 0.5 g product, nonrueduced: lane 3: blank: lane 4:2.0 pg product, non-redued; lane 5:blank: lan' 6: 10 pg product. non reduced: lane 7: Novex Mark 12 wide range protein standards (1 0 p1; Lane 8: 0.5 pg product, reduced.; lane 9: blank; lame 10: 2.0 pg product reduced; lane i b ank; lane 12: 10 pg product rduced I054] Fiur 2$. shows se exclioprod uc 5 de of Ie finale bivlei aH120.6 LC0ShK.R15. QofIA product, decrbe FK anipkici.

h teeted ontoaPhenamen RiSep SEC3000 colton 8 x 300 cnnin An. NalP 250 ict NaC p a a mOki dcuthesorbanee a 280 nun [0055] Figure 29A -B shows nonreducing (Fiue2A)adrduig(iue2B MA LI-MS mass spectrA analysis of the final sample of bivalkt aTH 120,6 LC ShIK[ -35, Q16K] Ab product described in Example 4, uing a Micromass MALIDl micro NIX mass spectrometer equipped wi a nitroger laser. The sample was run at positive linear mode. The instrumental voltage was set at i 2 kV and the high mass detector was set a5 kV. Each spectrumn was produced by accmuating data fOmn about 200 laser shots, External mass calibration was achieved using puritied proteins of' known mol euar masses. [0056] Figure 30 shows a Cooassie brilliant blue stained Trisglycine 420% SDS-PAGE of the final trivalent aK i 120() LCShK[b-35, QI6K] Ab prduet, described in Exampie 4. Lanes 1 - 12 were loaded as follows: lane 1 Noe Mark 12 wide rang proein standards (10 g lane 2: 0,5 g product. nn-reduced; lane 3: blank; lan 4: 2,0 pg product, non-reduced; lane 5:lank: lane 6: :10 pg product, nonv reduced; han : Novex Mark i2 wide range protein standards (10 1); lane 8: 0.5 pg product, reduced; lane 9: black: lane 0: 2.0 pig product reduced laned 4: blank; lane 12: 10 pg product. reduced, [00571 Figure 31 shows sie exclusion cbnonatography on 25 pgt of the ntal invalent aKLH 120. LA -ShNkI Q16K] Ab produce, dese tbed i Examp 1 e 4, injected onto a Phenomenex io\ep SEC (000 cotumnr (7. N 300o cmm) m 50 mM Nad>0.Ps 25 mM. NaCI, pH- rd P 1 mL. min detecumw the absorbcance at $80 nmc (00581 Ficue 32A~B show&s non-rieducing (Figurc 12 t and reduemy Wig~ure 32B1 MAL D-MS massspectral analysis of the final sample of triaent aK L8 1 20.6 t ShK[1Ki5, QI6Kj Ab product, described in Example 4, usmng a Micrormus \MALDI micro MX mass spectrometer equipped u ith a citrogeni Laser, T he sample was run at positive linear mode, The instnunenfs 'mlage was set at 12 k\' and t hgm detector was set at 5 k\ ,ach.speetrum wa produced r accumulating: data from about 200 las ots ternal ,asswalibraion w ached sing uied protons of knon molelar ma [0059] Figure 33 shows a Coomiassie brilliant blue slained Tris-glycine 4-20% SDS-PAGE of the fina2 onovaken aKLH 20h.6 Hg2 lC-$hk -35 R IA, 14A, Q1 6K A product, described inE 4. Lanes 1 wer e loaded as fillows: ane I: Novex Miark12 wide range protein standards (10 pl); lane 2: 0,5 gg product, non-reduced; lane 3: blank; lane 4: 2.0 pg piUct, non-reduced anc 5:bank; lane 6: 10 pg product, non-reduce, lane 7: Novas Mark12 wide rane protein standards (10 np) lane 8: 0.5 jpg product, reduced: lane 9: blank: lane .10: 2.0 pg product, reduced. lane l : blank: ode 12: 10 up produce t, reduced [0060] Figure 34 shows size exclusion chromatography on 25 g of the final monoaent aK L 20.6 igG H C-Shk{-3i k A, 14A, 16K] Ab product, described i Example 4. injecd onto a Phenomenex BioSep SEC-3000 colun (7,8 x 300 mm) in 50 rS M Nal PO 250 rmM NaC Ip 6 9, at I mnmin detecting the absorbance at 280 nmn [0061] Figure 35 shows reduced LC-MS mass speetral analysis of the heavy chain in the final s'ampic of monovalent aKLH 120.6 LgG2 H SK[1-35 R1A 4 A, QI6K) Ab product, described in Example 4. The product was chrmatographed through a Waters MassPREP micro dsahing column using a Waters ACQUITY UPLC sytem. The column was set at 80C and the protn elutecd using a linear gradient of increasing acetonitrile concentration in 0,I % foricn acid, Part of the column effluent was diverted into a Waters LCTI Pre'mier .ESJ-TOF mass spectrometer for mass analysis. The instrument was run in the positive V mode. The eapilUary voltage was set at 3200 V and the cone voltage at 1 V The mass spctrum was acquired from 800 to 3000 m z and deconvoluted using the Max En software provided by the instrument man ufacturer. SDSPAGE of the fOna aKE. 20. G -tbC6 Ab pouct, described in Example i ., 1..ae 1 eelaeda > os ae1 NvxMr 2wd ag protein standards (10 p); lane. 2: 0,5 pg product non-reduced; lane 3: blank; lane 4: 2.0 pg product, non-reduced; lane 5:blank lane 6: 10 pg product, non-redu ced; an 7: Novex Mark 2 widt range protein standards (10 pl): lane 8: (0,5 pg product, reduced:. lane 9: blank; lanc 10: 2,0 pg product, reduced; lane I : blank; lane 12: 10 ig product, reduced, (0%3] yng ue 37 shows We exeluion chromatography on 25 p; of the fnal aPaL 120) Ig~ H (CM b product, described m example i L incted onto a Phenonrne 1Boscp SL(-3000 column (7S s r10n) in 50' mM \alPO.

4 250 mnTI NaCL p1H Wa 1 i nmin detecting the akorbanec at 2x0 m, 00641 Fliurc 3xAt sows non-reducimg (igure 3} and reducin (Fiaure 38') MA D-\MS mass special analysis ofthe fial sample of aol. H 20 6 1G2 1W> (62M product, described in Example II, using a \/icromnas M A LI Dicro \MX muass spectrometer equipped with a nirogen laser. The sample nwa run at positive linear mowde T1 i u t' voltage w as set at 12 k\ and the hi.h muas detector was set .a 5 k\ E'ach spectrum was produced by acmulnatng dat from about 200 laser shots External maas cahbration was achieved uing purified proteas of knwn molecular masses. [0065] Fig ure 39 shows size exclusion chromatography on 50 g each of aKL H i gI(N297Q), AMP5~HC aKIL HlgG2 HC-AM P5 a L H lgG2, A MP5-LC aK ~LH igl and LC( AMP3 aKLH 'gG1) products described in Example 9, infected onto a Phenomenex Biobep SEC-3000 column (7. \ 300 mmt) in 50 mM NaHJPO 4 , 250 mM NaCL pH 6,9, at inrmLm detecing the absorbance at 280 nt. [0066] Figure 40A~E shows analysis of antiodies (described in Example 9) aKLM, 1gl N297Q (Figure 40A) AMP5-IC a L H gG2 (Figure 40B), LCAMPS aKiLH iG2 (Fuure 40C), IC-AMP5 aKLI IgG2 (Fgrowe 4OD), and AMP5LC aKLH igG (Figure 401 ) on a1 0 mm Triauglycine 4-20 SNLS-PAGE (Nove} deveoped at 220V u s nn - r ucg loading buffer ani uaing with QuIlu (B oston Biologicals). Lanes 12 were loaded as follows: lane I: Noves. Mark I 2~ wirange p'roei \tadard (10 p): lane2 0,5 pg product non-retduced; lane 3: blank; Ian 4: 9 0 pg pmda anreduced; lane 5 bhnk;an 6 prduc0 ronddted:ane SNovcx Mark 2 kdc ge. protin stndads (10 ph e S. 05 ooue rexmuced;n ane b la ne 0 2.0 ig 'prodt nod agp du Ireduced [0067] Figure 41A-D shows mass spectrographic analysis of reduced samples of LCAMP5 aKLH IgG2 (Figure 4itA AMP5AIC aKL H gG (Figur 4 B), HC AMP5 aKLA igG2 (Figure 41C), and AMPS-LC akLH gG (Figure 41D). described in Example 9. Each sample was chromatoraphed through a Waters Massprep micro dsalting column (2.1 x 5 m) si Acquty UPLC system then introduced into a Wates timr-of-fightLCT premier manss spectrometer for malss mecasuremrent, and the rass spectrian was dconvoluted using the MaxEnt I softw are. 068] igte 4i w \'eai ap of the &ndla4( lI] kaKd 6 LC fusioni construct.described int Example I0. [0069] Figure 43 shows size exclusion chromatography of 25 gg of the final Ex4 kG-aKLH 120,6 LC antibody fusion, described i Example 10. nected onto a Phenomenex Bioep SEC3000 o um (N 300 mm) in 50 mM NaHPO 4 250 mM NCL, pH 6.9, ati 1 mmn detecting te absorbance at 280 nm. [0070] Figure 44 shfw alyi of on .Tis-glyci.e 4-20% SD~PAG E (Novex) developed at 220V using redung and non-reducing loading buffers and staining with QuickBlue (Boston Biologicals). Lanes 1-10 were loaded as follows; lane I: Novex Mak wide range protein standards (10 p); lane 2: 0,5 pg other protein; lane 3: 0. MgA E KLH 120,6 AN, non-reduced: Lane 4: 2.0 ig other protein. Line 5: 2,0 pg Al 1206 Ab, non-reduced: lane 6; Novex Mark12 wide range protein standards (10 pd); lane 7: 0.5 pg other protein; lane 8: 0.5 pg Ex4 aKLH 120.6 A, reduced; lane 9: 2.0 pg other protein, tane 1.0: .2,0 pg E-aKLH 120,6 AN reduced.

45 d Mo 001 F w of Thoo ani scetierers aiinofNcr"aiad dra DUIATET) DESCRIPI OP M BODI MENs [00721 1e section headings used her n are far ganiationl ptposes only and are not to be contred as iinOg thesbjecnin described. [00731 etu [0074] L'nless other is defined h erein, scientic and technical terms uased in Conncctinu with the present applieation shall have the m ntat are cominoni y understood by those of ordinary skill in tbe . FuNter unless other ise required by eODtt AKinga terms shall include pursinies and plural terms shail include the singular hu as used in this specificaton and the appended claims the singular trrs a an :nd "the' melude plural referents utles the ent ceryndiate otherwise I or example, reference to "a protem" ine ludes a phuralivy of pio t eins: retence to Ca cell includes populations ot'a piertahty of cell1s [0075] "Polypeptide" and protein are used interchangeably herein and include a m lecular chant of two or rnore amino acids linked covalentl through peptide bonds The terms do not refer to a specific length of the product Tbus. peptide' and "ohigopeptidcs," are included within the definition of poi lpetide. The terms include post-trnslationa! modifications of the polypeptde, tor example, glyvcosylations, acetylations phosphorylations and the like. In addition protein fragments, analogs, utated or variant proteins, fusion proteins and the like are included within the meaning of polypepide, The tenms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included as can be expressed recombinant y using known protein eninecring techniques. in addition, fusion proteins can be derivatied as described herein by well-known organic chtemistry techniques. [0076] The term isolated protein referred means that a subject protein (1) is free of at least sornie other proteins with which it would normnaly be found in nature, (2) is essentially free of other proteins from the same source, e. gK. trom the same species, (3) is expressed recombhinantly by a eel I of a heterologous .species or kind, (4) has been separated from at least about 50 peracemt of polynucleotids, ipids, carbohydrates, or other ma terials with which it i s associated in nature, (5) is operably associated (by covalent or noneovaent rnctio[ wvithV a poypeptide with which it is not associated in nature, and/or (6) does noT occ in nature. Typicaly, an1 isolated protein" constitues at least about 5%, at Last about 10% at least about % east about 50% of a given sanpie, Geni DNoi A, cDN A , mRNA or other RNA, of synthetic origin, or any combation thereof may encode such an Preferably, the isolad pre from proteins or poly peptidies or other contaminants that aire found in its naitura} environment that would interfere with its therapouc, diagnostic, prophylactic, research or other use 00 A na 1a peptide (e an ung binding protein, or an atbodyi Comprise tan amino acid sequence wihormne or moe anuo WciU rsidues are inserted ito, dd-ed from and/or substied ino the amino acid caguene relav to anmor polvppide sequenem ais~eue tr"niortrs [00'I The torm 'fusion poti indicates that the protenm ddes polypeptide components derived frot more than one parental protein -o poh peptide. ypmaIly, a fusion protem is epresed from a fusion gene n which a nucleotide sequence e dm oddne eeci-omnepotm p e m e w ithiand optionally separated by a linker from, a ue leotde sequence encoding a polypepnde sequience rrom a Jtftcrent protein The fusion gee ca then be c-reedbya recomimant host eelI as a single protem [007 IA erdeted" protein refers to those pr otems ctpabk 0 being directed to the FR, secretory veidle or the curaellular space as a result of a secretary signal peptde sequence, as w,41 als tse proteas released into the euracelhukt space without neeess-y Uottning a sgnalsequence lithe secreted protein is released in the exraceliula spae. the secreted protem can undergo extraaellulo processing to produce a "ature" protein Release into the extraccilular space can occur by many mechatms. mehidmg eSoc toi and proteoltie cleavage In some other embodiments of the in\ventiv e composition, the toxin peptide analog can Lbe stesied 1 he host cell as aated pruteA who hp f f0080) A u tSed heremo "olutde'' n hen mt reerwc to a proAem produced by rceumbbnant DN\ teelmoog in a host cell is a proteim that exists in aqueous solution; if the protan contains a twin-arginine signal amino acid sequence the soluble protein is exsported to tihe peripltasrmic space mn gramn negative bacterial hots or is secreted io the culture medium by eukaryotic host ells capable of' seretion or y bacterdal hOst poswesing the appropriate ges (eg., the til gene) hus. soule protem is a protein unich is not found m an melusin body mnide the host cell ,\ltemnatinel dependog on the COMtext, a soioble prote. is a PrOtei.nUch not tound integruted in cellular membranes: in contrast, an insoluble protein is one w hich exsts in denaurder t'orm iside ertopl asrhe granules (cx lled an inclusiom bodyj in the hot cell, or again dependmg on the context, an insohible protein is whic bis present i ein membrmes including but not imited to, eytopiasmic memrbranes, mitochondria menibranes ehtorop Last mlembranes endoplasnuc reticulumn membranes, etc. [o8i] The term "recombinant" ndimcates that the material (eg. a nucleic acid or a poypeptide) has been artihcially or synthetically i normaturaly) altered hy human intervention. The alteration can be performed on the material within, or removed from, its rtMr environment or state For example a recombmam nucleic acid" is one that is rnade by recombining nuceie acids, during cloning, DNA shuffling or other wael known molecular bioologcal procedure. Examples molecular hiological procedures are found in Maniats et aL Moleular Cloning, A Laboratory Manual, Cold Spring Harbour Labora tory. Cold Sprin Hatbour, N.Y(1982) A "recombinan DNA molecule" is compised of >egrnets of DNA joined together by means of such molecular bioogical techniques. 11 torn recombiimi protein" or "recombiant polypeptide as used herein refr to a protein molecule which is expressed using a recombinant DNA molecule. A "rctombhinaint host cel " is a cell that contains andor expresses a recombinant nucle e acid, [0082. The term "poiynucleotide or "nuclicacid iudes both single-stranded and doubl e-sninded niuceotide polymers containing two or more nleitOtide res'iducs. The nuckeotide residues comprising~ the polynucleotide can be riboniucleotdes or deoxyribonucleotidcs or a modified form of either type of nuceoutide Said modiiations incOude base mo dificatons such as bromouridi and inosine derivatives, ribose modifications such as 2',3'-dideoxvribose. and intermnecotide linkage mondifications such as phosphorothioatc, phosphorodithioate, phosphoroselenoate, phosphorodisclenoate, phosphon an ilotioate, phosphormadate ard phosphoroami date. [0083] Tbe term "OionclIetid" mean a polynucleotie comprising 200 or fewer nueleotide residues, in some embodiments olgoneeotides are 10 to 60 bases in length. i other embodiments oligonueleotides are 12, 13. 14, 15, 16. iT 1 i 19. or 20 to 40 nuitcotides in length. (iNonucleotides may be single stranded or double stranded, eg r use in the construction of a mutant gene, Olionucleoides may be sense or antisense oligonucleotides. An oligonucileqtidc can include a label. inchiding a. radiolabeld a fluorescent label a hapten or an antigeni label fr detection assays, O1ligonuecotides mnay be used, for example, as PCR primiers, cloning primers or hybridization probes, [004) <\ "polynucleotide squene' or "ueleoude sequence'' or nucleic acid <quitee " as used uierebhangeahlyhlv"in, is the prir TV Regnenve Of rileleotide residrc in a pol vnudleotdc, tne udogn a'run ohgonucietide a D'A. and R' NAa nucleic acid, or a character sirimg representing the prnmarys equence of nucleotide residues dependich on context, Prom any Npeeid pose ictude sequence, eithe the riv en nuckie acid or the complementary polynuelteotide \eqluce can be deermoned included are DNA or RNA of nmic or synthetic mugm wluh nay be ingle, or double-stranded, and represent the sense or anisense strand. Unless speedied other wse, the letlhdnd end of any s ngleastanded tolheo eleotide sequence discussed herein is dhe 5' end: the left-hand direction of double-stranded ponnuel eoide sequences isren frred to as the ' d cetnn The dir ton ofS to addi on of nascent R\A. transerpts is referred to as the trdanrption dretion sCquence regions on the DANA strand haung the same sequence as the RN A transcript that arc' to the end of the RNA ruscr are referred to a upsrear sequences:> sequence regions on the D\ A strand han h sequence as the R \ trumsript thai are 37 to the end of the RN transcript are etrred to as [005] AS sed heren an ioated nuc icid n4MMei" r'onted tuclei acid sequence i a neii. acid olecule tt eher< idetied and sepated rom t ast ne conltamnuat fuclejc acidnrdcciCewit which t is rdhia associated in he natural source of the nucei add or l owned, amified tagged, otherwise distguished r background nuec acids suc tha sequence o the nuceid acid of ierest can hdeemnicd n ted nclic acid moeule ia . Oet in the forn orstg n hhd is lound n a lovc a listed nucleic acid molecule includes a nuclici acid moace onta rOdincels that ordinari e ipres th ntigenbinding protein . a deot efample Me War e o "'m n bo on Mv PA the nucleic acid molecule is in a chromosornallocation ddiudeteni ibat 4 natural [Q086I As used herein, the terns "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the orde or Otsequencee of doxyribomiucotides along a strand of deoxyribonucleic cid The order of thcse duoxyribonucleotdes~ determines the order of ribonucleoh d s a ong the mRuNA chain, and also determines the order of ammno acids along' the polypeptide (protcin chain The DNA sequence thus codes for the RNA sequence and for the amino acid sequence. [087] The term "gene" is used broadly to tr to any nucleic acid associated with a biologicali function, Genes typically include coding sequences and/or th regulatory sequences required for expression of such coding sequences. The taom "gene> applies to a specific genomic or recombinam sequence, as wel as to a cDNA Aee> AN'' coieio hat or miRNA encoded by that sequence, A"fsion gn"ctasacdnrginha encodes a toxin pepde analog. Genes also inehude non-ca~pressed nucleic acid segment thar for emmol recgution entSrf othe protAre.nslan .4 KXpiKessed a ador gettvtence strain sengdptionanrl eletuto which regidstogw proteins, sch as tirnscdptiontfactors inad resulting in trancritnion of [0V j xpresstin of a gen or "expression ot nuceic agiaf means transeriytin of DNA tlo R (optinay inehidit mod ificatirn ot their NAet licng translation of RNA iboo a polypeide (possby in ding subsequen post tanslational inodification of he poiypepttdel orbot transcription adanataion. as indicated by te contet [00891 As used herein the term "coding region" or coding sequence" when used i reerence to a structral gene refer to the nucleotide seunces winch encod, the amnino acids found in the nascem po lyeptide us a result of trrslation of au mRNA molceule. The coding region is bounded, in eukarvotes on the 2d by 1 the nucletide triplet N 1NT" which encodes the initaor methione and on We Y' side by one of the three triplets which specify stop codons (i e.. TA A, TAE G C [0090] The term control sequence" or "control signal" refers to a polynucleOtidC sequence that can in a particular host cel, affect the exprssion and processing of coding sequences to which it is lighted. The nature of such control seq enees may depend upon the host orgaisni i particular embhod imens control sequences for prokaryotes mray include a promoter, a ribosomal binding site, and a transcription termination sequence. Control sequences for eukaryotes may inc tude promoters comprising one or a pluality of recognition Sites for transcription factor transcription enhancer sequences or elements, polyadenylation sites, and transcription termination sequences. Control sequences can inc lude leader sequence and/or fussion partner sequences. Promoters and enhancers consist of short arrays of DNA that interact specifically with cellular proteins involved in transcription (Maniai, et at, Science 236:1237 (1987)) Promoter and enhancer elements have been isolated fr a variety of cukaryotic sources incliding& gens in yeast, insect and mammalian cells and viruses (analogous control elements, i.e, promoters, are aho fond inl prokary otes). Ihe seltetion oft a partiub~r p)romotear and 'nhancer depends On What -c tY pe i to be used to express te Pot 0 l'irT Sone eutarot. promoters and enhhcers hae broad host nmge while others are fumtional in a hnircd subset of eel types {ior reew see 1 a al , A trends ihee Sei, 1 1:87 (1%M) and ManWus et .L Scence .' 17 K19M7in [(00 1| ]'Te ten 'ector" means any molecule or entY (et, nuclei acid, plamid. bacterophage or ius) used to transfer protein codig iformatum io a host ce [00921 The term expressionr vector or expression construct" as used herein raers to a recombinant DN A molecule containing a desired coding sequence and appropriate nucle1e acid ntol tO sequences neces for the expression of thc operably linked coding sequence in a particular host ec LL An expression vector can include, but is not himited to, sequences that affeet or control transcription, translation, and, if tmrons are present, affect RNA splicing of a coding region operably linked thereto. Nucleic acid sequences necessary for expression in prokaryote\ nicude a prornoter, optionaly an operator sequence, a ribosonme binding site and possbl other sequences, Eukaryotic cells arc known to utilize omoters. enhancers, and termination and polyadenylation ignals, A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operable linked to the ouence of nterest, so that the expressed polypeptide can he secreted by the recombinant host cell for more Ladle isolation of the polypeptide of interest from the ce f desired. Such techniques are well knn in the art (g Gooday, Andrew R.; et aL Peptide and DNA sequences, U,, Patent No. 5,30267: Winer et aL Ompsi;tions and methods for protein secretion U.S. Patent No. 6O,22,952 and U S. Patent No. 6,335,178; Uemura en at Protein expression vector ani utilization thereof U.S. Patent No. 7,029,909: Ruben et al, 27 human secreted protns US 200;O 1400 Al) [0093 The icons "in operable cornin n opera korde ad "opera bI linked"as ued heei reer to theiikagc of ncce a sequence such a anne that a nucleie acid neocutle capable (Wf diflttn the transcrimton of a gnen gene MrdYin the synthesis of a desired protein oktcule a produced The term alo reters to the linkage ofammo acid sequences in such a manner so that a functional protein in produced. For example a control sequence n a vetori at is "operably inke" to a pitem cod ing sequence is h acted thereto so that expression of the protem coding sequience is acev\ed under conditions compatible wi5thle transcriptional acivi of the control sequences. 10094] the trnm 'host ec means a cell that has been. tamsomed, or is capabe of bemng transformed. with a. nuclei acid and thereby apresses a genc of imterest h term includes tie progeny of the parent celt uhelber or not the progeny is identiA i morphology or in gencei makcp to the orWgINS parent ell, so long as the ieno fin ies preo Av 'a ar ge number oi ia lie and wMeknowrn hos ceL1 may be used in the practice of this rnvennon The selection of a particular host i dependent upon a omber of fatos r ecogized by the art These ieltie, for e mple, compatibty with the chosen spression vector, toxiity of the eptdes encoded b' the D\NA molecule, rate of ransfrmation, ease of recovery of the peptides expression characteristics hi-sa fctv arid eost A balance of t hese factors must be struck with the understanding that not all hosts may be equally effective for the express won ot a partiuuu D\ N equence Withmn these ;zenerai ;mdelinaes useful micobial host cells m culte include bacteria (such as echeichia coli spt yeast (such as Sacebtionwes sp. and other Onga cells, insect Clls, plant cells, mammalian (icluding human) cells. eg. ( cells and 1WK~23 cells Modifications eam be made at the D\' level an ell The ptde-eoding DNA sequence may be changed to codons more conatulm u ith the chosen host cellt For F. col, optimired >odons are known m the an Codons can be substituted to hnaate resmeuon sites or to inlde Sllem restictio stes wineb may ami procsming of th UN \A in the selected host celL Nest the transformed host is cultued and pun hed lost cells Ia e knhurd under omeional fennaon conditions s that the desired compounds are expressed, Such fermentation condtons arc ell known in the art [00951 The term "transfetion" means the uptake of foreign or exg enous DNA by a cCll, and a cel has been "tansfcted when the exogenous DNA has been inroduced inside the cell membrane. A nunher of traction techniques are well known in the art andI are dicoe herein, See, e. Graham et al. 19~73, Viroiogy 512:456; Sambrook at al. 2001, Molecular CAmng: A Laboratory Manual supra4 Davis et al 1986, Basic Methods in Molecular Biology, Elsevier: Chuet a. L I Gene 1 3:197 Such techniques can he used to introduce one or more exogenous DNA mloieties imo( suitable host cellk [00%] The term transformation" reers to a change i charactesties, md a cell Ihs been transform ed w hn t has been modified to contain new DNA or RNA. For example, a cell is transorme where it is genetcaly mnodifiedl from its native state by introducingr.V new geetc material via trnstection, transduetion, ort other techno iques Foilowing! transfection or tranlsdc&tion, th ransforming DNA may recombine with Mat of the cell by physically megrig a chromosome of th cell, or may be maintained transiently as an epnomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have ben stablyy transformed" when the tran forrmn DNA is replicated with thre divison of the cell [OtN7] BY phvsooi'ally acceptable salt' of a conmosition of matter, for example a sail of the angien bmdig proiin. ch as ao untbody, i meanay salt or suat that are known or later discovered to be phartacuticalb acceptable. Some nonimitmg examples o pharmaceuticals acceptable sls are: acetate; -idluoroacetate, hydrobabdt, such as hydrohlorde and hydrdoromide, sufIat; citrate, nmalcate, tartrate: glvcoate, gIoceonate: stie mate; miesvlate: besy late salts of gaulic aid eter (gAlhe acid !s Aso known as 4 triby droxybcnoc aci ch as PecntaballovM hucnse I PG) i d cpigallocavtecrbo dalae (LOGO), salt of hltolei elfl ate parnoate, annate and oxalasah [0098 A kanan o "tre (usedierchnbly herei) of a proii us any poron of theinre pron. up to and i gete protein, bt typeagi 0y enlprSing kes than the cnpict rote A dOmai nt hut need not fld dependedytthe res oftheprtin cin andho be eKt d wih partonar biooical bochenta. or stur facion or location (0 a higand finding drmin U o y CtOSOl Ai tn emubinme or extrmaelr donantr [0099] "reatmnc"i or "treating" is an intervention performed with the intention ot prevening the development or altering the pathology of a disorder. Accordingly, "treatmem" refers to both therapeutic treatment and prop byactic or preventative measures, TIhose in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented, "Tfreatmnei" includes any indicia of success in the anelioration of im injury, pathology or cond ition, including any objectve or subjective parameter such as abatement: remission: diminishing of symptoms or making the inury. pathology or condtion more tolerate the patient; slowing in the rate of degeneration or decl ine; mak ing the final point of degeneration less debilitating; improving a patient's physical or mental wellbeing. The treatment or amelioration of symptoms can be based on objet or subjective parameters: including the results of a physical examination, self reporting by a patient, neuropsychiatrec exams and/or a psychiatric evalution [00100) An "effect Ae amount is genera y an amouu sutf eNt to reduce the seen\ty and or frequency of symptoms elmmate the svmptomsad/or under ing caGuse prevent te occurretiee 01 syrnptotes andt or their wmderlyinu cause, ad/or improve oi remcdiate the damage that result from or is asi sehd ith rntgramt headache In some embodiments the etfective amount is a therapeutically etiectwe amount or a prophylacties ity efftve amount. A "therapeuticauli' effetime amount'^ an amount sufficient to remedy a disease state eg., transplant rejetion ao GAM. inflammation mulipe $etrosis, eanc diabetes neuropathy pain) or symptom(s), pMrtic~dlaiy a state or symptoNi s soia t ed wih the disease state. or othlerwse prev ent, hinder, retatrd or rev erse the progrewion of 'the disease state or ainy other undesirable symptom associated with the disease i any ay xvhatsocver (ie, that provides "therapeutic efliey'. A Tronh iActaly etieem amount is an amlount of a pharmnaccua tis omposition that wh en administered to a suibicet w i have the imended prophylactic effect, c . preventing or delaying the onset (or reoccutrrenjcc) of rungtraine headache or mutdl ple e4erois rymtoms, to reducing the likelihood of the onset (or reoccurrence) of mgrame headache, migraine headache svnitptoms, or multiple sclerosi symptoms. the taul therapeur or prophy ltetne effect does not necrily occur by administration of one dose, and may occur il after administration of a senew of dose1 Thus a therapeutically or prophylacticaly etceiNe amount man be admatered in one or more admmistiatons, [00 IiR "Mammal for purposes ci reatment r tes to an animlass mifed as a mnamniatneding hmans~ domestic and titrm animals, ad oo. spns or pet animal such a dogs.lovs;s. cas. cos ratsne mo nkeys etc. Pcbiee mamma is hnman. [00 1 etr naturally occurring abused throughout the specificaion in [00 he term antibodyd u or itechangeably "oW ^ a used Ia the b oad sense and includes fully assembled antibodies monoclonal antibodies (inc luding humn, humtauized or c ei'ic antibodieh p lylonai unnbod ies, maulinspecific anibodies (eg. btspccific atntibodies} and antibody' tragments that can bind dantse (eg. h. Fa, iFrab' t, Fll single chatnaainubodies, thabodset cornsmy co'mpl':ntlemarity determlnn rentons ( CDRs) of the loiegomg as long as they eiubit the desired biologal activity \ttimers or ggrates of intac molces and/or fragments mludbng chemily deina d nibodies, arc contemplated, Antibodies of any isoype class or subclas, ieluding 1e.1IN Ig, ThA, and 1 InG I 1gG2, LgG, lgG4, Ig\l and igA. or any allomtipe, arc contemplated, Different tsotvpes hae di trent etector timetons; tr e'xmple, lel anmd IgO3 iSoty pes hav e antibody~dependent eiular cvtotouecitv (A0CC activty. [ot)01 041 The term antigen biding prote (ABnP) udde antibod s or annody coin eas dices derive iid tro Q haingt te Orothe bng ds oth - 13 oo01 05 1 in general.an antigen binding protein, e.g., an antibody or antibody fragmet speificallyv binds to an antigen (mg keyhole limpet hemocynin (KL 1 or dinitrophenol (DNE) when it has a significantly higher binding affinity for. and consequently ts capable of distinguishing, that antigen, compared to its a ffinity for other unrelated ptein, under similar binding assay codt ons Tpialy, an antigen binding protein is said to "'specttifia bind its target antien when the dissociation constant (K is <1> M. Th antIiody spccaly binds antign with h igh affinity" when the Ka is i5x 10" M, and with "very high iity" when the

K

0 is : 5x 10*~ M. In one embodiment, te anbodies wi bind to Ki L or DNP with a K) between about 0> M and 0 M, and in yet another embodiment the anibodies will bind with a K 1 Sx 10. H1(1T " Antigen bAn dmg regm"n or -anbgc n bdNg i' means a prison of a protein, that speeinealy binds a specitica angen, eg . kehoe lranpet hemocynin (L i) or dinitrophenol (DNP1 For example, that pormon of an antigen binding protein that contains the anino acid resaies that tract with an angen and confer on the antigen binding protein is sp ciiitty and affiny for the antigen i referred to as "antgen bothi ng regumn. Ane antigen bimdmg regon typk alil includes one or more "complementary binding regions iCDen) i twid antiier d regions also mcude onie or more 'ramew ork" reions A-FR ") A C DRI" is an aino acid sequence that ennr o antigen binding specfn and affinity "Framewor regions can am mamning the proper conformation of the CDRs to promote bnding between the an tigen binding teison and an aigen, [001(U?) An "isolated' anybody is one that has been identied and separate from one or more components of its naturd environent or of a culture medium in which it has been secr eted by a produneing ed L "Contaumamt components of Its natur al environment or nmediurn are mamneudk tht w ould inter fere w ith diagnostic or therapeutic uses for the aunbody and nmaymchude enzymes hormones and other proteinneous or nonproteinacatis soluteM n sone mbodiments the antibody will be prl aled i1) to greater than by weight ot'anbbod; and most preterably mo than 9% by neigwht, or (2) to homogeneiy by SDS-PA under reducing or nonredueingt conditions, optionally using a stain, emg. Coomnassic blue or silver stain. Isolated naturally occu\ rring antibody includes the antibody in situ within recombinant c'ls since at least one component of the antibody nature environ mn vil not he present. Typically, however. isolated a y w b prepared by at least one putrificaition stepn [001081 The term "naockmoa ant ibody as used hee refers to an nobody obumed from a population of subsuamtialhv homogeneous ,mnodes ie., the individual antibodies comprising the popuiaton are identical except for possible namuralh occurria mutations that may be present mn minor amionts Monockmnai antlbodies are hhly ;ApeeIe, beneg directed aga-ist an mda Rhial antigme ate or epitoe. in contrast to polyclonal antibody prepaunons that typicallv include Mifrenuit antib dieted ayat dhfl'erem eritopes \onog examp lea of monoclonal anti bodies nmch d' n' in , raoi rat chicken, chimeln, bumanki'ed, w barman antibodies fully aswmbl ed annb od6in ultispecie anmbodies (melcuding hoieie antiboteds Wdbody P moments that can bnd an anuigen including , F:a [ab'. [(a't Ev , smgle chamr antibodieN duabodies), muarnbodies namobodies arid ecombinant peptides comprsing (D s of the foregoing as long as the exhWi the desired Noo isa activy. or vrants or dem atSVes theret [001091 The nmodifier monuocional" indicates the charaer of the anybody as being obtained from a substantially homogenous population of antibodies, and is not to be construed as mrequring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may he made by the hybridomia method first described by Kobler et at., Nature, 236:4 95 [1975], or may be made by recomibinant DNA met hods~ (see, e.g.

U.S. Patent No. 4Y16567), The "mhonocltonal antibodies" t may also be isolated from phage antibody libraries usilng toe tech niue described in (iackson et at Nature3S2:624-68191 and Maks et atJ Mo.t Biot, 222:581 -5711991), for example.

[00 0 AmispeCific binding .g.aatiagn ina pron r anybody [(0111 l A bipcfc"dual-1specific" oY 'bilimetinal" binding agemt or antigen binding protein or antibody is a hybrid having two different antigen bAin, Biantigren binding proteins, antigen binding proteins anid atibodies a re a species of m ulO nig nb nd n roen ani e bindin protein or m ulispecific antibody and may be produed by a variety of methods including, but not lim ited to, fusion of hybridomas or linking of Fab fragntrs See, c. Songsivilai and Lachrnana 1990 Clin. E x imunol. 79:315-32: Kastyt W. 1992, . ImNunoL 148:1547-1553. The too bAdig sites of a bispeciic anigen bain mrin or anibody will bid to two different epiopes, which ma, rewide on t same or different protein targets, [001 12] 1he term Immunogobun encompasses ful antibodies comprising two dinerized heavy chains (HCi each covalenty linked to a i chain (LC): a sine undimerizOd immunoglobulin heavy chain and covalemly inked ight chain (HC + LCh or a chimeric innunogAIobuin (Ught chain +heavy chain}'c heteotrmer ja so-ealled "hemibody"), [001131 An "antibody" is a fetrameric gcoprote in a naturally-occurring antibody. each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "ligt" chain of about 220 amino acids (about 25 kDa) and one "heavy" chain of about 440 amino acids (about 50-70 kDa } The amino-terminal portion of each chain includes a "ariab e"("V") region of about 10 to 110 or more amino acids primarily responsible for anigen recogntion, The carboxyerrmal ponon of each chaIn defnes a constant region primarily responsible tbr effector function. The variable region differs amnon different antibodies, The constant region is the same among different antibodies. Within he variable region o each heavy Or light chain, there are tre hypervariable subregions that help determine the antibody's specificity for amigen, The variable domain residues between the hypervarabeV regions arc called the framework resdues and generally are somnewa homologous among different antibodies inmunoglobuhis can be assigned to di ferent classes depending on the amino acid sequence of the constant domain of their heavy chains, Humran iight chins arc classified as kappa (i and lambda (A) light chains, Within light and heavy chains, the variable and constant regions are joined by a "J region of about 12 or mor ami acids, with the heavy chain also including a "" region of about 10 more ammno acids, See cenerahy, F undamental immunolog Ch. 7 ~ (PuW ed2n ed, Raven Press N.Y. (19891). Within the scope of the inenton, an "antibody" also eneompasses a recombinanty made antibody, and antibodies that are lacking glycosyiaton [00 14) The termi "lght chain" or "immunogiobuin light chain" melde a hul length light chain and fragmemsthereof ving sufDicient viabL region sequence to confer binding specificity. A fulU-lcngt uiht chain included a variable region domain, Vi and a constant region domain, C The variable region domain ot the light chain is at the amino-terminus other polypeptide. Light ebains include kappa chains and lambda chains. [ol 15 The term "heavy chan' or "itnmuinogdobin heavy chain" includes a tL cngth hev chai and faments thereof having suffcan variable region sequence to confer binding specificity. A f Filength hea vy eban includes a varable region domain, V 0 . and three constant region comains, C(, Cu 2 , and C1>3, The V domain is at the amino-erminus of the polypeptide and the CQ domains are at the carboxyh terminus. with the Cq3 being cosesi to the carboytrmis of the polypeptide, Heavy chains are classifed as mu (p) deha (A), gamra (y), alpha h and epsilon ), and define the antibody' isoype as g M,' IgD, Igt, jgA, and lgE, respectively In separate embodiments ot the inveion, heavy chains may be of any sotyp, including Lg (including IgG, IgG2, Igf3 and fgG4 subtypes), IgA (including IgA and IgA2 subtyps IgM and igE, Several of these may be further divided into subclasses or isotopes. eg. IgG I, QgG2, gG3. gG gA and lgA2. Different lgG isotypes may have diffirent effector functions mediatedd by the Fc region) such as an tibdy-dependem cellular cytotoxicity (ADCC). and complement-dcpendent oytoo'y (CDC) hi ADCC, the Fe regon of an antibody binds to e receptors (FYCRs) on the surface of iune effector cells such as natural k in rs and mr ophage, tending to the Qhagocytosi o i ofthetageted ee s i CD the antibody ik u oe targeted e I by tine te coine its&ad he et surface. 0016 An egeiorouedintechaneably herei dotmo nor tindglobinhFe domadneontinstwo hev hai n fnagmeis, hich Ina fdt unody todeth Q and CI domaw of the attibod. he 1o heavy chain fragomwts are held togthe b two or more n tde bond and by hvdrophobic interactdons of the 01dnna. [0011?) The tem "safag receptor biding epitope" fart an epitpe of th rin~)0& of 1gG molecule (cgu g Ig s.gG14gk.or lg~ohtis resposfble for riereasir the na voerm hal i bf the c olecule [0011 j "Alotype" are variant in antibody Sequence, ofen mn the conshum ruion, that can be mmunogenic and are encoded by specific askleN in hunans Alloypes hane been idenified ior five of the hImman ICHC genes, the lGU 1, I0G12, [CR03 \AM and [It' genes. and are Jedunated as G ti GtM C3mt A2m, and 'm allotypes respctnyeiy At leat $ Gm alotypes are iknoww no oi( t I G i mQ), G lm (1, 2, 17) or G i m .t 0, itm(231 m G m (to Km(, A6 10. 1, 1. 1\. It6, . 24, A 2-. My or 63% ( ', h5, 0, 14. s, g1, cS, , vv, g51 There are toc Altn ailotypes A 2m 1) and A 2m(2 [001 19) For a detailed desenrpion of the structure and gnramion of anMibodies, see Roth Di.B, and Craig, NJ . Cl, 94:4 1[414 (1998), herein incorporated by reference in its entity B\lriefly, he process for generating DNA encoding the heavy and ulght chain immunoglobulin sequences occurs primarily in developing Bcels. Prior to the rearranging and joining of various immunoglobuhin gene segments, the V. D, J and constant (C gene segments are found general in relatively close proximity on a single chromosome. Dung B-cc -differetiation, one o eac of the a ppropriate famuyL members of the V, £5, J (or only V and A in the case of light chain genes) gene segments ate recombined to form fmionally rearranged variable reTgons of the hv nitm oglobin genes, Ti gene segment re.rrangement process appears to be sequential irt, ha cham D-o- points are madce, 'ilow0ed by heavy chaino-D joitns anJ light chain 'V-to-J joints, In addition to the rearranement of V D and E xgments further diyersity is in the primary repetotre of mmamoglobulm heaV and lght chams by way of vaable recombation at the lo s vs h the Ane htIi chain are min d" and where the D atd J segments of the heasvecain are loined. uch v ariation in the light chain ty pically occurs wtihin the Lust codon of the V gene segam and the firat codon of the J segment, SimiN i mreesnn Aining oceus oi t hles y han chromome between the) and n Ymemnu and may vend oer as man q s 10 nucleoides. Furthermore. ses Dral numeotde my be insned between the ) and J and between the YV andi D gene segments which are not encoded by cenomic DNA. ' he addition of these nueleotides is known a regon divetsity The net ekect of such rearrangement in the liable region cne segments and the variabe reconbmaton Wich may ocer durnu h om a is the producuon of a prnnary antibody repertoire, [020) The term h region refers to the amino acid residues ot sn antibody -h ich are rponsible for antigeninding, The hypervarnabe reion comprises amino acid residues tron a co"pementariy determining region i CDR [i.e., residues 24-34 (Lj SO-56 (L2) and 8997 (L3) in the hlt chain variable domain and 3 35 (H I Y 50-5 (H2 and 95-02 (H3) in the heavy chain variable domain as described by Kabat et aL, Sequenceso f Protins of ImnuogicI interest 5 i" Ed. Public health Service, National institutes of H ealh, Bethesda. Md, (1991)], Even a single CDR any recognize and bind antigen. ahhough with a lor. affinity than the entire antigen bindg site containing alt of the CDRs. (001211 An alternative definon of residues fron a hypervariable 'loop' is described by Chothia et at el Ma.io.ici / 96: 90)1AP917 (1987) as residues 26-32 (IL ) 50-52 (L2) and 91-96 ([3L in th oight chain variable doman and 26-2 (1), 53~5 (82) and 4- 1(1 (H3) in the heavy chain variable domain [00i 124 '1nTiwork or -FU residues arehose variablegindrside aher than tehypervriabl rgon esidues [00123) "Antibody fragments compise a portion of an intact ful le ngth antibody preferably the antigen finding or variabe regon f the intact antibody. Examnls of anmibody fragnments include Lab, Fabi Fhb) and Fv fragments; diabodies; linear antibodies (Zapata e at Protein En g1 0):57-1062 (1995)) smgle-chain ntibody molcule acnd uipcifi arntibodies formed frm antibody famns [00124) Paain digestion ofantibodieoduces two identical antigen-binding fragments called "lab" fragns, with asingle antigen-binding site, and a residual "Al' flnumnt witch coanins the constant region. The Fab fr gmemn contains al of the variable domain, as wel as the constant domain of the light chair and the first constant domain (CH Of the eavy Chain. T Fe framet d a carbohydrates and is responsible for many antibody effccior ftmetions (such as bn ing c ioent and ce receptors, that distinguish one class of atibody fror another. [0012 Pepsin treatment yields an RaQ! fragment that has two m -ch {in or Fvmuanibod t'ragments comprnwg the NA and VI domains oVau1iodv w herein these donmms are presemt in a single polypeptide chain Lab fragments dJer frin Fab' ftagments by the inclusion of a fbn additaoal residues at the curboav terrnmus of the heay chain (H ! dman mn diding one or more cYsteines troni the antibody hinge region~ Preferahly, the x pux peptide further comprises a polpeptide linker between the \H and \ N domas that enables the [y oan, the desired structure for antigen hindig Or a rt ia of seh ee Pluckthun in 'The Phravnaeohogv ort Monoelonal Anbodies vo1 31 Rsenbot and Monore edst Sprogin v ' erug, New ork, pp. 26 1 n a b c,1 00i 61 i6 A ab fragment" is conpsed of ne Oh chain and the (A and diblie bon 0h0c ohey caiavy ehhaia oa adisutfIe bognd"" with anthrhevcanmoeu.

4t (001271 A "' a' frngmtemot contains one light chain and a ornson of one heav chap that eomtunn the Vn domann and the t. domam and also the reion beteen the C l and W t domams snb th-t an e r'chn disulfide hnd canbeormed between the two heavy chamis ot'iwo f ab rents to tie an flab: molecule [0012&1 A "FtiN fragmenC comtn\ tuo ight chains and two bea chains containing a porton of the constant reion bet ween the Qo l and 2 domains such that an iMterham diulfide bond is ruined between the two heavy chins AF ab b fragment th us is composed of tw ith ftnients that ar e held together by a djsuitide bond between the two heaiy enas-s [001291 "v'i i he mimmu aotibody fragmem that contains a complete antigen r togmon and bindig site, his regon conssts Of a dtier 01'one heav and one iight-chaie variable dornai in Right. non-condent assoetation t is n this contmzm ation. that the three C DRs of each variable domain ieract to -definema antin N bndin ste on the surlice of the Vii Vb -dier A suglte i auie domain (or half of ar Av comprisng only three C DR wpei tI n fo antien) has the abil iy to rcongnie and bind antien. although at a lower afini) than the entre bardmg [00130} "Siegle-ehain antibodies" are Fv molecules tn which the heavy and tight chain variable regions have been connected by a flexible linker to form a single polypoptide chaie, which form an antigen-binding region, Single chain antibodies are i-scussed in detail in international Patent Applican Publication No. WT 8101649 and United States Patent No. 4,946T,7 and No. 5:2,3 the disclosures ofs wich are incorporated by reference in their entireties [00131 "Single-chain v" or 'cFv" antibody fragments comprise the Vn and V 1 domains of antibody, wherein thse domains are present in a single polypeptide chain, and optionally comprising a polypcptido linker between the V and V. domains that enables the v to thrn the desired structure fr antigen binding (Bird a a. Sciee 242:42.3426, 198., and HMston at au? Proc, Ad, Acad Sc. UA40 85:5879-S883, 188) An "Fd" frugment consists of the V and Cal domains.

[001323] The term "diabodies"? refers to small antibody fragments with two antigen boing sites, which fragmnts comprise a hev-hi v domin (VH) connected to a light-chain variable domain (VL) in the sanme polypeptide chain (V H VL), By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are fored to pair with the complementary domains of another chain and create two amigen-binding sites Diabodies are deserted more fully in, for example, EP 404,097, WO 93/ i16 and Holinger e aL, Proc. Natt Acad. Sci. USA, 90:6444-6&448 (1993). [)0133] A t"domam antibody" is am immunologically functional imnmunogobulin flaragment corntaining only the variable region of a heavy chain or the variable region ofa light chain, in. som instances, two or more V regions are covaiuty joined with a peptide linker to create a bivalent domain atibody, The two V regions of a Mancn toamte or diffream antinenas bivaient domain antibody miay taret<h aeo ifrn nies [001343 The term compete" when used in the context of antigen binding proteins (e. neutralizing antigen binding proteins or neutraiizing antibodies) that compete fi the same epitope means competition between antagen binding proteins is determined by an assay in which the antgen binding poin (egt antibody or immunologtica lly fnctional fragmeina dhereof) under test prevents or inh ibits specific binding of a reference antigen binding prten ("g- a lan or a referene antibody) to a common antigen (e.g. K1;H or a fragment t fhvof, r NP. Numerous types of competitive bindingt assay\ c-n be' used, for example: solid phase direct or indirect radioimmunoassay (R]A. ),>otid phase direct or indirect enzryme immnuinoassay (L A>L sandwich competition assay (see, e.g, Stabli et aL. 1983, Methods in Enymology 9:242-253); solid phase direct bion-avidin EA (see, e.g. Kirkhad et al, 1986, immunoL 137:3614~3619) solid phase direct labeled assay, solid phase direct labeled sandwich as a (se e.g. H arow and Lane, 198 Antibodies, A L oratory Manual. Cold Spring Harbor Press); solid phase direct 2a-bl RIA using 1 -25 label (see, eg. Morel et aL 1988, Molec. hmmunoi 25:7-) solid phase direct biotn avid in EctA (see g, Cheung, et ak 99, V0rovogy 1 76:546-52); and direct labeled Ri A (iMoidenhaueret aL. 1990 Scand. I, inimmuno 32:77.23. "yically, such an asay invoves the use of purifie antien bound to a solid surface or cells beaing either of these, an uniabeled ts ain inding proti and a labeled reference Intgen binding protein. (ompentte inhibitin is measured by deternning the amount of labed bound toH told surface or ells in the presence ot the test antigenn bindingt protein, U~suallfy the test annigen bindng protein is presemi in excess Antigen binding proteins identified by' competition assay (competing antigen bindi proteins) include antigen binding proteins binding to the same epitope as the reference antgen bd rotin and atigan bindigprotins binding to an adjacent epitope sufficiently prox imal to the epitope' bound by the reference antigen finding protein for stoic hindrance to occur. Additional details regarding methods for deterring competitive binding are provided in the examples herein. Usually, when a competig antigen binding protein is present in excess, it will inhibi specific binding of a reference antigen binding protein to a common antigen by atlas 40%4 41% 50, 55% 60%, 65% 70% or 75%. in some instance, binding is inhibaed by at least 80%& 85% 90%, 95%, or 97% or more. [00135] The trm nign refers to a molecule or a portion of a nmolecule capable of being bouid by a selective bindag agent, such as an antigen binding protein (inciding, e~g, an antibody or immunological functional fragment thereof and additionally capable of being used in an animal to produce antibodies capnole of birnding to tha antigen. An antigen. may possess one or more epitopes that are capable of interacting with diterent anigen binding proteins, e. antibodies. 01 6} The trins DNP" odinitophenoe are ed iterchangeablyherin and dene the antgei 24-din itrophenoL .)NP" orDNN or 'aDP arese interchangeably herein to rer to an anigen bionig protein. eg. an anybody or antibody fragent, that pecifiealg binds DNP [00137 The trs A F or yitole impet lengan& are used interchangeably herein and denotete n ect Mariture Keyhle ipet hemocyarn (ineKN dLIPe ac ot oogv Roc'd )Aord t manufctuern nK i s harveted from select populanons of the mollusk A3 Megathura crenuiata (keyhole Wipet} that are~ grown in mariculture, rather than being xtracte om wid popuhtions K H has a high molecular masQ0(4.5x 10 3 x 10 Daltons of mixed aggeates of 350 and 390 ka subunits) and eliCits a stronger~ immune response an SAu h% r ovalbuin. "niKHor "aKLU"r aKL H" are used interchangeably herein to refer to an anigen binding protein e g an amibody or antibody fragment tit specifically binds KLIM [00!i 1 The term epitope is the portion of a molecule that i bound by an antigen bindig protein (for example, an anybodyy. The term mcludes any deterrmtant capable of specifically binding to an aigen omnding protem, such as an antibody or to a -Cell receptor Ar eptope can be cotighiS or non eO itouS ie p in a sngcehain poly peptide, amino acid residues that are not conigous to one another in the poli peptide sequence buat that within the context ofl thnolecule ave hound by the anu=n bindi protein n cran endodmienis cpuopes ray he mimet in that they comprise a three diensional structure hat is \imdai to an epitope used to generate the amnsen nding protect or sonec the amo acid residues found m that epiope used to genera' the antgen bindg protein. Most often, epuops riode on protems, but in son instances may reside on Oiber kinds of modules such as nucleic cids. pirope determinants may mnlude bemically active surt'ee groupings of molecules such as amo acidssugar side chamw phosphoryt or sd fanyl groups and may have specific three dunensional structural charac terone' 'ndor SPiti Charg charactenRtis Generally, antibodies speific for a partaeultr tur amigen will preferenaaly recognue an epitope on the target antigen in a complex: mature ot proteins anmor miacromnoteuines. [001 The term "identity" refers to a relationship between the seuCeces of two or more polypeptide molecules or two or more nucleic acid molecules as determined by aligning and comparing the sequences. "ret identity means the percem ot identical residues between the amino acids or nueleotides in the compared molecules anti la calculated based on the size of the smnallest of the molecules being compared. For1 these calculations, gaps in al ignmnents (i> any) must be addressed byv a particular mathemtical model or computer program (L, an "'agorihm". Methods that can be used to calculate the identity of the aliged nuclic. acids or poilpeptides include those described in Computaional Moleculr Biology (Lesk, A. Mi d 988, New York: Ox ford University Press; Biocompui &nratcs anad Genomne Projects, (Smith, D. W. edt 1993. New York A cademi P'es Computer Anaysis of Sequence Data. Part L (Griffin, A. M, and GrifMi, H. G. ts) 1 994 New Jersey: HumIana Press; von Heinje G, 1987. Seqnceu- A n M York: Academtic Press: Sequence Analysis Primer, (Cribskov, M, and Devereux, L< eds 1991 New York:'M, Stockton Press ad Carillo et aL 198 8, SIAM . Applied Math 48:1073. For exam , sequence identity can be determined by standard methods that arc commonly used to compare the similarly in portion of the amino acids of two polypeptides. sing a computer progr suet as BLAST or FASTA. two polvpeptide orwo polynuceoidu sequences are aligned for optimal matching of their respective residues (either along the ful lnth of one or both sequences, or along a prdetermined portion ofone or oth sequences The programs provide a default opeong penalty and a default gap penalty, and a scornn matru such as PAM 250 [a standard matrix; see Dayhoff et aL ida ofPotein Squence and Structure, vol 5, supp. 3 (1978 q can be used in conjunction with the compute program. For example, the percent identity can then be calculated as: the total nuber of identical matches multiplied by 100 and then divided by the surm of the lenth of the longer sequence within the matched span and the number of gaps introduced into the longer sequences in order to align the two sequences. in calculating percent identity, the seNuences being compared are u igned i a way that gives the largest match between the sequences. [001401 The GCG program package is a computer program that can be used to determine percent identity, which package includes GA P (Devereux; ct at, 1984, Nul Acid Res, 137; Genetics Computer Group, University of Wisconsin, Madison, Wit The computer algorithm GAP is used to align the two polyvpeptides or two polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of ti respective amino acid or .45 ruceoti de (the "matched span", as determined byteagrtm.A gap open penalty (which is calculated as V the average diagonaL wherein the "sveraen diagonaf is the average. of the digonal of theomparion matrix being used the "diagomd" is the score or number assigned to each perfect amino acid match by the prilar cotmrsor matrix) and a gap extension penalty (which Is usually 1/10 times the gap opening penalty) as well as a companion matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithn i certain embodiments, a standard comparison matrix (see, Dayhoff et aL!. 97 At las of Protein Sequence and Structure 5;45~352 fOr the PAM 250 coxmparAon matriW enkoffet al1 1992, ProeNatiL Acad. Sci, U.S.A 9:10915- 10919 for the BtOSLM 62 companion matrix) is also used by the algtorithmr [001411 Reconrended p maets tedemining percent idemna t polypepdeor me eotdde sq ewse es s nu the GAPpra ne od he folog. [00142) 0ioih;Ne lia ta,170. , WI. MRu 45443453 0143 emarson tri )S 6 foniofe at, 1992. su, [001441 fap Pnalty: 12 (but wth no penat fr end gap) [001$ Gap Length Penlty4 [001461 hwesho kiof Sned aity: 0 [001471 'erin ahymnent acemes tor ab m ing o auino acid sequences ma; readt matching ofonl a short region of tie two sequences and this small lined region may have e ry high sequence identity even though thee is no sigiict relatoIoship between the tM wASuength sequences Aceor di lv the selected abgnoment method (0AP? program) can be adusted if so desired to rest n an alignment tha~t span at least 50 comcuous anuo acds of the target polypertide [00Ij ' he term "'Nodit ation- when ised in connection with anttigen baldin proteins, meuding antibodies and amibody fragment of the inventon, mnude, but -46 are not limited to, one or more 4mino aCid changes (inedoddg s\u ttiot0 insertions or dJenuds Y cbenu odj ito Ior covaleNn madItton hw conjegaon to therareutiS or dtiSanstk agents: ibehig (ea with radionuehdes or \ anmouA etiVYe COVMent 3ol\IVncr att ewtnt such as N I- id1 (Kderi\ativano w ith r10o\ ethvlerie gidcoi anld insertici ut suhstdon hia cheiiu s\ntiCle 0 ofOiW natural <nmo ads ilodified agntAg bindmg pot ems of the invention will retain the bindlia proptiert of aunodtfied no emes of the tU\ VtOtioni 40 149 The term "derivative" wiha use n connection with antigen handary proton> (induding atibodies and antibody fragmentsoOf the ivention refers to 301301hin10gproteins tat ar e covalentlv rnutcid bY cotuluga~tionl to theanentice antigenbindi, of ragnotl agents leling (e g. with radonundes or various en mesl. ernD polymer attachmt e asd PEvIation (deri\ati/ati0ni w ih polyethylene gi) wOO and narion of suAstu n b\ chemicaI synthesis of non-natural amino aeds Deriyautives of the invention w il retain the binding properties of underivatized okecules of the invention. [001 50] nnuolbai ihdtet FAdcoBnigPoen [0015 h ; In idi-length rmmmgiohahn Itlit adl heavy cms he variable andu content regions arc Qined by a 'T region of about w elve or more amino acds with the heavy chain also melhdiA a " regio n bout ten more ammo ands Sec. . , normal Immunology 2nd ed. M h I Pl. W. ed I 19W New Yorl Raven Pr'' thereby incororated by reference iti its entrety for all purposes} The variable region of e d bghtmheavy chain vt ypialyorm the tg d [001 52[ One example of a human IG2 hev chan(C onsat doana the aino acid seqpcnee A STIKGPSVPPLAPCSR STfS FSTAALO(TVKDYVRUKPS.G f11W SA TOVT PAY LQ\SGIbY S LCNVV PW UN lS NKVDK'J RKUCY F (PPCPAPPVAGPSVFLPPKPKDTIM SR TPF:TCVWVY DYSIEPE %QFN WY -47'I V F1 NAKWPREEQ STFRVVS VIVyQD L YKKXSNKG PAPI KTFSKTKGQPPREPQYYTLPPSREE$NIKN QVSLIUL KG bY PS DIAE\ ESN CQPEN N KPPM LOSDGSF ULYSKLT DOSR WQQG NV FSCS V HE Al IN , QKSLSSP G Q. iD N 6 [0015 31 Const ant region sequences of other lgG isotopes are know n the art for making recombianjt versions of the inventive antigen binding protein having an lgG 1. LTG2, gG3, or IG4 immnunoglobuin isoTe if desired. In general, human WgG2 can be used for tarts where efTetor functons are not desired, and human EgG I in situiatiotns where sch ettector tfetions (c.g. atibtOdy-dependent cytooxiitvy (ADCCp) are dired, Hunan gG3 has a reatively short half tTbam hanum lgk4 forms antibody "half-moeules Ther are om known alotype\ of human lgG LThe preferred alloype is referred to as "higGlz also known as the "KEEM" alotype. Human IgG alotypes "hG Ua (KDEL ) "hgDG1 (REEM) and '"hlgG lifa" arc. also usefUl; all appear to have ADCC effetor option, [001541 Humian h! I# heavy chain HC) consat domain has the ano aid ASTKPSV FPLAPSSKSTSG GT A A L(CLV K DY PEVTVS WNSGA LTSGVUT FPA V LQSSG LY YSLSSV VTVPSSS5 LGTQTY K'N VNHKPSNTKXV DKKV EPIKSCD KTHTCPPCP APEL LGGPSVFL FPPKPKDTL N S R TPiTCVV VD) VS UED)PE VK UNWY VD6V EVHNAKT KRE EQY N ST YRV VSVL TV L HQD W LNG KE Y KCKV SNKAL.PAPI EKT ISKA KGQPREPQ VY T L PPSREF MT KNQVS LTCLV KG FY PSD AVE W ES NGQPENNYKTTPPV L DSDGSFFLYSKIL VDKSRWVQQGNVPFSCSV MHEAL HNH YTQKSL SLSPGKH SEQ ID NO:7. R0 1551 Human higGIaheay chain (IC) constat main ha Athe amino acid A STKGPSVFPLAPSSKSTOGTAA LC D 1 KDVF PiP VTVSV N SCOATISC VI I UIMAQSSG YSLSSVVTVPSSSLCW ITY IC N NF KPSVLKV DKKVLPKSOI) KTH PCEAPE.I.NGPSVIPPKP K~ LMSRTEVTCV VDVSH E)PEVK FAA AVEESNOQE EN NY KAI PPWJVLD)SDCSFFL-"YSKLiVYK RWQO VJCV 001561 It maqn hIg(Ii f heavy Chainl (0 convtant doawlid zs thc aminp acid A 51KGPS VEPLA PS5K Si:SGTY~AEGC L VKDY , :[, FPP VVV' ,SWNSGAETSO WHYl EP V~.SSL S SSVVTVPSSS [OTQTYl N VNNKPSNTKVD)KKVEPKSCD) KTHTCPPCTPEELEPSV l FEEPPKPKLAI [ SKIPIEVI r(NV VVSH DHV PK FNWV DWVE1 ' VNKTKPR' ,:fE EQ YN STY Ni V-VSV [TV [riQ AVEJ N COK E Y KG-, KM, 4 [A [N: YTQSt SPGOKS SEQ ff)' N(09, VP AVEQSSOEY SLSSVVT VIPSS SEGTQTYY UI{I MKS NYKVI) KR VE PKSCI) KlUfit PECEAPEE LAOSML VP KP hWYt EN] SUPEVI (IYM5 UEL)PEVK ITA LNH[ITQKS LS SSPOKI SEQ )M9' ('1 13VTTPS K Q SNNKA SSYE IP ElT-:QWwKSFIR S V SCQVTUEGSTVEKIAP VEICSi I SEQ II DNt>) " [0 T59 . I is nsfu to inrease te c o ntibodie and is conenient her Y CL 62 and CLwh c anaso e sed ithn the scope the resen [00160} C..2 hou hgt 3 hain (E) constant doman has teami acid sequence GQ KAA.PSVT I 8PPSSE EQANKATL V KADSS KPVP i V TTPSKQS NKAASS SLTPEQ WKSHRS S CQV HEIT SK. NAPT E S S Q K) NQKSQ rooIfI 'CLA- human Ccntndoanhsteaiocdseuce CQ), t< SAP III S'PSLAKT VC S N h VIA W SKAT)SV PYAG A1 [001621 C-W hunaon ULcntn doma-in a t i~o ai eune GQPKAAPSVTL FPPSS EL. QANKATE VCIVS DFYPGAVTVAWKADGSPVKV G KIKPSKQSNK AN) SSV L f $RSY S(R UITVAP ECS SEQ I N oiera structre, conisn e nse We e oreins t edb reaons orCRa ThD CRs'o th tw chins ol eac hev1 hi/itcar rO's qrau 1ra iOh&Ga pair mentiond above typica a ned y thetamok r eions o fonm strctre ta ns spec tcaI wha epitope or omnon te tetet> K L ortYK). ~rcp~ -te~mn;.to Wdonvinal, natur-allv-occcurring lgt: ani dheavv chin riabl eicf bot tynic on t the fe emnts: R C& R DR3 and FR4 A mhra s mte has been devisd for asiein nvmprbers to uano Ads thOcp omii in eac of hese domain K s punieg syste is dined in KaSequnc of rote n of n kie I t 87 nd N W NI add MD) or Choti a & eW.98 96 9 Chothia V t. M. Aaita i 42 , [00 164] Specific examples of som~e of'the fai length light and heavy chains of the anibodies that arce provided and their correspond ing amino acid sequences are sunnarized in able i 1A and able 1 B below, Table IA shows exemplary gt chain sequences. all of' which have a common constant region lambda constant regio*un - CL- S) I[ NO:9) fr all lambda light chains. Table I B shows exemplary heavy chain sequences, all ofwhich include consent region human 1C2 (SEQ 11) NO861 However, ecompassed within the present invention are imu uins wi sequence chances in te cntaml, or f ore those isned in Ta l and/or TabhlB (eg.tIgG4 vs 1gG2, CL2 vs CLIi Alo, the signal peptide (SP) sequences for al of the sequence in able I A and Tal 1B are the same, Ei. the V11 SP signal petde: MDMRV.PAQLLCLLLLWLRA RC (SEQ ID NO:103; sige undermined) t i used in the bigh throughput clonn process, but any other suitable signal peptide sequence ay he employed within the scope of the invention, Another example of a usefid signal peptide sequence is VH2) SP MEWSWVFLTFLSVTTGV S (SEQ ID NO:95Y Other exemplary signa peptide sequnces are show in Table 1 A-Rl. a dob unde MDR xeg a de dated b single undee and f ouw and eoatn econi e riat underld SE DeSIgra C ntaintd Sequence D tn o ino(1Ine( QRKPGZAPKLYASL QSGVPSRP SIS U-De--ISSQPD AANCOOASS P- -- OQGTRVEI KET AX~virpso QIPnE K&OC NSQES(VT EQDSKDSTYS LSSTU LSKA DY KH KVYA CE VTHQG LS PVTKSFNRG C M\RV\QLC.LLLKQRCDIQMTQ O 3A4 S PSSVS ~sVCDRVTUPTCRASQC3sRRLAwY QQK PGKAPKL L AYA ASSL QSGiVPSRFSGSG

S

5 GTDFTL ' TISS L-QPE DEATY Y COOAN$FPFT FPGPC'[KV )iKRTV AAPS VFIFPPSD)EQL KSGI TASVVL U.NFPYPRE AR KVQ WKVDNA E.QS YKADY KR HK VV A EVTF-HQGLSSPVT.KSPNRG EC MDM K? AQ.LLL.LL A RDIQM KT 2 I SPsstSASEGDRVmCVRAsQG RNDLU\wY GSQC L P P kl AASSL QSGVPLRF S SGTEFLT ISSL.QPEDFA TYYCLQYNS\ PWI GQGKVEPIKRTMAAPSVYFPPSDEQKU 1A SM C LLNNFYPREAKVQWKVDNALQS GNSQES VT EQDSKDTYSTLSSTL T LSKA DY EKIIIIVYAE 1'GLSSPVTKSE'NRGEC DKCi Qf I tit"I-UD Q MTQ 125 4 C22 SPSSLSASVNRITCRASQGMSNYLAW SGTDFriirTISS LQPEDVAT YYCKES si E FGPGTKVMDIKRTV AAPS>VFP PPSDIEQE..SG I SVCL. E. PR RVQWK VDNALQS (INSQESVITQDSKDSTYSLSSTLTLSKAD)Y EKIKVYACEVHQOGLSSPVTKSNRIC R PSLASGDVT1144SLQY

Q

t KPCK AKRR LIVYAASSLQSGCV PSRFSGSGS5 GiFTLTISSLQPEDF ATYYC£LO(YtNS\SPWTFv GQGTEVEIRTIVA A PSVEIP\SDEQL K SGT ASMC INNFYPREAKVQW KVDNA\LQSG NSQESVT 'EQDSK DSTY SLSSTi SRkADY E KHKVY A C V EVTYHQGLSSPVT K SFN RGEC1 kntt MUM 1 P 3 RSSL S, GD RVT1T G ID NNWYQ G QKPGsKAP KL _SI- A FOPSRFSOSOS (3 FT CETSSiQG) G JNGQ NITOQ SL E KRV AA PS IPPDEQKSAS VVCLNN YI>RLXKQWKVNALSCNS QESVT QDSKDSTYS SSTLT LSKADYVE KVPAl'1 ~I&LQG LSSPVTKSFN RGEC IQL IL1'Qy QELVLTQSPGT 3 7 hL2 LSLSPGERATLS(tlASQNI TNYLAWYQQK PGQAP RFU YKASSRATILPDR SGSGSOTD) FiURSR EPEDFAV YYCQQVGRPSPGQ GITKLLiIKRTVAA PSV F FPPSDEQLK SGTAS VVCLLN NFY PR(EA KVQ WK VDNA L QSGNS QESVTEQ/)DSKDSTYSL SSTLTL.SKADY EKL KVY AV LYT [HQGLSS PVT KSFN RGEU M....L.L .LPDTGE yLTQSPGT 137 LSLSPGERA TLSCRASQ ISTNYA A WYQQK NQ PQAPRFLIYASSRATGPDRFSGSGSOTD C FLTfSRLEPEDFAVYYCQQFGRSPRSFGQ GTK LEIKRTVAAPSVF1FPPSDEQLKSGITAS VCLLN:NFYPRELAKVQ WK VDNA LQSGNS QESVTEQDSKDSTYSLSSTLTLSKADY EKH KVY AC EVT HQGLSSPVTKSFN RGW 14 L 9 1206 PSSLSASVG TCRASQGiRNDLGW QKPOKAPKR LIV ASOSGV PSRFSGSGS GTFTL' F SSt QPED 1, RYYCLQNSYPLTF (XGGTKVE KP RTA XAPSVFIEFPPSDEQL KSGT ASVVC LNNF P LREA KVQWK VDNA LQSG; NSQESVT EQDSK DSTY SLSST TLSKAD EN' KI k N\ FVUQLSSPV

