Antibodies directed to the deletion mutants of epidermal growth factor receptor and uses thereof

The description of ltybiiti^fLti^^‘^a^>ody sequences is coded as follows: “AB”refenring to antibody, “EGFRvIir'-refers to βίο antibody’s binding specificity, X refers to XenoMouse mouse derived, “Gr-rsfers fo IgGI isotype or “G2 refers to IgG2 isotype, “K” refers to kappa, “L¹ refers to lambda. Tte-last three digits referring to the done from which the antibody was derived, for example: AB-EGFRvEH-XGlK-13.12 “Label” or “labeled” as used herein refect to the addition of a detectable moiety to a polypeptide, for example, a radiolabel, fluorescent label, enzymatic label cbemjhnnioescent labeled or «.bfofinyl group. Radioisotopes or radionuclides may include *H, ^UC, ^1>N, ^MS, *°Y, ”Tc, ’in, ^IJ3I,. ^IMI, fluorescent labels, may include rhodamine, lanthanide phosphors or FTTC and enzymatic labels may include horseradish perqxidasey £jga£*ctosidase, luciferase, alkaline phosphatase.

The term ’phaipUqeS^^'igi^^ ^g£j?\used herein refers to a chemical compound or composition capable of indtici^^^Sjpg^^cripeutic effect when property sdnrinisteral to a patient Other chemistry terms herein are used according to conventional usage in ti» art, as

<img file="IN313DEN2012A_D0001.tif" />

exemplified by The McGnwJftK DtefioxOry qfChemiqal T«mr (Parker, S., Ed., McGraw-ΗίΠ, San Francisco (1985)). ..: :

As used,herein,^-*«u ¹ ‘ah 'object species is the predominant species present (i.e., on a molar I than any other individual species to the composition), and preferably a subetjmtiaDypurified fiaction is a. composition wherein fee object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular spedes present in-fee composition, moreprefeiably more than about 85%, 90%, 95%, 99%, and 99.9%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected tn fee oomposition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species, r ' . ·

Ίΐκ term “patient” includes human and veterinary subjects. .

The term “SLAM* Technology” refers to fee “Selected Lymphocyte Antibody Method (Babcook et al., Proc. Natl. /t&amp;xL· -^^'^ίέ^^:7843-7848 (1996), and Schrader, US. Patent No. 5,627,052).

The term XenoMai^^WemttraFSwof SLAM Technology with XenoMouse* mice (as described below). ; '

Antibody Structure . > '

The basic antibody -to comprise a tetramer. Each tetramer is composed of two identical each pair having one light^- (about 25 kDa) end one heavy* chain (aboid' 5f^cn^^(^^^^&amp;p4enmnal portion of each chain includes a

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¹ . .

variable region of about amino acids primarily responsible for antigen recognition. chain defines a constant region primarily responsible for effector fopotiorfc. chains ate classified as kappa and lambda light chains. Heavy chains are classified » mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, JgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions en joined by a Tregion of about 12 or more uninoaoids, with fee heavy chain also including a D region of about 10 mwe amino acids. See generally, Fundamental

Immunology Ch. 7(PauI,W.,ed., 2nd ed. Raven Press, N.Y. (1989)). The variable regions of each

Thus, an intact antibody hastwo. binding sites. Except in bifunctional or bispeeific antibodies, the two binding si tes are the Same.

The chains all eXhibitthesanwgeneral structure of relatively conserved framework regions . ' * · ; · *·«»>

(FR) joined by three hyper called complementarity determining regions or

CDRs. The CDRs from arc aligned by the framework regions, enabling binding to a specific epitope. ^^tfj^wi^W'fe-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2.FR3, CDR3 and FR4. The assignment of amino acids to each domain is in

Interest (National fostinites\ofH,e

Biol. 196:901-917 (1987);^^»

A bispecific or bifimtrndnai anl

<img file="IN313DEN2012A_D0003.tif" />

ns Of Rabat Sequences ofProteins of Immunological Md. (1987 and 1991)), or Chdthia &amp; Lesk J. Mol. 3&amp;8784$3 (1989).

is an artificial hybrid antibody having two different heavy/light chein pairs and two different binding sites. Bispeeific antibodies wm be produced, by a variety of methods including fusion of hybridomas or linking of Fab* fragments. See, e.g, Sungsivilai &amp; Lachmann Clin. £xp. Immunol 79: 315-321 (1990), Kostelny et al. J. Immunol. 148:1547-1553 (1992). Production of bispeeific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower far bispeeific antibodies. Bispeeific antibodies do not exist in the form of fragments having a single binding |jfe («.g., Fab, Fab'. and Fv).

hi addition to the general structural wpeetj ef antibodies, the more specific interaction between the paratope and t^.^tt^^nwyj^.examined through structural approaches. In one embodiment, .the strubtu^o^^^^^^^^pe, through which an antibody is able to bind to an epitope. The structi^^^^^tu^^^to'pc may be: determined in a number of ways. Traditional structural examinrticnapproachesmaybe used, such as NMR or x-ray crystallography.

<img file="IN313DEN2012A_D0004.tif" />

jy be.gcncratod in stflcb. A structure can be gtSiwated arcial package, such as InsightE modeling package $toan use the sequence of the antibody to be examined structures, such as the Protein Data Bank. Alter

These approaches may examine the Structure of toe paratope alone, or while it is bound to the . ... :.^v . W ,1 ζ epitope. Alternatively, moje ...... ' *--·- · - .. .ta'cS through homology pti from Accehya (San to search against« database of proteins of lent

<img file="IN313DEN2012A_D0005.tif" />

one identifies homologous proteins with kx modeling templates. Each of the' •^5 sequence alignments amoung*^ . MA

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i, these homologous proteins ere used as

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. In one embodiment, the entire epitope is icqucnce of the antibody with flic unknown Structure can then be aligned with ewset’^m generate a molecular model for the antibody with the unknown structure. As will be appreciated by one of ddE in the art; foere aremany alternative methods for generating such structures in silico, any of which may be used. For instance, a process similar to the one described hi Hardman Ct aL, issued U.S. Pat No. 5,958,708 « employing QUANTA (Polygon Corp,, Walfoam, Mass.) and CHARM (Brooks, B. R., Bruccoleri, R. Olafson, B. D., States, D, I, Swamtoatheo, S. and Karplus, M,, 1983, J. Comp. Chem,. 4:187) may be usod. . .

Not only is in determiningwhether and how Well a possible paratope wiU.bmd’m^'e^p^w'&e interaction itself, between the epitope and the paratope is a source of great information in the design of variant antibodies. As appreciated by one of skill in the art, there are a variety of ways in which tins interaction can be studied. One way is to use the structural model generated, perhaps m described above, and then to use a program such as Insight!! (Accelrys, San Diego, CA), which has a docking module, which, among other-things, is capable of perfornting ? Monte Carlo search on the conformational and orientational spaces ^r · J ·,;ι ,ii J ' >

between the parrtope and. its one is able to estimate where and how the epitope interact· with thcpaij pg&amp;oniy a fragment, or variant, of the epitope is used to assist in determining used in the modeling of foe interaction between the paratope and the epitope; As will be appreciated by one of skill intheart, these two different approaches have different advantages nd disadvantages, For instance, using only a fragment of the epitope allows for a more detailed examination of the possible variations ofeach side chain, without taking huge amounts of time. On the other hand, by using only a fragment of the epitope, or ehnply the epitope instead of the entire protein, it is possible that tiie.chj^uctip^^wrof the epitope fragment may not be the same as foe . characteristics for the increasing foe risk of being mislead during the computational modeling/’ approaches are used to a limited extent, in order to cross check the results. Ina pre ment, if a variant of an epitope is Used, it will be optimized so that the variant of the epitope comprises the most important residues of the epitope. The identity of the nwst important residues can. be determined in any number of ways, for instance as described in Examples 4 and 14 of foe present specification.

Through foe use of these generated structures, one is able to determine which residues are the mast important in the interactim^j between epitope and the paratope. Thus, in one embodiment, one is able to read ' es to change in order to alter foe binding .«»· ra*dn I _s · &lt;

characteristics of the ’antibody. ίϊ ipparentfiom the docking models that fee side chains of certain residues in'%i^pwS^M^^^oaUy hinder the binding of foe epitope, tin» ··· ¹ . ' I * ,

<img file="IN313DEN2012A_D0008.tif" />

<img file="IN313DEN2012A_D0009.tif" />

altering these resjduoa to residue»· with smaller side chains may be beneficial. One can determine this in many ways. For example,, cnemay simply took at the two models and critmatc interactions based on functional groups one may perform repeated pairings of epitope and paratope, as dMjq^^^h^^^eriOzobtato more favorable energy interactions. One can also variants of the antibody to determine alternative ways in which the the epitope. One can also combine the various models to determine how one should attar the structure of the antibodies in order to obtain an antibody with the particular characteristic* thatare desired.

The models detemtiped. above, pan be. teeted thrpugh various techniques. For example, the . \ ’ j · tp*‘'$7* ‘ ’ interaction eaargf tan deterite^.w^frr^'^bpanu discussed above in order to determine which of the variants to forther examine. tXlib, ctoilumWc and van dec Waals interactions «re used to determine the interaction energies ef the epitopo and the variant paratopes. Also site directed mutagenesis is used to see if predicted changes to antibody structure actually result in the desired changes to binding characteristics. Alternatively, changes may be made to the epitope to verify that the models are eorreet or to determine general binding themes fruit may be occurring between the

As will be aporocfetod bgAC^J9&amp;

paratope and the epitope.

The above methods for modeling strictures can be used to determine what changes in protein structure will result in particular desired characteristics of an antibody. These methods can be usedTo deteunfoe what changes hj protein stniqture will not result to the desired characteristics, the pt, while_: these models win provide tin k'i.'·.’ ».· ¹ _____________t , &amp; the P*®⁸®¹¹* embodiments, it may still be desirable to perform models, perhaps through In vitro studies, h iddition, as will he apparent to mw of skill to thean, any modification may also have additional tide effects on the activity of the antibody. For inrtance, while any alteration predicted to result in great» binding, may induce greater Wndtog/ittony also cause other structural changes which might reduce ot alter the activity of top ani lie tictenntoation of Whether or not this is the case is routine in the tot and can' bWotiS ' way*. For Example, the activity can be tested ' ’ ' ' » * v;'. v 'ttn frrrwgh an ELSA test, is in Example 21. Alternatively, the samples can be tested through the use

<img file="IN313DEN2012A_D0010.tif" />

>f a surface plasmon resonance device.

Antibodies Pbidinp and Variant AntibadjeaforSimcrior Binding tn one embodtmeto, the models described above are used to tosrease the binding Ability of he antibody to its epitope. The antibody can bind to the epitope more readily, and thus have a ighfcr association constant Q&lt;i). Alternatively, the antibody may dissociate from the epitope tower, and thus have a lower dissociation'constant Got), or the Ko ot the epitope-paratope ntcraction can be smaller to value/thus making toe extent of the binding between the epitope and •aratope higher. ·'· · ‘......

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•· ' ^! * ..IT..·. ♦*

*.··ί· ·’ ' ••‘•tf*· »···· • h -..· ;£.»;·?'· ,··Γ1 ί?2 is; .

<img file="IN313DEN2012A_D0012.tif" />

In some eiauwTO^na^w&lt;,¥p$TO;'antibodice are designed to bind with the opposite characteristics. That is, the antibodies do notbtod as tightly crpcrhape as qahtidy.

In other embodiments, the variant -antibodies are not different in their Kp from the wildtype antibodies» but the variant antibodies are more specific for a particular epitope. This may mean that the paratopes of foe design»! antibodies have a lower risk of binding to other epitopes. The antibodies oetn have other characteristics fat have been altered. For example, a variant may bo more immune to nonspecific antibody g x may stay solvated m edtokm even when the antibody is present to hisrf> ?^oh a variant may be present in fa discussed ' ~ ν^ν;Α’· · antibodies. For instance; wt EG^eroodcoitrations of some variant antibodies exammed below resulted m slower binding obaganfate to Biacorc experiments, for instance 13.12 mAb, certain variants did not. exhibit thia slower component, even at the same concentrations, L217N-2.1, for example.

The variants predicted by foe models determined above can be crested and fan tested to

<img file="IN313DEN2012A_D0013.tif" />

determine if they actually bind with fa desired characteristics. Mutants with a greater total interaction energy with foe epitope can be selected for further testing. The interaction energy can be determined in a number qf^ajx,-Orjiiof which is described above.

These variante.c^^foft^^^^jmbet: of ways.. Exemplary options include and are not limited to KinExA (e.g^Ij^^^i^|&lt;^,'Np. 5^72,783, Dec 13,1994) (Sapidync Instruments Inc., ID, Boise), surface plasmon resonance (SPRX&amp;g,, BIACORE™ Biacare, Inc., Pistcataway, NJ.), stepped-flow fluorescence, resonant narror, and fluorescence polarization. Many of these options are able to not only record the data,but also provide ready meets for fitting fa date to various theoretical curves, and fops detw^ine.thek,,kd, and Kd, as well as other properties, It is important to note fat fa fitting of thcsc curvc? to fa resulting data is not without fa possibility for some variation. Berawg^jfoj^^e'f^kvant association, dissociation, and equilibrium constants can be looked; fitting mechanisms, but also to direct comparison with each other, an^’m E^'d^fa/kpOwlcdge of one of skfll in fa art.

Human Antibodies and Humanization of Antibodies

Human antibodies avoid some of fa problems associated with antibodies fat possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to fa rapid clearance of the antibodies or can lead to the generation of an immune response against faantibody by a patient, to order to avoid fa utilization of murine or rat derived antibodies, fully human antibodies can be generated through fa introduction of human antibody faction into a rodcnt scfoatthe’^Jwi^fr^wes fUlly human antibodies.

The ability human foci to YACs and to introduce them into the mouse V’ powerful approach to elucidating fa functional components of very large or orudely mapped loci as well as generating useful models of human disease. Furthermore, the utilization of such technology for substitution of mouse loci with their

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<img file="IN313DEN2012A_D0015.tif" />

human equivalents oould provide unique insig^xt» into the expression and regulation of human gene products during development, their connmnricatien with other systems, and their involvement in disease induction and progression.

An important practical application of such a strategy is the humanization of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated offers the opportunity to study die mechanisms underlying program^ of antibodies as well as their role in B-cell development provide an ideal source for production of folly ·* * ^utefe¹** human monoclonal afrtiqomes

<img file="IN313DEN2012A_D0016.tif" />

milestone towards fulfilling the premise of antibody therapy in Jiuinta disease.'PUliy .human antibodies are expected to minimize die immunogenic and allergic responsesUng to mouse or toousederivatized mAbs and thus to increase the efficacy and saf^^f d antibodies. The use Of fully human antibodies can be expected to provide m the treatment qf chronic and recurring human diseases, such as inflammitioii, and dnaer, which require repeated antibody administrations.

One approach towards this goal was to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig^loci in anticipation that such mice would produce a large repertoire qf human antibodies in the 'absence of mouse antibodies. Large human Ig fragments would preserve fee large variable gene diversity as well tt the proper regulation of antibody production and expression. By exploiting foe mouse machinery for antibody diversification end selection and the lack of tmraunological tolerance to human proteins, the reproduced human antibody repertoire in time mouse strains should yield high affinity antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen-specific human specificity could be readily produced and selected, tieiction with Otir generation of the first XcnoMouse mouse strains, as publis .^SbeGreen et al. Nature Genetics 7:13-21 (1994)) The

XenoMouse strains were eo^nceredwifr ^cqst artificial chromosomes (YACs) containing 245 kb and 190. kb-eized genriHqe of the human heavy chain locus and kappa light chain loops, respectively,, ^^qvwiableandconstant region sequences. Ά. The human Ig containing YACs proypdib^bbfWBnpf^lc with the mouse system for both rearrangement and expression of antibodies and were capable of substituting lor the inactivated mouse Ig genes. This was demonstrated by their ability to induce B&lt;ell development, to produce an adult-like human repertoire of fUIly human antibodies, and to generate antigen-specific human mAbs. These result? also suggested that introduction of tager potions of the human Ig loci containing greater ‘1 numbers of V genes, additional regulatory elements, and human Ig constant region* might recapitulate substantially the full repertoire that^s characteristic of the human humoral response to infection and tanunizatian. The Work of Green ctal. was reoenfly extended tothe introduction of

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This, general strategy W|

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greater than «CTroxniiatciy’^i^^i^^^^iiitflxxiy repertoire through introduction of megabase sized, geiraliue configuration Wfe ftagn^fi'of foe human heavy chain toed and kappalight &lt;4·^ loci, respectively. See Mendez et al. Nature Genetics 15:146-156 ¢1997) and U-S. patent application Ser. No. 08^59,620, filedDec. 3,1996. .

The production of the XenoMouae mice -is forther discussed and delineated in U.S. Patent Application Serial Nos. 07/466,008, filed January 12,1990, 07/610,515, filed November 8, 1990, 07/919,297, filed July 24, 199¾ 07/922,649, filed July 30, 1992, filed 08481,801, filed March 15,1993, 087)12,848, filed 145, filed April 28, 1994, 08/376,279, filed

January 20, 1995, 08/430,-93^^^^^464^84, filed June 5, 1J9S. 08/464,582, filed June 5,1995,08/463,191, 0^^62,837, filed June 5,1995,08/486,853, filed June

5, 1995. 08/486,857, filed June 5, 1995, 08/486,859, filed June 5, 1995, 087462,513, fifed June 5, 1995, 08/724.752. filed October 2,1996. and 08/759,620. fifed December 3, 1996 and U.S. Patent Nos. 6,162*963, 6,150,584, 6,114,598, 6,075,181, «nd 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See also Mendei et al. Nature Genericr 15:146-156 (1997) and Green and. JakcbcMts/. Exp. M. 188:483-495 (1998), See also European Patent No., EP 0 463 151 Bl, grant publi^icd&lt;j|jne742; l996, International Patent Application No., WO 94/02602, publish») pebr^^^^^^^i^s^atiiwial Patent Application No., WO 96/34096, published October 3l. l9S^WO_r98/M8^pubtished June 11, 1998, WO 00/76310, published December 21,2000, WQ 03/47336. ' bi an alternative approach, ofoen, inrinding GenPharm International, Inc., have utiltoed a

<img file="IN313DEN2012A_D0021.tif" />

“minilocust* approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual gen#) from foe Ig focus. Thus, one or more V» genes, one or more D« genes, ope or more J« genos, a mu constant ji^ion, and a second constant region (preferably a gamma constant region) are f« for insertion into an animal. This approach is described in U.S. Patent X. end ?^atenl Nos. 5,545,806,5,625,825,

5,625,126, 5,633.425^ $66¾)^^ 89,650, 5,814,318, 5,877,397, 5,874,299, and

6,255,458 each to Loaberg and Kay* (J.S. Patentfab. 5,591,669 and 6,023,010 to Krimpeafort and

Berns, U.S. Patent Nos. 5,612,205, 5,72),367, tod 5,789,215 to Bout et ah, and U.S. Patent No.

5,643,763 to Choi and Dyim, sod GenPharm International U.S. Patent Application Serial Nos,

07/574,748* filed August 29,1990,07/575,962, filed August 31,1990,07/810,279, filed December 17, 1991, 07/853,408, filed March 18, 1992. 07/904,068, fifed June 23, 1992, 07/990,860, filed December 16, 1992,08/053,131, filed Aprfl.26, 1993.08/096,762, fifed July 22, 1993,08/155,301, filed November 18, 1993, 0^^$9^l^.i>ecen*er 3, 1993, 08/165,699. filed December W, 1993,08/209,741, fti'ed'Kia^^^S^^Si^^l^ropeanPatentNo. 0 546 073 Bl* International Patent Application Nos. WO 92/22647, WO 92/22670, WO

93/12227, WO 94/00569, WO 94/25585, WO 96/14436, .y/O 97/13852, and WO 98/24884 and U.S. Patent No. 5,981,175. See further Taylor et1992, Chen ctaL, 1993. TuaiOon eta!., 1993,

Choi et al., 19,93, Lonberg et al., (1994), Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild ct al., (1996).

Kirin has also demonstrated the generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced. See European Patent Application Nos. 773 288 and 843 961. Xenerex Biosciences is developing a technology for the potential generation of human antibodies. In this technology, SCID mice are reconstituted with human lymphatic cells, e.g., B and/or T cells. Mice are then immunized with an antigen and can generate an immune response against the antigen. See U.S. Patent Nos. 5,476,996,5,698,767, and 5,958,765.

Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and a murine variable region, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly in chronic or multi-dose utilizations of the antibody. Thus, it would be desirable to provide fully human antibodies against EGFRvHI in order to vitiate concerns and/or effects of HAMA or HACA response.

Antibody Therapeutics

As discussed herein, the function of the EGFRvHI antibody appears important to at least a portion of its mode of operation. By function, it is meant, by way of example, the activity of the EGFRvHI antibody in operation with EGFRvHI. Accordingly, in certain respects, it may be desirable in connection with the generation of antibodies as therapeutic candidates against EGFRvHI that the antibodies be capable of fixing complement and recruiting cytotoxic lymphocytes thus participating in CDC and ADCC. There are a number of isotypes of antibodies that are capable of the same, including, without limitation, the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgGl, human IgG3, and human IgA. Also, it may be desirable in connection with the generation of antibodies as therapeutic candidates against EGFRvHI that the antibodies be capable of activating antibody-dependent celluclar cytotoxicity (ADCC), through engagement of Fc receptors on effectors cells such as natural killer (NK) cells. There are a number of isotypes of antibodies that are capable of ADCC, including, without limitation, the following: murine IgG2a, murine IgG2b, murine IgG3, human IgGl, and human IgG3. It will be appreciated that antibodies that are generated need not initially possess such an isotype but, rather, the antibody as generated can possess any isotype and the antibody can be isotype switched thereafter using conventional techniques that are well known in the art. Such techniques include the use of direct recombinant techniques (see e.g., U.S. Patent No. 4,816,397) and cell-cell fusion techniques (see e.g., U.S. Patent Nos. 5,916,771 and 6,207,418), among others.

In the cell-cell fusion technique, a myeloma or other cell line is prepared that possesses a heavy chain with any desired isotype and another myeloma or other cell line is prepared that possesses the tight chain. Stall cells. ram.-thpreafter, be fused end a cell fine expressing m intact antibody can be isolated, j. . ./γνο

By way of βχ*ηφϊβ,._:ε*ώίί(ρ .·»^Ε£^ένΠ1 antibodies discussod herein are human antiEGFRvUj IgGl antibodies. If jsufcfi antibodyjpeteeased desired binding to the EGFRvfflmolecule, it could be readily isotype switched to generate a human IgfO, human IgG3, or human IgGA while still possessing the same variable region (which defines the antibody's specificity and some of its affinity). Such molecules, including IgGl, would then be capable of fixing οαπψΙαηΒπΙ and participating inCDC, and, if comprising and IgGl or IgG3 constant region, such molecules would also be capable of participating m antibody-dependent ccUular cytotoxicity (ADCC) through recruitingcytotoxic lymphocytes.

Accordingly, a* antibody candidates we generated that meet desired “structural attributes as discussed above, they can generally be provided with at least certain of the desired “functional’’ attributes throughisotype switching.. ..·, Design and Generaxionof ^&amp;'jrE^rap^tifiik . Based- on the actf&amp;tyMr^xtffiwiei that are produced and characterized herein with respect to EGFRvIH, the d&amp;sigh of otiMT^tiierapeutic modalities beyond antibody moieties is facilitated. Such modalities include, whhout litatation, advanced antibody therapeutics, such as bispecific antibodies, imnnfflp^j^j-ya^^'li^olabeled therapeutics, generation of peptide therapeutics, gene therapies,yWtic^ari^|!^^^e^.antiscnsc therapeutics, and small molecules.

In connection with das Bgxra^on'jafiadranccd antibody therapeutics, where oranplement fixation and recruitment of cytoxic lymphocytes is a desirable attribute, it is possible to enhance cell tailing through the use of bispccifios, irnmunotaxins, or radiolabels, for example.

For example, in connection with buptaific antibodies, bispecific antibodies can be generated that oomprise (i) two antibodies one with a specificity to EGFRvIH and another to a second molecule that are conjugated together, (ii) a single antibody thathas one chain specific to EGFRvIH and a second chain specific to a second molecule, or (Hi) a stogie chain antibody that has specificity to EGFRvIH and the other molecule. Such bispecific antibodies can be gcncrated using ' ! '· techniques that are well known for example, to connection with (i) and (ii) see &amp;g., Fanger et al.

Immunol Methods 4:72-81 (1994) and Wright and Harris, stqtra. and in connection with 0ti) see

<img file="IN313DEN2012A_D0022.tif" />

&lt;s BfTE technology) or CD89 (j»e Valerius « e g., Trauneckcr et al, 7:51-52 (1992). In each Mae, thesecond specificity can be made to the Pc cbafeagtifftky ' including, without limitation, CD16 ar CD64(ree «.g„ Deo et al. 18:127 al. Blood 90:4485-4492 (l9$7j).' Bispemfio antibodies prepared to accordance with tin foregoing Would be likely to kill cells eepitastog ΕΘί&amp;νΠζ and particularly those cells in Which the EGFRvIII ttntibodies0fthtf.inVait8op:ftC wSgrtivte··

In connection wiw tom^ctw^^OTnwh&amp;s can be modified to acres immunotoxtas utilizing techniques that are «g, Vitetta Jmmtmol Today 14:252 (1993).

I

I ¹ . . , , . ₄ , ₄ ‘

See also U.S. Patent No. 5,194,594. -'Wcanueewi with the preparation of radiolabeled antibodies.

. . ..iMd,” fart-lif-ii'· . .

such modified antibodies ^prepared utilizing techniques that are well known in ··. * the art See. e.g.. Jungharii etnSW ’ '1*4

Chafiier and Longo, eds., Lippmcdti^,RaS>ai^!'^:(l996)). Set also U.S. Patwit Nos. 4,681,581, 4,735^10, 5,161,827, 5,10^^3^^ 5,648^71, and 5,697,902. Each of ihmmhotoxms andradiolabcled inolccule^^^a ji^p^^l^ii wd js expressingEGFRvlII, end particularly those cells in which the antibodicsd^cStiJeffective.

The antibodies can be designed to bind more quickly, or to dissociate more slowly from the epitope, and firns the antibodies themselves can be designed therapeutics. Ths altered characterisitics of themtibodieeoan be used, for example, in the admin istration of a therapeutic to a patient

Therapeutic Immunocos^ugate»

As will be appreciated, antibodies conjugated to drugs, toxins, or other molecules (immunocopjugates Or immunotoxins) are highly uaefiil in the targeted killing of cells that express a molecule that can be specifically bound by a specific binding molecule, such as an antibody. As

I discussed above, EGFRvIH is not known to. be expressed on any normal tissues. Further, EGFRvEI shows significant expression in^nu^ejoyshurwn tumors. Accordingly, EGFRvni is a highly attractive molecule tor

Many repots $«3^ monoclonal antibody-drug 1987); Ghose ct al, in T Mediated Delivery Syste

<img file="IN313DEN2012A_D0023.tif" />

-apy and Biotherapy 655-686 (2d edition,

<img file="IN313DEN2012A_D0024.tif" />

specific targeting of tumor cells with ^^(Se^efjSL. in Immunoconjugates 189-216 (C, Vogel, ed. Bjg GoMbcrg, cd. 1983); Diener et al, in Antibody

<img file="IN313DEN2012A_D0025.tif" />

ed, 1988); Pietcrsz et al, in Antibody Mediated ' •i. ;

delivery Systems 25-53 (T/lt Buraol et al; in Antibody Mediated Delivery

Systems 55-79 (J. Rodwell, ed, I988),tytotoxic drugs such as methotrexate, datmorubicin, doxorubicin, vincristine, vinblastine, meJphalan, mitomycin C, and chlorambucil have been conjugated te a variety of murine monoclonal antibodies. In dome cases, the drug molecules were linked to file antibody molecules through ah intarmediaTy carrier molecule such as serum albumin ^Garnett et al. Gance* Kes. 4612467-2.412 (1986); Ohkawa et al. Cancer Immumol, Immunotber. 23:81-86 (1986); Endo et al. Cancer Rea. 47:1076-1080 (1980)), dextran (Hurwitz et al. Appl. Biochem. 2:25-35 (1980); Manabi et id. Biochcm. Pharmacol. 34:289-291 (1985); Dillman et al. Cancer Res. 46:4886-4891 (1986); Shovel et al. Proc. Natl. Acad. Sci. 85: 8276-8280 (1988)), or polyglutanrio acid (TsUkada et al, J. Natl. Cano, hist 73:721-729 (1984); Kato et al. J. Med. diem. 27:1602-1607 (1984); Tsufada et'al. Br. L Cancer 52:111-116 (1985)).

’ ♦ ’··/ t. t ·_4 £.··£*· *

A wide array of linker ..tew been employed for the preparation of such _t · _t .. '*·**-’·****·

I imiminocor^ugatea aiid'bcieS cases, file foil cytottetio p nblecules could be released from'thT^onJul^teifin unmodified fonn at the target site.

.t..uf..t5n.irf/'ϊ&lt;22.;. .

