AU2016202118B2 - 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

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 ofU.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-PCTSeqList031810-368_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.

[0006a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[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 activity- '.'diminishing chemical modidcations of the phaxmaco log!ca.lly acitve chemical moiety, or to reduce ixmal clearance, to enhance in vivo iia|Mile;or #her pharmacokinetic properties of a therapeutic, such as increasing the rate of absorption, reducing toxicity or immunogeniciry* improving Solubility, apd/of increasing .manufacturability or storage stability , compared to an unconiugated form of the pharmacologically active moiety, [000b] Esnmplos of such carrier moieties that have been employed in the pharmaceutical industry include polyethylene glycol (see, e a., Burg et al, Erythropoietin conjugates oith polycthy lene glycol WO 0; 0201 '0, immunoglobulin Pc domain (see, e.g,, Feigeci ah, Modi fed peptides as therapeutic agents, US Patent Ko. 6,A60,843h human serum albumin (see, e,g.. Rosen et ah. Albumin fusion piotesuv US Patent No o,92o<84b ,md US 2005 0054051, Brtdon ah, Protection of endogenous therapeutic peptides from peptidase aeth tty through conjugation to Mood components, US 6,887 4/0), mmstbytedu (see, e.g., Walker et ,u„ t se of tranatity toon pcpodc'proiem fusions to nwieasc the serum ku!i~hle of phaftnacologically:active peptides/protesns, US 2OO3/019S1S4 A:l; 2003/0lff0S6 A1), or thyroxine-binding gkVhulin, or a combination such as umminoglobminCiight cham-Hicavy chain} and Fc domain (the heterotrimerie eombination a so-called ‘'betjubody'l, for example as dcacrihed in Sullivan et ah, Ύολίη Peptide Therapeutic Agents, PC 1 US2007 022831, published ns WO 200s OSS-122. Phannacoiogscally 'aodve'iBbidti^:'K|w.'.8feo been conjugated to a peptide or small molecttie that hna an? a ft in it) for a long half-life set am protein. (See. e.u.. Rlaney et ah. Method and compositions for increasing the serum half-life of pharmacologically active agents by binding to transthyretin-xeieeme ligands, t IS Patent, Mo. 5,714,142, Salo et ah, Serum albuminMnding moieties, US 2003/0069395 Alt Jonesct ah. Pharmaceutical acme lonjng.stcs, 1 s Pauut No 0,342.725} [0009] Fischer ct ah dcscBhed a papideAmrriunoglobtdin-cor^ immunoglobd!t«-i^kM;fi'^i|[^^:o|tmps orfeolieavy chains and twdftght chains, in which the immunoglobulin. was not a functiontthie immunoglobulin (Fischci et ah, A pepnde-imtnunoglobuiin conjugate, \\ (i 2007/0454M \ I > [0010] The present invention provides carrier immunoglobulins yielding exceptional uniformity and efficiency of recombinant expression, in vitro stability and non-aggregation, resistance to photodegradation and oxidation, non-cross-reactivity with human antigens, and good pharmacokinetic properties.

[0010a] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

SUMMARY OF THE INVENTION

[0011] The invention relates to antigen binding proteins. The inventive antigen binding proteins, including antibodies and antibody fragments, have reliable expression and purification characteristics, resulting in products that are stable and relatively uniform, and have outstanding pharmacokinetic (PK) properties in rats and cynomolgous monkeys. The inventive antigen binding proteins are found to specifically bind to dinitrophenol (DNP) or keyhole limpet hemocynanin (KLH), but have not been detected to bind to human proteins, cells or tissues. These antigen binding prioteins can be used for many purposes, including, but not limited to, quality control or analytical standards for antibody-based drugs and as controls for biologically relevant isotype-matched antibodies.

[0011a] According to a first aspect, the present invention provides an isolated antigen binding protein comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs) designated CDRH1, CDRH2 and CDRH3, and the light chain variable region comprises three CDRs designated CDRL1, CDRL2 and CDRL3, and wherein: (a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 188, SEQ ID NO:189, SEQ ID NO:190, or SEQ ID NO:191; (b) CDRH2 comprises the amino acid sequence of SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, or SEQ ID NO: 195; (c) CDRH3 comprises the amino acid sequence of SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO:200, or SEQ ID NO:201; (d) CDRL1 comprises the amino acid sequence of SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, or SEQ ID NO:205; (e) 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 ID NO:209, SEQ ID NO:210, SEQ ID NO:211, or SEQ ID NO:212.

[0011b] According to a second aspect, the present invention provides a pharmaceutical composition comprising the antigen binding protein of the invention; and a pharmaceutically acceptable diluent, excipient or carrier.

[001 lc] According to a third aspect, the present invention provides a method for enhancing the pharmacokinetic properties of a pharmacologically active polypeptide comprising conjugating the polypeptide to an antigen binding protein of the invention.

[001 Id] According to a fourth aspect, the present invention provides a pharmacologically active polypeptide when produced according to the method of the invention.

[0012] In some embodiments, the antigen binding protein of the present invention is used as a carrier for pharmacologically active chemical moieties, e.g., small molecules, peptides, and/or proteins to enhance their PK properties. The pharmacologically active moieties can be conjugated, i.e., covalently bound, to the inventive immunoglobulin by a chemical conjugation reaction, or through recombinant genetic expression, they can be fused to the antigen binding protein.

[0013] The invention also provides materials and methods for producing such inventive immunoglobulins, including isolated nucleic acids that encode them, vectors and isolated host cells, and hybridomas. Also provided are isolated nucleic acids encoding any of the immunoglobulin heavy and/or light chain sequences and/or VH and/or VL sequences and/or CDR sequences disclosed herein. In a related embodiment, an expression vector comprising any of the aforementioned nucleic acids is provided. In still another embodiment, a host cell is provided comprising any of the aforementioned nucleic acids or expression vectors.

[0014] The inventive immunoglobulin can be used in the manufacture of a pharmaceutical composition or medicament. The inventive pharmaceutical composition or medicament comprises the immunoglobulin conjugated with a- pharmacologically active agent, and a phatptacga#ca% acceptable dildenb earner or excipient. t#B;j Numerous methods are hivemion. for example, a method In provided involx lag culturing the aforementioned host cell ovr.rnis.mg the expression \ color of the invention such th n the encoded antigen binding protein is expressed. A method ts also provided, ms oh ing culturing the aforementioned hvbridoma in a culture medmm under conditions permitting expression of the antigen binding protein by thehybpdoma; Such methods can also comprise the step of recovering the antigen binding poteiniromibe host cell -vulture. In a related einhoditneni, an isolated produced if tie aforemunt toned -metiiod Is provided..

[00161 Π>.: foregoing summary h not intended to define every aspect of the invention, and additional aspects am dciCTbcxilfe.-d^ miid'Dbtstil&amp;i:.;,

Description of Embodiments, The entire doctuh.^t|sxtit^hd;#%#Mbd:M:s unified disclosure, and it should be understood that till combinations of features Jewi bed here.» at e «.umempl atod, ,w en me eo. ibincvion of foulmes are not found together in the -xnc sentence. or paragraph, or sec turn of this document.

[001T] In addition to the foregoing, the invention include*. as an additional aspect, all embodiments of the bn ernion narrower in scope in any way than Ihe variations defined by specific paragraphs above. For example, terrain aspects of the invention that am described vs a genus, ;uid b should be underload that every member of a genuses, hide, sdnu A, ae aspect of the imerwou Also, aspects do.set dvd as a gums .or.selecting - t u w o i mentis should X t t s, kV to emh,t> e -.ογοί jUi^n o* too or moiv me nlvis of the gem,,-,, \Ithough the applicant* si un anted tie fnli xopcofthv uwemnm Jesrr.bod heron, the applicants do not intend to claim subject matter desenoeda' da p to tr vu«r\ e* othe.s Icereto.e, in t >e event *h< t s. mtosy prior art within the wupe of .¾ el mm w brought to the uttetriion of the applicant* by a Patent Office m ofhet era us or indie ideal, the appUcantmt reverse the nght to ettemiso amendmeip nuhts under applicable patent laws to redefine the subject matter of such a claim ίο sperifteully exclude such statutory prior an or Ob'. sous variations of statutory prior an from fie scope ofsuch s cl aim Yanshoru of the invention defined h-> such amended datms also are intended as aspects of the invention.

BRIE!' DESCRIPTION OF riiB ORAWl^GS

[00! x I figure IA-N dam s seh etnatio some tut ex oi sente embodiments of a com position of the imenuon that include one o? room asms of a ph arnia-.o topically dele e toxin peptide analog t sop and el mscd. via an comma! pcpttdd Imkev moicrv a IK n ux bat not limited to 1 5 or l 10 J.scHPed here in. a ith rate Or more domains oi an immunoglobulin. I heso .schemata, χ show a more typical IgG 1, although they arc '.intended to apply as well to btGTv. w hul, wd hox e 4 disulfide bonds in me hinge and a different arrangement of the dknlfide bond linking the heavy and light chAirt, and luGds and lgG4x figure ! A represents .¾ monox a lorn hctcrodimerie Fe~to:xiu peptide analog fusion e ith the io\m pepode analog iWd to due < 'terminal sad of one of the immunoglobulin K vlom.un monomer*. 1 tgure IB represents' a bivalent hwuodimene Fe-to\m pepu.T .m i.op Css.mx a ah hap eeet.de analogs (used to tV Λ -terminal ends of both of the mnmtnodubami be dome-η monomers, Fmure 1C leprexentx a monovalent hole-id i marie tosh; peptide analog-Fe fusion with the toxm peptide analog based to the N-termmu! end of one of the immunoglobulin Pc domain moimmem fsg tre 5 D represents a bivalent homodtmone toxin peptide- analog-fc fusion. with toxin peptide analog', fused to ise N-ietmmal skids of both of the immunoglobulin Pc domain mpnomers, Figure hetetonuuetic Fc-toxin peptide analog Ah composing an muntmogiokhio heavy chain t HC) + immunoglobulin light chain y.,C} t an immunoglobulin Fe tnonomef: with a toxin peptide analog fused to its C-terminal end. Figure IF represents a monovalent hetcrotciramerie (HT) antibody HO-toxin peptide analog fusion, with a toxin peptide analog fused to the C-tenmnul end of otto of the H C -monomers.· Figure !G to pres cuts a k valent HT ant? hod> Ab 1 IC-ioxm peptide analog fussou having toxin peptide analogs on theC-torminal ends of both HC monomers. Figure Hi lepiesentsa monovalent Hf toxm peptideanalug-CC Ab. uha the toxm peptide analog fused to the N-terminal end of one of the I..C monomers, figure 11 represents a monu\ aiem IΠ toxm pepoec aria log-4 K \k vsah the tome pepede urukw fused io the N-terminal cud of one oi the HC' monomers, figure Jj represents a monovalent HT Ab LC-toxin peptide analog fusion the., 1. C-toxin peptide analog fusion -r 1.C " HHCih with the toxin peptide analog fused to the C-terminal end of one of the LC monomer;». Figure iK. represents a bivalent HT Ah LG-toxin peptide analog fusion (i.e., 2tI i'-toxin peptide analog fusion) f 2<HOk with toxin peptide analogs fused to the € -terminal end of both of the LC monomers. Figure It represents a tri valent HT Ab LC-ioxin peptide uuulog;H€»toxm peptide analog tee., 2(LC-toxsn peptide analog fusion) «· HC-toxin peptide analog ftnuon i FIG), with the toxin peptide analogs fused to the C-iermmal ends of both of the LC monomers and one of the HC' monomers. Figure 1M represents a bn a lent antibody n ith a tosin aoaldgimdioty iaf^:.anjat#Sai loop of the imrmmogiobulm Fo domain of each HC monomer, f igure IN represents u monovalent antibody *,ith a toxin peptide analog moiety insetted into an internal loop of the immunoglobulin F'e domain of one of the IK' monomers. Dimers or t rimers will form spontaneously in certain host cells upon expression bf a deoxyribonucleic acid (DNA) eoostract encoding a single chain. In other host ceils, the cells e&amp;fs be placed-in conditions favoring formation of dimers’{rimers or the dthnet!^fe^iff'^;''!be'.fenned in ylho. If more titan one HC monomer. LC monomer, or immunoglobulin Be domain monomer is putt of h suK/ie embodiment, the individual monomers can. .be. tf desired, identical or different from each other. | bit 1 f; figure 2A--B ikmon^trutes by B.nch.\ptvss w elect rophysiology that the mouoYukmi alvl H Πί'-ShKt 1-35 Q 5 eh. "> Ve ;SFt) 1DNO ?N 3°, 32). as describee v. txamplee 4 ,md e. n> more potent t' Koev!^ lit. van κλ l * e. a rent < Fk.uc 2 Λ: than human Ky i. I gurtent {Figure 21.¾). f00.2b| i tgom 3Λ sH>v»s u LoomaMe b Omni blue stained 1 s s-glyt.sno 4-30*'. SDS-P \GF. of me fi ual tnonovalem I e~l s 0~Shkj i - G, Q Udcd uuti-k.Li 1 Ab pmctueh d,.sunbed in ['sampled he; cm 1 mes M2 nere kuoed a-· rbilows; lane i Nos-'< \Luk'2 v. ide mugs protein sea Cards {10 gig lane 2 0.5 μ a. ptoouet. non-ι educed, iaae 3' Hank, lane - 2 0 ug product, non ’'educed; l ce 5'bt mk. lane <r M pg produce neu-reduud, lane Noses ΜόΜ2 aioe ruugc p»otem standards {10 nib kmc 8; 0.5 gg product, reduced: lane blank; lane 10: 2.0 eg product, reduced; lane U: blank; lane 12: 10 pg product, reduced.

[00211 Figure 3B show* size exclusion chromatography on 50 ug of the final monovalent E«M..:t0~ShK[i-35, Q10R|Mnh-K.LH Ab product. described in Example 4. injected onto a Fhenomenex Bio Sep SEC· 3000 column (7.8 x 300 mm) in 50 mM NafbPO.f, 250 mM NaCly and pH 6.9 at I mUmin observing the absorbance at 2H0 rtrr>.

[0022] Figure 3C shows an LC-hfS analysis of the final L10-ShK.{ 1-3$. Q l6K}.anti-KLH Ab· described in Example 4. The product was emomatagiaphcd through a Waters MassPRET micro dcsaltmu column nsinga Water-» AC'QUH'Y UPLC system. The column was set at 80°C and the protein eluted usmg a l inear gradient of increasing acetonitrile concentration in 0.1 % formic acid* Pan of tire column effluent wax diverted into a Waters LET Premier ESi-TOh mass so. enonwtef toj mass analysis The instrument was run in the posh is e V mode, The capi liary voltage was set at 3,.200 V and the cone voltage at M0 M. The mass spectrum was acquired from M00 to XHXht nv> and deconvolved using the MaxEntl --cU'xarc pnw wted hy the instrument manufacturer.

[ 0023; figure 4A shows a Coosnaxsie brilliant blue stained Ercvglyctne 4-20% SDS-PAGiBof the final.monovalent anti-KJLH HC-LIO-ShKj' I-35, QJOK] Ab product described m Example 4. Panes 1-12 were loaded as follows; lane 1' Noves Mark! 2 wide range protein standards <10 pi); lane 2; 0,5 pg product, non-reduced; lane 3; blank; taste 4; 2 0 pg product, non-mil;teed; lane >:biank; lane 6: 10 pg products non-reduced; kmc “ Nox ex Maskl2 w ide range protein standards f 10 pi); lane H; 0.5 gg product, reduced; lane 9: blank; lane HV 2.0 pg product, reduced; latte, 11; blank; lane 12; 10 pg product, reduced.

[0024] Figure 4B shows size exclusion chromatography on 25 pgof the fittal •.monovalent anti-Kt.H 120.6 BC-L10-$b&amp;[ 1 -35. QioR] antibody product* described in Example 4, injected onto a Fhenomenex BtoSep SEC-3000 eohnnn (7.H x MM) mm) in 50 mM NaHvPCb, 250 mM NaCk and pH 6.9 at 1 roL/roin deteteting the absorbance at 2H0 mu. The deflection observed at about 11 min is an injectiot-related artefact. (0025I Figure 4C show?» a MALDI mass spectral anuJ\ms of the final sample of monovalent anii-K.LH HC.VLHF$hK[l~35. QlMvj Ah, described hi Fxantplc 4, analyzed :usms a Niteronwhs \J \Llfi mivv v\ ms'·1' \pcc*uM*uxc,‘ .cupped 0 uh nitrogen laser. The sample was run at positive linear mode. The msfrumemA vohage 0as set at 12 l\ and the high mass deteuot was set at 5 kV Pauli speehum ms produced by adsumolatmg dam from aid#200 laser shots. External mass: calibration was achieved using pm died pmtcms of known molecular masses. jtKOt'j Figure 3 \ slams a Ooomassie brilliant blue stained Tris-glycme 4-20% SDS'-FAOh of the final bivalent aKLH HC · L Itl-ShK [ 1-35 QlMv] Ah product, described in Example 4. Lanes Ail were loaded as follows lane L Notes Vhnkl2 wide range pattern standards 110 pi); lane 2: 0.5 ug product, non-reduced: lane 3. blank: lane 4: 241 gg product, «οη-reduced; lane Srblank. lane h; 10 pg, product. non-· reduced, lane 2 No\c\ Mark 12 wide range protein -standards (10 fill, lane *05 p.g product, reduced; lane 9: Hmk; itee 10: 2.0 p.g product, reduced; lane 11: blank; lane 12: 10 gg product, reduced.

[0022! 1 a tic^lt shous ,-uae esdusam chroioatographv on 25 ug of the tmal bivalent anti-KLH HC-L IO-SbNp-35, Q!fiK| Ah product.describedat.Example4*.·· hijeeu a ouio : Fheeomen.es BioScp SEC-5000 column (? K x .300 nut)} in 50 mlVf Nall .po4. con niM N.1CI. and pH 6.0 at I ml min detecting the absorbance at 2B0 nia The deflection observed at about 11.5· min is an injeeiiors-rehtied artefaet.

[o03B| Figure 5C show-, a MAI DI mass spectral analysis of the final sample oi bivalent amt-RLH EiC-L10~ShK| 1-35. Q1AK] .Ab. described in Example 4. anaK/ed using a Mieromass MALDI micro MX mass spectrometer equipped with a nitrogen Jaser, Fbe sample η as run at positive linear mode. 1 he instnuneuFs voltage was set at Γ2 kV and the high mass detector was set at 5 kV. Bach spectrum was produced by accumulating data from about 200 laser shots External mass calibration was achieved using purified protei ns of known molecular masses.

[00211 Figure 6A shows a Coomassk brilliant blue stained I ris-glycine 4-20% SDS-FACE of the final monovalent K.L111IC-1 JD-SMvjOMS. Q16K.J Ab product. dvworthed in Example 4 l anes 1 12 were loaded as follows' lane 1; Novcx Mark 12 wide range protein standards 110 pit; lane 2: 0.5 fig product, mnweduced; iane 3; b’ ϊπ,ν:. me -I 2 o is. produe*. on radioed bane o blank lane o 10 pg product, non-reduaxi; lane 7: Μ\λ Mark! 2 wale range proton* vandards (10 pi); lane H; 0.5 pg pmduet n' ecu L. o gv v Uneio 2 ug p ixW, ra i,ad, Lo Hank lane 12; 10 ug product, reduced, [0010] Figure 0B .«how a size exclusion ebromatoara pby on 20 pg of the Huai monos alem ant-.-Kl 11 lit'4 iO-\Mvj2~55, QlOw. Ab pfoe.aJ, described ·η I sample b injected onto a Fhenorncncx B-oScn SEC-3000 column ί 7.8 x 300 ram} tn 50 mM \.dl POi 2Ή toM \ (.1 «it a < * i rl ! ή iu m fVie abstrbmxi _t 2h0 nm. The deflection observed at about 11 min is an in section-related artefact. (0031] Figme bC «hmv* ar l ( -MS mass spectral analyst of the final sample of monovalent4mi-KLi 1 HC-l. 10“$hK.[2-35*QI6fCf Ah, described in Example 4, Ibd ; product wp elwdlatograplicd through a Waters MnssPREP micro desalfisg coien: : using a Waters ACQUiTV UP IX system. The column was set ut &amp;0°C and the protein eluted using «ι linear gradient of increasing acetonitrile concentration in 0.1 % nmrae acid Part of the column effluent was diverted into a Waters LOI Premier ESI-TOF mass spedrametcr fbi· mass analysis. The instrument was ran in the positive V mode. The capillary voltage was· set at 3,200 V anditbc eonc Aoliage at bfb V, 'OiS mass spectrum was acquired itom 800 to 3000 m&amp; and da'onvolnted rising: the Mas! Mi xoftwate piosided h> the rastuimcnt manufactures (00321 Figure 7 shows results of pharmacokinetic studies |single- subcutaneous <&amp;Β&amp;^$Μ0ίφ ^erjifcmied in Ipidguc-Dawicy rats. Open squares represent data fdr: monos a lent f'cf'e-l JO-ShKt I -35. QHdCi f heterodimer of SEQ if) NO l and SEQ ID NO:26> closed circles represent dine for monovalent ami-KLH antibosly-$h&amp;{i - *Q NO- 2E, SEQ ID NO:29, S EQ ID NG:28, ami SEQ IL> NO:32); and closed triangles, represent data for monos alent umiTCLB antibody (loopf-ShKl 1-35. Q10K) genomet of SEQ ID NO 28, SEQ1DXGX: 5ΓΟ ID NO:2H; and SEQ IB; Example Ahnl-Tdhlcilll:, [0033] Flgyre Ifstawrmtatsof studies (single- subcutaneous dose ::: 6 mg/kg dose) performed hi Sprague-Bawlcy sals for bivalent (open squares) and monovalent (dosed circles* anti--KL H antibody-Slta 1--33. QI6K) (respectively, tetmmers of [81¾IP MD: 2K, SEQ ID NO;32, SEQ ID NG:2K, SEQ IDNOD2] and [SF.Q ID NO 2b, SEQ ID NG:20. SF.Q ID NO:2k. SBQ ID NO'32|), as farther described i n Example 5, and Table ?.). 1005^ 1 Πcmc0 - how s results of pharmacokinetic studies t single- subcutaneous dpse ::: 0 mg kg? performed in Sprague-Dtaiey rats fist bivalent (opensquares)dnil monovalent tclosed Circles) nnti-KLH anribodv i loopt-ShKit I -33. QiON.) fmxpcemeh. run an ms of (SFQ ID NQ 38, SEQ ID NO 15, SFQ ID NO 2k. SEQ ID NO-33] and [SEQ ID NO 28. SEQ 1.0 NO:34. SEQ ID NO:28, SEQ ID NO:351t as further described in Example 5, and i able 71..

[fdlSJ Eigorc 10 shows the results of phannaMkftpi^ > subegtitneous dose) in SD mis of monos alctu taShE/Fc hetcrodunor .(.open squares), monovalent Fe-8hK./KLB Ab (heterofrimer or hemibody)(open triangle) and the |s(\ a lent ShK-Fc, ShK-Fe homodimer (dosed circles), 'Fhc-aioaovaieij.t'lieicrodj^|'-;:'·' aud hctcrotrimer provided much greats exposure than the bivalent homodimer. Eunher details on tln-v sUs,h, a-c p.oxided m Example .5. P03fj| Figare I I shows analysis of antibodies on a 1.0 nun Tris~g!yeine 4-2()% SDS-PAQE (Ntsvex) developedM 220¥ using reducing tading buffer and staining wnh QuiekB: sc iBoston Biologicahd, I anes were loaded as follows (left to right), lane .1, Novex Mark 12 standards; tad 2, 2 ug aDNP 3BI A b taro transient cell culture; lane 3, 2 pg aDNP-3 8.I Ab from stable ceil culture; lane -1 2 ug aDNP 3H4 Ah tarn transient cell euiture; lane 5, 2 ug .iDM* 3114 Ab from stable cell culture, lane bi2 pg aPNP iAf Ah tabs trahtassf cell culture; :&amp;DNP 3C3 Ah taro nanoonl ccd culture, tnul Fuse N 2 ug aDNP 3Ad Ab bent transient cell culture.

[0037] figure 12A 8 shows analysts of amtbodtes on a 1 o mm Irts <j\eme 4-20% SDS-PAGF: (Noses) developed at 330V' usrog nou-redueuvg loading .buffer and stam&amp;g with QitiekBIue (ilosloo: Biologicak). Lanes were fe&amp;d as follows (¾ to right): (Figure 12A): lane 1, Novcx Mas k i 2 standards; lane 2. 0 5 gg aDNP 3A1 Ab; lane X 0.5 gg aDNP 3A4 Ab; lane 4. 0.5 gg uDNP 3C2 Ab; lane 5. 0.5 gg aKi.R 120.6 Ab; lane 6, No vex Mask 12 standards; lane 2. 5 rig aDNP 5 AI Air; lane k 5 eg aDNP 3.A4 Ab; lane 9, 5 g.g aDNP 3C2 Ab; lane 10, 5 gg aKJJ-1 120.6 Ab; (figure 12BA lane i. Novcx Mark 12 standards; lanc2, 0.S gg aDNP 3BI Ah; lane X blank; lane 4, Nov ex Mark 12 standards; bine 5, 5 ug aDNP 381 Ab. job AN Figure 15Λ slums ,i;ut!\sjs of antibodies on a t 0 mm 'Dk'-gBemc >*-20% SDS-PAOb tNovexl developed at 220v iwmg non-reducing loading butler and staining with QtsiekBlue {Boston Biological^ Lane-' wen.' loaded as follow-< Bell lo nku). kmc L Nosex Mark 12 standards; bine 2. blank: lane y 0 2 va a DM·* 3B1 Ab; base 4, 0.2 gg aDNP 3Λ1 Ak lane A Hank; Due A 0 o ng aDNP 3B1 An; lane X 0.6 gg aDNP LA I Ab. Line 6, blank kmc 0. Lx ng aDNP 3ΒΪ Ab; lane 10. I n gg aDNP 3A.t Ab. 100501 Figure ! 5B Mow >< .maiy-is of antibodies on a 1.0 rnnt Bis-Iris 4-;2% NuPACiP >;Nose\) developed at 220V using tfou-redncitsg loading buffet and staining V'. itb QinekBlue t Boston BiolngteaNy Lanes uere loaned as lollows {left to neks:, ianc L Moves; Mark 12 standards; kin A. blank; lane .3, 0.2 ggaDNP 381 Ab; latte -1. 0.2 gg aDNP 3Λ1 Ab; latte 5. blank lane 6. 0.6 us aDNP 3B1 Ah; lane X 0.6 μη aDNP 3Λ1 Ab; lane H, blank; kite 0. I 8ggaDNP 3B! Ab: lane B.k I KggaDNP 3 A1 Ab.

[00401 F igure 14A*R shows analysts of anti bodies on a LO mm Tns-glyetne 4-20% SDS-PAGE (Novcx) developed at 220V using non-reducing loading buffer and staining with QuioLBlue {Boston Biologies lsk Lanes were loaded us fallows (left to right): {Figure HA: with 0.1% SDS in running bn fiery lane 1., Nov ex Mark 12 standards; lane 2. 0 5 gg aDNP 3BI Ab incubated ai room temperature for 10 min, lane 3.: 0.5 gg. aDNP 3ΒΊ Ab incubated at 85%' for 5 min: lane 4, 0.5 gg aDNP 38! ,Ab incubated at 100 (5 for 10 min, lane 5, blank; lane 6, I gg. aDNP 3B1 Ab incubated at room temperature for 10 min; bine χ I gg aDNP 381 .Ab incubated at 85:::0 fur 3 min; Lute 8, 1 μη aDNP 3BI Ah Incubated a? iOfNC fen !0 min; (Figure MB; 0.4% SDS in running buffer; 85VC treatment Γογ 5 mint: lane L Nos es Mark ; 2 standards, Due '3. blank; Lute 3. 0.25 ug aDNP 3BI Ab; lane 4, blank; Lmo 5, 0.5 ug aDNP .03 i Ab; lane (\ blank; Due 8. 1.0 gg aDNP .0¾ 1 Ab; lane 8, blank, Due 0, 2.0 pgaDNP8Bf Ab. (00411 Figure 13 shows. analysts, using two size exclusion columns i TSK-GEL. GIOOOSWXL. 8 mm parliesc size. 7 * s. 300 mm, TosobBioseience. 1)85411 in series with 4 100 mM sodium phosphate, 250 m.Vl Nut.'f at pH b.8 mobile phase flowed at 0.5 ml mom. of antibodies: a.ONP 5A! CM \". darker trace with post shoulder); aDNP 3BI iiBi”}; aKU-J 120.6 ("KUD: a DAP K'7 C'X'TX and aDNP 3Λ4 PGA 4'3. (1042] Figure 10 shows analysis of antibodies aDNP 3Λ! t"3AI% aDNP K'Z pl3C2'A and DNP-3A4 before and after 3 weeks of light exposure, using two size exclusion columns H'SK-GFL G3000SWXL. 5 mm particle size, 7.8 x 300 mm. TosohBioseiettee, 0854;} in series with a 100 mM sodium phosphate, 250 mM N»CI at pH 6.8 mobile phase· Bowed at 0.5 ml./min.

[0043] Figure 17Λ-Β .show analysis, using two sice exclusion columns H'SK-GEl G3000.SWXL, 5 mm particle size, 7.8 s 300 mm, loaohBioseieuce, 08541) in series with <i 100 mM sodium phosphate, 350 mM Nail at pH 6 X mobile phase flowed at 0.5 mlermn, of anti Oodles aDNP 3A4. uDNP 3A4-V {'AVIOfdY"), aDNP 3A4-F F'WIOIFM, aDNP 3.A4 VSS i'AVlOl VCCSS'A and <jDNP-3a4-F$$ ("WfOlFCCSS'O before {Figure PAi and after (Figure- MB} 2 days of Sight exposure.

[001?! Figure 18 shows ion. exchange analysis of uDNp antibodies (aD'NPMAd, aDNP~3A4~Y, &amp;DNP-3A4-F, aDNP-3A4-YSS and aDNP«3A4~F5iS'>. They were analyzed tor homogeneity using, a iosohaas KP-5PW column iKLpm particle, 7.5 rnrn ID X 7 5 ern long} uabtg Bui lev A 110 mM sxiitmt acetate, pH 5.0) and Buffer (¾ (10 mM stadium acetate, 600 mM NaO, pH 5,0} flowed at I mFmin with a programmed linear grad tern (I own 10 mm D' >' B, 50 mm 701.. B, ) mm 90%S and 5 min Ο'ΆΒΟ 10045] Figure 10 .sisow., an analysis of aDNP 3B1 (Figure 19 A), aDNP .1A4-T (Figure 19&amp;K and aDNP 3A4-FSS (Figure 19C) antibodies by non-reducing C.F-SDS with dense to·* of abse'C mee i 230 nou A bare filled -emeu eapdl try ro am '· ?<0 5 M W&amp;$ used for the separation analysis,· [0046] Figure 2C) shows an analysis of aDNP 3131 iFigure 20Λ). aDNP 3A4-F (Ftgjgre 2013), and ;aI3NF 3A1-FSS (Figure 20C) antibodies by reducing CE-SDS with dot., non ο» ...wo 1: rue r 22«'»! m ^ basc~*use 1 s2 ce cap 4 buy Ά) urn \ K) 3 om 'was used for the separation analvsis, [0047] Figu#::2l2^o#§' Mi-ipaiy^ curve), aONP-3A4« 1 SS (s«mi curve) and aD\!P-3FB (dusked curve) anttboduw were analysed by DM' nsin.g a MierCal VP-DSC where the samples were heated from 2(ΓΓ to 95'T at a rate of l°C per mmube, The pfstein in !0mM sodittm acetate. 0% sucrose. pH 5.0. (:004-8 j Figure 22 shows serum eonceotranott.s of aDNP 3A4-F, aDNF 3A4-FSS, and ul)\P 3b 1 antibodies in rats receiving a single subcutaneous injection of 5 tog'.,;, .s deteu need in FLPsA. Blood sampleswete collected at (k 0,25, K 4. 24, 7-4H. 12. 96. 1330, 504, 672, 840 and 1008 hews posNdpse.

[0040] Figure 23 shows plasma concentrations of a.ONP 3A4 or aJCLH 120.6 in male s.'ynomolgus monkeys receiving a bolus intravenous injection aDNF 8A4 (4 mg kg) or aKLll COo (3mg kg) amshouies, a-meetweiy. Serum samples were taken periodically and plasma concentrations of die ummadko was determined by ELISA. The data for aDNP 3A4 was normalmed to 3 mg kg for comparison purposes [OO'-Ol 1 tgure '24 shows a ( oonussic brfLam blue stained Iris-glycine 4 20l <-. SDS-PSt'))- of the final monovalent. aKI Π 120 A 1 ('-ShK| 1-35« U 1614) An product. .Example M2 wore loaded as follows: lane 1: Nuvex Mark 12 wide inuge ptolem standards i 10 pit; lane 2' 0.5ng punum,;, nou-reiium ck Me 1 blank: lane #: 2.0 Mg product, non-redneed; lane 5 blank, lane O' 10 pg product, non-ieduced, lane 2 ko\e\ Mark!2 wide tango ptolem skimiuds (SO id), lane h 0 5 pg product, reduced; lane 9: blank; lane 10k2;0;ggprodt^ 12: 10 pg product, reduced. 100b 11 Figure 25 *how a st/c exclusion chromatography' on 25 ng of tbe %ud monovalent aKIii 120.n LG-ShKj 1-35. QlMvj \b pooduct. described m I sample 4. irpeted onto a fbenomonex BbSep SEC-3W0 : column (7,¾ x 300 rostj in SO mM ΝαΗ’ΡΟ». 250 raM NaCl, pH 0,9. at I roL-'mm detecting the absorbance-.at '280 nm.

[0052! f igure 2oA~B .shows fion-n'dueing I Figure 26A} and reducing (Figure 20Bi M ALPl·- Vlk mass spectral analysis or die final sample of monovalent aKI.H !20.o l b-ShKj i - L\ Q 6k" prod-eg described m F\. rro'c 4. using a Mteromass MAI PI micro MX mass spectrometer etp.dpped with a nitrogen laser. Thepsmplo was runs! positive linear mode. The instruments voltage was set at 12 kVjadiihie-hi^h'tta^··'

Jefoctm sW". set -.· 5 kV. Fa..', spectrum was produced b-> ace m mi tat mg data from anou? 200 1 ,ser snots h“'n mus ci. b’.t'km ^ achieved usingpunfied proteins of f i town molecular masses |G(G > : Fs<;m\ 22 skews a C oom.isssc bnlbant blue stemed Ϊris-gheme 4-20% SPS-FAGb ot the rnul bivalent aKI.H 120.0 U"-ShK[i-35. QlOK] Ab product, described iu Example 4, Lattes 1-12 wore loaded as follows: lane 1: Noyex: Mmifl wide range protein standards (10 pi); lane 2: 0.5 pg product, nomreduced: lane 3: blank; lane 4: 2.0 ug product, non-reduced; lane 5 blank; lane b: 10 pg product, non-reduced, lane 2. Noses Vlad. 12 wide mnge piotem standards {\0 pH, lane 8 0.5 pg product, reduced: lane 9: blank; lane 10: 2.0 pg product, reduced; land i: blank: lane 12:10 pg product., reduced, 00054] loguro |8 shows sike uxclubioh P: 25 pg of the final biv&amp;lem aKLH 120.6 LC-Shk| l-35, Q16&amp;! Ah product described in Example4, injected onto a Phenomencx BioScp SEC-5000 column (7.S x 300 mm) in 50 isM Nal-BPO.}, 250 mM NaCl pH 6.9,, tu 1 ml /rein delecting the absorbance nr 280 nrn.

[0055 : Figure 20Λ-Β show* ηοη-ivducmg ( Figure 29A) and reducing {Figure 296) MALDi-MS mass spectral analysis of the tin ill sample of bn/dem aKLH 120.6 10-8hk'[ 1-35. Q1 Ms \ Ab product, described in Example F using a Mieromass MA 1,.D! 'micro MX mans spectrometer equipped with n nitrogen laser. The sample was run at positive· linear mode. The hiMfu mom's voltage w as sea m 12 fcV and the high mass detector was set at 5 is V. Each spectrum was produced by accunnhatmg data from about 200 laser shots. External mass calibration w as achieved using purified proteins of known molecular masses, [0056! figure 30 shows a Ooomaasie brilliant blue stained Tris-glycirso 4-20°A ADS-PAGE of the fund invalenl aKIH 120.6 LC-ShK| 1--35, QI6K.) ΛΙ> product, -described in Example 4. Lanes M2 were loaded as follows, lane ! · Moves Mark 12 wide range protein standards 110 id a laue 2: 0.5 με product, nosi-reduced; lane 3; blank; lane 4: 2 0 p.g product, vion-reduced; lane 5:hlank; lane 6: 10 fig product, non-reduced , lane 7: Moves Mark 12 wide range protein standards {10 μΙ>; lane S: 0.5 ug product, reduced; lane 9; blank: lane Hi; .2.0 gg product, reduced; land 1: blank; L-ute 12; 10 fig product, reduced.

[00521 Figure 5 1 shows sire exclusso.it chromatography on 2.5 ug of the mud invalent aKLH 120.6 LC-ShK.[ 1-35. Q'lbK.] Ab product, described in Example 4, injected onto a Phenomencx BioScp 8EC-3000 column P.8 x 500 mm) hi 50 r.nM Nal l/PO}, 250 mM NaOF pH 6.9, at 1 ml mm detecting the absorbance at 260 nm.

[0056! Ftgme 32A-B shows non-reducing (figure /2A.s and reducing {Figure 52Bi MAI..DI-A1S mass spectral analysis of the final sample of m\Tern akXR 12.0.6 If -SlsK|l~F5, QlPkj Ab product, described nt i.cantpic 4. using a Micromuss MAFDi micro :VL\ mass spectrometer equipped w ith tt nitrogen laser, i he sample was run at posit'd c linear mode. The inatruiucrsiM utliage was set ai 12 kV and the high news detector was set at 5 kV, Each.spectrum ecus produced by accumulating data from about 200 laser shots.. Bxtomal mass eaibmiom was aeMeved ttsfeg purified-proteins of known molecular masses [0059: Figure 3.3 shows a {.oorna.vsic Gilliam blue stained Trlx-glycine 4-20':« SQS-PAGE of the final monovalent aKLH 120.6 lgG2 HE-Shkp-35. R1 A, 14A, QI6K) Ab product, described in Example 4 Lanes 1-12 were lorded as lollops, lane I; Novo. Mark! 2 svide range pro-ent startdurds U0 μΙ): lane 2: 0.5 ag product, non-red is cod; lane 3: blank; lane P 2.0 s.;g product, non-redaeed; lane $:hlank. Innw 6: K? eg product. rsori-tedueedk lane 7; Novcx Mark12 wide range protein standards (10 μΐί}; lane 8: 0.5 μ$ pi^uet,rred.ueedjlls«€<f::SkdMb^®;:^l -,0 p.g: product, educed; land ;' blank; lane 12: 10 ug product, reduced [iOCGOI Πω«ν shows size exclusion chromatography on 25 ugofthe final monnvjjem uKLH 120.0 lgG2 HC-.Sbki ( -55, ill A, 14A. QlftKl Ah product described in Example 4. injected onto a. Phenontenex BioSep SEC-3000 column (7.5 x 300 rrsrnl in 50 rnM NuR-.PO,. 250 mM Nad, pi I 6.v\ at I ml. rum detecting (lie abxorbarice at 280 nm.

[Odd I Figure 35 shows reduced LC--MS mass spectral analysis of the heavy chain in the :-u: -'•'upe of s x;$n.w a ert sKl Η '70ο lgG2 IH ~ShxG-5\ l\: 7, GA. 016K’i Ab product, described in Example --1, the product was chromatographed through a Waters MassPREF micro desalting column using a Waters ACQUITY GPL.C .system. The column was set as. HOT and the protein eluted using u linear gradient of increasing acetonitrile concentration In 0.1 % formic acid Part of the column effluent was diverted into a Waters LCT (Vernier ΡΑΙ-ΤΟΕ mass spectrometer for mass analysis The instrument \\a< run in she positive V' tnode. The capillary voltage was sot at 3,200 V and the cone voltage at «0 y. The mass spectrum was acquired from 500 to 5000 nve end decoswoUued using the Max Em I software provided by the instrument manufacturer [005:21 Figure 30 shows a Ooomassie brilliant blue stained "fns-giyeme 4-7.(5:(. SOS-PAGE of the final uKLH Ι2ΘΑ lgG2 HG-Cbbl Ab prexiuet, described In Example 1I I anus GS2 were loaded as follows, kmc 1. Nov ex Mark 12 wide range protein standard* ΠΟ μι): kmc 2: 0.5 μρ product, ποη-redueed; kmc 3: blank: lane 4; 2 0 ug product, yon-reduced:; lane S'blank, lane 6- 10 pg product,. non-reduccd; lane 7: Nov ex Mark 12 wide range protein standards f 10 μ A; lane 8; 0.3 eg product, reduced; bine 9: blank; hue HI: 2.0 pg product, reduced; bine)); blank: lane 12; 10· pg prod u c b reduced.

(006.}j Figme 3? shows size eKdlusion chromatography on 25 ug of the firrai MCMI 120.6 lg<;2 HC-COhi Ab product, described in Lxantpie I L injected onto a Bdienoruenex BioSep 5*ΙΧ'~30ΠΟ column (7.8 s 300 mm) In 50 mM NaH^PO^ 250 mM bluCI. pH 0.0, at 1 mL-min detecting the absorbance at 280 nm. (00641 Figure 3M-V-B .shows non-reducing (Figure 38Λ) and reducing (Figure 3bB) ΜΛ1.101-MS mass .spectral analysis of the final sample of a HI. Η 120 6 igG2 HC-COXj product, described in Example 11, using a Miens muss MAID! ntkto MX mass spectrometer equipped vt ith a nitrogen laser. The sample was run at positive linear mode The instrument's voltage was set ut 12 kV and the high mass detector was set at 3 kV. Each spes'tnirn. was produced by .accumulating data from about 300 laser shots. External mass calibration was achieved using purified proteins off; sown mo I c e a I a r m a ss es. jOoob] I igute 59 -4-ow -> st/'C vs elusion chronvm\graph\ on 50 ug each m akd H )gilltX3'>X», AMO'-IK' ski. U k>G2, ΠΓ-Λ\10'~ Jkl h Igiil AMPV) ( aki H IgCn and L( -Ml05 akt H )g(i 11 products, described in Example A injected onto a Pheyometws BtoSep SIV-500U ^ohnun ( An \ 300 mm) in. eb mM V.d BIX A, 350 m.M Nat. b pH t\9, at Ί ml.mun detecting the absorbance at 2k0nm; (00661 figure 40 A-f shows analysts of antibodies (described in Example 0) a ΚΙ. I! tgCs I N297Q (Figure -10A).. AMP5-HC itKLH lgG2 if my re 408), 1..C-AMP5 aK.LH !gG2 {Figure 40Ck HC-AW5 aKI.il )gG2 (Figure 40Dk and AMP5-LC aKL H IgGl {.Figure iOE) on a 1.0 mm (Vis-glycine 4-20% FDS-PAGE {Novw} developed at 220V using non-reduemg loading buffer and staining with QuiekBtue (Boston 8<ologicab}. Lanes I -12 were loaded as follows: kme I: Novex Murk 12 wide range protein standards (10 ttd); lane 2; 0.5 pg product, tsati-redueed; lane e: blank; lane 4: 2.0 ttg product, ntm-reduecd; Kme S:bi;mk; kmc n: to gg product, ncm-ieUueed, kmc ~· Moves Mark!2 wide range protein standards (10 pi); lyric f. 0.5 eg product, reduced; lane o: blank; la.no fO: 2.0 gg product, reduced, lane! H blank; lane 12: 10 pg product., reduced, [000?] Figure 4IA-D shows ntuss spcctrograpmc analysts oi reduced sample'· of LC-AMP5 uKf.H lg;G2 {Figure 41 ΛK 4MP5-1 K.' aKI H lgG2 (Figure 41(¾). I lt'« AMPS aKlH lgG2 (Figure 410. and AMPS4.C nklH IgGl (Figure 4ID). described tn Example v. Each sample wan ch?onunoe.rufkcd through a Waters Massprep micro desalting column (2.( x 5 mm) using an Ac quay trial C system then introduced nao a Waters :nra>o A flight LCT premier mu as spectrometer lor mass .measurement and the mass spectrum was decern vole ted using the MnxEntl so!metre. (006S] F igure 42 is a schematic map of the Excmdio-4 C‘Ex4'>{kG-*KLH 120.6 L€ fttsion eotmtrttei, described it Example ICl, [0069] Figure 4 5 .«hows sine exclusion chromatography of 25 -tg o.l the fim-d E\4-i kii-aKI.H 120.6 }.(' antibody fusion, described m 1;sample 10, nqeetcd onto a Phettomenes BioSep SEC-3000 column (7.x \ AGO mm) in Ml rnM NaH;POi, 251) ruM NaCl, pH 6 *>, at I mL ntin detecting tits: absorbance at 2<s0 nm.

(0070! Figure 44 shows analysts of on a 1 0 nun Tris-giycme 4-20';SDS-PAGE (Novex) developed at 220V using reducing and non-reducing loading buffers and staining with Quick Bine (Boston Bkuogieaixs. Lanes )-10 were loaded as fo! loses; lane !; Novex Mark; 2 wide range protein standards (10 μ!); lane 2. 0.5 ng oihes protein: lane 3: 0.S ug Ex4-aK.i..H 12(1.0 Ab, non-reduced; lane A: 2.0 pg other protein, lane 5; 2.0 pg Ex4-aKLH 120.0 Ab. not!-reduced; lane 6. Novex Mark!2 wide range protein standards (10 μ!); lane 7: 0.5 ug other protein; lane X: ()..5 ug Ex4-uKLH 120.0 A0, reduced, lane 0. 2.0 pg other protein, lane 10, 2,0 ug IMdmKj. H 120,6 Ab, reduced.

[007 Jj Figure 45 \ho\ss u schematic representation of N-ienmnai and i'-tcvnirnai fusions of pharrnaeologicaily anise chemical moicucs ssiih the UC ma t 0 -mo-nomers of ;.-m arm bods of the mwmiora as further exemplified in b sample v.

m (All fci> 1H SC 'RIP I ION < H F\tBODiML\iS

[00721 The sceuoo houdmgs used herein me tor ornanhmtional purposes only and me Koi to be coustmed as limning the shined manor described [0071] IlgtoMSSl i : )1074] Unless otherwise defined herein, scientific and technical toons used in eonneemm o nh the present .."tpiieutior sisJl ha\e the meanings that are commonly understood by those of ordinary skid in die art. Further» imams mheru iso required by eomext, vngular terms sh,dl r\l >de m»ualitics and plural ν,ιηχ vuui metude the singular. Thus, as used in this speeilkatmn and the appended claims, the singular forms '"iC\ "an'' and “the” include plural referents unless the context dearly indicates otherwise for example, tetc:owe V Ά pa-teitf momdes a pin,· ajirv of pum-un, reference to "a cell” Includes populations of a plurality of cells, [tKPS] “Polypeptide" and "protein!> are used imerehmtgcably herein and include a molecular olndn of o.u> or more ammo acids imked covalently lb tough pepiide bonds. Theierms do hot refer to a speetfie length of the ptoduob Thus, ’'gejhfdes, * and "oligopeptides,'' are included: within the definition of polypeptide.: The terms include post-translationa! modifications of the polypeptide, for exsthpie, glya>syiuiunw, <n elylahons, phosphorsUmon*> and tin hke In uddmou, pmtem fhsgmepts» analogs, mutated or variant proteins, f usion proteins and the like are included within the meaning of polypeptide. The terms also include molecules in which one or more amino acid analogs or nomcanomeal or unni^i^ included as cun be expressed foeothhtnantiy using known protein engineering techniques. In addition, fusion: proteins cart be demmtiited as described herein by ''>yell^^ra'ofgMie;phOTishy· techniques. jlCi7b| The term “isolated protehiT referredmeans that a suNect protein (Ills free rjf at least some other ptotoihs^d%;Whiidh.:tf: would hormaliy be found in nature, i2} is essential ly free of other ipmtdms iom thesame source,, e.gdfrom the same species, p) is expressed reeombiinmtly by a cell of a heterologous species or kind, ¢4) has '•'.been sepahded ff6p .atleast about 50 percent of polynucleotides, lipids, •':OiSEfeohydt^espwofter':t»aterial,s with which it is associated in nature, {5) is oporahly Msociated (by covtdeut or noneovalent mtemetion) with a polypeptide with which it is not associated in nature, amt or (ό) does not occur in nature. Typically, an isolated protein"1 constitutes at least abopl: 5%, at ieast afeoui 10%, at least about: 'M%! or at least about SP% of a given saotple. Gentplie !>ΝΑ, cDNA, otRMA or other RN A,-of synthetic origin·;, or any combination thereof may encode such an isolated protein, Preferably,the isolMed proteitt is sitbstanuaUy free iAtn proteibs oi' : polypeptides or-other eontaminnoK that ate found in its natural environment Shat would interfere with us therapeutic, diagnostic, prophylactic, research or other use [00771 A '"variant" of a polypeptide t'e.g., an antigen binding protein, or an antibody's <. omprises an amino ac-d sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid ,-<quenec relative to another poh peptide sequence V annuts include fusion protenw (007b'; The teen ''fusion ptotobv5 indicates that the protein includes polypeptide components derived from more than one parental protein of polypeptide. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide scqimneejfbm one protein is appended in fmrae wiilg and optionally separated by a linker Rom, a uueieoideAe<|uenee encoding a polypeptide wrnem, 'tom a dift 'ant pioteui. The -fusion gene can then he expressed by a recombinant host eel! as a singleprotelui (OtT-a; reted" protein refers to those proteins capable of being directed to the PR, vx't eto-y x esi, les, or the extracellular space as a result of a secretory signal peptide sequence, as well as those proteins released into the extracellular'.space vvithoi tt necessarily containing a signal seqiei#e, 1 f the secreted protein is released into the extracellular space, the secreted protein cuts undergo extraedkthtbpstiddlshfg;; to produce a "'mature" protein. Release info the extmcei hilar space can occur by many mechanisms, including exocytosts and proteolytic cleavage. In souse other embodiments of tie inventive composition, the toxin peptide analog can be ^ by the Best cell as a seetvtcd p rotoim which cun thenfee further purified from the extracellular space and/or medium.

[0080] As Uttvd herein "soluble” whoa in rosereua· m a protein produced by recombinant .DNA teJrnulogy in a host edi w y protein that exists in aqueous solution; if the protein contains a twin-arginine signal am;no amd sequence the soluble protest; d exported to the penplasrme space in grant negative baemrial hosts, or is secreted into the culture medium by eukaryotic host cells capable of seeredon, or by bacteria! host possessing the appropriate panes se.g., the kll gene·. Thus, a soluble protein is a proven; which is not found m an inclusion body inside the host cell Alternatively, depending on dsn cotUcvU a soluble protein is a protests which ts not found integrated in cellular membranes; ht contrast, an insoluble protein is one which exists in denatured form snsidc ·, ytopuisrmc grannies seabed an inclusion body) m the host ceil, or again depending on the context, an insoluble protein is one which is present in cell men;hr,u;es, including but not limited 10, eyevplasmic membranes, mitochondrial membranes, ehloroplasi membranes., eudoplasnue reticulum membranes, etc. }00K]j The term ‘ recombinant"’ indicates that the material (e.g„ a nucleic acid or a polypeptide} has been andlc. sully or symbolically B c.. rson-uaturally 1 altered by human intervention. I nc- alteration cun be performed on the material within, or removed front, its natural environment or state For example, a "recombinant nude»; dcsd" is one that is made by recombining nucleic acids, e.g., during cloning, DMA shuffling or other well known rnomalar biological procedures. Examples of such molecular biological procedures are found m Vhmatli et u!„ Moleenlur {honing. A Laboratory Manual, fold Spring Harbot.tr Laboratory, Cold Spring Bur bo or, N. Vi 1982). A "recombinant DNA molecule," Is comprised of segments of DNA joined together by means of such molecular biological techniques, ihe term "recombinant protein1' or ’'recombinant polypeptide” ns used herein refers to a protein molecule· svhicb is expressed using a recombinant DNA molecule. Λ "recon tbit taut host cdB is a eel that contains and or expresses a rccombioasd nucleic acid.

[00*0] Tut· tents'“polyst ucicotido or “nudeic acid" includes both single-strapdsd s; id donble-atnutded rnscleotldc polymers eruhaming two or more nucleotide residues. The nucleotide residues comprising the polynucleotide can be ribonucleotides or deos.yrihonudooudcs or a muddled form of either type of" nucleotide. Said modifications include ba.se modifications such us hromourid I ad. M$· inostne derivative-*, ribosc modifications such asdbe'-dideoxyrihosc. and interuueleotkU: linkage modifications such as phosphorotluoate, piiosphomdithioaie. phosphoroscienoato, phosphorodiselcnoatc, ph o sp i nwo a n i 1 ot hi oa t e. phospho-arh fade to and phosphoroarnidate.

[OORl i 1'he term ‘'oligonucleotide' means a polynucleotide comprising 200 or fewer nucleotide residues. In some embodiments, oligonucleotides are 10 to 60 bases m length In other embodiments, oligonucleotides arc 1.2, 13, 14, )5, 16, 17, lb, 19, os 20 to 40 nucleotides in length. Oligonucleotides may be single stranded or double stranded, e,g.. tor use in the construction of a mutant gene. Oligonucleotides may be scn.se or antisense oligonucleotides. An oligonucleotide can include a label, including a radiotahoi. a fluorescent label, a hapten or an antigenic label, for ••detection assays. Oligooucleotides may be used, tor csamplc. as PCI* primers, clomng primers or hybridization probes.

[0084] A ''polynucleotide sequence" or ''nucleotide sequence" or ’’nucleicmeki senueuc·;·.,'1 as used is;rm\ hangeabb, herein, is the primmy sequence ohouch'omie residues in d poh nucleotide, including n!'art oligonucleotide, a ΠΝΛ. and ΚΝΛ. a nucleic ae;d, or a character string representing the primary sequence of nucleotide residues, depending on contest, brum any specified polynucleotide sequence, either the gn ers nucleic acid or the complementary polynucleotide sequence can he determined. Included ate DN A or RK of genomic or synthetic origin v, hicli may he single- of doihhe-mnmden, and represent the sense or antisense strand. Unless .specified otherwise, the kdl-hend end of any single-stranded polynucleotide sequence discussed herein is the 5' cud: the left-hand direction ol double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5’ to .V: addition of nascent RNA P'anscnpis is referred to as the transcription direction; sequence regions on the DN A strand having the san® dequeued af t|a RhiA : transcript that are S' to the- 5’ end of the RNA Irxnseript are referred to as "ppshnaio sequ-mecsf' sequence regions on dvr D\A strnnu do mg flu- same sequence as the RNA umtserlpt that u*e <* to the 3 mdofthc RN A ;tanvx«ft are o Anal to as "dow ton'eam sequel \. -," [OCdS | As used hetem. an "isolated nude® acid molecule”' or isolated nucleic acid sequence" is u nucleic acid molecule that is either (1) identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid or Oi cloned. amplified.tagged or otherwise distinguished from background nucleic toads such that the sequence of the nucleic acid of interest can fee determined- An isolatqdpucieid acid molecule is other than m die form ot setting m which n is Sound m nature t tones er., an isolated trueietc acid momeule includes a nucleic acid molecule contained tit cells that ordinarily express the antigen binding protein (o.g.. amibody} where, tor example^ the nucleic acid troUxnh' is m a chromosoto d locution different from that of natural cells. (OOxoj As used hcrcUh, the lenn.> 'nucleic acid tnolecule encoding^ 'BH.A sequence encoding,'* and “ONA encoding’' refer to the order or sequence of deoaiyribonneleotides along a strand of deoxyribonucleic acid. Thrordbr df s^ikj'ribonuelcotides detenoincsihe order of ribonucleotides along the raRNA: eMn, and also detcumnes the order of ammo acids along the polypeptide (protein) chatu. The DMA sequence thus codes tor the RNA sequence and for the amino acid sequence. f 008 7 j The term "gene" is used broadly to mi or to any maleic and associated with a biological function. Genes typically include coding sequences and or the regulatory sequences required for expression of such coding sequences, Thu term "gene" applies to a specific eenomic or reeombouuu sequence, as welt as to n c,l>\A or m 11NA encoded by that sequence, \ "fusion gene'' contains a coding region that encodes a toxin peptide analog. Genes also include non -expressed pueleie aetd segments that, for example, form recognition sequences for other proteins. Non·· expressed regulatory sequences including transcriptional control demerits io v-bich pgufatory proteins, such us n'an-vriprm t tactor^ bind, resulting m transcription of aljaeept or nearby sequences.

[OOdhj ‘'ExpresMon of a u ne" or ''ovmro'.snm of a nucleic acid'" menus transcription of ON Λ into RNA (opt endU -neludiov modification of the RN \, e.g, . 'translation of RNA imo a polypeptide (possibly including subsequent post- translational modtfieano, ot "he pod pepodet, ot hrh uansvfipuon end imrseUio.i, as indicated by the context. ]0089| As used herein the term "coding region" or "'coding sequence** when used m reference io a structural gene refers to the nucleotide sequences vouch encode die amino acids round in the nascent polypeptide as a sesuU ot translation of an mliNA tBolcbblp. The coding region is bounded, in cwbnvtcs on the 5' side by the nttolcxitide triplet "ATG" winch encodes the initiator n\tdbiotS«b'a»#Ottdbe.3’ side by on e of the three triplets 'vvliich.:¾¾::e<^ AO, TGA). 10090] The term 'Aontrdi sequence” or "control signal” refers to a polynueicotidej sequence that cart, in. a particular host ceil, a fleet the expression and processing of coding sequences to which it is ligated. The nature of such control sequences may depend upon the host organism In particular embodiments control sequences for prokaryote* may include a promoter, a ribosomal binding sue, and a transcription termination sequence. Control sequences for eukaryotes may inetmie promoters comprising one or a plurality of recognition sites for transcription factors, Transcription enhancer sequences or elements, polyadenyiation sites, and frimsetipfiou termmgtmn sequences, Crmlm! sequeneet^ leader sequences and/or.fusion partner sequences. Promoters and enhancers consist of short arrays of DNA that interact specifically with cellular proteins involved in trankcripiiOn: (Martians, ef nL Science 236: .1237 (1087)1. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeas!, insect and mammalian cells and viruses (analogous control elements, i.e., promotersvam also found in prokaryotes*. ilk' selection of a particular promoter and enhancer depends on whui *.eU type is to be used n.> express she protein of interest. So tec eukaryotic promoters end enhancers have e broad host range e hdc others are functional in a limited sublet ofudi types {tor review see Voss, et a I, Trends Biochem. Sci. ‘ Mb? (jOKbs and MamutN. et a)., Science 1 M\ \ 137 i t 987s), [:0091] The term '"vector'' means any molecule or entity (e.g., nockne acid, plasmid, bacteriophage or vires) ased to tjnatsfei; p$teuv kitoa

host cdL jlKbC; ‘Hie tern; "expression sector" ot "expression eons;rear' as used herein refers to a recombinant UK A molecule containing a desired eodsng sequence and appropriate midem acid control sequences taxessarx for the expression ot the ope raids- linked coding sequence in a particular host ceil An expression sector east include, bin is not I:mined to. mquenecs that atTeei or control transcription, nuusianom and, if mnons are present, effect RNA splicing of a codusg region operably linked thereto. Nm:km: acid sequences necessary lor expression in pmkm'vom-s indude a promotes. optionally an opemior sequence, a ribosome binding sin.· amj possibly other sequences, tmlmryubc cells arc knourt to utlime promoters, enhancers, and termination and pobaadenybhon signal ~ A secretory signal peptide sequmme can also, optional Iv. be encoded by rhe cops cm ion sector, opemhiy linked to the coding sequence of interest, so that the expressed polypeptide ean bn seem-cd in the recombinant host eelk for more incite isolation of the polypeptide of interest mom the ceik if desired. Such techniques are well k non n in the an i log , Cioodcy, Andreo Rn et ah. Peptide and ON A sequences, ITS Patent No. 5, Hie,OP7; Wemet ei ai . Compositions and methods tor protein secretion, U.S. Pa lent No. 6,011,75.1 and U.S. Patent No. ^,335,)7^: Uemura et ml.. Protein expression sector and utilization thereof, U.S. Patent No. 7,019,000; Ruben et id.. !· human secreted proteins. US 100a 0i04-700 A?) 100931 ilic lovms "its operable combination’*, "in opens bio ruder’’ and "operably Jinked" m used herein refer to the linkage of tmeioic acid sequences itvauch &amp; mahuer thata nucleic acid molecule capable of directing the transcription of a given gene md the as others of a dcvied p micm moleude i- produced, The t, no awo refem to the linkage of amino acid sequenees in such a manner so that a functional protein o mod λ , Ο Po , sample, a ensmil -w,pierce m e see to: mal ^ "opmabix dukef u> a piotein coding sequence is heated thereto so that, expression of the protein coding sequence is achieved under conditions compatible with die transcriptional activity of the control sequence,·*; [0094] l no term 'host eeif me.i-w a cch that has ivui ujuslomieci, or is capable of being tran.stonned. with a nucleic aetd nod thereby expresses a gene of interest The germ includes die progeny of the purem ^ clL whether or not the progeny ss Identical in morphology or in genetic make-up to dm original parent celt so long as the gene o: lutescst ;·- pi, scut. Any m a huge numbero·'asababtc ami veli-snowu host cell· uvtay be used in the practice of this invention. The selection of a particular hosfis: dependent upon a number of factors recognised by the art. Tlrcso.siiciudo. tbr example, compatibility with the chosen expression sector, toxicity of the peptides encoded by the 0ΝΑ molecule, rate of imnsfommdon, case of Recovery of the peptides, expression characteristic*, bio-safety mid cost* A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of &amp; particular DMA sequence Wuhm these general guidelines, useful microbtal host celts in culture include hacteiia (such as Escherichia eoh sp,>, yeast, (such as Sacchuromvces smi and. other fungal cells, insect cells, plant cells, mammalian (including human] ccllA e.g., CflQ cells andflEE-294: cdllA MtultUv8t>ons can he made at she ON A lev el, e« w ell The peptide-encoding DM A ;-sequence may be changed to codons more compatible w ith the chosen host cell. For H. coli. optimised codons are known in the an. Codons can he substituted to dim cure restriction sites or 10 induce -u lent restriction si a a. which may uni in processing of the DMA in the selected host edi. Next, the tran-formed host is , ,dt ued m,i p ndted Dost edUrtuy he et s, , cm \1,\ com·,' omd fenn.e 'on conditions so that the desired compounds are expressed. Such fermentation conditions arc well known in the art. 10095] The term 'transfection'* means the uptake of foreign or exogenous DNA by 5:.a cell, and a cell has been “ttanstectetT when the exogenous DMA ha* been introduced inside the cel! membrane. A number of transfect ion techniques are well known in the art and are disclosed herein. See, o.g.. Graham et ah, 19? 3, Virology 52:456: Sambrook et ah, 200 L Molecular Ciomsig. A Laboratory Manual, supra, [3 avis et al.s 1986, Basie Methods in Molecular Biology, Elsevier. Out et a 1., I OX 1, {lene 13;.197. Such techniques can he .used to introduce one or more exogenous DNA moieties into sutable host cells.

[0096] The term Mrast.stbrmailore' re tors to a change in a coifs genetic eharaeu sou s, ere a i. el1 has btmrs o A,>ossed o hi ss e,s\ l\\' > vdstse * so sotssam new DNA of ;RMA, ifor cxample, a ceil is transformed where it is genetically modified from its native state by introducing new gesseisc material via transfection, basivi <_uon, oi other echsu.ji.es I o use ennsme one ts «sue. asw >. i transforming DNA ma> reeosobino u ;h; that of site cell phy stcMA integrating into a <J. onososoc of the c Ί1, os mav be maintained tninMentiy as as: epwoma! eltniem w ahotd: feeing feplfcatsd, of muy mpheate mdependenfly as a plasmid, A cell is considered to hare boost ".«Uabk trastsforsnod" xvhest the tmsssfonnmg DA A ss of the cell, a [609¾ -acceptable salt” of a composition of outlier, for: example a salt s.sf the antigen bmdsiK. prtjtem. -men as an antibody, w meant any salt or salts that are known or later discovered to be phannaceuj&amp;cally acceptable: Some non~Ssmmng examples of phntmia ecu tic ally acceptable salts are: acetate: triflooroneetnte: hydmbaiidbs, such as hydrochloride and hydsobromMe; soifete; citrate: mabaie; tartrate; glyeolate; gluconate; succinate; mesylate; ibesylab: sabs of .guide acid esters (gallic acid is also .known, as 3A 5 trihydroxy ben £cnc actdfsuehas PentaG-ttloylGiU^ose s PGG) and cpigal kx,u e-Ann gallate (hGCG). salts of icholcfeteryl sulfate*, tsarrsosite, tenato a,pd oxidate :safts. 100**8 j A "domain" or "region" (used interchangeably herein) of a premiss is any portion of the entire protein, up to and including the complete proietst. but typically • comprising loss than the complete protein. Λ domain can, but need non fold independently of the real of ihe protein chain atid'or be correlated vvifh a particular biological, biochemical or structural function or location (c.g.. a ligand binding domain» or a cytosolic, trim,smembrartc or extracellular domain).

[bOO')j "'Treatment-" or '"treating" m »m mterveniiot; peH'ortned u hh the nUemmo of faex coring die development or allot my -be pathology of a eh sot dot. Accordingly ’'treatment" reters to both therapeutic treatment and propin luetic or prexeutarKe meoroires Ί hose ns need id treatment tiududc those A read) n sth the disorder as n eh as those tn x\ hts.lt tite dts<>rder is to be presented. " 1 tea·mem" mchtdex anv indicia of suei ess in the ammivianon of an min··)» rmihokmy or eemdttiom including mo. ohiecttx e or suhjeem ·,.· pammetet such as abatement: reoosmoo, diminolmie, of sy tnpfoms or soaking the injure» pathology or eondihon more mn'rabk' to the patient, sloo nm- in dor rate ofdegeiienumn ot deemo,, making the final point of degeuerauon less debilitating; improsing a patient's pbxsteal or memos o ell-being. The treatment ot amehosetioa ot sx m poos os can he basket on obsevtoe ot subject!', e parameters» no. lading, the results of.;, phymmi examination, self-reporting b) a patient, nentopsxeiuatr-e exams,, and or a psxeisiuine ex al nation jOOHHi] d; i "effect o e amount'- is generally ast at noma sy fib sent to reduce ha s>.;\ ct'itx and or frequency of s> sttptoms. eb.rooooe the m, momms and or underlvine »aus»\ pre\ »mt the occuneuce of symptoms amt m- their muioHymy cause, and'or uaproxu or ietnediaic the damage mm results fro so ot m associated o uh migraine headache Ut some embodiments, she cffceiixe .annum is a therapeutically eslecttxe amount or a piophyLsrOeady effeeuxe amount Λ 'Hher.tpeuttcailx efteeuxe amount'- is ;tn ;onourn .sufficient m remedy a disease state tern transplant rejection ot fl\ HO, inflammation, midnpm selerosts, cancer, doihetes, ttemopathv, paint or symptonus), psalieaUirix a state or m,soptorn:s) associated vx nh the disease Oare. or oths'i xx is»· prex cot, hits; let, muird or sex erne the progression of the dtscast siuic or any other undesirable sxoiniom assixtumd wtrh the dt,sea,se tn any xxay o ha momcr u,o that proxhhm '-hes tpett-ie efficacy";. \ 'ptxmhx larncaih etVeefx c amount-' ss at; ataotipi of a pharmaceutical composition, that, when miaou > ism sod to a subject, xxsli have the unaided prophylactic ciTeet. c,g., preventing or delay mg theor^et (or reoet urem\) ummgreme headache or multiple re is ores or teem mg th, likelihood of the onset (or reoccurrence) of migraine headache, migraine headache (symptom^ or multiple sclerosis symptoms. The full therapeutic or prophylactic lf_a dov.s ! *t v< occur bv door strata i o' o v nose. a no nuv 0\. v it o? b after administration of a series of doses. Thus, a therapeutically or prophyluaically cllectivo amount may be administered in one or more administrations.

[00101} ‘'Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo. -«ports, or pet animals, such as dogs, horses, cats, news, rats, mire, monkeys, etc Preferably, me mammal is human.

[00102) I he term "naturally occurring" as used throughout the snccblcatidh is connection o iih biological materials such a*» noiypemides, nucleic acids, hosteellSy and (he like, refers to materials v< Inch me found in nature. |4 The term .emU-eh' or utesehangenbl> " \h“',is treed m the houckM sense and includes fully assembled antibodies, monoclonal antibodies {including: hmu n huntauvA'd e.r.iuk autibodiesg polyclonal ant'hodK's, muflispeeifn anithodios (o.g., bispeeiik antibodies), and antibody (e.g., Tab, Fab% Fi&amp;b'Js, Tv, single chain antibodies, diabodies), comprising compkntentarity detemtinlngmgkins (CITRs) of the foregoing as long as they; exhibit the desired biologicaf^ytty,. Multimersor aggreiates of intaff molecules and or fragments including chemically demao/cd antibodies, arc contemplated Antibodies of any isotype class or subclass., including IgG, IgM, IgD, IgA, and IgE, IgGI, IgG2, IgGih IgG4, IgAl and lgA2, or any allotype, are contemplated. Different iso types have different c free tor functions; for crumple, IgG 1 and lgG3 isotypes have autibodyvdepemdeni cellular cytotoxicity (ADO'.'} activity.

[1» 104] The term' antigen binding protein" i ABF) includes antibodies or antibody ffagmesita, as defined above, and rceombinapt peptides or other compounds that contain sequences denied horn CDRs having the deemed antigen-binding properties [00103| In geberalyah; antigen binding protein, e.g„ an antibody or antibody fragment, “spec?fiealh hm&amp;O' to an antigen (e g,, keyhole limpet hemuevnm fK l It? or dmitropheno! (ON!*)) when it has a significantly higher binding affinity for, nod eonseqneritjy capable of distinguishing, that antigen, compared· Ιό;:ϋ other unceLueu p,ote aw urow ^near otnduty assay conditions Typically, an „,m binding p or ·' )- , id o “-ναίΑ V h o>f n\ nn get antigen when the dissociation courtam {K:.) is · ΙίΓ' M, The antibody spcdfteahy tends antigen whh “high affinity” when the K*» is <S\ !0'v M, und with "vary high affinity" when the K»is<5x ΚΓ’Μ. In one embodiment, the antibodies will bnid to ΚΠ1 or DNP with it Ki>bfbetvveen about ΗΓ M and I0‘!'’ M and in yet another embodiment the ,an?U'od\s wilth'-nd cuhek^ - 5\ to * [:00¾¾ "Antigen binding region" or "antigen binding sac”' fries ns a portiptrofa protein, that specifically binds a specified antigen, e.g., keyhole limpet hernooyhltt ί Κϊ Ml or dirnfiopi« n,e tiV\Pt for ample, rtu* reason of an antigen binding protein that contains the ammo aeul tesidsics that intent^* on the antigen binding protein its specificity and affinity for the antigen is retorted to as' aoitgen binding region " An mmger hmdmg region typically tndndes one or hiote "complementary binding regions' Certain antigen binding regions also include one w more " Ion nets or k" regions fTRA) A ‘ COR" ss an annuo acid sequence that contribute?*to antigen Mndingnpeelficify an# affinity, 'Tfamework" regions can aid in mainbilhlng the proper conformation of the €DRs to promote binding between, the pnllgen: binding thglonnnd pi antigen, {0011171 An “isolated” antibody is one that has been identified and separated from one or more components of Its natural environrnctn or of a culture medium In which if htia been secreted by a producing cell. "Cmuuminanf components of its natural environment or medium arc material'; that would Interfere with diagnostic or therapeutic u.<s for the antibody, and may include enzymes, hormones, and other proteinaceous or tionproteinacoous solutes, in some embodiments, the antibody vt ill be pme < d i)} to gw, te* Α.η- °5'*, g wcsgln of atudn«Κ, and mast prefi'.ahU nvw than 'fry lw veLht ot iZi. to homogeneity by SDS-P HIV intact icoucingor nonrcdueing conditions, optionally using u stain, e.g... Ccoirassie blue or si Ivor Mam. isolated naturally occurring antibody inch,idea the antibody in situ within recombinant cells since at least one component of the antibody's natural environment Mdl not be present. Typically, however, isolated antibody will be prepared by at leasi one purification step.

[00! Obi 1'he terro ‘'monoclonal antibody'' as used here;a refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual aniiboilles comprising die population are Identical except for possible naturaliy occurring mutations fhai may be present tn minor amounts. Monoclonal antibodies are highly -wee;be, being directed against an sndo ulus! antigenic site ot epitope, in contrast to pdyciomd antibody preparations that typically include d;0ercr:i antibodies directed against different epitopes. Nouhrmting esinupies of monoclonal antibodies include murine, rabbit, rat, chicken, chtmct ic, htnnani/ed, ot human muibodics. fully assembled antibodies, muitispeeim: antibodies (including bispeeific antibodies), antibody fragments that can base an antigen (including, Tab. Fah\ F(abf};, Fv„ single chain antibodies, diabodies), maxibodies, nnnobodies, and recombinant peptides comprising < DRs of she foregoing as long as they exhibit the -desired hiolugluaf activity, or sarumKor derm aris es thereof.

[001091 Tbo modifier %s®Rdcl0mr* indicates: tliechameier of die antibody as being o blamed from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of tits, antibody in an> patttcuUu nietliod. For example, the monoclonal antibodies to be used in accordance oith the present IftvCtition may be made by tlic hyhidmuu method first described In Kohlei et ah, 'Nature. 256:495 [1975], or may be made b> xvcomhinanr DNA methods isee, e.g„ O.S. Patent No. 4„HI 6,567). Tile 'monoclonal antibodies” may also be isolated bom phage antibody libraries using the techniques debefibeddn Glaclisoii etui..

Nature,352 4>24-628[ 1991I ami hbn Ks et ,5 , j Mo5 Biol, 22.2 5M -5°7 i ; 651) fo; example.

[001 10) A "raiihispeeiftc'' binding agent or antigen binding protein or antibody is one that targets more than one antigen or epttope: [001 11] A 'biapoeificA "dual-specific** or "bun oedema i'* binding agem or amsg‘~u binding protein or antibody is a hybrid having two different antigen binding sites. Stand gen binding protei antigen binding proteins and antibodies are a species of ranMa|0igen binding protein,: atpigen binding protein ortpobispecilk antibody and may be produced by * vaSeiv of iTsethoda including,^bni not liP-iited to, fusion of hybridomas or Unking of Eab' ftagments; See, e,g.< SongkMiai: and Lachmann, 1990. Qin. Exp.1nnrtunoi, 79:315--321: Kostelny et al., 19¾ If tomnnol 148; 1547-1553. lire two binding sites of a bispeeMc antigen bittding protein or antibody will bind to two different epitopes, which may reside on the same or different protein targets.

[001! 21 The terra "immunoglobulin·* encompass^ full asttbodies comprising two inovrwed hea%y clams- SIC h each covalently linked to a light chain (1.0, a single undimerized immunoglobulin heavy chain and eovatemiy linked light Pham (il€ t LCh ora chimeric itriutuvioglobnlinffigbi ebnin'^trheayy ellnirO-Fe heterotrimer fa so-called “hemlbody").

[00ί H| Air "antibody'1 ra a lelraruesic gjycopnoem. In a nauualfe -uceumng ,yip^bi^yi.e^;t00»m#^::^pipo,sc<l of two identical pairs of polypeptide chains, each pair having one "light" chain of about 220 ami no acids taboos 25 kDa) arid one "heavy" chain of about 440 amino acids (about 50--70 kDa). ' f he amino-terminal portion of each chain include^ a "variable" ("VI region of about 100 to 110 or more -amino acids primarily responsible for antigen recognition. The eatbow-lenmnal portion of each chain define^ a constant regum primarily responsible for effector ftnefibn. The sable rag ton differs among different antibodies The constant, region is the same among different .embodies. Wuhm the x «triable region of each heavy or light chairs, there are three ! open at table subregion- that help deterrmne rhe antibodyys specificity for antigen. The variable domain residues between the hypiWvuri&amp;bic regions are called the framework residues and generally ere somewhat homologous among ditTerent ant shod tea. Jmmunogiohulins can be assigned to d * ..ut elates detv n 4f dcei no aed segue <.ec* nreow it doma t pi i:.-.,n heavy chains, I Iviues 'svhi ciruiis tire clavsiikd as kappa! κ ! and lam',\k, χλ) baht cnams Within nd ' , >.i Am. chums me \) aok ar.d eon o. ant icguva ate joined by a M" region of about 12 or more amino acids, with the heavy chant also including a ’YD* region of about !0 more ammo acids. See genetslly, Fundamental Immunology, Clh, 7 (Paul, W., ed., 2nd ed, Raven Press, N.Y. (19*9». Within the scope of the invention (;ab “antibody" also encompasses a reconthinanUy made j^Sbedy* *n<i:&amp;sttb^dtes that are inching glycosylation 100114} The term Dtgnt mam' or mmo'ucvgiobuiirj bght chain" «iclndes a bub length light chain and fmgmoTtta thereof has mg suifn nan \ a ruble region sequenc;..... to confer bindingsjjecificity. A full -length light chain includes a s aruHc region doman , Yd, and a constant region dom'd'.. ( The \ unable roguv {Ionian; of the oglu chain, is at the annno-temiimm of me polypeptide. Light chains include kappa s • chains and lambdspltama.· -= [00 i 15] The term “heavy chain" or “immunoglobulin hcayy^cllum” IncIttdM- a; |di!r length jaeavy chain and fragments thereof having sufficient variable region sepebee ; to confer binding specificity. A full-length heavy chain Includes a variable region domain, V\j, and threeconstant region domains Cal, Cu2, and Cp3, The Va dotnasn is at the amino-terminus of the polypeptide, and the-C» domains are br ibe carboxyl-terminus. with the < 's,3 bomg closest to dieearOocj-iermums of the polypepodo Meavy ehams are classified as mu ig>, deha (A), garnrna <yY alpha (a). and epsilon m>, and detlnc the antibody's isotype as fgYl, IgD, IgG, IgA, and fgf, respectively.