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VHQDWL.NOKEY KCK.VSNK GLP API EWTIiSKTKGQ PREPRQVYTL PPSR EE * \MTK NQVS SLTC LVKCGFYPS DIMA VEWE SNGQ i PENNYK ITPPDSDGSFFEYSKLVVDKSR VQQGNVFSCSVMHEALHNHYTQKSLSISP GK ME Vt y QLLQLLLAWmtQARC(QVtQLVE E4 0A SGGGTVVQPGSLRLSCAASGFTFSGMH (WX10 Y) WV RQAPGK GL E[WV A\VWYDG.S.N.YYAihD SV KGR F ISDVRN SKT MNSLRAEDT AVYVYCAS \ N\YGMD\ WG QGfTT VSS ASTKGPSVFPL4A PCSRS'ISES >AA LCCLVK DYFEPVTVSWNSGALTSGWTFPA VLQSS G LYS SSVVTVPSSN FGTQ Y TCN VDKPS NT'KV DK TV ERKCC VECPPCPARRY AGPSV FLFPPKPKDTLMISR FT PEVTCVVV DV SH ED P EVQFNWYVDOV EHN A 'KP REEQF. QNS Ti RV VSV LVV HQDWLNGKEY KCKVSNK * ~~ GPA\ PIEK T I SKTKGQ PR EQYTL11PPSRiEHE * NIMTKNQV SL TCL VKGF YPSD lAV EW ES NGQ P RTENNYK T TPPMLDSDGISFLY SKLTVDKSR * WQQONVFPSSMH EA NHYTQKSI.SLSP \II/ APC!fl K L. .Vj.j.RU*ARQ111 F 117 H5 3A4-FSS \GGGVVQPCORSLRLSCAASGFTFSSXGM.H wyRQAPOKuILEW\VA VIWYDGSNKYYAD U SVKGRSRONNT YLQMNSLRAE DT \VYYCARYN NYCMDVWGQCTTV VS A\ST KGPS V FP L AkPCS RSTSEST1 A ALGOC L VK DYPEPRVTV swNSGALTSOVHTFPANVLQSS G$LYSLSSVVT VPSSNFG TQTY TCNVDH K PS NTKVDKTVERK VPrCPAPPVAGPSVF F..PPKP.DL. MISRTPEVT .. VVVDVSIHEDP E VQFNWY V DGs VLEVHN AK KTKPR EEQFNS\I t PAS EK RKTKQ PQV VTLPPS REEN T KNQVSTE VKGFYPSDI AVEW XESNGQP NN TSM]SDGSLS VDKS 1 WQQGNVFSCSVM HEA 1.H NI] VTQKS LSSP (,K \I1U .MIWPAQ~~ QE I. VJE I (O 119 116 SGGGVVQPGRSL L SCAASFTFSSYGMH X , VE SRDPS'KM EWYAI QDGS\K Y Y D \VKGRE'TISRDNSKNT LY LQMNSLR Ei \DT AVY CARVN NYGMVWQGT TVVSS AST&GPSVFPLAPCS.R STSTAALGCLK DYFPPVTV SWNEGALTSGVHITFPAVLQUOSS G LYE SSS VTV PSSN FG TQT YTCN VDH K PS EVQFNWY VDGVEVHNAKTKPREEQFN\S FRV VS VLT VVHQDW LNGK EYKCKVSNKG 1PAPIEKI I SKTKGQPREPQ VYT LPPSREENI TI KNQVS TCE V KGFY PDIA VE WESNGQP WQQNVECSYHEALNH YTQKSL SLSP \MDMNIR VPAQKOL.LLRciAgC RCOQI QE 28 SG14 NPGL VK PS ETILE LITCTYVSGGS SSY W XS\VI TISVIDSKNQFS KLSSVIA ADTAVYYCAR T YY DSSGY YYRAFD WGQGTMVTVESSA ST KGPSV FP[L APCSRSTSESTA ALGCLVKDYP I<EIPv kWNCATSVPT PA LGL YSLSSV VTVPSSNFGTQTIYTCNVDH&PSNJ KVD)KTVER KCCVECPP>CPAPPV AGPSVFLF PPK PKDTLMs~ 1111iCW V OYEE E Q NV i N KP EQFNSTER \VSVVHAQDWI NGKEYKCKVSNKGLP \PI EKTISKQR KE KQ PQVY TLPPSREETK NQVLI'L *-**-'A VE\VF.SNGQPEN N DGSFLYSK LTV DK*SR.WQ v QGNVFSoV E Ht NHNH YTQKSLSLSPGK 19 18 4 P NXIRQKP(I G GTSREN P& ____ _____* ______ CRTGYSEQWYPFDYWGQGRTVSVSASTI KGPSVPPI \PCSRSTS AST ALGCLVK)YF PEP'VTVSW\kNSGALTSGVH MTFPAVLQSSGL YSLSSV VTV PSSN FGTTY Yi TN VDHKPSNTV KVD)KTV[ER>KCCVMECPPC(IPA PPVAGPSVFLFP PPKKDI fI MHSR T PE VTC VVV I\S HED PE V QF~NWTE V TUM VFIAKTKPREFNSEWR MYRM TMRQD NuPN CKVSNKASLPI AEK .SK D KGQPREPV T LEPSREEM K \NVSL'TCLV"KGFYPSDtIA

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VEESNCQPEN NYKTTPP LDSDG N YK LTVDKSRWQ QGNFSCSVMriEALHUNHYT~QKSL.SLSPGK 144 H9 3 SOPGLVKPSETLS.CTVSGGSISHYYWSW VTISVDTYSKNQFSL[KLTSVT AADTAVYYC NYP IWQGTM VSSA STKGPSVFPLAPCSRSTSEST A A LVKD YFFEPVTVSWNSGALTSCVH T P VVLQSSG L\ YSSV VTVPSSNFCT QYTN VDHKPSN F PPK PKDTLMISRTPEVTCVVV\DVS\HEDPE MQFN W V DOM .: XKTKPREPEQ FNSTE RMV\?LT'VHDW1LNGKEYKCK MSNK GLi I APICK 1ISWKYPIRPUN YTLPPSREEMTI IKNMSLCLKCEN SP MEESNGQPEP N YKTTPPML DSDOSFFLYSK LTV KSR W * QGNMVFSCS VMHEA LH NH YTQK SL SL SPG MMRVPAMQ 1.(iL .. NVIJARQVQ L V 145 H 3A4 SGCGVVQPCRSLRL SCtASGFFSSY I 3C WVRQAPOKWLE WVA VW Y DCSNKYYA D SMAER F11 S R 1) N SRT INSLRAEr A MY YCARY NM NYG MDVGQGTVTVSS ASTKG PSVPLAPCSRSESTA ACLVK DYFPE PVTVSWNSGA LTSG VHTEFPAVLQ(SS GLYSLSS VVT V PSNETQTYTNV DH KPS NTKVDKTV ERKK . VECl' PPCPAPPVACPSV P LP.K P.K iE .lISRTPEVTVVDVSHE PEVQFNWY VDGVEMHNAK TKPR EQE S TTVVV f Q G KCF D KEN K''KVSNK GsLPAPIK ISKT KGQPREPQ VYTL.PP SREE VIFKQM LCEV U V S AVMEWESNGCQ PENNMYTTPPMLDSDGlSFFLEYSK LTVDKSR 77 H A- JRyQ~glilhgVQ V W11) SGGVVQPRS. .R ..SCAASGEFSSYGMH WVRQAPGKGL EWVAVIWYM)SNKY WAD SVKGR'TISRDNSKNTLY LQM SL RA EDT * XVYY CA RYN FNYGc M t DVWGQGTTVT VS ASTKGPSVFP L A PCSRST S EST AA LGC iK DYFPP\VTVSi WNSGAL TSGVH TFEPA VL QSS GLYSLSSVVTVPSSNFGTQTTCN VDHKPS NTK DKV VERKC VECPPCPA PPV AGPSV LFPPKPKDTM ISR TPEV'ICVVV DVSH ED * F PVQFN WYV DGV EVHN AKFKP REEQFN S I'RVVSVLTVVHQDWL NGK EY K(IKVSNK G LPA PIEK TISK TKCGQPR EUPQV YL PPSR EE MTlK NQVSLTC LVKGFYPSIIAVE SWESNGQ PENNYKTTPPML DSDG0SFFLYSKLTVDASR. tQQGN VFSC VEA L HN H Y QKSL SL cP \IDRVPQLLOI I I W KAR%VQ V[ E 181 F2 l\M x SGGGVVQPGRSLRLSCAA SCFTFSSYGMH A 01 N WV RQAPGKGLEWY VVWYDGSNKY AD SVKGCRYTI SRDNSKNTIViYLOMNSLRA EDT * \STKGPSVFPLAPCSRTSIESTAALGCILVK DY FPE PV TVSWNSGA L T SG VITPFPAY VLQSS GI Y SLSSVVTVPSSNFCTQTYTCNVDHKPS NTKVDKTVERKC(CVECPPCPAPPVAGPSV FLFPPKPKDT.LiSRPviCVVVStHED II [YE WYM IAI T N "IlK PREE WQFNS TFRV VL T QDWLNKEYKCKVSNK * GLPAP LK' '4SKTKQP REPQVY TL PPSREE * YFEMKNQVSLIICL VKGF~Y PSI)IAVEF WESNQ * PENNYK TTPPMLDUSDG)SFFLYSKi.TVDKSR WVQQGNVFSCSVMHE3ALHMNHYTQKSI SL SP G MIDMRVPAQO G*I.W tRGiARCQV QI..VE~ P2 Ei A IS SGGGCCVVQPGRStRL SCAASGFTFSSYG(MH WVROAX PGKGL. EWV? AV WYDGSNK YYN AD NVKRFFIlSRDNSKNT i YLQMNSLRAII * XVYYCARYVNY&'Mi)WGT TVI TV/SS A\STKOPSVFPLAPCSRSTSESI AN ALGCLVK DYFEPEPVWFVSWNSGA LITSV HTFIPAN VLOSS GC YSLSS VVTVPSSNFGTQTYTCNVDH K PS _____ ____________NTKVDKTVERKY'VECPPCPAPPVAG \PSVF LFPPKPKDTL. MISRTPEVTCVVVDVSEDP QAVFN WYVG VEVHNAKTKPR EEQFNST FRV'VS VL TVVHQDW LNGK EYKCKVSN KG P P1FKTISKTKGQPR!EQVYTLPPSREEM I KNQVS~LTCL VKGFY PSDIAVYEWESNGQP E NNVYKTTPPMALDSDGSEFL YSKXLVDJKSR WQQCN S S VM H'c DAL HN HYTQK. SLSAP I O HX A4 YS Sl GGVVQPRSR.SCAASGF TFSSYGMH WVQPSG AV MY DGSNKY YAD A VY YCARYNY NYCMDV WCQGTTVTVSS A\STKG PS VFPLA P(CSRAST SEST AA LOCLV K DYFEPE PVTVS WN&GA L[TSG VHTIFPA V LQSS tL YSLSSVVTV PSSNCJ E TCGNQVD H KPS N TKVDKTVRKS VECP(PPPVGPSVF LFPKPD~..ISR TPEV TCVVV. DVSH EP E VQFNWYVDG\VEVHNAKTKP REEQFNSI F RVVSNVLTVVIHQDWLNGiKEYKCKVSNKG L PA PIEKTISKTK GQ PR EPQNVYTL PPSREEM I YKQVS . LVKG FYPS DI AVE WE S NGQP NNYKTTPPMLD)SDGSFLYSK TVDKSR \v(?RN PQLG 1 ' R GAtCQVQLQE. 4 3 GPG VKPSET LSLTCTVSGGSISSYY WSW T!SV DTSKNQFSLKLSSVTA ADTAVYY(CA\R TYYDSSGYYYAFD 1 WQTM VTVSSAST KGPSVF[PLAPCSR STSESTAALGC LVKDYXF PEP VISNSAi OVI PN QS * YSLSS VVTYPS SNFGTQTYTC NVDHKPSNT N'DKTVERKCCV ECP>PCPAPPVAG(PSNVFLF v I 'KPDvTAIsfTCV VV VSH EDPEKV QF N WYVDGVEVNAiKTKPREEQFENSTERA V VSV. . VVHQDWLNKE YKC KV SN KL P A PEKTISKTKOQPREPQVYTLPPSRE FNMTK NQVSL TCL VK[FQYPSJDI ANV WESNGQPE N NYKTTPPMLDSDSPLY'SKLT VDKSR Q QGNVFSCSVMHEA UNYTOKSLSLSPG MIDMRVPAQ.LLLLLMARCQVQQ 18 j6 L4 GPGLVKPIQTLS TCVSGCS ISSGGWn SWVIRQHPVGKGLEWIGNIYY SRSTY YN PSI RVTISVDTSKNQFSLKLSSVTAADT A VY C ARIG(IYSiGYI:DYWVcGGTL V VSSAST KOPSVFPL APCSR STSEISTAA LGCLV DY F\ PEVV SWNSiA L TSGVHTFp.AV LQS\SGL Y S LSS VVTvS S NCTQT TNVDHIKPSNT KVDKT VERK CCV E.CPPCPAPPVA PSV LF PPKEPKDT LMISR TPEMVVVDV S HEDPEV QIFNWVYVDGVEVHNAKT TKPRE'EQFNSTE'R VVMSMVMVHQDWLNCKEYKCKVSNKCL P A PIEKTSKiKGQPR FPQVYTLPPSR E FMTK NQVSLTCLV K 'YPSDIAVEWESNGQPEN * ~~ NYKT l PPML.DSDGS FFL Y SKLTVDi)KSR WQ Q GN FSCSM EA HNYTQKS S LS LP At KiLl M;33 W; NQW SLQVQC EVQLVESGG 3 17 L ( VQPGGSLRLSCAASGTFSNYDMYVRQ TT0KGLE WVSALGTLAGDIYY P0SVKGRF' ISRENAKNSLYQM NSXRAGDVAY YCAR KSSTSEI)YWGQGITLTVSSASTFKGPSVF PLAPCSRSTSESTAALGCL.VKDYFPEPITV NWNSGA L SGVHTFPAVLO9SSGLYSLSSVV TVPSSNEGTQTYTCN VDH1KPSNTKVD)KTM RKCCVECP PCP PP AG PSVT LFPPKI) STXMISRTPEV TCVMDVS EDPE VQNWY VDGVEV HN AK TKPRE EQFN\STIF RVVSVLuT V VHQD W LNGKE NXKCKV SNKGL PA P EKT \KTKGQPRE PQVYX TL PPSREE MTKNQVSLIT CLNVKEYPSDIA V W. ESNGOPENN YTTP P MLD)SDGFFL N SKL T VDK SR\WQQGiNVFS CSVM E AL HN TQKSLV SL S PK \KIX FLEXsD VA W Q )LQ LQQ ESGP 139 H18 GLXMKSETLSLTCTVSGGSCDYSSSSYFWGWI RQPPG KGLEWIGSlYn YSNTFYNPSL KSRV I SVD)TSKNQL S L L NS MTA ADTAY FC A RQQGjAREY WY IWG R G TTVTVSSA STKGPS\VFPLAPCSRSTS'STAALCAK D XTET~ VS WNSGA LTSGVMHTFPA VLQSSG LYSLSSVVTVPSSNVGTQTYTCNVDH KPSN TKVDKT V ERKCCVECPPCPAPPMAGPSVEX F PPKP KDTRLMSRTEVTCV VVD-VSH E DE VQFNWY VDGVMEV HNAKTKPR FEENSTF ____ _____ _____RVVSVTWV VHQDWINCK EY KCK VSNKGLF 'PAP IEKTTrSKTKG(QMP FEQ VYTL'P PSP EEMT KNQVSLTCL VKGFYPSDIA EW ESNOQPF N NYTI'PPMiLDSDGSFPL YSKLTDKSR\W W}QGNVFSCSVM lEANHYTQKSLSLPG 1K 143 119 2, 1116AT QVQLVQ EVNKPGASVKVSCKASGYTFTGYHIMHWV RQAPGQG L WM W N PNSGGNYAOKF GRVTMTRDTiSIS'AYMELSRLRSDDTAVY YCAR DR.SY.Y WF D PWGQGT L.VTVSSAST KG PSVFEPLAPCSRS S ESTAALGCLVKDY F PEPV TVSWVNSGAL' TSGV HTFP AVLQSSGL Y'SLSS VVTVPS SNF GTQTfl CNVYDHK PSN T KV)KTV E RK((IV EPPCPAPPVAG PSVIL F P PKPKDTLMISR T PEVTCVVVDVSHEDPE V QFN WY VD V EVHlNA KIKPR{EENSTIR SV L T V 1VHQDWLNGKE YKCKVSNK( P A\PIEKT ISK TKQPR EPOVYTFLPPSRE EMT K NQVSL TCL VKGF YPSD AV EW ESNGQPEIN NY K TTPPM LDSDGSFFL YSIKLT VDIKSRWQ ( QGNVESCSVMHN EA LN YTQKSL SI SPOK 4 1120 20.6 \MDMRVPAlF . RARCDVQLVQ SGAEVKKPGASVK VSCKASGYTFTGYHM I WVRQA "PGQGLE\IWG\INPNSGG'i'NYA QKFQG RVTMTR>DTS ISTAYMEFLSRE RSDD) TAVYYCA( RDRGSyywFDPwGQGTLVTVS \ASTKGPSVFPLAPCSRSTSEST AALCLVF KDYF PEP VT V SWNSCA&.T SGV HiTFPA VL~Q ~SL YSLS SVVTYPSSN FGTQTYT N VDOHK PSNKVK:NERIOCM~TPPCPA PPVAGPS * ' ' EL PPKKD~M ISRTPEVMT VVDVSHEF PB VQFN WYyyVDGV'EV HN AK K KRE EDEN \TFR VVZVLTVV HQDWLN NGKEY KCK VSN KGLP A P1 l'IS KTKGQPREPQ VYTLPPSREF EMTKNQVSLTCLVKGFYPSDAVEWESNG QPEN~NY K T TPPMILDSDGSFEFY SKL TVDKS RWQQGN VESESY VMHEALH NHYTQKS LSI LjVT3 E.. EVQLV ESGGOJ TOK OE WS AIA DTYPSKRFI ____ ____ _____ VRBN KNSLYQ----------YCA I Y3STSAFLIYWQCVTSN'FGV PLA PC RSTS EST A ALGCL KDYF PEPYT V SWNSGALTSGVUTFPAV VLQSSGL YSLSS VV TVPSSNFGTFQTVTCN VDHKPSNT'K VDKiV .. HMAFLTVVVIEDPE VQFNWY DGflVEVINA KY K PREEQ)FNSTFR VVSV LIT HQDFUWL NGK ElYKCKVSNKGLPAPIEK TJ CLVKGFY PSDAY VEWESNCQPEN NY KT P 2DKSR WQQNVFS S VH FEA HANHY TQKSL S SP MKH L FFLVA APR W LSQ. &QESGP 1T V2 %+8 GL W MKPE NLSLTVSIGSISSSSYFWGW KQPPKLEW01YYSGIT'YNP>SL KSRV T% SVI! SKNQFSLKL NSMTADTAVYFCA> K ( Nks V \RC \'WY ED ,.' \VR>VTSS STKG PSVFEL.APCSRSTSE STAALEGCLVKD SF VWNSGALT S VHTFPAVLQSS LY SLSSVVTVPSSN FGTQTYTCINVDHKPSN TKVDKTVERKCCECPPCPA PPVAOPSVFL F PPKPKrFLiSRTPEVTCVVVDVSHEDPE VQFNWYVMDG VEVMNA KTIKPREEQFNSTF K VMS LI ThD -LN KKVSNKGL PAPH EITISKTKGIQPREPQVYTLPPSREE 'l K\NVC'LYKOFYPSDIAVEWE SNGQPE N NYK 'TTPPMLDS DGSFFL YSKL TVDUKSRW QNV SCSV MH -EA LHNIY- QKSLS SP 363 12 -- \-W - I-- - - ILL-A-I:--QVQLVQSGA VKKGA MSV KVScK ASGYTFEGYHMHWV RQA PGQGLEWVWNPNSCTGN AQKIQ G RVTMT RUTS ISTAYM ELS RE RSDUTAVY YCARDRGSYYWDPW'F G Q VVSAT IKGPSMFPL A PCSRSTS EST A ALOCLVKDYF PEPYT VSWNSGAL TSGVHTF PAVLQSSOL YSLSSV VTVPSSN FGTQTYMTCNVDKPN' KVDKTV ERKCCVECPPCPAPPV AG PSVFLP 1-PRERD.,M ISRI PEVTCVV DVSHEDPEV QFNWYDCVDGVEVNAKTK PRLEQFNSTER V SVLTVVHQDW N KEY KCV PSNK LP API EKTISKTKGQP REPQVVTLPPSREETK K NQVSL TCL..MVGY PSD1 AVE\VESNGQPEN N Y K T TPPMLSSFFLY SK LT VDKSR WQ GNVFSCSVM ..... EA L. ... N...Y'.........SSIk SPG 367 24 120 MDMVPAQLLGULW RGAREQVQLVQ SG AEVKKPG ASV'K VSCK[ASGYVITTGYHM WV RQ A PGGLE W MGWIN PNSGI N .KEQ.GR\VTMTRDTS 1STA YMELSRtjkRSDD IAVYY CARDRGSY Y WEDPWGQ iTL VTVS SASTKGPSVVPL APCSRSTSEST AA LGiCL KDYF PEP VTVS WNSCALTSGVH{TFPAVLQ SSGL Y SLSSVVTVPSSN FGTQT YTCN VDH PSNTKVDKTVERKCCVECP PCPAPPVAGPS VFLFPPKPKDTLM MISRTPE VV VV DVSH E D)PE VQFN WVYVDGIiVEVN ART TKRE EEQEN STFRVVL TMVVHQDW..NG KEY KCKVSN KGLPA PEKT SKTKGOQP REPQ VYTLPPSR E EMTKNQVSL'TCL VKG[YPSDIAVE WESNG QPENNY KIT PP MLDSDGS FELYS RE.TVDKS RWQQGN VFSCS VMHPEA JIHNHY TQKS LSL NPGK 100165 ) Some Imodime'nts of the isolated anti-DN P antigen binding protein comiprising an antibody or aibdfrmetcomprise. [00166} (a) an immunoglobulin heavy chain comrising the amino acid sequence of SEQ ID) NO:77, SEQ D NO:107. SEQ ID NO:1 iiL SE I) NO: 13. SEQ ID NO:1 1S, SEQ ID NO: 17, SEQ 1D NO: 11f SEQ If) NO 123., SEQ If) NC:129, SEQ ID NO:144, SEQ ID NO:145. SEQ ID NO; 15. SEQ UD NO;182, SEQ ID NO:1 83, SEQ ID NO:184, or SEQ ID NO:i85, or composing one of the foregoing seNquences fom which one, two, three, Wour or Ove amio acid residues arc lacking fromt the Ntermninai or COtermina, o~r both: [00167] (b an immoglou.in light chain comprising the amino acid sequence o SEQ IL) NO:105, SEQ ID NO: 109, SEQ ID NO: 121 SEQ ID NO: 125, or SEQ ID NO:i27, or comprising any one of the toregoing sequences from which one, two, three, four or five amino acid residues arc lacking from the N-trmina1 or C-terminai, or both; or 00681 (e) the inunoglobui heaxy chain of(a) and the frmmuuogonuhn light Ih~ ofv (hi. - ', "- [00 69~ Sonmceebodiments of the isolated wtiu antigen binding proci comprising an acnibodyantbodyagment c [00170) (a) an immunoglob uin heavy chain comprising the amino acid sequence of S EQ 1D NO46, SEQ ID N0:133, SEQ ID NO:139, SEQ NI N:143. SEQ iD NO: 1 86 or SEQ ID N :187, SEQ I) NO:366, or SEQ ID NO:367, or comprsing any one of the toregoing sequences from which one, two, three, four or five amino acid residues are backing frm the N-erminal or C-terminal, or both; [00171) (b) an immunogdobuln ai chain comparing the amino acid sequence of SEQ ID NO2 8, SEQ D) NOt 3 1 SEQ ID NO:1,' SEQ ID NO 137; or SEQ HD NO:14 or compistng any one of the foregoing sequencesfrom which one, two, three, tou or ive amino acid residues are lacking from the N-termninal or C-terminal. or balo 1001 e i ne i i a chin f (at and dtenmmnogo bu in i h caWin oft bY [00173) Again, each of the eveinplary an tiDNP hvy chains (H 1, H2. ["3,. et. listed in Table IB can be combined with any of the exemplary ani-DNP Pight chakins shown in Table 1A to form an antibody, E>:amvples of such combinations nchide H1 combined with any of LI through LS H2 combined with any of L through L 3 c- combined with any of Ll through Li 114 combined with any of L1 through LS, and so on. in some instances, the antibodies include at least one anti DNP heavy chain and oe anti-DN IgN chan fromn those listed in Tabl IA and 1B. some instances the antibodies comrise two different anti )NP haiv.y chains and two different MDNP light chaiws isted in Table 1 A and Table B In other instances, the anmibodies contain two identical ligt-l chains and two identica' heavy chains, As an example, an antibody or immunooially functiona fragment may include two H l1 heavy chains and two LI light chains, or two H2 heavy chains and two L2 light chains, or two 1-13 heavy chains and two [3 light chans and other simiar combina tions of nairs of anti-DN Pight chains and pairs of anti-NP heavy chains as listed in Table i A and Table IB.

[001 41 Agto, eain of the semplan! ani6*0 heany Sam (4 HAi H .., etc i oted in Table i B can be combined wih an of the exemplary atiKL I ight chams shown in iable A to tAnn an antibody, amples of such combmanons include i combined w ih any of L tbtluh I I H;12 combined nih any 'of L through I 5 I3 Fenvuid oit any of L I though 15 t 4 combined n sm.t of L I through LU and so on, in som instances, the antibodies include at least mnc ank I K 1A bav chuAM and one antiK I W ght chan frot thoe hlsted in abe I A anc I13. in somn' instances the antibodies comprise two different anti-KLH hemyv cham and to difrent ann-KUi lbght elims listed in Table IA and Tab e 1i hi ot he instance, thef antibodies contain two identical light ebana to identical heasv hims \ an example ainD antibody or nnumunologicallk un tion fragment aiy include two H hbeay ebain~s and two LI light chains, on t.o 1 heav chains and two L 2lght ehdna, or two 1R heavy chains mad two B 3 hi eh imts and other aijar comrbmanons of pair of antMi i bht chans and pairs of'antPKLH heavy chains as listed n Table A and Table I. [0017 5 Other antgen binding proteis that are pro'ided are iants of antibodies tormed by combination of the heavy anJ thi chains shoum tables I A and tbe 1$ and comprOe hght andor heavy chas tht each have at lest 70% at least 7% at least ";1%, at least S5% at east O0' at Iast 95% at leat 97% or at least 99% ideni to the amino acid sequences of thse chans in some stances, Wuch antibodies include at leat e heavy chatn and one lght cain, d i other instances the arint tornis contain WO N ldemie ght chains and two denmal heavy chams. is with the scope of the in enton ft th i hcaxn chainN and o light chai(s) mna have one, tw o, three, tour or Kive ammo actd tsiuiet "d a >tacky' from the terminal or (-termmnat, or both, in relation to any one otthe heav y and light chans set forth m Tables 1 A and Tale 1B. g , due to po rnalatonal modcatims For example, CHO cells typically cleave oiT a (-telminal Ipm.

[001761 Vaiab omai\f AntiodwI\ [001771 The various heavy chajn ad ht chain variable regions provided herein are depicted in Table 2A-BEachof ths variable regions may be attached to te above heavy and light chan contimt regions to form a complete antibody heavy and it chain, respectively, Further, each of the sot gencrad heav and light chain sequences may be combined to form a comprlete'3r hniocdy structure. It should be understood that the heavy chain and light chain variabe regions provided herein can also be atad to other constant doMain having differem sequences than the exemplary sequences listed above. [00 17 ] Also provided arc antigen binding proteins, including antibodies or anti body agents, that contain or include at least one immunogiobulin anti-DN P heavy chain variable region selected from V1. V&dY V Q31V 4 . V& 5 and VQ6 and/or at Leas one immunoglobulin ami-DNP light chain variable region selected from V I V2 VI 3. V4, and V5 as shown in Table 2A below, and imnmunologically functional fragments. derivatives .mteins and variants of these ight ehain and heavy chain variable regions. [00179] Also provided are antigen binding protein including antibodies or antibody fragments, that contain or include at least one immunoglobuin anti-KLtH heavy chain variable region selected front V7, Vst and Va9 and/or at least one immunoglobulin anti-KLW Hiht chain variable region selected from V 6. V7, Vi , and V, as shown in TMbe 2B klw, and immunoiogically functional fragments. derivatives, muteins and variants of these eight chain and heavy chain vaiable region [00018) Antgen binding proteins of this type can generally be designated by the formula Vnx/V Vy, whre Wx corresponds to the number of heavy chain variable regions included in the antigen binding proteji and y" corresponds to the number of the light chain vaiOble regions included in the antien binding protein (in genera, x and y are each I or 2) TWa 2A E2anqgry ad)NP VY{nd Cy(husns: areR udade uideimnxand traiwxdorkrcions atc nutmderined. Opntiona Ntermiin SgS1 eue esenot swne (Se< able I B dmmeX Ae 4d4 DIQMNT QSPS SVsSASVGDRVTVTCRAxNSJWL WY~ ~QRKC PKA PKL LIY ASS( Q~ SGVPSFSGSC 232 4JGTDFf TSSL QPEDFA; Y( OOA SS'FP WTFGQ GTRNVE1K DIQMTQSPSSVSA SVGDRV TITCI<V3)SQ JRL~A WYQQKPGKA PKI..YAASS.LQS GVPSRFSGSGS5 IL Xl ~234 (GTDFTSSLQPEDEATYYtC1OOANSP GPG TIKVDIK DiQ MTQSPSS SA S EGDRViITCRASQIRNDLG WYQQKPGKA PKRA L ASSL. S VPL-RSGSGS 236 TFTVLTISSLQPEDFATYYCUYNSYPWTFGQ) GT KVEIK. DIQ!MTQSPSSESASVGDRVTITCRA SQONMSNYL WY U QQKPRKV PKLLIY AASTLOSGVPSRFSGSG 34 W SG TDFT TVSSLQPEDVATYYCQKFNSPFjFGP GTKVD)IK DIQMTLSPSSLSAS WY>QQKPGKAPKR LAY A ASSLOSCV PSRFSOSCS L W5 240 GT FTLISSLQPEDFATYYCLOYNSIWTFGQG TEVETIK QVQLQESGPGLKPI SFTLSLTCTVSGcSISAYY WS\.RQPPGKG LWIGY YYSGSTNYN PSKSR H*H9 VH e 2 NVlS\VDTISKNQFSLKLT. SVTAADT'AVYYCARRAR (4D)YNYVPDAFDIWVGQGTMVTVSS QVQL VESGCVVN)PQPRSLRESCASGFTFSS Hi m y 2t2 A vRQA PG K (I wvAv yYDsNKYYADS \ KRFTlSR DNSKNT LYLQMNSL RAEDTAIVYY C AR _N D - V W- QGTTVT VSS a- inSE RIeac D A3mo Acid Sequence QVQ \ EN V EG VQGRSL R SC WAsGTISS M. : WVA 2P( KGEVAV Y GSN fY S kS NW ~4 V.KGRFISRNSKNTLYL QMNsLRAEiDTAV;YY (cA~RY\ 'NyGMoyw'ooQ(TTV TVSS QV Q L\VTSGGGVOPG RS R L SCA ASGFTI SSN YG H E N .:IWRQAPGKGSLEWVA VIYDSNK YXABS Hi N 14 'm VKG.RFTISRDNSKNTLYLQMNSLRALDTAV'YY (CARNYN ~NYGDMDvwooiQoT1TVvS S WXSWXI RQPPGKGLEWVICY YYSG NTNSNPSLKSR Hi, W 5 VISVDT[KNQFSLKLSSVTAADTAVYYCAXRTY YD~SSGY VYR AFPI wOOTVvSS QVOQLQESO GLVKPLQTLS TCTVSGGS ISSG xm. t~ NNYWNSWIROHPGKGLiFWHiGYX3RSIYYNPSL FXH3 > KSRVTiSVDTSKNQPSLiSYAA NYYXC

RTCSSGWY.PFDYWGQGVLVTVSS

'abi 2E3. Exempary anti tiR Vi ud n hain: C egionsaindicaed by undeiine and mnrwork regions are not der nad. Optiondtemral sig. equenes rnot shown (See Table 1A13 BREQ ID) N9 and SEQ NO: I0s) des igns R . Andno Acid Sequence Di QMTOPSPSSLSVSV'GDRVTITCOAGODJRN YELN V si6 PV42 WYAQQKPGKA PKL LYDASANLE T SRFSOSGS (YTA V FTISSLQPED IATYYCOOYDN LTFOQOIK E IVLTQSPGTLSLS PGE[RAT LSCIASiQ"ItNXtA 244 WYQQKPGQ APR FL YGAtSSRATGIPDRFSGSGSG0 E IV \TIQSPGTILSLS PG ERATL LSCI ASQMlSNXLA via 246 xYQQKPGQ APRFL YGASSRAT PRFSGSSG TFL TISR EP E DF AVYYOOFORSPRSFGQGT MKI . USX l1 R v----- 11IQSPSSLSASVGDURVITITCASQG N S WYQQKPGKAPKR LI YAASLQS_ VPSRFSSGSG VL9 24Ij I1TE TLISSLQPEDFA TYYCLOHNSYPL TFGGCK VEV'' EV\QL VESGGGLIVQP(G$RLSC'AASGiFTFSNYU ii H 7 26' M YWV RQTTG KGLE WV SAIGTAGDTYYPGSV&G > K FI SRE N AKN St YLQMNSLRAGDT A VY YAR KSx T t\ED XuuGQGTL. VTVSS Q1LQLQQESGPG L.. MK P; IL*SLTCTVSGGS ISSSW[ 8-l \ 2$ 64 WX(A RQPPCKGLEWIGSIYSGNFYPSiLSRV ZZ SVDT SKNQFS&L.KL NSM T A ADTA VY PC AROGG A kk RTGY WYF DLW WROTI VTV SS 4:1 *QV QL VQSGAEVKKPG AS VKVSCKASGYTIFTOY HMHW VRQA\PGQGLEWMyGWAINPNSGiGTNYAOK i-3 f9 6 FQGRGVIMTRDT S ST AY MELSRLR SDDT AV YY C 24 ~ A R DRGSY YWFDHPWGOOTi.VTVSS [00 181) Som e embodiments of the isohited antigen blinding protein that comprises an anti-DNP antibody or antibody fragment comrprising an immnog'iiobuInn ev chain variable region ad an immunobuin light chain vaibhle region: [00182] (a) the heavy chain variable region comprises an amino acid sequence at Last 9% 96% 97% 98%, 99%, or 100% identical to th sequence of SEQ ID NO:25W S EQ D NO:252 SEQ ID NO254, SEQ ID N0256 SEQ ID N025. or SEQ ID NO:260; or 1001831 (b 1he liob!a a tcr mneupee nmiDai eunea NM 232, SEQ I D NO234 V Q NE N D 6 SEQ D - oSQ3D or 240 or Cd [0 0 j 5 Sonc u -njodimorents uJFbe isolatna nie inigpo htcmoe CA aiQPntidy or a tbod fgmn. com0risIn an nnu o i oh iin [00 18. (u) t Oe heavy chain vai:A eincopie n inoai eunea leAT 9 6%. 97% 982KA. ;914~ or ) id OMa to tesqec fSQU NO:62 ~& ShQ If') N\O 24 ,A r SEQ MII >1:21k;'-- or [00 1 S71 1b the~I at Chaiata 0RIC ug reto niuP'Ae art amn acid sCyiCim cast 957" 906%, WY 41470 99% or IN,0% idntcaotesqeneoSE I N(1242, SEQ ID TWO044, SEQf, It) N24iorSEQ I)N{):248 or [00188 ) cAtS heavy Chain V-shoab> rjmo ()gdIcQi Aai mzgion a F J) f00 891 Each of the havy chain vriabe regionslted m IbL 2. A whether or not it i ided in a larer heay chan any be combed Wth any othe hight chai varnable reAons shown in abe 2 A to form an antien bmwig protem flamtplet of such cor ombmatnor nnlAe \* I combmred with any of \l \ V 1 \3. V!4i.o 1 m\ va combined with any ot \ 1, Vj L V 3, \ 4. r V 'I V V.oinid with any of' \, V V2 V4\3, V\t or V45; \ combined with any of )k'2, Vi V or and so on, [00190} Each of the heavy chain variable regions hsted n a 2 B, whether r ornot it is inchded in a larger heavy chain, may be combined wi any of the fight chain variable regions shown in Table 2R to form an antigen binding protein Examples of such combinations include Va7 conmbinied with any of V6 Vn7. V, 8 or V 1 9: VA8 cominired with any of 6, Vt 7. VR or V9; VH8 combm with An of V6, V7 Vi8 or V.9; Vn9 combined with anyV of V6 V7, V8 or V 9 [00 191 In some instances, the antigen binding pnteiP includes at feast one heavy chain variable region and/or one light chain variabfe region from those listed in Table 2A. In some instances, the antigen binding proten includes at least to different heavy chain variable regions candor light ebain variable region from those listed in Table .2A. An example of such ant antigen binding protein comprises (a) one Vol, and (b) one of V , V3, or VI4, etc. Another eampie comprises () one V 0 2 arnd (b) one of V 1 I , V 1 3, or V 11 I4, e Again another example comprises (a) one Vj3. and. (h) one of V' I V\2, or V4, etc.. Agai another example compr'ises (a) one. Va4, and (oe o' V 1 V or V , etc. Again another example comprises (a) one Vn%, an b) one of V 1L V , or Vn3, etc Again another example comprises (a) one VO, and (b) one of V 1 4. V2, or N3. etc. [00192 Againranother examp le of' such an antigen bimding protein comprises (a) one \U I, and (hbbme of \WI or \ 1 3, etc. Apan another eumnple of such an antigeni bimdm protein comprises () one V) 1 and AN one of V f or V , etc. Agamn anter example of sueb antangen bodng protem compte(a) one \' i anid (b one of Vf . o ec ndso on.

[0093 ibe vaous combhinanons of heavy chain varnable regions set f n able 2A ay be coo ined o n he various of aO nl t t ocan vnaabe renions se fort in Tabl 2 9019] i oter G~tnCO, tC atgen bindiflg protein contains tWo identiCad iht Utl 194] in other instanees-\ theI~ ant chain variable regions and/or two identica heavy chain variable regions. As an example, the amigen bindin protein na be an antibody orimmunologicaly funetonal fragment that includes two lght chain variable regions and two heavy chtain. variable regions in combinations o pairs of light chain variable regions and panrs of heavy chain varabie regions as listed in Table 2A, ['00195] In some int ances, the. amien binding protein includes at least one heavy chan variable reiot and/or one light :hai vab reion from those li-td i Table 23. In somev ntan~1cet5 the antgaen binding prte'in includes at teast two int heavy chai varIable regions and/or i chzin variable regions from thos itbed in Table 28. A n pie of such an antgen bnding protein comries ) one V7, and (b) one of Vi7, VIA or VIA Anoter example comprises (a) one V 8, and (b) one of V7, V11 or Vq9. Again another example composes (a) one V 9, and (b) onle af . V7 , or %H9, 100161 Agai another example of such an aigen binding potein eapse antigen binding protein eompnizs one , ad (bine ofV t 6, 7 A or 9 Agin another eampe of such an antig binding protein ompriesane 8 and (b) onef It T Agan another ample of such an angen bind ng Poiein eornprise S (a) one Vi9, and (h) one f V ? 8 or ) [0017) The vaous eomhrn-sons ofhewav ~" cha vattle regijons set to rut i Tabl M1 nuay he eo nondo an of the Y-ariOUN coNVnat ions of I i Qrh cha in NC'8q i fi r taned th antigen finding protein Contains two identica livgh ehai varableregions a ot identical heavy huan varhe iegiis A an mpl~e. the anigen bindn protem may be an anybody or imnnnolopicaly fnedonai fragnent that haluodes two Ight chain vannbli region and two heavy cham varable regions in comations Of pairs of ihtm chain vanable regions and pais of heavy cham variable region s stedt m Tabl 2f [001 | Some antgen bmdmng pr otein n that are provided comprise a heavy chami van abe domna comprn ng a sequence of amno acids hat ihis tium he asquence of a heavy Can v variable domain selected from \' i \ ' n a , \i 't \ A and V 9 at only 1,I 2. 4, 6 7, , i 10, 11, 12, 13, 14 s 15 :min ac ir\&iues wheroi each such sequence difference is independent V enr a deletion I, rntio or substIutio of one anmmo acid, v ah the de-etams miKrtion, and/or substitutiors resruin no maorc than e 'tmino acid changes re lative to thc. oregong VrMSe domA seqnces The teavv chum vanable rem m sorme angen bnd ngprotemn composes a sequence o W no ac ds that has at least '% at l lat5 a, a east a8t 0 at last 45%, at Vast Q% 4" east t%, at least 97 or at least 9e su'nce identity to the ammo s id quancs of the heave chain sunalA: region of V, 3 V\'-\ \ e 5' il \ub. V\', or \'a 002001 Certain antgen bMdig proteins compe a Wiht ebKo Si anabe domain comparing a sequence of'ammo acids tha dl 'fr Nom the sequence of a hht chaim variable domain selected Amm \K1, \2 1\. N\ 4, \ \ 6. \ ', \ Nnd \1 '9 at on4 1.. . 4, .5 67 8, 1. 10, i I. 12, 13, 14 o n- amme acid reidaks where each such sequence dif eren e iidependertk eCihe a deletion, inwervn or substituuon of one amino acid, ith the d'letons Msernons and/or substiutions rew uing mI no more tan 15 amno acid n s relative to the foregoiog v arwh domain se-quences. The: light chain su rvb rgon mn sonmc antigen binding proteins Comprises a sequence of amno acid\ thao at east 70% at least '5i at Sean 80% at least 45, at let 0% at least 95%A at least 97% or at least 99% sequence idetity tthe ino acideuene oftei chain varable ren of V). in 2, 3 4,v v 6Vi V x8or 0020 i Sil Pwother anoigen binding roteip N. on antibodie o inoogiedly n. naaine ncde iant f o a v Wrat heay cai and a vara cwhnas descbed herein. [002021 CDRs 00203 The a antigen bin ding proteins disclosed herein are polypeptides into which One orrmore CDRs are grafled, inserted and/or joined. An antigen binding protein can have 1 2, 3, 4, 5 or 6 CDRs, An antigen binding protem thus can have, for haychan (DR1 h example, one hev hanCR I DRI 1wl), and/or one heavy chain CD)R2 (-C)DR 2"), and/or one heavy chain CDR3 ("CDRH3"), and/or o light chain CUDR ("CDR L l) and/or one Qight chain CD12 ("CDRL2T and/or one bght chain C )R3 ("CDR L3" Some angen binding protns inchtde both a C DR 3 and a CDRL3, Specific heavy and uight chain CDEs arc idcntified in Tabl 3A-B (anti NPI) and Table (C Mi A i respectively. [00204) Comlementarit detrmning regions (CDRs) and framework regions (FR) of a given anibody ma'be identifed using the system described by Kabat et at. in Sequenes of Proteins of imunological interest, Sth Ed., US Dept. & Health and Human Services, PHS, NiH, \IH Publication no, 91-3242, 199 Certain antibodies that are disclosed herein eomise one or more ammno acid sequencs that are identical or have substantial hequence identity to the amino acid sequences of one or more of the CURs proseted in Tabl 3A (anti-NP CDRHs), Tae 38 (anti DNP CURLs), Table 3C (ant-KLH CDRis and Table 3D (anti-K H CDRLs). Table 3A E remplary Anti-DNP CDRBH Sequencce lSEQ ID .inahed i HC 1Designation Sequnce Nt H 5H CDRH 1- HYYWS 8 WH 11 Yli 1(13" - 74 SEQ ID Contained in HC Designation Sequence NO: H8, H16 CDRH 1-4 SGGYYWS 191 Hi, H9 CDRH 2-1 YIYYSGSTNYNPSLKS 192 H2, H3, H4, H5, H6, H10, Hl1, H12, CDRH 2-2 VIWYDGSNKYYADSVKG H13, H14 193 H7, H15 CDRH 2-3 YIYYSGNTNSNPSLKS 194 H8, H16 CDRH 2-4 YIYYSRSTYYNPSLKS 195 Hi, H9 CDRH 3-1 ARGDGYNYPDAFDI 196 H2, H10 CDRH 3-2 YNWNYGMDV 197 H3, H5, H11, H13 CDRH 3-3 YNFNYGMDV 198 H4, H6, H12, H14 CDRH 3-4 YNYNYGMDV 199 H7, H15 CDRH 3-5 TYYDSSGYYYRAFDI 200 H8, H16 CDRH 3-6 TGYSSGWYPFDY 201 Tabk 3B: Exemplarv AntliDNP CDRL Sequvncs C ontainedti LC |IDesignlation SeueceSQ 10 N I CDR L 1--1 RASOGISNWLA 202 L2 CDYRL 1-2 RASQCISRRL A 203 L315 |CR I-3 !' RASQiRNDLG 204 b-------- i- - ------ - - ------- ----- TA |ms. CR-4 RASQuMSNY Ai 202: L L L L3L CD)RL 2-1 A SSQS 206 4A CDRL 2-2 AASTLO(S 307 CDRL 3~- QQASSFPWT 208 L2 | CRL ') QQ~ANSFPFTY0 L3 .. CD.RL33 - LQYN PWT 21 L 4 | CRL>34 QKFNSAPFT 211 I c ) I < -- - ---------------------- 1Tale 34: Exemuplary Aint KL- UDRH Sequences cornairmdun ikc gnp won sNtn H M H22 CDR. - SSSYF WC 214 -- ---- ----- y-n-- - - - --------------- HN19, H20 H23 H2 CDRH 67 YivH215 - - -- - ---- ------- ----- ---- --- ......... r. .. .... - --- . .................. ................ .............. MV. ~ W -1 CUR 2-5 LQ awICTYPSX 1 H1& H22 CURH 2-6 SXYYSONTFYNPSL(.S 217 H 9 H2 H23 H24 CD~RiH 2-7 IWtNPNSGGTNYAQKFQG 218 L! - (~h -- ? 2K'5$TS BUYMIS H H CDmURH 3S QO&IAART :YYFDL 220 9 2 H2 H2 CDRH 3 9 DRSYYWFDP 221 TabMe 3D E-mtnplary AntI-KLU CDRL SeqmNveS r---------------------------,--- -r -------- r --------- Contained in LU Designation | Squee SEQ o NO: L- -|-CDR L - - -A- QAG-RN YLN 22 LI (CDRL 14 I6| ASONISTNYLA- 223______ L8 CDRL Li7 | ASQQISTNYL A 224 L9. Li0 CDREL 14 P ASQGIRND.G2W L6R- 2 DASNLET L RL- OASSRAT 226 L9, L10 CIDR P2- AASL QS 2 -- -- - -- - - - -- - -- - - ... . .. . .. . .. ..- 4-- 1 ~ - -- - ------------- --- --- ----------- --------- L6 CDRL 3%6 QQYDONLT 227 I CDRL 37 QQFGRSPRCS-|- LS | CDRL. VS QQFIRSPRSS | 24 L9t L 10- CDRL 3-9 --- LQ.SYPT |.3 . 7t. *. "" f - [002051 Te- structure and. proportes of C wthinn a namray ccurrin antiboody have been deserved, s pra. fBrefly, i a tradidonal amtibody, the CD)Rs are enmbedded within a framework in the heavy and ig~ht chain vaia reion where the-y consitute the regions reposil for anign bi ending and recogition. A variable region cop iat least three heavy o'r nigh chain CDRs, ae, supmo (Kabat e (ial ' 1991,~ equencesf~rotein ofauooia /nrere'st, Public Health Se>rvice Nl H-1, Bethesda, MD; see ias Chothia and Les'k, l987 ii A/fo Bil J%:901-97; Chohi er01 1%, atue 42 87783), wihin a f rmework region (designated frarnework regions 1-4, FRI1, FR.2 FR3, and FR4, by Kabhat er al, 1991 supra; see also Chahia and Lesk 1987, spru), The CD ks provided herein, howe-v, may not only be used to define the antigen binding domain of a traditional antibody structure, but may be em bedded in a variety ofthe polyept trucursNa d2r.e [00206} Some embed emins of' theisolated antigen binding prtein conmpn ian anti-DNP anuhody antibody i ent. cononin agndamngeoba in heay chan vWiate region and an inmunoglouMbut ight aCin \laiae region The heavy chamn ariabk regIOn compris three cornplemnrtarirv determi~pnn regoons dtstinted (:DRHL CDRH{2 and C'DREIH ando eroe light chai n raNe region omprie three tDRS designated CDRI I CDRL2 ad D R ,I R herein [002071 (a) CDJH has the iniKno id su e of $EQ ) NO81$> SEQ 1D NO: 89. SEQ ) NO90. SEQ 1H) > 9 and/or [00208 {b) £Di1H2 has lhe amino acid eNuence of SF N Q R 19. SEQ D O 194 r SEQ ID NO:95 dor [00209: (w CDR3 tha de amino d sequence of SEQ 1) ) % S Q NC) 197, SE.Q In NC98 SEQ D Sr 0:199 SEQ I1 NOW2{X or SEQ ) N 20 ando [002101 d) ICARL has the amino acid sequence of SQ fD NO202, SEQ iQ) 0 SEQ I NO20 o EQ NO andor [0211 CDRE2 hathe amno acid s nce of SEQ D NO:206 or SEQ ID" NO :07; andor )02121 ( DRiL3 has the a nmio acid sequence otSQ HD NO:20, SEQ ID NO 209 SEQ ID NO:2J0. SEQ ID NO ', or2SEQ iD N2212. [002131 In other aspects, the CDRs provided are (A) a CRn selected from (i) a CDRH I selected fro SEQ ID NO:188 SEQ ID NQI9, SEQ ID NO:190, and SEQ ID NO:191; ii a CDRH2 selected from SEQ ID 0:192. SEQ 1D NO:193, SEQ ID NO:194, and SEQ U) W:195; (iii) a CDRH3 seected from SEQ ID NO:196, SEQ ID NO: 97, SEQ ID NOD98, SEQ i) NO:I99, SEQ ID NO:200, and SEQ I D NO:201; and (v) a CDRH of (1), 5ii) and (iii) that contains one or more amino acid substiutions, deletions or insertions of no more than five, four, three, two, or one amino aci ds (B) a CDRL selected fim (i a CDR I selected from SEQ ID) NO:202, SEQ ID NO1203. SEQ 1D NO:204, and SElQ I) N:20S; (h)s CDRL2 selected from SEQ ID NO:206 and SEQ ID NO:207; (iii) a CDRL3 selected from SEQ ID NO:208, SEQD N:20 SEQ D NO:21 SE 0 and SEQ NO>'2 and OY) a CI)RL of (, (6) and oii he cdins on n amW oe suhsttonts deetons or netiosat no more tha fbu three o one aan ac ds anino acdds [00214] Some embodiments of the isolated antigen binding protein comprise an anti-KH anti body or antibody fragment. comrnimg an ininoglobuhin heavy chain variable region and an immnmmoglobudin hight chain variable region. The heavy chain variable region comrpris three cornplementarity determining regions design~ated CDR H L CDRH2 and CDSH. and/or the light chain variable region comprises three CDRs designated CDR L C DRL2 and CDRL3, wherein: [00215] (a) CDRI 2has the amino acid sequence of SEQ ID NO:213, SFQ ) NO0:214. or SEQ ID NO0:215; and/or 0) (0 CDRII has the amino acid sequenc of EQ D NEQ NO0 ?, or SEQ HD NO L2 nd [00217J (d) CnRI Ia he a m no acid sequenof SEQ 1NO >021. S iD N I:220 or 5I 40.N221 And)or [00218 (d) CR L has the ainn aeid o SEQ I 0 NO 22 SEQ Iii NO:223 or SQ DI NO:224 ad/or [0021910 e CDRL 2 has he aninc acid sqoece of SEI Odd>0:0 SEQ 11 N=:225 or SEQ ID N 26: and/o [002201 ( CURL3 has the nino acid sequene of SEQ DD 022 S 028. SEQ ID) NO229, r SEQ H' N4:230 (002211 In other aspects, the (R3ks provided arc (A) a CDRH selected frn () a CDRH ! selected from SEQ ID N2O3, SEQ ID N0:214, and SEQ ID N0:215; (ii) a CDRH2 selected from SEQ ID NO Kl6, SEQ ID NO:217, and SEQ ID NO:218; (iii) a CDRH3 selected from SEQ ID NO:219, SEQ ID NO:220, and SEQ ID 29 NO:22 and iv a CDRH of 6iA (i) and (M that contains one or more am no acid substitions, deletions or insertions of no more than five, fr thee, two, or one amno acids; (VB a CDRL selected from () a CDRLJ selet from SEQ ID NO:204. SEQ ID NO222, SEQ ID N:223, and SEQ ID NO:224 (iia CDRL2 selected fron SEQ ID NO:206, SEQ H) NO:225, and SEQ ID NOo:22 i aCD L3 selected from SEQ ID N(:227, SEQ ) NO:228, SEQ ID NO:229, and SEQ ID NO:230: and (i) a (DRL of(it (ii) and (i) that contains one or more amino acid substitutions, deletions or sertions of no more than five, four, three two, or one amuneacids amino dads [00222 in another aspect, an antigen finding protein includes .2,3, 4, , or 6 variant forms of the CDRa listed in Table3A and Taiblc 38. each having at least 80%M at least 83%, at least 90% or at least 95% sequence identity to a CDR sequence bled i Table 3A and Table 3B. Some antigen binding proteins include 1, 3, 4,. 5 or 6 ofthe (Rs listed in Table 3A and Tabic 3 each differing b no more than 2, 4., or 5 anno acid firomnthe UDR ist uhes tabe [2231 aothe aspec an anten bind g p e ati ad . 2, , 6 varaait fons o f the CDRs lted in e 3 ud Table 3D, c having at least 80% t least 85%. at lest 90% or at eas 95% seuee denti to a CDR se ence listed in fabM 3C and TAb 3D. Some anigen binding proteins include 1. 2. 3. 4. v or 6 of the CDR istd Tab 3C and Table 31D ac d iffrinbynomrethan 234 orx anno ais rot6 the CURs listedithese tabls [002241 I vet another aspect, the (ADs dielosed rein include consensus sequences delvxed from groups of related mnoelcong I mnibodies. As described herein, a 'consensN sequence" reerN to atmio at d sequences having conerved ammno acids common among a number of sequence and iabl amino acids that Nars wt ihm a gnven srunno acid sequences Thet tDR rosensus scqaiknC provude include CUs corresponding to each of( DRH I 'DR2E CD5R13 DR 1, ( DRI-l2 andCDRI3.