<img file="IN313DEN2012A_D0026.tif" />

eavable linkers have been investigated. In most A· , A .:

ciould only be observed, however, if the drug'

1(0 mW/ • joltoX

One of the cleavabte Jinkeri that has been employed for toe preparation of antibody-drug conjugates is an abid-iabilo^&amp;Uoe^based on ris-aconitio add that takes advantage of 1he addle environment of such as the endosomes encountered during receptor mediated endc^^^^^^^aet. Shen tadRyscr introduced th» method for the preparation of corvugates^f^aS^b^iWith macromolecular carriers (Biochcm. Biophys. Res. Commun. 102:1048-1054 (1981)). Yang and Reisfeld tised the same technique to conjugate. daunorubiefa to an anti-melanoma antibody (j. Nad. Cane, fast 80:1154-1159 (1988)), Recently, Dillman et al. also used an acid-labile linker in a eumlar fashion to prepare conjugates of daimorubicin with an ariti-T cell antibody (Cancer Res, 48:4097-6102 (19'88)).

An alternative approach, explored by Trouct d al. involved Wnlripg daunorubidn to an antibody via a peptide spacer arm (Proc. Natl. Acad. Sci. 79:626-629 (1982)). Trie was done under the premise that free drug could be released from such a conjugate by the action of lysosomal peptidases, · ·-4.,

In vitro revealed feat antibody-drug conjugates rarely achieved the same cvtotoxit^^Cfa^^s^^iiCi unconiugated drugs. This suggested flat mechanisms by-which drug nd^l^ arif released from the antibodies are very inefficient, fa the area of immunotoxins, conjugates formed via disulfide bridges between monoclonal antibodies and catalytically active protein toxins were shown to be more cytotoxic than conjugates containing other linkers. See, Lambt^.^^^; Chem. 260:12035-12041 (1985); Lambert et aL fa Immunotoxins 175'?0^,(^^r^C^^’ |S^^^Ohetie Ct al Cancer Res. 48:26J 0-2617 (1988). This was attributed to the of glutathione contributing to the efficient cleavage of the disulfide bond between an' antibody molecule and a toxin. Despite this, there are only a few repotted examples of the use of disulfide bridges for the preparation of conjugates between drugs and macromolecules. Shen et al. described the conversion of methotrexate into a mercaptoethylamide derivative .followed hy corrugation with poly-D-lystae via a disulfide bond (J.

’ . I ·

Biol. Chem. 260:10905-10908 (1985)). fa addition, a report described the preparation of a conjugate of the trisulfide-contafaing toxic drug caticheamycin with an antibody (Menendez et ai. Fourth International Conference on Monoclonal Antibody hnmunocoqiugates for Cancer, San • < · >. ’· i .

Diego, Abstract 81 (1989)). Another report described the preparation of a ocpjugate of the trisulfide-wnteining toxic drug catijtoo*9^ri^fo an antibody (Hinman et al, 53 Cancer Res. 3336-3342(1993)).

. i . ·,^Μ·;

One reason for the li

Λ* wSWEBRSR’

J. » a¹ •-l cytotoxic drugs fort bear a sulfar atom oonteOi^moieQ' teat can be readily used to link fire drug to an antibody via a disulfide bridge. Furthermore, chemical modification of existing drugs Is difficult without diminishing their cytotoxic potential.

Another major dmv^j^l|fiS^I^$^totibody-4rug conjugates is their inability to deliver a sufficient because of the limited number of targeted ·’ . h y-drug conjugates is the unavailability of antigens and the relatively moderate cytotoxicity of cancerastatic drugs lflce methotrexate, daunorubicin and vincristine. In order to achieve significant cytotoxicity, linkage of a large number of drug molecules either directly to fee antibody be through a polymeric earner molecule becomes necessary. However such heavily modified antibodies often display impaired bhufog to the target antigen and fast in vivo clearance from the blood stream. ·

<img file="IN313DEN2012A_D0027.tif" />

' Kupchan et at. c cytotoxic than tincristins (U.S.

j^ES of maytanrfnol (U.S. Pat No. 4,151,942).

'Snilogues of maytansinol have also been reported (Kupchan et al. J. Mod. Chem. 21:31-37 (1978); ffigashide et at. Nature 270:721-722 (1977); Kawai et al. Chem. Pharm. Bull. 32:3441-3451 (1984)). Examples of analogues of maytansinol from which C-3 esters have been prepared include maytansinol wife modifications on the aromatic ring (eg. dechlOro) Or at the C-9. C-14 (e.g. hydroxylated methyl group), C-15, C-18, C-20 and C4,5.

The naturally occurring and synthetic C-3 esters can be classified into two groups:

(a) C-3 esten with simple carboxylic acids (UB. Pat Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4309,428; 4,317,821; .4,322348; and 4331398), and (b) C-3 esters with derivatives of N-rnofoyl-L-alanine (U.S. Pat. Nos. 4,137.230; 4,260,608; 5,208,020; and Chem. Pharm. Bull. 12:3441 (1984)).

Esters totoxic than esters of group (a).

<img file="IN313DEN2012A_D0028.tif" />

Maytansine is a been reported to result reported to demonstrate; a' toft

Comparative Leukemia RoSQarchj-j^/jL:

urchin eggs and clam eggs atment Of L121Q cells in vivo with maytansinb has

[ in mitosis. Untreated control cells were *d* * ' .

m from between 3.2 to 5,8% (Sieber et al. 43 dacmat., 495-500 (1976)). Experiments with sea fiiytansine inhibits mitosis by interfering with the formation of microtubules ,through the inhibition .of the polymerization of the microtubule protein, tubulin (Rcmillard et al. Science 189:1002-1005 (1975)).

In vitro, P388, L1210, and LY5178 murine leukemic cel! suspensions have been found to be inhibited by maytausme at doses of lO'^J to Ι(Γ* unu-g/narJ wife the P388 line being the most sensitive. Maytansine has also been shown to be an active inhibitor of in vitro growth of human nasopharyngeal carcinoma cells, and fee human acute lymphoblastic leukemia line CEM was reported inhibited by concentrations m low as IO'¹ mg/ml (Woipert-DeFillippes et al. Biocbcm. I Pharmacol. 24:1735-1738 (1975)), .

In vivo, maytansine has also been shown to be active. Tumor growth in the P388 lymphocytic leuksmia system was'shown to be inhibited over t 50- to 100-fold dosage range winch suggested » high therapeutic index; also rignificarit inhibitory activity could be demonstrated with the LI 2 ID mouse leukemia system, toe human Lewis lung carcinoma system and the human B-16 ntelanocarcinon'» system (KwKhanliPed>£Prote33:2288-2295 (1974)).

<img file="IN313DEN2012A_D0029.tif" />

modified with a cross-linking reagent such as N-eicoimmidyl pyridyldithiopropionate (SPDP) tn introduce dithiopyridyl groups into toe antibody (Carleson et al. Biochem. J. 173:723-737 (1978); U.S. Pat. No. 5,208,020). In a second stop, a reactive maytansinoid havinga thiol group, such as DM1 (formally tensed N³' -deacetyl-N³ -(3-mcroapto-l-&lt;ncopropyi)-mayt«Dsmc, as the starting reagent., is added to toe modified antibody, resulting in toe displacement of toe thiopyridyl groups in the modified antibodies, and toe production of disulfide-Hnked cytotoxic maytanrinoid/antibody conjugates (U.S. Pat No. 5,208,020), A ouO-Step process for corrugation of maytansinoids is described ip U.S. Patent No. ¢,441,163. Mnytarwmoid-based imnnmotoxin technology is available from Immunogen Corporation (Crafaidge^h^L·--J

Another impotent toxins, Auristatins are derived from Dolastatin 10 that was pb^^|mH&amp;to^&amp;t&lt;dton Ocean sea hare Dolabclla, as a potent cell growth inhibitory substance, See U.S. Patent Nos. 4,816,444 and 4,978,744. With respect to other Dolastatins, sen also U.S. Patent Nos. 4,414,205 (Dolastatin-1,2, and 3), 5,076,973 (Dol«tatin-3), 4,486,414 (Dolastetih-A (Dolastttin-13), 5,138,036 (Dolaststin-14), and

4,879,278 (dolastatin-15^ _;^K^ainrf^mMfe»d_;by Dr. Pettit and colleagues at the University of Arizona, a variety of auristetii^^^^^^^^been tested and shown to be highly toxic to ceils; See Pettit et al. Antineoplastic tqgcpts 337. Synthesis of dolastatin 10 structural modifications. Anticancer Drag Des. 10(7):529-44 (1995), Woyke et al. In vitro activities and postantifimgal effects of the potent dolastatin 10 structural modification auristatin PHB. Antimicrobial Agents and Chemotherapy. 45:3580-3584 ¢2001), Pettit ¢1 al. Specific activities of dolastatin 10 and peptide derivatives against Chyptococcus neofbnnans. Antimicrobial Agents and Chemotherapy. 42:29612965 (1998), WoykeThrce-dimensional visualization of microtubules during toe Qryptococcus neoformans cell cycle and toe effects of auristahn PEE cn microtubule integrity and nuclear localization. Submitted, Antimicrobial Agentaand Chemotherapy,

Recently, additional auristatin derivatives have boon developed that appear quite effoctivo when delivered as payloads on.monometoy] auristetin £ (MMAE) has been shown as a potent conjugated to tumor specific antibodies.

Doronina et al. Development of ρ&lt;ηφ£fiiSpctopel antibody auristetin conjugates for cancer therapy. Nature Biotechnology. (2003) (available online), Francisco et al. cAClO-vcMMAE, an anti-CDSO-nxmomethyl auristetin B conjugate'with potent and selective antitumor activity; Blood.

(2003) May 8 [Epub a^ toxicity of toe auristatir/

<img file="IN313DEN2012A_D0030.tif" />

Apr 24 (available online), hi addition to toe I'shown that peptide-linked corrugates are more ¢2 stable , and, thus, more specific and leas toxic to normal tissues than other linker technologies in buffers and plasma. Doronina et al. Development Of potent monoclonal antibody auristatin conjugates for cancer therapy. Nature Biotechnology. ¢2003) (available online). Francisco et al. cAClO-vcMMAE, an tati-CD30-nx»oructhyl auristatin E conjugate with potent and selective antitumor activity· Blood. (2003) May 8 [Eptib ahead of print).’ Epub 2Q03 Apr 24 (available online). Such linters are btaed cm q,bmgjed'peptide design and include, for example, mAbvabne-citnillnw-MMAE conjugates, Doronina et al.

Development of potent conjugates for cancer therapy, Nature

Biotechnology. (2003) et al. cAClO-vcMMAE, tn anti-GD30rnouomethyl auristatin Boonjug^yi^ij^^^ielectiveaiititurDor activity. Blood. (2003) May 8 [Epub ahead of print). Epub ^S83^|*^^Btoblc online). Such designs and conjugation techniques are described, for example,' by King et al. Monoclonal antibody conjugates of doxorubicin prepared with branched peptide Enters: inhibition of aggregation by methaxytriethyleoeglycol chains. J Med Chem. 45(19):4336-43 (2002) tad Dubowchik et al.

Cathepsin B-scnsitive dipeptide prodrugs. 2. Models of anticanccr drugs paclitaxel (Taxol), mitomycin C and doxorubicin. BioorgMed Chcm Lett· 8(23):3347-52 (1998). Auristatin E-based immunotoxin technology based upon tire foregoing is available from Seattle Genetics Corporation ' /·., ' (Seattle, WA). ,

There arc a largo number of novel microtubule effecting compounds obtained from natural

<img file="IN313DEN2012A_D0031.tif" />

sources-extracts, and senrisyntheticand syntheticanalogs that appear to possess potential as toxins for the generation of immunocQqjugntes,. (see the website at newmcdinc “dot” com). Such molecules and examples of drug products utilizing them, include the following.* Colchicinc-site ’ . r'1.4·,* Λ. _{v k} *. ' · . .

Binders (Curacm), C * * .........~ ’

Ciyptophycins (LY355703j)?^^ 10, ILX-65J, Symplostatin*^ KOS-862.ZK.-EPO), El&lt;

NPI-2358), Hemi raterhns.

&amp; Combretastatin A-4 prodrug (CA4F), Oxi-4503), tin and Analogs (Auristatin PHE, Dolastatin Uones 0KMS-247550, BMS-310705, EPO906, hondrinB,(E7389), Haliiuide (NPI-2352 and ,. Maytansinoids (“DMl^XBivatuzumab mertansinc, Q*¹*¹**¹^*? mertansine,huN9&lt;)l-DMl/BB-109piTAP, MLN591DM1, My9-6-DMl,

Trastuzumab-DMl), EC-SPES, Peloruside A, Resveratrol, S-allylmercaptocysteine (SAMC),

Spongistatins, Vitflevuamide, Molecular Motor-Kinerins (SB-715992), Designed Colchicine-Site Binders (A-289099, A-29362Q/A-318315, ABT-751/E7010, D-24831/D-64131, ZD6126), Other Novel Spindle Poisons (2-Methoxywtradiol (2-ME2), Bezunidazole Carbamates (ANG 600 series, Mebendazole), CPZ48/GP461, HMN-214/R440, SDX-1Q3, T67/T607). Further, additional marine derived toxins era reviewed in Mayer, A.M.S. Marine Pharmacology in 1998: Antitumor and Cytotoxic Compounds. The Pharmacologist. 41(4): 159-164 (1999).

<img file="IN313DEN2012A_D0032.tif" />

TheraireuticAdgtiniatrationaitoFonnuiation^

A prolonged duration of action win allow for less frequent and mare convwfcnt dosing schedules by alternate parenteral routes such as intravenous, subcutaneous or intramuscular injection. ,

When used for tn formulations described herein should be sterile. This is reafely 'ac^j^^^V^r*^faqjle, by filtration through sterile filtration membranes, prior to Or following lybphiliratien and reconetititiion. Antibodies ordinarily will be stared in lyophilized fora or m solution. Therapeutic antibody cornpositiotis generally are placed into a container having a sterile «cocas port, for example, an intravenous solution beg or vial having an adapter that allows, retrieval of the formulation, such as a stopper pierccable try a hypodermic injection needle.

The route Of wto'be^-admi^?trati&lt;X).is in accord with known methods, e.g., injection or infusion by mtravenou^l'-intjt^i^^Ji^iacerebral, intramuscular, intraocular, intraarterial, intrathecal, inhalation or ^:itto»l^pi^^^'.er by sustained release systems as noted below. Antibodies arepreferably administered continuously by infiirioa or by bolus injection.

An effective amount of antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, tire route of administration, and the condition of the patient Accordingly, it is preferred for foe therapist tD titer foe dosage and modify the route of

<img file="IN313DEN2012A_D0033.tif" />

administration as required to obtain foe optimal therapeutic effect Typically, foe clinician will administer antibody until a dosage is .reached that achieves foe desired effect The progress of this therapy is easily monitaredby.

Antibodies at desCri acceptable carrier. Therapeutic iaitesfecr by tire assays described herein.

Safe y ·>*<- ** · >*-»“>T ______________ ______._v ·. be administered intravenously· or through tire nose ar lung, preferably as a liquid or ppwder aerosol (lyophilized). Composition can also be administered parenterally or subcutaneously as desired. When administered systemically, therapeutic compositions should be sterile, pyrogen-free and in a parenterally acceptable solution haring due regard for pH, isotonicity, and stability. These conditions arc known to those skilled in

<img file="IN313DEN2012A_D0034.tif" />

the ut Briefly, dosage formulations ofthccpmpounds are prepared for storage or administration by mixing the compound¹, degree of purity With physiologically acceptable carriers, excipients, or stibihi ' ' ’ .' · '.'in * concentrations employed, aha. me are non-toxic to tire recipients at foe dosages and n such as TRIS HC1, phosphate, citrate, acetate and Other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (Jess than about ten residue?) peptides such as polyazginme, protwna, such as scrum albumin, gelatin, or immunoglobulins; hydrophilio polymere such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or- tog&amp;srie; monosaccharides, dteaceharides, and other carbohydrates including cellulose- or its eJerivatrves, gtaxjse, mannose, or dextrinr, chelating agents ' . · . . .: . · Ί · .

<img file="IN313DEN2012A_D0035.tif" />

such as EDTA; or sorbitol; counterions such as sodium atid/or nonionio surfactants such « TTOEJl.^rokONICS or polycthyleneglycol.

Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in.Rwnlngiun's PharmaceuHcal Sciences (18“ ed, Mack Publishing CompaDy.Eastoi), PA, 1990). For example, dissolution or suspension of the active compound in a vehicle such as water tt naturally occurring vegetable oil tike sesame, peanut, or cottonseed oil ar a synthetic fatty vehicle like ethyl Oleate or the like may be desired. Buffers, preservatives, antioxidants and die tike.can be incorporated according to accepted pharmaceutical practice. ,.·...· . .

Suitable examples of sustainbd^elease preparations include semipameable matrices of solid hydrophobic polymer?^^^^^^eptjde, which matrices are in the form of shaped articles, films or sustained-release matrices Include polyesters, hydrogels (e.g^ poly(2-hydroxyett^nic^fcryiate) as described \sy Langer et al„ J. Biomed Mater. Res., (1981) 15:167-277 mid Langer, Chem. Zbcft., (1982) 12:98-105, or poly(vinytalcohcl)),

<img file="IN313DEN2012A_D0036.tif" />

b acid-glycplic acid copolymers such as the LUPRON jd of lactic acid-glycolic acid copolymer and teuprolide polylactides (U.S. Pat No. 3,773,919) EP 58,481), copolymers of L-glutamic acid end gamma ethyl-L-glutamate (Sidtpa^A^,. £&amp;$$piers, (1983) 22:547-556), non-dcgradable ethylene-vinyl acetate (Langer «( al?. Depot™ (injectable micm^x^l acetate), and poly-D-{-)-3-hydroxybatyric acid (EP 133,988).

White polymers such w ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37C, respiting in a loss of biological activity and possible changes in pmrwTnngwTimty Rational strategics can be. devised for protein stabilization depending on the mechanism involved. For example^ if the aggregation mechanism is discovered to be ititennolecutar S-S bond fonnati^tbropgii^sulflde interchange, stabilization may be achieved by nwtifying sulfhydryl solutions, controlling moisture content, using appropriate additix^te^ffife^feMPolymer matrix compositions,

Susndnedrreleased ^po^tioq^, jji^pinClude preparations of crystals of the antibody suspended in suitable formulations capable of maintaining crystals in suspension. These preparations when injected subcutaneously or intraperitoneally can produce a sustain release effect Other compositions alto^fa^fe^^^ily entrapped antibodies. Liposomes containing such antibodies arc mraiMtredb^ptej^^^S^pyi^yr'-sei U.S. Pat. No. DE 3,218,121; Epstein et al.. Proc. Natl. Acad. Scj.USA, Hwang et al., Proc. Natl. Acad. Sci. USA, (1980)

77:4030-4034; EP 523¾ BE 36,^7fi; EP 88,046; EP 143,949; 142,641; Japanese patent application 83-118008; U.S. Pat Nos. 4,485,045 and 4,544,545; and EP 102,324.

<img file="IN313DEN2012A_D0037.tif" />

The dosage of the antibody fonnuhtion for a given patient will be determined by the attending physician taking into oonsidtfetiou various fiaotors known to modify die action of drugs including severity and type of disease, body weight, sex, diet, tune and route ofadnsnistration, other medications and other relevant clinical ftutors. Therapeutically effective dosages may bo determined by cither tn vitro or in vivo methode.

An .effective amount of the.antibody to.be employed therapeutically will depend, for example, upon tho toerapciitib · · ·. ' .- 'λ .’Λ patient. Accordingly, it is pre

<img file="IN313DEN2012A_D0038.tif" />

titer tile dosage Mid modify die route of »,>· ··*· · * administration as required to obtain die optimal therapeutic effect A typical daily dosage might range from about 0,001 mgfleg to up to 100 ttig/kg or more; depending on the festers mentioned above. Typically, the clinician will administer tho therapeutic antibody until a dosage is reached that achieves the desired effect The progress of tide therapy is easily monttored by conventional

<img file="IN313DEN2012A_D0039.tif" />

agents that arc incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulation» can be found in die formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sctcnoes (18* ed, Mack Publishing Company, Easton, PA (1990)), particulariy Chapter 87 by Block, Lawrence, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, Kpid (cationic or anionic) containing vesiples (such as Lipofectta¹*), DNA conjugates, anhydrous

<img file="IN313DEN2012A_D0040.tif" />

accordance with the present inventi&amp;n, providc4^;&amp;at the active ingredient in the fommlation is not ·. . .·' .&lt; r · · .· .

inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See alto Baldrick P. “Pharmaceutical excipient development: die need for preciinical guidance.” Aegwl. ' Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. Lyophilization «nd develt^^ae^qf.rojyl.protein pharmaceuticals.” /nt. J. Pham. 203(1-2):1-60 (2000), Charman WN'.^Ni^gky&amp;ji^ii^fr-md oral drag delivery-some emerging concepts. J Pharm Set /89(8):96^-78^^^¾^^^^^ aL “Compendium of excipients for parenteral formulations PDA J Pluihn&amp;^ecfoioi'. $2:238-311 (1998)and die citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists. EratsratigapfABtihadiw /

Antibodies, as described herein, were prepared through the utilization of tire XepoMouse® technology, as described below. Such mioe, then, ere capable of produciughurnan immunoglobulin molecules «nd antibodies, apd. are deficient in fee production of murine immunoglobulin molecules i ; γς>,Γ and antibodies. the same are disclosed in the patents, appHcatrans, add ' -In particular, however, a one embodiment of transgenic production Of rafob »id enVbOSes therefrom is disclosed in U.S, Patent Application Serial No. 08/759,620, filed December 3, 1996 and International Patent Application Nos. WO 98/24893, published June 11, 1998 and·WO 00/76310, published December 21, 2000. See afro Mendez ct al. Nature Genetict 15:146-156 (1997).

Through use of such technology, folly human monoclonal antibodies to a variety of antigens can be produced, he one ettibodfooMUt, XcuoMause® tines o f mice are immunised with an antigen of interest (e.g. EGFRvnj), lymphatic cells are xeooveced (such as B-cells) from the mice fate expressed antibodies, and such cells.«a fused witha my eloid-typo cell line to prepare immortal hybridoma cell lines, and such cell lines that produce- anti¹for the production of multiplelfy

<img file="IN313DEN2012A_D0041.tif" />

>are screened and selected to identify hybridoma j®ttigen of interest, Provided herein are methods raiabelj-lines that produce antibodies specific to EGFRvin.

Further, provided herein are characterization of foe antibodies produced by such cell lines, including nucleotide and amino acid sequences of the heavy and light chains of such antibodies.

\ Alternatively, instead of being fated to myeloma cells to generate hybridomas, foe antibody produced by recovered c^i^|b^^^)^^nnusnzed XenoMousc® lines of mice, are screened farther for preferably EGFRvUI protein. Such screening includes ELISA with binding to NR6 M cells stably expressing fall length EGFRvIH and interaatization of EGFRvUI receptor by the antibodies in NR6 M cells. Single B cells secreting antibodies of interest are then isolated using a EGFRvUI-specjfic hemolytic plaque assay (Babcock ct ah, Proc. Natl. Acad. ScL USA, 193:7843-7848 (1996)). Cells targeted for ysis are preferably Sheep red blood ceils (SRBCs) coated with the EGFRvIU antigen. In foe jreseuoe of a B cell culture secreting the immunoglobulin of interest and complement, foe formation of a plaque indicates specific EGFRvIH-mediated lysis of foe target cells. The single antigen-specific plasma cell in faewnter of foe plaque: can be isolated and the genetic information fate encodes foe specificity of from faesjnglo plasma cell Using reversexaoscriptase PCR, the of the antibody , secreted can be: cloned.

»uph efonM. DNA can thenod^^^^^^^^^tob’suitable expression vector, preferably a vector ’.assette such as a pcDNA, more preferably such a pcDNA vector containing foe constant domains >f imTnunglobulin heavy and light chain. The generated vector can then be transfected into host sells, preferably GBO cells, andcuitured in conventional nutrient media modified as appropriate for

I . f nducing promoters, sgltttin^ .^ggTymaote, or amplifying the genes encoding foe desired icquences. Herein, we multiple single plasma ceils that produce antibodies | rpecific to BQFRvffi^^^^^^^^^^xnaterial that encodes the specificity of the sntiSGFRvin antibody is isrda^, ffi&amp;bdS^d^to a suitable expression vector that is then transfected ato hostcells. .

<img file="IN313DEN2012A_D0042.tif" />

B cells from XcnoMousc mice may be also be used u a source of genetic material from which antibody display libraries may be generated. Such libraries may be made in bacteriophage, yeast or jn vitro viaribosoine display using ordinary skills in foe art, HyperinraunizodXeaoMouse mite may be a rich source from which high-affinity, antigen-reactiveantibodies may be isolated. Accordingly, XcnoMouse mice hyperiramunized against EGFRvIII may be used to generate antibody display libraries from winch high-affinity antibodies against BGFRvUI may be isolated. Such libraries could be screened jj^ffl^thc pep3 oligopeptide and the resultiugty derived antibodies screening ag^^^i^^^i^BgFRyin ^to confirm specificity for the natively display antigen. Full IgC^t^xty^a^^^be expressed using recambhmt DNA. technology, See e.g., WO 99/53049. ’'' ’* · > .

bi general, antibodies produced by fee above^nentioued cell Knes possessed fully human

IgGI or IgGZ heavy chains wife possessed high affinities, typl measured by either solid phaspii^a-sc^^^ lower affinitiesi from about 10* through about

<img file="IN313DEN2012A_D0043.tif" />

•tit flight chains. In one embodiment, flee antibodies ^Ksof from about Iff* through about I0^JJ M, when In other embodiments too antibodies possessed

As appreciated by One of skill in the art, antibodies in accordance With the present embodiments can be expressed in cell lines other than hybridan» cd] tines. Sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus for vector) or by transfection procedures known in the art, as exemplified by U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. The transformation procedure used depends upon the host to be transfonned. Methods tor introduction Of heterologous polyhucleotid^· dextran-mediated timtefistetic protoplast fusion, electropora microinjection of the UNA into nuclei. .

. . ; ‘ , J . i .u. :·

Mammalian cell lines available as hosts for expression are well known in the art and . ‘‘ />>3^ .. ' ' ' . ..

include many immortalized cell-',lines Jtom the American Type Culture Collection ' .. v ij: t -i . ; ..

(ATCC), including but not iinnfScto ovary (CUD) cells, HeLa cells; baby hamster kidney (BHK) cells, monkey iloSty human hepatocellular carcmoma cells (eg., Hep

G2), and a number of other cell iinei Cell tines of particular preference are selected through

<img file="IN313DEN2012A_D0044.tif" />

in cells -are well known in the art and include precipitation, polybrenc mediated transfection, of toe poiynucleotidc(s) in liposomes, and direct

<img file="IN313DEN2012A_D0045.tif" />

determining which cell lines have high expression levels and produce antibodies with coMtitutive EGFRvIII binding properties.

<img file="IN313DEN2012A_D0046.tif" />

<img file="IN313DEN2012A_D0047.tif" />

The following 3 conducted and fa results achieved are provided for illustrative not to be construed as limiting upon fa present invention. >;. ,_{; 3} ;

The strategy for generating BQFRvin-epecific antibodies initially involved immunization of XerioMoUsc mice wife combinations of antigens (peptide, Various soluble prote'ns, antigenexpressing cells) followed by isolation of antibody producing cells, either as ftroutfi fusions to produce hybridonas or isolation of B cell ceils through fa XcnoMax™/SLAM¹* technology. Antibody producing cells were subjected to a primary screen for specificity by PI TS a _B secondary screen for cell surface binding by FMAT and/or FACS, internalization assays were then conducted to identify antibodies feat would bcuscfii] for drug delivery. Affinities of fa antibodies were measured. Certain antibodies were selected for epitope mapping. In addition, certain antibodies were selected for in vitro and in vtw tests to analyze therefficaqy ef such antibodies for treatment of cancers. , , .&lt;! ·· » *!' ' Auriga gftPOTtron

A EGFRvIII PEP3-K . , _

In oonnoctioo wife human EGFRvIII PEP3 (LEEKKGNYVV

TDHCCSEQroN^;^¹· *’ptiiesizedby R&D Systems. ThePEP3 peptide was fan conjugated tokcybbleThn (KLH), as follows: EGFRv©PEP3 (200 mcg) (R&D) was mixed wife 50 mcg of keyhole limpet hemocyanin (KLH; Pierce, Rockford, IL) to a fmal volume of 165 mcl using distilled water. 250 mcl Of conjugation buffer (O.IM MBS, G>9M NaCl, pH 4.7) was added and EGFRyHI PEP3 tad KLH were Crosslinked by fa addition of 25 mcl of 10 mgfml stock aolutien of l-cthyl-3-{3-diinethylaminopropyncarbodinnide hydrochloride (EDC, Pierce, Rockford, IL). Conjugate was incubated for 2 hours at room temperature and fa unreacted EDC was removed by centrifugation through a 1 kDa filter (CcntrifUga] filter; Minipore, Bedford, MA) using PBS pH 7.4; , .

In connection wife Examples, the 14-rper human EGFRvIII PEP3 (L E E K KG N Y V V T D H C (SEQ © NO: 56)) peptide was custom synthesized. The PEP3 peptide was then conjugated to KLH, to follows: human EGFRvIII PEP3 (200 mcg) was mixed wife 50 mcg Of keyhole limpet hemocyanin IL) to a final volume of 165 mcl using distilled water. 2S0 ’mc]^J-t>f to^ugiri9^a>>l^:!^^r (O.lM MBS, Q.9M NaCl, pH 4.7) was added and EGFRvIII PEP3 and KLH were crosslnfad by fa addition of 25 mcl of 10 mgfml stock solution of · ' ·· 1 ·. .><:<·. 'MtW- · .

•1!