In separate embodiments of the invention, heavy chains may be of any isotype, including IgO {including IgOl, lg<>2, lgG3 and TgG4 subtypes)„ IgA (including IgA f and IgA2 subtypes), IgM and IgE. Several of these may be farther divided Into subclasses or isotypes, c.g. IgGL 1gG2« Ig03, IgG-h IgA I and lgA2, Different IgG isotype^ rag, kno dsAYiant elledo, functions ana,haled by tin1 ft reg.on), such a> antibody-dependent cellular cytotoxicity (ADCC) and eompi c men t - dope no c!;t cytotoxicits <;( DC) hi -VDCC, the 1Y region of ;m antibody binds Ιο ΓΥ on eplots ihc'tRs) on tb_ sarfauc o* immune o'fectm eel a *uen as natural killers and 'macrophages, leading to-the phagocytosis or lysis of the targeted cells. In Ci>C the antibodies kill the targeted cel ls by triggering the complement cascade at die cell surface. j'CKK Ιό] An “re region", or used interchangeably horde», "Fe domain·1'or “immunoglobulin Fe domain'5, contains two heavy chain fragments, which in a full iPkbods on msetheCjl md ( 2 donna ts ot 1», antibody Ivo wvs eh,un fragments arc held together by Iwo^of more disulfide bonds and by hydrophobic interactions of the C«3 doniaiai, |On pt ΐΛο e Pi 'salvage tccep -.; lundi n> >, ’.‘opt” \ -> so a- ep.tupeoi i .J t logtoiofar: fri» molecule tcg , Id > , IgU- leO„ o: IgG » feu o respor.s e'e to: i n teasing rite r, \ no setum h&amp;P'-hle ,o'the IgG mole, ,rie ji*0 is “ΑΓο vs i t \aoafion··* n a bom st < ..a <. it n 1κ , οη*ί» at region, that can no immunogenic and are encoded b> specific alleles m humans: Allotypes have been identified for five of the human 1GHC genes, the 1GHG1, IGilG2, KlEGi, 1GI 1 \2 and IGHE genes, and are designated as Giro, G2m. G3m, A2m, and Em allotypes, respectively. At least 18 Gra allotypesare knowit: nG lm(l), uG lro(2), Glm 11, 2, A 17: or Glm (a. \, f, /.), G2m (23) m G2tn (up G3m <5,6> 10, 11, 13, 14, 15, 1t\ 21,24, 2b, 27.28) or G5m (b 1, c3. be, bO, be, M, s> bgl, c5, u, v, g5). There are two A2m allotypes A2m( 1) and·. A2m{2)v. }001 lb] For a dcHuJed description of the structure and generation of antibodies, see Roth. D.B., and Craig, N.L., GV/ri 44:411-414 (1498). herein incorporated by reference in its entirely. Briefly, rite process for generatihg; encoding the heavy and light chain immunoglobulin sequences occurs priirsardy m developing B-colIs Prior to (he rearranging mtd joining of various immunoiriobuhn gene st-gments, tin.·. V, Π. J vine constant if1? gene segments me found genet ally in relatoco close proximity on a single eitromosumc. During B-ccH-differcntuition, one of each of the appropriate feu-ily rnetuheus oi the V, D, ,1 ipr only V and I to m>- case of hunt chain; genes) getv segments are rect.nnbiivd to form fenihomilly rearranged, variable mesons oi'riu heavy and bgln rrnrnn oglobtriin g, :vs Dus gene segment rearmngvrnem process appears 10 he sequential Kiist, homy chain Umo-j joint* arc made, lolloped b\ heavy chain V-io-Di joints and light chain Y-to-,1 joints. In addition to tin; resmmngemcrii of k , ί> and J segments, further dh ersity is generated in the primary repertoire of immunoglobulin neat \ .arid light chants by vay of % triable recombination at die locations where She V and i segments in the light chain are joined itnd where ite D and j segments of the heavy chain am joined, Such vacation in the light chain typically occurs within the last codon of the V gene Moment and the first, codon of the j segotehti 'Similar imprecision in joining occurs ; on the heety di<un chromosome between the D and ,b segmems and may extend over as many as JO nucleotides. Furthermore, several nucleotides may be .inserted between the D and .In and between dm Vp and D gene segments which ate not encoded by gnomic DNA. The addition of these mtdcoiides is known as H-regiott diversityt The net effect of such remmotgemcots in the variable region gene segments and |έ^ηΐ^ίΐ3^όβί ystbls&amp;pay occur duriitg'$dh:ft.iC^tt^ production of a primary antibody repertoire.

[00.120) The term '1t> pert enable"' region refers to the amino -s.n.t residues of an . t'bodywhd ate mvmns'hie *b' arugeu-b.nd.ng The hyrv \ a t deteg on comprises ammo neb residues from a complementarity determining region or CDR. (i.e.. residues 2-V-T4 <1 : ;·, 50-56 (1,2) and 89-97 (1.¾) in the light chain variable Jo train and el-3^ H(h, m>,65 ι H 21 - xf Ό2 (H3i m the hcew v.utn sarmb.c domain as described by Kabul a aL Seqncru.es of Proreins oHmmunologicai Interest, 5:i' fa. Fold ie Health Service, National Institutes of Health, Bethesda, Md, 1199])], Even a single CDR may recognise and bind antigen,,Though with a lower affinity than the entire antigen binding site containin'; ult of the CDRs.

[90 i 2 JI An ul lev ware de* moon T ; ess does 'mtn ,> hypers mu,mb ''bon'-s dc.wnhed b\ Chothta et id, be 5/ /Vh °0i -O mjOKb ns residues 2e 5 "* H t). 59-52 (1 2) and 91-96 tL51 tn the hght eiuhn variable domain and 2*’-32 (HI), 53-55 (B2) and 96-101 (H3) In theboevy:chain variable domain,.: [00122] "Franses odd* or '1- R" residues axe those variable region residues other than, ihehyncrv&amp;riable region residues.

[00123] "Armbou} mtgm an ^omprist ·. ronton <e uu men i ml! length antibody, preferably the antigen binding or variable region of the intact antibody. Examples of an; m.r ifagtnet > tnelade F -Λ 1 av, ΓριΕΥ, and F\ fragments; diabodw linear e.-; hoe.es t Zap.a. a ei ai. I'totem ί ;ν..Ή .0).ιϋ57-Ι0ο2 {1 α^5)1. single clt-m antibody molecules, and iauh'sneeri~e antibodies lorined tKnn antibody .memo;am [00)24] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab"' fragments, each with a single antigen-binding site, and a residua! '*Fc" fragment, which contains the constant region. The Fab fragment lOntams all of the \ unable domain, as ad! as the constant domain of the light chan* and the first constant domain (CH t) of the heavy chain. The Fc fragment displays carbohydrates and is responsible Ibr many antibody effector functions (such as : binding complement and cel Preceptors), iitafdistingttishonecrass of antibody thnoti another..

[001,?5 i Pepsin treatment yield- an Fi&amp;b'b fragment that has two "Sn'ide-chrin Fv" or st Fs" antibody fragments comprising dr YH ami V t domains of antibody, whet out these domains are present in u single polypeptide chain. Fab fragments difu m> ϋ' !> Itai .K'Us )y tb, m U emtio a ge ,vdui<> u nodes Ok. eurboxy terminus of the heavy chain (Ή I domain including one or more cysteines-! from the amt body hinge region. Preferably, die Fv polypeptide further comprises a polypeptide linker between 'the' VM 'and V L domains dun enables the l \ to form the desired structure for antigen binding. For a rex ice ofscFx sec Pitickthtm in The Pharmacology ofMonodonai Antibodies, vol. 113,

Springer-Verfag, New York, pp. 269-315(1994).

[00126] A compri sed of one light chain and the (¾] and; variable tegmnsofone heavy chum. The heavy chain of a fab molecule cannot toon a disulfide bond with another heavy chain molecule.

[00127] Λ 'Tab' tragraem" contains one- baht chain and a portion of one heavy cham uuu emnams the Vt korun" r,<l the ( , i domam and ako she tewon between the C«1 anti C ·,&amp;domains such that an interchain disulfide bond can be formed I imcee the out heavy vios <>t m<> 1 φ I ejrt*,".*- to lotto <m Fiat i t \φ\ k |0<t [ 2k] Λ "Ftab'V fragment' columns two light chains and two he-wy chains i.oniaf ting a portion of the uvi ,t:»ni ewoo K tween die (3 > l and G>2 domains, such that an aner- t atn disulfide hot d is ion 'ed between the two heavy chains A I (ab% ffagmem thus -s composed of two Fab' fragments that are held together % a disulfide bond between the two heavy chains, [00129] 'TV' is the minimum antibody fragment that contains a complete antigen ; s, (.ognmmi ,m,i binding s:u I h s\pou consists of a dimer of one heavy-and one light-chain variable domain in tight. non--epvalenf association. It is in this eo'ngur.trv» Tat the three i f >Kn of each vamble-domain interact to define an antigen binding site on the surface of the V^fVlyfiragp. A single variable domain for half of an Fv comprising only three CPRs specific for an antigen) has tlte ability to recognize and hind «mu gen, although at a. lower affinity than the enure binding site.

[00110] “Single-chain antibodies''arc F\ molecules in which the heavy and ligM chain variable regions have been connected by a IlcAtble linker to form a single polypeptide chant, which loans an antigen-binding region. Single chain anfibGi®#, are discussed In detail m hnomanonru Patent Application Publication No WO 88 01649 and t'nned States Patent No. 4,946,778 and No. 5,260,207, the disclosures oi'which are incorporated by red ere sec in their entireties, [001 hi ] '‘Single-chain Fv" or "sd-V antibody fragments comprise the Vr and Vs. domains of antibody, wherein these domains are present in a single polypeptide chum, and optionally comprising a polypeptide Sinker between the v(, and V: domains line e-t .mV tl e t % to lone s ,e dev ted sKivi ne Vr antigen bu'dtng - Bad d aL, Science 242:423--126, 1988, end Huston or &amp;l„ Prac. NutL Acml Sci. USA 85:5879-5883, 1988). An "Fd,? fragment consists of the V» and CrI domains. 100132) The term "diabodies" refers to smalt antibody fragments with two antigen -binding sites, which fragments comprise a heavy-chain variable domain (VI I) connected to a light-chain variable domain (VU in the same polypeptide chain (VH VI. j. By using a linker that is too slant to allow pairing bet ween the mo domains on the stone chain, the domains are forced to pair with the coi^piemghpE!>it'#h^uioiii of another chain and create two antigen-binding sites. folly in. for.example, FP 404,097; WO93 i Π6Ι. and Acad. fid. USA. So:6-144--0448 (1.993).

[00133] A “domain· antibody”Is an 'mmuttdiogktdly fonu tonal unrnunogfobtiHn fragment containing why the variable region of a heavy chain or the variable region of a light chain, in .some »Mhmeo|, two or more V!; regions are covalently joined with a pephde baker to create a bivalent domam antibody. The two Υ>} regions of a bo- < _vn dor as . t i a-, ί'ν si ueoi it ifoictn antigens [01*134] 1 he tenx * compete" when used in the context of antigen binding proteins (e,g.. neutraianig antigen binding proteins or neutralizing antibodies) that compete· ....•fer;-ib|;^atne· epitope tpeans competition between antigen binding proteins is determined by an assay in which the antigen binding protein (e.g.. antibody or Iptmunoiogically fintebonsj fragment ihemo0pnder test pfoyenis or inhibits specific binding of a reference antigen binding pmtctn(e.g., a ligand, or a reference antibody) fp a corhioon antigen (e.g,, KLII Or g fragment thereof or DMRh Mgoremns typi s of competitive binding assays can be used, for example; solid phase direct or indirect rads oimmti noassay (R1A), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; (see, e.g., Sfaih 0(01., :1983, Methods in. ingymology 9:242-2.53}; solid phase direct bfotin-avidin El A (see, e.g.. Kirkland et ai., 1986, ,1. Immunol. 137:3614-3019) solid phase direct labeled assay, solid phase direct labeL.1 sandwich assay (see. e.g., Harlow and Lane, 1988, Antibodies. A Laboratory Vtjunab Cold Sprmc I Labor lb ess i, so ut phase direct label Ri \ n.sing M 25 label (see. e g , Morel et al, '988. Mokx lnv.ru >oi 2e ' fob sobd phas dtrew Lvonn-: avid in EIA (see, e.g., Cheung, ct a!., 1990, Virology I7(v 540-552); and direct labeled R1A {Moldenhaue^ot al„ 10<Ή), Scone. J Immunoi 32^-82/ 1 \ picul U. .such an espy involves theuse of purrbedaotlgen bound to a sold surface or cells bearing eMSier tet^iigen blading protein and a labeled reference antigen binding protein. Competitive inhibition is measured by detennining the amount of label bound to the solid surface or cells in the presence#'· Cue test antigen binding protein. Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen : binding protein* 1 include antigen binding proteins binding to die same epitope as ihe reference antigen binding proteins and antigen binding proteins binding to a.n : adjacent epitope suffn icritly proximal to the epitope bound by the reference antigen binding protein for sr.rie hindrance m occur Addiuomd details regmomg methods for determining unrspciitive binding are pros ided in mo lusually,; when a eoropeting antigen binding protein is present in excess, it will inhibit specific 'binding ofu reference ami gen binding protein •^•«^mnSou-'anMgca by at least 40%» 43*; 1:, 50%. 55%, 6(1%, 63%, 70% or 75%. In some instance, binding is inhibited by at least K0%, 85%, 90%>955 », or 97% or more, r [00135] The term vVamigen*' refers to a molecule· or a of being bound by a sci'cciix·.' binding agent, such as an atitigetfltih^i$$$$&amp;· (including, e.g... an antibodv or ntmiunological functional fragment thereof), ami additionally capable of being used in an animal to produce .mi; bodies capable of binding to tbar antigen. An antigen may possess one or more epitopes that are capable of interacting with different umigen binding proteins, e g,. antibodies.

[001 >6] I he terms "ΌΝΡ” or "dmirrophenor am is me imerchangcably herein arid denote the antigen %-imhiutfophestoL "Aob -DNP” ot "oONP” or :'aDNF1 are Used interchangeably herein to refer to un antigen binding pmtein,. antibod) Oagment. that specsfieall) binds DNfr.

[0013?\ The terms "ΚΙ 10” or '"keyhole limpet hemocyanin"" are used interchangeable herein and denote the Imjcctitb .(^hintiiltufe &amp;eyholdy$p# henukxamn pmki 11. Pierce B oteehuoiogs. Ro. ..fold 11 ) \ceuH ny to the manufacturer, mekLR is harvested from select populations of the mollnxk

MeplteM: ercmuata. (kcyhole Umpsr) that are grown in ra&amp;sfcuUure, rather thud, feeing wild population», KAJI ba> a hvh :ηοΙνΜ'·α* mass t - ς > 0 «· t .3 \ 10 Daltons of mixed aggregates of 3x0 and 3(>0 hi>a subunit»} and ebons a .stronger immune \espouse Hun BSA ot oiaPmmm ''\51ti-KUr or "o&amp;d If’ or "a kid Γ cue used interchangeably heroin So reft-i to an antigen bmdmg protein, e.g, :m antibody or Ntifeody Iritgmem. that ^eeirically hindsKittt: |00l3fe| T hglerm ^epsop#: is the portion of a molecule that is bound fey an antigen ^itltjdiiig^noteislfdf'ex^bplc, an. antibody), Tbaten includes any determinant scapattfeofspedftc^ll.^lkding to an antigen binding protein, such as an antibody or So a T~eoli receptors An epitope can be contiguous Or noncontiguous (e.g., in a single-cham polypeptide, amino add residues that are not contiguous so one another in she polypeptide sequence but that whlnn the context ofthe molecule are feotmd by the antigen binding protein). In certain embodimeols, epitopes may be mimetic in that they comprise a three dimensional structure that ts sunder to an epitope used to generate the antigen binding protein, yet comprise none or o«iy some ofthe amino acid residues found in that: epitope used to generate the antigen binding protein.

Most odor, eptiopes >< side 00 piovms, but ai some instances may reside on oihei kinds of molecules, such as nucleic acids. Epitope determinants may include; ; sbemuedh aeo\c vu feu e g'oupmgs of molecules such as memo arid», sugcu side chains, phosphors! or snltbnyl croups, and may hasespeenh. three dimensional structural characteristics, nud^or specific charge characteristics. Generally, antibodies specific lor a particular targes antigen will preferentially recognise an epitope on she target antigen in a complex mixture of proteins and;o*' jp.act:pino.kx'iries. (001391 The term "identity" fetors to a relationship between lire: sequeneesipf two; or more polypeptide molecules or two or more nucleic acid mpfeealek as determined· I fey niigpirig and comparing the sentiences. "Percent id endty" means tise percent of identical residue··'; between the· amino acids or nucleotide in die compared molecules and is calculated based ort the sice ofthe smallest ofthe mokcuies being compared, bor these calculations, gaps sn alignments {if <m>) must I sc addressed by a pur! tenia r mathematical model or computer program <Le.. an Ahgiwhbm'h, Methods that can be used to calculate the identity of the aligned nucleic adds or polypeptides include those described in Computational Molecular Biology. (Less, A, hi., ed.h I98H. New York; Oxford IhilYershv Press; Biocorapabog informatics and Genome Protects, (Smith. D. W.. cd. P 1993. New York. Academic From; Computer Analysis of Sequence Dicta. Pari 1. (Griffin. A, M. and Griffin. H. G,. eds }. i add, Mew Jersey-. Humana Pres.',; von lidnje. G.. 1987. Sequence Analysis in Molecular Biology. Sea York: Academic Press; Sequence .Analysis Primer, (Ghbskov M. and De cere ox, 1., ods.S, 199 L New York; M Stockton Press; and Can Do ei af. 19x8. SIAM j. Applied Math. -IP: 1073. for example, sequence identity can be determined by standard methods that are commonly used lo compare the sindbrity in position of the amino acids of foo polypeptides. Usurp a computer program such as BLAST or FAST A, too polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a nrc-dciermincd portion of one or both sequences!. Tire programs provide a delimit opening penalty and a default gap penalty, and a scoring matrix such as ΡΛΜ 350 fa standard scoring matrix, see PayhotYet ah, in Arfas at Fnnoh; .Segue,· see end Sifttciww vqL 5, supp, 3 (3 978)] cap be used .Witt, tijife computer program. For example, the percent idem sty can then be calculated as; the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span mid the number of gaps introduced into the longer .sequences in order to align the two sequences, in calculating percent identify, the sequences being compared are aligned in a way that gives the largest match between the sequences [00)40] The GCG program package is a computer program that can be used to determine percent identity, which package includes GAP iDevcreux et as., 1984, Nuel. Acid Res. BAG7; Genetics Computer Group, University ol Wisconsin, Mathson. Wls, The computes' algorithm GAP is used to align the two polypeptides or two polynucleotides for which the percent sequence identivy is to he determined. The sequences are aliened tor optimal matching of their respective ammo acid or nucleotide itbe 'vmatcUK-<l:^Ba%:^..dctc}i«inctd by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the "average dtapnaP is the average of the diagonal of the comparison matrix being used; the ‘rdiagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually JfiO times the gap opening penaltyk as well as a comparison matrix such as P4A4 250 or BLOSUM 62 are used tn coniuncltou wdh the algorithm In cestam embodiments a standard comparison matrix (see, Dayhoffcl al, 197k, Aiks of Protein Sequence and Structure 5:345-)52 for the PAM 250 comparison matrix; Henikoffet ah, 1992,

Proc, Natl Acad. Set. G.S.A. 10919 for the Bl 0$t.M 62 comparison matrix) is also used by the algorithm. (001411 Recommended·parameters lor determining percent, identity for polypeptides or nucleotide sequences using, the GAP program include the following: 1.00142) Algorithm: Aeedieman eial,¥;Iffld Mol, Biol. 48:443-453; 100143) t ornpansotr matrix; BLOSi 51 62 from 1 kmikoif et at. 1992, supra, (OO144] r tig tkmi Ity: 12 (but with oo penalty for end gups) 100145' t lap 1 ei gth Penally: 4 (00146] T hrcshokl of ^tmlarity: ψ |Oil 14") Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned Teuton may have \ cry high sequence idem.n ewe though thete no samtieant relationship between the two foil-length sequences Accordingly, the selected; uhgnmesu method .(GAP program) can. be..adiirstcd. if so desired to result in an ; 1 w u' i. e; that spans at ie,w A) usnuguous me so acids of the target polypeptide jOiri-k, 1 he tern; 'modification' when used in connection with antigen funding proteins, including antibodies and antibody fragments, of theinvention, include, but are ύύί limited to, or more amino msorboas or deletions); ehcnueni modifications; covalent modi Heat ion by conjugation to tbempeutK: or diagnostic agents; labeling te,g., oath radionuclides or various enzymes); covalent polymer attachment Mich as PFGylation idurivatisrauon v- oh polyethylene glycol! and insertion or substitution by c here: cai synthesis of e οπή Mure I amino acids. Modified a no yon binding proteins ofthe invention will retain the binding properties of unmodified molecules of the invention. 100 oki] The term "derivative" when used in connection ή hit antigen bmcimg proteins (.including ami bodies and antibody fragments? of the invention refers to. antigen binding, proton is that are covalently modified by conjugal ion to therapeutic or diagnostic agents, labeling (e.g,* with radionuclides or \arious enzymes). covalent polymer attach mcsU such as PEGyiation (denvati/istion with polyethylene glycol) and insertion or suhsiitutfon by chemical synthesis of non-natural amnio acids. Derivatives of the invention will retain the binding properties of under; vail end molecules of the invention. 1 t>o: %>| iromiroagioMk^ [00151] In t nil-length immunoglobulin light and heavy chains, the v enable and constant regions arc joined by a "J" region of about twelve or more amino acids, with the heavy chain also including, a "'O'' region of about leu more ammo snds. ΛΤν, e.g.. Eundtnnenta! Immunology, 2nd ed,. Ch. ? {PomL W.. ed.) 1989, New 'stork. Raven Press i hereby incorporated by reference in its entirety lor ail purposes*. The variable regions of each debt 'heavy chain pun typically form the antigen binding site.

[00152] One example of a human igQ2 heavy chain tldC) constant domain lias the amino acid sequence:

a STKG PS Vf Pi..A PC S KSTS ESI A Λ.ΕGO. V KD Y FP FP VTVS W NSG A L7SGYI ΠΙΕ AY LQSSG L 5’ S LSS V V ΓΥ PSSN NTVQ f Y TON V DHK.PSN ΕΚ V DICEY FR KC CV E CPPCP APP V AG PSY FLFPPKPKDTLMISRTPE YTC V YY DYSR EDPFYQFNWY \ DOVKVHNAK 1'KPRtWNSTfRVVSV U VVHQDWINGKE V RCK. V S N k GL PAFIfXTlSKTKGQPRFPQYYTf PPSRKF.MTKNQVS! Π.Ί VKUFYRSDIAVnV PhNoQPK'Wt k) IPPMlOSDGSFH \ Ski Ϊ \ DkSRWQfK AYPSCSV MHFAL HHiiVtQRSLSiSPGKJi SEQ. ID NOA6.

[00! d3| Cossiani mglott sequmfees; of other IgG ;#ft· Ibr

Ending protem having u$

IgG I, IgG?, IgG3, or !gG4 Immunogloindin isotypc, if desired. In general, human lgG2 can be used for targets where effector functions are not destred, and human 'IgG! in situations where such effector functions (e.g„ antibody- dependent (APE ('11 are desired. Human IgG3 has a wlauveh short half hfe ant! human !gG4 terms antibody ‘Itulf-moleeulesT There are tour known allotypes of human IgG I, The preferred allotype is referred to as “hlgGl z'\ also known as the 'KEElVT'allotype. Human IgG 1 allotypes GdgG Da'(kDiLIA'hlgGir (REEMf, and VKgGIfiCaor also useful; all appear to have ADCC elKetdrftthettotr [00 i 54] Human h.IgG 1¾ heavy chain JpBCj constant domain has the athmb «did : sequence;

AsTKClPSX m \HSsksrSGGi vAIGt 1 YKDVEPFPVT\S\\\‘SGA! TSGYHT i PAVLQbdCd 5 ns >S\ \ 1 VFSSNU , i ό 1 YK'NYSHKPSN I kVDKKA fc'PkSt D KTHTCPPCPAPi f ? GGiSYFl 5 ΓΡΚΡΚΟΠ MlSRFPEYTCA \ \ n\ SHI DPI'Yk t N\\ YVIHA f \ H\ \k 1 kPRl KD \S Γ\ RVVSY LIU ΗΟΓηΛ { \{ kf > kt kv : SNK.ALPAPIEKT ISKAKGQPRFPQ\ YTLPPSREEMTK NQVSI.TC f. Y COPY PhD iAVEWESNGGPt:.NNlYKTTPPYinSDGSFELYSKLTYDKSR\YGQGYYl:SCSV MHEALFlNirvTOKSI.Sf.SPGK " SEQ ID NO:R7 [00155] Human hlgG !/;j item> chain {HO constant domain has the amino acid .sequence:

YSTKGPSVFPi MNSKSISGGI WFGCI YKDYFPt PV1YSANSGA1 ΓμϊΥΗΤ FPAVLQSSGlA- SLSSVVTWSSSLGTQTYIC YVNHKPS.NTKV DKK\ LPkSCD KTHTC’PPCP Μ>Π i GGPsYFl FPPkPkDTI MInRTPFX Η \ \ Y !)V s|H Dpi VK

FNWVVDfiV E V Η NΛΚ.ΤΚΡR EEQ Y NSIΎRVVSVi ΓV JLHQOWLNGK EVKCKV SNK ALP A PI E KTISK AKGQ PR f:TQ YY'I LPPSR DE LTK.NQVSI .TCL V'K GFYPSD I A YEW i:.SN< iQV ΕΝ N Y KT i FP V t DSDGSFF L V SIC C f V DICSRWQQGN V r SCS V M REAL HNI1YTQICSLSLSPGK.·' SEQ \D NO**. (00!561 I turskin hlgGlf heavy chain {!ICj constant¢¢:*βώιφ-actcl .sequence:

AST KG PS VT P LA PSS.K SI SGGTA ALGOL. V K.DY FFEPx TYSWNSG ALTSGV’H T FΡΛYLOSSG1 YSLSSVYTYPSSSi.01'Q ΓΥK N VNjΓΚPSNI KVDICKYFPKSi D KT H re PPC'PAPEL LGGPS Y FLFPPKP KU Γ L Μ ! SRTPE v Γ (Υ v V OYSH PDF BY K FKW Y VDGVI:· VHNAKTK.PRLEQ YNSIYR VVSV1. TVLHQDWLNGKEYKCKV SNKALΡΛPJEKTlSKAKGOPREPQYY iLPPSREEWI KNQVSi'I'CL VKGF'YPSD LAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKITVDKSRWQQGNVFSCSY MliE'NLHNHYTQKSLSl SPGK/· SEQ ID NONA

[00 ί §7] Human: higG i l -a heavy elmin ( 11C) eeuslaur deniaiu has ιόν ammo acid sequence:

AsTKCfPSYFPLAPSsKsT$GGTAAi..GCLYKDY'FPEPVTV'SWN$QAl..TSGYHT l: P A V LQSSG LYSLSSVVTVPSSSLGTQT YION V Mi 1KPSNTICVDK R VEPKSCD ΚΊ ΉΤΟΡΡΟΡΛ PE t LOOPS V E L F PPK PK D ’EM tSR ί PEG IT'v Y v DY$ HEDP E V K f'NYv'Y VPCVE"VI IN A K.TKPRE EQYN Sl'Y R V VS V ί. ! \ I HQDW ί NG ICE YK C'KY SNK.A LPAPIEKTiSKAKGQPK EPQ V Y'ΓLPPSRDEL IICNQYSL ΓCLVkGF Y PSD! AVEWF.SNijQPENNYKTTPPYLDSDCjSFH.Y'SKLTVDKSRU'QQGNVFSCSVM I\ΕΛIJIN ΠYTQKSLSi SPCjK/' SEQ ID NO:%. (00! 5R j One example of a human immunoglobulin ught eham ; LC) constant region sequence R the ihHouing {designated ~Y 1..-1 '') GQPK.ANPTVTL F FPSSEELQAKKATl VC !. ISDF YPGA VTVAWK ADGSPVKA G ΥΈΤΤ 1C P S K Q S N N 1C Y Λ Λ SSYI SI. TFEQWKSHRSY SCQVTHEGSTV EKTV AP TEGS /SEO ID NO:9l.

[OP! Cl Hs tmefu· to increase tic pi of embodies end is cmoenlent. there are three other human immunoglobulin light chain constant regions, designated "CL-2"', ''Cl.-.?'' and "CL-7'; which can also be used within the scope of the presold invenhon CL-2 and CL~3 arc more c ommon m the human population.

[001(101 £1,-2 human light chain (LC) cohstaht domain has the amino acid •sequence. UUPkA \PS\ U KPPsM i'i «X)V\KAH VO ISO! \ PtlVVI V AWKADSSPVEACi· \Τ.Τΐ ΓPSKQ$NNKYAASSYi.oi. 1'PF0YVKAHRSYSCQVTHEGST VEKTYAPTE C S. SBQ t!> N0e>2.

[00 UX] CL~3 huradp ECleoostant domain has the smipb acid sequencer

VEXTrPRkOSNNKYAASSYi Si TPF.QWKSHKSYSCQWHECJSTVEKTVAPTE

OS ' SFO ID NOSKI (10162| 01..-? human f..C constant domainia^ the amino acid sequence: (X)PfCAAP$V'n.JF!>PSSEELQANKA>n...VCl.VSDFYPGAVTVAWKADCfSI>VK.V G\ \ Ί 5 RPSkQSNK'kY AASS\ I Si I PFQW kSHRSYSC RV HlKiS'l VI K ΓΥYP \1« s 'St A7 it) NO 04 [00163 j \ anabV reg'ous ,>f nvrnmoglobuOn drains generally exhibit the same os er dl on.; cm re, compt'kine, cclativclj censors cd framework regions (PR) joined by three hs pcrvanable regions, more often called ‘votnpiememerity detertnining regions'' or C'DP.s. The CDRs from the two chains of each heavy cbam^il^iEcto*i|·: pair mentioned above typically are aligned by the imniework regions to lpn® a .-anicJurc tiiat bmds specifically with a specific epitope or domain on the target e.g.» RLE or DN’Ft From N-uerminai to C-tcntnnah nutumliy-oconrnng light and heavy v .am ' as,Τα. '< s both \ meads eon.one w.sn the fol owing ,·' dm' of these elements: FR1. CUP I, PR. CORA FR3. CD Lin and FRA A numbering system has Ivcn de\- wed t'or assigning numbers to amino aeuis that occupy sxwioons in each of these domains. I his numbering system is defined lo Kabat Sequences of Proteins of sud 1991* Nil h BethesdtnMD), or (fitothla<%

Lmk, M?$X ΜϋΙ Biol 196:901 -917; Chothia-eiaL 1989, Nature-$&amp;&amp;*7g«, [00164| Specific examples of some of the fall length Sight and heavy chains of die antibodies that are pan ided and their corresponding ammo sc id sequences am l A and Table IBbeldWc Table i A shows exemplary light chain sequences* all of which have a common constant .region lambda constant regain 1 ii'i ~L Nl.(> ID NO :91) for a!! lambda light chains, fable i R shows exemplar hca\> eham sequences, ail of which include constant region human lgO:2 (SBQ If> MC);Sfi}.. However, encompassed within the present mveniion are tounttnoglobulutp with sequence changes in the constant or framework regions of those listed in 'fable IA and or Table IB (e g IgG4 vs lgC.12. Cl 2 vs CL 1). Also, the signal peptide ISPl sequences for ah of the sequence m luWe I % and fable IB arc the same, he.. the VK.-1 SP signal peptide; MDMRVPAQIJ..GLI.U,.W[.RGARC t'Sf.Q ID NO: jOlb smgle underlined) that is used in the high throughput cloning process, hut any other suitable signal peptide sequence may be employed within the scope of the invention. Another example of a useful signal peptide sequence is VHP 1 SP MEWS W v F i Fi- tSVTTGV H S iSh’Q ID NONM. Other exemplary signal peptide sequences are shown m Table IA-B.

Table 1A bunnmogiohulin Ligln Chuin Sequences. Signal peptide sequences are indlvuicU by a double underline, COR segiens am wdieanxl by single unde*line, and IVamcuorf and constant m· net underlined

Tabic IB. fnimuno&amp;lobuiin f Icuvv Chain Nequeuce>. Signal peptide sentences »i« indicated by a d%*ib$c underline, CDR region* ere indicated by single underline, and Camework and eciKraut rcyn.'sv, are not underlined.

oOOJ 65 ] Some embodiment'' of the isolated- atvn-DM P «nttgen binding protein ; comprising ioi aptlbody ormiiixxiv fragment; compfke: juO.wq (am n umimogCohu.m h,.,^ > ^ ,u eo ' ms ug*he monoand sequence of SEQ ID NO: "7. SEQ ID NO: iO". SEQ ID NO: Mi. SEQ ID NOD S3 SEQ ID NO'! 1$, SEQ ID NO:11?, SEQ ID \'0:Mo, SEQ ID NO; 123. SEQ ID 00:129, SEQ CD NO, I- E SC 0 CD NOD-D Ό Q fD NO ,h SoQ ID NO. 132, SI Q ID NO I Nb SEQ ID NO 134, ot Sf Q ID NO > 3ς m , o.eervng any one o' die tbregoing .sequence,s from which one, iwo, three. Sour or five amino acid residues are; lacking from, the N-terminal. or C-iernvinaL or both: [00107J vmm unogldbulin light chain comprising the anti no acid sequence of

Si Q CD NO'105. SEQ ID NO'HR M Q !>' NO 12·, SEQ ID NO 12-¾. ot NhQ ID Μ* Ι2Έ o? conipmsne anvoae o'«to t*n <.-,*>« _ sc· »c'ces t»or« wh e' one tsu*, •three, four or five amino acid residues are lacking from the NMerrmnai or (Nicrrmnal, or both: or [00168] (c) the immnmigkibulin hem, v chain of tm and the nmouuoglohuim light chain of (1>>.

[00169] Some embodiment?, of the isolated Ι-ϊ ίώϊΐΐ^ίϊ: |3>isi#issp protein comprising m antibody-.or antibody frggmenb comprise: }00' "*'<] ias a a rmmmogiobtuiu hem y chain cot'.poking rise usmno and sequence ofSH) ID NO Am SEQ ID NO: i '3. SEQ ID NOOSfo SHQ ID NO: 143. SEQ ID NO in6 or St Q t Π NO ! >T, J>t Q H > NO 3m ot M 0 ID NO 3b7* m cotopnv m ιν λ e ot f i ο e^or *g xequcrxex ι -η^ΐι* 'κνο w\t 1 c Vi οι l ve in no ado residue'- we 'making from tbe 'vvnriinO or C-tennmO, m kwh; [00 ΓΊ ) t b> an immunoglobulin Iight chain comprising the ammo <wut sequence of SFQ ID NO:28s SEQ ID NO· 131, SEQ ID NO: 135, SEQ ID NOQ07; or M® ID NO'i4Q or comprising anyoneM the foregoing sequences from which one, two, three, four or five amino acid residues are lacking, from the N~formmal or CMenntnai, or both; or [00172] {e) the immunoglobulin liee% γ chain of pri and the immunoglobulin light chuin off b), [00)73) Again, one « m he cxerip <w'> ant I * vJ he oy chains till, H2, H\ . cm 11 ved n l ah _ Bulk uoieh nod w t a . of I e exenipuu ut -DNP right chains shown in 1 able E\ to form an .mtibodv Examples ol such combinations· include Hi comb men w nh any oft I ibtos.yh t 5; 112 combm-xt whh any of 1.1 through 10; H3 combined with any ot LI through Li.1M combined with any of L 1 through 1.5, and so on in some instances, ihc antibodies include a* foist one anh-DNP heavy chain and one utiti-DNP hah; risaie Pom those Usted in fob ίο 1Λ ami 1B. Id some mst.etccs, the ami bo die-, comprise two different anti-ON P heavy chains and two dOferen: anu-DNP light shams foted in 'foble 1Λ .md fable IB. In other instances, the antibodies contain two identical light chains and two identical heavy chums. As an example, an antibody or mnounoioutealK functional fragment may include two El 1 hem s chains and two LI light chains, or two H2 heavy chums and tv ο I f belli cbu'us, et *wo ID neuxx .1 cum and ' light chains eta. other simiUtt v.ombimu;yns of pair- of unto DMJ light chums and pairs of anh-DNF heavy chums as listed in Table 1A and Table IB.

[00174] Again, each of the exemplary cutfi*KLH heavy chains (Hu B2, H3____ on ) listed ή Table tv" Iv vOmhuvd \vtinan\ M -'Ue e\cmp\o} TsS'~ki 1! bglu chains shown in fable 1Λ to form an .uvtibody. l· samples of such combinations include HI combined with any of LJ through 1..5; H2 combined with any of (.. I through 1,5/ FlTcoMbined -with any df fil through L5, H4 combined with any of U and so on. In some instances, the antibodies include at least one anti-•5. piHt heavy chain and.-is«e::^i^LiH light chain trom those ilsfed in Table lAand IB. In. some instances, the antibodto eonvprisc two dlflbrintantLRLB heavy chains and two di{Totem atnARI.il light chains lifted in Table IA and Table 1B. In other instances. the antibodies contain two identical light chains «no two identical heavy chains. As an example, an antibody or immunologically functional fragment may include two Η1 heavy chains and two LI light chains, m two H2 hemy chams and two 1,2 light chains, or two FIS heavy chains and two L3 light chains and other StMlar eombinaiions of pairs ofanfkKMt light chains add paifs of anli-KLI l heavy chains as listed in Table 1A and Table IB.

[0():1751 Other antigen Bidding proteins (hat are provided are variants of antibodies formed by eombtnationofthe heavy and baht chains shown in fa him 1A ami fable IB and comprise light and'or heavy chains that each have at loam 70%, at least 75%, at least 80%, at (east 85%, at least 90%, at (east 9555. at least 97% or at lea-b 99% idenmy to the amino acid scuueni.cs of these chains in wwoo instances, such antibodies include at least one heavy chain and one light chain, whereas in other s Vances i o ' <e et". fomw l.. s, ,< 55 two 'denaca.' 1 de ehusas and two me\ sed he..\ s chains. It is within the scope of the invention that the heavy chain*»} and/or light C'hain(s) ma> have one, two, three, four or five ammo acid residues lacking from the N-terminai os C-temhnal, or and., it. relation to any one of the heavy and light chain.·» set tor*Is St faMes A and T. Me ' I >, e.g , due *0 pou-avmM'i on -J mod tfk ebons For example, CHO cells typical!} cleave off a C-tenninal Ksnv [00; '?] \ he v a onus hc.ivs chain ard light eh;; n \ enable regions provided heroin am dep'tc , ο h, I'abV 2 \4> F, , r os these varia sic vy mw mnv he a'd ached so the above heavy end high- chain constant regions to iomt a complete antibody heavy and light ehasn, respeesivdv. ) arisen ete.t of the so generated h av;-> ,uo baht eham weuenocs may tv combined to toon a eontplete antibody s true too h -dunhd be under\tood tints she heavy eluin and isght chain variable regions:pr0\dd^''herem:'<^»t viIao he attached to othet contend domains having dilTcrefit-S^aengesjfc^ith^i exemplary sequences listed above.

[001 ?Hj Also prov ided are antigen binding proteins, ineid#t^:8a€tedi.#s or am: body fragment*, scat contain or include at least one inmvnnoglobultn anri-DNP heavy cham variable tegion selected from VHt, \ h~, Vtp. VvA Ynd, and \'Φ mid or at least one immunoglobulin unti-DNP light chain variable region selected from V\!« Vi 2, V; 3, Vfr4< and Vt 5, us shown in Table 2A below, and tinrnonologieully functional and vartanfeipf itese fight chain and heavy chain variable begionx, [sVsV ~,>j ABo psov awl ate antigen modatg wmems, tududutg antibodies ot antibody fragments, that contain or include at lea-stone immunoglobulin ami-KLH heavy chain variable region selected from Ve~, Vn.8. and Vfr9 ursa/or at lews one s omunogloeium amt kri- hd * eh n vanae’e me on schetee from \ f ο, V 1 vs be and Vt9, as shown in Table 2B below, and tnununologlcalty functional hatgrneffrs,:. ieitxativca, s'mtetnA aci v,e annuo ueso ic't dixie ui,< heavy,, w* surahc regions, [001801 Antigen bindiry pro'cine 'vf this rv pe c,m genenhy Iv doveratocl bv the :b„o i i \'(J\ V >T vi tcK \ cu mspowsto the nt ml u o* no tv\oca, t vmutbk regions me Tided in the annecn bine mg pro" tin and 'V* corresponds to the no other ot the light chain variable regions included in the antigen binding protein (in general, >, and are each: i or :2),.:

Table 2A. Exemplary anrj-DNP Vn and V* Chains- COR regions are indicated by underline. and. framework regions are not underlined. Opdonal N-icnrdnsi signal sequences are not shown (See, Table I A-8).

Tabic 2B. Exemplary y»fi-KLH V'u and V- Chains; t DR regions arc indicated by underline, and framework regions are no! underlined. Opbomd NBcrnoual signal .sequences ace not shoson {Sec, Table 1 vB; sec, c g.. $E0 ID NO-'>5 «rod Sb’Q ID NO :103).

[0018.1 1 Some mo boon noma of the isolated antigen binding ptnvm mai comprise'" a-\ anU-DNP antibody or .ant*bods fragment. comprKinb an iimuuuoglohulio heavy chain variable region and an immunoglobulin light chain variable region: f001B2f (al the heavy chain variable region comprises an ammo acid sequence at least 9o%, %% 97%, 98% 99% or 100% identical to the sequence of SEQ ID NO:;:eOf S£Q ID >10:252. SfcQ ID NO-.254. SEQ ID NCV.256. ShQ ID NO:258. or SEQ (D NO :2001^ 1.00183] 0.>; the light chain variable region comprises an ammo acid sequence at least 9f«· . v~%, 0v>,. 00«, 0J %()% identical to the sequence of SEQ ID NO Γ *.\ M:Q ID NO 234 ShQ ΙΟ V > 23o ol 0 ID NO 25% oi Si 0 ID NO 240. or [PO t 84 1 {^ t t ie heav s chair % unable i c g'on of s eed tne light chain vanable region of (b).

[00185] Some · embodiments of the isolated antigen binding protein that comprises an ann-ΠΝΡ unlibodv or antibody fragment, comprising an immunoglobulin heavy chain variable region and an immunoglobulin tight chain v aoable region: j00'.S<»$ oil the-teat \ chain \,u; tela region comps -os an ammo acsd sequence at :east 45«o, On1',, v7% 9s-',, 991% or i u0% identical to the sequence of SEQ ID NO :202. Μ U ID NO 2nd, or SbQ ID NO 20% or [001871 (hj, the light chain variable reujon comprises an amino acid sequence at; least 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SfcO ID NO.-242,. SEQ ID NO 244. SEQ ID NO:24% or %Q ID NQ;248; m [00188] let the heavy chain variable region of in) and the light chain \ finable regtbiiof tbt, [00189 f Kwh of the heavy chain \ .triable icgions listed in fable 2Λ, w hethet or not it is included in a larger, heavy chain, may be combined with any of the light chain variable regions show run lable 2A to form an antigen binding protein Examples of such co.mbimn.Kms mclude Vyl combined with any of\\ k V, 2, Vjo, \ ϊ 4, or Vj 5, \p(2 combined with any of V\ 1, Vj 2. V,· 2, V'i.4. of V$ 5: vn3 combined With any bf V:ii;IfVc2? Vpb Vi4, or Vi.5; Vj{4 combined with any of Vs 1., VAT Vi 3S Vs 4, or Vi.5, and so on. | ()01c>01 bach of tire heav v chain variable turnons listed ui X able 21V n bother of not it is included in a larger heavy ehain$ maVfee combined with any of the light chain x arivibk tegimw show n tri Table 21¾ to fomt an antigen binding protein Examples of such combinations include VH? combined with any oi V< t\ V5 7. Vs 8 or V}9; Vs A combined with any of V\.6, Vi.,.7, V*.8 or Vj9; V»H combined with any ofYrtx V· ?*

Vs 8 m Vs o„ V>pt combined with any of V{ ft, V5 7. V, 8 of \ A

[00 ί 9)1 In sonic instances, the antigen, binding pro tent includes at least one heavy chain variable region an.d'or one light chain variable regionbysrh those listed in fab.e 2 In -xiry .no.ances. the am Seen binding proton tool ides at kast two h fierce· he,xx cham variable regto w a no or hght chum \,u fme muons bom konv lifted n Table 2A, An example of -> tek an antigen bmdmj pum tn comprises su one Vnk and ih) one of Vk-T. Vi}3. or YA4, etc. Another example comprises (a) one Volk and (b) one of Yt J, V yd, οί V }j h ok Agar, another example ,.ot opt tsu* id) ore \ y.k and (b) one at Vpl, Vp.2, ot V mb etc. Again another example comprises la) one ViC-b and (b) one of Vy k V„7> or VAd, etc. Again another example comprises tal one Vti5, and (b) one ol Vy1, Vy2,or VnA etc. Again another example comprises (a) Phe V„ft, and I B one of Vyl, V:y3, or VyA, etc. (000)21 Again another example of suck an antigen binding pruvin caniprKm in) one Vi k and (b) one ο*'V* 2 <» \lb etc A< dm another example '4’such an antigen binding protein comprises la.) one Y; 2, and (hi one of Y{ I or v,· 3, etc. Again another example oi such an antigen binding protein comprises la) one Y; V tnui (b) one of VLi or Vi.2, etc., and. so oh.

[00! 93] T he various combi nations of heavy chain variable regions set forth ίο Table 2 A may he combined with any of the various combinations of light chain variable regions set forth in Table 2A.

[00 I94] bt other tEstahoes. the antigen Binding protein contains two identical light chain variable regions and/or two identical heavy chain variable regions. As an. example, the antigen binding protein may be an antibody or immunologicaliy functional fragment that includes two light chain variable regions and two heavy chain carmine icgions in combinations of paim of light chain variable regions and pairs of heavy ehhin ya riable regions as listed in Table 2 A.

[00195] In some instances, the antigen binding protein includes at least one heavy chan \ural\e tegiott amhor one light chain -variable region· thorn those bsted m Table IB. In some instances, she antigen binding protein includes at least two vdifferent heavy ehaiB variable regions and/or light chain; vaflsbie mgions from those listed In Table 2S. An example of such an comprises |a) One.

Vn?. and {ΒΊ one of VTA VT8. or V;s9. Another example comprises (a) one and ih) one o* \ . -, VnK m V«,o \gam anothc'' ev»tnpk composes (a) *me Vp9, and tb) one of \ [ A V»*, or \ jdA

[09 j 96] Again another esample w men cm antigen binding pom-in comprises uu one Vs (\ and (To one ο1 V- n, \ - ~ V y \', o Ag.m; uuwlk i esvmpA of «och «η antigen binding pro tern comprises < f»one \ < / and * b' one m t ο, V > A V's or \ , 9. Again another example of such an antigen binding protein comprises fa) one V; A and ib) one o' Vf m \ A \ a ο* \ Again another ev-.rree o*\ndi an .an men binding ponem comprises ία) one \\λ and {b»one of V\ o, V< A V*HorV <f [00 S 92] The; various combinations of heavy chain variable regions act forth in l able 2B may be „ombmed volt am of the \ vinous eueto.nat.ors of Smiv en ms variable regions set fonh in Table IB.

[OOlOhl m oths,r instances he antigen b ne.ng orotc a coma >s iso identical light -chain variable regions sgid/or -'woidentical heavy chain variable regions. A.s an ;c$smplc, the antigen binding protein may he an antibody or immunological!;* it:netiu\a tmjmeut i uh n jJ, -¾ mo hgh. . be in \ .waive toguvs and *v> o hew.) chain variahk regions m combinations of pairs of light chain vtuiable regkms and: paid of heavy chain variable region*; ns listed in Tabic 2B.

[00W{ Some antigen binding proteins that arc pros ided comprise a hcavv chain variable domain comprising a .sequence of amino acids that differs from the sequence of a heavy churn \ arable Jwnam selected fum V >1. Vis?. \ 5 Yh-L V C\ \ j m \ uY \ · 8. and at enh f 2. 3. 4, 6. 7, 8. Y 10, U, 12, U. 14 or 1 > :nvad aeu: ro.v.dt ex wherem each -ocii sequence dtffc.e^ee ts sndepcuJmtfs either e deletion, insertion or .substitution of one amino acid, with the deletions, insertions and 'or substitutions* resulting in no more than 15 amino acid changes relative to the oxeunsig" enable don m sequences 7 bo V.j\\ <. I ,w van hie regam ' v some arm gen binding piotenv» . ompriscs a sequence of ammo acsls that has at least ?0%. at .o -.at $V at lea«t so1 >. .« least *5Ύ, at le is. °*0%, at least m5V at leas* (>?rt» or ot least 99% sequence identity to the ammo acid sequences of the heavy chain variable region of Yu I., Y„2> V„3, V„4, ¥,,5. V„ft, ¥,,7, VHtherVa9. | Odfolq C estain antigen binding proteins comprise a huh? chain v unable domain composing a sequence of ammo adds that differs bom die sequence of a light eham variable domain '-elected tmm V I. Vt 2, V* 3, Vrl, Y\ 5, Yj6. Vj ?, W 8. and Vj9 at om\ 1,2. Λ v o, ", s, 0, = i> 11,12. Id. 14 os 15 amino mud residues, wherein each such sequence dtflcrenee ts independently either u deletion, insertion or subMittnion ol om wmuo aeul. with she deletions, msenmns and ot sub*iimtkms resulting in no more than 15 amino acid changes relative to the foregoing variable dornhiti fbqheneqs,. The light chain, variable region in some antigen binding protems comprihcl a. sequence of amino acids that has 0t4basi:1Q%, ^ least x0fl <). at toast·· &amp;>%, at. Iciv-t' >mV , ^ least 97% or at least sequence identity to the amino aetd sequence* of die light chain variable region off 1 h V- 2, Vt.6, V·.?, V; 8. or V,f>. 10020J i Sail other antigen binding protein*.. e.gm antibodies or immtmologiealiy functional trag{rtem% !ncIotfe variant forms of a variant heavy cMsv and a variant light chant ;e< described herein. ]0Ml H>Rs [OU203i I no antigen binding protems disclosed herein ate polypeptides* mto sxlneh one or more CDRs am grafled, inserted and/or joined. can ha\e L 2, 3, 4. 5 orb CPRs. An antigen binding proietn thus can have, tor example, one heas \ chain C DR I ΓΤΡ8 ΗI "I, and or one hcasy chain €'DR2 r'CDRnr'l and or one beasy citato {'DR 3 ("C DR lib's, and'or t»w light chant CDRI rVORU"). and/or one light chain Cl>R2 il'CDRL2"A and or one light chain CDR 3 \"( DRL 3Ά Some an tiger» btndmg protein1» include both a i'DRI 13 and a CDRL3. Specific heavy and light chain CDRs arc sdehbied ih 4'able lA-B (gn?:R ObiP) and Table 3C-D (anti-ICl Bl, rc^octiveiy, f00204) C omplemontariiy determining regions CCDRs) andlmmcworl regions •(t \i i u* - g \cn annhods mas be·tdcnbfsea usmgthe wstem described by Rabat et at. in Sequences of Proteins oflrnmnnologteal Interest, 5th Ed., US Dept, of Health and \ human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Certain antibodies that are disclosed herein comprise one or wore amino acid sequences that are identical or have iniiadanhai sequence idesttity to the amino acid seqttencesdfoiie or more of the CPRs presented in. Table 3Λ (and-lXNF -CDRHsk I able 3B brati -DNP CPKt \), Table 30 ianti-KEH CDRHsT and Tabic 3D (anti-KLH C DRls).

Table34; ECBRHSequences

TM>k· 3R; Exemplary ArkM'iN'F CORL ikquviicos

T&amp;t)k 3C: ExetapJMy A&amp;ti-KLf! CDRH S«jai‘U£e$ ·

Tabic 30; Exemplary Anti-KLH COliL Seipsences

[00305] The structure and properties of CDRs within a naturally occurring antibody have been described, supra. Brieiho in a traditional antibody, the CDRs are embedded within a framework m the heavy and lie hi chairs vanish k- region where they couMitute the regions responsible for antigen binding and recognition. A 'vuriabSe region comprises at least thruu heavy or light chain CDRs, .sec, *upro (Rabat 'et αί, 1991, Sequences of-Pmteim qflmmunobgicaiintcmsh Public Health Service N.I H , Bcthesda, MD; nx also Chothia and Lesk. iRb?, J 1/o/. Biui. 196.901-917; C'hothia ct ai.. 1989, Suture 342; 8??-hb3h within a framework region (designated framework regions 1-4. FR.u F.R2, FR3, and H<4, by Rabat ef ai, 1991, supra: sac also Ohotltia and tesk, ivH'k supra). The CDRs provided berem, however, may not only be used to define the antigen binding doinain of a iradibonal; anbbdcly stoicmrey but may be embedded hi m variety of other polypeptide Structured,; as described, herein, [00206] Some embodiments of the isolated antigen biftiiiog protem comprise an ann-DNP antibody or antibody fragment, compnvhn? am irmnsmogiobsshr· hea\y chain \ariahic region andttechain heavy chain variable region comprisothisse· i&amp;sg^tcmcnrarity determining regions designated CORHK ODRH2 and G3RB3, and/or the light eham variable rngtotr comprises three CDRs designated ODRLI, CiBRt.,2 ^

{00207i (a) CBRHI has the atoino acid ΝοφοοΕοofSEQ ID NpBSScSEQIp NO: I HO, SEQ ID NO: )90, oh SEQ ID NQ:I fijMdM

[0020S! thi CDRH2 has the amino acid sequence of SEQ ID NO:t92, SEQ ID NO: 193.. SEQ ID NO: 194, or SEQ ID NO: 195; and/or [10209] tes CDRHl has the amino acid sequence of SBQ iD MOd 9K SEQ ID NO; 19", SFQ ID NO;I9)C SEQ IDBOrl», SEQ ID NO»e#i£Q ID NO;20l; and/or [00210] i d) CORE I has the amino acid sequence of SEQ i D NO:202, SEQ ID NO:203. SEQ IB NO:2(^0 or SBQ ID N0:20S; and/or [00211 j te) CDRJL2 has the amino acid sequence of SEQ ID hi0:206·or SEQ ID 240.207; and or [OQ2 3] rV'PRI t :as the amino acid sequence of SFQ ID NO20N SEQ ID NO:209, SEQ ID NO;2IO, SEQ ID NO;21 E or SEQ ID NON 12.

[00213 ] in other aspects, the CDRs prm ided are {A} a CDRH selected from 0) a C. DRH i .setecied from SEQ ID NO; i 8Q SEQ ID NO:DO, SEQ ID NO:190, and SEQ ID NO DM: tilv § CDRH2 elected from SEQ K> NO:!92. SEQ ID NO:103, SEQ ID NO' 194. and SBQ tD NO: 19.5: (iii) a CDRH3 elected Com SEQ ID NOH-h·, Μ O ID NONA'S SI O ID NO 198, SEQ tD NO SEQ ID N'ONOO, and v Q ID NO \J , di'H, { likll opd* it O- id < s r tιν -,unt<j ^ otic m tv-' ev’tie v c J \s vcj «tjon.v dei_jo ot msetners n, r«> mo,a fan I ^e, to, , Uu-e two, or one ammo aeid.N t Bj a CORE selected Com ti) a CDRf. 1 selected from SEQ tD No ?<C. M Q ID NO 203, B pm NO'.Etm, and SEQ H) NO 395, sum COIN 2 .'.elected frum SEQ ID NO;'2D6 and Si Q ID NO:307; (iii) a CDRI 3 selected Rom

Shi.) 10 NOTOS. SEQ ID N0:20>. SEQ H> ΝΟΤΙΟ. SEQ it) NOT) E and Shi) ID NO'2! 2; ;md (·γ) a C DRi of {is, Oi > and (hi) shui contains one or more amino acid sid.vsbroiiotm, deki ton,\ or irtsCiiiort,' of no more than hr e„ four, three, two, os'on·:/ arnino at kN amino acuN ( 00214] Some embodiments of the isolated antigen binding protein comprise an anti-KLH antibody or antibody iragmeut, comprising an mmmnoglohutht heavy chain variable region and an immunoglobulin light chain variable region. The heavy chain variable region comprise three eotopientetuarity dmermmimg regions designated CDRHI, CDHH2 and CORHS. and/or the light chain variable region comprises three CQRs designated OPR!. I CDRL2 and CIT.iRL.E wherein·. (002151 tat CDRHI inns the amino arid sequence td'SBQ ID NOC13, SEQ ID NOTK or SBC) ID N0:2I5; and or (00210( sbt<'DRH2 has the ammo acid sequence of SEQ ID NOT lns SEQ If) NOT 17. or SBC) ID NO:218; and-or [00217] (c} CDRH3 has the arm no add sequence of SEQ ID NO:210, SBQ ID NOT20, or SF.Q ID NO.22 L and/re [00218] id) CDRLI has the amt no acid sequence of SEQ ID 'NO:204, SEQ ID NO:222. SEQ ID NOT23. or SEQ ID NOT24; and-Or [00219] (e) ODEIT has Che amino add repotted of SEQ ID /NGTOh, S1Q ID NQ:225, or SEQ ID NO-226; and/or [00220] (f) CDRL3 toe the amino add seqnenee of SEQ IP NO'227; SEC) ID NOT28, SEQ IDNOT29, or SEQ ID NO:230.

[00221] in other aspects, the CDRs pro-aided are (Λ} a CDRR selected from (tl a

f DRH.1 selected from SEQ ID NOT! 3, SEQ ID NOT 14, and SEQ ID NQTI5; (it) a CDRM2 selected from SEQ ID NOT 16, SEQ ID NOT!?, and SEQ ID NOT IS: (ili) a CDRH3 selected front SEQ ID ΝΟΤΙΟ, SEQ ID NOT20, und SEQ ID 19EE221;aad Cib) iafeohDms one or more ammohchl substitutions, deletions or insertions of no more than live. four, three, two, hr oho amino adds: {B: a CDR.L selected from Cb a CURL i selected from SEQ ID N0:20% SEQ ID \*t t 222, SFQ ID V ?,Γ25 and SFQ ID \0:22% (id a COR1 : seh wed from SEQ ID NO:2ft6, SEQ ID N(>:225, and SEQ ID NO;226. fmi a t DR 10 selected from SEQ 11ΤΜϊι227,; SEQ ID 190:2¾ SiQ ID MD:229, and SEQ ID NQ:230; and (U)a CDRl of (sb (b > and (to) that commas one or snots; amino ac id substitutions, deletion', or insertions of'.to moo. m.o, toe, fotn\ three, %o, or one amino a, ids amino acid*, [:00222] In another aspect, an antigen Ending protein includes t, 2, 3, 4,5, m h variant fbnnsef the CDRs listed In .Table 3Λ addfl'sllle 3B, each having at least 80%, at least 85%, at. least 90% or at. least 9,5% sequence identity to a CDR sequence Listed m 'fable 3A and Table 3B, Some antigen binding proteins include 1, 2, 3, 4, 5. or 6 of the CDR* listed in Table 3 A and Table 3B, each differing by no more than I, 2,3< 4 onS amino acids Ifom the QDR* listed in these tables. (ft022*| lit another aspect, an antigen binding protein includes 1,2,3,4, S, qf h vaisant forms ol the C DR* listed in Table 3C and Table 3D, each having at least 80%, at least 85%, at least 90% or at lea*t 95% sequence identils to a CDR sequence listed m 'fable 30 and fable 3D, Some antigen binding proteins include 1, 2,3, -1 5, or 0 of the CDRs listed hi Tabic 1C and Table 3D, each ddVenrig by no more than 1 2,3,4 or 5 amino acids horn the CDRs listed in these tables, [002241 lit yet anothet aspect, the ('DRs disclosed lutein include consensus fern groups of related monoclonal antibodies. As described he e n a uvtsu'-s s sequence tele s < a tern ^e,d so ^ >v<. · n u ' t eonse,vee am no act Is cor u on ,i,stone uumKi of vo, settees a ,cl variable ami no acids that vary within a gh en amino acid sequences. Tlie CDR concensus sequences provided include ODRs corresponding to each of C DRH1, ODRH2, CDRH3, (' DRf 1, CDRL2 and CDRE3; [002251 Antibody-antigen intenwrions can be characterized by the associationmfe; son.xtam sn M s ikh w 'dv .K\e>. mm π use eo'wusn in s ! t k0 r or < hemal tveiy She 'dissociation equilibrium constant in Μ (Κιχ), 10022o| I ho ps »vat ins ο uimi provides a vai icty of antigen binding pfofoittk including bv \w murid to * \ " v i<at specified 1> bind DNP or kl U. respectively- that exhibit de#£bie chafoctemti|£:#^^ . < measured by Ko (dissociation equilibrium constant) fcriPp tp the Mngc of 10'" M pr lower, ranging down to iff u M oridwereoraviliiy M : ipeasnped; by ktS idissociation rate constum) for tospectivdy.. iffihe- range Of 10 ’ s 1 or lower, or ranging down to 10 5>'s 1 or lower. (See, Example .12 hercein).

[00227] In some embodiments, site antigen binding proteins (eau antibodies or antibody fragments) exhibit desirable oharacteristies such as binding ayidity as measured isy k<: (dissociation rate conattmtiforOiNP or KOI, respectively, of about 10'A, HI'. 10 \ 10 x i0\ 10’, 10k HTi?, :|;0'u,«^:or lower (lower values indicating, h elm o 'King avdnvb ane o* ns dntg affunty as measured by kn {dfssoenuton equilibrium constant) for DNP or KOI respectively, of about Ilk, 10‘!k 10'· -..ΙΟΆ. 10 1 \ to :0 ' \ 10 K VI or io'ACf (lower values indicating higher btnd tog ailinoyt Association rate constants. dksoas.iOou rate constants, or dissociation equilibrium I constants may be readily determined using kinetic analysis techniques soehr#; surface plasmon resonance ('BlAeorek e.g . FKcbei'ei at.. A peptide-immun.ogl.ohu)in-conjugate, WO 2007.045403 At, Example 10. which is «ioorporatej bet cm « refere tee i' n\ eutue.s t os Km! s \ uvtng general procedure^· outlined by the manufacturer or other methods known in the art The kinetic· data: obtained by BIAcore1" or KinF.xA max be analyzed lt> methods described by the manufacturer.

[0022b] In some embodiments, site antibody comprises all three light chain C rills, ail three hem.x chum kriRs. ui .d' k\ i 'DR'. In some exemplars eniboOtniem-, two light: elpftt CDRs from- m; .anti hods mav be combined \x ith a third light chant CDR from ο different embed', - AJterrnhws\, 3 C OKI I from one .mtibody can be vO'i -m ej. v\lib ,u PKl * from a f.'t>ereot armbody and a CDR.I l from yet root hot ami body, particularly where the CDRs are highly homologous. Similarly. two heavy chinn ('DRa from an anhbod) may be combined with a bind hr;n\ chain t.'PR. from a dPtu sit annbui), o. a C DRM bon o >, mUihodt v boil nod w ha UD^ID ron' a Ji lereof arnsUdv < nj a CDR*D Dor, -,c, anemc, 1 mbow, particularly where the CDRs are highly homologous. : {$02291 invention provides a vai icty os' compositions comprising one. kyo, and or three CDRs of a heavy chain „ .tmNc wgion and'or a light chain variable region o; ei, antibody n, h.omg muds*',euuos m t auves theicof Sub f^^posiddM may he gcncraJed.-’byiechuiques dc^eiibcd herein or known in she art. |0023(0 in some embodiments, the antigen binding ptotein rum bull rw as .(foodies tod: ;a|iiibod>· fragments) ca® fcuselisi as a therapemie molecule w}&amp;^b#:W # singularly or in combination with other therapeutics to achie^tltd.:-desi^|-:0#ae%;; hi such embodiments, thomyehtive ansigem binding protein (felMkgisiOtibiWi#. and .anybody fragments) further comprises one to twenty-four, one to sixteen, one to eight, or one to four, pharmacologically active chemical moieties conjugated thereto, Whether a small molecule or a pplypeplidc. The pbarmaeologiealiy active small Molbcule or polypeptide chemical moieties can be conjugated at or via the N-fernvmal or C-terrniiud residue of the antigen bind ing pmnein immunoglobulin monomers feat, :LC or BC mopomemh chemical reaetiops hnowg in dm art and: "fertfepr;d^rtbs<i. herein. Alreniadvdy encompassed by the invention, is coiijugabon of the pharmacologically aetitc chemical moiety, or moieties, at or via functional .fr#ip:O0-0^dr moife--#d^-e|hii.8S0f the amino acid residue(s) within the primary chain of the inventive anogen binding protein. Useful methods and interna! i> n. w, 'ton sues ιv u , \ .utte hi ev'teu e 'e^diusi wu η ιη^ m \J '<'\d,n chon's: are known in the art te.g., Gegg et ai„ Modified Fc Moleculess published hi WO 200'· 0220/0 and US 200?026936^':^|(kh:#o;iiigorpomted herein by mfcrencb m their entireties).

[0023 1 I in other embodiments of the im ention. in which the phut maeologieully active chemical moiciv is a polypeptide, a recombinant h'enen protein eats be produced with the pharmacologically active polypeptide being inserted in the primary ammo acid ηφιηκ e of the of foe unmnnoglobnUn heavy enact wifbm an interna) Loop of the Fe domain of the immunoglobulin ;>casv chain, fo-vead of a· Use N~ and or C-remhnus. as further described in foe Examples Itcrcin and in the an (e.g., Csccl vt ah, U.S Patent No. ~.442fo~h, U.S, Patent No. 7.ό55.”ο5; t N Patent No '.t -' N I. U.S, Patent No. “fo3,03ri l S Patent No 7,M5,6oi pub-whed US. Patou Applications US .2009 0281286; arid US 2009 0280964, each of which are incorporated k their entireties).

[002321 UYmn mated" means that the pharmacologically seto c chemical .nxneoes !>e eosahnstfs hrd cd. or bound, eiset fly to an amino ac'-d residue of the antigen bmdmg pujtem. ot optionelK. n< a pcptidyl or non-peptidyi linker mosety that is covalently linked to the amino acid residue of the antigen binding protein.

[002331 As stdted above, some embodiments of the inventive eosnposMohsimrolvb .active polypeptide moiety conjugated to the pharmacologically inactive antigen binding protein of the invention, for example constituting a recombinant fusion protein ofohe pharmacologically active polypeptide moiety eonjegatod to the pharmacologically inactive ant%en#»dsig protein of the niveufion The term *fohormae«i!ogical!y active" moans that a safestapeefo described is determined to have activity that affects a medical parameter | e.g., blood pressure, blood cell count, cholesterol level, pain perception) or disease state ic,g„ cancer, autoimmune disorders. chronic pate), < otwerseh. the term "pharmacologically inactive" means thru «ο activity aOcctsng a mefoeal parameter or disease state can be determined for that substance. I hus, pharmacological 1> auric peptides or proteuw comprise agonistic or mimetic and ee;..gor>n\ ivpuees as del mod below. The ow-sent uweruon en, emp«ws the ns,' of any pharmacologically active prorein, which has an ammo acid sequence ranging 0>n.bou ς o ai ; vo ni no neut wwuitn ^ m length, and v .sc t s m.rabe o recombinant expression, In some useful embodiments of the invention, ihe pharmacologically active protein is modified in one or more ways relative to a native .sequence of interest,, including ammo acid additions or insertions, atphto acid deletions peptide truncations, ,«mno acid substitutions, or chemical' derivatization of ammo uotd rasidues {accomplished in· known chemical tcchniqitcsi^'tot'li^^ ivqn me bsoaetiv >ts tsmamuwd, (002341 The terms •"-mimetic peptide,” "peptide mimene/' and "'-agonist peptideT refer to a peptide or protein h.o mg biological activity comparable to a naturally oeemmu protein of interest, m- example. om not I muted to, a Hum peptide :. molecule, c.g„ ShK or OSKJ toxin peptides, or pepbcie analogs thereof, 'lltes© terms further include peptides that indirectly mo me the aims fty of a naturally occurring peptide molecule, such as by potentiating die eft ecu- of the naturally occurring molecule.

[00c 35) The tenn "-antagonist peptide,"' ''peptide antagonist," and "inhibitor peptide" refer to a peptide that blocks or in some with the biological acti vt ty of a receptor of ipteresb or has blologfesf ifetmty comparable to a know» antagonist m inhibitor of a receptor of interest {such asphui-pot limited to, an: iott vnatmel or a O-Protein ( ouplcd Receptor (GPC’Rfe (0023bl Examples of pharmacologically active proteins that can he used within the present invention include., but are not 1 touted to, a mxm peptide (e,g„ OSKI or an OSkl peptide analogs Shiv or an ShK peptide- apalogl, art IL-b binding peptide*: s :: CHRP peptide antagonist, a bradykinin Bl receptor peptide antagonist, a parathyroid homumc t PTH} agonist peptide, a puiatbsroid hormone (Is Hi I antagonist peptide, an ang-1 binding peptide, an ang-2 binding peptide, a myostatin binding peptide, an: ciydhtvpoictin-mimetic {EFO-mimetic} peptide, a thrombopoleiin-mmaetic (TPO~ mimetiei peptide (e,g<, AMF2 or AMP5K a nerve growth factor (NOP) binding peptide, a B cell activating factor (BAFF) binding peptide, and a glucagon-like peptide (GLPl-l or a peptide mimetic ihcrof or GLP-2 or a peptide mimetic thereof.

Glucagon·· like pepnde I tGI P-1 i a,nd the related peptide glucagon are piodueed via differential processing of piOghteagoo and have opposing biological activities. FK>glucagQB itself is produced: in os-cells of the pMcreas aad in;: 1¾¾ enterocHdoeiisie L-edls, winch arc kx a led primarily m the distal small intestine anti colon in the pane·esx glucagon is selectively Gcttsed from progforagon, in the intestine, in conirest,, progiucagon is processed to form Gt. P-i arid glucagon-like peptide 2 (GLP~ 2k which correspond to ammo acid residues 7s~)(n and 126-158 of proglucagon, respectively bee, e.g., Irwin end Wong., Ι9*>5, ΛΛ>/. fekrat/i. -6267-277 and Bel! e/ ;/.. 19^3, Xaiiirr 504.368-371). By convention. the numbering of the amino adds of GLP-1 is based on the GLP-1 ί 1 -37} formed in>m cleavage ofproglueuyoit. Tbo biologically act is e (bn os are generated frotrt farther processing of t his peptide, which, in one numbering eotnemiom yields GLP-1 {7-37J--OH end GUM i --36)--MB'. Both GLP-i (7-37s-OH (or simply GL P-1 (7-37)) and GlP~i (~~36)-Ml· have the same activities. For convenience, the term MsIP-1M, is usee to refer to both of these forms. The first amino acid of these processed peptides Is His” in this numbering convention. Another rtnmhes mg com eniton recognized in the act. however, assumes that the numbering of the processed peptide begins with His as position 1 rather than position 7. Thus, m this numbering scheme, Gl.P-1 1 i-3 hi w the same as GLP- H7 -3 --), and GLP-1(! -30.) is the same as GLP-1 (7-36). baampl-m of GUM mimetic polypeptide sequences include; RGEG I'FTSQQSSY LEGQAΛΚEF!AWLVK.C>RG ·/ fSEQ *D NO;290); HGEGlTTSDQSSVMi.GQAAKmAWL QKGRG (SLQ ID NOAM); HGEGTn'SDX’HSYQFXiQAAKf'RAWI.VKGRfi ; tSFQ ID NO;2M); HGEG IF T ΫΟ VSSY L EGQ A A KEF f AQ L V K GRG-v' (SEQ ID NO:293>; I (GETΠ H'SD VSS Yt. FGQAΛK ΕΡΊ AQ LQKGRG/· {SEQ 1D NO;294); HGEGTFTSDYSSYLEGQA ΑΚΕΠΑ WL.QRGRG'V (SEQ ID RO:295k f!NET 1'i"TSf>YSSYi F.GQAAKEFIAW't. YKGRGe (SEQ ID NO.2%) HGfcGTF1 SDYSRYLENQT'AKEFiAWLYKGRG.·.·' (SEQ ID NO:.297); IIGEGTFTSDYSSYLFGNATKRdA WLYKGRGF (SEQ ID NO;298>; HGEG'IFVSDVSSY LEGQAΛΚEFiA WL VNGfGA (SEQ ID HO:299>; HGEGTFTSDVSSY I.EGQAΛΚ ERA VV1. VKNRT. / (SEQ 1D NO. 300», HG EGTFT SO \ SSY i. EGQAAK. F.E I AW LVRGRNGT 7 (SEQ ID Nf L301). HO EGTFT SD VSSY l E GQ AA KEF f AWL VK.GR GGTGNGT / ·' (SEQ ID \0:3U2k and HGHGTFTSDVSSYi.EGQAAK.ERAWl VKGRUGSGNG1 / (SEQ ID *10.203). f00237j Human GLP-2 and Gl.P-2-mimehc undoes arc also known in the art. (See, e.g , Prasad et uL Ghieagorhike peptide-2 analogue enhance?· irhesitnal mucosa! mass alter ischemia and reperfusion, J. Pedtalr. Surg. 2000 Feb;35t2}:357··· 59 (2000): Vesta et ah, Glucagon-like peptide-2 receptor activation engages had and glycogen synthase kinase-G in a protein kinase A-depcmlvut manner and p rev cuts apoptosis Γοί lowing inhibition of phosphatidyl inositol 3-kmuse, j. Biol. €hem. 277(26):2469{S'-906 12002)}, [0022hj 'Toxin peptides" include peptides and polypeptides having the same ammo acid sequence of a naturally occurring pheonacoloeieally active peptide or polypeptide that car: be isolated limn a venom, and also include mod tiled peptide amtlogs of sued riatu rally occurring molecules. (See, erne. Kalman et ah, SitK-Dnp22, a potent Kv 1.2-specific mowmiosyppres-o\m poiypcpiide. .1 Biol. 0 hem 272(49):22607-70' {1996), Kern ei al., US Patent No. ftuWWiWU; Mouhui el ah.

OsK I derivatives, WO 200ft'002*50 A2: Cbandy et al. Analogs of SINK toxin and their uses in selective inhibition of Kv], 2 potassium channels. WO 2000 042)5!.; Suilwan ct ah. Toxin Peptide thcmpvmie age-nrs, WO 2006- i 1615ft Λ2, ah of which are incorporated herein by teferenre- in their entirety }. Snakes, scorpions, spiders, bees, snails and sea anemone are a few examples o.t org&amp;nkois that produce venom that can serve as a rich source of small bioactive toxin peptides or "toxins'’ that potently and select is <hv target un; channels and receptors. An example of a toxin peptide is OSK I (also known as OsK I), a toxin peptide isolated from Orlhochirns sCiobiCitlosus scorpion tenotn. (o.g., Monhai ct al., K-t channel types targeted by synthetic OSK 1. a toxin from Orthochirns scroblvaiostss scorpion venom, Biochem, 2. 285:95-1(.0 {2005}, Mouhai et ah. Pharmacological profiling of Orthochtrus xcrobieuioxns toxin 1 analogs with a trimmed N-ierroinal domain, Molec. Pharmacol, 69:254- 62 (2009); Mocha? et a)., OsK I derivatives, WO 200ft 002650 Λ2). Another example is ShK. tsohited from tire venom of the sea anemone SOchodaqvla hcljunthus. ([:.' g.. Tudor et a 1,, ionisation behaviour and solution properties of the pomxMum-channel blocker khk toxin f or. ,1 Bioehem 251(i - 2 k lx34 1(kb 4p Pennington ef id - Rme of dkulfhl·,· bond- m She .orm lure ,md potux-sum channel Hookup act λ stv ofkhk toxin. Biothent 35(-14}- ! 45-;m5X i 1999). kero ot as , Shk to' sn cumpoxitsoux and methods of uxc» 1 h Patent \o οΤ'-'Τό'χΟ; 1 ebum et uL kenropeptulex ot mournne. us m otpion, l 3 Patent No Omkoj-hk Beaton ct -3 ,

Tares ting effa tor memory 1 seHs u oh a xefi.vm, o peptide Inhdutorof k\ 1.3 clutmtnekfm therapy ojTutominimie dtvLjwex. fifidae Pluumaeol 0~i4pi5mk.x | (24.)05)).

[00239] The toxin peptides arc usually between about 20 and about 50 anti no acids in length, contain 2-5 disulfide linkages and form a very compact structure, Toxin peptides {em„ from the venom of scorpions, sea anemones and cone snails) have been isolated and characterized for their impact on ion channels. Such peptides appear to have evolved from a relatively small numbe? of structural frameworks that arc particularly well suited to addressing the critical issues of potency and stability The majority of scorpion and i. onus toxin pep Odes, tor example, contain ! 0-40 arnino acids and up to five dkuifide bonds, forming extremely compact and conxlramcd structure unicroprotoinsi often remxuint io nvucoly-ux The conoiaxm and scorpion toxin peptides can lee divided into a number of superfamuics based on the;? disulfide connections and peptide foklv. The xohttlon structure of many of those has been determined by NMR xpecnoseopy, illustrating their compact structure uud verifying conservation of their family fold, iili.g , Tudor ot ;fi... Ionisation behiiviou; a»d solution properties of the potusMum-elututici bUtckct Shk toxin. fur. f Bio el tern. 25 H 1 -2k 133-41( 1 kef y Pennington ct ah. Role of disulfide bonds in the .’'trueiure end potasxium channel blocking achsuv of Silk home 8 sue hem. )5(44 V 1-15 ] C; _\x (: 1999); laravine ct ai., Three-dimcnfionnl :0 mo lore of moan OSK1 from Ormochins* serobicuiosns scorpion senorn, Bioehem. 56(h).! 225-52 (1997k del Rio-Poniilo ci ah: MvlR solution structure of On 17, a novel peptide from the Mexican scorpion C entn.uoidex noviux with a typical beta~K>xin ve·]nonce hut v, itM alpha·like physiological activity, bur. ,1. Bioehem. 271(12)'. 250··)· to (2004?, Firacbm'cka-Chalufour et ah, Solution structure of diserepin. a new K-t-channeii blocking peptide from the alpha-KTxl5 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, OSK1, 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.,

OsKl 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 ah, 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 term “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-terminus. 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-terminus. “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- terminal 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 - blocks, decreases, lmpedes<, or ihlbbits CGRPi reeept#; acby&amp;potvfey full length i&amp;C'GR.P.'O.r'^OltP under physiological αηκ!θιοη\ of temperature, pH. and ionic strength C.'GRP peptide unUigonsxts include loll am! partial antagonist. he detected Py lMyn ik vitro methods or m.vivo functional assay methods. (Sec. e.g . South et ai., MbdiBeaitdhs to the N^termlnus hut opt the CTtermioos of calcitonin gene-re Hied peptidet 8-37) produce antagonists with mere used affuniy, j ’vied Chem. 4η·2427-2455 (2003», Hxamplesof useful <'GW* peptide antagonists, are disclosed in Gegg et al*UGRP peptide antagonists and conjugates, W f>. 200? 048026 A? and l S. Serial No. 11 58 hi 77. filed on October 19, 2006, published as US 2008/0020078 Λ1. which is incorporated herein by reference in its entirely.