[00225 Antibodwantinen ineractionsca be characterized b the asatidonrae cottin > 5 (ka).oi the didcttin nie (znsteftin s (kX or alteninvely hi disoiation equilibriu constant in M ac KK), [002261 The present invenion provides a variety of antigen binding proteins, including but not imitd to antibodies that specificaly bind DNP or KI , respectively, that exhbi desirable characteristiu as binding affinity as measured by K 0 (dissocition equilibrium constant) for DNP or KLH, resp ctively in the range of 10I M or lower, ran ing down to 10 " M or lower, or avidly as measured by k (dssociation rate constant for D)NP or KLH,~ respectively, in the range f 10, 's or lower, or rangng down to 0 . or 1ower. (SeeExample 12 herein). 1002271 i sme embodiments the antgen binding proteins neug., antibodies or antiBody fragment) e\h. hit desirable char ceristics seh as od ng ,ruditv as measure e Kd i(dby ocraon rate constant for DNP or M. 1 1espeSI Al. of ahout 10 . 10 0 W0 W 0 10 10"' 1 o 0 " a s or 1oa 1slouer u the ain. hi gh#r htl no autdny) and or hmndmn affinity as rmeasured by Kn (di sui ation equl6i iumn costan. ir DNP or K]H respectively of shout 10 1ot 1 I 104 . 1 0 1 0 X\ or lower (hoer valus mdicatng higher bmdmg afiuy}. Asoeiation rate constant dissoctation rate constants, or isoeton eguhbr onsaus way ma be reail ' determined using kinetic analsiv teW ques such as .rfae plasmon reason ee (Bi\eore , g ,,iseer t al ,A4 peptide imrtunoglobul-n ingate, \ O 200' O4463 Ai 1E Fample 10 which is mneorporated herein l reterene in Os enrer). or Krf NA usag general proceeds oulned by the manutacurer or other methods know n the art The kinetic data obtamied by liAeore' or Kmn'\ may be unulyed by methods described by the manufacturer. 00228l itt sonie emoiodimenis the antibodyecmprses althreghtechinCDs three heav dan CDRs or al six CDORs. In deiy enbodungns to iight chain C0iss tfom an antibody may be rombioed with a third b chain frm a di fferent antibody, Afternatively, a CDR<Li from one antibody can be combined with a CDR L2 frn a different antibody and a CDRL3 fom yet another anmib~ody, particularly whner the CDA)s are highly h,' oooous Similarvy two heavy cAin CDRs from an antibody may be comined with a Third heavy chain DR fron a different antibody; or a C'DRHI I from one antibody can be coined with a CDR{2 from a different antibody and a CDRIH3 from yet another antibody particularly where the C DRs are highly hornmoogoup [O2291 Tba the inventon provides avlet f compodtaOne composing Ofe two, andor tree UIs ota heay chanaeriin ando a' ht chain be antibod inch. regon off a di ding medihon.ia det av hreo Such omposit ins may bI e ineraed by techniques describe herein or known i th ar 90O3Pl In Anc entodinienit, the atigen tlndmo protein (uidit antntodi-s and anybody agmeni can he usefu. as a therapeutic moleeuk which cn be used singudarly nIin m ta aonI with other therareuttes to achiee the desu d offrets n such embordiments the imenti\ve antigen binding protein ( ineh0ding nubodite and antibody fragnments) further con'praes one to twenty our, one to M teen, one to eight or one to tour, pharmacologically acive ehemkial nioiese oniugated thereto, whether a smallI molecule or a polypeptodc T he phaumnacol~ogstally actie smnall molecude or pohypeptide ehemieaal moieties can be conjugated at or ia the W teni a or (>teiromal resi due of the antagen binding protsm 'a gu noglobauI monomers eg 1 C or HC moncmerst chemical reactions kuon in the art and roher deSribed hevin Ahematively encompased by the im etion, a coiugauon of the pharmao a acte eheanc moety; or i, o antonal groups on one or tmore ide chains of the amio acid resid A AP) within the prmary cham of the inentivse antgen Hnmo protein seful methods and iemua conjugation sites e. pocrtiular cyteie reidues) w ithm immunogohulin chains are known i the art (eg g , ! eg , Ma dified "c Molecule, published in WO 200C/0220'0 and US 200'093A. nhich are incorporated herein by reference in then entireti eat [002311 inl other embodiments of the inventon. in whic the pirmacologically atiwe chemical moiety is a polypeptide. a recombinant fusion pnrotein can be produced with the pharmacologicaly active polpeptid being inscrted in the primary amino acid sequence of the of the immunoglobulin heavy chain within an internal loop of the Fe domain of the inmmunlobulin heavy chin, instead of at the N- and/or terinus, as fuhrx described in the xampes herein d in th ar (e. Goeg et al. U. Patent No, 7,44277 U.S, PentNo. 7,655 5 U.S. Pate No, 7,65,764, Paten N o/i,3; U S Patent No, 7145,861 punished US. Patent Applitons US 209/281286; and U 2009/0286%4 eah of wich are incorporated herein by ttreere in their entireties [00232] tonuated" meas thatte phmacologicah acdie chetncal mois are caetinkedOr bound t to an,- n acid resde of the an binding protein .or otiontally; to a peptide nopeptidy hker moieythat i eovaentp linked io the aminoacd esidue ofhne antigen bidin protein. [00233] As stated above, some embodiments of the inventive compositions involve at least one pactie potypeptide mfOety conjuated to the phaimacologically inactive antigen binding protein of the invention, for example constituting a recombinam tbsion protein of the pharmacologicaly active polypocptide moiety conjugated to the pharmnacologicailly inactive antigen binding protein of the invention. The term "pnarmacologicall active" means that a substance so described is oeternined to have activity that affects a medical parameter (e. blood pressure, bood el count, cholesterol leave pain perception or disease state (e cancer, autoimmune disorders, chronic pain) Conversely. the term "pharmacolomically inactive" means that no activity atecng a medical parameter or disCase stale can t dtermned for that Substan Thu parmacologically active peptides or proteins comprise agonisi or mimetic and amgonistic pep t idesi as defined belov. The present in vention encompalsses~ the use of any pharmacologically active protein, which has an amino acid sequence ranging from about S to about 80 aino acid residues in leth and which is amenable to recormbinant expression. In some useful embtodiments of the invention, the pharmacologically active protein is modified tn one or more ways rehtive to asnative. sequence of interests , finding amino acid additiors or nrtiOns, amino acid detions, peptide truncations, amino acid substitutions, or chemical derivatization of amna acid residues (accomplished by known cUheMa techniuesX so long as the requisite bioactivityw irmaintai ned, 002344 The utm' mimec ppde; ^4eride mumt and ~~ago 2 w ptd refer to a peptide or protein hmvog biological acti3t comrable to J natural ocewing protein of interest, mi umple, but not limaed to, a toun peptide molece, e ShK or O)SKM tomin peprAde, or pepide analogN thereof. Phese terms trtreld r1 epnides tbt wndirectl m ame th artmt,'0 oaturallx' ocunmig peptidc molecule, such as by potentatmng dhe det'et of the natualy oeccrumrm [002%) 1'he term 'anmagonis eptide~ ".'etie antagjmist/ and "inhibitor peptide" refer to a peptide that bIocks or in sone way iert'ees with the Nologial activi it of a receptor oi itere, or has biologal actiity comparable to a known antagonist or inhibitor of receptor f nierest (Such us, but not limited to, an ion c0annel or a 3-Protein a coupled Receptor (GPCR p, [00236l humoIple ofpharfracotogiesly acti\ve proteins that an be used W ithlin the present mention include, but arc not Iimuted bo, a toxin peptide (4g. ,COSIK or an~ OSM peptide analog: ShK or a Sh nentide muao0. an H -6 bindmg pepte, a CORP peptide atwtgomm a bradytinin it eeptor pelptie antaani pah hornmon '' P a uoms p optde, a paratmtoid hormone (P' I I antagomst peptide. an ang- I binding rntt de,n ang.g mndmg pepide, a myosim hiding peptOde. an eirthrpienn-mmetie I -(nmette) pepnde, a tOfm.otn-rmmetic Il'P~ mninmeici Prptide it., AMP2 or AMlP a nerve gron Ictor (NFi bindig peptide, a I sevl ietvatog fator {RAFFI bmu pude, and a guragon-iike petide iMk Pw or a peptide mimsetie dherof or (6 PC or a peptide mmetie thereof Cleage"t Ipe pepct d I L P. Ii and th elated epude glnagon a educed via Sffere a sing proglcagon and hiae opiposig bioogl a uAi Poglucagon itselfis produced in cells of the nav'r"u and inh 1h tmendoen no L-cls, which areloated primarily in the distal mall itetne and colon, in the pancreas, glucagon is selectively cleaved trom proghucagon. in the intestine, in contrast. prog o ; processed to f GL -" anid gluni pt ;i 2 (GLP~ 2), which correspond to amno acid resides 78-107 and 126 158 of progUCagon, respectively (ee, cc., irwin and Wong, 1995, MW doNrn 9:267-277 and Bel e. l1983 Naure :683 ). Byonventon, the nurnberi of the amino acids of LP is based on the GLP- (I-37) forme from ceav ageP- o rg' c n The biologically active forms arc geerted frm fther process sTing of this peptide, which, int one numnbcring convention, yields CLP- . (7-37)>-H and GShLP-1 (7-36)> N~ll Both CL P-1 (7-371-OH (or simply G LP- (7-37)) and CUP-i (7~36 )-NH have the samec activies. For convenienc e, the termn "CLP-B", is used to refer to both of these torms. The first amino acid of tse processed petie isH in this numbneri ng convention. Another nunbering onvention rcgnized in the art, however assumes that the munberin of th processed peptde begins with His as position 1 rather than position 7. Thus, in thisaumering' scheme, GLP-i 1-3 1 is the same as UPI(7-37 and GLP-1( -30) is the same asH LP- (7-36y Exanmpes of GLP- m imetic polypeptid sequences il ue: HCECTFTSDQSSYLEGQA AK EFIAW LVKCR&// (SEQ ItD ND:290); HIGEGTIFTSDQSSY LGQA AKEFIA WLQKGRG/ (SEQ 1) NO:291); HCEGTFTSDVSSY QEGQA AK EAW LVKGRC;/ (SEQ 'D NO092); HGEGTFTSDVSSV L EQAAKEFTAQ LVKCRG// (SEQ ID >1:293); GEGTFSD VSSYL ECQAA KEf IAQ LQKGRG/ (SEQ ID NO:294): HGCECTFTSDVSSYUEGQAAKEIiAW LQIKGRC//(SEQ IDN1:295): HNETTFISDVSSYj Q E AAKEFAWL VKGRG/ (SEQ iD NO HCETFT SDVSSY LENQTAKEFI AWLVKGR// (SEQ ID NO:297); HG ETFTSDVSSYL EON ATKEiAP WL V KGRG. (SEQ ID NO:298); HECTFTSDVSSYLEGQAAK EFIAWLVNGT&/(SEQ ID NO:299): CEGTFTSDVSSYLEGQAAKEF AW LV KNRT/(S EQ 1D NO:300); HCGTFTSDVSSY LECQAAKEFIAWLVKGRNGT/ (SEQ ID NO:301); HOEGT FT SDVSSY LEGQAAKEFIA WL VKOR GTONCT /(SEQ 1) 1:302); awd HGETFT SD VSSYLEGU:QA AKEFnA\WLVKGRGGSGNOT// (S EQ ID) NO :303)i. [00237] uinm GL-2 nd (LP-2-mieck analogs are also know pin te art. (See, etvg. Prasad ci ai Glcagonlike peptd~ analogue enhances inrestina muicosal mass after tithmia and reperfuion Peiatr, Surg, 2000 eb;3$(2):357 59 (2000); Yst t aL Glucagon-like peptide-2. reeptr activation engages bad and glycogen synthaxe kinase-3 in a protn kinase A-dependent manner and prevents apoptosis follow ing inhibiton of phosphatidyi noslitol 3-kinased. Biol1. Chen. 277(28)24896-906 (2002)) [00238] "Toxin peptides include pepties and polypeptides O, aithe s me amino ad sequence of a aurly occung pharmcoogcly aive pepide o pyptide that can be isolated icomn a6 veomnand also In modifie peotide analogs of such naturally OCcUrri'ng molecules. (See, e~g Kalm an et al.. SbK Dap22, a potent 1Kv L 3-specific inmmnosuppress ive1polypeptided B iot, Chem.n 23(49):32697J707 (198 Kom e ) t a US Patent No. 6,0776 Mouhat eI at OsK I derivatives WO 200025 A2; C dy et al. Analogs of SaJK toxin and thner uses in elect iv c inii taiOn oF Kv 3 potam channeIs, WO' 0 06/04 -215; Sulivan at i'oxn Popte h peutic agents, WO 2006/1016 56 A2, al of which are incorporated herein by refe'reincea in their entire - Sv nakesscorpionsspirs bea, snail nd sea anemone are a. examples o rgnimss tht produce venom fbat can serve as a rich source of small bioactve toxin peptides or "tns" that potently and \eiectively target ion channels and receptors, An example of a toxin peptide is OSK 1 (also known as OsK: } a toxin pepide isoladfrom Orthochrus scncnituosyA scorpion venom. (eag, \Mlad et a K+ channel types targeted by syn thetic OSKI. a toxin from Orthochirus scrobiculosu' scorpion venomi, Biochem, S38S:93-1(04 (2005); Mokuhat et at Phar macolog ia profil ing. of Orthochirus s'crobicutlosus toxin I ananlogs with a trimmed N-terminal domain. Molec, Phatnacob. 69: n4- 62 (2006); Mount al Os1K1 derivatives, WO 2006/002850 A 1 2I Another example is Shin. isolated f rm the. venom. of tbe sea anarmone Stichodatyla hehninus. (E. g. Tudor at al. lantsation beh aviur and solution properties of the potasinxmihanA blocker SiK toxin, Eur. A. 2lioche 5( } 34 Pei go te ttid Roleo disdde nds n the Stte and tcst ainine bockig atit of Sloin ochem38(44): 141.5. (999 Kom et SK Neiopeptides oginratinrin scorpion Sdatento 49 ei Targzetitaeffector memory cv cells with selectiv peptide inhibitor ot Kvl. channnelstr therapyof atotnnmwne diseases. Moee Phamnaco 4 694 (2005B [00239) the toxin peptides are usually between about 20 and about i0 amino acids in ienth contain 2-5 disulfide linkaes and form A very compact stuturc, Toxi peptidn eg fom the venom of corpions sea anemone been isolaited and characterized for their impact on ion charmels. Such peptides appear to have evolved fom a relatively small number ofstructural frameworks that are pacticulaciy well suited to addressing the critical issues of' potency and stability. The naority of scorpion and Conus toxin peptides, for example, contain 10-40 aiino acids and up to live disulfide bonds foming extremely compact and constramed strctucr (micmoproteins) ofen resistant to poteolysis. The conotox in and scorpion toxin peptides can be divided into a nunber of superfamiies based on their disufidle Connections and peptide folds, The solution structure Of many of these has been determined by NMR spectoscopy, illurating thicr compact strucOre and verifying conservation of their family told. ( Tudor et a, Lonisation behaviour and solution pmperties of the potassium-channel blocker ShK toxin Eur J. Biochem. 251(12):133-41(1.998); Penuington et aL. Role of disrdfide bonds in th Structure and potassIum channel bcigatv ofShK toxtn Biochem, 38(4 14549-5 (19 Jaravine et al Three-dimensiona structure of' toxin OSK from Orthochiu i crobidosus scorpion venom, Biochem. 36(6):122322 (1997) del Rio-Portllo t; NMR solution structure ofn a novel peptide from the Mexicain sorpion Centnruoidcs nox is with a typicbta-toxin sequence but with alpha-ike physological activity, Eur. A, Biochm 27 Q12): 2504-16 (20041: Prochnika-Chaufour et al, Solution structure ofdisccepin. a new K+-channel 87 blocking peptide from the alpha-KTx15 subfamily, Biochem. 45(6):1795-1804 (2006)). Examples of pharmacologically active toxin peptides for which the practice of the present invention can be useful include, but are not limited to ShK, OSKi, charybdotoxin (ChTx), kaliotoxin 1 KTX1), or maurotoxin, or toxin peptide analogs of any of these, modified from the native sequences at one or more amino acid residues. Other examples are known in the art, or can be found in Sullivan et al., WO06116156 A2 or U.S. Patent Application No. 11/406,454 (titled: Toxin Peptide Therapeutic Agents, published as US 2007/0071764); Mouhat et al., OsKI derivatives, WO 2006/002850 A2; Sullivan et al., U.S. Patent Application No. 11/978,076 (titled: Conjugated Toxin Peptide Therapeutic Agents, filed 25 October 2007, and published as US20090291885 on November 26, 2009), Sullivan et al., WO 2008/088422; Lebrun et al., U.S. Patent No. 6,689,749, and Sullivan et al., Selective and Potent Peptide Inhibitors of Kvl.3, U.S. Provisional Application No. 61/210,594, filed March 20, 2009 (corresponding to WO 2010/108154), which are each incorporated by reference in their entireties. [00240] The tern "peptide analog" refers to a peptide having a sequence that differs from a peptide sequence existing in nature by at least one amino acid residue substitution, internal addition, or internal deletion of at least one amino acid, and/or amino- or carboxy- terminal end truncations, or additions). An "internal deletion" refers to absence of an amino acid from a sequence existing in nature at a position other than the N- or C-ten-ninus. Likewise, an "internal addition" refers to presence of an amino acid in a sequence existing in nature at a position other than the N- or C-tenninus. "Toxin peptide analogs", such as, but not limited to, an OSK1 peptide analog, ShK peptide analog, or ChTx peptide analog, contain modifications of a native toxin peptide sequence of interest (e.g., amino acid residue substitutions, internal additions or insertions, internal deletions, and/or amino- or carboxy- tenninal end truncations, or additions as previously described above) relative to a native toxin peptide sequence of interest. [00241] A "CGRP peptide antagonist" is a peptide that preferentially binds the CGRPI receptor, such as, but not limited to, a CGRP peptide analog, and that antagonite, block, decreases reduces. ipedes or inhibits CGfR0 receptor einvattm by tii ergth 'amie human (CRP or iOR P under ph ysokinai ConfditumiS of ter perature, pH, and ionm strength. CGR P peruide ftTagomnis inalude Fu a.nd' partitd Jnueot&~ Such athdponist activiry can he detCted b) know a n nix rmthods A in ro Rimamna! asay mmOds (See . gl, Snhb e, A., lodiftiCunsto tuhe N inm but not the ( enmius of c:f itonm e tt peptide( 7 produce antagonis nith mereased allinity, 3 Med Ch , 4t 2435 12003) Fxanmpes of ufu ( GU ppdde antagonists are decohed in Ge, eA, C RP peptidc uagonin and eonjuga te, W\ 2007 048026 A2. n l S Seal N 1 1 584 71 fied on (ctober 19 2006 published as S 200 002020W Al, which is incorporated herein hy refdrenee in its entuelv, [00242'1 the terms "parath yroid hormone (PTW gonst and 'P'JTH agonistrete to a molecule that Inus to PIT - dlor P l k& receptor ndm as OF decreaSes one or more Fl H activirs assay parameters as doe. fullaength ut v human narathvroid hormone anmples of useth riP aronist perd are Jeoned Wi Tabe 2 of U S Patent No. 6''56480. tidled Modulators of recertors for parathyitd hor mone and parathpomid hrmoneriated groin, which i incorporated herein by reference in cntrretx An s>eapiar PIN actvm asay is dislosedr En ample I of U.S. Patent No. 6: 'h,480, [00243 The term "arathyro~id hormone (PT H antagonist 0 refeC.rs to a moleculec that binds to PT H or PT receptor and blocks or prevents the normal effhet on those parameters by full lengthi native hurnan parathyroid hormone. Examples of useful PTH antagonist peptides are disclosed in Table 2 of U.S. Patent No. 6\ 756,480, which is incorporated k'rein by reference in its entirety. An exemprlarv PIH activity assay is diselosed in Example 2 of U.S. Patent No. 6, 0 7.56A. [0024) he erm "badyini B1receptor antagonisl peptide" and "radykinin B receptor pep tide antagonist" mean a peptide with antagonist activity with respect to human bradinN 1 receptor (MB1). Uf bradykin Bl receptor antagonist peptides can be identrfed or derived as described in Ng et aL Antagonist of the badykinin 1 ecptr 20050215470 A, pbshed September2l 05 whih isured as U Patent No, 7,605.120 US. No 544A or 54463 A exemplary1receptor atti yassays ae selosed n ExamPes &A of S 2005/02 15470 Al. [00242] The terms "thrombopoietin (TP2-mmtic peptide and TP-mimetic peptide" refer to peptides that can be idenfied or derived as described in Cwira q (1997) Science 276: 1696-9 , U.S, Pat. Nos . 69 n 5c ar incorporated by reference in their entireties: U.S Pat App. No. 20Im3/17632 published Sept. 16. 2003. which is incorporated by reference in its entiretvy WC 033 5, publhed Aril 17. 2003; WO 0/24770, published a y 4, 2000; and any peptides appearing in Table of published appcmtion US 2006/0 140934 (U.S, Serial No, 1/234,73, tiled Septeber 23, 2005, i Modified Fc Molecuies, which is incorporated herein hbretrrrne i n ita tirey y. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually dicosed therein by following the disclosed procedires with different pept libraries. [00246 1Te terms "EPOminmetic peptide' and "erthropotin-mimotic peptde" refers to peptides that can be idenifed or derived as dcrbed in Wrhton et aT. (19960 Science 273; 458-63, and Naranda et at ( 999), Pioe, Nati. Acad. S.i, USA 96: 7569-74, both of which are incorporated herein bv reference in their entiretics Usefd EPO-mimretic peptides mcude EPO-mrnmetic peptides listed in Table 5 ofi published U.S. patent application US 2007/0269369 A land in U SPat. No. 61660043, which are both hermby ineorporaed by reference in their etietics [00247) 'inh term 'ang dido peptide' composes peptdes that can be identified or derived as described in U S. Pat. App. No 20030229023, published Dec.2 1i, 2003; WO 01057 134, ubbihed July, 17, 2003; U.S N00M S 93 published De25, 2003 (each of wich is incorporated here by reference in Ats entirety); and any pepudes oppearng in able 6 of published apphiation US 2006 0140934 IS. Serua No, 112343 1. fled Sptember 3. 2005. Utled Modified Fe Molecdes whichis incorporaed nerein yvrence its entirety Ti~os orordiary sil in te art apprxatg ha each ofleomgew' 'u to selet diferent peprtdes hn actually disdosed theeinby followiethediscosed pocedo wih d Kifrnt pAdepldlibna [0024K] The terms 'lerve growth factor (NGF) binding peptide" and "NGF hindn petie com prie peptides that can be iden tified or derived as decribed in WO) 04/026329, published. April 1, 200 4 aind aneptides identified in Table 7 of published applicaion US 2006/0140934 (U.S. Serial No, 23 1, flied September 23, 2005 titled Modified E Molecules, which is incorporated herein by reference in its entirety). Those of ordinary skll i the art appreciate that this reference enables one to select dierent pepides than actually disclosed thein 1y oilowing the disclosed procedures with d1iffent peptide iraries [00249J The termmn ostatin-hmdmg pepnde" ompr ppdes that can be identified or deived as desebd m U.S. Ser No. 10 742',39, fI'et Decembr Kl. 2003, bich is ncrporaed herei by rekrenee in its entiretyand peptide\ appearing in able n of pubished applicaton U S.2006 014093 ,, >S, Sevd No 11 2 '3 41 tiled September 2. 2005 titled Modified Fc Moleen k hc n is incorporated hereim b reterene ias emirt . Thse or ordinary All m the a appreciate that each of these references enables one to select diferent peptides than aetally disclosed there b fblowimy the diciosed procedure wth di ifeta peptde hbrates (00250] The terms "BAFFantagonist pepid and "BAFF binding nentide" comprise peptides that can be identified or derived as described in U.S. Pat.Appln, No, 2003/0195156 A 1 which is incorporated herein by reference in its entirety and those peptides appearing in Table 9 of published application US 2006/014094 (1 S Serial No. l U234,731, iled September 23. 2005. titled Modified Fe Molecules, which is incorporated herein by reference in its entirety). Those of ordinary skill in the art appreciate that the foregoing references enable one to select different peptides han ttaly ddilosd h erein hyoowi h dibey fed vroedre dffren ptd l~ibiesi. [00.25 3 'j heregomg me miended merely as :nonhnuun examples otfhe pharmacolottially active e poly peptides that can bec usefully conjmgated or fused to the mr entrve anigen bnmmg poei~ne indhudme amnbodi cs and antibody fitagementst Any hn idd phaentcologil y astxc polypeptide moiety can be used within te scope of the int ention,. meludog a polypeptide having a s-ale avimier structure (see. eu gK liKran ct aL., Novel Piotin wit. Targeted Bondiog ITS 2005/008992. Baker et :dit 11 imdmu Proteins US 2001002{1076; Stnonmer ct aL- Protein Seaffuids and U'ses Thereof, U'S 2006/02231 114 and l'S 2006 0231299x [ 00252] Usefbl preclit al anmal nodeh are know nm the attfor use m vahldatinei a drug i a therapeutic indication interest le un adopted ransfcr model of periodont al diesee by \ lverde et ,I. J. Bone \Tiera i e 5.5 (2S 004); an ultrasonic perascular Doppler flow mete-ased umal model of arterial thrombosi in Grumer ci a L Blood 105, 14099 (2005 I; puinmonary thromboembo ism models aorta occelusion model, and munn ne stroke model mn Braun Ctl. ,W) 2009/ 5100 Al). For example, an adopmie transfer experin al autoimmune cncephalomy chits (A.\TA M model of multiple selcrosn has been described for ivestigations eone eriap immne dise ase.m such as multiple sereosis ikReton et aI Immhmnmuo 4 or (n 9 (200 Betn et 'al, P\AS 98 1 04~ 2 0(1 o Sullivan et &l Lhxample 4 of \ WO Th0\5 08422 *' meuncrporated beremi by referenuee in its entirety). ha .he A 1- A''F mod swmniiamly reduced disease severity and mecreased sor a dh xr ~pected m'o aunams treated t an ff cinve amnoun~t of the ientnve pharmiaceu tica composition, w hile untreated aninia arc expected to develop sev ere dihae and or mornality F or rnnmg~ the ,\ f4Tl' model the cncephalomyedogcnie (74 rat I cell line. P,\S, specific for mvclh a basic protein \N'BP) origmnatcd frm Dr. Fely ne Berand. The maunmenance of these cell in vitro and their use in the.\EA model has teen described earlier [Beeton et al( 2001 IPN AS OS. 139474. PAS I cellIs are maiam~ied in vito by altcrnatmo romnds of antigaen stimulation or actiatiuon with MBP and irradiated thyrnocy tes (2 dlay. and propagation with T. coll growth tors (5 days). Act-'tiat of PAS T cenls (3 x 10/mI) involve es incubating the cells for 2 days wh 10 m N-IBP and 15 S10/m syngenoci irdiated (3500 rad) thymroc'ts. On day 2 aftrin vitro actton, !0- 1 4 iAb PAS T cells are injecwtd into 6-12 week old female Lwis rats (Chades River Laboratories) by tai IV Day subcutaneous injections 0 vehicle 2% L ewiA ra serum in PBS) or test pharmaceutical composton are gven frm day -1 to 3, where day -I represent I day prior to injecton of 1 PAS T cells (day 0). In veh ice rate d rats, acute EAE i expected to develop 4 to 5 days after injetin of+ PAS T cells, Typically, serum is collected by tai vein bleeding at day 4 and by cardiac puncture at day 8 (end of te study) for analysis of loes of inhibitor. Rats are typically weighed on days -,4, 6. and 8, Animals may ho scored blinded onco a day from the ay of cell transfer (day 0) to day 3, and twice a day from day 4 to day 8. Clinical signs are evaluated as the total score of the degree of paresis of coach imb and tailL Clinical scoring: 0 No s:,:: =dt limp tad, L. -::::: limp tail, 2 0 =mild paraparesis, ataxia, 3, = moderate paraparesis.5 = one lindt leg paralysis 4,0 = complete hind leg paralysis, 5.0 : complete hind ite paralysis and inconutincoc. 5,5= tctraple~gia, 6.0 =moribund state or death. Rats reachingr a score of 5,0 are typically cuthanized, [00253] PAnu n q'A!tkbtEm im. hlindingocus [00254] Polvejonalantiodies Polyclonal anhiodie\ are preferably raised in animus by multiple Subuaneous (so) or intraperitonea op) ivetions of the relcvan antigen and an adjuvant. Akerntvex, antigen may be injected dircly no the animals lymplh node (see Kilpatrick et aL Hybidoma 16:381 -3819K9). N improved anrtibOdy response may bie obtained by .onjugating the relevant antigen to a protein that is immunoeic in the species to be nu .g-, kheyhole lirmpet hemnocyanin. serum albumin, bovine thyroglobulin, or syantrypain inhibitor using a bifunctional or deriv'atiig agent, for e'xamrple, malim idobenzoyi sulfoauccininmide ester (conjugation through cysteine residues. N hdxysucciniidt (trog lysi. residues)t gilutaraldehryde , succinric anhydride or' other agents knownin tho art 00251 Animalare wnwized against ntigen numgerc igor. 1c (rs " X- bo and thes Aren s compte anna iccig me soo an nladomnuv at nt i rpksies Onnth atentharurrustie hoodaxiwit 15t 11 it. ra amount otp~A de Or Congatei [etdnd' ete auv mt b iutn ut 'i I o"' )nand nons [00256' ~ ~ m anto~x qib tier Awnhamb wyTAKord ad tese a isassayed an .o -ne am ar boostedani tee plaeaus. Prefhrabv the animal is boosted ith the conjgate ot th same antigen. Conjugates atso can be made inrecomabinant cel cultue as protein fusions. Also angegatin 'wget s ~ic ,~an a sutbt sec end e th imun respnn [ 02%5 6 Monirzi 'md tiois Th inentieatigen uding flfe ns o ant binding prot that are proved indwca n at ahbodieo that Wd to DNI or .l.respe ye onamdntsodies mybeo ed u si c known itn the art, g~ by imnmortW in splencel s harvested ftrn th transg1eni animal afler completion of the inuaation schedule The spn ells can inrnrtalireo usng any tedne known-e ar. oa byin 1V53ishW inyveoma eels Io prodoec hybridonma. For amp te onoceonal an ibodWs nm be ade usttehyrda nehode frstderbed b Kolr al. a .. 5649 ( 1975 V. or may he mad byrcohiat.DAmtos(g. hlLa0Neod of proUcing mungobl ns.vcors and trntrmdhs aclsfr u.se hren f tate the genea eimor produeton of lgh and havy chains optionay usCg naMOi ce inese D that can Qesyate the antbod ( elg.. Page, Antibody 1roduetio. EP048 1790 A and US Pamen No. &,545v10l ,s [00257 nhe hybtdonma methodanmuse anotherappopriatehsniamma suc'h ars Tth neror nicqemne.is ~tma~e t eentec'ioa ls lymphicys ihat produ or ae capable of producing anibodies that u emat' m ued in ito Lmpnocves the are hoed wgt rye Inna ells us i g suitali feusingagenituc aob~ s polyethylene gaeo.t foa hvfbridoma ceO(Goding Adoncdorr Amu~tg~ueene id Phyed 403 otrruche TOM Moncioas Andbodiey Priciie and Prace fpp5 03icaemcPes.j% fOO25) in some instances, a hybridona cel hoe is produced by imumzing a transgem adnnldm having huma iWliNnyoglouO 010 quece with a DNP or MAi immtunogen; harve estm sp een ecl from~ th muie am hi iomy the harvested spleen elln to a ravelomu c bne, there c encruting hybridorna c= sabishirg hybridomi cell lies fom the hybndoma ells and idenniYing a h bridona celi line that produces an atibody that bnds DNP or Kl resevy Such hybridoma ceel hnes and nionockmal anihodics produced b them, ar aspects ol th e preet inmenon. [(02309 Thoe pen ieon also etu zfnipasc o na th prdcesthe nventie antien bidi protehi tat is a noieknad tnttody Acordin , the presen gnventdon ei also directed to aometodi pl 026 (a) cttnn thehybridoma in a culture medum zunden editions penmtOng expression of the antigen iding proin b Otehy omaani 00.(6 ) reoenAe aig icen hdingm oten tot the cult re mediuo, whch hy k ,-,,v eans N he accomplhed by known and ody purficaton techniques suc as ineed to. rnoeonal antibody nneato enes' dislosed inExanoc herein. O}6I The hybridown cobs. toC prepared, are seeded and grown in a suitabe cul ture medium that preferablv etaid. one or more substances that ihibit the grouwth or survjaIa of the infused, parental oweloma cel l [or ciirnpl, if the parentad mveloOma cells lack the enzyme hyponthine guanme phosphonibosyl tranvsterase (I IPR' or 1-PR 1 the culture medium for the nytdomas typically I include hypoxanthme, amoptenn, and thynidine illAT medium, which substances pevent the grow th of H1GPRrTdefilient cells (00263] Preferred mycloma cells are those that fuse efficiently, suppOrt stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium. Human myeloma and ousc-uman heteromweloma cell lines also have been descrbed for the production of human monocloal antibodies (Kozbor, I. Immo. 133: 3001 (1984} ;rodeur at L. Monoclonal Antibody Production Techniques and A ppiieations -pp.563 (Marcel Dekker Inc New York, 19I. Myeloma els for use i hybridoma-oducing fusion procedures preferably are nonamibody-producig have high fusion efficiency, and enzyme deficiencies that render them incapable o gwg in certain seect ie media which support the" growth oftonly the desired fused cells (hybrdomas) Examples of suitable cell lines for use in mouseusions include S-20, P3X63/Ag8. P3X63~ Ag&653. NS1.Ag 4 y Sp210-Ag14, F, N So/, MlC-i V MPCI 1-X45.-1T V and Si94/SXX(O Bul; exarls of cell lines used in rat usions include R2I0ACY3 Y3~Ag 1,2. 1R983F and 4B1210. Other cell lines useful for cell fusions are U26 GM15300-GRCG2, LICR-LON~HMy2 and UC729-6, [00264i Culure medium in which hybridoma cells are growing is assayed for production of monoclonal ani 'is directed against the amigen, Preferaly, die binding specificity of monoclonal antibodies produced by 'hrdma Ces determined by imnroprecipitation or iy an n vitro binding assay, su\h as radioimmunoassay (RIA ) or enzymeiked immunoabsorbent asay ( LISA AA The binding affinity of the onoclonal antibody can, for example, be determined by BlAcoreC or Scatchard analysis (Munson ct al Anal Biochem, 107:220 (198M, Fischer et. al., A peptide-immunoglobuin-coniugate, WO 2007/043463 A l Exa~mple 10, which is incorporated herein by reference in its entirety i [00265J A fter bybridoma cells are identified that produce antibodies of thie desired specificirty, affinit and/or aetivity, the clne may. ~ be) subcone by imiting dilution procedures and grown by standard methods (Goding M'?vonoclonal Antibodies: Principles and Practice, pp.59-I03 (A cademic Press, 1986t}} Suitable culture media for this purpose include, for example, D>-ME M or RPMV-1640 medium, in addition, the: hbridomna eellIs may' be grown in vivo as asci tes tumors tn an anirnal [00266 Uhridonas or muAbs may be further sceened to identify vAbs with particla p ptez \Nuch as the abdity t inhibit .K fu d' K bxmpecs of such screens are provided in the ampnlcs Ie low iniondlona asnubedncssecreted bydv theubd~ones arntaolvb separat d to ~hccltued tredja asches fhuwt or sene PUh convcnuoi irminiinogdobur tmi hciao pmocedures such as~ for example, protein N~epharose, ydroxylapaite chrmatography, ci ceetrophoresis dia i flntcrmtgahy. or an ther stable puriftattn technkgue known in the artt [O0267) Roccomn ant Production of Antibodies. The invention provides isolated nucleic acids encoding any of the antibodies (potyclonal and monroconal), inud antibody fragments of the invetion described herein, optional operably enked to control! sequences recogmzed by a hos cell vectors and host cels comprig te nucleic acids, and recombinant techrniques for the production of the antibodies. which may comprise culturing the host cell so that the nucleic acid is expressed anld. optionally, recovering the antibody foM the host cel culture or culure medium Similar materials and mnethods apply to production of polypeptide-based antigen biding proteins. 00268 ~ Relevant armno aci sequences .man rnn gk i or polynto interest may be determined by direc prenisequencinsand stable encoding. ndewlede sa e car be d acordin in aeraldon c. Atemativel. genomc or eDNA encng thenonodona. abodie may be iolated and sequenced kiom cells producing su ch antibdie dang conventioal procedures (eg by ing ohgonucleotide probestd are capable of bdin specifially to genes encdingt the heavy and light chains of the tmonocioad antibodis [00269]i Cloning of DNA is carried out sing standard techniques (see, cit Sambrook et at (989) Molecular Cloning: A Laboratory Guide, Vols .- 3, Cold Spring harbor Press, which is incorporated herein by refrence) For example, a cDNA library may be constructed by reverse transcription of polyA+ mi{NA, preferably nebrane-asociated ruR , and the library screeed smg probes speecii 1or humam m~lunloglobul iniR> polype&) genieea quences. In one embodiment, howeer, the polmcrase chain reaction (PCB) is used to ampty cOD\ s (to pOronu of tid l-ingh cD\NAs) enicodmg ani innmoglobl ien segment of interest (g, a giht or heany chain triabsegmeno). The amphlied sequenescan be readily ClAned into ny suitable wSoi, e g, expreson wetors mTalnien e ctors o' phage disply vectors. t bw :4ap ciated that the particular method of cloning used is not entiel so long as it is posible to determine the selneQ of sonic potion ot tnit in toglobuUn wolv\ pept de of interei [270] Onc source for antibody nuileic acids is a hybridoma produced by obtaining a B celB from an animal immunized with the antigcn of interest and fusin it to an immortal cel. Alternatively, nucleic acid oa be isolated from B coils (or whole spleen) of the imnmnized animal Y et another source of nucleic acids encoding antibodies isa library of such nutclei~c acids generated, for exam pie, through phag d isp lay technology. Polynucleoides encoding peptide of interest, e.g., variable region peides with desired bindinteritis, can be identfed by standard techniques such as panning. [00271) The sequence encoding an entire variabne region of the im unoglobulin polypeptide may be dermined: hOve uwil sometimes be adequate to sequence onl ai portion of a vaial rein fort \. eaple the CDR3kencoding portion Sequencing is caribe out using standard techniques ( ee, e~g, Sarnbrook et al 1989) Molecular Cloning: A Laboratory Guie Vols 13 Cold Spring Harbor Press and Sang, F, et a (1977) Proc. Nat Acd Se USA 74: 546467 which is incorporated herein by reference). B~y comparing the sequence of the cloned nucleic acid with published sequences of human immunoglohin genes and cDNAs, one of skill will readily be able to determine. depending on th region sequenced, (q the germline segment usage of the bybridoma iunoglobulin polypeptide (including the isotpe of the ay chain and (ii) the sequence of the hcavy and i chain variable regions, including seduces resuming frn N-region add itiOn and the process 1 E omatice mutation. One source of immunoglobulin gene sequence infornadna is the National Cete for fBi iholot ind imtion. Nanoena V ~ib edie Nationl utitutes of ealte BtAhasd Ad. [0X2] solated DN an be opermbly iked to control seguences o placed io expression vectors, which are then transfected ino hostells tha donot otherwe produce inmnunoglobuln protem, o directhm snmhes ofmenoeional antiodic, i the necombi namt host cells. Receome nnt podueton of antibodies is wel known n the art. [00273} Nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequience. For cxample DN A for a presequence or secretary leader is operably linked to DNA for a poypoptid i it is exp prrssed as a prcprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if itvcts the transition of the sequence; or a ibosonme binding site is operably linked to a coding sequence if it is positioned so as to facilitate translaon, Generaly operably HOked mans hat the DNA sequences being linked are contains and, i the case of a. sccetory leader, contiguous and in trading phase. However, enhancers do not have to be contiguous. Linking is accompished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or fliers are used in accordance with conventional practice, 00274] Many vectors are known in the t. Vector cmpomnt may include one or more of the following; a signabsequence (that now for exae direct ecetion 04the antibody e.g.. A TGC'GGGGCGTCGTCTGGC COCT OTGGCT GAGAGTGCOCCTIGT SEQ 1D NO: 102, wich encodes the VKM signal peptide sequence MDMRVPAQLLMLL LLWLRG ARC : SEQ ID NO:03= an origin r on, one or more selective muaer genes (that may, fO exAmple, confer antibotic or other drug resistance, complement auxotrophic defcinces, or supply critical nutrients not available in the edia), an enhaner element, a promoter and a transcripton termination sequence, all or which are well known in the art [002 75 Celt cUel na ccll cuttre are ofen used mterchargabi nd all such Jcsignatin> hcreain A telde pmogeny fransfortuunts and trunsformed cel mi alude the primary subject cel and cultres derived therefrom without regard for the nnber of trse t is ande d h al progenwmay nr be precisely iW AA W DNA conte to ddiberate o uadertent mutations Mutam pr ogeny that h ae the samae timcion or bmkoloial actiry as screened lor in the or~gin ally transforrmed reU. are mecluded. [00276} Exemplary host eel s include prokaryote, yeast, or higher eukaryre cells Prokaryoic host cels include eubacteria, such a\ Gram-negative or Gnrampovive orgrarn iss tor example, Enterabtkceria(cea su ci h s P'~Q Eshrca Cg R gr' i, Enteobacter, E'rwinia, lebsiella. Proteus, Salmnonea'ige'W Samu~ila iyuhirn milmur s"rrii c yRega ntia riacsas an Sunggltd as~ wd lacRilus such as &subtiis and . lChcoiis lisadas. and oeltomyces Eukarvoic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for recombinant poyeptides or anfbodimes, Secharomvces cervsae or common baker's yeast, is the most commiony us am~ uong lower cukaryotic host microorganisms. however, a number of other genera, species, and strains are commonly available and useful herein, such as Pihia, e.g P gasjoris, Sehioscchamyvcs Romnbc Klvronwccs artri ; imW ida; fnchodena resi a' Neuiosera casa Schwannionmvces such as Schwaniniomyces oceidentalis and filamentous fungi such as, e g. Ncguospagri Pggi.,ja p .gl m and \argijj busts such as A.iulans and tA gge [00277 Host cells for the expression oftgiycosylated antigen binding protein, including antibody, can be derived fromn multicellular organismns. Examples of' invertebrate cells include plant and insect cells, Numerous bacuiov iral strains and variants and corresponding permissive insct host cells from hosts such as 4Sdoptera fragijArda (cterpilar) . ge aeyt .mos. to.Ades albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified, A variety of viral strains for transaction of such eellIs are publicly avidOa, I, tnhek Wovrint of Auokngaion NX d h 3n5srano [00278) Vertebrate host celNs a also s4uable hosts, and recombinant production f anigen bindig protein (including antibod6) f-om such cells has become routine procedure. Examples of useful mammal ian host cell lines are Chinese hamster ovary cells, including CHOK cells (A TCC CICL61 DXB 1, DG-44, and Chinese hamster ovary cells-DHFR (CO, Urtaub et al, Proc. Natit Acad. Sc USA 77' 4216 (1980); monkey kidney CV he transformed by SV40 (CS-7, ATCC CRL 1651; human embryonic kidney line (293 or 293 cells subcloned for grow in suspension culture, [Graham at, Gn Virol. 36: 59 (.1977)]; baby haimser kidney cels (BH/K, ATCC CCL 10): mouse sertoli cells (TM4, Mat, Bio Reprod. 23: 243-251 (1980)); ronky kidney cells (CVI ATIC CC 70. ; Al fican green monkey kidney cells (VER -,76, ATC( CRL-157%; human cervical carcima cells (IELA, ATIC CCL 2); canine kidney cells (MDCK, ATCC CCL 34) buffalo rat liver cells (BI A, ATCC CR L. 1442) human lung cells (WI 38, ATCC CCL 75) human hepatcma cells (Hep G', HB 8065: mouse mammary tumor (MIM1 060562, ATC CCLS ) TR 1 cells (Mather e al Annals N Acad. Sct 33: 44-68 (1982 MRC 5 cells or FS4 ells: or mammalian myvloma cells ['00279 Host cell are transformed or transfecte w th the above-deanbed nuclei acids m vectrs ir prodheio nane hmdnig prowtm and are nltured im coMMentionai nutrient media rnodied as appropnatc or mnduem promter s seleting trantrmits or amphyingme genes ened ing the desiWd equencec in additon, novel vctors and transfected eeli mL's x th muhtile copies <f trnscurpton umts separated by a slective marker are partiularly usefl i fur the expression of' anti ent ngp proteins [002801 The host cells used to produce the antigen binding proteins of the invention may be cultured in a variety of media. Commercially available media such as H am's F 10 (Sigma), Minmal Essential Medium ((M EM), (Sigma) RPMl -1640 (Sigma), and Duibecco's Modified Eagie's Medium ((DMEMl Sigma) are Siable for culturing the host cens. In addition, any of the media described in Ham et al MeTh Enz, 58 44 (1979), Barnes et at AnaL Biochem, 102: 255 (1980 US. Patent Nos. 4,767704: 4 ,657866; 4.927762: 4,560,655; or 5,122469; WO90i0343T: WO 87/009; or US. Patent Re. No. 30,985 may be used a' culure media for the host cella Any' of these media may be supplemented as necessary with horrnones and/or other growth factors (such as insidin, iransferrin, or epidermal growth factort salts (such as sodium chloride, calcium magnesium, and phosphate . bufrs (such as HE PE S), ceotides (such as adenosine and thymidin. antibiotics (such as Glentamxyci 0 M drag. trace elements (defined as inorganiAc compounds usually present at final concentrations in the mitcroroar range), and ghtcose or an equivalent energy source. Any other necessary supplement ma al 'so be incauded at appropriate concentrations that would be known to vtose slhed in the art The culture conditions, such as tempoerature, pH. and the like, arc those previously used with the host cell selected tor expression, and wll b apparent to the ordinarily ski lled artisan [002811 Upon culturing the host cells, the atigen binding protein can be produced imracellularly, in the periasmic space, or directly secreted into the mnediurm. If the bintrneJllularlv, as a antgn~ inding protein is produced itaeulryasafirst step, the particulate debris, eit host cells or lysed fragments, is removed, for example, by centre fuga~u tio or ultrafltration. [00282I The antigen binding protein (e. an antibody or antibody fragment) can be purifid ung, for example hydroxylapatite chromatography, cation or anion exchange hronatogranphy, or preferably affinity chromatography, using the antigen of interest or protein A or protein G as an affinity igand. Protein A can be used to purfy proteins that 6iclud polypeptdes are based on hmnan . 12., or 14 beav chaina (Lindmark et aL S. inmmunol. Meth. 62: 1 - (190 83 Proin G is recommended for all mouse isotopes and toe buman 7 (uss et at. EMBO J 5: 15671575 (18)Y The matrix to whI the affinity ignd is atachid is most often agarose, but other mnatrcea are available, Mochantcally stable matrices such as controlled pore glass or poly(styrenediviny)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the protein corm prises a (> 3 domain, the Bakerbond ABX 1 .urein (. T. Baker. Philipsburg, NJ,)is useful tor purification, Other techniquCs tr protein purification such as ethanol precipitaion, Reverse Phase HPLC, chromatofocusing, S ~P)3AG E, and ammonium suliate precipiation are also possible depending on the iantbody to be [00283] CI'inierie.gmanized and tHmnan Engineered ' mnlonl antuibodies. Chimeric monoclonal antibodies, in which the variable Ig domains of a rdem mnon~oclonal antibody are fIsed to human Constant i domains, can be generated using standard procedures known in the art (See Morrison, S .L, et at (1984) Chimeric Human Amtibody Molec &ul es; Mouse A ntigen B inding .Domain s with Human Constant Region Domains Proc, NattAc~ad.N Si. USA 81, 684 .- 6855; and, oulianne, . O, et at Nature 312, 643 (1984)) A number of techniques havc been described tor humanzring or mody nt bd i y I eqe ce to be more hun-t like, for exmple, by (1) grafing the non-human copLementarity determinmg regions (CDRs) ono a human framework and constant regon (a process referred to in the art as humanizing through "CDR grafting") or (2) transplanting the entire non human variable domains but "cloaking" them with a humanlike surface by replacement of surface residues (a process referred to in the art as vencring") or (3) modifying selected non-human amino acid residues to be more human, based on each residue's likel ihood ot participating in an tigen-binding or antibody structure and its likelihood for immnunogenuicity. Se, e~g- oncs et al, Nature 321:522 525 (1986); Morrison et al, Po. Nat .Acad, Si, U.S.A. 81:6851 6855 (1984): Morrison and Oi Adv. Imnunol, 44:65 92 (198); VWhoeyer at A Senuc 239:1534 1536(1988): Padlan, Molee. fmmun. 8:4 498 (1991 .)Padlan( Motec. Inuno. 31(3 169 217 1994); and Ketteboronugh CA et a. Protein Eng, 4(70:773 83(1991) Co. W S. et at (1994 J. mnmno. 152., 2968-29 7 6Y Studnicka et at Protein Engineering 7: 8 .- 81 4 (1 994; each of wichI isincorporated hereim by reference 4in its etwrety, [00284 A nmber of tcniqes heben descrbed fbr inmuzng or odifytng antibdy .equecv to be. nme hnnan-ldrc. Ibr example, byv (1) ing the non-~ human eompeentarity determining regions (CDJ s, onto a human framework and conistant region (a process refrrred to in the art as humanizing through "CDR grafting") or (2) tras2)plating the entire non-human variabic domains. but "cnoakingK them wth a h nand ike surface by repaeentt of surface residues (a process referred to in ti art as *vncering" or (3) modifyng selected .nonhuman arino cid residues to be more human based on each residue likelihood of participants in antgen-inding or antibody strumre and its likelihood a immunogenicit e~g Jones etaf, Nature 321:522 525 (1986); Morrison e Proc. Nati. Aca. c. U .SA 81:6851 6855 (19 8; Mor rson and Q4, Adv. Imnui. 44:65 92 (1988) Verhoeyer eta., Science 239:1534 1536 198) Padlan, Molec. lmnmun 28:489 498 (1991; Padiam, Molec. Inmnol 31(3):169 217 (1994 and Kettleborough, CA et at, Protein Eng. 4(773 83 (1991 KC, ( R, t LA (94k 1 1 Immnot 152. 296$-' 2976); Studnicka et al. Protein Engineering 7: i05-8 (1 994); each of which is incorporated herein by references. i1 its entirety, [00285] hn One aspect CoRs of the light and heavy chan variab e regions of the antibodies provided herein (see , Table 2A~) are grafted to framework regions (FiR) fRm antibodies from the same. or a difTerent, phvlylogenec ecies, for exampIle, the CRs of the heavy chain variable regions (eg V \ V u3 Vu4. VS5, VU6, V&7, V 8, N V9) and/or lih chin vaiable regons (.g V; I, Vi2, V34 VV5, V, V7 V orVt9)anbe grafted to consns human FRs. To create consensus human FRs, FR from several human heavy chain or light chain aniuo acid sequees may he aligned to identiy a consensus amino acid sequence. In other embodints, the FR of a heavy chain or light chain disclosed herein are replaced with the F Rs from a different heavy chain or light chain, in one aspect rare amino acids in the FRs of the heavy and ligh chains of the antibody are not replaced. while the rest of the FR amino acids are replaced, A "are amino aci" is a specific amino acid that is in a position in which this particular amino acid is not usually fund in an FR Alternatively, the gra fled variable regions from the one heavy or light chain may be used with a constant region that is different from the constant rein ot duat a' Ucar he o ih chiaasdadhmd herin 'other ebodnent the gUndva rib n pat of a Mngi chan 1 antnod. [00286) Antibodies can also be produced using transgenic animals that have no endogocnous nimmtmnoglobulin production and are egineered to contain human imtmunoglobulin loci, For examei, WO 98/24893 discloses transgeni amals having a human ig locus wherein the anvimak do not produce fHnctional endogenou imnumogobtdlins due to th in alon ot endogenous heavy and light chain loci. WO 91/10741 also discloses transgenic non-primate mammalian hosts capable of m nimun resp to an immunogen, wherin the antibdies have. primate constant and/or vaNabl regons. and wherein the endognouti immnunoglobuin encoding loci are substituted or inactivated. WO 90(O498 discloses the use of the Crc/Lox system totmodify the irunogoloun locus in a mammal, such as to repace aA or a potion f the constant or variabLe regio tfm a modified antibody molecule. WO 94/02602 discloses non-human mamma lian hosts having nacti vated endogenous Ig loci and functional human IA lotl U.S. Patent No. 5,939,598 disposes methods of making transgenic mice in which the mice lack endogenous heavy chains, and express an exogenous mmunoglobin locus comprising one or more xeuoencic constant regions. [00287] Using a transgenic animal described above. an imnmne response can be produced to a selected antigenic molecule, and antibody producing celis cam be removed tnom the animal and used to produce hybridomas that secrete human~ derived monoclonal antibodies, immunization pr otoos, adjuvants and the like are known in the art, and are used in immunization of! toe example, a transgenic mouse as described in WO 96/33735. The monoclonal antibodies can be tested for the abiltv to inhibit or neutralize the biological activity or phvsioocal effect of the corresponiding protein. See also Jakobovis et at, Proc, Nail Acad. Scit USA, 90:2551 (1993); jakobovits et al., Nature, 362:255-258 ( 3Buermann ei aL Year in Immuno, 7:33 (f193); MOnde et al. Nat. Genct 5:146156 (1997; and U.S. Pat No. Z59 1,669, U.S. Patent No. 589,369, U.S. Patent No. 5545,807; and U.S Patent Apiplication No, 20020199213. U.S, Patent Application No, and 20030092125 decribKs mahod biag the tununr e a animal to te deited epop" Human tbOdis nay aloe ge nctned by ur v cvatend B el see U.S, P Nos. 5567S40 and 27 [0288 tAibody rwdne ion b hc is a tcnicues 0C9 t1he devtelopmnent of teelnolognes fo rnukimg ieretouire of recombinant hurnmant iody genes and the displav of the encoded antibody fragments on the surface of ttmitcntous baeriiophge ha s prouded another neans f or' genettng humah~nle\ de.rxd antibodres Pia\djledl Is deserrbed in e g., owe of a, W() 0171, \ afbrt' et al , \ <' 0 04 imd Caton and Koptowsk Proc Nai Acad. SeJ tS 87:N4uu4 (1990). each of which i incrporated herein h rerence i i entirety The anbodies produced by phage telogy are usually produced as antgen bining niem\ e a, L\ or Lab fragment, 'An " a lack ciffeor limnetions lffector functions can be introduced y one of uo strategies The f agents cn be engineered eithr into complete antibodies for expression in numa hara cll r io Ubispxenmtibody fragments with a second bindio ste capable of triggering an fftcor linction. [002901 Tyypically. the 'd tragment ' t 4 y I) and light chain (\ -CJ of antboth are separately cloned by PCR and recombined randomly in combnatoal phage display lbri, which can then be seleed Or inydn to a particular amigen Th anybody fragmems arc expressed on the phage surface, and selection of L or Fab (and therefore the phage conting the DNA encoding the atibody fragment) by antgen LAnding i accomplihed through several rounds of antigen bindn and ce amplification. a proceed re termed panning Antibody fraanmemt snecitic for the anti aen are enrnehed an in al lv slated. [00C19 Phage diplay technique can also e used in an approach fo, the hunanization of rodent monoclonal antbodies called "moed \elecdon' (see kaspers el S, et at.Bio/Teebology 12. 890-903 (1994A For this, the Fd agent of the mouse monoelonal antibody can be displayed in combnation with a human bght chan library, and the resltimg hybrd Fab library munsa heseecd with andgen The nouseyd hagment thereby poides a to grideihe seki Sbsequid he N cited haman i h a ae Ombinedwit a mn Ed ragmen library. Seieon oNhe reung library eid endirdy human fat [00292] A variety of procedures have been described for deriving human antibodies tfmm phage-display libraries (See, for example, Hoogenboomn et. l. Mob. Biob, 227:381 (1991 );r Marks et aL 1. Mob, BioL 222:58SV597 (!991 US, Pati Nos, 5,53,332 and 5,73,95 Cackson. '. and Wels, 3. A. T IBT ECH1, 173 184 (1994. In paticular, in vituo selction and evolution of antibodies delved from phage display libraries has become a powerfuD too. (Sec Burton, D R- an Btarbas 1,11 C. F, Adv. Immunol. 57. 191~-2M (i994i and, Wimter, G t al Anna Rev. immrnoL 12, 433-455 (94) U application no, 20020004215 and W)92/0 1047: U.S. patent application no. 2003019031 published OtobrN 9 2003 and U.S. Patent No. 6054,287; U.S. PatentNo57,93 [002 Warin "Scedenintg of' Page-Expressed Antiody Libraries by Capture Lift Methods in Molecuar BiologyAnibody Phag Dipa: Method nd rotocol ?1 ST 9 and US. Parent Application ublcadon N 20010044772 pubihed March 6. 2003 describes a thods for screening agexprsed anibod immobihrntton Ohe Can didate biding. Ioecules on a sold supot [00T94, Other Enbodimerts ofAnnen bindin moein Antibodv framents 00295)3 As noted bove, antibody fragments comprise a portion of an imact full tegth antibody, prefe.rabty an anigen binding or variable region of the inact antibody. and include linear antibodies and multispecific antibodies formed trou antibody fragments. Non limiting examples of' antibody fragments include Fab., Falb Ffab'j2, Fv, Fd, domain antibody (d0, cormplemnentarity deterining region (CDR) fragments, singl-chain antibodies scFvh singIe chain antibody fragments, maxibod ies, diabodies, triabodies tetrabodies minibodies, linear antibodies, heating' recombinant anibodies, tribodies or biOies, intrabodies. nanobodies, sunall modular imnviopharmaecuticalis (SM IPN) an and tenindsng~domain 3 Oimimoglohbain fusion pro tin, Q can lized antibody, a \iHH cntaing antibody ut nmte ins or dernv'tixt thcreot' and polpaptldes ttum at eas a portion of an immunoglollbin that is soicient to center specific antigen hindiag to the pupeptid, such as a CIR sequpence, as tou as the antibodW etams he died hiolottead actis ity~ Such antige'n tiagm-ents inay he produced by the mnodrfication of whole <mtibodies r syotnemezd oW novo using r ecombinant DN A technologies or petide ntho s 29 Addiona anbo f anis in ni a (Wad i a., aceu 341P:54-640. 19 %9) Whic nis oa dmin a, ia i of snde n sen (V; u C1 I which torn a pa, - A gioen bindiro L a bod a be hispeci or aonospec (apata cm a- Ptin F. 6:100-0 (1995)1. [00298 A nibo consisting of scmv sed to (0 via p epide jnker (hingeless) or Via an Igo ne hashen e n s et al Proten Ene De Se4. 2004 Apoe 7(4) 3 15N3 .0 [002991 The recn, 'aihodx"' rufcs in hinaen mcv con a imr attchbed to Lhe Pctn ot an inunocobmin see, for campoe Frersci a.roei Elgineern Design (2004 hnnrn~oi 'cai Mhds 251211V35 (2001) [000 Funiona heavy-chin bodiesdoid h as ae naa GVa W sauch as eames dmedares, alpaas nhe an idmg ite i reduced t a m Vi omain ntibodies frm antigetid inKe n sng on 0 heav 6c-t vhbhrmodcc. hese ncunal antbodisa are homonners of heavy chains only having stotue H rered to as e"hevy-chain :iiods e -i a died repo recm inesw 2andh const n ons tat ontin Age. CST and (7113 domains and lack a (11-1 dormin. Mums!ca VW 6 -00nugiT 1'rff6et5 difficult to poduce in solubl form, but improvements in solubiity and specific binig can be obtained when framework residues are tered to be more VIHeike, (See.gu.. Reichman, etL Im3nmunol Method\ 199 31:25-3 Cameized v6an domains have been found to ind ~to antgen with high affnity (Desmyter a at, . Bo.Chen.. 276:26285-90 2001{ and possess hiN stAility i solton (Ewen et aL Bkochumlrtry 41 :3628-36, 2002). Methods for generating antibodies having cameci7zd heavy chains are described in, for ex ample in UIK Patent Publication Nos. 2005/0136049 and 2005/0037421, Aternative scaffods can be made from human variable-like domains that more closely match the shark V-NA R scaffl arnd may provide a framework for a long penetrating loop structure. [003011 Because the variable domain of the heavy-chain nbds s t smalst fully functional antigerpbinding fragment with a mnolieuar mass of only 15 kDa, this entityis referred to as a nanobody (Corte-Rtamozo et aU Cancer R -eder 64:285357 20 A n body hbrary may be generated from a immunized dromedarv as described n Conrath et a, AenA s A C ?Kmha er 45 28R7 12, 2001). [00302] intrahodies are sitgle chain antibodies which demionsrate intracel Mnar expiressin and can manipulate i tracellular protein imcton (B iocca, et a EMB130 9:101 -10x 1990: Colbyt atl Prc NatiAcad c' . SA 101 :176621, 200 4 Inrabaodies, which comprise ell signal sequences winch retain the antbod contract in intraelu lar regions. may be produced as described in Mhashilkar et an (EMBO3 J 14:1542-5I, 1995) and Wheeler cc al (FASEB t 7: 7335 2:003. Transbodies are ceb-permeable antibodies in which a protein transduetion domains (PTD) is fused with single chain variable fragment (seFv) anibodies Hing et aL. (Med2 Hypth 64:1105 82005). [00031 Funher encompassed by the invention arc amibodies that are SM Pa or binding domain immnunoglobuiin.fso proteins spcfi o tagt protein. These constructs are sides comrprisigM antigen binding domains fused to immunoglobutin domns necessary to canry out anibody effeetor functions, See I09 egw 00301000, LS. atent pubitRa 20030 93and US aten Pubicatin 2003011592, f00304) Various techniques have been developed for the production of antibody fragments Traditionally, these fragmnents were derived v it prorcotytie digestion of intact antibodies, but can also be produced directly by reombinant host ecLs See, fr example, Better et al. Scene 4: 104 ~1043 (1988): Skea et at Scnce 240: 1038-1041 (19\0); Carter et ali. BiTecAmology 10:163-167 (99). [00305 1 Other Emsbodidcnts of Anthee Bindi naProteiin v lu in'n t ibodies [00306) In sonme embodinments, n itty be desirable to generate nmulntivlm or even a miseit ic (cig. bispecc trsecific, e monoclonal antibody, Such antibody may hve binding specificiies for at least two different epitopes of the tagt antigen, or al temnatively it may bind to two different molecule etg to the target antigen and to a cell surface protein or receptor. For example. a bispecifie antibody may include an arm that binds to the target and another arm that bi a triggerng molecule on a leukocyte such as a T-ecll receptor molecule (eg, CD2 or CD3), or F c receptors for 'gs (FerR), such as eyR i (164), Ecyfil (CD32) and Feykil (CD 6) so as to focus ciluarr defense mechanisms to the target-expressing cell, As another example, bispecitc antibodies may be used to localize cytotoxic agents to celis which express arget A i agen, These antibodies possess a targe-binding arm and an arm which binds the cytotoxic agent (eg., saporin, anti-intrferon-0. vica alkloi, ici Achain, mnethotrexate or radioactive isotope hapten). Multispecific antibodies can be prepared as full length antibodies or anuibody h agents, 100307] A d ditional l the anti-DNP or anti-KLIJ antibodies of the present invention cin also be constructed to fold into uRltivalent forms, which may improve binding affinity Specificity antd/or increased half-life in blood. M ultivaient fooms oft ami-DNP or anti-KLFH can be prepared by techniques known in the art 00308X Bpecifie or ntipecifie antibodies include cross-inked or erocojugate antibodies For arnp on of the antibodisi th heteroconiugate can he coupled to aWidn the other to Noti Hetemeoniugate atbodies ma- be made Asng any conenient erosmking methods. Stable croshnkm& agents arc wei known in the art and ae discosed mn ob Pat. \o. 4o67.9tt a long with a numhber of etossiinkimgte i ov' Avmqe knibe ncrwthod is designed to myake tetraners by g a sreptavudimeodm sequence at the C terminus of rte sen> Streptmvdmn is composed of four subunis so when the sehw streptadhn is tolded, tour subunits associate to fonn a Ietramert lupiy anm et al iHum Antibodice Hbbidomas 6(3): 93>01 (199 , the disc sure ofrwhich is mnorarted herein by reference unit, entery L. [00309] According to anT approach for making bispecific antibodies, the interface between a pair of antibody molecules can he engineered to maximize the percentatge of heterodimers wh ichi are recovered frm recormbinant cell culture, One interface comprises at least a part of the Co3 domain of an antibody constant domain In this method, one or more small amino acid side c n h o irst antibody molecule are replaced with larger side chains (e.g. tfyrosineor tryptophan) Compensatory "ai ies" of identical or similar size to the large side chains are created on the interface of the second antibody molecule by replacing Iange amino acid side chains with smaller ones (g. ainine or threoninex This provides a mechanism r rasing he yid of t heterodimner over other unwanted end-produets such as homodimers, ~See WO 96/270 1 published Sep, &t. [00311 & Technique for genemaing bispecific or muispecdie antibodies, front antibody framuent, have aho been described i the iterature, For example, bispecific or trspecifie antibodies can be prepared ung chemcal linkage. Brenam et aL Science 229:51 (195) deserbe a procere wheenir antibodies arc proteolyticaly cleas ed to generate f(alb fragments, fuese fragments arc reduced in the prewece of he: dAhiol complevin anon n d ursnie a' stabhhAe Siinal dihio and parent, interolecular disulfide formation The Tab' fragments generaeed arc then converted to thionitroenzoate (INR deriisi es One of the Sab' lB derivative es is then reconverted to the Fub'tdio I by r eduction nith mereaptoethylamine and is mixed wih an equimolar amount of the other FabtTNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobhiizatio ot enzymes, Better etlaScience 240 10411043 (1988) disclose secretion of functional anitibody fragments from bacteria (sea ig Better et a, Skerra ct at Science 240: 1038 1041 (1988)). For example, FzabtSHi fragments can be.drcl eoerdfo .cliadceial coupled to fom hispecifie antibodies (Caner aL.. Bio/Tfechnology 0:1 63W (1992); S aby et at, J Exp. Med. 173 2175225 19920) [003 01 Shalaby at. a xp. Mcd. 72 2 9 escibe te production oi a ~ uar ed be0iS3 e111 Sltbody Ib~: nlcxic EdachP fragsment was separately seceteed from Ecu! and subiecteto directed ceica couplinga m to "tete RispeAic arnttbod v [003121 Various techniques for making and isolating bispecific or multispecific antibody fragments directly ftom recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine i s, g. GCN4. (See generally Krste1ny ct at A. immoto. 148(5)147-4553 (1992) The Ieucine zipper peptides from the Fos and Jun proteins w i d tF Portions of wo different antibodies by gene flionw The antibody honodimers were reduced at the hinge region to torm monomers and then re-oxidized to form the antibody heterodimers, This method can also be utilized for the production of antibod y homodimers 003131 bodies described above are one exope of a bispecc antiody Seefor example, Honger et Proc Na. Acad. Sc. USA, 90444448 (993' B ivaicr dieabodies can be stablizedEb disulteI in age. [0314 Stable mnonospei ficor bipeci F temiamers can also be gene by nnovalent assocation in (eNybconfiguraion or as bisdt abodes. Alenaieyto ifretstv anb oie i .fltmtotrmabs-cv [003151 Another strategy tor making bispecifie antibody framnets by the use of single-chain Fv (sv timers has also been reported. Se rubberr e, J. Immnl O, 5368 (1 994 On approach hs been to link two sMx antibodies wtn liners or disuide bonds (Mal ender and Voss Biot Chemn. 269: 99~2061994, WO 94i 1380 and U.S. Patent No, 989330, th discosur of which are incorporate herein by reference in their entireties). [003161 Alternatively, the bispecific otibody may be a linear antibody produced as described in Zapa et at Prowin Eng, 8(10:1057-1062 (995. Briefly, these anmibodies comprise a pair of tand.em Ed segnts (Vu -CH NVa -C;l which form a pair of antigen binding regions. Linear anotibodies can be bispecic or nonospeifi [00317] Antibodies wth more than two valencies arc also contemplaed. For example. trispecific antibodies can be prepared, (Tutt er at £ Immunol 147:60 [0031 8 A 'heating rec umbinant antibodies bispecitic antibody tha cognizes adjacent and non-overlapping epope of the target anten, and is 0Oexibl enough to bind to both epiopessitulanensi (Neiet ud, 3 td 246i6:: 7997 [00319] Production of bispecifi Fab-seTv (bbody") and trispeci tic Fab-(seFv)(2) ("tribodyv) are described in Schoonj ans et at (Jnmmuna/. 165:7050-5 7, 2000) and Willems t a U Chromagr B Anati T hBomd 1f Sc N 6676. For bibodies or tribodies, a sehv molecule is fused to one or both of the VL-CL (L) and VH-CH (EI) chains, e., to produce a tribody two scFvs are fused to >termn of FAb whle in a bibody one se is fused to Cterm of Fab. [10004 i yet another method, diners timiers, and ternmers arc produced anter a tre esteme is introduced m the parental pnoen. Apepdesed cros tinker wth variable umnhers (two to four) of maleimide groups was used to cross link the protein of interest to the free eyees (Cochrn er a, LImmunity 12 241-50 (2000) the disco e of' nich is mirpomatd herein in it entirety> [0032i Omrtbodimentsofnien 1dino rotins [00322] Inventive antigen binding proteins also include pepubodies, The term ir a mo ule comprising ati antibody Fe domain attached to at least one peptide. The production of peiodies is gnraly described in PCT publication WO 00/24782I published May 4, 2000. Any of these peptides m be inked in tandem ('i, sequentially with or without liners, Peptides containing a cysteinyl residue may be cross~inked with another C7ys-containing peptide, either or both of which may be linked to a vhic, Any peptide havin rnore than one CS residue may torn an intrapeptid disufe bond, as wet Any o these peptides may be dervatized, for example the carboxyl termnus may be capcd wih an nmino group, cysteines may be cape. or amno acid residues mayv subtituted rby mifictiCS other than amo acid reskdues (e .g, harnagar et 4 Md C 39: 38 4-9 (1996), and CuthQrtson et A . Med. hiem. 40: 2876-82 (1997), which are incorporated by reference herein in their entirety) Tie peptide sequences maybe optimized, analogous to affinity maturation Ior ant ibodies, or otherwise altered by aManine scanning or random or directed mutagenesis followed by screening to identify he bet binders. Bowman. Ann, Rev. Biophys. Biomot Struet 26: 401-24 (1997),\ vaious molecules can be inserted into the antigen binding protein structure. cit.g within the peptide portion Itself or between the peptide and vehicle portis of the antwen binding protein, while retaining the desired activity of arisen binding protein. One can ready insert, for example, nolcuies such as an Fc domain or fragment thereof polyethylen glycol or other related molecules such as destran. a fatty acid, a lipid, a cholesterol group, a small carbohydrate, a peptide, a dtectable moiety as described herein (including fluorescent agents, radiolabelt such as radioisotopes\ an oligosaccharide, o ligonucleotide. a polynucleotide, interttrence (or other) RNA, enzymes, hormones or the like, Other molecules suiable for insertion in this fashion will be appreciated by those skilled i thea. and are encompassed wthin the scope of the tn ti on Th incis irtot ornmd a desired molecl in between to cnsecutve anonoaida ptionauyjoined by asuiib 114 [00323] Linkers. A "linker" or "linker moiety", as used interchangeably herein, refers to a biologically acceptable peptidyl or non-peptidyl organic group that is covalently bound to an amino acid residue of a polypeptide chain (e.g., an immunoglobulin HC or immunoglobulin LC or immunoglobulin Fc domain) contained in the inventive composition, which linker moiety covalently joins or conjugates the polypeptide chain to another peptide or polypeptide chain in the molecule, or to a therapeutic moiety, such as a biologically active small molecule or oligopeptide, or to a half-life extending moiety, e.g., see, Sullivan et al., Toxin Peptide Therapeutic Agents, US2007/0071764; Sullivan et al., Toxin Peptide Therapeutic Agents, PCT/US2007/022831, published as WO 2008/088422; and US Provisional Application Serial No. 61/210,594, filed March 20, 2009 (corresponding to WO 2010/108154), which are all incorporated herein by reference in their entireties. [003241 The presence of any linker moiety in the antigen binding proteins of the present invention is optional. When present, the linker's chemical structure is not critical, since it serves primarily as a spacer to position, join, connect, or optimize presentation or position of one functional moiety in relation to one or more other functional moieties of a molecule of the inventive antigen binding protein. The presence of a linker moiety can be useful in optimizing pharamcologial activity of some embodiments of the inventive antigen binding protein (including antibodies and antibody fragments). The linker is preferably made up of amino acids linked together by peptide bonds. The linker moiety, if present, can be independently the same or different from any other linker, or linkers, that may be present in the inventive antigen binding protein. [00325] As stated above, the linker moiety, if present (whether within the primary amino acid sequence of the antigen binding protein, or as a linker for attaching a therapeutic moiety or half life extending moiety to the inventive antigen binding protein), can be "peptidyl" in nature (i.e., made up of amino acids linked together by peptide bonds) and made up in length, preferably, of from 1 up to about 40 amino acid residues, more preferably, of from 1 up to about 20 amino acid residues, and most preferably of from 1 to about 10 amino acid residues. Preferably, but not accessaris the :enino acid residues in the hinker are from among the twenty cauoluai ammo acids more preterahy ,eysice, ylycine, alu e Proline, asragine, glu n, and for sne Esen more preleruhv, a peptidvx inker is made un ot'a majnrity of amnino acWds that re sterwalv uiuudereJ, suc asz gdycme, senne and alai ni inkd by a pepOde bond. l 4M desirable that, i present a pepidi hn! be selected that aids rapid proteovtci tumover in cireumtion i 1W Scan at these a rniu acids may be wlyecoUMsL, as ts vell understood lw thos in the tai l' Or eumph, a usefulimker sequence constuuting a salylaion site is X~N~\t SEQ 110 Ni0:' ! her"in N\: and N arc each mdependen ny amino acid residue [00326 in other embodiments, tue i to 40 amino acids of the eptdyl linker moiety are selected from gl ycinme, alarnine, prolinme, asparagine, glutamcine, and I ysine, Preferably, a linkcc is made up of a majorny of amino acids that are sceically unhindered, such~ as glyine and alanine, Thus nreferred sinkers include polVAy ines. plysernMes, and polyalanines. or comOinaons of any of tWsc, Some exemApary peptidy! linkers are poly(Gly), parcularly (G , (Gly (SEQ D NO: I 4), (Gly (SEQ 'D NO:150) and (Oly) (SEQ it) NO-15 ) as well as, poly((l cr (SEQ ID) >0:152. ~poly(GV-Aa and poly(Ata) 1 Other specific examples of pcptidy bikers include (ly ILys (SEQ I) NO:154). and (GlyhysArg (SEQ ID NO: 155) Other examples of useful peptidyi linkers are: Other examples A setid peptidy! liners are: [0032 (Ciy99Lvs(ly (SEQ ) NQO 9~ [00328) (Ol y>AsnGiySer(Oly (SEQ ID NOt 1506 [00329) (Oiy>Cys(ly (SEQ Df NO: 15knd [003301 0lyProAsndlyGi y SEQD N>40155 - i N~~ ~ ~~~~ 3 GS 8 1S GSQ N4 n [00331 1I e expin be a e en fo exarn e it i} eas rd la e aduetuL ah Eh03321 fohk e n i e (00 o (SEQ D NO 6 i ID ", E ny ilnkers usedsin th oKing eArte. [00333 n som embodiments of AnC . conmouinn orI' ti S i ntiio ich wrwoa pbd War Tookt ac dersde. h xape Iitraeo asprtte esdue, rc laedI Inc anion said secpmnca of the biker noiety. Examples inude the fWHOwi, tp citrsqecs [003341 00 EGOG.- ( ) NO:0 1 0 [0033] 00EE00 (SE 0I :16 5 [003361 GED (SEQ ID NO:162t [003373 G (GGSE 1GD N G$ E16 [003391 Q A0090 (SEQ 11D NA;1 65 4: [0034] 00100 (SQ DNO: Il 6$.) [00344 S' [00342 00 SDS'DEC iIOOOO0S ((3 QNi) [003433 WEWE (v i sE ID IC1 [003443 EEL ' (SE Q IDNO:1 [003451 E F WWW (SEQ ID) NOU 17 1.

[00346l EEEEF (SEQ I0 NOpI2h [003471 VW E:W (SEQ ID NOI 73);r [003481 FF EN F S EQ 1t)N 0: 17 4 [00349) in other embodiments, the Jinker constitutes a phosphoryhtion site, eg. XOXJXAXS (SEQ ID NO:175), wherein X , X, N and X are each independent any amino} acid res'idUe XLXiSXXG (SEQ E)DI NO176%hri X, XsX and Xj are each independentlyoay amino acid residue: or X7T X4XG (SEQ 1D NO: 77), whereint Xs X X, and X are each independendy any anino acid neide. [003501 The linkers shown here are exemiphiry: pepidyl liners within the scone or His invention may he much longer and may include other residue& A peptidyl linker cani cointan eg a cysteine, another thio. or ncleophile for conjugation with ahalf Pie exending moiety, in another obodiment the linker contains a cysteine or homroevsteine residue, or other 2-amino-ethanthiol or 3amino-propanetbiol moiety for conjugation to maleimide, iodoacetaamde or thmoeste, functionalied halflife extending moIety. [00351) Another useful peptidy linker is a Iarge, feile linker comprising a random Glyv/Ser/Thr sequence, for examnple: OSOSA'TGOSGST ASSG SGSAT H (SEQ Il) NO 7) or HGSGSATGOSCSTASSGSGSAT (SEQ ID NO:1791 thai is estimated to be about te ize of a 1 ka PEG molecule Alternativelya usefu peptidyl linker may be comprised of amino acid sequences known in the art to form rigid helical structures e Riid linker: AE AA AKEAAAKE.AAAKAOG-)(SEQ I) NO: 1 0) Additionalty, a peptidyl linker can also comprise a nonpeptidyi segment such as a 6 carbon aliphatac moiecul~e of the ftormula -CH;rCHrC7HtCbr CA The peptidyl linkers can be aterd to forrn derivatives as descrbed herein. [003521 Optially. non peptidyt linar moiny i alo sef for wonjugning te Ilf i IZb x'ndn oeyt h e tc~fin ttehlF tet~dn oev 118 conjugated toxin peptide analog. For example, alkyl linkers such as -NH-(CH 2 )s-C(O)-, wherein s = 2-20 can be used. These alkyl linkers may further be substituted by any non sterically hindering group such as lower alkyl (e.g., Ci-C 6 ) lower acyl, halogen (e.g., Cl, Br), CN, NH 2 , phenyl, etc. Exemplary non-peptidyl linkers are polyethylene glycol (PEG) linkers (e.g., shown below): [00353] (I) 0 N 0 no H wherein n is such that the linker has a molecular weight of about 100 to about 5000 Daltons (Da), preferably about 100 to about 500 Da. [00354] In one embodiment, the non-peptidyl linker is aryl. The linkers may be altered to fonn derivatives in the same manner as described in the art, e.g., in Sullivan et al., Toxin Peptide Therapeutic Agents, US2007/0071764; Sullivan et al., Toxin Peptide Therapeutic Agents, PCT/US2007/022831, published as WO 2008/088422; and US Provisional Application Serial No. 61/210,594, filed March 20, 2009 (corresponding to WO 2010/108154), which are all incorporated herein by reference in their entireties. [00355] In addition, PEG moieties may be attached to the N-tenninal amine or selected side chain amines by either reductive alkylation using PEG aldehydes or acylation using hydroxysuccinimido or carbonate esters of PEG, or by thiol conjugation. [00356] "Aryl" is phenyl or phenyl vicinally-fused with a saturated, partially-saturated, or unsaturated 3-, 4-, or 5 membered carbon bridge, the phenyl or bridge being substituted by 0, 1, 2 or 3 substituents selected from C -s alkyl, C 1 4 haloalkyl or halo.