<img file="IN313DEN2012A_D0048.tif" />

l^thyl-3-[3-diioethylaminopn5>y '1 Conjugate was faubated oentrifogatioa through ail

<img file="IN313DEN2012A_D0049.tif" />

4^J^/4rochloride (EDC, Pieroc, Rockford, IL), re and fa unreacted EDC wu removed by tint Miltipcre, Bedford, MA) using PBS pH 7 A

<img file="IN313DEN2012A_D0050.tif" />

B, B300.19/EGFRvni Jkmsfebtpi^ in order to prepare foe B300.19/E&lt;3FRviH tnmsfoctaato, wild type EGFR was foftiaHy cloned from A431 cells and EOFR gene was modified to code for BGFRVHI to delete foe codons encoding residues 6-273, with a codon encoding a Glycine residue created at foe junction of the deletion. Ths deletion occurs wtititafoe codons surrounding the deletion GTT (Valine) and CGT (Arginine), such that the fife deletion is GGT (Glycine). (Wikstrsnd et al. J

Neurovircl. 4(2):148-58 '

1. Cloning of Wild tvde'EQFR Cagstruct!

PolyA+mRNA was extracted finm A431 (ATGC) cdls usingMicro-fast RNA kit (InvitrOgen, Burlington, ON). Total oDNA was synthesized from polyA+ mRNA with taodam pdN6 primers and M-MuLV reverse transcriptase (NEB, New England Biolabs, Beverly, Mass.). A 2.3kb PCR product wm amplified fromA43l oDNA with the following primers:

63) sense 5’-GGATCTCGAGCCAGACCGGAACX3ACAGGCCACCTC-3’·, (SEQ ID NO : 62) anti-sense 5’-GGGATCTC^^^ (SEQ ID NO.

using Pfb DNA ' ·

The PCX product was digarte^MmS*6v6i, gel purified and ligated into plasmid pWBFNP (see International Patent Application No. WQ 99/45031) Ihieartecd with Xhol to yield plasmid WtEGFR/pWBFNP-

2. Genyatiortof EGFRvlH Construct:

PCR products amplified^^fro^jil^^id Wt-EGFR/pWBFNP template with primer pairs C13659/C29538 wk} International), in which the C29538 and C29539 were nhosnhorYlatedwilh.Tj^>ly^^qtid^kOT»e (NEB, New England Biolabs, Beverly, Mass.):

C13659: 5’&lt;GGATGAAITCCCAGACCGGACGACAGGCCACCTC-3'(Sense) (SEQ

ID NO: 64); · '

C29538; 5'-CTTTGTTTTCCTCCAGAGCC-3’(Anti-Sense) (SEQ ID NO: 65);

C29539:5’-GTAATTATGTGGTGACAGATC-3’(Sense) (SEQ ID NO: 66); CUZSSiS’-CGGATCTCGAGCTCAAGAGAGCTTQGTTGGGAOCTG'fAnti-Sense) (SEQ ID NO: 67). · · · · · ' ¹ . ' i .. r ” ¹ were ligated to introduce a deletion in. the. sequence encoding amino acids 6 through 273 of fire EGFR extracellular domain and^Jkrf^d^^ij^isression vector pWBDHFR2 (see International Patent Application No. ΦΟ

A 232 bp fragment foe deletion was generated with primer pair

C13659/C29S38 from Wt-EGFR/pWBFNP template amplified with Pfo polymerase (NEB, New England Biolabs, Beverly, Mass.). The PCR product was digested with EcoRl (NEB, New England ,'7

Biolabs, Beverly, Mass.) and gd purified. A 1273 bp fragment representing foe 3‘end of the deletion was generated with primer pair C29S39/CI4288 from Wt-EGFR/pWBFNP and the sl template amplified with Pfa polymerise. The PCR product was digested with Xhol (NEB, New England Biobibs, Beverly, Mass.) and ge] purified. Fragments were ligated into EcoRl/Xhoi digested pWBDHFR2 with T4 DNA ligase (NEB, New England Biolabs, Beverly, Mesa) to yield construct EGFRvUI/pWBDHFR

The intracellular domain ofEGFR was introduced into the resulting construct as follows: A I566bp DraM/XhoI fragment was isolated from plasmid Wt-EGER/pWBFNP and ligated into Draffl/Xhol digested EGFRvIH/pV/BDHPk to yield EGFRvIH-FL/pWBDHFR..

3. Transfection of B300J9 cells with EGFRvIH-FL/pWBjDHFR.'

B300.19 cclle (8x10®) were used per transfection in 700 μΐ DMEM/HI medium. 20 pg EGFRvIH-FL/pWBDHFR and 2 pg CMV-Puro plasmid DNA were added. Cells were electroporated at 300 volta/960uF with Bio-Rad Gene Pulser. Following electroporation, cells were cooled on ice for 10 minutes and, thereafter, 10 ml non-selcction medium (DMEM/HI Glucose, 10%. FES, 50 μΜ ΒΜΕ,^ ., L?/p^Bmpe, 100 units PeniCillin-G/nd, 100 unite MCG Streptomycm/ml) wasa&amp;jedfdr48hrs at 37*C 73% COj.

Following fiaqubajl^^^^^^^Viritd selection medium (DMEM/HI Glucose, 10% FBS, 2 mM L-Giutamm^^,4^>B^^4w0 units Penicillin-G/ml, 100 unite MCG StreptomycinAnI,2ug/Wpuroniycinj,at2xlb*, 0.4x10*'and 0.08x10* cells/ well ip 96 well plate and were selected in selection medium for 14 days to generate stable dienes. Pftro resistant clones were stained with E752 mAb (an anti-EGFR antibody, described in Yang et al., Crit Rev Oncol

Hematol., 38(1):17-23 (2001)) and Stot anti-humim IgG PE then analyzed on FACS Vantage (Becton Dickinson). C. (VwwlTiirtinn of I (c.HTaTh .RbFoBxpr^v^COTstnMt^ h> order to generate tire EGFRvUI nibbit Fe fiisiou, protein, we first constructed a vector oontainmg DNA encoding rabbit Fc. T£is was ligated with DNA encoding EGFRvUL This approach is described in more detail below:

I. Construotion ofRbFc/bcDNASAHygU'.

Primers 1322/867 (below) were used to amplify b 72 lbp fragment encoding the HingeCH2-CH3domain of rabbitlgO. ,..<img file="IN313DEN2012A_D0051.tif" /> #1322 (sense): (SEQ ® ^°⁵ **>

#867 (antisense): S'-A^fe ^N0:®> ;

The resuItingJPClt.pr

ΚρπΙ/NotI digested pcHNAXll

RbFc/j»DNA3.1 Hypo.

<img file="IN313DEN2012A_D0052.tif" />

<img file="IN313DEN2012A_D0053.tif" />

ATTTACCCGGAGAGCGGOA-3 ’ (SEQ ID

3» · ’ '

I with Kpnl and Noll, gel purified and ligated into ivitrogen, Burlington, ON) to yield plasmid

<img file="IN313DEN2012A_D0054.tif" />

<img file="IN313DEN2012A_D0055.tif" />

2. Construction of EGFRvIB-RhFc/DCEP4:

Primers 1290/1293 (below) were used to amplify an I165bp product from EGFRvIHFL/pWBDHFR plasmid template with Pfu polymerase #1290 (sense): 5'-CTACTAGCTAGCCACCATGCGACCCTCCGGGA-3’ (SEQ CD NO: 70) #1293 (anti-sense); 5’-CGGGGTACCCGGCGATGGACGGGATC-3’(SBQIDNO: 71)

The PCR product was ftgested with Nhd and Kpnl, gel purified and ligated into NheI/ΚρηΙ digested RbFc/pcDNA3.1. Hygro·tfryifeiid plasmid EGFRvUI-RbFc/poDNA3.1Hygro.

A 2170 bp SnaBIDQfe^^aji^^|^|lated from EGFRvUI-RbFc/pcDNAS.lHygro and subcloned into SnaBI/XhoI digeS^i/ttTEPiiffnyitrogen, Burlington, ON) to yield plasmid EGFRvIII-RbFc/pCEP4. ’ ... ‘

3. Generation ο!293Ϊ ]Β3ΡΚν£^Ρο stable Cell Lines;

Plasmid BGFRvlil-RbiFc^CEP# was introduced into 293F cells (Gibco, Grand Island, NY) by Calcium Phosphate transfection, as follows: one day prior to transfection, 1x10*293? cells were plated on a gelatin coated 100mm tissue culture petridish and incubated at 5% CO2,37C. Cells wap fed with 10 ml of fresh non-selectiyp media (DMEM/F12, 10% PBS, 2mM L-Glutananc, lOOU/ml Penicillin G, lOOU/ml MCG Streptomycin) 2-3 hours before transfection. Transfection reagents were prepared in a microfUge tube, as follow: IQpg of DNA (BGFRvIII-RbFc/pCEP4) was mixed with 62μΙ of 2M Calcium Phosphate and deionized water to make the final volume 500μ1. In another tube pipette 500μΙ of 2XHBS is drawn and used to transfer the transfection

<img file="IN313DEN2012A_D0056.tif" />

reagents.

The solution in the tube pipette was added to the cells 'drop by drop, while maintaining proper pH by leaving cells fa a 5%CO2 incubator until transfection was perfonncd, 15-20 hours * ¹ 't · . * ¹ · ’ after transfection, Cells were washed with PBS and feed with 10ml of fresh 293F non-selective media. Expressingcells w^e giatyesthc 48-72 pott-transfcction and cells were plated at 0.08x10“ cellsiwell in ay?J^^fectivemedia(DMEM/F12,10%FBS, 2mMLQlutamine, lOOU/ml Penicillm^^V^myMGG ^trBptomycin, 250ug/ml Hygromyctn) for 14 ' . · , ? V'G^-.r’aAH;·- ____ .

Hygromycin resistent clonei weft screened by ELISA using anti-EGFR. antibody E763 (US Patent No. 6,235,883) u the capture antibody st lug/ml and detecting with a goat anti-rabbit IgG

HRPQ (CalTag) at 1:100 dilution.

D. Conjugation of EGFRvUI PEP3 to OVA viaMaleimide Conjugation

The EGFRvinpeptidB-OVA used for titration of antibodies (Example 3) was produced as follows:

207 gg of EGFRvUI PEP3 was reduced using, pro-weighed DTT from Pierce (#20291). One vial of 7.7mg of pre-weighed DTT was dissolved using 100 pL of de-ionized water. The DTT

<img file="IN313DEN2012A_D0057.tif" />

Stock Was added to the EGFRvIII PEP3, The volume of the reaction was brought to 600 pL «sing PBS pH 7.4. The reaction was rotated for 30 minutes at room temperature,

A GiO oolunm was prepared by weighing out 5 grains of GIO sephadex beads and adding 40 mL of PBS, mixing and leaving at room temperaturefor 10 minutes, and then eantrifiigipg the beads at 1000 rpm for 10 minutes. The supernatant was removed and an additional 20 mL of PBS was added. The beads were centriftiged at 1000 rpm for 10 minutes. The supernatant was removed and enough PBS added ώ,φ&amp;Ίΐ 5S%etany-ofG10 sephadex beads. 5 mL ofthe 50% shiny mixture was added to a 5nu&lt;s®^9lS0U^^dte'colunip was placed, in a 14mL polypropylene tube. The column was cottr^ig^Mtij^^So for 3 minutes. Another 3 mL of PBS was added and the column was cenrifoged again.' at 1000 rpm for 3 minutes. The polypropylene tube was replaced with a new tube and the columns were now ready to use.

DTT was removed from fee reduced peptide. After the 30 minute reaction time far reducing the peptide, 300 pL of the reduced peptide was added per column. The column was centrifuged at lOOOrpm for 3 mlquWu>An iid0iwal 250 pL of PBS was added to each column and centrifuged agabvat 1000χ4^^τΒ.ήύ&ΐή^ίί^^6.'reduced peptide was collected in the 14 mL polypropylene tube. , ·

The reduced peptide was conjugated to maleiinide activated OVA and collected in an eppendorf tube. 2 mg of the maleimide activated OVA was dissolved (Pierce: 77126, Rockford IL) with maidmide conjugation buffer to make a 10 jngftnL stock, 414 pg of the maleiinide activated

OVA was added to the reduced peptide in the eppendorf tube. 500 pL of the tnddmide conjugation buffer was added fo the reaction. The reaction was allowed to incubate for 2 hours at room temperature and then 2mg of cysteine was added to-quench any active rnaleimide groups that might have been‘present The cysteine was allowed to react for 30 additional minutes at room temperature.' The conjugste-yri^^tbeo* pH 7.4. Hus removed anyfre?{^^ conjugate was removed f

<img file="IN313DEN2012A_D0058.tif" />

jwjfo a 10K centrifugal column 3 tiroes using IX PBS ejr3id hot conjugate to the OVA and free cysteine. The iTcolumn using gel loading tips and transferred to an eppendorf tube. Finally, the conjugate was brought to the desired concentration using IX PBS pH 7.4. Ths conjugate produced had a molar ratio of 14.5:1 (peptide:OVA)

Example 2

Production of anti-EGFRv^A^^ttoics ThroughHybridQmaGcnerstiQR

Eight XenoMouse niqe''foto_J un^uc^lnntibDdies with a gamma-J constant region ' ' ί* ϋί ·ζ^.·ιοία·ΐηΐ^ϊηί32:3ϊ·,«: ‘ ··’ ·.. '

[XenoMouse G1 mice) were imniuriizcd-pri.dayOend boosted on days 11,21,32,44 and 54 for this protocol and fusions were perfonned on» day 58. All immunizations were conducted via subcutaneous admfatistrstion st the base of tail plus intraperitoneal administartioh for all injections. 1 The day 0 immunization was done With 1.5 x 10⁷ B300,19/EGFRvHI transfected cells (Example

IA) suspended m pyrogen free DPBS admixed 1:1 v/v wife complete Freunds adjuvant (CFA) Sigma, St. Louis, MO). Boosts on days 11, 21, and 32 were done with 1.5 x IO⁷

Λ

B300.19/EGFRvHf transfected cells to DPBS admixed 1:1 v/v with incomplete Freunds adjuvant 0FA) (Sigma, St. Louis, MO). The boosts on day 44 was done with 5 pg of the PEP3 (EQFRvin peptide) - KLH conjugate (Example 1) inDPBSadmixed 1:1 Wv with IFA and final boost, on day 54, was done with 5dg PEPS (EGFRvUI peptide) -KLH conjugate in DPBS without adjuvant

On day 58, mice were euthanized, Mid then inguinal and Lumbar lymph nodes were recovered. Lymphocytes were released by mechanical disruption Of the lymph nodes using s tissue grinder then depleted of T.qqfl^by ^^fl^gidiYe selection. The fiwitm was performed by mixing washed enriched B c^s-a^:iu^^^^j^ioma P3X63Ag8.653 cells purchased from ATCC, cat # CRL 1580 (Kearney 123:1548-1550 (1979)) at a ratio of 1:1. The cell mixture was gentiy pelleted by centrifugation at 800. g. After complete removal of file the cells were treated with 2-4 mL of fratBae solution (CalBiochem, oat # 53702; 0J mgM in PBS) for no moire than 2 πΛήΡ^,^Τ^ρ, FBS was addod to stop .the enzyme activity and the suspension was tdju^S^^^t^j^i^iitoig.docfro «11 fusion solution, ECFS (OjM Sucrose, Sigma. Cat# S7903, O.ImM Magnesium Acetate, Sigma, Cat# M2545, 0.1 mM Calrium

<img file="IN313DEN2012A_D0059.tif" />

Acetate. Sigma, CM# C4705 (St Louis, MO)).

The supernatant was removed after centrifugation and the cells washed by resuspenrion in 40 mi EGFS. This wash step was repeated and the cells again were resuspended in ECFS to a concentration of 2x10* cellsftnl. Hleotro-cell fusion was performed using a fiiskn generator, model E CM2001, Genetronic, Inc., San Diego. CA. The Alston chamber size used was 2.0 ml, and using the following instrument settings; Alignment condition: voltage: 50 v, time: 50 s, Membrane ' ‘U . · breaking at: voltage: 3000 v, time: 30 μβ,Ρόβί-ήίβϊοη holding time 3 s. After fhston, the cells were re-suspended in DMEM (JRH BfosM«ices),15% FCS (Hyclonc), containing HAT, and supplemented with L-glu rtro,.PPI (oxaloaoetate, pyruvate, bovine insulin) (all from

Sigma/Si. Louts, ^40) ar^jL^fBo^ '^Mwwihoim) for culture at37“C*nd 10% CO² in air.

Cells were plated to tissue culture plates at 4x10* cells per well.

Cultures were maintained in HAT (hypoxanthine, aminopterin and thymidine) supplemented media for 2 weeks before transfer to HT (hyppaarrtirip· and thymidine) supplemented media. Hybridomas were selected for by survival teH^lmq^^ad.SUpcmatants were screened for antigen reactivity bv ELISA. The ELISA formatejfitaijed.....ng'stgjematatt on antigen coated plates (EGFRvUI <img file="IN313DEN2012A_D0060.tif" /> peptide-QVA coated plates and wil^t^pe EGpyteptide-OVA coated plates as a oounter screen) Mid detecting EGFRvM-spwifie binding using horseradish peroxidase (HRP) labeled mouse antihuman IgG (sec Table 2.1).

.1

<img file="IN313DEN2012A_D0061.tif" />

<img file="IN313DEN2012A_D0062.tif" />

TABLE 2.1

PlataWeU

13.2 DIO

133 C)2

133 Ell

13.6 Bl 1

Hybridema 1**0Ρ 2nd OP.

., , . Jforionplate . muEOFr EGFr ' ' ' ^{0 &lt;βα} 0.051

0.049

0.049

Q.052 γ4Μ * ι· i^i

Qdlcdoxubi .’c’UiSmj.e 5'4?

• ···.·“.* · M AV .133 ¹ ’*·

134

2.633

2.443

1.081

1311 •r· jji,.

13.1.1

13.12

OD#1 OP ‘ ·* ^1^'.

'ά.5β^2^ώ'0^Λ.6Ϊ4 ; ’ 0.5c/wF5 2248

Hate

2.586

1272 ♦f· · s

0.042

0.041

As will be observed, at least four antigen specific hybridomas were detected; 13.1,13.2, 133, and 13.4. These hybridomas feat were positive inthe ELISA assay EGFRvIU specificity were confirmed by FACS on stably transfected 300.19 cells expressing EGFRyin versus 3QO.19 unttansfectedparental Cells. ’ ' '

Clearing was perfta^rlg^ta^^^faearPOsitive wells using; limited dilution plating. Plates were visriaHy'-taspem^gS^^^^f^ single colony growth, and supernatants from single colony wells then screerwp^^s^nt-^w^iic BLISAs and FACS confirmation as described above. Highly reaotive clones were assayed to verity purity of human gamma and kappa chain by multiplexELIS A rising a Lumincx instrument. Based on EGFRvIII specificity in the ELISA and FACS assay, Clone 13.12 was selected as tire most promising candidate for further screening and analysis. The nucleotide and ammo acid sequences of the heavy and light chains of 13.12 antibody . are shown in FIG. 3L and SEQjDfiQ; 1.3^1 139 for heavy and light chain nucleic acids and 138 and 140 for heavy and^!li^^ati^^( oes., In addition, a comparison of the 13.12 heavy chain and light dtei^^^^^ti^^gcnnline sequence from which they were derived as shown in FlGs4and 5.

<img file="IN313DEN2012A_D0063.tif" />

/I/·:

* » t·

EXAMPLES

Antibody Generation ThrooyhTJse of XannMax Technology

Immunization of XenoMouse animals

Human monoclonal antibodies against human EGFRvIH were, developed by sequentially immunizing XenoMouse mice tat produce antibodies with a garama-1 constant region (XenoMouse G1 mice), X«i0Mo^e^^^^;^oduee antibodies wife gamma-2 constant regions

<img file="IN313DEN2012A_D0064.tif" />

Al ¹^! id* .Λ'^ξί; * (XcnoMouse XMG2 mice), end XenoMouse mice that produce antibodies with a gamma-4 constant region (XenoMousc G4 mice).

To generate mAbi by through XcuoMax technology, cohorts of XenoMousc G1 and XMG2 mice were immunized with EGFRvIH PEP3 (Example 1A) rad BCTRvm-farprnwing 300.19 cells (Example IB) or withbactcrially expressed extracellular domain of BGFRvIH protein (EGFRvHl-ECD) (Dr. Bigner, Duke University) and EGFRvin-cxpressing 300.19 cells or with EGFRvHI-Rabbit Fc fusion,?&&&^GRfc^teRbFc) (Example IQ and EGFRvEI-expreasfng 300.19 cells or with EG^^-kbWW|^?o«‘pad (FP), or via base of fl» tail by subcutaneous injection and intrapcritoneum'(BffX >'' '

For footpad immunizations, the initial immunization was with or without 10 X 10* EGFRvUI-cxprcssing 300.19 cells and with or without 10 μg of EGFRvUI PEP3 or EGHRvIH-ECD or EGFRvIH-RbFc mixed 1:1 v/v with TjtennaX gold (Sigma, .Oakville, ON) per mouse. The subsequent boosts were performed, with half of Ore ambunt of immunogen used in the initial immunization. The first four boosts ^re^^e^-takrng thc imimmogcn mixed with shun (Sigma, Oakville, ON), adsorbed in the Table 3.1 below. This was followed by one infection With ti**m«|e^^ititimn0mfb Tftotmx gold* one injection with alum and then a final boost of the immunogen te PBS as shown in Table 3.1. In particular, animals wore immunized on days 0, 3, 7_t 10, 14,37,21 and 24. The animals were bled on day 19 to obtain sen and determine the titer for harvest select on. The animals were harvested on Day 28.

Table 3.1

Footpad immunization schedule

<td colspan="2"> Group #</td><td> 1</td><td></td><td></td><td> .4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td>

<td colspan="2"> # of animals</td><td> ' 6</td><td></td><td></td><td> $</td><td> 5</td><td> 6</td><td> S</td><td> 6</td>

<td colspan="2"> Mouse strain</td><td> XMfi?)</td><td> r&amp;rt3^r</td><td> F.XMgjJ</td><td> ;xMsc4</td><td> VMG2</td><td> XM3C-3</td><td> XMG2</td><td> XM3C-3</td>

<td> rest #</td><td> Adjuvant</td><td colspan="2"> lrhrnurii^.:_?;;</td><td colspan="2"></td><td colspan="2"> immunogen</td><td colspan="2"> Immunogen</td>

<td> jtr-</td><td> Titermax gold</td><td colspan="2"> EGFRvlll-30019 cells + ΡΕΡ3» KLH</td><td colspan="2"> EGERvfll4oO.1S cells+ EGFRVIIIΕΘ2</td><td colspan="2"> EGFRvlll-300.18 cafe + EGFRvlllRbFc</td><td colspan="2"> EGFRvlIFRbFc</td>

<td></td><td> Alum</td><td colspan="2"> EGiOi4«m5 cells</td><td colspan="2"> EG^FMil-300.10 'cells</td><td colspan="2"> EGFRv))l-300.19 cells</td><td colspan="2"> EGFRvlil-RbFc</td>

<td> r”¹</td><td> Alum</td><td colspan="2"> PEP34Q.fi</td><td colspan="2"> EGERvllkECb ¹</td><td colspan="2"> egfMi-ecd</td><td colspan="2"> EGFRvlil-RbFc *</td>

<td> 4* </td><td> Alum</td><td colspan="2"> EGFRvHi-300.19 -Gaits ·-&gt;&lt; &lt;ύ&lt;</td><td colspan="2"> EGFByllHWS</td><td colspan="2"> BGFRvII1-300^ 9 cells</td><td colspan="2"> EGFRvlll-Rb^c</td>

<td> 5«·</td><td> Alum</td><td colspan="2"></td><td colspan="2"></td><td colspan="2"> EGFRvill-ECD</td><td colspan="2"></td>

<td> r~</td><td> Titerrnax · sold</td><td colspan="2"> EGFfe/'inW.^f . . .critariwfc.ifrti</td><td colspan="2"></td><td colspan="2"> cells</td><td colspan="2"> EGFRvilt-bbFc</td>

<td> yir-i</td><td> Alum</td><td colspan="2"></td><td colspan="2"></td><td colspan="2"> EGFRylil-EGD</td><td colspan="2"> EGFRvllM^bFc</td>

<td> B^w</td><td> PBS</td><td colspan="2"> EGFRvlll'WJb cells + PEP3KLH</td><td colspan="2"> EGFRvill-30q.1&amp; Odle + EGFRvUI· ECO</td><td colspan="2"> EGFRvill~300.l9 cells + EGFRvlllRbFc</td><td colspan="2"> EGFRvllt-RbFc</td>

<td colspan="2"> Harvest</td><td colspan="8"></td>

<img file="IN313DEN2012A_D0065.tif" />

The initial BIP immunization with the respective immunogen, as described for the footpad immunization, was mixed 1:1 v/v with Complete Freund’s Adjuvant (CFA, Sigma, Oakville, ON) per mouse. Subsequent boosts were made first with the immunogen respectively, mixed 1:1 v/v with Incomplete Freund’s Adjuvant (IFA, Sigma, Oakville, ON) per mouse, followed by a final boost in PBS per mouse. The animals were immunized on days 0,14, 28,42, 56, and day 75 (final boost) as shown in Table 3.2 below. The animals were bled on day 63 to obtain sera and determine the titer for harvest selection. The animals were harvested on Day 78.

Table 3.2

Bip Immunization schedule

<td colspan="2"> Group</td><td> 9</td><td> 10</td><td> 11</td><td> 12</td><td> 13</td><td> 14</td><td> . 15</td><td> 16</td>

<td colspan="2"> # of animals</td><td> 5</td><td> 5</td><td> 5</td><td> 5</td><td> 5</td><td> 5</td><td> 5</td><td> 5</td>

<td colspan="2"> Mouse strain</td><td> XMG2</td><td> XM3C-3</td><td> XMG2</td><td> XM3C-3</td><td> XMG2</td><td> XM3C-3</td><td> XMG2</td><td> XM3C-3</td>

<td> Boost #</td><td> Adjuvant</td><td colspan="2"> Immunogen</td><td colspan="2"> Immunogen</td><td colspan="2"> Immunogen</td><td colspan="2"> Immunogen</td>

<td> 1^st</td><td> CFA</td><td colspan="2"> EGFRvlll-300.19 cells + PEP3KLH</td><td colspan="2"> EGFRvlll-300.19 cells + EGFRvlllECD</td><td colspan="2"> EGFRvlll-300.19 cells + EGFRvlllRbFc</td><td colspan="2"> EGFRvlll-RbFc</td>

<td> 2^s3</td><td> IFA</td><td colspan="2"> EGFRvlll-300.19 cells</td><td colspan="2"> EGFRvlll-300.19 cells</td><td colspan="2"> EGFRvlll-300.19 cells</td><td colspan="2"> EGFRvlll-RbFc</td>

<td> 3*</td><td> IFA</td><td colspan="2"> PEP3-KLH</td><td colspan="2"> EGFRvlll-ECD</td><td colspan="2"> EGFRvlll-ECD</td><td colspan="2"> EGFRvlll-RbFc</td>

<td></td><td> IFA</td><td colspan="2"> EGFRvlll-300.19 cells</td><td colspan="2"> EGFRvlll-300.19 cells</td><td colspan="2"> EGFRvlll-300.19 cells</td><td colspan="2"> EGFRvlll-RbFc</td>

<td> 5^1fr</td><td> IFA</td><td colspan="2"> PEP3-KLH</td><td colspan="2"> EGFRvlll-ECD</td><td colspan="2"> EGFRvlll-ECD</td><td colspan="2"> EGFRvlll-RbFc</td>

<td> gth</td><td> PBS</td><td colspan="2"> EGFRvlll-300.19 cells + PEP3KLH</td><td colspan="2"> EGFRvlll-300.19 cells + EGFRvlllECD</td><td colspan="2"> EGFRvlll-300.19 cells + EGFRvlllRbFc</td><td colspan="2"> EGFRvlll-RbFc</td>

<td colspan="2"> Harvest</td><td colspan="8"></td>

Selection of animals for harvest By titer determination

Anti-hEGFRvIII antibody titers were determined by ELISA. EGFRvlU-RbFc (2.5 pg/ml) or a control RbFc (2 pg/ml) or EGFRvIHpeptide-OVA (2 pg/ml) (Example 1) or control OVA (4 pg/ml) were coated onto Costar Labcoat Universal Binding Polystyrene 96-well plates (Coming, Acton, MA) overnight at four degrees. The solution containing unbound antigen was removed and the plates were treated with UV light (365nm) for 4 minutes (4000 microjoules). The plates were washed five times with dH₂O. Sera from the EGFRvIII immunized XenoMouse® animals, or naive XenoMouse® animals, were titrated in 2% milk/PBS at 1:2 dilutions in duplicate from a 1:100 initial dilution. The last well was left blank. The plates were washed five times with dH₂O. A goat anti-human IgG Fc-specific horseradish peroxidase (HRP, Pierce, Rockford, IL) conjugated antibody was added at a final concentration of 1 pg/mL for 1 hour at room temperature. The plates were washed five times with dH₂O. The plates were developed with the addition of TMB chromogenic substrate (Gaithersburg, MD) for 30 minutes and the ELISA was stopped by the addition of 1 M phosphoric acid. The specific titer of individual XenoMouse® animals was

<img file="IN313DEN2012A_D0066.tif" />

determined from the optical density at 450 am and Is shown in Tables 33 and 3,4, The titer represents the reciprocal dilution of the serum and therefore the higher the number tta #eafcr the humoral immune response to hEGFRvXH.