[0024.21 The terms "parathyroid hormone (PTH) agdniM^^d^Wtt'agoniM^rcfer to a molecule that hinds to PTH-1 Of PT1I-2 receptor and increases of decreases one m -.cere ΡΊΗ actum rssay parametersmdoes full·lengthnatnehumanpar,uhytwd hom-one. Examples of useful PTfi agonist peptides are disclosed in Table 1 ©fibSt Patent No 6*756.480. titled Modulators of receptors lor parathyroid honnone and jabnuone^h^^ y?hich is incorpordt^ilt^ip· by refeiremxGh%5 ertoet' \n oAe.np.uiy ΡΉΙ actmty a.v^o, is disclosed at 1 sample 1 of UN. Patent. No. 6,756,480,; [00243] 1 he term "parathyroid hormone (PTH) antagonist” refers to a molecule that binds to PiTI-i or PTH-2 receptor and blocks or prevents the normal effect on those, parameters by full kngtfi hatiy«..tem«n.;p8mhyK5id hormone. Examples of .:n^fnii%Bi;'^ita^t)nist peptides are disclosed in Table 2 of ti,S. Patent No. 6,756,480. which is incorporated herein by reference in its entirety. An/exenlpfary Ρ1Ή activity assay is disclosed inTNmnpfe :2 of UTb Patent. No. 6,756,480. P0244| The terms “br&amp;dykmfn Bi receptor antagonist peptide” and ‘Nradyktaih Bi receptor peptide antagonist’/ mean a peptide with antagonist activity with respect to human bcadykinin B1 receptor (hB!>. I iseibl bradykinin 8.1 receptor antagonist pcptldds cab be identified or defiyed as described lb Ng et al,, Antagonist of tbe

Imsdykinin B1 receptor, US 2005/0215470 Λ1, published September 29, 2003,. which issued as U.S. Patent No. 7,605,120; U.S. feitstt Nos. 5,834,431 όΡ5,449>463> Bis exemplary 8 I receptor activity assays arc disclosed in Examples 6-4 of US 2005/0215170 Ai. f00245; The terms ‘hhmmhopoictift (TPOUmimetic peptide" and 'TPO-mlmefic, peptide" iot'er to peptide* that can 1¾ idenhime m dcOxcct as described m CM hla et ah (1077), Science 276; 1606-9 , U.S Pat, Acs. 5,869,451 and $,432 o 16, which are incorporated b\ refemnee in then entireties, l N Pat App No 200c 0P6352, published txcp' lo, 2003 which w 'ueoipowe’c: bvteterenee re w ;rm;en, AO 03 0' I '~49, puhnsl-cj Άρη 1 70t)5 WO 06 2a"70, published May 4, 2000, and any peptides appearing in Table 5 of published application US 20Cn< 0( 40034 (U.S. Serial No. ! 1 23-1,“3 L filed September 23. 2005. tilled Modified Pc Molecules, v Imh s'' operated terem by mterenee -n os cat net) s Usov of ormnarx Adi m the art appreciate that each of these references enables one to select different peptides thm acthdlly disclosed therein h> fol lowing the disclosed procedures with J; fien.. cr peptiA hbmnev [00246] Ί he terms M-PO-mimetic peptide" and "crx thropotetm mimetic pep tide refers to peptides flat can be identitled or derived as described in Wrighton et al. fl996lfSeienee 273; 454-63, and Narands et al. (1999), Pone, Natl. Acad, Set. USA :96: 2569-74, hbth of which are incorporated herein by reference in their entireties. Useful EPO-mirnctie peptides include EFO-oumetlc peptides listed in Fable 5 of published ITS, patent application US .2007/0269369 Aland in U S Pat. No. 6.6o6,44 h which are both hereby uworoomted by refe-.enee in then entireties [00247 ] T he term ’“ang-I-blndine pepude" comprises peptides that can be identified os d-nxed ;,s clewn sed in l 's. Pto App, No 20*13 tm'ΜΟ,Ά published Dee. 11,2003; WO 03:057134, published My, Π, 2003; U.S. 2003/0236193, published Dee, 25,2003 (each of which is incorporated herein by reference in its entirety);· and any peptides appearing in Table 6 of published application US 2006/0140934 s'U.S, Serial No. ϊ4/23431A pleelScpfember 23,2003,rilled

Modified be Molecules, which is incorporated herein h\ reference m its entirety! Those of ordinary skill in die enables one to select different peptides tte ^aally ^tseiosaa'^efetisiby following the disclosed? Itfoeedures with different peptide libraries.

[003.4KI The terms Mien e growth factor (NGF) binding peptide'* ami vkNGF-7¾¼¾¾¾ that can be identified or derived as described in WO 04/02633¾. published April 1,3004 and any peptides identified in Table 7 oT published application X1S 2006/0140934 (U.S. Setdait|^^4tlT2'M^3ij::filed SeptemDer 23, 2006. titled Modified ic Molecules, which is incorporated herein by reference in its entirety). Those of ordinary skill in the art appreciate that this reference enables oneio select different peptides than actually disclosed therein by following the disdosod procedures with different peptide libraries [3302491 The term eotppdses peptides fhai can: be iArofied ot donved a» deseobed m U.S. Sen No, 10/742.379, filed December 19, 2003. which h incorporated herein by reference in its entirety, and peptides appearing in fable 8 of published application US 2006/0140934 |U,S. Serial No. 11/234..731. filed September 23, 2006. titled Modified Fc Molecules, which is hu>"vf>u *J ivsen h\ ie οΌ'λ,* m so. ent'reis ,> Paise of ordinal} skdl m the art appreeiaie that each of these references enables one to select ditfemti ,al a K <h „ h'wd t serein !·' to k>" m- be ih^i. o-e 1 piU'-edmc-* with dillewnt peptide libraries.

[002601 t he terms "'BAH-'-antagonist peptide" and "BArF binding peptide" comprise peptides that cum be identified or derived as described in U,$, Pat. Applm No. 200 * 019515b A1, which is incorporated herein by reference; in its entirety and those pegud-w , rp,.swing m I able α oi ρ',.ΚΝίκΜ mphejnon l 8 300*·' 9: '00 3-1 ^ g :.Serial No 11--234,731, filed September 23. 2003. titled Modified Fe Molecules, which is incorporated herein by rcferen.ee in its enuiety}. 1 nose ot ordinary skill in die ait appi, cuiie th.: the huegebw w\ r, neev enable one to sCKi t ml IcserU pepodes tte actually disclosed teem bf following Ids disclosedlproced^res wick different peptide libraries. j 002511 I ho foregoing arc intended merely as non-iimOing examples of the pharmacologically active polypeptides that can be usefully conjugated or fused to the inventive antigen binding proteins i I m: led lug r tail bodies and antibody fragemenis) Any include phamutcoloyicaHy act Ac polypeptide moiety can bo used within the scope of the in\ enfion, ineludmg a polypeptide having a so-called avhncr structure (see. e.g., kolkoiun el aL Nos el Protfciwith Targeted Binding, 5 :S 2005 0080952; Baber vU aL it -O Binding Proteins, lIS 2008 02HI070; Mcmmer et ah, Protein ScafLkls and Uses Thereof, l » 2000-022 .¾ 114 and US 200» 0254209) [00252] Useful preckmcal animal models ate known iu the an for use in snlidaimg a drug in a therapeutic indication of interest fe.g„ an adoptiyeAfanefov model of periodontal disease by Vuivcrde el uh, J. Bone Mineral Res 10:155 12004): :.ea ultrasonic peri vase unit Doppler flow me to?-bused animal model of arterial thrombosis in Cirnnerc.t ah. Blood 10$: 1492-^9 (200$); rnnmonary thrombocnibolisro model, aorta occlusion model, and murine stroke model in Braun et ah, WO 2000 115000 AI). For example, an adoptive transfer expeOmennd autoimmune ericcphidomyelins {AT-i'AFi model of multiple seleroAs lias been described for investigations concerning immune diseases, sued as multiple sclerosis • Breton sit ah. .1. Immunol 100:950 *200;), Beciort et al , PNAS OS:) 0>42 12001r Sullivan e; ah, i'Aiample 45 of WO 200s 2)00422 \2, incorporated herein by reference in its entirety i. in ihe ΑΎ-BAf: mode I, x I guinea rely reduced disease severity and increased stir\ s\ai me expected tor animats treated with an effective a mourn of the in.vcn.tivc phanrsaecnh'oul composition, while untreated animals are expected to develop set ere disease and or mortality. 1 or running the Λ LiI AO. model, the encephnlmnyclouenic (1>-L mt 1 cell line, FAS, spec ilk lor myelin· basic protein l \IBP} ovigi meed from Dr Kvelyne Beruud flic mshnenanee of these cells in vitro and their u*c in the AT-b.Ah model has boon described earlier (Beeton et id. (200! i PN AS 9s, I $942j. PAS T celU are maintained hi \ iuo b> ultornaang rounds of antigen stimulation or aetn soon with 01 BP and in,abated thymocytes (2 clays), am! propagation with Ύ cell growth factors (5 days). Aetivabon of PAS ! cells (3 x 10"4ril) involves^ tM cells fer 2 days with lOgg/mf MBR and 15 10‘ ml v* ngenew sn.idiatee t5:nX) rad) :liym<x\;es * >) Jh\ 2 anct n \ mo aeOvsbon, 10-15 - Iff'uabk P \5 T cellsarc mjeeted t no»f-12 weeso'U temafe Leys rats (Charles River imboraferiyV Ly Mil IV, Dally subc«t^d#iad&amp;t|et#dS #' vehicle i 3Ά Lew rat serum in PBS) or tesi pharmaceutical composition are given from days · I to A where day - I represent ! day prior to injection of PAS Γ cells Ula\ 0), in \ chide treated nus, tjeute EAE us espetted to det el op ) to 5 days after injection of PAS T cells, Ts picul iy, serum is colleen d by tail vein bleeding at day 4 ,md b> e utl ac puncture at dr·- n t md of th.e '-mdv) tin analysts oftcscK of mmbtior Rate are typically weighed on days -1,4, 6. and 8. Animals may be scored blinded once a day from the day of coll transfer (day 0)ΐο day 3, and twice a. day from day 4 to day 8, Cltntcaf signs are evaluated m the total score of the degree of paresis of each limb and tad. Clinical scoring. 0 No signs, 0.5 distal limp tad, 1.0 bmp tail, 2.0 - mild paraparesis, ataxia. 3,0 - modern to para pares is, 3.5 :::: one hind kg paralyses. 4.0 complete hind leg paralysis. 3,0 -complete hind leg paralysis and incontinence, 5.5 - tetraplegia, 6.0 --mo Abend staid or death. Rats reaching a score of 5.0 are typically euihmksd,

[00255 j · Froduglioii<>fA!iiibr>dy Embodinteak af.thc. AtpiR^iLBiadinrdPi^jciM (002541 Polyclonal antibodies. Ihm dona! antibodies v. p clew >6 > ed iu .mnvnds l"· .nulhp e \t e^maneoas tv ) os mtiap^ mock (mi s oeuiotu. of no relevant antigcdt ahd an adjutant Alternatively, .mtoren may be injected directly into the annual’s lymph node (see Kilpatrick et aL Hybndoma, 16:381-389, 1007). An improved anttbody reaponse may be obtained fey coujttgattttg tbe relevant antigen to a protein that is immunogenic inthe species to be unmanly hemoeyarnn. scrum albumin, bovine thyroglohnlm, or soybean trypsin inhibitor using a bifwocticroal or derivatiziug agent, lor exatrtplc, ntaleinttdobemtoyl sidfosuccmimide ester (conjugation. through cysteine residues), N-bxdaossva i mm, < it uough sm v codecs) < ylurah.d ' de, s. ew w hsCoCem Other agents known in the art. (00255] Animal* are immunized against fin.· antigen, immunogenic conjugates, or derivatives by combining, c.g , i00 ggofthe protein or conjugate ilbrmieo) vAth ? volumes of Freund’s complete udnmmt end miceting the solution inbudetmally at multiple sites. One mouth later, the animals are boosted ά ith 1/5 to 1 10 the unginal amount of peptide or conjugate tn Freund’s complete adjuvant by subetrtaneous injection at multiple sites. At AI I days post-booster injection, the unimats are bled and the serum is assayed for antibody rite?. Annuals are boosted until the titer plateaus. IVeierably. the -mimal is boosted with the conjugate of the same antigen, bur conjugated to a different protein end "or through a diuerens cross-iiukiug reagent Conjugates also cun be made In recombinant cell culture no protein fusions. Also, aggregating agents such as Anns are Mutably used to enhance the immune response. j00050] I be bnenuve antigen binding protom' or antigen btndtng proteins that are provided include monoclonal tombed res that bind to DAP or KS..R, respeeuvciy, Monoclonal antibodies nt.ty bn produced using uny teehtttque knuun to the art, e g., by immortalizing spleen celK harvested front the transgenic animal alter completion of the intmnnnnuion schedule. Tfte spleen cells cun be immortalized using any technique kuoo o in the art. e g.. by fusing -hern vs-tb mveioma cells to produce hybridomos. for example. monoclonal aAioodles may be made ttsutg the hybridomn method first dem-ibed by Kolber ct ah, Nature, 250:44,5 {1*575), or may be made by reeumhinam ORA methods Key., (' abslK ct ah. Methods of producing intmunoelobulins, vectors and transformed host cells tor use therein,

IhN Patent No. mo : ,415), Including methods, sued as the ''split 004104 method., that facilitate she generally equimohrproduouon of It gin and heavy chains, optionally using matnmuhan cell lutes (e.g.. ( HO ceils) that cun glycosylate the antibody «See. c.g.. Page, Antibody production. EPOlb PvO A2 and IdS Patent No. 5,545,105). (00257] in the Itybridomu method, a mouse or other appropriate host mammal, such as mfs, hamster or macaque monkey, is immunized as herein described to ehen lymphocytes that produce or are cap-able of producing antibodies fitut nob specifically bind to site protein used for immunization Alternatively, lymphocytes may be immunized in vitro, i.ymphocytes then ate fimeo w lib myeloma cells using a .suitable fusing agent, such a-- polsethslene glycol ίο tbsm a hvhodomu cell (ifodmg. Monoclonal AnuKkhes Prmmpk's and IbucOce, ppd(V]03 tAiadennc Pass, 1%M> ΓΟ0258] In some instances, a.hybridoma cell Utw is produced by immunudng a transcensc animal having human immunoglobulin sequences with a ONP or KLH immsmogen; harvesting spleen ceils from the immunized animal fusing the harvested spleen ceils· to establishing hybridoma cell lines front the hybridoma ceils, and identiiying a hybridoma cell line that produces# antibody respeetiveiy,:;

Such hybridoma cell lines, and monoclonal anibpdies produced by titem, are aspects of the present invenhon.

[00250] The present inveuiton also encompasses a hvhridoma that produces the invents e antigen binding protein that is a monoclonal antibody. Accordingly, the present invention:# also directed to a method, costprisiitg' [00260] (a) culturing the hybridoi$£%^cdiguifc medium under conditions, permitting expression of the antigenMndingpmiein by thehytedatuat hud: ; [00261 ] ib) recos enuu rite antigen binding protein from the culture medium, which cm be accomplished by hnosin antibody purification techniques, suchas but not hnnied to, monoclonal antibody pinifi cation techniques disclosed in Example 1 herein.

[002621 I he hybridomu edis, onto prepared, arc seeded and grown in a suitable - culture medium that preferably contains one or more substances that inhibit the .growth or survival of the un fused, parental myeloma cells, '.for cx«m$>le, if the parental mseloma cells lack she enr^bb:y^xauthiee''^^ne:'pfi.baphdftbb#i tiansfem,w MldPRI or IfPR \). the culture·medium tor the hybodomus typically vuit include hypoxanihine, ami.nopte,rins and. thymidine (ΚΑΤ' medium), which substances present the gross# of [002(>3! Preferred myeloma cells arc those that fuse efftefortK, support -WiNc high-level production of antibody by da selected asst food','p-odueisy· ceil·-, end ut, sensitive to a medium. Human myeloma and mouse- human heieromyeioma tel· lines aiso hm , bees* described fos the mode..,on ot'heme.' wmtoefonx vedeeiise-t.fxoxbor, ) Immunol., 16' rtlOl (19S4) Jimdeo* e: -I, Monoclonal Anunod* yfe'ddiatetft»» Techniques and Application*», pp 51-63 {Marcel Dekker, Inc... ^ew York, 1987)). Myeloma. cells for use in by bridoma-proik icing fusion procedures preferably are no n - a s u i bo dy ~ p rc> ch t ei.n g. have high fusion efficiency, and enzyme deficiencies that render thetn incapable of growing in certain selective media which support the gam tit of only the desired fused cells (hybrt domes}. Examples of suitable ceil ‘sues tot use in mouse fusions include Sp-20, P^-XtVpAgh, P3-.X03-\>Α*3ΧΝΜ I A a i XMMlb-,\etdjn,\S01 .MPifoti VIΚΊ ί-Χ-5-G iit t ' and SUM sXXO Bui; examples of cell Itnes used ut r.e fusion* include R210 RCY3* Y.'AAg 1.23,1X983' ami -t R2Hf cither ceil lines useful for cell fusions are 13266, 0Mi5bb-ijR07.1 lCR~t.€>VUMy2 and UC729-6 [00264] Culture medium in which hvhridomu cells arc growing is assayed for production of monoclonal armbodies directed against the antigen. Preferably* the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by mmnmoprecipiufoon or by an in vitro binding assay.Midi·as· radioimmunoassay ιΒΙΛ) or enzyme·linked immunoabxorbem assay fHUSA). fhe binding affinity of the monoclonal antibody can, for example, be determined by B1 More* or Scatsb.n J anal' sis (Munson ct ah, Anal. Bfochem., 107 120 {1980κ Hsdume, a,, Λ o mhoe-inm ,n '·3·.fouI'n~eott innate. VHO 7007 015 163 A I, Example 10. which is incorporated herein by reference i« it* entiretyI.

[002651 After hybridoma cells arc -dentifted i|gt produce antfoodiesipf the •’specificity, uffiniry, and/or activity, the clones oiay be subcloned by lipiftlng dilftiipp procedures and grown by standard methods tCipdfogi. Aiottocloftal Antibodies; Principles and Practice, pp.59 103 {Academic Fres&amp;u^bB- Suitable etdfofo media fty this purpose inchtdc, fes csau'Mc, D-YlkM or RPMI-1640 medium ht addition, the oHMomo. cal stray bepnrxn n so as useAe.s tnmom m an ummaf \ 00206) Hvbridomas or tnAbx may be further screened to identify mAbs with par Ocular properties. such as die abifitx to ns bib'5 k! ilnx though K\ l ,x ehaunek bxamplex ofsuch screens ate provided tn the examples be Ion 1 he mo notional antibodies secreted by the snhclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, tor example, protein A-Sepbarose, hydroxylaps tit e ehaunatograpln, pel electrophoresis, dialysis. aiHmn clmomMogtaphy, oi any othei suitable pun fkunon technique known in. the art [00207] ReemnMEitf The invention provides isolated: .nucleic acids encoding any of the antibodies (polyclonal arid monoclonal), including am feed Y fragments, ofthe invention described herein, optionally operably liniedlo ; coni'o! '-c.p.jTices tecogns/ed by <i host eeb, \ coitus and host cells cotupnMng the :· nucleic acids, and recombinant techniques for 1 he production of the antibodies,: which may comprise culturing the host ceil so that the nucleic acid is expressed and, f optionally, recovering^a&amp;tbodyfrom the host cUS culture or culture medium. Nintbar materials and methods apply m pros!notion of polypeobde-besed antigen binding proteins. |0026b) Relevant amino acid sequences bom an immunoglobulin or polypeptide of interest may be defeMned^rd^i^/pofop'Seqnendngs and suitable encoding nucleolide sequences can be designed according to a universal codon tablef Alternatively, genoniie or eOMA encoding the monoclonal antibodies may be isolated and sequenced !mm cells producing such antibodies using coiweniional procedures te.g., by using oltgosmcieotidc ptobes thaf are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). (002001 Cloning of PNA is carried oprfusing standard techniques t see, e.g., Sambmok et. ah i 19hy> Molecular Uonutg A Lahot,story mode. V ols 1 m, ( old Spring 1 larbor Press, xvhich iaincorporated herein by reference),. For example, a el>\,\ lihraty may be constructed by reverse transcription of poly -V- mRNA, preferably memhrano·associated mRNA, attd the library screened using probes specific :or human immunoglobulin polypeptide gene m quenee^ in one embodiment, however, the polymerase chain reaction (.FCRj is used to amplify „D\ \vs (its pmrnuts id rifi-te yt , (i\ \s) ^'kodmy 3 ' emnuno Jo ndm gene sey.ueru o :. net eel (e e. „ a he u o hc.o ' eluir sun able ^cgtneui) 1 he umph' , e seejofkva< u be ream \ clone i ode ·. e, suuaf e wuot, e g. express η vs sc· *oo, mmige se \e<,k> . or wuge dnp ,n wxtoo U a -f, he apotee\ned dtui the p.uheela:r method of cloning used is not critical, so long as it is possible u.> determine the sequence of some portion of the immunoglobulin polypeptide of interest.

[00270] One source for antibody nucleic acids is a hybridomu produced by obtaining a B cell from an animal immunized with the- antigen of interest and fusing it ίο an immortal ceil. Alternatively, nuelcie acid can be isolated from B cells (or n h»< „ spkx ! o he tmmum vd am n d \ e: aaotne: -ousee ol nueVie <n Jv encoding antibodies is a library of such nuUeie acids generated, for example, through phage display technology. Polynucleotides encoding peptides ol‘interest, c.g„ vur.able notion pepudes n ith desired binding characteristics, can be identified fev standard techniques such as panning.

[00271 ] The sequence encoding an entire variable region of the immunoglobulin polypeptide liiiy be determined; however, it will sometimes be adequate to sequencer only a portion of a variable region, for example, the

Sequencing is earned out using standard techniques (see, e.g., Sarnbrook et al (1089} Molecular Clonings A Laboratory Guide, Vofs 1 -3. Cold Spring Harbor Press, and Sanger,Jk ct al. tM977) Proe, Natl, Aeud. Sci. USA 74: .5402*5467, which is rncorp0ra^;h^ttih by retbreneef By comparing the sequence of the dotted niteleic acid with published sequences of human immunoglobulin genes and cDN \s, one of skill will readtlv be able to determine depending on the region sequenced, {i} the gercr.il itte segment: usage of the hyhridomu immunoglobulin polypeptide (including the isotype of the heavy chain] 'and (ti) the sequence of the heavy and light chain variable regions, including sequences resulting from N-rcgk>n addition and the process o; som.me mutation. One mume of intmunogiobukn gene sequence information is»· tho National Comer for Biotechnology Information. National Library •of Medicines National Institutesof Health. Bothcsda. Md |OU272j Kouneii DN-Λ c an !x' op·;·;ably linked ίο control sequerKV1· o; placed into ok pros ;>;o:n vectors, which arc turn trunsfeeu'd imp host telD that do not inhere ise produce imu'mimglohmm protein.:; direct the m nines is of monoclonal antibodio in the meomhinum host ceils. Ikecoruhinunt production of antibodies is e di Luo-a e at the art-.

[#02131 Nueleie acid: m opbrably: linked Alien it is pliieed into a fbnctiosial: relationship with another nuclei, at id sequence for example. DTs.A iter a presequent-e or secretory leader is opirraHy linked to DNA for a polypeptide If it is expressed as a prepmtem that participates in the secretion of the polypeptide, a promoter or enhancer is operable linked to a coding sequence if It affects the iron script ion of the sequence; or a n bo sente binding site is operahly linked to a coding sequence if it is positioned so as to facilitate translation. Generally, oneiahly linked means litre the DNA sequences being linked are ccuuiguouv and, trt the case of a .secretory lender, contiguous unci ι-t reading phase. 11 ox ever, enhancers do not have to he contiguous. Unking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional pfactice, [00274] Many vectors are knovn in the art. Vector components may include orator more of the following; a signal sequence (that may. for example, direct secretion of the antibody; mg.. ATOOACATCAGOCTGrCCCiCTCAOCTCCTGOOOCTCCTOCTGCTGTOCCT • GAGAGGTGC'GCCKIGT. SF.Q ID NO.102. wind* encodes the VK-I signal peptide sequence MDMR VPAQLLGl 1 U..WI.RGARC ' SEQ ID NO;103), an origin of replication, one or more selective marker genes (that may, tor example, ··confer antibiotic or other drug resistance, complement auxotrophic deficiencies, or supply critical nutrients not available in die media), an enhancer element, a promoter, and a transcription termination sequence, ail of which are well known in the an. (00275] Cell ceil line, and cell culture ure often used intetthangeably-Md all such designations herein include progeny. Trans fo-manls and n-erislbrrnod cells im lode the primary subject cell and cultures derived iherefmm without regard tor the number of transfers, It is also undersfood that all progeny may not be precisely identical in DMA content, dee to deliberate or Inadvertent nudatlons. Mutant' progeny that hare the same function or biological activity ns screened for in fire originally transformed ceil are included, 100271] Ilsemphu'y host eeiis include prokaryote, yeast. or highes eukaryote celts. Prokaryotic host ceils mdude eubacteriu. such as Gram-negative or Oram-fosmys organisms. for example, Enh-mhachriijt. <.uti' such as Esrberiehju, e .g., ill coli,

Mm· Kjobekik, Mg&amp;i&amp;ik- *.&amp;, MmmM ?Vp|u.ninnupy.Sorr;ida. e.g\, Scnaria rnareescans. ami SjUgejla. as vved as Bgcdius stteb as 8. xubhus and B. iichemfomys, fo^tdmnonas. and Sg;mgotnyces. Eukaryotic microbes such ns filamentous fungi or yeast are suitable cloning or expression hosts for recombinant polypeptides or antibodies. Saecharomvces eerevisiac. or cosnmon baker's yeast., is the most commonly used among lower •eukaryotic host microorganisms However, a number of other genera, species, and strains arc commonly avail-dale and useful herein, such as Piehia. e.g. P. pastoris. blhllPldl.lPdfPI?.iyces gombe; jv|uyg;erone'eey. Y.aQO.g;ig; Candida, Trtcjgidctyna mihlfo hour^om.crassa; SchMailromvckS such as KdMddPimcos omdem&amp;iS: and filamentous fungi such as, e p., Neumsportu McUjium, Toiypoekdkm, and Aspergillus hosts such ns A. nidulans and fo nlgcr.

[:0277] Host ceils for the expression of glycosylated antigen bntdntg protein, including antibody, can be derived front m uUiceliular organ isms. Examples of" invertebrate cells .include plant and insect cells. Numerous baculoviral strains land variants and corresponding permissive insect host cells from hosts such as •Spodoplcra trugiperda (caterpillar), Andes aegypri imosqnuo), Aeties albopictus (mosquito), IMSThkild (fruilflyi and Bombvx. mod have been idMtifisbl A variety of y|ml straips available. o.g., the L-1' variant ofAttiograpfocaliBmIea::

Bonibyx moo NPV, |'002?81 Yenobrate host cd Is arc also suitable hosts, and reeombmusit production .oflmtie.cn binding protein (including antibody) from such cells has become routine procedure. Example* of useful mammalian host ceil lines are Chinese hamster ovary ί^^''^^{Μ0Κ 1 cells (ATCC CCL61), DXB-i i. DC-44, and Chinese :ltota^is^;OVtay t»l,isADHPR (CHO, Urlaub et. a!., Proe. Nail Acad. Set. USA ??: 4216 (108051 monkey lidhey Cyt line transformed by SV4fo(COS-7, ATCC CR.L ioM u eum <' > abivomc keine> hoc t >08 os „0 > to N suoe rated for growth m .suspunoon . ukase, jCirals.ur d ah, J lieu 1 '·-. /. do 50 (1077)1, baby hamsie*· kidney -cells (ill ik, A1U 1(1 10) s muse "oh aci s (TM t Msthes, Bto Reprod. 23: 243-251 (»80)); monkey kidney cell* (ί Ύ1 ATCC CCL 70), African green motley kidney ceils (VERO-76» ATCC -CRL* 1587); human cervical earentoma ceils (1 ill,A, ATCC «. C 1 7), canine kxlncv cells (ΜI)(, N, M ( C C (Ί 34); buffalo rat liver cells (BRL 3A, AT< ’<'('RI. 1442); human lung cells (W138, ATCC CCL 75): human hepatoma cells (Hep G2, HS 8065 s; mouse mammajy tumor (MMT 060562, ATCC CC 1-51); TRt ceils (Mather et aL Annals N Y Acad. Set 3b8 11 ·;> (,082r·. MbT 5 edis or F81 ce K os im-mmastiii inxeFuw ceb- [002701 Host cells are mm-.formed or sram-Cuco χχ itb the a box c-described nucleic adds ot \actors for production antigen hmdoig punt sos and 'em as him d os conventional nutrient media modified as appropriate for inducing promoter*,··;., selecting trao-uhrmant*, or amplify ing the genes encoding the desired sequences, hi addition, rove- ^octt-ss sum uansCeted eel :ma« wish multiple ,oases -if o.ewer φίsee umts mar-tod ex a -elccm,. masks s are paui-ml-eA usefal tos the expr woon of antigen bindnm piotems, I0O2S0 The host e, IN use-' m e odme t'u arm = t, esedseg ptoienu- ttt s'w invention may be cult tired in a xariety of media. Commercially available media such as Ham's F10 (Sigma), Minimal i-ascnual Medium {(MEM), (Sigma). RFM1-1640 (Sigma), and Duibecco's Modified Eagle’s Medium ((DM'EM), Sigma) arc suitable I or culturing the host. cells. In addition, any of the media described in Ham et ah. Modi Fme. 5fc; 44 {19?'», Barnes et a).. Anal. Bloehem. 102’ «55 (1980), U.S. Patent Nos. 4,"'67;704· 4,65",866; 4,9: /,702: 4,560,655; or 5,1 ΙΜ,ΊοΜ WQ9OI03430; WO 87 00195; or U.S. Patent Re. No, 30.9*5 may be used as colt are medw for the host cells, Any of these media may be supplemented as necessary w itls hormones and or other growth factors (such as Insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, 'magnesian!, and phosphate}, hniters «such as HEPES). nucleotides {such us adenosine and thymidine), antibiotics fsuch as Gentamyein™ drag), trace elements (defined as inorganic compounds usually present at feed concentrations m the micromoUtr range), and glucose or an equivalent energy source. Any other necessary supplements tuny also he included at appropriate • concentrations dun would be known to those skilled it: the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily 'skilled artisan.

[00281) Upon culturing the host cells,, the antigen binding protein can he produced huraeelhharly, in the periplasnsie space, or directly secreted into the medium. it the antigem binding protein is produced intraceliulady, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or altrailUration. |60:S7] Ί be antigen Hading piotein >e g„ an antibody or antibody fragment) can be pm b um using, v>; example, fy:ho\} lapame eiux'mamgraphy. canon or muon exclnntge chromatography. <.>r prefer,:hi', affinity ihromatographv, using the antsgotx of interest or protein A or protem G ,m an affinity ligand Protein A san be used to pa a by proteins that me mde polypepud.es ate based on human yL yd. or >4 6010 > eltmns i landmark a el, J. Itutnunol. Merit 0 7 imp; toygyg Preaem G is recommended tor ah mouse wowpc., and to; human re iGuss el ah, FV1BO 1. 5' 156" Go t19x6)), '1 he matrix to wlwfh iheaffuuo, ligand w attached w most often ,ig.uoss\ but other mm; ices are ;e, atlahle. McciwmeniK stable nucriees sueh -.s controlled pore guts- or poly mix retted niin Ubctwcnc allow Ice tVuer floe rates and shower pfocessusp limes than can he ,;ch fc\ ml wnh mem iso \\ here the unman cob:) prises a €>; 3 domain, the Bakerixmd ABX'raresin Ο. T. Baker, Phillipsburg, N J.) Is useful for purification. Ofitertoekmqncs tor protein purification such as; ethanol precipitation. Reverse Phase EIPLC, ehromutofocusing, SDS-PAG E, and ammonium sulfate preeiptration are also possible depending on the antibody to he recovered. I On'-* g 'in 1 u t hirum1^ mdlbr* i .M..ic.sic 0 ..T.P.lAh M:A.AMihodies, Chimeric mu: me Iona! auo bodies, i» which the variable Ig domains of a rodent monoclonal antibody are fused to human constant Ig Anna ins, can he generated using st,usdard procedures I uoh η n the ait (nee Mona vat, S L. e; ah {I °84} Chimeric Human A no body Molecules; Mouse Antigen. Binding Domains with flttrnan Constam Region Doma-ns, Proc. Natl Acad. Sen USA SI, 6841-6855, and, Boubunne.G t. , emu, Aciure c'2, 6A).u,m A number of teeb nones Ino e beat described for humani/osg or modifying; autiho,h sequence to be nvn he;nan* like, for example, bv (I) gran in g the non* hunt an complementarity determining regions {CDRsl onm a human framework and eonoam region (a process referred to m the art as humanizing througlt "('DR gtafting") ot (2) transplanting the entire non* human satiable b>u\<ens, be' ‘ cl on I 'eg” to, rn wuh a kunnoi-hhe surface by K'plaeeraeni of surfaee residues or process referred to in die art as ''veneering") or (3) modifying selected non-human untnu acid residues to bo more human, based on each residue's likelihood eA participating in antigen-binding or antibody structure and As likelihood tor immunogentvtij See, e.g,, Jones c: ,u„ Nance 311 >32 523 ί I °So)„ Monism, ei ,n , Proc Natl Aeac, Sex, t\S A , 81 68' 08's {'9X4), Morrison and On AeA. Immunol., 44-65 92 t I9XX): V'erhoeyo J , Science '239:15)4 15)6 A v88k Padlan, Molee fmrnna. 28:489 498 (1991V, Pudlan, Molee, Immunol, AAfA 2Γ’11304). ana Kelix .vrmigh, ('.A et m , Protem t eg 4i7) "?3 8c (1991); Co, M 8 , et at j immnnol 152 >*o8 29?6k StnOmeka et si Protein Engineering ” 805-814 (1994); each of which is incorporated herein by mferen.ee in its entirety.

[90284! h*v.· been described for humanizing or modifying antiboti^'Mtl^ebtb'lbbr^dpg human-like, for example, by (i) grafting the nun- f&amp;otsrtcorapleraetuaritv determining regions (CDRs) onto a human framework andri constant region {a process referred to in the art as humanizing through "CDR : grafting"') or (2} transplanting the entire, non-human variable domains, but ’'cloaking" thorn web a hvn w- do mu Ur ·. hv κρΙ«» omens of surface ' ..'Udncs (a p.oees* referred to p the art as "veneering”) or (3) modifying selected eon-human ammo stand residues to be more human, based on each residue's likelihood of pametpemic in antigen-binding or«^body:#mcthm:;sad:itis ikcl;l^^;Mlmmanogenicity. See, e,g<, Jones etai, Nature 321:522 525 09Sb)t Mon#&amp;^#:lk;Ptrievl)tgtl. Acad. ScL* U.S.A., 81(685! ^|5.5 (1984); Klorii son and 01, Adve.lnipihriol.,:·^;^· Ό2 (1088); Verhoeyer et a!., Science 230:1534 ? 536 (I OSH); Padlan, Molec. fmmusi. 28:489 4$jj| (1901); Pudkn. Molce. Immunol !U3)'l69 2!7tl 9°M); and keitieborotigh. C.A, et al.. Protein ring. -67):773 83(19915; Γο„ M S,. ct ai, s feMh 3 Immunol. 132,2%8-2976); SmddiSkfrei il '^tdiri-Bngincering 7: 805-814|I994);-e4eh#>yhtbfe% incorporated herein by reference in its entirety.

[00285] ;in am aspect, the CDRs of the light and hea vy chain variable regions of the antibodies provided herein (see. Table 3A~B> are grafted to framework regions (FRs) from antibodies iron! the same, or a different, phylogenetic species. For example, the CDRs of the heavy chain variable regions (e.e. Yul. Y;;3, Vu3. v„4. \ s5. \f m V Γ \ v m \ t ij < ΐ ig in a \iMuhe rj.ous u g ,\ \

Vi.3» Vi4. Vs 5, V?ή. VCR V- H, or Vt.9)can he grafted to consensus human FRs. Ίο create eonsevtsus human FRs, FRs from several human heavy chain or light chain ammoae-d segueu^es nun '.v a ιη,.κ,-α to teomify a consensus ammo acre sogeeree ht other u mod rents tv Rno* heav. chum r bub" ' n e s, .,ηοα Itemn te replaced with the FRs from a different heavy chant ot light chain. In one aspect, rare ammp acids in the FRs of the heavy and light, chums of the antibody :irc not replaced, while the rest of the l;R annuo amis are replaced, A '’rare amino acid" is a spec iff ammo acid that is m a posit*·. *r. in -a at oh una rwrueiJet' amino acid ts not usually Found m an FR, Mt< mans eh, 1 a grafted v. ruble regions fix on the one heavy or lighten, n nu> be ooe h im , v 0 rum teg on ns u re An cm m rt tee von^u* t region M that puriieulas heavy ;of: lip t eMia M-d^Jo^ilv ifaefn;;; Id plhei embodim, nix, 'he mn ted %-at able tpymr<« yv ran of a smgte chans Fv asvtsbody, 1/302161 Anybodies cars. akso bp produced usirig tmn^gemb arbmafe tltai have no endogenous immunoglobulin production and arc engineered to contain human iramtnoglobulln loc i. For example, WO §8/2489 3 discloses transgenie fnlmals having a human Ig locus* wlKsrei.u.thd/#^όϊfrp&amp;dtet? immunogbbwlina duoιό the inaedvariba of endogaroox heavy and light elmln lioei WO 01/10741 also dixebses;transgenicnon-primate mammalian feoxtsegFaMopf mounting an immune response to an Immunogen, wherein the urttihodiestraye primate constant amfot variable regions,· and vvUadn-.the endogenous lummnoglo a m encoding Iocs <ne sobxtitnted or *nuem atod \\ t * ’h> ^0498 discloses she use of Hie t Ye 1 ox system to modify the irnmuoogbbnhn locus in a mammal, such ax to replace all m a ^^l0n:.of variable region# fdrri a modified antibody rpoiecule, WO ^/OSbOS diseloxeauon-bnman otatntnaiian hosts has mg ieecnvated iodogenonylg Iocs and bnetional human ig loci. 11.¾

Patent No. 5,939,59s disclose* methods of making transgenic snipe. mice kick . a, logo· mux ho -λ v , h,dnx, ami expiexs au exogenous rmnismogtobulm locus comprising one or more xenogeneic constant regions.

[00287] l Nmg a transgenicanimal described above, un urnnunc response ohtibe pit finest! :o «» x. k<, see ,u ngcmierraokni . a -d antibody producing eelN i an he removed from the mi ire a: ami used to produce hybridontaa that secrete human-de'txeJ monoclorn ire md e-- inxrom v, on piotoeo *, adiux mm n j the I'be are know η ό the att, as e a,^ used .a mem. , . on ok tor evmonie, a karxgen ,. mm Mi as described in WO op 337e5. The monoclonal antibodies can be tested for the .ability to inhibit oi ‘'euruhee me h'Oiocma! a cm uv oi phys.ologieJ a Keel ,ethe corresponding protein. Sec also Jakohovits et el.. Free. Nad. Acad. Set. USA, 90‘2'O s I‘>9 3 k li.koboMt'* et ,.1, \an.ec. 5o2‘5 Y'-.?5\ ; Ieo3); Rmjsvonain* of at, Year in Irnmuno., -:33 td'Y-O); Mender etAcs Ncue/. 15:14b· ;56 Π99Ό; and tj S. Fai No. 5.591.669, O.S, Patent No, 5,589,eWh U.S. Patent No. 5,545,81)7;. and 1 X W U \ tpl cation No 59020 NAVI χ t x 9, ^ t ApH.culto- No dad .'00W·' \2~ de&amp;cr > no,bxl^ to: buisi :¾ the immune i espouse ut .m annual to tlm desired epitope. Human antibodies may also be generated by m viim^gcdTaled B cells ('-cv L< v- Pat. 'Vw :\5o mMQ end 5,220,2751 [0028H1 Antibody production by nhaoedisoky techniques j002*4i the J :· \ el· ϊ p i -: ei:: of te. b no I o g scstor nuking repertoires of recombinant humee uKw \ genes. and the dwpkn off he encoded antihod} fragments on the ph'face of filamentous bacteriophage, hasp foyidnd another incaas fecgederatldg hum m-derro d mtibodm* Ph ige display is described in e g , Duwct et id , WO dl'i “271 v Met 'efferty et at. W () 02 '0 1 04 7. and ( a ton and kopmwak u Pnsc. Natl Acad. Vt. I o \ S7:M$0*M5t (MdO). each of which is incorporated herein by reference in its entirety 11 he amibodifes £fod«ced% ;jp|iige .technology arc usually produced as antigen binding thtgntsntx e,g. Fv orFlibiragmeirts, m bacteria and thus Sack effector luneherw ΓΠΜ tor fnnerxms van be introduced by one of two strategics. The fragments can be engineered either:&amp;jn.-po«itf 1¾adMbodfe-iiti· expression in nunnnabao cells, or into bispeeiilc antibody ffapttents with a second binding she capable of o kgering an effector function. (00200] f\p\ J \. th. FJ imgniem V c~iι. * 1 1 b_’M i bam (V{-C; )of anObrnhes are separate!} cloned by I’f'R and recombined umdomly tn combinatorial phage display libraries, v\ Inch cm: ihen be sMemod tor binding to a particular an is gen. The tip|ibody irsgmunts arc expressed on the phage surface. and selection of Fv or Fab (and me; wore the phage containing the DIVA encoding the antibody fragment) by antigen binding is accomplished dirongh several rounds of antigen binding and recur d v.atse a nroeeujre ;e ; a J mir nr>e \ "Λ ; · erw -ί i V \>i the ·· anugei» a;c enriched and finally =*okted.

[00291] Huge dtsph'.v techniques van also no uvd u' an approach Im the burn animation ofrodetu monoclonal antibodies, called "unuied select ion ’ (sec .kspers, L. S., et ah. Bso'Technology 12, 899-903 {1994η. For this, the Ml fragrheiW o* the mouse monoclone, .mtihodx c.m bt displayed sr combination wh a human 1 gin cha n Tbmry, m d Pv iesuh'np hy’end Fab ubnnx mas "hen be w eeti J oPb antigen, t he mouse Fd fragment thereby pros ides a template to guide the sdeetiofe Subsequently, tiv selected human light chains a c combined with a human Fd fmgmen ibmn N- ee* oa of the em.t ng iibta ^ yoc 4- 0Ή t ;4> human bah [00292] A variety of procedures have been desert bed for deriving hitman antibodies from plmge-displsy libraries (See. tor -example. Hoogenboom et al, )< Mol. Biol.. 227:5*1 11991}. Marks et al.. .1. Mol. Biol. 222:581-50? f 199 D; U.S. Pat. Nos 5.565,332 and 5,573,905: Clackson, 1., and Wells, .1. A„ TIHTKCH 12, 115-184 ϊ Ν04» In particular, m vstm si. lection and curiutme of mm xsdics dented from pnage<.;"-p ,n ubniOON nas xv --m.. i raxe tu *ooi (Nee button, D. R.. and Barbas III. C. F., Adv. Immunol, 57, 191-280 (1094); and. Whiter, 0 . et al,, Auou Rev. Immunol. 12, 433-455 (1994}; U.S. patent application no. 20020004215 and W002/01047; l/.S. patent application no. 200 HH 90317 published October 9, 2003 And li.S. Patent No. bjOSdSt?; 0,8, Patent Nos 5,877.293, [00293] Watkins, '“Screening of Phage-Impressed Antibody Libraries by Capture

Lift,” Methods in Molecular Biology, Antibody Phage Display; Methods and PsotocoblfN v Patent hpp.ic non Pn niea ou N.' 200^0044772 published March 6, 2003 describes methods for screening phage-exptvssed antibody libraries or other binding molecules by capture lilt, a method involving immohilimboi· of the e.m Jicktc binding molecule- on a -olid support, : [002941 Other Bmhooi intents of Antigen .bimtmu nroteins: Anilbodv. Pmfflients: [002951 \s noted ,mo\e. antibody hagmeuis eomptise a portion id an intact full length antibody, preferably an antigen binding or variable region of the Intact antibody, and include linear antibodies and multixpecific l|§a|$d irorn

antibody examples of antibody Itagments Inelhde Fab, FabV 194092, Pv, Fd. dorhaln antibody (dAhJ, eopipIdmeptqAw detcrinining i'egirni tCDR) fragments, single-chain antibodies i.seFv), .single chain antibody fragments, maxlbodies, diabodies. tifahodlex, i^abd^^'hsiftibpdics, lineaf antibodies, chelating recombinant tMtibodies, tribodies dr bibodics. intrabodies, nanqhodieS, small modular immunmris. urueeurit {νΜΙΡχ, an a nixo-hm i.oj-eoimm

Anion protein, a:o4i:»#kcd::ainii>ody, a VHH containing antibody, or route t«s or derh utiyes thereof, and polypeptides I'mi contain at least a portion of an immunoglobulin that is sufficient to confer spec;in antigen hind mg to the polypeptide, Midi as a ('DR sequence, a,·» long a\ the amthody retains the desued biological actB%,: produced by the tpodtiAcailhhof whole antibodies or synthesized he nova using recombinant DNA technologies or peptide synthesis, [00296! Addibosmi «uiib0|y;fiagrodnes:|M«de a domain antibody fdAb} trngmeat |t¥ard et a!., Aatutv 341:544-540, 1989} which consists of a-Vu domain.

[002971 ^idnenr antibodies^ comprise a pair of tandem Fd segments <Vt} -Cu 1 - V o · Cid t winch lotro a pmr of uotigui lord toe legions I meat iumbodn"*«. an pc bispocific or monospecific (Zapata of ai Protein Eng. 8:1057 62 (1995 )).

[00298] A 'htsinihody" consisting of sold titled to CH3 s tu a peptide hnkev thtngckss'i or \ ia an IgG hinge has been described in Oki'sen. ct ai, Protein Eng Des Set. :(K)4 Apr;r?{aVJIf-23. {002991 The temt "maxilnniy'’ refers ιο bivalent scf'vs coxaienify attached to the 1 c me or omm m hu,i>_k> tt n >v<. ΐη·*Λ* ip e, 1 eocrs.V'ct t iboten Engineering, Design &amp; Selection. 17:95-100 t20()4) and Powers ct ai„ journal of imnnmoiogicai Methods. 25 s;123-135 (2001 )♦ [00300) Functional heavy-chain antibodies devoid of light chains arc naturally 0 Ui”! i * m <. stt t pc <.s dm t'ts uh*on s, '-h hs > ve^ongs vs a•'d (VurA/ os such as car: so is. duimedanes. uipacus .ash llamas. The aoitgen-blndmg site -s reduced to a --regie dorr A re the VH-.j domain, in these animals. These ammo dies form antigen-binding regions using only hears -, ch^in \ enable region, i.e.. .these functional antibody are homodimers of heas'x chains only having the structure H;:tv irefeoed to ns “heavy-chani amibodies1' or ’*HC. Airs'"). (Vm-elhred Vuu 1 reportedly recombines o uh DG2 and GG3 constant regions that contain hinge. CH2. : and CM3 domains and lack a (.’Hi domain. Classical Vmoob i augment- am difficult to produce in soluble form, but improvements Insolubility and specific binding can bo obtained when framework residues arc altered to be more VTifi-hke (Sec. e.g., ileichman, etal, j Immunol Methods i 900,23!:25--3H.) Camelmed Vhh -domains have been (bund to bind to antigen with high: afinity (ISeam^r et nbs <b AM/. Che»’. 27t\2e285-90, 2001) and possess high stability in solution (Owen et at. 8hchwih:$y'3 1:3628-36* 2002), Methods for generating antibodies baving camelked heavy chains are described in, tor example, mUS. Patent Poblicaddn Nos. 2005/0136049 and 2005/(1037421, Alternative seaftdids can be made fplm In e-b\i, Oomarns unit rntnv elovh match the shark V-NAB .scatlbldand may provide a framework for a long penetrating loop structure.

[ 003011 Because the variable domain of the heavy-chain antibodies Is the smallest, inlly functional antigen-binding fragment with a mblectdat mass of only IS ·®η,; dtls: entity is referred to as a uanobody -btdi,*. 64.28S3-37, 2004). A nanobody library may be generated from an immunised dromedary as described in Conptb etab, piniimteroh Agents CkmitHher 45:280?-· 12,, 2001). 100302 ] iniruhodks are single chain antibodies which demonstrate intraeel ltdai expression and cars, manipulate intracellular protein function (Bioeca, et a I , EMBOJ. MO! 10M 1090; Colby ct si., Fn*. \«?i Acad Sci V S A. 101:17616-21, 2004b intrahodies, which comprise cell signal snqnenees which retain the antibsssly contract nuntraeetlular jegions. mav be produced as desetshed m Mhashtlkas etal {LMBOJ 14:1542-51,1995} and Wheeler et el. (FASEB J. 17:1733-5. 2003}. Transbodies are celt-permeable antibodies in which a protein transduction domains (PTD) is fused with single chain variable fragment fseFv) amibodies llcng ct al, (MuiHypothese-si 64: Π 05-8, 2005).

[003031 further encompassed by the invention are antibodies that ate SMIPs or binding domain immunoglobulin fusion proteins specific for target protein. These; constructs are single-chain polypeptides comprising antigen binding domains fused tb to cany out. antibody effector functions. Sec WC5p3/lMX!&amp;C>p;: :οομπ ivm:. )00304 \ .> m, -< ue’numn -I axe bee \ dm- cloned Fv Tse povuetsoa of emduKiy itegme<'\' I s format^ . Hies, fragments xxcm derncd x.e me'coiyttc digestion of intact .mhbomos Jmt can also be produced directly by recombinant host cells See, for example. Belter el a!,, Science 240; 1041-1043 i lOSS s; Skesra et a!. Science 240: 1 038-1041 uOxk); Carter et ai., Bio/Technology ID: i 63-1 o? <;1602}. !P3#J liliSLjiiMfc [00306} In mme embod:mvm.% it may he desirable m eeru-mte unbox., ;em ot oxen ά mulnsmecfoc to g, bispm dux inspect tic, etc \ monoclonal am: body. Such antibody may have binding specificities for at least txvo different epitope* of the target ami gen, or ai tentatively it may bind to two diucreni molecules, e.g to the target antigen and to a cell surface protein or receptor. For example, &amp; bispecitte antibody may include art arm that hinds to the target and another arm that binds to a triggering moiecade on a leukocyte such as a. T-celi receptor molecule {e.g., CD2 or CD3), or Fc receptors forlgO (FeyR), such as FcyR.I (€064), Fc^Ril (€032) and FcyRJO (€016) so as to focus cellular defense nieelanlsrns to the target-expressing cell, As ; another example, bispeetde antiibotlks may be used to localize cytotoxic agents to cells which express target noiigen. These antibodies possess a targoi-bmdmg arm and an amt which binds the eyfoloxie agent (e.g·* saporin, anti-imerfcronfoO, vinea alkaloid, ricin Λ chain, methotrexate or radioactive isotope hapten). Vluitispecific antibodies can be prepared as fall length antibodies or antibody fragments.

[1)1)307} Additionally. the anti-DNF or anti-KLH antibodies of the present invention can also be constructed to fold into multivalent forms, xliieh may improve binding .rfon'.s. μκ„γχο^ and hr increased hutl'-nfe m cloud Muitf\a«etu townx of hnii-DlNP or ami -K ι H can he prepared by techniques known m the art.

[0Θ3ΟΗ] Bispecific or nmltispecifie antibodies include «,mss- lucked or ?Telerocor:jtsgfoe!t antibodies. For example, one ot the antibodies in the hetesocosiugatc can be coupled to the other to hmizm Beteoicooliigete antibodies may bo made using .any .convenient cross-linking methods. Stumble crovs'h'ih'ne agents ate λ til ho mt tr. do art o d ate doJe^nl m t ,S, Pat No. 4,676,9*0. along with a «\nrbet of. .osv» taking technic. os Wlhet method tt* dessg'Xi o ii. o .hi i\ by .κχ n ^ *. o q-ee·. >d.n~ecv n. -.equence at the l · terminus of the sc Fv Sueptavidin is composed of four subu nIts, so whpttthc scF\ -•strepur·...1 n .v iolded, iou; vidimus associate to form a knarnot {Ivgnyunm _t a! Hum Anobodio. Hyb.ieotr.ts 0(31 ,->7-Ι0Η ί*#5k oivhxurc otViueh H incorporated herein by reference in its entirety).

[00709] According to another approach for making bs specific antibodies, the ioterfhoelietwecn a pair of atdlbody tttolocoles can be engineered to maximize tie percentage oflciemdouers widen ate ivcovet ed horn recombinant ceil culture One interface comprises at least a pm of the Cu'3 domain of an-antibody constant domain. .In this method, one or more small amino acid side chains .from the interface of the first antibody molecule are replaced n ith larger side chains le.g.. tyrosine or ;; tryptophan j Compensatory ''cavitlef - of identical or similar size to t he large side chain thd arc creeled on the imereace of the second .'embody molecule by replacing large amine add side eh,.mv> with smaller oncx te.g , alanine or threonine}. This provider a mrvh.nn'ao sot mm easing the weld of the hekaodimer over other unwanted end-products such us horaodimera See WO -b 2 ?0I 1 published Sep o 1996.

[00310] Technique for generating bispecific or multispecific antibodies from antibody fragments have al.vo been described m die literature. Fot example, '"bispecific or trkpeeifie antibodies can he prepared using chemical linkage. Brennan >_: J . Semrx. 33 t st ! HxfT dcsetxbo a ptoeedate where n mam ...n bodes an. pro.eok ted v c w <. J m gem.» e I (ah')·- fie ament·. I se uagne-tv am *e ceJ i r the presence of the dithiol completing agent so-,bum arsunite to stabilize vicinal dtthiois and prevent intermoiecuUtr disulfide terms bon. The Fab' fragments generated arc then converted to thtoniuobenzoate (TNft) derivatives. One of tkb I ah!-ΓΝΒ derivatives us then reconverted to the Fun-thiol by reduerson with tKe^aptoetiyiaaiine and is mixed wlh as equimolar amountoffdb-TNB derivatke to form the bispecific antibody. The bs specific antibodies produced can be used as agents for the set active immobilisation of enzymes. Better ct ah, Science 240, iu~t BIO-3 { B>k^ ) dis<. fo.xe see-unm of Fnnetvmd anfiboJ) fragments fonn buetcod [stt, i\g, Beum e- ah. Sect a et ,fo Scienci. 240. Ϊ03b-5041 t >>NnV» Tot example, EabbSB fragments can be directly recovered from £. cob and dsermeaity;:;| coupled to form hispeeific antibodies (Carter et ah. Bio/Teidmology 10.163-lb? (1992); Shalaby et ah, i. Exp. Med. 175-217-325 (1992)).

[00311] SMiahy et ai i, E\p. Med, I ?5:2P 2251 ί092> describe the-production-of a fully humauiited bisptxiik antibody Ffah’l,· molecule... Each Fab* fragment was separately secreted from f2<sd/and subjected to directed cteotiealfooitpling in vin o d0ijfe^Ote;Sispec.fle-at>dbody.

[00 312] Various techniques for rhafeing uud isolating biapecitio or myliihpcelBe a n body +i ^merts ducufo bom tceombinuni cell culnae fuse eKo been descnbed For esampk. Hspceifte rippers, e.g UUN4. (See genet ally Kfoytdm· et ab. j. Immunol. 148(5 y: 1 547-4 553 {1992t.} The leucine ripper peptides from the Eos and Jun proteins wore Imbed to the Fab* pomows ot two diffcicnt auobod-es by gone fusson The mmbodv homodimens wore reduced utfhe hinge region to htm, monomers:-anftt|M#-#xidizcd antibody hoterodimers. This method nan also be utilised tor the product ion ot a ntibod y homod i mers, [00313 j Dmhodtes, described ahos e, are ope example of a bispecific antibody,

See, forexaPipiefifoffmger eta!,, Proe. biaib Acad. Set. USA, 90:6444*644$ : Biyaient diabtuhe·, cun be stah||iued by dihitlidd; linkage., [00314] Stable monospeuik ci nupemfe Fs tetmmei\ c.m also be gaimtort b\ ihopcdvulepiassociation in (scFv ,4-, configuration or as bA-tetrabodies.

Aite.oatSxely, two J-tv c >' scFs ·>, an be ,ο,ηο 1 to tandem to toon a hts-scFx [003151 Another strategy for making bispeciftc ^ntii'ociy fragments by the use-of 1 sirrjc-riiumt \ ml \; dm, ms ini-- ,nso been repo* Ad V, snubes et J ' tnnmnot. 152: 3368 (1794;, One Approach 'has 'been to Sink two seFv ami bodies with linker or dw JAJe 1ό\ΝΛ1>.4 enda: and Yus*, ,f Bud Chen* '«<·) s09~2u61°a., WO 94 13x06, and U.S. Patent No. 5,989,830. the disclosures of which are mcorpofated '•herein by reference in their entireties},;: [00316] Λitemarively, the bispccific anuhodv may be a 'linear antibody" pmdobed: as deaern'ect in Zapata et al. Protein Hug. Hi 10;·: 1057-1062 i 19s>5). Briefly, these • antibodies comprise a pair or tandem Pd segments fW a'o!»Yh 4'Vl> which pmt of assLasP b'nd'ng tepmos ( !ui am so ln> t an -, bispt m \ ot monospecific.

[003tT'· Antibodies w,;J, note tu.m ou" Jeuetes r< cko cum, mplated For example, trispecittc antibodies can be prep:',red. ffou ot at., J. Irmnonoh H?:60 1IWM >}.

[00318| A “dtdaiijag recombinant antibody" is a bispccific antibody that teec>gdpes adjacent and non-overlapping epitopes of the target antigen, atti is to bind to both, epitopes simxiltamxuwly (Neri et al.../ Mot Biot. 246:36^-73, i sOs s [003 i 9j Production of bispeeifie Fab-seFv ("bibody") and uispedfie Fab-fscFv )(2) {'"irihody" s arc described in Schoonjans et at. {JJmmmot. I op :7050 -5 7, 2000) and V\ iileois ct at. U ('hmmaiocr B Athilyi T<?vhw>i Bhmed / h be?', 780:161-76,2003). I os bibodies or tr 1 ndies, a \ch\ molecule ;s fused to one or noth of the VI A 1 (Π a-'d VH-CH, (Fd) chains, e g„ to produce a tnbodx ore w fss are fused te> C-tcrm of Tab while in a bibosiy one seFv is fused to C-term of Fats. {00320] In yet another method, dimers, t? biters, and -etramers are produced after a tree eystem * o introduced »n the pan mat psmvm Λ peptide-baaed cross tinker with: satiable omubem (two to fours of nmleimide groups was used ns <. ross link the prpteisi of interest to the free cysteines (Cochran et ah. Immunity ; 2(3>: 241-50 (20000 .he J'v ovum o*'vri u a ,, os, orpof ted Ινκ ns m is en*n, o) m$mi |av£at&amp;i antigen binding pmtelp^1^^ pcpwbodie^. The term; “pcptihody*'' refers ro a molecule comprising an antibody Fc domain anached to as least one peptide. The production: of peptibbdics is generally described in HfT publieationvW6''#/24I^^ ihiHS^ May T, 2000. Any of these peptides may be linked in tandem ; e, sequenmdB ), w tth or -a itho,e linkers Pepnoes containing a otsteinyl residue ms> he ctO-s-fuked \sit.i anofhe! Cys-eontainmg peptide, eitheror both of wInch n,e he mhed to a vch,e e Vas pen ve ' m ng ,ro.e than one C>s "residue ttbay fepi an intrapeptide disulfide bond, as veil. Any of these peptides may be derwatiyed, tor example the carboxyl terminus may be <. upped with an amino groupeeyetetnes may be eappuyor aminp adM residues may substituted by moieties other than urnino acid residues (see, e.g., Shat eager ct ah, j. Med. Cbom 30; 3h 14-9 (! 996), and Cut Phan son et a!., j. Med. ('hern. 40 2H 70-82 i1997 ), winch are incorporated by .reference herein in their entirety ). The peptide sequences may be optimized; analogous to affinity maturation lor antibodies, or otherwise altered by alaninespanning or random or directed mutagenesis followed by screening to ddtenttfy.ihe best: bride* \ logman, \r * ike* BmpK's Bsomui Sued 3e AM-2-* (1997). Various molecules can be inserted into the antigen binding protein structure, c.g,, within the peptide portion Itself or between the peptide and vehicle portions of the. antigen binding proteins, while retaining the desired activity of antigen binding protein. One can readily insert, for example, molecules such as an Fc domain or Iptpnmt :ihensic>f,-;p0lyetityl.e«e glycol or other related molecules such as dcstrun, a fatty acid, a lipid, a ehdleaterdi gmup, a sntail carbohydrate, a peptide., a detectable moiety as described hereby (mcludlng fluorescent: agents, sadiolabels such as mIioi^d0pe^w^%o^pdteid0g0%o«ucl<»lide,-8 polynucleotide, interference {or·-Other); EM A, enzymes, honnones, or the eke Otises molmdes sunable toi insertion in this fashion will be appreciated by those skilled in the art, and are encompassed within the scope of the invention. This includes insertion of, for example, a desired molecule in between two eonseotdiyo amino acids, optionally joined by a suitable I Inker.

[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 ah, 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.

[00324] 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 necessarily, the amino add residues in the linker are from among the Aveng, canonical ammo acids, mure preienibly, eyases;sc, glycine, alanine, proiine. asparagine. glutamine. and or serine. Even more preiernhiy, a peptidyl linker is reside up of a masm'lA of son;rso adds that are ate-maliy unhindered, such so- glycine, .serine, and alanine linked by a peptide bond, l! is aKo desirable that, if presents, a pepridyi linker be selected shat, avokls rapid proteolytic turnover at circulation m ΐ'Λ'ο, Some of these anotio acids may be glycosylated. as sa well ossdes'asOfKt by those i?; the art. For example, a useful linker sequence constituting u «ahyknion site is Xi X >NX,;X; Q {Sl\Q ID NO' 14s), wherein X i. X;.X ; and X * arc; each independently aiy mpino aeid residues [00326) In other embodiments -ifec > to 40 amino acids of the peptidvi linker moiety are .selected front glycine. alanine, proiine, asparagine, glutamine, and lysine, Preferably, a linker ;s made up of a majority of amino acids that arc Dentally unhindered, auel; as glycine and alanine. Thus, preferred linkers include pisJygXeines, polysermes. and polyaianines, ot combinations of any of these. Some - exemplary pent idyl linkers are poiy{ Giy); x particularly tOlyh, tGlyg (5EQ ID NO: 1401 iGiy,k (SE.Q ID Ν(>· 15(0 and (GlyMSEQ ID NO· 1511. as v\dl as. poiylGlyuScr (SlfQ ID NO; 152h poiy{Gly- Alu.j.'.; and poiyt Alwh.,. Other specific examples of peptidyl linkers include (Giy gLys ID NO; 1541, and tCly td.y.sArg CSEQ ID NO; 155). Other examples of useful peptidyl linkers are: Other examples of useful pepiidyi linkers are:; [0032“) (GiygLyxtGiy}.: iSFQ ID NO:159s; [003215) (fjiy} < AsoOlyScrtOl y}.· sSEO ID NO: 156); [00329) {GiybCysiGly).; (SEQ ID NO: i57): and [00330) GiyProAsnGiyGty <SF.Q ID NO: 15k). 1003 Ml So explain me above nomenclature, for example, (Gl\ )d,y\ftih },, means (jiy-Gly-COy-l. ys-Gly-Gly^rtly-Gly (SFQ ID NO;15Q> Other ivtabinaHons ofCry and Ala are also useful.

[003321 Commonly used linkers include those which may he Identified herein Ί.3'' sGGGGS; or “GilT; SEQ 10 NO; 152k Ί10" iGGGGSGGGGS; SFQ 1q NO. 153k *125" (GGGGSGGGGSGGGGSGGGGSGGGGS;SEQ ΙΟNO: 146 > any linkers used in the working examples hereinafter.

[00333] In some embodiments of tV compositions of thm moem-om winch comprise a peptide hnl ermmeiy. uefdu resumes, for example. nhuarnun or aspartate residues, ;nx placeo m the am-no acid sequence of ftt: lather moiety. Examples include the ίο How mg peptide imker sequences; 0$MI GGEGGG iSEQ.ID NO: 1003; [005551 GOEEEOGG tSEQ ID NO: 161 k [0055ft] GF.EEG fSEQ ID NO; I ft.2k 163); : [003.3k] GGDGGG (SEQ ID NO: 164); [003391 GGDDDGG (pQ B 140:105¾ [00340] GDDDGCPQ 1010:10¾ [003411 GOOD (SEQ ID NO: 167k [00342] GGGGSDDSDBGSDGEDGGGGS (SEQ ID NO: 168); [00343] WEWBW (SEQ ID NO: 169); [00344] BEFEE (SBQ ID NO; PG k 100545] Eft ft WWW (SFQ ID NO. ml), [00346) ;plE6l# :(1¾ IDΜθΜ% [0031¾ : WWEEB^W [SE§ p NO: t73); or [0034k] EFEHi EF (SBQ: p N Q; 174¾ [00349] in other embodiments, the linker constitutes a phosphorylation site, e,g.. X \ 3 Y»XA> OBQ ifi \ό 1 ), Vvherei" X', Λ X, ,r-d X, are vie" ι Λ · s.rd,. e > any srnj.no acid residue, X.,X;-SX ;XA I )Sf.Q ID NO:170h wherem X-, \.>JG and X> are each mdependentty any amino acid residue: or Xpv 'TX.5X\G (SKQ ID NO: 177), whet cm Xu X», XX end X^ aie each independent!) am amino add residue.

[00 >50! The linker·» *hovui here ere e\uup»u?y, pcpodyl hnko:-< within rise vooe of this invention may he much longer and may include other residues, A peptidyi linker can contarm eon, a ey'vte/ie, auuil .*» ruo> «rv »>, eeeh :e V eorjugatu», vuth u halt-life extending moiety. In another embodiment, the linker contains a cysteine or hompdytddind mptlng, or other Tammo-etlumcthioi or Isamind-pPspanethlolmoicty Ibr ponjngntifin to |dalehnides iodoneetaam Ide or -half-life extendin g moiety.

[00351 ] Another useful peptidv I linker is n large, flexible linker comprising a example: GSGSATGGSGSTASSGSGiSATB pEQ MaXO:i7ld6r GGSGSATGGSGST'ASSGSGSA1' (SEQ ID NO: !'?<», that is ; estimated to he aland die as re of a I ki>a PEG moieude Ainu namely, a useful peptidyi linker may be comprised of amino acid sequences known in the art to form rigid helical structure* te.g,, Rigid linker: - A E: A A AX.E A A AX FI A A AK AG<X}(S E Q ID ]NG:iS0)< Addaidoally, a peptidyi linker can also comprise a oon-peptkiy! parhdn aliphatic molecule of the formula -CH yit^o'^^^dyilipkei^ cm be altered to toiro derivatives ns described herein.

[ 003521 Optionally, a non-peptidvl linker moiety n also nsetuHor conjugating the half-life extruding moicty-'fo the peptide port sou of the halGltth extending moiety- conjugated toxin peptide analog. For example, alkyl linkers such as -NH-(CH2)S-C (0)-, 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., C|-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. Exemplary non-peptidyl linkers are polyethylene glycol (PEG) linkers (e.g., shown below): [00353] (I)

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 form derivatives in the same maimer 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-terminal 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, Cm haloalkyl or halo. - [003571 Tldeteroary Γ? is m 5,0 Or 7 kembered monocyclic or partiaHy~5>^tate4W ««datur&amp;ieiiS~*7A:&amp;>'^·|0*-ox.· il niembcrcd bleyclic ring, wherein attiast oa^Sng:tS''e^lira.tf^die monocyclic and the bicydie rings (. oniasioug 1, 2, 3 or 4 moms selected froni N, O and S w hi u m the * ay 5-, substituted by 0. 1, 2 ot 3 subsoments selected Arm. t' s JkyL 1' t hukxbssl and halo.

[0035b] Non-peptide portions of die inventive composition of matter, such as non-ppptidyi linkers or non-peptide hal Alife extending moieties eonvesiOonai organic chemistry reactions.

[00350] the above is merely i llustrative and not an '.exhaustive treatment of the kinds of liakets that eagfoglioM in accordance .with the presedf invention.

[ilOaOOl As noted above, recombinant PNA- .md-or RN A-mediated protein.·expression and protein engineering techniques, or any other methods of preparing peptides, me applicable ίο the making of the inventive compositions For example, polypeptides ean be made m *r*ms,o,'i\\i ns cells Bs <<r'- . unombmatU DX A mm 1 ak\ 01 construct,, coding for tite peptide is prepai'cd. Methods of preparing meh DNA molecules arc wed known m the ad, l or instance, sequences encoding the peptides can be excised thornIMA using suitable restriction enzymes, Any of a large number of available ami well-known host ceils may be used in the practice of this invention. The selection of a pamenlu host is dependent upon a is umbra of laetos s ucognircd by the ark These include, for example, compatibility with the chosen expression vector, toxiuty of the peptides encoded h\ the 1ΜΛ 5 so c^u mie u t urs'o n us er ease o*' recovery of the peptides, expression -,. .emu wrevscs, bio-safety and costs λ balance of these faoiots must fed dtruck with the understanding that not all hosts rn&amp;|-:%.θφδί% effeetiivl'"^^· the 'exprepion of a particular ONA-^equence. Within these general guidelines, useful microbial host cells'in culture include bacteria (such as Eseherlehia-pIiajllyyeMt:(such as ||e0||roniyoes $p,>#d r>ibe|impi: cells, insect 3' 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 maybe 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 Fc 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 ah, 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 incoiporated 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- .Vdrtafits are prepared with the intern to modify those amino acid residues which are directly involved m epitope Unking. in other mnbodirrenia, rnodincntiori ofyoudnes which arc not uitvaK involved 1st epitope binding or jesidues not involved =n epitope binding in an\ o a), is desirable, for purposes discussed herein AlnragenevU w Ohio any of die C'DR regions arui or framework nektons o contemplated. i "o\ ananee analysis techniques can be ere,ployed hy the skilled asm son to design useful modifications m the nrrnno add sequence of the antigen binding protein, mein ding an antibody os' antibody fragment. (E g., Chottlieo e? ad Covananeu Analysis of Protein f am dies. The Case of the Variable Domains of antibodies. Proteins; structure, Function, and Oeneucs 4 1 :·-Ρ5--4κ4 (2000); Demurest cl ah. Opuoiigutio» of the Vntibody if; A Domain by Residue Prctjucncy Analysis oflgG sequences, i Mol. Blob 305:41-48 i2004); Hugo el ah. VI position 34 is a Ley determinant for the engineering of stable antibodies with fast dissociation rates. Psotdn L'ngtneering 1ό(5);3Χΐ Βίο 13003 s, Aurora ct ah, Sequence covanunec networks, ntetisofls aisd uses i hereof. US 2008 '031 N207 A11 (a las or et. af., Stabilised poly peptide compositions, US 21)003)0-48122 Λ1; Uredt et ah. Sequence based engineering and optimization of single chain antibodies. WO 2008-110348 Λ i: Bonus ctaL Methods of modify mg antibodies, and modified antibodies with improved functional properties, WO 2000-0000^ Λ2). Such modifications determined by covariance analysis can improve potency, pharmacokinetic, pharmacodynamic, and, or matuifaeiumbility eharattenstks of an antigen fund mg protein.

[0()3:,2] Nucleic acid molecules encoding ammo add sequence variants of the anngen binding protein or antibody are prepared by a variety of methods known in the art. Such methods include niigcniucleotide-mcdiated (or site-diteeted) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant: of a ooiuvarianf version of the antigen Binding- protein, [330363] Substitutional mutagenesis within any of rhe hypervadsble or CDR regions or intniework regions is contemplated. A useful method for identification of eertain residues or regions of the antigen binding protein that an; preferred location* far-mutagenesis is called 'Viamne scanning mutagenesis,'1 as described by .Cunningham- and Wcl Is Science, 244:1081 S 0H5 (1080). Here, a residue or group of target residues are identified te.g,, charged residues such as arg, asp. Im. iys, and gM) and replaced by a neutral or negafiyely cMrged amine add (most preferably nlaMne or poiyglanme} ίο affect the inipmehon of tfe fepipo adds with antigen Those amino acid locations licmrmslnuing functional sensitivity to the safextii utions then are refined by idtsajdneing further or other wants at or fop^tbe siiea of .substitution. Thus, while the site for introducing an amino acid sequence variation sx prexietetmi ned, the nature of the mutation-per se need not be predetermined. For example, to analyse the performance of a mutation at a given site, ala somnmgor random mntageneaid isconducted at The target codon or region and the expressed variants are screened lor the desired activity [003641 Some embodiments of the antigen binding proteins of the present invention can also fee made by synthetic methods. Solid phase synthesis is the vprstfmod technique' dftmaktng individual peptidessince it is the most cost-effective: method of making small peptides, for example, well known solid phase synthesis techniques include the use of protecting groups, lintel, and solid: phase supports, as I wed as spec die protection avid ueprolecfeon linker cleavage conditions, use of scavengers, and other aspects of solid phase peptide synthesis.

Su cable techniques are well ! now o in the art. t E.g , Morn Held (i 623 h Chetn.

Pols peptides, pp. .fo>.o ; ;Kaiso\ annU and Panayons cds.). Mernfidd 1190.1), j.

Am. I in.m. Sec. sf, 2149; Davis et ,4. (,1983). Bioehem. 1ml. 10; U>4-414; Stewart ,iitd ) oeug (lonOj Solid Phase Peptide Synthesis; l N Pat. No 3,941,70.)·, Finn et at. {P*"6), The Prod:ins (3rd ed.) 2. 105-253; and Erickson et al (1 Q~61, The Proteins (3rd ed.) 2' 257-52?; ’"Psofedin.g Groups In Organic Synthesis," 3rd Edit fern T. \V. Gre-nn. ,νκί P. G. M Wut.s, Eds., John Wiley 5¾ Sons, Inc.. 1999, NexaBfoehern Can log 1Ό00; "8s i tlvtie Peptides. Λ User’s Guide," G U Grant, fed , V\ H Prcentv.i ά t o v.pans, \.m 3 or^, \ 3 , 'u2, " Vetoed C e ate.

II r dfeook off ot'(bm..'oi al &amp; So'.d Phase On suv Uhem's-'ts, Μ O Bonnet J W Ob'istensen. t , K H a maker, \ I L Peterson. Μ R Rhoces and Η H banco. Us, Advanced Chemroeh. W98; "Principles of Peptide Synthesis, 2nd ed.,” M .Bd&amp;mssky, Ed., Springer-Ycrlag, 10¾; ’’The Practice of Peptide Synthesis, 2nd ed.T M. Bodanseky and A Bodar^/ky, Eds., Springer-Vertag, 1 bv-E ’'Protecting Groups," P. J. Koeienski, Ed., Georg Thic-me Veriag, Stuttgart, Germany, IAN; 'Tmoe Solid Phase Peptide Synthesis, A Practical Apprcf<n:h,’! W (2. Chan and P. B While, Eds,, Oxford Proas. 2000, G. R. Fields ei ui, Sy Whet it: Peptides: A User's Guide, i SPO, 77 -! H3t. For iiutiicr examples of synihenc and pund.eation methods known in the art winch are applicable ιο making the inventive compositions of matter, sec. e.g . Sullivan etak Toxin Peptide Therapeutic Agents, US2007 007176-1 and SuHiYitri el ak. Toxin Peptide Therapeutic Agents, PCT.ThS2007 02263 i, published as WO 20(18 (188422 A2, which are both incorporated herein by reference m the:r entireties. jOCOtG] In flirt he! describing my of the antigen binding proteins hetein, a-' noli m wtoaufs, a one-letter ah dies muon -0-. stern rs heuueuby applied to designate the identities cd The twenty 'V.monieai" ammo acid residues generally hwornciutfod ntto nntu tally occurring pepwies and protects (1 able 4 s. Such une-leiiet uhbres miioes etx entirely irm: wh a nge. hide nt meaning v, oh titree-letter abbreviation's or now abbreviated arnino acid names. Wiihm the one-letier abbreviation system used herein, m upper case letter indicates a. L-amino acid, and a lower case letter indicates a D-arni.vto ac td. for example, the abbreviation TC’ designates 1..-at gin me and the abbreviation Ύ' designates D-argmme.

Tabic 4,. Qrtgdgtfer ahhreviaikms for foe eikioniektimilnb addsc

Thwe-ietrar abbreviations arc In parentheses.