[00357) 'Heteroaryl" is an unsaturated 5 6 or 7 membered nonocych or partiay-saturated or unsaturated 6 9, 0 or 1 I membered bicclc ring, wherein at last one ring is unsaturated, the monoeyclic and the biccic rings containing 1, 2,~ 3 or 4 atomh selected from N, 0 and S. wherein the nis subsuttd by 0 1. 2 or 3 substituents selected from C a dky C A haoalkyi and [00354 Nonpeptide prons of the inveniteconposti natter such as ron pgply n r ononp id hS~esetending monciees can be synthesze by petdI lineso nonIpptid half-i ife 1, es I convention organ chemistry reattons. r00390] The abonis m l ustative ndnt an xhaustv treatment of the kinds of lnkers Hut it mWionb be w -rpy d 1 Nn aud n wih ihent inenton. (00360] indg ti4gui ttg Bidijjg Poten datnji: As noted above, recombinant FIN A- and/or RNA-mediated protein expression and p-rote-in engineering ehnqus, or any other methods of' preparing peptidesi are aie to the making of the inventive compositions. For examnpe, polypeptildes can be made in transformed host cs Briefly, a reconant DN A molecule, or construct coding for the peptide is prepared Methods of preparing such DN A nmolecules are wel known in the art. Fot instances sequences encoding th pepttdcs can be excised from DNA using suitable restricton enzymes. Any of a ,ag number of available and well-known host cells may be ued in the practice of this invention. The selection of a particular host iV dependent upon a number of factors recognized by the art These include, for example, eom patib iity wvith the chosen expression vector, toxiity f tepptdes encoded by the DNA molecule rate of transfotimation, ease of recovery of the peptides, expression characteristics bio-sa fety and costs. A balance of these factors must be struck with the understanding that not all hosts mayv be equally effective or the expression ofa particular DNA sequence, Within these general guidelines, useul microbial host cCls in culture include bacteria (such as Escherichia coi sp> yeast (such as SaccharonWces sp,) and other fimgal cells, insect 120 cells, plant cells, mammalian (including human) cells, e.g., CHO cells and HEK-293 cells, and others noted herein or otherwise known in the art. Modifications can be made at the DNA level, as well. The peptide-encoding DNA sequence may be changed to codons more compatible with the chosen host cell. For E. coli, optimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. In addition, the DNA optionally further encodes, 5' to the coding region of a fusion protein, a signal peptide sequence (e.g., a secretory signal peptide) operably linked to the expressed specific binding agent or antigen binding protein, e.g., an immunoglobulin protein. For further examples of appropriate recombinant methods and exemplary DNA constructs useful for recombinant expression of the inventive compositions by mammalian cells, including dimeric Fe fusion proteins ("peptibodies") or chimeric immunoglobulin (light chain + heavy chain)-Fc heterotrimers ("hemibodies"), conjugated to specific binding agents of the invention, see, e.g., Sullivan et al., Toxin Peptide Therapeutic Agents, US2007/0071764; Sullivan et al., Toxin Peptide Therapeutic Agents, PCT/US2007/02283 1, published as WO 2008/088422; and US Provisional Application Serial No. 61/210,594, filed March 20, 2009 (corresponding to WO 2010/108154), which are all incorporated herein by reference in their entireties. [00361] Amino acid sequence variants of the desired antigen binding protein may be prepared by introducing appropriate nucleotide changes into the encoding DNA, or by peptide synthesis. Such variants include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequences of the antigen binding proteins or antibodies. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the antigen binding protein, such as changing the number or position of glycosylation sites. In certain instances, antigen binding protein variants are prepared with the intent to modify those amino acid residues which arc directly involved mn epitope binding, in other emboodiments, rnodi fication of residues which are not directly involved in epitope binding or residues not involved in epitope binding in any way, is desirable, for purposes discussed herein. Mutagenesis within any of the CDR regions Mdor framework regions is contenrlated, Covariance analysis techniques can n eCmloyd by the sille aisan t design usefu modifications in the ammno acid sequence o the atgaen binding protein including an antibody or antibody fragment. (Eg. Choulier e ( ovariance Analysis of Pmixn Families: The Case of the Variable Domain's of Antibodies ProteiNs: Structure, Function, and Genetics 41:475-484 (2000); Demnarest et ad LfOtmization of the Antibody C13 Doman by Residue ruency Analysis of iG Sequenees. kvot Kit 33541-48 (2004) Hugo et aL, VL position 34 is a key deterimnmt tr the engineering of stable antibodies with fast dissociation rams. Protein Engmering 1451:38 V~6 (2003); Auron ct at. Sequence covariance network, methods and uses thereof us 2001318207 A l; Glaser et a!, Stabilized polypeptidc compositions, US 2009/0048122 A 0; Ureheta, Sequence based engineng and optimization of single chain antibodis WO0 2008 1 10348 Al; Borras et aL MeVthods of modifying antibodies, and modified antibodies wth improved functional properties, WO 2009/000099 A21 Such modifications determined by covariance analysis can improve potency, pharmiacokinetic, pharmaceodynamic,. anid/or manufacturability characterstics of an antigen bi nd ng protein. [001 2 2 NuAei acid molecules enodig amino acid sequence varimts of the antgn inigprotem or anybody are prepared by a vaiety of methods known m the art Such methods incude ob couci eotidc-mediatedt (or site-directed) mnutagenesis P mageness and cassette mutgenesiS of an carler prepared variant or a nonKarimt person ofthe antigen bncmg protein. [00363) Substitutional untagenesis within any of the hypervariable or CDR regions or framework regions is contemplated Auseful method for identification of certain residues or regions of the antigen biding protein that are preferred locations f-or muwtagenesis is called "alanine scanning mutagenesis " as described by Cunninghamad \\ d S&ience, 2.44 106 05 (1M#), Here. a rene or group ot target resdues arc ideunied (eg, euharged resdues such as arg asp, us, lys, and and replaced by a neutral or negatmve charged ami acid (most preferably alaorue or poly larine) to afket the ineracruon of the ammo acids n uh antigen 'hose amin acid locations demontning funconal sensitiit to the substitutions then are rained by irodurms f'urher or other variantst, or for the stes ot sutulen, 'tlt Thius nlhle the site for ontroduungr an amino acid setpuence varution Predeterim ned, the nature of the mutation per se need riot he predetermined. For eumple, to aimlye the p t ot a mnutatmo at a. ynen sne, ala sand or random muttgencss is conducted :at the target codon or region and the expressed v ariants are eened ror the desired acuvmtv [003641 Some eunbodimnents of the antigen bnd ing pro ei n of the present invention can also be made by synthetic methods. Solid phase. synthesis is the preferred technique of making individual peptides since it is the most eost-ofeetive mthiod of making small peptides. For example, wel know solid phase snthesis techniues include the use ofmroecin groups, lnkers, ad soiid phase supports, as wcill as specific protect ion and deprotection reaction conditions, linker cleavage conditions. use of scavengers, and other aspects of solid phase peptide synthesis. Suitable techmiques are well known in the art. (E~g , Merrifield ( 9~3), Chemn. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis edict Merrifield (i13)1 Am. te. Soc. 85: 2149; [avIs t at (1985) Biochem. In. 10 394-.414: Stewart and Young (1969), Solid Pha Pepde Synthesis; U.S. PatNo. 3,941;763: Finn at at (1976, The Proteins (3rM ed, 2 1 md Erickson et at (1976) The Proteins (3rd ed,} 2: 257527 "Pmoteeting Groups in Organic Synthesis," 3rd Edition, T. W. Greene and P, G. M Wuts Eds, John Wiley Sons, lne- 1999 Novaliochem antaog 200; "Synthetic Peptides, A User' Ga ide G-. Ai Grant, Ed. WAi Frcemn & Conmy, Ncew York, N.Y. 192 "Advanced Chemotech H-landbook ofCombiinari & Solid Phase Orgnuic Cheistr* W, h ennet. A Christensen, L K. Hanmker. , L Peterson. M. R. Rhodes and H. H. SanI, Eds. Advanced Chemtech, 1998: "Principles of Peptide Synthesis, 2nd ed" j B3odanszky, Ed Sprnger-Vorlag, 993; "Th Practice of Pcptide Synthesis, 2nd ed." v1. Bodanisky and A. Bodanszky,. sxrigr-Lag, 994; " Groups) P. 3. Kocienski, Ed., Gceg TPhieme Verlag, Stuttgart, Germany, 1 994; mc Soid Phase Peptide Synthesis, A Pact ical Approach." W. C, Chan and P, D. White N, ds. Oxford Pess 200, G . MCids' ynfhetic Peptides: A Ur' Guide, 1990, 77- 183) For t examples ofsynthetic and purification methods known in the art, which are applicable to making the invenmve compositions of matter, see, e.g, Sidbivan c &a Toxin Peptide Therapeutic Agents, US2007/007V764 and Sullivan et a. Tiw PpId 1herapeutic Agns, PCT/US2007/022831 published as WO 2008/088422 AT. which are both incorporated herein by reference in their entiretics, [00365] 1in rather describing any of the antigen bmding protins herein, as well as vOaants~ a once-ectr abbreviation sys temn isfeunlpplied to designate the identities o the twnty 'canonica" amino acid residues generally incorporated into natu ally occurring peptides and proteins (Table 44 Such one-letter abbreviations are entirely intecrchangaeable in mueaningr with three-letter abbsreviationa, or non abbreviated amino acid names. Within the one- etter abbreviation system used herein, an upper case letter indicates a L-anino acid, and a lower case letter indicates a D-amino acid. For example, the abbreviation "R"' desigunates L-arginine and the atbbrev it rt designmrs uarginin Tbe4One-leternbbheviatins thy the eanotical amnina o a. Iht etter Lbb Mv arem ndarent Ahatine (Aka A (dutamineG n I ~~1, et';'4cmI. Agtnine A uha tamic Amid E Shrine (1110 Asparaic (A Mvthinine Mi\et) NI All stpidind () U [003661 An amino acid substitution in an amno acid sequence IS typicaly designated herein with a one-etter abbreviation fr the amino acid residue in a particular position, followed by the numericL amino acid position relative to a original sequence of interest which is then 0l lowed by the oneetter symbol for de amino acid residue submitted in, For example I30D" symbolizes a substitution of a threonine residue by an aspartate residue at amino acid position 30. relative to the orignal sequence of ineet A.noher eamleh W 10IF' symbolizes a substitution of a trypophan residue by a phenylalanine residue a a mino acid position 10 I. relative to the original sequce of interest. [0036~ Non-canonical amino acid residues can be incorporated into a polypeptide within the scope of the invention by employing known techniques of protein en ginering that use recombinantiy expressig ells. (See, eg, Link et al, Non canonical amino acids in protein enineerng, Current Oninion in iotechnoiogy, 14(6):603-609 (2003)). T he term "non-eanonical amino acid residue" refers to amino acid residues in D~ or L-form that are not among he 20 canonical amino acids general incorporated into occurring proteins, for example. P-amino acids hoimoamino acids ecdic amino acids and amio acids with denvaized side chains. Examples include (in the L-frm or D-fhm'nh-!lanine, aninopropionic acid, piperidinic acid, aminocaproic cid, aminohcptanoic aci inopimetc acid, desnosine, dianinopinelic acid, N:ethylglycine. N"-etlaspauine, hydroxy ysirae allo-hydalroxylysine, isodesmnosine, allo-isoleucine, as-mithylrin ine, NK. nmethyiglycine, Ntrnetylisolucine, N"-methyiva Iine, , ycarboxyglutamate. ii NNN-trimnethyl lysinegcN -aec-tyl lysine, (Vphosphoisacrie N"-acetylserioe.

Nk- fnrrmmethionine, rnethvlhistidine. 5h dro lysine and other sAnae anra acids, and Thoe hted in isbe S hckw :md derivaized tems of any of these as described herein. ale contains some eemplary non-eanoical ammo acid rWnd th are aseti in accordance wo; the present vwntor and as:ated :thresistas s epiet ued ere athougch the skdied praetncnei d i understand that di fe rent Thbrcvtaiofns and flomienh13res may lbe app heatble to th sIae substance and appear intercant tbW herem, Tpabe . Useful non-canonical amino acids for araino aid addition, insertion, or substituton into peptide sequences in accordane with te present invention. n the e:venft an abbrevitation bste fri Table 5 diffetrs from ant~uher abbevitio f- or the same substance disclosed elsewhere herein, both abbreviations are understood to be appleable. Thbe amino acidsisted in Table 5 can be in the L form or D-form kmno AMid Abbrevadonis) IAcetmnrdomethy Awm Acetylargininc acetlar a amwit10dipic acidAd amiiohutyec acid Abui 6 &nubehxanio e ac d Ahx ~h 1 Ktnie- ydro\-n pnuneh And-a).oinyanc- .. bv.a A...... -- - -- - - -- - -- - - -- - -- - --- -- q a 4 anzniantaie ran nox acidh A . .... b.......d.y..p.p.. h nidn m hagmnelaite.4x~ h~rg - -Ni -- si -I N --educed -------------- -o- ----- -- filo~~~no1 vsn deAMpk -------------- --- r: ----- ------------- the__ _ __ __ __ _ __ __ _ h~~~ -np ~f ------ ( . l l --- --- --- -- ----- ---- ---- -- C , 'l -tU O -------------------- ------------------- --4 ----------------U--- ---------------- .. . ......... .. ........... dia p ent du 4 r diuo prulin Ot aP ...... IQj j~ }1 j l ..................... n oarin hA > ;hbR~tov I m5Vphe s aflehh: hK1O }1h SIdrt~ylglybci (csrhldriyylglyinc5) dutyrinct Tyr~c ci Ly s{CHtNH reduced amxide bond d ys ---------- _ ___ ----- - - - - - - - - - - - - - - - - - - - - \ lthylvr n ne N'vkR N { CH NRI C1LN HN't )subst tue N A~'~ meht utane N> clQ - ~------- - - - .. ............. ~jj1jl{ N N~ti~ 1T - -s N net veuc n N' .MeL;NMeL NMe eu; upropy-4 mIn \ ejJ7>t - I \ ruthyinovucia:NM"'Ch~ -~ IZ - n---inuh N -- N- n ap~vjhthtige N 00 rwty-vha Ny aiNvSa Ornith() Ornnoch ) lpao8 r1naCH2NH teuced amdI n ~ Nd rip' f' a4PipA N ci 4 pyn nylna amne d meihlymie Qv~ 2a pydmolahoe-caol &4O - - - - - - - - - - - - - - - - - - - -[par amino hnyt ard____e__ 4AmP ____4__A__n____he --- -- --- Ipara -- odopheny-a- ani- c (-r 4- p-Phe ---- ------------------ - ' Ts 4-phenttpbenwrx?~ c 4k ' bp ivalanine ________________ ___________________ * ppcci'i acid Pf 2 4m by- quit a drxp - ---- -af h vdrot-------e --- - T- UPAJUB'aJ~oitommsio on'C. P Bchmalromevl re(CB-Thv be 56'9364 &Aminob Acids and eptde_198, 1, 38410 Bicheic 1Nome*ncareI an Relate Documents, 2n dto orln rs, 92 ae angl anthn4 cpa0bxvii i0e0 Inld mn cdh dilnau neiasu iou dewhet prtietcasae aId saituadoreeia der9\'365miont ncatureo dad SvToest for dem cids n n by nuneem' ethme Uioc\mm. i9. 10 thu3l; mod8fi 152O 1;e19d eleg 'mducr ttt I edrt o .Res.0 19cid re4h. meorted nr 84; ted (therm, r9am 0 to the ammo Api tenenme 1984 5a6. 5954.'Qule OA 1 Acrid and P etiel 1985f.ete Or. 4U h0;~ ilchtnoide d [06'he oned a comrea uelctodhi fie o~ ectophib tid ie ainfte ina greoupv Fo eanih the tpmdsi odiaied tmio acidnkeune inaldes eteaot oet in aci rc -1n.u -n-ed -------- t-------------re--ive-to the -am no-- ci- seque ce--- -a~ se-ec ot ineet -) -hc th -isre or su siue a-n aci residue------ ------ has~~~~ ~~~ a- sid chain ---- co -rin ---- a -- ce-----c-re-c-o-i-c-eat-efuc-onl--r by~~~~~~~ ~~~~ ---- ich-- t-' -etd i - -oiwae to a -ike an/o -at f e-t -o accordance w ith dhe inve mion, useful ean'pe of such a nuecophic or electrophile reaci re timetonal group inide but are not Aouid to, a Biod a primry amine, a 'cleno a hydrazide an ddehyde, a eatovv'i 'cid, a keone, an amInooV. muzlSked (fprotwd0 wdehAyda or a maskd (proeeted) ke tuiuonal gnmp. Wxmples of amino acid residues hasing, a side ehuin compremy a nueicophiie reactive fimetional group itNclde, but are not limited to, a lysine residue, a homnolysine, an K~diaminoprooneacd re sdue, an inydsuaminobutydre acid residue, aunomithme residue eteme, a homnocysterne, a glutame acid residue, an aspartie acid residue, or a seienocmseie residue. [00370) Amino aid~ residues are comnmonly eatego ized according to different cemriceal and/or phy'siceal characteristecs. The term "aciic amnino acid residue"C refers to am ino acid residues in D - or Lform ha ni si ec an o"rsi g ai i r u s Exemplary acidic residues include aspartatic acid and gutamnatic acid residues 'The term "alkyJ amino acid residue" ref&s to amino acid residues in D or -frm having C 6 alkyJ side chains which may be incar branched, or cyclzed, including to the amino acid amine asin proline, wherein the (Cauy iM s substAted by 0. . 2 or 3 substituents seiec te frm( Chaoalkyi, halo, eyano, nitro, -C(=0)Rb -C(=)R t C(=0::.::O)NR"RlJ ACNR")INRK .NPU R*C(;= RIN RYR -O -OC( )R OC(=OJNRR X alky NR"RK -O(,akylORh -Si -S(=0)R S ()R SONRR , -NRi -N(R{)C(=O)R N(4)C()OR -N(R) (=) (ARR" ~NiR)C=Nf)NlaRC -N(R4)S(=OhRP NC(R")S(=ONR R' -NR"CuaikylN"R and ~NRCal tkyaOR w erein R is iunedent'y, at each instance, I or it and P" is independentlyiat each instance Casakyl substiuted by 0,1, 2 or 3 substituents selected orom halo C Oak. C 1 'baloalk, -O alk ~NH AHCWaik, and -N(C malk)Cda'lk; or any protonated fom thereof including alanine, valne, leucine isoieuc~ine, prolieserine, threonine, lysine. arginine, histidine, as partane, guntanma te, asparagi ne, gtamne, cysteine, methionine, hydroxyproline, but which residues do not contai an arv or aromatic group, The trm "aromatic amino acid residue" refers to amino acid residues in D)- or L~brmi having. side chuins comprising aromatic groups Exemplas rraa reddnues nrelude trptophan, trosine,3 nhthylbaianinc, or phenyladanine residues, The term "basic amino acid residue" refers to amino acid residues in D or Lfom having side chains comprising basic groups. Exemplary basic amino acid residues include histidine. lysine. homolysine. orni thine, arginin, N-mcthyl-arginme, m-aminoarginine, m-methyt-arginine, ~ methyl -histidinc, 3~methyl-histidin, and homorgininc (hR) reidues. The terrm "hydrophiie amino acid residue" refers to amino aciesidaes inI D, r Lform having side chains comprising polar grus. Exemplary hydrophiic residues include cysteine, shrine, threonine, hitidin.e lysine aspargi apl, gnfla lutamfalte, gutamine, and citNHne (Cit)rsidues The terms "iophihi amino acid residue" refers to amino acid residues in D or L-fon having sidechains comprising uncharged, aliphatic or amticit groupsI Exemplary lipopnhli sidechains include pheny ala~n in i.soleuci ne, leucine, methionine, valiine, trypopban, and iyrosinc. Alanine (A) imphiphiic------it is capable of acting as a hydrophilic or lipophilic residue. Alanine. therefore iN included within thc definition of both "iltihi li residue" and "hvdrophilic resiu." The term "nonfuanctional amino acid residue" refers to amino acid residues in D- or L-orn having side chains that lack acidic., basic, or arornatic groups. I emplary nouitraol amio acid residuesinclude tmethionine, gycinem alanine, valine, isolene m, kucine, and norleucine (Ne) 100il I Additional useful enthodiaents of canh ren from rouservaine modification of the amino acid sequiences of the >olnetles disclosed herein. 'onsen vJUN modtifitaui w dil pretice ha-be extending mnoiet -corgugated peptides hi og t'mntional phyialt and ceical chuacternstics simiur to those of the comuataied te g, PEGconegnedi) peptide from whih such modiicabins are made, sch consenvavively mnodiled fA ms of the coanugated pobreptides disclosed herein are also contempated as bein cmbodment of the present vention. t00372 )n comrast, substantial modi-ianons in the functions and/or chemical cbarttristics of peptides may be aconphshed by seletaug ribstiiuurs in Kh: amino acid sequence that ditfr signitiantv in their Cfct on maitaning (4 the struerr ofl he noleeulat bacllme im the reion of the substam, 5 or example. as an rnhelial conformsion Q! teb rhh or ydrophemv of the molecule a tnget siA.t o ) the sie of th nolecu e [00373) For example, a "onservaive amino acid substtution" may involve a saubstiuti of a native ammo acid residue with a nonnlativet residue such that there i ile or no effect on the polarity or chargof th a acid rsiduc at that position. I'4rthermore. any native residue in the polypepntide may also be substituted with alanie, a~s has been previously described for "alanine scanning mutagenesis" (see, iOr example. Macennan et aL, Acta Physiol Scand. Supp.. 643:5547 (i998); Sasalk ct aL 1998, Adv. Biophys 35 .2 (99l which discuss adanine scatming oiuagenesisi 003741 Desired amino acid substitutions (whether conservative or non conservaive) can be determined by Those skilled in the art at the tuie such su6stitutions are desired, For example amino acid substitutions can be used to idenitify important residues of the peptide sequence, or to incre'aseo eresh i~~ ~ ~ ~ i ileeroel o'-k asec affinity of the peptide or vehiclrcon jugated peptide molecules described herein. [003751 Naturaly occurrig resdues maybe di ed nto sse asked on omn sidechan propeortmes' ii hydrophobic: norleucine (Nor ore, Met AaVt Leu Hie 2) neutral hydrophili C S 11 Asn Ghn 3) acidc:Asp GI; 4) basic: FhIs Lys. Args 5i residue that influence chain orienation: Gly. Pland t4xarmatie 'Trr, Tyr, Phe. 003761 ch'osern ae amino acid suhstmSutons ) tin ohe exha.e ot a member of one o thye classes with another member o' the. sam' acid subtituuns may encompass non-natmuraill) occurring amino acid residues which are typically inorporated by chemical peptide synthesis other than by sy nthesi ino hiologial stems. These include peptdonin'etics and other re\ersed or inerted forms of amino acid moieties [00 N7 Non'consentative substions amy inoil the exchange of a member of be induced into regions of thelx in peptide anaIog [00378] In making such changes, according to certain embodiments, the hydropatic index of amino acids may he oInsidered. Each amino acid has been assigned a hydropatic index on the basis of its hydrophobicty and charge characteerstis They are:'slecn (+4.5); vahine (+4 2); leucine (+3.8): phenylaanine 1 M Ystene/cysine (+2.5); mehionac (+ L9; alanine (41 8 glycine (-0A): theone (-0): scrno &S0X tryptophan (19); tyrosino -li3 prohi ( b h istidAn (~ 31 ghaniate (5); glutamine (V 5) aspartate ( 3,5) asparagine (3.5): tysine (-3.9): and argine (-4.5). [00379] The importance of the hydropatic anino acid index in conferring interactive biological function on a protein is understood in the art (see.fohr exam ple. Kyteratl 19822/. B13j/, 15:105~ 31 ). It is known that certain anino acids may be substituted far other amino acids having a similar hdropathic mdex or score and stil retain a similar biological activity, In making changes based upon the hdropatic index, in certain embodiments, the substitution of amino acids whose hydropathic indices arc withni2 is included, in certain emnbodimenats those that arc within il are included, and in certain embodimens, those within *0.5 are included. [00380] It is also understood in the art that the substitution of like amino acis can be made effectively on the basis of hydrophiliity, particularly where the biologa nctional protein or peptide thereby created is intended for use in irnunological embodiments, as disclosed herein, in certain emhodiwmts, the greatest local average hydropilicit oa protein, as goveed b th ydropiicity of its acen amino acids, correlates with its immunogenicity and antigenici ty, Ai., with a bio logical property of the protein. [00 e Ths c allna g ydPhiii al ave been asinedto h s amr aid rsI dues: a Cainno 03 Am0tV &sno (4 3.) 1 sait A ±3,0± 1; glutamateI(BA) 1) scminea (403) aspmraua (02.A gluts-ninc (±P0.21; lyie(0) ihrconie (-04) police (1.5 - 1 K Raine & 5K histidine )e & Ot. methioninc 1 , vahne (- i) tleuine (-I olemucie (~14 i rsme (-2.A) phenylmaan u (2.5) and tr\ ptophrin (3.4). lI nmgkt enanges based upon simiwidrophi eirv Alues in certum e.mboduines, the srnti ot'amino aculs whose hvydrophdi.eny v abies are wi n2 im meludd i certain embodnents those that arc n mi Iy ate includdcd, and mn certam embodiments those w thin ±65 arc meluded~ One may also idepit opes t'roma prioar, amino mid sequences on It basis ot'hydropihety These regions are also referred to as "epitopic core reions " [0038 2 ) i.xampies of conservative substtutons inclue the substitution ot on non-polar (hydrophobic) anmno acid residue such as isoleuck ine, valine, krancine noreucin' aanine. or methionine for another. the substitution of one polar (hydrophilic) amino acid residue for another such as between rginiine and lysine, between glutamine and asparagine, between glycine a series, the sbttution of one basic ammo acid residue such as lysine, arginine or histidie for another, or the substitution of one acidic residue, such as aspartic acid or glaimc acid for another The phrase "conservative amino acd substitution" aiso includes the use of a chemicaly derivatized residue in place of a non-derivatized residue. provided that such polypoie displays the requisite bioactvt, Other exemphry amino acid substitutionthat can be useful in accordance with the prese nnvention are set forth in Tale 6 below. Tble 6. Some Useful. Amino Acid Subtitutions. - - -T----- ri (~jg Exemn'plary ResiuesSubstitutions Ila Vat Leu,le Arg [ys, Gin, Asn G v Po, A

--

A---G---Ly--Arg - --- ------- I (ito ValMetA Wa Pbc-, Lys Argo, W,4-Damino buyrc A idsGn Pro A A . Ser T hr.-t Ala. -------------- -- - - - APia Norleucine wthth egial naenbiL hs prt nt Inbd umitn no ai euncso ne the eag r th ligt chm riabe reIonut~, ralat (% ora las 0 ora ' ml3 kU , 86e ~i0 Phe. n [yr ^IJ 8c V' \a y CM We, 44's 9acid"sq4c. %wigs and the% adnty or h omy it T ., ps r tN ahi Cet' 0 dnie h r a the pec ng ol'ammd c id i gwal es the~~~~y Nav totr zChwt s, 65% Su tMs 70 n prihthe. oriat AM a0% why ncmom prraoodvbl ams Wat &4 NCiiflC oA either MYt examp*. NON, 81%A 824, 83%A 44A SA. 96%A X74, SSA 10% 904, TV% 92%- 92"n 94% 95%V %%", 97%, on". 9M% and 10ow". MAWnt or homology with the cadidate sequence Mt are idn With te og u the sequen es ad introducing gaps if necessary, to a ldeve the muumm percent seuienec: dentary, and not Considering any conservati\e aubstiloias part ofthe steiee identity. None ot NAcrinimat '-ternumLI or mntesul~ exienSnmrs, delethoUn or mnserttungs into the antigen bhimdia protein or antiody seuneshall he construed a affeeing sequence identity or homioiogy [OO4J Armno acid sequence mnartions melude am no- an or carboxy-termial luMions raitUg in l>noth II'O due 0 peptides eontaion a hundred or mlore rewdes a a xil da intrcqseuene isertions of smqe Or multiple amino cid resiues Exan ples 0t tetmraal inlsertionS inchude an ant;gwn bn'hng poaemr vith an Ntemniin methionvl e>'due or the artigen binci gproten' (hehidnma annoodv or antibody fragment } f'used to am epitope zag or a salvage receptor b'.dmhg epitope. Other nscrtonal arnants of the antiyen binding promi or mtibody molecule mclude the fom to a polyptide which icaw's the serunthalf-litbofthe antigen hiodm protein e g at the Ntermmuz or ( term nut [00315 j Fxaples of epitope tag, mlude the fl t A tag olypeptide and its antibody WCA5| f'ied at a, Mol Cell BI 8: 215-2165 ( O) the c-ty tag end the WFA RC, 6F 10 (14, It' and GF 9 0 amibodies thereto [fvan et al , Mol Cell Biol 5(A 361(-30 16 0945,1 and the Herpes inmplex Virus glycoprotem D fD tag and its antibody IPalte t A Protem Fnecrmy M6 W753 (199)1 Other eemplay tans are a po"uhsAdin equence, general around m !ustidine residues that permit l.5la&tion e1 ii cotmpoilnd so labeled usimg nickel chelation. Other labels and tags such as the F At tao iEastman Kodak, Rochester, N are wel known and routnely used n the art. [0O061 Some pacu ar- nonl ng entodnents of no acid substin variants 1 othe anvdrn ant bdng proein. icluditg ide antibody trIniRt -ire exmbfedels* [0387 An y eenc resdue n ~nyoled in maintaning the proper conformatio of the antigninna preien ao may begsbstitued, eneraliy wit s-i1 rb to improve theovidatd ie stability offh'e nm"Akeu and pretaberan iedng CnuvrseIy cysteinebndik) may 1e added to thea en b ng prti to re is stability partiularly wherehe antigW indig peini an antibody gnent such as an Av furagmnt [0038K h. certain instance, antigen binding protem vnniams are prepa red w ith the mtem to modu n those amnmo acd residues which are direly ins o cd m pitope bmdogw In other embodimvents modification of'residues vwTh are not dbreetiy mnvolved in epitope hInor residues nlot tiosKed mi eoro wmdm; in :mny waty s dtesirabl, for purposes discussed hrem, Mutageness'shtin an\ f t. DR regon,' ad/or frmework r egions is contemphied. [00389] in Order to determine which atnten binldingQprotei~n ami;no ac4id residues are important for epiTope recognition and binAing, alanine scanning mutagenesis can be performed to produce substitution variants See, for example Cringham ct at, Science, 244:105 1085 (1989), the disclosure of which is incorporated herein by reference in its entirety, in this method, individual amino acid residues are replaced one-at-a-time with an an nin residue e and the resulting anti-DNP or anPiKLH antigen binding protein is scroned for its ability to bind its snecific epitope relative to the unmodifoid polypcptice. Modihed antigen binding protein with reduced bindingcapacity arc sequenced to determine which residue was changed, indicating its signii chance in bining~i or biologi Cal properties, [00390] Substitution variants of antigen binding proteins can be prepared by affinity maturation wherein dndom amino acid changes are introduced nto the parent polypeptide sequence, See, ior example, OuwebandetaL Vox Sang 74 (Suppl2t223-232, 199$> Rader et a Proc. NatI. Acad, Sci, USA 95291-8911 1998; DallPAequa et at., Curr. Opin. Struct. Biol. 1998, the disclosures of which are incorporated herein by reference in their entrees, Af Y mn involves preparing and screening the anti-DNP or anti-KLH antigen binding proteins, or variants thereof and selecting from the resuhing variants those that have moditied biological properties, such as increased binding affinity relative to the prent anti-DNP or amiKLH antigen binding ptovein. A convenient w :y for enerating substitutional vagrants is affint masuration using phage dislay. Bordy. several hypervariable region sies arc mutated to generate al possible amino suibstdiutis at each site, The varianti thus.geneA4ted a~re expressed ini a moniovalent fashion on the surface of ilamentous phage particles as fusions to the gene 111 product of M! 13 packaged within each particle. The phagedisplayed variants are then screened for their biologic activity (eg. bmding atffnity) See eg. WO 92/01047, WO 03/,123,66 WO 95/1388 and W) 93/19172. [00391) Current antibody affinit maturation methods belong to two mutaenesis categornes: stochastic aid Constochastic. Error prone PCR, mutato bactil rn (ow at AL. I Mi. B. 359-, 1996 an iaturtion mtagenesis (Nishimiya et at, NU Bo/, Chem, 275:12813~20, 2000; Chowdhur, P. S, thmde o iol 178 269-85, 2002) ar typical examples of stobuhstic mutagenesis method (RRypal et at Pro NAM? A ScW U S.4 102 :846A 2005). Nonstochastic techniques often use aiani.ne-scanning~ or sit-directed mutagenesis to generate limited collections of specifi mteins. Some methods are described in further detai below. [00392] Afinity mwati viapnigmtoeAfnt auaino recomabi nant antibodies is comm on ly performed through several rounds of panning of canlatc antibodies in the presence of dcreasing amount of nantge, Decreasing tQ Smunt of antgen per mund selects the aiboies with the highest affinity to the antigeni therevby yielding antibodies of 4 hgafiiyrma rge pool of starting mteria Ainity maturaion via panning is wel known in the art and is described, or exampL in iM at A (Anocr huncma Imo ther, 50:16071, 2001). MUethod of affinity maturation using phage display technologies are described elsewhere herein and known in the art (see eg.. Daugherty t al Proc' Ni ad WS S 9710293 200 [00393 1ok through tnutagenes o through utageneis (T ) (Rajpal et Pl., r Nati Ac SiC U S A. 10284661 20 ) ovides a ethod raidly mapping the anibodvyinding sit. For T nine amino aciad repreeniveothe major ade-cham chemistries prove ided by the 20 natural amno acids are selected to dJsecr the tiftenual utdechamn umtritiousj to hiting at evecry posmton mn all sa 1s of an anthodty. LF1 generals a pontonal sees of single mutatoms thin a CDR where each id type" rsidue is systematicalv subsmuted by one oie seleed ammo acids mutated 'DRs are combined to generate contutoral 5Agie cham i arabi fra gment eF ih brn, of measig com px Ity and e utbout becoung probhi ny to the quanttam d' dl a of all ine iun \et posiiM e section, dlons wnith improved bimdmg are sequencedamd beneficial mutations are rnappd [00394] Erorprone PCR -Error-pron PCR evolves the randomiation of nucleic acids between different selection rounds. The randomization occurs at a lot rtne by the intrinsic error rate of the polmerasc used bout can he enhanced by erro prone PCR (Zaccolo et A,. Mol Biol 285:775-3, 1999) using a poymrase having a high intinsic error rate option (Hawkins et A., J Mol Biol 226:889 96. 1992). After th mutation cycles, clones with poed anity for the antigen are selected using routine methods in the art. [003951 Techniques utilizing gene shuffl ing and directed evolution may also be used to prepare and screen anti-DN? or anti-Kf!1 antigen binding rtino d i 4) N PpI t1-AnnI i or variants thereof. for desired activity, For example Jormutus et atL Proc \atl Acad Sci U S A- 9():24 (2001) showed that tailred in virroeleition sPtees based on ribosome display were combined twitri virro diversificat by DNA shuffling to evolve eihr the off-rate or thermodynamic stability of scPvs: Fermer et at lurnour Biol 2004 Jan-Apr;25(l~2):7-13 reported that use of phage display in combination with DNA Shuffling raised affinity by almost three orders of magnitude Dougherty et a, Proc Nat Acad Si U S A. 2000 Feb 29; 97{5):2029-2034 reported that (i) functional clones occur at an unexpectedly high frequency in hypermutated libraries (i) gai)-of-ftumtion nutants ar well represented in such libraries. and (iii) the nmaiority of the sCFv mutations leading to higher affinity correspond to residues distant froms the binding site.

|00 ) Alternativ eyv or in addition, it mnv be benefic d to ana y/e a crystal structue ot'the anugen-annbody complex to iiental' contact points between the ant hody and antiyn, or to use computer softwre to model such contact pois Such contacesi dues and acigthorhtm residues are ednci dutes nir substi t iOn according to the techmanhn elaborated here. Once such viiamts are generated, they are subcted to screenmg as described herein and antibodies th superior Properties one or more relevant assays may be seteced 14l further dev ernIet, [0038) Antigen bndng protein variats can also be produced that have a modified gvv cosyaton pattern relative to the parent polyneptide. for example, addin or donMing one or ure oftthe carbohydrMie moitiee bow to te aier bNndo protem, andor adding or deleting one or more gWcosyhition siea in the antigen bmndmge protein, [00399) Glycosylation of polypeptides, in cluding antibodries is typically either N linked or O-linked, N-linked refers to the attachment of the carbohydrate coety to the side chain of an asparagine residue. The toieptide sequences asparagine-X scrine and asparagirne-X-threonine, where X is any armino acid except probine, are the recogniton sequences for azymatic attachment of the carbohydrate moiety to the asparagine side chain, The presence of either of these tripeptide scquences in a polypeptide creates a potential glycosylaion site, Thus, N-binkd gvcosylation stes may be added to a antigen binding protein by' altering the aino acid sequence such that it contains one or more of these t rippt ide sequences O-bnkeNd glycosylation refers to the attachment of one of the sugars N-aceylgaiactosamine, galactose. or xyoua to a hydroxyamino acid, most commonly serine or threonine although 5 hydroxyproline or 5-hydroxylysine may alo be used. 0-linked glycosyla tion sites may be added to a antigen binding protein by inserting or substituting one or more serine or threonine residues to the sequence of the original antigen binding protein or antbod [00400)unto (0040O4 Cysteine residue(s) nmay be removed or introduced in the Fe region of an amib ody or Fc-c~ontairnng poiypeptide, thereby eliminaiting or icresn inechi disuide bond formation this regon. A homodimeri antigen binding protein thu geneanted may have improved itrnalization capability and/or increased compoen't-meditcd cell kidlng and antibody-dependent ce dar cytotoxiety (ADCC). See Caron et a. J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. 1. Imnmunol. 48: 29'8-2922 (1992 Homodimeic antigen binding proteins or antibodies may also be prepared using heterobifunctional cross-inikers as described in Wolff t al( Can Research 53: 2560-2565 (199)A Aiternatively. a antigen biding ptowin can be engineered which has du Fc regions and may thereby have enhaned complemeni lsis and ADCC capabiliie.Se Stevenson et at, Anti (Canerikug' Desi gt >19t230 (1989 1004021 it has been \hown that S enceS n a th (DR can cause an antibody to bimd to NHC Class i armd trigger an URant hlper cell response A conservatie substulnc can allow the a.nti ndig protem to ietam Nbi u actovity vet reduce it, ability to mer an unwanted -ecl raqptmsc, A is alo comemplated that one or more of thee heavy or light chan are removed [004031 Modifleations to increase serum half 1e also mra desirable, for example, - meorporat of or addoe of a sal vae receptotimm epoby mutatIn of the appropriae region or by oiporatong the epiope io a neptde that i then tised to the antigen binding p rotein at either end or i the mddle, eg., y DNA or peptide synthesis e.g. W)7 or adding molecules such as Pol or other water soluble po mers melud ma relysaccharide polymers [00404 The salvage receptor binding epitope preferably constitutes a region wherein any one or more arnino acid residues &rm one or two loops of a Fe domainu are transferred to an analoaous position of the antigen binding protein or fragment. Even more preferably, three or more residues frm one or two hoops of the Fe domain are transferred, Still more preferred, the epitope is taken from the CH2 domam of the Fe reWon tag., of an 1i) and trunsfbrred to the CH 3Il or MR 1ion(iOt nuiP hmn one Much regom, of the amten buxdmg protem or antibody Aheaeiviy, the epitop is taken fron the C'H domain of th Fe regon and transferred to the C tegion or V iilor bi, of the Lmoeno m prtein fragmem. Se ako Iateruntional applications W{I 9746 aund \\) 6224 '' w hich describe Fe variants and their interaction with the salvage receptor. [004051 Other site and amino acid residues) of the constmt region have been identified that are reponsbte (or complemnent dependent t totOxilty (CI)C} sitcb as the (l q bindig site, andor the antibody-dependent eellular cytotoxvcy ( ADCC [see, e g., \folee, hmunol 24 d) $391 09 Shiel et al J .H& Chm, 2T(9)t59a1604 2008; ,a-a er al , Proc. Nati Acud. Set 10 1 W:4005 (2006, which describe the ef:et of mutations at sreci he rositons each ofwhneh is mcorporated by refenene herin m its entiretyl J \utation of tesidus y tin 1 c recpt-or binding sien can resul in aOred (i C.e, eased or deetased) efcto function, such as altered atfnty tr I' receptorNs altered ADCC or ( D C acutv or ahered hal E e descrbd above po t 'ntial mtaions include incruon, dejection or subsiution ofone or more residues unehding substutna with a. conservati e sustitunon, a. non-conservative substitutir or r replacement with a coresponding amio acid residue at the same rusmon iom a it uhelass (e replacing an lIG residue with a corresponding 9 ' residue at that postiont. [004061 The ivention also encompasses prodmction of antien binding protem molecules imeludc g anbodie <md anibody fragment, wi t al ered carbohydrate structure resulting in altered eletor aetiy umeluding anybody moleculea with absent o reduced tucosylaton that exhibit impi rcJ Al MW ivity. A variey of ay aie known in the art to acomphsh this. for 'xampie. ADIXC efector activitQ is mediated by nding of the anybody molecule to th evc R receptor, w hich ha been shown to be depended on the carbohydrate msust of th NO inked glvcosvlation at the AWNa-297 of the CII donai, \on-fucosyhited antibodies hind this receptor wth greased affinty od inggar Fe vRi1-medated effector fimetons more efieientlv than native, fuosykNved antibodies. For examp, recombinatt production ofnon ucosylted mtibody in CHO cells in which the apha-1,6-fuosy transerase enzymae has been knocked out results in antibody with 100~Old increased ADCC activity (Yarnan-Ohnuki et al. Bioteebn B-ioeng, 2004 Sep 5;87(5):61 22) Similar efteets can be accomplished through decreasminthe. activity of this or other enzyms in the .fucosylation path way, e. g, through siRlxNA~ or antisense RNA lteatment engneering cell lines to knockout the enzyme(s), or culuing with selec&tive glyceosylation inhibitors i(Rothmtan et ali Mo I immurnnot 1989 Dec;26(12):i13-23). Some host cell strains, e~g. Lee 1 3 or rat hybridoma YB2/0 call line natally produce antibodies with lower fucosyaition levels, Shields et l J Biol (hem. 2002 Jul 26;277(30 2733-40; .Sinkawa et a Biol Chem 2003 Jan 31;278(S):3466-73, A ices h leve ofbsctdcrbohydrate, e~. through recombhinantly producing anybody in cOls that overexpreGs G it enzyme, has also been determined to increase ADCC activity. Uana et a Na t BotechnoL 1999 FIeb; 17(2)17600. it has been predicted that the absence ofonly one of the two fucose residues may be sufficient to increase ADCC activity. (Ferrara et at, Biol Cemin. 2005 ee St [004ti7) Other Covalent Modifications of Antigen Binding Proteins [00408) Other particular covalent mod in cations ot the anti-DNP> or anti-KLHI antigen binding protein, are also included within the scope of this invention, They may be made by chemical symhesi or by cnzymatic or chemical cleavage of the anien binding protein or antibody if applicable, Otter types of covalent modifcations can be introduced by reactin targeted amino acid residues with an org~anidv n agent tat iO capable of reacting with selected side chains or the N- odd:-errmn ralesidnes. [004091 CysteinVl residues most commonly are reacted with oa and corresponding aminesh such as chloroacetic acid or chloroacetamide, to give carboxvmethyl or carboxyamidomethyi derivatives, Csteinyl residues also are derivatized by reaction with bromoihfloroacetonc, ,alphi-,-bromio$~(5 imoidozoyl~propion icac id, chloroacety I phosphate, N-lymliie,3nto2 pyridyidisfide methyl 2pyridyl dis fide ch2romeribe te.t2 1004101 ThtAjv! rtds~t are dcnvAnzd by rec ithd tvpocrbruet pH 5N-0bcauetisara.is atvly pcific f the hitidA side chVA RaON bromrophcnacyi bromide also is useibl;the raction iselrefitbly perfored in (L MI odi vm acodaIe at pa hA). (904) Lysiny and amino-terminal rescues arc reacted with succinie or other carboxYlc acid anhdrides. Derivatization with these aents has the ettect of reversing the charge of the lysinl r sO uitae reagents for derivatizing .alpharamtino-ceontaining residues include imtidoesters suichI as methyl picohinmidtoe pyridoxad phosphate, pyridoxa]t corO hide rd, O methylisoure 2,4-pernanedione. and trnsamias-catalyzed reaction with glyovIate. [042) A rginyl residues are modifed by reaction with one or several conventional reagents, among them phenylglyoxai, 2 3 utanedione, J2cvclohexanedione, and ninhydrin. Derivatization of argininc residues requires that the reaction be performed in alkaline conditions because of the high pK, of the guandine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginne epsilon~amnino group. [00413) The specific modification of tyroayl resiches may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazoniumn compounds or tetranitro methane. Most commonlyNace tvimidizole and tetranitromethane are used to fbrm Gamceyl ty rosyI species and -nitro derivative, respectively Tyrosyl residues are iodinated using 9l or "I to prepare labeled yrote nrs tor use at fidioimnn~ unoassaa. [00414] (aboxyl side grus partt gtuamyI) arde octiveivrnodihed by racto wth carbodunides dbd. where R and c aMre different ethyb344zoniued-itnehyipent) carbodi mide Futh ere asard hand gJ;tafv resitduerc nrverted to asparaeinyA and ghuaminvi e.ta by reacio Wth iWmmOnIit u int5. ~00 Yn l utanminvimad aspar-aginy residues re feqoetr dienimkdaed to the carrespontng ghtaangd asparrylrsidutn ,rsnectie y These residue arc deanidated under neutral or basic conditions The dandated firm of tese residues rails within the scope tthis tnenti. [004 1 6] Other modifications include bydroxylation of' proline and lysine, phosphorylation of hydroxyl groups of seryi or threonylI residues. miethylaton of the ,alphanmno groups of lysine, arginine, and histidine side chains IT E Crighton. Proeins: Structure and Molecular Properties, WV H- Freeman & CJo San Fr ancis o, pp. 7086 (1983)1, acetylation or tbceiNterminai amine, and amidation of any C terminai carboxyl group. [001 7 l' Another type of covalent modification involves chemically or enaymtatteausy coupling glycosides to the antigen bindmng pr otein (eis. anti body or antibody frent) Tbh'w procedures are adv antageous in that they do not regeire production of the amngen handing protein in a host ccll that has gl vcosylatamn capabilities for N or C)blked 0y cosylanon, ependong on~ he coupling mode used, the supmrs) anry be atabed to ia rmeand hiN'tidm, ' fb ree carboxy l groups (e) free sudthiwdril groups \uch 0 fhose of cysteine, (dl free bv droxyi groups such a thoew of setme, threonine, or hydroxyprohine. ' "romati residucs such as those of kilola hlt t\ 0te or t tptrl or4 (t he annide groun of u utamine, T hese niethods are described in \\ OI 050 published ii e and i Aplin and Wriston. 'RC' Grit. Rev Biochemd. pp 759 (1 I) [0048} Removal of any earbohydrat moietie preem on the atigenbin protein may be acecomtplished chemically or enzymatically, Chemical deglycosltinrequiires exptosure of the antigen bi ning protemn to th omon tritluoromtethanesulfonico acid, or an equivalent compound. This treatment results in the cleavagte of most or all sugars except the linking sugar (Naeyguoamnine or 4 N- acetyigalaetosamrio while leaving the antigen binding protein intact Ch emical degycosylation is described by H akimuddin, et AL Arch. Biochem. Biophys. 259: 52 (1987) and by Edge ot at Anal Biochoem, 11 13 (981. Enzymatc cleavage of carbohydrate moieties on a anttgen binding protein can i achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura etal Meth. Enzymo. 138: 350 (1987). [0049 Anodler typ of cvalent -'iooiahol of' the antigen bl d1g pof Otins o the invention including atibodies and antibody fragrn compues linking the. amien biding protem to one of a vieatv of nonprotemseeous polymers et polyth W lene gyot p1 ypropyiene glycol polyoxyethlwated po yols, poiyoxy cthiyated sorbitol, poly ouyethy cited glucose, po lyoxy ethylated giyeront poiyo:yalky le. or poly acchande polymers such as denran Such methods are known in the art, see, .8Pt1n o 4N=40 0g. 4S49No9 4,391,144 46703. 7? 4391.192. 4 193?.429206d4 l 7A330 7.495:285 4.699.46 or P 315456, F01 AnAotherl. a fsrec encoecs an antigen binl protein of the invtat'don, such as, but !1tt iMimttd to, ai olaed nuclic0 acid tha encodes an antody or aibody frdalmnt of the mnnuo0, Su1ch nuclei acids are made by reoniaa technqus koxvn in the art andor dClosed herein. [004221 5r example the isolatednulcacdeoesnatiebnigpren coniphnmg an irntolbinheawy chain variablhioncnpilga mn acid sequence at east 95 idticato SEQi) NO2'50 SIQ iD NO:.252, SEQ I NO:254. EQ DNCL2S SE ID N 258 c EQ Ii NO [0042g i other or isnents, the isolad nuic cind ncoda esgan ngn c init a i um nglbh ight chain vraergo compmintau n amo acid sequence at Ioea 95 % idetcal to SEQ )F NOW23, SEQ if) N23 SEQ ff) >42 EQ) >02Kon SEQ A NO2W [00424) Oter exampes ofe isolated nuleic acd AM& sch codes an dsinted CDR nd DR and whe [004251 () (IMR1 compises the amino acid selcg.o E D>01WSEQ, D N1 .Q SEQ ID 1 or S EQ DNOl9a [004 26 14 WM13 comprise the amoo acid 5cqtc cc of SE 14 1)D NOA I9L SEFQ ID O:I 9. EQ0 >0:94 orSQ ID N0 9 ansQd N 0 n ID NO: 203 SEQ 1) NO: i 9 SE Q 1) >40:199. SEQ ID >40:200. or SEQ D [0042 Sl other eamres o ne iuec id ic. QD Na 2 S an tin- eumprisinn michan vari re hin e pated ni ad [EQ429a ( DIL 0I s SE ,Q H SE Q ID >4:0,SEQ t) N00,orSQ1 >025 [00431 W cCDRIJ cmprises the, aumno ac id seuneof SEQ 0DMZS E 1.) SE20 ,l-QIt) NCUD:210. SEQ ILYN:2i1,c SEQ U1) W0212. I004321 Inl other emoietheisatd:tcecaidnc esnnig .bnig pomein. comprising ant immunoglobuli heavy chain e msiJ h I noaci sewec fSQ10":7 E I NO:' 107 SEQ ID NO: I' H) 51"' D ) 13 SEQ ID NO: 15, SEQ ID NO)7, SEQ ID NO:19 SEQ ID NO:123, SEQ ID NO;29. SEQ IfD NO44, SEQ D Nt145, SEQ ID NO:i81, SEQ DNO:182, SEQ ID NO:83. SEQ ID NO:iW4, or SEQ ID NO 8 5, or composing any one of the foregoing sequeces fvom which one, two. three, tour or five amino acid residues arc lacking from the N-termin t or C-termii n or both. [004331 And in soe embodimens the isolated nueiC acid encodes an attigen binding.pronopnrsng an iummmoglobulin lightd chain compriing the amino acid sequence of SEQD NO S N: 109, SEQ iD N0,2,1; SEQ ID NO:125. or SEQ 1D NO:127, or comprising any one of theO freoi sequeces ftm which one two, tmee, four or live ari no acid residues are lacking from the N termia or C-erminaI or both [0434 For another exual. the islated nuclei acid ecae anlnigntnnz prorcin cornprisrg~ an irnrnem abrrliri hencavMI varl region comrlolr anI amnoacd se mec a es 95 STb A idnia o Qh sequenc of'SEQ ID) ?0062, SEQ f DNO:2 or SEQ IDNO:266. [W04351 i Othe Cn~O~ s teed isltdncei cdecde nat ampng. an amino aid sequence ate 9 % decan tSi 24 S ) NO:244 SEQ ) NO:246 or SEQ D NO 4 [00436J te xmlso h sitdtlit inin&esit htecdsa rnamoo n h eaavc a e reon, ween thes ed nucle accd designate C DRI. (DR 12 and CENV[3 and whin [004371 (a) (ThIIIuarie the amino -,- acdsqune f SEVQ H-D NO:2 13, E 11DbNO : 214 0S, orEIDNO\:0 2I15 148 [00438 W5) CDRH2 comprises the amio acid sequence SEQ D O10 SQ i) NO2 7 or S EQ D N 218 and [004391 Q; (DR H3 compso te aUntO ac ezeo SEQ D N 9 SEQ ID NO20, r SEQ I) N20:221 O0440 Si other eam les of a isolated ce ac include such hat encode anim6nogobuin lght chain vibl region 4 yhrein the isolad nulic addl corrie d n enees for tre cn id nent Itming regis designed CIRIL OR and C LDRL3 and wheein' [004411 (at CORM comprike he amino acid sene oO O) N4:222. SEQf) NO23 o SEQiD N [004421 (l) C1R2 comp o e aino d euence o SQ D 10.206. SEQ D0 NO 22 or SEQ N O Q2,6 00c443 I) CDMR compiss thenam acid sequence oSEQ If) N 0:227, SEQ ID NO:8 SEQ I) N:229, or SEQ D NO:30 [004441 I other embodiments the isolated nucleic acid encodes an aigen binding protein comprising an immunoglobulin heavy chain comprig .th: amino acid sequence of S EQ 10 NO:46, SEQ ID N:33, SEQ I) NO:Mt SEQ WI .N:143. SEQ ID NO: 6. or composing any one of the foegoing sequences from which one, two, three. tour or five amio acid residues are lacking from the. N-termina or C terminal. or both [00445] And in somc embodiments the isolated nucleic acid cncodes an antigen hiding poein compnsing an i chain comprising the amino acid sequence of SEQ ID N:28, SEQ ID NO: 131. SEQ I) NO: 135. SEQ if) NO: 137: or SEQ ID NO:141, or comprising any one of the foregoing sequences ron which one two, three four or five amino acid residues are lacking 6om the N terminal or C-teroma l or both.

[004461 The presen invention is s direted to vecos dncdi aepsson ceiithat compriseeepresionetori lso neonp e ssbythe prsn ndenti w s nade b Oe biological te eiques known iet and ao disclosed h Tan1 T ention s so di cted to a method nvoving [00447 4a) etuin t host cell ine etne aredium under e iions e t expesio o te ntt~nbindig protei encode by"h xrsio etr n [0044 (A recovering the antgen binding tenftonn the cUlture dun. R e u the antigen dnp otein iS eeorinps'ecd by known ehods anThody puniation, such as bnot not WiNtie to. antibody u ieto ecnqe discosed E arp d esewhere hen [004491] eeI u i [00430fl Deliv ery in a therapeutic antigen bmndimg protein to appropriate cells can be effeted va ua heray ex io, in si or. in io by uze of at suitabk aipnroaeh known\ n the 3rt For example, kor in ivo\ thCeapy, a nucleic acid enleodina Fondnm prm 'inin the desed antigen &ing prote in antibody eher alone or in congieton wth a vctor, hposomte, or precipitate maybe injected dir ectly into the subjet and in some embodimiens may be mjnected at the ste where the expreson of the ougen bdm.g protein compound is desired. For cx iv o treatment, the subject's Cells are removed., the nuclc aid is produced no these cells, and the mnoddaeJ ell\ ar rtnned to the suIject either directly or tot exatnp encapsuled w thin portuu min Mbranes are impnted io the patient See e L S P'Ns. 5 and 34 187 [00451] Thure are a variety of techniques available for introducing nuclic acids into viable cells. The techniques vary depening upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cels ot she itended host. Techniques suitable for the tansfer of nucleic acid into mammalian cells in vitro inchide the use ot liosomes, ceetroporoun mcroijection, cel fusion, chemical 0 traimentsDEkLbdentran and c ciumr phosphat precipitaomter hrg vto nucieicacid transfer redmiques include tnsfection with viral vectors (such as adenovirus, Hrpes simplex ViN s, adcao-associated virs or retrovirus) and pidbased sySimS The nuclei acid and tmstion agent are optionaly associated with a micropartice. Exemplary transfection aents include calcium phosphate or calcium chloride co-precipitation, D EAE-dextran-mediaited Lransketion, q uaernary ammonium amphiphil oITMA ((dioeoyloxyvpropyi) tiethsylammonumn bromide commnercialized as Lipofetin by G 1BCO-BR L)(Fegner e i a(97) Proc Nalt Acad. Sci. USA 84, 7413-74 17 Malnkw at al (1 989) Pro N t t Acad. Scn USA 86 6077-6081A; lipophii glutama era with pendent trimethylammn heads (Ito at a. (1990) Biochan Biophys. Act 1023, 24-3) the metabolizable parent lipids such as the cationic lipid dict adecyhamSdo gycylspormine XXGS, Trnasfet-amn, Pomega) and dipalmtoylphosphaid ethanolamyispermin (DPPES)(J P. Behr (1986) Tetrahedron Latt 27, 58.-86; P. Behr et at (19 9) Proc. Nat, Acad. Sc. USA 6, 69824986); mctabolizable quaternary ammonium salts (DOITB, N 41 .[ 2 t3dioleoyloxy ]proyl-NNN-trimethylamonim methylsulfta (DOTI c AP(ochringer Mannheim polyethylencimine (PEP. diolaoyi asters, AChT, ChoSC, DOSC)(eventis eat 1990) Biochm Inter 22, 235-24"90 3beta[N-(Ni N' dimethylamintothaneV-earbamoyicholesterol (DC- Chol) diolcoyliphospa tidyi ethanolamnine (DlOP E)/3bhent(N~(NN dimnthylaminoefhane>y carbanmoylIchtesterolDC-Choi i one to one miItures (Go e at ( 1991) Biobhin. Biophys, AMta 106, 8-14) permine, spermdine, lpopolyamines (Behr et ali., Bioaonjugate Chem, 1994, 5: 32389) lipophiic polylsines (LPLL) (Zhou et al. (1991) Biochn. Biophys. Aeta 939, 81 [{. Il( 1,1,3-tatamethylbutyl)cre soxyjetnoxyJ~ethylldimmethylbe'nrlammonium hydroxide (DEBDA hydroxide)with VM al t W.1 9)3oc excess phosphatidylcholine/cholestero (Blase at 98 inim Biophys, Acta 939 8~i), etyvrimnetihyaammoniurm bromide (CTAHB)/DOPE mitue (Pinnaduwage. et al (1989) Biochim, Biophys, Acai 985, 33-37), lipophille dieste of glutamic acid (IMAG) with DOPE. (TAB, DERDA, didodeclammni bomide (I)DAB). and stearlamine i admixture with phosphatidletbanolamine (Rose at al. (1991) Biotechnique 10, 520-5251L DDAB/DOPE (TransfkcIA CE, GIBC B RL Q 151 and oligogalaetose bearing lipids. xemplary transfection e enhance ans that increase the fficiency of tran.sfer include, for example, DEAE-dexirat. po1ybrene, NysOsome-diruptive pep~ie mor N d cia, Biochem Biophy Res Commutn Jun 27 1997:235h(3 i:726-9, chonidroitan-basedt proteoglyc-ans, sulfated proteoglycans, polythyleniine, poyysine (Polkr H et at, j Bid Chent 199 273 (13):7507- I) integrn-binding peptide CVGGRODTIP (SEQ U D NO:235Y linear dextran nonasaecharideni gryerit c holesterylI grups tetbered at the 3Wterminal in ternucieoside link of an oionucleotide (Letsinzr, R. L 1989 Proc Nai Acad Si USA 86: (I7:6553-6), lysophosphande, lysoposphatdylchoine, iysophosphatidyl ethanolamine, and -oenyi lysoposphadyldcholine. [004521 In some situations it may be desirable to deliver the nucleic acid with an agent that directs the niuceic acid-continurng vector to tage ceWls Such targetingg" molecules ilude antigen binding proteins specific for a ell-surface membrane protein on the target cell, or a ligand for a recepttor on the tart cell Where iposonmes are employed, proteins which bind to a cell-surface membrane protein associated with endoeytosis nay be used for targeting andor to facilitate uptake. E xamnpics of such proteinls include ce paid proteins -and fragments thereof tropic for a particular cell type, antigen binding proteins for proteins wh i udergo interna tlIitioni in cycling, and protei ns that target intrace-hul am localiation and enhance intracellular half-life, In other enbodimtents, rec t or-mediat enoc-ytsis can be used, Such methods are described, for example, in Wut et aL. 1987 or Wagner et at. 1990. For review of the current known gene mark ing and giene therapy protocols, see Anderson 1991. See aso WO 93/25673 and the~ reeen cited therein. For additional reviews of ge~ne therapy tehnoiogy, see FriedmnnScene 244: 1275-1281 (1989); Anderson, Nature, supplement to vol. 392,. no 6679. pp. 25 30 (1998T); Verma, Sc ienti fic American: 68-44 (1990): and MI ilcr, Nature, 357: 455460 (199 2).

004531 Adinistratioonand Preparation o harmaceutical Formudtions [004541 The ati-DN P or antbiK LH antigen biding proteins or antibodies used i the practice of a method of the i anruon may te ormuated huo pharmceutical comapositons anod medic~aments compring a~ carrier suitable for the desired delivery method, Suitable carriers include any material which, when combined with. the anti DNP or antKL LH antgen binding rotein or amibody, reains the highaffinity binding of DNP or KL H respectively, and is nonreactive with the subjectS immune systems, lxamples include, but are not lirnited to, any of a number of standard ph armaceutical earniers such a\ \trink phosphate buffered saline soltutions, bacteriostatie water, and the like, A variey of aqueous cariers may be used. c, water, buttered water, 0,4% salbne 0.3% lycie and the like,nd d may include other proteins fr enhanced stabiity, Suc as ahlui, lipoproten, giobu1in, etc, subjecred to mild chemical modifications or the like. [004551 haemplary antigen bind ng I3IotenI concemtrutions in) the tforml iion man range iorm about 0 1 rngiml to about 80 r:mel or from about 0 1 mgml to about 50 mg mL, from about 0 o ng n to about 25 my/niL or alterntu.lv from about 2 ribm to about JO my mnl \ n aqu.eou r~ m ulation of the antigen binding protein may be prepared in a pl-buller ed Noluon, fon eample- at ph rangmg front about 4.5 to about a.5 or from about 42 lo hoot 5.5 or aternatively about 50 Elamps of bulffers that are suitable m a piH wihm th range MCude acetate e g. sod iumn acetatJJ, sucemrnate (such as sodiumo suecrete luusnt hinu. citate and other orgmi acid bumffer, The buffer concentratin n b e from about .1 mM to about 200 mt or Bom about 1 mM to about 60 nWA, dependwna, for eutml, on. the buft'r and the desired isotamniir ofth e zormukition. [004561 A tonicY agent, which may also stabilize the antigen binding protein, may be included in tie formulation. Exemplary tonicity agents include polyois, such as mnntol, sucrose or trehalose, Preferably the aqueous formulation is isotonic, although hyperton ic or thypotonic solutions may be suitable, Exemnpl ary cone andons of the polyo in the frmuatioa ra e fronm abottoabout 5% Wv [00457 A surfate may so be added t e agen btnding protun orration to reduce aggregation of the fbrnulated amigen binding protein and/or minimize the fomaon of particulates in the formulation and/or reduce adsorption. Exemplary surfactants include noninc surfactants such as polysorbates (eg polysorbate 20, or polysorbate 80) or poloxamers (ed. poloxamer 188 . Exemplary conce"nWnons of surfactant may range frorn about 0,001% to about 0,% orip rom about 0.005% to about 02% or al.enuativey from about 0,004% to about 01% wv [00458] In one embodiment, the formulation contains the above-identified agents (i.e, antigen binding protein. buffer, polyol and surfacant) and is sseniay Wfree 0 one or more preservatives, such as benzyl alcohol phenol. m-c~resoL~ chiorobutanol and benztomium CL in another embodiment a preservative may he included in the formulation, e~g.. at concentrations ranging frm about 0.1% to about 2K. or r i.om abu 05 %. One or more other pharmaceuticany acceptable carriers, recipients or stabilizers such as those described in Remington's Pharmnaceutica Sciences' 16th edition, Osol. A. Ed. (980) may be included in the ormu latiorn provide th at they do not adversely affect the desired characteriAues of the formtul ation. .Accentablec carriers. excipients or stabil1Zizers armoxc t ree pints at the dtosageN and concentrations cmoye.d and inhie additional tuffering 'agent co-solvents: antox idants includingt ascorbic acid and methiionine; cheing agents such as EDTA: metal. complexes (e.g Zn-protein comNpexest biodegradable polymers such as polyester; and/or sah-fbrmng countertions such as sodinn [00439] Therapeutic formulaions o the1 antigen bindin 1 protein are prepared for storage by mixing the antigen binding protein having the desired degree of purity with optional physioiogically aceptable carriers, exeipients or stabilizer (Remingzton s Pharmaceutical Sciences 16th edition, Oiol. A, Ed. (1980) in the torm of lyophilized formulations or aqueous solutions, Acceptable carriers.

recipients. or stabiiers are nontoxic to recipients at the dosages and concentration employedh and include buffers such as phosphate, Citrate, and other organic acid; atioxidants including ascorbic acid and ethionine; preservatives (uc a octadecyldimethyHbenzyi ammonium chloride; hexamonethoium chloride tbentalikon ion chloride. benzethonium chloride; phenol, buty or be~nzyl alcohol: alkyl parabents such as methyl or propyl paraben; catechol: resorcinah' cyclohexa nol; 3~pent anod; and mcrosoli; low mtolecular weight (lss than about 10 residues) poloypeptides; protein such as serumn albumin, gain, or immunoglobulins; hvdrophilic polymers such as polyvinrylpyrrolidone; amino acids such as glycine, glutamine, asparaie hisidno arginine, or Ivaine; monosaccharildes disacecharides. and other cartbohy drtes flinclin glucose, mannfose, rmaltoise, or dextri as; chel ating agett such as EDTI A: sugars such as sucrose, muannitol trehalose or sorbitol: sat torming counter-lons such as sodium; mcta conmplexes (ecg., Zn-protein coplexes anid/or nonf-ionliC surfactants such as TWEEtNN 1 , PL URONICS'M or polyethylente glyco (PEOY [00460} In one embodiment, a suitable formiuation of the claimed invention contains an isotonic butter such as a phosphate, acetate, or Tris buffer in com ination with a tonicity agent such as a polyot Sorb dio sucrose or sodium chiotide which toniicifies and stabilizets. One exarmple of such a tonic ity age~nt is 5% Sorbitol or sucrose. in addition, the formtulation could opticonally include a surfactant such a-s to prevent aggregation and tor stabilization at 0.01 to 0.02% wt/voL. The pH1 of the formulation may range from 4.5-6.5 or 4,5 toi 5. Other exemplary descriptions of pharmaceutical formulations for arnbodics mtay be found in US 2003/Q01 13.316 and US patent no, 6,171,586b, each incorporated herein by reference in its entirety. [00461) The formutation herein may also contain more than one active compound as necssrytor the particular indicatnon being treated, preferably those With comaplemientary activities that do not adverselyv affect each other. For example, it may be desirablec to further provide an inmmos uppressive agent. Such molecules are sd abl present tn combwatin amountthat are efeie for the purpose [00462] the act igredients may also be t erap el for extnpl. by oacervaton techniques or fy ineftcia polymerzato, for exampleydrogmnetylccllhdose or eneroepsu and po nethhne thacyLn to) erd a respetetly. in cE ida drug delivery system (Ir example iposomes almi mrosphers micrenuions a parieleid nanoeapsu lesi or in macroenneIsions Such techniques are disclsedn Renngtors Pharmaceutical Sciences 16th editon, Osol.. Ed.I %} [004631 SuspeMins and crystal fors of ntigen bindng proteis ar also cowtcntlated. : .. adcytlfrsaekont n skilli the at [P0464] The formulations to be used for in vivo administration mstbe sterile The composition of the invention may be sterilized by conventionaL well known steriization techniques. For example, sterilization is readily accomnplihed by filtration through sterile filtration membranes, The. resulting sok utions may be packaged fOr us or filtered under aseptic condition ns an d lyophilized, the KyophiUized preparation beitg combined with a sterile solution prior to administration [004651 The process of freeze-drying is often employed to stabilize polypeptides for long-erm storage, particularly when the polypeptide is relaivl unstable in biqud compiositions, A lyophiliaincycle is usually composed of three steps: freezing, primary drig an eodr rig W ias and Polli, Joumnal of ParenteralI Science and Technology, Volumie 38, Numbher 2. pages 4Th$9 I 1984). in the freezing step, the solution is. cooled until it is adequately frozen. Bulk water in the solution forms ice at this tage The ice sublimes in the primary drying stage which is conducted by reduc i chamber pressure below the vom pressure of the iWc Using a vacuurn FinaHy, soIrbed or bound waler is rm oved at the secondary drying stage under reduced chamber pressure und an elevated shelt temperature, The proes produces anterilknown a a iyoplbhed cak herceafterhe cake caan reo united pio to us9' [00466) The standard reconsatution practice for lyophilzed matter is to add back a volume of pure water (typically equivalent to the volume removed durng lyophilization), a.1louh dilute sohutions of antibacterial agents arc sometimes used in the producton op nc ils for parental admination Chen. Drug Development and Industrial Pharmacy, Volume, 18Numbers I I and 12, pages .131 1~ i354(OWN [00467) Expients have been noted m som cases to act as stabilizers for freeze dried products: Carpenter et al, Dcvelopmems in Biologicai Standardization, Volune 74. pags 225-239 (9 O FOr example, knownt d (including tanmniol, sorbi tol id glycerol); sugars findingg glucose and sucrose.); and amino acids (including alanione glycine and glutamic acid), [00468) poiyois and s a a o often used to protect poyepuotides from fieczaing and di ng-induced damage and to enhance the stability during storage in the dried state. in general sua, in particular disaccharides, are effective in both the frez-dyig roes and during storage. Other classes of molecules.inclIuding mono- and di-sacehaides and polymers such as PVP, have also been reported as stabilizers of lyophilized products. [00469) For injection, the pharmaeutical formulation and/or medicament may be a powder suitable for reconstitution with an appropriate solemn as described above, Examples of these include, but are not limited to, reeze dried, rotary dried or spray dried powders, amorphous powders, granules 4 precipitates, or particul ates. For injcion, the formulations may optionally contain stabilizers p n odifiers, surfacants, bivailabiiy moditiers and combinations of these. [04Th 3 $Sutained-release preparations may be Prepared. Suiabe exampiles t sustained eae preparations include serainenneieNe atries of so lid hydro habi polrnte.ang ndatie badn whee ebinharices are id thformi ot shaped articles, e.g. films, or microcapsule. ESxamnples of sustained-release matrices include polesters hybdrogwels (for example, poly(2-hiydrox yt-mthacrylt) or poly(vinyacohol),k polylactwdes (U.S. Patent No.773 19> copolymers of L glutairc acid and y thyl-U -glutamate. non-degradable ethyleinyl actate, degradable lactic acid-giycolic acid copolymers such as the Lupron Depots (injetable microspheres composed of lactic acidglycobic acid copolvmer and euprolide acetate), and poly~D-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycobic acid enable release of molecules for over 100 days. certain hydroges release proteins for shorter time nerods When encapsulated polypeptides remain in the body for alone time, they may denature or aggregate as a reUlt of exposure to moisture at 37C, resultin in loss of biological activity and possible changes in mmunogenicity. Rational strategies can be devised for stabilization depending on the m bchanism involved For example, if the aggregation mechanism is discovered to be intermolecular S-S bond forrnaton through thin-disufide imterchanmge, stabilization may be achieved by mod ifying sul fhydiry residues, lyophilizingfliom acidic solutions, controlling moisture content, using appropriate additives, atnd developing specific polymer matri compositions, [to4l 11The tfrnadations of the invnion 4nay be designed to beshorteie, at~ eeasng.togactingo sustaned-r Seig a nasacred herenh Thua the phaimaceutical fbrnaiuatins may aso be fornudated for controled release or fbr [00472) Specific dosages may be adiuisted depending on conditions of disease, the age, body weight, general health conditions se N andt diet of the subjet, dose intervals, administration routes, excretion rate, and comnbinations of diugs, Any of the above dosage torms con tainn effective amoinut are well within t bounds of routine experimentatrion and therefore, well within the scope of the intt in vention. [00473O The atntigen binding protein is tdniinisted by amy suitable means. iOe dchng parenteral, uncutaneottu trarpiatoincal, iramionars. and iitatisa. and. if desired for local treatment, intalesionad ad~iintation arenteal intusiots includ intravenous, intraarterial inraperitooeal oamruclar, intradermal or subcutaneous administration, in addition, t antigen binding protein is suitably administebred y puhs infusion Poarticuarly with declinig doei S of the antigen onanuibody. Prefb I yl by in' ' Ietos TTIOSI binding protein or m P the doini is giv preferably intravenous or subcutaneous injections, depending in part in whtear the admnnistration is brief or chronic. Other administration methods ar contempated, including topical. particularly transdetmaL transmucosal. recta oral or local adminitrtioni e.g. through a catheler placed close to the desired sit. Mot preferably, the antien binding protein of the invention is administ ee intravenously in a phslog ica solution at a dose ranging g betwon 0 0 mg/kg t 00 m kghat a frecquenICy raing~u from daily to weCeklyv to mronthly (e g. every day, every other~ dlay, every third day or 2. 3. 4, 5, ort6ttimes per week). preferably a dose ranging fiom (.i to 4 mg/k 0.1 to 15 mg/kg or 0 to 10 m/, at a o or times per week, or up to 45mg/kg once a month. [00474] The inentio i iustrateud by the f u1 owinag eampie, whch oarc not intended to be limi ini an way [0(475 EXAM4LES [004761 LaMle I [00477) Generatin of antibodies to DNP or KLH aiid sreeoing [00478) ounizuvations Anti-DNP antibod ics were geneIraed by immunizing XenoMouse'ince with ONP-KLD, over a period of 4 weeks, and by scroning for those antibodes that bind to DNP- ysine More particularly, XenoMouse XMG2 strain of mice were generated generally a described previously Mendez t aLc Nat' Ae. 15:1 46156 (1997. published Intemaionai Patent Appliation Nos WO 98/24893 and WO 00/76310, LI discosurcs of which are hereby incorporated by retrence) and ininaunizd 55ith 24 D~inietrgenvi-eghnvle 'npet Hemncvan (DNP -KL H conjugate: BioSearch. Technologies, Novato, CA L using a range of 10 30 p/ouse f immunogen emuKifed in TiterMax Gold adjuvain (Sigma-Aidrich, Oakville, Ontario) fo the inil immunization of the XMG strain of XenoMouseT according to die methods disclosed in International Patent Applicatio No\, WO) 98/24893, and WO 0076310, tHe disclosures of all of ich are hereby incorporated by reference, Following the initial immunization, subsequent boost of inim unogen~f (5 21 yg/mouse) were administered on a schedule and tor the duration necessary to induce a suitable anti-DNP titer in the mice, Titers were determined by enzynme imnir ioassaiy using rimobilized D)NP~BSA (BlioSearch Technologies, N'(vato, CA), tim conjugate was prepared such that the final DNP:B1SA molar ratio was 30:1, [004 79) Immunizations to raise anti-KL H antibodies were conductd over a period of 4 weeks, usin imject® Mariculntre Keyhole Limnet hemocyanin meK.LH Pierce Biotecnology Rckford, I: ea# 77600, ot#1 440951M. Immunizations were conduted using I1pg of KL H per mouse in Aumnium Phosphate Ge Aduvant (II Biosector, Frederiksund, Dentark; Catalog # 1452250); delivered via footpad injection. The inia i hmmunizaton of t MG 1K \train of XenoMouse was according to methods previously disclosed (Mendez ci at. NA Genet 15:146-56 0 9974; published international Patent Application Nos. WO 98/24893, and W() 00/763 10. the disclosures of which are hereby incorporated by reference, which are all herehy incorporated by reference . Following the initial imuzation, subsequent boosts of immunnogen (5-,10 pgmus)were .admnistered on a schedule and for the duration necessary to induce a suitable~ anti-KiLl titer in the mice. TIters were determined by enzyme immunoassay using inmmobilized KLH FPierce Biotechnology, Rockfr IL). [004891renain01 nooc na nthd e.Mie exhbiting suiable iters wer e identified, and lymphocy tes and svneneytes were obtained trom draining lynmph nodes and spleen, then were pooled for each cohort. B Rells were dsociated iOm the tnue by grmding m a stable medim (for example, Duibeco's Modified Eage \tdi in; DMF.4: nitrogen, Clsbad, CA) to release he eelis rn t he tssue nd were suspended in DM[NI. B ces were selected and ex handedd unme tandrdd - 16 0 methods and fused i suitable fusion paneur eamd nonsetioary lntoma P3X63 Ag8 653 c Amcic a Ty Cture olecton C.R c580C Keane end humwnu. 12314 1550 (197914 usn techniquesknown n the [00481] I celts were mixed with fusion partner eells at a ratio of i:. The cel mixture was gentvqly ted by centrifugation at 400 x g 4 minutes, the SUpernatant was dcanted, and the cell mixture ws gently mixd by using a 1 mI pipette. Fusion wxas induced with PEG/DMSO (polyethlene glycol/dimethyl sulfoxide; bind fi-m Sigm-Adh t. Louis MO; ml per ino of lymphtocytes)tPEG/MSO was slowly added with gemie agitation over one minute followed, by one minute of mixing. 1DMEM (DMEM wahot glutamine; 2 ml per million ofLB cells), was then added over 2 minutes with gentlk agitation, followed by additional DM EM (8 ml per million B-cells) which was added over 3 minutes. [0041 The fsed cells were gendy pelleted 100 x g 6 minutes) and resuspended in 20 ai Seecaon nedonu fo example, DIiEM contamnn Aaseime and Hypoxianthine [HIA] iand other supplemental matcrnals as necessary) per million S cella Cell iere imehbated for 20-30 minutes at 37'C and then eie resuspended in 200 i Secion medium and eulured for three to four days in Ti 75 asks prior to ~6well plaumg 100483] C cs nwre distiuted io 9~nell plates usmn standard tcchmiqes to m &Ami cOwnaht" of the rutino colonieS A fer seeal days of culture, the hybridmon sup:raants were collected and subjected to screenmg assays as detaled in the cmples belo imnuding confrmation of'bindg to Kill or DNP, respectively Posiinve cells were further selected and subjected to standard cloning and subeonming techniques (lual hies were expanded in uto. and the \'Cited human anbodies obtained tor analysis. Sevecral cell lines secreing DN P-specific antibodies w ere obta med, and the autnbudie were turther charade iire T 1he sequence Thereof are pressed heriAn and in the Sequence Listii, ad results of various ts usmg these anbodie are pro ided.