For the mice immunized via base of file rail by subcutaneous injection and intreperitoneum, the titre was determined exactly as aboveexccpl the plates were coated with EGERvUI-RbFc ¢2.0 pg/mi) or a control RbFc (23 pgtal). '

<img file="IN313DEN2012A_D0067.tif" />

t

<td> Group *</td><td> Imnumtzatibn (site and Immunogen)</td><td> and sex</td><td></td><td> Sfeivin ^RbFc®' -'2jugtal</td><td> Centre! RbFc® 2.0ugtaL</td><td> EGFRvIH peptideOVA coated at &amp;0 petal</td><td> OVA coated at AOpg/ml</td>

<td rowspan="6"> I</td><td rowspan="6"> FP EGFRvIII. 300.19 cells + EGFRvIII PEP3-KLH (set Imre, ’ Scbed.)</td><td rowspan="6"> XMG2</td><td> 0748-1</td><td> 330</td><td></td><td> 13549</td><td> &lt;100</td>

<td> 0748-2</td><td> 237</td><td></td><td> 7635</td><td> &lt;100</td>

<td> 0748-3</td><td> 109</td><td></td><td> 9824</td><td> &lt;100</td>

<td> 07484</td><td> 714</td><td></td><td> 8014</td><td> &lt;100</td>

<td> 0748-5</td><td> 1β5</td><td></td><td> 9421</td><td> &lt;100</td>

<td> Naive</td><td> &lt;100</td><td></td><td> n/a</td><td> n/a</td>

<td rowspan="6"> 2</td><td rowspan="6"> FP EGFRvlll· 300.19 cells + EGFRvIII PEP3-KLH (seebnm. Sched.)</td><td rowspan="6"> XM3C- 3</td><td> 0741-1</td><td> 388</td><td></td><td> 347</td><td> &lt;106</td>

<td> 0741-2</td><td> 327</td><td></td><td> 240</td><td> &lt;100</td>

<td> 0741-3</td><td> 385</td><td></td><td> 330</td><td> &lt;100</td>

<td> 9W-4 ’</td><td> 589</td><td></td><td> 227</td><td> &lt;100</td>

<td> 0741-5</td><td> 273</td><td></td><td> 626</td><td> &lt;100</td>

<td> NalYe '</td><td> &lt;100</td><td></td><td> n/a</td><td> n/a</td>

<td rowspan="6"> 3</td><td rowspan="6"> EGFR vl II300.19 cells + • EGFRvlllECD (see Imnr. Scbed.)</td><td rowspan="6"> XMG2 i'.⁴ U .·' * &gt; :..,U ..j</td><td> 0749-1 .</td><td> . 552</td><td></td><td> &lt;100</td><td> &lt;100</td>

<td> 0749-2</td><td> ’ 477</td><td></td><td> &lt;100</td><td> &lt;100</td>

<td> 0749-3</td><td> ~ IS)</td><td></td><td> &lt;100</td><td> &lt;100</td>

<td> 07494</td><td> ϊδδ</td><td></td><td> &lt;100</td><td> &lt;100</td>

<td> •©74%5^i</td><td> l«31</td><td></td><td> &lt;100</td><td> &lt;100</td>

<td> ’ 'NtofcS</td><td> few.</td><td></td><td> n/a</td><td> Wa</td>

<td rowspan="6"> 4</td><td rowspan="6"> FP EGFRVIII·300.19 cells.+. EGFRvlllECD (sea Ixnm. Sched.)</td><td></td><td></td><td> |#372 '</td><td></td><td> &lt;100</td><td> &lt;100</td>

<td rowspan="5"> w 3 V.</td><td></td><td></td><td> I ,</td><td> &lt;100</td><td> &lt;100</td>

<td> :</td><td> 1/-57484 .</td><td></td><td> &lt;100</td><td> &lt;100</td>

<td> WiBPf ··</td><td> 1 '-530</td><td></td><td> &lt;100</td><td> &lt;100</td>

<td> 9742-5</td><td> 270</td><td></td><td> &lt;100</td><td> &lt;100</td>

<td> Naive</td><td> . 100</td><td></td><td> n/a</td><td> n/a</td>

<td rowspan="6"> 5</td><td rowspan="6"> FP EGFRvlll300.19 cells+ EGFRvlllRbFc (see hnm. Sched.)</td><td rowspan="6"> XMG2</td><td> 07504</td><td> 5399</td><td> 175</td><td> &lt;100</td><td> &lt;Ϊ00</td>

<td> 0750-2</td><td> 3072</td><td> 151</td><td> &lt;100</td><td> &lt;100</td>

<td> 0750-3</td><td> &gt;6400</td><td> 358</td><td> &lt;100</td><td> &lt;100</td>

<td> 07504</td><td> 5845</td><td> 196</td><td> &lt;100</td><td> &lt;100</td>

<td> 0750-5</td><td> 5770</td><td> 196</td><td> &lt;100</td><td> &lt;100</td>

<td> Naive</td><td> 100</td><td> 100</td><td> n/a</td><td> n/a</td>

<td rowspan="6"> 6</td><td rowspan="6"> FP EGFRvlll· 300,19. cells + EGFRvIllRbFC (see Imm. Sched.)</td><td rowspan="6"> XM3C- 3</td><td> 0743-1 ,</td><td> 1220</td><td> &lt;100</td><td> &lt;100</td><td> &lt;ioo</td>

<td> 0743-2</td><td> 1183</td><td> &lt;100</td><td> &lt;100</td><td> &lt;100</td>

<td> 0743-3</td><td> .645</td><td> &lt;100</td><td> &lt;100</td><td> &lt;100</td>

<td> 07434</td><td> 759</td><td> &lt;100</td><td> &lt;100</td><td> &lt;100</td>

<td> 07.43-5'</td><td> 1260 ’</td><td> &lt;100</td><td> &lt;100</td><td> &lt;100</td>

<td> Naive</td><td> 100</td><td> &lt;100</td><td> n/a</td><td> vfe</td>

<img file="IN313DEN2012A_D0068.tif" />

i * ·*.>· > <

<img file="IN313DEN2012A_D0069.tif" />

<td> Group #</td><td> . w ··« Immunization (rtteand Xmqnmogea)</td><td> t-WitWX&amp;l Strata and sex</td><td> '^'CuOlUC^</td><td> r EGFRvXn -RbFc@ 2.Suj/ml.</td><td> Control RbFc@ 2.0ug/ml,</td><td> EGFRvHI peptideOVA Matedat 2.0Mfal</td><td> OVA coated at 4.0pgftnl</td>

<td rowspan="6"> ' 7</td><td rowspan="6"> FP EGFRvIRRbFc (See · hum. Scbed.)</td><td rowspan="6"> '· ; i</td><td> 0745-1 .</td><td> 1897</td><td> &lt;100</td><td> &lt;100</td><td> &lt;100</td>

<td> iffiMSAirp</td><td> ... &gt;6400</td><td> 323</td><td> &lt;100</td><td> &lt;100</td>

<td></td><td> ^1225</td><td> &lt;100</td><td> &lt;100</td><td> &lt;100</td>

<td></td><td> £ 4047</td><td> &lt;100</td><td> &lt;100</td><td> &lt;109</td>

<td></td><td> i :.'-852</td><td> &lt;100 .</td><td> &lt;100</td><td> &lt;100</td>

<td> 'Ww'</td><td> 100</td><td> &lt;100</td><td> ηΛ</td><td> ηΛ</td>

<td rowspan="6"> 8</td><td rowspan="6"> FP EGFRvillRbFc(see hnntScbed.)</td><td rowspan="6"> XM3C- 3</td><td> 0744-1</td><td> 362</td><td> &lt;100</td><td> &lt;100</td><td> &lt;100</td>

<td> 0744-2</td><td> 807</td><td> &lt;100</td><td> &lt;100</td><td> &lt;100</td>

<td> 0744-3</td><td> 479</td><td> &lt;100</td><td> &lt;100</td><td> &lt;100</td>

<td> 0744-4</td><td> «31</td><td> &lt;100</td><td> &lt;100</td><td> &lt;hOQ</td>

<td> 0744-5</td><td> 1112</td><td> &lt;100</td><td> &lt;100</td><td> &lt;ϊόο</td>

<td> NaJVe</td><td> 100</td><td> &lt;100</td><td> u/a</td><td> π/»</td>

All OieXenoMbUseaniraals from group 3 «rid XenoMouse animals 0743-5 from group 6 from Table 3.3 were selected for XenoMex harvests based on the serology.

<td> Group 1 *</td><td> ImmtutizatfoU (site and Imnmnogen)</td><td> ’^ral.^r*^|l,rt ’htw and sex</td><td> IJ?s</td><td>^!BQFRvIH -RbFc@ , LOug/ml.</td><td> Control RbFc@ ZSug/ml.</td><td> EGFRvIB pepttdeOVA . coated at 2.0 pg/ml</td><td> OVA coated at 4.0 pgfatl</td>

<td rowspan="6"> 9</td><td rowspan="6"> BIP EGFRvtll30039 eon» + EGFRvlll PEP3-KLH (seelmm. Sched.)</td><td rowspan="6"> : - ^x</td><td> 069341.-</td><td> ;- 2921</td><td></td><td> &gt;128000</td><td> 472</td>

<td></td><td> tU 2219</td><td></td><td> 30504</td><td> 379</td>

<td></td><td> U-4B09 .</td><td></td><td> &gt;128000</td><td> 608</td>

<td></td><td></td><td></td><td> &gt;128000</td><td> 368</td>

<td></td><td> 1580</td><td></td><td> 1975^</td><td> 269</td>

<td> ’’ NW-xe’</td><td> &lt;100</td><td></td><td></td><td> 242</td>

<td rowspan="6"> 10</td><td rowspan="6"> BIP EGFRylll- 300.19 ceiB + EGFRvlll PEP3-KLH (seelmm. Scbed.)</td><td rowspan="6"> XM3C- 3</td><td> 0700-1</td><td> &lt;100</td><td></td><td></td><td></td>

<td> 0700-2</td><td> &lt;100</td><td></td><td></td><td></td>

<td> 0700-3</td><td> &gt;6400</td><td></td><td></td><td></td>

<td> 0700-4</td><td> 5342</td><td></td><td></td><td></td>

<td> 0700-5</td><td> &gt;6400</td><td></td><td></td><td></td>

<td> Naive</td><td> &lt;100</td><td></td><td></td><td></td>

<td rowspan="6"> 11</td><td rowspan="6"> BIP EGFRvWP 300.19 cells + EGFRvlllECD (see 1mm. Sched.)</td><td rowspan="6"> XMG2 ' &lt; ·</td><td> 0696-1</td><td> &lt;100</td><td></td><td> 56Ϊ</td><td> 240·</td>

<td> 0696-2</td><td> &lt;100</td><td></td><td> 788</td><td> 326</td>

<td> 0696-3</td><td> &lt;100</td><td></td><td> 604</td><td> 266</td>

<td> 0696-4</td><td> 143</td><td></td><td> 444</td><td> 263</td>

<td> 0696-5</td><td> &lt;100</td><td></td><td> 303</td><td> 254</td>

<td> j</td><td> *4·&lt;ίθθ</td><td></td><td></td><td> 242</td>

<td rowspan="5"> 12 </td><td rowspan="5"> BIP ’ EGFRvtll300,19 Cells + EGFRvIllECD (see fam.</td><td rowspan="3"> 4 ·· .5</td><td> Igsjgw·!</td><td></td><td></td><td></td><td></td>

<td> 107¾½)¹</td><td> • ’ 469</td><td></td><td></td><td></td>

<td> EWjH</td><td> 401</td><td></td><td></td><td></td>

<td rowspan="2"> —</td><td> 07te4</td><td> &gt;6400</td><td></td><td></td><td></td>

<td> 0702-5</td><td></td><td></td><td></td><td></td>

+rir ibas »l·^'

<img file="IN313DEN2012A_D0070.tif" />

v ‘-/J

¢)0

<img file="IN313DEN2012A_D0071.tif" />

<td> Group #</td><td> Immunization (site and Immunogen)</td><td> • 1’ Mouse Strata and sex</td><td> Mouse LD«</td><td> EGFRyHI -RhFc® 2,0ug/ml.</td><td> Centre) BbFe® 2Jng/mL</td><td> KGFRvm peptid»· OVA coated at XOligAnl</td><td> OVA coatedat «Jpgtari</td>

<td></td><td> Sched.)</td><td></td><td> NtSve</td><td> &lt;100</td><td></td><td></td><td></td>

<td rowspan="6"> 13</td><td rowspan="6"> BIP EGFRvlll300.19 Cells + EGFRvlllRbFcfsee hnm. Sched,)</td><td rowspan="6"> XMQ2</td><td> 0694-1</td><td> &gt;6400</td><td> &gt;6400'</td><td> 250</td><td> 243</td>

<td> 0694-2</td><td> &gt;6400</td><td> &gt;6400</td><td> 296</td><td> 309</td>

<td> 0694-3</td><td> &gt;6400</td><td> &gt;6400</td><td> 736</td><td>^r 60S</td>

<td> 06944</td><td> &gt;6400</td><td> &gt;6400</td><td> 739</td><td> 1111</td>

<td> 0.694-5</td><td> 3710</td><td> &gt;6400</td><td> 517</td><td> 465</td>

<td> Naive</td><td> &lt;100</td><td> &gt;6400</td><td></td><td> 242</td>

<td rowspan="6"> 14</td><td rowspan="6"> BIP EQFRvlll· 300.19 eMs + EGFRvlllRbFc (see Irani. Sched.)</td><td rowspan="6"> XM3C- 3</td><td> 0703-1</td><td> 2740 .</td><td> &gt;6400</td><td></td><td> •</td>

<td> 0703-2</td><td> 408</td><td> &gt;6400</td><td></td><td></td>

<td> 0703*3</td><td> 1406</td><td> &gt;6400</td><td></td><td></td>

<td> 0703-4</td><td> 1017</td><td> &gt;6400</td><td></td><td></td>

<td> 0703-5.</td><td> 455</td><td> &gt;6400</td><td></td><td> ·*</td>

<td> Naive '</td><td> &lt;100</td><td> &gt;6400</td><td></td><td></td>

<td rowspan="6"> 15</td><td rowspan="6"> Bff : EGFRvlll-¹^ RbFc(see, , Imm. Sched.) &lt;</td><td rowspan="2"> ί •.r</td><td></td><td> &gt;6400</td><td> &gt;6400</td><td> 340</td><td> 348</td>

<td></td><td> &gt;6400</td><td> &gt;6400</td><td> 642</td><td> 1793</td>

<td rowspan="2"></td><td></td><td> 6242</td><td> &gt;6400</td><td> 319</td><td> 346</td>

<td></td><td> 1766</td><td> &gt;6400</td><td> 133</td><td> &lt;100</td>

<td rowspan="2"> -rl</td><td></td><td> &gt;6400</td><td> &gt;6400</td><td> 685</td><td> 448</td>

<td></td><td> Γ &lt;100</td><td> &gt;6400</td><td> 243</td><td> 242</td>

<td rowspan="6"> 16</td><td rowspan="6"> Btp EGFRvllJRbFc (see ’ Itnm. Sched.)</td><td rowspan="6"> XM3C- 3</td><td> Ό701-1 ·</td><td>¹ 592</td><td> &gt;6400</td><td></td><td></td>

<td> 0701-3</td><td> 11IB</td><td> &gt;6400</td><td></td><td></td>

<td> 0701-3</td><td> &gt;6400</td><td> &gt;6400</td><td></td><td></td>

<td> O7014</td><td> &lt;100</td><td> &lt;100</td><td></td><td></td>

<td> 0701*5</td><td> nA</td><td> nA</td><td></td><td></td>

<td> Naive</td><td> &lt;100</td><td> &gt;6400</td><td></td><td></td>

XenpMousc animals (0695-1, M9S-3 and 0695-4) were selected for harvests based on the serology deta in Table 3.4.

Selection of B cells. ..... ' B-eells from foe Bbove-diecussed. annnals were htrvested and-cultured. Those secreting

<img file="IN313DEN2012A_D0072.tif" />

EGFRvni-pcptidc specific antibodies were isolated as described in Babcook et al., Proc. Vail. Acad. Set. {/S4, 93:7843-7848 (199f). ELISA was used to identify primary EGFR-vIH-pcptideOVA -specific wells. About 5 million Boells were cultured from XcnoMouse animals in 245 96 well plates at 500 or ISO or 50 ceils the antigen-specific representative sample of Wp

<img file="IN313DEN2012A_D0073.tif" />

were screened cn EGFRvHI-pcptide-OVA to identify «-.*·· · showed ODs significantly over background, a

Table 3.5

<td rowspan="2"></td><td rowspan="2"> Total ' plates</td><td colspan="4"> &lt;r --- a - :¾ .. 4^11</td><td colspan="3"></td><td colspan="9"> abev i cutoit OD of:</td>

<td> 0;0</td><td colspan="2"> ai-fiMh</td><td></td><td colspan="2"> HBBfloLXt</td><td> ,Λί</td><td> 0.7</td><td> .0.8</td><td> 0.9</td><td> 1.0</td><td> 1.5</td><td> 2.0</td><td> 2.5</td><td> 3.0</td><td> 3.5</td>

<td> 2«nsera 500 · ;ells/ well</td><td> 12</td><td> 11 52</td><td> 63 4 !</td><td> Jt'u ; i</td><td> ·· *' 3 .5$*·</td><td></td><td> w</td><td> Ux</td><td> 32</td><td> 29</td><td> 26</td><td> 25</td><td> 18</td><td> 11</td><td> 4</td><td> 1</td><td> 0</td>

<td> Sigma 100 «Hs/ veil</td><td> 13</td><td> 12 48</td><td> 77 3</td><td> » 5</td><td> : » » 13 9</td><td> 11 7</td><td> 99</td><td> 80</td><td> 73</td><td> 58</td><td> 53</td><td> 49</td><td> 21</td><td> 9</td><td> S</td><td> .1</td><td> J</td>

<td> lignja 150 t«nw reH</td><td> 20</td><td> 19 20</td><td> 13 04</td><td> 47 8</td><td> 17 8</td><td> 91</td><td> 67</td><td> 55</td><td> 47</td><td> 45</td><td> 36</td><td> 33</td><td> 19</td><td> 9</td><td> 5</td><td> 2</td><td> 0</td>

<td> Total</td><td> 45</td><td> 43 20</td><td> 27 11</td><td> 75 4</td><td> 37 3</td><td> 25 7</td><td> 21 1</td><td> 17 3</td><td> 15 2</td><td> 13 2</td><td> n 5</td><td> TO 7</td><td> 58</td><td> 29</td><td> 14</td><td> 4</td><td> 0</td>

244 of EGFRvIH-peptide-OVA-EHsa positive wells of OD > 0.5 were screened again on EGFRvIQ-pcptide-OVA and on OVA to confinn that they were EOFRvHI-peptide specific, A rcpresentativeexampleof these results is shown in Table 3.6.

. Table 3i6

<td> Plate j</td><td colspan="2"> 1V.· 4»</td><td></td><td> Z’EGKRvHI peptide-OVA OD</td><td> OVA OD</td>

<td></td><td> f</td><td></td><td></td><td> , 1.4085</td><td> 0.135 , J</td>

<td> I 121</td><td> A-</td><td> k·</td><td> feiiw</td><td> 2.1491</td><td> 0.126 8</td>

<td> 121</td><td> D</td><td> 0</td><td> ' 0.0743</td><td> 0.4179</td><td> 0.153 1</td>

<td> 121</td><td> E</td><td> 8</td><td> 2.0415</td><td> 2.6965</td><td> 0.149 8</td>

<td> 121</td><td> H</td><td> 10</td><td> 0.8611</td><td> 0.4288</td><td> 0.159 5</td>

<td> 121</td><td> C</td><td> 12</td><td> 2.1455</td><td> 2.5443</td><td> 0.140 4</td>

<td> 122</td><td> H</td><td> 1</td><td> . 1.8890</td><td> 2.5987</td><td> 0.115 4</td>

<td> 122</td><td> H</td><td> 5</td><td> . 0.5943</td><td> 0.8321</td><td> 0.157 2</td>

<td> 122</td><td> F</td><td> 8</td><td> 0.8834</td><td> 0.7715</td><td> 0.145 0</td>

I Limitedantiyen aaaav and anatwjis , The limited apti present in B-celf

<img file="IN313DEN2012A_D0074.tif" />

{hat affinity-ranks the antigen-specific antibodies al) other antigen-specific antibodies. 1a the

<img file="IN313DEN2012A_D0075.tif" />

.

. ·· Mirs presence of a very low to any detectable level at 03/48730) ’ ’

<img file="IN313DEN2012A_D0076.tif" />

he highest affinity antibodies should be able to bind p, e,g., International Patent Application No. WO

EGFRvin peptide-OVA Was coated fo plates at three ooncentrations; 7.5 ng/ml, 13 ng/ml and 0.03 ng/ml for overnight at 4^s C on 96-welI Elisa plates. Each plate was washed 5 times, with dHjO, before 50ul of 1% milk in PBS with 0.05% sodium azide were added fo the plate, followed by 4 μΙ of B dell supernatant added to each Well. After 18 hours at room temperature &lt;m a shaker, the plates wore again washed 5 times wife dH»O, To each well was added SOul of Gtanti-Human (Fc)-HRP at 1 pg/ml. After 1 hour at room tenper*turc,the plates were again wadted5 times with dHzO and 50 μΐ of TMB substrate were added to each well. The reaction was stopped by the and the results shown in Table 3.7.

Table 3.7

<td></td><td colspan="2"> •J Jf</td><td></td><td> ggjgji</td><td colspan="4"> fe* ’_; Urnfted An</td><td> Hiflh Antigen</td>

<td> Culture Plate</td><td colspan="2"> Welf ft</td><td></td><td></td><td> KjL\ ..</td><td></td><td></td><td></td><td> (Ι,ΟμβΛηΙ)</td>

<td></td><td></td><td> . .*</td><td></td><td></td><td></td><td> j/rnl '</td><td colspan="2"> 73ng/ml</td><td></td>

<td></td><td> •</td><td> • · .‘.I</td><td></td><td></td><td> j^JOD .</td><td> Rank</td><td> OD</td><td> Rank</td><td></td>

<td> 133</td><td> B</td><td> 2</td><td> 0.7670</td><td></td><td> 1.189</td><td> 54</td><td> 1.871</td><td> 86</td><td> 2.050</td>

<td> 124</td><td> G</td><td> 12</td><td> 0.7400</td><td> ' 2</td><td> 1.885</td><td> . ..1</td><td> 3.101</td><td> .1</td><td> 3.463</td>

<td> 145</td><td> C</td><td> 1‘</td><td> 0.716</td><td> 3</td><td> 1.552</td><td> 7</td><td> 2.671</td><td> 10 </td><td> 3.194</td>

<td> 129</td><td> G</td><td> 10</td><td> 0.6720</td><td> 4</td><td> 1.367</td><td> 22</td><td> 2.692</td><td> 8</td><td> 2.977</td>

<td> 156</td><td> B</td><td> 6</td><td> 0.657</td><td> 5</td><td> 1.842</td><td> 2</td><td> 2.859</td><td> 3</td><td> 3.411</td>

<td> 143.</td><td> F</td><td> 12</td><td> 0.653</td><td> 8</td><td> 1.677</td><td> 3</td><td> 2.741</td><td> S</td><td> 3.166</td>

<td> 136</td><td> E</td><td> 3</td><td> 0.8340</td><td> 7</td><td> 1.488 ·</td><td> 15</td><td> 2.683</td><td> 8</td><td> 3.280</td>

<td> 137</td><td> C</td><td> 11</td><td> 0.595</td><td> 8</td><td> 1.582</td><td> 5</td><td> 2.84</td><td> 2</td><td> 3.444</td>

<td> 139</td><td> A</td><td> 11</td><td> 0.582</td><td> 9</td><td> 1374</td><td> 18</td><td> 2.282</td><td> 47 </td><td> £255</td>

<td> 174</td><td> F</td><td> 1</td><td> 0.573</td><td> 10</td><td> 1,677</td><td> 8</td><td> 2.776</td><td> 4</td><td> 2.384</td>

The results generated from Umitod'antigUn analysis were compared to the total OD obtained ♦

in high antigen assay. A relative ranking of affinity was done by taking the ratio of die OD obtained in limited antigen assay Vs that obtained in high antigen assay. Antibodies with higher ratio will have, the highest afHnity, . Table 3,7 shows the sample ofB-cell culture supernatants that: : ·&lt;·* ' were ranked based on Ιϋτ ited?

0.03 ng/inl) Vj thehigh έή Ϊ

Native cell binding assay V ¹ » ~n jtjm

EGFRvlH peptider.OWc

<img file="IN313DEN2012A_D0077.tif" />

kT a^Op (for file lowest antigen plating concentration of lr supernatants were analyzed for their ability to bind to the native form of EGFRvIU Mably «presaed on NR6oelIs (NR6 M bells) (See, Batra et al. Epidermal growth factor ligand-independent, unregulated, oeU-transforming potential of a naturally occurring human Growth Differ. 6(10):1251-9 (1995». NR6M cells

Were seeded at 8000 cdl^t^eft^^vbatod over night in 96 won FMAT plates. Media was then removed leaving 1$ μ] in the well. 15 μΐ B-cell culture supernatants were added and 15 μΐ anti-human IgG Fc Cy5:« 1 pg/ml final concentration added to wells. It it then left incubated at 4° C for 2 hours. The cells were washed with 150 μΐ PBS, and fixed before reading on FMAT. The results were expressed as total ftuuresoenttensity (Table 3.8). Hunan anti-EOFRvEI mAh 13.1.2 was used as a positive ocu^M^^ hjmlml final concentration and negative control was PK 16.3.1 at the same oonce^te^^^^^^^^^’ Samples tested bound to NR6M cells of which 62 had a total fiuoresceoce of these 134 binders were false positives.

The same type of native buiding assay , was done an NR6 Wt cells (NR6 cells expressing »

EOF receptor) (Bee Bafta et al. Epidermal growth factor ligand-independent, unregulated, celitransfonningpoteatial of a nsturaUy occurring human mutant EGFRvUI gene. Cell Growth Differ. 6(10):1251-9 (1995)) to eliminate the binding is due to binding to Wt receptor (Table 3.8). ABXEGF was used as a positive control and PK 163.1 at the same concentration was used as a negative control antibody. 3 out the 134 NR6 M binders were bindtag strongly to NR6 Wt cells. 190 of the 244 wells bound EGFRvpT^ptidnih^ta^wercalso bound to the native form on pells. Examples aregivenfoTWteX* .