[00366] An amino acid substitution in an amino acid sequence is typically designated herein with a one-letter abbreviation lor the ammo amd residue to a particular position, followed by the numerical amino acid position t clause to an ononud .sequence of interest, which is then followed by the one-lcuor symbol tor dee amino acid residue substituted in. For example, “ V30D" symbolises a substitution of a threonine residue by an aspartate residue at anti no acid position 30, relative to the iuu-mni sequence of nOcre-n Another example, " W101Γ” symbolizes a substitution of a tryptophan residue by a phcnyLbuninc residue at amino arid position 101, relative to the original sequence of interest

[003610 Non-canonion; ant «no acto residues can be incorporated into &amp; poiypepride within toe scope of the invention by ero pi ovine known techniques of protein -engineering that use recombinant|y expressing cells (See. e.g., Link ei at, Non- canonical amino acids in protein engineering, Current Opinion In Biotechnology. 14(6):603-600 (3003)). ’the term "non-eanonieul amino add residue" rotors to amino acid residues in D- or L-tbrm that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins, for example, [hamtoo road-., homoamino acids, cyclic am«no a,c:ds and annuo acids with denvati?.ed side chains. Fxamples Include (in the t-form or t>-form) p-hamne, jfourenopt'oplonie acid, piperidiuie acid, aminocaprioie ae;d, a:rsmohcpU;noic acid, amirsonimehc acid, dosmosme, diaminoplmciIc acid, N" -ctnyigive iue„ N''-et!ryiaxpargme, hydroxy 1 > siuu, stfo-hydroxvivaete. hodesrnosirtc, aifo-tsoleuchtc, (U-mothylargimne, N“-methylglycine, N'l-methyKsoleueine, Nf'-mi'thyMUmg, y-carboxygiutamatc. ^ Ν.Ν,Ν-trtmctbyllysincy u-No.icctyllysittc. O-phosphoserine, Nv-&amp;cetylf>ei»ttc. N‘'-formy!mcthiomnc, 3-methyl histidine. 5· hydroxy lysine. and other sirnilur amnn^' eudv aud those bated in Tabic 5 betew . aval dematired fbmts of any of those as described herein. 1'able 5 contains some exemplary non-canonic»I amino acid residues bub arc use ini in accordance with (he present itnetbiou and associated abbreviations as typically used herein. aitboifcji the skilled practitioner will understand dm? different abbreviations and nomenclatures may be applicable to the same substance and appear imercbao^eabl) iserein. fuble b l Vfnl non-‘..unome>ti amino acids Urn ammo ac;d addition, insertion. or substitution into peptide sequences in accordance vote -he present -mention in the m.-nt at; abhrerLmot! h-bed m Table 5 dbVem from amithet ubbrestatton tor the '-vene vubsbnn c disclosed cKcnhcre heroin both abbreviation^ are understood to be applicable, I be amino aesds hsiec! m Table 5 can he tn the t -torm or L)~fomu

[0036H] Nomenclature and Symbolism for Amino Acids and Peptides by the UPAO-lUB Joint ( onumssion on Biochemical Nomenclature (JCBN) bas e been published ία the following documents: Biochcm. .L ION. 219. 3-15-373; Bur. J. Bioehom., S9mh ) 38. \ΜΆ 19x5- 152. I; 13193, 213. 2; {menut!. J, Peps Pro?. Res.. 19H4. 24. following ρ 84; J. Biol. Chum.. 1985. 200, 14-42: Pure Appi, Chem.. 1984. 56. 595-624; Amino Acids and Peptides. 19X5. 16. 3S?-4 j0; Biochemical Nomenclature and Related Documents. 2nd edition, Portland Press. 1992, pages 39-69.

[003691 The one or more useful modification* to peptide domains of the inventive aBligett biiidiBg prdtMO can include amkto add additioM or insertiods, atpibo acitl deletions, peptide trutueuions. amino add subsHuulons. and/or chemical dcrivati/atton of amino acid residues, accomplished by known chemical techniques. Fnr example, the thitsiy modified amino acid sequence includes at least one amino acid residue inserted or substituted therein, relative to the ammo add sequence of the native sequence of interest, in which the inserted or substituted amino acid residue has a side chain comprising a nucleophilic or electrophilic reactive functional group by which the peptide is conjugated to a bakes und-'or halt-1ito emending moiety, in accordance with the ;Ih vsatiq®, Mefu! £xam PI os of such a nudeopbu ic nr electrophilic reactive functional group include, hut are not muitod to, a thiol, a primary amine, a selooo, a hydrazide, an aldehyde* a carboxylic acid, a ketone* an., armnooxy, a masked (protected} aldehyde, or a roasked (protected) kelo functional group. Examples of ammo acid residues has mg a side ehaih eeinprixmg a : nucleophilic reactive functional group include, but are not limited to, a lysine ks, h low 'so ar«d m uno tpionn* t* dies die it u , i ( >' no s iR ue acre ϊ Ik > * o* m tne te'-ufo , a s' xte r*e* * ho nomstene a gtuUmm (- d .residue. an aspartic acid residue* or a sefenoeyshnne residue. jQOlTD] Amine» acid re*sidus\s are eommonK eatsgonzodtiieeordmg to differem chemical and/or physical characteristics. The.term 'hieidiiaaitti«o acid residue" refers to amino acid residues in D- or l*-form having side chgins eontprisiBg acidic groups* Exemplary acidic residues include gspartatic acid and ghnarnatfe acid residues. The term "'alkyl amino acid residue*’ refers to amino acid residues in D- or L-fomt having Cj.saSkyl side chains whfehOiayM or cyclizcd* including to the (.menu acid cm mm *is m prointe, s\ herein the (\ yilkvi is substituted In 0* I* 2 or * substituent^ ^efoemj from Cmchaloalks'* f*o, eye to, nitro, Ct nlRh, -('t OtOR\ -Cl OiXH Is'* t‘{ NR')NR*R,-\PV* MONR’RM-ORM R i CAR'’’* -OiMR'Rh iX\< c-ft Iky fob; Re, ΟΓ·· alkslORA ^R·’.-StOiK , -SfeOpMrRf -MR ‘R \ -be; frit < OtR'y -NiR^Cb 0}0h\ -N(R**)C<: 0}NR*R\ -N('Ra»C{-NR MNK'K®, -NiRJ>St-0>>R!\ *N(Ra)SfoOKNR Ή ', ART^alk) lNRaR“ and -NRa(\f.;dks lOR'b wherem R" is mdenendewK, at each instance, H or Rand R1’ is jodepondont'\ * at each :urn,nice ( -. ftalkyl substituted by 0, 1* 2 os » substituents selected t'(sr >*uo t ** * I < In feds, -OC oik *M' MIC jalfeund -Xt( ; iulkK'f mlkorany protouated form thereof, including alanine* valme, leucine, isoleueme* prohue. sere re. threonine, lysine, arginine, histidine, aspartate, ghnemate. asparagine* glutamine, .ystcine, methionine* Isydroxypmlinc, but winch residues do not contain an utyi or aromatic group, fisc term "aromatic amino acid residue" refers to annuo ace! residues in D- or !,-form having side chains voumrisino aromatic groups. Exemplar) aromatic residues include tryptophan* tyros me* 3u I -naphthyl ndanine, or phenylalanine- residues. I'he term "bask amino acid residue” refers to amino aenl residues in D~ or L-furm ha\ ing side chains comprising basic groups. Exemplary basic axmnoacid residues include histidine, lysine, hunwlysinc. ornithine. arginine, N-methyl-arginine, mmrnmoargmmo, w-'methyl'-arginme, 1~ methyl-histidine, kmethyl-hixlldmc. and homoarginme ilsR» residues. The term "hydiophilie amino ae;d residue" refers to amino aetd tesidues in P- or L-form having side chains comprising polar groups. Exemplary hydrophilic residues include cysteine, serine, threonine, histidine, lysine, asparagine, aspartate, glutamate, glutamine, and citrudinc <Cii) residues. The terms “lipophiHe amino acid residnif relers to amino acid residues in D- or t -form having si dee hams comprising uncharged, aUpluitie or aromatic groups. Exemplary lipophilic -edeihams include phenyhdiiuine, kotcudne, leucine, methionine. valine, tryptophan, and pmoxine AI an me (At is amphiphilic it is capable of aethig as a hydrophilic or hpophihe residue. Alanine, therefore, is included within the definition of both "lipophilic residue" and "hydrophilic residue." The term ''nonfunctional ammo acta residue" relers to amino acid residues in D- or L-form having side chains that lack acidic, inode, or aromatic groups. Exemplary neutral ammo ucid residues include methionine, glycine, alanine, valine, isoleucine, leucine, and norleuci.no (Nle) residues. j 00:171] Additional useful embodiments of can result from conservative modifications of the amino acid sequences of the polypeptides disclosed herein. Conservative modifications will produce half-life extending mowty-corsjugated peptides having functional, physical, arid chemical characteristics similar to those of the conjugated (e.m, PEG-conjugated) peptide front which such mod ilka lions are made. Such conservatively modified forms of the conjugated polypeptides disclosed herein arc also contemplated a-, being an embodiment of the present invention.

[00372} In contrast, substantial modifications in the functional and or chemical characteristics of peptides may be. accomplished by selecting snbsbmdons in the ammo acid sequence that differ significantly in their effect on maintaining {a} the structure of the molecular backbone in the region of the substitution, for example, as an oohehcai conformation, {h) the charge or hydrophohieity of hie molecule at the target Nile, or te) the size 01' the molecule, P03T3| For example, a "comervutive ammo add suhsUtut ip# mm involve a Mbsttp.iii0n of a native amino acid mediae with a nonnatfve residue Sbeh that there is littieor oo effect on #tc polarity or charge bii#ant^ position.

Furthermore, any native residue in the polypeptide may also he substituted with alanine, as has been previously described for "alauttie scanning mutagenesis5' (see, for example, MacLerman. et al, Acta Physiol. Sound Suppi., 643:55-4? (1998); Sasaki eta!,, .i998, Advv Bipphya, 35:1824 (1991), which discuss alaptpe scanning mutagenesis). (00374] Desired athhio aoM safostittttiOBS (whether conservative or non* conservative) can bb ^glfc^lBpd by: the art at the tipid sndli: substitutions arc desired. For example, amino add substitutions can he used to identify important residues of the peptide, sequence, or to im*iea\e or decrease the jffimty of the peptide or vehicle-conjugated peptide molecules described herein. #0375} Nairn <{IK oecnrrme -esi dues may be divided into classes bused on contmon side chain properties; :1) hydrophobic; norleucme (Noror'NIe), Met, Ala, Y«ii, Leu, lie; if netnral hydrophilic: Cy.v Ser, t hr, Ami, Gin, 3) .icidie: Asp, <ilu, A) basic; His, Ly.v, Arg; 51 >cn dues tnut< Ouenc eba a one 'u,t or oh the, and 6) aromatic: Trp, Tyr, Phe, \0D37P1 (\>5S'-o -,,urte urnino ueid substitutions may in\oi-.e <,\s,h,„sue of u ntend -'ιοί one of these classes with another m,-, mber of the same class t ,v„scn am, e ten no add substitutions may encompass non-naturaily occurring amino acid residues, which are ty pi tail;, incorporated by chemical peptide synthesis rather than by s} nthesis i hsiloyhcd '-yxter.s. Tne.-e indede pi pxbonumeites and older mwo-ed or inverted forms of amino aeid ptoietlos;.: [003771 Non-conservative sufesttunions may involve the exchange of a member of one of these cla:oiex fern rnember imm mrnffierclass. Such fee intmduced mtq regioos of ibe tosm p^mfle analog.

[003781 fn making «eh changes, according to certain embodiments, the feydrdpgfbic index oiarmnp acids may be considered; Each amind geid hayfeeen assigned a hydropathic index on the basis of sis bydrophobietty and charge ebaracterisilcs. They are: isokueinc (·*·4.5): valine {*-4 2); lencine (4:3,8): phenylalanine (p-2.8); eysteine/ev&amp;iine {t2.5); methionine (11.0); alanine (* 1.8); glycine (-0,4); threonine (~0J7)cserine (-0,8); tryptophan ( 0.9); tyrosine (- i .3); prohne {-! be histidine (-3.2); glutamate (-¾ 5); glutamine (-3,5b aspartate (-¾ 5), asparagine (3.5): lysine (-3,0): and argmioe (-4,5), [00) Ό t he importance of the hydropathic amino acid index in conferring interactive biological fdnetfOh on a protein is understood in die art (see, jfc>r examples K\f. if,;/,. Ic>82,d. l&amp;>/. Bioi. 357:105-)31), It is known that certain ammo acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hyinopvithe index, in certain embodiments, the substitution of ammo acids whos*. hydropathic indices arc v ithin el is included In certain embodiments, those ihat me within 5::1 are meludeds dod in eertain embodiments, those within K),5 are included.

[00380) Itthe substitution of like amino acids can be made effectively on the basis of hydrophilic!?)', particularly where the biologically funetio ial protein οi peptide thereby created is intended .for use m immunological embodiments; ardisolpsed herein; pettiest local average hsdrophilieit\ of a piotem. as goserned by the ivydrophrtteny of its adjacent amino acids, correlates with be immunogenieity and antigenicity, /.<»., with a biological property of the protein.

[00381:) The tbikming hydrophiUcity values ba\e been assigned to these annuo acid residues: arginine (4-3,0): lysine (- 3,0), aspartate (·) 3 0 ? 1); glutamate i *3.0 t 1); serine (t 0.3), asparagine it 0 2), glutamine 1 ’· 0 2); glycine (0), threonine MM); proitnc (-0.3 * 1 κ alanine (-0.5)¾Mstidine (-0.5): cysteine (I ,0p methionine (·· I .5): valine (-1,5); leucine (-1.8.s; Uoleueme.H); tyrosine (-2.3); phenylalanine (-2. O and tryptophan (-3.4). In making changes based upon similarhydrophthetiy values, ip certain embodiments, the substitution of amino acuis who«e hydrophilierty values are withm ^2 is included, m certain enihodmtenK those that ate within *) are iBpihded, and !» certain embodiment^ ttese piihin 2W).5 are included. OneMay also identify epitopes from primary ammo acid sequences on the basis of hydrophilkdty, Xted as ’’epitopic core regions,* (00382] t&amp;amp !es of conservative substitutions include the suhstituilop of one uoo-pola1' (liyd'ophobie' are.no acid -residue such as luUoucun, salute, ieueuse ttorieuc ino, alanine, or ntethiouinc for another, the snhsdtuiion of one polar tlwdiophiUe) annuo acid lesnhie Us anothot such a - K ou on c-Ursine and lysine, between y S utamme and asparaeiue. be; wee a glycine end serine, the substitution of one basic amino sold residue such as lysine, arginine or Instldme .for another, or the substitution of one twichc residue, va.it as aspartic acid or glutamic aea! for another The phrase “conservative amnio acid substitution^ also includes the use of &amp; d iemiealiy derivafired residue in place of a non-denvaiteccl residue, provided dun such polypeptide displays the requisite bioact tv ity. Other exemplary arnino acid substitutions that can be useful in accordance with the present invention are set forth in "fable 6 below.

Table 6. Some Useful .Amino Acid Substitutions.

[6(1383] Ordinarily. amino acid sequence sauauts of the antigen binding protein .Milltteve an amino add sequence bas ing at least 60% ammo acid sequence identity -λ nit the ongioat antigen binding protein ot am bods amino uetd sequences of c inter the heavy or the light chain variable region, or at least 65%, or at least 70':3, or at least %% or at least 80°·« identity, more preferably ec least haS.. identity. eson more preferably ai least 90% identity, and most preferably at least Odd:· identity, mdudtng for exantpk, 80%, 81%, 8;3%, 83%, 8-13..,85---, 863,, 8~%, 883s, 80«;,, 90%, 91%, 93' 9i%, 94«.. 9$%, 97«y .?h%, 99'... ard 1 (Kb o idenr's or homolog) v tth respect to this sequence Is defined herein as the percentage oi ammo acid te si rates ;n the candidate sequence that are identical with the original sequence, after aligning the sequences and introducing gaps* if necessary, to achieve the maxismmt percent sequence identity, and not considering any c misers, di\ c substitutions us part of the 'sequence identity None t«'\‘-ienuinat. t ’-fetnunai, o>' internal extensions, deletions, or insertions into the antigen binding protein or antibody sequence shall be construed as affecting sequence identity or homology;: {:1)(13841 Albino acid sequence insertions include amlhps and/or caThoxylAeBrnou! te^oto ranging m length from t)ne residue to polypeptides comannng a hundred or rune didoes \ oe‘J as urn sequence insertions of single or multiple ammoseid » -, .a s f^ roles nt tot nun 1 insertions include an aofuxn bmdmg mxgeorwitJh an N-terminal methionyl residue or the antigen binding protein (including antibody or a;mbody 0 am cm} fused \> an epeope tag or a sab age iceeptor bugling epitope. Otbet ' risen,on d sanants of dv ant ren binding ptotem or antibody molecule include f"e t'e.-non to a polxneptidc which increases the serum half-life of the antigen binding protein, e.g. at the N-terminus or G-terminns, [:003851 Example's of epitope tags melude the flu HA tag polypeptide and its antibody 12CA) [Field et ah. Mol Cell. Biol. 8; 2150-2165 (1088)]; the c-mye tag md the KFO, 80 A off JO, G4, B? arc; OHIO antibodies thereto [Evan et a!, Mol fell. B ol r; 1 s V .0 3o161 .md the Herpes Simplex tints gKeoptotem O ui>) tag and Us antibody jPahm'Ay et al. Ihotem Engineering he)' ^47-558 t 14P0J| Other exemplary tags are a poly-histidine sequetcey^n^.lytaiipu^d-htx histidine residue'^, that pi rtnifs isolation of a compound so labeled using nlekel chelation. Other lihoN and tags, such as the FIAO/ tag 11 ashman Kodak, RoGie\ter, \ Yt are well known and routinely used In the art; [603861 Some particular, non-limiting,-embodiments of amino acid substitution i ariants of the nmmtive antigen binding protos.nv including antibodies and antibody tragntents am exemplified below, [00 1 ,'\a\ es steinc residue not invoh ed in maintaining the proper conformation of the antigen binding protein also may be substituted, generally with serine, to improve she oxidative stability of the molecule and prevent aberrant ero&amp;dmkiog. Conversely., cysteine ben<i(s) may be-.added todhe antigen binding protein So improve its stability tpameularly where the antigen binding protein is m antibody fragment such as at) f v bagmen?).

[003HKj In eerie in instances, antigen binding protein variants are prepared with the those amlrto acid residues which afodlmetiy involv^lh epitope binding in other embodiments, modification of residues w inch are not directly involved in epitope binding or residues not involved in epitope binding in any way, is desirable, for purposes discussed: herein. Mutagenesis wiibin any of the CDR regions and/or femow-orii^^0ns:#::.^ftfomplatied·, [00389] In order to determine o iueh antigen binding pan cm annuo and residues are important for epitope recognition and bidding, alanine scanning mutagenesis can be performed to produce .substitutionvariants. See, foueknmple,.:Cbbdmgh^::#;ab··^ Science, 244; 10¾ 1-1085 (1989), the disclosure of which is incorporated herein by reference in ds ettfirety; In this method, iudiylduai amino acid residues are replaced: oue-ai-a-tirae with an alanine residue and the icsuhing anh-DNP or unU-ΚΛ1 i antigen binding protein in screened for its ability to hind os specific op dope relative to die unmodified polypegitde, Modified antigen binding proteins with reduced binding capacity are sequenced to determine which residue was changed, indicating its significance in binding rrebiological properties.

['003901 Substitution, variants of antigen binding proteins can be prepared by affinity maturation, wherein'random ammo acid changes are introduced into the parent polypeptide sequence. Sec, for example, I baseband et at.. Vox Sang74 (Suppl 2).223-23:. IW, Radct etal., Pruc. Nath Acad. Sci. 1 SA »»5:^10-8915. 1998; IMbAequaei al, Curr. Opm. Struct. Biol. 8:443-450, 1998, the disclosures of which am incorporated herein by reference In then entireties. .^®atprntt«#4tion··· involves preparing and screening; the anti-DMF or nifo-KXH antigen binding protcnis, or variants thereof and selecting from the resulting» variants those that have modified iStdldgieal pmparfies, such as Increased binding affinity relative to the parentbinding protein. Λ convenient way for affinity maturation using phage display Briefly, several hypervariabtc region sites are mutated to generate all possible amino : substitni]bns at each slip.:: lire variantatlina generated are Oppressed in a monovalent fashion on the surface of filamentous phage particles as fusions to the gene III product of Μ13 packaged wiihm each particle. The phage-disphtyed variants are then screened: for their biological activity (e.g., binding affinity), See;e;|dSO 92/01047, WO 93/112360, W09#iB8S and WO 93Ί9!72; [00391! Ltnrent antibody alrinity rnatoration methods belong to two mutagenesis categories; stochastic and nonstochastie. Error prone PCR. mutator bacterial strains (Lowet al.,,/, MM. Bmt 200,359-6¾ 1906), apd sattnsrion mutagenesis (bUshimiyg ct ah,,/. BioL Omm. 275:12613-20, 2000; Chowdhury, P. S. Method* Mot. Bu.>L 176, 269-63,2002} are typical example» ofstoehMfe otethods/fRigpai et alp

Proe Nail Acad Sci UEJ. 102$4662¾. ic ieclmtqnea bllehmse alanine-scanning or site-dneeted mutagenesis to generate knitted collections of seecdii mutesns Some methods are described m ftnther detail below [00392] A(fmky mmmnhn v/u pxtwing mefhtjxi*- Affinity foaiut^tfon of resu'ub'nant antibodies ts common!} pet formed through ses end sounds of panning of candidate antibodies is the presence of I ten easing the amount of antigen per round selects i he antibodies w ith the highest affinity to the antigen thetehy yielding antibodies ofhigh affinity from a large pool of starting material.. Affinity maturation mdtika&amp;wxt hi foe art and it described, for example. in Hals et al, Κ ΐ»<τ<· hnmmoi 50:165-71., 2001), Methods of affinity maturation using phage display tecbublogies are dcsenhed elsewhere herein and known m the ait (see e,g., Daugherty ct ah,/boo A(:n/ -AvMSd V S;l 97:2029-34,2000).

[00393] Look-Through fo Look-through mutagenesis (l I'M) {Raj pal et ah, Pros' Natl Acad Set I'A A. 1026466-41, 2005) prowdes a method for rapidly mapping the antibody-binding site, for LTM, nine amino acids, representative of the chemistries provided by the 20 natural amino acids, arc selected to dissect the functional side-chain contributions to binding at every position its all sis CDRs of an antibody. LTM generates &amp; positional sm ies of single mutations within a CAR where each "w nd type" wwiue is systematically substituted by onoofnino selected amino acids. Mutated. CORs are combined to generate combinatorial smg!er chain variable fragment (scFv) libraries of increasing complexity and size without becoming prohibitive to the qoamvtaUve display of all tnutems. Allot posmw sdoctiom clones with improved binding are sequenced, and beneficial mutations are .1 mapped, [00394] Error-prone OCR......Error-prone PCR involves the randomization of nucleic acids between di fferem selection rounds. The randomisation occurs at a Sow rate by the imrmMC enot rate of the polymerase used bin can be enhanced by ostot-prone Pi'll (/aeeolo et aL I. Mol Biol, 385:775-783, 1W) using a polymerase bav s >g a high norm- c error rw during transcription (I law kins et al., j Mol Biol 336 884.%, 1042' Alter the wunnum < \Des, clones vuth ungloved afnnov fot the antigen arc selected; using rumble tueiliods ln the arb: [003-)5] Todfunques utilizing genu shuffling and directed evolution may also he used to pi, pare and screen auo-DNP or anu-KI 11 antigen binding proteins, or variants thereof, for desired activity, for example, Jermuros et sL Proe ΜΙ Acad Sci U S A.. 98f I );75-80 *2001) showed that tailored u? vim* selection jdrategies based on ribosome display worn combined with m vstn< diversification by ON A shuffling to ew>h e either the off-rate or thermodynamic stability of seFvs; Fortner et aL Tumour Biol. 2004 ,hm~Apr;25t 1-3) 7-13 reported that use of phage display in eo.mbimit.ioii with ON A shuffling raised affinity by almost three orders of magnitude. Dougherty et al., Proc Natl Acad Set U SA. 2000 Feb. 29; 47(5) :2(129-2034 reported that it} functional clones occur at an unexpectedly high frequency in hypermumted

UfeAveil represented insueh libraries, and (ini ;ffee,ma|or|iy of the scFv mutations leading to highegkfintty cpiiPppond to residues distant Sum the binding sue. jlOpObl Edition, it may a-brutal'; of the anUgcT»-atmbotiy complex to identity contact points between dm; antibody and antigen, or to use computer software to model such contact points.

Such contact iCMdiK'N end neighboring ivudnes arc candschnes tot suKlmatsun 4cc0MMgtdft^e::.t!6ctiniquei>. elaborated herein:«Oboe fttch v&amp;riaots are generated, they aremtb|ceted to screening a# described herein and antibodies with superior properties in. ode or more relevant assays to^y bo selected for hidber de^loptHent. j00398] Antigen binding protein variants can also be produced that have a tuodified glycosyiation pattern relative to the parentpolypeptide. for example, adding or deleting one or more of the carbohydrate irioietics bound to the antigen.; binding protect, and or adding or deleting one ot more gheosyhniou sites tn the antigen binding protein. 10U399] Gl> cos> leoon of poly peptides, including antibodies as typically esther dv» linked or ί Mtitled, N'~linked refers to the attaehntent of the carbohydrate moiety to the Je chase of >m asparagine residue. The tripe pride sequences asparagine-iX-^ans, rad iianamgme-X-thnionhn.% vbere X any ammo acsd except nroline, am the recognition sequences tor enxymatu. attachment of the carbohydrate moiety to the L.evuagme side a .,rn. The prasene, <n enher of these tntvotsde m qneuces rit « polypeptide ere-nc'- a potential gh eoxx Uhon site ΓΟην \ n lee c ycosx la eon - dcs .may be added to a antigen binding protein by altering the annuo acid sequence such Uautv-osku \ one or mo,e oi ties rpeplide w\ jo !v>> 0~ iwce ghioxsl on refe's ο K1 nt<\ ' t ent o* one of t X saga,'* .ce^Lasaetttvn nrc, gala, \c ot xylose to a hydroxyamino amd, most commonly serine or threonine, although ,> hydroxyptolihe or Sdiydroxylysine may also be used: O-linked ghxxxyvhuion sites may be added to a antigen binding protein by insetting or substituting one or mom serine or threonine residues to the sequence of the original antigen binding, protein ot antibody.

[00400] Altered Effector Function Ϊ 00-mU Cysteine rcsidue(s) may be removed or introduced in the Fe region of an mtuvd) os I * -v mnamnig poy.repttde. thereby ei.mmatmg m mcreaamg snseich'un disulfide bond torniution in this melon. A honitxiimerie antigen blndinu protein thu^ generated may have improved interna hzation capability andor increased comnlement-mcoiated eel; UIU ug χκΐ an t i bod v ~o e pe n dent cel near cytotoxicity t A.DCC), See Caron et ah, J. Exp Med. I ?6: 1 Ιοί -1195 (1992s andShopcs, B. J Immunol. 148. 2918-2922 Cl 992), Homodmterie antsuen binding proteins or antibodies may also be prepared using heterobifunctional cross-linkerx as described in Wojffet al. Cancer ReseatchSa 2560-2565 O'-G.G Alternatively, a antigen binding protein can be engineered which has dual Pc regions» and may thereby have enhanced complement !y ms and ΛΓΗ X' capabilities Sec Sievem>on el al, Astts -CancerDrug Design 3: 219-23011989b [00402] It has been shown that sequences xvrtfifftdhe £I9R' osti'eayge :φ·^ϋ&amp;0$¥ i&amp; bind to MHC Class \\ and trigger an unwantedhelperlUeell response, /V conservative sabstimikm can allow the anttgen binding protein to mtain brnding: .activity yet reduce Its abdny to trigger an ww anted T-cclI response. It is also contemplated that o v «a i xne of the N-iotmm *. 2(t amino acids of the heavy or light chairs are rcmoyc.<|. (:004031 Modifications to merense -rentm half-life also may desirable, for example, by meospoonso \ of or addition of a v I v age rcivpiot bmdsug epitope te g , by hmtation of the incorpomiing the epitope info a peptide tag that is then fused to the antigen binding protein al either end or tn the noddle,d-gy % DNA. or peptide synthesis) /see. e.g.. W096/32478) or adding molecules such as PEG or other water notable polymers, incktding polysaccharide polymers.

[0040-11 The .salvage meeptorbinding epitope preferably constitutes a region w he* cm any otic or more ammo acid residues bom one or two loops of a Fc domain arc transferred to an analogous position of the antigen binding protein or fragmpit Even rnom prefctaddyythree or rnore residues from one or two loops of the Fc -doorblti ard^ preferred, the epitope is taken htim lhg iCfli domain of the Fc region se.g., m an IgCi) and tmostbrred to me CHI, C H3, or VH region, or more than one such region, of the antigen binding protein or antibody. Alternatively. the epitope is taken from the CH2 domain of ike FT regum and Srwivien'cd to the C, region or V region, or both, of the antigen bin-ding protein frugroent. See also iniernutiotw! tionheattoux WO 97.-34631 and WO \H>'32478 which describe Fc sarlynts and riwtr mieraebort with the salvage receptor, [00405i Othersites and amino acid rcsidtscls} of the constant region have been identified that are responsible tor eomplernem dependent cytotoxicity [ΠΧΊ such as rite flq binding site·, and or tite antibody· dependent ecdidar cytotoxicity t AOt.T s [see, mg., Mo ice, bnmuno). 2L> (5}- 635-9 (1992); Shields >.d a! , j Bros (.'hern , 2 ?6{ 9):669Ι-·όό04 {2001 g La ear oi a 1., Pox. ΝαΠ. Aeed. Set. 10311} y 40()5 (2006) which describe the eilcct of mutations at -specs He positions, each of which is incorporated by t'cleressee herein m its entirety]. Mutation of residues withm Fe receptor binding sites can ro.suit in altered (i c. increased or decreased} PTocim-function, such as altered affinity tor Fe receptors, altered ADCC or i. DC' acto sty. or altered half Fie. As described above, potential mutations include insertion, deletion or substitution ofonc or more residues, including substitution v> sir olsiune, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same post lion from a d liferent subclass ie.g replacing an IgGt residue with a corresponding in CM residue at that position y [00400} The invention Jso encontpa-mes prodneuou ofanugeit binding proiem moiecules- ineindutg antibodies and antibody IragmeuK with altered carbohydrate structure resulting m altered efiector unto Uv including antmed-, molecules with absent or reduced fucosylatiott that exhibit improved ADCC activity. A variety of ways me known in the art to accomplish this. For example, ADCC etfectnr aeli\ its· in mediated by binding of the antibody molecule to tite FeyRIU wept or. which lias besiu shown to he dependent on the carbohydrate structure of the C-tinfvd glycosvlatton at the Asn-297 of the C H2 domain. Non-fncosysated antibodies bind this receptor with increased affinity and trigger FcyRlH-mediated effector {auctions more efficiently than nutve. lueosyiated aatutodies. For example, recombinant

production of\aoo--fueusvlated antibody in OHO ceils in which the alpha- Ul-lueosy! tom.sfera.se enzyme lets {seen knocked out results m antibody with HXM'old sues eased ADCC activ ity (Yara&amp;neddhnukl or al,< Bioteeimoi Biocng; 2004 Sep 5:87(5):614-22). Similar dice is can be accomplished through decreasing the activity of this or other on/ymas in the fucosylation pathway, e.g.. Unongh siRNA oruntiscuse RNA eeiFlmea id hhoeltonr the ettxymeG}. or culturing with selective giyeosylahon inhibitors f Rothman et ah, hltsl Immunol, I9p> 1)λ dot 12):1113~2iO Some hov cell ^ oat ns, e.g l ee 13 >n mt hyhaoomu 5 B2 0 ceil Hue naturally produce antibodies with lower fueosylalion levels. Shields d al. j BoI ( hem 2002 ini 2m27 o 30; 2n"*3* Ί0. Sh inlaws et d . j bid c bent 200¾ Jen ^ 1,2“S{st 5460-7t \n mc>e.w< 't the d of b'seeted . atbobuitate, c v tlnuitgh recombinantly producing antibody in coils that oserexpress Go Pill enzyme. has also been determined to increase ADCC activity, uremia et at., Pat Biraecbnol. 1900 I e ^ 17(2) ~o 'Ό1 M as been piedictea t at he . vecee .PonF once .he u\ 1 fucose residues may ice suffteicut to increase ADC'C activity. (Ferrara et al, J Biol Chem. 2005 Dec 5), 100-UP J Other Cos a lust Modifications of Amman Binding Proteins [00408] Other particular covalent tnodlfeations of the aml-ONP or anh-Kiil: antigen binding protein, are also included within the scope of this invention. They muy.be made by chemical synthesis tw by mmymatk o* eheimeal deavape of the antigen binding piotcm or antibody- if applicable, Other type's of covalent modifications can be introduced h> reacting targeted ammo uad residues with an oi game dement zing agent dial js v a gable oOeauutg u dh «ejected suit chains οι the N- or C'-tennina! residues.

[1104991 Gybteiuyi residues most '^fe^h|yv^.:'^aetcd with u-haioacetates (etui antespomimg atnutes). sueb a* Pdotoaeeno and of t-hkuoacetauude, to gne carboxymethyl or earboxyamtdomdhyl derivatives. Cystemyl residues alxo ere ddfivalized tty reaction with .alpha.-hrovnd--p4|Si- tdMoxoyiipropionic add, chlormcetyl phosphate, N-ailyknaicijmdes, 3>ntiro-2- pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromerctirihenzo&amp;te. 2-chlorotnercuri-4~riitix>pher«ols or ehk>ro-?-nitrobon$!:o-2-oxa-1«d-diazolc. 100410] Hisfidyi residues arc derivaficed by reaction w iih diethylpyrovasixmate at pH 5.5-7.0 because tins agc-Rt relatively specific lor she histidy! side churn. Pan*·· bromophenney! bromide also is useful] die reaction is preferably performed in 0.1 M sodium cacodyiate 8l pH 0,0. (0041) j LysiUyl and amino-terminal residues are reacted with succinic Of either carboxylic add anhydrides. Ocrivatixatior· with these agents has foe effect of rctersing the charge of the tysinyi residues. Oilier suitable reagents ibr derl\ enemy ,;dpna.-mm!no--comamisig residues include imidoesicrs such as methyl pteolmimidate, pyridoxul phosphate, pyridoxin, ddoroborohy dride, mum r o hm tee nest; i tons c acid, O-md by 1 iso urea, T4-penmnedione, and trunsamiitase-catalysed reaction with giyoxykfo; [004 1c] Argioyl residues are modified by reaction with one or several conventional reagents, among them phcnylglyoxaL 2,3-bt.uunedione, 1 ^-eyelidiexariedione, and ain.hydnn. Donvatiectiou of arginine residues requires that the react ton be performed in alkahue conditions because of the high pK„ of the guamdme functional group, furthermore, these reagents may react with the groups of lysine as well as the ar gi n ir: c epsi I on~airri no group. (00413] The specific modification oftyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic •di axon turn compound* or tcinautroructhane. .Most, commonly, N-ycetyirandixole and fetrauitromethane are used to form O-acetyl tyrosyl species and j-srn.ro derivatives, respectively Tyrosyl residues arc ioditnued using *'T or ! 'Ί to prepare labeled proteins lot use in radioimrnunoass&amp;y. (00414] f arbor y! side groups (asparis I or glutamyl) are selectively modi lied by reaction with earbodiimitfos tR-N.dbd.C,dhd,.N-R0, where R and R! ate different alkyl groups, such as l-eyidohexyd-.342-fnorplKrfiriyb4~etl.iyl) earbodiimlde or 1- I> v a rhodiumde. Furthermore, asparty; «M: gluSarnyi residues are converted to ayuimyinyl and glutannnyl residues by react tot! with ammonium ions.

[004151 f nutammjl and asparugut' \ ic-'da> arc fteqnentSs to* seated to to. eo; respuse' mg glutamyl and as party! residues icspeeove v Thov. teas cues ate deamkbted under neutral or basic conditions 1 he deamidated hum re these resumes falls w ithin the scope of this invention.

[004 i ο i Other modifications induce :vs dmo.y union of ore:me and lysine, phosphorylation of hsdroxyl groups of sc- > I or threonyl re mb s?>. methylatum of the , alpha.·-amino groups of lysine, arginine, and histidine side chains ff, £. Creighton. Proteins; Structure and Molecular ihopeides, W H Freeman &amp; Co., Sun Francisco, IP <o po icety Union of th \ ft τ'* ,. mru, mvi amur-no > > ! my C - terminal carboxyl group, [004P| Auo'her typeof covalent n a Iduauon muxrtes chemically or •term maticaily coupling gi>eo.suies to the antigen binding ptraein (e.g.. antibody .or antibody fragment). These procedures are advantageous in that they do not require production of the antigen binding proud» In * host cell that has giycosySatkin capabilities tor M- or O-linked glycosyiaiop. Depending on the coupling mode ttsedy the sugar(s) may be attached to (a) argkine and histidine, sb) free carboxyl groups, (c) free sulfhydryi groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydraxyproline, to) aromatic residues such as those of phenylalanine, tyrosine, or tryptophati, or (0 the amide group of glutamine. These methods are described In WO87/0533Q published 11 Sep. 1087, and in Aplin and Wsiston, CIIC C'rit, Res. Biochem.. pp, 250-5()6 (1981). } 004! 8 { Removal of any carbohy mute moieties present on the antigen binding protem htay be accomplished chonsicatt^.jpr^zynmticallyi^g^c^ deghiosylatmn icquuev* exoosute of the antigen bnuhng ototem to the compound trifluoromctlu-mesuffonie acid, or an equivalent compound. This treatment residtyM: the cleat age of motor all sugats except the linking suga? (N-acetylgiueosarnmeor N-aceiy IgaluCKM&amp;roiftek while leaving the antigen binding protein inract. ( hemlcal •dcglycosylation Is described by Hakimuddsn, ct al Arch Biochern Bionhys. 259· 52 (1987)and by Edge et al. Ana! Bioehem., 118; 13; i 1981 j. Enzymatic cleavageof carbohydrate moieties on a antigen binding protein can be achieved by the use of» variety of endo- and extvglyco.Mdascs a* described h\ 1 hotukura ei a! Mctk. Eneymoi. 138; 350()987). P04 )9) Another type of covalent modification of lire am (gen binding protein* of Ore invention {including antibodies and antibody fragment*} comp rives linking the antigen binding protein to one of a variety of non proteinaceous polymery e.g., polyethylene glycol, polypropylene glycol, polyoxyethykiiod polyols, polyoxyethyla ed sorbitol, poly ox vethy kued glucose, polyoxyethylated giyeerol, polyoxyalkylones. or polysaccharide polymers such as dc strain Such methods are known in the art, see, e.g, t !,S. Patenr Nos. 4,040,83.4; 4,490,689; 4,301,144, 4,670,41 ?; 4,791,192. 4,179,337. 4.7*6,106. 4, i 79,.537, 4.495.285. 4.609.546 or BP 5I5 4S6, 100420] [00421 j Another aspect of the present invention is an isolated nucleic add that encodes an antigen binding protein of the invention, such as, but not limited to, an isolated nucleic acid that encodes an antibody or antibody fragment of the invention. Such nucleic acids are made by reccurdhna-U techniques known m the art and/or disclosed herein.

[004221 Bor example, the rwoumd nucleic ucid encodes an antigen binding protein -comprising an immunoglobulin heavy chain variable region comprising an amino acid .icnuenee at least 95 % identical to SF.Q ID NO.250, Sh'Q ID NO.252, SfiQ If) NO;254. SBQ ID NO:2S6, SEQ ID NO:258, nrSEO ID NQ;260.

[00423] In other embodiments, the isolated nucleic acid encodes an antigen binding protein comprising an immunoglobulin light chain variable region vomnriiirtt* so airs mo eeid sequence at load 95'N ;dentkal to SEQ H * NO 232, SEQ |pNO:234, SBQ ID NO;236, SBQ ID NO-m or SBQ ID NO;240.

[00424] Othe. example'' 05 diu sso! , 0 \k ’.e\ <4t .o 5h tuoo meh due isu eef ^ ar immunoglobulin heavy chain variable region, wherein the Isolated nucleic acid comprises coding sequence» for three complementarity determining region». Jc*igmitea VDRMl { DkH2 and CDIND and okuesn («>04251 las OMOil comprise» the umioo acid sequence of SBQ ID NO-1χχ, SBQ ID No- [ m, SBQ ! D NO: 190. or NEQ ID NO: 1 !·> 1; :(00420] tbK'OOHN comprise ^ .he ..τιην acid seex-mee of SBQ NO '42, '“I Q ID NO: 193. SBQ ID NO; 194. or SBQ ID NO. 195. arid [00427] (cs ODRH3 comprises the amino acid .sequence of SEQ ID NO;196s SEQr ID NO;197, SBQ ID NO; 19k S.BQ ID NO; 199. SEQ ID NO;20D. or SEQ iD NO;20I.

[004281 Still other examples of the isolated nucleic nc id include such that tfocodes an. immunoglobulin light chain eariahforngiofo«femin the isolated nucleic acid comprises cocling sequences for forceeomplemenfo^^^ regions, [0Θ429] (a'i CDRLI econp'rise» the amino acid sequcstCe ol SBQ ID NO202. SBQ IDNO;203, SEQ ID NO;204jr SEQ ID NO:205; : [004301 (h)'CDRL2-comprisesfhe-arnino acid sequence of NBQ> ID NO,200 or SBQiDNQipTiand [0043If (cl CDRB3 comprises the amino acid sequence of SEQ ID 240:208. SBQ ID NO;209, SBQ IDNO;2I0. SBQ IDNO:211, of SEQ ID NO;2!2. (00432 ( In other enfooihmeni'' the isolated nucleic acid encodes .m ant icon binding psoiefo composing an imucjnogkfoulin heuvq chain Ciunpnsmgt te ammo aeD seque me of Si Q ID V K" M U ID M» 07 SBQ ID NO Π 1. M Q 10 NO 03, SEQ ΙΌ NO: I 15. SEQ ID NO: 1 17, SEQ ID NO: 1I 9. SEQ ID NO: 123, SEQ ID NO; 12« Sl'Q ID NO; 144. SEQ ID ΝΟΊ45- SEQ ID NO: 181. M:Q lONOibD, SEQ ID NO; 183, SEQ ID NO; 184, or SEQ ID NO; j &amp;5A or comprising any on? of the Tore got eg seguenees from «. hi J- one, tw o. three, four or fh c amino acid re-Ahe $ arc lacking from dm 'NAemnnal or C-terminai, cn both.

[ί10433| And m some cplibodimems the Isolated nucleic acid encodes an aougen binding protein .comprtling an immunoglobulin light chain comprising ihc amino acid sequence of SEQ I D NO: 105, SEQ ID NO: 109, SEQ ID NO: 121; SEQ ID NO;i25, of SEQ .ID NO; 127, or eoraprismgany one of the ioregoingsequences from w hich one, two, three. four or five amino acid residues arc lacking from the N-termmal or U·· terminal, or both.: (00434j f or another example, tte;|^^iod>sttpcl$Ic acid encodes an antigen binding protein comprising an immunoglobulin heavy chain: variable region comprising an ammo aod sequence at least 9"~ C> identical to the '-conenee ot SH> \D NO:262 SEQ ID NO;264, or SEQ ID NO:266.

[004351 (e other embodiment1" the isolated nucleic ac:d encode^ an antigen binding protein comprising an immunoglobulin light chain variable region ,/comprising an amino acid sequence at least 95 % identical to SEQ ID NO:242, SEQ ID NO:244, SEQ I'D NO;246, or SEQ ID NO:24S.

[00-Do" Other examples efthc ism eed "ackue aetd -oelnee ntel: in*. encoder an immunoglobulin heavy chain variable region, wherein the isolated nucleic acid comprises coding sequences for three complementarity determining regions, designated CD Rib, CDRH2 and CDRIB, and wherein: [DO44” t 11 DRDI cot q \ -we the ammo wd sequence ol Ά Q ID NO gΠ M Q ID NOD 14, or SEQ ID NO:215:/ jOO I "Ή (b> CDKH2 eomprN/s the ammo acid sequence of SEQ ID ,NQ:2U> hi Q ID NO-21 or SEQ ID NO:21 X; and 1004 V>] (c} CDR H3 comprises the ammo acid sequence of SEQ ID NO;2!0, SEQ ID NQ:220, or SEQ ID NO:221; }OO440f ^n)' other examples of the laoUtcd uicL\c uem include seen that enemies an immtmoglobuiin light, chain variable region.· t herein hie Isolated nucleic aetd comprises coding sequences for three complementarity determining regions, designator; CDRIE QBlD&amp;dad CDR 13* end t herein; [00441 ] Cut CDKl I comprises die amino acid sequence of SEQ ID NQ;2d4t S1C|:; ID NO-2'22. SEQ ID NO;223, or SEQ ID NO.224,

! 004421 (h) CBEL2 comprises the amino acid sequence of SEQ ID NO;2()6. SEQ ID NQ:225, or SEQ ID NG:22Q and [00443] (c) QDlliLS eompriscs the amino acid >^-c{ud»c^::0t^iQ:#·BQ.:222?:$:EQ·:.* ID NO:22H. SEQ ID NO:229, or SEQ ID NO;230, [00444! In. other embodiments he :.--0heed nucleic acid encodes an antigen binding protein comprising an immunoglobulin heavy chain comprising the annuo acid sequence of SI Q ID NO H\ SEO U> NO * O. SbQ ID NO' I SEQ IQ NO D SEQ ID NO: ISO. or comprising any one of ihc foregoing sequences from which one, two, three, tour or Eve ammo ado residues are lacking from the N-terminal or € iernrhuh or both [0()44S| And in some embodiments the isolated nucleic acid encodes an antigen I tii'ug ptuten - o ep t^tu-s an mu in os oh 1 tie e'> ts eer^Ms.ng rite mime acid .sequence of SEQ ID NO:2h. SEQ ID'NO: I 31. SEQ ID NQ:i35. SEQ ID \*) 132; ot SEQ ID \u ί41, οι <-o vpt acu* <«n one ot 5i,e hnegmng .sequences nom which 055-. Ονο, * >vt m -> foe - t «μ ^ d>es h-. a at- >a horn +hc V ierorma! or C-tunmnal, ot both [OOsgot Ihe piomοι mw t«on ><ι Nomteetee ,e \_cio”s, me ad ng» χρίουν. Ycetofs, (hat comprise any of the imentive isolated nuclei*. acids Λ» isolated host cell that comprises the expression vector is also encompassed by the present invention, which ts made by molecular biological techniques known m the an author disclosed hcrem. The invention is also directed to a method involving: [004-4? | is) eultm mg the host vcl: nt a culture medium under eondstiotw p-.·-mating expression of the antigen binding proa-in encoded h the expression vector; and [00448] ;:-attn^.cri' btifee·-feuitltirij·y

Recovering the antigen binding protein is accomplished by httown methods of such as hid not limited to. antibody -purification techniques discidsed in Example 1 and elsewhere herein: [0044b] GencThcragy [00450] Delivery of a therapeutic antigen binding protein to appropriate coils can be efieeted \ ia gone therapy ex s tux tn situ, or m vivo by use of any .\uitablc .approach known in the art Tor example, for m s wo therapy, a nucleic acid encoding the dCM.<. c an tger uudn ^ pro+..η ot a cubed'·.. uthet atone or tn conjunction v mi a r ector, hnusome, or pwmpiiutc may be injected discctly into the subject, and m some embeds centv iw f«* uj., ,cd ,<, the stfe oc.v m da explosion o*, be a Imdmg protein compound >s demred. I o« m, vuotua trent '-uoieet ^ .el ^ -. e enoved fit* μ,\'„ «η id *i fodtii e-t n these eelK and U n< H. tied u K arc untuned o titcsubject mi hoi dimed\ or. for example, encapsulated v-uhiu potous membranes which ate implanted !nt0:|hq:$&amp;tient. See, e.g. L.S Pat. "Nos. 4,892,538 and 5,283,1b?.

[1)0451] There ait d variety of teehmqties avaitable ior introducing nucleic acids into viable :^ilxV’%e:|^hmqu.eS'-V!&amp;fy depending Opon whether the nucleic acid Is transferred into cultured cells in vitro, or in vivo in die ceils of the Imeruk'tl host. Techniques suitable tor the transfer of nucleic acid into mammalian cells in vit ro include the use of liposomes, cleetropmuium. microiojeedou, cell fusion, chemical treatments, DimUhiksit'an. and calcium phosphate nuxunuaiion. Outer in viu) nucleic acid rom-dcr ledmiques include frimsiertiou ohh viral vectors (Midi us adenovirus, Herpes simplex f vires, adeno-assoit-ucd vuus or icn-mims} and hpui-based systems The nucleic acid and transfection agem are optionally associated wnh a micro pare-do Exemplary transfection agents include calcium phosphate nr calcium chloride eo-precipitation. D 1:: AF- de x s ru m- m e d iaϊcd imrisieesion, quaternary ammomum amphiphtie DOT ΜΛ όdioleoyloxypiopyl! irmiedolantmoimmt bromide, oommercialDod as Lipotbefiu by CdRCO-BRl )}(Pelgncr et ah i 1 9:sr·t p^ec Natl Acad. Set. ISA ad, 7413-74 I"; via lone el al ; ;aM9i Free, Nat; Acad, Set ISA 80 όΟ" 7-6081 j: lipoohdic gjuSatmu·,- ciomiers ouli pendent frimeihyin.mmomum heads tho ctaf f 1990) Biovhvm Biophys .-\eta 1023, 124-182):, the metabolizable parcel lip-ids such as the catii.;nic lipid dioetadecGamido glyeylspcrotine (DOGS. Transieetam. Promege) and dipalvnitoyipnesphaddy! elhanobmn 1 sperm;no (DPPFSil J. P, Belir 11 9X0} Tetrahedron l eft 2~, 5001 -5864, J- P. Bohr et al 11 Ass-) Free. Natl, Acad. Set. USA 8o„ 6vh?--<s9X6); mcdahoUtrablc quaternary umroonitmt saits i DO T B, N~{1 2.3-dioleoylos\ 1propyi)-N.N,N-tnmelhylantniotmmi methylseifatc (DO iAPuBoehringer Mannheim), nolycUtyk-neinime (RBI), eioleoyi ••ester's, ChoTB. ChoSC, DOSOi 1. eventis et ah i 1990) Biochint Inter 22, 23,8-241 K 8heta[N-(N', N'-dirnethylatninoedianey-eurtumoyi jcholesterol (DC-Choi), dloieoylphosphtoddyi ethunolarnine 1DOPE)· .>be!a[N~(NhTsf~di/Yicfhyi3nthtoeihanel-eari>amoyi )eholestcroliX'-Choi in one to one mixtures (Gao et al.; 194))) Biochim. Biophys, Acta .106.5» 8-141 spermine, spermidine, lino poly amines (Behr of. a.l. Bioconjugatc Them, 1994, 5; 382-589), lipophilic polyKsines (hid. j.) (/hoc et ah. 11991? Biochim. Biophys. Acta uThe 8-!9), {lii,iJ,.>-toL^nK*tbyibtityl>cre-soxyiedmkv jedndjdlnmthyihenxylHmmoiaorn hydroxide tDl.BDA hydroside) v\ id; excess phosphaodyIchohno/At ο 1 csi e re; (Balias et al, ί Biss) Biochim. Biophy-x Acta 939, 8-.18), eclyhrirneriiylamrnooiuo· bromide (CTAB) DOPE mixtures (Finnaduvvaev.' et a], (1989) Biochim. Biophys. Aeia 985, 33-5-1 lipophilic diesiev of glutamic acid; IT Vi AG) voth DOPE, CTAB, OEBDA. didodcoylammonium bromide (ODAB), and stearx i a male in admixture vs ith pbosphatnlylethanohimine (Rose et ah, (1991) Biolechniquc 10, 520-535), DDAB DOPE ;Tr;rnslectA('F. GiBCO BRi.)„ and Mmogaiaaose bearing lipids. Exemplary n'nijsieefion enhancer agents that increase din efikiencv of transfer include. km example, DFAP-dextmn. polybrene. iysoxome-dlsruntive peptide t'Ohmori A I ct al Bioehem Biophys Rex Commun inn. I?A I 99~;2 25t 3 1:720-9), elnuvdrohan-based pivteogKcsas, ani fated nroieogiyenns. puh etln lent mine, polylysme {Pollard s I et ah j Biol ('hem. 1 9*A 273 t 1 3 3. “507-1 I}, mtegrimbinding peptide C YGGR.GDTP (S1:'.Q ID R0:235p linear Jextrao nonasuceharide, glycerol, eholesicryl groups tethered a5 rite .V-terntinui iniernuelcoxido link of an oligonucleotide {Letsinger, R. 1.. W89 Free Natl Acad Sol USA HR: (1 7-:6553-6), lysophospnatide, lysopliosplunuNleholme,. lysopbospbaddyldthanolaraiftiv and I -oieoyl iysophosphatidy idiolme.

[00-152) In sonic situations it may bo desirable to deliver the oueiete acid with an agent chat directs the nucleic acid-containing vector to target cells. Such "targetingf molecules include antigen bmdmgprotdus specific for a cell-surface membrane protein on the target cell, or a ligand for a receptor on the target cell Where liposomes arc employed, proteins svhkh bind ?o a cell-surface membrane protein associated with endocytosis may be used Kir targeting a.tul 'or to facilitate uptake. Bssrnples of such proteins include- capsid proteins and fragments thereof tropic fora particular ceil type, antigen binding proteins tor proteins nhicb undergo internalization m cyding, and rente ins that target intracellular local mat ion and enhance intracellular half-life, hi other embodiments, receptor-mediated cndoc> utsis can bo used Such methods arc described, for example, in Wu ct ah. 1987 or Wagner et d., I 990. for review of the currently know u gene marking and gene Ac ropy protocols, see Anderson i 992. Sec also WO 03/25673 and the references cited therein. for additional reviews of gene therapy technology, see Friedmann. Science, 244: 1275-1281 (iyfh)p Anderson, Nature, supplement to vol. 392, no 6679, pp 25-30 (1998)1 Verms, Scientific American' 68-94 {19903: and Miller, Nature. 357. 45546011992), [104531; Administmiou atfo [:00454] Γη. η od)NP m ant M II o ntgeu b 55 «tote ^ , s antibodies uw'd i the practice of a methoe o 'die n,\emiou uu-λ U .o.m\ L, <j , uo phtniracumeaJ > compositions and m-.,dieu meats comprA;og a carrier suitable for the desired delivery method Suitable carriers include any rattened which, a hen combined with the anti-ONP o'- aru -klJl anugen binding piofemor ami hod>, retains the high-affinity binding of DNP or k 1. R, respectively, and is nmuvactko u tth the subject's immune xvsteres i'atnp.es jJuee. bur are not .sendsa t<> ,w ,d a "j.W's of xtarutare pharmaceutical caniers such as sterile phosphate be; re red saline solutions, bacteriostatic ouvr. and the like. A variety o*'aqueous cm-ncr-·· may be used, e.g., lyaiord'buitered kater. 0.4Α> saline, 0.3°« glycine and the Uke, and may inelddbolfe; proteins for enhanced stability, such as albumin, lipoprotein, globulin, etc., subjected to nuld eh enured modifications or the like.

[00455] I se opu,y am'gen b.odmg s' oicw conccmru* ,ons m the humiliation nux range from about 0 1 rog/mi. to about. 180 mg'mi or from about 0,1 rng/mi. to nboiti 50 mg'mt, of Umu about 0 5 mg ml to about 25 mgniL, or aitermdoely ft om about 2 mg-ml. to about 10 mg/mim An aqueous formulation of the antigen binding protons use;, be prepared in a pH-butTered solution, tor example, at pH ranging from about 4,5 to about. 6,5, or mom about 4,8 to about 5.5. or alternatively about 5.0-,. kvunpies of buffers that are .suitable for a pi 1 vviibin this range include seefowde.g, sodium acetate), .succinate (such ns sodium suecin.uie). gluconate, hsstsdiue, curate and other Prgahipmdhuffer^.· The buffer concentration can be from about 1 mM to about 200 m M, or from about 10 mM to about 60 fflM, depending, for example, on the butter and the #sifed isofonieity ofthe formulation.

[004561 A tonicity agent, which may also stabilise the antigen binding protein, roes lie included in the formulation. Exemplary tonicits agents include polyols, such as mannitol, sucrose or trehalose. Preferably the aqueous immolation is isotonic, although hypertonic m bypofdpfe solutions may be suitable. Exemplary concentrations of the polyol from about !%lo about 15% w/v. P0457] A be added to the antigen blfidmg io#duee aggregation ofthe Ibmtulatedunogeu bmdmgprotein and/or tMnttiizd thd idrotation 6ffs.adA>r 'reduce adsorption. Exemplary surfactants include ttonionte surfactants sac!; as polysorbates te.g. poK-Norbatc 20, or poiysorbate HO) or polosamers (e.g. poloxamer frib}. Exemplary concentrations of surfactant mayiangelmt! about %00i% to ahom 0.5%, or from about fr.005%|o about 0.2%, .or dliemaiMy-fktm-Mtottt 0.004% to about 0.0! % w/v.

[004501 in one embodiment, the formhlation contains the above-ddentifretf agents (no. antigen lauding, protein- buffer, polyol and .surfactant) and is essentially free of one or more preservato es, such as benzyl alcohol, phenol, m-cresol, ehlorobuta.noi and besnzethonium Cl. In another embodiment» a prcaon?afryebtay be ibehtded ih lid ; formulation, c.g., at concentrations ranging front about 04% to about 2%v or alternatively from about 0.5% to about 1%. One or mom other pharmaceutically: acceptable earners-, excipients or .stabilizers such as those described ut Remington's Pharmaceutical .Sciences 16th edition, Osoh A. Ed. {I'^O^·=ΐ5πρϊ3?··t^ie mmf .. ton p.o\ teed that rhe> do not adx emely afreet, the desired eharaeiemtses ol tlte formulation. Acceptable carriers, excipients or stabilizers amnonidgie to i,;,plants at the dosages and ioueeno alums employed and melude; additional bnfieriitg agents; co-solyents; antoxidants including ascofrnc acid and chelating agents such as EEfrA| me|ai completes |C;g. Zn-proteut complexes^; biodegradable poismess such us polyesters, and Or suh-fruoung vouuteuons such as .sodarm.

[00459| Therapeutic formulations of the antigen binding protein -are prepared for storage by mixing the antigen binding proteui haying the desired dbgme of purity with optional physiologically acceptable carriers, ex.eipi'^^:or4taMii5ep' {Remington's Pharmaceutical Sciences Ifrth edition, Osoh A, Ird, 11900}), in the term of lyophibeed formulations or aqueous solutions. Acceptable carriers. excipients, or stabilisers are nomoxie ro recipient at the dosage* and coneemratiohss employed, anti include buffers such as phosphate, citrate, and other organic: acid?'; antioxidants including ascorbic at Id and roetmomne; preservatives (such a* 0 c I rid ec y I d i m e t h y I See ηνγ i ammonium chloride; heK&amp;rneihonium chloride; iamzalkomum chloride. hen/ethoiiiuro chloride, phenol, butyl or honey 1 alcohol; alkyl parehevis such as methyl or propyl pa radon; catechol; ro'ordnol; cyclohexa nob; 3-pcniarinl, and m-cresolK !οά molecular weight bless than about 10 residues) polypeptides; proteins, .such as serum albumin, gelatin, or imrmtnoglohuUns; hydrophilh. polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaeehandes, and other carbohydrates including glucose, mannose, maltose, or dexirimt; chelating agents such as IDEA: sugars such as sucrose, mannitol trehalose or sorbitol; salt-forming counter-Ions such as sodium; metal complexes ic.gr. Z tv protein complexes.!; and or non-ionic surfactants suelt as [WHEN!M, lR.URON(CS!M or polyethylene glycol (PEG),

[ 00-160} In one embodiment, a suitable formulation of the claimed invention comas ns an isotonic buffer such its a phosphate, acetate, or I ns P-dler in combination vvith a tonicity agent such as a polyol. Sorb no!, serose or sodium chloride v> Inch tonicities and subtilizes One example of such a tonicity agent is 5%i Sorbitol or .sucrose, In addition, the rormuiaum could optionally include a suriacnav? such as to prevent aggregation and for stabilization at 0.01 to 0.0UU 01 vol. Thu pH of tlie formulation may range from 4.5-0.5 or 4.5 to 5,5. Other exemplary descriptions of pharmaceutical formulations tor antibodies may be fotmti in US 20054)1 133 IS and US patent no. 0,171,580, each incorporated herein by reference in its entirety.

[ 00-Us 11 Ί he formulation herein may akt contain more than one act tv·.* compound as necessary for the particular mdicatson being treated, preferably those v ;th compivtnemary aettviries that do not adversely affeu each other, for example, it may be destmbk* to further provide an tnirtm-ioMipptvssh,-.- age;it, Such molecules are saltahly present tn cpmjbmaiStie» m amounts thai#&amp; elective for fhe purpose intended.

[00462] The derive ingredients may also tv entrapped ut tmcrocaptulc prepared, tor example, by coacervation techniques or by intcr&amp;dal polymerizMiori, for -example. hvdmxyroeihylcellulosc or eclatm-roieroaipsuic and poly-(roclhy Imethacylate) rnkfocapsuic, respectively, in colloidal drug delivery systems (for example, liposomes, albumin mierosphents, microerottisious, nano-particles and oanocapsuics) or in macrocmuhious. Such techniques are disclosed in Remington's Pharmaceutical Sciences lOth edition. Owl. A. td. U9X0). f00403} Suspensions and crystal forms oi antigen binding proteins are also contemplated. Methods ίο make suspensions and crystal forms are known to one of skdi In the art (604m( I The. formulation» to be used for in vivo administration must be sterile. The -compositions of the invention may be sterilized by eon eon banal, well known sterilization technique:». f or example, sterilization Is readily accomplished by filtration through sterile 111 matron membranes. The resulting solutions may be packaged for use or filtered under aseptic conditions and lyoululized, the iyophiimed preparation being combined with a sterile solution prior to ad.ministrat.ion.

[00-1651 The process of freeze-drying is often employed to stabilize polypeptides ibr long-term storage, particularly when the polypeptide is relatively unstable in liquid compositions. Λ lyophili.oitiors cycle is usually composed of three steps; freezing, primary drying, and secondary drying; Wlhbnss and Fold, Journal of Parenteral Science and 'Technology, Volume vV Number a. pages 48-5° ί 19^4}. In the (feezing step, the solution is cooled until it is adequately frozen. Bulk water in the solution forms ice at this stage. The ice sublimes In the primary drying stage. vvhii.ii is conducted b> reducing chamber pressure below the vapor pressure of the ice, using a vacuum, f inally, sorbed or bound vy.aicr is removed ar the secondary drying stage under reduced chamber pressure and an elevated shelf temperature. The process |5:mdufee$ a; material linpwn as a ©act: be reconstituted prior to asm 100466] The standard reconsunpion practice for lyophiUxed material is to add bach a volume of pure water (typically equivalent to the volume removed doting di^ufev^ltnionA. of antibacterial agents arc sometimes used in the production tU'pharmaceuticals for pas enteral admimstnitiom Chen Drug. Development and Industrial :fe&amp;$emacy. Volume lb, 'Numbers i 1 and 15, page* K>1 1-1354(1002!.

[0046?) I emp-ems have been noted n some eases m aot. -· stabm/eis for bee ; -dried product;»: Carpenter er ah, Developments in Biological Standardization \ oi one 74, pages 27ς-2,ν,' <109|) fc, example, kerns " v,v ψ seats me luce polyols. ( 0., Hiding mannitol '<orhud and glycerol): sagats p. κ\αο ng ek-co-m and sucrose}, and amino adds (including alanine, glycine and glutamic add), |P046B] In addition, polyols from freezing mid drying-induced damage and io enhance thednyii^duringodoruge in the dried state. In general, sugar*, in particular dmccharikfel,^«tBctiyem-fybth the freeze· .i»irg ptoi ev* and dating storage Ot.hei els^ev id molecules, me Hiding mono* and dksaccharide?» and pels mors such t$ PYf. have alboBeen reported as smKlver* of iyoplti i/ol produce ! Omf-m, for injectmj\ me piiarmseenn, a'. formulation and 01 mean ament may he a poo/der suitable for reconstitution sv dh an appropriate solution as described above. Examples of these include, but. are not limited to, freeze dried, rotary dried or spray dried poo ders. amorphous pov, de;s anomies. precipitates, or pat Ululates I or injection, the fonmdahons may opnon'dh contain stabdmers, pH modifiers, mu facta its. bioas anabdu. ntodtficu, a* d eomlunune;···, of these [{,1047:0) cuMu'nad-ri lease preparations may Iv pm-pared SmtabO examples <u sustained- release preparations include serntpenneable matrices of sohd ?ndiophubic polymers containing riccn are in the form of shaped «nicies, e.g.. films, or mieroeapsulc, Examples oi sustained-release nvnfsces include polyesters, hydrogels t for- example. polys2drvdrr.nyeihyl-n';efhuefy)<ne}, or polyfviny [alcohol P>, polyluaides tU.S. Patent No. 3.773.419,). copolymers of Ιοί ui anile acid real y etiivi4.-glutamate, nun-degradable etlg,dcire-winyf acetate, degradable lactic acid-glycolic add copolymers such as the ί .apron Depot;M 3 injectable micnwpherc» composed oiluctic acid-glycolic acid copohmer and leuprolide acetate}, and poly-CM · )-3- h\ droxybufyric acid. White poly on era such as -ethy lenc-vinyl acetate and lactic add-glycolic acid enable release oitnoieeuks for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated polypeptides remain in the body lor a long time, they may denature or aggregate as a result of exposure m moist arc at 37"'C.. resulting in a loss of biological activity and possible changes In immunogenic!iy Rational strategies cun be devised for stabilisation depending on the mechanism involved For example, if the aggregation mechanism is discovered to he interntojoetdar S~S bond ion station through thio-dtsulildc interchange, stabilisation may be achieved by mod dying sulfhydryl res idem, lyophilDing horn acidic solutions, controlling moisture eonteut, using appropriate additives, and developing specific polymer matrix compositions.

[004711 The formulations of the invention may be designed to be short-acting, fast·· releasing, long-acting, or susfamed-rcleasing ms described herein Thus, the pharmaceutical formulations may also be formulated for controlled release or tor slow release, [0047c] SpcctOc dosages may be adjusted depending on conditions of disease, die age, body weight, general health condemns, sets, and diet of the subject, dose intervals, administration routes, excret ion rate, «mu combinations of drugs. Any of the above dosage forms containing effeetn c amounts «αν well wnhm the hounds of routine experimentation and therefore, '.veil * itlnn the scope of the instant invention.

[0Θ473] The antigen, binding protein is administered by any suitable means, including parenteral subcutaneous, intraperitonoal, min;pulmonary, and ImravuisaI, and, if desired for local treatment, mtraiexiotnd administration. Parenteral infusions include intravenous, intraarterial, intraperltoocak Intramuscular. imradomtal or subcutaneous administration. in addition, die am naan binding protein m subsidy administered by pulse infusion, particularly with declining do*es of the antigen binding, protein or antibody. Preferably the dosing is given by i raccoons, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic Other administration methods arc contemplated, including topical, parucuUrk fKawdcnnai, uansiruscosa!, rectal, oral or local adrnnnsirmion e.g. through a catheter placed dose to the desired site. Most preferably, the antigen binding protein of the invention is administered iiitritvencnislf in a physiological solution at a dose ranging between 0,01 mg kg to 100 mg kg at a frequency ranging from daily to weed} to monthly (e.g. every day, os cry other day, every third day. or 2, 3. m 5, or U times pet week), preferably a dose ranging from 0.1 to I'5 mg’kg, 0 I to id tog'kg or 0 1 to 10 nag kg at a frequency of 2 or 3 times per w eek, or up to 45mg kg once a month [fK}4M] The invention is dlust-ated by the follow mg examples, which are not inieuciedte fee limiting 'id 'my way, [00475] EXAMPLES 1004 76 j Exam ph: 1 [00477J Generation of antibodies to DNP or KOI ami screening )00478] Immunizations. An*i-DNP antibodies were generated fey immunizing XenoMousc^ mice with DNP-KtH, over a period of 4 weeks, and fey st reciting for those' antibodies that bind to DNP-hsine Mon.1 paAlcuiarSy, XcnoMouscKMG? strain of mice were generated generally as described previously {Mender et ak, \<ji Gencf. 15:146-.156 {19971; published International Patent .Application Xos WO 9H/24893, and WO 00/76310, the disclosures of which ace hereby incorporated by reference) and OTArunized with .;2?4-Pmifr0phfeuyl-K«yht% sM^pcfeBemdcyaBib {DNi'-KLH conjugate: Bk:>,Search Technologies, Novato. CAb usings range of Kb 30 ug-'mouse ofimmu nogen emulsified in 13 ter VI ax Goid .adjuvant {Sigrnu-A Idrich. Oakville, Omrambi for -ho inn-ad immunization of the XMG.7 -anan of Xeno Mousellv; according to the methods disclosed in Imoraaturnai Patent Application Nos. WU '*b 14 no A and WO 0076.00, the disclosures of all of which arc hereby inootpo rated ho reference. Following the initial imnnmization, subsequent boost of Immtmogeu (5 -10 gg-mouse) were administered on a sc hod ok and rot the dotation necessary to induce a suitable anti-DNP titer in the mice Titers were determined by enzyme, immunoassay using immobilized DNP-BSA t BioScurch Technologies, Novato, <3 A), this conjugate was prepared such tltut the final DNP:BSA molar ratio was 30· 1, (00479] Immunizations to raise anti-K.l.B antibodies were conducted, over a period of 4 weeks, using. trnieeGS Manet;here Keyhole Limpet hemocYsnin trneKI. ft.

Pierce Biotechnology. Rockford, II.; catb "?b00, loXB114035144 immunizations were conducted using lO.ug of KLH per mouse in Aluminium Phosphate Gel AdjuvanMiK I Biosector, t'reden ksnund, Denmark. Catalog i 1452-130); delivered <·, la footpad injection. The initial immunization of the XMG1K. strain of XenoMousc*»’ was according to method* previously disclosed f Mendez et ah, .Vo/. Gena. 15:146-156 11907); pubis sited hiicmariomd Patent Application Nos. WO 98 14893. and WO 00-76310, the disclosures of which are hereby incorporated by reference, v, hich are all hereby incorporated by reference). Following the initio] immunization, subsequent boosts of immunogen (5· 10 nghnouse) were administered! on a schedule and for the duration necessary to induce a suitable ann-KUl liter in the tniee. Titers were determined by enzyme immunoassay using immobilized K1.H CPierce Biotechnology, Roektbrd, 1L), (004sib Preparation of monoclonal antibodies. Mice eshlbiUng suitable titers were 1000014.0,1, and lymphocytes and splenoeytcs were obtained from draining lymph nodes and spleen, then even.· pooled for each cohort B cells were dissociated ifom the tissue by grinding in a suitable medium B’or example, Dulbcectbs Modified Eagle Mddi 1.1.0:; OMEM; ho itrogen, Carlsbad. CA) n> release she cells from the tissues, and were suspended in DMF'M. B cells were selected and or expanded using standard methods, and fused with suitable fusion partner, for example, nonseereiory myeloma P3To2 \gS o53 i ells t Amortem 1\ re i 'i.hare Collection * '81 1580, l\eunv> et a a «Λ fmmmol. 12a 1 PdSufoSO (W'd'o. ass no techniques known in the art, [004811 B culls a cre mixed with fusion partner cells at a ratio of 1:4. The coll mixture was gently pelleted by centrifugation at 400 x g for 4 minutes, the supernatant a as daeaumd, ,md the ml mixture w as gently mixed by mote a I Oil pipette. Fusion, was induced w ith PEG DM SO {polyethylene glycol-dimethyl sul foxide; obtained from $lgma~Aldtich, Sr, Louis MO; I ml per mi Ikon of lympbocytes), FEG/DMSO was slowly added with geode agitation over otto minute followed, by one minute of mixing. iDMEM t DM EM without glutamine; 2 nd per million ofE pells), was then added over 2 minutes with gentle agitation, followed by additional I DM EM (8 ml per million B-eells) which wax added over ,> minutes.

[00482] The fy*ed cell* were gemlv pelleted {400 \ g 6 ms notes} and resuspended in M ml Selection «tedium (for example, DM EM containing Axasenne and Rypoxanlhtne [HA] and other supplemental materials as necessary) per million B-celG, Cells were incubated for 20-30 minutes at 3T'C and then were mMwponded in 200 ml .Selection medium and cultured for three to four days In T CD Basks prior to 00-well plating, [00483] Cells were distributed into 06-wcll plates using standard techniques 10 maximize cionaUty of tbe resulting colonies. After several days of culture, the hybridorna supernatants were collected and «ubjectcd to screening assays as dehitbd : in the examples be.ov, m. luding cmdu ounce n; b n.l m to M1! ot DN!\ respeem ely Positive celh wire furthe- selet ted. and vabmeted to si mdard etmnm; and subelonmgtechniques, Ootraf hoes were expanded xkro. and tbe seen.red human antibodies obtained for analysis. Several cell lines sec ret mg DNP-sp'.ctde antibodies were obtained, and theautihodieswere further characterized. The sequences thereof are presented herein and in the Sequence Listing, and results of various teats using these antibodies Me provided; t

Ck>nmg,MlEn&amp;^ AntM^.Mtlijyua.ap.<j..fenti4.>NP.

The sequences for the .Kersomoaso derived human an?i~K.LH antibodies were obiumed bv me polymerase wum reaction (PC'R» amplification technique known a* ?' R \i ( b pm uph ^ut o*' <u ν.Π^ V ends) Tm ί RV\ was wmateb t on tb.ee Its brnton-a-' exp? easing Ki H binding monoclonal antibodies. I o. 1 L I Ob 1.2 and dm n us TRLv* reaver. {bat hsot t u) fo lowed O' mt he p. * λ a bon us ng +bc R\ Mn K i lO ajj.cnϊ Aimed? i loo . idol a cl ο ι sk t ’V she d lb f rcadv cDNAs were prepared using the GoneRucer Kit {Invitrogcn), PCR a np'b v a urn ,·> of fh. <, DMAs were pe' toroed o eh Advantage HF2 D\ \ polwcetev (Ckuuceh) vxiththc forward primer. GcneRaeer''5 nested primer: J00-1S5] S'- CP4iAC TOA €Af GGA CTO Λ AG GAG ΙΛ -V-7 (SEQ ID •'NOprimes; I'OOdbb) 5'. C re CTG GO A GTi AC C CO A TTG -3'.'/ (SEQ !OilGi2:|kApri{hm light chain, ηικί 5 -1 iAT GGG ί ( G ΓIT.} OTG GAG GCT GAGOAi5iA]C00TO'· ACC. GIG G GrV (SEQ ID NO:2?3 b for the lumvy chain. The PCR reaction cycles consisted of a 30 second deuaturation of the eDNA# ^4°C, followed by three cycles of-aimplifieMipo with each cycles consisting of 20 seconds at 94s€’: 30 seconds dt 5;e€: and 90 seconds at 7>TN plus an additional 27 cycles consisting of 20 seconds afM'TA 30 sceonds-M 03°C; and 90 seconds at 72^0. The reactions were then incubated for 7 minutes at 72 C following t|u« last PC'R cycle to msuie complete elongatiop. The RAGE FCR products* were dotted into pC'R4-TOFO < Irottrogen) :ahddc%Wtincd using AB1 DMA sequencing urstn intents f Perkin Elmer). Gonitet-sus sequences were determined using Vector NTI 8,0 softwate ilnupogenunv used to <.xvon p iru > iw tu'l-lcng.h antibody chair. I\ R amplification.

[:01)4871 To obtain the complete coding region sequences for the expression of ami-kl 1! mrmod .¾. v vnu 16,3 '. as at' c* ample, ^ 'R w-.s .mat t used t he hub* chum .V PC'R prints^ encoded the j-nino terminus of the signal sequence, a Sail restriction enzyme site, and an optimised Kozak sequence was: [ 004*81 5AAG Cl C GAG G) C' GAC'fAG ACC’ ACT1 A) G < *AO A'I'G AGG -Tu (SEQ !D NO:274K trod the 3' primer that encoded she carboxyl terminus and termination eodmoas well as a Not! restriction site was:

f 004*0] s'. Λ AC CGT Ή A AAC CiCG GCT ΟΓΓ (ΛΛ CAC l Cl C'CC CTG TTG \ A "3 ·' iSB3|ONO:275K

[004901 The heavy chain 5' PCR primer encoded die amino terminus of the signal sequence, a Sail resTieriqtf en^ypie sifevtf Knnati Sbqtieqee was::: [0049 M S'- A AG CTC GAG GTC GAC 'IYU. M 3 ’ ACT' ATG (ΪΛΛ Ή G C4 ί A C IG AG 44' (SfcQ ID NOG76), end the 3' prime? encoded theea-rteyHeimtmJS· sind komhutm' xodom as -a all as a \otl w-t-tumm s>\ a as 100492} 5\ At COT 1 Γ A A At' GCG t XT G( Ϊ C A T ΓΤΑ C'CC GOA GAC . AGG GA -37/ t$EG 10 NO:277).

[00493} The PC Rs were performed using and

Pv w tenw cycles von-fv.ed ev second deoatur.mou of the cDInA a? *MV\ OGoacii b\ 40 cyek"- eomusong of 2(¾ seconds at 04 <’. 30 seconds at 65°C; and 90 seconds at ~2 C . The wv.ctions vwn. then incubated for 7 minutes at "Tf following the last PC R cycle to insure complete elongation. The restdf mg PCR products: wend gel isolated, purified using QIAquiefc spin columns (Oiegeni digested with Sail tNFBI ί snj Vet {M HI y v s iso o and p rd , d uv.n> qd \qtneh s nn colas uw, and then ligated into die mammalian expression vector pTT5.

[00494] The sequences for the X an o A louse15: -densed human a nil-DTP antibody variable region» were obtained by sequencing reverse transcription PGR products. PCR. was then used to adapt the variable region .sequence ends to make them ;·compatible with the ends ol'pTTS vectors containing a VK.I signal peptide and the. appropriate antibody constant region. As example, untl-DNP 3A4 light chain was cloned mto pi ic- using the unique BasHlI site at the ere <V.t Vk I -¾ enal peptide atm the unique BsiWI site at die beginning of the human kappa constant region. To add tb&amp;IiMiil i aM ifseBsiWI sfe to ilK end<5 of the 3A4 variable mgimr was mnpliied: by PCR using 5’ primer: [00495] 5' 111' H'T TTG ('(.if.' GCT GIG ACA TCG AGA TGA CCC AGT C 3’vTSEQ !l> NO:2"·>;}.

[00496] <md o' primer 5' AAA AAA CGT .AGO TXT GAT ATC CAC ΠΤ GGT C C' X' 0Sl:.Q ID >40:279}.