[00484} ( lonin and Engineerin of Carrier Antibodies antiLH and anti-DNP, TIe s qun e s for the X1nomouse ved human anti-KL j antibodies were otaine~d by th p'olymrase chain reaction (P1CR amplificaton technique known as 5' RACE (rapid amp ifctiorn of cDNA ends) Total RNA was isolated from three hybridomna expressing K L binding monoclonal antibodies; 16, , KL2 and 120A using Tizol reaget ( nvitrogen) followed by a further purification using the RNeasy Mini Kit (Qiagen). Mixed random and ohigo-dT primed first strand. RACE ready cDN As wer preae usn th e GezneLace Ki (i ,nvitrogn PCR amplifcations of the cDNAs were performed with AvnaeF2 DNA no lymerase (Clontech) with the forward primer, GenefRacer" tested primer [00481 5 GGA CAC GA C A GA C AMG GAi TA 3$(Q 0 [00486) 5'~ CTC CTG GA GTT ACC (CGA TTO -3N (SEQ ID NO:272, tor the liht chain, and 5- GAT GG CCC TT GTG CAG G CT GAG GAG ACG GTG ACC GTiG C ~3' (SEQ ID N:273t for the heavy chain. The PCR reon eyceks consisted of a 30 second denauraion of the cDNA at 94C, ol owd 'by tee cycles of amplification with each cycles consitng of 20 seconds at 94"C; seconds at 55 *C and 90 seconds at 72 * pus an additional 27 cycles consin ; of 20 seconds at 94WC 30 seconds at 65 0 C; and 90 seconds at72*C, The reactiinr tben incubated for 7 mteS at 72'C fl lowing the last PCR cycle to insure complete clongation. T[he RACE PCR products were clonedt into pC.R4-TOPO invitrogen) and their sequences determined using ABI DN A sequencing instruments (Pari-tn Elmer), Consensus sequences were determined using Vector NI 8, software (invitrogeni anid used to design primers fortnt ful-iength antibody chain PCR armplificaton. [00487 To obtain the cornopink coding region sequecs fr the epresion of ard K antibodies, using 16.31 as an sampe PC as again used Thelght chain 5' P tI pim coded e annd ermiuofe StgNa s equenc a SaP restri enzymesite, and an, onrzed Ksksnunews SE NOY 1)=741 do can 3'isn NO}4 K 5> AA OCT hA AAC 00 CC MC CAA (A CTOT CC Ct [T10 A (S Q C I D A: A, f00 4 9 0 Th heWa vy chai 5' Pk prViC0er ec"o te aio termius o f t sina seuece Sai retiortG' nv ra site and an nptmwned Kan snuenee vwat [004 ]- 5> AAG C10 GAG CYPOT CY C TA G ;\C'\ ACV AJ AA JIT " G ilA. 011 YAG X v (SE NO N01276), and the A' Prierenoedth etxy tomu atma on, as wl a rs [004921 5V A.AC COT' 1-1A AAC CG GC GO(T CAT- IFTA COO CEtA GAO AG A E7 [00403] e r R re per dug a H 2 DNA re and the fkioI 0, le consssce of a 30 send u of o OM 9407 Onl Y 30 cvD- o n of20 seconds at 9417; 3t0 seconds at (C Pnd 90 seeoial re 72on The rachons were then ea er tnrt PC odowIn th las P( Rh sdt insueadapl etfie eonlto. ht~te c'ds 16 Pke protuts er gal islaei. purthed ung Q cpTk spin cons aVena estide Stl (NEI3L and Nul (NEI3 14 Isn~ted andpuiedungQqik p cuns and then ligad ino Owe niannuan exrcssin vectr p1 5. 0041Te nquencestat tngion, Ao oumed antDNP ntino vurtabie SecIng reretacitonPC i Rodc P~k was then used to adapt the aibergo eunc ust is:he counpaddbe with the en of pITS vecorscotingaK sgnupttadth owned imo plI I e unique El-l site at he end of a VkI signal peopde and the unique BlW1 site at the begt ut of the human kappa consutmaN ren To add the sA and th Mi u ste to the 3ndas A an sampld 00495] 5 TT ITT TTG CG CT G A CA TUG WTA (CC AG C 3V (SEQ M) NO2"Q [004961 dnd 3' yrimer 5' AAA AXkA CC ' AG I GA7 AG fAC IT'( SC 3 I (SQ " L) NO 9h. [004971 Th DNP ntwed a pai i on of t G 11TW YGGA.,IT ('j TAk11 ANK0 GG 3 V9 SF DudQwaljqmar A h A CA A D d ane by ng stand prioer [005001 5' CT1 TOT AT AC TO GT AT AC AA ACT AG CA TOO AG TG GO:1. (1A ) nd strnd Wrr 10050 5CCAAG OTG GAT AC GTA T AA GTT ATA (CT CG ACA (11k l CAC Ail 3+SL 0I U)N2 ian oawoin yFuing(. strand3 primr: [005001 A CT T T AT AAC GTO GGA GOT CATA AI' AGA GAO [005011 Y c'GG GoGI A AUG ( 1 A&OTT CIA OTT AA nd COG AGA G&A (SEA GAG AGU AM, (SE ra)c Smu... nmerOy W~i A hv o"tan If hond primer:s1N [005(12.~~~~b 5 API A 17CAC GACAG R'_';( 3(~ 1005014 GOA CAA GAG ACT7AI % COO (AA AICT I' T ' CON7n CM A.CC GTG CCC GG # (SEQ D M).286I and Q Aand prm: [00501 5" TA GGCC AGOI (31 COG ACT CGA coA AAG ATT OO OCT (AAII C AG W I WI (V 3 n {SLQ "i) 05} .fCif it hZh"V hi 5 nd proner [00506) 5' AA Cm: GAO GTC GA TAG ACT' A lt GAO AT AGO (11 CC 3G 0 (T1100 (C1( 30 V4 (SEQ IT NO:25 and 0he heay chain 3 pimer [00507 5' AMC CG A AA 0CG GCC GIT ('T'ITA CC (iCA GA AO GA Vi (EQ ID) NO:891 [00508) 1 ransiceet ression to generate regoibanots moionalantjjodies Transient transfections were carried out in HEK 2 93 cels as foows. The human embryonic kidney 293 cell line t e e Epstein Barr virus Nue lear Antien-1 (293-6 F celi) was obtaind frm dhe National Research Con cil (Montrea C anadt Cel were m airme tas senf-ree suspension cultr esusig F17 medium (Invirogen, Carlsbad. CA) pplemntced with6 mn -gutamine (Invitrogn, Cadsba 4 d, C A ), 1J1% F--8 FVonie (nvirogen, Carlbad, CA) and 250 ~g/u l Gneicin (In vitrigen, Carlsba, CAY rTe suspension eel I cultures were maintained in Erlennmeyer sihake ask cultures, 'The cunture flasks were shaken at 65 rpm at 37 "1 in a hunidi ld, 5% 0C1 atmosphere. A stock solution (1mg/mi) of 25 k~a linear PET (Polysciences Warrington, PA) was prepared in water, acidified with HCl to pH 2 0 until dissoLved, then neutralred with NaH, sterilized by "ihation (0.2 pmt alKuoted, and stored at -20C t unti used, Tryptone N was obtained from OrganoTechni S A (Tekniseience, Q C Cnada). A stock solution (20 w/v) was prepared in Freestyle medim Inmitrogem Carisbad, CA), sterilize by nation through 0.2 un fibers and stored at 4 * until use, 'Typicaly, transfections were performed ao the IL scale, (elks ( 6E) were grown too a viable cell density oft L1 IX 106 cells/mA then transtection complexes were prepared in 1/10th vdlme of the final culture volume. For a I-L trarsfection culture, transfer tiotn co lexes were prepared in 100 o F 7 basal medium, and 500 g plamid DNA (heav chain and light chain )NA, 1:1 ratio) was firs diluted in 100 m F17 medium, After a ninute incubation at room tempnratrure, L5. i &f PUi solution was added. T he complexes were vortexed ilY, then incubated for 15 minutes at room temperature The cells were transfeeted by adding the s cornplex mix to the cells in the shale flask culture. 24 hours post-ranseeton.Tryptone N1 was added to the transketed culture to a final concentration oF 03% and the tra'nsected cultures were main.ained on a shaker at 65 rpm at 37C i a humidified, 5% CO atmosphere for another 5 days after which they were harveed, The conditioned rmedim was harvested by centrifIgtiion at 4000 rpm, and thensteriiteredthrough 0,2 pm fiter (Coming c [00509) The stably expressed aMKLd 120.6 control antibody pool was createdQby rsteting H('O d- host cells with expression plasmids pDC3 2 3 anti-K a l.2t,6 kappa LC and pDC324 anti-KLH 120&1gG2 HC using a standard electroporation procedure. After transfootion, the cells were grown as a pool mna sern free. 41HT selective growth media to allow for selection and recovery of the plasmid containing cells, Cell pools grown in -GT selective media were cultured until they reached 85% viability. The selected r ecU pools were amplified wth 150 an and 300 ni ocethotrexate (MATNT Upon reaching >85%, viability the 15 ni M pools were then further re anmplifed in $00 no MIX. When the viahility of he MT X amplified pools reached >85% i te pools were screened uing an abbreviated six day hatch production assay with an. enriched production media to assess exprression, The expression of the amplified pools ranged from 120400 pgm. The best pool was chosen based on the six-day assay and sealedup using a ten-day fd batch process, The conditioned media was harvested and purified to provide protein tor analysis. [00510] The stably expressed aKL H 120.6 antibody pool was created by transcting Hd(10- hos cells with expression plasmids pDC323 aniIKLII 12,6 kappa. L and pDC324 anti-KLH 1 2 HC using a tadar lcetroporationi procedure, After transfection, the cells were grown as a pool in a serum free -GHT selective growth media o allow for selechon and recovery of the plasmid contaiing 66 cells. CeIl pool grow in Kn GH 1 selective media were cultured nuV they reached > 85% abiity. The selected e pools w ere amptbied Wh 1 m m tand 300M l X Upon aching '8% viability the I50nM pools we then further re anphfied n 300 mrn MTX. \\hN the viability of the \MTX amphfed pook reached 'N' ahty, the pools were screened using an abbrevated s day batch produntIon asstv with an e enriched production meda to BVss expresion The expression of the amplified pools ranged tn 1 2400 p .<ml [he best pool waS chosen bsed on the mx day assay and scaled up Ang a wen ty fed batc process the conditoned meda as harved and purified to prone point f ilnaly, [0051 1 The aDNP 3A-F and aDNP 3BI antibody stable expression pools were created by transfeting CHO DHRS) host cel with corresponding heavy chain and light chain expression plasmid sets using a standard electroporaion procedure, Per each anibody molecule, 3-4 different transfections were performed to generate multiple pools. Afer transfection the cells were grownasapoo in a serum ree GHT1, select e growth media to a low for selection and recovery of the piasmnd containing cells. Cell pools grown in (4fT selective media were cultured until they reached >85% viability, The selected cell pools were amuplifid with I 50)m methotrexate. When the viability of the methotrexate amplifed pools reached >85% viability, the pools were screened using an abbreviated sx da rdc assay with an enriched production media to assess expression the best pool was chosen based on the six day assay titer and correct mass confirmation, [00512 .. hody.p...ationn.. jc.j. The antibodies were purified by Mabt Select Sure chronmatography (CE Lif Sc iencest using 8 column volurn% of Duibecco's PBS without divalem nations as the wash bffer and 100 mM acetic acid pH 3. as the elation buffer at 7oc. The elation peak was pooled based on the ehromatogram and the pH was raised to about 5,0 using 2 MI is base, The pooi was then diluted with at least 3 volumes of waler. fiklred through a .22 cellulose acetate filter and then loaded on to an SP--HP sepharose column ( i Life Sciencesl and washed with 10 coluom volumes of S-BuRr A (20 mM acetic acid, p .0) followed by el Uton using a 20 column volume gradient to 50% S Be B (20 m M ace Acid, 1 1 NC p1 50) at 7 C A pool was made based on the chromatograand SDS-PAG E anayi\ts, then the material was concentrated about 7 told and diafiltered against about 5 volumes of 10 WoM acetic acid, % sucrose, pH 5i using a VivaFlow TFF ca-sette with a 30 a membrane. The dNiale material was then filtered through a 0:22-yin cellulose acetate filter and the concentration was determined by the absorbane at 280 nm. [00513] The lead adidates were theneted basee or the pro dct behaor b S)SPAG. he aoN B .3d Ind 3A4 antibodies from both transient and statble expression mamnmaian cell lnes were analyzed fr prodt quality on a 1.0-rom Tris-gjycino 4-2.0% SDS-PAGE (Novex) sing reducin loading buffer (Finure I 11 Using these data, the aDNP 34 antibody produced a heterogenous product from the stable cl line, which indicated that it was not a good candidates as atarier antibody, sine a homogenous product is dsirable. Thre aDNP 3A 1 A , and 3B1 and aWK [H 120.6 antibodies were analyzed for product qldity on a 1.0-mre Tris-glycine 4-20% SDS-PAGI E (Noves using non-redutcing loading bufe (Figure 12A-l~ The aDNP 3C12 antibody produacd a beterogtenous product with exceptional high molecular mass mnateriaew cting it was not an ideal candidate as a carrier antibody, since a product containing high molecular mass matcril is not desirable, in addition. tbc DNP 3B1 antibody showed a doubt under these conditions. lTe aDNP 38 and aDNP 3A I anti;ois wero then compared using both Tris-glycine SDS-PAGE as well as bis-Tris NWPAGE systems under now-reducing conditions (Fpure 1 3A-B). it was toand that the aDNP 3B1 antibody clearly produces a double not observed with aDNP 3A on the Tris gleine SDS-PAGE; howev, the aDNP 3M1 anotody appare more homogennus than the aDNP 3A antibody when anatyzed.by i NTis NuPAGE, indicatn that the double may be an artifact of the method of analyNiN Whnt caDNP> 3B13 anthiody as analyzed b ris-glycie SDS-PAGE after treatment wit non reducing Nsamples butter at room teporature 85 C or [0 XC the double was not eliminated (Figure. 4A). However, when the ai NP 3131 antibody was examined by Ts-glycine SDS-PAGE usinA OA% SDS in the gd running buffer rather than the usual 0. %,th dautib ws aneNredud dFigure 541 offaring~a additioa exdece that he doublet was an at adet of thentm0 anna [00$54v Antibodies werefurther analyzed fr homogenety using {wo swze exclusion coluoms (TSK-GEL (3000SW XL 5 mm patch siz, 7. x 300 mm, TosohRioscience, 08541) in series with a 100 mM sodium phosph 20 mM NaCi pH 6. mobile phase flowed a~t (,5 nmLmin (Figre 15). The aDN? P3C antibody displayed a substania postpeak shoulder, which was deemed undesirable, so this amibody was demoted as a candidate earrer anbody In add iton it wa observed that the aDNP 3C and aDNP 3A4 antibodies clured later than expected indicating a potcei al interactions with the stationftry phase of the chiromatograp~hy% cohnnn. [00515] Antibodies (aDNP 3A, aDNP 3C2 and aDNP 3A4) were tested for resistance to photodegrradation. T he antibodies were either exposed to fluorsct light at 4 o>c for 3 weeks or were protected from light by covering samples of each with aluminum foi. The antibody samples were then anayed using two size exclusion columns (TSK-GEL G3000SWXL 5 mm panicle si 7>1 x 300 mm, Tosohtioscience, 08541) in series wt 100 rnM sodinm phosphate, 250 m)M NMa(L, pH 6 8 mobile phase flowed at 05 mn min (Figure 16) The aDNP 3C2 and a NP 3A4 antibodies showed substantial peak broadening fter light exposure, which is consistent with oxidation of a susceptible uryptophan 'o reduce the oxidation susceptibilty of the aDNP 3A4 antibody, several varian[s with it CD)R3 tryptophan mutated to either tyrosine or phenylataine were construted (aDNP 3A4, aDNP 3A4-Y aDNP 3A4 , alN 3A4~YSS and aDNP 3A4-SS). These antibodies were then evabutted by SEC for resistance to photodegradion aller two days of light exposure (336Wfm2 UV light and 331 klux fr fluorescent light) at 6 C y analysis using two size exclusion columns (TSK-E L G3000SWXL1 5 m particle size, 7,8 A 300 mm, Tosohl3ioscienee, 08541) in series with a 100 mM sodium phosphate, 250 ,M NaC at pH 6,8 mobile phase flowed at 0.5 ninba (Figure I7AtI [005 161 All four of the aDYNP 3A4 variants showed substantially less peak broadening than the wild type mioleue, indicatin g that the CDR)13 tryptophan was responsible for this undesirable phenomenon, Furthermore, the retention time extension on SEC was also greatly reduced with the varians indicating less interaction with the stationary phase of the cohaurn., Amti-DN P 3A4 antibodies with various mutations (aDNP 3A.4, aDIN P 3A4-Y, aDNP 3A4-Fz aDNP 3A4-YSS and aDNP 3A4-FSS) were analyzed for homogeneity using a T o obaas SP~5PW column (1 0-pm particle, 7.5 nm1iD) X 7.5 cm tong) using Buffer AX (0 mM sodium acetate, pH S.0) and Buffer: 13 (10 mM sodium acetate, 600 mM v Nai , pH S,50) flowed at I nmt'min with a progranmmed lincar gtradiet (I moin 0%B3,. 10 moin 35% B, 30 mn 70% B. 3 min 9{0%B and 3 rmin 0%B3 (Figure l18}, The aDNP 3A4 antibody with the CDR<3 trytophan converted to phe'nylalanioe produced a more eirable narrower elution peak than the wild typ or tyrosine variant: therefore, the aDNP 3A4-F variant was deemed to be the superior mnoeue. The MaP 38, aDlN P 3A4-F, and DIN P 3A4-FSS antibodies were analyzed by non-reducing CVSDS (Figre 19A-C). All CE SDS experiments were performed using Beckman PA 00 CE systw~em (Pulerton, CA 1 equipped with UVY diode detector, 221 nim and 220 nm wavelength were employed. A bare-fused si lies cipilltary 50 pmn x 30:2 cm was used for the separation analysis, Muffbr vial preparation and *oading as5 well as Install Capillary CIartridge were described in the Beckman Couoter manual for IgG Purity /Hete'roge"nciy The running conditions for reduced and non-reducetd CEF-SDS) were similar to those described in Bseckman C oulter manual for igG Purity/Heterocgeneity with some modifications whinc r briefly described below. For non-reducing cionditions, he antihbodyvsmple (150 pg) was added 20 pl of SDS reaction huffe and 5uy of 70 rmM N-ethylmaleimide. Water was then added to make final volume 3$ i an the protein concentraton was brought to 4.3 mge/nI The SDS) reaction buffer was made of 4% 8DS, 0,01 Mt citrate phosphate buffe~r (Sigama} and 0036 Nd iun phospihae diha. e oerepma aaee epaatot wanore thoroughly and hMatd a 45( (hr S min. t t prepnartin was then added additinal II5 gI of 4% SUS After being vortexed and centrfinged, th preparation was placed in a. 200 pi PCR val nd he loaded onto the PMOG intmnear The samp le was injected at the aunode th reverse polaitvsing I 0 kV for 30 see, and w as then separated at -153 k' with 20 psi preur: at both ends 0 f capitary during 35 nun seprion 1he aNP 31 anmibody produced the most desirable: profile with the highes level of uidbrmitw under non-reduring conimons The aDNrP 3B L aD\P A~F, and aUD\P 3A4-FSS antibodies were anahyed bV reducing L~SDS (Figure 2Fo-C here m 'o ducig condit the antibody sample as diluted to 2A4 mg/mt by addog puntid t,(0 and 95 p] of the anubodv was added 105 pl of 8DS snple buf'ker (lHeckmanr with S .6 beta imereaptoethanol. The preparation was then onreed toroughly and then heated at 7 0'1 fOr 10 Qmn A fer beMp centrifuged, the supernatant a plakcd i a 200 p! FCR 0a0 and then loaded onto the PASO t nsment. The sample was injected at the anode with revrse oArity using -5 LV for 20 sec, and as then Nsepad at 15 k\ wuth 20 ps pressure at both ends of capillar dunn 30 rmm weparmon T he aDNAP 3A4i prodded the most desiable umnirm p akst under redutmy tondWtons [0057 7? The aDNP 3A4-F aDNP A4FSS and ADNP 3111 antibodies were analyzed for thermoresisance y DS C uing a Micral V P-DSC where the samples were heated from 20&C to 954C at a rate of I"C per minute The proteis were at 0.5 mg/md in 10 mM sodium acetate, 9% sucrose, pH 5. (Figaure 21). The aDNP 3(11 and auDNP 3A4-F antibodies produced the most desirable mAi prof11&dies, with a higher temperature for the initial transition, Th 0DNP i3 and aDNP 3A44F antibodies were differentiated by the presence of a single melting transtion for the aDNT 3Bi antibody and a double transition for the aDNP 3A44F antibody, [0018 T;LI aI\E S assays ka~i' were conducted as follows, Costar 3072 medium bnding 384 well plames (Comin L twife v Acence were coated with DN BS (BiozSearch Tcnlge Novao ( A) at 5 ml in IXPBS/005% Azide, (40 p A The plates were incuked at 4 C ov enght T he plates were then vased using 3-cyclec waash on a Titertek M34 plate washer..Ti..rek,.Husville, AL). The plats were blocked with 90 pl of AXPS/t% milk and inated approximately 30 mites at room temperature. The plates were then washed using a -cycle wash on a Tiertek plate washer. !Ol antibody samples were added to 40ul 1 XPBS % milk.

he pttAs were then ic-a te I u at room e u N ates then washed using 3-cKe wash an a Titertek N384 plate washer {Titertek, Huntsville AL, Goat anmi Human IgG Fl HRP was then added 00 ng/mI (I :4000) in 1 XPBS/ %milk/10mM (a (50 p/well) was added to the plate and wa incubated 1 hour at room temperature. Ih pates were washed once again. using a 3-cycle wash. The plates were then patted ir wih paper towel Finally, 1 step TMB (Neogen. L exington. Kentucky) (50 pl/el) wtas added to the plate and was quienched with 1N hydrocAoc aid (50 pwA ) ar 30 minutes at room temperature. 01D's were read immrediate y at 450 nm using a Titertek pl ate reader. F0OS19] E amiec [00520] Pharmacokindtic (PKC) & Phaermarodvyenmi (PD) Studha of Andi DNP Antibody Embodimnats of d mention [00321 The pharmacokinetic pofile of the aDNP 3A4-F nDNP 3A-FSS and aDNP 3Qantbodies was determined in adult Sprague-Dawicy rats (8-12 weeks old) by injecting 5 mg/kg subcutaneously and collectng approx inately 250 pL of blood in Microtainer serum separator tubes at 0 0,25 1, 4, 24. 48, 72, 6, 168, 336, 504, 672, 840 and 1008 hours post-dose from the Ler tail vein [gere 20, Each sample was maintained at roomi tmperature following collection. and following: a 30-40 minute clotting period, samples~ were cenrifuged alt 4" at 11,500 rpm for about 10 mnutes using a calibrated Eppendorf 5417R Cei System (Brinkmtann instrumen'ts, Inex, Westbury, NY). The collkected serum was then transferred into a pre labeled tor each rat), cryogenic storage tube and stored a -60*C to 480*C for- analysis. T o masure the serum sample concentrations from the PC study samples, the following method was used: A area black plate (Conmg 3694) was coated with 2 pg/mi ofAnti-bu FC. A !35 in ix PBS and then incubated overnight at 4"C. The plate was washed and blocked with lBlck (Appled Biosystens) overiht at 44C. if samples needed to be diuted, then they were diluted in Rat SD) serum, The standards and samnple we"re' dluted 1: 20 in P- LOc + 5% BSA into 380jp of dieting buffer. The phte was washd and 50-9 samples of pretreated standards and samples were inmserred into an Ah I 35, 1 coated plate and incubated for 1.5h at room temperature.c plate was washed, then 50 pi of 100 og/ml of anti-ho FC Ab 21. RP conjuga in -Boc + n5% BSA wr e" added and incubated for 1.5 h The pate was wshed, thn 50 g1 of Pico substrate were added, a er which the plate was immediately analyzed with a lumnomneter. The pharmacokentic profe was good for al antibodies, but the aDNP 3Bl i!showed the best overal profile. f00522 i the pharmacokneti protiie of the aD\Pm 3A44 antibody was deternuned in 0 Male eyt101ologous monkeys ( by kg) Vh n tchng 6 mTg k bolbs dose ina venously and taking blood sampkat 0 and 30 minutes and 2. 7. 1 21, 2K 35. 42, 49. 56 and 63 days (igure 2. The phatmacokine profile of the - ll 120.6 antibody was drenniwd in 4 nale yninologou% moikeys ( Ag) by imjeting 3 mg/kg bolusN dose iranenousv and t aking blood sampleN i 0, 0 1i L4 S12. 24. 72, 16 240. 3. 408, 504. 576, 62. 744, 440, 1008, 1 ^6 ai 1344 hours (Figure 2> To measure the serum sampl cn'tmrations from the PK. stody samNples the name lthod as Toentiontlad aEhe tot th" rat hatUtnokinetic sud\ w&a emoployed. the pharnmokmuete profile for both antibodies in enomobogous monkey us wasgood, ut the doWe normalixd pronle for the a 1IA-i 120.6 was margnalIi better than that of the aDNP 3A4 - , (00523] Exam de3 V00524] lHuman tissue cross- reactivity assessment [005251 ht £:eneral accordance with the guidance laid out in Pois to Consider ia the Amanue and. Testiun of MononlAabdPou tfrilmntw (48 Department of Heailth and Human Serviees Food and Drug Adminstraton, Center 1kw r Biol ogie IN Eahation and Researc h (I OW9)) 4 prelain-ary nond U study was caided out to determine erosreanaity of invenut n onoes with a unrety of human tissues If an antibody is iended for drue dev elonment a more extensive testing under OiLP conditions is required. Th tissue~} cross-reactivity of antibod~ies aD\P 3A4P d ni a K Li 120.6 was evaluatd (Cha ive Laboraoies, Preclinical ServieS, Reno, NV) wth cryosectis of slectend human tissues using Alexa~ Flunor 488I Labelcd form s of the test artcles Normnal human tsue f trnm two unique individuals hmess otheirwsi\e indicated) were obtaited from the Spcial Pathology Services Human Tissue Bank collected by the National Disease Research Interchange (NDRIL Philadelphia, PA) Cureline. Inc. (Bur ingame. CA L Cybrdi (RockviiIe, MD), or Rocky Mountain Lions Eye Rank (Aurora COY Tissues tested included huxman cerebellum, htng, cerebral eatex:, ovary (fro maure femnale' eye placenta, gastrointestinal tract (small ittineotm skin (1 individual heart, spleen, kidney (1 individual tLhrid, iver. estis Sectuns o t fresh-frozen human tissue' and control bead blocks (D NP[31]bovine ir album [BSA] beads [postiv] and human serum albumin [HS A beads [negatve] were e at on the cryostat and thaw mounted o:n capAinay gap slides. The tissue and control head slides were fixed in cold acetone for approxiately 10 minutes at W0"C to -25'C. The fixed slides were allowed to dry for a tlest one hour (to overnight t fored frozen, fixd slides were removed from the freezer on the day prior to an oxpermeat and allowed to thaw overnight prior to use, AIll the~ followingt steps were performed at room temperatre unless otherwise specifiedT slides were incubated with IN Morpihos"vemrfor approximately 15 inuteLs to preserve tissue mnorphology then washed two irms tOr approximately 5 mnutws each in IX phosphatelbuffered saline (PBSy To block endogenous peroxidase; the slides were iubated i a glucose oxidase solution for approximately 1 hour at approxi matehy 37"C The slides were washed two tmes in 1X PBS for approximately 5 minutes ehndogenous biotin was blocked by sequemial incubation (approximately 15 mnoues each) in avidin and biotin solutions. Following the incubation in biotn the tissue sections were blocked with a blocking antibody solution Or approximately 25 minutes, Aexa Fluor 488'-Ah 3A ' 101 F (nti-DNP), and AlA F Iuor 488 anti L H (anti-KLH Ab} were applied tO se ins ahthe otnna concentratior (2. g) or t t optimal concentration (0.0 pglnhIs tot aproximately 25ninutes.dusere washed 3 time Wh wash buffr and the icubated w ti econdaranibody rabbita Alme or 488 for approximately 25 minutes, ~oowing incubation with the sQc tIodaatibody, slides were washed 4 times with wash buffer then incubated with the tertiar antibody (horaeradishi peroxdasc conjugated goat anti-rabbit gG antibody)I for approximnatcly 25 rminues and bindingt visualized with a diaminobnidine (D AB) chromogen substrate. DYNP(31 I BSA beads were used as a positive control in all experiments, HSA beads were used as a negative control. Tissues were qualitfied as adequate fr immnunohistochemistry via staininng with an ambody against C131 (anti-CD3 I) i.e., platelet endothelial cell adhesion molecule (PECAXA I), There was no speci fie staingn any human tissue examined at either 2.0 or 10.0 pg/m concentration fr any of the tested anibodies, [005271 xpression and Purification of Monova1ent or Mutivalent Inmmnoglobulin- and/or Fe domaitoi n Peptid Analog Fusions [{0528] An assortmem of monovalent, bYalent and trvalent structures were expressed and nuri tied for comparison, including ex emplary embodiments of the invention. Those inleltWd AK LH IgG2/Fc-ShK varnts (see schematic represennaon of Figure 1E hemibody" configuraYi and antiKIL H ig62-ShK variantb. (.see Figure JF-L). For examptle bivale'nt Fc-iL 1h[35]. monovalent anti-K.ol I impet Iemnocyanin (KL H) inmrnunglobuin heavy chami-[Lys6jShK fuston antibody (designated "aKLH HC-[Lys16]ShK A b; see Figure I i nd mnovalent aniti-KLUH imunogtrloubn slih chnain [Lys6]SbK antibody (dsignated "aKh~ L LC-{Lyvs16]ShK Alt" see Figure lvfPgG F/F-K variastnts (sec Figure IA), bivalent e-L 0 -ShK[2-35], monovalent F e/Fe-L 10-ShK[2-3] wjere ~ made for comisotn, by recombinant methods as described in Suidvan e aL W() 2008/088422 A2, and in. particular Examples , 2, and 56 therein incorporated by reference in it\ entirety, or as miodified herein, [00529] iansient expression ystem used to eneraten eide anage fusions Qpeptibodi )or either maogiobuin fusion enbodimen HEK 2R3 E cells/ni in F I 7 medium suppleeed with L-Goutamine (6 tM) and GuaNticin (25 pg/ml) at 37C, 5% C) and shaken at 65 RPM. At the time of tnsfection, crils were diluted to L I x A0t celltm in the F 7 medium mntionedabv t% of the final culture volume. DNA comp lex was prepared :in Freesty 1e293 medinn at. 0% of the final culture volume. DNA complex includes 500ug total DNA per liter of culture. and .5ml PElmax per iter of culture. DN A complex is bify shaken once ingredients are added and incubated at room temperature for 1) to 20 n utes before being added to te cell cudture and placed back in the incubator. The day afer transfection, ryptone N (/L) was added to the culture frmi id 20% s'tk. Six days after transfection. culture was centrifuged at 4,000 RPM for 40 minutes to pellN the cells and the cultured medium was harvested thouh a 0.45um filer. [00530) In preparing the DNA complex, the ratio of plasmids was proportional to the des4ired molar ratio oft th peptides needed to generate the imnended product, The ofthe IG2 F/Fc-ShK include [gG2 Fc and FgG Ec-ShK at a : i D uringx expression these assemble htto lgC2 Fe homnodimers. IgG2 Fc/e-ShK heerodimuers, and IgG2 Fe-ShKIhomodimers. The lgG2 Ec/Fc-ShK heterodimer (monova lent form) was isolated during purification using cation exchange chGromaography. 100532 IgG2c FShKf ~ 35aG2 K k12-3xOKIaGC e .F-Si h 5 02 immunog aobul n Fe domain of' rnan (gCi MEWSW FFL SVTTV i iSER KVECPPCPAPPVAGPSVL F P P KPKDTL M SR T PEVTCMVVDVSHAEDPEVQFN WYV MDGVEV [N AKTKPR EEN STF RV/VS VL TVV4HQDW LNKEYKCKVSNKG LPAP EKTISKTKGQPREPQVYTLPPSREEM TK.NQVSLTCLVKGFYPSDIAMEWESN{3QPENNY KTTPPM LDSDGSFFLYSKL TVDKSRWVQQGN'VFSCSV/MHEALHINHIYTQKSLSLSPGK// (SEQf )NO' U fused in-frame to a monomer of the Kv L3 inhibitor peptide ShK[2-351 or a mutated ShK[2-35, Q16K] sere constructed using standard PCR technology. The ShK[235] or ShK[ 2-35, Q 36K] and the 10 amino acid linker portion of the molecule were generated in a PCR reaction using thW original Fe-2xL L-SK[2-35 in pcDNA I W)CMVi as a template (ae Slivan t a WO 2008 0S8422 A2 Example 2, Fignre 15A-B therein) The ShK -35] was generated in a PCR reaction ing the o nal Ex-ShK[1-35] in pcD)NA.'(+)MVi as a template (Sullivan t aL. W\ 20 08/88422 A2, Example b Fiur l4AAB therein) These ShK constructs have the followingu modify id \H2! Signal peptide amino acid. sequence of MEWSWV FE LSVTIVHSERK VEC CP// SEQ H) N):2 generated from a pSe leaxis-Vb2-IgG2-etemplate with the tfolowing olis 5' CAT CAA 17 C7 ACC AG GAA TG AGC TGG -3' (SEQ UD NO:3); and 5' (A CGW TGG GCA CTC GAC TT ( CG CTC GGA T GACO ACT -3' (SEQ lD NO:4t [00532] Wild Type ShK[2-35] with N-termin4 Baler exansion aino acid sequence GGGGSGGGGSSCIYTIPKSRCT A QOKHSMKY'RLSFCRKTCGTC/ SEQ ID NOT:) was encoded by the DN A sequence below: OGAGOAGGAGG ATCCGGAGGAGGAiGA AGCACTOCA AWGC A CCA C CCCAAGAGCCCTGC ACCGCCTTCCAGTGC(AAGCAC AGCAWT AAGTACC GCCTG AGC0TTCTGCCGCAAG ACCTGCGGCACCTOC//(SEQ ID) NO:5. A fragmrent containin this coding seque'nce (SEQ U) NO-5 was generated uning the olgos bLow (SEQ ID NO:7 and SEQ ID NO:8 -and the original FeL 0-ShK[2-35] in pcDNA3. (+ CMVi as a template (Su Iivan et A, W) 2008/088422 A2, Example 2, Figu re 15A-B therin, incorporated by preference: 5 47]V A ('AC T (AG ( AAA G1n (lA C f CA i C SE ID NO:;7 and 3' TOO TCC TOO TT ACC CGG AGA C'AG GG A GMAG 3//(SEQ [D NO:) [005331 Mutant ShK[235 Q16K] was generated ung "site directed mutagenesis with Stratagene's Quik(hanI Mlt site-Diretcd Mutagenesis kit *cat# 200531 per the manufacterrs instrucion. Oigos used to generate the nmtagenesis were: CT (WA C0G TC A\G O AGO ACA GO 3C (SEQ ) NO:9 and C- l TG CT G T T A CMI' (IA (1GC7GT (:( -3 (SEQ 1D NO: ); ad uin; the original Ee I 0-ShKj 215] i peNA3. IN )CM Y!as a t at (SOdknan ut aL W() 20OIH;O*422 A2, xape2, Fiue Atb thri) euiag inlh N GCACGAG GOAGTC GAGGAGCAAGCA CAICCACAd N which encode the amino acid ene Sh 5 6 w, h nu l xen t.o: G SGC K C S$SCiI PK SR CTIMK: K$ YR RL S H'R 1, I1C [005341 ShK[ 3jT get w A-F as enred sigheoriginal lFe,-,,2xL-,SihK4 1 Ct; MY as tp (SnUivan et W. W) 2008 4M2 A2, Example 1, figi 4A3 tein) and c 5 -(TO (:AC( TONG (X& ANAA (00 GAOI TG OA CAO OO(>3 (SEQ s >40t and CTG TOOG AO CS AA GG AC AGAG GTA G(S D G SUMGG NA GA A AAA-TG AG G-A A C GC GOT ONAA A QC ( G GNO:C GGa DNAWA coding equence 0(1ACA( /(!T'*-'::i C A T(T-C CCC CIA AMM&AC Q(i WAC'CO ('CYOTCTW(G A(AG ACAM ACTOGC;T ACTCGTOACOGTT 4CTCACAGACTCO C ATOOMA G itGC' WAGGE V3CA(OOC (AGGACTCCC \it ACTG(UV1iGAGAOCAT(I iCG COOC A A M AA M1TGCOK ACAOTO(' AC AA GAGCAGGTOGGCAGCAGGGG AACOTCTT CTCA TGCTCCGtATGCA TGAGOCTCTGC ACA ACCACTA.CACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAA A S SQ I) NO: 5. which encodes the amino acid sequence APPMAGPSVFL FPPKPKDTLMIS PEVTCVVVDVSHEDPE VQNWY VDG VE VHNAKTK PREEQF NSTPFRVVS V LTVVHQDW LNGKE YKCK VSN KG LPAP EK I SKiKG QPREPQVYT LPPSRZE EMTKNQ VS LTC LV KGFY PSD [IA VEWESNGQ PENNYKNTTPPM [DSDG S ELYMS KLTVDKS RWQQG NVFSCSVM HEAL UN[RYT QKSLSLSPK SEQ iD NO16), [00536] The PCR fragments were generated and the poduct were ran out on a get After gel purification, the DN A fragments were put together in a PCR tube and sewn together with ouWtside primrs: 5'- CAT GAA TIC CCC ACC AST GAA TG AGC TOG ~3' (SEQ ID N:) 3; and 5'~ CAT MGC (K'C GT CAT TAT CAG GG-3' (SEQ ID N:4t [00537] The PtR products were digested with EcoI and Not (Roche) restriction enzymes and agarose get puri fied by (Get IPurifieation Kit, At the same time, the pTT14 vector (an Amgen vector comamin a CMV pror Puromyvcin resistance gene) was digested with ERi and Nat retriction enzymes and the laMe tragment was purified by Gel Purification Kt Each purified PCR product was gated to the large fragmnt and tramsforme inob OneShot lop 0 bacteria. DNAs from transformed bacterial colonics were isolated and subjected to Eco~L and Not restriction enzyme digestions and resolved on a one percent agarose get DNAs resuting in an expected pattern were submitted for sequencingt. Alxt though, analysis of several sequences of clones yielded a I00% percent match w it the above sequience, only one clone of each construct was selected for large scaled plasmid purification. The final iT 14-MN I SP-IgG2-Fc construct encoded bgG2-e~-L. I0-ShK(i2-35) ifusion polypeptidie having the followmg sequene:~ MEWSWVF SVTTGVHSERKsECPPCPAPPVAC SV FPPKPKDTMI.SR TI M V)N ISECI VS I QFNW Y MMDGVS M I-N fI(VK PT FEQE NSI TF RWS MI TVVUQDWL NGREYKCK VSNKC P API EiKiIS KOQEPQ VYL PPS LMM 1KNQVSUCI \'iK('EMP SDIAVEWETSNCQPENaNKWTPML.DSDOSPEiSn.

KSR WQQIN SCSVMH EA N IYTQKSLSLSPG KGGSOGMGS SCU KSRCTAQCKHSMKYR. SFCRKTCT( (SEQ ID NO17). f005338 The pT I4XH2SPi1g2-Ec-hI 2 >Q6K) condneczencoded IgG2 1.. 10 S0K(-35) Ql1K) tugion pype hdd sequence: MEW SWVFLFLSVITGVHSERKVEC PPCPA PPVAGOPS VFFPPKPKDTL.MISR TPEV TC VVV DVSH P P EVQFWYV DV YE VI HNAKTKPR IEQE S1hR V V E'E Q. TVVHQDWLGEY KCKV SNKG PA PETI S KTKGQ PRPQV YTLPPSR EEM TKNQVSLTCLVK(GFY PSDIAVEW ESNGQPENNY KTTPPMLDSDGS8FF LYSKL. TV DKSR WQQG(NV FSCSV N EA LHNH Y TQKSLSLSPGK(GGSGGSSC D TIPKSR(TAFKC'KHJSMKYI RLSFCRKTCGT(C SEQ ID NO:I8; and pT 14Vi2SP- gG-F Sh( -5 consruct conaied a coding sequence for igG2 Fc-L41-ShK( ~35) fosio poaypeptide having the followi meenc: MEWVSW VF LFFLS VTOtGVHSERKVECPPC.PAPP>VAGP>SVFLFPPKP KDTL MISR TPEVTCVVVDVSH EDPEVQFN WYV DGCVE VHNAKTK PREEQF NST FRV VS L? IVVQDWL NGKEYKCKVSNKGLPAPIEKISKTKGQPREPQVYTLPPSREE NI TKNQV SLTCLVYGFYPSDLAVYEW ESNGQPENNYKTT PPM'LDSDGSFF LYSKL4 TVDK SRWQQGhN VFSCSVM E11BALHNHYTQKSLSL[SPGK GGGGlSGGGGiS RSC

DIPKSRCTAFQCKFISMKYRL

4 SFCR KTCGTC//(SEQ ID NO: 19. V0391 Giencadne the VH2 ISP gG2 Fenh contruct in pYNI 6 (an Amgen ve:in Ce ltIng[t CMV prolter POI A O~ .ia ni-Inrgogneh resince geTY necured as tfows:'The V02 sigymda peptide was gaenetated usng the foiloing z5-CAT AAG CTT C1CC ACC AlTG GAA TOG AGC TGG-3' (SEQ ID NO:20}; and 5 CA C1{G'TG GCIA CTC G1AC TTT CCC G GA GIG GAC ACC) -3' (SEQ 1D NO:4}, and using the pSelexis template as noted above. [00540j] The Fe region was generated using the pSelexis temphJte described above and following oligos: 3 i7( GAG TOC GAG (CCn AAAN iC GAG GIC(CA 000 GC C- (SEQ ID) NO<I and 5' CAT CiA TCC WA TTT ACC CGG AA CA GG G -3 '. (SEQ D NO:ll). [00541) The PCR Or ents were gel prified and sewn together in singe PCI'R reaction using outside primers SEQ ID NO:335 and SEQ iD NO:336 the resulhing POR fagmrent was gel pufed and digested by HindIll and BamH I . Conrmrentlvy pYD16 vector (an Amgen vector containing a CM V promoter, Poy N tAi and a Hygromycin resistance g"n was also cut by Hindu and BamHi and the lare vector fragment was purified by Qiagen's Gi Purification Kit The parfed PCR product was haed to the large fragment and transformed ino OneShot Top 10 bacteria. DNA fron transformed bacterial colonies were isolated and subjected to HindlIN ande BamPH restriction enzyme digestions and resolved on a one percent agarose getSr DNAs resulting in an expected pattern were submitted rot seqiuenicing. Althouhanalysis of several sequences ofdcones yleided a 00% percent match with the above sequence, only one clone was selected for 2rge scaed plasmid purification. The final pY9 D16WHllSP-hrgG2-Ec construct encodedi human hgG2AFe (SEQ ID NO:! above}. [005421 And AiK L , .... ......... -------.--K------tlaneaiesio sig h DNA 15 M INIM 2.N IVPgG2dl0 (> Shlif W5 Q 16111 onstruh, e fgment containing the DN A eodingi sequence CCATCCCA (N (A AG IAICA GCCf CCT0CATCGACACCATCCC AGA GCOCTGCAC CCC(TTC AATCA ACCACACCAIT AAGTACCCCTGAG CTTCTGCC GCAGAC(ITCGCC ACCT T AATGGC GC AGOC TCGACUC GGCAAGCGG lTC//(SEQ ID NO:22) was cut out usig B Daml FIBamH This coding sequence (SEQ HD NOI23) encodeYS ShKi T-35, Q6 ) h n IN-terminal linker sequence: (SGGSRSCIDTi PKSRC ITF KHSMKYRLSFCRKTCGTC// (SEQ lD NO: 23. [00543) At the same time, p'T l1 4-h!g2-e-ShK[1 -35]WT construct, was also digested by BamH/Bam~Hi, thereby removing the Shk[l35J coditg region to yield the coding sequence iAT(IGGAdeCTGT5Ydi SC'I"dY' dT' AGTAACOAOT'OOT$ (CACTCCONAGCGXlCAAAGTCGAGTG(XCCA (TCTGOI AGOAU ACTFo GCAO CC CAGTCT OCTCT TCCCCAAAACCCAAGGAA CCTRCAT GA T(CCCiOGA(ICCCiG AGQTCA CG TGOGTGGTiGGTGG A C~I)TAGOAG GA ACOdCGAGeK(A (A('u VA CGGC GA C T& ATAATCCAAGAC AAA CCAGOCAGA AGCAGTTCAACAC ACGTTCCG 'iGlNlAGl('ClI~lCCOl l1'CTG lAXOAGGAI'G '1GAACGOCA AO GaiACTAC AAGTCAAGGTCTCAACAA.(tCCCCAGsCCCvCOATCAGA AAACCATCCAAA ACC AAAGCAG(CGA GAACACA GOTOTACAC C TGCCCCCATCCCGGG AGAGATOACCAAAACCAGoTAGCTGAC TGCCTOGTC AA AG(I)TCTACCOCA (dGACAT(GCCGTGOAGTGGGA GCAAIGGCAGCCGGAGAOAACT ACAAG ACCA A('CCOCATGCTGOA CTCCGACGGCTCCTTCTTCCT CTA CACCAAGCT CACCGTGGCAC AA CAGCA CACAACCACACACGCAGAACAVCCCCI dTCTCCGCT AAACGAG GAGIGA /.!(SQ II NO:2A ecoding the &IlC naci seuence MEWS " "LS, V ERVEC( PA PV AGPSV PKP KIA 'SR TPT.VTCVVVO DSH1EDPEVQEi DG'VNI E I NAKTh PREEQFNSTFRV VSV. TV OVHQDW OK IY KECKV SN KG PA PIEKW KTKGQ PRlEPQVT PSRE M TKNQVSGTCLVKGIWYPSDA YE ESNGQPENNY aZTPM LD$DOGK LSKE ILQ 1D SR WQQGN SCS IMH FALI{NL C Q SN NE N9EG fN ( S EQ G S NO:2.$ [005441 The p'TT 4hG2+Fe vector wit the ShK renTved was treated with CalfC intestine Phosphanase (CIP) to remove the T Phosphate oup and Phennl/Chloroform extrated to prevent religation of the vector upon itself. 'The insert ShK[1-35, agent as gel puriea fromt up with Qingen Gel Purification Kit, The purinied insert was ligated to the large vector fragment and transformed into OneShot Top10 bacteria, DN As from transformed bacterial colonies were isolhned and s'ujected to~ Bam I restriction enzyme digestion and resolved on a one pceent agarose gel DNAs reutigi an expected pattern were submitted for' sequence ing, Although, analysis of several S0eXUnelCs of "bk\n gi idad00% percntem \1teh wih th aboeag ene only (W O C' \VUS eS tV f u scahed plaTnu ei.The yia IiT 1 gG RA hK ( Q6Kc onstrct encoded te ftowin 1gG2 Wi0-ShK(135 MEWSWVIFF L SVTTGVHSERKVP FP PA PPVAOPSVFL FPPKPKDTiL MISR TPE VTCV VS' PEVQFN WYV DGVEVHNAKTKPR EEQFN STFR V VSV TVHQWNGEKCKV SN KG L PAPIEKIS KTKGQPREPQV YTL PPSR EEM TK'NQVSLTCLVKGFY PSDIAVEWESNGQPENNY KTTPPML DSDGSFF LYSKL TV DKSR WQQGNV FSCS MH NUE ALHN H YTQ KSLSLS PGKGGGSGGGGSRSCI DTIPKSRCTPAFKC KH SMKYR LSFCR KTCGT(>I (SEQ ID) NO:26). (00545J Mhamain Lxpression of anti -KLH imnoglobuin eavy chain (HO components of the. AKL H Ig2F-b shmtclepresented by Figure. I E) 0056. (a) aKLH 20.6 kappalC SEO ID :28, beow hich incorpo Nermninal VK? SP sngad peptide seqtiencceSFQ 1) Nt 103): MDMRVN PPAQL. LG L L L L W LR GARCDIQMTQSPaSSLSASVG DRVT FTC RASQG I R NDLOWVYQQKPGK APKR& L7VAASSLQSOV PS RFSGSGSTE FTLl TSSLOPF ATYYCLQHNSYPLTFGGGTKVE KRTVAAPSVFIFPPSDEQ LKSGT ASVV CL L NNE YPREAKVQW KVDNALQSGNSQESVTEQDSKDSTY

T

SLSSTL TLSKA DYE KHKV KY ACEYT HQCLSSPVTKSFN RGEC//;(SEQ ID NO:2.8\ (00547J (b) aKLi 120.6 QgG2 HC (SEQ ID NO:29, belov) whi incorpas a N-terminal Vk-1? Sp signal pepide sequence(SEQ ID NO:1034: M DMRVPAQL.LOLL LLWLRGARCQVQL VQS GAEVKKPGA.SVKVSCKASGY TUTG Y; MH 11W VRQA PGQGLEWMOWINPNSGG'NYAQKFQORVTMTi'RDTSI STAY ME LSR LRSDDTA YYCARDROSY YWFDPW QR T VTVSSASTKG PS VFPLA PCSR STSEST A A LCLVKDYUPEPVTVSWNSGA L TSGV TUPAVLQSS GLYSLS S VV VPSSNFMTTYTCNV DHNK PsNTKVDKT VERKCCV ECPPCPA P PVA GPSVFLFPPKPKDTLM ISR TPEVTCVVVDVYS FlEDIPENWYVDGVEV NNAKVKPREEQFNSTE KVVS NIT VHQL)WLKG YKCK VSN KGb P Ei' IS KTKGQPRPQVYT.PPSR EETKN QVSETCM AK"E SD(AVENGQP EN$ K I iPPMLDSDO SFIL SKET YDKSR QQGNV Sf5 VMH EALHN$HY QKSSL SP Yd7(EQ DNO:29L [00469 eiE2 ict10.h( i $ MEWVSWVFLF FL SVfTGCVHSERKVEC{PPC PAPP VAGPS VFLFP P KKDTL N SR TEVTCVVVDVSHEDP EVQFN WYV DCVV N AKTKPRZ EENSTF RVVSV L TVVHQDWL N KEYK (KVSNKA LPA PIEKTISKTKGQPREPOVYTLPP EEM TKNQVSL TCLVKGFY PSDIA VEWVESNGQPENNY KITTPPM LD)SDGSFFL YSKL TVDKSR WQNVFSCSVMH .EALHNHIYTQ iSLSL SPC KCGGSCGG GSRSC DTtPKSRCT AFQCKHSM KY R LSFCREKTCGTrC//(S EQ ID NO:30OY [00550} The desired aKLH IgC2/FWShK product eontaind one copy of each of components (a)-(ck immediatev above, congwured as in [ure 1 E, Because o this, the ratio was i:1: L This product can be described as hal aiody and half Fe fusion~ ("hemibod'' coupled together at the Fc d.omaim Addtiona peptide assemblies that had to be removed ronm the culture wee The aKLAI Ab and the PV SiIK homodiracy [0055 p. '' ~~faos [00552 Th oponets Wk 1 C S0 fAson aIbody [0553 ) I H 20. k LC (SEQ )N. above [00534 ib at I 1 'SQ D NO a bov A 00555 l aK 0 G$SK fusion hang the g 1. sequence MIDMVPAQI.ELLEA IR-ARQVQ QGAE VKPEAS KASGY FTC W I NI I yV QA PG? EWM OWIN NSO V UAQKEQORI ITRTTS F STA YM E' LSRL RSDDTAVY YC ARDRGSYY WFDPWGQGT LV TVSSASTKOPS VFPLAPCSRSTSESTA ALGCLVKDYFPEP VTVSW NSALTS TFPAVLQSS GL YSLSSYVVPSSNFGTQT YTCNVD\ KPSNTKVDKTVERKCCVECPPCPAP PV AGPSVFLF.PPKPKDTLiiNJSRTPEVTCVVVDVSHNED PEVQFNW\ YVDGiVEV HNAKTKPREEQFNSTFR VVSV LTVVHQDWVLNGK EYKCKVSNKGL PAPl EKT ISKTKGQPREPQVY TL PPSREEMfTKNQ VSLTC: LV KCFYPSD IAV EWESNGQP ENNYKWTPPMNLDSDG;SFFLY SKLTVDKS RWQQGNVES(CSVMHELALHN HYT QKSLSL SPGGGOGGSGGG1S RSCIDTIPKSR CT AFQC KHS MKYR LSFCRKITG TC (SEQ ID NOG1). 005571 Thieoponents otanovraintaKLH216 0g2-SW 5 Q5 ftision mntibdy (shentad H reg-sented in Nunr I F) indtided Tfnonfltefl [005581 (a) aRIAH 1206 kappa C (SEQD I NO ahovce [00559] (b) al H 120 Ag02 FRC (SEQ ID NO 9,bove and [005)) (c) aLH 1206 lgG2-ShKI 05. QinM]fusion hav ntheIlong sequence; MDMRVPAQLLCLLLLWL RGARCQVOLVQSGAEVKKPGASVKVSCKASCY TTGYIHWVRQAPGQGLE.WMWINPNSCTNYAQKFQGRVTMTtRDTSI STAYMLSRLRSDDTAVYYCA RDR GSYY WFDPGQT VTVSSA TKGPS VTPAPCSRSTEST AAI LCLV KDYFPEPV TVS WNSGALTSGV TFPAVLOSS GLYSLSSVVTVPSSNFOTQTYTCNVD HKPSNIKVDKT VERKCCVECPPCPAP PVACPSVFLFPPKPKDTLM SRTPEVTCVV VDVS HEiDP EVQFN WY VDGVEVZ HN AKTKPREEQFNST FR VSV TV V QDLNG KEY KCK VSKLAPET I SKTKGQPREPQ VVTLPPSE EMTKNQ VSLIT'CLVKGFY'PSDLIA VBWESNCQP ENNYKi TPPMLDSDG SEFLYSK TV DKSR WQQNNVFSCS M EA[NYT QKSLSLSPCGGGGSCGGGSRSCIDTIPKSRCT AFKCKHS KYRLSFRKTCG TC// (SEQ ID NO:'32).

005611 The oponents oe'ltheianovaent aWL.N 206 HShf~l M-5. fA Q rh f,.naody (e Wna esentedin FHmreI)inuehd the Y ]owiong monomer : 00562i (1) WKUI 1206 kapp EC (SE d NOU28 [0563 ( HW. 1 02 HW SEQ ) ) [00564 1 JaKlH 1206 1YO2 HC-'ShMj 1435, IA 4.06 1tsmhv h MD) MRV PAQL LGLL LL W [iR GA RCQVMQ LV/QSG AEVKK PGASV KVSCKASGY TFTGYHM WVRQAPGQGE GINPNSGGTNY AQKQGR VT MTRD 1I STAYMELSRLkRSDDTVA VY YCARDRGSYY WFDPWGQGTLVTVSSASTfKGPS VFPLAPCSRSTSESTA AL(i(LVKD)Y JFP VT VS WNSGALTSG VHTFPAVLQSS GL YSL S SV VTV'PSSN FGTQT YTCN VDOHKPS NTKVDKT VEPRKCCVECPPCPAP PVAOPSVFFP PKPKDT LJISR TPE VTCVVVDV HEPEVQFNWYVDVMEV HNAKTKPREEQFNSTFRVVSVLTVV QD W NK EYKCKVSNKG[ PAPIEKI' ISRTKGQPREPQVVTL PPSREEMNTFNQ VS LTCLVKG FY PSD0 AV EWESNGQP ENNYVKITPP MLDSDG;SFFLbYSKLTVDKS RWQQGN VFSCISVM H EAW LHYT QKSLSLSPGGGGGSCGGGSASCAD'TPKSRUTAFKCK H$MKY RLS FCR KTCG TC (SEQ if) NHO34) The desied mooalem aKLtH 1206 l gGZ i:{-ShK anal ogne prodnet w as a fallI antibody wih the SK peptide fused to the (Mermius of one heavy chan With two dioffrent heavy chain sharing one variety of light chaI, the rao of heavy chain' chaing 1 ht chm heax vchv -ShK w as 1 T 'he expeed epsi produes are aKLH 120 6gG anfbod\ mn aent 0K2 WI 0.6 Qg2 HCShK peptide analg and bi1 alen aki Ct 10 n b(tb1 peptide analog. The tmnovalen a.K LH 120/i 1i HfoON~f >ple iorontolnmmibhody wasisolated from the mim usmg cntion exchange ciuom tograph1y, as descrbed hereim 00565 1 he componet oflte monovaent aKEE ($ IIA ShK~ 15 RIA Q I , K30E] f2R6 anody (sMheWKa30 R Aeseti FgreFnehe toIlowig monomens: fPO66] (a) aKLH1 20ZS kappa U(SEQ ID NO:8: 0056 (1) AKti 12 ig-Sht A, Q 6 K3OI-iI fuion having the fo1owing sequence MD MRV PAQL LGLL LL W L R GA RCQVMQ LVQSG AE VKK PCASV KVSCKASGY TFTGY - M\ H WVRQAPGQL EW VVGWINPNSGGTNY AQKFQG R VTMTRDTSI STA Y ME ELSRL RSDDTIVAVV YC ARD)RGSYVY WF 7 DPWGQGT L VTVS SASTKGPS VFPLA PCSRSTSESTA ALGCLVKDYP FP VTVS WNSGALTSG VHTFPAVLQSS GL YSL S SV VTVPSSN FGTQ'T YTCN VDOHKPS NTKVDKT V ERKCCVECPPC PAP PVAGPSYF LPPKPKDT LIISR TPE VTCVVV DVS3 HEDPEVQFZNWY VDVEV NAKTKPRE EQFNSTER VVSV LTVVHQDWL NGK EYKCKVSNKG LPA P EKT ISKTKGQPREPQVVYTL PPSR EE MfTKNQVXSLTC0 LV KGFYV PSIAV EWESNGQP ENNYK WTPP MLDMSEFLYSKLTVDKSRWA QQGNVFSCIS VMEALHNHYT QKSLSLSPGGGGCS GGGGSASCI D l PKS RCT AF KCKS NI KY R LSFC RETCT C'. SEQI ) NO305 The dcsircd monovalent aKLE 120> IgG2 HCShK analogue pmduct was a full antibody with the ShK peptde fused to t Cuterminus of one heavy chain. With two difeent heavy chains sharing onc variety of 2 ight bhain, the ratio of heavy chain: chainlight chain:heavychain-ShK was 1: The ex pected expression products are aKL I 120A IgG2 anibody, monovalent aKLH 120 6 gG2 HC-ShK peptide analog, and biilent k LH 20.6 gG:? HC- asK peptide analog. The monovalent aKLH 120.6 VgG P C-(toxin peptide fusion-contaiining antibody was isolated fmm the mix usi ng cation e.xxhae ehroma togniphy as described~ herein.

[0405694 live components of the monovaen. aKtHiJ20.6 HC (IgG2Y~hK(1.5 RIH. HA, Q 16K< fusion antibody (sehornade a c.seented tn Pigure F) inldd mGonomers [00570, (c) aKL 1 2(16 kappa hC(SEQ 8DN:) [OO571 (b 1 g (SEQ D 9 ); an [00572, (c aKI 1K 206 H IgGISnK 135, 1 QA, fu A&sio having We fol owini amino aci sequence MD MRVPAQL LGLLL LW R GA RCQVQ LVQSG AEVKKPCASVKVSCKASGY TFTGYHM HWVRQAPGQGL EWMGWINPNSGGTNY AQKFQGR VTMTRDTSI S'TA Y M ELSRL RSDDTAVY YC ARDRGSYY WFDPWGQGT L VTVSSASTKPS VFPLA PCSRSTSESTA ALGiVKDYP FP VTVS WNSGALTSG VHTFPAVLQSS GL YSL S SV VTVPSSN FGTQTi YTCN VDOHKPS NfKVDKT VE1RKCCVECFPPCPAP PVAOPSYF LPPKPKDT LMISR TPE VTCVVV DVS3 E DPEVQFNWY VDVEV HNAKTKPRE EQFNST FR VVSVYLTVVHQDPWL NCGK EYKOKVSNKG L PA PiEKT' ISRTKGQPREPQVVYTL PPSR EE MTKNQVXSLTC LV KGF PSDKi AV EWESNGQP ENN YI TPPML DIS QKSLSL SPGGGGSGGGUSH SOADT PKSRT AFKCK H$MKY R LS FCR KTVG TC// (S EQ 0D NO:306). The desied monovalemt aki l 1201. l gGZ NI-ShK analogne product w as a fallI antibody wh the Shk peptide fused to the OMtermius of one heavy cha With two JAffrent hay Chains\haong one vaety ot ight cha the raio of hea chai ha ight chamnteavcal-ShK w a T12 Ihe e-ctdexreson produe A H 120 Ig6h b mon aent I hI 2f &gb2 HCQ-ShK peptide analo and bis aLet akI H 016 N 2 H(\hK pepaide analog. 'h taonovalen aK LH 20A ts H n t peI ptid rior-sotomm ibody was isolated from the mis usmg cntion exchauge ehuoma tography, as descrbed hereim 33 bhe cmnmtpolnftS of l;en vnoae Ka&L 1 CHShK1 Q1 OK. 130E.] fuo cantihody(scdcmntcati WpremedQT i itr icudedi t monomer: 004 a aKLH 1206 kappa W (SEQ ID NO:8) [00575 ) 1 02 EQ 9NO:); ad [005761 (&) aKL 20,6 [gG2-ShK -35, R QI 6K, K301 E ion having the MD MRV PAQL LGLL LL W L R GA R.CQVQ LV/QSG AEVKK PGASV KVSCKASGY TFTGY4\HM HWVRQAPGIQ{LEW VNGWINPNSGGTNY AQKFQGRVMTRDTSI STAY M ' ESRL RSD DIT A Y YC ARD RGSYY WF DPWGQGT L SV T SASTKGPS VFPLA PCSRSTSESTA ALGCLVKDY JFP VTVS WNSGALTSG VHTFPAVLQSS GL YSL S SV VTV'PSSN FGTQT YTCN VDOHKPS NTKVDKT V ERKCCVECPPC PAP PVAGPSYF LFPPKPKDT L M1SR TPE VTCVVV DVSH EDPEVQFZNWY VDGVEV HNAKTKPRE EQFNSTF R VVSV LTVV HQOWL NGK EYKCKVSNKGLPAPiEKT ISKTKGQPREPQVVT L PPSR EETKNQ VSL:TC LV KG F? PSDAV EWESNGQP ENtNYK TPP MLDSDG SFFLY SKLTVDKS RWQQGNVFSC1SVMHEALUNUYT QKSLSL SPGGGGCSGGGGSH S~l DXI PKS5RCT AF KCKHSMN KY RLSFC RETCOT C'f NBS Q 1D NO:30t) Tihe desired monovalent aKL H 120* 1gG2 HC-ShK analogue piduct was a full aintibody with the ShK pepide used to th C terminus of one heavy chain. With two difeent heavy chains sharing one variety of ight bhain, the ratio of heavy chain: chainwight chain:heaveyhain-ShK was 1:1 The ex pected expression product are aKI H 120A Ig12 antibody, monovalent aKLH 1.20,6 gG2 HC-ShK peptide analog, and bivaent aKL [10.6 lgG HC-ShK peptide analog. The monovalent aKLH 120.6 (G 2 H C-toxin peptide fusion-eontaining antibody was isolated mm the mi, usi ng cation exchange chromatography as described herein.