<img file="IN313DEN2012A_D0078.tif" />

Table3.8

<td colspan="3"> Plate</td><td colspan="2"> 1’Vlll-pepOVAOD</td><td> rvilbpep• OVAOD</td><td> OVAOD</td><td> FMAT native binding to NR6 M cells</td><td> FMAT native binding toNRBWt cells</td>

<td> 174</td><td> F</td><td> 1 ,</td><td></td><td></td><td></td><td> 0.1900</td><td> 138373</td><td> 1688</td>

<td> )87</td><td> A</td><td> 4?</td><td></td><td></td><td></td><td> 0.1920</td><td> 128826</td><td> 202459.8</td>

<td> 132 -</td><td> D</td><td> *' gw?</td><td></td><td></td><td> MfWwo.· </td><td> *•0.1649</td><td> 109379</td><td> 0</td>

<td> 142</td><td> C</td><td> 11&gt;</td><td></td><td></td><td> n£»81M</td><td> 0.2239</td><td> 94944</td><td> 0</td>

<td> 129'</td><td> A</td><td> 7</td><td> 2.1«</td><td> I?™</td><td></td><td> 0.1615</td><td> 84024</td><td> 0</td>

<td> 127</td><td> E</td><td> 1</td><td colspan="2"> 2,6923</td><td> 3.1986</td><td> 0.1219</td><td> 82031</td><td> 0</td>

<td> 124</td><td> G</td><td> 12.</td><td colspan="2"> 3-2929</td><td> 3.5834</td><td> 0.1456</td><td> 73080</td><td> 0</td>

<td> 141</td><td> C</td><td> 6</td><td colspan="2"> 0.7512</td><td> 12557</td><td> 0.1547</td><td> 60816</td><td> 814.5</td>

<td> 173</td><td> C</td><td> 1</td><td colspan="2"> 2.5728</td><td> 2.5714</td><td> 02134</td><td> 58702</td><td> 2523.4</td>

<td> 128</td><td> Θ</td><td> 8</td><td colspan="2"> 0.6293</td><td> 0.7483</td><td> 0Λ62Ο</td><td> 49831</td><td> 0</td>

<td> 129 .</td><td> H</td><td> 8</td><td colspan="2"> 2.9370</td><td> 3.0952</td><td> 02582</td><td> 0</td><td> 0</td>

<td> 183</td><td> E</td><td> 11'</td><td colspan="2"> 2.3460</td><td> 2.7717</td><td> 0.1050</td><td> 0</td><td> 0</td>

In Table 3.8, svpem*tant’^‘frteS'_!ⁱ>>’^HW^:Jis identified u a Wt binder and 141C6 was a false positive for NRfiM.^elis-^hK^fefrgfe|s<&g9H6 aoti 183EU are strong peptide binders with no native binding. ’

<img file="IN313DEN2012A_D0079.tif" />

The top . 60 native binding B cell culture supernatants were father assayed fa their ability to internalize thereceptor, NR6 M cells were seeded at 8000 oells/well into 96 well FMA.T plates and incubated overnight. Media,was removed and 10-15 μΐ B-Qell culture supernatant in a total volume of 30 pl media, in duplicate was. added. Next, 15 μ! of secondary antibody (SS Alexa 647 anti-truman IgG Fab at 1.5 Was added and the mixture was incubated on ice for I hr. An was used to see the effect of the culture media. Human anti-BOFRvIIl inA> 13.2:^ vfcfca used as a positive control starting at 1 pgtal (final concentration) and negative control was FK 163.1 (human anti-KLH IgG2 antibody) at the samc concentration. After incubation, toe cells were washed withooM PBS, 50 μΐ media was added to all of the wells, one of toe duplicates were incubated at 37 °C for 30 mins while toe other duplicate

<img file="IN313DEN2012A_D0080.tif" />

remained on ice. After the incubations media was removed, 100ul of cold 50 mM glutathione was added to the set incubaied at 37 “b’d^iWuilof.cold media added to the other set, both sets wore then left on ice for 1 hr. TH«ee^i With 100 μ) cold PBS and then fixed with 1% paraformaldehyde and read iniw. were expressed as % internalized, calculated as total fluorescence to toe presence of glutathione/ total fluorescence in toe absence of gfutetoione X

100. Representative, information is given in TaMe3.9.

Table 3.9

<td> Wellno. , .&lt;‘1 ι-if fiiit*</td><td> NO giutathfone ΐ iEftllif es/ftsfaftt - ΪΜίΡιΙίήΐώδ^</td><td> with glutathione iFLtXcount i* ' </td><td> % Internalized, (glutt/giut-) X100</td>

<td> 124 00..,</td><td>^,ij **1</td><td> 1394</td><td> 74.3%</td>

<td> 124Gt2</td><td></td><td> 8959</td><td> . 37.6%</td>

<td> 125 H1</td><td> 14008</td><td> 3086</td><td> 252%</td>

<td> 125D10</td><td> 2342</td><td> 1238</td><td> ’ 52.8%</td>

<td> 127E1</td><td> 15050</td><td> 1318</td><td> 8.7%</td>

<td> 127 B0</td><td> 12444·-</td><td> ·'· 7109</td><td> 57.1%</td>

<td> 127E.11</td><td> . 6623 .</td><td> . . 0</td><td> . . 0.0%</td>

<td> 128G9</td><td></td><td> 1851</td><td> 184%</td>

<td></td><td></td><td> ^-'3708·</td><td> 31.5%</td>

<td> •ffsd©^</td><td></td><td> ^.'4354 ·</td><td> 95.5%</td>

<td> TiS¹</td><td></td><td> %' '2658</td><td> 28.7%</td>

<td> 132D8</td><td></td><td> 13293</td><td> 37.1%</td>

<td> 133 F9</td><td> 9773 ‘</td><td> 3821</td><td> 37.0%</td>

<td> 136 F10</td><td> 2392</td><td> 0</td><td> 0.0%</td>

<td> 137 ©6</td><td> 6104</td><td> 1021</td><td> 204)%</td>

<td> 137G10</td><td> 3451</td><td> 0</td><td> 0.0%</td>

<img file="IN313DEN2012A_D0081.tif" />

EGFRvUl-specific He

A number of

<img file="IN313DEN2012A_D0082.tif" />

prepared as fellows. ' :

1. Biotwylailon tf &lt;Sheqp nd blood cells (SRBC). SRBCs were stored in RPMI media as a 25% stock. A 250 μΐ SRBC packed-cell pellet was obtained by aliqUoting 1.0 ml of SRBC to a fresh eppendof tube. The. SRBC wefe pelleted with a pulse spin at 8000 rpm (¢800 ref) in

<img file="IN313DEN2012A_D0083.tif" />

tube, 2.5 mg of Sulfo-NHS biotin was added to 45 ml of PBS pH 8.6. Once the biotin had completely dissolved, the 5 ml of SRBCs were added and the tube rotated at RT for I hour. The SRBCs Were oentriftjgcd at 3000ipm far 5 min and the supernatant drawn off. The biotinylated SRBCs Were transferred to an eppendorf tube and washed 3 times as above but with PBS pH 7.4 and then made up to 5 ml wife immune cell media (RPMI 1640) in a 15 ml falcon tube (5% BSRBC stock). Stock was Stored at 4· C until needed.

<img file="IN313DEN2012A_D0084.tif" />

ZOmin. The washing Stope were repeated and fee SA-SRBC were re-suspended in 1ml PBS pH 7.4 ‘5% (v/y)).

<img file="IN313DEN2012A_D0085.tif" />

4. Determination of the qualityof EOFRvfflpeptide-SRBC by immunofluorescence (IF).

.0 pl of 5% SA-SRBC and 10 μΐ of 5% EGFRyEB peptide-coated SRBC were each added to a operate fresh 1.5 ml eppendoxf tube containing 40ul of PBS, A control human anti-EGFRvni ntibody was added to each sample of SRBCs at 45 pg/tnl, The tubes were rotated at RT for 25 tin, and the cells were then washed ferae times- with 100 μΐ of PBS. The cells were re-suspended i 50 μΐ Of PBS and incubated with 40 mog/mL Gt-anti Human IgG Fc antibody conjugated to .lexa488 (Molecular Probes, Eugene, QR). The tubes were rotated at RT for 25 min, and then 'ashed with 100 μΐ PBS andtbebeils^Wi^pia^cd in 10 pl PBS. 10 μΐ ofthe stained cells were potted onto a clean with a glass covcrshp, observed under ( .uorescent light, add scored

5. Preparation of plasma ce&amp;. 'The contents of a single microculturc well previously 'AptiSed by various assays as containing a B cell clone.secreting the immunoglobulin of interest

Λ· wctc harvested. Using a 100-1000 μΐ pipotnwn, the contents of fl» well were recovered by adding 37CRPM1 (10% FCS). The cells were re-auapeudod by pipetting end then transferred to a fresh 1.5 ml eppeudorf tube (final vol approx 500700 pl). The oeI18 were certtrifiiged in * ndcrofoge st 2500 rpm (660 ref) for 1 minute at room temperature, end then toe tube war rotated 180 degrees and spun again for 1 nrniute at 2500 ipm. Thcfrocae naediawu drawn eg and toe fammne ndiy in 100 μΐ RPMI (10% FCS),ttel_fcajtrifti^i This, washing wife RPMI (10% FCS) was repeated and foe cells re-suspended FCS) and stored on ice until ready to use.

6. slide (2 x 3 inch) were prepared in advance with silicone edges and allowed to cure' s&amp;rnight at RT. Before, use, the slides were treated with approx, 5ul of SigmaCoat (Sigma, Oakville, ON) wiped evenly over glass surface, allowed to dry and then wiped vigorously. Ton 60 μ] sample of cells was added 6Θ pl each of BGFRvinpeptidecoated SKBC (5% v/v' stock), 4x gttmj$ pjg.'am^lcmcat (Signa», Oakville, ON) stock prepared in RPMI (10% FCS), and in RPMI With 10% FCS). The mixture WU spotted (10-15 μΐ) onto the'^p^^^^^^^^e'spots covered wife undiluted paraffin oil. The slides were incubated at 37° C for a ttdninhitin if AS minutes. The EGFRvIH-specific plasma cells were identified from plaques «nd rescued by ndcronsmiputatfan (see Table 3.10).

TlMaaiO

<td colspan="3"> Well ID</td><td> Single-Call Number</td><td> Total number of Single cells picked</td>

<td> 124</td><td> G</td><td> 12</td><td> EGFRvHI-SCX-1 Q5-116 (LL) .</td><td> 12</td>

<td> 129</td><td> A</td><td> 7</td><td> EGFRvin -SCXtS1^.(P^ ·.</td><td> 12</td>

<td> 174</td><td> F</td><td> 1</td><td> EGFRidu .</td><td> 8</td>

<td> 182</td><td> A</td><td> 5</td><td> EGFRvUl 382-169 (OP)</td><td> 20</td>

<td> 125</td><td> D</td><td> 10</td><td> EGFRvIII -SCX-17M81'(OM); 194-201 (U)</td><td> 20</td>

<td> 127</td><td> B</td><td> 9</td><td> EGFRvIII -SCX-182-183 (LL); 2Q2-209 (OP)</td><td> 20</td>

<td> 190</td><td> D</td><td> 7</td><td> EGFRvIII -3CX-210-2» 'LL)</td><td> 20</td>

<td> 130</td><td> B</td><td> 4</td><td> EGFRidll</td><td> 20</td>

<td> 138</td><td> D</td><td> 2</td><td></td><td> 20</td>

<td> 145.</td><td> C</td><td> 1</td><td> EGFRvIIIΛ '· ·</td><td> 18</td>

<td> 172</td><td> Θ</td><td> 12</td><td> EGFRvIII -SGX-93-104(LL)</td><td> 12</td>

<td> 187</td><td> A</td><td> 4</td><td> EGFRVIII -SCX-270-281 (LL)'</td><td> 12</td>

<td> 173</td><td> C</td><td> 1</td><td> EGFRvIII -SCX-282-293 (BC)</td><td> 12</td>

<td> 127</td><td> E</td><td> 1</td><td> EGFRvIl Γ -SCX-294-3O5 (LL)</td><td> 12</td>

<td> 142</td><td> C</td><td> 11</td><td> EGFRvIII -SCX-306-317 (LL)</td><td> 12</td>

<img file="IN313DEN2012A_D0086.tif" />

<td colspan="3"> Well ID</td><td></td><td> Total number of Single cells picked</td>

<td> 141</td><td> A</td><td> 10</td><td> ESFRytii -</td><td> 12</td>

<td> 132</td><td> D</td><td> a</td><td> EQFRVIII '</td><td> 12</td>

<td> 124</td><td> D</td><td> 4</td><td> EGFRvIII -SCX-342-349 (BC)</td><td> 8</td>

Single cell PGR, Cloning, Expression, Purification and Characterization of Recombinant antiEGERvUI Antibodies. .

The genes encoding tfre viriabte regrans were rescued by RT-PCR on the single micromanipulated extracted and reverse transcriptase PCR was conducted to generate the variable heavy and light chains was specifically amplified using goiytoeraatf’tfosin reaction. The human variable heavy chain region was cloned into an IgGl expression vector· This vector was generated by cloning the constant domain of human IgGl into the multiple cloning site of pcDNA3. l+/Hygro (Invittogcn, Burlington, ON). The human variable fight chain region was cloned into an IgK expression vector. These vectors were generated by cloning the Constant domain of human IgK into the multiple cltiuing site of pcDNA3,l+/Neo Qhvitrogen, Burlington; OJQ. The heavy chain and the light chain expression vectors were then of 70% confluent human embryonal kidney 293 cells and the trraafcc^j^^aB^fegKK'j^ete a recombinant antibody with the identical specificity as the original hSiirs. The supernatant (3 mL) was harvested from the HER. 293 cells and the secretion of an intact antibody Was demonstrated with a sandwich ELISA to specifically detect human IgG (Table 3.11). Specificity was assessed through binding of the recombinant antibodty to EGFRvIII using JUXSA (Table 341).

Table 341

«.·♦

<td rowspan="2"> mAb ID</td><td rowspan="2"> ------i-----------r1 t id . » </td><td colspan="2"> * Titer</td>

<td> £. Total antibody</td><td> Antigen binding</td>

<td> 129A7</td><td> SO EGFRvij^^S^.^^;·:</td><td> p.-V &gt;1:84</td><td> &gt;1:64</td>

<td> 13SD2</td><td> SC- EGFRVtH--XSⁱf-2^^ⁱ^</td><td> ’ &gt;1:64</td><td> &gt;1164</td>

<td> 174F1</td><td> SOEGFRVII)-XG1-131</td><td> &gt;1164</td><td> &gt;1:64</td>

<td> 182A6</td><td> 80 EGFRVIII &gt;XG 1-139</td><td> &gt;1:64</td><td> &gt;1:64</td>

<td> 19007</td><td> SO EGFRvIli -XGt-211</td><td> &gt;1:64</td><td> &gt;1:64</td>

<td> 125010</td><td> SC-EpFRy1l.l.-X&lt;^*170 .</td><td> &gt;1:64</td><td> &gt;1:64</td>

<td> 182D5</td><td> SO EGFRVlI^-XG^Ififtazir</td><td></td><td> &gt;1:64.</td>

<td> 141A10</td><td> SO EGFRVpmaiatfEfe^;</td><td></td><td> 1:64</td>

<td> 132D8</td><td> SC-.EGFRvlji^i^^^^</td><td></td><td> &gt;1:84</td>

<td> 124D4</td><td> SO EGFRvIII-XG1-34? ‘</td><td> . &gt;1:84</td><td> &gt;1:84</td>

i8

The secretion as follows. For Ab secretion, 2 ggfaiL of Goat anti-human IgG H+Land furanhgm^ihdin&amp;ufpg/nil of EGFRvIII-Rab Ig Fo fusion protein was coated onto Costar Labcoat Universal Binding Polystyrene 96 well plates and held overnight at four degrees. The plates were washed five times wife d%0. Recombinant antibodies were titrated 1:2 for 7 weus from the undiluted mmilipofectian supernatant. The plates were washed five times with dHjO. A goat anti-human IgG Fc-speoific HRP-conjugated antibody was added at a final concentration of 1 pg/mL for 1 hour at RT for fa secretion plates and binding plates detected wife I pg/fnl Rb anti Hu Fc for 1 hour at room temperature. The plates were washed five times with dHiO. The plates were developed wife fa addition of TMB for 30 minutes and fa EUSA wbs stopped by the addition of 1 M phosphoric acid. Bach ELISA plate was analyzed to detennmc fa optical density of each well at 450 nra.

Sequencing and sequence analysis

The cloned heavy and light chain oDNAs were sequenced in both directions and analyzed to determine fa germlinc sequence deri vation of fa antibodies and identify changes from genritine sequence. Such sequences arc FI&amp;.;3A-3K and (SEQ © NO. 34-55), A comparison of each of fa heavy and 4ighiti|in™M8M™8ad fa gennline sequences from whioh they are derived is provided sequence of fa hybridona derived 13.12 antibody is compared to its. gteti^ero^^i^wIOe. 4 and 5,

As will be appreciated from the discussion.herein, each of fa 13! antibody end fa 13.12 antibody possess vvry high affinities for BGFRvIH, are internalized well by ceils, and appear highly effective in cejl killing when conjugated to toxins. Intriguingly, each of fa antibodies, despite having been generated in different immunisations of XencMoUse mice, and utilizing different technologies, each , we derived from very similar gennline genes. Based upon epitope mapping work (described herein), each of fa antibodies, however, appear to bind to. slightly different epitopes on fa EGFRvIII molecule and have slightly different residues on EGFRvIII that are essential for binding, These results indicate fat fa germline gene utilization is of importance to generation of antibody therapeutics targeting EGFRvIII and that small changes can modify fa binding and effects of the antibody in ways that allow further design of antibody and other feerapcuncs based upon these structural findings, Binding of Anti-EGFRyHT rpAbSto qqpvB ΕσΈΚνΙΕ expressed cm cells

In this eximplc, binding of inti-E^TtviH -antibodies to NR6 M cells was measured. Specifically, unquantitated suptr^tjft^|p^gg^frx derived IgGl recombinant antibodies were assayed for their ability cells. Cells were seeded at 10000 / well and incubated overnightMedia'was removed and 40 μΐ mini lipo supernatant (titrated down) vraatoaM^m»pwnwcra incubated on ice for I hr. Thehuman 13.12 EGFRvIII antibodies and ABX EOF (E7.6.3, U.S, Patent No, .6235,883) antibodies were added as positive controls. The PR 16.3.1 antibody was used as a negative control. The ceils Were washed

<img file="IN313DEN2012A_D0087.tif" />

with Cold PSS. secondary antibody was added (SS Alexa antihuman IgG Fc) at 1 jig/ml,40 ul/well aud incubated on ice for. 1 hr. The cells were then washed with Cold PBS and fixed *nd read by FMAT. All antibodies were tested for specificity for binding by counter screening against NR6 WT cells. · . ·

Purification of Reoombim ' ' ·

For larger scale :hain expression vectors (2.5 pg of each chain/dish) were lipofecte .‘were 70% confluent with HEX 293 cells;

<img file="IN313DEN2012A_D0088.tif" />

The transfected cells Were » replaced with 6 mL of fresh media. At dsy 7, the supernatant was removed and pooled with the initial harvest (120 mL total from 10pistes), Each antibody was purified from the supernatant using a Protein-A Sepharosc(Amertham Biosciences, Piscataway, NJ) affinity chromatography (I mL·). The antibody was eluted from the Protein-A column with 500 mcl, of 0.1 M Glycine pH 2.5. The eluate was dialysed in PBS, pH 7.4 and filter-sterilized. The antibody was analyzed by nonreducing SDS-PAGE to assess^r^^rfo Concentration was also measured by U¥ analysis fotemalizationof BGFRvIB^p^^^^^ftiSTitahti-BGFRvni mAbs

XenoMax derived IgGI recombinant'antibodies were expressed, purified and quanliialcd as described previously; Antibodies were farther assayed for their ability to internalize the EGFRvIII receptor in NR6 M cells. 250,000 NR6 M cells were incubated wife primary antibody (SC95, SC131, SC 133, SC139, SC15.0, SC170, SC211, SC23Q, SC250 and human 13.1.2 as a control) at

<img file="IN313DEN2012A_D0089.tif" />

of tho celts incubated at 37 C were treated with glutathione (Ss previously mentioned) for ] hr on ice. Then the cells were washed and resuspended in 100 pl of cold 1% FCS in PBS and analyzed by FACS. The % internalization was.calculated from foe geometric mean obtained from foe FACS analysis [(mean at 37 *C with glutathione - mean at 4 *C with glutathione) / (mean at 37 °C without glutathione - mean at 4 C with glutathione)]. NA means that a FACS analysts was performed but

<img file="IN313DEN2012A_D0090.tif" />

j.

<td rowspan="2"> mAb</td><td colspan="2"></td><td></td><td rowspan="2"> % IntemaMzatkm</td>

<td> wifoout' 4 glutathione s 37 *C ·· “</td><td> f- .1'. &lt;4 tiSMkwi· ·..</td><td> pift) flhrtathlone 'r 4 °C</td>

<td> 13.1.2</td><td> 22.12</td><td></td><td> 5.38</td><td> 82-5%</td>

<td> sc95</td><td> I 22.58</td><td> 17.75</td><td> 5.13</td><td> 72.4%</td>

<td> sc131</td><td> NA</td><td> NA</td><td> NA</td><td> 72%</td>

<td> 50133</td><td> 23.39</td><td> 18183 '</td><td> 824</td><td> 722%.</td>

<td> sc 139</td><td> 22.64</td><td> 1923</td><td> 4,88</td><td> 80.8%</td>

<td> sc150</td><td> 2029</td><td> 7.78</td><td> 4.86</td><td> 20.0%</td>

<td> scITO</td><td> 19.97</td><td> 7,75</td><td> 4.67</td><td> 20.1%</td>

<td> SC211</td><td> 20.76</td><td> 823</td><td> 4.78</td><td> 21.6%</td>

<td> sc230</td><td> 2068</td><td> 7.97</td><td> 5J&amp;</td><td> 18,8%</td>

<td> sc250</td><td> 24.13</td><td> 8.07</td><td> 4.84</td><td> 16.7%</td>

tafe foe .presence of two subsets of antibodies,

13.1.2 is an antibody that was generated through hybridoma generation (Example 2) that Was directed against the EGFRvm epitope previously and Was uied as a positive control in this experiment. These those that: are efficiently inti

<img file="IN313DEN2012A_D0091.tif" />

Aafl BCTRyJMAgffiwfflw « that arc not (22% or less).

In older to determine certain of the antibodies oi the present invention bound, the epitopes of 6 human and I murine monoclonal antibodies (mabs) against EtMcvHI were mapped using synthetic peptides derived ftom foe specific EGFRvUI peptide sequence. The antibodies mapped were the human hybridoma derived anti-BGFRvIII 13.12 antibody, the human XenoMax derived snti-BGFRvin 131, 139, 250, 095, and 211 antibodies and foe murine antiEGFRvJB H10, Y10, andBS antibodies (from Dr. D. Bigner, Duke University).

The approach that was used was a custom SPOTs peptide stray (SigmaGenosy s) to study the molecular interaction of foe human anti-EGFrYIDantibodies with their peptide epitope. SPOTs technology is based on foe Solid-phase synthesis of peptides in a fomt suitable for the systematic analysis of antibody epitopes. Synthesis of custom arrayed oligopeptides is commerically available from Sigma-Gcnosys. A peptidearray of overlapping oligopeptides derived from foe amino-acid sequence of foe SGFr VIII variant was ordered from Sipna-Genosys.

A series of nine 12sheets. The peptide array..' ·. •.IWrTBW-.rys deletion of amino aside 6-27^¾^ glycine (G) residue at the iynthesized as spots on polypropylene membrane 5£η50 -ο£ foe BGFrVUI sequence, representing foe x domam of wtEGFr, and the generation of a peptide was offset by 1 residue from foe

<img file="IN313DEN2012A_D0092.tif" />

~?l previous one, yielding a nested, overlapping library of arrayed oligopeptides., The membrane carrying the 9 peptides BOFrVni antibodies (I gg/mt). The binding of foe mAbs to peptides was assessed by an enzyme-linked immunosorbent assay using *· secondary antibody followed by enhanced chemiluminescence (ECL). The array utilized is shown in Table 4.1,

I. ¹ I - r '«.7

<img file="IN313DEN2012A_D0093.tif" />

Hence:

_____VT(SEQE>NO:72) (SEQ ID NO: 59)

3. EEKKONYWTDH (SEQ ID NO: 73) , 4. BKKONYWTDHQISEQIDNO: 74)

5. KKGNyWTDHGS (SEQ £DNO; 75)

6. KGNYWTDHGSC (SEQ ID NO: 76)

7. GNYVVTDHGSCV (SEQ EDNO: 77)

S, NYWTDHGSCVR (SEQ IDNO: 78)

9. YWTDHGSCVRA (SEQ ID NO: 79)

In addition, functional epitopes were mapped by Combinatorial Alanine scanning. In this process, a combinatorial Alaninc.»scwiitirtg»-stoegv was used io identify amino acids in the EGFrVUl peptide ^tl-EGFRvtn mAbs. To accomplish this, a second set of SPOTs 8rrtya.w^^j!^^^^|nine scanning. A panel of varian ts peptides with alanine substitutions in each of foe 12. residues was scanned as above, Spot M. the unmutated sequence, is a positive control for antibody binding. The array utilized is shown in Table 4.2.

‘ · T . . f

<img file="IN313DEN2012A_D0094.tif" />

jifSID (SEQ ID NQ; —59)

2. AEEKKGNyVVTDfSEQ IDNO: 80)

3. LAEKKGNYWTD(SEQIDNO: 81)

4. LBAKKGNYWTD (SEQ ID NO: 82)

5. LEEAKGNYVVTD(SEQ IDNO: 83)

6. LEEKAGNYWTD (SEQID NO: 84)

7. LEEKKANYWTD (SEQ Π) NO: 85)

8. LEEKKGAYWTD (SEQID NO: 86)

9. LEEKKGNAVVTD (SEQ. ID NQ: 87)

10. LEEKKGNYAVTD (SEQID NO: 88)

11. LEEKKCHTYVATD(SEQIDNO: 89) -lg^^lfe^^AD(SEQbNO:90

<img file="IN313DEN2012A_D0095.tif" />

Epitopes of alJ 9 mAbs to the human EGPrVHI were mappod and identified by SPOTs procedure. AlJ 9 antibodies were reactive wife file peptides. The results obtained with 3 natriry, antibodies and 6 XenoMouse mouse derived human antibodies are presented in Table 4.3, Highlighted residues ate those which.,we .nested to alanine and abrogated binding by ths test antibody. These are

<img file="IN313DEN2012A_D0096.tif" />

<td> EGFR</td><td> A</td><td> T</td><td> c</td><td> V</td><td> K</td><td> K</td><td> C</td><td> P</td><td> R</td><td> N</td><td> Y</td><td> V</td><td> V</td><td> T</td><td> D</td><td></td><td> G</td><td> S</td><td> C</td><td> V</td><td> R</td><td> A</td><td> SEQ ID NO: 92</td>

<td> EGFRvlll</td><td></td><td> ♦</td><td></td><td> L</td><td> E</td><td> E</td><td> K</td><td> K</td><td> β</td><td> N</td><td> Y</td><td> V</td><td> V</td><td> T</td><td> D</td><td> H</td><td> G</td><td> S</td><td> c</td><td> V</td><td> R</td><td> A</td><td> [SEQ ID NO: 83)</td>

<td> 13.1,2</td><td></td><td></td><td></td><td></td><td> E</td><td> E</td><td> K</td><td> K</td><td> P</td><td> N</td><td> Y</td><td> V</td><td> V</td><td> T</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> (SEQ ID NO: 94)</td>

<td> 131</td><td></td><td></td><td></td><td></td><td> E</td><td> E</td><td> k</td><td> K</td><td> G</td><td> N</td><td> Y</td><td> V</td><td> V</td><td> T</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> SEQ ID NO: 84)</td>

<td> 139</td><td></td><td></td><td></td><td> L</td><td> E</td><td> E</td><td> K</td><td> £</td><td> ft</td><td> M</td><td> fl</td><td> w</td><td> V</td><td> T</td><td> D</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> (SEQ ID NO: 95)</td>

<td> 250</td><td></td><td></td><td></td><td> L</td><td> E</td><td> E</td><td></td><td></td><td></td><td> 1?</td><td> ft</td><td> &amp;</td><td> v.</td><td> T</td><td> D</td><td> }</td><td></td><td></td><td></td><td></td><td></td><td></td><td> [SEQ ID NO; 95)</td>

<td> ¢95</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> £</td><td></td><td></td><td> Ψ.</td><td> $</td><td> V</td><td> t</td><td> D</td><td> H</td><td></td><td></td><td></td><td></td><td></td><td></td><td> [SEQID NO: 96)</td>

<td> 211</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> L *</td><td> J-k</td><td></td><td> Y</td><td> V</td><td> V</td><td> T</td><td> D</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> (SEQ ID NO: S7)</td>

<td> H10</td><td> ♦</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> Y</td><td> V</td><td> V</td><td> T</td><td> D</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> (SEQ ID NO: 87)</td>

<td> V10</td><td></td><td></td><td></td><td></td><td></td><td> E</td><td> K</td><td> K</td><td> G</td><td> .N</td><td> Y</td><td> V</td><td> V</td><td> T</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> (SEQID NO: 88)</td>

<td> 39 -</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td colspan="2"> GjN</td><td> Y</td><td> V</td><td> V</td><td> T</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> [8EQ1DNO:99)</td>

The shaded amino acids shown fa Table 4.3 art the most relevant reriduM fa the eiptopc for s,

<img file="IN313DEN2012A_D0097.tif" />

antibody recognition. The πώώηϋ. lengths pf epitopes of all ten of the mAbs were precisely mapped using peptides of overlapping. and the tolerance fw mAb binding to mutated epitopes was determfaed.-by each residue in the epitope with Alanine.

fa Table 4.4, ad$tf ^F the antibodies are summarized. SpecdfciaDy, a subset of the antibodies were tesied/fbf efeifhfading of to lysates of tumor cell tinea in Western plates of polyacrylamide gal electrophoresis under either non-reducing or redyeing conditions. Purifcd recombinant protein is. also included. Antibodies binding fa both reducing and nonreducing conditions suggest that tits epitope is linear. Sample identifications:

EGFRvUI - the rabbit Fc fusion protein

H1.477 - H80 hl

These cells express bo

EGFR - purified

Λ431 - human tumor cell tineexpressing only wild-type EGFR A549 - human tumor ucll line expressing Only wildrlypC EGFR H80 - human tumor Cell line expressing only Wild-type EGFR

EGFR BiaCore - mAb* wart tested fa Biacdre for binding to purifcd EGFR as * highly sensitive test for specificity ’’ '

<img file="IN313DEN2012A_D0098.tif" />

I .

e transfected with EGFRvUI expression contract.

‘i

<td> mAO</td><td> EGFRvIII Western</td><td> rEGFRvlll Western (reduced)</td><td> EGFRvIII FACS</td><td> H1477 Western (native)</td><td> Η147Ϊ Western (reduced)</td><td> pep3 KlriExA</td><td> EGFR Western (native)</td><td> EGFR Western (reduced)</td>

<td> 13.1.2</td><td> ♦</td><td> +</td><td> +</td><td> + .</td><td> ♦</td><td> 25 pM</td><td> •</td><td></td>

<td> 131</td><td> *</td><td> +</td><td> +</td><td> +</td><td> +</td><td> 0.05 pM</td><td></td><td></td>

<td> 139</td><td> 1</td><td> +</td><td> +</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td>

<td> 095</td><td> +</td><td> +</td><td> +</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td>

<td> 211</td><td> +.</td><td> +</td><td> +</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td>

<td> 250</td><td></td><td> +</td><td></td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td>

<td> lAb</td><td> EGFR Biacore</td><td> A431 FACS</td><td> A431 ·’ Western (native)</td><td> (NW®</td><td> w</td><td> Western (native)</td><td> A549 Western (reduced)</td><td> ΗβΟ. FACS</td><td> Hao Western (native)</td><td> ΗβΟ Western (reduced)</td>

<td> 1.1.2 '</td><td> -</td><td> •</td><td> - .</td><td> Λ-U-An⁸ • 1 .·.,»« »'·</td><td></td><td> -</td><td> •</td><td> V</td><td> .</td><td></td>

<td> 131</td><td></td><td> ♦4-</td><td></td><td></td><td></td><td> · ..</td><td> •</td><td> -</td><td> •</td><td> -</td>

<td> 139</td><td> N.D,</td><td> ND</td><td> ND</td><td></td><td> FiN.ftw</td><td> . ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td>

<td> 395</td><td></td><td> ND</td><td> ND</td><td> ND</td><td> W</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td>

<td> 211</td><td> -</td><td> . ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td>

<td> 250</td><td> -</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> ND</td><td> NO</td><td> ND</td><td> ND</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

The results Showed that most of these mAbs have essentially the same binding specificity, seven of the mAbs were shown to bind specifically to the EGFrVlH variant, while 2 mAbs cross reacted with wildtype EGFr (murine H10 and human 211) in Western blots of purified protein and rn lysate of A431 cells. Note, however, that while antibody 211 binds to both native and reduced purified EGFRvIII in Western blots, it binds Rightly more strongly to the non-reduccd protein. In tests against a lysate of A431 cells, antibody 211 .binds Wrongly to a band of the size of wfld-typc

<img file="IN313DEN2012A_D0099.tif" />

EGFR in the non-reduced sample but there is no signal in the reduced sample. This suggests that toe binding of antibody 211 is.dwPjid· 8.¾¾¾ represented differently in flje 2-12 spanning the EGFRvIII (including H10 and 211) spi EGFr. Antibody 131 b _____ for expression of EGFRvItt;jM(jh^jS non-reduced or non-reduced purified E mrfonal epitope present in wild-type EGFR and ^epjtdpcs of 5 of the mAbs are within residues Mrresiduc, whereas the epitope of 4 of the mAbs Inch are commo to the EGFRvIII arid wijdiype ^SSJelK iti FACS.. These cells are apparently negative jgGFR expression. Antibody 131 does not bind to El or to reduced or non-reduced lysates df A43 and A549 cells in Westerns suggesting that antibody 131 may be binding to a variant of EGFR expressed on the cell surface .of some human tumor cell lines. This variant would be sensitive to denaturation.