[004071 The miU-DAF 3A4 contained a tryptophan in the variable region of ihe heavy chain. The tryptophan codon sv&amp;h mutated to u phenyUdanie by PCR using {} strand primer;: [0049b] 5' ('TG TGT AiT Α(Ί GTG CGA GG T ΛΤΛ AC' l ΤΓΛ AG 1 ACG OTA TGCXACG TGT GG 3 V/ CSBQ IP NO:;2Mf} and (-] stmswl primer; 100499] 5' CCA GAG GTC GAT ACC GTA Gil' GAA GTT Λ ΓΑ (. GT CGC ACA GTA AT A CAC' AG 3'/ t$EQ ID NOC8U and to a tyrav.no by PCR using (f) strand primer; [00500] 5' CTG TGT ATT ACT GTG CGA GOT AT A ACT ACA ACT AGO GTA TGG A< G TGT GG itSF.Q ID NO:2H2) ana t-; strand primer.

[005011 5' GCA GAC GTC CAT AGG GTA GTT GTA GTT AT A C CT CGC ACA G J A AT A CAC AG S‘::CSEO iD NQ:2K3> in conjunction with the heavy drain 5’ end primer [00502] 5" AAG C TC GAG GTC GAC TAG ACC ACC ATG GAG Alii AGG GTG CCC GO' CAG GTG CTG GGG CT 3' (SEQ ID NO. 284» and the heavy chain 3! primer: [00503 [ S’ A AC CGT IT A Λ AC GCG GCC GCT CAT TTA CCC CGA GAC AGG GA 3'·' (STQ ID 00:247;. Also., to reduce disulfide scrambling in the in.nee region of the 3A4 igG3 heavy chain . as example, the hinge cysteines 2N .md 3.70 I EC numbering) were mutated by PCR using O } .strand primer.

[00304] 5' GGA CAA GAC AGO TGA GCG CAA ATC TTC FGT CG.\ GIG (. ACC GI G €CC AG V·/ (Sf.Q ID Ν0.286» and {-} .v.nind pnrnor: [00505] 5' CTG GGC ACG GTG GGC ACT CGA CAG A AG ATT TGG GCT CAA· CTG ITT TGT CC 3"A (SEQ ID NO:2S7> in conjunction with the heavy ehsin 5' end printer: {005001 5' A AG ΓΓΓ GAG GTC GAC TAG ACC ACC ATG GAC' ATG AGO GTG ax GCT CAG ere CTG GGG CT 3'· (SfiQ ID NO. 288) snd (he heavy chain .V primer:; [00507] 5' AAC CGT ΤΤΛ A AC GCG OCX' GO CA Mi \ C CO GGA GAC AGG GA 5' · (Sf: Q ID NO:289>.

[00508]

Transient transfections wore carried out in HER 2°3-6E ceils as follows. The human embryonic kidney 293 cell line stably expressing Eipstein Barr vines Nuclear Antigen-1 (293-61: cells) was obtained from the Nut tonal Research Council i Montreal, Canadas, Ceils were mairaamed as serum-free susnensiun cultures using FIT medium (’tomopen, Carlsbad, CA) sypplemeffifed with AmMiL-gliitaffiisje (Invitrogen. Carlsbad, CA), CFG E-f-A Plutonic (Invlrrogog ( aiisbueL CAt and 250 pg-ul Genciicin (Invitrigcn. Carlsbad, C A) The suspension ceil cultures were maintained in iiclciuneywr shake flask cultures. The cohere flasks were shaken at 65 rpm at 37 C in a humidified. 5':·« (, 0.> atmosphere A stock solution ( Irrtg mi) of 25-kDslinear FO (Polysciences, Warrington. P.A) was prepared in water. acidified w tth ITClTo pH 2 0 until dissolved, then neutralised with NaOH. sterdiaed by filtration (0.2 prop ahquoied, and stored at -20A" tanti used. Trvptotie N’t was obtained front OruanoTechni S.A iTekntSeience, QC, Canada) A stock solution (20%. w v) was prepared in Freestyle medium (ho itrogem, Curishud. (. A). slerUtr.ed by filtration throneh 0.2 urn filters, and stored at 4Τ' nurd use. Typically, transfections were performed at the IL scale. (. ells (293-61:.) were grown sou a viable cell density of Cl ,X .106 cells an! then translcetion complexes were prepared in ί /10th volume of the final culture volume. For a Id. transfection Cithtnv, transfection complexes ά ere prepared in 100 ml h 1' basal medium, ami 500 pg plasmid ΟΝΛ theavy chain: an# iml-t cUam DNA, 1 ;1 ratio | wax fn>t diluted in 100 mi Π7 medium. Alter a 5* minute incubation at room temperature* ί<3 ml- of PisLsohftton· wm added:. The v.otrolexs.a hok' \m ed n, ldl>, then mculwlod te? IS minutes at room tomperanm' Rie cells w. >e s.urxw ted i \ acmee the uunsfeeiton complex rm\ to ihe eeih m the abate flask culture. ,M hours po^-tranaifectid^,f#J waa added to tlte transfected culture n> a final concentration of 0.5%. mamtamed on s> ,c ait' pt ii}"( < t.. numiditied. teR Ui> atmosphere lot another 5 days attef w Inch they w c« c ban ested ihe conditioned medium was harvested by centrifugation at 4000 rpm. and rhea sterile'filtered through 0,2 pm biter {Coming Inc.), i tate"*.)] Ttie v/oK ' \pw\w, J Jsl Η 1 SO P vontroi anubods pool was ueatui by twins vetirw C'HOd Post ceils w d. exp, vision .plasmids pDC.'323 ams-teI H 120,0 kappa LC and pDC32i anti-Kl.H 120«Idl^.:dcctroporaddd procedure. After transfection, n»_ sells were grown as a pool in a serum free -GH'i selective growth 'media to allow" for selbeiibtr and recovery bfdie plasmid cont|iim;% •cells. Cels pools grewn in Gif Γsclecdye moiha were cultured until they reached S5% viability. The selected cell pools v> ere amplified w ith 150 nm and 300 nM methotrexate (MTX) Upon reaching >85% viability the 150 nM pools were then further .re amplified m oOO nm ΜI \ \\ hen th„ v whsI»tv ot the VI1 \ vnol fed pools reached >85¾ viability, the pools were screened using an abbreviated six day hatch ^^lietten attehftelted produeion media to assess eaptession. The: ej^^otepfAd.^mplified'pools ranged fern 120-400 pg/mL. The best pool was tboxen based ou (tic x*\-da> a,v>as and scaled-up using a ten-day fed batch ptocoss The conditioned med<a was hancsied and pur died to prot tde protein for analysts [005 m The stably expressed a&amp;llf 120.0 a®f body pool was created by transfecting C’HO d- host cells with expression plasmids pD023 umi-KLU 120.0 kappa LC' and plX'324 <«ui -KX.H 120.6- lgG2 11C using a standard eicettoperation procedure. After transfection, the cells were grown as a pool in a serum free -GHT selective gmw;iJhpedte to allow for edteetidtt'ea^teipvcry cells. Cell pooh grown in GHT selective media were cultured until they reached > 85% viability, The selected cell pools were amplified with 150 mrrand 300flM MTX; Ppotr reaching >85% viability the !50nM pools were then further re -amplified-in 500 nvn MTX. When the viability of the MTX amploled pooh reached. " 85% stability, the pools wore screened using an abbrex taicd <n\ day batch production assay w uh ao enriched ptoduedon media to assess expression. The expression of the amplified pools ranged from 120-400 pg/ml. The best pool was chosen based on the six day assay and scaled up using:!· ton day ted hutch process. The conditioned media was fun·., -. ed ' d p't ο*oo h) provide p*ovm ,' u,\nyst\ [00S i i j Tne aDNP 3A4-F and ul>M 1 annbody stable expression pools new created b\ transfecting C'HO DliFRi ? host -,eds with coiTespenchng heavy chain and baht::eham exptsvs«iSu » plasmid sets \ mi t v. \j ed eles.nope.a,so i pms, duo Pet each am Tody molecule, 3-4 different nausSechons were performed to generate multiple pools. After transfection tie cells were grown a.\ a pool nt a scrum free -Gifl selective gross th media to ai loo for selection and recovers of the piasrmd : containing ceils. Ceil pools grown hi --GHT selective media were cultured until they readied >85% viability, The selected cel! pools were-siiripiI4:50. viability oTthe methotrexate amplified pools- reached >8S%; \ vnbthiy, the pools w eie wrecned using an abbrm ntted day hatch production assay with an enriched production media to assess expression, The best poo! was chosen hased on the six day dbdi.pr^.0ny' IPS laj AatMy.PMifieation and sclecuons. The antibodies were purified by Mah Select Sure einomatogiugly KIT Life Sciences* using 8 column volumes of

di\<dcm cations its the wash buffer and 100 mM acetic acid. pH 3.5, us the do non buffer at 7\' The elution peak was pooled based on iha dimpmtpgram and the pH was raised to about 5.0 using 2 Vt |j is base, Fhe poo! was then diluted with ai. least ί volumes of water, filtered through a 0.22-pm cellulose acetate, filter and then loaded on to asv SP- BP sepharose column (CIE Life Sciences) avid washed with 10 column volumes of S-Buffer A (20 mM acetic acid, pH 5 0i followed by elution using a 20 column volume gradient to 5(1½ S-Buifer B C20 taM acetic acid, ! M Nad, pH 5.0} at 7 'Ό, A pool was made based on the chn.miaiog.ram and SDS-PAGf analysis, then the material «as concentrated about ?-· Ibid and diafiltered against about 5 volumes of 10 tnM acetic acid- 0% sucrose, pH 5 0 using a Vn allow ΤΓΡ cassette with a 70 LDa membrane. 7 he dials/ed material « as then filtered through a 0,22-p-m cellulose acetate filler and the concentration was '&amp;e;ab»ori>»nce at 280 nro.

[00513} The lead candidates were then selected based ott tbc ptodtiei b^avior % SDS-PAGE I he aDNP 3BL 5114, *Γ2„ 5ΛI and 3Λ4 antibodies from·both transient and stable expression mammalian coll lines were analyzed lor product: vitality on a I u-nnn TrN-glyeui.' —70* <> SDS-PaGF (Nov ca } UMug teduemg loading buffer (Figure 11K Using these data, the aDNP 3H4 antibody produced a heterogenous ηκΗίικι from the -nehle cell line, which indicated that n was me a good candidate asm carrier antibody, smee a homogenous productis desirable. The aDN:P "\t, G\4, 3C2, <md 3B1 and eKd H 'ft* 6 antibodies «ore ana iv red for product .mul t\ ort a 1 0-iotr Irv-glyem.. * 2Uwo SDS-PAG£-.(Noy^:i::i$mg non-n^ucing· loading butYcr (Figure 17 A-B} The aDNP 3C2 antibody produced a heterogenous product w ith exceptional high molecular mass material, indicating it was nut an ideal candidate as a. carrier antibody, since a product containing high molecular mass material is not desirable. In addition, the aDNP 3B1 antibody showed a doublet under these conditions The aDNP 3BI and aDNP 3 AI antibodies were then compared using both Trus-glycine SDS-PAGE. as well as bis-Tris NuPAGB systems under non-redueing conditions (S:rigurc 13A-B}. it was lound that the aDNP 3B1 antibody clearly produces a doublet not observed with aDNP 3A1 on the Tris-glvcme SDS-PAGE, however, the aDNP 5B1 antibody appealed more homogenous than The aDNF3A 1 antibody when analysed by bis-Tris NuPAGE, indicating that the doublet mas be an artifact of the method of analysis. When ihe aDNP 38! antibody was analyzed, by Tris-giycinc SDS-PAGE aftet treatment with non" reducing sample buffer at room temperature, K5 ”F. or >O0 A', the doublet was not eliminated (figure MAh However, when the aDNP 3BI antibody was examined by rri^-glseinc SDS-PAGE using 0.-1% SDS in the gel running buffer rather than the usual 0.1%, the doublet: was greatly reduced (Figure 14B), offering additioBdif evidence -bat the doublet e, us an artifact ofihe system of tnudyHs [00514] Antibodies were further analysed for homogeneity using two ssxe exclusion columns (TSK--GEL CGQOOSWXt. r> turn panicle size. AH x 300 mm, ToaohBioseiencc. 08541) in series with a i 00 r»M sodium phosphate, .:50 ruM NaCh pH 0.8, mobile phase flowed at 0.5 ml. nun (Figure 151. The aDNP 3C2 antibody displayed a substantial post-peak shoulder, which was deemed undesirable?, so this antibody was demoted as a candidate came? antibody. In addition, it w as observed that the aDNP 3C2 and uDNP 5A4 antibodies eluted later man expected indicating a potential interactions w ith the stationary phase of die chromatography Column.

[00515] Antibodies {aDNP 5AF aDNP H.'2 and a DAP 3Λ4) were tested tor resistance to pbotodc'crsdaiiom The antibodies were cither exposed to fluorescent light at 4 %' for .1 weeks or were protected from light by covering wimples of each with aluminum foil. The antibody samples w-ete (hen analyzed using two size exclusion columns (TSK-GO. G3000SWX!..» 5 mm particle size. 7.8 x 300 mm. Toi'ohRioseienee, 08541) m series wttn a 100 ntM sodium phosphate, '350 ntM Na< '1 pH 0 8, mobile phase flowed at 0.5 mi. 'sum (Figure HO. the aDNP 3C2 and aDNP 3A4 and bodies showed substantial peak broadening after light exposure, w inch ts -eonsintent with oxidation of a susceptible tryptophan, lo reduce the oxidation .susceptibility of the aDNP 1A 4 antibody, seveoti van an is with theCDR.3 tryptophan tmuated to either lyrosuie or phony lain nine were constructed (aDNP 3Λ4, aDNP 3A4-Y, aDNP 3Λ4-Ρ. aDNP 3A4-YSK and aDNP 8.A4-FSS). These antibodies were then evaluated by SIT' for resistance to pifotodcgmduuon alter two days of light exposure {33hWTn2 UV light and 331 k~utx foi fluorescent Huhtt at 6 (Λ by analysis using two size exclusion columns (TSK-GEL G 5000$ W NX, 5 mm particle sire, 7.8 >; 300 mm, ToxohBmseierec. 08541 s in screes w eh a 1^0 tn.M .e.xlnun phosphate, 250 mM Nad at pH N8 mobile phase dewed at 0 5 rel. min (Figure ΠΑ-Βί.

[005! 61 All four of the aD.N'F 3Λ4 variant;, showed s u Ira ran ti ally less peas broadening dram die wild type molecule, indicating dun die CDR5 trypsophan was responsible tor das imdeairubk phenomenon, furthermore, the retention time extension on SI:. 6’ was also greatly reduced with the* sanatsts huh eating less interaction w ith the stationary phase of the column. Ληη-ΠΝΡ 3Λ4 antibodies with λ r sous rreeauionv (a DTP 1 V-k up\p 3 US uDNP U. -Γ, aONP *A I 3 SS and eD\P Udders St v, etc λ a w ed ioi bomrayrara ot ukog Twoh-as 54-5 PV\ column (10-pm particle, 7.5 turn ID X. 7.5 cm long) iron a Butler Λ (Ί0 m.M sodium acetate, ptl 5 (1) and But let B ( K> :n\f sodium acetate. r()0 mV: ViCT pi 1 5 0) flowed at > rabnun with a programmed 'incur gradio -* (1 mm 0* * B '0 mi" kb B. 30 turn O'D B ' t\ is ‘ΚΜ,,Β and 1ra n O'* B) tPgnre . M 1 he aD’vl'* 3 A4 m.Tixxh with the CDR3 tryptophan v,oo\encd to phem tabs rune ptoduecd a mots' desirable narrower clunra', reek dsrm toe wdo i\p-e otpra-w to s count, theu'*ore Jte uDSP 5 V'-F variant was deemed to be the superior molecule. The aONP 3BI. &amp;DNP 3A4-F, and uDNP 3A4-FSS antibodies wem analysed by non-reducing CT.-SDS (Figure l9A-€)> Mi OF SDS experiments weie pertbimed using Beckman ΡΑΗ00 <h system i Fullerton. f'A ) equipped with 15 V diode detector, 221 ran and 220 ran wavelength were employed. A hare-fused odea capillary 50 pm a 30.2 ctn was used for the sqpuratiosi u&amp;slyais. Bn Bor via.! preparation and Jpding as well m Inblall Capillary

-id the Beekmand.'otilier manual for IgG

Purttyd-ktemgenehy. The running conditions for reduced and nou-redueed ( E-SPS Wero sin.rila.rdo those ddsetibod in Beckman Coulter manudi for lg<3.· PtnitViBetemgcneitv with some modifications which arc briefly desenbed below For non-reducing conditions, the antibody sample (150 pg) was added 20 μ! of SDS reaction buffer and 5 μί of 70 rnM K-ethy 1 male: nude. Water was iheu added to make final wduroe 35 ,ul and the protein concentration was brought to -1.3 mg ml The SDS fcuetloP buffer was made of 4% SDS, 0.01 M citrate phosphate b:uJTk (Sigma) and 0.036 M sodium phosphate dibasic. The preparation was vortesed thoroughly, and heated at 45”€ for 5 min. The preparation was then added additional 115 gf of 4*3kSPS. Alter being vonesed and centrifuged, the preparation was placed in a 200 μ! FOR vial and then loaded onto the PAS00 instrument. The sample was. injected at the anode with reverse polarity using * !0 kV for 30 sec. and was then separated al -15 kV with 30 pat pressure at both ends of capillary during 35 rein sep nsninn Hie J >M 3B1 antibody produced the trod destrabL- ’'rofile wtth the highest iesel of nnribnimy under non-reducing condition^ The a DTP 3BL aDNP 3A4~l% and aDNi> 3A4-FSS antibodies xsere analyzed by reducing i'E-SDS (Figure 20A.-C herein),: Fortedbeing conditions, the antibody sample was diluted to 2.1 rngd-td by adding purified IFO, and 95 μί of the antibody was. .added 105 μι of SDS .sample buffer fBeckman 1 with S.6% beta mercaptoethattol The preparation was then vortexed thorough!y end then heated:at TfPC' tor f0:irftin. After being oentrtfttged, the supernatant was placed in a 200 nl PC'R vial sod then loaded onto the FAbOO instrument. The sample was injected at the anode with reverse polarity using -5 kV for 20 sec, and was then separated at --13 kV with 20 psi pressure at both ends of caps l ,t\ drur ^ Ό s' n vpiration * he aDNP 3 A ' F produced 11 mov des'iiableijumforrn peaks under redadiig conditions, i p51?j TheaOTP 3Λ4-Γ. aDNP 3A4~F$5und aDNP 3Bi adObodtes were analyzed for thermoresistanec by DSC using mMierCal VP-DSC where the samples were heated from 2if!C to 95 at a rate of 1 (" per ntinuto. The proteins wore at 0..5 mg%ti in 10 mMsodium acetate. 9!9> sucrose, pH 5.01Figure 21). Ϊhe aDNP 3B1 and a DTP 3Λ4-Ρ anobodew produced the most desirable melting mo ides, with a higher temperature for the mitt»! transition 1 he a 1>NP 3Bi and .ιΟΝΡ 3Λ4- F antibodies were dlfferdhtiaied by the presepe of a single melting transition fprtbec a DTP 3B! antibody and a double transition for toe aDNP 3A4-F antibody [00515. t j ISA essays. PUNA as.wix \ xxcre conn-ieuxi as VAoxs v t os tar 3073 medium binding 3x4 well plates fC'oming Life Sciences) were coated with DNP-BSA (BioSeareh'I'echnologies, Notate, GVs at 5 μη. ml in 1 XPB8:0.05% Azide, (AO uIVelB. Λ he plates we-e incubated A 4' C ox etmyle, Ihe plates \\ cm then \su\had ustog 3-ex clc xx ash or a 1 ttenek M fbe p{ ue -aedict t t .milch. I hmis\ d re. A1k Foe plates were blocked with 90 μ! of l.XPBS 1% milk and me aba tec approximately 30 minutes at room temperature. The plates χ-xere then washed osmg a 3-eyde wash on a Tderlek plate washer, I0ui antibody sampkts were added io 40ul IXPBS PA milk

The plates were then ineuhated for I hour at room temperature. Next, whites were then washed using 3-cycle wash on a Titertek lkf.384 plate washer {! iieriek, Huntsville, AXK Goal .oni Human IgG he HRP was then added at 100 og ml {1 '4000? m 1XPB\ '* ,-suik lOosM (V (50 id'welh λ a* .wiled to id. okr\ and w'us met, nned ' flout .a u«»\ temperature I he plate- wee s%a<hud once again, us mg a 3-cycle wash. The plates were then patted dry with paper towel. Finally, 1 step TPB peogeti. Lexington, Kentucky) (50 μΐ/well) was added to the plate and was quenched:with 1N hvdrochlorle;acid (50 uiAvell) after 30 minutes at room tejnneraiure, CD's wore read ippiedintdy at 450 rnn :ush)i dtfiteftek plttc reader, PW4aii!li' [00520] PfenrmacoMnettctPK)^ Phsmnacodyoamie (PD) Studies of Anft-BIN P Antibody KmboiMtnents of the Invention [005311 The pharmacokinetic profile of the aDNP 5A4-F, aBNP 3A4-FSS and aBNP 4Π am I bodies was devnrined to add? >y agne-Dav ley rats (5-12 weeks :··ο,d > 1>> eyeding 5 rag kg subcutaneously earn col eettoe ..*fp'ο» r ueh 5e0 at of blued ju M , totamer-a .serin ' separator tubes at 0, C 25, 1 4, 24, 4b, 72, T\ fiA 33o, :>01 u 540 ,i\d lOtts hours post-dose fern t -e lateral bit! wtr: t Km m 25} Each -ample was maintained at room tempera umo following collection, ansi following a 20-40 minute clotting period, samples were centrifuged at 240X1 at 1,500 rpm k t abo a Id mu' nos using w , ahhruteP L foumorf 54 ”K ( one Page S'sierUh td o ns 1 'bun its Ik \3vsft„w NVi lue Ή u wttni wether! transferred into a pre-labeled {tor each rut}, cryogenic storage tube and stored at wire ίο -MbT for ar,a!vsis '1 o measure the «.rum sample eoneemmt-on« from the PK .at idy san ml,--,., il e *. of lowing method was tsed1 ' ,n'< a black pkne tCorrmig 3604i »sas cebee wit 2 gu ml of \m ;u K hr 1 X i m \ *BS and then incubated o’.. nmdst n -'C The nhee was washt. J ano dudo'd wdb }-Bbv\U (Apnked Blow si on si me night at * {' Ifsa.-uple- needed v be dtlmed thee tbcy were diluted in Knt ol> serum. Hie standards and sample*» werechined t T ni 1

Biuck>?>i r 5% BSA into 380 μΐ of dilutmg bilfe. 1%e: ppe% samples of pretreated standards and sansptes wete transferred into an Ah I 35,ΐ coated plate and incubated for 1,5 h at mom-temperature, 1 he plate was w ashed, then AO ui of 100 og'tnl of aod-fut l:€ Ah 21 I-1IRP eouiugaie in I~Bk>ek5M < 5% BSA wet ο added and mounted fbt I 5 h,. Ί he plate was washed, then 50 μ I of Pico substrate were added, alter which the plate w&amp;sbumediaiely analyzed with: a luniiiionteter.·^ The pharmaeokentic polio was food for all: antibodies, Inst the sDHF 3B1 showed the best overall profile,: |0tt5221 'The pharmaeoiipe&amp; pmtie ofihe ttDNP 3A4-F anfbodf was detennined: sn o iu.de esoomologous monkeys {.hi kg) hs nrjecoog h my. kg bolus dose intravenously and taking blood samples at 0 and 30 minutes and 2, 7,0, ! 1, 14, 21, 28, 35, 42, 49, 56 and 63 days (Figure 23), The pharmacokinetic profile of the aKlUl 120,6 antibody was determined in 4 mule cv nos no logons monkeys (2~4 kg) b> injecting 3 mg kg bolus do.se:|pr«v^u&amp;%.:«n4;^-km§-bl.^ samples at 0,0.25, 1,4, 8, 12, 24, 72, 16k, 240,336, #8,5<%·37%ι§?2<. 744,^4©^ 1176 and 1344: hours (Figure 23). To measuretheserntn sample conccnteidiiiiii^rii^tFKstudy samples, the same nvtbod as mentioned above ibi the rat phanuaeokisteue study was employed. The pharnuwokinehe profile for both antibodies in eynomokpous swnskep was good, De the dew normalized profile foi theuKI II I20.t> was marginally better than that of the aDNP 3A4-F. i^523| Seailel: 100524 i 11 n man tissue cross- reactivity assessment [005251 lit eenetal.w\e <1, λ. o tth the g t oancc ado * t --V uts v t. enssaer m nte M^Oida^urgjm^ Tesiuigμ-,Γ Monpeignm Andb&amp;dy Prod.tg is for I Inman.! A; (5 .S. Department of Health and Human Services, Food and Drug AdnnnAtmton, C.-ntoi 0« Biologies To Ammon and Reseat eh (ΙΟοΆμ d prclntnnafy non4 4 0 sut-K was carried out to determine crow-reactivity of in\entlve antibodies w ifn a \ariety of human u.wnes, If an autibodt In intended for drug development, a more extensive tcspnva under GLP conditions s-, required } ho tissue οιο^-ΐΌ-ι-ΐο it\ of ami hod ms iRXP f '\4-Γ you ,*Κ1 IF 120 Ο evaluated nThas'Ies Rom ! snoiaHfoew Preummai Sete wey Reno. Ν\ ) with u'wseet so os of -elected hurnan tissues using tleoi -iSh kikded h'y ms of foe H-st loo Normal human 0--010-. flout two niuijne todo muem >, uukss mheiw ise mdwated i w οι o obu sited Irom die Spec ut i iXViboaigs bom tee- Rumor Fo-ue Rani cshtecred b) the X-rhomb Disease IXasean.h 1 mom Hart ge ί \I>R i. Philadelphia., PAF Cm cl me. urc. iRarhngaroc, ( \i, Cybfdt ί Ruskc Ore, \!P j, or Rock;'. MouuXtin I -osos l ye Bmik s Aurora. CO) Tissue- resie-i included human cco-bcilnnu hemp, eetemul coftcv u-,:00 {hon; mat mo FemaleF 0}0, phn.eou, o„ououuo-hool tract f sited! uo0,-4dec -km (i individuals. bear;., spleen, koinoy r 1 indis-dualr, To food, Over, testis $<λ Lons of fiesh-frv/en human Fssno-and euimv-l head bloclm iRAPL-I b bovine scrum Chumm jBSAj heads |pov;io 0], and hum -,n -ernnr albumin j HSA; bead- L mean-eh v,es0 cos ou rho cry oxer! and thaw mounted onto capt'darn gup hide- F:»·,· tissue and eonuot dead slides woo need in '.oU a. crone fur approMirtarely 10 nunuteh at -10 C ιο-25'C TW iked si id on ivoro allowed to dm tbi ,0 loan 000 hour {to ov uumht; if sioi od Ooeen, Oxui slides ',v>„ro removed front On. froc/·.-!' on the da v poor to an experiment and allowed to thaw oxesoodn puo· hi u-,u \h the ιοΠο-ν irm slops emu pekocmcd at room tentper.rmjv mhos- o-hoovLsc specified Fho slides worn incubated with LX Morphosav0;^ ids .ippfoxmiyteiv 15 minutes a present, twsue morphok-gy Ρκη 0 ashed two ποιο λ Fin apptosmtaieU 5 rmtmtes oath 01 i X phosphatcdmlfemd saiine (PRb 5. 10 block >,.m dimes η hi- peroxidase, tho and-.0 v-ere uteuhako 01 a pju-oae oxubhc sointioo lor appotcutintoK 1 bon; at apniosnoateP, d( 1 he slides vscio oa-aard too notes in 1X PBS for appsOMirnueh “· ηνΐπηΐο- e.wb Fnd>po.enoos biotin s\ as blocked Fry aapioana: sncubatuin iappiVicmiutol·. 15 mmats- raoh i in .0 idea and biohn solutions Follow mu the istcnbation in baron, the ns-ee sections ere blocked 0 nh a F-ica inny eudhoda. -.oiicion id; appro-io;5 1 canutea Xicoa; FFaoi dkd-Xb 5ΛΊ \\ 101 F irmr DXFF and Χίο-a 1 iuordsS am··K.LH (:.,ηπ·Κ1 R Ab) 0arc ippbed Hr set uons m the optim-d eoaoemralu.m (2 0 pa sol } tr 5 times the optimal coHceunvaion S10 0 toy rn 1,1 tos uppmcanaToic 25 snotutes Slsdc-· wore washed 5 Fane- 0 nh w ash buFFer and then incubated w nh she secondin') .rmibn-jy 1 ns bn-it anti-

Alex&amp;Fiuor 488; for approximately 28 minutes. FoUowing Incubation with the secondary antibody, slides were vsuxhed 4 limes with wash bailer then incubated with the tertiai-y antibody (horseradish peroxidase conjugated gout anti-rabbit igG antibody] lor approximately 28 minutes and binding vtstn-hzed with a diamuiohen/idlno (DAB) ehroniogcn substrate. DNPt21 f-BSA heads were used as a positive control in all experiments, HSA heads were toed as a negative control. Tissues were qualified as adequate for immunohistoehemKtty via staining with an anohody against CD21 imwwCDs!) i.e., platelet endothelial cell adhesion molecule { PEC Λ M~ l.p There was no specific staining in any human i iso to exanii ned at either 2,0 or 10.0 ugm t concentration for any of the tested and bodies.

[00527] Expression and Purification nt' Monovalent or Multivalent 1 rornamigiohu It ?w and/or Fe domain-'Toxso Peptide Analog Fusions [00528] An assortment of monovalent, bivalent and rnvalent structures were expressed and purified lor comparison, including exemplary embodiments of the invention. Those Included aKLFi igG2.TC~Sh.lK variants (see schematic representation of Figure IF. "hemibody" configurations. and *a?n-Ki R lgG2~ShK variants {see Figure ) F-L}. For example, bivtdcrvr Fc-L lO-ShKj 1-551, monovalent anti-Keyhole 1.impel 1 lemoeyamn (KI.R) immunoglobulin heavy cham-l t ysl6)$hK fusion antibody (designated AiKLR HC-[tysl6]ShK Ab'h see Figure IF), arte monovalent anti-KLH immunoglobulin light ehaiti-|_Lysl6]ShK antibody (4emanated •TK.LH Lf dhysHViShK Ab"; see Figure 1.1.1. lgG2 Fc/Fc-ShK variants tsec Figure l AT bivalent Fc~l. 10~ShK j2-55j, rnouavalent re Fc-t. Kl-ShKI2-88| were made for comparison, by recombinant methods as described iu Sullivan et ah, WO 2008.08842.2 Λ.2. and in particular Examples 1.7, and 56 therein. incorporated In reference in its entirety, or us modified herein.

[00529] Transient expression system used to generate toxin peptide amtlog-Fe fusions fpeptibodicO or other immunoglobulin fusion embodiments. HFK .TE -6E ceFs vs ere maintained in 21. fernbach Frienmeyer Flasks betwem 2e5 and 1.2or- ••cclla/ml in FI 7 medium .supplemented with 1.-(31 usmome (0 mM) «md Geac-tiem (25 ug/mi) at 37,;C, 5% CO;, and .shaken at 65 RPVf At -he time of transfection. cells were diluted to 1.1 x I0*! eel 1« 'ml in the FI 7 medium mentioned above at 90¾ of the final culture volume. DNA complex tv» prepared in Fwe>tylc£93 medium as s0”b of the final culture volume. DNA complex includes 5{)0ug total DNA per liter of Culture and l .5m) PEhnax per liter of culture. DMA complex is briefly shaken once ingrcdiem.x are added and meuhaicd at mom temperature for 10 to 20 minuses before being added to the ceil culture anti placed back in the Incubator. The day after transfection, Tryptone Ml (Sg't.i wax added to the culture front liquid 20'W> stock.

Six days after transfection, culture was centrifuged at 4,000 RPM tor 40 minutes tope! let the cells and she cultured medium was harvested through « 0 45am fiher [00530] In preparing she DM,A complex, she ratio of plasmids was proportional to the desired molar ratio of the peptides needed to generate the intended product. The -components of the- lgG2 Fc'Fc-ShK include Ig02 Fo and !uG.2 Fc-ShK nt a Is 1 ratio During expression these assemble into lgG2 F'e homodmicrs. tgG2 fe. Fe-ShK Jtetcrod inters. and IgG2 Fc-.$hK..hdtnodhnora The lgG2 Fc. Fc-ShK hetesodiaier (monovalcoffbraf) was isofaied during pushOediiotrnsihg eatiop exdhadge -chromatography.

[0053 Π

Fc-ShtCf 1-.35, ΟΙ6Κ.Ί mammalian expression, DK‘A socjuout'es coding iyr the immunoglobulin Fe domain of human lgG2:

MEttWVTLFFISVTTGVHKERKVECPPCPAPPVAGPSVFLFPPKPKDTLMiSR T PF.VTC V VVDVS HEDPEVQFN W 3' V DG VP V HN A K.TKPR E EQFN STF RVVSV1. TVVHQDW PNG K.F V'KC'KVSN KG LPAP11IKTISK TKGQPR I : PQ V YTLPPSR FEM TKNQV$LTCLYKGFYPSD{AVF.WE$NGQPFNNYKTTPPML.D$DGSFFlYSKf.. TVDKSRWQQGNVFSti SVMHEAi..HNHYTQKSLSLSI>('jK·/ {SHQ ID NO: 1 d fused hvfrarac to a monomer of the K.v 1.3 inhibitor peptide ShK [2--35] or a mutated ShR[2~35. QI6K.I were constructed using standard PCR technology. The ShK.p-.35j or SbKp-35, QloKj and the id amino acid linker portion of the molecule were generated in &amp; FOR reaction using the 0dgkalrFc*2kL»$|K.f2T3-S|.^il· pcDNAil. i(f}(. MV? as a template. tsec SuHAan ct a!., WO 2008 088422 A2, example 2, Figure Ι5Λ-Β therein*, the ShKf 1-35] wigs generated in a POR reaction using the original Fe-2>:l. -ShK[K>5] in pcDNA5. if +)CMVi us a ten-plate (Sulhvao cl ul.v WO 2008088422 Λ2, Pxamplc I, Figute I4A-B therein). Those ShK constructs have the following modified V.H21 Signal peptide amino acid sequence of MEWSWVTLFFLSVITGVHSHRK VF.< FPCP- SPQ \0 NO:2 generated from a pSdexis-Vh2 C-hi.gG2-Fc template with the following oligox: CAT GAA TTC CCC ACC ATG G A A TGG AGC TGG -3' I SCO 10 NOG); and CA ( GG TGG GCA CiC GAC l i t GCG ΓΤΓ GGA GTG GAC ACC -3' (SbQ ID AO.4).

[00532] Wild Typo ShKj2-15j with N-temiinai linker extension (amino acid sequence OGGGSGGGGSSCIDT1FKSRCT AFQCKRSMKYRl.SFCRKTCGTCC SIIQ ID NO:6) was encoded fey the DN A sequetice bellow: GG A GG AGG A GG A if ‘CUGAGGAGG AGGAAGCAGCI GCA) C(j AC ,AC( 'ATC CCCAACA G€CG< TGCAttXK TTT< ‘CA< iTG<. A AGC AC AG C A TG AAG'I'ACC GCC rGAGCITCVGCCGCAAGACCTCCGGCAC'C KiCf (SFO ID NO;5s. A fragment containing this coding sequence iSFQ ID NO'5} \s as generated using the oilg.os below 0SF.Q ID NO:? and Si:Q IP NO;8}~*nd the original Fc-L lO-ShKp-35] in poDN A3. H OCMYi as a template (Sullivan et ai , WO 2008 088422 A2, Example 2, Figure· 15 A A) therein,. incorporated hy referesicef: 5:~GTC CAC TCC GAG eGG AAA. GTG:GAG TGG GCA GCG. TGG C4 (SCQ ID NO:?); and 5'- TCC TCC TCC TTT ACC' CGG AG A GAG GGA GAG -3'-' ;SHQ ID NO :81 [00533] Mutant ShK [2-35, QlOK.j wa* generated using site directed mutagenesis with SOatageneN QuikChangc Multi sito-Dircctcd Mutagenesis kst eata 200531 per the manniaetercGs instruction. Oligos used to generate the mutagenesis were: 5:-GCT GCA (TO CC V TCA ACT GCA AGC ACA GC 3' fSEQ ID NO;9s; and 5'- GCT GTG err GCA CTT ΟΛΑ GGC GGT GCA GC CC iSEQ ID NO: 10); and using the original Pe-T IO-ShKI2-35| In peDN.\3.'u OCMYi as a template * Sullivan ei aL WO 2008/088422 A2: Example 2* Figure ISA-B merei.n) resulting in (be DMA coding sequence

GO AGGAGGAGGATfCGG AGGAGGAGO AAQC AGC'TGCATCGAC ACC A i'C CCTTAAGAGCCTXTGCVXXTXX'TTCAAGTGCAAGCAC AGCATGAAG'LACC xx:rTGAGCTTCTG{.'CGCAAGAcnxxGG<.'AcrrGC/' cseq m no·, i ) κ which encoder the amino acid sequence ShkRL 35, R 16} with a N-ierrninal linker ΰχίαωοη: GGGGSGGGGSSC IDT! PKSRC.T AFKC K i IS MR \ R (..SI C R KT C< ΠΧ '.· SEQ ID NO:12).

[00524] ShK[ R25j\VT fragment was generated using Use original Fo-2xL-Sh&amp;[ i -25'j in peDNA2.it -X' MVi as a template (Sullivan ot <X, \VQ 200R OHM 5 : Λ2, Example !, Figure Ι4Λ-Β therein) and ohgos: 5'ΤΓί€ CAC TCC GAG CGC AAA. ClTC) GAG TGC CXiA.GCG TGC CAT 011¾ ID NO;?}; and S'- IT C TCC TCO TIT AC C CGG AG A CAG GG.A GaG -2' (SEC) ID NO:8> [5)05.351 The tgG2Fe region was generated usmg oligos; 5'X ( G GG 1 AAA GGA GGA G'GA GG A (( C GGA G-2' IS 1:.0 ID NO: t2}, and S'- CAT GCG GCC GCT ( AT LAG CAG GΙΌ -2' (SIX) ID NO: MX and the pSoleGa Vhe GIOgGE-Fe template resulting, in a (ragmen? containing Ok; folio wing DNA coding sequence:

G( ACX ACCTGTGGC AGGACCGTC ACfiCTTCTTCTTCi CCCCAAAACGCAA GGACAC CCTCA'lGATCTCCCGGACC'i. (TGACXiTCACGTGCGTGGTGGTGG ACG'TGAGCC'.ACGAAGACCCCGAGGTCCaG'TTCA ACTGGTACGTCIGACGCj CGTGGAGGTCX'ATA ATGCCAAGAk AAAGi '(.'A( GGGAGG AGCAGTTCAAC' AGCACGTTCCGTGTGGTCAGCCfTCCTCACCGTTOTGi.'ACCAGGACTGCjCT G 6 AC/GGCAAGGAGTAC A.AGTGi AAGGTC"ΓίΧΑΛΟΛA.AGGCC i'CCC'AGi C ((( A' i’CGAGA A A ACCAI ( I ( C ΑΛ A.\CCA AAGGGC AGGCCX (s AG AACCA.C SGGTGT AC AC C CTCC'CC. (.'CAT C CCGi.iG kGGAGAT (.1ACCAAG A AC t.'AGGT Γ AGCC ΓΟΛ CC I GC CTGG i'C ΛΛ AGGC f ICIA CCC CAGCGAC AIX GC( O' CGG AG I ΟίΧΐΛΟΛΤΚ. A.Λ1GGGCAGCC GG hGAAC AAC ΓΛ( XAGACi. A(. AC C IC (. CATGCTGGACTCCGACGGrt (X ΤΊ Π TCCTCt ACAGCAAGi TC'ACC G FGG

Af" ΑΛ GA Gf AGO' rGGCAGC AGGGG A ACO'T'C'.'TT CTCA'FCCTC CG TGA'TOGA TGA.(XKi<:TGc;AC'AACCA{:'‘rAcrACG(:'AGAAGACK::{:"ri:"i{:T.T:i'GTcrccijG GTAAA ' SbQ ID NO: I 5, which encodes the amino acid sequence AFPVAGPSVFLFPPKPKOTLMISRTPt'.VTCVVVDVSHHDPHVt^FNWYVDGVIi ΥΉΝΑΚΤΚ PREEQE NSTFR VVS V LTVVHQDW LMG KE YKC K VS N KG LPAP f FK 1ISK.T KGQPREPQVYT LP PS REE MI K NQ V'S LTCI..V KOFY PSD1AVE \\ ESNGQ FENNYKTTFPMLDSDGSFF LYSKLTV DKSRWQQG N VFSC'SV Ml I FAl HNi U YT QK8LSLSPGK. SF.Q ID NO: 16) [00536] The PCR fragments were generated and the prod:lets were run out on a gel. After gel purification, the PNA fragments were pur together in u PCR tube end: .sewn together with outside primers' < AT G A \ TTC CCC ACC ATG GAA 1'GG AGE TOG ~.V (SBQ ID NOG), add: e ~ t \ 1' GCU GC.'C GOT CAT TAG CAG GIG -3' %EQ ID NO. 14).

[005371 The PCR products were digested with RcoRI and Not! (Roche) restriction enzymes and agarose gel purified by Ge! Purification Kit. At ?hc same time, the pTH4 vector run. Amgen vector containing a CYIV promoter. Poly A tad and a Puromycin resistance gene) was digested with FcoRI and Nod restriction enzymes and the large fragment was purified by Gel Purification Kit. Each purified PCR product was itgated to the large- fragment and transformed- into OneShot TopJ.0 bacteria. DMAs front transformed bacterial colonies were isolated and subjected to EcoR.1. and Not! restriction enzyme digestions and resolved on a one percent agarose gel. ON As resulting tit an expected pattern were submitted for sequencing.

Although, analysis of several .sequences of clones yielded a 100% percent match with the above sequence, only one clone of each construct was selected for large scaled plasmid purification. The final pTT 14-VI·! 1 $P~igG2-Fc construct encoded IgG%Fe~L iO-Shfsi2-35} fusion polypeptide having the following sequence: MEWSWVFLFFLS VTiGVHSE.RKVECPPC PAPPV AGPSVFt PPPKPKDTLMISft TPEVTCV V V DVSH EDP EVQFN W Y V DO V ΕΥ Η N A K ΓΚ PR EOT N S' i'FRV VSV L TVVl-fQDWLNGKEYKCK.VSNKGLFAPlFK'HSKT KGQPRFPQVYTLPFSREFM TKNQVSLTCLVKGFYPSDiAVEWESNGQPF.NNYKTTPPMLDSnGSFFl.YSKI. TVDKRRWQQGNVFSCSVMHFALFtXFtYTQNSLSLSPGKGGGGSGGGGSSClD TiPKSRCTAFQC:KHSMKYRLSFf;RKTC'GTC// (SEQ (O NO:) ?}, [0053S] The pTT 14*VH21SiMg<52*^c«-LI0»ShiCi2~35sQl6K} con&amp;imct eiccsied^ IsiCfS-Fc F i0~ShK(2T>5, QloK) fuOou poh peptide vxjnence- ME VYS\V VFLFF L SYTTG VHSERK V EC' PPC PA PP V A GPS V F l FPPkPKDTLMISR 1'PF VTCV VVDYM1 F.PPHVQf N \YY V PGVHVI I NAR' ΓΚ PR Γ EQf"N STFRVVSVL TVVKQOWLNCfK FY KCRYSNKo I. PA FIE KTfSKTKGQPRf.PQV YTLPP5R EE VI TRNQVSI 'H'FYRGi 3 PSIH W F\\ FSNC YWNVVkn FPYH OSlXiSITLA Ski TV DKSRW QQGN V FSCSVMHFALHNHS 1 QK.SLS1 SPukot >Gi iSt KKKjSSC 10 HPkSkC T-M KCKUSMkYRI sfcrktyvk ’ SFQ ID \i * lb and p H ; 4Λ HYSF-lgGNFe SHK.K33 const met columned j ceding sequence tot IgG2 l v-l Y}-\akx l-'Ϋ ih-oou po’y peptide o eg the \> Kv urn e , no MEWSWYFLFFI ^VTTGVHSEIUKVBCPK PAPPY AGPS VFLFPPKPR 0Ή MISR TFEVTCV VYsDVBHEDPEYQFNWYVDGY BVMNAKTK PR EEQF NST FRY VS VL r\ \ HQDW 1 \GRF5 ki K\ SNKul PAPIFk 1 N\ 1 RGOPRt FQVY rtPPSREFM TK.NQYSLTC1. YkGFYPsDiAVEWESNCQPENNYKTTPPM-EDSDGSFI'EYSil:^·· ) VDKSR'YQGGW ί \YW ΜΙΙΓΑΙ HMD iQkAI SI SFGRCRIGGSGGtsGSRSCf PTSPKSRr ΓΑFQCKHSMR YRLSFCRKTCGTCV? (SEQ 10 NO:If), [00539] Generating the VH21 SP-tgG2 -Re-only construct iipYDIE (an Amgei vector containing a CMV promoter, Pok A tail and a Hygromyeisi resistanceg@M) occurred as follows: Hie VH2I signal peptide was generated using ilieFMi»Y%Jg; : YYos: 5'-ΓΑΤ AAOGTT CC C ACC ATG GAA TOG AOC 100-3’ (SEQ ID ΝΟΤΟ): and <'~i U'tKYRYGi \( H GAC m'(rt'(K'iY GGAG Ki GAC U GY iSEQ ID N« 1 4), and using the pSdexis template us noted above, i [00540s The Fe segum λ as m e, :ated using She pSo cm- ter rotate 0 --,-0¾ ,1 utxne and tbUmviag otigos: s' u re ( m ivc gag cgc λαλ grc" t.ag igy t ca ygg rc*cκ 5' γτο ID NO-7}; and 5'·· CAT GO A TCC TCA ITT ACC CGG ΛΟΛ (AG GGA G -3' (SBQ 10 NOG I). 10()541 ] The PC R fragments were gel purified and sewn together in single PC.''R reaction using outside primers SliO 10 NO:335 end SCO U'.> NO:53c. Hie resulting PC R irygnicnf whs gel purified, and digested by BmdHl ana Bann-i!. Concurrently, ρV"Γ> i 4 vector fan Amgen vector containing a CMV promoter, Poly A tail and a Hygromydn resistance gene) was also cut by Hindi!! and BoroHl and die large vector fragment vvas purified by Qiagen\ Gel Purification Kit. The purified PCR product was I i gated to die large try groom and transformed duo OncSbot Top 10 bacteria DNA from transformed bacterial colonics were isolated and subjected to HindHI and Bum HI restriction enzyme digestions and resolved on a one percent agarose gd DMAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sen nonce, only one clone was selected tbr large sealed plasmid purification. The final ρΥΓΗΑΛΤΤ 1SP-IgGTFe construct encoded human fgG2-t:c (SEQ (ID MY I. above).

[00542) Anti-KLH leGTTc ShKp-33., QlOKi n-uinruaten: expression. Using the DNA pTTSmKi. H! 20.6-VK I SP-lgG24IC4.10-ShK| I -35. 016K.1 construct the fragment containing tire DNA coding sequence

GG A rCCGGAGCAGGAGGΛΑGCCGCAGCfGC ATCGACACCA IT CTCΑ.ΛGA GCCGC TGCACCGC CUT AAGTCC A AGCACAGC A FGA \GT ^CCGOCTG 3G CTTTTGC COCAAGA( CTGCGGC. ACT 1(,.1(3Γ1ATGAGCGGC (IKKX.IAGGCG GGTA AGGCCGG AICCA (SEQ ID NO:223 w&amp;s cut put using BarohO/BamHl) This coding sequence |$BQ ID MO:23) encodes ShK( 1 -35. QtoK) with an N-terrotnai linker sequence: •GSGGGGSRSnDTIPKSRCTAPKCKHSMKYRLSFCRiaCGTCCCSEQ ID NO:23i.

[005431 At the same time, p7T14-h!gG2-Fe-ShKiI-55jWT construct, was also digested by Bam HI Bum HI. tliereby removing the Sltki i -35) coding region to yield the coding sequence ATGGAATGGAGCTGGGTtTriGTCTiCTiXXTGTCAGTAACGACrGXiTGT ^AfcfrfcCGAGCGCAAAGTCGAGTCKXXACCGTGCtT/AGCACCACTTam GC%GGACrG'rCAG'i'rrTC'CrrrTCXXTCXAAAACa'AAGGACACt'C ICA V MTCTCCCGC*ACX:CCTGAG<rrCACGTGC0TGGTG<nGCi:AfiGtOAOCM|·: G Λ AG AC C rt'GAGGTCXr AtiTTCA ACTGG'l ACGTGGACGt XX ΠΧ iG M *G I'GC * ATAATGCCA AG AC A AAGC'C ACGGG A GO AGCAGTTC A AC ACC ACGT TCX G t< r rc »< nc ac k xn ex t< 'acx x rr re πχκ v\a agg actggoc ϊ a a a ;g< λ ac ; gagtacaagtocaaggtctccaacaaaggcctcccagcccccatcgaga AAACCATCTC^AAACTAAAGGGCAGCCCCGAGAAa/ACAGGTGTACAC ^•CCrGCCCCCA'rCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC 'TG'C30T0C;^rCAAA€IGCTrCTACCCCAGCGACATC‘GCCGTCiGAGTCjGGAGA XlGAA'rGGGGAGGCGGAGAACAACl'ACAAGACC’ACAC'CrGeCAXGCTGGA ; CTCCGACGGCreCTTCrrCCTCTACAGCAAGCTCrACCGTGGACAAGAGCA GGTGGCAUC \G(;GGAACXXrnrT( Λ ΙΧΧ'ΉΧ'(ϊΤΟΛ RIGA rGAGGCRTG ( -\CAACC \C ΓΑί ACGCAG A AG AGCCTCTCCCT G ITTCCOGGT A AA GG AG GAGOA..· (SEQ ID NO:24 X encoding the antino acid setpenee I Μ E W S W V1' L. F F LS V 'Π 'G V H S X R k VBCFPC PA PP V A G PS VFLFF P kP kP" II, ΜIS H TPPViCVf YPYSHmi'i \ Of XA'i'VfXWrVGNAKrKPRFFOrXSTI'RVVSVL ! \ YHQDW t \GkbA KC k\ SAkof PAPIER USkmGQPREPQVYTi PPSRtJbM IKNQVSd H LVKCJFYPSDIAWWFSMiQrFNNYKrTPPMI DSDGSFFt VSKL l\ PkSRWt&amp;JGWI scs\ ΜΗΓΛΙ HMD IQKSI SLSPGKGGG idl'Q ID NO :2 5).

[005441 Du. ηΐ l {4 hlgiif t on witft tht Sltk amoved was treated 'wrihduaft .Intestine Phosphatase fCiP) to remove rhe 5’ Phosphate group isd Phetun t 'hbrotorm can acted fo pre-vein religation of the vector-upon itself;. The insert ShR.fl X. Qlok] fragment wts gel purified away from its veetor&amp;nd cleaned up wnh Qumo > tsel Pie rtVanuu Kit The funded insett was bgused to dm large vector fragment and transformed Into OncShot TopiO bacteria. DNAs from transformed bacterial colonics were isolated and subjected ίο Band H restriction;. ::enzyme digestion and resolved on u one percent agarose get, DMAs resulting mart:

Although,, analysis of scveotl sequence of clones yielded a 100% percent match with the above sequence, only one clone was selected tor large scaled pi asm; a purification. The ttnal aTTiddgGY fe~ShK.[ 1-35, QI6K.) construct eixoded the following lgG2 Fc-LIO-ShKH-ii, QinK.s fusion protein sequence; MFAYSWVFI.FFL SYTT6VH SERK.VECPPCPA PP V A GPSYF l FPPKPkDTLM I SR TPFYTCYYYDYSnFDPEYQf NWYYPGVF Y11N AK'ΓΚ PR fi f.Qi'NSTFR\ VSYL TVYHQDWi..NGKI:;VKCK\'SNK('jLPAPlEKT!SKTK.GihPRFPQYYTlFPSRI::FM TKNQVSLT{-I.YKGFYPSPPyYI:,\YF!sN(iQPi':\;NYK,-?TPPMi.DSOGSrFLVSKL TVORSR WQQGN VESCSV MHbALHNH \ \ ()Kn| 815PGRGGGG SGGGGS Ρ5Π DTiF’KSROTAFRCKHSMkYRI. SFCR KTC. GT(Y {SEQ ID M>2a·.

[005451 a.=,\G.. Li ρ)ολ aflid λ y i. I;: ?. i .i Y.y .i y .F: yp. ?j yD:..(. tiv.Y a .i .s i.Y.i .0..1 ia :. υτλ y i λ ). .i λ i.f ia .vi .10 Πιο components of the aKI.l I IgGa I'e-ShK. f schematically represented by Figure IF.) included: (00546] fa) uKU-l 120.6 kappa LC i$EQ ID NO:28, below), which incorporates » N-terminal VK-) SP signal, peptide sequence)SPQ ID NO: 103): M D M R V PA Q 1.. LG I. LLLWUKiARa^QMTQSRSSlSASVGDRYTnVRASQGIR NDLOW YOQNPGK APKRLIYAASSL QSGVPSRFSGSGSGTFFR. 1 ISSi.QPfc'OF AT Y YC I. Ql IN SY Pi. IFGGGTK V FIK RYY A APS V R FPPSDEO I. kSG'F ASVYCX L NNF YPREAKVQvVK VDNALQSCiNSQES V FEQDSkDSTYSl SSTt 11 SKADVF KHkVYACE YTHQGLSSP VT KSFNRGEC (SEC) IDN0.28K (005471 ihiaKi.ll 120.0 lgG2 IIC{SEQ ID NO:20, below), which incorporates a N-terminal Yk-i SP signal peptide sequence) SK> ID NO: 103).

MDMR VFAQL LG). 1.. 1.. LWl.RG ARCQYQl YQSGA ΕΥ )< K.PG A 8 YKA'SCN. ASGY TFTC iΥΪ1 MilWYROAPCi<,)(.;I.EWMGWtNpNSGGTN V AQRPQGRVTMTRDTSi STAYMELSRLRSUDrAVYYCARDRGSYVWFDPWGQGTLV'fVSSASTkGPS VFPLA PCS RSTS l:\ST A A1. .GCLVK.DY PPP PVTVSWNSGA LTSGYHTFPAY1 .QSS O LYSISSVVT VPSSN FG1 OTYTCNVD HR PS N TKVOk Γ V FRNCC Ύ ECPFCPA P PY.AGPSVR..FPPK PK.DTLM t SRI PlfY'i OVVVDVSH 1::DPI:.VQFN\YY VDGV EY

HNAKTKPREKMWSTFRVVSVLTVVRQOWLNGKhTKOiVSNKGLPAPIEKT ISKTKGQPREPQVY T LPPSR EEY1TK NQVSLTC LV KGFYPSDIA V EW ES'NUQP EN.NYK) fPPMLDSDGSFFLYSKLTVDKSRWOQGNVFSCSVMHEALHNHYT QKSLSi.SPGK / (SEQ ID NO:29i; [005481 and [005401 (a IgG2 Ec-i.10~ShEO-55f: ME\\'S\VYFU:R.SVTT<iVHSi:RKVIin>RTAPPVAC(PSVn.rPPKPKD'n.MiSR TPEVTCWVD V$HEDPEYQEX WN YDOYfr.VHN AK.TK.PREEQEXSTFR.Y’VSVi. T VVi (QD WLNGK.EY KCK YSNKG 1..PAPH'.KT? SKTKGQPRfPQYΥΠ EPSRFILM TXNQVSL'Vr LVKGEY PSD) A Vb\VhSNGQPEMXYΚΎΊ PPM t DSI>GSEF 1 \ SKI. TVDK.SR WQQGN VFSC S VM H (LA t. Μ N R YTQKSLSLSikiKGGGGSGGGGSRSC I DTIPKSRCI Λ(Χ)ΓK.HSMKYPLSFCRR'f CG KY; {SEQ (L) NO;30}.

[00550] The desired aKLH lgG2 Fe-ShK product contained one cons of each of . components (&amp;Mck immeduitely above. eornktured ns in Figure IE. Because of this, the ratio v% a λ (;!;(. This product can he described as halt' anybody and half P e fusion (Rsennbody G. coupled together at the f e domain. Additional peptide aosombhuc that bad fu be removed from the eakure earc the aK ί H Ah and the he-ShfC hpmodtfrtef.

[00552] t he components of the uKi.JH 120.6 )gG2-Shlv fusion antibody (scberndtically represented in Figure IF) included monomers.

[00553] (a)aKXH (20.0 kappa LC {SEQ 10 NO:2K above); 10O554] (b) uKLH 120.6 (gG2 (1C (SEQ ID NO:2'A abo\ eIt t.md [00555] (¢) akf H (20,6 lgG2~ShK fusion (saving the to (towing 11C sequence: M DM KV P AQL (-01.(.. (- i .WLRG ARCQVQLYQSG AE VKKPG A S V K V SCKASGY TFTG V Η Μ H W V RQA PGQGLEWM GW) NPNSGGTN VAQKLFQOR V (MTRDTS) STAY.Mi;:lSRLR.$DDTA V'Y \ C'ARDROSYY WFDPWi iQG 11 v l VSSASTKGFS VFPI Λ PCSRSTSFSTA ALGCLV K DY P PO> YTYS WNSGAlTSGΥΉΊΤΡ A VLQSS GL YSLSSv VI'VPSSNFGTQi VTCNVQHKPSN.'TKVDKTVFRKrC'VpC'PPC'PAP PVAGPSVFLFPPKPKDTLNUSRTPESTn'VVDVSHFDPEVQFNWYVDGVEV IIN AKTKPREHQFNSTF R Y VS V l TV VI lQDWi NG K ΓΥ KGKVSN KG I. PA PI F.KT iSKTKGQPRP.PQVYTLPPSRE.ENiTKNQVSLTCLYKGn'PSDIAVEWF.S‘>iGQP ΡΝΝΥΚΤΤΡΡΜί.DSDCiSFFi.YSK 1TVOKSR WQQGNVFSC SYMMl'iAL ί1NRYT QKSLSLSPGGGGGSGGGGSRSGDTIPK.SRCT AFQC KilSMKYR LSFCR KTC0 Tc-(S£Qmm:>r> i 00556) (00557) T|« components of &amp; monovalent aKLH l 2CK6 lgG2-Shk( I -35, QJ6E) fusion GRiloiH \ schema*scaily:represented in Figure IF| i neiadeb monomer: [005581 (DaivLR 120 6 kappa LC (BEQ ID NO:2K above); [105591 (h) aKLH 120.5 lgG2 HCcSEQ ID N<>:2'>, above);.and [10560) uo aKL R \ 20.6 fgG2*SKK( G35, Q16K [ R-Αοη having the RTknving sequence: MOM RVPAQL LOLL L LVYLRG AECQVQLYQSG A FVK KPGASYKVSC K ASGY TFTG YHΜH W\f RQAPGQGi FWMGW ;NPNSGGTN V AQK.FQGR v Ϊ ΜΤΚΙΪFSl ST AY VIFLSR.LR SDDTA V Y Y C A RDRGSYY WFDPXVGQGTf. VTVSSA ST KG PS VJrPLAPCSRSTS.FSTAALGCJLVKD YFPEP V I VSWNSGAL TSGYRTFPA VLQSS G LYSLSSY VT VPSSNF GTQTYTCNVD R.K PS NT K YDKT YF.RKCC V FCPPC PA P PVAGPSVFI .FPPKPK DTI .MiSRTPFVTC VVVPYSi IEPPEVQF N WY VDGVFV HNAKTKPR BF.QFNST FR WSYLTY VHQDWL NGKF.YKCKV$Nrk'.G!..PAPIFK ί I SKTKGQPREPQ VY YLPPSR F.HVH' KNQVSi TC (. YKGF Y PSDIA V R W F.SNGQP ENN YKTTPPMLDSDG SF H..Y8K LTV DKSR WQQGMY FSCSY Vi IT FA L Η NH YT QKSlSLSPGGGGGSGGGGSRSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTeG TG/V (SEQ ID NO;32).

[0056]) The components »>{'the monos a km uKLH )20.6 ΗΓ-ShK[ 6-36, K)Λ. M.Y. Q16K] fusion antibody ίhe-v«av;caΠy represented m Figure IF) included the to I love i π g monoat ers; [00562] (M &amp;KLH ) 20.6 kupp&amp; LO (SEQ I0 NO:2h); [00563] (b) uKLH 120.6 lgG2 HC (SEQ ID NO:29): and [00564] (c) &amp;KLH 120.0 lgG2 FlC-Sh&amp;j i~35, ill A. I4A, Q16K j tnsion nosing die following amino acid sequence: MD MR V PAQU..G I..U..I W 1.. R Q A R CQ VQL VQSGAEVKK PC AS V K V SCO A SGY TF\ GY HM HWYIIQAPGQOLEWMGW) \PNSGGTN't'AQKFQGR YTM TR i.Ti'Si STAYMb'LSflLRSDD'VAVYVCARDR.GSVY WF'DPWGQG ft v i'VSSAS FKGFS VI-P1 A PCSRSTSFS1A ALGC LY K DY F PEPVTVSWN SGAL TSU VHTFTAV LQ5S GL V SISSY' VTV PSSN PCs IQ i V TC N V OH KPS Ν' I'K YDKT V F 1¾ KCC Vfcl P PDFA0 PVAGPSVFLFPPKPKDTLMISRTPE\TCV\'\-OYSHEDPE:’VQPNWYVDGVEY IΪΝ AKTKPRF.HQPNSTER VVS'V1TV V! 1QI >Wl NO K f ,'Y KC1CYSN KG i. PA PI Γ.ΚΤ tS&amp;TKGQPREPQ VYTLPP8REFMTKNQVSI..TC 1.. VK.GFYPSD!AVF\\ F.S'NGQP F.NNYICiTPPMi DSD<iSFFI.YSK1.1'YDKSRWQQGNVESC '$ VMl 1F A1 HNi021' QKSLSLSPGGGGGSGGGGSASCADTIFKSRCTAFKC K HS MR Y ELS PORE l CCi TC··' {SEQ IP NOCOd).

The dcfeitvd monovalent aK.FR 120.6 lgG2 HC-ShK analogue product was a full antibody with the ShK peptide m.sed to the C-terminus of one heavy chain. With two different heavy chains sharing one variety of light chain, the ratio of heavy chain' chain;light cham:iic&amp;vychain-ShK was 1.2:1. The expected expression products are aK.LR 120.6 lgG2 antibody, monovalent sKLH 120.6 lgG2 HC-ShK peptide analog, and bivalent aKLFl 120.6 ?g.G2 HC-ShK peptide analog. I ire monovalent aKLH )20,0 lgG2 HC-toxin peptide fusion-corny i n tng a mibody wav isolated from the mix using cation exchange chrormtogmphy, as described herein [00565] The component of the monovalent ukl.H 120.6 HC-ShKf l -35, KI A, QlbK, K30P] fusion antibody (schematically represented In Figure i F) included the following monomers: }0056n | (aUKLH 120* kappa U' i ShQ I L> NG:2h): }005o?j (b) uK2.ll 00.6 lg<iZ 11C iSEQ ID RO;29); and [0056ft) (c) aKLH 120.6 fgG2~ShK] I -35, RI A, Q!l 6K. K30EJ fusion having the: following sequence:. MDMRYPAQlI.Gi.l2J.,WFRGARCQYQbyQSGAFVK.KPGASyKYS€RASGY TFT GY Η Μ H W V RQAPGQG ϊ. Γ W MG WIN PN S GGTN Y AQK.FQG R VTMTRDTS! SlAY.MF.LSRLRSDt>rAVYV<'ARl>RGS¥TWFDIAVGQClltVTVSSAS'ilCGFS VFP! APCSRS1'SE:STAALGCRVKDYFPEPVTVSWNSGALTSGVUTFPAVl.QSS Gl \ 51 SYY VI \ Py-SN fU f'O UHN\ DH KF8\ ΓΚ V Dh ϊ V ERkC i Y'LCPPCPAP PVAGPSVFl.FPPRPRD11.MISRlPFVTCVWD¥SilteEVQFNWYVI>GyF¥ flNAK'i'KPREEQFNSl'FRVVSViTVVHQOWLNGKEVRCfKVSNKGLPAPliKT ISKTKGQPREPQ ΥΎΤΙ.. PPRRFF MTK.NQ VSLTC L V KG FA’ PSDIA V EWFSNGQP HN'N Y KTT PPMLDSDG SH'I. YSK i TVDKSR WQQGNYFSCSVMHF AU INI IYT QkSl SLYPtiGGGGSGf It>( A \SC1 D11 Pk-RC 1 AhRCkHSMk> R> SPCRBTCGT C {SFQ m NOGOSh Τ/ο- Pcsircd monovalent ukt H 120,6 kG2 HC-ShK. analogue fmclue? tv as a Ini) antibody with the ShK peptide fused to the C-terrnimss of one heavy chain. With two different heavy chains sharing one variety of light chant, the rat to of heavy chain: chain:light chah:heavycliain-ShK was. 1:2: t. The expected expression products are .< k t ¢( \ 20,t wG2 untilxuh. vtonos a lent a KI II 120 o igti2 IK'-sbk peptide analog, and bivalent. aK.tR 120.6 lgG2 HC-ShK peptide analog. The monovalent aK.l.H 120.6 |gG2 HC-to&amp;in peptide fuskni-contuiruog antibody was isolated from the mix using cation exchange chromatography, as described herein.

[00569] Thu components t>i‘the mummaienr aKLH )20.6 ΗΓ (lgG2)-S«K|1-35,, R ΓΗ. )4 A, QibKj fusion antibody Gchernaiically represented in Figure HA included monomers: [005701 («Π *KIH 1 20.0 kupp* LO i$BQ 10 NO:2h); [00571 ] (b) aKI.H 120.6 lgG2 HC {SEQ ID NO:20), and [00572] (c| &amp;ΚΛ H 120.0 HC lgG2-5hkv -G, Ill Η, 14 A, Q*6K j lesion ba\ in$ die following amino acid sequence: MDMRYPA<X.LGU..l.l WLRGARCQvQLY\)SGAEVK.KPGA$VKYSCK,ASGY TP'TGY HM H\YY RQAKKXdLFWM GW! NPNSGGTN Y AQKFQGR YTMTR DTS1 STAY V1F L SRI.RSDOYAVVY CAR DRGSY Y WthdpWGQG Η. v I VSSAS 1KGPS Vi- Pi AK'SRSTSF'ST.A ALGCLYKDY[TM:dAGVSWNSGALTSGYFnTPAViJ,>SS GL 'V SISSY VTV PSSN FC» IQ f V TC N V DH KPSNTKVDKTV HR KCCVHl m‘'PAP PVAGPsVF-'LFPPKPKDTLMISRTPE\'TCV\fYDVSH£:DPE:VQf:NWY VDCiYEV I IN AKTKPRE FQFNSIT R VVSV1TV V! 1QDWI NGK iY KCICYSNKGi. PA 10 Γ.ΚΤ 1SICTKGQPREPQ VYTL PPSREE MTKNQV Si..TC 1.. VK.GFY PSD! AV f.'W fi.SNGQP F.N N Y KTFPPMi DSD<ISPFI. YSK1 TVDK.SR^QQGNVFSC '$ VMiIP A11 INI 1YT QKSLSLSPGGGGGSGGGGSBSCADTi PKSRCf'AFKC K HSMK Y RLSFORK I CCi TC.'.·.·' (SGQ ID NO:300i.

The desired monovalent alCLR 120 6 i yG2 1 IC-ShK analogue product a as a full antibody with the SsK. peptide rosed to the C-tenyumw of one hem y chain, with two different heavy chains sharing one variety of light chain, the ratio of)-envy chain' chain:hght ehaindteavydiatn-ShKi was 1.2:1. The expected expression products are ak.LH 120,6 fgG2 antibody, monovalent aKLH 120.6 IgG2 HC-ShK peptide analog, and bivalent uKLH 520.6 lgG2 HO-ShK peptide analog. Tire monovalent aRLH 120,0 lgG2 HC-toxin peptide uwiotwconfaining antibody was isolated from the mix using cation exchange chromatography, as described herein [105731 Tbs cotBpooettid of die monovalent atCLii 1201' HClShlCf 1 <-35> RIB, Q16K, KJOb.J fusion an6 hody(s e h en νί O c a H y rcprocfHwd in Figure IF) included ike monomers: [00574} (it) αΚΙ,Η S 20.6 kappa IX' i7if() ID NO :2k); [00575] (b) uKIJ-i 120.6 lgG2 HC iSEQ ID NO:29); and 10057«»! < o i aKl 11 120.6 %G2-ShK {1 -35, R1 IT Q1 6K, KJOIG fusion havmg the oHosx ing scummcr MDMRYPA<X.LGLLi.l Wi..RGARCQvQLYQSiTAEVKi\PGA$VKY$CRA$G3' TPTGY HM HWY RQAPGQGLEWM GW! NPNSGGTN 3' AQKFQGR YTMTR DTS1 STAYMbTSRLRSDDTA V3'VCARDRGSY 3'WFDPWGQG Π.. v Γν'ΝΝΛΝ I'KGPS VTP1 APCSRSTSFSI.A ALGCLYKDY fTMIlPV'TYSWNSGALTSGYVnTPAYLQSS GL V SLSSV VTV PSSN FT» IQ ϊ V TC N V DH KPSNTkVDKTV fc’R KCCVfct m'PAP PVACPNVFI..FPPKPKDTLM!SRTPEVTCV\'YDV!5>HEDPEYQ!:NWYVDCjVEV I IN AKTKPRF.HQFNSTF R V VS V l TV V! 1QDWI N<3 K ΓΥ KCKYSN KG 1. PA PI F.K.T 1SKTKGQPREPQ VYTL PPRREE MTK NQ V Si.. TC L V KG FA' PSD! A V F\\ fi.SNGQP ENNYKJTPPMI DSDGSITIA'NKLTVDKS RWQQGN VFS< NVMHFAUINI 131' QKSLSLS PGGGGGSGGGGS HSO DTI PKSRCT Ah KC KHS Μ K Y RL NFC R F' FOOT € i'SFQ ID NO:30")

The desired monovalent aKLH 120.6 IgG? HC-ShK analogue product was a full sndhody with the SKK peptide fused io the € -terminus ofone heavy chain. With two diffeten; heavy chains sharing one variety of light chain, the rat to of heavy chain; chain: light chmnthe&amp;vycitain-ShK was 1:3: t. The expected expression products are aKLH 120,6 igG2 antibody, monovalent aKLH 120.6 igG2 11(1-51112 peptide analog, and bivalent aKLH 120.6 lgG2 HC'-SKK peptide analog. The monovalent aKLH 120.6 !gG2 HC-toain peptide fusion i-comairung antibody was isolated from the mix Msitig cation excimge chromatogmphyy as dewribedkereis. I 005 "m} The components of the munov a lent aK !.. H J 20.6 HP- ShK [! -36, R 1 K, ΙΊ A, QlOKj fusion antibody {aehcrrfaoeaily represented in Figure IF) included the monomers: [00573] (a) ;,KIH 1 20.6 kappa LC CSEQ )D KO:2K>: [0O579] (b) aKOi 120.6 !gG2 HC iSEQ ID NO:29); and [005R0] {¢) &amp;KI Η 120.6 HC UgG2)-ShK! 1-35. RiK. 14Λ, Q16K] fusion having .the following .sequence: MDMRYPAQU..GLU..I WLRGARCQVQLVQSGAEVKKPGA$VKY$CKA$GY TfTGY HM H\YY RQARGQOLFWM GW! \PNSGGTN Y AQKFQGR YTMTR DTS1 .STAYMb'LSRLRSDm'AVYYCARDP.GSyYWFDPWfiQCiavrvSSASl'KGPS VFP1 APCSRSTSF'SI.A ALGCLVRDY fTM:!PVlY?SWNSGAL12$GYH1TPAVLL>SS GL V SISSY VTV PSSN FC» f’Q ϊ V TC N V PH KPSNTKYDKTV fc’R KCCVfct m‘'PAP PVAGPSVFLPPPKPKDTLMISRTPEYTCVYYDVSHEDPEYQFNGVY YDGY.EV IΪΝ AKTKPRivHQFsiSTF 1¾ V"VS'V1TV V! 1QDW1 NG K fY KCKVSN KG i. PA PI F.KT 1SKTKGQPREPQ VYTLPPSREEMTKNQVELTC 1.. VK.GFYPSD!AVf.W ESAiGQP F.NNYΚ7ΪPPMI DSD(iSR'LYSK1 TVDKSRWQQGNVFSC '$VMi1F Al RN!1YT QKSl,SLSPGGGGGSGGGGSRS('5'\D1'lPKSRi'"r,AFKC KHShlKYRLSFCRKJCG TC.· ' fSFQ ID NGr.Wj.

The desired monovalent uKLR 120 6 IgGE 1 IC-ShK analogue product was a fall antibody with the ShK peptide msed to the C-tcrnwnus of one heavy chain. Vvijh two different heavy chains sharing one variety of Hem chain, the ratio of heavy chain' chaum light ehaindtcuvyriiam-ShK was 1.2:1. 1 he expected expressiofi prod urns are aK.LH 120.6 ?gQ2 antibody, monovalent nKLH 120.6 lgG2 HC-SltK peptide analog, and bivalent aKLH 120.6 IgG2 HO-ShK peptide analog. The monovalent aKLH 120,6 lgG2 PIC-toxin peptide thsion-eontaining antibody was isolated from the mix using cation cod:arum chsornmograpitv, as described herein [005Η1) Thu components of the mono v idem aKl.H 120.6 HC-ShK] 1-30, R\K. QIOI-G K30EJ fusion antibody (schematically represented in Figure IF) included the monomers: [00582] (a) &amp;K.LH 120.6 kuppa LG i$EQ \O NO:2b>: [005831 Φ) aKLH 120.6 lgG2 HC iSEQ ID NG:20); and [O0584] ic) aKi 11 120.6 1 gG2-ShK{I-35, R1K, Q1 6K, KJOIG fusion havtng the following amino acid sequencer MDMRVPAQU-GLU.lWLRGARCQVQL.VQSGAE.VK;KPCA$VKy$CKA$GY TF\ GYHMH\YYHQAPGQGLr-WMGW?\ PNSGGTN3'AQKFQGR VTMTRDTSi .STAYMb'LSRLRSDm'AVYVC'ARDP.GSyVWKDPWfiQCirLvi: VSSASTKGFS VI-PI APCSRSTSFS'I.AA.LGCLVKDV[YM:P\G'VSWNSGAL128GYhnTPAViJ.>SS GL V SLSSV VYVPSSN FG-IQ ϊ V TC N VPHKPSNTk V'DKTVOR KCGVK PPGPAP PVAGPi.vn..FPPKPKDTLMISRTPE\TCV\'\-OY?iHEDPE:’VQFNWVVDGVEV I IN AKTKPRHKQFNSTF R V VS V l TV V! 1QDWI N<i K ΓΥ KCKVSN KG I. PA Pi F.K.T ISKTKGQPREPQ VYTL PPSREE MTKNQ V SI..ΤΟ. V KGFY PSD! AV FW fl.SNGQP FNNYKTTPPMI DSDGSPFLYbK 1 .TYDKSRWQQGNVFSi YYMHFALiIN!IYT QKSLS t S PGGGGGSGGGGSΚΝίΊDTIPKSRCT AcKCKHSΜKYRl SFCRGI'CGT € iSEQ ID 180:300).

The desired monos;.dcni aKIH 120.6 igG2 HC -ShK analogue product was a full antibody with the SKK peptide fused to the € -terminus ofono heavy chairs. With two diflereni heavy chains sharing one variety of light chans, the ratio of heasy chain; chain:light chainrheavvchain-ShK was 1:3: 1. The expected expression products are aKi,1:1 120,6 igG2 antibody, monovalent αΚΙ,Η 120.6 igG2 HC-Shfv peptide analog, at\d bivalenutRLH 120.6 lgi'j'2. HC'-SKK peptide analog. The monovalent aK.LH 120.6 lgG2 HC-roxin pepude iuMon-eontaimng antibody v-as isolated from the mix using cation exchange Grromatogruphv. as described herein.

[005851 The components of a. monovMeat aiGLIT 120.6 !gG2"Sb'K|2<-3$. Q16K] fusion antibody I schematicallyrepresented hs Figure IF) included monomers: [005861 (a) aKU'i: 120.6 kappa U.' (SEQ 10MO:2S, above); [00587; (fe) «XI I ? 120/: lgG2 HU (SF.Q tL> NO:2X above); and [00588) (c) a Kil l 120.6 igG2«ShKp.GSs 1:.61¾::HE' set|uence;

MDMRVPAQL LGLL LLWl.RGARCQYOLVQSGA EVKKPGASYKVSi K ASGY TFH)VIIΜHWYRQAPGQGLBVMGWtNPNSGGTN VAQRFQGRV [ MTRD i'SI STAYMLl.SRLRSDDTAYYYCARDRGSYY WTDPWGQGTLVTYSSASTKGPS VFPLAPC SRSTSLSTAAi G<' i .VKDYFPFPVTVSWNSGALTSGYi ΠΤΡΛVLQSS GLYSLSSWTVPSSNPCIOTYTCNVDHK PSN i KYQX ΓVLEKCCVPCPFCPAP ρ\·'Α€θΥ5νΓ)θΊ>ΡΚΡκ.ί>·ίΐ2ν·θ8Κΐ!Α:·:γτονννρ?ν^ιο::(:)ΡΡνορκ\\'ν vdgvpy HNAKTXPRbPQiNS ίFRV YSYLTV VHOOW LNGKPY RCK VS NRGtΡΛΡILK I ISKTKGQPR EPQVYTLPPSR PFMTKNQYSf. TO YKGf YPSDIA YEWPSNGQP ERNY K' PiPPMEPSiX.;SET l. YSK O'V DRSRWQQi iN YTSCSY Μ ί Η· Λ Li-ί N Η Y T OKS LS L SFGGGGGSGGGG6SC1 Dll PR SRC 1A F K U K H.sM K Y R.l. SFCRKTCGT {'. i'SEQ ID NOGS).