I09 The comnnts of tle moanovaent a~H16 HShifl Ri K A, A161K1 usion autbody {secmatiahy orseted n FhrIF inchudkdthe Mcnptonekr [00575 (8] KU120. kappa C SE D NO028) [00579] (b) as. 1 20.M6l - ICt ,, (SEQ i) 'NO(:29.); a [00580) {e aK 2 HS W[ . i K. 4A, 1 in havirg the fos ini g sequence AD MRV PAQL LGLLLL W LR A RCQVQ LVQS G EVK PCASV KVSCKASGY TFTGYHM yf.HW'VRQAPGQGMVNGWRNPNSGGTNYAQKFQGRVTMTRDTSt STA Y M ELSR RS D AV Y YC ARD R'GS YY WF DPWGQGT L V TVSSASTK(PS VFPLA PCSRSTSESTA ALGCiLVKDYP E PVTVS WNSGALTSG VHTFPAVLQSS GL YSL S SV VTVPSSNFGiTQT YTCN VDOHKPS NTKVDKT VEFRKCCVECPPC PAP PVAGPSVF LPPKPKDT LMSR TPE VTCVVV DVSH HEDPEVQFZNWY VDVEV HNAKTK, REEQFNSTFRVVSV LTVVHQDWL NGK EYK(I'KVSNKGIL PAPILEKI ISRTKGQPREPQVY T L PPSR EE T NQ VSLTC LV K(GFY PSDAM EWESNGQP ENN Y KTT PPML DSDGSFFLYSKLTVD)KSR WQQGNVSCSVMH EA L NYT QKSLSLSPGGGCSCGGSKSCADYT I PKSRCTAFKCK HSMKY R LS FCR KTCG TCL/ (S EQ 0D NO:308. The desied muowwacnm aKN I 1206 l gGZ NC-ShK anal one prodnet w as a fallI antibody wih the ShK. peptide fused to the (Mermaus of one heavy chan With two JAffrent haXy Chains\hrng one vaety ot ight cham, the raio of hea chai \tham ight cham 'heav\. enshK wTs12 he egete ex\Ssion4 produces are aL W 120 Q02 KIMnJbod mn aet 20 gH h peptide analo and bis aln aKi H Li 1 c bs(I ( hK pupaide analog. 'he taonovalent aKELi 1( 01 NC H iUn Nepdi 016 iorsontdmIGg antibody isolated from then mH img action echauge eoma tography, as deribed hereinm [0$ T1 no ciannt& tflnhcnoalen aLH 6 K LShl d 1 I K Q 6K, K30E] fion antibody (schemail reqeseted are Hc ined monomers: [0052 K 206 kappa LC (S N28) [0058$) (b) A.k Lli 1.4&ll(- L2: HK 'SEQ N' O2§ "); an [00584 (1) IJ KI 12, 6 If6,2,'-ShK 1 R 1 Q QI16KW 13 0IEQ fusion having 1 he fol owing amino acid ecquenwd AD MRV PAQL LGLL LL W LER GA RCQVMQ LVQSG AEVKKPCASV KVSCKASGY TFTGY HM M H WVRQkPGQGL E MGCWNPNSGGTNY AQKFQGR VTMTR 1RDT STA YMEL SRLRSDD9TA VVYCARD RGSYWFDPWGQGTVVSSASTKGPS VFPLA PCSRSTSESTAALGCLVKDYPP VT VS WNSGALTSG VFITFPAVLQSS GL YSL S SV VTV PSSNFGTQT YTCN VDH KPSNTKVDKT V ERKCCVECPPCMPA PVAGPSYF LFPPKPKDT L. MISR TPE VTCVVV DVSH E DPEVQFN \WY \ 2 VDTVEV HNAKTKPREEQFNTF~ ERVVSVLTVV[HQDWILNG KEY KCKVSN KG LPAPHSI' ISKTKGQPREPQVYT L PPSR EEMTFNQV XSbIL:T LKGFYV PSD[I AV EWESNGQP ENN YKITPP MLDSDGSFFLY SKLTVDKS RW QQGNVFSCS VMHE[ALH[NH[YT QKSLSLSPGGGGCSGGGGSKSLDIPKSRCTAFKCKHSNIKYRLSFCRETCGT C) (S EQ KD NO:309), Thve desired monovalent aKLN 120.v iG HC-ShK analogue product was a fl antibody with the S'K peptide fused to 1t C teumi nus of one heavy chain. With two dflerent heavy chains sharing one variety of ight chain, the ratio of heavy chain: chainrhiht chain:henvchain-ShK was 1:1 The expected expression products are aKl E 120A Ig(2 antibody, monovalent aKLl .20,6 igWG2 C-ShK peptide anaog, and bivalentaK LH E 120.6 IgG2 HiC-ShK peptide .analog. T1he monovalent aKLH 120.6 1 gG2 HC-toxin peptide fusion-containingt anibody w as ioloated~ from the mix> using cation exchange chromatograiphy, as described hereimt 005 'he components of'a nonovalent 16 t1k20 - Sh215. QI6K] fusion wit ody aheTnat y F'rsent edO g Fin cednicider: 19l [00586J a) aKW 124 kapa iC (SEQ ID .NG28 above: [0 0$58 6 (a a.KLH 120.6 (SEFQ U)N&2Ya c n MDMRVPAQL.LGLL LWLRG ARCQVLVSGAEVKKPGA SVKVSOK ASGY TFT Y M NI HWVRQAPGQG LEWM GWINPNS GTN YAQKF:QCRTT iRDTS ST AVM ELSRhRSDDT)1AVYYC A RDRG(SYYWF DP WOQGTLVTVSSAST1KG PS VFPLA PCSRSTSESTAA LG CL1V KDY FPEPVVS T WNSGALTSGVHTFPAV LQSS GLYSLS S V VT VPSSN FGTQTYTCNV DHK PSNTKVDK VE R t (KCCIV ECGPPC PAP PVA GPSM LFPPK PKDTLM I SRTP EVTVVDVSHEDP E NWYVMDVEV HNAKTPREEQFNSTERV VSVVV V Q)DW L KEYKCKVSNKLPAPIEKT ISKTKGQPREPQVVTLPPSREEiMTK(NQVSLTCILVMKGFYPSDIAVMEW ESNGQP ENNYKTPPMLDSDGSL SK LTVDKSRWQQNVFSCSV : iA LHNHYT QKSL.S LSPGGGGGSGGGGSSGI DTWPKSRCI TKGKHSMKY RL.SFORKTGT C/ (SEQ ID TW3). [00589] The desired aKLH M 20.6 Ig32-ShK anal product was a fl. antiody with the Sh peptide fused to the (terminus of one heavy chain, contgured as i Figure 1F. With two different heavy chains sharing one variety of liht chain. the lato of heavy chan:light chan:heavy chan-ShK was 1:2:t The expected expression products are aKllH 12.6 1gG2 monovalent 'KL H 120.6 gG-SK, and bivalent aKLH 126.6 1gG2-SK. The nonovalent aKLH 1206 lgG -toxin pepide (or toxin peptide anaog) usion antbody was isolatedd from the mix using cation exchange chromatographx, as described herein. [00590) Ati-L H IUiacoorA bK. The aKLH Igloop-ShK als had a single copy of the ShK peptide sequence insened into one of the heavy cbains, but in this caso it wax inserted imo an interna conjugtion tn the Fe domain instead ofat the C terminus, (Sec. g., Cegg c' A U S. Patent No, 7,44 7; US , Patent No, 7,655765; U, Paten. No 7,655,764; US, Paten No 7,62,31 U.S, Patent No, 7,645,861 pubMished U.S. Patent Applicaions US 2009/0281286; and US 200) 0286964,each of wich a incorpoad herein b refen in e ei ccs. Ithv ponI=wens tofheK W I kdhK Laibody nduhe [005911 (a) aXKAM 20.6 kappa (SfEQ IDN)ae [00592 bL) aH I 2 0. I( 1 HIC MOMRVPAQI.LLL[LWLRGCARCQVQLVQSGA EVKKPGASVKVSCKASGY TFTG0 Y I MHW VRQAPGQG LEWM OWINPNSG GTN YA QKFQORTMiTRDTSI STAM 'ELSRLRSOTAVY CARDR(S YY WDWOQGTLVVS SASKG PS VFPLAPSSKSTSGGT A ALGC(LVKDIY PVTVSWNSGA LTSG VHTFPAV LQ SGLYS LSSWVTV PSSSLGOQTY CNYVN HK PSNT TKVDRV EPKSCDKTHTCPPC PAPEA LGGPSV FLP PPKDTL N ISRTP VTCVVVD VS ED PI" EN WY VD VE VH NAKTK PREEQY NSTYRVVSV LIV L1 QDWLNGKEY K K VSNKA LAP SEKTISKAKGiQPR EPQVYTLPPSR EEMTKNOVSLTCL VKGFYPESD) AVE W ES N GQPE NNY KI TPPVLDOSDGSFFLYSK LVDKSRWQQGNVFSCSVMHEALUNH YTQKSLSLSPCKA (SE £9DT NCO:34); [059n and [00594) 1c) aKI 26 igO I oon hK: DMRVPAQALCLLW GARCQ VQ QSGA VKKPASVKVSCASOY STAYNiELSRRSDTf YCARDRG SY WFDPVGQGTI VWSSASTKGPS MUPA PSSKST SCOT AALGC'VKOYEPM'VSWNSnAT SC VHWEPA MLS SaLYSL SSVVTVPSSSLGTQ iINVIIKPSN IKVOKRVEPK SCI KTHTCPPC PAP UGOPSlFPKPKDTL SRTPEViTCM DV SH1INPN LVKEN W MDC WE "IN AKTK PREQYNSTR VRVS [TV HQWL NKEY IC, KYVNKA EAP EKCTISKA KGQPRE PQVYTLI PSRDEI 2 OTIRSC DTIPKSRCTA KC KM SMNKY 1 L NFCRKCGTCGTKNQOVSLTC IKONFYPSD [A VEWESNGQ PENNY KTTP PV DS DOSFY YSRil DKSRXWQQGN VFSCSYMMAHOYTQKSLSLSPOK (SEQ D0 O35).

[00515J With tvo difeeintheavy chais shoing liht cha th aio of he canightichin:ieay ichainSSbK i Thn cxpeded expreao RductS r aKd l{h6igb l tanovalent di LI 0 6 { igGlI10 >opihk. tn. biv;n ed aKLH 1206 lgG op~Sh t notovana 1 120 g A op-Sh usion anithoy (tepreneahemacally bFg Nr 0 t isolate rom the bi FsIn canon exchange d matographyas descred herin. [05%] Monovalent sKi H 120 karia I.CShK{ 35. 0K] fsitn The component of"the monaent aKL 1206 kappa WS-ShKi-3, Q16[< frsion anboys emavicai represented i gre . J n ie the monome [0097 KL 2( g2 1C S t N0:29 [00598] () K Li 120.6 kappa CF (SEQ I9 NO:2f and [0099 (c)aKI 120 kappa QC SK -5 1 6K fn WhaA the ol fowing sequence [0O600] MDMRVPAQLOLLWLROARCDIQvTQSPSSLSASVCDRVTITC RASQC RN DLG WY QQKPG KA PK R LY AASSLQSGVPSRFSGSGSGTET L IS SLQPEDFATV YC 1LQ HNSY P LTFGGGTX KVEI KR' V A APSV IFPPSDEQLtSOT A SVVCI.LNNFYPR EAKVQWKVDNA, QSONSQ ESVTQDSKDTYSSSTLT LSKA DlY EKHK VY A CEVT HQOLSSPWVT KSFNRGECGGGGSCGGSRSC IDUTI KSR.CTAFKCKH SM KYRLSFCRKT(:C// (SEQ ID NO:267 I. Ths embodiment o mofnoalent aKLH 1 206 Ig) I (C~ l 1- QI OKj product as full anybody Wth the ShlK pqpde fused to the itermnus of one Ight chan as shown in Figure 1 \\nh two duiernt hlgt chains h g .e vaney of heavy chaim, the ratio of light cta hairhv chai~nlhght en mn-ShK[1 Q r (k1 ni3as 1:2.1L The expeed expresion products are tK I H a< 0 6 lgC monov alent aKLil 120.6 IgGIt -Shl 35. Q16K|- and i uhi U '0 206 Q2 1 CNShK 1-31k Q 1(j Te monovalent aK!t.H 1 20.0 Q g62 L \i peptide usior-contain antibodv w a isolated f rom the nos tcva eatiou exchange chromatography as deebe herem.

col ln13 sit OF OWe monxi {aIf 1201 okappa I Sh K{2-35, QIOK Ifhsion anbiode (seemat cally presented maui g i de udedSt 1 nonrs R}0602-f g K > W 1 1)~ QI NAI'4 [OU031(hi aYLIE 120AS kappa IT (SEQ 1D NO.2H4 and 00664 (c) tKLH 1206 Skappa C002051 ilitsioi h S KQPED T LQ Y G STK K VS P RIQLKSG asvy sho in ""-' vo chase s shog mayofstssa This mb7odinn5 of aent aKlHI2QS 1W20 6 SL O h2, Qi 6K product s adytlb SEK ppade (sed to the enii ot oneQ h chain as sW in ICe I th di n ChS \ a of Using Won he Mapne es 1, kappa ' hK 5 o6K I Mim l IgG2 E(SK23. 6j n N~aes dKI1 W1( IK * mniod W. isehemail HY represed i) JF' . I K (1 inclue th.mnoes [006071 a M Mil 12046i1012 f K: (SEQ ifD NO 2 ), anwd:. [006$ 1 (b KI ul 20. kappahLK[ 1-35 1 O~lIin(SEQ I'D N(>267v' shove, This embodiment of bivalent aL 120.6 gO LC-ShK[15, Q16K] antibody product was a fuh antibody with the ShK peptde hused to the ternkus of both eight chains as shown in Figure 1K. The ratio of hoay chainght chain-ShKi1-35. 116K) was 1:1. The expected expression product is bivaem K 1 120,6 Qg2 L ShK[-35. Q16K. The bivaent aKLH 1206 igG2 LC-bKI5 Q .16 K] peptide fusion-containn antibody molecide was isolated from the mtix usingz cation exchange chromatography as described herein, [00609] Bivalent aKLH 120,6 kappa LQCShK2-35. 016K) htsion. The components of the bivalent aKLH 20.6 kappa LC-ShK[2-35, Q6K] fusio antbodios (schematicaly represented in Figur K ) ,n ded the monomers: [006 10] a) aKW ii20 1 2 A SQ i S Nt 29 and [Q0 06 b (b) a1 f.120.6 kappa IC-Sh K[25 Q3 I Kvsion SEQ tL NO268) above. This embodiment of biviaent aKLH 120.6 igG2 L h - Q1 6K] product was a ful anibody w ith the ShK peptide fused to the CItenninus of both light chains as shown in Figure 1K. Th e ratio of heavy chainlight chain - 35. Qi 6K) was 1:1. The expected expression product is bivalem aK.LH i 240.6 1g2 L C-ShK[2-35 Q%6K] antibody, The bivaen aK L 20.6 >g2 LC-ShK[2-35 Q16O] toxin fusio-contanig antibody was isolated from the mix using cation exchange ehromatography as describedi n. compnnts otf the a civt a J<tb 120. kappa C.h [ 5 K]Iio Qa!tiod is schemnata repsedi Fu 1) in cuded the m no st :0f0)6 13 a) aKSi1 Q20 IgZ 1(17 (S EQ 11) W29. above o [006141 (b);aKH 12. I it 0k 2 HQ&Sk[V 3 5. Q 1 6K] WbON hNving t he anow acid of SIE If) N:32 -,sowe;a.

[006151 lI206 kappa Shk 3 Q16 un havt theamto icid sece enceo MSNQ D NO:27 abo This emubodimem of trivalen aKL H l 206 1g02 lC-ShK product was a full antibody with the ShK[ 35. QI1K] peptide f of both light hains and o heavy cain a, shown in Figure L With two differ heavy chains sharing one vaety of ight chain ,he ttiok of heavy chaight chan-ShKl[ 1 -35, Q 6K]heavy chain-ShK[ Q 1 ws : L The expeed expression products were a bv alent aKH 1,20 )gG2 LC -ShK[ 1- 01 6K] antibody. tivalem aKL H 120s6 JgG2 LC-ShK[1-3h Q I K] antibody, and tetravalent aKLH 12026 9g2 LC ShK[ K35, Q616K anybody, The trivalent aKLH 120.61gG2 L(toxin peptide fusion -contaiIng antibody nmoecule was isolated from the nix using cation exchange ch romtograph ats described hetrin [006161 irjvin KL.FI 6 a .CNSK295 ktQhIJM he mpoent of theaAle i 1216 kappa insh1g3 Qi K n anybody {chemaily eoeseted inre i incded the monomers [00617 a) AM 2 0 SEQ RI) NO:29) [0061 (b) ALI 120 C62 H SQk 1 0161<] fusion {SEQ ID N023), above and [0(c19 akiLl Q06 kaup 1,ShK2C5 Q 1 Kulion SEQ D ND 2W)68 above. This nodtmet of trlalent aRLAII 120+ Q62 11MK120AVO lCAl2dtlh product wa a uill ambody vith the ShK[^ QIK] peptide fhed to the C> tenmuwf both ight chains and one haxy chamn as smho% i a iu an L With two different heav y chins harigm one aey of cight e Aln he rako of eavy eba n ighlt ch hAin I -35 QI 1 fheat \ vy. n l,2- a Qa & 3 0X 6< a I 1 'Ph' expected expreion products were A i a i a Hi 1A6 1 g) 6 t C hKU 3$5 Q6K] antibody trivalen aKf 11 120 6 1gG 2 L(CSbK[2-3i Q I 6Kj anybody and tetmrava enta KHll 120% LgOCth K 6K) atiody. The trivalent aKdH 12( .titoxn eptide t incntaf nt (UrflOdy moleule Wai isolated trien tja u ting ca On edumgeC cronatograpnv as eeserie eeina [00620] Anmi-KL H 120.6 Antibodyv Light Chainnmamrnalian exp VrOsfon T he noMouset hybridomna expressing KLH monoclonm a l aibody 120.6 was used as a source to isolate total RNA. usin TR o® reagent (invitrogen irst strand cDNA was synthesized usng a random primer with an extension adapter 5 GCC CG ATA GGC CT( CA NNN NNT3(SEQ jD NO:3) and a 5' RAC (ad amplification of CDNA ends) was performed using the GeneRacer Kit (invitrogen), For the hight chain sequence determination, the forward primer was 5> TG OTT GAG AGG TGC CAG ATG TGA CAT TGT GAT GAC T[CA GTC TCC -3 ,SEQ H) NC:37 and the reverse primer was 5' A AC CGT TTA AAC CG CC GCT CAA CAC TCT CCC CTG 3TT AA \ (SEQ iD NO 8). The RACE product was cloned into pCR4-TOPO (Invitrogen) and the sequences determined. Consensus sequences were used to determnne probable framework and signal peptide sequence and design primers for full-length antibody chan.PCR amplification. [006211 The expression clone for the anIiKLH 120.6 kappa hght chain was prepared by PCR. The 5' PCR primer encoded the amino terminus of the signal q ,an Sal restriction enzyme site and an optimzed Kozk sequence S'-AAG CTC GAG (T C GAC TAO ACC AC> ATG GA A AG GC CCC 0 3' (SEQ ID NO:39). Tbe V primer encoded the carboxy! terminus and termination codon, as wel as a Nt restricion site 5 >AAC ( ( 1 A A C CCC OCT CAA CAC TCT CCC CTGTG AA W (SEQ ID N:38). Ti resuming product wvas clond into pCR4-01P) (Invtrogen) and the sequences determined, After the insert as connrme, the pCR4~TO product was cut with Sal and NAL the insert gel isolated and Qgen puified, ad then lighted into the mammalian expression vectorp (00622 A C ain to ehan the ignai eptide nom then C-peed dernv se tro the hybddoma to the K 1/1 nentid The pimers ued iM the VK /012 fragment were ( AG CTC GAG GTC GA AG ACC ACC A GA( ATG AGG GTG CCC GCT 3' (SEQ 1D NO:40) and S-C TC G1 A TGT CAC ATC TOG CAC C 3' (SEQ ID NO:41 The priers used for the mature light chain peptide were 5GGT GCC AGA TOT GAC ATC CAG AG A - f3' (SEQ kD NO:42) and (SEQ I) NO:38A. The resulting fragmna\ were joined by overlap POR VsNg primers (SEQ D NO:Ao) and (SEQ ID NO:3t The equece ofthe rehing clone encodes the following inunogohbuin kappa LC sequence MDMRVPAQL LGL LLLWLRGARCDIQM TQSPSS SASVGDRVITCRASQIGR NDLG WY QQKPG KAPKRUL YAASSLQSG VPSRFSGSGSGTEFTLT7ISSLQPEDF ATVYCLQHN SYPLTFG(GGTK V QIKRT VAAPSV F PP PSDEQL K SK TA SV VCL NNFYVPR EAKVQWKVDNALQSONSQESVTEQDSKDSTYSLSSTLTLSKADY E KUHKVYACEVTHQGLSSPVTKSFNRGEC//w(SEQ ID) NO-28). jv&.3 i'L6.5106 At ntibojl' 14god Cihiin-Seli pedeado i maninmmm a CVgavg. ie S4I14-5. 16K fragmnen as gen'rnted bi PC R using the pITT 4~huicG2Ec ShiK 1-3Q 1K] encoding (SE5Q 1D N026 described above. as a tmplate anid the ol ios 5A4.('AGO OGA OAO TOT 0-GA G0.4 GGA. 0 7CC 0 0 -3 SEQ ID NO: 691: and - VAT 0 00 0CC OCT '(+A1' T\ AO (+ 0 3' (SEQ ID \O:2'0i. [006241 The bicht chain fragment and 5lK POR podule uere then amp.fed by PC R usirig the outside onimers CA\T TC'T AG A. AC 4C tC A1 TGO A7 400A OTOG (SE Q 1D N0343t and SEQ 1D NW.2'0 The PCR product w as then digested by Xhail and Noti and POC c lean up kit (Qiagen) purified At the same time, p\t D1f6 w as c ut by Xbdal ad Notl The pY D1.6 vector wa mun out on a 1% stgarme get and the larger fragment wis cut out and gel punfid li Qiagen's Gel Purification Kit. The pun tied POR product t s higated to the large vetior argennd tumsfonued imo OneShot T op1 0 baceria, D\ As fiom transformed bacternal colonies were 1s0iated and subjcted to \bal and Nodt restroeuom &B2ym dtg&ttonm and resobved on a one percent agarose get. D'N A resulting in an cxpected pattern were submitted tot sequencin Althg alM of aevral Sequences of'on es k ad S 100% percent match w ith the above sequence only one clone w a cted for larke scald plasrmid pudicati The AN'm pYD16 Ki i.'K ISPA .: 4 bK 15 Q OK] construct eneoded an At H I 2L 6 i 1YShK[ 11 Q 1 6Kj poly peptide (S EQ 10) \O: 2&7} r006251 The Shk[2-35 Q 6K fragment s encred as described aboe usn fIaKTl20.6 HShK ~t Qn Q13e5.eand the oh onucleoide prners SEQ U NO269 andSEQ ID) N0270. [00626] The light chain and ShK PCR products were anTlified by PCR using the outside primers SEQ ID NO:343 and SEQ M) NO:270, The PCR product ws ther digested by Xbai and Not] and PCR clean up kit (Qiagen) purifed. At the samte time, pYDi6 was ct by Xbal and Not1. The pYD16 vector ws run out on a 1% agarose el and the larger fragment was cut out and gel pmuifie by Qiagen's Gel Purification Kit. The purified OCR product was gated to the large vectorfg and transformed into OneS hot Top 10 bactera. DNAs from transfonmd bacteria colonies were isolated and subjected to Xa a!nd Not resriction enzyme digestions and resolved on a one percent agarose gl DNAs resulting in an expected pattern were submitted for sequencing. Althought, analysts of several sequences of clones yielded a 100%ercent match with the above sequence, oy one ckme was selected for large scaled piasmtid purification. The final pYD)16-aKLH 120,6VK ISPIL Li0-ShK[2-35, Q16K] constructiencoded an .igQ2-LCh l 0'ShK{2-35,QifiKi fusion polypeptde monomer (SEQ D NO:268), OI0.hbK neptide analos in mamm alian expresatn. C TCT AA Ccc A(C ATU GAC ATC AG(i GTG-3' (SEQ U-) NO;43); and GGA TCC ACC TOC TCC CG COG AMA CAG kG GAG 03 (SEQ i) Nti:44), 00 ~ ~ o reur N. id h "RnE~ 20-0K I S0g2 W avy Cai(n C su cotr ata ing the codin (SEQ H) N :4; beMO. n cod ng a 2O4AKS gO2 Heay Ch Q D) NO 64 below) G A AETCCAG IR CGTOUAG G3CGATC1hGGG TG AGOTer ACAC(T A CXACAGAAGTCACGCACGCuCACC ATCACCAGGGACAC1 C:C ] CCNAGAACCTGGCACTCTCCACC CACGWMGFAAGGCC(ANO C ATCCOGAGCA-CAGTCGCATACT CAG A G CCTO()GACCCTCCrtAC(:CTCCTCCTCACCGGTAC CCCTCG AOCICA GCTCAA CCCCAA G rCGGCC CTUGCAG CAA( COCACCCAAdCTACACCAACGTA NTCACANOCC(AI? AG A ACA CCA (GTG GACAAGACA iT GAGCCAA TTI- i'CYCAi(CI CACCGTOOC CACACCACCTCTOGCACCACCCTCNO A(VICTCFTT(CCC U1'AAA CiCCeAAGC CACeCiCTCAGAX'TC CCGACCCTCCTCACCT GeCCTO TooCACTG ACAAA GAG 0C'CC A;060AGTTrACTC (YEAEYGGA CGCTGOAGCTCiATAATGC AAGA(AAAGCCAOGGAC C~AGCCACrCAACACCACGTTCCGTnGTCTC ACC TCACCnT 0C A (71'AGACTCGCTCA ACCGACGAMl AGAA GCAC (tTC CA A:A G(CCTQCA(CCCCCA CAGAAAACCAICTCCAA CCAANGGGCAC CCGC AMC TAACATCA! ACCTCTCA6CG G TCCCO CGGACAGA C CAACAA CC C IAOC'CYGACC 0C0TG9iTCAAACG(1TCiCCCCACC (ACACGCCTOGAITCGAGAC(1'AAT 1 GGCAd (i(OG A ACA ACTAC CIAA )CrCACCrtG ACA AGACCA )cFG CooACCACGA mAGrCaT TOCfCCTOATCATOAGGCTCT0CACA ACCACTA CACOCAOA A A GCC TCTCCCTGT CTCCGOT// (SEQ ID NO:4), encoding the amino acid sequence MD MRV PAQLU LLL LLW L RGA RCRQVQ LVQSG AEVKK PGASVK VSCK A.SGY TFT GY HM Hf WRQAPGQ L' W MG VW NP NSGG ANY AQKEQGRVTMR' 'DTSI STA YM E LSRL RSDTA VY C ARDRGSYYWFDPWGOGT LVTVSSASTKGPS VFPLAPCSRSTSESTAAL C LVKDYFPEP VTVSWN SGALS VFPTPAVIQSS GLMSL.SSV TV PSSN FOT YTCNV DH KPSNT KVDKTVE RKC C VECPPCP A? PM AGPSVF MLPPKPKDT LMISRT PEMTCVVVDVS ED DPE VQFWYVDVEV HNAKTKPREEQFNSTERFtVVSVLTVVH QD2WLNGKEYKCK VSNhKG LPAPIEKT' ISKTKGQPR EPQVYT LPPSR EEMTENQVSL TCLVKGFYPSD AM EW ESNGQP EN NYKTVPPM LDSDOSFFLY SKLT VDKSR WQONVPSCS VMH EALHN HYT QKSL SL SPG/ (SEQ ILD NO:46) [00628] Tb ShK[.Y~35]WT lragment wse generated usmg the originaU Ec-Li10~ SbK[ 35i in pcDN A3w ( as a te (described in Example Figure 1144B~a in ulivasn et at, Toxin Peptide Therapeutic Agents PCT/U5200}7/02283 I published as WG) 2008/088422 which is inorporated herein by reference in its entirety) and the oigos: 5'-TCC CTM TCT CCG GGT GGA 0A GG.A GG A TCC GGA G-3' (SEQ D NO:47K and 5' CAT (C CC C CAT T AG AG GIG -3' (SEQ it) NO:14) The PCR products were run on a 1% agarose gel. The bands were pnched for an agarose phug and the plugs were placed in a resh PCR reaction tube, The arose plugs were then amplified by PCR ung th outside primers SEQ 1D NO:357 and SEQ ID N"330. he' PCR product was then d by IXba and Not and PCR clean up kit (Qiage purihed, At the same time, pTT5 Vector (an Amgen vector containing a CMV promoter and Poly A tail) was cut by Xbal and Nod. The pT15 vetor was run out on a 1% agarosceel and the harger fragment was cut out and gel purified by Qiagen's Gel Purification Kit. The purified PCR product was lighted to the. lage vector ragnment and transformed into OneShot Top10 bacteria. DNs frorm transformed barial cie were isolated and subjected to Xbal and Notd restriction enzyme digestions and resolved on a one percent agarose got DN As reshin inf ar epected pattern were submitted4 for SeqecCtfling. Ahhough analysis ot several sequee ones yielded a 100% Percnt match with the above squec'e, ordy one cone was selected fo arge scaed p lasmid puficaJco The final pTT5-aKLti H 20NKISP-IgG- C-L 10-ShK(I35] construct encoed an lgG2-RFC-L 1 0-SLK[1 -351 fsion polypeptide with the amino acid sequence: MDPM RVPAQL LOL L L L WLRG A RCQVQ LVQSGA EVKKPGA SVKVSCKASGY TFTGYFHM HWVR>QAPG QCL EWMGWX INPNSGGYTN YAQKFQGR VT MTRDT SI ST AY Mi ELSRLRS SDDTA AVVYC A RDR GISYY WFDP WGQGTLV VSSA ST KG PS VF PLA PCS STSST AA LOCLV KDYPEPV T VSW NSA L TSGV TF PA VLSS G LY SL [SSVVTVPSSNFGTQT'YTCNVD)R-KPSNTK VDKTVE3RKCCVEC(NPPCPAXP PVAGPSVFLFPPKPKDTN ISRTPEVTCV VV DV SH EDPEVQFNWY VDGVEN PN AKTKPREEQFN ST FR VV SV LTV VWQ) DWLNG KEY KCK VSNKC L.PAP E KI JSKTKGQPR E PQVVYT LPPSR E [MT KNQVS LWT LV KGFYV PSIA V [BW [SNGOQP ENNYKTTPPMLDSDYG SFFLY YK LV DKSR WQQCGNVFSCSVM [EA LHNH YT QKSLSLS PGCGGSGGGGSRSCIDTIPKSRCT AFQCK H1S MK YR [SFCR KTCG TCW: (SEQ D NO:44 [00629]1 To generate the SbK[ -35. Q 61K] mutant version of' tis construct, site directed muta"enesm was performed using the Stratagene Quikehange Multi site Directed Muta-genesis Kit (Cat#20053) I pe r manufacmurers instructions, and 01ig0s: S>-OCT GCA CCC CCT TCA ACT GCA AGC ACA (IC 3' (SF9 ID NO:9); and 5'~ CT GTG (111 GCA CTT GAA (IC GUT GCA CC ~3' (SF9 ID NO:0il The final construct pTTS-aK LH20f VKI SP-Ig2-HC-L 10-ShK[-35, Q16K] encoded IG2-H(CL10-SbK[i-35, Q16K] fion2 pKoiypepbde with the following aino acid sequence: MDMR\VPAQLLLLWL RGARCQVQLVQSGA EVK KPGA SVKVSCKASGY TFiG N M H WV R QV A PQGL EWM OWINPN SG N Y AQKFQRV TMTRDT Si STAY M ELSRLR SDDTAVVYCARDR VYSYY W DPWQGWT. VTVSSA S7KG Ps V F PLA PCSRSTSEST AA LOCLV KDYFPEPV VS W NSOA LTSGV H1 PA VLSS S[Y SL S SVVTVPSSNFTQTY TCNVDHKPS'N T KVDKT VERKCCV ECPPCPAP - 203 PVAGPSVF LFPPK PKDTLM (SRT PEVTC VV VDVSHEDPE VQFEN WYVDG VEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNKEYKCKVSNKGL P APIEKT ISKTKCQPREPQVYTLPIPSREEMTKNQVSLTCL VKOF YPSDIAVEW ESNGIQP EN:NYKTTPPML DSDG SFF. YSKLTVDKS RWVQQGN VFSCSVMIH EALR[NHUYT QKSLSL[SPGGGGSGCGOSRSCfI DTiPKSRTXA FKI'KH SMNKY RL[SFCRKXTG T/ (SEQ ID NO:49t [0063(0) iA41 tvvtt iSK235 1(Kmmain ~resasn. Using DNA construct pTTS-aKL H 1 20,6-VK I SP-gO2-HC-L I0-ShK 1-35]} as the vector the ShIK[ 135] was cut out using BamH UlamlI. The vector fragment hom pTT5-aKLH 120.6-V K SP-IgG2-C without ShK[ .35 contained the coding GAG AGGCCAGATGTCCtG CACT CG GCV1 AI(TrGGCTGTGI; AAGAAGCCTG "GCCTCAGTGAA 0GTCTCCTGCAACCTTCTGkATACA (XMTCA(1'C&3C fUI ACT( 3 iAG(i '71 (OFA -,C Cit A( GCTTGAGTGG ATOG00ATGGATCAACCCTAACA.G~TGTGGCACAAACTAT GY AC CAAGTTTCA(G1AGNSIAG A (-CAT( tACT GG AC'ACT ATCA CIA CPT CATT(Y YXA(AACTC GTAT TGTQCCTG GTVCTTCCCCCTCGCGCC'CTCCTCC A(GAOC)ACCTCGAGCAGCACAGC(0GC (7AGTCCTCA~~~ (43YM AC MACCE'lA ICO1G(C CCAI'RCC( 1''1'CAG? CAACTTCGGCACCCAGACCTATA C TC G ATCACAAGCCCAC AcAOCAna CC GT(ACAAGACAGTTGA(sAc AAaTTGTCA C CTeCC G TACOTCCOC OAGC AA Ai CAAGAAA CC ACCO AG GACCAGT(ACA CACCTTCCdTCTCCTC ACCCTC CCACdCGNEGI NaA (CAGCACTGCCTGA AcCCAAG~iAaTAcA At ~C ACOTeTCaC XAA GOCCCTCOC OCCCAIC GACAAAACCA TC'TCCAAAACCAANGGGKCC CCGAGAACCAC AGGT(CACA CCCTGCCCC{ ATPCCCGOACCACATGA C CACAGACCTCACTGACGCT IGCAAGCGCACA C ACAC G&CCXCCTC(dIGOA ;iGAAcIdGG{AIxONOACCTCTCA~'TACAC AYAGAC ACAU It( A W OCIMAdCA 0,G,TONP "Cl CtiA C AACCTCACCCTGCGACAAGACOCAGOCTGCCACCACdCCAAONTCTTCIA TGCTCCCTGATCCATGAGGT CA CA.ACC ONCACGCACAAGAGCC TCTCCCTCTCTCCGGGTiGQAGGAGGA i SEQ iD) NG:SQ} encodin tbe amfinlo acid sqIuecel MD) MRV PAQOL.LGLL.L LW LRCARCQ VQ LVQSGAE VKKPGAS VKV SCKA SGY TFTGY HM HWVRQAPG OL EWM GWTNPNSGTN YAQKFQGR V TMTRDT Si STAY N ELSR L RSDITA VYYCARD R GSY 'Y WFDPW(QGT VTVSSA STKG PS VFP LA PCSRSTSESTAALGCLV KDYF PEPVT VS WNSA LTS I VTFPAVLQSS (G JYSLSSV VTVPSSN FGTQT YTCN VDHKPSNTVKVEKCVCPPA P VA GPSV FL PPKPKT LMI SR TPE VTCV V CV DSH 1DP VfQFNWY VDC N HNAKTKPRE EQFNSTERVVSV LTVVHQD WL. NK KEY KCK VSNK LPA P EKT tSKTKGiQPREPQVY T LPPSR EE MTKNQVSL TC LVKGFY PSDIA VyEW ESNGQP ENN YKTTPPML DSDG SFL YSK LTVDKS R WQQGNVESCS VM [REA LHNH VT QKSLSLSPGGG/ (SEQ 10 NO:5 IL Tbe vector fragent was then treated with Calf Intestine Phosphatase (CIP) to remove the 5' Phosphate group and Pbcnoo/Chorofmi extracted to prevent religatio of, th vector uon itself .h insert came fromn pT - 4,V H2 SPtgG2Fe Sh K[2~35EQ6K] encoding igG2 eL I0~ShK(2-35, Q16K) MEWS V v F LFFSVTTGVHSERKVECPPCPA PP VAGPS V LFP KPKDTLM SR TE VTC"VVVDV SF1 EDPEVQFN WYV DGVE VHI NAKTK PR. EENSTFRV VS VL TVV HQD)WLNECK EY KCKV SN KG L PA PIEKT IS KTKGQ PREPQVYTL PPSREE M TKNQVSL2TCL.VKEGFYPSDiAVEWESNGQPEN NYKTCTPPM LDSDGSFF LYSKL TVDI'KSRWXQQGNVSCSVMH EfAL HN4HYTQKSSLSPksKGGsGGtSGGGSSCID. T VPKSRITAF KCKI SM KY RLSFCRK TCCTC/' (S EQ ID NO:1 S and the inset wvas abo digested out using BmH/BamH L Tinet ShK[-35, Q16K] fragment was gel purified away fro t vector and cleaned up with Qiagen Gel Punifeation Kit, A purified DNA insrt containn the coding sequence GGA TCC GGA GA ( A, ( AGA GCGC \GC TtC AT C UACC ATC CCC AAG AC CGC TGC ACC GCC TTC AAG TGC AAG CAC AGC A AAG TAC CGC CTG AC TC TG CGC AAG ACC TOCGGC ACC TCC TfAA TCA V(SEQ ) NO:52 encoding the amino acid sequen ce OSGGGSSCI DTIPKSRCTAFKCKHSM KY RLSFLRKTCTC (SEQ ID NO-53), was ligated to the lag veotor fragment and transformed into OneShot Topi10 bacteria, DNAs frm transloried acterNal conies were isolated and subjected to BamiW rcstritAion enzyme digestion and resolved on a one percent agarose goL DNAs resulting in an expected pattern were subm'~itted for' sequencing. Altough, analysis of several sequences of clones yided a 100% percent match with the above sequence., only one clone was selected for large seated plasmid purification, The final construct pTT5-aKL igG2 HO-L10-ShK[235,Q I6K] encoded an lgG2 C L i0-ShK[2-35916K] fusion poly peptide: MDMIARVPAQLLGLLTLLWLRGCARCQVQLVQSGAEVKKPGASVKVSCKASGY TFTGYHMM HWV RQAPGQCGLEW\MGW xNPNSGGT' NY AQKFQG RVTM.TRDTS I STAYMEL.SRLRSDTAVYYCARDRGSYYWFPWQGTL VTVS S ASTKPS VFP LAPCISRSTSESTA ALGiC'LVKDYF:PEPvVVN SGALTSG VITFPAVLQSS GLYSL.SSVVTVPSSN FOTQTYTCNVDH KPSNTKVDKF VE RKCCVECPPCPAP PVAPSFLPPK PKDTPLMISRTPEVTCVVVDVSHEDOPEVQFNWYVDGVEV HN AKTKPREEQFNSTERVVSVLT VVHQD WLNGKE YKCPKVSN KGLFPAPIEKT EN NYKT PPMLSOC SS LY SKL VDKSR WQQGNVT SCS VMH EALNH YT QKSLSL.SPGGGGGSGGGGSSCIDTI PKSR CT AFKCKHSM KYR L.SFCRKTCGT C// (SEQ N) NO:54). [006 3 1] The Shk[ 1 35. RI A, 14A. 16K] fragment was generated using pTT5 aK H 1206~VK I SPI 2-1HC 10 ~PShK 1-35 Q 6K] as a template and the obios: 26 [00632] 5> AGO AGO AGO AAG C6C (AGCG CG GA 'AC CAI CC C -Y. (SRQ Mi WEf 3b 1Atn [006 1 S. COG OACT OCO( iC C (WA OT- G(0 OCT C-" GO TtX 'T S~T 10 1D5,0 37 (SQ IL)NO:3 ) Site-directed mutagenesis was performed using the Stratagzene Quikchange Multi site Directed Mutagenesis Kit per manufacturer s mistcions. The final pTTS aKL V 1206~1 ISP-IgG2 -HCL 10 ShK[ 635 t! A, 14A, Q 6K] onstruct encoded an Og2-HCL10-ShK[135, RIA, 14A 016K] fusion poAypeptido (SEQ HD IKE:300). [00634) T ho S hf 1 -35S. RI A. Q 16KQ KI3QF) nar n was go nvated a v deoc6iVed aLbov'e Osng the toflAMing four chaos: [0063 51 Y GAG GAG GAG 6AA (iC CA OC C TCG AC I ii (SEQ [00361 G (AG TT Ok 0CC CGA GAC TO CO CAC - (SEQ iD NO:313', [B0637l 5n ('GA TOC COPTIC:G& C CIT (10 0 f (SEX HD [0063h~~T CCO ('CG1 G ( 00 ('Ci AAGi CTC37 SE j Th a a ,, o 20.6- ViK S P- H I 0K A 5 P. 1 A , Q 6K. K 30] C1t~C Cflcot m an2 -z1 (-T 1 0-StiKR 1-35, R. IA. Q I16K, 1K0h F 1in [006391 The ShK[1-35. RIAH. WK, QI6 hapm ioatnt wapeneratd uslgTS aK~] 206-VE ISP- 0G21 RC-Lt0 -ShQjiW35 QIK asi ahc tepea d eoips.

a00640 5> GA sA OA AGJC G A O 5 ,S F 3, I [)* N 0 : [06641 5' GG GA TO GC GA GOT OTO G TOO "(I TO1 AW 'SEt-Q I t) >:3 17% Sie-directed mnutagtesis wt ateronned usine the Stratagene Qukehane Multi si Directed Muauemis Kit (KatI#0031. N penufactue mstructims. The final pTTMK N 120A-VKI SPgC24C1-1 I1ShK[I- RI tA, QI6KI construct e noded en IgC4 - -hK[ R H 14A QI6KI vision polypeptide (SEQ ) pq 0: 306)M 100642) he Shk[ I -35. RM. Qi6K K30I frag nt onatd as daMed etbov using lho tolcwing fournt ijos: [006431 5 GGA 00A 00A AGC CA AGC TOO AT( GAC $-3( 9 NOJI}Y n ~ LQ D NO: 3 13. [0064415 GTPC COATC GC0 04J( GOGTTC1C NYQ d F V i f20'$ I 0SK[ I35 R 5K cstct eneCded an igG2- HO-Li 145h K. .15. ROt-, Q16K- K ThE n polpefic(SF EQ H1 >4,,0:3071 [00645) The Shk[ 1 -3 5, RI K, 14. Q 1 6K] fragment.. pa 4eeae usn 5 WK LA i 216-VK IS&P-1§1 2I-HG 1 0-ShKoi -35 ()1 41K] aoavitpe n h irz [006461 5'- C0 GAG GGGAG (,,AA NC AA(16 l OWL O(1T3 C -OG A A Tp CIA AGA A3"' (SEQ 7 NO320e [00647 5> TT TG GGA TOG TOT CG ( AG TC T Oc T C 1-lC 00 (SEQ D >40:321)H Ste-directed mutagene a1 Performed umg th \tratgene Okhange NIU soite ueed \huagensis Ka ((JCO>'05 ,, pr manutaetr'r ; mYU:onS. The final" p11M K LH I 0 VK K1SP-gG'-H( A. 10 ShK[1 V R35 1K 4 OA QIN conduct ecded art gG2UC10 ~S h 11 RIt 1,4 Q6K] uson p0{ypepIde (SEQ 11 NO nIP [0648 lh e hk[ \Y433 R iK01 K frJ agajnr as'~ g tede as described above 1ing the fo3owint, four OIOW [00649 Y - CGG AGO AGG AGG AA GA A 1T CAT OGA ( ( AC CA K (D N022) and SEQ ID NO 00650) ' GG TOT C&A TOC A "TM C TTC CTC C T CC (1 SEQ i) NO2 and E i) NO the inal pT'5-aKLH 120k-K1SP gG&' i 4-hK $5 IN Q6X. K3O] conduct enoded ag24 W 19 $ 5, Ra K QK 6k K30E 1ion popeptide (SE Q I) 309. Trivalent Ab L(Toxin Peptide Analog Fusions initial puriication of the conditioned media was don' by affinity fast protein l1iqu id chrom Stography (PP LC) capture of the Fe region using Protein A Sephsarcse (01E Heatihcare) flow oed by a Solumn~t w5%ash with DuibJcco's PBS wtthouit iidiale cationv~ itrogen(S) and step cluUton 'with 300 mvA acoe acid, pH 3.5 at a flow rate of' 2, cm/'min. Pr otein contain ing fractions were pooled, and the pH1 was adjusted to 5 0 using 10 'N N'a.H and furt her di lu ted with 3 volumes of water. The matercialI was filtered though a 0.45 pmn cellulose acetate filler (Corning) and further purfe by cation exchange FP L (S P Sepharose HIi gh Performance; OF Healthcaret Sampl es were loaded onto at colaumn equilibrated with 100% hbuffeTr A (50 mMl acetic acid, pHN .0) and eluted with a gradient of 0 to 80% buffer B3 (50mM acetic acid, I1 M NaCG pH 50) over 30 column volumes at a. flowrate of L5.2 cm/min. Peaks containing target spcies were pooled and totrmulated into 1 0 mM sodium acetate, 9% sucrose, pH 15,0. Exemplary purifications of monovalent. bivadet and trivalent imuolblntxnppide anlo ilson proteins arc shown in Figure 24-26A-B, 27-9A-B, 30-32A-B, and 33 35. The non-reducing SDS-PAG analysis 2iors 24 30 and 33) demonstrate that the fully assembled antibody can be ferned, and the rching SDS-PAGE analysis demonstrates that the desired c0omp3onents are presenTe size exchttain chromatograms (Figures 23, 2, 31 and 34) show that the majorty of the punfied product is in the desired non-aggregated state. Final th mass spectral analysis (Figures 26A-B, 29A-B, 32A-R and 3nstrates that the desired fusion products are prent. Taken together these cx naples demonsuraer that the aKL H 120.6 antibody can accept. tusions in a wide variety of coigurations including species containing an even- or odd-numbered valence of at least one to eight pharmacol ogivcaly active polypneptie moieties p DA al sequenceoding a onomer of e K inhibitor peptide ShKf j fi1sed in-frame to the Nterminal Fe regan f nn g 1as coansucten as deSWe beow. [0065) [o 00construction of \'H2) SP--hK( O-35) 10 V C evpression vector, I PCR strtegy was employed to generate the \'V121 signal repude Shki -35) gene lbnked to a tur gelycie and one serme amino acid fanked 1y mdI t lan BmHI reietn sites and a tour glycme and one scne amino aWid hMacd lo 1§31 Fe trmen: flanked by BamiH and Notl restru n ites was generated in a KR reaciusing the I'e-[ 10-OSK1I n peDNA43 l (MVi as template (described in ample 4 1 and Fiure 42k-B of SAl in et aL WO V 0o; m422A2, meorporated by refitence~ [006541 Totgenerare MH2L SP-ShK0i-3> -O4 two oligos with the acuec as depiced bo we ued in a PC reactionf wi the o kotStart DNA polymerase (rat ne) at S -3se. o30n ' ecf ce Hind! I (atgotnd Iarn ggate resrictionste a denied: forward primer: CAAOTACT ACGA OTACH NTNTAATGOAG CTCGAC C AAGC(GC GCACCGC (TCCAGll SEQNO 5 and Reverse mruer CTCCGGATCCtTCTC1CTCCGCi(AGG(TCCGCAGGTCTTOCGGCAGA AGCTCAGGCGGOTACTTCATOCTOTGCTThC ACTGGAAOGCOOTGC AC G3CTCTTCGGATOGT&TCGAT//(SEQ ID NO:56). [0065] The resuming PK products were resolved as the 202bp bands on a two percent agarose gel The 202bp PCR product was purified using PCR Purification Kit (Qiagen) then dies ted with HlindHUl and BamH i)Rche) restriction enzytmes, and agarose sge was purified by Gel Extraction Kit (Qiaen). flw Weligos Wh f the sequence as denictdbeo were used in a R reaction th urbo tta DN ponerase (tene at 95 C305sec 5 30se 75 minar 30 c les- ai e an o (gcggcnge)restnton sies are under ned Forward prminer A dGGQNF ix f;>.c \iA A AAGOGACAAA ACACAC SEQ Q Reversepruier (XiAGC G GGTTACT A CCCGAGACAGGGAI! (SEQ ID NO 5). [00657] Thu resulting P R produtCt were reached as the 72hp bands on a one percent agarose gel The 72-b C o R product wa ifie using FOR Purificaton Kit (Qigen), then digested wvh am l and Not (Roche) restriction nzymes, and agarose gec was puriied by GelExtraction Kit (Qiugen . [00654] The p cDNA3.,+)Mi-Ec-L10-OSK1 vector was digested with BamHI and No! restrition cnzyms ad the Lge fArment was purpfed by Get Extraction Kit. The gel purified 4GS-gG1 E ragncct was iigatd to the purifid large fragment and transformed into tne S hot Top ' 10 (0vitroger) to cate a pCMViFc L 10-gG I F vector, Subsequen0ly, pCMVi~Fe-L10-lgG I Fc voetor was d digested w'ith HiIdJU and BamH I rsitetion enzymes und the 'ae fragment u as purifed by (l Fxtraeo 'it T gnuTijed \'1121 SP-ShK( -35 4(S rent uas liguted to the purified large ifrutment and trnstoined inmo On' Shot" I onp10 (Inautrogen) rexulting int a. pC M\KV H2 SP-Sh KU 35NL I0g U Ie .on4Ftrt DW from01 tuormed bacterial colonies were soluted and digested wu ith Ratd II and Mu reswteio enzvnmts and resch ed on a one nereeat agarose ge L.DNV resulting~ m an expeciced patten were submiuted for sequencing Ahhough, analsi of seve a sequences of clones teded a I 04 % percent natch w ith the aho& esquences tMnx one clone from each gene was selected for large sealed plamid purificton. Thr DNA 41frm VIlP I SP-ShK(125 10$)-L19Gl l' in pCM\I vrctor wa reseqtaenced to confirm the F- c and linker regions and the sequence 5 10(0% identical to the abowe S, -S, ~ Iewe Frge Vnilt \-2I SP-ShK(IYr.4 JdgGtO 3F conined the coudng ATaaG A t croWGiGCTTCTrCT (1'(tTCAcioTAACOACTQOTaT TCC AOTGCAAG CXACCATOGAATIA CC CTGAGOiTTC TCCCAAOA CTGCCOGCAUCC CGAGGAC CAGO AC CGOAOGACCAAOOAOGCOA\A AAC'TCA CA.AIUCCCAbaQGCC:AGCACCUdAAC CCCCiGGGACCGi TCACETCCTCTTCeCCCC AAACCAAACA CCicTATGATC olCC GOACAA\AC CCGGGCAGGAGICAGC CAACACGTACCG~TV 0GGTAG COTdCCCACCCTCCTOCACAGGCACdOCIG AATGCAACGACJACAMG 'TOAAO~lflCTCCAAOlA AAGCQl'CTC(:ACG ('C CTGAdAAAplCATir C AAAGCCAAA IGCACICCCGAG AA CCACACCrICTAACCTGCCCCCA TeXCn$(10ATCA~l GACCAACAACCAGCIXACC(1'GACCTC(1CGiC GA (IaCOGAGA ACAACTA(CAAG ACCAGCCTC(IGTCGCnACTCCGACC (I 0GGGGMACGTC"TTCTCATGCTCC'GTGATGCATGAGCGCTCTGCACAA('CACT encoding VFI21 SP~ShK(1 -35)~L. 104gGu hc Fermno acid sequence MEWS WV F LFFLSVTTGIVHISRSCI DT P7 KCTAFQCKHSMKY RLSFPCRKTCO T(:CHGSGGGGSDKTH TCPPCPAPELLGPSVP PP PKPKDTL MISRTPEVTC VVVDVSFEDPE VKFN V D G )C V VH NAKTKPREA1EQYNS'Y RVVSV L TL HQi DWLNGK EV KCKVSNKA LA PAPIEKTISKAK QPREPQ VT LPPSRDE LTK NQ S TCLVKGFY PSDIA V EWESNGQPEN NY KTTPPVLDSDGSFFLYSKL T VDKS RWQQONVFSCS\VMf EA LFNHtYTQKSLSL SPOK/ (SEQ if) NO:6U}. [00)659] Mgmalufiareregi~on ofN-termrlinu S' hKI -35. ) 1KJI-aXLU C' aind N-terminus ShKfI1-35016KL-aKLi HO. Using at contract encoding N-teinus ShK[ -35] Wid Typ- 1 4O-F e, site directed mtag genesis was perR rmed using the flowing oligos to produce a Q6K mutation Wi e ShK region: 5'~GCT GCA CC C TC AcGT GCA AGO A(C'A C-3Y. /SEQ FD NO:9T and 5'- GCT GTG CTT GCA CTT GAA STC OCT GCA GC -3' (SEQ ID NG: 0> The Stratagene QuikChange Nulti Site Directed Mutagenesis Kit was used according to the manufacturer' s instructions The final construct for pM M~i-N iermpinus-ShK[ I~35Q I6K]-LI 0~1gGOF enco ded the follIowing Signal peptide (V2m SPl-ShK 1-35, Q16K- 104gG IFc fusion poypeptidc: ME WSW\VFLFLSVT-TG VHSRsCiIIPKSRCTAFKCKHSMKIXYRL[SFCRPKTCGi TCGGGGSGOGGGSDKTHTCO~PCPAP ELLGGPS VFLFPP~KPKDITLMiSRTPEVT-C VVVDVMSH EDPEVKFNWVYVDGV EVHNAKT KPRE EQYNSTYRVVS VLTVLHQ DWLNGKEYK CKVSN KAL PAPEKT ISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKOFY PSDI AV EW SNGQPEN NYK'TTPPV L- £5 DSE FL SK LV MDKS R WQQGNVFZSCSVMHUEALHNMYTQKSLSLSPGK//(SEQ ID NO:61 . [0066t1Th generate the N-terminus ShKV 1-35, QI6K -a KL H HOC construct a PCR t ah ptd-ShK[ I -35Q 16K]IL 10 n was produced using the following oligos: 5CA i TO A> (CA (CAG A) A -3 (SEQIi) N:Q 69 C('AG G CAT GC WT- TC(C TOG iC0 GG-3' (SEQ ID NO:63) a dten Qate N ennimuShI2 K O 004< reslt edi a t gment containing ht coding scquece ATGOA ATGA GCT(GGGTCTTCTC ITTCTICTGT0CAGTA IACGACTGGTGT (CC1A CTCGC(IAGCOCATCGACACCGATCCCCAAGAGCCGCTXCA CGCCT TCA AOTGCAAGACAGC:ATGAA GVT ACGCC TlGA GCT(TCTGCCGCA AGAC TGOCGCACCTCk'A GAGO AG A C CCAGGAGG AGGAGG AA G /(SEQ I) NW:4X encoding h VH21SPhK( 13, Q I 6K). CHOW an acid sequcte NMEWSWVEINSv TTGWHRSODIRCTI FKKSMKYSECRKIGi IGGGGCSGGCG S1 ( SEQ N E061 to curete.AKEH-j fnwmcm. a MIT prodact ws crete usma, 5-OGA G,X GA AGO GAG GTG AC (7AG GSE iD NO:6} 3WI( l (JOG OG GOT (AV FTIA ('C( -3'S (S,! O: andte rpiate p 5-KLH 1 0. H7 resii naDNA yrgu n utin the GCA.GGTG CT 6(16(' GOJ G AAA'OT GAGAG (7 A TGA CG1GGAOAOA C0GC00M WAO AGOGGT AC GTGOGA I GTG A A GG G AA6AAA A C c> cGG .. EaTCAGAoocOAGA(Aec Ce AeGGAG ACGGTOCOCAGCAOCGGIdTGGddAGOO(AICGAATOOCA(71CA(7( AACTACICTOAGAOATAGCCAGGAIG7l7AA (AAGOGTGdACA GAC ulGAGGCOICTAA?(1((TAT rGOG101 V(IAO NCOCOAGA(I7 AX (GTAOGC DCTGAA C'OA CAPI(7A ACA CO G GA-FCOA(A (( AAICO AA AOACAGTdACGCA r GTGAdTGC A6OTG CCAGCACC A pCTfGG(iCAGG ACCGTCAGTCTTCTTTC (PCA.AA.ACCCA AOiACA CCTCATGATCTCCCGG ACCCCTGAGGTC ACO OGTOGCGTGACGTG AGCCACGAAGNM CCGAGCT(W AGTCAMTOGT OGACGGCOTG GIG GATAAT OCAAAAAA CIAC GO A IAC C A ACAC &ICAGTGidCAGCGTCCYCACCnGTTGTGACCA GGAC"OGIGAA Cc OCAA (GATOl CAAGO GAAGC (T f AACAAA N)i ( C('A G CCOC CGAI3 AAACCATCTCCA AA ACC AAAGUGCAGCCCACAA(C ACACOTO TAC AC CCTOCCC TCCXEGOAGGAOAr.AC A' ACCAGGTCAGCC TGACCCTGiCAAAGGCTTCTA(ACCO CAG ~CCON (GACYGG G AGhAGCAf ATGGQCAGCCGAGA ACA ATACA AGACCA CACCTCCCATC TGGA.CTCCG ACGGCTCCTCTCGCTiACAGCAAGEiCAOGTOGACAAG AGCAG TOOAC AGGGAACGT ITCTCA TCTCCGTGATUCATGAGO CTTCACAACCACTACACGCAGA AG GCCCIUGCTCCXGGGTAA TGAQ (SEQ ID NO:6$fl encodin amio acid sequence Q VQL VQSGAEV KKPG AS VKVSCKASG YTTGYHM H WVRQAPGQGL EWM GWINPNSGOTCNYAQKFQGRVTMTRDTSISTAYM E LSRLRSD3DTAVYYCAR D ROSYY WFDP WGQGT LVT VSSASTh GPS VFP LAPCSRSTSESTAALGC LV KDY FPEPVT VSWNSGA LTSGVHTFPA VLQS5(1 VS S SVVTVPSSN FGTQTYTCN V DHIKPSNTKV DKTV E RKCC VECPPCI'PAP PVMAGPS VFLFPPKPKDTIL MI SRTIPE VTCVVVDVSOHEDPE VQFN WY VDGV EV'N AKTKPREEQFNST FRN VS VLTV VHQDW LNI KE YKCK VSNKLPAPi EKTSKTKGQPREPQVYT LPPSREEMTK NQVSLTCLVKGFY PSIAV EWESNCQPENN YKTTPPM LDS DGSFEFLYSKI TV DKSRWQQGNVFSCSVM[AL MNHMY TQKSLSLSPGK/(SEQ MD NO:69). [0062] o PR products were run out on a gci and the appropriate sized band was punched for an agaose pug, The aose phis were piLaced i a singe new PCR nation. and the fragments were sewn together usn outer most prmers (SEQ ID NO:6) a (SEQ ID NO.67, The PCR fragmem was cut using Xbal and Not and cleaned wivh Qiagen PCR Cleanup Kit At the same time, pTT$ vector was also cut by Xba Imd Not and gel purifed. The purified insert was igted to the argc vector fhigmnent and transformed in OneShot TopI 0 bacteria D As from transformed bacteria coloncs were isolated and subjectd to Xbad and Nod restriction enzyme dincst. and reoed on a one percent agarose get NAs resting int an expected paittern were submttd fo tmequyencing.. Although, analysis of several aequenes of clones yielded a 00%P pece'nt rnatch with the above sequence only one clone was selected for large scaled plasmid purieation, The inal construct pTT5-N-terminus ShK[1-3QI6K)L10aKLFH120&-HC encoded a VH2I SP-ShK[ <35, QI16K ]- 10 -aKL H20.-C fusion potypeptide: MEWS V VF LFFLSVTTGV\H SRSCIDTIPKSRC"THFKCKHSMIKY RLSFCRK TC TCGGGGCSGGGGSQVQLVQSGA EV KKPGAS VKVSCKASCYTFVTGY HM HWU\V RQAPGQGL EW MGW iNPNSOGTN YAQKFQGR VTMTRDTTS STAY M ELSR L R SDDTAVYYCARDRGSYY WFDPWQGTA VSSASTKGPSVFP L APCSRSTS ESTAALGCL VK DY FPEPV TS WNSCALTSGV TFPAVLQSSC LYSLSSVVTV PSSNFGTQTYTCNVDHKPSNT KVDKT V RKCCV ECPPCPAPPVAGPSV FLFP PK PKDFL MISRTPEVTC V VD VSW EDP EVQFN WY VDG V EIN AKT KtPREEQ FN STFRVVSVMLTVVHIQDWLNGKEY K(:KVSN KG LPAPIEKTIS KTK GQPREPQ VY TLPPS RE EMTKNQ SL TC LVKGFYPSDA WESNGOPENNYKTPPM L DSDOSFF LY SK.LTVDKSRt wQQON VFSCSVM E1IAL HNHYTQK SLSLSPGiK/ (SEQ ID N:70) U00663 Laiy th terminus bihK t K, 4ik-I M 6 UghtChain 5MCAT TCT AGA CCA (CCA TOG AAT GG3' (SEQ ID NO:62); and 5- CAT CG G T OCT TCC TCC TCC TCC GO -3' (SEQ D NO:71i and template pCMVi-N-tcrminus-ShKi1 )35Q f6KtL 10Ig -siitin in DNA fragmen containing the coding sequence ATG ATGG(IAGCTGGTCTTTCTCTTCTT CCTGT CAGTAA CGA CTTG CCACTCCCOCAGCTGCA TCGA CACCATCCCCAAGAGCCOCTOCA 'CCGCI (CTGCGC(ACCTGCGGAGGAGGAGGATCCGGAGGAGGAGGAAGC// (SiEQ U) NO#4}. QrtC&UQtheaumowaid sequevcc tbr a Oal~w peptic (VA21 SP>)SOK( 435, I 6K>Louiner TCGGGCGCGSII(SEQ D) NO:65) Usine template ad dens: 5'-COA CC GGA AC ( C ATC C ACT 01% MY (SEe? U.) NOW and 5 (i~ (IC AO CCM? (X :VV -3' SE Q IF) NO0:73V. The restdting coned PCR fagn ent.cnied he ding sequence ATGCAAT1;G1}AGCrGVA GROfCRMTC CA0IACC CCA ( CT (CCACT3iCAWK6'J CACACCCCAQC6YkAt:C INTC.ACTC (711C( AACA1 AG 1ACC V C 'FlGA AGETTCTGCCGC AGACGG (11OCCC(CC 116 AG -AOAGUO IA 'CCACk]AG AC A CCAA''GACCCA TCTCCATCC CCCTTI'1 ATTCT AGGA GA CA'C TCA' TCUACCCGC CAAGTICGGCATfAGAAATAETCAGGiG AY (11 C CCA CTATT(C. T ATCAGCAGAAACCAGGA G AAGCTO A C AETO C AAALGGOCQATC AAOCTTA~GCcACGOATcTGC CACAO ArA1 rT ACAATCACAehC-~CCCAC-CCTC AAGATrTCAA (CTTTACGTOCAGCCA 'AATACCGTCACTTCCGOA G A CAACCA AMTAAA ITCCGCC CCCCATCTGATCACCAGTTGFAA ATCTGG AATGCTCNGTTGTiG" C' AGCAAO ([ACAGC AC(TACAGCTAG (AGCA(CUCI(;AGGOI( 'A CC AAOG CAA NCTEA(AGA AAC ACA A A-rdTACGCCTOCCAACTC At)CATPCGG (fC G( ICOCCCCTCAXCAKNAA TCAACA GOCOAOAGTCITAI (SEQ E) NO'Vwas :e' KIM W I iCht (c ( M, h)An MSW FI V. SVTTGVUSRSC DTIPISRCTAFKCKIM SNCRKC TCOGGGSGGGOSDIQMTQSPSSLSASVGDRVTTCRASQG RNDLGW YQQKP GKAPKRtA YAASSLQSG VPSRFSGSG TE TLTSSLQPEDFEATYY VLQNSY PLTFGGG6TKVYAKRT VA APSV HF PPS DEQ LKSGTASV VC L LN NFYPREAIKVQ WKVDN A LQSGNSQESVTEQ11DSKDSTYSLSST LT LSSKA DY EKFUKVYA CEVTFH QG LSSPVTKSFNRG EC/ (SEQ iD NO:75v [006641 Both PK R rg t( DNA gnent conuting Ne coding sernea SEQ ID Nd64) andK i 20 6IN Lion Chain K tg a cofltaung te odit seg-ence (SEQ tD NO 2Y er na out on a ge and the appropdate sized banv wa punched for aagan a ose pngt Te agarose pg we e en a se P(R reao andthe agents sre sen together ua oer aost re (SEQ 0 NO:62) and (SEQ ID N73) Theteduna agmen a u nsng Xba and (00665] At the same time pTT 14 vector (an Amgen vector containing a CMV promoter, Poly A tail and a Puromycin resan gwas alscb ban Noti and gel purified, The purfed insert was gated to 'the arge vector fragment and transformed into OneShot Top 10 bacteria. DN s fronm transformed bacterial ctoonics were isotecd and .sbjected to Xbal and Nod restriction enzyme digestion and resolved on a one percent agarose g. DNAs resulting in an expected pattern were submitted for sequencing. The fina consucet pTT 14-'N-terminus ShK[ 35Q16K[-L10-'aKLIt 12-0.6-LC enkodin a St\ inal Peptide-ShtiW-35, QI 6KV.L10 aKiL 120.6-L C fusion polypeptide scquence (i.e.. SEQ itD NO7), [00667) Mak ing of Plasmid pITST - aDN P 3A4 (W10 Fl VgG -Shkt-3016K]1: DNA sequences codiWng for the heavy chaun of human anti-2dinit rphe) DNP} antibody fIsed in frame to a monomer of the K L 3 ihibitor toxin poptide anaog ShK[l 1 QI6K] (SEQ ID'NO:76) w cnstcd using standard c.oning technology, Paid pTT - aDNP 3A4 (V10 1F) PgG2 ~Shkf~35 QI.6K was generated by 3 way Nigation of te p1 T vector with a portion of an an tiDNP 3A4 (W0 I gG1 He av.y Chain (pDC324 aDNP 3A4 HC (WA4 IF) having the amino ald sequence MDMRVPAQLLLLLLW LRGARCQ VQLVESGGG0VVQPOR SLRLSCAA SOFT FSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFT SRDNSKNT LY LQM N SLRAEDTAYVYCARYNFENYGMD1)VWGQOTTVTVS SASTKQPS VFP LAPCSR ST SESTAALCLVKDYFPEPVT VSWNSA LTSVTFPA LQSSG L VSLSSVVTVPSSNF(TQTYTCNV DHKPSNTK VDKTVERKCCVEPPCPAPPV AGPSVFLFPPKPKDTLSRTPEVTCV VVDVSHEDP EVQF NW YVDGVEVHN AKTKPR EEQFNSTFRVVSYVTVVQDWL NGKEYVKCKVS NKG LPAP IEKTISK TKGQPREPQVYkTLPPSREEMTKNQ VSLTC LVKGFY PSDJAVYEWESNGQPENN YKTTPPMLD)SDSFFLYSKLTVDKSRWQQGNVFSCSVMEALHFNYT QKSL SLSPG/ (SEQ I) NO:77 and a portion from Vg2e-Shk[I-3i QI6K]. The pTTR vector was cut with Sall/Notd releasing the multiple elonings ste, The vctor wa s the'n treated with Calf ntetine Phosphantse (CIP) to reduce background. The fist is'rt came from pDC32.4:a!NP 3A MOC (WI0F)by cuttin i Sal/Stut resuming in te a DNA fragment continuing the coding sequence ATGGA CATG AGIjG CCC4GCTCA GCCCTGGGCTCCOCTGCG C[ GAOAOGTXbGCG CTT CA GGTCAGCT G GTGM AGTCTGGGOG AGCT1 GTCCAG(CTGGG AGGTCT AG ACTCTCCTGTCAGCCTCTGGA TTCAC CT TCAGTAO /TATGGCATGCACTGGTCAGCTCCA AGGGG CTGG AGTGGGITGGCATT ATA1 TiTATGATGGAA -TAATAAATA CT ATG ClAG ACT'CCG'TGAA GGGCCGA TTCACTA1TCCCAAGACAA1TTCCAAGAA GGEGTA M GAAA UAAAGCT AMA CCGAGGA O(( AGG GCJiT TA Tl AGGTCA.GGT AATA AAGTATOGAMWCTGOGOGCA ACCCGGCGGCAC (GOCGTAGCTOC GAGCAAGGC1GGGCV TGCGGCTGGGGGG ATCGA AG ceGACGGCNO NGAG GGGT ATG AGGGCTTACAT OGGt G AC OCTCA AA IGN 'A ACTA GCTC AA GCA( G A M AACOGGACAAG ACAGTTOAGCO(I'AA AT T GTCGAOTGCCGA C caracce( AG(McUeaaMXeercaae ce e SHII)NX7onaiymn cre!asrocao ct,:o~ GACTtO TG AG CCAG A AGACCGAGGT CAGTTCAAGGT GTOGACGGGTGGA GflGT rAATCAACACA6AGCAdd'GGAG(O0AG (PAT'V AAAG A GTTCGT TC OCGTCCT AGVGACA GOAAA CTGAGG A AAC A as( Q)N71 eaaera deoL aio acid eqauenrcz 60 MMR GA A Q L"LGA QC SGG QPORSll CAA SOT FSSVGMHWVRQAPGKG E3WVA'YD~tGSNKYYAD)SK0RPTSRDNSKNT LLQM N SLATAV WYYCARYNE Y iGML)WGQGTI VS SASTKGPS VP LAPCSR STSESTAAGI OCLKDYEPEPYT VS WSGA TVSOVUTPAVLQSSG VSI YSSVV V'SNEOTQTYTGNVDUIKPNTKDKV RK (XC P(I'PPV AGESYFU PPKPKDThMISRIPEVTGVVVDVS $EDOPWFQFNW VDGYLVHN AKTkPEEFNSTER VVSLV QO E NC V$CK VS KG (SEQ 0 The second inset was digeed out usBMg Sttd ot a contained the codingi sequence CTCC2CAGCC2 CCCATCGAGAMAACC ATCTCCAAAACCAA.AGGCAGCCG ATCGC(TIOG ACT OCGXGAIAATGGC(AG(I'I'GAGAACAACTACI'AAG ACGACACCTC'CCATOCTOGGCTC(PGACGGCTCCTTCTT CTTA 2CAA GC"CACCGTOGACAM3AGCA GGMOCiCG CI AGCA 6T d C C TCO GATGCATMAOG('CVTGCACA A{A T ACAAGAAGAGTCTC CTGTCT COGT AA\NCAGC CGAOGOAYCCOC ACGOGACGAACCC (PAOTC AM ACA CATCCCA ACACCCTGCACCCCTTCA AGTCCA A(IXAC(AGO ANA(YACCC(I~;GV CF 00(K'AAGACCCCCCK CTGCTAA GAI (SEQ D NO0O encoding the 1 kiing runcated lIgG 2 F I (- ShK I Q;5 QI (6K. amino aicd LPAPEKTI SKTKGQPREPQVN PPSREE MTKNQVSCLVKGFPSD AV WESN GQPENN YKTTPPM UPSDGSW LY SKL ND 1SRKWQQGNV PSCSVMMHE A LHNH YTiQ K5S .SLPCKGCGGSGCGGGSRP.5DTIP KSRO AFUKC KNSMKYR SEC ?KTCCG/ (S#EQ ID) NOt i [00668} The vector and insert fragments were gel purfed and cleaned up with Qiagen Gel Purification Kit, The pifieh d inns were gated to the lare vector fragment an transfer m" into OeShot Top10 bacteria. DNAs frm transformed bacterial coloni es wee isolated and subjected to Sail/Nodl restictioni enzymne diestion and resolved on a one percent agme getN DNAs resultn g in an expected ptPtern~ were submitted for sequen~cing. A clone yieadingia)100% percent match with the above sequence was selected for Iare scae plasnid pufaon. The final. - aDNP 3A 4 (W0F) 1g02 -Shk[ 3 QK] construct e a aDN PA4 (W01 F) IgG2 111 10~Shk[ 5, Q161K having the fooin amino acid sequenee NDMRVPAQL LOLCL L L W LRC ARCQVQLVESOVVQPCRSLR LSCAASGFT FSSYGMHWVRQAPGKGLW WVA VIWYDGSN KYY ADS VKGRFTISR DNSKNT LYLQMNSFRAEDTAVYYCARYNFNYOMDP VW QTVTVSSASTKGPSVFP L APCSRSTSESTAA LGICLV KDY FPEP VTVSWNSGA LTSG VW TPAVLQSSGL Y$ LSSVVIVPSSN FGTQTYTCN VDH KPSNTK VDKTVER KCF(fVEC PCP APPV AGPSVF LFPPKPKDITLM ISRI PEVTCVV 'SHED PEVQFYVDV [V AN AKTKPREEQF NST[RVVSVLTVVHQDWLNCK YKCKVSN KGLPAPI EKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCL VKG FYPSIAVEX EWS NOQENN YKTTPM~4LDS DGSFFL YSKLVDKSRWVQQGNVFSCSVMNIHEAL HNHYTQKSL AS PGGGGSGG GGSRSCI DIPKSRCTA PKCKHSMKY RLSFC RKTCTIC/ (SEQ 1D NO:82). [a66] mgrahiagxpeiotn fai:DNP 3Anibod ightChin. The XenoMouse@t hybridoma expressing a.D.NP mnonoelonai antibody 3A4 w 4 as used as a source to isolate total RNA, One step R'T-PCR with multiplex tetpecifc primers was done to obtain a variable region product, This product was ramplified with a forward primer to add a 54 BssHM restriction site 5' -TT IT TITTC CGC OCT GTG ACAT CC AGA TGA CCC ACT C -3 (SEQ I) NO:3) and a reese primer to add a 3' HNiW restrition site 5' - AAA AA A CGT ACG TT GA T ATC CAC TTT (oT CC- 34 (SEQ 1) NO:4), The resuming PCR product was cleaned by Qiagen PCR clean -u digneszed wit BssHil and BsiWi restriction enzymes, cleaned by Qiagen nucleotide removal and gated into a mammalian expression vector pTTS containing a 5' VK 1/012 signal peptide and a 3 human kappa constant regon. T h amino acid seuence of the resulting anti-DNP A4 Antibody Light Cai is th tolowint MDMRV PAQ .LLLLLLWL RGARCDIQMTQSPSSVSASVGDRVTITCRASQGIS Rt RLA WYQQKPOK APKLU LYA ASSLQSGY\PSRFSGiSCGSGTDFT LTISSLQPEDOF ATYYCQQA NSFPFTFGPGFKVDKRT&VAA P ESVF FPPSD EQLK SGTAS\VCL 1 NNFY PR EAKVQWK VDNALQSONSQESVTE~QDSKDST V SL SSTL T LSKA DY E KHKVYACEVTHQOLSSP>VTKSPNRGEC// (SEQ ID NO:I191. [U0670}C Rht s i ll id' A'na1 a nd Aed A: Ad b L-Toxin Pentide Analos Fusions. initial puiiatiaon of the conditioned mia done by affinity fast protein liqui chromoNgraphy (FPLC) capture of the region using Protein A. Seph ars (GE Hcalthcar) tolowed by a column wash with Dulbecco's PBS without divalent nations nitrogene) and step elution with 100 mM acetic acid, pH 3.5 at a flow rate of 2,5 cm/min, Protein containn fractions we pooled, and the pH was adjusted to 5.0 using 10 N NaOH and further diluted with 5 volumes of water. The matcria was filtered through a 0A5 gm ellulose acetate filter (Comiag) and further purified by cation exchange PLC (SP Sephrose High Performance: GE Heatcae). Samp were loaded onto a column equilibrated with 100% buffr A (50 mM acetic acid, pH .0 anl d elated with a gradient of 0 to 0% buffer B (5OmMt acetic acid, 1 M NaC pH 50) over 30 column volumes at a flowrate of 1 5 cmmi. Peaks confainn monovalent species were pooled and fornmalted. into 10 mMr sodium acetate, 9% suerose, pH 5,.rExemplary purifications of immunoglobuhin-toin peptide analog fusion proteins are-shown in Figrc 3A-C, Figure 4A-C, Figure 5A C Fure 6AC and Figures 2:4-35 [00671} MethodforgelautieN oo a t4_( ~~ on&ovatengt.~ atndg Trivalent Ab> LC-Ton kPfe tidoAalo Fusions. lniial purification ofthe conditioned mcia was done cb fiity fast protein liuid chrmtography (FPLC) capture of the Fc regon uing Protein A scpharos (GE Hea tcare) followed by a cohimn wash with Dulbecco 's PBS without divalen't cations (Iv-iirogen) and step elation with 100 mMt actics. aci d, pH 3,5 at a flow rate of' 2.5 ern/mwin, Protein contains fractions were pooand th pH was adjusted to 5.0 using 10 N NaOH and further dliuted with 5 volumes of watr The materal was fitered Hthuh a 0,43 pm celulose acetate filter (Corming) and further purified by cation exchange FPLC (SP Spharose High Perfoann ce GE reathcare Samls were loaded onto a column equilibrated with 100% buffer A (50 mM acetic acid, pH 5.0) and clted with a gradint of 0 to 8% buffer B (50mM acetic acid, I NaL, pH 5,0) over 3.0 column volumes at a flowrate of L5 cm/min, Peaks contain target species were pooled and forulatemd into 10 mM sodium acetate, 9% sucrose, pH 5.0. Reducing and non-educin (+odoacetamidl analysis was done on 4-12% or 4-20% SDS PAG Triis-glycinc gels (Invitrogen) with 0.5 pg. 2 pg, ad 10 pg of potei, stained itnh (uicT.lu (Boston Biogicals) Anaytical SEC was done usi'g a Bisep SEC-S3000o col umnc (Phenomene-x) and a n isocratic ehutiont of 50n mMt so]iumn phosphate, 250 m 4 NaCl p11 69 over 18 min Exemplary purifcations of immunumogobuin -toxin peptide analog fusion proteins are shown in Figure 24-36 A -8. 2 9A-B, 30-32A-R, and 33-35.