Example 5

<img file="IN313DEN2012A_D0100.tif" />

EXAMPtsS . firos.-rewtiyity yith waalflM BOFR

Antibodies directed to variant BGF receptor? have been shown to cross-rcact with subsets of wild type EGF receptors on cell? in which gene amplification has occurred (Johns et aj, Int J. Cancer. 98: 398, 2002). To determine whether foe human EGFRyffl antibodies identified had simitar properties, they were tested for their ability to recognise wild type EGF receptors on a variety of cells in culture. Antibodies were incubated With toe indicated cell lines at 4’C. After washing in FACS buffer, a secondary antibody corrugated with phyoerythrin was added and the incubation was continued, All cell lines analyZod expressed wild type EGFR. A subset of wild type EGFRs was recognized by the antibody XGl-131 on both A431 and SF-S39 cells but not on A498 or SKRC-52 cells. Another antibody to EGFRvIH, 13.1.2, did net recognize tids subset of

<img file="IN313DEN2012A_D0101.tif" />

with subpopulations of wild type receptor? may be determined by both the specific epitope within the junction of the mutant receptor and the affinity of the antibody for this unique epitope (See the results of the epitope mapping and affinity determination section herein).

Characterization of Specificity ofAnti-EOFRyin Antibodies to vitro·. Binding of tbeAatibodies to Ptilfcfogi

The specificity of the purified antibodies was ascertained by performing FACS analysis on ι panel of chit lines. H8O, a human glioblastoma line, and H1477 (H80-EGFRVHI) that expresses high levels of EOFRvm, A431, a human, epidermoid careinotna line, and A549, a human hmg rarcinoma cell line were used as toe cell lines. All ccH lines wen from Dr. Bigner except A431 and \549, which were from ATCC (Rockville, MD, U.SA·). Celts were incubated on ice with 10 rg/ml of the respective antibody for 30 min;, Washed in FACS buffer and subsequently incubated vith PE-conjugated goat Miti-hurnanJIgG‘.fropi Jackson ImmundResearch (West Grove, PA, istUgrwn indicates cells stained wife an

<img file="IN313DEN2012A_D0102.tif" />

its foe staining of foe relevant antibodies.

The anti-EGFRvUi antibodies 13.1.2,131 and 139 bind to the EGFRvUI protein on the transfected cell lines. A graph summarizing seme of the results is displayed in HGs. 9M-9P.

<img file="IN313DEN2012A_D0103.tif" />

Antibodies directed to variant EOF rcccjHnrs have been shown to cross-rcaci with subsets of wild type EGF receptors on'cetls in which gene amplification has Occurred (Johns et al., fat. J. <img file="IN313DEN2012A_D0104.tif" /> Cancer. 98:398,2002). Hi this exajnpk, A451 and Λ549 stained by XGl-l3band XG1439. FIG. 108,and FIG. 10 C shorty * j Certain cross reactivity with the wild type EGFR instead of justrceogniritigg iti H80, A431 and A549 line. However, this cross reactivity is only at 10% of foe lei) FJE7.6.3) staining on these cell lines. The results arc provided in FIGs 9A-9P an^liW^ jfes. ··

Antibodies- directed can be used as delivery vehicles that specifically transport drugs or toxins tirto «Μ If the antibody stimulates internalization of antigen.

the drug or toxin can result in death of tile ceil, perhaps after the drug or toxin is cleaved from the antibody. Speh a mechanism can beutilizod to specifically kill rumor colls in animals and in patients. One way to select antibodies that can deliver drugs to cells is through secondary ‘ cytotoxicity assays. hi these assays the primary antibody binds to the cell surface and* secondary antibody dial is conjugated with a drug or toxin is added. If the primary antibody stimulates antigen internalization, foe secondary antibody wiU be co-intcmalized and upon cleavage of tin· drug or toxin result in cell killing.

Swfflfav. CytPtox’wlY Assays

In the following studies, EGFR^IH-specific antibodies were used to direct toxins Conjugated secondary anti^^^^j^jmji^^cma cell line (H80) bind EGFRvHl transfected glioblastoma Cell line (H.IgG (cat # 555784) from Pharmingcn (BD Biosciences Phanmngen) toxins AEFP (Seattle Genetics. Inc.) and maytansine (DM1, bimiunogca^t^iO^^^^>-AEEP (murine anti-human IgG-AEFP and mab-DMl (murine wtiAimpiri^gG^S^^^s^p^to conjugated goat anti-human IgG, Hum-ZAP (TM, cat # IT-22-.25Q, affinity-ptii^ed^^ril^^ujnan IgG-saporin) is from Advanced Targeting Systems (San DfegO. CA, U.SX). H80 and ΗΪ477 cells were plated out in 96-we JI pistes with lOQO cells in 100 μΐ growth medium per well. After 24 hours, primary antibodlcswcre mixed with conjugated secondary antibodies at 1:3, serially diluted #! l:S over 6 wells. 100 μΐ of diluted primary and toxin secondary antibody mixtures Were added into wells of cells at final starting concentrations of 0.1 pg/ml of primary antibodies and 0.3 pg/ml of secondary antibodies. ’Πιο plate was allowed to continue to culture far force days. On the fourth day, CellTiter-Glo reagents (pal #07571) from Ptomega (Madison, WI, U.S.A.) were added and luminescence was read. FIGs. 11AΠΙ, 12A-12I, and 13A-13I demonstrate the results from this experiment. Hum-ZAP mediated antigen specific tailings in Hl477 (filled circle) comparsd to HBQ (filled square) in most EGFRvUI specific mAbs tested. MAbs XG1-131 and'XGl-139 generated antigen specific secondary killings

<img file="IN313DEN2012A_D0105.tif" />

<img file="IN313DEN2012A_D0106.tif" />

with mah-AEFP, at less extent wife mah-DMl, Among the antibodies tested, XGl -131 performed at least one log better than 13.1.2, XG1-095, XG1-139, XGl-150, XGl-170, XG1-250 and XGl-

<img file="IN313DEN2012A_D0107.tif" />

211. IgGl was used as a negative ocn^rpl and antigen positive cells (H1477) were compared tn antigen negative cells (H§0)^ , ·

The amount of sp£qfl ‘ «η vary depending upon the particular use. la one cells is sufficient. For example, a redaction of 0$^0-70,70-80, 80-90,90-95,95-99, or 100% cf the the desired reduction in target cell nuaber is

1.1 *3^ embodiment, any reduction in

1,1-5, 5-10,10*20, 20-30^ target cells will be sufficient also a function of the nonspecific lethality of toe antibody combination. For example, antibody/toxin combinations that only have a 10 % decrease in target odl number may be sufficient;

if tilers is very little nonspecific targetingand lethality by the antibody. Far example,, the antibody toxin combination kilts loss than 1.0% of a non-target population. Again, the particular amount will depend on the particular need and situation. Particularly usefbl are antibodies that axe highly selective; for a target ceil (e.g., 131477) and bind well tolfhetarget cell, or proteins associated with the ceil, bi one embodiment, fee target is fee EGFRvIH protein, or a fragment thereof, hi one embodiment, antibodies that arc human, or humanized, Efficient at being internalized, specifio to tite EGFRvDI protein or fragments thereof associate tightly with tile BGFRvIII protein or fragment, and are associated with an effective toxin, an taught from these examples.

IJ

<img file="IN313DEN2012A_D0108.tif" />

mftgfrgs ity Cl wuggnig Amvs city assays, EGERvIH-specific antibodies were tested ή. j ' .' υΙΠ antibodies direct toxin conjugated secondary cell line (H1477), toxins are releasod inside the .ft proliferate to form colonies. Thus, the application in addition tojbese in the clonogenic sssays^Sgg antibodies into EGF^vE^ cells and eventually reduced ‘fee' of these EGFRvIII antibody^ttxxms g^ptwed'reduced number of clones when cells were re-plated after primary and secondary toxin antibody treatmen». In this example, H80 and H1477 cells were plated out in 6 well plates at 30,000 cells per well and incubated overnight. The primary antibody and secondary toxin antibody were mixed at a ratio of 1:3. This this antibody mixture was added into-the proper wells at a final concentration of primary antibody at 0.5 ggtail and secondary toxin antibody at 1.5 pg/ml. This was the incubated at 37 *C overnight. After incubation, the toxin mixture was disposed of properly and thocells wore detached from the wells With IX trypsin solution. The cells were counted and plated at 200 cells per well into new 6-well plates. Triplicates wells were plated for each treatment group. These plates were incubated in a 37 °C incubator for 23 weeks until the colonies formed and could ba identified by eye or under a microscope. The medium was aspirated and 5M Methylene Blue in methanol was added for 1 hour. The plate was rinsed in water and the colonies were counted.' FIG. 14A and FIG. 14B show foe results from this • ' ·'? ·'

<img file="IN313DEN2012A_D0109.tif" />

expenmem. As can be seen, mab-AEFP secondary toxin antibody inhibited colony formation· with the three EGFRvni antibodies tested.

Example 10

<img file="IN313DEN2012A_D0110.tif" />

EGRvLII antibody 1112was directly ConjOgafcd with aurisiatin E MMAE and AEFP conjugates

<img file="IN313DEN2012A_D0111.tif" />

H80, were only killed when exposed to very high concentrations of the antibody. Results from this, cxperimentareshfwn m FIGs. l SA-15 C. ’

Direct conjugation of the EGFRvlff antibodies with the drugs or toxins is a particularly advantageous method for therapeutic use. Thus, this initial experiment showed that such conjugates do result in specific killing of EGFRvHI- expressing cells.

. 11

Znvf poAnti-BGFRvUI AntibodiesCharac terization

An optio nal method to determine if au antibody is capable ofdelivering a cytotoxic drug to a cell is to evaluate the effect of the opnjugatedjmtibody on the growth of human tumors in vivo. This example presents, one au^^^^^^^^^.glibblfetQnw Cells we cultured in vitro, harvested by trypjinis^ticai bnS^^^pto^^^edded in Matrigel as explained below. Five million cells were mjccted subcifeepusfy i^rouemale nude mice and tumors allowed to develop

<img file="IN313DEN2012A_D0112.tif" />

groups and treatment with fte^adiaj^OQncertratiat of conjugated antibody intravenously every 4 ^r ·.· ΉΓι/η VtSJto'te a⁴· S9«ft it

MMAE). If tin antibody is administered with an equivalent amount of unconjugated drug, (Group2), titere is no effect on tumor growth proving that targeting the tumor cells in vivo requires the antibody conjugation with the toxin.

The animal model used above was developed by injecting HI477 cell xenografts into nude mice. Various amounts of fee cells were injected with er without MATR1GEL into 8 week old nu/nu female mice and the ftimor implantation over days analyzed. From this analysis, the number of c ells that would allow for an appropriately sized tumor was identified as 5 million celts in MAWGEL for approximately 22 days. Group 08 was included as a control to show that the killing was antibody specific. Group G7 was included a$ a negalive control.

<img file="IN313DEN2012A_D0113.tif" />

Thus a protocol toy as follows:

. .· • · ;.·· ·>π *

Day I: tumor implantation'of 5 million cells in MATRIGEL into 8 week-old uu/nu female mice.

Day 22: antibody pro-drag treatment* every 4 day», via LV., as shown in Table 11.1

<td> Group</td><td> Number ofhdice</td><td> Treatment</td>

<td> G-l</td><td> 8</td><td> 13.12-DM1 250 pg every 4 days, via I.V.</td>

<td> G2</td><td> 8</td><td> 13.12-OM1 75 pg every 4 days, via LV.</td>

<td> 03</td><td> 9</td><td> 13.1.2-MMAE 7$ pg every 4 days, via LV. '</td>

<td> 04</td><td> 8</td><td> 13.1.2-MMAE250 ug every 4 days, via IV.</td>

<td> G5</td><td> 9</td><td> 13.1.2-AEFP 75 pg every 4 days, via LV.</td>

<td> os'</td><td> 8</td><td> 13.1Λ-ΑΕΗΡ 250 pg every 4 days, via EV.</td>

<td> G7</td><td> 8 · :</td><td></td>

<td> 08</td><td> .0' Vf' &lt; '•f-</td><td> ^jfraccwiighted) 250 pg+Maytansme 4pg</td>

. · .

The results are showiJiPlG’.-.l^jwifit'tije arrows indicating the addition of drug. Groups • •V. · · ·*·. , ₍. . j · ·

G1,05, and G4 showed effecb'yerkilling? iQrth5»'G3 showed a lesser amount of killing. Groups 08 and G7 stowed no killing. Certani-teSiciiJi may have been observed in the high dose vc-AEFP group. Group G8. These animals received. 2 treatments at 250 pg and I treatment at 125 pg.

Bameuliz .

Exaresrioq of gGERvIR ip Cancer Patienta'Human Turnon;

The expression ofEGFRvUI on humin tumors was determined by naming frozen tissue sections from a variety of cancer patients with,» combination of 2 murine monoclonal antibodies (B9, IgGl and Y10, IgG2 (Dr. Bigner, Duke University)) known to bind specifically to EOPRvUl The same sections were Stained with iwtype-matched control antibodies. A summary of the staining results obtained from all patient Samples is presented in Table 12,1. /

Table 12.1

<td> Turner type '</td><td> Sample Size (N).., '</td><td> EGFRvIH&gt;+</td><td> BGFRvO+H·</td>

<td> Glioblastoma</td><td> 8 '.MwAfe</td><td> .100%</td><td> 100¾</td>

<td> Breast Cancer '</td><td></td><td></td><td> 24¾</td>

<td> NSCL cancer</td><td></td><td></td><td> 3555</td>

<td> Head &amp;. neck Cancer</td><td> 21</td><td> 42¾</td><td> 38¾</td>

<td> Prostate Cancer’ </td><td> 22« wftAlip’«►tipifell?»’'</td><td> 45%“ ~</td><td></td>

<td> iij</td><td></td><td></td><td></td>

..___ · '7*τ’«.·ϊ· iTyiit? fr? , ___

EGERvIKH-: include all tumors that express EGFRvIIl EGFRvrn>++: include Only those tumors that express *tteast lO% or more EGFRvffl

<img file="IN313DEN2012A_D0114.tif" />

<img file="IN313DEN2012A_D0115.tif" />

The expression was found primarily on the. cell membrane and/or cytoplasm. Significant numbers of breast (31%), NSCL (47%),· and head φ neck (42%) canCcr specimens stained positively for EGFRvIH. In oettafo mria High quality IHC staining, the use of two antibodies oan be butter tija@ , Frozen tissue specimens were superior over fixed tissues. .. . -:X

As appreciated by one ofsfelfItfWaiV’iFmay be advantageous to test patients before using therapeutic antibodies to ensure that the tumor which is being treated expresses EGFRvIII.

sawruia

In Ww Anti-EGERvIH Antibodies Characterization.

The method of Example 11 will be used to treat lung cancer and gliomas. This will be broadly examined by producing animal models. Animal models for glioblastoma and lung cancer are developed as follows: lung cancer cells that express wt-EGFR are transfected with EGFRvIII. The cells are injected into the lungs of nu/nu mice and tumors allowed Io progress to a comparable stage to that above. Anti-EGFRvIIIcpnjygalea^yglLthen be injected intravenously as ubovc every t W 10 days as needed. The sitotoi pp^yti^^^ppession of continued growth Of these cancer cells will then be monitored, to detcniiinefac'.efE^Btiveneas of these Anti-EGFRvUI antibodies and antibody-toxins combinations; As apprcciated’by one of skill In the art, this ean be done for any of the antibodies discfosed^iCrcm.,.

<img file="IN313DEN2012A_D0116.tif" />

• · . RVa* *

In, order to farther resolv ajminp acid residues that are indispensable fur binding within the EGFRvIli 'analyses &lt;jf the amino acids in the epitope peptides were perfonnedi The staining point Was the sequence that was derived from Example 4, LEJEKKGNYWTD (SEQ ID NO 59). In this example each amino acid of the mapped epitope was substituted οηβ-at-a-time by all 20 L-amino acids, thus, all possible single site substitution analogs were synthesized and sweated to provide detailed information on the mode, of peptide binding. Discrete substitution patterns were identified for mAbs 131 and 13.1.2. Tire results from the substitutions arc summarized in Table KI.

¹³¹ <se^{q ip NQ: 57}>

h3.1.g WK gWVVl (SEQ ID NOT 57)]

It appears that for mAh 13.1.2, 5 residues are important for binding (bold), while only 4 residues ere essential for the binding of m^b. l31,;>Th.e rest of the residues were replaced by various nemo acids withouVsigraficant legj^g^^^^^^> the.i3i and 13.1.2 epitopes are identical by sequence and length, the binding: pattern foreach appears different. Binding of mAb 131 is strongly dependent on the residues EKNY (SEQ ID NO: 60). On the other hand, the data revealed that residues EEKGN (SEQ ID NO: 61) are involed in binding of mAb 13.1.2.

<img file="IN313DEN2012A_D0117.tif" />

transfected into 293T cells. Seventy-two hours later supernatants were collected and essayed for secretion and binding to EGFrVIII antigen by ELISA.

The results demonstrated that antibodies derived from expression of 131 heavy chain with 13.1.2 kappa chain, and vice versa were expressed well but binding activity was reduced

<img file="IN313DEN2012A_D0118.tif" />

by 75% probably due to the di (data not shown). This demon;

• · Γ I .· · <T - > I

13.1.2 m Abs, again suggesting that the structural characteristics of the epitope selected for between the two mAbs are different.

Example 16

Molecular Mbdelaag of 131 andits Paratope

<img file="IN313DEN2012A_D0119.tif" />

antibody 131 was generated through? modeling approach using the InsightH modeling package from Accelrys (San Diego, CA). The model was built from the variable region sequences described: below, Table 16.1. The residue numbering starts with the light chain amino acids, and continues to heavy chain amino acids.

. Table 16.1

Light chain variable region ,,.. ^erent binding, pa- .

<img file="IN313DEN2012A_D0120.tif" />

WLQQRPGPPRLLIY

RISRRFS (CDR2)

<img file="IN313DEN2012A_D0121.tif" />

(SEQ ID NO: 100) (SEQ ID NO: 101) (SEQ ID NO: 102) (SEQ ID NO: 103) (SEQ ID NO: 104) (SEQ ID NO; 105) (SEQ ID NO: 106)

QVQLVESGGGWQSGRSLRLSCAASG1

NYGMH (CDR1) wvrqapgkglewva (SEQ ID NO: 107) (SEQ ID NO: 108) (SEQ ID NO: 109) (SEQIDNO.· 110) (SEQ ID NO: 111) (SEQIDNO: 112) (SEQIDNO: 113) if ώ;' V-t-rh/·,

I ,

VIWYDGSDKYYADSVRG (CDR2) rftisrdnskntxylqmnslraedtavyycar DGYDILTGNPRDFDY(CDR3) '

WGQGTLVTVSS .

Antibody 131 sequences' w^^SS^SS&feainst the Protein Data Bank to identify homologous antibodies and their stfiuctures.^as^d on the homologous antibodies' sequence similarity to the 131 antibody, several structures were selected. The structures selected for modeling samples from the Protein Data Bank had the Protein Data Bank identifications of 1HEZ, 2H1P, 1AQK, 1DQL, 1MF2 and 1FLR. These template structures were then aligned by superposition and used to generate structure-based sequence alignments among the templates. The sequences of antibody 131 ’s variable region were then aligned to the template sequences. The structure and sequehceaEgniments were used to generate the molecular model for the variable region of foe^s?W^liahliW|y;. The sequence for CDR1, light chain was: RSSQSLVHSDGNTYLS (SEQ Π) NO 101). The sequence for CDR2, light chain was: RISRRFS (SEQ ID NO 103). The sequence for CDR3, light chain was: MQSTHVPRT (SEQ ID NO 105). The sequence for CDRl. heayy chain was: NYGMH (SEQ ID NO IQ8). The sequence for CDR2, heavy chain was:'^^fepKYYADSVRG (SEQ ID NO 110). The sequence for CDR3> heavy chain^^^^^^^BbiPRDFDY (SEQ ID NO 112).

The interaction surface for aiitibody'T^l'Was calculated from the structure model and shown in FIG. 17- The various CDRs are identified as follows: LI (light CDR1) 10, Hi (heavy CORI) 20, L2 30, H2 40, L3 50 and H3 60. A prominent feature on the predicted antibody 131 interaction surface is a deep cavity. The cavity is mainly surrounded by heavy chain CDR2, CDR3 and light chain CDR3, with a small portion contributed by light chain CDR1. The cavity is probably the binding pocket. Within 5 Angstroms of the binding cavity are residues 31, 37, 95-101, 143-147, 159, 162-166, 169-171, 211-219, 221 and 223. These residues are likely to comprise the paratope and make key contacts in the binding of EGFRvIU epitope. It is also likely that the residues provide important structural features to the binding site in general. ^uencealignipep,.. · ·

Site-directed model for antibody 131

This example demonstrates one method by which models that suggest residues that are important in binding may be tested^ The Example also results in several antibody variants. Antibody variants of the 13 lcloncwe^e .generated by single residue mutations introduced to the heavy and the light chain Of Cyjariants were then analyzed to determine how the altered side chains *i contributed to antigen binding.

if*· f·'·* ’ .*4' · **?.·/*

Changes were made in the heavy and light chains of mAb 131. On the heavy chain L216 was changed by site directed mutagenesis to R. On foe light chain, V99 was changed to F. Both

<img file="IN313DEN2012A_D0122.tif" />

<img file="IN313DEN2012A_D0123.tif" />

<img file="IN313DEN2012A_D0124.tif" />

mutations affected the expression end secretion of the variant antibodies compared to the wildtype sequence. BothnnnatioMresultedbii tong of toe mAh variant to toeEGTRvIIliotigen.

This demonstrates L2I 6 since substitutions of thCws re&amp;S

<img file="IN313DEN2012A_D0125.tif" />

^^^igni&amp;ent contacts with the EGFRvUI antigen y resulted in reduced activity. Of course, it is always an option that these substibitioasiie tosmptiveco the antjbody’s geaeral structure.

Example 18

MglwdirMadafat tf IStLlmd iO ftgtepg

The three-dimensional structure model of toe variable region of the 13.1.2 antibody was generated through homology modeling approach with the fraeigfixtll modding package fipm Accetrys (San. Diego, GA). The mode) was built from toe variable region sequences, shown bdow in Table 18.1, using the x4^^^^-0uatoB8. as templates.

Table 18.1

Light chain variable region (ΙΊ13) DIVMTQTPLSSPVTLGQTASISC

RSSQSLVHSDGNTYLS (CDEl) WLHQRPGQPPRLUY K1SNRFS(CDR2) GVPDRFSGSGAGT MQATQLPRT(CDR3) . . ,,.,

FGQGIKVEJKR ·» ?rt^-

<img file="IN313DEN2012A_D0126.tif" />

X.,· .

(SEQ ID NO: 114) (SEQIDNChlOl) {SEQ ID NO: US) (SEQ ID NO : 116). (SEQ ED NO: 117) (3EQIDNO: 118) (SEQ ID NO; 119)

Heavy chain variable region (114-234) QVQLVESGGGWQPGRSLRLSCAASGFTFS SYGMH(CDRl) , ,. .

,. t uc i no&amp; jl was be · wvrqapgkglewva &lt;·· ;

*«»»f»raed x-ray cntoU/i .

VIWYDGSNKYYVDS VKG_;(CDR2)

RmSl©NSKNTLYU2Mi'J^^ DGWQQLAPFDY (CDR3) wgqgtlvtvssa (SEQ ID NO: 120) (SEQ ID NO: 121) (SEQ ID NO: 122) (SEQ ID NO: 123) (SEQ ID NO: 124) (SEQ E> NO: 125) (SEQIDNO; 126)

The sequence for CDRl, lig^it chain was: RSSQSLVHSDGNTYLS (SEQ ID NO: 101). The sequence for CDR2, light ehaih wm:KISNRFS (SEQ ID NO: 116)· The sequence for ODR3, light chain was: MQATQLPRT (SBQJjg. NQ; 1(8). The sequence for CDRl, heavy chain Was: SYGMH (SEQ ID NO* :· ’tKS

<img file="IN313DEN2012A_D0127.tif" />

- for CDR2, heavy chain wm:

8i

VlWYDGSNKYYVDSVKG (SBQ ID NO: 123). The sequence for CDR3, heavy chain was: DGWQQLAPFDY (SBQ ID NO: 125).

Antibody 13.1:2 sequences were used to search the Protein Data Bank to identity homologous antibodies. The structures with the Protein Data Bank identifications of 1IIEZ, 2HIP, 8FAB and l’AQK were selected as modeling templates, based .on their sequence similarity to antibody 13.1.2. The template structures were aligned by superposition and used to generate structure-based sequence alignments among the templates. The sequences of the variable regions of the 13.1.2 antibody were then aligned td the template sequences. The structure and sequence alignments were used to generate the' molecular mode) for antibody 13.1.2 variable reg i on.

The interaction .swrftcejyteP^^^^^ the model and is shown in FIG. 18. A major feature of the 33.1.2 mode) w

<img file="IN313DEN2012A_D0128.tif" />

jf&amp;ve on the surface of th? CDR region. The --:-.1 ,* -Ί .

groove.is outlined by heavy όί JR3 160, and light chain CDR1 110, CDR2 130 and CDR3150. One end of thegroove tottehesti» rest of light chain CDR3 150, and the other end opens to the wM«· area ^^^ai|i£D$3fj.6O near the heavy chain-light chain interface. The groove is probably the binding-pocket'for.jbemitigcn. Within 5 Angstroms of the binding groove are residues 31, 33, 35-39, 51, 54-56, 58-51, 94-101, 144-143, 160, 163-166, 172, and 211-221,

These residues arc likely tocqmprise the paratope for the binding of EGFRvIH epitope. It fa also likely that the residues provide important structural features to the binding site in general.

SzssBklS

Docking Models of a Peptide to pn Antibody

The epitope rnappingstudiea in Example 14 revealed that the relevant amino acids required for binding of the eptope to the paratope of 13.2.1 mAh reside in the six-residue peptide EEKKfjN (SEQ ID. NO: 127), Therefore, docking models of this six-residue peptide* complexed to the CQR region of 13.1.2 structure model were, generated. First, a model of the peptide EEKKGN (SBQ ID NO:. 127) was produced. This wa&amp;done, similarly as described before, except this time using the xray crystal structure of 1181, as M

<img file="IN313DEN2012A_D0129.tif" />

lleln Data. Bank, as the template. Next, this peptide structure was manuall^·^ ^ove. to form an initial assembly complex. A

Monte Carlo search rieritational spaces were then automatically performed with the Docking modulo In InaigtUlI. The peptide conformation was allowed to be flexible by giving each οϊ ώβ'ΐ^^ίϊι an^Cm angles full rotational freedom. During the docking process, the residues ^thin’^^^ww^lta^indmg groove were allowed to move while the other residues of foe antibodj?v^fc^xed.‘-*^1ie plausible configurations found by Monte Carlo search were subjected to simulated annealing and energy minimization to reach the final comptex structure models. For each docking model obtained, the -interaction energy between tile antibody and the peptide was calculated with the Discover^ module of Ihsightn package. The interaction energies for all docking models were assessed and the model with the strongest overall antibodypeptide interaction was examined and is shown in FIG. I9Aand I9B.

In this docking model, there, are six hydrogen bonds between peptide EEKKGN (SBQ ID NO: 127) and antibpdy 13,1.2, as shown in FIG. Γ9Β. Tho peptide residue number it labeled farm N-tennihus to the C-terminus as I through 6, Six hydrogen bonds are indicated by green dashed lines. The six pairs of amino acids fanning hydrogw bonds are: R2..,Yl.72,K3,..H31, K4...H31, N6...D33, N6...Y37, and N6...K55. In this docking model, tho peptide is bound to the groove in an extended β-strand oonfixmation. Residues in the peptide alternately face the solvent and tiw antibody surface, The residues facing the binding groove with the most significant contacts to the antibody are E2, K4 and Nd. Thia these farce residues may be important to peptide binding, consistent with The interaction energies for each of flic six peptide residues with the 17^^^^^WtcaIoUhKed with the Disocver_3 module and the’ results are shown in Table 19,1. Table $.1 shows the interaction, energies far each of the six peptide residue? with tbe l3,T2.peraWd. /Aft energies are in the unit of fcaJ/mol.