[005R}) The cleared aK.LH ) 20.6 lgG2-ShK analog product was a full antibody with die S.hK peptide fused to the C-ierntinu*ot'onc heavy chain, configured as in Figure IF. \Vi;h two different heavy chains sharing one variety of light chain, the rado of heavy chain;light ehaurheavy ehain-ShK was l '2 ;. The expected expression products are aKLH i 20.6 lgG2, monov al-mt aKLH 120.6 igG2~ShK, uadi bivalent aKLH .:20.6 IgGd-ShK. The monovalent aKLH 120.6 lgG2-toxin peptide (or io>.in peptide analog} fusion antibody wa\ wo Leal from die mis using cation exchange ehttumatogranhy. as described herein,

[005901 Anti-KLB IgO 1 -loon-ShR. The aKLH lgGl-ioop~$hR also had a single copy of the ShK peptide sequence inserted into one of the heavy chains, but in this case it was inserted into an internal conjugation in the Fe domain instead of at the €-lermirms. (See. e.g., Gegg el al, U S. Patent No. 7,442,778, ITS. Patent No. 7,655,765; U.S. Patent No. 7,655,764; U.S. Patent No, 7,6o2.'GU U.S. Patent No. 7,645,86;; published U.S. Patent Applications US 2tKH;0281286; and US 2009.;02R6%4* each of which arc incorporated herein by reierenee in their • entireties), 1"he components of the aKLH IgGI-loopSSiiK antibody include [0059 I ] \ a s a KLH 120 0 kappa L C (SEQ ID NO-.20 above)· [00592] (b) aKLH 120.0 IgGi HC. N'iDMR VFAQLLGLLLL vVLRGARCQVQl.VQSGAEVKK.PG,'\S\'K.VSOKASGY TFTi iVHMHW Y'RQAPGQGLEWMGWiNPNSGGTN YAQKFQGRV i MTRDI’Si STAY M ft l. SR LR SDDT AV YYC A R PROS YY WF DPWGQGTLY TYSSA ST KG PS VTP( AP5SKSTSGGTAALGCi.VKD\'ΓΡΕP YTV S WNSCiALTSGVI|ΤΓΡΛΥ1 .QS SiiLYSL SSV YT\ PSSSLG i0 i\i('NYNHK.PSN ! KYDK.RYPPKSt 1>K 1ΗΤΓΡ1 *C PAP ELLGG PSVf LFPPK ΡΚ.Ι.ΤΪ ΙΛ1fSRTPEVTCV V VDVSH EDP EYRFN WY VDG VEVHNAKrKPKEEQYNSTVRVYSYLTYLHQDWl NGKEYKC k VSNK.A LPAP 1EK7 ISK.AK.GQPREPQV YTi. PPSRFE.MT KNQVSl TCLVKGf YPSDi AVEWGSN GQP EN NY K77 PP V LDSDGSF FLY SR l TV DKSR WQQGNVFSC S VMi ί EA L DNR YTQKSLSLSF6KA(SEQ ID ΝΟ:34.κ [00593] and [005941 OlaiCUl 190,6 IgGiAoop->ShlC;: MOMRVPAQLLGU. LΙ.ΛΥ(..RGAECQVOI.YQSG 5TVKkPGA SYkYSC KASGY TFTG YHΜ H W VRQAPGOGUOYM GW INPNSGGTN \ AQK FQGR v l MTRD ('Si STAY VIΠ.SR! R SDDT A V Y Y C A RPRGSYY WFDPWGQGTI YTVSSA ST KG PS VTPLAPSSKS1SGGTAALOFT VKDYFPh'PVTVSWNSGAL i SG VH i FPAVLQS SOLY S l SSV VTV PSSS l Q'l OTYIC NY N HK PS Μ K V DKRY EPK SC DKTHTCPPC PAPE!. 1 .GGPSVR.ΓΡΡΚPKDTI MlSRTPliV ΪGY V DVSi (EDPPV KFN WY VDG \! E VH N A KTK PR f£QY NST YRY VS V LTV 1HQDW1. NGK E Y KCKVSNKA L PAP i F KT ] S fCA (CG QP R E PQY Y ΐ ί ,PPS R Π f. 1 GG RSGI DTt PKSIU ΤΛ FKF'K) ISM KY R LSFC'RKTCGTCGGTK.NOYSLTCLVKGKYPSDUVEWESNGQPfN-NYK'rTPPV LDSDGSFFLYSK 1,TVT>K SR WQQGN V FSC S VM Η EA i Η N HYTQKSLSl .SPG K.' (SEE) tU NO:35).

(005¾) With two different heavy chains sharing one light chain, the ratio of heavy chain:light chairwheavy chain-ShK is ! :2: i The expected expression products arc aKLH 120.6 IgGL monovalent aKLH 120,6 igGI-loop-ShK. and bivalent aKLH 120.6 IgGI~!oop~ShK. The monovalent 120 6 IgG i-loop-ShK ίη-·:,-'. antibody (represented schematically by Figure IN'» was isolated from tie ors using cation exchange bhramaiogmphy as described Itereia.

[005-)6) Monovalent aRl.il :20/- kajgj: j::C:ShR? ΚηΥ.ΟΐΕΚ LMion. The components of the mmtoveient aKLH 120.6 kappa LiVShKJ 1-25, Q16K) fusion antibody {schematically represented ut Figure Π1 included the monomers: [0059?] i a s aKLH 120m j gGΙΗΓ t $ HO 11> K0.2'K: [0059K] 0» aK LΗ 120.6 kappa LC (SFQ IO NO:28>; and 1005T>j (cVaKLII 120.0 kappa FC-ShKj IMS, Q16N1 lesion having the following segue; tee: (006001 MDMR V PaOLI. G L U..I W 1.. R.G.A RC DiQMTQS PSSI. S AS V GO R V ϊ i TC RASQGJRN Oi GWYQQKPGK.APKR L1V AASSl08( iV PSRFSGSGSGTi:FΉ TlS S LQPEDF ATY YGLQHN SY PLTFGGO'I K YE! K R TV A APS VFIFPPSDEQL KSOT AS VVC(.LANFYPRΕΛKVQ WK VT)NAl.OSG'NSQFSVTEQDSKDSTYSt.ySTi T L$KADYFKHK VYA(B [HQGLSSP VTKSFNRGHOGGGGSGGGGSRSC1ΟΊ 1P KSR.CT AFKCkl ISMK.YR1..SFCRKTC GIT· i SEQ IO NO.267 s.

This embodiment of monovalent aKLH 120,6 O.G2 IX -ShK (1-.15, Q16!.<j product \\its a full anti body with the ShK peptide fused to the (.'-terminus of one light chant as shown in Figure hi. With two different Oglu chains sharing one variety of heavy chain, the ratio of light chaituheavy chain:light chain-ShK(1-35. QI6K) was 1.2.1. The expected expression products are sK 1..H 170.6 lgG2, monovalent aKLH ! 20.6 !gG2 l( -ShK[!-.(5. 0i6Kj. and bivalent aKLH 120.6 IgG2 1 < -ShKj.S-35. Q16KJ, The monovalent aKLH F20.6 !g('12 LC-toxm peptide (nsiowconiaming antibody was isolated from the mis using cation exchange chromatography, as described herein. Γ006011 Moteite :aki K g20.fi kn ηpa. .1C (AG K [2 G5,01 6H],histen. TM voniponents of the nsosun b. sn ok! H 120 .o vtppx I € ~S d<[?-3>, Q1»fK j suooss antibodies {seheotatediy represented in Home U) included the monomets: [00602] (a) aKLH 120:6 lgG2 HC (SHQ ID NO:29): [00603] (b) aKJLH 12().6 kappa 1C (SEQ IO N0.2B 6 and } 00604j (c) a Kill 120,6 kappa L( Yhk| 2-.0. Qtfk, hisso;. tuo mg in; iolk'musg sequence:

[006051 MDMRVPAQi.UK. U..LW L ROARCDIQMTQS PSSl SaSVOORVTITC

RASQi i!RM)I GWYQQKPGKAPKRi |YA ASSLQStA'FSkFSGSGSGTEFT! TIS StUPtO) \s>H! QKNS> PilM luG l KVEIkR ! \ VV’SX JMFPPSIR QL kbG 1 ASVVC LLNK FY PR EAK VQWK VD\ At QSGNSQF.SV1'EQDSKDSi"V SLSSTLT L.SK.ADYEKHKVYACE\'1 HQGI.SSP VI KSFN RGFCGGGGSGGGGSSCiDT IfK SRC'IAFKCKHSMKYRLSFGRKTCGTC^mSEQ ID NO:zm),

This embodiment of nxmm 3.:01 ah.I) { 120,6 igG2 LC-Shk}3-35, QlbKj prodnes v*a.x a full antibody with the Shk peptide fused to the C-termmns of one light chain as xhoxtn in Figure U. Wirh two different light chains sharing one \ anesy of heavy v ,u"> t !. '.oo'P'hght nr heu\) .b.un bght v. .<on-ShK[ G G'c\' eu- 1 2 F the ex|\.mvd exp'esMcr. pseeaets die 'skill 20 6 lgG3. i'vnosa _ r ,G2. G >20 6 tgG2 UYMfKj:- YY Q)6kj- and bhweci ski If ;20 6 h?G2 I C-Mikj2~TY QlokJ The monovalent aKLH i20.o !g02 LC·toxin peptide fusion-tontainutg antibody xml isolated from the mis. using cation exchange cbsxn.nuography. «s described herein. 100606] Bivalent nklil 120.6 kaasaT Γ-SnRl !«35, OI6RI fusion. The components of the bivalent aRLH 120.6 kappa Lt -ShKf 6-35.. () t < <K) txsxioa ant-bodies {schcinatieaily represented m Figure IK) isteiuded ihc monosTserx; [00607] (as aKLH .120.6 lgG2 HC (S6Q ID NO:29>; and [OOOOSf (h) aKLH iiOGiiippa ^ above. '1 bi'i embodiment of bivalent aKLH 120,6 lgG2 ΙΧ-χηΚ,Γ i-35, 0 1OK 1 antibody product ws> a fyil antibody with the ShK peptide fused to the C-ternunus of both light chains as shown in f igure IK. The ratio.of heavy •chanr. light chain-ShKf I -2b. Q16K.) was 1:1, The expected expression prodtn t is br% a I out .akU i I 2().0 did 1.( ShK[ UK QloKj. The hb dent aKUl )202- |$G2 LC-ShKj) -35, QS6KJ peptide fusion-containing antibody molecule n as isolated nom the mis «sing cation exchange chromatography, as described herein. ίψφί Biggem^^ The components of the his ah uf eh. I H ) 20 6 kappa !,(.'-ShK [2-25, GlbK I fusion antibodies tsehsmutieuiK *cpresented in Figure IK) inchded themorenters [00610] <»mKLH 120.6 IgG2 HC (SbQ ID NO;29); end [006!ij COlPBII 1211.6 kappa LC-ShKp-65, Q16K] hasten {SEQ ID NO:268¾ above.

This embodiment of bivalent aKLH )20.6 IgG2Lt-ShK [2-35, Q16K] produefwas a fui! antibody with the ShK peptide fused to the (.''-terminus of both light chains as shown in Figure IK Ibe moe s ee,} J\ m O ·,\ n Q oK'vv is l I

Pm expo* ted m pre-ssion pox*a, t o 'male5 abJ : I ' 20 o f;C )2 11 ~Shk{2-o b Q16K] antibody. Thebivaiem aK 1..Η 120m igG2 LC-ShK[2-35, Qlt-K] toxin iKtidehiCdr^ was isolated 'ftpttl die mix using cat ion exchange- -chromatography, as described herein, [006)2] Trivalent aKLH 120.6 kappa I.i'-Sh|^.^iil6KlMion I'he components of the trivalent aKLH 120.6 kappa Li -ShK i ' -35., U i 6.K ] fusion antibodies iwberaatice :¾ reptcscuted oi Genre Π ) ineluacd the monomers; 1006U | at u.ki H 120 o kG2 UP {SEQ 10 \0.2G u'oo^ei, [00614] |b) nKl.H )20.6 )gi.<2 WC-Shk[ 1-55, Q16k] fusion having me amino add of SEQ ID NO:32, abOveytim! [10615) (a aKi-H \ ~0.6 kappa fimo» feavmg ίΜ sequence ©f U'D ID \U U7,. *© e

This ;mthadhtvnt ©Finx-in-in aid Η 120.6 igG2 1.0-ShK product was-a lull antibody with me ShKf 1 -35. Q16K j peptide fused to the C-ternunus of both light chains and one heavy chain as shown in Figure 1 1.,. With two different heavy chcnhs sharing emc variety of light chairs, the ortio ofheavy chain,· light ebatn-SltK[!-3S, ::Ql6K]:hea\y chain-ShKf ί -35, Q16K] was t :2:1 The expected expression·product were a bivalent uKl.H 120.6 ig<!2 L€-Shk i \ -35. QlOKj antibody, ti ivalent aKUl 120,6 JgG2 LC - ShKf 1-35. Qlt»K] antibody, a ad tctravalent »KLH 120,6 igG2 LC-ShK [ i -35, QI6K) antibody The bivalent sK L Η 120.6 f gG2 l,€-toxin peptide fusion-containing antibody molecule was Gokued ihsm Oto thix using ptiob •exchange el»\>snatogm^ltys §s described herein:.

[00616} Irh.<tjohr.a.KI j:f J.20,6 kMPpa. LC-SbK. 12-33, Q16SC| fusion; 1¾ components ot the bo, Tent aKUl 120 6 kappa LC-KhKj2-35, QiOKj fusion antibody (v,'hennasen!iy s'eprescnted in Figure 11. ) included the monomers: [00617! f dKLH 120.6 lgG2 HCf.SEQ tD NO:29); :: [00618] (b> aKtti 120.6 igG2 HC-Shki2-35, Ql6K] fusion {SEQ XP NO:33}, #oyeta«d [00610] ic) aKLH 120.6 kappa LC-ShK!2~35. QI6K] ftmoniSBQ ID 610:268), above. 'Thisembodiment oftrivalent aKUl 120.6 lgG2 LC'-ShK|2~35. QiOKJ antdxxh product was ,i tub antibody with the ShKf2-^5. Q16k] peptide fused-to the C-lemtinus of both light dteos iod one lieioy chant as show n in Figure 1¾ Wih b|p different heavy chains sharing one variety of light chain, the ratioof heavy chamdigbt chuin-ShK[2-35. Ql6Kj:hea% y chain-ShK[2«35. QI6K| was 1:2.1. The expected expression products were a bivalent aKLH 120,6 fgG2 1 C-ShK|2-35. QlOKj antibody, tri valent aKU l 120.6 lgG2 LC-SbK[2~35, QI6K} antibody, and tetrasulent aKLH 120,?· 1gu2 LC*ShKf2-,G, 016K] antibody. The rnvalent aKJ.H 120.0 h.:.(.>2 I.C-toGn peptide fusion-coniifmmg antibody molecule was isolated iron? IPe ess ism v eatsv cachenet cNo n you mb' is descuheJ bet cm. I'ooo20| Λεμ^*1.cms&amp;m- V, «(Mouse nbmh> ua evpres>mg Ki H r ·. Iona inHw> ·-fi 0 w < asm vuu'ee to iso.atc total RV\ evng TRI/oiC icancel i!n\it roe cm). hao stemd cDNA wa\ syntht**i'/ed using »random primer w"h ,m extension adapter 5 -GGt CGC Vi'A GGC ί.Τ(' ( \N\NNNM 3'{SFC ϋϊ NO Mo and a 5' R AOGramd ampiiticdtsor o, , DA A e xR v% »s *v hu re 1 us.n», 'be Ocnckae lx it i Invitrogen}, For the light chain wqaencc determination» the forward primer was 5-GTG GIT GAG AGO TCjC CAG ATG TGA CAT TGT GAT GAC TCA GTC TCC -35 (SFQ ID NO:3?'> and the reverse primer was 5'·· AAC CGT ΓΤΛ AAC' GCG G( C GC Γ t A A CMCTCT (XT CTG TTG A ^ -3' -St <3 iD\O: 3ft The H \t.T product χ λ e mted ι sto pi RJ* t (>PO t las ns ogeul .rut t ό sequence^ determr ed .> Consensus sequences were used to determine probable framework and signal pepudG sequence and design primers for full-length antibody chain PCR amplification. 100621] The expression clone lor the anti-lit Η 120.6 kappa light chain was prepared by PCR. The 55 PCR primer «encoded the amino terminus of the signal;: sennem., an ΛΧ/1 tests x non enzyme site» and ms opimu/ed Ko/ak sequence S'-AAG C IC G \G G 1C GAC SAG ACC' ACC A TG GAC A TG AGG G 1C CCC G -3' iSl'Q ID NO:CD 1'he 3' primer encoded the carboxyl termmes and termination codon, as well as a Noil restriction site 5' AAC’ CGT ΤΊΆ AAC GOG GCC GCT C AA ('AC TCT C CC (' TG 1 TO Λ A -3' t SFQ ID V) ,T} f he tesuhing pt odnet was cloned into p€R4TOP0 {htviiro^getfl aMlTbo seqnonces determined. Alter the insert was co«ftrmai: the. p€R4*r0ROSail and /Vb/I, tbe insert gel Isolated and CHagen puriBed. and vector p?T5.

[006221 A PCR was done to change the signal peptide from the native peptide deri ved from the hybridoma to the Vf£1/012 peptide. VK.I ·ΌΙ - fragment wore Y ΛΑΟ CTC GAG G IT GAG TAG ACC ACC Λ IT (.IAC ATG AGG GTG CCC GCT 3' (SI:.Q ID NQ:40) and 5'-TCA 7O' GGA TGT ΓΑί Λ IT TGG ("AC C 3' (SEQ ID AO: 4 ί v The primers used for the mam re light chain peptide were 5-GGT GCC AG A TGT GAG ATG GAG ATG A -3' (SEQ ID N0.42> and {SEQ ID NOOK). The rosehip Gagmens were joined h> overlap PGR using primers (71:.() ID TOGO; mid (SEQ ID NOOK; The sequence ol the resulting clone one*Kies rite following irorouttoglobulm kappa EC sequence: MDMRVPAQl EGl LCiA\TRGARCDK)MTQSPSSLSASVGDRV77T€RASQGlR NO LG W V QQKPG KA PE RLIV A ASSLQSG v PARE NGSGSG' f LET LOSS LQPEOF AT Y YT LQH N SVPL ITGGGTKAT. I E.RTVA.A PSY RFPPSDEGLESGTASYYCLL NNFYPRt'AKVOWKVDNALQSGNSQCSVTEQDSKasTYSLSSa'fLSKADYfc' E BEVY AC irVTMQC>LSSPVTfC.Sf NRGIr.(.'7 (SEQ ID NOGY) [00623) Anti-K.LB I20.E Antibody Light ChaimSh.E. pepEdc analog mammalian ••expression. The Shk( t-35. Q (6K.| fragment was generated h-v PC'R using the pTTM-hulgG2-Fe ShK [1--35., QlC.K; encoding (SEQ ID NO..Y). described above, as a template and the oligow 5a AAC AGG GGA GAG TGT GGA GGA GGA GGA TCC GGA G -3' (SEQ ID NO:269); and 5'·· CAT GGG GCC GOT ( A Γ I AG GAG G -3' (SEQ ID NO;270).

[00624] The light chain fragment and ShK PGR product were then amplified by PGR using the outside primers CAT TCI AG A ACC ACC ATG OAC ATG AGG GTGv (SEX) ID NO:343? and SEC) ID EO.2?0. The PC.'R product was then digested by λb;J and Noll and PC R dean up kit {Qiagert} purified. At the same time. pYD 16 was cut by Xbal and Noll. The pYDIO sector svas run out on a EG agarose g.cl and the larger fragment was cut out and gd purified by QGgonA Gel Purillcatiort Kit. 'The purified PGR prodtscl was ligated to tits' large vector iragmem and trr-rt.sformed into OneShot Top 10 bacteria. DNAs front transformed bacienal colonies were isolated and subjected to Xhal and Nod restriction oneyrne digestions and resol ved: on &amp;ΐ)Μ percent agarose gel. ON As resulting m m expected pattcr&amp;YttfePS φ^φϊΜύ-lor sequencing. Although, analysis of several sequences of clones yielded 0 £00%· pereent match with the above sequence, only one elone was selected for large sealed plasmid purification. The final .pY016^K.LHi20v60VK 1 SP4X'4. HTSh.Kl.J~35*. ||16Kf eonstmet opcodedAo aRLB 120.6 LC4.10-ShK[)-35, Q16KJ foslob polypeptide (S EQ ID NO:267>. p0625| The Shk[2~3S. Q16R1 fragment was generated as described above using: gTTS-aKLH 120.6 HC-SbK}.: -15, QioK] us a template and the oligonucleotide· pnmetx si g 11 \0 2~* « <' v υ ID NO ' /0 |0002ο] The sight chain and Shk RCR products xtere smphfied by PC R using the outside primers SEQ ID \OvG > and SEQ ID NOIDO, The PCE product was then digested by XM and Not! and RCR dean up kit (Chugc-ns penned. At the same time, pYD;6 w as cut b\ XKi and Not!. The r>VD; 6 veetm xt as nut out on a I % agarose gel and the larger i sue sues it was out out and get purified by Qiagerf x Gel Purification Kit. The purified PCE product was ligated to the .faigoYeptPr l»p|i^|n and transformed into DneShof TopiP Eactpria. DMAs from transf»p#cd|}|aetpc^il'' polonies were isolated apd suhjeetod to X|mi and Notl restrictionsenzyme digestions and resolved on a one percent agarose get. DNAs residing in an expected pattern were submitted for sequencing. Although analysis of several sequences of clones yielded a !00% percentmatch with the above sequence, only .one done was selected for large sealed plasmid purification. The final pYDIo-aKLH i 20,0-VK. ISP-fT.'-IJ0-ShK|2d5;5QI6K! cousiritet encoded M lgG2-l.C-L! (kShKf 2-35, fhsioh polypeptide monomer (SEQ ID N0::26ly

160627¾ A »* manma|lau.exjMas|M·

Using oligos t'-CAT TCT AG A CCC ACC AEG GAG Al'G AGG G'EGG' (SEQ ID NO;4J): and s'-gga icc icc ire ire act: cog aga cag go a gag cta (Seq m NG:44),

stu; ;.il\LH- igG2-Hcuvy Cham reglun amplified by PCK from a p iTS-ukLR ; 20.C-V K I $ IMgG? 11 Cii vv ClubrH Hi? i construe! conia insrig iho α π buy sequence i$m ID NO:45: below), encoding uKLH i:0.6-VkiSP«igG: Heavy Chain {SHO ID NO:46; below): ATGGAC' ATG AGGG'ΓίI( C'GGi Ί' C ACiC?TGCIGGC ΓΓ'CTGCTGCTGTGGC Ύ

GAGAGGT GCCAGATGTCAGGT GCAGCT CGTGC ACTTCTGGGGCTGAGGTG AAGAAGC )'I GCiGGC OX(FAG IGAAGGHHC C IGC A AC;GC TXC.IGGA I ΛΟΛ CCTTCAtXGGCTACCACATGCACTGGGTGCGACAGCXXCCTCXJACAAGG XX'TTGAGT'GGAIXiGGATGGATCWXa'AACAGTGGTGGCACAAACTAT OrACAGAAG'nTCAGGGCAGGGTffACCATGACC/U'iGG/vCACGTCCiVK'A GCAC’AGCCTAC ΛΊ GGAGCI G.ACX. AC1GC I GAGA K' i GAC’GAC ACGCC.’C G1 Gf AIT ACVGI'OCGAGAGATCG I'OGGAGC”i'AC 1'ΛΓI'GCVf 1'CGACCCC IOGG

GOC aOGGAACCCT GGTCACCOIOTC CTT ACXXTCCACCAaGGGCCCATC G "GTCTTCX CCrrCGCGCCTTGCTC CAGGAGC. ACC.T('(XAGAGC.ACAGCGGC‘ (:‘CTGGGCTGCCTGGTCAAGGACIACTTCCCXNGAACCGGTGACGGTGTCGT

• GGAAt'Tt'AGGOGCTCTGAtXFAGC GGGGTGGAi ACXTTCXX'AGtTGTCCTA 'rACnTT'TC'AGGArrrrACTCrCTCAGCAGCGTGGTGArCGfGCCCTCCAG CAACTrCGGCYXCCAGACCTAC ACX'TCCAACGTAGATCACAAGi'XCAGC AACACCAAGG iGGACAAGAC'AGTTGAGCGC'A.AA’VG'H'G FG rCGAGTGCC CAOCGTGC CC AGO AC C AC CTGTGGOAGG AC C GTC AGTCmX'TOriTTX' C iCAAAAGCC AAGGAC'AtTT'TOATGAT('T(XCG(jA('(XCTGAGGTCA(?GT GOGTGGTGGTGG AOGTG AGCCACGAA GA COCT G AGGT C C AGTT C A AC TG

GTACG1 'GC '> ACGGCGTGGAGGTGC Λ T A A'l Gt'CAAG AC'AA AGCG ACGGGAG XjAGCAGTTf.'AACAGCAf?GTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCA {'CAGGACTGGOTGA A< GCX 'AAGG AC iTAC AAGTGC ΆAGGTC TC ΓAACFAA A GGrerCCOAGCCrCX'ATCGAGAAA ACCA ’ G ! GC Λ ΑΛ ACCAA AGGGCAGC XCCGAGAACO\CAGCn'GTAC?4CCX:'TCXXX:<: catcccgggaggagatgac •CAACiAACCAGCVVCAC'}CCrGACCrGCXvrGG‘rCA,V\GGrriT"CAC<:-CC-AGC G AC. ATCCXXG7 GG ACT GGG AG AGO A A TGGGC AGCTFGG AG A A C A ACT' AC .A AG AC '(.'AC ACC TC C C VYi GCTGGAC TC.C GACGGC'IX TT'IV H'C X? IX? TACT AG C AA GCTC aOCGTGG ACA AG AGO AGGT GGCAGO AGGGG AACGT CTT CT CA TGCTCCGTGATGCATGAGGCTCTGCAt aaccactacacgcagaagagcx TCTaXTGTCrCCGGGTv (SEQ ID NO-451 encoding Hie amino acid sequence MD MR V PAQLEGL I.ITAV i. 1¾ G A RC QVQI. VQSG AE VKK PGASVK VSCK A SCO" TFT GY ϊ M 11 Vv \' RQA FGQ< 11 F WMG Wl NPNSGGTN Y AQKFQGR VTMTR DIN I ST Λ V M EI..SRI RS DDT A VV VC ARDRGSY V WFDFWGQi 37 L V'l VS$ASTK OPS V!"PI ΛPCSRSTSFSTAA(.Gi''I VKDYEPFPNTVSWNSGAl7SGVIΠΤ P A V I .QSS GLV Si. S$ VYTV PSSN FGTQT YTCN V DM KPSNTKVOKTVERKCC VECPPCP AP PVAGPSVT L ΓΡΡΚ PKDT LM1SRTPEVTCVVV DVSHB DPEVQFN WY YDGVEV HN'AKTKPREirQFNSTFRWSvLTVVHQOWLNGKfc'VKCKVSNKGLPAPlEKT ISKTKGOPREF'QVVTL.PPSREEMTKNGV’SLTCLVKGFVPSDiAVEWESNGQP ENNYKU-PPMLDSDGSFFLVSKLTVDKSRWQQGNVPSCSVMHEALHNHYT QK.81.XLSPG,·'·' fSEQ ID NOAM. (OCRGkj The ShKD~n5]WT fragment was generated using the original Fc-LfO-ShKiFTb] in pcDNA3.H-t-)CMVi as a template ^described in Example U Figure 14A-1-4R in Sullivan et aL 7’oxm Peptide Thempcrunc Agents, PCTVCS 3007/0jliKs1. pnbiishedus WO 200&amp;08842¾ which is incorporated herein bv reference in sts -entirety) and the o'iw<\ S'-T€€ CTG TCT CCG GGT GGA GO A GGA GO A TCC GGA G-.T (SEQ ID NO:47): and 5'- CA T CCG GC C GOT C AT TAG CAG GTG -5' tSEO ID NO: 14} 'The PCR products were run on a IN, agarose gel. The bauds were punched tor an agarose plug and the plugs were placed in a ftesh PCR reaction tribe. The agarose plugs were then amplified by PCR using the outside primers SEQ ID NO:35~ and -SEQ ID NO;330, The PCR product was then digested by Xbal and Moll and PCR clean up kit lOiaucns purified. At the same tune. «ΤΤ5 Vector (an Amgen vector containing a CMV promoter and Poly A tail) was cut by Xbal arid Noil The pT75 vector was run out on a 1% agarose gel and the larger fragment was cut out and gel pur died by QutgcnVs Gel Purification K.H. The pur died PCR product was ligated fo the large vector fragment and transformed imo OneShot Top 10 bacteria, DMAs from trunsfortned bacterial colonies were isolated and subjected to Xbal and Noil resuictio» &amp;B2vmo dictions and resolved on u one percent agarose gel. DMAs resuluiig in an expected pattern were submitted (or sequencing Although, «niiKsis of several .sequences of done» yielded a 100% percent match with the above .sequence, auh one cion·,· was selected for largo ,scaled plasmid purification The 'final ρΎΊ'5-uki Η 12tU- VICI SP-lgi>2-1!C4. I O-Shki 1 -35] constructencodedan lgG2-HC4.10-ShK|‘i -35 j fusion polypeptide with die an tin© add sequence:

M.DMR.VPA.QLL<3LLLLW.LRGARt'QVQL\'QSt,s U'VRKPGASYkYSC KASGY TFTGYHMIIW YRQ*PGQGL.B\TOUW!NPNSGGTM AOKFQCRVTMTRDTSI STAYMf 1 aid WVYCARnRGS^ VWiOdUgHd YTVSSASTKGPS VPPLAPCSRSTSb'S ΓAALGCLVΚΟΥΚΙΆP\ ! VSWNSGAITSGYHTFPAVLQSS (.11 VS LSS VVT VPsSN FG1XJTYT<'NVDI IK PSNTK VDRTVFRRA CYECPPCPAP P\ AtiPSVHFPPkPKD't 1441 SRIΡΕΛΊCY\ VDVSHEPPfcYQPNWYVDGYEY RNAKTKPREEQFNSTFRVYSVLTVVHQDWLNGKRYKCTCVSNKOt-PAPiEKT Lsk I'kUQPREPQX Y n.PPSRELMTKkOVSi.TC’{.YRGR PSIHAVPWPSNGQP ENN V K.TTPPMLDSDGSF14..YSRlfrYDKSR WQQGNYFSGSVM HEALHNHYT QkSLSLSPuiiGGGSGGGGSRSnDnPRSRG rAFQC'Ki ISMK YRt.SFX ’RKTCTJ TC (SEQ ID NO.4%.

[OtU.2oj To gene- do Is,· shkj i~ %, Qi okj muvui vctsion of tins construct, sne-::·'directed mutagenesis was performed using the Stonagene Qnikcbange Multi site Di reeled Mutagenesis kit {CmAcOOeik 1, per manufacturer's instruction?-, and ohgdsgnt 5'-GOT GCA CCG CC'T T( A AGT GCA AGC ACA GC 3’ (SEQ ID NO:% and 5'~ GOT GTG CTT GCA CTT GAA GGC GOT G< A GC AT (SEQ ID NO: 10),,

The final construct pΓΤ5-uK LH120 o-YKISP-1 eG2-HC-L10-ShK[Ϊ-35, Q16K] encoded id 12-1 it'-L 1 O-Shki i -35, Q ink j fusion polypeptide v, ith the following amino acid sequence;

YIDMRVPAQl 1 GO 1.1 \\ 1 RGARCQVQLYQSGA EVK kP<> A SYkVSCK ASGY rFTGYHMH\V\ RQAPGQGLHWMCiW INPNSGGTm AOKFQORv 1MTRDi'St STAYVin spi rsddi u yycardrgsys wt dpwi nun \t\ ns VPPLAPCSRSTSESTAALGCLVKDVEPEPY 1.V.SAVNSG>\LTSGVHTEP.AVEQSS·, GLYS1,SSVYTYPSSNPG01QTYIGNYD1WSN7R:YDKT\,ERRCCY13C:FP(:PAP

PYAGPS \Ύ L FPPK PK ΌΊI MISRT Pfc V ί CWVDV SHBPFBVQFN \V V YDG V fcV HNAKTKPREEQFNSTFRVVSVTTVVW^DWLNGKEYKCKVSNKGLPAPn'KT lSKTK.GQPRfcPQVY i 1.PPSR B'HMTKNQ V$t. I CLVkGF YPSOt A YEWESNGQP bBiNYKTiPPMLDSDGSFM. VSR LTVUKSRWQQGNATSCSYMHGALHNHYT QK S LS LS PGGGGGSGGGGS RSCi OTIPKSRC ΎΛ FKCKi ISM RY RLSPCRKTCG TC·· {SEQ ID N0:-19i.

[00630] aKLH-laCG Heavy (ham-Li0-ShRf2-35, Qi6K) numinialiun expression. Using DNA construct plT5-aKI.H \ 20.f-VK I $PTgG.?.~HC4. lO-ShKfMS] as the v ecs or, the ShK[!G5j was cu? out -.using Banal·! i.'Barmil. The vector h'se-ruera irons pTTS-aKLH 120.0-VK j SP-sg.G2-HC without ShK| 1 - j contained the coding. .sCQUonce:

ATCKiACATGAGGCrFGCC<XXTCAGCTOCrCXKXKna:TGCTCA'TGlOGt:T {jAG AGO ϊGYX.AYiA TG s OAGG 1 GCAYX 1 GGl GC.'AG 1C' i’GGGGC.' iYCAYX.! i'G . AAG A AGCC Γ GGGGOC T C A O'? G A A GOT C T COT GC A AGGCTTC’TGG A T AC A CCTTCACCGGC1' ACC ACATYX.' AC KiGG' fUCG A( .AGGYX X X. TGY i,\CA AGG ••GCTTGAGTGGATGGOATGGATCAACCOTAACAGTGGTGGCACAAAC TAT GCAC'AGΛ ΑΥΠ I Ti.'AUGGC'AGC.fGTCACCA 1 GAC't.'.AGGG AC Ai GTC.X'A ICA GC \ί"Λί'ΧΧ'Ί AC Λ IX'iGAGC iGAGCAGCTlGAGATCTCUCGACACGGCCG ί or vn.\CTG1' ί iCGAGAG ATCGTGGG AGCTACT .ACTGGTTCO AC‘CC C'TGGG Tit CAGYsGAAYX ( KiGKAYXXili ; ( YTY'ACXX !( ( ACC AAGGCCCX A IT Yi •GIVTTf CC (X ΓGGC OCCCTGCTCCAGGAGCAC CTCCGaGaGC ACAGC GGC ί ("ΊΥ X ft X ΊΧX '<ΎΥΧΓΓΓAAGGA( ΊΆΛΊTYXX ( GAA(X GGTi iA( (XUi iTCG'i -GGAACTCAGGCGCT C TGACCAGCGGOGTGCACAC C TTCC CAGCTGTCC ΤΑ ( As..* S ( G 1 ( AisGAC i ί I Αί. i Y ( (.) ( AYi( AG{. Y> ICsG I YjAY Y G ί YX it. It. Y c\G ( AAC IT CGGC ACOC AG AC CTACACC TGC A ACGTAG ATC AC Λ AGCOOA GC A AC ACC A AGO Ϊ GGACAAG ACAC- l I GACiC YX. A A N I (> 1 ) G t GI C GAG I GC Y CM'CGrGCCCAGCACVACCl'GfGG( AGGACCGKAGIC I !'CC ΚΊ KXXT CCAAA.aC CC AAGG AC A (XT TC ATG.AT(ST(XXOGA<XX CTGAGGTC ACGT GCG i'GGTGG' f'GCi ACG CGAGCC AC (>A A GAY C CCGAGG' CCC Λί V f'TCAAC i G 'CTA€GTGGACGGCCiTGGAGCTGCATAATCiCCAAGACAAAC>CC ACGGGAG GAGCAGTICAACACX'ACGTrCCGTGTGGTCAGCGTC’C'TCACCGTTG'J'GC'A (T' AGGACTGGl" Γβ A AC (JGC A AGGAGTAC A AGTGCA AGGTC TCCAACAA A GGCC rfXXWGC CCCCATCGAGAAAACCA i'CTCOAAA ACCAA AGGGCAGC Γ(Χ {i\{ ΪΛ rc ACAi unGTAc hoc tgcccc vatccc irk jaggagatg \c CAAt .A \0 'AGGTC At ,ί t'TGAC'C R X X' FGCΠΧ'A \ ACiOC 11Cl AC YYTAGC ’ GAC \R Q C GTGGAG FGGGaGaGCAA i'GGGCAGC GGGAGAACAACTAC' λ ag ac '(.'ac Acrrax'AiGCi'GGAcrrccGAcxK'jtTt'CTTrnx'rrcrACAi» i AAGi Ti Hi GTGG AC A AG AGCAGG TGGC AGC AGC «GG AACXtTCTTCTCA AGC 7( (ίϊΤΟΛ fGCATGAGGC f'G f'Gt'ACAAC (.ACT AGACGCAGAAGAGCC in a C \ GTCVrCGGGl'GGAGG ACiGA mSEQ ID NO:?Oj. encoding iho amino acid sequence MD:MRVP.aCH..IXiLLLLW.LRGARCQ.VQLYOSGAE\ KKPGASVKYSCKASGY TFTGYHMHA VRQAPGQGU-WhlGWrNF\SGGTN\ AQK.FQGRVTMTRDTST SI VYMI i s!U RSDD1 U 1 \ ( AkDRGSYl WtOPWCGGH \ i VSSAS'IkGPS VTP L A PGSRSTSFST A A1. GCL V RDYF PHPV'T VSWNSCALTSGVHTFPAVI.QSS G L V S LSSY VT V PSSN VU Γ Q i YTCN VDHKPS NTK VDK V V ER KCCV ECPPCPAP PVAGPSVH FPPkPKDU MRU DM V'K Y\ YDVsDi DPFYQFWn YDGYE\ HNAKTKPRFFQfNSTFRWSYL n-VHQDWLNGKRYKCKYSNKGi PAPIEKT iskrkCiQPRt iha γ π ppsp f: \u r\q\ sl igl y rcr y psimav l w esngqp EKAYK'rTPPMI.DSDGSFFLYSKLTYDKSRWC^QGNYFSC/SWIHFAFHNHY'r UKSl SI SPG* RiG CNl.Q iDXO.51) then treated with Calf Intestine Phosphatase (OP) to ret no vc the 5* Phosphate group and PhenoP'i 'h loro form extracted to prevent reh'gtnfon of the vector upon itself The insert came fsora ρΤΤ 1 1 ME! ISP-lgUP-l'C-encoding igG2 Fe-L iC>~ShK{2-35. Q16K). MESVSWVFLFFLSVTiGVHSfcRRVBCPPC’PAPPVAGPSVFLFFPRPKDTLMlSR : TPIYTCV VYDVSI-iEDPEYQF^SiWYV DGVEVG N AKTK PR ERQFNSTFRV VS YE s TW HQDWLNGK FY KCKVSNKG lpa piektiskt kgqprepq vyteppsr fem TKNQVSLT CLVRGFYPSDFA YPAVESMilQFFNN Y RTTPPM EDSDGSFFLY SKI, IVDKSRW^QGRYFSCSYMHEAI i INtiYl'QKSIAlAPCIRC}GGCISGGGGSSC;II> AFlPRSRCrAF^^ ID NO:i% and the 18¾¾¾¾ also digested out using BaroHf/BamHf, The insert ShKj2-35, QI6KJ fragment was gel purified away from iis s ector and cleaned up with Qmgen Gel Purification Kit. A purified D'NA insen containing the coding sequence GGA fCC <IGA GOA GiiA GGA At X At »0 it »C AT0 GAC ACT ATX Ci( AAG AGC CGC TCiC ACC GCC TTC AAG TGC A AG CAC ACC ATG AAG TAG CGC CTG AGC TIC TGC CGC AAG ACC TGC GGC ACC TGC TAA IGA .// (SEQ 1BNQ:52), encoding t.he amino acid sequence GSGGGGSSCilJTIPKSRCTAPKCXPlSMkYRLSPCRKTCGTC fSHQ IDNCVSi). ‘Λ as ligated to the large vector fragment and iransten tned into OneShot 1 op i 0 bacteria. DMAs iron} franYormed bacterial colonies were isolated aval subjected U> BamHi restriction c?-.cyme digestion and. resolved on a one percent agarose gd, \ A,'\s requiring in u>" eweted pattern v\ i re sakmeed tor νχαο^χι Although, analyst'» of set oral sequences of clones % i aided .. s 00° « percent nunCo -a Uh the abme sequence, only one ehme vtas select} fir large a led piav-mo ρηπ 2» at on The final construct pTT5-aKUTIgG2 HC-U0-ShKt2G5tQ 16KJ encoded an igG2 UG-.L 10»ShK{2-35.Ql6K] fusion polypeptide: i^MRVPAQlTOtLLC^LRGARCQVQLVQSGAFVMKPGASWiVSCICASGV TPfGYHMHWVRQAPGQGLEVVMGWINPNSGGTNYAQKPQGRVTM'TRPTSl STYtYMEi.SRlRSPOTAYYYCARGRCiSYYWFDFWGQGTLVTVSSASTKGPS Vi:FLAPt'SRSTSESl'AAI..GC'LYKPYFPRPVTVSWNSGALTSGVHTFPAVLQSS ΟΕΥ§Ε$$ννΐΎΡ85ΝΕΟ^«ΤΝνΒ^1ΚΡδΝ1'Κν0ΚΤ¥ΐΛ€ΤνΕ€ΡΡ€ΡΑΡ PVAGPSVFLFPPK.PKDfl.M1SRFPE VTC'A VYDYSi 1FDPFVpI NWYM)t 1Y 1 V HNAkTKPRFFQFNSTFRY' YU \ \ HQ'0\vt NGKEYKCkN ‘SNK.Ul.ΡΛΠΙΎΤ ISk Ϊ RGQPRFPQVY1 LPFSRI FMIKMdVFl I Cl VKGFA PSIXAVEW! FNGQF fcNAYkl t PFMl OSIXRTH'} ^kt f VDRGBt fiX/GM Fsf SS YUlfc U,H\H> 1 QKSLS1 SPGGGGGSGGGGSSCID1' 1 PkSRCTAFX.CK.KSYIKYR LSECRXT <. G'T C" <SFQ IP .NO:54}. j OOF > 1' 1 n,;· S ikj ι-C. ICA, 14 G>bkj Gnunont n as generated using ρΠ"5~ aRLH 120,6-VK lSP-iqG2~H€~F K)-ShK(l - C Qi6K,j as a template and the oligos.

[0063:] 5'·· AGO AGO AGO ΛΛΟ CGC ( AG Cl G CGC CGACAGCATCCC (. ..Γ·' {SI5Q ID NO.310); and 100633] 5’ - GGG GAT iX*T υTC GGC GCA GO' GOT GO' TCC TCC TCC T 3'ViSOQ ID NO:3 H >.

Siic-direeiod mutagenesis was performed using the Srrutagenc Qmkek&amp;uge Multi ssite Directed Mutagenesis Κή. per rnanutacMrrr's instruction*» The ilnal pTT5-aK l.H 120.6-V K] I SF-SgC.sO Η -I = OAGK {1-36 R ] \, 14Λ, Q \ OKI cousin set encoded an lgG2-1KM. 10-ShKi 1-35, R i A, 14 A. 0I6KI fusion polypeptide (SEQ ID NO: 304».

[006341 "Tits Shk{; 13-3:5, R! A, Q16K, K30'E| t:-ίΐ^'^-ιρΐΐ^ί^·· above using the following four olsgos; [006551 5G GAG GAG GAG GAA GGG CCA OCT GCA ICG AGA 475 |SEQ IDMV.313):

{006361 5'-GAG err CTG CCO AGA GAG CTO CGG CAC 4’e fSEQ ID NOG 13 H

{006 C/j 5'- CGA TGC' AGC 1 GG CGC ΤΓ C CTC CTC (T<' -5' <SEQ IO NO :314); and [0063k] 5'- GIG CCG CAG GIT TCG CGG CAG A AG CTC -37·' (SEQ ID NO-315) ίhe final pTH-uKI H 120.6-VK ISP-IgGADC-i ID-ShW IG5 R! A. QI6K. K301:1] e* instruct encoded an IgCG-IK -LlO-ShRl 1-35, RCA, Q16K. K30R] lesion polypeptide tSEQ ID NO:305) [006301 The ShKj 1 -35. RI Η, M A. Q i OR] frogment v as generated using «ΤΤ5-aK.LH 120.6-VKI SP-IgGd-HiΊ.. i 0-8ήΚ{ 1-35 Q16K | us a template anti the oligos; 3 [00640) GOA GO A GOA: AGC: 'GAO MtjmW£&amp;mS£€ ATCCCC- 37 TSFQ ID NOG 16); and j 006411 5GGG GAT GGT GTC GGC GCA GCT GTG GCTICC TCC TCC - 3 W (SHQ ID JsiO:3! 7).

She-directed reutagcncak was performed umhl* ihc Simiagcne Chtikehatige Multi Mfe Directed Mutagenesis Kit (CWrOOSD), tv; n.<-'nuG<. jeer \ .n&amp;l racoons The One' ρΠ'5-aK.UI Ote<A Kih!MgG2-H<'-l 10-ShR[j~3S R11 L 14A. QI6R) construct encoded an IgCC-HC -UG-$hK[ 1 - '5* R 1H, 14A.. Q16K] fusion polypeptide ($BO ID j_ NO: 306).

[00642] The S i\[!- C. k II. Q16te, R30FJ innocent Oio generated a-, .k "v-pteid ahdye ntdng the ioHowing four oii&amp;o.s:

[006431 - UGA GG \ uGA \G( 3 AC AGC 1(4' \!CO u ϊ jSLO ID NOG i ) end SBQ ID NO:313; [00644| '|^:GTCGATGCA:G^'<TO;#CT^O-i#fCC -37/(¾¾ ID NOG 14) and SBQ !D NOGi 5.

The final plTS-alv 1 if j20.6-YK lSP~G.G2~fIt ~L j0~ShR[ 1 -35 Rill. QI(>KS K30Γ] emsirm encoded an IgG2 -MC-t 10 -Sh K[ I - 35. R1H, Q16K. R.30B1 fusion, polypeptide (S FQ ID NO;307).

[00645] The Shkj 1 -35, RI ky 14A, Ql6¾] lf%o3eo( was genemted usmg pTT5-aRLIi 120.6-3/K! RP- lg( i2- H(A f !0-Shte[ f-35 Q16K.) as a template and the oliyoi'· [006401 5*- COG GAG GAG GAG GAA GCA AGA GCTGCG COG AC A CCA TCC CCA AGA-37/{SEQ ID»320); and [006471 5 C TOT SG GGATGG TGT CGG CG€' AGC TCTTGC TTCCTC CrCClVCGG-3' (Sfr'DIDNO^n

Stfcdireetcd mutagenesis was performed using the Siratugene Qtukchaogc Multi site Directed Mutagenesis Kit {Cate'd 0(;5 >: g per rnanuiaceirer's loam rations The final pTC-aKLH t:0.6-VKlSP-lgG:-HC-L10-ShK[i-35 RiK, I4A, QlhK] construct encoded an lgG2~BC-L!0~ShK[ 1-35, RIK, 14A, Q16K.J fusion polypeptide (SEQ iD NO. 30 8) [00648] The Shk[ 1 -35, RiK, QI6K, OOfij fragment was generated as described afebyg -using the followi.ng four oligos: ! 00639] 4'- CGG AGO AGO AGO AAG CAA GAG CTG CATI®A OACrCA -37/(SEQ ID NO:322} and SE<| ID NO:3li

[ 0CR50] 4 TGG rc JT f C ΙΑ INK ’ \C( TCT Ϊ GC Γ ΓΟ CTC CTC (‘ rc Gi I -3' |SEQ ID NO:323) audSlQ ID MO-MS

The final ρΠ'5-aKLH 120.6-VKISPAgG2-HC-Ll0-ShRi I-35 RIH,QD»K. K30Ej construct encoded an igG3-HC--i.Ul-SbK[ 035, RIK, QI6K.. K30F) fusion polypeptide iSE Q ID NO:309).

[006511 MediodMlsOktinc Mermwdcnt \h UC-andMpnosMent BAatcnl.and InydemMLUA^ Initial purification of the condition, d ηκηί» was cone H affinity last pro\ m unde ehnimuuvraphy if Pi (.) capture of the Ft region using Protein A Sepharose (GE Healthcare* follow ed by a eoIunut mash with DoilvccoA PBS without divalent cations Ho vlt.ro geo) and step elution with 100 mM acetic acid, pH 3.5 at u How rate of 2.5 ero/rom. Protein containing tractions were pooled, and Re pH was adjusted to 5.0 using 10 N NaO; \ and further diluted with 5 volumes of water. The material was filtered through a 0.15 inn s Aiuiuw acetate filter (i emingt and further purified by i.awm exchange EPIC (SP Sepharose High Performance; GB Healthcare). Samples were loaded onto a cohtrnh equilibrated with .100% buffer A· <50 prMacetic acid, pH S.-0) and eluted with a gradient of 0to80% buffer B tSOmlVf acetic acid. 1 M NaCI, pH 5.0} over 3(1 column volumes at a flowrate of 1.5 cm/min. Peaks containing target species were pooled and firm in luted into f 0 mV! sodium acetate, 0% sucrose, pi I 5.0 Exemplary purifications of monovalent, bn stem and tri valent ίιηοι^0^οΜΙ»«ίόχίί·η $dptid£--analog fusion proteins arc shoo a in Figure 24-26A-B, 2?>·29Λ~Β, 30k?2A-B, and 33-'e Ιό on re lues „Si>SM\ul mb,«> s migauw2·1, 53 h\v ddsUletw.suai© thai the fully assembled antibody can be formed, and the fedneing SDS-PAGE analysis demonstrates that the desired components are present. The mz.c exclusion chromatograms (Figures 35, 23, 3 1 and 33) show that the majoriis of the purified product is in die desired j ion-aggregated state. Finally, the mass -peeirai analysis (Figures 26A-B, 2AA-B, 32.A-B ami 35} demonstrates that the desired fusion product's are present Taken tuuedv? 'haw example *> ,1, nv.wtrate that the akr I I 120 A antibody van accept fusions in a wide variety of configurations me bud mu see*, am i oseeru ,g an e\i - or odd-unm 'wed \ alen-.v oi n leas' «re to e>gh' pl.au >te ol \vuih a cox. pob vouiL rosettes [00052} VH2 ISFrlSAermiaus ShKj 1 rit$l.XVild..Type4^(jl~Fc...mammultun expression A Phi A sequence ceding ior a monomer of the K\ 1 3 ί debitor peptide ShK[ I-351 fused m-W.-mw to the N-icru nui Ϊ c region of human luG 1 was constructed as described below.

[00653] lor construction of YH21 SP-ShKC 1-35)--1. 10-IgG I Fe expression vector, a PCR stratog} was empioxed to generate the VM2I signal peptide Shki 1-35) gene linked to a four glycine and one serine amino aeid Hanked by HmdJil and BamHS restriction sues and s tour glycine and one serine amino aeid linked io IgG i Fe ri^gmobf: Ishked by Baml-H and Not! restriction silos was generated i«;· a POE reaction: using the Fe-LIO-OSK lm pcDNA3 It nCMVi ns a template fdescribed io Example 41 and lugiire42A-B ofSuHivasi et ah, WO 20t)8/t>83432A2binemparafe4? by reference). f 0005-31 To generate VTi.2i SF-Shh'i 1-35)-Οα$χ two oligos with the sequence as depicted below were used in a Pt R reaction with PfuTiubo HotSurt DNw fob,mere'-e (btratagenm at 45 i .msec, 55 C- T)sec, 75 C 4sv\ee for 35 cycles, llsndtli wwgvtf) end Baud H ipgatee) rust net· tin wbw are under! med

Forward primerr TGCAGAAGCiTCJ AG ACT ACΓΛIGGAAIGGAGC 5OGGTC T! IClCi it'ΓΓ Cn't:nX'Af.iTA^CGArrGGTG'rCC At τα CGC UX.'TGCATi. GAiAACCATCC ΓΤ'ΛΛΟΛΟΓΓΟΓ IGCACXXTCCTTtCAGT·· iSJbO 1L> and

Reverse primer: i"Ti TTa.IATCCTO' TrCTCCGCAGGTGCCGiPAGGTCTTGCGGCAGA AGCU/AGGCGG'i Ai.TFt A fGC IGTGC I HK. AC. 1 GGAACjGC GO VGCAGCG GrrrriXiGGGA7GGTGTCGAT-<(SF.Q ID NO:5f.>. (006351 The resulting FOR products wore resolved as the 203bp bands on a two percent agarose gel, The a02bp Pi R pre-due: yes punned using. FCR Pun Beanos'

Kit (Qiagen), Gen digested with Hind Hi and Bam.Hi (Roche) resnicuon enzymes, and agarose gel was purified by Gel Bx traction K.h (Qiagon) jOOufO] To generate G.->S~lgG 1 Be. two ohe.os with the sequence as depicted below svero used in a PGR reaction w;ds ΡΑΠ urbo UmStart DNA polymerase fStraiagette) at v5 C'-cOsee. 55 C-diKec, '-5 € Grass's (or 30 cycles; Bum HI s ggaicct and God •geggeege) restriction sues are underlined.

Forward primer; GTAGGATCGUC»AGGAGG AGO Λ AGCGACA AA AGTG >\ C. AC / iSPQ 1 D NO:S7); and Reverse primer: xxiAGCGGaxxTrACTArrrAc: ccgg ag acaggcja/· csbq id no. 58*.

[006571 The sestdting PCR products were resolved as the 72Gbp hands on a one percent agarose gd The 721-bp PCR product was perilled using PCR Punfication Kit (Quigcnk then digested wuh Barn HI and Not! (Roche) restriction enzymes, and agarose gel was purified bs Gel Ox a action Kit tQtugens. (0065K i The peDG A3,1 (a- jCMVi-Fe-L 10-OSK.I vector was digested with BantH! and Not I restriction coevrries and the (urge fragment was purmed by Gel Extraction Kit, the gel purified 4GS-igGl re trugmem was ligated to the pun Ik’d large fragment and transformed into One Shot' 'Pop 10 t hivumgen} to create a pOMVi-Fc-L10-!gG 1 Pc vector. Subsequently, $CMVi~l:c-L!0-IgG I be vector was digested with Hindlil and BiimH! restriction chymes end the large inge.rncnt was purified by (id fbitrachon Kit The yd nun find VH21 SP-ShKi I-35t-4US trayment was Is gated to the purified largo fragment arid transformed into One Shot topiO (insitrngen) resulting in a pi Vt Vf-YR.l:1 SP--ShK(i -35)--1.10-bgG I Fc construct. DNAs trout transformed bacterial colonics were isolated and digested with Barn H I and Not! real sec bon enzyme,1-» and resolved on a one percent agarose get. DNA:» resulting in an expected pattern were .submitted for sequencing A(though. analysis of sev eral sequences of clones yielded a iOO'V percent match with the chose sequences., only one clone from ouch gene was selected for large sealed plasmid peri flection. The DMA front VI \21 SP-ShKg 1* Cm 10-lgGI Fc m pCMVi vector was reset] act teed to confirm the Fe and linker regions and the sequence w as I 00% identical to the above secuencc. Fragment VI12 i SP-ShK.fl -35)-1., 10-hi.G 1 Fc contained the cod me. sequence

Al'GGA,A1'GG.NGn'GGGTrrn'n'ni'(;TTC(.l'i:iTCA<iTA4<X}ACTGGTG‘I f.X.'Ai IX X.X.'Gi AGifTGX'AlTTiAf Ά< 'CATC CCCAAGAGCCGi TGCAO.XX X.T TfXAGTGCAAGOACAGCATGAAGTACXGCCTGAOt TTCTGCCGCAAGAC C'TGi.‘GG(.'A<‘i.‘TGC'GGAGG.:\GGAGGAT(. CGG AGGAGG AGG A AGCGACA A aac-tc-acacatgcccaccgtgcccagcacctgaactcciggggggaccg t < act ct ί (xi cttccc ax; a a a ac c: caagg acacxttcatg atctcccg

GAf CCC.1 G AGG ΚΆΓΛ ί GCG ί GO ί GG1GGACG t GAGGC ACGA AGAC (TT GAGGTCAAA.G ΓΓ('Λ A< ί GG ί Af.'G IGGACGUCG FGG.hGGTGi. ΛΊΆ VfGC (. A AGAf. AAAGiXTiCGGGAGGAi sC AG I A( 'AACAGCAGG .1 AC CO t (* 1C.IG Ft AG (G T t' CTC A C CCnc ti TGCAGC'AGGaCTGGCTG AATGGC AAGGAGT A( Ά AG ΪGCAAGGTCTCOAAC A A.AGi VC Γί XX ACT ('C'GG.A fe GAGAA.AA( (Ά1 Cl C CAAAGC'OAAAGGGt:AGf.'CCX'GAGAACCACAGGTGTACACCsCTGCCCXNCA 'T<XXXj<IGA'rGAGrrGAfX'AA(fAAi:X'AGciTCAG<.X'TC5ACCTG(’{.' i'GGTi'A AAGGOTTCf A 1 CCTAGCGACATCGCCG iGGAGTGGGAGAGCAATGGGCA GCCGG AC A AC Λ ACl'ACA AG AC C AC GCCT C C C GTGCTUGA CTCCGACGGC TC CVIXΎI (X' 1C 1 '.AC AGG A AGC fCACCGT GGAC.A AG.A GCA.GG! GGCAGC Λ GGGGA'hCGTC'nciiC ATGCT'C CGTG ATGC'ATGAGGCrCTGC AC A ACC ACT ΑΓΛ< GCAGAAGAGaTrCTCCC ) CTVCrCTGCiGTAAATAGTA#/:($EQ ID NO: 59}, •encoding v'H21 SF-ShRO-.FSM. KMgG t Pe amino acid sequence MIGA'S W V r LFFLS YTTG V HSRSOID' OPR SRC' ΓΑ FQCKHS MR V R US F ORK.' fCG TCGGGGSGO(jaSDKTHTCPK'PAPEU.GGPSVFlFPPK.PK.DTLM!SR'TPf.VTC VVVDVSiC.DPFV&amp;F"N\YYVDGYΡΛΊ IN AGTRPRFFQYNSTYRVVSV}TVS 1 IQ DWLNOK EY K CRVSN R Λ 1.. PA PlEKTf SK AK GQ PR EPQVYT .1. PPSRDF LTK.NQ V SI TCLVKGFYPSDIAVΓΛΥΈSNGQPFNNY KTTPPVI.OSrX.sS.FFLY SKI T VDRS RWQQGNVFSOSVMHbALHNMY TQKS1 SLSFGK, (SIS) 10 NO:60h (09650] SG?ASAiPAI.SSPASPPPSiAl.NARPR10PiP..ShRl.l.r:SS^.G.L0NR;;QijiFHS.f;;;Q N:|onnmuShELG.LL t '«mg a construct encoding Ndemtinus ShRji-35]Wild Type-L\OHgG 1 -Fc, site directed rmiiHgcucM>s vtas penbrrned uMtig the foilosvsng ougos to produce a Q I 6G mutation :n Use ShK region. 5:~G( T G< A VVQ OCT TCA AGT GCA AGO ΑΓΑ GOG'··'. t'SF.O ID NO:9). and 5got gtg err gca err gaa ggcgotgca gc cf tSFQ idnogop

The Su'atngenc Q ink Chance Multi Site Directed Mutagenesis Kit wan used according to the numuiaetnrer's instructions. Fhe Seal construct tor nCMvi-N-scrnnnuv~ShR} IGCQ16K |·9.. 10~lgC MY encoded the ibl lowing Signal peptide (V Η-1 SP'f ShK[ I -35. Q1CR]-L10-1 gG I -Fc fusion polypeptide'. ΜE\VS >Y VFLFFΪ.S YTTGVH SRSC IDT iFKSRCTAFK.CK.HS.MK.YR.I..SF( *RJtf«. TCGGGGSGGGGSDKTinCPK-PAFFU.GGPSYS'LFPPKPKDTi.MSSRTPFMC Y V VDYSH EDPRY K.FNW Y VDG V ΕΥΗΝΛ JU R PREEQY NSTY R V V'S VI..TYL HQ DWI .NGKFVK CRVSN R A1 PA PI F.K TiSK AK GQPR FPQY VTt. P P S R OF. I. TKKQV $ LTCi.. V K GFY FSDIA V PAVF.SNGQPF NN YK.TTPPV 1. DS DGSF R.. YSK LI' YD.K S !UVQQGN\TSCSVMHICAmNf!YTQkSLSI.SPCiKG (SF.Q ID NO:M I. (00660] Tu generate the N-temhnus ShKί 1 -35, Q: r?K]-aKLH HC construct a PC R product containing the Signal prpiGc-ShRj I G5G 16Kj~L i 0 linker's as produced ;gsmg the following oligos: 5--CAT' TCT AGA CCA CCA TOGA AT GG-.C (SBQ TO RO:63g ;Γ- GAG CFG C'AC OR) GC'F ICC* FC'O FC'O Η C’G'G -3' (SfcO lONGXG). and template pC.MVl-N-iermirraa-ShK(R.>5, Q1 OK )-L 10-lgGKFe, resulted in a fragment centalntny the ceding sequence

ATGGAATGGAGCrGGGTCTTrcrcncrTCXrrGTC'AGTAACGACTGGTCT OOACTCOCCK'AGOTGCATeGACACCAri'CCCAAGACXXXKrtXXAOCGCCT TCAAGTGGAAGCACRGCATGAAGTACOGCiCTGAGCTiCTGCiCGCAAGACG {IX XXIGCACCTGCGG AGGAGG AGO ATOCGGAGi J AGGAGG AAGC7/ (Sl'.Q fDNO.ed), encoding fhc ΥΠ21 SP-ShK(l~ >5, QiOKO-l 10 ammo and \cqecuce M E W S \\ Y F L1' S' 1.. S V FTG V RS R SC ί D 3 IP KS RCTAF KC&amp; Pi 3 ΥΪ K > RLBPCRK1 C G TCCXGGGSGfiiGCaS' ( SiiQ 10 NO:65). | O06M | I o generate the aKJLH HO fragment a OCR product was created using edges: ft-GGA GGA GGA AGO GAG GIG CAG CFG GTG € AG-3" fSEQ ID NG:00); S'- 0Λ1 Gt G GCC GC(' OAT TTA CCC -3 ISf.Q 10 NO:67); and template pTTe-ekLH 120,6-1 K., ivsddng tn a 0ΝΑ l^gmm.amisdamg.the coding sequence ('AGO Hd \u<' IXX i Μ X \G J Ο J OCX>G( Ί G AGG1 ί. ίΛ Ai» \AC a s' i ui XXXX 1 OAGTGAAGG1X TOC TGOA \GGCT ΓΟΤ GG ATAC AC'CT TO AC. O.XA.TaOOAC \\U AC 1GGGKX GACAuCt CrCKXaAt'AAGGCX 1 K. \eH>uAi^(d\ i GGAtt \ XCCC1 \ XCAGRXdOGCACAAAC ΓΛ FGC AC AOUtd T H AGnG OAGCXG X \( OY!G 'X'CAGGGACAt 'GTCCATCAGCAf Άί ΧΌΊ At ΆΊ GGAG CTGAGC AGGCTGAG A TCTG AOG AC ACGGCCGTGT ATT ACTGTGCGAGAG AHXFFGCiGAGt ΊΛΟΓΗ UiGl HXiAt (XX'FGGGGCO \GGG X At t t'lVXiit' At tX-R ICC Π \GCC1\X ΑΓΟΑ \GGGtXTATtXXiTi 1 It t Kt KdX t XX'

(. ί Gt"Ft'ts \< 10At X'AC Cl'CX'G AGAGCAC AGC'GGt' CCTGGGt' TGCC 11 «GTC AAGGAOTArrrCCC'CGAACXXXdGACuGΓΟΗί 1GGAX0K Cut G( R 1 GACCAGCGGt;GTGO \( ROOT I'CCXAXGC'TGTCCTAOAo FOn't; AGO Ai'TOT AC FCCCTO\GCAG('tVi GuTGACCOTGCCCTCCAGCAAC FTOGGCAOCCAG ACCTACACCTGCAACGTaGATCACAAGCCCAGCAACACCAAGGTGGACA AGACAG Π.ΟΑί X'GCAAA K'iH GTGTCGAGTGCC("AC('GTCC CCAGCAC'C ACCTG’I GGCAGG ACC'GT <' ACT CTTGCTCTTCCCCCCA A A ACC. CAAGGAC A CCCTCA I'GATC 1 C CCGGAC CCCTGAGGTCACGTGCG rGGTGGTGGAC GTG AGCCACYhAAGACCCCGAGGTCC AGTTCAAC FOCI ACGTGG ACGi iCGTGG AGGTGCΑΤΑ ΛΊ GC Γ AAG ACA AAGCCACGGG AGGAGG A( 1TTCAACAGC AC GlirC^TGTGarGAGCGTCCTGAiGGFl'GTGCACCAGGACTGGCTGAACG GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGC'CTCCCAGCl'CCCAT CXJAGAAAACCATCTGCAAAACCAAAGGGCAGCC/CCGAGAACCACAGGl'G TACACCCfGCGGCCATCCCGGGAdGAGATGACCAAGAACCAGCiTCAGCC TGACCi CCCTGG'K'AAAGGCT 1 Cl ACCOCAGOGACA i'CGC'CGTGGAG FOG G AG AGC A ATGGGC AGCCGG AG A AC A ACT AC A AG ACC AC ACXTCCC ATGC 'rGGACTCGGACGGmCCllT-rrCGIGTACAGCAAGCrCACXXilGGACAAG Acc AGGTGGC ^GCAGGGGAACGTCTTCTCAlGCTCCG'niATGCATGAGG V I CRK ACA \( { AC Ί Af 'ACGCAGA AGA< RTI CCCC C' Vi 1 ΐί' t'CCGi iG V\ \A TGAC(SEQIDNO:6C}. encoding arm no acid .sequence Q\CcLVQS<^AEVKKiK.iASV'KV'SCKASGyTFrGVHMHWVRQAPGQGCEWM GW INPkSGGTNY AQRFQGRYVMTRDTSISΓΛVMΠ SULRSDOTAVW CARD RGS Y Y WCDPWGQGTLVXVSSASTK GPS V FP LA FC 'SR S' VSES’I AALGCCAICDY FPEPYT VS WNSG A t.TSG VHTFPA VLQSSGL VS LSSV VTVPSSN FGTQTYTCN VDllKPSN'rkVRK1Ti£RK<rVI<CPP{'PAPPVAGPSVm:PPKPKiri LMISR'iPl· \?TC VAAWSaBDiTA'QPMWY VDG V EVHN ARTKPREBQFNSTPR V VS VI.TV YTIQDWLNGKFY&amp;CKVSNKGt PAP!IX1! SKFKXFPQ ΥΎΤi.PPSRV.fiMl'K NQVSLTCLVKGFY'PSD1AVEWESNG0PHNN\'KTTPPMLDSDGSFFLYSKI.TV DkSRWQQGN VFSi'S\ VI! !! A1 HAIG I QKSi SI SPGk' *SVQ ID NO m [0O6E21 Che two PCR products were run out on a gc!the appropriate band was punched for an agarose ping The agarose plugs were placed in a -so Je new PC'R reaction, sod the fragment.' were rev- π together using outer most primers C-VQ ID NO (Gt and < M 0 ID AO o?) The Pt 'R fragment was cm using Xkd uud Not! end cleaned with Q;agen PCR ( ieatiup kn. At the susae time. p'H'S sector was also but by Xbal. and Not! and gel purified. TI^-puti-iodLiaserltwdigt!^.#dho-large vector fragment and tranGorme-j into OncKhot Ί op 10 baomnu. UN As from ivamsfonned bacterial colonics were isolated and snbjoWed U> Need and N\n i restriction enzyme digestions and revised on a one percent agwosc gel ON As resulting in an expected pattern were suG'uUen to- scduc'tctng Ahboinb·, unaiy-fs of several sequences of clones yielded a 100“a peuvtu match « uh the -foose sequence, only ortc done was selected for 1 tree sealed plasmid puntlcadon Ida. final construct pTT5“N4crminus ShKj 1 -A5? DbKH ίθ-akl HOlki K' encoded a VH21 SP ShK[ 1-35.. Q16KJ-I..10-afv.t RLTO 6-RC fusion polyponode: MHVVSWVi·UT LSVfTGYTiSR8C1D HPRSRCfAFK.CKHSMKVRLSK' 1<K.I CO TCGGGGSGGGGSQYQI.VQSG A ffV k K.PGA S V KVSC K ASGYTf TOY 1!M1AYY ROAPGOGffc'YVMG W1NPNSGCTΓΜ\ AQKFQGRVTMTRm SIS 1ΛYMi:!.SR LR SDDTΛΛΎ YC AR DRGSYYWFDPWGQGTLVIVSSASTKGPSVFP1. APCSRSTS f.STAAl .GC1. Vk DY ΓΡ1-PYTVSWN SGA! TSG v i ΠΊΤΑ V LQSSC i 1.. Y S I.SSV VTV PsSNFGTQTYTCNYDHKPSMTKVDKTYP.RK.CCVFCPPCPAPPVAGPSVT!..FP PkPklJfLMISR rPi:V1X'\'V Y'DYSl iLDPFVQFNWYVDGVfiVHNAKTKiPRF.FQ FNsTFRVVsVlTVYROD^lNGKP.YRCKYSNKGLPAPiEKTiSKTKGOPRFPQ VYTLPPSREt.MTK.NQVSLTa.VKGm^DiAYFWFSNGQPFNNN'KTITPML DSDGSPf L YSKL f VDKSRAV OQGNVTSCSY.MHEALHNHY IQKStSt SPGK. ’ iSHQtDNO-70). 1:00663) Lastly, the Ndcrmrous-ShKf 1 -33. Q(f»K.]-l 10-&amp;KLH 120.6 Light Chain 11(' > was generated in the sense manner as above, A PGR product containing the signal pepside-SiiKj 1 -35. 01 Ok|~1.10 was created using oligos: 5‘-CAT TCT AGA CCA CCA TGG ΛΑΤ GG-.F (Sf.Q ID NO:6'}; end S'- CAT GIG GAT GTC GCT TCP' ICC lift.' TC G GG -3' (SEQ ID NO:71 r and template pCMVi-N-k'rmnius-ShK{ M5QI6Kj-l HMgGl-l'e. lesnUoig w a DNA fragment containing the coding sequence

ATGG AATGG AGCTGGG'TC i'TTCTCTTCTl (. GTGTCAGTA * CGACTGGTG I rT'ACTCTT'GCAGCTGCA FCG.ACACCA 1 OCCCAAGAGCCGCTGCACCGCCf TC A AG'f GCA AGC AC oGi A TO A AGTAC t.'GCCTG AGCTTCTGCf. GC A AG At C s Cf(. (.IGC.'Ai ί. 1 GCGGAGGACR 1AGGA1 ί CGG YOGAGG AGCfA AGC (SP.Q

ID NOFGK •encoding the arnino acid...sequence for a Mgna). peptide {ΥΠ2! SPLSbKi 1-35. Qli'VKMJf} linker: MEWSNWFLFR-SYTTOVHSRSC I071PKSRCTAFKX. KMSMK YRI. SFCRKJ CO TCGGGGSGGGGS ·' (SLQ ID NO:GG. I Cine temp late and oligos: 5YGiGA GGA GGA AGCGAC AXG:GAG AXG AC€ GAG lYAY (SLQ IP NO;7.:>; and 5'~ CAT (TC GAG CGG CCG CTC A AC -3' {SEQ ID NO:73).

The resulting cloned PCK fragment contained the coding .sequence Λ TOGA ATCKiAGC TGGGTC.TTTC TC TTCTKXiTGTCAGTAACGAn'GGTGT c t: ac" rcct gcagctgc a t cg acaccatcc c caagagc cgctgcaccgcct r c a agtgc a agcac agc at o aagtaccgcctga gcttctgccgcaagac CTGCGGC.ACCT GCGG AGGAQG AGO A f COGGAGG AGGAGG ΑΛ GCGA CA I C'CAGATGACCCAGTerCGAirCTCa:TGT(.TGCATt.T{:iTAGG4GACAGAG TCAAC'CATCACTTGCT TXKiCAAGTCAGGGCATrAGAAATGATi'TAGGCTGG T AIT A GC AG A A ACC' A GGG A A AGCCCf 1A A A CGCCTGA TCT AT GCTGC AT • (Γ AGTFFGCAA AGTGGGGTCCCATCAAGGTTi WGCGGCAGlGGATC TGG •GACAGAATTCAC TC TOACA ATCAGCaGCCTGCAGC CTG AAGaTTTTGCA A C'n'ArrAC'TGTCTACAGCATAATAGITACCCCiCrCAnTrCGGii'CiGAfJGG ACCAAGG- CGG AC i A T C A AACGA ΑΓ I'GTGGC I'GCAC C A' I'C" f'GTC T i CA TOT {. CCGC'C AT Cl' GATGAGC AGTF G ΑΛ AT CTGGA AC TOCCTCTGTTGTGTGCC TGC'TGAA CA AC ] re iATCTC'AGAG AGGCCAAAG CACAO CGGA AGO CGG/\ TAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGrAGGAC AGCAAGGAC'AGC'ACT TA<7\GC75TCY\GCAGCACCCTGACGC PC', Λ (3(' ΑΛ \<, CAGACTACGAGAAACACAAAGTCTACGCXTGCGAAGTCACCCATCAGGG' {'<;']TJAGCTC(i{X'CG1X'A<'AAAGAGi.'n'C'AAC'A(:iGGGAGAGTGTrGA,V (SEA) ID NQC4} was generated, encoding she amino acid sequence For N-terminus Vile i SF-ShRJ l~?5, CGdR.J-t. M}-aKCH i 30.6 Light Chain {CO with an N -tenn mat signal peptide:

M FAVSW VTL.F Π. svn G VHSRSCI DTfPKSRCTA h ΚΓΚΪ ISM KY RLSFCRKTCG

'TCGGGGSGGGGSQ Ϊ QM'VQSPSSLSA S VC DR V ΠΤ< R.ASQG IRN Dl C \-\ Y QQKf GK.A PK III 1Y A ASS I..QSG VPSRESGSGSG TE FT 1 .TISSLQPEDT AIT YCI. QHNSY PUT GGG'VKYEi K.RT YA APS V R P PPSD EQLKSG' FAS V VC U..NN PV PR CAK V Q WKVDN4JLQSGNSQES\TEQDSKD$TYSLSmTiSKADYGKHKVV*CEVTH QGLSRPVTKSFNRCFC ' (SPQ I DM); 75). |i>0Cf-4 i Both PCR fragments (DNA fragment centsinlug. the coding sequence iSI-Q ID N(}64 ? arid aKJ.Ii 120 6 Light Chain LC fragment coniutnmg tVsc coding >equenec tSEQ IP NO:~4> were run out on a geh and the appropriate sized hand was punched for an agarose plug. The agarose pings were placed m a single nan PCR reaction, and the fragments were ssvn together using enter most primers (SPQ ID NO:ha) and rSPQ fD NOf/31. The resulting PCR. fragment was cut using Xual and Noil arui cleaned wifhQiuuen PCR Cleanup Kit, [00665 j At the same time. ρΤΤ 1 d s eetor Fan Amgen vector containing a CMC promoter. Poly A tad and a Pure my cut resistance gene i was also out by Xbni and Noil find gel purified. The guvs he d insert was is gated to the large vector frag mein and try ns Formed into One Shot Top 10 bacteria, DNA-s from transformed bacterial colonies sucre isolated and subjected k> Xbcl arid Nod restriction emu. rne digestions end resolved on a one percent agarose gel, DN.As resulting in an expected patient were submitted for sequencing. The final construct pTTld-N-tertrsiniH ShKlC 35Q16K)-L 10-aK.S.H120.6-1 X' encoding a Signal Peptidc-ShRl I -35. QI6Kj-l.lO-a K 1,. H i 20.6 - FT Fusion polypeptide sequence u.e.. SEQ ID NO'7S).