00I OO7~Pharnankineiic/Pharmatcodynamic E vakaion of Monoandent Fel U AhahK[24$lHeterodliners and lionovalent or Uivalent e/ EhK( 2$ Q16K)(gG2) Hleterodiners and hnmwnogobi u sion Pro i f we. [00674] Embodimens of the antigen binding proteins of the present invention, used as immutnoglobuin carriers for phamnacoiogicall active polypeptides were demonstrated to provide tavorable pharmacokintic andS pharnnacodynamiic properties, Monov aent or bivalent Ec-I LWSK{235] monovalent or bivalct Fe Li 10~ShK[i-S) NmTlaalnt or bivalen Fe-L ( 0ShK( 1-35. QI6K), mnowvalent or baiaent anti-KLt WSHIC-Sh(1-35, 0106 Ab. mONaent or bivatet antiKLU AbLoop[Lys16]fShK fusion prot'ins, movJzent Fe-Shk{-35 Q16K VKLH Ab heterotrimer, and other exmlary amTodiments hited in Tabe 7H wre expressed isolated and purified bry methods desribed in E~xample 4. PEGylated and un PEyIated toxin peptide comparators in Table 71 wre prepared symfhteally as follow: [00675] Pftide Synthesis. N(&dimoc, side-chin protected amuio acids~ ad H Cyvs(Trt)-2Cl-Trt resin were p urchased from Novabiochem,1 Bacem, or Sigma Adih, Th oLl owJing Sidchain ptotection strahty ws employed: A pOtuY A rgPbh CyTrt), Glu(Otiu His(Trt) L~ ys('NEoc), Ser()tE4), Thr(t0 u) and Tyr(Otbu). ShK (RSC3DTWPKSRC CTAFCKHSYNVR LSF4RKT(CGTC/ SEQ ID NO:361) [Lysi6]ShK (RSCI DTIPKSRCTAFKCKHSMKY RLSFCRKTCOTC/I SEQ ID NO:76), or other toxin peptide analognamin acid a equences, were synthesized in a sewise manner on an CS B$io peptide synthesizer by SP PS using DIC/HO~t coupling cmistry at 0.2 mmnoi equivalent scale unm H-Cys(Trt)~2l- Tr res'in (0. mo 0.32 mmolg loading), For each couphLg cycle, inmo N' Fmoc-amino acid was dissolved in 2.8; mL of O,4 MI 1-bydroxy benztrnizol (HO~tI in N-dimorthylformnamide (DMF At Tothesoluion Wats added L imL of I.0 Mt N N'-ioprpyltca ~rboimide (DMC) in DMIF, Thre solution was agitated with nitrogen bubbling for 15 min to accomtpish pre--activation and then added to the rmin. 'The mixture was sten Nbr 2 h.I he; resi was GitRed and whd three Cunes with DMW, twI' e with dcichloromethane (DC\1L and 'ace ties with DM\W Fimoc deprotections were Carried ot by treatmem with 20 pipardie in 9Ni (5 ord. 2 x 1 mm). The firs. 23 resdues were smuge auled rough repemmon of the i Nle&dflinO acid cOUmitg and Fmoc remma steps described e he rmamnn residues were double coupled by Performing the couplmng step twice before proceeding With Fmo c-remova-l 100671 iFolowng synthes, the resin a then drained, and washed sequenally with lX'M, DNF M, MO . and then dAe m 'vaeuo, The peptde-resm was tranferred to a 250-d plastic rmand botom tlaih The peptde nas deproected and releauwd fromn the resn by treatment with trntsopropvisiane ( S n L i diox- l M otane ditiol (DODT. 1. A ), waterI j j t influomoacete ac id (TF 20 miL and a st ar, and the mixture uas stirred for 3 h The mature waos ftred through a 150 m. siraerod ga unnel into a 250-m plstic round boom lask. The mixture was ilered through a 15n0-n smtnercd glass funnel into a 250-o. plantic mund ottom flsLk and the Itrate was concentrated iacuco The crude periide was precipitated with the addition of cold d othyi et -c!! t ere by cemrafialon, and di ed under I Penide l'oldin & te dor crude Inear peptide tou 600 rg for .'ur~f1l jI a sih]hk peptide KSEQ ID NO46f or [I ysZ6VShK-Ala (also known as (1 1A AXu Ve \hk, SFQ 1)11 ' UQ) peptide, an dissolx ed n 16 mlt aettic acid, . ml water, and 40 ni aceonmin The miauar' was stirred rapid for iA nm to complete dhssolution. fue perptde solution w as added to a 2-L plastic bottle that contained 1700 ml. of wattr and a large sr bar. To the thus diuted soluAn was added 20 ml of concentrated ammonmum hydroxide to raise the plil of the solution to 15 AThe pH was admted with smal amouns of acetc acid or N as necessary The solution was stirred at 80 rpm o vermight and notre by i'-\S. Widin; w a usualy jadged to he complete in 24 to 44 h, and the solution was quenched by the addMtion of acetic acid and TA (pi 2.5) The aueous soinoa w as i ltred (M.45 nit cellulose memthrune %.

[00678]I Rcv ersed-P hase FPLC7 Purification, Reversed-phase hig~h-performance iqid Chromat.ography was performed on an analytica (CW1, 5 n, 0,46 mr; 25 cm) or- aprearaive (Ci8, 10 gm, 22 Cm 25 cm) co umn. Chromatograhic separations were achieved using inear gradients of buer 13 in A (A = ,1% aqueou TFA: B 90% aq, AC(N containing~ 0,09Q% TFA) kmpcally.595 ov er 35 mnat a flow rate of f mL/rin for anmytical analysis and -65% over 90 min at 20 nlLmin for preparative separations, Analyical and preparative HPLC fractions vere characterized by ESMS and photodiode array (PDA) HPLC combined and [00679] 1 s Spetmry Mass spectra were acquired on a smgle quadrupole mass spectrometer equipped with an Ionspray atmospheric pressure ionization source, Sampes (25 pT) were injected into a m ing Wvent (10 p~tmin; 3030:20 ACN/M& HI contdaining 0.03% TFA) coupled directly toth ionization source via a fused ilica capilary interface (50 pmi. Sample droplets were ionized at a positive potenti of 5 kV and ctered the analyzer trfae plate and subsequent through an orifce (100-120 pm diameter) at a potential of 60 V. Full scan mass spectra were acquired over the mass~ orange 40-2200 Da. wih a Seanf sit p size of01 Da. Molecular masses were derived frm the observed m/ values, [00680] PEJvlton Purification and Analysis Peptide, e.g. {Lys 16] ShK (SEQ 10 NO76) or [Lys16]Sh-Ala (SEQ 10 NO:362 was selectively PEGylated by reductive alkylation at its Ndcrminus, using activated linear or branched PEG. Conjugation was performed at .2 mg/m< in 50 mM NaH HP0 4 pH 4.5 reaction buffer containing: 20rnM sodium eyanoborohydd and a molar excess of 20 kDa monomethoxy-PEu-adeyd (NO, Japan) Conjugation reactions were stirred for approximately 5 hrs at room temperature, and their progress vasn mentored by RP HPLC,. Completed reactionsnvere quenched by 4-old dilution wit 20mM NaOAc. pf 4 arnd chilled to 44C, The PEG-.peptidenvere hen purified chromatoraphicaily at 40C: using SP Sepharose HP columns (OE Healthcare, Piscataway NJ) cled with linear 1~!M NaCI gradients in 20mM Na , p H 4.0. E-lated peak fractions were analyzed by SDS-PAGE and RP-HPLC and pooling determined by purity >07%. Principle contaminant observed were di-PE ylted toxin peptidn g Selected pools were concentrated to 2-5 mg/ml by crifuga fixation Ca t3 k1a MWCO rembranes and diayzdR iito 10 mM NaOAc pH 4 wi 5% \Orbit, DiaWyzed tools were then steie fiteed through {, micron liters and puriy dtermhined to be >97% by SDS-P AGE (data not howvn) Revers-phae H P 'C was performed on an Agilent 1 100 mode HP LC running~ a Zorbaxst 5pm 300SBCt 4.6 a 50 mm column (Agilent) in 01% TF\I20 at! in1 mmin and column temperature maintained at 40QC. Samples of PEG-peptide (20) pg) were i nje and eluted in a inear 6-60% gradient while monitor ing w aItih 215 nm. [0068 1 Fusion Proeins. Gneraily, Figwure. IA and igure i ow a schematic represenaton of mtonovalent and bivalent Fc-toxin peptide (or toxin peptidc nlg fusioni proteins (or "peptibodies"), respetiUvely, The hi valent Fe-ShK molecule is a homodimer containing two Fc-ShK chains, The monovalent RShK toxin peptide (or toxin peptide analog) molecule is a heterodimer containing one Fe: chain and one Fc-ShK (or analog) Chain, Since the mAnovalent Fc-ShK moecule contains jut a single ShK peptide per diner, it is considered monovalent. Construw or chains referred to as Fe(toxin peptide analog conitain a Nerr Fe, region and an optional flexible linker sequence (e.g. LIP peptdy hiker GGGGSGGGGS: SEQ iD NO:!53 covalently attached to the toxin peptid or taxin peptide analog, such that the orientation from N- to C.-terminus would bte F-ker- toxin peptide or toxin [O08T) in Examples I and 2 ofSulivan ea at WO 2008/088422A2, were desenbed the euivhtyofb biuaent i cbhK epribodies Fell 0~ShK( 1 05$ and FE~ L I-Shu ) cxpresse~d from mammnali~ani cells inEnmple l of W) 200'8422.A wa a lso described iolation of a monovalent Fe-L -ShK( I35) molcule, formed as a small bjproduct durmn expression The bident Fe- 10 ShK( 1 35 and El' 10~ShK(235) conjugates prodded pote blockade of E 1.3 and 1 cell cvtokme secretion in human w hole blood (see, Yiabe Th. By whole cl patch clamp electtophysiologw the bihale. Fed 10.ShK( 1=35) moleule had about 8-told sreater Ks U . active ity romptared to the hn valemt c- ed 1Sih.K(2-135) miolecule that is devoid of Ar. of Sh K. Like N-terminal PEG conjugates of native SbK (' Examples 4), both bivalent Ec~ShK conugates showed little selectivity ifor Kv .3 versus 1Kvl J . Thus, N-terminal conjuti~on of native Sh K alone (with either PEG or Fe-linker) does notsinificantly imrove its Kv1 3 versus Kv1J selectivity, Pharmnacokinetic (P1K) stud ies in rats 'wee pe rformed on brvaen t Fe-L 10~ShK{ 1<1) and Fe-L1 I0-ShK(2-35) peptibodies to examrin~e their stability and half-life in vivo.. As a cnotol, P1K was also performed on CH D-dCerived recombin ant human Fe Igl). All molecules were delivered as a single, intravenous bolus dose. [00683] P1K assavs [00684] Antibodies to ShK. Rabbit polycional and mouse nmonoclonal antibodies to ShlK ( SEQ U) N:36!) were generated by unmmunization of animals with the Fe ShlK peptibody conjugate. Anti-ShK specific polyclonal antibodies were affinity purified from antisera io isolate only those antibodies specific fora the ShK portion of the con jugate, Following fsion and screening, hyjbridomnas spcii for ShK were selected amd isolated. Mouse anti-ShK specific monoclonal antibodies were purified from the conditioned medui ot t clones, By. ELISA analysis, purifiedt anti-ShK polyelonal and monoetonal antibodies reacted only to the ShK peptide alone and did not~~~~ cK rosratwt3 [0068.5) Pharmnacokinctic (P) studies on 2{)kDa-PEG-ShYK (SEQ ID) NO:3) and 20 k~a-PEG-[ Lys16]ShK (SE2Q ID NO:364Y peptide conjugates in rats and mon-keys, Single subeutanco us doses were delivered to animalIs and serum was collected at varions time points atter injection, Studies mi rats muvoived two to three animals per dose group, with blood and serum collection occuring at various time ponsover tecourse of the study Male Sprague-Dawley (SD mIas (about 0,3 kg) and male cynonmolgus monkeys (about 4I kg) were used in Ihe studies described herein (n =: 3 animals per dose group), Approximately S mzne CDL-I mice were used per dose and time point in our mouse pharmacokinetc studis, Sertum samples were stored frozen at -80tC, until analysis in an ceazyme-inkcd imunosorbent ssay (EL ISA 100861A brie f dzcitof t te ELAApooo o eetga eeso [006SlY and Pmbda EPEG~6SShK i sMnvdPdEG [006S7] ( I) Prooco 1. (?)4) beow, deas PU>SIK ad PPG4Lsi 6ShK, as Yw as the Mh W and [Ls iSK pepfids alone: [O\'008)()Stetvdn reorc phltes wercoatd w'ith 2A0 ug/'niionvte anushK mouse monoclonal atibody (mAb2 10, Aim en) in b bock uffer [per ltr: 1000 m !X.PBS without (Ca Q \CUI 5 m5 'fwecn 20 Thermo Scintick 2 block reagent (irona)) at 4, mneulated overm ithout shakmg. [006891 (b) Plate were washedthree te with KP wash buffcr I rkegaard & [00690) Qc) Stnars(11)1.qultcotls( )ansarleduinswe prepared with 100% pooled sera, then diluted 15 (pretreatmerd in I ock butfer Pretreated Sifl, Q(s and samples were added to the washed plate and incbted at root temperature for 2 hour& eal dmons of S'T Dt QNu were prepared i 100% pooled sern. Samples needmg dilutin were also prepared with I00% pooled rA, Ie pretreatment was done to hoth ads, Qs and aunp)e to imamie the maths efe ) [006911 (do clates werost e three tie ihKLt tbnr [00692 ( \ HA P aeed rabbit nIShK poteonal Ab at 250 ngjl in b blok bWE was addd and nbed r o perature fr P hadking, Femto [Thermo Scientifi] snbstrat was adt [00694 ( The platewas read ih a a 184 (Molecud Device) [006i4 (g) eter.

iand [0069 51 Pharmaceokinetic (PK,) studies on Fec. Lg. or Ab coniuattes of Sh iK and Lys 161hK were performed in me SD rats. S vucutaneous doses were delivered to animals and scrum was collected at various time points after injecuan. Three animals were used per dose group. with bood and serum colectiOn occuring at varous time points over the course of the study Serum samples were stored &rozeni at 40*CLmtil analysis in an enzyme-liked immunosorbent assay (ELISA I A biefdecritio o tb ELIS prtool ordetecting serum levels ofUe EcWg or .A. bhownw~ OW Ll 9 M Nrthe WAS If \c puvco itt Abconjugaes of ShK and ([ILs 16]ShK in provided below, Protocol 2 bclow detects both the human g, Fe or Ab potion of the molecule, as wll as tshe Sh ptide portion, Protocol 3, below is an candy assay tat e the human Fe region alone and was used 1o early issessmet of serum levls of Fe-ShK pnpibodis in rodet pharmacokineticstudies A brief description of thesn E LA pmtoccl is provided: [00696 (2) Prtocol 2, (ag) bciOW, detects both the hma 1 Fe or Ab portion of the molecule, as well as the ShK peptide portion: [00697 (a) Streptavidin ioier plates w coated ih 20 I otinylated an h mouse monoclonal at ady ( ab 2. An b k bf fe [er liter 1000 ml IXPIS without CaC nl een 210 (hermno ieMe 2 1 ro~ ]at 4 erih without shki,, [00698) (b) Plaes were a w td tie time w it w hicrlegaard & Pery Laboratoriest [006) (el Standurds (STD! quality cdrols (QC) and sam mle diltiols Wi l 100% pooled sera were prepared, then were dilnted .1/ (pretreatmentiniblock butter Petreated S'i sk QCs and samples vwere added to dthe w asbed plate. ncubation w at room tempertmre r 2hrs Serial dilutions of lTDs. QCS wore repaed 1 l 00' p oled snL SamNple vleed lution w ere aso prepared w th 100% pooled ser 'The treatment was done to both stds QCs and samples to rnnnzae the matrix effet. ; [00701, (cIA IRP-,-laekd Ab3S (against human Ig Qc at 150 on/tn in! SVoC h thyew was add ad and plate weerwte6.a om crl ralr b lhuXt [00' 7 0 2 i (') lae VO stoed theetis Win th k.ws ufr n h:Prt [Thmoy Swceat lid utrt wasxade [007031 (g) The plate was read wt m~ i3 Ml(ta eie tiut nornete' [00 7 04] 13 PntIjj 3.( bI) olow, ~is an ealc ssytatdtct h hmmF return alon and wsueto? eanly assesmn of serum levels of .4SK peptiocns i a Tdetopm ruoinn tdjs [00705) (a) Costar 3590,' )f J It R MA late were coated W 01 n~yL/l of [007061 j Pa)f~~an "wr iWve e h ti e"'," wth PST (PBS + 0.% T euO and blockd hy addition of 0.3 l ao lto IPM, VAU I i Tee20 NO% noialr Wuik) to each wel arid incubated Obr I h a fom tempeature (RT) with shwknwg. [0070 7 (eQ) Plates werashe wih a" K? Wash Souion-cat40#-0. f0070S! (d) pilue sewrum sampesan onrosradad "i Dilutio tul (PBS. . Is d~.n~ as Mu pmucH, kb~c ro teprature with".. shbaking Ibr hor; [007091 (a) Plates were washedd With a K Wah Soluion (at /031.K (tithe s .0) [00710 if() A. HR labele 14 i y~\ (iPex, ff3 F 1WGa d l" k i ~diue 10) in. P11 nd en 10 i'w is adde an ~nn atcd at Iwith shakng for hour fO0l 11 ( nsUi were washed wit a KY WashSoluin Cat #50- 3 00 KPL Gaithenrburg, MD) and 100) pAll of ABTS substrmte (AB rS Microwel Substnae NCo nonent (9#r51Y-66I)fl .. K. wa s added: [007 A (ht) appropate nes afr substra adoio and shakiN the plate as read widi a SpecraMax34O [Melecdar DevcA] pl [00713] Whereas the original monovalent Fed-LI0-ShK( I-35) nmolecuie was isolated as a smalli by-product during maaiarin ami expression of the bi valenm moltecuk, Eanple 4 herein Aso dcwribes cloning and mnmnlian expression of the monovalent F-L 10-ShK[23 be'rdier. B~riv, to produvc recominant monovalent e- 1TShK[2-5) two recombinant polypepides are co-ex pressed in the same cell, those being a human Fe (gO) chain and Fc-L 10ShK[2-35 chain (also with hurnan IgG) Fc1 region). Under these conditins it is possible to ftrmi three distinct dimers. vhich include n Ec/F homodimei a F&-L 0-ShK(2-5/Fc Ll SbK(t35) homodiNer and a Fc-L1-h) hetcrodimer, By optmizing expression conditions the monovalent Fc/Fc-L 10-SK(2)n tc eterodimner (also referred to as just monovalent Fc-L 10-SbK(2~35) wa produced clfficiently and was readily prified ~ to homogeneity (Exanmple 4 herei). The nmonovalent Fc-L1 0 ShKT35) molecule had an IC50 of 2 1 1M (Tlbk 7b) in blocking iL- secreton frnm human whole blood. The monovalem F-ShK/Fe heterodirner ha an extended half-l in vivo and exhibited significantly greater exposure than the hivradnt homodimners ShK-Fc/Sh K~Fc (Figure 10) and Ec-ShK/Fe-ShK, Since the poteney of this construct was about 10-fold less than the PEG-ShK conjugate and conjugtes of native ShK had poor Kv L3/Kv, 1 selecivity, we developed additional rnovalent peptibodies and foed conjugates of ShK toxin peptide analogs that were id ed to have improved Kv 1 3 versus Kv 1 Aselectivity The examples that follow provide additional details of monovalent peptibodies with improved selctivity and in vivo phamacology the i rds fon these si indicated tih monvaient hKt danalogtmkoleaues exhibitedgeate s oevelsand 1xpsp r rasxven compared to the IinT brms of the same mole t rinthe slow eliination [00714] Monoxvden Ic/F\i kj iQM Lhedimger g1> ShK[.Lvy16] C toxi ep'tiaalog (SE 1)D1 NO:76) shows~ significant 1Kv L3\electivity over neuronal Kv I (Tabe 71). To increase me stability of tis toxin peptide: analog in vivo, :we. gentd a mnovalent Fe fusion conlstmet that from N~ to Caermnus contaned:. human F(IgG2)- L0 linker4[LysI]ShK moice, that was co expressed with the human EdgG2) chain alone to generate a monovalent heterodimer (se, Example 4). A schematic renresntation of this monovalent construct is provided in Figure I A. The mnonovalent Fc/Fc-t 0-ShKi'-35 Q 16K heterodimer [also referred to as monovalen Fe/Fe-ShK( ItS35, Q6K)] potently blocked T cell inflammation in whole bod suppressing 1L-2 secretion with an IC 0 of WJ 6 nM (Table 7W). Unexpectedly, studies to examine the Kv 13 versus Kv l 1 selectivity of the molecule, revealed thatL the monovalent Fe- E-K1-33 0(16K1), conugate had signicantly better Kv .3 selectivity than the [Lys16]SbK peptid alone. Whcras the [Lys16]ShK (SEQ iD NO:76) peptd alone, showed about 18 fold selectivity for Kvi. 3 versus Kv . 1 (Table 7H 7 at onov :ent Eu/F- 1 I 0 ShK( 1-35 Q 6K) heterodimer was about 1225-fold rore active in blocking Kvl 3 versus 1Kv 1 L. Therefore, the [Lyxs I6]ShIK pep tide w hen ionjugated sihowvs a unique pharmacology of enhaneed selectivity. Since the N~20kDa-Pt EG-[Lys6]SbK conjugate (SEQ ID NO:364 also showed enhanced K"v3 selectivity Table 7H-i) relative to the peptide alone, the combined data suggests that the y b ShK (SEQ ID NO:'76) peptide when iused at its N-terminus with cithe PEG o r c-lnker e bts a dinc a cologyonioved Kv 3 veNus Kv activity. [00715) To assess the pharmacokinetics and stability of the molecule in vivo, as a basis of comparison for the inventive molecules single-dose PK studies were performed in rats, After a single 6 rmg/kg subcutaneous dose, t monovalent Fc/Fe Li1-Sb1(01.35, Q 16K) heterodimer (of monomers SEQ ID NOS:i and 2) exhibited an extended hafylife in vivo (Figure 7) Sinc the sandwc ELISA used to measure serum levels of the molecule " 'rotocol 2") requ irs binding of to antfiod, one an anody specific to human Fc rgion iand the other an antibody recognizing [Lys16]ShK (SEQ ID NOo76 d here indicate that th conjugate had prolonged half-if and remained intact in vivo as a Fe-L0-ShK( 135 QI6K) fusion protein (Figure 7. open squares; TIbe 71 below). T he monovalent Fe/Fe-.10 SK(I-35 Q16K inmoleec exhlii an extended half-ife of about 56 hours, that was about 112 times longer an the SIK (SEQ ID NO:36I) peptide alone that was reported to have a half-life of2000 min (C aeton e , PA S 9W8:13942 (200 14 [00716] intlend shK&35 Q6K)hom i. QgG) Th hi valent Ec' ShK(P35, Q1K) homodimer contains from N- to C-treminus: human F MgG2) L 0 linker - [Lys16ShK (SEQ ID NO:26) A schematic representation of this bivalent construct is Njrovided in Figure 113, The molecule (homodimer of SEQ 1D NO:26) was cloned, expressed and prfd as described in Eixampc 4 'wrein. T he purified mo leeu e was tested for activity in the human whole blood aso inflammation and found to have an IC50 of I850 nM in blocking 1L-2 secretion (Tabe 7H), The acivity of this bivalent form was abot I times less th' te monovalent torm (above) which had an 1C50 of A 6 nM in this same assay. The reason why the bivaleNt flo was Iss active than the rmmovalent is unknown. It is possible that the bivalent molecule containing two positvely charged VL ys 6IShK (SEQ 11D NO:76) peptides at its end, is less stable and/or interferes with Kvl 3 channel bind ing to some extent. [00717) Monovaentan iv -jagg tK 1111 t Q I - ~l'ga 16 ] Nb The monovaent anti-KLH Ieav Chain (HC) fusion anuibdy (Atb) construct efrnbodiment of the present inverion contained, frm Nta termis human anti KHt Ab Hevy h ain peptdyl Minker-[LsAlShK molel (SEQ ID N: 32, that wa~s -c\pressed wirth the humn a L H Hcary Chan alone (S\EQ ID NO:29) and the human aKLH light chain (SEQ D N0:20) to tK [Lysi6]ShK molecule (heterottramerof SEQ i) NO-2; SEQ ID N:29; SEQ NO:28: and SEQ ID NO:32). A schematic rApresentation of this monovalent 234 constrmt is provided in Figure F The monovaent aKAL HlCShK(3 Ab potenty blocked T cell inflanation in whole blood, suppressin IL secreton with an Iso of 0.274 nM (Table a Unexpectedly, studies to examine he KvL3 versus KvU . selctivity of the rolecue, reveaed that the movaem 1KLH -I' ShKA -35 QI6K) Ab (heterotetramer of SEQ ID NO:28; SEQ UD N:2 SEYQ ID NO:28; and SEQ ID NO:32) had signiicandybetter KvL3 selctivity than the [LysShK (EQ I NO:7 6 pcptde alone, This monovalent Ab-ShK conjugate was aboutt I 458-foid more activ in u blocking Kv1.3 versus Kvl . (T able 7TH and Figure 2A), [007 1X1 To assess th pharmacokinetics and stability of the molecule in vivo 0ingle-do0s PK studi weo, re performed in rats. Afte single 6 mg/kg sbeutancoos dose, the monovadlent aLH HC-ShK( 1-35 Q16 Ab conugate exhibited an extended halfhiew nvO (Fiure 7 closed circles) Snc- the sandwich ES SAused to measure serum levels of the molecule (protocol 2") requires binding of two antibod es, one an anybody specific to human Qgregon and the other an antibody recogniring [Lysl6ShK (SEQ ID NO76), the data here indicates that conjugate has prolonged haf-life and remains Iiact in vivo as a monovalent aKtl-H IC-ShK(1-3S Q6K) Ab fusion protein (Figurc. Figure 8, and Table 7 T'he ivaent aKLH HUC ShKf1-35, Q 16K A b molecule schematically represeted by Figre D 1 giVen at the same 6 mg/k dose, showed a similarly slow limination rakt (ige 8)M but provided about 37 times less exposure (as measured by AUA Table 7J)relative to the noovaent molecule (Figure 8), The potent and selectise monovalent anti KLI~Ab-[Lys 16]ShK molecule exhibiehd very slow clearance n rats (L/F = 0.9 mfL hk ok (Tabie 7J). (007 191 oqqovalent aKENJJL-Sh!Ki2-_Qlle1j b. 'Tbi monov alent aK LIH Heav Chamn (HCO) fusion antibody (At) constmect enbodument of the piresent hv a enen contaned rrom N- to ('-terminus~ human amtiKtl 11 Ab lensvy Chann haker i'desdrg L Lys alSnhK molecule (SEQ ID) NO;33 thias cno-expresed ith the huan aKi flea vv Cham ESQ 1D NO;2) and ibe human aKL-H bght chain (SLQ -0 NO:2) to fon a monovaent al] AP4 desAr-K J1L L fliShK molecule. A scthematic representation of tis monovalent construct is provided mt Eigure l ' rhe monovalem aKl 411CShK(2-s5 QinK i Ab terotenramer of SEQ 10) NC:28; SLQ N) NO:29; S~IO tO1 NO 2$; and SEQ IID MO:3) potent bloc ked I cell inflammation mt hole blood, sppressing i1 -2 secretion u uh an 1(00 of 0.570 nA (able 71I and une speedway was about 15b ld more potent n locking the T-cell potsium chanel Kv 3 than the neuronA channel Ky b [00720 Monovalent Fe-SbK( -35 O6K. iSKL H Ab Heterotrimer The monovaleut Fe-ShK1 M5, Q MK e or ben e n the present v ention contained (rom N- to C-termius: human R (gGl Li W imker [tyI yljhK iuolecule (SEQ ID) N\:0 that w u5 co-eprene uA uh i the hone aK LI Heay hain (1g62)(SEQ ID NO2)ad the human'tR U Lght chin (SEQ ID \O85 .A schematic rerreentatno t'is monmalent COUi5WUQe is prouied m bimue iML The monovalnt Fc-ShK(13> Q K4/KLH At hetrcotuimer (SEQ 11) NO "t SQ I) SEQ W 10 ? 0rV paontiy blocked T eci on~flamma'tmin - human whole blood, suppressmg 1~ Necretion w ih an 1.50 of 0245 ral Table oH). Smpi0smgly, sudies cuimamng he Kx L3 versus Kv 1 . selectiwt of the molecule revecaled that the mnonmoalent Fce.hK(I -35, Oi6Ki l Ab heteronmer had significamty better K 1 3 slectvity than the fLy 6eShK peptide am t ISEQ i) NlOQ ) Tis monvalen heterotuoner was about 1935 Iold more atie in blocking Ky 13 verss Ky L 1 (Table 7- Hi (0072 II Although w e hav e nor examined the phamacoinetncs IR I of the Ky 1 3 selective monov alent FE-ShRI 1 -35 QI 6 Kl H At h eterotrimeir or hetmbnody w e have examined the PR profile of a sdim lamhody, that being the Fe-ShK(2 35) KL H Ab heterotrimer. A schematic of'the struture ot' this mool ecle is provided in'gur 1T, and the molecule from \- to Cterminus contasb human 'e (g1 1 2) ShK(-35 W which is coexpresed with the human aktH hav chan and ight chains A fte a sogle 2 Tmg kg subcutaneous dos the monomaen ' eFhR K2 3)SkLH At heterotrimier (also referred to as m2onvale)t Fe ThaK L Ab hetennmnmetr exhinted an extended half-it. mn rats (Thgme 10). Sme-theandnueh EL SA used to measure serumn levels of the moeule ("ptotocol "'I requires binding of two antibodies, one an antibody specific to human Ig region and tbe other an a ody r gizin ShKU235 the data here indicate thot conAjugt has roonged half-life and remains intact in vivo (igure 10. Table 7K.). The large about 103 kDa monovalent Fe-ShK(2-35)KL Ab betrOtrimer or hemibody showed greater eaposureo and about 2-fold es clarance than the about 56 kDa rnonovaent Fc/Fc> SMK heterodimer (Figure 10, Table 7K) The very smalL about 4 kDa ShK-L5 pptide was cleared much more quickly, hav ing a clearance valuc in rats (CUF= 2052 raL. 4 kg' Example 3) that was about 91 times faster than the large monovalent FcHShl(2~35)/KLH Ab heterorriamer (CL/P= 22.6 ml b k molcule [00722] Mov alut and bivalent antKill AhoopslJMhK afus irtein< Reconmbinant monovalent and bivalent ati-KL H AboopiLys I ShK fuson proteins embodimnents of the present invention were conlsrtactd as describedv in Exampie 4 and 1S Patent No.742,778 32 to produce fu antibodies wit [Ly,1 6iShK toxinynptd analog inserted into ioop region\ o the Fe domain in one mionoval ent) or both (bivaent) HC enonerners. The nmnoaw e Wn aKL H(Coop ShK( k35, Q6K) Ab contained three chains a human KL H Abt beavv chain, a human aKLHAb light chrin and a human aKIH Ab heavy chain where the [Lsl]ShK peptide was inserted into ao lop within the Fe region ofthe heiav chan. The [Lysl6ShK pepide within the e loop contained a flexible linker sequence attached to its N and C- trmnus to allow for independent foYding and extension from the loon. A schematic representation of this molecul is provided in provided in Figure i Unker \equences of die rng amino acid compostion and length were examined. The monovaent abni-KLN AbLoopy s 16 ]SK fudon protein was a select inhiitor of K v 13 activity ovr Kv 1; 2 2oId mor' selective ftor K v L3; Tabl 7H and Figure 2A-13) T he monovalmt KLH-AbLoopItys16]ShK mnolee ex i hhibited the slowest cleared, in rs of all the novel t x in-conjugates that we nave exam ied (Fiure 7 and [igure 9 and Table 7L . [007231 The hivalen aKt [CAHop SqY 1h-33 Q6K) Ab contained two chai a. Moan YK.L. Akblihhi and a human aWKlH Ab heavy chin w here the [Lys16ISh petitde was inserted into a loop witin teeei:nfh ha chain.

A schematic r'pr euat1n of this moleule ls provided im provide in bhue I M In c ompare the phianusiokmieties and stand n. i mo of thns bndtaL molecule to the monovalent form, sincle 6 metkn suhetanmeous doses of each molecule w ere delna credh to rats. Despite show ing a slow elnnahon rate, the hivalen: ak i- HC.10 loop-ShK -35. Q6K) Ab gav profoundly less esrosure in rats than the tnonovient trm of the sme molecule (monovae ak I H C-loop-ShK( -35, Ql} Ab)(see Figure 9). exposure as measured by A M, was about 161 te less for the bialen: aKtH H(l Ioo.ShK( K -3 Q ik) Atb moleul e compared to the monovaulent a.KLI HC.t-loop.-ShK( in .-Q Ih . hmlet 7 a l). Ther eforce, our nove d ono eni Sorms shtow an unexpected and vastl better pharmacotinee PrOlIC mi vivo compned to typkal Na]lent forms of the same molecule. 107^24] Monovalet c hc~k e ted odb ner. The monovalent ShK( 1-35, Q h ok)~ 16 heterodimer comais two chams, one hema a human Fei't cW1 ham and the other b gShIK(!.-S, Q n)pnde iimod to Fce that ontam\ horom 'N- to C-ermNus: [L 6iSShK LD 1l nker - human Fe ( jG T : his peptid'-tirmn protein contained from N- to C-terminus: the 35 amino acid Ilys SbK pepide, a ten armo acid GGSCIStJrtIIS (SEQ MD N') 5) I 1) liker sequence and the human Pc (1g02 sequence, Therefore, the linker-Fc region was attached to the Cteminus ofi' I CiShK tblow'mgp6 sS Th[ mole ule s also referred to as monovalent S:(1 -35' 014k I F heterodimer A schematic representation ot'thinmonovalent construct is pnsided igure I C The molecule was Chned expressed and puritid as desnbed example 4 herein The purified molecule w as highl putent hivm n~ 't( 50 o'0 u I nM in bloeing i002 seeeion in the human whole blood asta of udlad n (Tphle 'l1 Despie its exellent potenct the monovalent Sbhi! (11 Q1140-k.T F. bmteadimer showed only a modest -10 (bld selectvt uthino Kr) .3 versus 1Kv 1,- I (Tl 7I- tl1 Theref'ore, u n ould appear that thi linker-Fe fason partner attached the C-termmus of i LysI61NhK dows iot result in a further enhancent A K1 [3 selet i tis contrasts s h N-temnna fusions to Lys ShK, such as the monovalent cR shK( I -3 ( l ki heterodhmer T able ~' ) whbieb show ed -15 b ' d seleciv ity and had the F e- n er sequence attached to the N-terminal ArgI resIduc of [Lysj6]ShK. An important and notable exceptions, howeveM is the [ys1Sh K-Al pepide (SEQ 1D N:362) which contains a. single (tenina Ala, residue adding lowing Cys35 ot [Lys16]ShK. This moleue exhibited an enhanced 262 fold imroved seicetivity for Kv L >versus 1Kv 1 (TCable 711 Therefore, we envision that the specific amino ac-id residue added after Cys35 at the C-terminus of [Lyslfi]ShK, can alter the selivty pro~e of the fusion protein. For example, the monovalent ShK( 1-351, Q16K) -i 0- f 1 molecule describedmi this example contains the linker (ly residue~ ade after (Cvs35 of' [Lys16ShK. If an Ala residue was added instead f yong Cy1051 an enhameed Kv 13 selectivity might be observed, Indeed, we do see 2 told improved KvlJ selectivt by thme [Ly.s1 6]ShK-Ala peptide, Thts, we anticipate that speCciamn acid resdue at the fusion junction woud alter the selective profle. Theso residue can be ready incorporated into te linker sequence between t [Lysl6]ShK peptide and the human Fe domain or immunoglobulin light cham or heavy ebain to inprmve the con jugates 1Kv 1 3 selectivity. [0072<1 Monovalent ShKI 135 Q InK\PHC ai I H Ab The monovalent ShY( I-3> Q 16K)i u1t sI H embodiment ot the reesemt muvemion contams three chams, one beig the hman aKYH Ab light chai, another beme the human akLT Ab heavy dim and H the tid beig a pertie-aKI I A v chain fusion that contained from to C-terminus: 1s1 y IIShK 1 0linker human aKLH hcas chain. Therefore, this ttsin ton tamed the linker-hea y chain r gion attached to the 6-~tenniuus of{ F(s 6 lShK fto ing Gys5 A shemcata r epteentati1on O 'th monovalent ShKt 1-3, Qli VlK :d KI.H Ah molecuis o rided in Figrt i The purified molecule was highly potent hang an IC50 fA 14 aM in biockmn It 2 aretion in the human whole blood anay of inflammation Wale 7H\ Despite beM very large m wec and fused to a hOnan i heavy cha the mnono valett s 6aKH At molecule retained hih potency in blocking F cell responds. f0072] MvonovatraDN? HOtthYV-3 E i6 ) Ab he monovaent aDNP -any Chain (T) Ksio anybody (Ab consrict enmodientohe opaesent inano contanud fro N enrninu< human antiON P. Navytan inr- (LylShK molecule, at was co-expressed with the human &DNP Heavy (Chain and the human aDNP li chain to ormu a mcnovaleni aDNP .Ab-Lysi1ShK molecule. A schematic rcprcsentation of this monovalent construct is provided in Figure f. The monova In TDNP H C-ShK( 1-35. Q 16K) Ab potetly bokektd T cell inflammation in human whole blood, suppressing L-2 secretion with n I'5 of a,278 nM (Table 71H) Studies to examine the Kvi .3 versus Kvl.1 selectivity of the molecule, unexpected ly revealed thtat the monovalent atDNP HC4ShK(1-35 QI6K) Alb conjugate had significantly better Ky . \elecivy than the Lysl6]ShK peptide alone. This monovalenlt AbShtK cong ws >86 fol more active in blocking 51 tZj ns ... t- $~~ ----------- ~4s - its, - - -- -- - -- -- - -- -- - - - - - - - -~ - -- - - - -- - -- Cal Cal w!3 A A) 7.'"-~ -~ ------ _ -- ~ to I\* -' -- -- - - - . . . . . .. .. . . . .. .- - - - -- 241 0~~t 0 O t enn Cy 1 --- W - ------------ - --- --T tot N -.... e e s.. .. e r t.. T ' > 2 Z O 4 n 0o 0 e- N 4 4) t V Q N t (C SQ C C) o Z 2 0' (C SQ C oil - - - - A A 9"£ 2C 'C I>' a 0 -Q Nt E Cot to P -E E he c . .. w : m -s -eN 2Zs _5 ~< ~1 ~s ' -> '9E > :9 o ot * N- z (e e - N -e' CS 0 U d Q ' G ir CC 'ccya 'aT (c) Cl ~~ 'y ' &It C----r r C f di as e f s ca1. 112 3(. '1 4it C o e''s CC ' ri <' v e ' ed e T441e 7 Phnimnokainetics of monovARent FAI [Lys6 inprague Dawl rats (p 1,~ uN Camn A (M 4 AuuMa C 11 ( WzD It (as; fun amm MO- rlh " I W"m'hS ' H Tab: 710. Phlnacokinetic daa. for ecombinantmnoaentnd bivalt nmiKU A .LsI ] h3iOn proteins adminited by subctaneouinj ect on dose 6 mg&g to lt .k 4us QpaeQ w Icy rat Ai 'R: 3).o I-7M 64Ro1 UK 71K Phrac data r recombinant mono vient FeFe-%K heterodmaer monovalent Frs hK lUi A.b heterotrimer and b talent ShKle %K-Fc homodimer uion proteins. admmnnied by subcutaneous iniect ion tdosce = 7 mg kg topragu-Do:e rats {n Tm co AtiC0. tfLU C &MPOI jt (3u al) t b~r~mt oN rmt' (mnLohr 3 IY MR T (h ShK;KLU < 6t 2< 2/5 t 53 54 ISS ::745 - I 632) 2 2 3Isk4e flN 00D 'able 2L Phur maohk ic data. fbr recombinant monovalent and invalent untiKL H A bioop-[ I ' 6]ShK Rion protemns adnnmitered by wuiaeous mipeetintoc mg/ka) to SimiueucDa ce rat (n 3) ________ A_____ I C m n U fl- .s (25 if (1L f' XfD 1 on (3a371 abr~mL) J ahramtP 'l I mLrM^< 3 Hi Lo (0012 Ln I [0s8] 13 ad iV loirophysioogy [0029i Cellns:~:"\v ho~ e 7 .HO Kces wee a "inaseted \vith hwma .3 o for counter eens see. Eampl i hcrnsl in th h~E 4 ik it, O hKvl . ^',HFK~3 cells were stable Onpresnmg human K 1 3 or with human Ks I I Cell lines w ere trn Amgen or BoFocus P (A alapagos Compant MCO M K eels stab expresing hky i1, ir counmersereyns were puchased 'rom \illipore (Catt.CY 30l5 1 |030] Whole cell patch clamp, eltroihy'noogr, Whole-eel I currents w ere recorded at romi tempemat'ur usig Multi(lamp 700B amlhner fron M ocular Deices Con, iunnyvale, (A v. suth- 3C\1 p ites pulled from boroy hese; <a (World Precision hnsuumes Oun [. D nm dan egimrmon, caracitre can sere canceled by analogue subtraction. no Ner3es re state compensation w as used, andIall currents w ere 'ittered at 2 kia The cells w e bathed in an esacellular SOuto Vonvaimin L. 4m t' t' 5 ' mM QC1. 135 aiM NaCL 5 m\1 Glucose. 10 m! HJUK% pl 7 4, 00 m Osmf The Miternal solution containn 90 mM KC1 40 mi El 10 mMl NaC t Iml Mgt 1 10 M FGTA. 10 m l H-PE'S, pH 7:. 2=000 musm. fbe currents w ere evoked It applying depourimg Stage steps frorn 4.0 rW\ to 30 mV every 30 a KA L 01 10 iKs . for 200 rs minervalS at holdos potamiai f~ m\' '1 determme Wt'50,np or pe-ptide convugate coneni tton at 1:3 dilutions were made mo extracelluia~r solution w ith 0. 1 ? B and delin'ered hoca ll to eel I with Rapid soltuon Oi.Changecr RSc.- 60 I~io ogi Science nstuments), ( urr-mm wer. achieved to steady state foi ech concetnration, Dta analyst was pertnied using pCLAMP ( ersion 9. 2)and (rginAro (v version 7) and l pecn crents before and aftet each test article application w ere used to calculate thte perecntane of current inhibiton at each coxncnriron.. [00732 Pawtpngrg'ghjarawvgjang tgletronk tg o o lics were bathed in an extracell uar solian on onm ining 13 mM Ca(? 5 mM KC I m onM NaM, 5 mM Glucose, 10 mM HEPES pH 7A 210300 mOsm. The internal suction contained 90 mM KL 4)0 mM KF, I0 mM Na lt 1 mM Mpg>l 10 rmM ECT A, 10 mtM HElES, pH 72 290000 mOsm. Usually peptride or pepide conjugate concentrations at 1:3 dilutions re made to determine die 1C . Tb peptido or peptide conjugates are prepared in extracellular solution contamng 0.1% BA, \Dendrotoxi n-k and Margatoxin were purchased fronm A lomone Labs Ltd, (erusalem, I ShK toxi was purchased from Bachem Bioscience, Inc. (King of Prussia. PA): 4- AP w as purchased from Sigmnad~idrich Corp. St. Louis \0> tmntms n ert recorded a roomreiperature USyg a PatehXpreW 7000A ecrophiology system from Molecular is es Cor,. (Sunny ale, CA The \volatc protocols For hK\'l and hkv 1.1 arc \ in l I'a 04 in Eainple 5 herei n extraeel ulalution wn RSA was apphed frt to obtain 100% percent of control ( POC1, then followed by 5 different concentration of i> peptide or peptde conjugate dions fm' eery 400ms mnubation tir. At the end, an of a specific benhmark ion channel milnuorable TM n K example K) was added to deine tall or 100% blockage The resdual curent present ater addition o benchrnk mhibitor, was used in some cases for calculation of zero percent ot control he benehark inhibitors iar 1Kv 1.3 and Ks; 1 1 are decribed in Table ~M in Exanmle K Each mdivdual set of traces o trial were visually vpec ted and either urecbted or rejected The [0072 I.Baseline curient us be stable [00331 . Ial pak curn must be pA [00741 3 i nual km and final Rm rost .00 Ohm [007351 4 Peak cunent nust ahev a steadstate pir to flat conmpound addition [ 6 Te X POC w caleiatd fon the average peakcuntrofthe ast 5 sweeps befloe th next concentration pound addition and epoed to Excel frIC50 lcuaton [007371 !gig Ji m ggbpit. Ymiitpseh..elji nhys t rleetrophyiolog was pertmed on (I) eal expressing 3 and HEK2. cels stably expresin hKv L 1. The procedure r prepr(nn of The 'YAs Piate' contains Sh. analogues and conpugatel-or 1WQ electroph log ws as follows: all anogues nweirc disolved in extraeecllular Pufft PBS'i "uh 0 0m Cr'M and 05 M Mg1 it 03% S..A aneddisp in the row £1 of 6eil polpvropyew pL us at the concentraton of 1.00 nl from column 1 to column 10, Colun i 1 and 12 were reserved for negative and postive control then serial diluted at 1:3 raio to row A., lon\orks Quattro I \Q) eiectoph~sroioy and datamnals were aecomphshed as tblow resuspended celts (m ext'acelular buffer), the Asay Plate, a Population Patch (Pl) PatchPlate as well as appropriate iracellulay to0 mK potawsum glucona.te, 20 .ml IE, 2 mM NaCL 1 mMN MC I, 10 mMluCIA. 10 mALI EPES, p14 73 and extracellular buffers were psiuoned on on orS QU atqro. Whe 0 te analoue lwe n0re, adAded to0 ra tthplate they weore Qfbdher dibited 34old trin the assa plate to achuee a final tes coraientrttion nmge t'rOm 333 uM to 15 pNI w ith 0. ISA.. lectrophviolog recordings were made from the C0 fI d-nd s ceJ l using an anten11 iri1 hed pertiw pateliip methodsinm the Voage-clamp circuitry of the lo r Quattro, ells W ere held a t a membrane potential of --- 0 m\' and rohlag.eeat ~ated K irrents weres oled by stepping the ieirnbran~e potential to 930 m\ for 400 nxs. K currentsa wer evote ind1r control conditilons i e, in the absenee of inhibiur at the henno of the peaement and after 0-mnne icubato i the presence of the ai1alogues and controls I he mean V. current anilitude- was nasured betw en Wt) and 440ms and the data were exposed to a \icrosoft Fxcl spreadsheet Thanplitude of the K- current in the presence of each concentation of the analogues and control as ex pressed a a percentage of-the K-P current of the pre compound current amplitude in the ami: weall. \\hen these % of control values awere plotted as a uncUon of concentration, the K5 0value for ench coa round could be cakuhted umg the dose-response fit nodel 201 in cel it program which ules the fl1WOWn equaunon. %.- of control m: cone wee n is he min im alueoierc 'yinma h e ixanmn- cx of the re. conc. i h test concentration ad enh isth U iaoe of tecure [c073] Measurig BioctIiy in nan ne Bo-ed (0(740] Es Lsio assa\ to euamine impact of toxin perntide snalos 1Kv 13 inhibtors on secretionjof------j--- AMC potency of ShK analog and coniugate in blocking I cell nflamation m human whole lood was examred O an on x No asavs that has been described earher (\e [sample -46 of W D 201044422 A mnrported herem by reference in ts entirety. in lf, 50 human whole blood s tuul-ateJ nith hapvgagin to nduce store depletion, caleium mobilication and cym tonc secrition, In a -st dhe potency of 1n0ocie in blocdhg T cel cytokme seemton, various centratons ot Kr-i 3 hbeek peptides and peptide-coniugate were pre-incubated w ith the human whole Nood saple for 30-60 nin prior to addition of the thapsiargin simulus. A fer 48 hours at 37"C, 5% CQ conditioned medium was collted and the level of cytokne ecreion was determined using a 4-epot eetroehemhlumines.cemtl imm~funloaSSay tromt MesoSeale Discovery, Ulsing the thapsigargin stimulus. the cytokines I L2 and IFN-g were secreted robusily frn blood isolated fronm multiple donors. The iLK2 and I EFN-g produced in humtan whole blood following thapsigargin stimulaion were produced from ' cells, as revealed by intracelular cyok inc gaining and fluorescence-activated cel sorting (F ACS) Wlyis. [00'74 I Kv 3 is the major oitage-gaed potass um channel present on T ces- Allonwin for K .ffhn, K vl 3 prxides the drintr for hm coinined Ci influx w which is necessry for the ~'suaned eles anon in irael luhlr calcium needed for efficient I cell acivation andi evtokime secretion Ks 3 inhibitors has e Xnshow n arher to Supprew this Calcium flux induced by TCR iamion (WC( Koo e tl, 09. ( . l Immunol, 197. 99-107)1 Thapsizargin induced stoae-deletion and ICR pHation eheitnuAr patterns o C 2 mobilization in iUslated]? eels j E Dhnnadieu et aL, 11,4 ioL CLm26'7 '564 'IA hut w(e hav e found thapsigargin gives a more robust response in whole bood. T therefore, we devedoped a bioassay whereby the bioactitv of Ks ! inmhibitors as assessed by examimmn their ability to block thapsigagin-nduecd eytokinc wecr<en fRom I cells in human whole blood Since whole ood is a complex kluid contaming hgh protein lesOK, the activity of pptides and peptide conuugates m this nwhole blood aswa has an aditonal adv antage in assessnmg the molecules stability over hours in a biologically relevant fluid. The whole blood assay provides important confirmation of th K\ I potency of molecules de0m3 med by electrophxsiolog icPhs since cPhn as as are enerlh of short duration -2 , hours and use physiologial s meine containkng no proicin The lmger duration ch the whole blood ussay my allow for more efdctw e determmaion of equiibrim binding kmetis relauive to ePhys studies which are of short duration [007421 Exmpe 10074 ge3 n channel coun tersereena f0.Kv2 K Q i . Ky I A and Kvj. Pmnch nae nanar vatlam eiect r iioWieo han currents can be recorded at room merperaare usg eatch prss 7000A elecropihysiolog system IfommMDC using methods and cels cribed 'ap 6 above. TIe vot proo0 Or each ChUnd hoWn in a 71.below. I ~ICt~ V~ aw rotocols and r eodng cond in. km omeSp Puhe Time Holding enchmark Channel DmuratAn between Potential inhibiu On M liKvI, 2 Faon80uV 4Orna 30s 80n \ M1 M 4MX Nt S I7K h.Kvt mm-k0 to SO00ms 58 4V1m ~tP wOVI.7 rom "trm to Amwhs", ji1 (Or mV MrN Nh h 4,A Q107451 Cardiac ion channel cMnesten hRS HKvLQribnt K.haL. "i 1007461 fjyij inca. H10293 calls stably trnfce jhKQTI/inn M and AFRO wer frm Amenor (ivtn) v. W i. 1011K?)' l\ \tw~ ibL a~s.octd nith hznow [Nay .5 w ere purchsed from Cv om Inc, HFKO3 cell, stably exprosng KU 3 and UH1 cells stable expresg hKv 1 .5 nre hom Chanest CHO I C eH staNY axp m the human E tpve calcmm channel Cay 2 w ere from Chan 1at and conained the hmnan C \CA A iC gene encoding bKIs 12 and coepreswed the beta 2 s'whu encoded by ha(m 1A3 ad [00747 FAS'aehs studies were performed a ('aneat to examne the impact of pepatas and conigaes on. the cloned human r aype cium channel hav 2, cloned hk43 and McAned KI . in o ved Pateh~press (Model 7000A, \Molecular Denies Unaion City. CA lcrophv'iology at room tepeaur Tb etacel our recordimg solution (HB ('c A) eetr eprtr.Te PS) conatned I.3 mi NaCI Wl K~l, I < MAI CaC l> 1 WM \<g1 2 10 WOl l.FTS and 10 mn Glucose adlusted to pH 7 40 with NaOl. The iNtraceihuar mecording soluaouitP hKv?3 and hKv15 contained 1SO mW potawsMa aspartte 5 mMO \gCi., rnX 1 i A W$AT P a ad 10 wMRPSa4utdt pH it KOH, The intaefula soutont hCaV 1 2 contained 30 M cesiam aONatate, S mMl \igCl:, S trM ESGT A, 4 triM \TP rnN LD1T \, Xh(.m a 1 01 mM toP and 10 mAl }I IPES djttted to pH t.. with rv' inmbh vL-lucamre. in preptnWu I'm recordig, intracelular solutao s loaded into the iracellular compartments of dhe Yaebip a planar electrode. Cell suspensions are p iretted min the extiacellukarcompanmntue of the :alchtp plauar elecAtrde Afte estahlihhng a whole-celi configuration, renmbrne currents are recorded uow dualchannel patch clamp amifuIers in the PatchNpres s'vten. Bel'ore din nation, the c urr ents were low-pass P hoved at onet tt(b of the samphngv tieq'ucncv Icwe concentration of pQptdc coniugatcs (test article) diluted umo 1(HIlS wth 1% BSA are apphled at fIve mirute ntcrials to n\ie ellis Solution e2ldfh mw ere performed in quadrupl icate and the duration of exposure to each test artlel concentrations was lIve n1utes vehiclee cottrols were also apphed to ,a'e cells andtler a solution exchange p0itivC Con1trot are pied to er sensitvity ton channel bloetade All positive controls are diluted into HB-PS with 0.3e DM5. Positi e controls tdr blockade or chanels i tidd. ifedihpiute (0.01,M) whlich produced about 751 hCav 1 .2 current bockt fecainide 10. i m ) Which produced about 750 mnhhition of the bl43 current and 4-aminop\ridin (2 irlM) which blocked abot 90% of the h.Kv 1 current vahd whole-cell reor dgi must meet the followirngcriteria: I00 membrane resistance (Ru m) :200 \l. , 2) lak current :125 channel current. The test procedures ft' hCav 1 .hin 3 and hKv 1. w ere as follows: d007441 n y hasp r Onset nid stead) state block Wh av 1. 2412123 channels ee measured using stimulus volage pattern consing of a depolarizing tetU pulse (duration. 200 ins avelirutde. 10 mV\ at. 10-s intervals f~rom a. -40 mt1 holditg potentiaL Ist article concentrauins mar be applied cumulativel i ascendog order without wadshoni ew een apphicatiors. Peak curremt w as mleasued ditriing t step to 10 1M\ Saturaurie conce~nultion of'nit1d1ie. (10 5 tM A is added at the end of' eah experinheat to block hCav i 12 cunet. I ak current wa digital subtracted trom the total membrane curt record. [0)0749] hi hK v43 estaroedure Onset and steady state block of hK v4.3 current were measured using a POulse pattern with fixed amplitudes depolarizationn: 0 mV for 300 s) repeated at 10-s intervals from a holding potential of -80 mV.Pak and sustained test puise current amplitudes were measured during the step to zeroy mV.

[007501 c3 hKy v 5 test procedure. Onset and steady state block of bKvl L5 current were measured using a pulse patten wtlh fixed ampli tudes (depolarzation:+20 MV amplitude, 300 ins duration) repeated at 10-s intervals fror a holding potenial of -80 mV. Current ampinude was measured at the end of the SeP to +20 mV [007511 .. optersc.ref'n aj.a.t.thjen cl dj uman .a .... ljd.um.hamegjusmg...e PacgjluA\stem The extracellular (H-PS2) rcrding sou conta ned 70 mM NaCL 67 mM N-nmethlK-Dgucamine, 4 mM KCL lY aM ( a(C1 I mM Mgl 10 mM JE PES, 10 mM Gucose adjusted to pit 7A with [T Tb internal recording solion contained 130 mM CsF 10 mM Nat , 10 mM EGTA, 2 mM MgC . ,10 mM HEPES adj uste d to pH1 720 with CsOH. Stock solutions ot reference standardd or tesarties were dihned no HB~PS2 prior to appicaton. Test articles included either peptides or peptide eon iugats described herein. lidocaine (1 - 30 gM) was the reference standard. A standardized step protocol is used to elicit ionic current through the hNav L5 sodium channel Cells are held at -80 mY Onset and steady state block of hNav L5 sodium current due to Test Aictle vas measured using a pulse pattern with fixed amplitudes (conditioning prepulse: -120 mV fr 50 ns; depolarniIng test step to '0 nV o 20 ms repeated at I0-s intera. Curemns ar futerOd at 3 kHz and acquired at I0 kz in episodic mode. When a Cood reording a estabishedlls were washed f'or ' mUtC tolowing by apping control vehicle tor 5 mInaes Then corttul and each concenvtition of test articlk was applied tor 5 minutes. There were 3 additons for each concentraton th 1-rminte' intervat Dipense speed was 40 plA xxth suction on To Jetern't Tet Xrele at I p.