The residues wifo_;the._ssfrQpg«l interaction energies ere in the order of N6, K4 and Έ2, confirming that these residues toe key contributors on the antigen side in the antibody-antigen interaction, again consistent wifa experimental data. These date provided strong evidence to support the docking model, fa this embodiment, fae paratope is defined as foe residues wittata 5

Angstroms of the docked peptide. The 20 residues conqrtising the paratope as so defined are residues 31-33, 35,37,55,96-101,148,163, 165, 170,172,178 and 217-218. To evaluate, cm an individual residue basis, the contribution of each of these residues of the antibody fa foe antibodyantigen interaction, the interaction energy between the paratope residues and the peptide EEKKGN (SEQIDNO.· 127) was calculated for each of the above. 20 residues, The results are listed in Table· 19.2. Table 19.2 shows the interaction energies far each of the 20 paratope residues with foe peptide EEKKGN (SEQ ID NO: 127). All energies are in the unit of kcal/mol. The residues with

<img file="IN313DEN2012A_D0130.tif" />

<td> Peptide Residue</td><td> Coulumbic</td><td> VdW</td><td> Total</td>

<td> ei</td><td></td><td> -3:738</td><td> -5.751</td>

<td> E2</td><td></td><td> -0.817</td><td> -11.278</td>

<td> K3 1</td><td> -9.816</td><td> -0.493</td><td> -10.309</td>

<td> K4</td><td></td><td> -0.968</td><td> -12.091</td>

<td> G5</td><td> -1.241</td><td> -1.488</td><td> 3^55</td>

<td> NS</td><td> -16.504</td><td> -0,181</td><td> -16.885</td>

<img file="IN313DEN2012A_D0131.tif" />

<td> 13.1.2 Residue</td><td>^r ittrmeOa?</td><td> I h</td><td> VdW</td><td> Total</td>

<td> Hfe3l .'</td><td> r*t ,_t..... ;i2te35S</td><td> •Α--ΓΊ</td><td> 3.033</td><td> -9.801</td>

<td> Ser32</td><td></td><td> y ’</td><td> -1.062</td><td> 1.794</td>

<td> Asp33</td><td></td><td colspan="2"> -0.698</td><td> -4.679</td>

<td> Asn35</td><td> 0.253</td><td colspan="2"> ' -1.009</td><td> -0.7S8</td>

<td> Tyr37</td><td> -2,058</td><td colspan="2"> -2.463</td><td> -4.521</td>

<td></td><td> -l4463</td><td colspan="2"> ' Tsee</td><td> -12.794</td>

<td> Aiase</td><td></td><td></td><td> 0.696</td><td> -5.182</td>

<td> Thr97</td><td> . 5.· ^.#33%</td><td></td><td> --1.431</td><td> -4.171</td>

<td> Gln98</td><td></td><td colspan="2"> 7:;'.· -1.548</td><td> -4.09</td>

<td> Leu99</td><td></td><td> •A *.</td><td> -2.779</td><td> -4.286</td>

<td> ProtOQ</td><td> . 0439</td><td> I- -.’···</td><td> 0.379</td><td> 0.061</td>

<td> Aral 01</td><td> 3.992</td><td colspan="2"> -0.549</td><td> 3.443</td>

<td> Hls148</td><td> ’ ο,ιόΐ</td><td colspan="2"> -0.083</td><td> 0.018</td>

<td> Vail 63</td><td> -ο.ιο4</td><td colspan="2"> -0.237</td><td> -0.342</td>

<td> Trp165</td><td> . . 1,368</td><td colspan="2"> ' -T.122</td><td> • 0:236</td>

<td> AsnltO</td><td> -2.102</td><td colspan="2"> -0.487</td><td> -2.589</td>

<td> TyrtTg</td><td> ^8.7</td><td colspan="2"> 0.896</td><td> -7.804</td>

<td> Lys178</td><td> ; . .-318141</td><td colspan="2"> -0.03</td><td> -3.644</td>

<td> Leu217</td><td> 0.761</td><td colspan="2"> -1.426</td><td> -0.664</td>

<td> Ala218</td><td> -0.071</td><td colspan="2"> -0.281</td><td> -0.352</td>

. ' iBpitY-ImproYgd Mftedws

This Example 'dern&lt;mstnrtiaCTw^^w pocking model can be used as the basis of rational design for affinity-in^rOved'anS^difti«^directed mutagenesis. Each ol the 13.1.2 paratope residues was mutated to all 19 other imino acids in silica, resulting in a total of 19x26 or 380 virtual mutants. The mutation Was do^p by residue replacement followed by 50 steps of energy minimization to account for. any.Joc^i^Or^iidDal changes (hat could be induced by the side chain change. The’ interaction'. ^^^^^^F^yiVhOle peptide and the. whole paratope was calculated for each mutant biutaS^^S^-^B^fetcraction energy stronger than the wild type .13.1-2 could potentially have a higher affinity for the peptide EEKKON (SEQ ID NO: 127), and perhaps oven the whole EGFRvIII protein·' These mutants mostly have stronger coulumbic interactions than the wild type 13.1J2, but so®⁶ ^em have weaker van der Weals (VdW) interactions than the wild type antibody. Considering that in fee wild type 13.1.2 antibody, fee VdW interacting energy is -9.689 kcal/rtiol, mutants wife VdW interaction energy weaker than -8.5 kcaVmol were filtered out The rest of themutants that have stronger, total interaction energy, than the wild type 13.1.2 are listed in Tabic 20.1. The wild type data ate listed a&lt; the bottom for comparison. All energies are in the units of fcalfaaol.

<img file="IN313DEN2012A_D0132.tif" />

<td> Mutant</td><td> Coulumbic</td><td> VdW</td><td> Total</td>

<td> TyriTZAre</td><td> -93,004</td><td> 3/7(52</td><td> -101.706</td>

<td> LeuflSGlu</td><td> -79,897</td><td> -8.506</td><td> • -86.403</td>

<td> ArglOlGlu’</td><td> -77.984</td><td> &lt;833</td><td> -86.817</td>

<td> Leu217Glu</td><td> -78.124'</td><td> -8.898</td><td> -84.123</td>

<td> Leu99Asn</td><td> ' -7G.G37</td><td> ~ &lt;8§T</td><td> -83.231</td>

<td> Leu99His</td><td> -73,631</td><td> &lt;008</td><td> -82.639</td>

<td> AqjlOIAsp</td><td> -71.983</td><td>¹ &lt;877</td><td> -81.861</td>

<td> Leu2l7Gln</td><td> -70.263</td><td> -9.795</td><td> *80,058</td>

<td> Leu99Thr</td><td> -69.682</td><td> -10.153</td><td> -80.035</td>

<td> Gln98Glu</td><td> -70.651.</td><td> &lt;257</td><td> -79.908</td>

<td> Leu217Asn .</td><td> &gt;70.989</td><td> -8.789</td><td> -79.758</td>

<td> ArqWlGtn</td><td> -89.432</td><td> -10.164</td><td> -78.596</td>

<td> Leu217Asp</td><td></td><td> -9.643</td><td> -79.578</td>

<td> Asn35Gly</td><td></td><td> “ -10.191</td><td> -79.207</td>

<td> Tyr172H»</td><td></td><td> Γ &lt;509</td><td> -78.820</td>

<td> Val163Aen</td><td> . ¹ IS</td><td> F^- -9.944</td><td> -78.784</td>

<td> Tyrl72Asn</td><td> -681886</td><td> -9.871</td><td> -78.767</td>

<td> Ala218Lys</td><td> - -70.024</td><td> . &lt;570</td><td> -78.594</td>

<td> Asn35ArQ</td><td></td><td> -9,604</td><td> -78.593</td>

<td> Trp165LYS</td><td> &lt; *.X</td><td> &lt;7eb</td><td> • -78.344</td>

<td> Trp165Arfl</td><td></td><td> &lt;216</td><td> -78.030</td>

<td> Leu99Tyr</td><td> · · ' -6W6&amp;</td><td> ΠΓ -10.484</td><td> -77.517</td>

<td> Tyr172Thc</td><td> -68.146'</td><td> -9.225</td><td> -77.371</td>

<td> Ala96Thr</td><td> -67.534</td><td> &lt;823</td><td> -77.158</td>

<td> Ala96Ser</td><td> -87.222</td><td> &lt;822</td><td> -77.045</td>

<td> ProlOOTrp</td><td> -67.398</td><td> &lt;498</td><td> -76.894</td>

<td> Leu217Ser</td><td> -66.676</td><td> -10.133</td><td> -76.810</td>

<td> Ser321le</td><td> ^88.700</td><td></td><td> -TS.TH</td>

<td> Tyrt72Ser</td><td></td><td> &lt;146</td><td> -76,734</td>

<td> His31Glu</td><td> -67.070</td><td> -9.481</td><td> -76.531</td>

<td> Leu2l7Tyr</td><td> -85.605</td><td> -10.726'</td><td> -76.331</td>

<td> Vall63His</td><td> -67-.236</td><td> *·#♦·</td><td> -76.300</td>

<td> His148Ser</td><td> -66.780</td><td> &lt;405</td><td> -78.274</td>

<td> Hls!48Vai</td><td> -60.834</td><td> . -9.628</td><td> -76.263</td>

<td> His14BAIa</td><td> -66.770</td><td> &lt;473</td><td> -76.243</td>

<td> H1s148Gly</td><td> '' -66.782'</td><td> &lt;456</td><td> -76.217</td>

<td> His148Thr</td><td></td><td> : -8.508</td><td> »76.209</td>

<td> Leu99Ser</td><td></td><td> X -10.006</td><td> -76.132</td>

<td> ProlOOAsp</td><td></td><td> Γ’’ &lt;0.787</td><td> -75.940</td>

<td> rrp165Glu ’</td><td></td><td> F¹·’ &lt;267</td><td> -75.932</td>

<td> Hls148Asn</td><td> · ’ aAnrfHA*'</td><td> &lt;.889</td><td> -75.899</td>

<td>^srolOOGln</td><td></td><td> k &lt;871</td><td> -75.745</td>

<td> eu217Thr</td><td> . - ..’.'•^etewra'</td><td> &lt;672</td><td> ~75.7l7</td>

<td> Ser32Val ·</td><td></td><td> Im· -Jr·.·? &lt;854</td><td> -75.689</td>

<td> 5er32Pro</td><td></td><td> 1-. &lt;813</td><td> -75.620</td>

<td> ’rotOOGIy</td><td></td><td> t.-fc &lt;774</td><td> -76.615</td>

<td>^srDl OOAla</td><td> -651869</td><td> &lt;712</td><td> -75.601</td>

<td> 3er32Ala</td><td> ' -65.407</td><td> -10.089</td><td> -75586</td>

<td> 5er32Thr .</td><td> -85,7^3</td><td> &lt;861</td><td> -75.584</td>

<td> Mutant</td><td colspan="3"></td><td colspan="2"> VdW</td><td> Total !</td>

<td> Ala2l8Thr</td><td></td><td colspan="2"></td><td colspan="2"> t* -9.505</td><td> -75.560 !</td>

<td> ProlGOSfer</td><td></td><td></td><td></td><td colspan="2"> Γ -9,899</td><td> -75530</td>

<td> VaH63Gly</td><td> * , I ¹¹ '3Λ</td><td></td><td></td><td> '*</td><td> -9.536</td><td> -75.529</td>

<td> Gln96Thr</td><td colspan="2"> ’•Λϋνί</td><td></td><td colspan="2"> $.277</td><td> -75.438</td>

<td> ProlOQMet</td><td colspan="3"> -65.811</td><td colspan="2"> -9.602</td><td> •75.412</td>

<td> Ser32Met</td><td colspan="3"> -68.252</td><td colspan="2"> -0.153</td><td> -75.406</td>

<td> Ser32Gly</td><td colspan="3"> -65.509</td><td colspan="2"> -$.891</td><td> -75,399</td>

<td> Pro 100As n</td><td colspan="3"> 45.729.</td><td colspan="2"> -0.555</td><td> -75.384</td>

<td> Tyr37Phe</td><td colspan="3"> -86.283</td><td> • «</td><td> . -9.020</td><td> -75.272</td>

<td> Va 1163Ala</td><td> • ·’ ·»$?©</td><td></td><td></td><td colspan="2"> -9.543</td><td> -75255</td>

<td> Leu2171ie ’</td><td></td><td></td><td></td><td colspan="2"> -8.759</td><td> -75;238</td>

<td> Wild type 13.1.2 · ·</td><td></td><td></td><td> 992</td><td></td><td> $.889</td><td> -75s20S</td>

<img file="IN313DEN2012A_D0133.tif" />

The mutants Hated in f able20-1 Coufd tie candidates for engineering of affinity-improved antibodies. For the top 14 candidates In the list, per residue contributions on the antigen side and on foe antibody side were farther analyzed to examine the impact of the proposed modifications. The 10 mutants selected for tri vttro site-directed mutagenesis were Tyrl72Arg, ArglOlGlu, r«u99Asn. Leu99His, ArglOlAsp, Lcu217Gln, Lcii99Thr, Leu217Asn, ArglOlGIn and Asn35Gly. The results can be. seen in Example^ 1.

<img file="IN313DEN2012A_D0134.tif" />

firming themodclfor 13.1.2 ^U>y.which the above models, which suggest residues Example also results in several antibody variants. Antibody variants of 13.1.2 were generated by single residue mutations introduced into the heavy and the light chains of the 13,1.2 mAl?. Thc'variants were analyzed to determine foe contribution that the various side chains .had (η binding. A list of the mutations introduced by site directed mutagenesis arc summarize

This example denwst that are importantin hindingfcaT

<img file="IN313DEN2012A_D0135.tif" />

Table-

<td></td><td> Chain - *</td><td> MutStioh··'</td>

<td> 1</td><td> Light chain (CDR3)</td><td> ArglOlAsp</td>

<td> 2</td><td> Linfatchain (CDR3)</td><td> ArglOlGln</td>

<td> 3</td><td> Light chain (CDR3)</td><td> ArglOlGlu</td>

<td> 4</td><td> Light chan (CDRl)</td><td> AanSSGly</td>

<td> 5</td><td> Heavycham{CDR3)</td><td> Leu2l7Asn</td>

<td> 6</td><td> Heavy chain (CDR3)</td><td> Lcu2l7Gln</td>

<td> 7</td><td> Light chain (CDR3)</td><td> ^eu99Asn</td>

<td> 8</td><td> Light chain(CDR3)</td><td> Leu59His</td>

<td> 9</td><td> Lightchain (CDR3&gt;</td><td> l««9j8Thr</td>

<td> 10</td><td> heavy chain (€0X21«^^</td><td></td>

<img file="IN313DEN2012A_D0136.tif" />

Each Of the ! 0W

13.1.2 mAb. Each mutated' diii&amp;rfyiir was introduced into the heavy Or light chain of foe sfectod with the complementary wild-type chain in

<img file="IN313DEN2012A_D0137.tif" />

293 cells; Supernatants were then tcstedfcr expression and secretion efhun^m wiibodies, and for binding to EGFrVHI antigen. Thc-nsn^.iudctenuined by en EUSA, are stnmaarmedin Table

2ia · ,’*Z »;::

Table 21.2

-t*i . f I.S'»» · ·· «

<td></td><td> Mutation^'</td><td> Bltx&amp;ng Energy</td><td> Expression</td><td> Bindinfl</td>

<td> 1</td><td> Arg 101Asp</td><td> -81-881</td><td> Yes</td><td> No</td>

<td> 2</td><td> Arfl101Gln</td><td> -79.596</td><td> Yes</td><td> No</td>

<td> 3</td><td> Arg101Glu</td><td> -08.817</td><td> Yes</td><td> No</td>

<td> 4</td><td> Asn35Gly</td><td> -79.207</td><td> Yes</td><td> Yes</td>

<td> 5</td><td> Leu217Asn</td><td> -79.758</td><td> Yes</td><td> Yes</td>

<td> 6</td><td> Leu2l7Gln</td><td> -80.058</td><td> Yes</td><td> Yes</td>

<td> 7</td><td> Leu99Asn</td><td> -83.231</td><td> Yes</td><td> Yes</td>

<td> a</td><td> Leu99His</td><td> . -82.638</td><td> Yes</td><td> Yes</td>

<td> 9</td><td> L®u99TtyJ:</td><td> At 7-80.035</td><td> Yes</td><td> Yes</td>

<td> 10</td><td> ..WcIt^WSi</td><td></td><td> Yes</td><td> Yes</td>

<td> 11 -</td><td></td><td> ί&amp;&amp;&amp;205</td><td> Yes</td><td> Yes</td>

<img file="IN313DEN2012A_D0138.tif" />

ExamnlB22 fapOTtaa alEGERYffl/pELAG yaowt wnsfcwgt

This example demonstrate*· how a· variant to EGERvM con be made. A lO92bp fragment encoding the cxtfacelhilar domairi'ttfflS^BWt^WM generated with primer pairs 9712 and 9713 (Qiagen, Valencia, CA): · . ,γ. ξi.

Primer# 9712: S*^tn^ic^^^$uMtguiiggt8atta-3' (sense) (SEQEDNO 128)

Primer # 9713: 5’-T7^r5^A^^GG&lt;»ATGGACGmATCTTA- 3’ (antisense) (SEQDDNO129) from plasmid template EGFRvIH-rbIgG/pCEP4 (as described above) amplified using Pfu DNA polymerase enzyme (Stratagene, La Jolie, CA). Primer# 9712 introduced a Hindd site and primer # 9713 introduced a ΚρηΙ Site. The PCR product was column purified (Qiagen column purification Ιός Valencia, CA) digested with tpadHJ and Kpul (NEB, Now England Biolaba/Bevcriy, Mass.) and gel purified (Qiagen gel iju^^rtfon.kivValencia, CA). Fragments were ligated with T4 DMA Ligase (NEB, into pFLAG-CMV-l (Sigma, St. Louis, MO) linearized With Ηί^τπ apd Ragland Biolabs, Beverly, Mass.). The resulting vector was designated EGFRvEVpFlAd^ShiV-1 # L

EwnpieM

PrcparationOf EGfRyEI/pFLAG Betxmbmant Protein

This example deraonsteates howa variant' EGFRvIH protein can be made. First, 500 pg of EGFRvUI/pFLAG-CMV-l# 1 plwTirid ijNA. wa· resuspended in 25 ml of Opti-MEMI (bmtrogen, Burlington, ON) and combined wit^^ⁱ‘‘‘^^93fectm’(lnvitrogcn, Burlington, ON) resuspended in 25 ml of Opti-MEML Tile fricubatcd fct-jSO xrin at room temperature then mixed with 293T cells (lXlCr) le media (brritrogen, Buriington, ON),

<img file="IN313DEN2012A_D0139.tif" />

W T supplemented with 2% FBS and 50 pg/ml 0418 (Invltrogen, Burlington, ON). Cells arc grown for 7 days at 37*C in 8% CO» ‘with shaking at 125 rpm.

EGFRvUI-PLAG ftision protein purification was earned out with Anti-I-LAG M2 Affinity Chromatography kit (Sigma. St. Louis, MO) according to the manufacture's protocol.

Monomeric fusion protein was produced as follows. First, purified protein (1508 pg), was reduced with DTT in a final concentration of Ϊ0 mM for 30 minutes at 55 *C, Then IAA (iodoacetic acid) (Sigma. St Louis, MO) wu added to 22 mM and incubated 15 minutes at room temperature in the dark then dialyzed against PBSat 4 Ό in 7k MWCO dialysis cassettes (Fierce, Rockford. Π1.).

<img file="IN313DEN2012A_D0140.tif" />

A )l

The following. i KinExA experiments. ’ These?# pics 24-30 ^^fidatortr toeperiments (surface plasmon resonance) and vie how one can test the various antibodies and variants thereof produced by the above examples to determine if they have the desired binding characteristics; All of the variants examined woe variants in the 13.1.2 background.

All surface plasmon resonance experiments were performed using Biacorc 2000 optical biosensors (Biaoore, hie., Piscataway, NJ). Al! Kinetic Exclusion Assays were performed usings KinExA 3000 instrument (Sapidyne Instruments, Inc., Boise, ID).

SMggPte.

Pep-3, NH,-LBEKKGNYWTDHGOH (MW - 1590 DS) (SEQ ID NO: 130), was custom synthesized and purchased frbm Anfttech, Inc. (San Jose, CA). All mAbs were prepared in-ttouse. The antigw EGFRvUIpflag (iodoucetic acid reacted in order to block aggregation through free sullhdryl groups), MW 39,907, was prep’red in-house. Bovine scrum albumin (BSA) fraction V (#BPl$05-l00) was purchased from Fisher Scientific (Pittsburgh, PA). All other general reagents were purchased from Sigmi-Aidrichrlnc i^cLoUis. MO).

AU antigen and _LSndJHaExA analysis wen prepared in vacuumdegassed HBS-P .NaCl, 0.005% surfactant P-20. Biacorc Inc.,

Uppsala, Sweden) amine-coupling reagents, l-e|hyl-3-(3dimethylaminopropyl) csrbodiintidd (pXlJ.N-bydroxysuccininudctNHS), and ethanolamfnc Were purchased from Biacorc, ho. Biacorc surface regeneration was with a 12 second pulse of 26 mM NaOH for the pep-3/mAb 131 experiment All ether mAbs dissociated to baseline within 20 minutes; Research grade CM5 biosensor chips, were purchased from Biacore, Inc.

The KinExA detection antibody was CyS-Iabeled goat tati-human IgG. Fey specific (Jackson ImmunoRosearch Laboratories, Inc., West Grove, PA, #109-175-008) and was diluted 1000-fold in HEPES buffer (0.01 M HEPES, 0.15 MNaQ, pH 7.2) from a 0.5 mg/mL stock (1 X PBS, pH 7.4), The solid phase particles, used fo· the KinExA experiments were NHS-activaied

<img file="IN313DEN2012A_D0141.tif" />

Sepharose 4 Fast Flow beads (Phannaoia Biotech AJ0, Uppsala, Sweden, #17-0906-01). Prior to reacting the sepharose beads with «tigeu, ahead stock aliquot of 1.5 mL in a nticrooentriftige tube was spun down and washed ar least six times wifo cold deionized H,O. After rinsing the beads

<img file="IN313DEN2012A_D0142.tif" />

a CM5 sensor chip. To unturaree man tmnsport and crowding mAbs ware wnmnhffiqgd at levels chat gave a maximum antigen binding response (R^ of no more than 50 -100 R.U. A reference flow cell on each chip was activated and blocked with no mAb immobilization to serve as a control,

All Biecore kinetic experiments were conducted at 23 °C. For each experiment, a series of six to. eight antigen ccncentrttiens (starting with 1.01 ipM pep-3) was prepared using 2-fold dilutions. Antigen samples were randomly injected over tew biosensor surface in triplicate at 100 pL/min. Several buffer blanks were ityeqted intermittently over the course of an experiment for double referencing. Each pep-3 concentration and blank were injected for 90 seconds. Dissociation was followed for 13 to 180 minutes. Dissociation.data for pcp-3binding to mAb 131 were acquired by alternating three additional injections of 251 nM pep-3 with throe additional blank injections and following the di

All Biacore season . awes

<img file="IN313DEN2012A_D0143.tif" />

jfomg Scrubber software (Version l.lf, BioLogic

Software* Australia). Sfitgcjrteiijn» beginning of the injection.', · reference flow cell resporue'JU.'nfe.W analyte and blank senewgran^^j t zeped tin foe y«cis and then x-aligood at foe . changes were removed by subtracting foe whrrifcTcroarise of all blank injections was subtracted from all life artifacts between the experimental and reference flow cells. CLAMP bfosenspr data analysis software (Version 3.40, BioLogic Software, Australia) was used to determine k, and ka from the processed data sets. Data from all flow ceOa were globally fit te a 1:1 bitoclecular binding model that included a mass transport terra. For several of the mAbs foe injections corresponding to foe first or second concentration of the pep-3 series were excluded in the nonlinear kinetic fit where ft was obvious that the seusorgrems were not described well by a 1:1 interaction model. The Ko was calculated fromthe quotient kA· KfofixA gqqiiforfrm MtaritnyfeaSl·

All KinExA expcrimetits were conducted at room temperature (-23*0). For all eqtriEbrittm experiments, antigen was serially diluted into solutions having a constant mAb binding rite concentration. For foe first 10 titration pointe foe dilutions were 2-fold and the 11* and 12* serial dilutions were IQ-fold. The sample flow rate for all expe^itieats Was 0.25 mLAum and the labeling antibody flow rate was 0.5·¹'tiiiy&amp;taX p^^pnZantibody samples were then allowed to reach equilibrium, which took -48-72 hr to reach. For the pep-3/mAb 131 KinExA experiment the starting concentration of pep-3 in foe Kp-controUed titration was 352 nM arid the constant [mAh binding site] “219 pM;. for foe riiAb-controiled titration the starting [pep-3] « 251 nM and the (mAh binding site] »11 nM. During the Ke-controlled experiment with pcp-S/mAb 131, 1.25 ml, of each sample was drawn through the flowed). A sample volume of 250 gL was analyzed for ihc antibody-controlled experiment. Two or three replicates of each sample were measured for all equilibrium experiments. The equilibrium titration data were fit in a dual curve analysis to a 1:1 binding model using KinExA software (Version 2.4, Sapidyne Instruments).

The EGFRvfflpflag/m^bu)i?i **⁵ Studied with KinExA under K_o-comroJlcd

Was 198 nM and the [mAb binding site] was 150 ujgb the flow cell. Duplicate measurements were jjjlM.data were fit in a dual curve analysis to a 1:1 ,’-u. V - . _ .. . . . _ . . ,.

<img file="IN313DEN2012A_D0144.tif" />

conditions only. Tbesteftplig^ pM. A sample volume dfll^ collected far all samples. Thc eqtrili binding model using KinExA software (Version 2.4, Sapidyne Instruments). See Example 28 below for results and predicted equilibrium .constant

For the KinExA titrations of the EGFRvJUpflag/mAb 13.1.2 complex the starting concentration of EGFRvUI was 5.26 μΜ (mAb-oazurolledX 230.1 nM (Kc-contralled) and [mAh binding site] » 9.59 nM (raAb-contrplled), 498 pM (Kc-COntrolled). During the Kn-controilcd experiment, 1,30 mL of each sanple was drawn through the flow cell. Asample volume of 250 μΐ. was analyzed for the antibody-controlled experiment Two or three replicates of each sample were measured for all equilibrium experiments. The equilibrium titration data were fii in a dual curve analysis to a 1:1 binding model using KinExA software (Version 2.4, Sapidyne Instruments).

EssaBlS-ii ' In Vitro determination ofbinding constants for antibodies

The hading.kinetics of the wild typ? mAb .l31 was observed by using a Surface Plasmon

Resonance (SPR) kj -and a rapid k.. .Estijn^ejfog ^-2246^0^61^./0^/

In one embodimem»-^P^J improved kinetics, it is meant thif ^le’s^tf

Ko was very low, 380 pM, owing to the very slow ffrictic, parameters, derived from curve fitting, were

<img file="IN313DEN2012A_D0145.tif" />

odies with improved kineiics are taught. By Grie^'i.'ctemcnls of antibody binding to an epitope is superior to the safoe element in previously known antibodies for the same epitope. For example, an antibody that binds to pep-3 with a K_D of greater (in binding abili ty) lhan 1.3 * I O’* M wpiiW be an improved antibody. As such, antibodieswith a Roofless than 500 nM, 500-300 nM, 300-lOOhM. 100-lnM, 13 nM, 13 nM to 1000 pM, lOOT pM foJOO pM. WO-SOOpM, 500-400 pM, 400-300 pM, 300-100 pM, 100-50 pM, 50-1 pM, or smaller Kp are contemplated.

<img file="IN313DEN2012A_D0146.tif" />

&amp;S£D)G£&amp;22

ZaviteoJetehninatioc ofbmdfag omMtants far antiboflea

Similar to Example 24, te binding kinetics of mAbl3.1.2 to Pep-3 (BGFRvEI epitope) were examined. The estimated Ko wu 67nM, bit varied sKg^xtty between experiments. Estimates, for the other kinetic parametos, derived from mm fitting, were 1^*2,835*10* and krO.01922, ' * ώ^ίίώ fiarfttffcodwg

Similar to Εχβπψί^^ί· finetios of mAb 095 to Pep-3 (EGFRvHI epitope) were examined. The estimated KowutStinU. Estimates, for the kjiwtio paranneters, derived from curve fitting, were k.-l .491*10’ and ^.927*10^ ..

• ./ . ' &lt;*“ ‘ *»·· ·* ' Ά* »’ '· , «\· · V * ’*·,

Similar to Examplt 24, the binding khtetica of mAh 139 to Pep-3 (EGFRvHI epitope) were examined. The estimated Ko was 290nM. Estimates, fin* foe kinetic parameters, derived from curve fitting, were K*1032S and gxamplegS

In vHr? dctemfeffttM pf Wntftir owtoBiafor artftsdfa bi order to more folly analyze the binding characteristics of the antibodies, KinExA experiments were performed to determine foe binding ohaiacterisitics of foe mAh 131. The K_odetermined from a dual curve analysis was L74* W^w. In a KinExA experiment, the Kp for EGFRvHIpflag to mAb 131 was 62«·ΚΓ'&lt;

<img file="IN313DEN2012A_D0147.tif" />

<img file="IN313DEN2012A_D0148.tif" />

<img file="IN313DEN2012A_D0149.tif" />

taring

In order to more •IJ4

KinExA experiment was perfonned to di jfjjjffitY fa'™*™* «rtfrndte.