[00666) o^ivor,a:0HF;3:A4;:j,,351 ~ [00667) MhMxgMPIg^ DNA. sequences coding for she heavy chase of human ami- 2., 4 ~d i η; ΐ r ο n h aa y 1 (DNP) anybody fused in frame to a monomer ut the Κλ 66 inhibitor toxin peptide analog ShKj 1-35. Q1R< j f SL'Q ID NODbt were constructed using standard cloning technology. Plasid pTTS - aDNF 5Λ4 AYtOiF) igG2 -Siikj. 1 ->5,Q!6Kj \sas generated by 3 nay ligation of the pi le vector with a portion of an amd-DNP 3.A4 (WiOIH iuGd Heavy ( haiti ij.dXT24.uDNP 5A4 ΠΓ {WIOIH [saving the annuo acid sequence; MOMR'V PAQ tLG 1..LU. W LHGARCO V Qί_ V LSGGG\ VQPGRSLRl.SCAaSGL T P SSYGMfOVVR QA PG KG L EVY V A V i WYDGSNKY Y A DS VKGRFTi SRDNS KMT L V LOM NSU< A EOT A V V YC AU Y N EM Y {idl) V WGQC > Γ Γν Γ VSSA.S! KGFS Y r F LAPC$RSTSE$TAALGCLVKDYFPEPYTVSWN$GALTSGVR'FFPAYI..Q$SGl. YSLSSVYTYPSSNFGTQTYTCNVDHKPSMTKVDKTVERKCCYfXTPCPAPPV AGPSYFLFPPKPKDTLMl$RTPEVTCYVYDVSHF.DPF\'Q!:NWYVDCi YE.VHN AKTKPR KEQFNSTFR V VS V1.' ΓΥΥ11QP W1. N’GK ΓΥ KCKVSN KG 1 .P API F.KTiSK •TKO^PREPQVYTlPPSREtWrKNQVSLTCLVKGR'PSDIAVfcWfcSNGQPfcNN YKTTPPMLDSDGSPR. YSKLTV PKSR WQQGNVFSCSYMHEAL HNHYTt^KSL SLSPG·' tSEQ ID NOD'7k and a portion front lg.G2Fc-Sh.k| G35. Q16K], The pTT5 vector was cist with Sail /Not) releasing the multiple cloning sue. The vector was then treated with Calf Intestine Phosphatase ICIP) to reduce background. The first insert came from pD(324:aDNF 3,·\4 110 < WIG IF) by cutting with Sal I''Stub resulting in the a DNA fragment containing the coding sequence

ATGGAGATGAGGGTGC ( CG< TCAGi.TCXfTGGGGC; ΓΓΓΊΌί. TGCTGTGGC Ύ GAGAGGTGCGCGC i'GT C AGGTGC AQCTGGTGGaG'I ( LGGGGGAGGCG'i'G t.rrccAG(x: i ggg aggtcc c TGAGA(.T(.T(;r.TGTG<;AGCGT<: tgcattcac nTCAGTAGCTATGGCA rGCACl'CC'iG fCCYiCCAGGnX'CAGGrAAGGGG C'TG{.iAGTGGGTGGC.AGTTATA1Y.K'i1'ATG-M'GGAAGTAATAAATACn'ATG ('AG AC." i'CCGTG A AGG< ICCGATTC Λί ,ΤΛ’ ΓΟΓΓ AG AG AC ,ΥΛΤΤί C AAGA A t'ACGt Hi ΓΛ KiGi ΑΛΛΚ?ΑΛί AtsCC > GAGAGi Cti.AtiGAi 'At (>t K ΗΠ(ΐ r ah actgtgcgaggtataacttcaac'f ACGGTAiOGACGTCiGGGGCC a AGGGACC ACGGI €Αί CG K K LAG 1 (;CC t CtVSCCA AGGGL OCA l t'GG IV i TCCCCCTGGC GCC CTGOTCCAGGAC.KLACC'TCCGAGAGCAC AGCGGCCCTG GGrrGi.'CTCiGT{.'AAtiGACTAnT{.'CC'C'(iAACCGGTGA<:GG'rGT(.‘{.i'rGGA ACTCAGGCGCTCTGA.CC-AGCGGCCVlGCACACCTrCCCAGCTGTCOIACAG T< X ICAGGACTCTACTtX CT< AGC AGGGTGGTGAC X 'GTG( 'CCTCGAGi "AA CTTCGGC'ArCC'AGArCTACACCTGCAACG’TAGATCAC AAGCCC AGCAAC ΑίΓΑΛΟΟΙΙΧ^ΛΓΛΑΟΑί' AGTrGAGCGCAAATGTTGTGITGAGTGOCi' \C Ct i'VGCC G AGG AGG At CI GTGGCAGG ACC G IT AG VC Π VC ΓΓ PTCT'Ct CCA AAACXTAAGGACAC‘<;'<TCArGA‘irrt;t'C'Gt.iAt:rt'rrGAGt.iTCA(.'GTGt:'G'F GGTGG ΓυΟΛΓΟ \ GAG<'CACGAAGACCCOGAGG 11C ACTϊ(‘A ACTGG LAG •GTtXiACGGC'GTOGAGCiTGCATAATGCCAAGACAAAGCCACOGGAGGAG •CAGTTCAAC'AGt ACGTTCtTnTnXAVCCAGCGITCK'ACCGTTGTGCACCA GO At l GGt 1GA AC GGC AAGGAG ΐ AC A A(j i GC A AGG K It C A AC A A AGGC (SF.Q ID GO:78k encoding ammo acid .sequence GO MR-V PAO t. LG It, U, W L H GA RCQVQLV LSGGG \ V OPGRSLRLSC AASOFT l: SSYGMHWVR QA PGKG l EWY A VI \YY DGSNKYYA DSVKGRFHSRDNS KMT LVLOMNSLRAE'DTAYVYCAR^'NF\YGMDYW{iQ{>rrYrVSSASTKGPS\'FP LA PC SR STS IrSTA AIXX LVKDY FPF PVTY'SWNSG. I LTSGY HTFPA V L QSSG L YS i AS V Vi VPSS NFGTQ' rVTCNV Dl 1K PSN7 RV DKTVF RK CCV F.CPW 'ΡΛ PPV AGPSVFLFPPKPKDTLMISRTPEVTCYWDVSHEDPEVQFNWYVDCjVEVHN AKTKPR LRQFNSTFR WSV1 TV VHQDWLHGKLYKCKVSNKG / {SF.Q ID HQ; 79),.

Tde second insert was digested wt using SddASett aod coffiaiiiM the coding sequence

CTCCCAGC CCOCATCGAG AAAAtCATC7CC A.A AACCAA AGGGC.AGC C GC GAG A ACC AC AGG TGI AC ACCCTGCCCCC A' PCCt. GGG At iG AG ATGACCAA G A ACCAGGTCAC jCCrGACCTGCCTGGTCAA AGGOTCTACCC C AGCGAC AITGi XIGTGGAGTGGGAGAGi' AATGtiGt '.AGC ‘CG< iAGAACAAtTACAAG ACC aCACCTCCCATGCTGGACTCCGACGGCTCCTTCTTtC TCTACAGCAA GL K ACC Gi GGA( AAGAGt AGG i'Gi jCAGCACjGGCAACU Κ 1 1( IC.AfGC lX?CG'rOAl'GCAl'GA<KKl'CTC(‘ACAA<:(: AC-'rAC’AC'GCACiAAGAGt'CrrrC CaORnTCGGGTAAAGGAGGAGGAGCLVTrCGlIAGGAGGAGGAAGCCG C AGCF GC AT CO AC AOCATCCCCA AGAGCOGCTGC ACC GCXT TCA AGTGCA AGCArAGCATGAA<n>,rCGCf.TGAG(.TlT<TGCCG{.'AAGACCTGCGGCAC CTGCTAATGA.·' (SEQ ID NiO HOk encoding the following truncated IgCG Fe~L 10~ShK( i -35, Q16K ί amino acid sequence. LPA PI EKTIS KTKGQPREPQ V Y7LPPSR F.FMTK.NQVSLTC L VKOF YPSDJ A Y F WESNGQPENNYKTTPPMLDSDGSF FLYSKITYDKSKWQOGNVFSCSX ΜHfc A 1.. HN HYTQKS LSLSPG RXXXXiSGGGGSRSCIlTil PKSRC3 A P RGKJ1SY! K Y RL SFCRK.7C.GTC7/ {SEQ ID NO* I).

[00668] The vector and insert fragments were gel pun (led and cleaned up with Outgo n Gel Purification Kin The purified Inserts were legated to ihe large vector fragment and transformed into OncSitot Top 10 bacteria. DMA-* from transformed bacterial colonies were isolated and subjected to Sale Noil restrict ton eusryvne digestion and resolved on a one percent agatoxe gel DNA> resulting in an expected pattern were submitted for sequencing. A clone yielding &amp; 100511 percent match with the above sequence was selected for large scale plasmid pursfioatient. Tito final pTT5 - aDNP 3A-1 (WOOIP) IgGO -$kk[ 1 GY Q16K] construct estcoded a uDNP 3Ad {W i 0 i F) lgG2 11( -1.. 10-8hk[ 1 -35, Q16KJ having the following a anno add sequence: MDMRVPAQLLGLLLLWLaGARCQYQLVESiGGGV\-QP(iRSLRlSCAAS(iR· FSSVGMHWVRQAPGKGLEWVVWiWYDGSNKY^'ADSYKGPFnSRDMSKNT IA' LQM NS LK AEDTA V 3' V( ’ARY N FNYGM Ο V VvGQGl 1 YTYSSAS’ f KG PS V FP 1. APCSRSTSESTAALGCLYKD Yf FFPVTYSWNSGAI.TSGYGTFPAYLQSSG 1. YS1.8SV VTY PSSNr'GTQTATCN V D11 KPSNTKY DKTYF R KGGY HCPIX "PAPPY AGPSVTLFPPRTR.DTLMISRTPEYTCVVVDVSHEDPEVQFNWYYDGVFYHN A KTK PR f.FGi' NST PRY VS V1.7 V"VI f QD W l .NGK.F YK C'RVSN RGI.PAP 1 PR HSR TKGQPR EPQY YTI.PPSR E EMTKNQVS LI Cl VK.GF V PSD LA V E W ESNCQPENK y K.1" ΓΡΡMl DSDGSFFL YSK L TV DK.SR WQQGNVFSt SV Μ ΗEA LΗ NH V'TQKSl. S1 S PGGGGGSGGGG SRSCIDTIPKSRCT AIKOKHSMKY R.LSPC RKTC'GTC / (SEQ ID NO;82}.: [00660] Mturonajum expreys^^ The

XenoMouserK· hybndonua exposing af>NP monoclonal antibody 3A4 was used as a .source to iso hue total RNA. One step RT-PCR with multiples gene-speebk primers was done to obtain a variable region product. Tins product was marepldicd with a forward printer to add a 5' BsaHII restriction *ire 5' · TIT TIT TIG CGC GCT OTG AC A TCO AG A TGA CC C AGT C 5' tSFQ ID NO:x.*> mid a reverse primer to add u .V BsiW· i restrict bit situ 3' · ΛΛΛ AAA COT ACG ΪΤΓ GAT ATC CAC TTT GOT CC ,r {$PQ ID NO-.MI The resulting PCR product tv 8¾ cleaned by iduigen PGR clean-up, digested with BssHj! and BsfVU restriction enzymes, cleaned by Qsagen nucleotide removal and ligated into a mammalian expression vector pTT5 containing a 5' YKJ 012 signal peptide and a a human lappa constant region. The amino acid sequence of the resulting arnhDNP 3A 4 .Antibody Light {. ham is the following!'

MDMilVTAQLLGLLLL\YLRGAECD!QMT{,)$PisS'VSAHYGDRY11TCRASQGiS RRl A W VQOKPGK APK.LLI V A ASSLQSGV PSRFSOSOSCIWΓL11SSIQPFDF AT YYC QQ.A NSFPFTFG PGTKY DIK.RTVAA PSV FfFPPSOEQLK SGI' ASY VCLI NNFYPRFAKVOWkVDN ALQSQNSQF SVTEQDSKDSTYSLSS 1TTI.SK.AQYF KHKVYACF.YTH<XU.SSPVTK.$FNRGEC tSEQ ID NO: KOI 1.00670)

Ab IC-Tosin Peptide Analog Fusion^. Initial purification of the conditioned media wav dene by affinity fast protein mmul chromatography (FPLC) capture of the Fe region using Protein A Soph arose iGb Healthcares followed by a column wash with Dulhecco's PBS wit I tout divalent cations t invinogerA and step elution with 100 mM acetic acid. pH 3.5 at a Ottvv rate of 2.5 envrnin, Protein containing fractions were pooled, and the pH was adjusted to 5.0 using 10 N NaOH and farther diluted wish 5 volumes of water. The material was filtered through a 0,.45 Aim cellulose acetate filter (Coming) and further purified by cation exchange FPLC (SP Senhurose High

Perthi ounce; 0 1- Healthcare) .snmpis, -> w e: e ioudee onto a soamm equ hhrateJ w 'th 100% buffer Λ t ίϊΛΐ uvs'tle acel pi 1 5 0s end eheosi w on a gra-Item <" 0 to N0!) i< buffer B ;50mM acetic acid, 1 M NaCI, pH 5.0) over 30 column volumes at a fiowTtte of 1.5 CTftmfo. Peaks e«stt#nihg mhphvaleflt species were ppolediftid formulated into 10 mM sodium acetate, 4% sucrose, pH 5.0. B.semphtry imrifteshons of tnionntoglobulin^toxm peptide knalpg tftdbu pfotefts are #0½ ft Figure 3A-C, Figure 4A-C* Figure 3A-'Cs;FigureOA-fi and Figures 24-35. j 006? 1 ] Method for iso in mm Monovalent Ah IK- and Monovalent, Bivalent and initial purification of the sondmoned tnesna was dons* b\ at Unit \ fast piotem lupud sinotnatogmphy (FPI C) cupftft ef the Fc region using Protein A Sepharpse (CIF Healthcare) followed 1ft a column wash with Dulbccco’s PBS without dfv&amp;ient cations (Invilrogon) and step shut tort with 100 mM acetic acid, pit 3.5 at a flow rate of 2.5 eta'min. Proiem containing factions were pooled, and the pH was adjusted to :5,0Using 10 N Na<3H a, a hwhet dduted w ult 5 solumes ol warm, fhe material was ΠIteted tbrough a 0,45 μτπ cellulose acetate filter sCorning) and Further perilled by station exchange FPI O f SP ^ ft esc 11n- P<! lorrun s,, OF lie 1th,, vs ‘samples were Netted onto a column equilibrated with 1 Olr·4,- buffer A (50 mM acetic acid, pH 5 0) and eluted woh a gruJvi" ΜΌ M ft1 <. builm B rftmM ,veOe as id. 1 M Net. L nil 5,0) mcs 50 column sofontes at a flowrate of 1 „4 cm ram Peaks containing target species were pooled and formulated into 10 m.K1 sodium acete: *, 4 *« sneross pH 5 si Reducing and nun-reducing {-Hodoaeetanuds'> anahstx was done on 4-12%· or 4-20% ft ft·· P \i A I ns >, mre geN i 1 wocer j with 0 5 git,~i _ ή j is' eg o‘ woion, stamen with QnickB! tie (Boston Biological s). Analytical SFC was done using a Biossm \ "t -o 1000 col tmrs (Ph * torn »s i and an su. dtw clime o 50 r Λ1 sod tut' f'i'ovm ,te, 5% aM Naftf '41 Λ over IN mm Fvcwplurs pnreft .ams ot immunoglobulin-toxin peptfte analog fusion proiems are shown in Hew; 2 F264-B, !T->U-B, 30-32A-B, and 33-35.

(00072j ExrnmWS

[00f>'v51 FfearniacokhitHc/Phartnacodyfiamtc Fvaltmiioit οί Memo valent Fe'Te-UO-Sf*Kj2-3$j Heiermlimers and MournGent or Bivalent FezFe-ShK{ 1-35 Q.16&amp;.}(Ig<52) fietmulkm;r§ and fomun.u>gk>buiin Fusion Proteins of the

Investil»» [O0674] Totbodtntcnis of the antigen binding protents of Use present invention, used as mummeglebolin carriers for phunnacologically active polypeptides were demonstrated to provide favorable pharmacokinetic and pharmacodynamic properties. Monovalent or bivalent Fc-L 10-Slt.K12-Γο], monovalent or bivalent Few LI0~ShK[i -35j, monovalent or bivalent FwB 10~ShKf I-e5, QlbK). monovalent or bivalent attti-KLH ΗΓ-ShK-t 1-55, QI6K.) Ah. monovalent or bivalent unri-K.l H AbLoop-(Lys16j$bK fusion proteins- monovalent Fe-ShM i-35 QIUKVK.LH Ab hetorotrimer, and other exemplary embodiments listed in Table ?H. were expressed, isolated and purified by methods described In Example ·* PEGyiated and nn-PFCiylated rosin peptide comparators in Table 711 were prepared symheUeaUy as follows'

(00ό751 Peptide Synthesis. FH-Pmoc, side-chain protected amino acids and H-€ysnVt}-2Cf~lVt resin were purchased from Novahtoeherm B&amp;ehcm, or Sigma Aldrich, The following side-chain protection strategy was employed: Asp{ Of Bit}. AtgiPhfk (ystlTtl GhuOtBuh HisiTrO, LysiN -Boo}- SoriOtBu). TnriOtBu) and T> rfOtBu). ShK j RSOD ilPKSRf'TAFQC K KSMKA R LSFT'RK iC GTC ' SfcQ 10 NO:361). {Lys I A j$hK {RSCIDT1PKSRC! AFKCKH5A-1KVRI.sFCRKI'CGTC; SFQ ID N(};76), or other toxin peptide analog amino acu:i sequences- were synthesized in a stepwise manner on an CS Bio peptide synthesizer by SFP5 using DIG HOBt coupling chemistry at 0.2 mmol equivalent scale using HA ysl TtH-ACH Tn resin i0.2 mrnoL 0.32 mmol- g loading}. For each coupling cycle. I mmol N6· l: ntoe-amino acid was dissolved in 2.5 ml., of 0,4 Μ i -hydi'oxybenrooiazolc (ΗΟΒΠ in N.N-dimeihyUbrmamide (DMFs. To the solution was added B0 ml. of BO M N. N V wsoprop)dc;rrbοdi;;rsIde {PICA in DMF. I he solution was agitated w-tth nitrogen bubbling for 15 min to accomplish pre-ueiH ution and then added to the resin. The mixture was shaken for 2 h. 1 he resin w-es filtered and c ashed three times with DMF, twice wnh dichtoromcthane (DCM), and three times with DMF. Fmoc dcproicctioas were carried out by treatment with 20% pipeline in DMF (r> ml.., 2 x 15 min) The first. 23 residues worse single coupled through repetition of the Fmoc-amioo acid coupling and Fmoc removal steps described above. The remaining residues were double coupled by performing ihe coupling step twice before proceeding w ith Fmoc-removal. }O0o uY; Following synthesis, the asm w as then drained. and washed sequentially w ith IW'M DMK DCM, and th-.-n dried in vacuo. ? he pepode-resm was transferred to a 2 50s η I. plastic round bottom Ibsk. The peptide was ueprotocied and retoasrd iront the resin b\ treatment wdh tnssopsvpvlsiiane Π.5 ml. k ,U>-dio\n· KF-oetune·-dithioi {ΠΟΠΤ, 1.5 ml }. water (1.5 mi k trinuoroucetic ;u hi ΟΤΛ. 20 snl.). and a sin bar, and the rmxture was aimed for s h The mixture was filtered through a 150-mf„ snttered glow funnel into .= 2.0Frof. plastic mood bottom fats!·;. The mixture was filtered through a 150-mil sintered glass funnel tnto a 250-tol. plastic round bottom flask, and the ilhrate was concentrated m xacuo. The crude peptide was preeipiuiiod with the addition of cold dsethx I ether, collected by rentniugadon. m id dried under vacuum.

[006?:] IVnlidc Folding. 'The dry crude linear peptide (about 600 mg), for example [I. ysUfjShK peptide tFFQ ID \0~M or [Lysl6]$hK-Aia talso known as (i.ystP, AlnMj-ShK.: SFQ ID NO:362) peptide, was disseised in 16 ml, acetic acid. 64 nil, water, and 40 ml. acetonitrile. The mixture was stirred rapidly tor 15 min to complete dissolution The peptide solution was added to a 2-L plastic bottle that contained l TOO ref. of water and a large stir bar. To the thus diluted solution w as added 20 nil of concentrated ammonium hydroxide to tai.se the pH of the solution to 4,5, The pH was adjusted with small amounts of acetic acid or NFHOF1 as necessary. The solution was stirred at HO rpm overnight and monitored by LC-MS. Folding was usually judged to be complete in 24 to 4b h, and the solution was quenched by the addition of acetic acid and TFA (pH 2.5 k The aqueous solution was filtered 0).45 pm cel I those mem bran e k [OOe Μ Reversed Phase UP! c Punrtoation Hoveled phase high perflumanee i ,μ, 2 ¢. 'noon ogw'm\ was ee formed ,·η ad a 1i A tea* {( Its, 5 gin 2 4e ^ u 2^ emu>r i prep.; ram e ιί'18, lu pm,2,2 cm x 25 ·,re}column. Chumutographie v paiat <>ns were «is sieved name, hnere gradients ol huiVei B v X { X 0 % uqueo i\ Π X, B - 90% aq. At N eorummig 0 tin% l BA* tvg'cally 5~<!y\-> owe mm at a How rate of 1 mL/min lor analytical analysis and .>65% over 90 min at 20 mL/roin for preparative separations. Analytical and preparative HP1.C fractions were-characterized by ESMS and photodiode array (PDA; .HPI.C, combined and lyophiHrecl p>i>:“0| Mas* Spedex; mem Mas* spectra w ere acquired on a single quadrupide mass spectrometer equipped with an loos pry. atmospheric pressure ionization sruace Santp«es(25 Mb w<. ,e nsjeenu Into a mmmg solvent {10 n!,'nru, 50 50,20 'XCN'Mei ΗI containing 0.05' > Π X ϊ s-ouplcd directly to the sons/atson source % ia a fused si lie. i eapillaty interlace (50 pro i.d }. Sample droplets were ionized at a positive potential of 5 kV and entered the analyzer through an interface plate and subsequently through an orifice (1 00-120 put diameter I at a potential of 60 XA JFnl! scan mass spectra were acquired over the mass range 400-2200 Da with a scan step size of 0 I Da Molecular masses were derived from the observed m/z values,

mm] PBOvHti^..^^ Peptide, e.g„ [LyslbJShK fSEQ ID At t 76) or [Ly.MbjShK-AIn iSEQ ID 610:162), was selectively PEGyiated by reductive alkylation at Its N-terminus, using activated linear or branched PEG; t o" legation w ,* performed at 2 mg ml m 50 mM ΝοΙΓΡΟη pH 4.5 reaction baiter containing 20mM sodium cyanoborohydridq apd a 2 molar excess of 20 kDa rnonomelhoxy-PEG-aldehyde 1NOF, iapim)i: flonfugatioh reacdottk were sdrredlof: approximately 5 hrs at room temperature, andthlelr ptvtgresa was nlOnltored b;> Rf-HPLC. Completed reactions were quenched by 4-fold dilution with 20 mM ,NaOAc, pH * and ,Ή, d (o ) t Ha PI G-peptide*· v*ev 'Vn put tiled ehrunatogtapiucallv at Ai^O; using ><P Sepnaowc HP to. turns tCh HeMme.av. Pise.noway. XU eluted w ith linear OH M NaCl gradients in 20mM KaOAe, pH 4 0, Bated peak fractions we v anah -t. N Mrs-P XC4 ane RP-HP1 0 are poole g dctefmnwdb\ purity >97%. Principle eoetamiiiants observed were di-PEGy bated toxin peptide analog, 'sdected poohs were concentrated to 2-5 mg/m! b\ centrifugal fdisanorr agumsl 1 kDa \t\\ CO mc'r* end m.krce mto *t> m> 1 Vd>Ae pit 4 λ in 5(,<> ^ubiro. Dialyzed pools were then sterile filtered through 0.2 micron idiots end purity determined to be >97% by S0$-PAGE (data not shown). Reverse-phase IIFLC was performed<m-&amp;-A§p3®$£ .11(10 model HPLC running a ZorbaxtK* Sum. 300SB-CK 4.P x 50 mm column (Agilent) in 0J%12fA/ll:>O at i ml/min and coluntn maintained at 4QeC. Sample* of PEG-pepdde (20 pg) wore injoeidJ^dJld^#'|i' linear o-6t>% grad'd' vvh I r <m *ormg was-, ore ' 2t' nx [OOoKt j fusiet* Fromms. ikueidly, Figero 1 Λ and Ftgute G> show a sclvmutk: representation of monovalent and bivalent Fc-toxin peptide (or toxin .peptide analog) fusion proteins {or "pepiibodies'"}, respectively, The bivalent Fc-ShK molecule r- a hornodimei containing two F’e-ShK chains. The monovalent f 'c-Sblv toxin peptide (or toxin peptide analog) molecule is a heterodimer containing one Fe chain and one I'c-ShK. tor analog) chain. Since the monovalent Fc-ShK molecule contains just a single ShK peptide per dimer, it is considered monos ak-nt, Constructs or chains referred to as Fc-( toxin peptide analog), contain an Vkumirtal Fe region and an optional flexible linker sequence {e.g„ L 10 peptidy! linker GGGGSGGGGF, StO ID NO 15Gs'ova)eudyatt^ u e h to\m pi ptidc oi tox,n pepoeo analog -,.κ tk>i tse orientation fiom N Ot in. -> < ο, Ό he Fc uwe» tooi v„pT'de ,u u>'· i peptide Muidg..

[Q0682] In Examples 1 and 2 of Sullivan et. ah, WO 2008 08X4 22 A2, were described the ucilvtn of uvutent I e-Shld pepnbodics, i e-1 lO-Shki 1 - w s zed F,~ t j.0-8hK.{'2-35) expressed· §om mammalian edlx. In Example I of WO 2008(Gn422A2, was also described isolation of a monovalent Fe-Lt0-ShK.{ 1-.)5) molecule, formed as a smut! hv-product dunng expression. The bivalent Fc-JL 10-ShK( 1-25) and Fe~l. iO-ShKi 2-55) conjugates provided potent blockade of Kvl.) and I cdWytokme secretion m human whole blood (see, table TFB, By whole csll patch damp ekrottoplivsknogy, the boa lent Fe-1 10-SbK(t ->5) molecule had about 8-lbld greater Κ,γ 1.3 activity compared to the bivalent Fc-UO-»ShK,(2~35i molecule that is devoid of Are Ϊ of ShK. Like bo terminal s-RiG conjugates of nato e ShK t see. li \;iv«pies 4'l bod* bba Cm Fe~$hK conjugates showed ICC setecus Uy Ibr K\F3 versus K.vs J . thus. N -terminal conjugation ol native ShK tueme in ith either PbG or Fc~!inimD does not significantly tiupmne hs Kv! 3 sowns K\ IJ sokvtivh} . Phannaeokineue (PK) studies in rats were petihrmed on bivalent Fe-I RFShKt 1-35s end Fe~L lO-ShK.(2-35} peptibudiev to examine their stability and hall-life in vivo.

As a control. PK ws? also performed on Oh keensob meomhmant human Fc ί IgG 1}. AH molecules were delivered us a single. auruvenons boles dose. i.00bh31 PKjmms |O0bH4] Antibodies to ShK. Rabbit polyclonal and moose monoclonal antibodies to ShK (SKQ ID N();3b!) were generated by inmumirauon of animals with the Fc-ShK peptibodv conjugate. Aati-ShK specific polyclonal antibodies were affinity purified from antisera to isolate only those antibodies specific for the ShK portion of the conjugate. Following fusion and screening, by ini do mas specific fo? ShK were selected and isolated Mouse ant I-ShK. specific monoclonal antibodies wore panned from the conditioned media of the clones. By ft.ISA analysis, purified anti-ShK polyclonal and monoclonal antibodies reacted only nr the ShK peptide alone and did not ·. ros.s-roac? with Ft: [006851 Pharmacokinetic i PK > studies on 20kDa-PIHG-ShK ISF.Q ID N<>:36.*> and 20 kDa-PEG·[Lys16]ShK (SbQ ID NO:3b;D peptide conjugates in rats and monkeys. Single subcutaneous doses were delivered to animals and .scrum was collected &amp;t various rime points after injection. Studies m rats involved two to three animals per dose group, with blood am) serum collection encoring ut various time points osertfee course of the study, Male Spruguc-Da w ley (SD) rats ^aboui 0.3 kg) and male cynoruofgus monkeys iabout 3 kg) were used in the studies described herein (n :: 3 annuals per dose- group). Approximately 5 male CD- > mice 'were used per dose and time point in our mouse pharmacokinetic studios. Serum samples wore stored ifemen a.s -80 C, until analysis in an enzyme-hrmed immunosorbeni assay (BUS At, [00686} A brief description of the ELISA protocol for detecting serum levels of PEG-ShK. and PEG~S'l.ysl6]ShK is presided below; [0068?] (!) Protocol L uoug) below, detects PEG-riihK and PLG^lW'SkjShP, us w ;-J as the ShK. and [ 1 vs > OiShK, pepbdev alone 100688* (at Srepvwe n tneio.r_, plutec wue coated wdh 750 ng ml hiotmybtiink a;ni-$hk mouse monoclonal antibody (mAh.?. 10, Amgen) in 1 block buffer fpcf.itet· 1000 raLJ\PBS without C at k \kC5 mi 1 ween 20 t Themio Scientific k 2 u I block, reagent {Ttopt' 1' at -'· C, incubated o'.croigbt wit mm shaking.·.

[006801 (b) Matek: wore whsb&amp;d. fhree times with K.P1. wai); bdidt:flCf^patd <&amp;

Perry Laboratories)* [OooCi'i; ici Standards (STDj peaks controls (QC) and yeuni a dilutions were prepared with ]0O\> oooled sera, then diluted 1 ;$ (prcuoatment) in 1 block buffer. Pretroated STDs, Q€s and samples were added to the 'cashed piste and hum baled at rod'll lempciature ha 7 \mra, (benal ddunons of SI IK QCs were psepared in 100k pooled sera. Samples needing dilution were also prepared with 100% pooled sera. Hie pretteatmeni wa^ done to hot it aids. OCs mid samples to minimi At the matrix effect.) [006011 (d) Plates were w ashed three times with KPL st ash buffer.

[00692] (e) A HRPdahdcd rabbit ami-Shtv polyclonal Ah at 250 ug.'ml in 1 block buffer was added and plates were incubated at room tempos store lor ! hour with shaking.

[00693} (ft Plates were again washed three times with K.PL wash buffer and the Pernio [Thermo Scientirieptibkhwre was added.

[00094] tg) The plate was read with a 1..max ii 384 (Molecular Devices) iummometer.

[00695] studies on Fc-, lg-> of Ah eoi^ugates^of^S;® usd [LysI6)ShK wore performed in nude SD rats. Single subcutaneous doses were delivered to animals and serum was collected at various time.poi«^;4fe:!iftu^cip<.f Three animals were used per dose group, with blood and serous collection oecuring at various time points over the coincse of the study. Serum samples were stored frozen at -80~'€. until analysis in A boot de^enption of the FLIS \ piotoeol bn detecting serum bxels oft c-, lg~, ot Ab-eonjugat.es of ShK and |Lysl6|ShK is provided below. Protocol Z. below detects both the human Ig, Fe or Ab portion of the molecule, as uell as the Shiv peptide portion. Protocol 3, below is an early assay that detects the human Fc region alone Md waso.ised lor early assessment of senn'n levels of Fc~Shl< peptibodios m toddtd pharmacokinetic studies, A brief description of these ELISA protocols is provided: [00696] (2) Protocol 2, (a)-(g) below, detects both the human lg, Fc or Ah portion as well as the ShK, peptiderpodtott:; [006971 tal Slreptavtdtn micromer plates were coated aoti-S'hK mouse monoclonal antibody (mab 2. iO, Amgen) in 1Mock buffet* [per litcA 1000 ml IXPBS without CaCh, MgCb, 5 ml tween 20 (Thermo Scientific), 2;gl block reagent ffropix H at 4% , overnight without shaking; [00698] dw Plate-, v,., re w ,-, *,<, iKee ernes w a . KPI x^aeh butfei (10:4 egaard A Peny La bo ratones 1 [00090 ίοϊ Standards I'Di peal S controls (QO and v-ennle dx uno,w wtin 100,;<> pooled sera xxere prepared, then χχ e:o dinned I 5 (pretreutrneru i m I block buffer. Ttctrcatcd $TDs,^ QCs aad,samples were added to the washed plate, Incubailpu was si: room tempumtum btr dhonrs. (Serial cli Unions of STDs, QCs were prepared in 100% pooled sera. Samples nceding dilution w ere also prepared with 100% pooled sera. The pretreatment was done to both stdx, QCs and samples id: .minimize the matrix effect); [00200} (dj FIates were washed three 11mes wdh KPL wash buffer; (00703 ] {cl Λ HRH-tabclcd Λ be 5 Ug&amp;irot human IgO Pc) at o0 tig mi m i block buffer wex added and plates %ere itu Gutted 'it loom temperaturo tor iwne sviro shaking, 1:007¾] (f) Plates were washed three times with KFL wash buffer and the Fcmto [Tltsgttp Scientific) substrate was added:; [00702] (g) The plite was read with a 14¾¾ IT 3K4 (MolebMlar Dmdees] him urometer. 100704] m mu#) below, is an early assay that detects the human Fc icgto t alp Ό r^d was used for early assessment of serum le\eh of be Shk 'pepiibodks ip rodent pharmacokinetic .studies:· [007071 (a) Coswr 3590 90-well ΕϊΑ/RIA plates were coated with 0:1 mL/well 00 2 pg'mt. Goat imibHufe, Fab2. (Sigma 1-3391} diluted m ix Coating Buffer {I0x i mating Bullet; I 50 ;\t ( 0.. ? 93 a OtHCCC in 100 ml H,0) l>ioe\ were scakd and incubated at 4°C' osermg^t («'ifV’oe :h> Plates x.j.e washes dire*. t r>w wOb PRS'l (PBS j 0 i% I weon-201 and blocked by addition of 0.3 ml of blotto (PBS, 0.1% Tween-20, 5% non-fat dry to ilk I to , ech v.e'S ard meCsatcu for I h at room lemneraune (R Ϊ} w ith shaking, |O070?1 tci Plates were washed wiih a K.P Wash Solution (Cat 050-03-00, RPL, Gaithersburg, ?vll>): [0070b] (d) Diluted serum samples utid controls standards in Dilution Suffer (PBS, 0. BSA, 0.1% Tween-20) plus rat scrum, if needed, were brought to 10% rat setum final, and 0.1 ml sample waf added per w e! I. PI ales were incubated at room Mnpembtte with sMcing ibr f hoirr;; [00709] (ei Plates were washed with a RP Wash Solution (Cat '00-03-00, KPi, Gaithersburg, VID); [007JO] u) A HRP labeled seeuad&amp;$ antibody {Pierce r:3;4S6-HRP Goal w-Hu sgG Pc} wha diluted : .5000 in PRS7 and tiicu 100 ·η well is added and -neahmeu a; RT w ;h shaking for 1 hour; [00?I} 1 (g) Plates were washed with a KP Wash Solution {Cat PSO-Pd-CM), KFL. :·Gaithersburg, MD1 and 100 μΐAvail of A.BTS substrate (ABTS Miaowell Substrate 1-Component, Cat#S(MstvG (Η, KPL) was added; [007121 (h) At appropriate times after substrate addition and shaking, the plate wits tead with a SpectraMmGdO [Molecular Devices] plate reader.

[00713] VvheieiR, the original monov a lent K·! 10-ShKt {--35) molecule was isoFtvd as a small by-product dunm: mammalian expression of the bissden) molecule, 1 xampk -1 hvivin uKo describe* Uonmg and mammalian expression of the .immovaiem 1 c-t Id-ShKj 2-55 j hcte-odimer ftnetls. to n-oduoe teconihintna monosment Pe-t SO-Shkjf- C- L two oxo-nbrneni pots peptxle-. ace eo-esporssed m the same coil, rlmse being j. human Fc t le.C« 1} eham and =: F·.. 1 10-40 kj ?- -tel eh.tin (also with durum ly/i 1 1 s regain), t ii;k; ihese conditions si is possible to form three ciistine: dimers, winch include an Fc Fc homed tmer. a Fed.IlfShKi?,όο> Fe-L10-Shk(7.4 5) ho-nodmiu andoF*' Fe~t ik-\hk{?.G51 notcmdunct Fs opimn?5ng expression conditions the monosGem 1 e Fed. lO-ShkkFhe4hetenidimer {also referred k> n-= pwt monos alem Fc-i Kf-Nbki2~*5>) s\<j- produced eifcscrslK <mj vwi-readily punned ro homngeneds tFxumpD = herein) Ihe monovJem fc l ;0-ShKte-35} molecule had un H 50 of? 1 nM (Tabic '"HI m blocking U A secretion from human whole Mood The memo aiom FV--ShR Fc heieiodmset hod an octets j-.d half-life in so o and exhibited sigmfeautls gtemer exposure than {he hie mem hornodimem Mskd e Shkd >» if iginc 10) and 1 e-fhk Fo-Shls. Niece the pomses of this construct w «,-= about Kt-fold k -= s th m me PuG-fhk conjugate and mmjnyotcs of iishsc ShK had pom k\ 1.7 Ks l 1 seh, ·„ to, uy, we dm «hoped uddmonul monos teem peptibodies and mrmod eomugates or Shk toxin peptide analogs ban note identified to have impressed k\ i = sctvis ks 1 1 screens as 1'he « samples den iblkm pomdc additional details of monovalent pepithodies withiniprovedtiteleetmtyaad in vivo pharmacology. 1 he result from these studies indicated that monovalent ShfC Jbfod peptide analog molecules exhibited greater serum levels and exposure m rats ween compared to the bivalent forms of tbs same molecule, yet retain the slow elimination rate observed in the t?rigitutl iprite|:^cptibodics.

[007141 «μ $mmm toxin peptide analog (SEQ ID NO :70) shows significant Kv 1..1 selectivity over neuronal Kv 1.1 (Table 7H1. To increase (he stability of this toxin peptide analog in vivo, we generated a monovalent Fc fusion construct that from N~ to C'-icrtnmus contained.' human Fe(lgG2hL 10 lather-|L>slo]ShK molecule, that waxco-expressed with the huma» FciIgG2) chain alone to generate a monovalent heterodimoj (sec. Example -I ί Λ schematic representation of this monovalent construct is provided in Figure i A. The monovalent Fc/Fc-LIO-ShK(1-.15 Q16K) hevetodiRiei [also referred to as morKw .Fern K 1 v-ShKt I O, 16K>| potently blocked T cell inflammation in whole blood, suppressing 11.-.7. secretion with an IC50 ol'O.lP'tM flahle 11). 1 '.expected.'. stud cs to ex...rme the ks 1,3 veisus-KvLl selectivity of the molecule, revealed that the monovalent. Fc-LISvShKf 1>$5' 'Q.l. conjugate had significantly better Kvl ,.1 selectivity than the jLysIbpbK peptide alone. Whereas the [LyslbJShK (SEQ ID NO:76) peptide alone .showed about Ifo-fold adeem isy for Kvl 3 temns K\ l.t (Table 11). the monos alum Fc Fc4J0~ SHKCI-35 Qlbk) hetcmdtmor was about 1225·-fold more sctivc-.M-bMking Kvl 3 versus kv ί 1. Therefore, the f LyslPjShK pepndc when conjugated shows s unique pharmacology of enhanced seiectiuly. Since the No~20hDa-PFG«jl ysIFIShK conjugate (SEQ ID NO;364j also showed enhanced Kvl.3 selectivity (Table 7H) relative to the peptide alone, (he combined data suggests that the (LvsIbj-ShK {SEQ ID N0.'7fi} peptide when (used at its bi-terminus with editor PEG or Fc-!inker exhibit x a dwinet pha:min<. elegy of a '.prosed Iv- ί 5 , i-mx Κλ 1 1 selei tv ity, 100715] To assess the pharmacokinetics and stability os'ihe molecule ;n vivo, d·- a basis of cofoparfeon forfbc inventive moideojes, sihgle^oseFilfe^dl^AV'erd. performed in rats. After a single ό rog/kg subcutaneous dose, the monova let» Fc/Fc-ElfoShK(l-3S, QI6K) beietodimer (of monomers SEQ ID NOS: 1 add 26) exhibited an extended half·life in vivo (Figure 7). Since the sandwich ELIS A used to measinsS senur let , N e* in melee.. is' T'nroto, oi 7'i teepmes kndutg ul in o a 'C'OstkK one an antibody specific tc> human Fc region and the other an antibody recognizing [Lyal6JSiiK {SEQ ID NO. 76}, the data here indicate that the conjugate had prolonged half-life and 'enumod intact in as a (e-L 10->hk{ i-35 Q16K} fusion protein. (Figure 7. open squares; Table 71 below). The monovalent Fc/Fc-L 10-ShK( I -35 Q16K> molecule exhibited an extended half-life of about 56 hours, that o,:s about s 17 tunes fumes than the SuK (>t π ID M >061) peptide alone that was κ rvred to heso a ho Τ'-He of 70-30 run (t 16 eton ei e . PM 3S % 13942 (200! >1 100716] Bivalent Fc-ShK.d-35 Q16R) homed imer (fuG2). Thebivalent Fc-Shkf I -35, QI oh. j homodimer contains from N- to C-tcrroinns; hum»» Fc {1|6;2} · 1,10 ! inker - {1 ys j 6 |ShK {S BQ 1D 610:26). A schematic representation of this bivalent construct is prewided in Figure IB. The moiecide (hombi&amp;mef$g|§ NO:2o) was cloned expressed and purified as described in Example 4 herein- The pi e c,i moiv-vt s' s' w η,-ied tot ucm tt\ ,o tbs' human-whole blood ussa\ of inflammation and found to haw: an 1C50 of U5Q nM in blocking 1L-2 sceredoti (liable 7Hk The activity of this bivalent form was about 12 ibef monovalent form «abovel which had an I CTO oi 0.16 nM in this w-cme assay. The κ ason why dtu b-\,rient form was css ..¾. n\e Tun ibe snoHosaieuf m nnsnowSi If o-possible thm the Inwhom molecule co;wdninv oso posTneh charged [LyslolShK {SEQ ID T\f):76 i nepdditifit its end, is less stable and/or interferes with Kv|;J : channel binding to some extent, 1.00717) M™gyaioitan&amp;.fa O I f-K),Ah. (he monovalent anfi-KLH Fl.ea.vy Chain (RC) fission antibod} fAb) comtrna embodiment of the present invention contained, front N- to (.'-Terminus: human anti-k> H \"l> ntf Mi t-peptfJs I nkes t ^ i ]Shk molectn- (StQ ID SO 33) f ^ -oas eo-expto-weo with the human nki R lie, \y ( bain alone (61 Q ID MO 2d) ved the human. aKLH light chain (SEQ ID NO;28) to form a monovalent uKLH Ab-[LysIbjShK molecule (heterotetrarnerofSEQ ID NOCK; SEQ ID NO:29; SEQ ID NO:28; and SE.Q ID NO;32), A schematic representation of this monovalent construes is provided in Figure IF. Themonovalent »KLH HC-ShM 1--35.QS6K.) hb potently blocked Γ cell uftlnirouation m whole blood. sapp*exsmg 11 -2 secretion with an 1C30 of b.X?4 n\l ( Idble 7fh. Inexpectcdly. studies ίο examine the Ko 1.3 v corns K\ 1.1 selectb u.y of the molecule, rev etded that the monovalent aK I 111K7-ShKi 1-35. QUiK) Ah ilteteredcfxamer of SF.Q ID NO:2N SEQ ID NO:29; SEQ ID NO Ά enei SFQ ID NO 3?) had sjgmftcanth betiei k v I 3 «doetnti} than the }1 yslbjShK. tSFQ ID NX) 7e> peptide done Vhis monovalent -Yh-KhK conjugate wax about I'158-fold more acuve m blocking kvl 3 versus k\ U Oreble 7H and Figure 2A-B), (00718 J To assess the phunnucoknudies ami stability of the molecule ns s reo„ smgk-doxe PK studies were peripimecl is mix. After a single b mg/kg subcutaneous dose, the monovalent a!v! 11 HC-SbM 1-35 Qlok) Ab conjugate exhibited an extended half-life in vivo (Figure 7. closed circles). Since the sandwich ELISA used to measure serum, levels of the molecule (“protocol 2”) requires binding of two antibodies, one an antibody specific to human Ig region and the other an antibody recoghiring (lyslb)ShK. (SFO 1¾ N0'76), the data here indicates that conjugate has prolonged halftlite and return u^ intact m vivo as :·. monovalent aKLII IK -Shkt 1-35 iJlClCTAlyficsau· protein (Figure"·. Figure 8. and Table ?J). The bivalent akl Η ΗΓ-ShK.fl-35, QlbK) Ah molecule (schematically represented by Figure TGr given at ire vrere bmg k_ t se, showed imwi ulysleo oArewes tre G moeM h. provided about 37 times less exposure res measured by ΑΙΑΝ*. Table ?jj rd.ttive to the monovatent oo.re.u e {Fret e s* Hie pored are scks.oe c<vmaiem aej-KLHsAh-{Lysl6]S:hK molecule exhibited very slow clearance m rats tCL/F ::: 105) mb. h * kg'5) (Table 71).

[00719} Mojja^ This monovalent ak£H

Heavy Chain (HOj &amp;stdn antibody (Ab) construct embodiment of the present invention cOtlMibgd fror? N~?o C~femmms: human anti-KLH Ab Heavy Clnnn.....

Fnkcr :desArgl. I vslojShk mo.^caietSFO tD NO;33)> that ’-v;re vo expressed ws h the reunao uKI Η 11. a\y t ha 5 t^FQ 1F> NO 20) and f.v F.unreu aKl II l.ght . cam {Μ O ID NO 2K) form .1 reonova on: ~K3 H \b-( Cc\ krgl. l.sNOjShk. molecule. Λ schematic representation of thin monovalent construct in pros idea m Elgin o IF. I ho monox «dent aKI i l HC>Shl£{2-35·. Qlo&amp;:) Ah ilKdeKHotramer of .SEQ IP NO:28; SEQ IP NO:29; SEQ ID NO:28; and SHO ID NO;33> potently biox kod T xvd inflammation tn xvhole blood suppressing 0-2 secretkm; \x oh an If 50 of 0 570 nM i Table 7Rt and unexpected lx was about 157p toM mom potent at blocking the T-cell potassium channel KxT.3 than the ncurcmal channel K.vLi. :$0?20] Μμμ1«!ΙΜ!^ 1'be ntonocalcttt Fc~ShK( 1 -35, Q16K VKl Π Ab heterotnmer or bentibnd v embodiment of the present invention contamed:front N- to G-tcrminus: human Fc (Ig02) L10 linker··· (LysIbjShK molecule (SEQ ID NO;26}„ that was co-ex pressed with the hittmm aKLH Heavy Chain (]gG2> (SEQ ID N0:20) and the human aKLH light cl tat η {SEQ ID V'K!8s. A schematic reproxeotanou of this monovalent avnxtiuct is provided in figure IE. The monox a tent Fe-ShK.{l-35. QN>K) Kl II Ab heterotnmer (SEQ ID NO:28; SEQ ID N0:29; SEQ ID NO:2b> potently blocked T ceil Mflammntion m human vxhole blood, suppressing 11.,.-2 secretion with an 1C50 of 0,2.45 n.M {Table 7H). Surpmngly, studios examining theKvifl^ yemts Kvl,l sekvtsx dy of tl^ nolceule revealed that the monovalent Ee-SbKt 1-35, Qlok) El Jl Ab haermmuer bad significantly better K\ IJ selcctivity than the iLysln]ShK pept be alcro sSFQ ID NQ;7o) This monovalent hcViotruner was about ED5 sold more aetsv e in blocking kx l ,3 versus K\ I. I ( fable 7H). (1)07211 Although \x e Stave not examined the pharmacokinetics {Pk) of the Kv 1,3 selective monovalent Fe-ShRt 1-35, QlfcK} Rl H Ah homrotiimer or hcmihxxiy. we have examined the PK profile of a similar hemihody, that being the Ec-ShK.12-Abi/RLEf Ab-heUtrotrimer A schematic of the structure of this molecule is provided in: Figure IE, and the molecule from N- to C-ierminus contains: human Fc (fgG2) -bhKt2'3o), x\ Inch is eoexpressed with the human aKLH heavy chain and light chains. After'a single 2 mg/kg subcutaneous dose, the monovalent Fe~Shk(2~ 35};KLH Ab heterotnmer iaiso referred to as monovalent Fc-ShK/KlH Ab heterotnmer} exhibited an extended .half-life in rats (Figure 10),: Since the sandwich El.ISA used to measure scrum levels of the molecule t‘"protocol 2*') requires binding "of two antibodies, one art antibody specific to banyan tg region and the other an antibody recognizing ShKi~-35), the data here indicates that conjugate hies prolonged half-life and reonnns intact in vivo i Ho are 10, I a He ?K). The large, about 103 kOa monovalent Fe-$hK.(Πv5 jR LB Ab heterotriitvr or hemibody showed greater exposure and about 2-lbtd less clearance than the about 30 kDa monovalent Fc/Fe» $bK. heterodimer (Figure !0, Table ?K}, The very small, about 4 kDa ShK~L5 peptide was cleared much more quickly, having a clearance value in rats (CL/F · nf \»„ Example :>) Hat was a ve; ';i' urn - \>v,\ t' a the ' trge monovalent 1 c-HuktO-hS; ΚΙ H Ab heteroA-one* KT I 2? 0 mi h Hg B molecule [&amp;Γ22! Mope;, a Hag and ΤΑ Hem anti-KL (I AH ooHi .yb I Recombinant ntotwulem and bw.d.mt unt: RtH A.noif jl vsWvjShk. fusion pfOidlsB embodiments of the present" invention wem constructed ns described in Example 4 and U.S. Patent Ho.7,442,778 B2 to produce huh antibodies with [tysldjShK toxin pcptkle analog inserted into loop regions of the Fc domain in otto {monovalent} or bodi(bivaletit) HO monomer*». The monos aknt akl H HC-loop-$hK< i -35. Q16K0 Ah contaiaed Hree chains; a human- aKLH Ab heavy chasm h human aKI.fl Ab light chain and a human afCLH Ab heavy chain where the [Lysl djSliKi peptide w as inserted into a loop w ilhin the I e region of the heavy chant The [LyalbjShK peptide within the Fe loop contained a flexible linker sequence attached to Its N- and C-tcrminus· to allow lot independent folding and extension from the loop. AAbhematie repavsentation of this molecule is provided it* provided in Figure HirClsk^sequePdes of dstfermg amino acid composition and length 'were examined The monovalent atUuRLH Ah! oop-1i ys16}ShK fusion protein was a scieetwa niHHioret K\ 1.3 ucthttv truer R* El. Ί2’,-loin :eor. sekHive fot Rx 1.3: Table '41 and Figure 2A-B). The monovalent Kl. R-Ahl.oop-jl. yxlbjShK r»ok‘v, le ,' isfuted tlv -low ,'st <„ e, umee .n tats o* 1 L tv* e. to> to-, o pugotss Hat o e have examined (Figure and higftre 0 and "fable ?U.

[:007:131 aKi,B HO-loop-Shl&amp;C-i -35, Q16K) Ah ooiiitdittedi1^^ chsi^lg, a human aKI H Ab light chain aud a human uK 111 Ab heavy chant when: the (Lyj»1b|ShK peptide was inserted mto a loop within the Fe region of the heavy ehdim A schematic representation of this molecule is provided in provided in Figure i M To corn pure the pharma* okmeucs and stability iu \no ot'thw bw a bn it molecule to me monovalent form, single (> rng/hg si'Nijtan.vus iiov> of each mol mule \^ere t«! nN Deep k show mu: a s t »m \ < kor < e dvbwe,, Λ HI 10 loop-Sb Kb' -^kQJhKt kb gave profoundly kw- exposure in rats than dtc raonovaknt kum of the vane molecule t monos.deni skt.H HC-loop-ishkG-35, QMs Abl {see Figure M, \ sposure as measured by ΛΙ L\ was ahum lol tmsc-· less fo4 'he b” dew nKUl Hi aiv-v$hk-: t-35, Q.ok) A ' molecule comesn.,1 v* me mo u owe eni a K l Id FK Moop-ShK{< - '< ?Q i 6K s Ah molecule if able ? ( ) Themfoi c, on iose *1 o xwatem kn η--.how u mespeMcd a a >,wtk better phsrrsi,.oi, k\ v pro rile its vivo compared to typical bivalent forms of the same molecule. j 00724] Monovalent ShK{ 1-35. ΟI (dO-Fc'/Fc heterodimer. The monovalent sh\ll~5p Qk'ki'l <, IneLms, i »„* umia ,' w,, chants one berm a nunur l et lg02i chair and 0-.·.' other bane SisM 1-35, Qlokt peptide fused to Fc that contains from k- to ('-terminus: | LyxlrijShK, - Ltd tinker - human be |1gG2). This peptide-tux urn protein contained from N- to C -terminus' the 35 amino acid }Lysl6)ShK peptide, a ten amino acid GGGGSGGGGS (SHQ ID NOT S3) L10 linker secgienee and the human he (lgG2f sequence, Therefore, the linker-Fc region was attached ?o the C-terrain u«* of [I yslOjShk following C\\35. This molecule is also referred to as monovalent ShK{l -35,01bK.)-Fe hetmdimer. A schematic represehteMtn; pfthis monovalent construct is provided in Figure 1C.. The molecule ο as cloned, expressed and purified as described f sample 4 herein. 1 tic purified pot^it,having an It oft) i! oVt m hiockon» H -? scctcfion m the human whole blood assay of mflaimnanon ι Γη hie ~1 h I tcspite Os excellent potency, the monovalent Shk(i-35, QtbK)-Fe/Fe heierodimer showed only a modest -Hi told selectivity tor Kvt.,3 versus Kvl. t {Table 7H), Therefore, it would appear that this linker-Fc fusion partner attached the C-term in as of [Lysib]Sh&amp; does not result in a further enhancement ofKv 1.3 selectivity, This contrasts with N-lermimi! fusions to [Lyal&amp;]ShtT, sdelt as the monovalent Fe/Fc-$hK,{ 1-33, ()1614) hetcrodirou (Table 7H> which showed -12.25 fold selectivity and had die Pc-linker sequence ailsMssgd Ιο die MTertrimai Argl residue of [I^ys l efSiafC. A:aJ«i|>orfgift end notable: exception., boomon i>oL' jf,ysK'}Shk.-Aia peptide (SiilQ ID NO:362) whnh contains a single C-iermifud Ale residue adding follow mg Cys35 of [LysltpSnk. This molecule exhibited an enhanced 262 ibid hnproved solemn ity for kv I 3 \ e.rsnx Kw 1 \ { i’abie ?Hk i fierafbre, w c ere ismn that the specific arm no acid residue added after C'\x,3> at the C-terra in us of f {.λνΚ<ΊΑΙ0\, can alter the selectivity profile of the luxiou profem. For example. fee mourn a lent ShKhivm. QtoKt-i.K}-Fe moiccule 'described in dux example contains the imker Coy residue added after OyxivS of [Lys I bjSUK. If au \ia residue o ns addled instead following Γν,ν?5, an enhanced Κλ 1. * a el ecus ity might be u Oxers ed. Indeed, w e do see fob lold improved Kv1 .3 select is ;t\ by the II y skgjShK-Alx; pcplick thus, no anticipate that specific amino acid residue at the fmmm junction would niter the selectivity profile. 1 hexc tes-due can be readily incorporated into the linker sequence between site [l>slb]$bk peptide and the bureau Pc don lain or imemmogkhmhn light cham or heavy chain to improve the ceitjiiguie# Kvl ,3 selectivity,: [00725) Monovalent ShK( 1-35, Ο ΙόΚ pHC αΚ.ί,.Η Ah The monovalent Shift k-35, QIPKVHi ak? 11 Ab embodiment of the present irreemion contains tiucv chains, one being die human uKAH Ah light ehmm another being ihe human wKLH Ab heavy chain and fbc third benny a peptidmakt. 1 ί Ab heavy chain fusion that contained front N-, to C-terminus: ItvsUoShk t.10 linker human uKi,H beary eiiain. Therefore, titso fusion contained the mmer-heavy chain region attached to the { 'terminus of iky-- I PISItK. lefdow ing C > s3:k \ schematic representation of ike rnouovaient ShKi h-35, QioK eHC aKLH Ah molecule Is prot ided in Figure 11 The pur-lkcl molecule was highly potent It tying an K AO of 0.2 hi nM in blocking if.,-2 secretion in the human whole blood assay ofinflammation (Table 7Hh Despite beiriu wrv lame m sure and fused to n human be heavy cham, tire monovalent v.· : v.,. ... .. >· flysK'd-uKAH Air molecule retained higlt potency m blocking T cell responses, [00 'Aho Monovalent aDNP (ir-ShK-t 1 -33, Ο; 6Kj Λο, I he monovalent a DAP Heavy Chain (HO) fusion antibody {Abo construct eml'mchmanl of ihe present invention combined from A- to {.''terminus: human anti-DNP Ah Heavy Chain linker - (Lvsl6}$hK molecule. that was co-ex pressed with the human aDNP Heavy (.ha in and the human aDNP light chain to fonts a monovalent aDNP Ab-[I..ysl(V|ShK. molecule. A schematic representation of this monovalent construct is provided in figure 11:. Use monovalent aDNP HC-$h&amp;{ l -35, Q16K.s Ab potently blocked T cell inilammHtion In human λ hole blood, suppressing 11..-2 secretion with an 1C50 of (feed n.M {Table ?Hb Studies to examine she K.vl A versus K.vf 1 .selectivity of the molecule, unexpectedly revealed that the monovalent aDNP Ht'-ShKi I -15, Q16KJ Ab conjugate had significantly better Kvf3 selectivity than the |Ly\sl6|ShK peptide alone. Ibis rnorsovalesn Ab-ShK. conjugate was -·5Ν0θ fold more a cove 1st blocking Κλ be versus Ks 1.1 (Table "Tit. (able 7f I Data demonstrating * a no us conjugates off Ly> 1 ojsfck having improved Kv (. t selectivity. Fox in pep?kk> and toxin pepiide analogs were PiXly la-cd as described in f sample -4 herein, Imrounogtobutin-eoniainirig compounds wore rcoombb-anby expressed arm purined as described m example 4. Fieeirofnv>loiocy ua.s by FatcbXpressne IPX}, except asterisks indicate data from whole cell patch clamp (see, Kxamples and K hereinj. Human v%-hole blood f'VvB'b essays of II. -b and micriefor;-gamma hdFXg'h were conducted as described in Example ? herein I.

Tabic 7H comlriaad

I able Ί AKimimukumoes ofmo-.Mix Jem I c be--! to stoKhK. in Spragueby rats (n 3).

Τ^Ιϊίέ; Tl. Tharntac$k|ncitc data for recombinant monovalent and jbw&amp;feiitant?*i£i>H Ab? |Ί, vslojShK. fusion proteins administered by subcutaneous injection tdose - 0 mg kgi to Spragnc-Davviey rats (n - 3)

T able 7K. Pitarmacok mode data lor recombinant mm a? valent Fe Fe-Sbfs hefoioduner monovalent TVSfikf &amp;XH Ab hetenotrimer and bivalent $b&amp;.~f\ ShK-Fe hontodinter ttonon nrofein* administered bv subcutaneous injection (dose ::: 2 raglfcgj to Sprague-Dawley rats (n .=== 3).............................................'.....................:.............

I ’ ............λ......... 1 able 7L. Fharmaeoksneiic data for recombinant monovalent and bivalent anti-KLH AM oop-{ I \slojS b\ U>»on ptoietns administered by sa be «mucous injection tdose 6 mg/kg) m SnragnoDawtey raisin ::: 3).

( [OO'GKj KvUaed KvlJ Bdeetimplmtoiogy |0CD39] .CdUMh^ Π Ι0-Κ.1 cell* were stably tranced vyith human Κλ· l.3. or tor eoyntmefeens (see. Example b herein), with hKv! A* HIM ! .0. or UK.vl.7; HLK.393 cells were .stably expressing human Kvl 3 or with human Kv! 1. Cell lines ocre from Amgen or BtoFoeus DPI i A Galapagos Company). GRO K.1 cells stably-expressing hK.% 1.2, tor eonntetrsereens. were purchased front MHlipore (C ar#.CY 1.50151. jOCMOj Whole cell parch clamp vdeemiphysiology. Whole-eeli ei.uTerrs were recorded at room temperature using MuHiC'lamp ' 00B amplifier from Molecular Devices C ore. (Sunnyvale. CAP with e-5Mil piper-ex nulled from borosilieare glass (World Precision instruments, Inc). Dm ing dam acquisition, capaem'v c currents were canceled by analogue subtraction. no scries resistance compensation was used, and all currents were filtered at 1 kllc. The ceils were bathed in an extracellular xo hit ion eomaining I ,b rnM Cail.·.. ? ntM KCl 135 mM Nyi.'j. 5 ntM Glucose.. 10 ntM REELS. pH ? A »0-3UO mOsm. The intern ai solution containing ’HI mM kC1.40mM DM tOtnM KaCI, I mM MgCb. Kt mM EGTA, 10 mM HI: PlaS. pH '13. 39(1-300 mOsm The current were ex ok cd by applying depolarising vohage steps from -M> mV to t 30 tnV every 30 », t |<v t.31 or lost Ko t, i) for 300 tns inter, aix at bolding potemial of -80 mV. To dot or nm to K'50. 5-b peptide or peptide conjugate concentration at I '3 dilutions were made in eximcelluDr solution w hit 0.1 % BSA. and delivered locally to cells with R.u'tid Solution Changer RSe~ 100 < Rios ogie Science Instruments). C ufronts wore achieved ro steady state for each eoncemratton. Data tm.dyxiv was performed using pCi Λ.ΜΡ {version 9.3) mtd OrigmPro (version 7), and peak currents before, and after each test article application were used to calculate the percentage of current inhibition at each concentration. 1.993311 IDKJlXnWMiCells were bathed in ;m extracellular solution eomaining I A rnM CaCH, 5 mM RCL 135 mM AuCl. 5 mM Glucose. 10 ntM RBPfc'S. pH 7.-4. 390--300 mOsov The imcntul solution contained 90 mM DO. -40 ntM KM 10 ntM Natl. 1 ntM MgCK 10:01 EGTA, It) mV? HEPBS. pH A3, 390-500 mOssm Usually 5 peptide or peptiuc conjugate concentrations at 1:3 dilutions are made to determine the IC50s. The peptide or peptide conjugates are prepared in extracellular solution containing 0.1% RS.A, Dendroloxin-k and Margatoxin were purchased from Alomone i aM Ltd. (Jerusalem. Israel); SKDioxio waxpttehaseef from Bachcm Bloscienee, b-e. (Ding of P s\v PW.* \P λ»&amp; pur* h troro Mdtteh Crop dt U ^ MO) Ci „<< s v. (‘re reciwdvd a room temp. : rouse rosing a P.nehXpruxv*·4'' "OQOA elueuop'm srolu< \ \vstem for1’ Mmecuku l>c\ con ( oto :m, emaie t'A), I he wdt&amp;e, protocols ιοι ST <.5 3 ane Jw 1 I arc showi'' Table ~M in t sample x bet on Λγ exw-oel uk» seen turn with 0 1’ «

Bi \ was apphed f r-4 to obtar '(Hi1 o peace' of control ι POO. tOn !b lowed bs o er'feront concentrations of 1:3 peptide or peptide conjugate dilutions for overs 40t)m\ incubation time. At the end, excess, of a specific benchmark ion channel inhibitor {Tabic "M in Example· M wax added to define lull or 100% blockage The residua! current present after addition of bettdhmsrk inhibitor, was used in some cases for calculation of zero percent of control. I he benchmark inhibitors tor l\i 1.3 atal blv I I arc described in Table “M in Fsample x Bach indifid ttal set of traces or trial were visually inspected and either accepted or rcicctetl The general criteria for acceptance were: [007331 1. Basel ine c u rrc n t rdu#i be stable

[00733;]: 3. initiaI peak current must be >300 pA

[00730] 3. IrfotisI km and final Rm must >300 Ohm 00 '351 l ITstk ro cm mtot a nroc < χ »i b ο uv p^ot to ,,at eomponnd addition [00736] The POC was calculated from the average peak current of the last 5 sweeps before the nest concentration compound addition and exported to Excel for 1050 calculation.

[00" 5 71 jonBTTsJiwhwha;ueh£m^ 11 eemmhwoiog) wa- paifro ts.v or (in) *e,h xtabk e\p, ew% fowl t UTk303 edN stubb expressing hKro 1,1 1 he p-m.edv:e tor preparation of the ''As>u\ Plate" containing ShK analogues and coni agates tor IWQ efocirophysiofogy wax us follows: all ahalggubk weiv J.sM'Wei in estiaec it ‘a-, u'let tPBv with 0.° roM C. a .rod0 '< m\l Mg' j with 0,3% 13 S3 and dispensed, in the 11 I poly prop, lone plate- at the concentration of 100 hM from column I to column 10. Column 11 and 13 were reserved for negative and positive controls, then serial diluted., t I 3 retro to row 3. ion Works Quattro fiWQ) eiecbojfoyssoiogy and data analysts were accomplished as follows; re-suspended cells (in extracellular buffer), the Assay Plate, a Population Patch Clamp (PPO PatchPlatc as well as appropriate intracellular 190 mM potassium gluconate, 20 roM K.F. 2 mM Nad. 1 mM MgC'ia, 10 mM EGTA, 10 mM BEPES, pfi 7.35) and extracellular buffers were positioned on lonWorks Quatrro. When the renboniwx were added to patch plates, they were further diluted 3-fend worn the essay phife to achieve a final wm eonivnmition range -Von; ?.V3 oM to 1 5 p.\1 with 0. 1¾ BSA Eleen'uphy.sioiogy recordings were made worn the CHO-kv; .3 ask! Ι ΙΓΚ-Κ,νI, I ecu lx axing an lenphoicnein-baxed perforaicd paich-ciarnp triemod. Uxinn the vobayc-danap circuitry of the loftWori··* Quauru, cells were held *ta. membrane potential of HO mV and vohage~actA ated cenouk were evoked by slopping the membrane pnicrukil to -‘MO mV for · 100 no. Kw currents were evoked undercontrol conditions be., in dr,' absence of inhibitor ai the beginning of the experiment and after 10-mimiio incubation in the presence of fee analogues and cot pro Is, ihe mean K e current amplitude w ex mease· od between 440 and 4400¾¾ and the dara were exposed to a Mu; rum ft Excel spreadsheet. The amplirnde of the l\r current in the presence of each concentration of the mod owner and corncob wax en p:\oxed ;* ;i perccmape of ihe Ko enrrem of dn; pre-compound current am pi n ode m -he xantc well When these oi control values were plotted ax a lunetiou of concentration, the K'50 value tor eiwh compound could be calc eluted using the down-response ftt model 20: th 1/xeel tit program which uhlizex the following equation*

where ymin lx the minimum y-s-alue of the curve, ynuix is the maximum y-valt-e of the curve, cone, is the teat concentration and n is the Hill .slope of the curve, [0073kj Example 7

[CIO'TVJj Medshrieg Bioaeihity in fi urnan Wlniio Bhlotii 100740] foiAhilMcEvjfimmmM secretion of 112 and IFN-u. The potency of ShK analogs and conjugate* in blocking T cell, irdlarnmafion hi human whole blood was examined using an ex \ ivo assay that has been described earlier (see Example 46 of WO 2008/088422 Λ2. incorporated herein by reference if? its entirely). In brief, 50¾ human whole blood is stimulated, with fllapxigargm to induce store depletion, calcium mobilisation arid evtokinc secretion. To assess the potency of molecules in blocking T cell eytokme accretion, various concentrations of Kvl3 blocs.mg peptides and peptide-eoniugaica were prc-incnbatc-J with the human whole blood sample f»« 30'60 non prior to addition of the thapsigargm stimulus. After 43 hours m 3?°C, 5% CO*, conditioned medium was collected and the level of cytokine seer chon was determined using a 4-spot electroebemillumincsecm immunoassay from MesoSeaie Discovery. Using the thapigargin stimulus, the cytokinhs 1L-2 and were ssd’eted robustly test Wood isolated from multiple donors The 11.-2 and IPN-g produced in hitman whole blood following tnapstgargm siirrmkuiou vvete produced from Γ cells., ns scsealed by niftaeeiiular cytokine Gaining and fluonesccncc-uctivatcd ceil sotting (FACS) analysis. 1007411 Kf 3 U the major voltage-greed potassium channel present on T cells. .Allowing for K efilnv K\ 1 t pi ox ides Pte dr is ing dace f'e” columned f V influx, which is necessary for the .sustained d ova nets m imracelluUu ealcsnm needed for efficient T ceil activation and cytokine x»\ tenon Ks 1 3 inhibitors have been shown earlier to suppress this ealcunu flux imimed by Ti'R ligation t(».( boo e>. al. v>!<!, t. dh Immunol. A7.99-107). Thapsigargin-mdused store-depletion and ITT ligation d;e4« M-odar patterns of CaA^ mobilWation in '«dated FeeiKiJ !>onnadjui el W , le>x>] i BUi Chem 267 25864-2^872). but we have found ihupstgargm go us a mo 1 obusr i oomwe te w ho e blood, Iheretbre. wo developed a btoas^ax whereby the bioacux'tx of K.\' ΐ ndWmoss .s assessed by evenmunc their ability to block thapMgui giu-mduced e> tokine secretion from ‘1 cells in human whole blood.· Since o, -ole Wood ss . eon de\ \ne >„o' \u img high piotem levels the activity ot peptides and peptide conjugates so dps whole Wood essay has au additional udxuntage in assessing the molecules stabddy over 4b hours m n biologically relevant fluid. The whole blood, assev prox ides important confirmation of the Κχ I 3 potency of molecules determined by etectrophxsiologv tt-Phys), since ePhys assays are gene rally of short duration h |-2 hours) and use phy siuiogieW sal Inc containing no prorein The longer duration olbhe w hole Wood assay may allow for more effective determination of equilibrium binding kinetics reiauxe to ePhvs studies which arc of short duration, [007431 ioo channel eounfersereens [W744] Αό1.ίνΚνΙ,2, K.v 1.3. Kv 1.4. Kvi,6 and Kvl.7. PatchAntes sT planar eaich-damo eleotrophysl.o 1 ogy. Ion channel currents can he recorded a.t room temperature using PdtchXpicsS'S· 700b A oleenephysiolony- system non MDC using methods and cells described in Example 6 above. '1 he voltage protocols for ouch channel are shown in Table ?MS below.

Table. ?M„. Votings protocols mi recording eoodiOodb

[00? 451 Cardiac ion channel conn terse mens IhFRCj, hICd.Oi I b-mnK., hNar 1.5. hKv f 5, uCi-iY!..?, hKv4.si.

[007-46j CdHinos. ΗΠ(2')Ι ceils stably transfected with hisYLQTI hmiriK. and hb'RG were from Amgen or Cviomyx. Inc. HE&amp;3M ceils stable trausieeied with human hNav.15 were purchased from C vtorovs., inc. RFK2':M cells stably expressing hK.vd.f and OHO cells stably expressing hKv 1.5 were from Chars Test. CHO cells stably expressing the human L-type calcium channel Cat. 1.2 were from ("hanTest and contained die human CACXA 10 gene encoding hC'avl .2 and coexprcs-sed the beta 2 subunit eneoded by human f' \CNB2 and aiphtCdciial eneoded by the CACNA2DI gene,.

[007-I?) FASTPateld studies were peHormed at Ciuut 1 ewt to es.amme the impact of peptides and conjugates on the cloned hnm.ns 1. -type calcium channel ItOiv! 2, cloned hfvv4 J and cloned hho t,s involved beicb.Xprevs (Modd "000 A, Molecular Dev ices, i cion City, f'At dectrophysioiogY at room temperature. The cstraceilnlur recording solution (H8-· PS t comamed 1Λ7 mM Mail, 4 mM KCb FS mM Cat Is i ruM Mgdi , 10 ntM HbPLS and 10 mM Glucose adjusted to pH 7 40 with NaOH. The intracellular rceotdmg solution for hfCv4j and hK\ t.5 contained 120 mM potassium aspartate b nsM MgCid 5 mM Ι.,ΡΠ'Α, 4 mM Λ TP and 10 ιπΜ HfcPES adjusted to pH 7.2 with KOHL Thu· intracellular solution for hCavl.2 contained 130 mM cesium a&amp;partate, 5 ηι'νί Mg€l·, 5 mM EGTA, 4 mM ATP, 2 mM EDS'A, 1 mM CaCi?, 0,1 mM GTP and 10 mM HEPES adjusted to pH 7.2 with N-meihylT>sglucarnme. .1» preparation So? reeowbng, mtmeeUnbr solution ss loaded mto She intracellular compartments of the Se<?/ehtp!6 planar electrode Oail suspensions are pipetted mu> the extracellular compartments of the A« vrielupμ, planar electrode. After establishing a whole-cell configuration, membrane currents are recorded using dual-channel patch damp amp! diets tn the PatchVp'ess v.stem P<rka Ss^ul mo' the i unenN woe lev.-pa-.s likened at one-fifth of the sampling freqnawy. fincc eoiteemmtions of peptide conjugates (test atm Hi dialed uno HB-i'S with *% Bn A are apuh.ee <4 live rmunn mtes\ jK to nut's e cells. Solution exchat tge were performed in quadruplicate and the duration of exposure- to ukMes eoss« tmtas όas Den ua s \ of u e Kittol-ae tKoapploe o .me cells and after a solution exchange poster e conk ok are applied to serdy sensitivity to ion channel blockade. AH positive controls 'acre diluted into HB-PS with 0,3% DMSO, Posiitvs-eoruols tor blockade of ehamrels nie.'u.k J uskshpur. <0 rd pMl v\ inch produced ai on" "5° > hCv-' 1 e< ,τογ* blocs ties ntde·, 1 I r\l > <,\ inch r> earned Sort ;5% < Tubman of the HKA~ ^ vti’vf and 4·· axn 'opvridme *2 m\!> which tdocked about 80% of the hlevES current. Valid whoie-cd.1 recording* must meet the following criteria: (Π membrane resistance (Rib) > 200 MO . (2) leak cummt < 25% channel current. The test procedures for. hCa v 1.2., bids 4.3 and h.K.v 1,5 were as follows: ίΚΓ4\| a } h( ay. Γ test procedure Onset and steady state block of hi In 1 2 jl2 u2$ channels were measured using a stimulus voltage pattern consisting of a depolarising test pulse {duration. 200 ms; amplitude, 10 mV) at 10-s imemtkj from a -40 mV holding potential. Test article £S5poeptm:tions i-a^·· washout between .explications. Peak current was measured during the step to 10 rnV. Saturating eoneenisuhourif nifedipine f!P of each experiment tor block hi as, ? curia-·· 1 Ή. current uss digitalis subtracted from the total membrane current record : iOfrid'g lu NRv-gx He" procedure Onset and steads stme Hock <u *tKx4,3 u >teut sseis' measmed wong a mhe p., t m with f.xed a nplttudes .Are- ,u leaf me 0 m3' for 300 nisi repeaed a- I0~s iruerv.ih from a holding potential of - Hi mV. Peak ami sustained test pulse ear on arnpV.nda·· veO measured dating t% step to r>. o .n\ [00750] c.) hfCvi .5 test moccdnrc. Onset and steady state block of h&amp;v1.5 current were nnwstu ed using a nuhe pattern \-vnh *p ed amplitudes {depoienrabou' * 70 mV atnpbfude. 500 ms duration! repeated at 10-s Intervals from a holding pew„nhel of SO m\ , Current amplitude was measured at the end of the step to n2C) mV;, [0075) | cU>nd!yiMt:fe^5 sodium

PatchXpoAs" s\s?cm 1 he extracellular {Β.ΒΤ*$2) recording solution contained 70 aill Nail, 67 mM K-methyCD-ghteatnine, 4 mM ΚΠ. IT mM CaCS.s 1 mM Mgt"f, 10 mM M|sPI3> 70 0¾ Glucose adjusted to pH 7.4 with HCT The internal recording solution remained 130 mM CsF, 10 mM NaCk Ϊ0 mM FGT.A, 2 mM MgCk 10 mM Hf.PFS adjusted to pH 7,20 with CsOH. Stock solutions of reference standard or test articles were diluted: Into HB~PS2 prior to appbeatiPSi Test attlcles in eluded either peptides of peptide conjugates described herein, tklocaine {1 --30 pM) ms the reference standard, A standardised step protocol is used to elicit icmle cuO'ent through thobKavl .5 sodjgm channel Cells are held at -HO mV, Onset and steady stete block: of hfsavl .5 sodium current due to 1 est Article w :<s measured using a pulse pattern'with fixed amplitudes (conditioning ixemdse ) 20 mV to; '*0 ms, dex» at*, mg test step at 70 mV for 20 sepcated at lO-s intervals. Current are filtered si 3 Ilk and acquired at 10 kHggUi episodic mode, W:hpn a good recording wax established, cells werewaxhed tor 2 minutes, following by applying control vehicle for 5 minute*». 1 hen control and each concentration of test article w as applied lor 5 minutes. There were 3 additions tor each concentration with 1-minute interval Dispense speed was 40 pi x wsib suction on To determine ! 17.), Test Article at i μ ML, t μΜ. 10 μΜ and 30 μΜ was applied to cells in 3 cells) cumulatively {without washout between TOM 'Article concentrations) m axeembng otdor. to each cell π. 1 where n nrimher of cells) Each concentration of test arrtcic was applied for 5 minutes. There were 3 additions tor each concemmtion with a i-tnipuie interval, Electrophysiologieal data acquisition was performed: ovtn Patch\ reset mrenanderwi 0¾ \V)u huesa events. 1 '«on C t>. I'M aud uraKsoswas performed none Dar-tXprexs vi .4 iAson Instruments, I'mon City, CAT The b peak current before and after test article application were used to calculate the percentage of current inhibition at each concentration. Acceptance criteria for a good recording include: {1) seal resistance e 300 Mil, Mi access resistance 10 Mil (3} peak tad current200 pA. (4) leakage eurrem < 25V. of the peak tad current. (5) rundown v 2,5V> minute in control s chicle.

[00752) Countemcree^ ΙΚ.ν fhKyl.Qi f : hmlnK) pnnwsmnpdymncj .'.Sv .... ... Λί^ίί.νλ^ Ah.v. 21:’'V. 12i/.v.2 /nv/22 The extracellular recording solution was HB-PS. The internal recording solution contained 20 mM KF. 00 mM ΚΠ. 10 mM "Ned. 10 mM fc'GTA». 5 mM KeVTP. I mM :vig(.1:<. 10 mM HOPES adjured to pH 7.20 with KOH. Snack solutions of reference standard or tea: a rides were diluted into Ηβ-PS prior to application. Test a ο idea iecitsdod either -peptides or peptide coniugufes described herein Chrornanol 2°3B iff 3 10 pM) was the reference standard. .A standardised step protocol wa.e used to dice ionic current through the IKs potassium channel Cells were held at -HO mV. Onset and steady state block oflKs potassium current due to Test Article was measured using a pulse pattern with fixed amplitudes (depolaritiing test step to - 30 rnV for 5*.) repeated at 10-x intervals. Cun cuts is filtered at 3 kfie and acquired at 10 file, in episodic mode. When a good recording was established, cells were washed for 2 minutes, tbtlowing by applying control vehicle for 5 minutes. Then control and each concent· ution of test article were applied tor 5 minutes. There were 3 additions for each concentration with 1 minute interval.