3 L 1 xM\. 10 p and 30 1a was apphcd to cells (n - 3 cells) cuulus e (wihout washout between test article concentrationin ascendhng ordr, to eact ell i ; where a number of el Each concentrauin of tet arucle no s aplbed for 5 minutes There wr ce 3 additiors for ch concentration w ith a 1 -mine inter x al. fllectroph\ysiol ogid al a arusto w as performed using PatchXpres (Iomrnander v . 4 (Axon Inunts. Uninn City. CA)anud analy ses was performed using DarWress vI 4 (Axon Instrumens, Union City. \E The c peak currents before and after tst am le appltaton were used to calculate th percentage o curent inhibtion at each concentration. Acceptance criteria for a good recording nclude, I } s~eal ac 200 M (2) access resisance 10 \la Q pa +ail errent 'N3; PA0 p 4) euke current , '25 of the peak tail current (5) rundown 25%nmmute in control I vehicle 250 [07532] (ounterscsreens aaamst the human IAs (hKv LQT I- hminKl potassium channel ting the Patch.Xpe system. The extraceliukar recording solution vas 1-13 S.l The internal recording solutm contained 20 uM KY, 0 mM iKCL 10 mM N'l 10 muM I3TA. 5 i Q> \TP,! inmM\dgCI 10 mM l il' adputd to pH 0 with KOI. Stock solutions of reference stndrd or teot articles we re diluted into 1 Fo PS phor to anplicaion. Test aruccticlt eue either peptides or peptide conjugates denied herein Chromianol 2038B 0,3 10 ptM) was the reference standard A standardized step protocol nas used to elicit ionmecunee through the IK-s potassu channel Cells were held at 80 m\ Onset and teadI stae block of Ks potasum current du: to fest \rile wa measured usmng a pulse pattern wih thfed amplitudes (depolariing test e to m for 5a repeated at 1 inferi a]s Currents is filtered at 3 Mla and acqgied ag t 1 l-a in episodic mode, When a good rewording was established, cels nere washed fOr mandn talov wn by applx ing control vehicle to 5 mmiute ' hen contal and each concen antov of tesi .tilcle w er applied for minutes Ther e uere 3 addition for each concentration wIth minute itervnL Dipense speed was 40 d m with sucton on te t artiLe ai p M l, 10 pM and 30 p.M were applied to ells n 3 cellIs cmuulniielr without washout bnween test rictle roncentrations) in ascending order, to each Cell. ( 3 whbre n number WoeUtsE Fac concentratbon of test article was apphed for 5 mmutes There were 3 additons or each concentration u ithi a I minute mierL El ctrophyolotical dam capegoion was pertrmcd usang Patch~pr evs ( Commandei \ M 4 \so inthiruniuts (I n on Cit ' \ and anaynses is performed using Daw~aprs A xon Intrumnies, union Ct \ Tlhe 5 peak currents before and fter tet article appbraloa sor n used to calculate th percentage ot current inhibiion at each conccmranon Acetanc cnteria fQr a good record Wnchldes: (I) seal resistancs e 10 \fl ( 3 peak tail current 200 pAl) leakaw current , 25% of the peak cunrenti5) rundown < 2. 5nnumte in control vehicle. [007531 CIontsereis against the human ir (hERG or bvo I .) potassiumn channel by conventional whole cell patch clamp electrophysiology, One to 2 drops of the cell suspension s added to a 35 mnmpoiy-d-vyine coated cover slip for overnight incubation before electrophysiology experiments. Whole-cell currents were recorded from single cells by using tight G seal configuration of the patch-clamp technique, Ao35 mm cover slip was transferred to the recording stage after rinsng and replacing the culture madiem with extracellular recording buffer c n 13 m. N ., M K, " CCl 10 mlM H EES, and 5 mM Glucose (jpH was adjusted to 7.40 with NaOH and osmolarity was set at 300 m~snq Cells weore conunousKy Perused w Ali the catrAciUar recong WHO Vr vw oe of the glass capilanes arranged n parallel and attached to a rwooa roJ, bieh places the glass capillan directly on top of the cell bein recorded. For hE~t prothna the recording ipette solution conitame~d 1 30M KF, 2 rmM \igCs 10 mG FGTA and OJ 10 reM HUFPIFS adjusted to pH 7.40 wt KOH and osmrolardy t at >0 mOsm, Experunengi ere perforned at rom temperature amd recorded usng \ht tetam p 700A amplifier (Molecnlar DecS Wn r W1Pipette resistances w ere tY pical '- \Q Ck e r l aa n S0 m\. 'oaci ese a baselme or reference poit ""O te peak outnard tad eentm atep to 50 o wr $00 ms was used his n f followedd by a deroiariai. step to ,-0 'W foi 2 s to dn ye the aannels to the inaetwtied state. Astep back to -0 P\ tr 2 allowed the inactivation to be reheved and peak hERG currem to be measured Pukes were one every 10 . Total hINR current was Onmasored as the difference between the pea cunret at the repolarimg 10 n\' step and tie bashne current at 10 o\ I st ta uls (up to 10 pMI which included the peptides and peptide conjugates described heroin, nre mixcd into the etracellular recording buftbr contaimng R 01 bovine serm albunu (13SA an subsequently transferred to glas pertusion re en otrs Electronic pinch vahs controlled the flow of the test articles 'rom the resrvoirs onto the Ceel hemg r I'50: % b anat curSe fit were estimated usng te fr chanel inhibtor cisapride wyas used to validaOe the isay, [0(754 Couantebrsreens agamnt calcium-activ acted potassium chatmel human Ca 1 and BK( a by consventional w hole cell patch clamtp eleetrophy siology. (~ HIIK.Ca and BK('a cell lines wer e btamned from Biofocus DPI (A Galapagos Company Onc to 2 drops of the idKCa l or 13KCa eal suspension is added to a 35 mm pol 4yine coated scover slh 'or overni ht incubation before elcctrophvvolo?~ cxperrmtents \\ ole~elt currents were recorded from single cells y ustn tit (f seal cion of the patch-clamn techltue. A 3$ mm er slp w as trnsered to th eordiog tag itr rising and replacing the culture medium with the eLxtracclluia recording buffer contn 5 m M ta S mM KOL:, n 8i mM CaC l 10 mM i LPl'S and S mM hlucose i Il w as 'vd to 740 n i Na1 and osmiolartv wa se t at 300 mosm Cells w ere continuouol ncrfue V =iw the tcr'acellar recording butr via one of the glass capilare arranged in pairleI and acttashed to a motored rod, wh ich places the glass capillay diectly on top of the ell hems recorded The recording pipete sohition contamed 130 mM potassium aspartate. 1 oM Mg%, I A nl CaC , mM VA. ,\ mM N M tA P and 10 mM Hihl IS adusied to pH 7.40 with KOJ and osmoarit set at. '80 mthm Expements wer performed at room temperature and recorded usHMg \l twlamp Y0A \rlhticr (Molecular Devices tui Cells were held at potential of 80r mV Bo0th BK and 1K. currents were active ated as calcium ion diffused into the cell frour recor ding pipette sohuion. Antvanion of the es cram dependent outar~d potassium current b) calcium dif'uion cenerallv takes 3 to 5 mn for full acuuti on, Outward urrents were coninously motored at holding ptentai of , 0 .mV befrte and during drug exposure hemnat el>, 400 ms votage ramps fronn -12O to W60 m\'n e ieyated once ev ery 10 a to earitne the Currant Voltage relaion foi both chaimek before and dunng dru, exposure Test artck Iup o 10 pML whca in eluded the peptide and. pepude contigates deser ~"i hermn r "m ed into the eutraenlular preceding bfthl containin 0.1 bovime serum ai BSAum) {B,\ and subsequently tranferred to 'iss perfusion reservoirs lectrom pMuch VAiT sontrolled the flow of the test articles Atn the resenoirs onto the call be"ig recordedi Pipete revitanees were t pically 2~3 M2 I(^0 values and curve Aits were eatimated usmg the four parameter logistic iM of N LfQ soft are A IKC a and BKn pptide nhii, charyndoto a 1 n. w a a ppied at the concun n of the assay procedures for phormacologica vldion of the assay. [ 00755] Exanirk 9 [0075( AMP5aK LII Fusiow [0077 he ,\AMPS TPfAmiete peptide was genetically fused to anti.LH antibodies of the intion m a l l Go Possible Cernivrd foson tipAaon3 rprnted t m Figure I K i i rc 45, he. Nverminali\ fused and tei hnally fused to both nimumglobhein lizhg chain monomers and to bUob imnoglobuln heav chain monomers and was expressemn in nammahan (K 10) cells. The (ons were then purtied by protein A chromatography (GE ife sciencell using 10 column aiouma of Duibecco\ PBS t without dialent eatzons as the wash hef'er and 100 mWl acetne acd as the elation buler at , The clution peak ws pooled baseJ on the chromitogrm and the pH was raised to - using \M Tris base. The pool Ow then dilued with at least a obumes of water and hn loaded on to an SF i epharosa cohlum i- d(G e Sciencss and \ashcd with 10 colunm a inues of SRuffer A (20 mM acetic acid, pHA 5.0 followed by elatin una a 20 column, volume gradient to 600 Stuffer B (20 mI acetic acid, 1 \N \CI' pl 5,0) at 7'C A coo1 wa made based on the chroratogr am and the Oaterial was dialNzed against n2 v volumes of 10 a acetic acid, 9% sucrosew p 50 using 10 kDa Slide-A yPierce) at. 4A The dialyzed material was then filtered thrKuh a 0.22 pm celujose acetate ter and concetrto wa detemined by the absorbance at 280 nam Injected 50 g of each anybody along with an unused control on to a Phenomenex SEC 3000 column (7, a 300 mm) in 50 mM NaB2PO4 pH 65 250 mM NaC at developed at 1 nmin observing the absorbance at 240 nm (Figure 39r Each antibody was analyzed using a 1.0 m Tris-glye 4~0 SDS-AGE (Novx ) developed at 2'20V using reducing and noneducing loading buffers and staining with QuickBe (Boston Biologicals)(Figare 40A-Eh and the masses were detemnnind by LC-MS (Figure 41 AD [0075S) The comnponents of Wh vaious wKI 120h) QG0-AMP.A SaENI2(. I g0. aKN 1:0.6,~. ~..NJ~' 4(0B) oop hMsoy, andl AMPSMYkit! 120.6 app embody ts clud e too popetde mon [00759] a) JKLH 120. kappa iA (SEQ ID NOW. above): [007601 baL 1.20.6 IR(3 MA(EQ) W20129. bove): [ 00761] ) aKEH 120g I HC (SEQ i1) E 0 34, aove): [00762] (e} aKi 120 4O2 H (Amp5 hig the tom ar no aid syuncvz M1 I PAQL L ROARQ Q) V QS S AEKKPAS VSUK AS z 1 11M MWV 1.CG E.WMCW VV0iNPN SOOG h it AQFOG F? V !T RP D!N S! STAY M1 ELSE LAW 0DTAVYCARDROSYYWDPWGOQOTLVNrSSASTKGFVLP'%?SRSTS ESTAAM CIALVD Y FP V WNS6 Al T S6HITP AV LQS S 1 SS VTYP\SSN FGIF RPSNTKVD TVERKC N VV IN NAKKPR IEQEN STPRVYSY 1, VVHQDWLNOKEXK.CKVSPPCPA/hASVFLFPKPKDTLM SPJPEVTOi VV EjHEnDEVOQ NNKOLMPAPI KTISKTKG1 I GQ REMT QVKG[V PS DRA kE SEN OQPENN KIT PM LSODSF YSKLTVDKSREWQNNSVMH E A [11N TQKS[LSSPGGGGQGCSSCO PT RE WQ QCR RAQHSK (SEQL) SENfON:31 aia h oP'm [76 (d Amn pS-iKU 1 2 WO ( i.g. ac.id sepr'.-,-'eec MMRVPA QL LGLLLL WLRGARCQGCSSGPRE WQCRRAQHSGGGGGQVQLV QSGA EVKKPEGAS VK VSCKASGJYTFTOY H MH WVR QA PGQG L EWM GW INPNSGGTN YAQKFVQGRVTM RDTSISTAY M ELSR ERSDDT AVYYCAR PROSY Y WFDPWG QI' L VTVSSASTKGPSiVPL APSSKSTSGGT AA LO(I.VIDYFE PEPYI.S NSGA LTS VHfT PAYVLQSSOLY SLSSVVT VPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHITCP CPA PELLGPSVFLFPPKPKDTLM SRTPEVTCVVVDVSHEPEVKFNWY VDGV NAK' KPRE EQYQSTYRVVSVETi VLHQDW LNGKEYKCK VSN KALP APIEKTISKAKG QPEQYLPSEMKQSLTLKFPDLAVEWE£SNGQ}PENNYKTTPPVL DSDGSF FLYSKL TVDKSR WQQGNVFSC SVMHEA LHNHYTQKSLSLSP (SEQ ID NO 3 [001641 to) CJ 1I 121 hgI N297Q40 ,,. fmp Eo( (,-I43) Lop.- 6,kjuL,, tOIQ ~~hD i d scuno NM~KPAAi I QR CQQL MQSGAE\/KPGAS SMIMCKAS(' I Y MIH WARQAPCQOLE£WMGWMtNP NSGGT NY AQKFQGRMTTflRDTFSISTXYM EESRL RS DI)TAMYYCAR RRG YWePNPCQC L \T!SS AST KUPM FP L APSS KSTSGCAA 1 GC VKIPP TiSWNSGAL VG VITFPA LQsSGSLSS MT VPSS, SCYIP NVNHUKPSNTK VDKRVEPK SCDK THTCPPC PAPELLGGPSVF LPPKPKDLM ISATIPE DVS HEDPEYKN DV \ HNEVUNAK f <£5 EQYQSTY KIM SM LE fQ YLNOKE KCK SNKALPAPEKT1SKAKOlQPREPQVYTLP PSREEM6GQGCSCGPT PRUNW'QCRRA QH SGGTKNQV> LThM G PSDIA.VEWESNGQPENN YKWTPP fV SG YK 6 MDKS RWQQGN FSC II EALNNYTQKSLSLS C(SEQ 1 [00165< '0 .m p 5a KU1 1251 Aa ppa It" polypa ptide ASK.o Wain hctfwgaiNO ad scqucnco: MDMXNRVPAQL LGLL L LW LRGA RCQGCSSGCPTLREWQQCRRAQ HSOGGGOD)IQMNT QSPSSLUSAS VCDRVTITCR ASQG IRIND LOGWYQQKPOKAPKR LIV AASSLQSGV PS RFS OGSOGSGTEFTLTISSL QPEDFATYYCLQHTNSYPI TFGGGTKVEPIKRTVA APSVFIFPPPSDE5 Q LKST ASVW LNN FYPR A KVQ WKV DNA LQSCN SQ'S VTEQDSKDSTYSL SST L TLSKA DY E1KVY ACEVT HQLSSPVTKSENGEC.(SEQ )D NO:342 desied aKIA- 120gG 2 lDcaKtAI product is af0l antibody With the AMP5 pepde fused to the Cdermninus of one heavy efham, eonfig&ured as in the schenmatc representation of Figure 1F and was embled by two separate rounds of iymerase Chai Reacuan (PGR) tiarig PFV High Fdelity UMmra, b Srataene. The frst rund of PGR renerated two rpuntas. \' K 1 sp-uKL Hi20h IgG2 HC DsK-G5 and G-AMP5 frgmente Tbe obgos Wnd PCR mparnthay er d to general t netc SEQ ID NO:32S and dow Pokhmerase Chamin ReatIn IPCRlienerated the \ p -K IpKLIJ 1 20. 6gG IHC Desk-Gs fragment and exiitg DN A that coded for the VKI jpaKL H LO 120, 2 DesK Speti wassed as uipla ws rOO7 Forwardy prier eguge wa [00768] AA CTC GAG GIC GAG TAG C AC ATG GAG AGTh < iF G &CC CCI'GA CY CGIG C (SH) D =251 ad [00769j Reverse Pimer sequnce was 00-7701 GCC C CI GCA GCC GG A' AG MC TCC ACA C 0(; AGA CAG GGA GAG (SEQ ID N03261 10U77tl Theamino acid sequence encoded by tV spaLH 2 IC De IWagsumZR :ueratd 03o0i PCR was: NIMMRVPA QLLOLL-L L WL RGARCQVQL VQSGA EVKKPGA SVKVSGKASGYTE TGYMH MH WV N dN RQPQGL EWMGOW NPN SGGTN YAQKCFQG R.VTMTRDTSI>5ST AY ME LSR.L RS DDT AVYYNCAR DRGSYY WFDP WGQGTL LVT VSSASTKGPSV FP LAPC"SRSTSFSTIAA L G CLVKD YFEPV P TVS WNSG AL SGVHVTPAV LQSSGQL YSLSS VVTVPSSN FGiQTY CNVD HKPSNTKVDKTV. RKCCV ECPPC PAPPVP S F LFPKP KDTL M I SR TP VIC V VV DVSH EDPF-VQFN WYVDGVEVHNAKTK E WEQFNSTFR VVSVLTVVHQDW LNC KE YKK VSN L PAP! KI SKTKGQPR [EQ VYTL PPSR E- MTKNQVSLTCL VKGF Y SD IA VEW ESNGQPFNNYKITPPML D1G S DFFLYSK LT VK SR WQQGN VFSCSV NH E AL HNHYTQKSLSLSPGCGGGQGCN (SE? ID NO:327. [007721 Po lIerase Chain Reaction 2 ( CR2) geainated ti-c G 5-AMPS fragment (SEQ D1) NO33t and eis DNA a oded for the A -MP5 poyeytide ws used aoempilate wih [00773) owrd r enee a TCCT CG GOT OCT iGA GOT OGT (GCAG T GGCTOG"CAGO COO (SEQ Dt N428 and Vl4] Reers prno~ seqence t (PT 0ThG GMC GT GAG OVA C TOO 'VA CGO COG CO t't(SEQ ID NO 9) The amino acid sequence encoded by ne gtenerated from PRH was: [O0T75 S LSPGGGGQGGSOGPiTREWQQCRRAQMS7 (SEQ ID) N(}330} [Q07'61 T'he products veor run on a 1% arose gel The bands were punched for an agarose pug and the phl s were placed in a resh P(R reion tube The agaros plugs were then amplified in PC03 using the outside primers SEQ ID NO 325 and SEQ ID tO:3 The tina PC R product was rn on a i arose gl The correct size product was cm out then gel purdh'd by (agena Gel Purittiol Kit The and gel fragment of YVKkp, aK H10 igG. DSR f4C-c\P ws digeste vth rtwion nv mnes Sail and ANMd and then the dwaed product was pun by: Qigen s P( R Purification Ki At the same time. pTT5 \ector (an Amgen vector containing a CWV promoter and POy A tai) was cut by Sal and No. The p T5 ector was un out, on a M% agnuoNe ses and the largr fragment was cut out and gel purified inb QiNe's Gel Puriiation ri The VI sp-uKLH 120(, lgG? Desk. H-GiAMtP5 product n ligated to the large vctor fagneont and transformed io OneS hotJ Top 30 bactenal cells, The D\As om transformed bacteial colonies were isolated and submitted for sequence .alsis One correct clone was selected for large scale plasm J pm li cuion. [00FT) The fnal pTTSV:K IspuaKLH i0if6tGDesK H(CG5 AIMP5constuct encoded the flowiing QG Desk HCAM pA pqudCe MDIR PAQLLGLLL L LRGA RCQVQLVQSGAEV A SVKV 5 1 VISKA SGY FGYH MHB WVRQA POQOL EWMAGWINPNSGGTNY AQIKEQGRVTMTRDT'SISTAYVMELSRLRS DIDT AVYY CARDRGSY V WFDPWGQOTL VTVSSASTKGPSVFPLAPCSRSTSESTAA L GCLV'KDYFEPEP VTVSWNSGALTSG VHTFPAV LQSSOLY SLSS VVTVPSSNFOTQTY T NSV DH KPSNTKVDKTVERKCVEPPUPAP PV AG PSF LF PPK T S T EVT VVVDVSLEDPEVQFNk: WY DG VEH [IN AKTKPR EEFNSTFR VVS V QT-V V KHQ LG IKEYkCIKVS NK(GiLPAPI EKTIS5KTKRGQPREPQ VY TLPP-SREE MTKNQ4VSL TLVYKG FY P S A E ESNG pENN V PNK.DSDGSFLY SKL LKSA GQ SCSM ; I AL IN YTK~tI..P(iC(iGGQGSSGPTRFVWQQCRRA QHSP(01 WQ~ (>331. [00s s] o:goHaYscarauiwopresss on [00770] Ihe dired AIP&KLH 120.6 #2 DeK HC. prduct (SEQ 10 M:3 abo e mingljjh te monomer is a ful anybody with the AMP3 peptide used to te Ntenmiius of one bas cham, contoured as in schematic reresexuato igure L and Yas aameid by two sepatrate roumds of OCR usmng PEW Hgh Eidely bitra by Stratagene. Th rtu imround of P(k genrtd three framens: VK I sp-AMPS M A 5S an d C15 -aKt Hi 1206 1g§12 DesK C fareme, I he. olig's and P(IR tenmplates that were used to generate 4 hes; fragments are sted hon rolertnase Chainm Reaction i (PtR I) generaed th \ K inA\MPS and exjsim DN A w hieh coded for the '1 01 was ds template. Note tis fragment was also used i construction of the \1 ~AMPS~ LH 120.Q Kappa 1&. [0U7/0] The forward pr nier sequence was [00Y7811 AG CAV7 AG 61(C (3AC7TA ACC7 A(C KGi .f VHTJ AG(,., 01f C G(' A ( T(C PC ' 17 (SEQ NC. D and 100732] T he revere m e seqencwS: [00782]G( 6(7( I( WT9 OfA UK7( (77 ;W" MA IT'1 60( ACT' IC COON W77 (3) M) >40:33). The am i l ae squenc enoded by Te fagmnt jnnrated froni OR was: I E0 [00P5 Forword Primer sequence wns: [00736 A GAG 070 (7.OT0( AGO O 0(07.( NE 335; and [0 074f7] The revrs. oor sequence ws f 0078_] CA 716 ('ACV MG1 ACT" AM'( AMt777ACOC I '1 At?07 SEQ Ws) SWA33M.

[00789] The amtnacid sequenene eoded by the fragment gaented m CR2 was: \]L.RGARCQGCSSG PT.RE WQQCRRAQHSoccGGGQVQ 1/ (-SEQ II) IN 37 [00790] PCR generated (35-MIA 20.6 IgG2 DesK [,C aud e in " hccded firthe a I 2Ok IgC HW (SEQ IDNme> D t [179 The irward primer sequtc wa COCOT CAG OANT AGO OCT CCA CGT OGT CSTAG GTO LAG OTG>' S ID NC:3384 and [00792] The ees mrsqec loss' 'T A GCOOCT CAA CC A GA AGO GA , SEQ ID NO 9) [007931 The amino acid. sequence encoded bni .ramn gaentd from PIR 3 ow RAQHSGGGGGQVQLVQSGA EVKK PGASVK VSCKASGYTFTGY I HWVRQAPG L EWMGWXI NPNSQGTNY AQKFQG RVTMTRDT1 SI ST AY' MELSR LRSDDT AVYYCA RD .RCGSV VWWGQOT LVTVS SA STKCPSV FP LA PS SKST SOG TA AL GL VKDYF FPEPV TVSWNSGAl TSGVHTFPA VLQSSGLY SLSSVVTVPSSS GQTYCN VN H KPSNTKV DKR VEKS(DKTHI CPPPAPELGPS V FLFPPK PKDTL MIS RTPE VT VVD VSHE DPEVK NWV DGVEA HN AKTKPR EEQYQSTYRYVSV TVLHAQD WL' NGKEYKCKV SiNKALPVA PIEKT11SKAsKG3Q PREPQVYTL1.PPSREEMITK NQVS LTCi .VKGFBY PSDIAE E SNCQPE3NNY KT TPP VLDSDOSFFLY SKLtTVDK SR WQQCN VFSCSVM IE AL N HY T QKS IPLSPGr SQD N:) 0 [00794] The products were run on a % agaros. geL The. bands were punched fo' an agatwo plug and the plug were placed in a fresh PUR actionn tube The agrse nnu were then amplified by PCR4 using the outside pnmers SFQ ID NO )> and S'Q I M) )3 'he final PcR prodAuct was ran on a 3. %aarose gd. The correct se product was cut out, then punrfed by Qiagen's Ce Purillation KitM T pmnfid el ti-agmen of VK Isp AMIP-G$-uKLH 0, 1g2 DesK HO was digestel nh restnetion enzymmes Sal and NotL and then the digested produce was purified by Qiagen s P0 Rntication Kit At the 5IC time, pTT Vector (an Amgen vector contaung a CI \ ymmate and Poly \ ta) w as cut by Sal and NotL The p1 T1 vector may run out on I M'' mroxe goc and the larger fragment was cut out and gel pulled by Qiagen's Wce Purification Kit The V' Isp-ulPS-41-aK LI 20.6 gG2 DesIK HC product was lighted to the harge vector ragrnent and transtormed into OneS hotly 'Top 10 bacterial cells. 'The DNA 'a from {ransformed bacterial colonies were isolated and submined for sequence analysis. One correct clone was selected for larte scale plamid purification The fmnal pTT5AKI!sp-AMP5-GS-aKLH 120,6-4g02 Desk H C construct encoded the AMP5-1gG2 DesK HC polypept ide (SEQ iD NO:33*2, above. [00795] ~t 2. gj jgggaacle s taldhQ&Al~if)iI jigayvx htinli(I ntBR~Ltigxprig4ji The desired aKL H 120.6 IgG!' aglycosyvted O (N97)-AMPS-Fc HC product comprising IC fusn monomer SEQ D NO:3 tabovel i tub antiody with the Amp5 peptide inserted nto tho (3 domain of the igGs (NO97Q) Fc DesK hwavy chain confgucd as schemahcally repes d in Figure I Mi. Th \ I sp- KIL 120.6 if I 7QAMPS 'Fc Desk HC product was ordered by the synthetic gw company. Bme Hern, The fAN product was gcnemted by digesting the VK sp-aKLH 120A6 G (N297T AMP$- e DesK HC with its corresponding restriction enzymes, Salt and Nodt The digested product was run on a % agarose gel. Thbe fragment was cut out e purfi ia Gel Puification Kit. At the same time, pTT5 Vec\t (n Anmen vector containng a CMV promoter and Pol a was cut by Sll and No. The pTT5 vector was run out on a 1% agarose gl and the larger fragment ~was cut ot and gec purified by Qiagen's Gel Purification Kit. The purified. gel fragment of aKL H 120.6 hgG I (N297QA MP5- Fe DesK VC was gated to the large vctor fragment and transformed into OneShot® TopI0 bacterial celL. T he DNA's from transformed bacterial colonies were ioated and submitted for sequence analysis, One correct cone a lecte for large scale plasmid purification. The final pTIT5:VK I sp-aKL H 120,6 igG {N297Q)-A MPS$ Fe DesK MC construct encodes for the aKL 2 120 IgG1 (N297QNAMP~DesK oypi Mfinonomer SEQ ID NO' 41, above. [G07% :tj\ 1PS-G-aLU I 120A Kanra L inbt Chainfbt L)Nlmmalian hEpresson. 'The desie MPS -aK 1206 Kappa L(" product is a full ATibody with AMP3 pepude fused ;o the Ndeminnus of one ight chain usnion tmonomer (SEQ ) NO:34 above), configued as schematically represented i Figure l R and was assembled by two separate rounds of Poermase Chain Reaction (PCR t sing PFU Hih Fidelity M'trs. by Stratagene. 'The fir round of PCR generated three fragments which included. \'K l sp-:MP5. AMPAYi and GA. alkiLH 120.6 K appa EC, The oigo's and tenplates used for PCR reactions to generate the fragments are listed below The fragment that generated the \K KpAM P5 is the samte fragment that was used in construction of the AMP5-KLH 1206 QgG2 DisK H C, and is described in that section. Polyetrmase Chain Reacion 2P0R2) generatd dhe AMP5-G fragment and existing DNA that coded for the APS peptide was used as template. Forward primer sequence was (SEQ ID NO:335, above and reverse prer sequence was CTG 'GGT CAT CTC GAT (iT C ACC ACC ACC TCC ACC CT ATG GG AG (CX(SEQ ID NO:344 The amino acid sequence encoded by the fragment generated from POR2 was: \ LRARQCSGTLEQCRAMGGlMQ(E D NO:34.5y [00297 PCRI genod rh CA aa freA that coded tom aKH 12016 Kappa.", (SEQID NZ)wsue.a epae 100794] The Aorad puner sequence wVas: (c' COT cAC CAT AGMCT CG OT CTGO GAOJ AltC IT C A CX A SAQ) NOO and [00799 the rvose primer sequence was AA' COT TTA. AMO CC COO GUC . AA (FCAO CO OTO 'G A SLQ 11) Che peptd squence e t edc CR RA QU 5000000 Q I

T

PSS LSASVCDR- Vt ITCAQJNO VQP AK EVA'$1 QOVSP SOOSTE T s ESSLPWTYOQUNSYMM~ LTOCKVEj I PIQLNKD\IY SESST LTES KA DATI EKUKVY AEVHQCES VK SUN R(C EO"'k/ SE [00001 The product were ftn ona1 grs.gtTebnswr uce b'a iwaos Ws'and the plugs were placed in a fresh PCR recintnIb qao luswr then wmpnpi ad by PON uOng theousdprmrSE DN:V>adEQN:3. the a a P ' pouc w s no aIagose rgelh corcts w irdcwa cu ot then10 purited by Qag1s GA PUrea Kit o puritieto s [MPSAS]aIH 120 6 Kappa on as Wydiesd "i hebandtin e'Nmeshe fo and then the digested product was purified by Qiagen s PA Puriicauon K. At the t me time, p T5 \Vector (an Aagen vector contammg a tMV promoter and Poly A il) was cut by SA and Nod. The& pTI5 vector was run out on a I agarose eel and the larger ragment w as cut 26V out and g uifi y an's Gel Purification Kit. The VK :1p-ANMP%5-5aKLH 120.6 Kappa LC product was ligated to the large vector fragment and transformed into OneShoti To 10 bacteria. DNAs fromn transformed bacterial colonies were isolated and submiuttedt fr qce analysis, One correct clone was selected for large scaled plastid puriication. The final pTT S:VKsp-AMPt-GS-aKLH L 20,6-Kappa LC construct encodcd an A NPS-Kappa, LC polypeptide fusion monomer (SEQ z) NG:342. above), 0O1801 j 'l'ransient transfeet i wa carried oit in 29 .i cells i NRCC usn PHI Polvethivlenimne, linear, t ha I mg mni series \te solutmen, pH 'A0 Polysciences) The D O cell dens' was N 10 before trasftcucsi, theunusmg 500 microgams otF DNA Tohev chaMin anad h qht chaim DNA, 1:-1 roa par hir of M s tMafeeted. The DNA was added to 50 ml 29 k reeSt le media (nv itrogen) and combin0d with 1,5 ml of P solUoN, \ortexed fmildi and trhn neubaied 15 minutes a room temper atue The cells wer transtected hr adding the wiol> PF-DA rmiture o the culture P Clls were the n incubated on a hakes (120 ippn) at 3 C cmaimning 5% Ct Or 24 hours Tryptonc N i JekmSeicnce inc. 200 in Freesttl medua wus then added to l ni concentaton of (15% and th' incubation was continued fri 5 days. The conal ton nmedii n was harvested at da 5 by certrifuge at 400 rpm followed t ration though a 0.45 am iter Corning I c.) [00502] The fusions were then purified by protein A cbmomatographw (OH Life Sciences) wng 10 column volumes of Dulbcccos PBS without divalent eations as the wab buffr ad 100 1ik acetic acid as the Cluion buffer at 7 ' ,TIhe eWtON peak has pooled baei on the chromutogram and the pH was raised to about 5.0 using 2 \A Ta bae The pool was then diluted whit at least 4 volumes of water and hen loaded on to an SP-ir sepharose cohmm WE L e Sciences) and washed with 10 column volumNes ofM fe A (20 l acec acid, pH 5. Ioued by 'bion using a 20 co amp volume gradiem to A= 'SBuffer It (20 m1 acetw acid, 1 M NaUCi pH 510) at TV, A pool was made based on the chomatogram and the wal ss dia W nod Aamo >20 volumes of 1 0 to K aceti Acd Q 0% sure. 5.0 name 10 kDa SIide- \Ly-er s (Piete) at 4 'The dialyzed material was then iItered though a 0.22 um cellose acetate filter and concentration wa detemmed by the absorbaree at 0 na Injeted 50 pg of each antihody along w ith i unused control on to a Phenomenex SEC 3:000 column (.8 300 inm) in 50 mA NalliPt pi (15, 250 mM Na(I, developed at I ml nin, detect the absorbane at 210 am figure 3). All fve antibodies showed te expected retention timec for molecules of the size showing tat v:ery' little aggegate w' a presm Each antlbody was analyed usmg a i 0 mm As-vcie 4-20" SDS-PAiGE (euvc) developed at 220\ usng reducing and noieducitg loadmg hatters end stuimg NWith Quickilue flBoton Biologicais Figure 40- F cad the mases were derfome by -LC \IS (Figure 4A N u a in a e xpti nflt 1.0 jg of the sample was reduced i 25 gi of S \M Odl i 30rnM Ul ric H5) for 30 min at 55", then the reduced material was chromatogaphed through a - ake s Manprqp nicro desatn colan 2 . x 5 nm) using an Acquity LPLO stem Wys em A was 0 1 femti acid in water and solvent B was 0. § fir mic id in acetontmi I The cobmn was eguhated w ith S .o.tB at a oate 012 l per n11n at 50 Nad upon aniple introduction, ithe coumn wa w9 -ihd I w ii5. for I mm beOore the protein was ehuted uing a inear gradient from S to 40' It over 10 rain Fhe column efflu'nt was introduced into a Waters ame-ob-Omht iLOT preiel ldaw spectromer for mass measurement a ions ( rg a er n mi m Sisopropanoh Was used as lock mass. The mas spectrum was deconvolhttd usios hek Mad'ut sofsire supplied wi(th the instrument. The SDS-PAG E analyis demonstrtad that all the mmected grarcruy .tmetures were lorned. and the mans spetral analyss JLnmn\u1tes thauhe expected fusions were present in the purified molecules. faken together these data indicate tha usions can be made winh any of thie tour toosible N-terminal or (O~termmal fusion eof~tpdiratio of the Mononc-ra of' aK I .2W1. anitood a wel a Fe domain internal lp nsero (see,Figure IF- N and Figure 4 schematic representations> [00803' Exam k .0 190y04 1 Ex4-aKLM A b Fusin |0(003I The Eiendmna reptile (FI0CE TFTSDI ShQEQEE~A\'RL FIE'Wh.K\GG PSSCA.PPPS/ SEQ 1) 'NO- 'l uwas geneticall It fied to N-termins of the light chain of thre amiKLi-l 1 204 antibody through die 1kG lmkTz. designatedd lax4- I LO-aK L.H I3 20.

Ab''and expressed in rnansmahan cellk Fugur e 42 is a sechematuc map of tme Exrendi~ QExl'kud-aK LH 124 t fusion construct 1008061 Theconoents of the (tI 1kG--KL I2.04b fusion included the.foing monoumers: [00507] (a.)Ex4- IkGaK.IT- 4. appa L thainc te tll-ni amnino acd sequnce DNIM RVPAQLLL L L LW RGA RC HG EGIT FTSDL SKQM EEEAVR LTIEW LKNCG PSSGAPEPSG SGSA TCGSGSGASSGSGSA T GS01 QNMTQSP 8SSLSASVGD)R V~FfCR(ASQC IRNDLO~WYQQKPGKAPKR Li Y AASSLQSGV PSRFSGSCG T'EFTLTI SSL QPEDEATYYCLQHNSYPLTF iGTKVEIK R TVAA PSVFI F PPSDEQL LSG TASVvCL LNNFYPREAKVQW KVDNA LQSN SQESVTEQDS KDSTYSLSST LTLSKA DYEKHKVVACEVTH QALSSPVTKS FNRGEC/ (S EQ ID NO:355 and [00804 1 WKll 12 04 2 14(SEQ A NO: bvW29. abv 0018091 The desired E-41 kCaKLH 12 -Ab rwduc was a ni anmibody configured Knh the 1E4 pepide fused to the Naernii of both light chains (see, wcheans. representation a Fiure k) Tbe atio of Ex4-ght chainhcav chain was i: .Inh scatiou. and sconing of'the genes ending Xenoious\ h biiJoma pressing aKLN 120.6 nowoeinal anybody 120.6 heavy and let ehans lae ben desenhed m Eample i and Example -4, bowe. Win naive sQmal peptides hav teen replaced by the \KPO 2 peptide \MD\1RVPAQLiGL.I 11 WRGARC/ SEQ ID Mfl10 I )l a' dI'eened above. DNA fugaents encoding aKIH 20,4 W (SEQ ID 'NON and alhlH 120,6 1K' W1t SI:Q 10 0291 monomers were individually ensued into amman expression s eo p1 -T (An rmen vector coninig a CMV promoter and Poly A tail) to venerate p T5waK 1 H120 6 VK ISPkppa Light ChaiL) construct and pTT5 :AKLh 120 6-VK ISP-gQ2 H Nov Chai (DCII construct respeceavel y [008101 A DNA frgent (S EQ 1 D NO:331, below) flanked by Sail (3) and BamH 1 (3') that comprises the Kozak sequence and the first part of an ORF that encompasses the VK l /012 signal peptide (SEQ ID N(13}, the Ex-4 (139) peptide (SEQ D NO:349) and the kG linker peptide was synthesized and cloned by OcnScript (Piscataway, NJ) according to standard ene synthesis techn iues. &aL K 1 vA A A PAT oT :IGV tiY w c1 T3 .'N~ ~ ~~4p$T A~~g 147N\l' Q5 GGWCN.N.'AC N.NP', 1 T~47 T' L" C'N AAM 1ES E A AG N T N~N AGA '. C TOT T\ CA T' .O'." T GG7\NN~~ Txl,: C A j &gGATC WATtTT MA i AGA> TO7. - ' NNO: [O0Uthe The WARndtil {I) to Not (3) t (SE a b at>36n ep covers the kcipart ofK' (n RE tat onit of anr maraKI 2.-bOwsapfidfo h eRdn 0 a O AemeC dciedabov (a.d down 14d d0\XKA S U11pe it i ChIi hO on0ruc t)C wi ]h pr n of hgo Prim vrso 'AA NTsb AC c AC s\on (SAG AlD MCO tA cd the ammNG:35 adqen of th GOA! WOT WAA TAC TOT MCA1 (SEQ7 AWSAC)G N : codn osadr ehius 10W W -.- J- No oft-CT anfnh 120jCL17 comaing TOAcCAAO YSE ID.Y~ 110:54)-utKK. VY<W- amn WdnYck oY Af 'W E ta rh p iticatio 11- nou'M12 siLi Iih human nb(SEQ W kin ,^3ei nesal xrsm Epstem Barr urun Nuclar Amnu-1 294A6 ws maimad amp ru-ie suspenonn alu rwm ujug F 17 mediuam 0 mutdogen, NOrWa, (0;\ inutinge, Calida , A)and '50 un Ulenwtic (I n irrign, ( lobad. CA) TO h suspnsin cll ultresw ee nunt inm FrWImOyer shake tlask eultura 'The culture fOaWN ei e haken at 65 rpm at u m "i a humid i Rd, %4 (A),:amoheeA t mo 26& (1 tw/ni) of' 2$i4 lihnear PL ( Polysceeca. Warrinigon, PA) wadi prepared in wadter, acidi1ied w 1h 1R 4 to pH 2 0 until dismoled, then neutralized with NaOH, steri zed filtration 10.2 pm a U and toI a ' nti u r A , i OrgntTectuu 8 A. (IQKmlSleufle, QL, C(analdif A Stock 5nOVton~Z .0% w!Y) w as prepared in freestyle medium (nIvhtrogem, Carisbad, CAt stenied by filtration through 02 pm tilters, and staed at 4(' Until use Tyically, transetions were per fonmd at the it, scale. Cela ( NtfD were. gron n to a vable cell dineit> of 1 \. t0' eeltinm then transfdtion comIplexe w ere prepared mi 10 volume f tn e final cu tu re w1ume, Foi a i-_ Ims " ItheCun culture. anst0etonl confexes wee trepared in 100 ml F 1 banmedbum and 500 pg pliamid DN A ln and light chain D\ \, 1I rati wS first dilaed m 100 mi mediumn \tLei a. 5minute incubation at room tenmperature, 1 ml ot'PF I Noution s added. The oa mplan were vonexed nily, then inebated for 5 nnutes at room tempenur She cells were transfected by adding the utasecuin complex ix to the cells i the shale lask culture. 'Tuentyour hours poor-transfection, Tryrptone N w added to the tranfected eultue to a final eonkcentrauom ot 0 and tie truansfected ltuen were mainnamed on a haker at 65 rpm at 37C in a humidie.d, 5 C0, atmosphere Or another 5 days ier whcb they were harvested. The condibioned iediumi was haes ted by etifintlon at 40(0 rpm,, and then stril futerA through 0.2 n filte orningI [0014l The lions were then punted by proein A chromatogaphy (Gi ciencesi using 10 colum " volumes of Dlulbeccis PBS without dai ra cadens as the wash buffer and 100 mM aec acid, pH 3.5, as the ehutio bufer at ' The pH of the fractons were increase by leading 0,025 volumes of 2 \ Iris base 0 the ihavion collector tues The el unon peak as po led based ai, the chromatogra m and then dialed aahd s '0 aol hms of 10 mNA acetic acid. sucrose, p1H 5 0, usiag 10 kDa SIde-A -L p s (P ier at roon tenmperamrie tar 3 hours. The daived material was then tittered hronga 0 U urm celhioee acetate filter and concentration was determned by the absorbance at sQ nm Samples of 25 of the antibody iuston were injected on to a Phenonmene\ 5f 3000 common = 0 \ 300 n mni m 0 mNJNaHYPO4 pH 6 i 0 m NaCI at de eloped at I nmin observing the ab2rbance at 20 rm ( igure 431 Smee the fusion nrotem elated with the expected retention te for a protein of is expeac(d size, this indictes that the protein was able to torm the expeted complex without exc \ie aggregatMon The tX4~aKRH 120.6 antidy was unaled using a 1 .0 mm isglycmne 4-20% SDS-PAGE iNovcx) developed at 22d\ usmn r ed emg and non-reducing loadsin buffers and stainna with Qucklu0e (Boston Bioloie41s (Eigure 441 The non reduitg SDS iM)E idictes tathe epected qaterngu compe of the~ ~~a ttsoiprli a bid and Saom tof the oxmidiri-4 pcptidi t3o he 1KL 21, antibody restt ts tn a product with the epected structe. [081 i]Eamplei O00N16] AvimttraKII Fusions [00817] The CANs polypepte i an I +1 ind ing poiyp'ptie with a socalled wime stracture, (Se e g tKolkman et atL \ov el Pkotemsl wth f arg eted Bindins:. 125 2005. 00831 Baker et at.. It-6 Rinding Probems I'5 2008 0 107 Stemmer et at., Poiem Seaffolds and kea Thereof Us 200 0234 a nd US U00 023499V [0081 1] The crpoiets ofthe CAN 1ab 1201 2 NC si M' n Mudced the [0089] (a) aKi[F 120.6 kapa l., G DEQ N 0 nd [00820] (b~ (VK-1 SPC68(GKS L 0iGN fusion having te fhowin amiino acid sequience: MDMRVPA QLLG LL.LLWL RGARCSCGSCLPDQFRCGNGQCIPLDWVCLX)VNDC PDD SDE EGCPPRTCAPSQFQCGSGYVCISQRW VCDGEN DC ED)GSDEANCA (YK' GSVPTCPSDEF RCRVNGRC IRA WRCDG VNDCALDNSDE EDCT EHTGOGGGQVQL VKQSGAENVKKPGAS V KYVSCKA SGY] FTGYH MH V'N'RQAPGQG E WMG WI NPNSG (GTNY AQKFQG RVTM TRDTSISTAYMEAL.SR [LSDDTAXVYYCARPDRGSYY WFDOPW\GQTL VTVSSASTKG PS VFPLA PCSRSTSESTA A LCtLVKDYFPEPVTVSWNSGA LTSGV HTFPAVLQSSGL YSL SS\VTVPSSNFGT QTY.TCN VDHKPSN TKV DKTVE RKCC(1VECOPPCPA PPVAPiS V FLFPy PKPK.DTLM STPEVTCVVDV Sc EDP EVQ NWYVDIVEVHNA KTKPR EEQF N STF RVVSV LTVVNQDWLN KEY KVSNKLPAPI EKTISKTKGQPR EPQVYTLPPSRE 2T KNQVSL TCL.V KG FY PSLA6 Y PVEESNGQPENNY KTT PP MLDSOSFF LYhVDo KS RWQQGNVFSCS515VMH EALNHN HYTQKSLSLPG// (SEQ ID NO:336 [00821 The desired 068 K -aNI 12. 1g02 HC pvoduea was a til andbod wih the Avimer fused to theNternus of both ecavy chains, h iod tdC 1-heavy ehaiiha 26 chain w J :. TO cexoecd i - I 120.. IM 32 WCfso rti wsioae.ch If Xhhehrmonfltogrtphy. d erbed hered [0022] The (64 1a.KUA 12.6 IZ va abe 11C fuin waodeedfon Bhue Herona a sn th ce i~c eedun bh toin are no ad I seutence [w M, p1) ) S 1 ) O PAQ L R ~ b W. RGAvCI'CSC PDQcFR CNGQCI P DW C)GV NDCPPDSDEEGFCPPRTCA PSQFQCG:SG3Y(:SQRWVV(IDGEN DCSD)EAN CAGSVP Ki PSD)F R C RNGRCI PP A WRCDO VNDCA D NSD EU EDCTIE H TGOGQVQ LVQSG AE V KKPG;A SVKVSCK ASGYITFTGY NM MHWVR RQAPGQGL EW MG W I NPN SGGTPN Y AQKF QGR VTMTiRDTESISTAYMEL SKRRSDDTAvYYCARD)RCSYYWFDLwCQ&GTLVTVSS5 ASK:./d (SQ 1D D350t [00K24] The fraent was digested with S2 and lhmb, run out on a I aarse gel and the corresponding fagment cut out and puriied by Qiagen's el Purifction Kit At the same time, a pTTVK ISP-aMKL 120 Qg02 HC DNA templat was digested and purified sumdarv , eldimg a p1 15 v elor bualsbone ruth the constamu NC( repon. I he ,\xjimer framint xvs ligated to the pTT$h02 NC consat region ad trsformed io OneSho Iop 10 bac eria, D%\s were shmit ted for sentencing -tbouna anal of seural sequeneenf clone wided a 10% rowpereet natch w ith the above sequence, only one eone w as kecd for largescaled plasmid purification The final pTIANK I SPIY 1 -aKH 1.20.6 g1H conatmAet encoded a. (C8 ( G5>aK.LN 120.6 1G? NC(Tion po peptide (SEQ 11D NO &} [00826 Th opponents otheaKN 120 H-C68 tin included the 2 K! P E 12W0 6 TG2? A 4 1 ffA isin in he fcin 1 a$i QI e CNVP KPSNTKV DKTVERKCC V C PPCPAPPVGG( PS VP UFPPKPKDTL N 'ISMP MEV-1 - 269 VVV DVSH1EDPEVQF NWYVMDGVEVHNA KTKPR4EEQFNSTFRV VS VLVVH-QDW\QNG KEYKCKVSN KGLP>APIEKTISKTKGQPR EPQVYTLPPSR EEMTKNQVSLTCLVKGFYP SDIAVEWE[SNGQPENNYKTTPPMLD tSDGSFFL YSKL'TVDKSRWQQGNVFSCSM IHE A L INHIYTQKSIL SL SPG(G(GGGSGGSCL PDQFRCGNGIQCIPL DWVCDG(IVNDCPDDSD) EEPRITC APSQFQCOSOCY C1lSQ RWVCDG3 ENDLCEDGS DEANCAG SV PT CPSDEFERC RN(CPRAWRCDGVNDCA DNSD EEDCT EH T/ (SEQ ID NO:357)h [O828] IT h derd aK .I 1i162 Qug2 (>CN I parem t was a fi an wody kith the ,Aximer funed to the Cerminus of both hem, chams (bhmatically represented m Fio ure 6). The ratio of hea chain4CY+I ight cbain a 1H Tbe expected aK 1 .20 igh G CM fuson proteinas isolated uing on exchange ehromatograuhy as described herein. [0 9 The '81 fragetdih ag an d Not restrit oites was ordered crmDn eo was a syn ienc gene encoding the {ikwing aino aci sequence [O0530 1 i TKN QVs I1CLV G FY PSDOIAV E SN GQ PENN vKT FP H os rx; S IF: SK V KSR WQQKNWSCSVN L H.1YTQKS LSPGGGGSGGSCPL P0MW (GNC QCIP DWVCDni VNDCPDD)SDiE ECPPTCA SQuQCdGGYCIQRWVClDGFND3 CEN[ DNANCAG SWPTC'P&IDFRC RNGR (P RA WRCOC PNNOCA ONSDEN LOCT V (SEQ IDYN358L [00831] T he fragmemn was digested w ith SexAl and Nott u~m out on a i % agarose gelbnd the correspondmg haument cut out and punftied bv Qlagen's (Get Purification Kit. \t the same timnea pTT T VK lSPaH 120 Th1 02 [IC DNA template nwa digested with Sail and Sex l and puriued %smiarivto genenue the DA 4coding sequence for' ak id 10) 6 IgtS2 'I( monomer (SL'Q 11D Nfl >? A ft 3 vector 'was cut w ith Sal) und \Nt, run out on a 100 agarose gi and he larger fragment cut out arnd gel purned by Qiagen's Gel Purakcatlon Kit. The Auier and uKEl H 12.06 1g02 [HC .fragments w ere Iligated to the n l ' fragment and transformed io One~bot TopI10 bacteria. DN% weare submitted t'r sequencing Although, analysis of .severa~l seqpuences of clones yielded a I 00t per cent match wnah the above sequence, only OneC clone w as seleted for large sealed plasm'id purification. The final p l'T VK I SP-aKL I 120,6.1g02il C68 onamnet encoded aKLIA 120. 1q0g2 HIRf5)4> I1 fusion 0:opcrtide (SE ID NO 535 [0032 ] b c6ot ppa I Ct LKH 2 n0 E n m ao d hequence Maa RDR NSD ',-J DG GOO.Q SVN SG moos:m g m snamos e mg m [00835 The AirNd 20aKM 120 lpp If' pruct u"as a tiM anhdf w"it \ha vmet Kid to rho Sta s thoh hgh s. binsTe rato of 814 thai ba ch)dn v i ('68 1.KIhe mxpctd (M kA f bik on pr the to t wing anos auted Kyng n 3 1ebn doMO Raph! nal decoe herem ao asu nhi vn ncd MDMRVKYPAOQL LGOLLL L WT LRCA RCSGGSCLYPDQF RCGNCQCIP>L DWVC DGVNDCPI)DD SDE EGCPPRTCAPSOFQCGSGYCISQRWVCL DGENDCEDOSDEAN&CSV PTCPSDEF RCRNG RCI PRA WRCICDGV NDA DNSDEEDCThl EHTGGGGGDIQM TQS PSSLSAS VDR TV TCR A SQG IR NILCWY QQKPCKAPK RIY AS ASLQSGV PSRFSOSOSGTEFTT SS LQPEDFPATY YCLQN SYPLTFGGGTY KVEKRTV A (SE D11F NO T V) Ih ors3pondm g desid met ot and p1 ba y QV p 'c Gal aPianatond Mith the smradyarc seldmg to th Tr s t bkot e w chai Thent rat ionih hane em chrainm was ghed , pte p I 5-kL106kappa n rionn art'onuwd ia ine Topi acina acid seun Ae r equenoes ofdjones Aded a n0%perentdatch wit the abov, sequenceA ou on clne as seleted Ator ac e d pasec plandpiaton. final T KSP(681 -aKTA 0 kpa Lconstruct noded a (8 - L 120.kappa LUion polypepid HSQ U)NO:35 0[002ll3:811M Eli il! iIM uapartlicatuan of the conditioned media was done by affinity nt protein hquid chromatography (E PLC) capture o the 1:e region. noe A. hr (G .br EoWOWued by a column wash with Tris-buit~red isne, I mM (. b (ITknova) and step eluton with 100 m M acetic acid 1 m' CaUL:, pl1 31S at a flow rate of i fmnmin Protemn conmaimng ti-actions w ere pooled, and the pH was adusted to 4.0 un 10 NaOH and further diluted W ith 5 volumeof Owater The material wn tiered through a 0 45 gm celhlose acetate 1i1wr (Corning and tWher purined by anion exchange FPL ( Q Sepharose High Pertormanek GE Healthcare), Sampls were loaded onto a column equilrated v0th 100% buffer A W0 m1 Tis 1 mM !H 1x 0) and elued with a Madiem of0 to 80% buer B PIN m ri4 m \ Na ' iA." mM t( a( b pH KG) over 30 column volumes at a tlowrate of I m cmin, Peaks contammL target species nere pooled and torrdlated into 10 mM Thi us. 1 50 Mr C, M o10 CaC pl k0 fEemplar purifications of -terminal iH and LIT and CGtueemal HC runsn rotems are Khow n sgures 3n-38. The non-reduema SDSM-PAGE analysis Fmigure 3() dnontrates that the Y assembled antilodo can be formed and e rewdueg SDIPAGl' analysis demonstrate that the desired .omnpone nts are present, The size excluson chromtatogram (Figure 3'? shows that the maoitn ofthe purified product a in the desred non-aggrgated state, finally , the mass spectral analpis (Figur e 3h demonates that ie sared fuson products are present Taken together these examples demonstrate 1bat the at. 111 0 A antibody can accept fMsions to ,\vimers frmNinag the desired product [008401 BfAorke Buding Assays of aDNP and aM. Anhtodies [00M411 Materials, Puihed unti-D\NP antibodies from either hy bridomaA (31, 372, 3A4 and 3R31) or recombinam (W) (3A4-F-G2 and 3I -02 expression nere rested, Aunhinmman Ib , Fey-specife antibody was from jacksou IuunoResearch Laboratories, Ine West Grove. PA. CD\P-BSA 12 -dinitrophenol conjugated to bo ine serum albuin W as from Bioscarch iTechnologies Inc. eNvato, CA) le lACore 2.000, research gad~ce sensor chip CMS. surfactant P-20 (pl yoxyetlvenccorbitan), HBSTP (10mM HE PS, 0, 1SM NaCL 3,4mM EDTWA, 0 % P-21 pH 7A aninc coplng reagents. 10M acetae ph 4, and 1I0mM glycin, pH1 L5 we're from B AC ore, a c (Pcaway N) Phoshate-buffred saline (PBS I X, no calcium chloride, no mntreMim chloride) was fOm lnvitrogen (Carlsbad. CA) Bovine serum albumin (BSA., fraction IgG fre) wi fon Sigm ( ouis, MO). [0084' Pnufied anti-ALPH antibody butnIG~oir L U I exr"sed from hbbridonu a tested. Muhmene high molecular weight key hole impet hemocyanin ( KL was from Pierce (Rock~foA so tn-human IQ, F 'e pecitic aitibody nas from lacson mnmnoR esearch Labor atornwe ln. "W\ Grst Br' It re 1FvC 2000. research grade sensor chip (M \5 surfatchaut2 f& pel noyethyvlen .~in i .R-PP (10%m.i viPEr, 0 l Nat'. 3.4ml EDA. 000% P20, pH 7) amine uouping reagents imt estate, P! 4& and 10mM leme, pH 1 5 were from BlArore, 1n. xPa tumay M) Phosphate buffered sahine (PBS, I . no calcium choride. no .magnetum ch nde u was fom himrogen (C arlsbad CA) Bovime scrurn albunnn (BS \,ion \ kG free) a from Sigma (st Louis MO). [00140 3 ethods. BRcre i lyc nore carried out ns allows -imobila of ant? human 1gG, me+pecie antibody to the CM5 sensor chip surae w as peRnromed accordhm to manuOctunref instructs ons, using a continuous (low ot' 10 mdd HFP iS. OiS\M Nat'L 3 4mI FDT 4. 005K P-20\ pH ^'.4 i 1BS.-lP bufer) y ofl cadoayi g pn enor chip surfaces wera activated by injecting 60 aL of a mixture contaiuung 02 NI \ 1-ethyl 3.dimethyamninoproTpl carbon iide hvdrochloride UFDCumd 0.0$ M N hydroxescinimide (N l-TSV Specific surfaces 'were obtained by mniechn og iM0 gt of imut human 1 gO. Fey-specitle anuibody diluted in 10 m\1 acetate buffer (for assay of aKL I anttbodies pli 4, at a concentraton of 30 pg/mi; for assay of aDNP antkiodia pH 4 0 m a conentration of 6s0 ugnil. E xces reactis e groups on the surfaces w ere deaeinvated by njecting 60 t o I At ethanobarine ial imnmobdiied levlsek were about 9,00 ('or assay off aKLI-I antibodte or- about 10,000 (:forassay of nDN P antibodies) resonance units ( Ri't A blak, mock-coupled reference surface wa also prepared on the sensor chip Antbodies and utigen w.ere ddlured in sample butler consisng of PRS 0 o005 PQF420 - 0,1 mght mL B1SA, [00844] -nti-dNP antibodies were captured on in nidual flow cells, followed by imecon of either sanple buffet r D'-SA\ ranging i conenrataon from 078 100 n. Two different DN P BSA samples ucre tested ftr aalinity to the antDi NP antibodies The D\P LiSA samples ditered Min the number of DNP movies coupled to each moecule of R SA. u ith one wmple containing 3 NP moieties per BSA and the other COntaming 31 DNP moiehes per iSA. Onl the DN P 31 WBSA at concentratonsfrom 3 Nfl u\) was tested for affmin to the recorbnant anti-DNP antibodies In each cycle, three indhidual antibodies were captued on low cells 2. 3 and 4. with fiow cell I leil blank to serve aa referene surface Following sample butihr or antigen injection, iach surfae was rynaed by two injectons of'10 tM glcine. pH to disvociate captured antibody from the onmobilwed anti-human Fe surface BAe aluation software was used todetermme apparent Lincee pa-ametersfor binding of DNP-S\ to captured ant-\P anthodies [00451 AnA I antibody uase captured on mdivdual flow cells follow ed by infection of eher sample buller or rL rangg in concentration from 0s 100 nt To preare di noons of the Rmulimerie hib molecular we-n KL It an average molecule nweigt of 500000 dahton was used F'ollowing samnpl' buffem or antige injection, ech surface w as regenerated by two injetons of 10) mM ie pH 5 to dis cie capturd anlbody W'r the immobdired untihuman Fe surfaces Ltaluation software was used to determine apparent inetic parmetersnkding of QEL tcap ued anti-Ki antiboe&d [0046 Aordtdrigaamat ut, Table S\ below summares the apparent asociation (1k nd dissociation Pa rate constants as w ell as equilibriumn dissociation constants (.Kf obtamned for the binding analyis of anti-DNIP antibodies binding to DNP IASA \The data in I abLe 8-\ demntte that the am) NP antibodies nind specitically to D\P, and that they hind more tihly to the hihe de PlNP 31 )BSA than to the tower density DNN3 It \, ts would be expcted. ,\pparent bidogw affmnites for DNPf3 1-LBSA are all single digit nanomolar or higher. [00847] Table 88i below sumnrizres the apparent association (kd and dissociation (Pa rate constants, as well as eqluilibrium dissociation constants (Kf a obtained for the binding analysis of anti-KLHI 20.6,1 antibody binding to K.LH. The data in Table xB demonstrate that this hybridoma-produaced anti-KLH antibody binds specifically to mnultimeric KLHI, with an apparent sulb-nanomolar binding affi nity.

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Claims (17)

1. 3. The isolated anige binding protein of Cdin I wherein the heavy chain varable region copri ses three complementarity determining regions desig nated CLDRI, CDREI2 and CDRIH werein: (a) CDRFHi comprises the aniino acid sequence of SEQ ID NO: 88, SEQ ID NO:189 SEQ ID NO:. 90, or SEQ ID NO: 191; (b) CDRR2 omprises the amino acid seque of SEQ ID NO: 92 SEQ ID NO: 193 SEQ I) NO: 194, or SEQ ID NO 195; and (c) (DRH3 comprises the amino acid sequence of SEQ1 I) NO: 196. SEQ ID NO 197, S EQ ID NO:198, SEQ ID NO 199, SIQ I) NO200.or SEQ IDNO:201L 4, The isolated antigen binding protein of Claim Icomprsi an immrunogobulin light chain variable region, tie ligh chayinr'iable region comprising thrc CDRs designated CDRIl. CDRL2 and CDRL3, wherein (a) CDRE.3 cornmises the anino acid seqence of SEQ 1) NO:202 SEQ - 279 IDN:203, SEQ ID NQ:204. or SEQ ID N0205; (CDR L o omrises the amino acid sequence of SEQ I NO06 or SEQ 1D NO20 and (c) (DRI,3 amnpises the amino acid sequence of SEQ I NO:208, SEQ ID NO:209, SEQ ID NO210, SEQ ID N:21, or SEQD NO:212. 5, The isolated. atigen binding protem of Claim 1 wherein the heavy chain variable region conprises thre complemlentarity detemning regions designated CDRICD a CDRFI' and the light chain viable region comprises three CDRs designated ODRO CDR L2 and CDR L3 wherein: (a) CDRHI comprises the amino acid sequence of SEQ JD NO: 18$8 S EQ ID N0189, SEQ ID NO:190, or SEQ ID NO:191; (b) CDRF-H2 comprises the amio acid seqence ofSEQ ID NO: 92 SEQ ID NO):193. SEQ ID NO 94,or SEQ ID NO195; c) CDRH comprises the amino acid sequwnce of SEQ ID NOI 96, SEQ I D NO:197 SEQ D NO198, SEQ ID NO 99, SEQ ID NO:200 or SEQ ID N0201; (d) CDR L1 comprises the am no acid sequence of SEQ ID N0202 SEQ IDNO:20I SEQ D N0:204, or SEQ ID NO:205; o) CDRL2 comprises the amino acid sequence of SEQ ID NO:206 or SEQ ID NO:207 and (F) CDRL3 Comprises the amino acid sequence of SEQ ID NO:208, SEQ II) NO:209, SEQ I1) NO210, SEQ I) N10,211, or SEQ ID NO:212. 6, The isolatedant.igen binding prtei f Clairn I, wherein the isolated antigen binding protein comprises an antibody or antibody ftagmntt 7, The isolated antigen binding protein of Claim 6. comprise an I I, 1g02, gG3 orIg04. 8 The isolated antigen binding protein of Claim 6, comprising a ionocional antibody, The isolated antigen binding protein of Claim 8 comprising a chineric or hiumanized a aitbody. 0 The isolated antgen binding protein cf Claim 8 comprising a human antibody. h1 The isolated antigei bnding protein of Claim 6 comprising (a) an nmmunoglobudin heavy chain comprising the amino acid sequence of SEQ ID NO;77, SEQ ID NO:l07, SEQ ID NO-.. I SEQ ID NOl13 SEQ I NO:1 15, SEQ ID NO] 117 SEQ I) NO,1.9, SEQ ID No23, SEQ ID N:129, SEQ ID NO:144, SEQ ID NO: 145. SEQ ID NO18 J, SEQ I) NO:182. SEQ DNO: 183, SEQ ID NO: 184, or SEQ ID NO:185. or composing any one of the foregoing sequences frm which one. two threeftur or five amino acid residues are lacking from the Ntermlinal or Terminal, or both; (h) an imnmnogobulin light chain comprising the amino acid sequence of SEQ ID NO S 05, SEQ ID NO: 109, SEQ ID NO:i2K; SEQ ID NO:i25, or SEQ I) NO: 27, or conprsing any one of the foregoing sequences from which one, two, three fur or five amino acid residues are lacking ron the N-terminad or Cdenaaor both; or (c) the immunoglobulin heavy chain of (a) andthe l light chain of(b) The isolated antigen binding protein of any of Claims I1 I., further comprising one to twenty-our pharmacologicaly active chemical mioleties cornjgaed thereto. 1. Ihe isolated antigen binding protein of Claim 12 wheein the pharmracologicaily active chemical rnoet is ap piarmacologically acuve polypepti de. 14 The isolated antigen binding protein of Claim 13. wherein the antigen binding peptide is reconbinantly produced, It The isolated antigen binding protein of Claim 14, wnrein the antgen binding protent comprises at least one .mnunoglobulin heavy chain and at least one immunoglobidin light chain and wherein the phannacologicailyactive polypeptideis inserted in the primary amino acid sequence of the of the immunoglobuin heay chiain within an iniema1 loop of the Fe domain of the immunoglobu in heavy chait 16 The isolated antigen binding protein of Claim 13 wherein the antigen binding protein comprises at least o1n iminanoglobau heay chain and at least one munoglobulin lghtehain, and wherein the phanmacologically active polypepAide is conjugated at the N-terrinal or Cterminal of the immunoglobuin heavy chain 17, The isolated antigen binding proen of Claim . 1 whrein the antigen binding protin comprises at least one imnunoglobuin heavy chain and at least One iimmngloidnin light chain, and wh in the pharmacologicaly a ive polypeptide is conjiugated at the N-terminal or C-terminal of the immogai~lobulin light chain
2. 18. The isotedangn bindindg protein of Clairn 13 wherein the pharmacologically active polypepti de is a toxin peptidean IL-6 binding peptide a CORP peptide antagonist, a bradykinhi B I receptor peptide antagonist a PT1 agonist peptide a PTH antagonist peptide an ang-i 282> bindingpeptide an ang-2 binding peptide.a myostati binding peptide. an EPOnirn ethic peptide a lPOnummeic peptide, a NGFT binding peptide a BAFF antagomnst pepide, a GLP-1 or peptide mimetic thereof or a GL P 2 or peptide .rnineic thereof 19 The isolated antgen bn ding protein of Claim 18. vherein the toxin peptide is ShK or a ShK peptide analog.
3. 20. A pharmaceutical composin comprising !he antigen binding protein of any of Claims 1 19; and a pharmaceutical acceptable dihuent. exipient or arnier.
4. 21. An isolated nucleic acid that encodes the antigen hiding protein et any of Claims -4. 22 An isolated nucleic acid that encodes the antigen binding protein of Claim 5. 21 An isolated nucleic acid that encodes the aPgen bnding protein ,f Claim i11
5. 24. An isolated nie acid that encodes the andgen binding protein of any of Claims 14-19
6. 25. A vector compising the isolated nuclie acid of any of Clains 21-24. 2& Te vector of Clanini 25, comprising an expression vector,
7. 27. An isolated host cell, comprising the expression vector of Claim 26. 28 A method, coniprising 28> (a) cuituring the host cel of claim 27 in a culure medium under Condilons permttg ex pression of the antige bi.ndi.g protei-n eanoed by the expression vector; and (b) recovering the antigen binding protein from the culture nmediun 29 A hybridoma wherein the hybridoma produces the antigen binding protein of Clain 1 .
8. 30. A method comparing: (a) L!turing the hybidanva of claims 29 in a ccure medium under co.onditions permitting expression of tne antigen binding protein by the hybridona; and (b) ecoverig the anigen binding pmtein from the culture nxedbum, 31 An isolated antigen biding protein/ comprising an inimunoglobudin heavy Chain vaia region and an immunoglobulin light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence at least 95 % identical to the sequence of SEQ ID NO:262; SEQ ID NO64,or SEQ ID NO266 32 The isolated antigen binding protein of Clairn 31 vherei. the light chain variable region comprises the amino acid sequence of SEQ ID NO:242, SEQ ID %0:244, SEQ D NO:246, ur SEQ ID NO:248 33 The isolated antigen binding protein of Clain 31. wherein the heavy chain liable region conmprises thre conplenentaity determining regions designated CDRHL CDRIH2 and CDRU.3, wherein (a) DR.H. comprises the amino acid sequence of SEQ ID NO:213, SEQ 1D IN0:214. or SEQ I) N 0:215; (b) (RH2 conprises the amino acid. sequence of SEQ D NO2 16, SEQ ID NO:217. or SE (D IINO :218; and (c) CDR)1<33 comprises tho amino acid sequence of SEQ I D N0,219, SEQ ID NO:22 or SEQ ID NO,221
9. 34. The isolated antigen binding protein of Claim 31, comprising an immlunoglobulin lig chan variable region the light chain variable region comprising three CD-s designated CDREL CDRL2 arid CDRL3, where: (a) CUR&cL eompiss the amino acid sequence of SEQ ID NO:204, SEQ ID NO:222 SEQ ID N00223, or SEQ ID NO:224; (b) CUR .2 comprises the airno aid sequence of SEQ 1) NO206 SEQ 11D NO;'22-5 or SEQ ID NO:226 and (c) CDR L3 comprises the amino acid sequence of SEQ ID NO:227, SEQ ID NO228 SEQ I D NO,229, or SEQ ID N0:230,
10. 35. The isolated antigen binding protein of Claim 3 1,,wherein the heavy chain variable region conprises three omieplementarity determining regions designated CUR1, CDR H2 and CDRH3. and. the light chain variable region comprises threc CURs designated CDRLI. CDRL2 and CURL), wherein: (a) CDRHl comprises the amino acid sequence of SEQ ID N0:213, SEQ D0NO;214. or SEQ ID NO:215, (b DR H2 comprises the amino acid sequence of SEQ [D NO:216, SEQ ID) N0:21o SEQ 1D 0218 and (c) CDR13 comprie anmio aci sequence of SEQ ID NO:219, SEQ ID NO:220 or SEQ ID NO:221 (d) CDRLA comprises the amino acid sequence of SEQ ID NY:204 SEQ ID NO:222 SEQ ID NO:223. or SEQ ID N:f224; (e) CDRl.2 comprises the amino aci sequence of SEQ ID N0:206, SEQ ID N;225 or SEQ ID NO;226; and (f) CD.RL3 conpries the anino acid sequence of SEQ 10 NO:227, SEQ ID )0228, S EQ D ) NO229, or SQ 1D NO:230, 285
11. 36. The isolated antigen binding protein of Claim l wher en the isolated antgen binding protein comprises an. antibody or antibody fngment, 3 7. The isolated anfgen binding protein of laimn 3 coX Mig an IgQi i2 IgG3 or IgG4 38 The elated ant gan binding protein of Claim 36,yomprising a nmonocionai antibody. 39 The isolated antigen binding protein of Claim 38 comprising a chimeric or humnized antibody
12. 40. The isola ted an.tigen binding protein of lain 38 comparing a himan antibody, 41 The isolated atigen hiding protein of Claim 36 p coinprisig: (a) an ini.noghulin heavy chain comprising h'e" amihno acid aequene of SEQ EN $EQ ID NOI 133 SEQ ID NOJ I39 SEQ ID NO 143; SEQ IE INO:186, or SNQ ID NO I87 SEQ ID NO66 or SEQ ID NO:367, or comprisig any one of the foregoing sequenes from which one, two, three, four or five aMino acid residues are lacking from the N terminal or C-termi natl or both; (b) an immunoglobdI light chain comprising the amino acid sequence of SEQ ID N: 28, SEQ ID NO: 131 SEQ I) NO: 1.35, SEQ ID NO,137; or SEQ ID NO: 141, or cmprising any on e of the foregoing sequences tent which one, two, three four or fve amino acid residues are lacking rom the N terminaI or Cderminai or both; or (c) the iminmioglobulin heavy chain of (a) and the immunoglobuli-n light chain of (b)
13. 42. The isolated antigen binding protein of anof Claims 3 141, futher comprising one to twenty-fmir pharmacologically activ chi cal moieties conjugated thereto.
14. 43. The isolated antigen binding protein of Claim 42, wherein the phar nacologically active chemical moiety is a pharmacologically active polypeptide.
15. 44. The isolated ant in binding protein of Claim 43 wlierin the antigen binding peptide is re~conbinarntly produced, 4 5 The isolated antigen binding protein of Claim 44, Wherein the antigen binding protein comprises at Least one mmunoglobulin heavy chain and at least one inmunoglobulingt t chain, and wherein the phannacologicall active polypeptide is inse.ed in the primary amino acid sequence of ihe ofthe imtrnunogobain heavy chain within in internal loop of the Fc. domain,-i of the inndounhev chan..
16. 46. The isolated antigen binding protein of Claim 43, wherein the antigen binding protein cornprises~ at least one imnunoglobulin heavy chain and at least one immnngobuin ligit chain, and wherein the pharmacologicaly active polypeptide is con.ugated at the Ndterminal or C-terminal of the mnulohbuln heavy chain. 47, The isolated anagen bnd ing proen or Clain 43 wherein the antigen binuing protein comprises at least one immunoglobuLci vy chain and at least one inianoglobulin light chains d wherein the pharmacologically active polypeptide is conjugated at the N-terminal or C-terminal of the immunoglou light chain.
17. 287. 48. The isolated antigen binding protein of Clain 43 wherein the pharmaeologicag atve peyptide is a taoxn peptide an .A6 bindidg peptid a (ORP peptie antagonist a brady'kinn I receptor peptide artagonist;ia P'lF agonist peptide a PTH atigonist peptide, an ang I binding peptidean ang-2 bnngn peptid> myos4ath binding peptide. an EPO-ninmetic pepdde 0 niene peptide, a N bindi pepide a BAFF arntgonistp pde, a 1P2- or peptide mrnic thereof ora GL-2 or peptide mimetitherto 49. The isolated anten binding protein of Claim 48, wherneint toxin pepide is ShK or a SPK peptde analog, 50. A phainmaceutical comoition comprising the antigen binding protein of any of Clainms 31-49; and a pharmaceuialy eptabe dituent, xcpient or caneirc 5 L An isolated nudtei acid that encodes he antigen binding protein of any of Chains 3134, 52. An isolatd nucleic acid that encodes the antigen binding protein of Claini 3 $3, An. isolated nucleic acid that encodes the antigen binding protein of Claim. 4] 54. An isolated nuclei acid that encodes the antigen binding protein of any of Cainms 44-49, $5. . vector compri sing the isohamd nuclete acid of any of Claims 51-54 $6. The vector of Claim 5$, cnpising an expression vector. 288 57. An isolated host cell, comprising the expressionVector of Claim 56. 58, A ieda comprising (a) cturing the host cell of Claim 57 in a culture medium umder condiutos peri in g expression of the anti en binding proton encoded. by the expression veto; and (bIrecovering the antigen binding protein from the culture rnediun 59 A hybridmas wherei te hbima produces the antigen binding protein of Claim 4 1 60 A method, comprising: (a) cuuring the hybridoma of Claim 59 in a culture medium under condition pi inug expression of the antigen binding protein by the hybidom; and (b) recovering the amigen binding protein fom the culture medium