Dispense speed was 4Q nil s with suction on lest article at 1 uM, 3 μΜ, 10 μΜ and .10 μΜ w-ere applied to cells in :: 3 cellst cumuktlw ely t without washout between test article con e.m ton ion m in ascending order, to each cod (o : 3 where u = mnoberof cells) bach concentration of test article whs applied for 5 minutes 7 hero were 3 additions for each concentration with a l minute mwrvul Eleetrophyskfloglea] data acquisition was performed using. PtitelrXptess^e Commander v t .4 (Axon instruments. Union City, CA) and analyses unperformed using DataXprcss v I .-4s Axon Instruments. Union City, CA). Tito 5 peak currents before and after test article application w ere used So calculate the percentage of current inhibition at each concentration. Acceptance criteria for a good recording include.*: (I) seal resistance ·-- 200 VET. (2) access resistance < It) ΜΩ, t.% peak tail current > 200 pA. (4) leakage current < 25% of the peak current, (5) rundown < 2,5%/tnin.ute in control vehidq. :00753] Countcrscrcen.s agamst the human ffCr fhERG or hKvl I.if potassium channel by conventional whole cell patch clamp elecnophysiology. One to 2 drops of vhc cell suspension Is added to a 35 mm poiy-d-lysine coated cover slip for overnight incubation before etecfrophystohgy experiments. Whole-cell currents were recorded from single cells by using fight GCi seal eon Sign onion of the patch-clamp technique Λ 35 turn cover slip was transferred to the recording stage after nosing and replacing the culture medium with extracellular recording buffer containing 135 m.M xuf 1, 5 rn.M KG, I .H mM Cai'U. 10 mM HEPES, and 5 mM Glucose fpH was adjured to 7.40 with XaOH and ostnolariiy was set at 300 mOsm). i'clis* were continuously perfused with the oxtracelkdat recording buffer via one of Ihe glass capillaries arranged m parallel and attached to a motorized rod, which places the class ceps liar v uueetlv on top of the cell heme wcotded I or hH<G mof,i no, the seconding pipette solution contained 130 mM Kid 2 mM MgCh, 10 πιΜ HOTA, and 10 mV! HEFES sdjusfeb to pH 7#0 wild Mid and osmohffty set at 280 mOsm, Experiments were performed sit raw temperature and recorded using Mu 11iclamp 700A amplifier (Molecular Devices Inc j. Pipette resistances were typically 2-3 MCI Cel iw were held at a potential of · NO m\ Fo acleeve a baseline or leferenee point for the peak outward tad current, a strip to ~ 50 m\ for 500 rns o;-ss used This was followed by a depo'arrmg step to -?-2fl mV for 2 s to drive the channels to the inactivated state, Λ step back to -50 snV for 2s allowed die inactivation to be relieved and peak hERG current to he measured Pulses were repeated ssure as v) 10 n fetal *1 R< 1 out tcat was r « ava λ! o\ the d’tfo -eree Ivwv con -he peak cures,t the repoLu’Kurv - Ό mv, \wp and t w ,\wJ ne cutter, at -c0 m\ Rst ,n„Jes (up to 10 μΜ t, which included the peptides and peptide conjugates described hefeiru were mixed into the cMiueeduler recording buffer coonumne, 0 1 A> bovnu serum albumin {BSA'> and subsequently transferred io glass perfusion resort oirs. Electronic pinch νύν/s controlled the flow of the test .irticlv trow the reservoirs onto the cell being recorded. If'50 s a lues and curve fits wete estimated using the four parameter logistic fit of XI lit software Hie hERCI channel inhibitor, cisapride, was used to validate the assay, •; f00?5dj; Cotmiersereetis a^tfof ealeiunnactivated potassium channels human IRC# End BKXa h\ con sen mm! whole edl patch damp eleetroplrysiologs. < HO IKC'a and Bkt'a cell Ones were obtained born BioEoeus DPI tA Galapagos Company k One to 2 drops of the MKCai or BKCtJ cell suspension is added to a 35 tom poly-d-lysiue coated cover skip for ovemsght incubation before electrophysiology experiments. Whole-cell currents were recorded front single cells by using tight GO seal configuration of the patch-clamp technique, \ mm covet sup xv^s wwoe'vd to the iovo'diug stage *,!♦,, a tsuw, and replacing the culture medium with the cxlmecllular recopdiftg buffer eontammg 135 mM NaCI, 5 rnM KC1, 1 8 m\t ί a( K ill m\f HI FEE. and 5 mM Ulneow ipi 1 was adjusted to 7,40 with NaOl I atul mOsrnk Cells were continuously perfused with the extracellular recording buffer via one of the glass cap! 1 lanes arranged In parallel and attached to a motorized rock which places Ηκ glass capillary directly on top of the cell being recorded.

The recording pipette solution voutained 130 rnM potassium aspurtaux I mM Mgt’fo I 2o mM CaCI>, 2 mM EGTA, 2 mM Mg-Λ If Arid 10 mM IfERES adtusted to pH 7,40 with EOM send-o^tohrity act at 230 mCKm tsperimenis were performed at room icmpemturo and recurο,ονί K,dng My I vie la rip 700 \ '· mudsc?’ t Mo lee u Lx Dev Ly- lix ) ( oils wen- held at potential of-SO mV. Both BK and IK currents were activated as calcium ion diffused into··

Ik cdl from leemeing pipette -mb. sen Acto./son ot the e<* ^ mm dependent outward xUassium euncm by calc?um d.fu ·* on general y takes 3 to c mm tor full ucuyuuock t ixvxd cm rents w ere continuously monitored at holding pokntial of -t-50 raV he tore and durmg mug explore Alternate eh, 400 m* voltage ramps from ~ 120 toxbO mV were repealed once c\m 10 \ to eiKuacxsu'e tlx current voltage relation tor both channels before :-aad during drug exposure. Test articles (up to 10 μΜκ which included the peptides and peptide conjugate·, described hewm. were mmed into die exlracelhdar recordim» buffer containing 0 ΐ 'M bovine serum albumm tBS-\) and subsequently transferred to glass "!< ? I isio' * vrvoirs flcctro no ρ x o valves , oofrohed the dow of the test mlK'le^ than the wxa 'sonio the cell being xamded Pipette leostanees were typical l\ 2~3 MO,. iCIfti-valu-.'S and curve fits were estimated using the four parathoteriogjstk fir. of X List software, \ IKCa and BK peptide inhibitor, charybdotoxm 1100 uVlh was applied ut tlx eonelnsum of he assay procedures tor pharmacologteal validation of the assay.

100755] BvampteJ :: :[0|175ft] AMPS-a&amp;LII FushMS

[00757j I'he AMP5 lTO»mimeix. peptideav.as genetically fused to ants-KUi antibodies of the i’ λ /upon m ail to?/" possible i/nor ,d fusion configurations i represented scbeniatienliy m Figue IF-IK: b?gum 15s.uc,,A icirnraliv ?, wc and ( -tetminaih fused to both immuiiGglohoim light chain monomers and to both immunoglobulin heavy chain motsoinets, and was expressed in mammalian ?(IK)) cells The fusions w ere then purified by protein A chromatograph·*’ (G£ Life Sciences» using 10 column volum·-·** of DaibcecoLs PBS \wtlx>ut divalent cations as the wash, buffer and 100 ruM. acetic acid as the elution buffer at /"€, The elution peak was iv-oVd based os f>„ ehrom dogr, m aid the f 11 was raised to - e,0 using 7 VI Tos base. The pool was then diluted with .it )e<u<t 4 volumes of water and then loaded on ίο an SPdHP scpharo'-c column iGF I ilc Sciences'' ,,m>,i washed with 10 column volume** of S-Buffer \ 130 mM av.etie acid, pi I 3 0, iolhmed l?y elution UMug a 20 column volume gradient to Ρ0Μ S-Buffer B <20 mM acetic acid, I M NaCI, pH 5,0) at AC. A pool was made bused on the chromatogram and the material was dblyred against >20 volumes of tO mM acetic acid, 9% sucrose. pH 5,0 using 10 kl>a $iide-A-Lyzers (Pierces at 4 C\ The dialyzed materia! was then filtered through a 0.22 pm cellulose acetate filter and concentration was determined by the absorbance at. 2H0 tun. Injected 50 gg of each antibody along with an uniused control on to a Phcnomcncx Slid? 5000 column (7.8 % 300 mm) in 50 rn.M NaH2P04 pH 6,5. 250 mM NaC! at developed at 1 ml/min observing the absorbance at 2S0 nni (Figure 391. Iliach antibody was analyzed using a I ,Q mm Tri«« glycine 4«20*a SDS-PAGE (Moves: ( developed a.t 220V using reducing and non-reducing loading buffers and staining with QuiekBlae (Boston Biologieals) (Figure 40A-Εκ and the masses wore determined by LC-MB (Figure 41 A"D), |00?58) The components of the canons aivIJ I 120m lgG2-AMP5„ AMPi-aKLH 120,6 lgG2, aKLH i 70.6 hlgG: (N20?Q>-\MP5- fct( He) Poop fusion, and AMP.vaKI.H 120.0 Kappa embodiments include the following polypeptide umno-nerm :00750] {a) aKLH 120.0 kappa Li (Shi) ID NO:2H, above): lOtrPO] (b) aKLH )..20.6 IgG2 H.G (SEQ ID N():20. above); 01076I) 1C)aKLH 120.6 IgG 1 HGiSEQID MO:34, above); [00762] (A) aKOT T20JIgGS HC'-AiupS fiavingfefolfowmg mmno acid sengenede M DM RVPAQLLGLLL LWi.RGARCQYQLYQSGA EVKKPGASYK.YSCK ANGYTF TOT' H ΜHVVVRQAPGQGLEWMGWfNPNSGGTNYAQKFQGEVTMTRDT$l8'! AYMSiiiARLRb DDTA\AAT;ARDRGSYYAFDP\\'GOCrrLVT\'SSAS'rKGFSVLPi..AP('SKbTSPSTAAI.. G(l..VKDyidH::iAH'V8\VNSGA)..1SGVH rf PAVLQSSGLYSLSSVV1 YPSSNFGTQTY Γ CNVOHKPSNTKVDKTVERKCWECWYA’IXiVEVHNAKTKPREEQF^STFRVVSVLT Y VI IQDWf. N(iK.EVKCKVSPPCI>.M>PVAGr,SVH..FPPKPKDTI.MISR‘1 PEVTCVV VDVS H F DP E VQ FNN KOI. PAP I If KT1S K.TKG QPIl E PQY Y TP P PS RE E k! TK.N Q V S LT CL V KG F\ PSDl^VEWESNGQPENNVKTTPPMLDSDGSm VSKi..TV‘DKSR\VGQGNVFSCSVMH EA UiN H 5' 1QKS1. SL.SPGGGGGGQGCSSGGPit. RLWQQCRRAQHS/. 18EQ 1D. NO: 324): j.00763] (d) Amp5-a.Kt.H )20.6 lgG2 Hi' (SEQ ID N'0:332) having the following annuo acid serpj.en.ee: MDMRYPAQLLOLLLLWLKGARf QGCSSGGP't YRHVVQQCRRAQHSGGGGGOV0L V QSG AIΓ V KK.PGAS VK VSCKASG YTFTG YH MBW VR QA PGQGL PPAiGW IN PN SGGTN YAQKFQGRV ΓΜΊ 'RDTSIS Γ A V M Hi SR LRSDDTAY Y YCAR DRGSY Y WFDPWGQGT i. VTVSSASTKGPSVFPLAreSKSTSGGTAALGC LYivDYFPEPVTY'SWNSGALTSGYHTF' PAY LQSSG f.Y St AS VVT VPSSSt .GTQT V TCN VN11 KPS NTKYDKRV EPKSCDKTi i TCPP CPAPELLGGPSYFLFPPKPKDTLΜ1 SRTPEVI{ VVYDYSHEDPEVKFAVVYVDG VEVH N A KJ KPRE EOYQSTY RV Y'SVt .1V LHQDW LNG KE YKCKVSN K ALP APIE KTISK A KG QPREPQVY'TLPPSREFMTKNQVSL'frLYKGF'S'PSDlAVLWESNGQPI'NNYK'tlPPVL DSDGSl· FFYSKLTYDkSE WQQGN V'FSC SVMHI: A I. HNBYTQKSLSLSPG ; tSEQ ID NQ:332). 100264] (c) aKLH )20.0 iilyG! N29?Q-Amp5 FciCH3) Loop having the tot lowing amino acid stajiiencc:

MDMKYPAQUAiLLLLWLRGARC'QYQLYQSGAEVKKPGASYKYSCKASG^TPTGYH MB\VVRQ APGQOLP\YMGWi NPC8GGTAYAQKPGijPAGAl ΓROTSI.STAYMP'LSRLRS DDT.-VV YYC'AR DRGSYY WFDPWG0G7L VTYSSASTKGPSYFPL APSSKSTSGGTAAL GCLVKDYFPEPVTYSWNSGALTSGVHTFPAYLQSSGLYSLSSVVrVPSSSLGTQTYiC NVNIiKPSN ί KV DKRYEPKSC DKTI n'CPPCPAPELLGGPSVPLFPPKPKDTLMISRTPE YTCVYVDVSHLDPfr.VKFNWAADGVLYHNAKTKPRFLQYQS'n-'RVVSVLTVIHQD WLNGKEYKCKVSNK. ALPAPtEKTISKAKGQPREPQYYTt.PPSREEMGGQGC'SSGGPT LRFWQOC'RkAQHSGGTKNQVSLTn..YKGr'YPSDtAVE\VFSNGQPf:.NN‘YKTTPPVLD $OCiSFFtYSK LT VDKSRWQQG NV FSCSY ΜΉ BALHNΗY TOKS fS LSPG -·; (SFQ (D NO .vi ί F

[01G651 (0: Amp>a K1 B 129,6 kappas LC2po!vpepi!:dP mate imteg. the following nteM acsd scejuentc. MDMRYPA^)U.Gi.LU.WLRGARCQGCSSGGin'LRE\V(X)CRRAQHSGGGGGDIQMT Q\ PSS ES ASYGDR YTVK'R ASQG ί RND LOW YiJQK.PGK APKRI. ϊ Y A ASSL. QSG Y PS RPS GSGSGTLFTL'nSSt.QPEDFATyYCLQHNSyPL'n'GGGl'KS'LiKRTVAAPSYnFPPSDF QLKSG'fASVYCLLNNFYPREAKYOWKYDNAL.QSGNSQESYTEGDSK.DSTYSLSSTL TlSkADYlHKHKVYACEiV'rHQGLSSPV'l KSFNRGLC : (SFQ ID KG:342).

[O0?6Pj aKLH Ι20.6~ΙΰΟ2 Hojvv ChamtHC)-ANlPS Mammalian Expression. The deteod aKLH 120,6 IgG2 Dtte-AMPS product is a full antibody with the AMPS peptide fused to the € term isms of one heavy chain, configured us In the. schematic- representation of Figure IF, and waj> assembled by two separate rounds of Polymerase Chain Reaction (PCR) using PEG High Fidelity Ultra, by Stratagcne. The first round of PCR generated two fragments. VKisp-sKLH 120.6 |gG2 HC DcsK.-G5 arid GS-AMP5 fragment. The oligo's and PCR templates that were used to generate those fragments were SEQ ID NODES and 326, below. Polymerase Chain Reaction I (PCR 1) generated the VK Ksp-aKLH 120,6 igG2 HC DesR-GS tragrnem and existing DN A that coded lor the YK.isp--aK.LH 120.6 !gG2 DesK Nil' peptide was used as template.

[01702] Forward pitmer,!se<|Ue»e4'W^: [00766] A AG CTC GAG G VC GaC TAG ACC ACC A 7 G GAG ATG aGG GTG CCC GCT CAG CrC CTG GGG CT / (SFQ ID NOG25); and [00769;]; Reverse Primer sequence was; [00770] GCC GCT GCT GCA GCC CTG A( t' ACC' AC C TC'C ACC' ACC CGG AGP, CAG GGA GAG 7 IS EG ID NOG26), [ 00? ? 1 ] The smfoo aeid aequenee etieoded. by the ¥Klep-aKCIil 20,6 igU2 HC DesK -Cm tragntenCi9Bdr^di,i0fin: PCitl was;:. MDMIU‘PAQLLGLLLL\YLRGARCQVQLVQ$GAEYKKPGASYKYSCKEA$CiYTF!OYH MHVVVR0APG0GO'WMGWfNPN.SGGTKYAGK.FQGRVTMTRf:vVSIS'i AYMFLSRLRS DDTAVYYCARDRGSYY\U;DPWGOGTLVTVSSASTK.GPSVTPi.APrSK9TSES7AAE CiCLYKDYFPiilPYTVSWNSGALTSGV H1TPAV1..QSSGLYSLRSV\”| YPSSNFGTQTY Γ CN νΤ>ΕϊΚ.Ρ8Ν’ϊ K VDiCrYERKCCVECPPCFAPPYAGPSYFLFPPKPKDTLYlhSRTPBVTC Y V V DYSH F DP FYQF N \VY VDG YE YH A A K.1 K PR f. F.Qf ASTER VYS VI TYVHQDWf .NG KH: \ K.CKVS'NRGtPAPIERTiSKTKGQPRrPQY Y D..PP\R El: A11 RNQYSi. 1 CLVK.GF V P SDIA YEWESNGQPE NN YΚΎΤPPM 1 DPDGSFFEYSK I TVDKSRWQQGN YIYCAY M HE' Ai.HNili TQKSLSLSPGGGGGGQGC '(SEQ ID NO.32/5.

[OOPE] Polymerase Chain Reaction 2 tPCRE} generated the G5-AME? fragment fSEQ ID NG:330i. and existing DN.A that coded for the AMPS pots peptide was used as template with the following primers sequence: ;007?3] Forward primer sequence was n C TOO ('TG TGT COG GOT GGT GGA GO T GOT GG1 GAG GGf' ICC AGC AGO' GGC'7 miQ ID NOG2K); and :0()074] RewTs.' pnPUT V'gUriX.e Wit St CTA CTA GCG GCC GCT GAG CTA TGC TGA GOG CGG GG (SEQ ID NOG2G The amino acid sequence encoded by {he fragment generated from FCR2 was: [00- :751 LS LSPGGGGCIGQGCSSGGPTL REWQQCRR AQHS'7 {SEQ ID NQC30).

[00776] The product w ere run on a PG agarose ge! The bunds were punched for an agarose plug and the plugs were placed in a fresh PGR reaction lube The agarose plugs were then amplified in PGR.) usmg the outside primers SEQ ID NO.'eps and SEQ ID NO;329.

The final PCR product was run on a i'G agarose gel The correct sice product vow cm ouh then gel pnrii'kd by Qiagen's Gd Purification Kb. The. purified get fragment of VK1 sp-aKd.i 1' 120 <7 GG2 Desk FKGG.G 4ΛΙΡ5 was digested wish resin».7km enzymes Sail and Noth arsd tltcn the digested product was port tied by QiagcnT PER Purification Kit. At the same time. ρ'ΓΪ'5 Vector tan Amgen vector containing a C'MV promoter and Roly A tub) was cut by Sad arsd Noth The pTT5 sector was run out on a 1"» agarose ge! and the. larger fragment was cut oni and gel purified by Qlagcn's Gel Purification Kit, The VKlsp-ai\l.H 120.6 jgG2 DesK. HC'-Cid- AMPe product was ligated to the large vector fragment and transformed into OncShot'to Top 10 bacterial cells. The DMAs from transformed bacteria) colonists were isolated and submitted for sequence analysis. One correct clone was selected for large scale pl asm id punfi ea dot:..

[00777] 'The final pTT5:VKiap-aKLH )20.h~!gG2 Desk HC-GS-AMPS:eonsmietaieoded the following igG2 DesK. iI(T-AMP5 polypeptide:

MDMRVPAQLl.G1. LL1.WI. RCARC'OvOi..\'OSGAEVK.KPGAS YKVSCK..ASG YTETG YH Mil W'VRQ A PGQG t. GVYMG Wj NPNSGGT NY AQK P QGKYTMT RDTS1STA VM F.i.SRLRS DPI AVI VGARPROSY YWFDPWGQGT LVTYSSASTKGPSYFPL APCSRS1SLSTAA 1.. GCL VK DYFPEP \ TVSWNSG AL TSGVH1TFAV LQSSG LI' SI AS YV T \ FSSNFGTO TY T CNVDHKlhSN"ΓΚYDKTYERKCCVR'PP( PAPPY AGPS VT mils.PKD1 L7USRTPFYTC VV VDYSH h OPE VQEN vv Y\; DG VPV RN AKTKPR I." EQENSTTR 7 VSVLTV YHQD Wh HC K 1:. Y KC'K.VS N R.C ί 1..ΡΛΡ1 EKT1SK1 KGQPREPQ VY FL PI foKP.E ΜΊΚ NQ VS LTCLY KG ΕΥ P SD1A V'EVVESNOQPENNYKTTPPMl.DSDGSFFLYSK’.LTVDKSR WQQGN VFSCSVMHh ALHNHYTQKSLSLSPGGGGGGQGCS5GGPTLR.EWQQCRR.AQiiS·/ (SEQ ID ΝΟΑίΓΕ [00778] AMP3-ygKjJ;I.120 ;0(}'?7O| {'be desired AMP.TaKLH 120.6 GG2 DexK HC product (SEQ IP ;\0;G2, above.) including the monomer is a full antibody with the AMP5 peptide fused tit Ihe N-terminus of one heavy chain, configured as in schematic renresematton Figure U, and was esse reined by two separate rounds of PCR using Pf U High Fidelity Ultra, by St rat age no The first round of PCR generated three fragments. VK.L\p-AMP5. AMPS-G5. and Co-aKLH 120.6 lg(12 DesK HO fragment. The on go's and PCR tern plates that were used to generate these, fragments are listed below, PoSyenmoe Chain Reaction 1 (PC R: } generated the VKisp-AMF? end <.\hiing DMA which axled for the V'K Isp was used as template. Note this fragment was also used in construction of the Vk | y-v AMPS-GS-oX El I S 2d.6 Kappa LC.

[007ί0|: The forward, pooler sequence wad:

Γ007Ηi] ACi CTC GAG GTC GAtTAG ACC ACC’ ATG GAG ATG AGO'OTG OX GCT CAG CTC CTG GGG Civ (SEQ ID NO.J25J: and [00782] The reverse? primer sequence was: :00783] GCC GCT GCT GCA GCC CT G ACA TCT GGC ACC TCI CAA CC\ iSHQ ID 180:533). The amino acid sequence encoded by the fragment generated from PCR 1 was: mdmrym;® (SBQ, ID NO'554).

[00784] PCR2 generated the AMPSXS and existing DN:A which coded. Idr the AMPS peptide was used us template.

[0i!7i5:| Forward Primer sequence was:; [00786] GGT IGA GAG GTG CCA: GAT' GTC AGG GCT GCA GCA GCG GCC (SEQ ID NO:3351; and [0078?] The reverse primer sequetteg was:. i 007SK j CAG C i'G CAC CTG ACC ACC ACC ICC ACC GCT ATG CTG AGC GOG// (SEQ ID N 0:336)-.

[00789] The amino acid sequence encoded by the fragment generated from PCR2 was: WLRGARCQGCSSGGPTLRKWQQCRRAOJ-lSGGGGGQVQi..V//(Sf:Q1DNO:337).

[00790] PCR3 genersied120,0 igG2 OcsK HC, and casting DMA which coded for the akd.H 120.0 lgG2 HC (SFQ JO \0:29) monomer was used as template. 1007011 The forward primer sequence was: vGGC OCTCiAOΟΆΤ OGT GGA GOT GGTGGTCAG GTG CAGCIXIAiSEQ iD NO:338); and [00792] Ί he (ΤΑ CFAGCG GC(. GCTCAA CU GGA GAC AGO GAG AYfSEQ IONOG39): i [00~OAj T1 e ammo new! seqtu ns o em odod fry tin* fragment generated from IT 'R 3 v,<*s n AQi f$GGGGGQ\ Ql VQSG A PYKKPG ASYK VSCKASGVTFTGYGMI3 WYlOAPGQG LB WMGW1NPNSGGTNVAQKFQGR\ TMTRDTSISTA YMELSRUGOTTAVYYCARD RGSYY'\\TOP\\OQGTLVTYSSASlXGPSVFFLAPSSKSTSd<#AAi:i6LfKaWM?y;· TYSW NSGA1 PSGV\ 1'ΠΤΑVI .QXSG LYSI SSVYTVPSSSI GTQTYICK\'kΗK PSNTK V DKRV'PPKSi.'GKTHICKPCPAPFLLGGPSVFLFPPKPKDI'LMISRTPEYirV'YX’DVSHfc DPEVK FN W Y VDGVE VH N? AKTtC PR F.EQYQST YR YVS V LTV l HQDWt \i *KEYICC|CV· SNKALPA PIF.KTISKAKGQPREPQVYTLPPSR EEMTK NQVSLTCLVRGFY PSfcflW^' ESNGQPENNYK'nPPVTOSDGSFPLYSKLTVDkSRWQQGKVFSrSVMHEAtHNin· Γ gKSL frl.YPG (SFQ ID NO.74()1.

[007941 The products were run on a FS> agarose gel. The bands were pnnehed for an agarose plug and (he plugs wore placed in a fresh PCR reaction fuhe. The agarose plugs were then ampli fied by PCRd using the outside primers SEQ ID 190:325 afrd SiQ ift 180:039:

The final PCR product was mn on a 1% agarose gel. The correct size product wMCMt ddh thengeipurified by Qiagen's Got Puri front ion Kit. The: purified gel tragmsnit. of YMisp-AViP.i-GS'-aKLH 120,9 !gG2 Desk HC was digested with restriction enzymes Sail and V?th and then (he digested product was purified by QiagenA PCR Purification kn. At the sa;ne time. pTTS\se(or tan Amge-t\eetor containing a CM \ pronto ter and Pols Λ mil t w as cut h'- sad and \oti 1 ό pfTh seevt η n ou onF ays rose gel U tse lame' nsgmem eont and g^t p. *r ed e\ Q ager ^ t Ad Pnrite . n· s )\n, The \ r\ I sp- \MP5~G '~uR t 11 \2V 6 IjG2 Desk 1 P' pjocroe: w m hg nee A> he i u„e scev* f ,w.nem am n u’sfotn ed uto CKii^i'o i I o' <'» ojcms a, eel-> The D\ ' s turn; u Mon'cv IvUewu' who ue>. weie isolated and submitted for sequence analysis. One correct clone '-'.as selected for large sea!# plasmidputniieatkMt, The final ρ'1Ύ5* Vk Isp-AMP5«G5-aKU-l l20.6~?gG2 Desk HC construct encoded rite A\!F5-lgG2 Desk HC polypeptide tSh'Q ID \OU32. oxwel, [00795] aK.lJ-1 120.6 aciycoayfatcri hl*Gl-AMP5 Fc(CH3) Loop Heavy ChaitdHC) Mammalian Expression, Tire desired aKLH 120.6 IgGI ^glycosylated i N297Q)-AMP5-Fc HC product comprising HCiuaion monomer SBQ1D N0:MI {ahpveftra fell antibody with: the Atnp5 pepridc inserted into the Clin domain of the IgG 1 (N297Q) fc Desk heavy chain, configured as sehcmatictdly represented in Figure 1 hi 1 he VKisp-aKlii 120 6 IgGI (N297QTAMP5- Fc Desk HC product was ordered by the synthetic gene company,. Bine Heron, The final product was generated by digesting the Yk Isp-eKTH 120.6 jgGUN247QV AMPS- Fc DcsK HC with its corresponding restriction enzymes. Sail and Noth rhd#gcAted product was nut on a FV agaro,w gel. The fragment was ent out, gel punbed by Qutgen\

Gd Puriftcsbon Kit, At the same time, p 775 Vector (an Amgen vector containing aCMV promoter ami Pols \ tail! was cut by Sail and Noth The pTT5 sector was ont out on a id* agarose gel and the larger fragment was cutout and gel purified by Qtagen *t*$;

Ktt 7he purified gel b tgr.e-u of «ΚΙΒ 129 6 igu I t\x>/Q}-AMP5 Fc DcsK Ht, was ligated to the large secror fragment and muKionned iuto OneShoU* 1<\Μ0 bacterial cells.

The DMA s from transformed bacterial iauqmcs were isolated «ml syhminedilbi'gecgHtmcc anal) so One correct clone was selected rorSurge scale plasmid pun fie Aron. The final pllo \ K'sp-aKUi 1206 igvi HMbV'Oi AMP*' 1 c Desk HC commad encodes tor the aKt H 120 6 IgGI {N297Q}-AYlP5~DesK polypeptide fusion monomeriMiQ ID UOfikil, above], 00?%] A-TiFyiro aKUi21X.Light ChatnfTCiManimallanExpression. The desired AMPf-akLH 120.6 Kappa L( pnxluct is a full ami bods with AMPS peptide fused to •Jm N-ten n u- «l one semi J us x bo mi more mu sM 0U>\O 142 ahoxch t-osifisjuredas scbc:rat,',alA represented η, 1 tear/ H, and was assembled by two separate rounds of Pols os mese Chain Reaction {PC 'R > using PF1 1 bglt fidelity Ultra, by Stratagene. The first round <>, PUR genetitted three fragments which included. Yklsp-\MP5, ΛΜΡ5-05. and G5-aKIJl 120 6 Kappa 1 C. The oligo's and ientphdes used tor PCR reactions to generate the fragments are listed below. The fragment that generated dtc VKlsp-AMPi is the same fragment dun was used in construction of the AMPfmKLH \ 20.6 !gG2 DesK HC. and is described in dee section. Poiyermaxe Chain Reardon 2tPCR2i generated die AMP5-G5 fragment end existing ON A that coded for the ΛΜΡ5 peptide ο as used as lentpUie. Forward primer sequence was (SEQ ID NO; 1 15, above; and rcvcr.sc primer -ieqnee.ee wsw C'TG GOT CAT C'TG GAT G'iT ACC .ACT' ACC I CC ACC' OCT ATG CTG AGC UCG · {SCO ID NO;344). Ί he amino acid sequence encoded by the fragment generated from PGR 2 a as;

WLRGA RC'OGC SSGGPTLR LWQQCRR AQHSGGCGGDIOM 'i'O iSb'Q ID AO.3-GI

[OtV'07] PCR3 generated the G5-uKLH I.)0.6-Kappa I.C fragment and existing ONA that coded for the aKU-i 120.6 Kappa I C (SEQ ID NOTtS} was used as template.

[00:708:] The forward primer sequence was;: GG(. GC Γ (..aG C. A I AGC GG I GGA GG I GG I GGI GAG 4 ΓΟ (. AG A7G ACC C AG ' SEQ ID NO:34os; and [00700 ] the reverse: primerxef uehee was;; Λ At' CGI ΤΓΑ Λ AC' GOG Gi'T GOT ΓΑΛ CAC 1X1' GC C (" CG TTG ΛΛ. ' i'Sii.Q ID NO :34'G, T he peptide sequence m’ the fragment generated from PCR3 was:

RAOHSGGGGGDIQMTOSPSSLSASVGDRVniCRASQOIRNDI.GWVQQKPGKAPKR 1.1Y AASNLQSG VP$R f SGSGSGT E FT L 148$ LQPE DP A TV YCL QHNSY Rf.TPGGGT KY El KRTVAAf^VFlFPPSDEQlKSCiTASVVCLLNNTYPREAKVQWKVDNALQSGNSQESV

ri':QDSKDS7Y'SLSS1’Ll'LEK.ADYEKJ IK.YYAC'EYTi It IGLSSFPG KSFNRGGi.':: (SEQ ID NO :3,41().

[OGKOii] I he products w ere run on a !.. agarose gel flic bands were punehed for an agarose ping end the plugs were placed in a fresh PCR reaction tube. The agarose pings neve ihen amplified by PCR4 using the outside primers SEQ ID NO' 525 arid SI..Q ID NO.eh". The final PC R produei was run on a !'*« agarose gd. The us meet sire product was cut oni. then gel pur-tied by QiagersN Gel Purification Kit Hie purified gel fragment ofYKisp* AYIPf-Gn-aRLH :20.6 Kappa LG was digested sGth festrsenon enzymes Sail and Nod... and then the digested product was purified by QG.gen's FOR Purification Kir. .At the same time. pTT5 \ ector i an Amgen sector ermtaimng a CM V promoter and Poly A util) w as cut by Sail and Noth The pTT5 sector was run out on a IN. agarose gel and the larger fragment was cut our and gel purified h> QtugenN Gel Purification Kit. The VK1 sp-ΛMP5-G5-aKi-H 120 ft Kappa LF product w us ligated to the large vector fragment and Trans termed into OncShotW' Top 10 bacteria. DNAs (rum transformed bacterial columns were belated and .submitted in·' sequence analysis, One correct clone was selected tor large scaled plasmid purification. The dual pTl'5: V'X.lsp--AMP5-G5--nKFH M0.0-Ka.ppa I.C construe! encoded an AMP5-Kappa LC polypeptide fusion monomer iSEQ ID NO;342. abovet.

[00801 j Iianskin t at <- <. < < v s < * ioT- * u'Ki\Rs t su-n^Pll ί lAdyeihyknlmine, linear 25 kDa, I mg ml s-enk stock sole turn. pH TO, Pulyseiencesk The .793-61-. cell density was I I X PT be I ore hvmsfectiun, than none, 500 tnierograms of DNA (heavy dune and Sight c I tarn DNA, I; I ratio! per bier ol cells transfected. The DNA was added to 50 mi 70¾ Free St) lo media (Invitcogen) and eorobnied with 1.5 ml of ΡΓ I solutiorg vortesed mildly anti then incubated 15 minutes at room temperature. 1 he cdH were transfeded by adding the whole PE l· DNA mixture to die culture, t el Is were then incubated on a shaker (170 -pmi at A C eon tain me 5% (O' Tor 24 hours. T rvntoue NS f IVAniScienoe bam 20% in f rceStsic media) was then added to a fuitd concentration of Odd. and the tneubation w an continued for 5 days. The condition medium w as harvested at day 5 by centrifuge at 4000* rpm followed by filtration though a Ode urn filter (Cuming lew. ). :00507] Tire fusions were then purified by protein Λ chromatography (GH 1..:fc Sciences) using 10 column volumes of Dtubccco's PBS w ithout diva.lem canons ns the wash buffer and 100 mM acetic acid as the eiut-on buffer at " A..'. The elution peak was pooled baaed on die ehronuuogram and the pH was raised to about 5.0 using 7 M Iris base. The pool was then diluted w ith at least 4 volumes of water and then loaded on to tut SP-HP sepharose column f G£: l.iie Sciences) and washed with id column volumes of S-B-iiier Λ Γ20 mM acetic so id, nl I TO, ibllowed by elution using a 70 column volume gradient to fd'G S-Buffer B )70 m\l acetic acid. 1 M NaCL pH 5.0) at 7°0. A poo! was made based on the chromatogram and the material was dialyzed against - .20 volume*; of 10 mM ncetk acid, 9”« sucrose. pH 5.0, using Id kDa Sbde-A-Lyzers (Pierce) at TT The dialyzed material was then filtered though a 0.22 um cellulose acetate fiber and concentration was determined by die absorbance ut 250 nm Injected 50 gg of each antibody .along w ith tut ui; fused control on to a Phenomestex SET 3000 column t 7.8 x 300 mm) tn 50 mM NaFFPO.'., pH 0.5, 250 rnhi N;.t( k developed ut I ml min, delecting the absorbance at 2h0 nm t Figure 39). Ail five antibodies showed the i:\pev ted retention time for molecules of their size showing that very tittle aggregate was present Each antibody was ynalyxed using a 1 .0 mm Tnv-giyewe 4--20% SDS-PAGE (keweM dev,doped a? 220V u.ung reducing and non-ivdnehm loading buffers end staining wifi; Quirk Blue (Boston Biologicals; Figure -VVV-Ei, and die masses were d-Gcrmined by 1C-ME (Figure A I A-Dk in a iypioei exponmenL 10 eg of ibe sample was reduced in 25 μ! of 8 VJ GdFH i 50m XI I ris (pH G5i tor 30 nun at 35 ( , then the reduced material wns chromatographed through a A iders Mursprep micro desalting column (2.1 x S ram) using an Aeqmty 1JPLC system (soKem A was 0 1 % formic acid In water end solvent B was 0. BE formic acid in acetoumdci. 1 lie column uas equilibrated with S % solvent B at a flow rate 0.2 mi per min at 80 °(E arid upon sample introduction., die column was washed with EE? B For I mm before the protein was eluted using a linear gradient from 5 to 40% B os or 10 rain. The column effluent was introduced into a Waters Umc-of-iiigb* LOT premier mass apectrometei for mass measurement. Cxi ions {3 mg Cxi per ini in 50% isopropanol) w as used ax lock muss. The mass spectrum was dcconvoiutcd using the ViioiEnti software supplied with the instrument. The SDS-FAGE analysis demonstrated that all the expected quateinaiv stme-ures weie formed, and the mass spectral analysis demonstrates that die expected fusions were present in the purified molecules. Taken together these dam. indicate, that fusions can be made with any of the four possible bi-terminal or (.'-terminal fusion configurations of the monomers of aKLH 120.6 antibody, ns well as He domain internal loop insens (see, Figure IF-IN and figure45 schematic represemaimns).

[0Q803J liampld IQ: [(h')KOdJ Ex4~aXLH Ab Fushmx 100805j The Excndm-4 peptide (HGFGTFTSDL SRQMFEEAVR I..FIEWLK.NGG PSSGAPPPS.·' S1-.Q ID NOG46) was genetically fused to N-temduus of the light chain of the anli-KLH 120.6 antibody through the IkG linker (designated 'i:\-4-lkG-aKLM 120.6--Ab"and expressed in mamma!urn ceils. Figure 42 is a schematic map of die Fxendtn-d {;15>%'> | kG-aKLH 120,6 LC fusion construct. Γ00Κ06] The eomuonems of the Ex-4- IkG-aXLH 120,6-Ab fusion included the iollowmg monomers: 100807] (a) Es-4-ikG-aRUi 1.20.6 kappa. LC having the toiiowiug amino acid sequence: M DM R VP AQLLGELLL WLRG Λ RC HG EOT ETSDE SKQM E EE A VR l R E W(, KNGG PSSGAPFPSG SGSATGGSG5GASSGSGSAT GSDIQMTQSP SSLSASVGDR VIn'CRASQG 1RNDLGWYQQKPG1vAPK.RU YAASSLQSGV PSRFSGSGSG TEFFiTiSSLQPEDFATYYCLQKNSYPLTFGGGTKViirKR TVAAPSVTII' PPSDEQEKSG TaSVVCEENNFYPREAKYOW KYQNALQSGN SQESVTCQDS KDSTYSLSST ΕΤΕΕΚΑΙΙΛΈΙΟΙΚΥΎ AOEYTif QGLSSPVTKS FNRGEC7/ {SEQ ID ΝΟ:3$5κ and iOOSOKI ib) aKl.H 120.6 tgG2 HC (SEQ ID NO:FA above).

[09809j The desired Ex-TikGmK.LIi 120.6-Ab product a full antibody configured with the Ex -4 peptide fixed to t.l ;c N-termini of both light chains (see. schematic representation its figure IK>. The ratio of Ex-Might chmmkeavy chain Has 1:1, The isolation and cloning oft be genes encoding X eno VI ousts i>: hybridoma expressing aKLii 120.6 monoclonal anybody 1 20.6 heav y and light, efndrss have been described in Example 1. and Example -f above. Its native signal peptides have been cep laced by the ΥΚΙ.ΌΓ2 peptide {MDMR VPAQLLGl. LLL WLRG ARC SEQ 11.) NOHO.G as described above. DNA fragments encoding aKLH i 20.6 EC tSEO R> NO:2M and aK.LH 120,6 HC lgG.2 (SEQ ID NO'29) monomers were individually clotted Into mammalian expression vector pTT5 i An Amgen veoto· containing a CMV promoter end Poly Λ mil ) to generate pTT5:.aK,LH,i 20,.6-V'K'ISP-keppn Eight f.'haln(LC) eonstruct and ρΤΊ'5'uK.EH 120.6-VK loP-lgG2 Heavy Chain i Hi ; const met, respectively. Γ00Η10) A DNA bagmen·· (SEQ ID NO:YH, below) Hanked by Sail (S') and BamHI O') that comprises the Kozak sequence and the f rsi part of an ORE' that encompasses the Vl< 1 012 signal peptide (SEQ ID NO: 103), the Ex—1 {1-39) peptide (SEQ ID NO.349), and iho IkG linker peptide was synthesized and cloned by GcnScnpi (Piscaiavyay,, Nj) according to standard gene synthesis techniques. ball STC9AOfAGAGC:AOCST00ai3ATSAGSST€O0:C0ClM.oGf;fCeTGOGS0TeC::®GCTATTsTG GTOOASAOGTQGCASATGTOATOOGSrAGGSAAGaTTTAGAAeOGATOTQADGAIlAClAATGS AG SAAS AGOGAST ΪASASTST TCAT TG.fATS-SA If O AAS AAOSG CD® A€GG ATT AOTGOT SC T C1.,· otcG ί t v (.: e> AGt.,: 9 eax iO ailAA) AOy:-S: AC· 'itSA rev

GAGGf GGAGDGGGDGCM #3 SECl;: ID EG:351) •V·: ·*·:·. *yv>>x>«>: 'S'·'

BdMfl 003· 11] Iiv Band 11 (3' Mo \oti t3 ) fr ijaiww tSI Q fD Xu 368, Mow} that rexπs tbe Litter part of an GRF that consists of the mature aKIH l20.6-.Ab LC was amplified from the iiKIJf imJ-# f .C DNA template described above fpTI S-aKXIiltt^ylfcl^k^ppa *--*ghi Μφ a pair of oiigo printers::

ΛΑ I CKJ A f< \l( ( At) \tu ACC C.xCi H .SCO ID NO 352b and A \I to G

GtV CK 1 C \ \ ( At ΊΧΤt C «SCO d> M>' o31, iaeoordmg:.^4toditrd. FCRAeeiinicitCs, ΒΜΒΙ:

i.;G3T(Xt'ACA?CbAG/0'AA':;CCAi?;b:CCCACCC!'CCX:-Tc;TCTGCATCTGTA-SGAGA(;AGoGT : CACCGTCACTTGCCGGGCAAGTCA6GGCAITAGAAATGA3T?AGGCTGG?AICAGCAGAAAC

AGGl'TGAGCGGCAGTGGATCTGGGACAGAATTG&amp;GICTCACAATCAGCAGCCTGCAGCCTGA

; " >.‘'‘ΓΑ ^ t v " 'C TiX'. a ^ _ ' ·? “ \A’ V" r ” ’ """ '3GX .VM CM

GGAAGGTGGAGAECAAACGAACTGTGGCTGCACCAGCrGTCETCATCGTCCCGCCATCTGAT

GASCAGBTGAAATCTGGAACTGCCTCTGTTGEGTGCCTGCTGAATAACTGCIATCCCAGAGA )Gr , ΑΛΑ'" ACS'-IG ΧΑΜΑΛΑ' A,V !'u’ Ί AAV, >s 'V\ ΆΆ 'X AOVCObG' ; VC 'λμ'Α'Ά n ' <"*0 "X Ά i,rt <. VC ^ xv " GACTOCGAAAMGliAAAGTCTACGCCfGCGAAGTCACCCATCAGGGGCSAGCTCGCCCGf 3 CACAAAGAGCTTCOACAGGGGAGAGTGITGAGCGGCCGC// (SEQ ED «0:368} tA.>t 1 100812} Fite .synthetic Sali-BamHI fragment and the POR-ampiitled BumHl-Noti (ragmen! were diiesded by corresponding restriction: enxprtexpiproso gel and llpted into the Sail and Nor! cloning sites of the pTTS mammalian transient expression vector nccotiihi id standard molecular cloning above in Example si to : a^LH ! 20.64iCX[Lys 1bjEhR Ab) resulting in the expression vector plTS: Ex-4-1 kO-&amp;KLH 120,0 LC' containing a clone iSEQ ID NO; 354) that encodes the amino acid sequence of the

Ex-f-IkGatKLH 120.6 LO monunicr (¾ hh f\ -terminal VKi '012 sign a I peptides t'ShQ H.> NO;355).

Sail C'fa'aeo.rse..:edi'navee..Ohe>:'S :.a.va\.:vneh\n.\:S/j':Pn.o.;a/neeshAra' aAe.ams.rs.O,;V.;.e. i.'av >ofi -.ia t ’jt:’ Cjr'X'· 'ί ΐ a- ai2 a;i ΐ jrC_^ La ΦΑ-ΑφΑΑί iCi£CA£-CCf;M^'SCwiEtiASGAS£i^ TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCCjCCCGTCACi^.A&amp;AGCTTCAACAGGGG ACAsGCSCT:SAQSSSGA-SC/ / (EBa I© MEo3S4) ,^0:T···.^ [OOKI '>·} 1 tansies:· expression wan conducted witls these pair of expression veetots φϊΤ5:Βχ-·-Α I kC.CnK.tH 120.6 Li' ami pTT5; aixLH 120.6 Hi') u> ye n crate conditioned nsednot: for the purificadon of Lam IkG-aKtH 120.6-.'\b fuMnn, 1 he: human ernbryoun. Kidney 2Ό ceil line stsblv expressing F paten· Barr viru« Nudear Antigen- i t>>.v6b cells) v% a a obtained not n the MaOnttnl Research Cnnneil fMo; bread (. anad.i;. Cells a ere misnamed as sextan-free susptanmon cultures ns i;ga f 1 ~ medium s Ins iiiOgetn Carlsbad. C A) steep let ner: fed with (an Vi f .-glutamine (hts irrogen. ( arlshad, (.'A;, 1 .ΙΈ FAG Rlurouk Unvirfojfcn. Carlsbad. ("A - and 250 tig til Genuliein i Gvnrtgca, Carlsbad. CAf The suspension cell eulnuvs were maintained in Frier mover shake flask cultures The culture flasks were shaken at 65 rprn at M'X in a hurtnditted, 3Gf COy atmosphere. Λ stock soiMton. i I mg mb of 23-k Da linear PE I (Polyseiences, Warrington, ΡΛ) was prepared in water, acidified wuh HO to pH 2 0 until dissolved, then neutralized with NaOH, sictHbvd by fiHrution (0.2 -imt, nHqtmted. and stored at -20'C until used Tryptone N l was obtained in mi OrganoTechm S.A (TektuSeinnee, Q€, Canada) A sueck wH-tbon (20'.··», w v > mas prepared ?n freestyle medium b m itrogeot, Carlsbad, C'A>. sterilised ip. filtration through 0 2 pm filters, and .stored at 4 C until use 1 vpkxbly, transfection'·* w ere peribrrned at the IL wale Cells {203-όΕ? mere proven too a vmhie cell density of 1.1 X iff ceils ml then transfection complexes mere prepared ;n 1 10!" volume of the fmai culture volume. Eo; a 1-L transfection endure, iransfcetion cony Hexes mere prepared in 100 ml Π ~ basal medium, and 500 p.g pi umnd DNA theem ehxm and light chain Π\ \, bl maim vra*» first di=nit J m 100 mi Π7 medium Vika a 5 numne mederetm „u toom iemecmmre, : ,2 ml eu PFf s·Hutton v a*- added. Tin- complexes vwre * one red oddly, dam ineuHteJ lot 1'' mmtnes nt '\\>m teoipommre ilk s\0s n ere inn-dected ;u .ukbng the Uanxfcethtn complex mm n.> the eeiw n- the shale ibtak multure loom;·. sour hour' pm-u tumHeetton. i'cvptork M v\„x added v- the transteeied etsb'nie to a dud concentration m'odd ^ .uni me tkmsi'eeted odiums wete mamuuned on a shaker at d> rpro nt 2"" C ;n a htmndisA-u, 5Ά CO at:eosphere· u>: -mother 5 da>s .ipe: η iu'-j they n are uamestO The conditioned medmm v as hemes led by eemTdue, v urn it -HKU) rpnu and then steido Titered through u 2 urn fdtei H'oinme die t iOOHid] 1 he fusions mere then purified by protein Λ chromatography <GE Idle Sciences» using 10 column volumes of Dulbeceo's PBS without divalent cations as the wash buffer and 100 mM acetic acid, pH 2,5, us the elution buffer at 7'C. The pH of the fractions w ere increased by leaving 0.025 volumes of 2 M Trts base in she traction collector tubes. The dm ion peak was pooled bused on the chroimnogmm and then dialyzed against >20 volumes of 10 mM acetic acid, 051¾ sucrose, pH 5.0, using 10 kDa Shde~.A~L veers (Pierce: at room temperature for 3 hours The dialyzed material was then filtered through a 0.22 :tm cellulose acetate filter and concentration was detcimined by the absorbance at IG0 nrvt. Samples of 25 ug of the antibody fusion were injected on to a Phenomena SEC 3000 column (7.8 v 200 mm;· in 50 mM N'aHjPO} pH 6,5. 250 mM Na.Cl at developed at 1 ntProm observing the absorbance at 2K0 ntn (figure 4.R. Since the fusion protein eluted with the expected retention lime for n protein of its expected a mm this indicates that the protein was able to form the expected complex without excessive aggregation The ExT-uKT R 120.6 untfoody was analyzed ustng a 1.0 nun Tris-glycinc 4-20°·« SDS-PAGIr. (Movex) developed at 220V using reducing and non-reducing loading buffers and staining with Quick Blue (Boston Biologicals; (Figure 44). Thj?>Roa*rcducing SDS-PAGB indioistc^ that she expected quaternary complex of the fusion protein was formed and fusion of the e>.endin-4 pep! see to the a&amp;LH ' 20 .6 antibody results in a product with the expected structure, }B08Qj fedmote 11 [ODdJ6] Av!hafef-alCLH Fusions [0081?] Thu C of 1 pels peptide is η» π -o binding polypeptide with a so-called sonnet structure. fSec, e.g., tsoiknun ei ai,, Novel PtotentN with Targeted Binding. I s 2dlG 008°° 1? stakes e' .il. Π -6 Binding INotems. 1 A 2008 02810>*. Stemmer et ai,

Protein ScailokK and Uses Thereof. US 2006/0223114 and 1 hS 2006 023-1209), 100818] 'Hie components of the C681-uKlif 120,6 igG2 HC fusion included the monomer#';.

[00819] i a > ,ίΚΗ I 120.6 kappa IX (SEQ ID NO:¾ and [008201 (h>iVK-1 $Ρ)Τ'681"(ό5;ΡηΚΟϊ: 12E6 fgG2 HCfMlau amino acid sequence: MDMRVP^OU.GLLLLWLaOAkCSCrCjSt'' (.PDQFRCGNGQC'IPLDWVCDCtVNDCPDD sni ! i A PPR t ( VSQl Qt GSU 11 ISQR W \ t IX ί ΝΠ( f DOSPF \M ACN\ PTCPSHl I RC RNGfH 1PR -\\\ R( i)CA NDt \l>\St>l I UCTFHTGGGGGQVQLX QSGAEYKKPG.as VkVM’K \St.V ] FHIVHMllW\'Rig \ PGQH.fi EW MGV INPNSGGTNYAGkFQGRYTM 1RUI Sib t \A \ tl t sR I RSI>0! W \ 3 t \ IlDK* JS3 t W) OPu GOG Π \ 1 \ SsAs 1'K.GPS \ ΓΡΪ APT SPsiSf ST Λ M GO \ k ΠΥΊ PI PV1 GSW \M 1 \t fSG\ Hit ^ \ Vi QSSi d 3'Si SS V VTVPSSNFGTQTYI ΟΝVDHKPSNTKYDKTVERKCC VPC PPC'PAPPVAGPS VFtFP PEPKDTi MlxRTPFXTt \ V VIA Mil OPrVQt NWYXOCA ; \ HN \kt k'PRfl 0- NSTE RVYSYL'milQDVVl.'NwK.EYkCKk WKGI P Xf'MBKTiSKiYaiQPRLPQy Y 11 PPsRld ΜΊ KNQVSLTCLV ICG FYPSD1A VEWESNGQPBN Ν'Y KIT PPM L DSDOSEFLY SKI .Γ U.) RSRWQQGKVFSCSYMiM (SEQ ID NO:356).

[00821 ] rite desired product was a loll antibody with the

Aturner fused to the N-ternumw of both heavy chants, 1 he ratio of ('68i -heavy chant: light chain was ΐ: I. The expected COHI-aKLH 120.6 lgG2 HC fusion protein was isolated using ion. exelmnge cftffinja.iqgmplif , as deshrlMd teem, [{l0S22j The C6HI~aKLH I 20.6 lgG2 variable 11C fusion was ordered fromBloc Itern a* a s> iuheOe gene eoeodidg the flowing; arhino: acid sequence: 100X22j MDMRYPAOU.GLU,l.WLPGARCSGGSa,PDQFRCGtelOCIPLQWYCDGY NiDCPDDSDOiiGCPPR I CAPSQF(X'GSGYClSQRVYy('DGl:NiX''M)GSDFANCAGSYP 1' C PSD E F RCENGRCIPR Λ \V RC DG V NIX' ADNSDP e: OCI'FH T GGGGGf) \'Q L VQSG A E VKK!KlASVKVSCKA$GYTHC«YHMH\YYRQArc;QGLFWVJGVVIN!'NSGGTNYAQKF QGiU'TMTRD'fSISTA^'MhLSRLRSDCHA V'YYC'ARDRGsYYVVFDFWGQGTLM'VSS ASTK.- (SFQ ID YC.U50),

100X2 I[ The fragment was digested w »h Sals and Bsmhl, run out on a FA agarose gd end the conespondhig fragment vid oat and purified by 0 teen's Gd Purification Kit. At the same time, a pT'o-VKi SP-aKFH 120.6 igG2 HC GNA template vs as digested and pun tied similarR. yielding a p’H 5 vector backbone with the eon.snuu K€ region. The Avirner fragment was ligated to the pT Te doG2 HC -ton si a at region and transformed into OneShoiX I op! 0 bacteria, DA As wote submifled for sequencing. Although, analysis of several sequences of clones yielded a i00% percent match s\ith the alxwe sequence, only one done was selected for large-scaled plasmid purification. The final pTTC-VKISP-f 'bhboKXH 120.6 lgG2 H(' con-4.: net encoded a CoX i a'GShaK LH 120.6 igG2 HC fusion polypeptide i SFO if> 6-0:256). :00X25] The components of the aKLH 120.6 !gG2 HC-COXj fusion included hie monomers'.

[00X26j fa? aKi H 120.6 kappa I C (6f.Q ID AO:2X?: and I00X27[ fbr nK1 H : 20 6 lgG.2 I 1120.%% s fusion having the following ammo acid sequence:

MnMRVf'AQi.i.Gf LLLWLRGARCQYQf VQSGAPYK.KPGASVKYXCKASGY 1ITG YH Μ H A' V RQ.-\ PGQG L F W YKIW IN PN SGG'l N Y Λ QK F OGR V ΓΜΤ ROTS ISTA5 MELSKLKS DDT' AY YY C -A RDRGSY Y WF DP WGQGTL YTVSSASTKG PS V FPL A PC SR STS PSTAA I. GCL VKDYFPFPVTVSWNSGALTSGViTI FP.AYLQSSGL YSLSSYVTVPSSNKGTQT YT (TKXITHKPSKTKVDKTYFRKi'CYFC'PPf'PAPFYAGPSvFt.FPPKPKDTt.MiSRTPFVTC V VVDVSH fc'DFEYQFN\VY V DOVE VH ΝΛ K1 K PREEQFNS'IT K Y VSV L. i \'VHQDWLNG KI:'N'KCKVSNKGU>APH:.KTi»Kl'KGQPRO>QVVTLPf>SREi::MTKNgVSUrCLVK.Gi:VT SD1AVE\VESNGQFENN VKTVFPMEDSIX1RFTLV SKLT VDKSRWQQGNVFSCSY.ΜΗE A I. i-l'Nf}y'rQKSLStSPGGG{.JGGSGC3SC'LPDQFR.ajNGi'XiPLDWV('DGV-'NDa>F>DSO FEGCPPRTC A PSQPQCGSGYC! SQR W VCT>Ci ITN PCEDGSDEA Ni AGS V PT C PSDEF R.C R N G R Ci PR AW KCDGY NΙΧΆ PNSD EEDCT HHT / iSFQ ID KO:S57> [0082^1 The desired aKLM ) 20.6 tgG2 (1( -(7681 product was a full antibody with flic Avimer fused to the €-terminus of both heavy chains (schematically represented in Figure IG). The ratio of heavy chain-C6fl I rlight chain was l·!. The expected «ΚΑ.Η 120.6 igG2 FIC-COHl fusion protein was isolated using ion exchange chromatography, as described herein.

[00829) The COS! fragment with flanking Sex AI and Not! restr; risen sites was ordered from Bine Hero·· as a synthetic gene encoding the follow mg amino acid sequence; 100870] M l KNQYS1..T< 1. VKGFYPSD)Λ VE WESNGQPENN YKfFTPPMLDSDGSFFLY SK LTV DK'SRV\‘QGGN VFSCSY ΜΗΕΛ LH'NHVT'QK^LSLSPGGGGGGSGGSCLPDQFRC GNGQOIf!.. D\V \!CDG VN DCPDDSDEIX iCRPRTCA PSQFQCG SGYC'f SQR W V'(7DG END ¢1: DGSDEANCAGSYPTCPSOHFRCR NG1U1PRAWRCDGVNDC DNSDELIXTFH I; (SEQ ID NO:35Si.

[00831 ] tire fragment was digested with Sex At and Noth run out on a l % agarose gel and the corresponding Augment cut out and purified by Qiagen's Gei Purification Kit At the same time, a pTTY'VK I SP-aKAEI 120.0 (gG2 H(7 DIVA template was digested with Sait and Sex.Af and purified similarly ret generate (he DMA coding sequence for akl.R 170.6 lgG2 Rif monomer (SFQ ID NO;29b A pTT5 vector was cut with Sail and Nod, run out on a 1S> agarose gel and rhe larger fragment, eat out and gel pari fled by Qiagen's Gel Puriikation Ida. The Awmcr and »KXH 120.6 lgG2 HC fragments were ligated to the pTT5 fragment and transformed into OneShoi Top 10 hnevena. DMAs were submitted for sequencing. Although, analysis of several -sequences of clones yielded a 100% percent mutch with the above sequence, only one clone was selected for large sealed plasmid pun beau on. The fired ρ'Π',ν YK.ISP-aKER 120,6 lgG2 Rff-CfAi construct encoded aKLH 120.6 lgG2 H(XG51-( 681 fusion polypeptide iSirO N0.357}.

[00832] The components of the {.'68 I -aXi-H 120.0 kappa L.C fusion included the monomers.

[00832] fa) »KLH 120.0 lgG2 HC (SEQ JD N'0:2<>): and [00034 ] ί h ; (308 ί -a RLE! I 20.6 kappa Ι..Γ fusion intv ing the follow ήtg armno ucid sequence.

MDMKVPAQLL Gl..i.LL.WLRG.\RrSC)GSCLPDQFR(‘GNGQCiPj.i.)\VVCDG\''NDCPDD SDEnCK'PPRTCAPSQFQCGSGYnSQRWTCDCfENDCKDGSDEANCAGSYPTCPSDEF RC RNK'tRC I PR AW RC DGV NIX'Λ DNSD13 EDC Γ EHTGGGG GDIQM1 QS PSSI. S AN V GDR S' TITCR ANQG IRNDL()\YYQQi\PGk'.APKRί.IVΛ .ANSI QSGVPisRPSOSGSGTEFTET 1$S (.QPEDFA'n'Va..QHNSVRi.Tr{jGGTKV£:IKRTVAAPSVnrPP.SDiiQi.KSfjTA.S\'VCi. LANE VFREAK YQW K V DN A 1..QSG N SQE SY7 EQDSK DSTY S 1.. SS Π. TLS K AD V EKHK Y VACESTMQG! SSE V fKSENRGEC’’ (SEQ ID NOGNOi [0083 5] The d cm red C68l.-ak'EH 130 6 kappa 1.(3 product was a full antibody with -he Avinto· fused to the N-terrmnns of both light chains. The ratio of (3081 -light eitaimheavs chain, wax 1.1. (lie expected (3681-a ICE H 120,6 kappa ΐ C fusion protein, nan isolated using ion exchange chromatographs-, ns described herein, [00830] TheiVK-4 SF}-C681-fGSi-aKLH 120.6 kappa variable IX fusion was ordered IVorr! Blue Henan with Hanking Sail Bxi Wi restriction sites as a synthetic.' gone encoding the fed low Ing amino acid sequence-. MDMRV!>AQiJ.GLLUAvVLROARCSG{jSC''LPDQi;R(3GNUOnPLDWVCtXVVNtX'!>DD SDH 13.CK" PPRTCA PSQFQCG S(3 VOSQR W VC IX ί ON DC EDGSDb Λ N CAGSYPTCPSDE H RC'RNG IX' 1 PR AW RCDGV N DC A DN SDI2 130(1' EH TCJGGGG D1QMTQS PSSLS AS VGDR VTITCR ASQGIRN DI ,G WYQQKPGH A PKR L 1Y.A ANSLQSG V PSRFSGSGSGTir.FTt.TiNS 1. QPED1; ATY\ a.QHNSYPLTFGGGTKYEIKRTY AY fSEQ ID NOG60)

[00837] The fragment was digested with Sail and SsiWi, rust out on a r.Y agarose gel and the corresponding fragment out out. and purified by Qiagcu's Gel Purification Kit. At the same time, a pTT5-VK INP-aKLH 120,6 kappa 1.,(3 DNA template w as digested and purified similarly, yielding a p!T5 vector backbone with the constant 1.,(3 region. The Avimer fragment was ligated to the nTTS-kapna 1..(3 constant region, and transformed into OneNhot lop 10 bacteria. DMAs were submitted for sequencing. Although, analysis of several sequences oft (ones yielded a 100% percent match with the above .sequence, only one clone was selected for large sealed plasmid punficaiion. The final p‘i T5-VK1SP-C6K I ~&amp;KJLH 120.6 kappa LΓ construct encoded a C681~{G5)--aKLH 120.6 kappa LC Fusion polypeptide iSli.Q ID 180:350?.

[00838| Met]M.kd.8oki^A foiual purification of the conditioned media w as done by affinity ifo: protein liquid chmnistography (FPI.O capture of the F<;: region using Protein A Sopharoso (GE Healthcare! followed by a column wash with Tnsdntfilmed saline, I roM Cud; (Tcknova) and step elution oath 100 mM acedc acid. 1 mM CaCH, nil 3.5 at a flow rate of A5 crrCmin. Protein com wrung traction*' w ere pooled, and the pH was adjusted to 8.0 uxmg 10 N NaOH and further diluted with 3 volumes of outer. F lie mnierial was filtered through a (8--15 pm cellulose acetate filter (Coming) and further purified by anion exchange EPIC if) Scpitnrose High Performance;; GE Healthcaref Samples were loaded orno a column equilibrated with UHhC buffer Λ fat) mM Tris, ! mM. pH 8 Of and eluted with a wadi cm of 0 to 80*8; bullet B 03 ntM Ί is, 1 M NaCL I mM f af 1.-. pH 8.0? over 30 column volumes at a flowrate of i ,5 envmin. Peaks containing target species were pooled and fortnnLttcd into 10 mM Tris, 150 mM Nd( ). I mM CaCf-. pH 8.0. Exemplary ivuri ft cations of N-tcrrnlna) HC and 1..C and fMeooirtal ΗΓ fuoon proteins a-e shown m figures 36-58. The non-reducing SD8-PAGf an ah sis t figure 361 demonstrates that the. fully assembled antibody can be formed and the reducing SDFDPAuE analysis demons-rates shut the desired components are present. The she exclusion chromatogram i figure 3?) *5tows that -he majority of :he purified product is in the desired norwwvrewuod state. Finally, the ntass spectral analysis 5Figure 58? demonstrates that the de.-eve-d fusion products are present. Taken together name example's demonstrate that the aKl.1! I Γ.0 o antibody can accept fusions to AvimerS: fomiing ike dost rod; pmdtief 100830] Simple 12 [008401 BLAeore® Binding: ,4¾%¾ Of kllMF ipd ιΜΙΤΙ Antibodies !00H-11 ] Materials. Purified auti-DNP anubodies from either hybridorna t5,-M. if 3. 3Λ4 and 3BI) or recombinant O10 (3a4-1'~G2 and 3BI-G2) expression were tested. Anfohnntair IgG, ley-spcdiie antibody was from Jackson hmn-tnoRexcareh Laboratories. kw, f A'cst Grove, PA). DMP-BSA f 2,4 Mini irophcnol conjugated ro bovine serum albumin) was front

Btosearch Technologies. hie. (Novato. C A). Bi Afore 2000. research grade sensor chip CM5. surfactant P-20 (£.K>)voxyethyle«asorbitan), HBS-EP (!0mM HHPLS, 0.I5M Νιιί.Ί, 3.4mM bf>TA, 0,003% P-20, pH 7.4), amine coupling reagents. lOroM acetate pH 4.0 and !0mM glycine, pH 1.5 were iron? BlACorc, hie. tPiscataww, NH Pho.spharc-hul leted saline (PBS. IX. no calcium chloride, no magnesium chloride) was horn Imitroucn A nrisb-td I AV, Bovine serum albumin (BSA, fraction V, IgG free) was horn Sigma (Si I oms, MOi [00842] Purified nnti-KI.H antibody (human IgG 1, done 120.6.1 s expressed from hybridoma. was tested. Muitlmeric high molecular weight keyhole limpet hemocy-mm (K.LH) was from Pierce {Rochtord. IL). Anti-human IgG, Ρογ-specilie antibody was from JacksoH-ImmurwRcsearch Laboratories, Inc. t West Grove. PA). BlACorc 2000, research grade sensor chip i. MS, surfactant. P-2Q fpolyoxyxtfhylcncsorbitan), HBS-BP GOm.M IIFPFS. 0. ISM NaCh 3.4mM El>7A, 0.005% P-20, pH 7.4), amine coupling reagents. lOroM acetate, pH 4.p, and tOmM glycine, pH 1.5 were from BlACorc, Ine. (Piseaiaway. Nj). Phosphate-buffered saline fPBS. LX, no calcium chloride, nr» magnesium chloride') was from Insritroacn (Carlsbad. CA). Bovine serum albumin (BSA, fraction V. IgG free) w ns from Sigma tSt. Louis, .MO).

[008451 Methods. Bl.keore^' amOy-ea were carried out as follows, Immohilimtioii of anti· human IgG. Fey-specific antibody to the CM? sensor chip surface was peribrmed according ro manufacturer's instructions, using a continuous flow of 10 mM HI:. PBS, 0 1>M NaO. 5.4mM LDTA, 0.005®« P-20, pH 7'.4 (HBS-LP butter). Briefly, carboxyl groups on the sensor chip surfaces were activated by injecting 60 μΐ. of a mixture containing 0.2 Μ 1 -ethyi-3-[5-dimeth>danuuoprepyl| earbodiimide hydrochloride H2DC) and 0 05 Μ N-iiydroxysuecinimlde (NHS), Specific surfaces were obtained by injecting ISO uL of ami-human IgG. bey·specific antibody diluted in 10 trJvl aconite butler l lor assay of aKJLH antibodies: pi I 4,5 at a concentration of 50 ug ml.; for assay ot'aDNP antibodies: pH -1 0 ai a concentration of (>0 ug.'tnl.}. Excess reactive groups on the surfaces were deactivated 0> injecting 60 μ 1.. of 1 M erhanolamfne Final immobilized levels were about 9,000 (for assay of aK LB antibodies) or about. 10.000 (forassay of aDNP antibodies} resonance units t R15} A blank, mock-coup led reference surface was aLo prepared on the sense- chip. Antibodies and antigen were diluted in sample buffer consisting of PBS i t).005n.> P-20 ; 0.1 mg'ntl. BSA.

[00844) AutbPNP oafcel)^· follows!]' of either sample buffer or DNP~BSA. ranging ίο conceit trie km iron·) 0,78 · 10() nVI. Two different DNF-BSA samples were tested-foi affinity to the anty^NP antibodies; Tie l>MP7 BSA samples· differed in die number of DNP moieties coupled to each molecule of BSA. with one sample containing 3 DNP moieties per BSA and the other-containing 31 DNP moieties pot B>A. On is the DNPf 31)-Bis A Sat concentrations front 0 3‘ί 50 u\l) was tested tot affinity to the recombinant anti DNP antibodies. In eaph |£M?bddie$ were captured on floss cells 2. 3 and 4, with flow cell l left blank to surface. Following sample bnffer ot' antigen injections cadi •attrfhbc. wi^/h^n&amp;rlfed feyTSsro mjecuons of 10 tnM glycine pi I 15 to dissociate captured antibody front the nmnobdh'ed antBbnman Fc surfaces. BIAevaluaiion srtffsv&amp;re was used to determine apparent; kinetic parameters for binding of DNP-BSA anf^odt#. fOOMS] Anil~bXH antibody \vuse captured on individuaiBow cells, followed; fey ipjeetiep of e id ter sample buffer or kLR, ranging m concentration from 0. H - IPO tAI, To prepare dilutions of the muUimcrie high molecular weight &amp;LK an average molecular weight, of „5.000.000 dahons was used. Following sample buffer or antigen, injection, each surface was regenerated by two injections of 10 mM glycine, pH 1,5 to 'dk^Me-captured.antibody fbbfn the irnrnob timed amt Tut man Fc .surfaces, BiAevai ration software w.-s used to determine apparent kinetic parameters for binding m KLH to captured antt-KI li antibodies. :00Mb] Bl A core A binding acv.m·, kmiK. labL Νλ belt to sutetrarj/.sw the apptnetU .Uisoeiation fk.,> and dissociation <;k, t r,u·, eonsnmts as e,eJ as equilibrium saasoei.ruw o>rwarts (Ms) obtanted for the b> wit w vtsjy *e>, o' ,χηίι-ΠΝΡ arufoodios bwdtr.e nt DNP HS \„ I he data m 1 ade 8Λ AermevK w that the w mRNP .mtd\>etes end speette nB to DNP, and that they bind more tightly to the higher density QNPOi $-SSA than nt the lower m would be expected. Apparent binding affiniftes for DNPtal )-BSA arc all single digit imuomoSar or higher. |00M "] (able 8B below summarises the apparent association ifot and dissociation fled rate constants, as wed m equilibrium dissociation constants tKr>) obtained fos the funding analysts ofami-KLH i 20.6,1 antibody binding to KLH. ,'T&amp;dat&amp;dftTdbl^iB demonstrat#f by br.dom ·, produced unfi-KI R antibody bntds specifically to nubimeriv K’ fI, with an apparent sub-nanomolar binding affinity.

Table &amp;A, BIAsorei) bimiiBg easap for aPW Mi&amp;odiP, IMP- fie*t detemisecl; apyl- η ή HP'·

Table 8B. SIAebpPbi^mg aasap for aKLII as.?ihbdip; se|j) « χ lip'

Abbreviations

Ahhre'. iatjorss used throughout this speeiftebriori are as deleted below,. labesS/etbeovrsei debited in xpt.viue oiivuntsfancCs

As acetyl iu<ed to refer lo uectyiuted residues) •AcBpa acetyl ated p - benroy I - L- phony him ri no ACN neeksnitnle

AoGH ·<χ:οΓ«· acid ΛίΧΧ asnihoeCokpeiusoni cellular oytoto.ueity

Aib uutinoBobnty tic acid bA ibefa-abotnc

Bps pbeoeov I- t-phem- kdanine

BrAe hrotnoacetyl iiMA-PCiOs BSA Bovine scrum alburni«

Hid Betszyl

Cap ("ape;it: acid CBC complete. h.kuai count CO P D Chronic obstructive pulmonary disease ('ll Cytotoxic fly topi messes DCC Bley h/oi)C\y SciU'boi Pirn ο\·.

Ddc i -(4,4 -di incrhyb 2,6- dhAo-eyeluhexy I idunctedhiyl. ON P 2 A -dsnhrophew.d DOPf' i Dioieoy ban· G l> ccro Aq >hosph<.vho!ine DOPE L2 -Dioieoyi ··;;; Glyccro-u phosphoefiniuotamtiig O PPC : A-Dips !m itoy i -s n-G f y ec ro - 3 phoxp b ooh o Ho e DS PC i .2- D is S ceray Pan-G iy c c ro - 3~pht.wphoehi>] "me DTI' Dlthtothrcitul EAE experimental autoinnnunc eocephalomyehns bbd.. enharie ed c bo aid unnsa.wcwve PSi-MS Ooclron spray ionization m<)ss spectrometry FACS. fluorescence-activated cell son;ay

Fo-irC tluoreoylmcdioxyearbonyl HOBt 1 -HydrOKyhcnzofriazole HPLC high perfonmnes: hquid chromatography HSL hosnoserine lactone IB inclusion bodies bXa calcium-activated potassium channel t ineiu· bog 1KC. a, BKh a, SKC'aj KI..H ke> in. Ίο \ nunc; Hcnmcpnom kv \ di3C0 SOtCs! pOSiteskim oh<:C:iid k:SO i..siUS30 ke,d I .PS Ppnpmyeai vharkle f. > ΜΓ1 t I'' m; >i ϊ-'.s MALDJ-MS· Matr: s-nm-kud laser iies:0r|1fios mdseppri. mass spedrsmdtry

Me medy!

MeO nicdov.

McOH methanol MHC- major h'nuueoinpattbilsty eotnpto ksNI?' matri.s me?alU>proictaase M:W Moleedar Weighs MWCCi Molecular Weighs Css? Off -Nap !. rnipthylalamne MEtIT neutrophils

Me storkm-de MME H-methyl-2- pyrrol idi none OAe ace! are

PAGE polyacrylamide gel eieokopismsisE EBMCJ peripheral blood roonomtelca.r· smil

PiiS Phoaphak-hs·iicrod \dde

Fbf 2,2,4 ,<k 7 - pc mi a«Kthyldlb>#0b|^0^P^iSa40ifobyl P€E polymerase chain reaction PD pharrmioody na rrue

Pee pspeecilic acid

Ps2,: Pdy; cih> lone gp v ed > pOiu p\ rouimmoie acid

Pis.; pk’db'ik: <0: >! I *K phoccsai. <4. 'me tic :p.V pho.sphti:tyTo.ain.e RBS ribosome binding die

ET Kwm temperature {about2.5sO S&amp;r \ο:\ίι\ϋ;θ SDS" ssklkstst iiedecyl ssdEsks STIv rmiict. iSirconlcc kinases i B: ό fos5 Bet·>\s c misuse, i din fconcctyt TOR T col) receptor- ΤΡΆ |Hilnorouoeric add

Ti l}·' ihvnvc humoral factoj:;

Tit: iriiyl

Claims (25)

1. An isolated antigen binding protein comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs) designated CDRH1, CDRH2 and CDRH3, and the light chain variable region comprises three CDRs designated CDRL1, CDRL2 and CDRL3, and wherein: (a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, or SEQ ID NO: 191; (b) CDRH2 comprises the amino acid sequence of SEQ ID NO: 192, SEQ ID NO:

193, SEQ ID NO: 194, or SEQ ID NO: 195; (c) CDRH3 comprises the amino acid sequence of SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO:200, or SEQ ID NO:201; (d) CDRL1 comprises the amino acid sequence of SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, or SEQ ID NO:205; (e) 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 ID NO:209, SEQ ID NO:210, SEQ ID NO:211, or SEQ ID NO:212.

2. The isolated antigen binding protein of claim 1, wherein CDRH1 comprises the amino acid sequence of SEQ ID NO: 190, CDRH2 comprises the amino acid sequence of SEQ ID NO:

194, CDRH3 comprises the amino acid sequence of SEQ ID NO: 200, CDRL1 comprises the amino acid sequence of SEQ ID NO: 204, CDRL2 comprises the amino acid sequence of SEQ ID NO: 206, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 210.

3. The isolated antigen binding protein of claim 1, wherein CDRH1 comprises the amino acid sequence of SEQ ID NO: 189, CDRH2 comprises the amino acid sequence of SEQ ID NO: 193, CDRH3 comprises the amino acid sequence of SEQ ID NO: 198, CDRL1 comprises the amino acid sequence of SEQ ID NO: 203, CDRL2 comprises the amino acid sequence of SEQ ID NO: 206, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 209.

4. The isolated antigen binding protein of claim 1, wherein the heavy chain variable region comprises an amino acid sequence at least 95% identical to a sequence selected from SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO:258, or SEQ ID NO: 260.

5. The isolated antigen binding protein of claim 1, wherein the light chain variable region comprises an amino acid sequence at least 95% identical to a sequence selected from SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:236, SEQ ID NO: 238, or SEQ ID NO: 240.

6. The isolated antigen binding protein of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, or SEQ ID NO:260; and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, or SEQ ID NO:240.

7. The isolated antigen binding protein of claim 6, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:258; and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:236.

8. The isolated antigen binding protein of claim 6, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:254; and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:234.

9. The isolated antigen binding protein of any one of claims 1 to 8, wherein the isolated antigen binding protein is an antibody or antibody fragment.

10. The isolated antigen binding protein of claim 9, wherein the antibody or antibody fragment is an IgGl, IgG2, IgG3 or IgG4 antibody or fragment thereof.

11. The isolated antigen binding protein of claim 9, wherein the antibody or antibody fragment is a monoclonal antibody or fragment thereof.

12. The isolated antigen binding protein of claim 11, wherein the monoclonal antibody is a chimeric or humanized antibody.

13. The isolated antigen binding protein of claim 11, wherein the monoclonal antibody is a human antibody.

14. The isolated antigen binding protein of any one of claims 1 to 13, further comprising at least one pharmacologically active polypeptide conjugated thereto.

15. The isolated antigen binding protein of claim 14, wherein the antigen binding protein is recombinantly produced.

16. The isolated antigen binding protein of claim 15, wherein the antigen binding protein comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, and wherein the pharmacologically active polypeptide is inserted in the primary amino acid sequence of the immunoglobulin heavy chain within an internal loop of the Fc domain of the immunoglobulin heavy chain.

17. The isolated antigen binding protein of claim 14, wherein the antigen binding protein comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, and wherein: (i) the pharmacologically active polypeptide is conjugated at the N-terminus or C-terminus of the immunoglobulin heavy chain, or (ii) the pharmacologically active polypeptide is conjugated at the N-terminus or C-terminus of the immunoglobulin light chain.

18. The isolated antigen binding protein of any one of claims 14, 15, or 17, wherein the pharmacologically active polypeptide is conjugated to the antigen binding protein via a non-peptidyl or peptidyl linker.

19. The isolated antigen binding protein of claim 18, wherein the peptidyl linker comprises glycine, serine, alanine, or combinations thereof.

20. The isolated antigen binding protein of claim 18, wherein the non-peptidyl linker is a polyethylene glycol linker.

21. The isolated antigen binding protein of claim 14, wherein the pharmacologically active polypeptide is a toxin peptide or peptide analog, an IL-6 binding peptide, a CGRP peptide antagonist, a bradykinin B1 receptor peptide antagonist, a PTH agonist peptide, a PTH antagonist peptide, an ang-1 binding peptide, an ang-2 binding peptide, a myo statin binding peptide, an EPO-mimetic peptide, a TPO-mimetic peptide, a NGF binding peptide, a BAFF antagonist peptide, a GFP-1 or peptide mimetic thereof, or a GFP-2 or peptide mimetic thereof.

22. The isolated antigen binding protein of claim 21, wherein the toxin peptide or peptide analog is ShK or a ShK peptide analog.

23. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1 to 22; and a pharmaceutically acceptable diluent, excipient or carrier.

24. A method for enhancing the pharmacokinetic properties of a pharmacologically active polypeptide comprising conjugating the polypeptide to an antigen binding protein of any one of claims 1 to 13.

25. A pharmacologically active polypeptide when produced according to the method of claim 24.