Ending characterisitics Of the 13.12 antibodies, a smirxe the binding Charactcrisitioe of toe mAh 13.1,2, The Kp determined from a dual curve analysis was 7.538*1(Γ^15ί. Additionally, the antigen in this ♦ « ’«· «· 2 »» *example was fee EGMvinpflagvariarfbajid jyasreected with todoacetic acid (IAA).

- ·· /·* ;);) _

Comr^sonof coreresu/tsand KipHxA results • .-· 11 J.·*·.·'·

The results of the previous Examples and the KinExA tests ate presented in Table 30.1 below. Numbers in parentheses in Table 30.1 are 95% confidence intervals. ND,” means not determined and denotes binding to EGFRvIIIpflag(iodc»xretioacid reacted), instead of pep-3.

As is evidenced by the rate constants, mAb 131 appears to have the greatest association constant and the lowest dissociation constant, thus jpving mAb 131 the lowest Kp.

<img file="IN313DEN2012A_D0150.tif" />

<img file="IN313DEN2012A_D0151.tif" />

TABLE 30.1

<td> MAb</td><td> —.,ΚύΜχ¹) ..</td><td> 5¾ I</td><td> Ko (nM) 1</td><td> KlnExA Ko (nM)</td>

<td> 1^1</td><td> 2.25 x 10^B</td><td> 8.50 X 1(T</td><td> 0.389</td><td> 0.174 (0.0827 on EGFRvIJIpflaq)</td>

<td> 13.1.2</td><td> 2.10 (0.58) x 10»</td><td> 0.01 Θ (0.003)</td><td> 75 (14)</td><td> 0.75 (on EGFRvlllpflag (IAA reacted))</td>

<td> 095</td><td> Ϊ.49 * 10^s</td><td> 9.90 x tO*</td><td> 68</td><td> NO</td>

<td> 139</td><td> 1.03 x 10*</td><td> 2.98 « KT*</td><td> 290</td><td> ND</td>

istantt-for L99T-5.3 variant antibodies fig itefri (EGFRvfll epitope) were examined. The

<img file="IN313DEN2012A_D0152.tif" />

fust step was to immobilize 5,W ceWts (RU) to 8,000 RU of mAh L99T-5.3 to two flaw cells (Fc) of a GM5 sensor chip and 5,600 resonance wilts (RU) to 8,000 RU of mAb 13.1.2 to one Pc using standard EDC/NHS coupling chemistry. This surface density yielded a binding signal i,' r*♦[/·*«Ai ^**34?with pep-3 of less than 100 RU. 'Two, CM?^s^ijpi^hipS were deed in total to immobilize both mAbs, With the previously ®)Hec^fcd»h^ti?i3w^iMjd a total of 5 independent experiments for both antibodies that allows the 95% confidence intervals to be calculated. Biacore 2000 optical biosensors were used for all studies.

Next, pep-3 was flowed across the. mAb immobilized biosensor surfaces, Tne starting concentration ·,of pep-3 was 1.25 μΜ, which was followed with eight two-fold serial dilutions in randomized triplicate infections. Blank infections were nm every sixth sample throughout the injection series for double referencing purposes.

Finally, the biosensor data was processed with Scrubber and the data was fit to curves utilizing Clamp With a 1:1 interaction model· with a term included for mass transport. The high concentration injections, 1.25 μΜ, were excluded from the kinetic fits because it was apparent that ! .··.’»·.

the data was not consistent ^^0St ^^,4Ce^* ^^s deviate» >^s caused by non-specific interactions be<img file="IN313DEN2012A_D0153.tif" />w^^^tofrations of pep-3. Al! the kinetic data fit a I;I interaction model satisfectorillf·^^·,?·

The estimated Kp varied from 54-70 nM. Estimates, for the other kinetic parameters, which also varied slightly between runs, werek^^.8HQ\eji)i Ιν·0.012)7. fiUSBbL&amp;ag _: . .

^dh^tics of the variant mAbs through the use of a Biacore device. The first 'st^ ^-^h^‘’«U^fiples involved the immobilization of 5,600 resonance units (RU) to 8,000 RU of each mAb tested to one flow cell (Fc) of a CMSsensor chip using standard EDC/NHS coupling chemistry. This surface density yielded a binding signal With pep-3 of less than 100 RU. Three CMS sensor chips were used in total to immobilize all mutant mAbs with a unique mAb immobilized to each flow cell. MAb 13-1.2 was included on one flow

LHMl

Examples 32-3.8 flutter te

<img file="IN313DEN2012A_D0154.tif" />

<img file="IN313DEN2012A_D0155.tif" />

<img file="IN313DEN2012A_D0156.tif" />

cell for two out of the three CM5 sensor chips. Biaeore 2000 optical biosensors woe used for all studies;

Next, pep-3 was run across .foe mAb immobilized biosensor surfaces, The starting concentration of pep-3 eight to eleven two-fold serial dilutions in randomized duplicate or injections were run every sixth sample throughout the injection serie* fordouWeroferchfeihg purposes.

Finally, the biosensor data was processed with Scrubber and fitted utilizing Clamp with a ): I interaction model with a tarn include^ finnan transport. Same high concentration ejections (4.98— 1.25 μΜ), deprading.'i^&lt;v 'd»;ip^'*hd its affinity, were excluded firms the kinetic fits when it wu apparent that flie data was notoofisistent with a 1:1 interaction model. Most likely, this deviation is caused by non-specific interactions occurring st high concentrations of pep-3. All the kinetic data fit a 1:1 interaction model.

y.frtwwla r?

fo vitro determination of binding constants for L217O-1Q.1 variant antibodies

The binding kinetics of mAb L217Q-10.1 to Pep-3 (EGFRvlll epitope) were; examined. The estimated Ko was 92 nM. Estimates, for the other kinetic parameters, derived from curve fitting) Were k,“2.04*l 0^s and krO-Ql 885.. , '' EsaSfflllS

In vitrodetermination nFbindjpy, for L217N-2.1 variant antibodies

Similar to Example 1217N-2.1 to Pep-3 (EGFRvlll epitope) were examined, ·ηΜ. Estimates, for the ofoer kinetic parameters, derived from curve fitting, jirerejivtad98*l(? and k^-C.04069.

In vitro deternnnationof binding constants for N35G-3.1 variant antibodies

Similar to tiTsiAb N35G-3.1 toPcp-3 (EGFRvlll epitope) were examined. The Estimates, for the other kinetic parameters, derived from curve ΙφΦ.03057.

gwn?»??

In vitro determination of binding constants fpr variant antibodies

S imilar to Example 32, the binding kinetics of mAb L99H-9.2 to Pep-3 (EGFRvlll epitope) were examined. The estimated Ko was 395 nM. Estimates, for the other kinetic parameters, derived from curve fitting, werek,»*83390 and k*0.O3293.

gampH ag

In vitro determination of binding constants for variant antibodies

Similar to Example 32, the binding kfaetios of mAb Y172&amp;-1.2 to Pep-3 (EGFRvHI epitope) were examined. The 927 nM. Estimates,* for the other kmetic parameters, derived from curve%t^^i^fe^^8Titidktf*0,O7622.

1P vitro for-variant «ntihndi™

Similar w Example &amp; foe bin^W^M^ L95N-4. i to Pep-3 (EQFRvIII epitope) were examined. The estimated Ko was 1Λ )1M/'* MAb L99NKI was fit using a steady-state (equilibrium) binding model in order to determine the Ko because the kinetics were too fast to be Ttted. '

Comparison of 13,.1.2 wftii designed variants

As can be seen in Table 38.1 a mAb with improved binding characteristics was developed. The 95% confidence intervals are shown in parentheses. L99T-5.3 exhibited an enhanced k,, a decreased ks, and foua a slower Kpoverail. While statistically there appears to be little if any significant difference in the equilibrium dissociation constants and kinetic rite constants of Pep-3 binding to mAbs 13.1:2 and U^^fovibB-j^joofideheit interval), there still seems to be an intuitive bias for a marginal increase .in.affinity fo^Pep-3 binding to L99T-5.3. Moreover, when the same biosensor chip was used, L99Tⁱ5.3 seemed to always have a higher affinity than 13.1.2.

<td colspan="2"> _ .1¾</td><td colspan="2"></td>

<td> MAb</td><td> Xa (M*Wn</td><td> Is”)</td><td> . K₀ (rtM)</td>

<td> 13.1.2 '</td><td></td><td> pjBSfegg)..··</td><td> 251141............</td>

<td> L99T-5.3 . </td><td> 2.-16</td><td> W0.OQ1)</td><td> 60 (10)</td>

<td> L217Q-10.1</td><td> 2,04 * 10* ’ i . :</td><td> W9 </td><td> 82</td>

<td> L217N-2.1</td><td> 2^0 x.10’</td><td> 0.040</td><td> 185</td>

<td> N35G-3.1</td><td> 1.50. * 10⁹</td><td> 0.030</td><td> 204</td>

<td> L99H-9.2</td><td> 6.34« 10*</td><td> 0.033</td><td> 395</td>

<td> Y172R-1.2</td><td> 8.22 x 10* '</td><td> 0.076</td><td> 927</td>

<td> L99N-4.1</td><td> t4b ’</td><td> ND .</td><td> 1,400’</td>

Additional Docking Models, and Methods of Selecting Models and Predicting Binding Affinity In other embodiments, the examples described above Can be performed with various length peptides rather than just peptides that are 6 amino acids, in length, as long as the key binding residues are included in foe peptide. of li re six amino acid'peptide, EHKKUN (SEQ ID NO: 127), a seven amjpp.§c$QQSQ, ID NO: 131) can be used. Any size peptide for foe epitope can.^'j^^^^^Hnbodimenls, the peptide is selected from the following peptides: LEEKJCGNYAWTWQfSEQto NO: 56), LEEKKGNYWTD (SEQ IQ NO: 59), LEEKKGNYWT (SEQ ID NO:132), and EBKKONYWT (SEQ ID NO:57). Any sized peptide between the short fragments disclcs^ herein,to the fall length peptide, or variants thereof, can be used.

AS-eppreciated by end off manner in which the peptide binds

<img file="IN313DEN2012A_D0157.tif" />

bfiddifipna) amjno acidscan alter the ‘only does the presence Of the additional

<img file="IN313DEN2012A_D0158.tif" />

amino acid allow for alternative and additional bonds to be formed between the peptide and the antibody, but the additional amino acid can change the structure of foe peptide and foe structure of foe antibody upon binding of foe peptide with ths antibody. Thus, in one embodiment, various lengths of the epitope peptide, e.g. EHKKGN (SEQ ID NO: 127) and EEKKQNY (SEQ ID NO: 111), can be examined far binding prupesttes and binding optimization. Not only Will the longer fragments of the peptide provide an accurate depiction of foe peptide-entibody interaction for longer segments of foe peptide, but an examination of the changes in binding strength and the residues involved in binding wfll allow additional information concerning longer peptides to be extrapolated from the data.

In addition, Ind perhaps complementary to the testing of longer peptide fragments, additional filtering steps can be 'pg^^gfodp-.foe various docking models in order to select a refined docking model, allow one to filter through numerous docking models to .find avajlsble experimental data.

ΐα ope embodiment; toti'fij^it^^d-Ίρο fine-resolution epitope tnapping data, e.g., the experimentally characterized individual residue binding profile, which could be correlated with foe Computed binding energy profile for pach amino add in the peptide. The binding energy profile of a seven amino acid peptide, for example, can be used to select docking models that contain similar binding energy profiles, A binding energy profile is an assignment of foe binding energy of each amino acid in a peptide to the particular ammo add to create a profile of the peptide to toms Of each amino acid's binding energy to that model. For example, in one docking model, given a peptide comprising amino acids A and B, Where A has a binding energy of -5 and B has a Wading energy of-20, one would havea profiled Al (at-5) and B2 (at-20). This profile could be used as a filter to select other docking models. For example, foe use of this binding energy profile « a filter or “template” would result in other dotting models being selected if the peptide in the candidate model had a relatively low value attributed to petition A, and a relatively high (lazger negative, higher absolute value) value attributed to position B, In an alternative erubodttnent, foe . , . . . A.' ' . .

template requires additional limitations; for example, feat foe value at position B is four fold higher * ·*«&amp; b’StVi? £^; than the value at position A, ' ^:- · ··- * W *. t· '

Template with theprofilee of foe peptide in the

<img file="IN313DEN2012A_D0159.tif" />

One can compare tracXg other docking models m a i desired binding energy profile, then the n can be used to pick out fovorable doclcmg nwdds for . ! · · i ι· ♦· · « 'f .»*.4 ‘ 1 .

further examination. If foe binding energy profile template is dissimilar to foe desired binding inding energy profile template is similar to the energy profile, then foe filter can be used to eliminate un&amp;vmable docking models. In one embodiment, the filtering process includes « template with both favorable and unfavorable binding energies and the filter is used to both select and exclude docking models. As appreciated by one of skill in the art, there are many possible different binding ^energy profiles, and tot» many different binding energy profile templates that can be used dependidg upon the situation.

$7

In one enibodSmcnt, energy profile template as a template that has a series of relatively high .bp^^®ft^0^^®^culi(r positions in the peptide. In a preferred embodiment, the binding the binding energy profile selected by the template, will have relatively Wri^^M^^afe^gsritiQn 2, 4, or 6 of the peptide, EEKKGNY (SBQ ID NO: 131). In energy profile template will have a relatively high binding energy at^!porition? 2, 4, and 6 of the peptide EEKKGNY (SEQ ID NO: 131). hi another embodiment, the binding energy profile template will have a relatively low binding energy attributed to position 3 of the peptide EEKKGNY (SBQ ID NO: 131). hi the above discussion, the positions lie assigned as follows: Bl, E2, K3, K4 OS, Nd, Y7.

In one embodimenh the filtering process first involves a comparison of the binding energies at K3 and K4. Docking models tint result in a relatively higher binding energy for K4 compered to K3 ore selected, While docking models that reeultin a lower binding energy for K4 compared to K3

<img file="IN313DEN2012A_D0160.tif" />

|t binding energy in the peptide. Thus, in this Could be summarized as follows: El can be any should be less than K4, K4 should be greater jfeMfi·· · bpgreater than the lowest value, Y7 can be are filtered out Thus, by “relatively high” ft is meant diet die binding energy for K4 is greater (mere negative value, larger absolute value) titan K3. Next, the dotting models are again filtered through toe binding energy profile template, fids time, those binding models with relatively higher energies at positions 2,4, and 6 are selected for, while the other models can be removed. Thus, by “relatively high,” it is meant that tfee binding energy at positions 2, 4, and 6 are higher (more negative value, Inger absptytp embodiment, the binding' value, E2 should be greater ft than the lowest value, G5 carf any value. Thus, El, G5, and Y7 cd^dBeHtiy yalfie/as tong as at least one (or KJ) is lower than al least one of E2, K4, and Nd. fit another embodiment, “relatively high can be set to a standard value as determined through modeling er experimentation. la One embodiment, that the docking models pass the first filter is more Important than the docking model pass the second filtering step. As appreciated by one of skill in the art, cue need not perform these two steps sequentially arid they . can be performed simultaneously.

. Additionally, these profile templates for filtering through results will vary depending upon the peptide, the antibody, and the binding conditions. One of drill in the art, given foe present disclosure, especially with reference to example 14, could determine the appropriate binding energy profile template, For example, as shown In Table 14.1, there are several possible important residues for peptide binding bothta foe 131 and in foe 13 ,1.2 antibody. In the 131 mAb, positions E2, K4, Nd, and Y7 are taportrat for the particular peptide tested. In the 13.1.2 mAb, positions El, .,. -1 . t »« · , ·

E2, K4, G5, and Nd are impQtWt^feft^jrticular peptide tested. Those residues that are wwpnftimt can be residues a binding energy profile template. As clear from tite discussion¹ beiow^^fi^W^^^^y. gspfilc template in Example 39 appease to be different from that suggestod'by|S^^^^^^^ri^letl4. Example 39 is a less stringent version of the. template that pit the number of models

<img file="IN313DEN2012A_D0161.tif" />

Ibugh die screening step. If one wanted to reduce rowing step, oue could farther add requirements concerning El and G5. ' The following example demonstrates both the use of a longer peptide, how it can alter Che results demonstrated above, what such changes am mean, as well as demonstrating the use of one of the above fUtersfor selecting particular docking models.

<img file="IN313DEN2012A_D0162.tif" />

-· . π . - -v .

demonstrates, methods for selefetmg orfc Booking model over another docking model.

First, a structural model far the seven-residue peptide EHKKGNY (SEQ ID NO: 131) was built in an extended conformation and energy minimised with Discover_3 module in Ihsigiitn modcling packagc. Next, the peptide structure was manually placed, into the combining site to form an initial assembly? A Monte Onto search ip the translational and rotational spaces was then

<img file="IN313DEN2012A_D0163.tif" />

Angstroms of the starting position. 'fhe pUmsibie configurations found by the Monte Carlo search were followed by simulated annealing and energy minimization to reach the final complex structure models. A total of 63 docking models were obtained.

For each dodang model, the interaction energy between the antibody and foe individual residue in the peptide was calculated with Discover^, The profile of individual residue

<img file="IN313DEN2012A_D0164.tif" />

energy for K4 is pronunent, and those for Nd and E2 are large. This binding energy profile template placed particular emphasis on the fact that K4 is greater than K3. This binding energy profile also placed an emphasis on the requirement that E2, K4, and N6 are relatively large, fa other words, the binding energies of B2, K4, and N6 were not the lowest (least negative or smallest absolute value) binding energies in the peptide.

Table 39.1. Binding dnwgy profile for individual residue in the seven-residue peptide to antibody

13.12 is consistent with epitope mapping data in Example 14.

E1 E2 . K3 , K4 G5 N6 Y7 Total -10,97 -19.34 -10.1 -18.19 -15.15 -111.45

For ths 19 model^on the binding energy profile, epitopeantibody binding energetics fcimSlwwwgre performed on each of the seven mutants with affinity data (TyrI72Arg, Example 36; Iieu217Asn, Example 33;Leu217Gln, Example 32; Asn35Gly, Example 34; Leu99Asn, Example 37; Leu99His, Example 3$; and Leu99Thr, Example 31). Smet the extent of electrostatic interaction in this complex had to be approximated, a number of different dielectric constants were used in a series of calculations. The mutation was done with residue replacement foUowed by 30-100 steps of energy minimization to account for tny local coufonuationel changes tint are induced ty die side chain change. For each docking model, the interaction energy between,^ 'seytp-ife^ldue peptide and the whole antibody was calculated for each mutant for foesdde^^^^ti^C’l^.eBch set’of 8 binding energies (7 mutants phis foe wild type), a linear fitting procdditfc Waili^s. on each set of the binding date, in comparison with the logarithm of Kd. A oorrelatibn 'coefficient' was calculated for each linear fitting. The best correlation was obtained for one model, foe model with the daa described in Table 39.1, with a dielectric constant l*r and SO step* of energy minimization. The epitope-antibody binding energies for this model ere shown in Table 39.2, The. Correlation coefficient was 0.80 With all the data. As the Ko for Leu99Asn was not mo^^^h^fegh degree of accuracy, see Example 37 above, a separate linear fitting data -for Leu99Asn. An excellent correlation coefficient of 0.91 was 20. The refined docking model is thus well represented by the above selected model. The model with space-filling peptide is shown in FIG. 21, and the hydrogen bonds are shown in FIG. 22. L3 150 is the lower section and H3160 fa the upper section on FIG. 22. H2140 fa to the right of foe peptide binding area. The peptide itself is placed into the binding site with El being positioned at foe top of the paige in a light shade, down through K3, K4, G5, Ν6» and Y7, progressively getting darker. The antibody residues involved in hydrogen banding are sbown in MG. St. The model produced from this example demonstrates that there are seven hydrogen bonds R4...Q95, K4...Q95, N6...Q98, G5...H31, Y7...H31, and . .. ,1» ) Y7...W165.

/0°

- Simulation,Of epitoj

<img file="IN313DEN2012A_D0165.tif" />

icS, in comparison

<td> mutant</td><td> ' ¹</td><td> otaf I</td><td> -n(Kd)</td>

<td> 172Atg··</td><td> -27,982</td><td> -47.065</td><td> -13.891</td>

<td> 2!7Asn</td><td> -28,715</td><td> -47.718</td><td> -15.503</td>

<td> 217Gta</td><td> -18.977 -28.73</td><td> -47,707</td><td> -16.201</td>

<td> 35Gly</td><td> -19.095 -28.431</td><td> -47.526</td><td> -15.405</td>

<td> 99Asri</td><td> -18.719 -28.778</td><td> •47.497</td><td> (-13.479)</td>

<td> 99Hls</td><td> -18.837 -28.719</td><td> •47.566</td><td> -14.744</td>

<td> -99Thr</td><td> -19.165 -28.704</td><td> -47.859</td><td> -18.475</td>

<td> WT</td><td> -18.961 -28.728</td><td> -47.708</td><td> -16.268</td>

<img file="IN313DEN2012A_D0166.tif" />

i^whichis consistent with K4’s importance in foe appears that N6 is modeled to bond to Q98;

V ’ ’» . ' ' to form H-bonds in foe model. One e key residues from the binding energy profile

As can bo seen from the model selected in Example 39, which is represented in HQ, 21, the docking model revealed some unexpected Tesdhs. One interesting result is that while residue» E2, K.4, and N6 are important residues in fire binding of foe peptide as a whole, not all of these amino acids are modeled as involved in forming H-bonds with the antibody, ft appears that K4 is involved in the formation of two H-bonds, both vdth binding energy profile and however, in this partjciilir rt interesting trend foutf ,«msw&amp;^8 template (e.g, E2, K4, and. ^® dre.m^fl^buried and thus in close contact Wife the antibody binding groove. Thus, this docking modei selection can account fcr the fact fast these key residues are important because of their close interaction with 0» antibody. Additionally, it is possible flat El is involved in a hydrogen bond with W214.

Example 39 also demonstrates fast the above described method results in a strong correlation between binding energy end Kp, suggesting that models created by this method win also allow optimization or at least a prediction ofthe K^ of the antibody-peptide complex.

As can be seen from * comparison Of Example 39 and Example 19, there are some residues that are important between foe two models, some residues that appear only in the seven amino acid docking model, as well as some residues that do not appear to be as important fa the seven amino acid docking model For example, the seven peptide epitope appears to create H bonds betwoeu K4...Q95, K4...Q95, N6...Q98, G5...H31, Υ7,.,ίΪ31, and Y7...W165. On foe other hand, foe six peptide epitope appears to create R bonds between fe...Y172, K3...H31, K4...H31, N6-..D33, Nd...¥37, andN6...K55. As can re4tj'fr^^l*?ebpye data, both foe six and the seven amino acid peptide models emphasiaB^^^^^^^^fEl . ar both models involve H31 forming two hydrogen bonds with the possible trends between foe two flats acts, it also appears that riuti^ t^^^o^Ctav^^^^&amp; havo changed from the six amino add model to ind N6) ire ,’*^! /©/ the seven «πίπα acid raodel,^ H^Wever,.fecsc examples demonstrate that variations due to epitope size can be detected wifif'<img file="IN313DEN2012A_D0167.tif" />the scaling up from shorter to longer epitope peptides should disclosure. The presence of amino acids feat consistently demonstmfeibifo jApfowfce tn various binding models allows one to bias the t^witaiwe of foe yarn so that shorter peptide models can be more representative oflcnger p

As appreciated' by art, any of the above discussion or examples concerning foe six amino acid peptidc.'EskKGN (SEQ ID NO: 127), can also be applied towards foe seven amino acid peptide, EEKKGNY (SEQ ID NO: 131), or any longer peptide. For instance, ExwnpleZQ can be repeated wife foe information from Example 39 for rational design for affinityimproved antibodies by rite-directod mutagenesis. Furthermore, Example lllean be repeated, using the results of Example 39, following an attempt of rational design for affinity-improved antibodies by site-directed mutagenesis , to test any new antibodies derived from Example 20.

In one embodiment, foe results from Example 39 are used to redefine the interaction «tea between the antibody and the peptide. For example, the paratope, for EEKKGNY (SEQ ID NO: 131), can be defined as including the Other residues .on the antibody that are predicted to Interact with foe peptide, for example, residue 95« Alternatively, as in Example 19, the paratope can be defined as all residues wifo^fe foe ached peptide.

In silico affinity maturgflgpjlg,

'.-j 1¾ 8?.; ?.'.9s

<img file="IN313DEN2012A_D0168.tif" />

<img file="IN313DEN2012A_D0169.tif" />

^: .f'M'preMsa r effing Sep successfolly done m vitro in a number of different studies, Typically, raiidontined ggipaji. peed to .be constructed by molecular biology methods and selection/ ' _: developed to enrich the clones with good * * ^{1 1}J' binding capability. SClecti

<img file="IN313DEN2012A_D0170.tif" />

purified to determine affinities. This process requires a series of lengthy and laborious £x is possible to accurately predict affinity maturation through ftr silico selection utilizing an antibody' nts. 13ie following example demonstrates that it antigen complex structure alone.

SsncteiS

In silico Affinity Maturation through Antibody-Antigen Binding Energetics Simulations

This Example demonstrates feat In silico antibody-antigen binding energetics simulations can be used for affinity maturation, bi particular, this example demonstrates that the: binding kinetics pf a Fab-12 (IgG form known as rhuMAb VEGF) to VEGF (vascular endothelial growth factor) cm be predicted through the above described in silico process.

The crystal structure of the VEGF-Fab complex used was located in the PDB database wife the accession number IBJ1, at a resolution qf 2.4 angstroms. Published experimental affinity data for a series of mutants pf«(«^»4iBw®W&amp;erc used to test fee concept. The 3-D coordinates of the VEGF-Fab struetur^^^wH^^^q^rag Outtn silico mutation for the following mutants: H97Y. SlOOaT, T28D, ^l?OlMS^^:ic«IaT, N31H, Y53W, 71I73K, 71V73V. The /oZ-

<img file="IN313DEN2012A_D0171.tif" />

<img file="IN313DEN2012A_D0172.tif" />

affinity dau were obtained from the paper by Chea, Y et al., (J Mol Biol., 293(4):865-81 (1999)), The energetics simulations were duffed out between ta-varioi» VEGF-Fab mutants, aa described in Example 39. The results arc The results from tiris example demonstrate that a significant con-elation brt and affinity ranking was obtained though this process. The linear fittingofi s logarithm (rfthorfclativ'c affinity is shown in

FIG. 23. The correlation coefficient of'-OJl'indicates tat the tn slUco simulation accurately captures the detailed interaction at the atomic level.

Table 40.1. Antibody-antigen binding energy simulation compared wife affinity data.

<td></td><td> Sequence Relative</td><td> Ln(Relative</td><td></td><td></td>

<td> Kabat Number</td><td> Number .· ' Affinity</td><td> Affinity)</td><td colspan="2"> BindlngEnergy</td>

<td> H97Y</td><td> 101^ J ^Λ-'^Α· ,. </td><td> 14</td><td> 2.639</td><td> -59.065</td>

<td> S100aT</td><td> 406¾^</td><td> 1.9</td><td> 0.842</td><td> -57.465</td>

<td> T28D</td><td> 280*’-U^:·</td><td> 1.4</td><td> 0.338</td><td> -57.647</td>

<td> 2BD31H</td><td> 28031H</td><td> 3.1</td><td> 1.131</td><td> -57.699</td>

<td> 28D31H97Y100a</td><td> I</td><td></td><td></td><td></td>

<td> T</td><td> 28D31Η1Ό1Y105T ’</td><td> 20</td><td> 2.996</td><td> -69.518</td>

<td> N31H</td><td> 31« L.x'·</td><td> 3.8</td><td> 1.281</td><td> -57.724</td>

<td> Y5SW</td><td> 54W ..,.</td><td> 1.3</td><td> 0.262</td><td> -57.504</td>

<td> 71I73K</td><td> ™74K||</td><td> pj?.</td><td> -0.105</td><td> -57.158</td>

<td> 71V73V</td><td> 72V74V</td><td> 0J3</td><td> -1.204</td><td> -57.314</td>

<td> WT</td><td> WT</td><td> 1</td><td> 0.000</td><td> -57.404</td>

As is dear .from the Examples above, the simulation ean be extrapolated to identify higher affinity mutants without the use of fr vitro experimentation. Additionally, it » clear that this approaoh is useful for different antibodies and for different peptides. This methodology tan be generally applied to perfoTm.affmityTnaturation pt riftco, using only a high-resolution antibodyantigen complex stnitau^^K^^M^^^erit, this use of in sffieo affinity maturation will save tremendous amounts

Determination of canonical claases of antibodies

Chothia, ct al have described antibody structure in terms of canonical classes far the hypervariable regions of eaph immrinogtobulin chain (J.. Mol. Biol. 1987 Aug 20;196(4);901-17).

The atomic structures of the Fab jutd Vll ftpgjpertts of a variety of immunoglobulins were analyzed to determine the rclatioa^j£$g$^ arid sequences and the taco-dimensional • * /03 structures of their antigen binding sites. Chotiria, et al. found that there were relatively few residues feat, through their packing, hydrogen tending or fee ability to assume unusual phi, psi or omega conformations, were primarily responsible for fee main-chain conformation» of fee hypervariable regions. These residues were found to ow *t sites within fee hypervariable regions and in the conserved beta-sheet framework. By examining sequences of immunoglobulins having unknown structure, Chotitfa, et al stew feat many immtaoglobuins have hypervariable regions that are intilar in size to caw of the known structures and additionally contained identical residues at the tes responsible for fee observed conformation.'

Their discovery impliod feat theSo hypwvtaiablc regions have conformations close to those n the known structures. For five jSf regions, the repertoire of conformations rppeared tote landed to structural classes. These commonly occurring main-chain cc

<img file="IN313DEN2012A_D0173.tif" />

regions were termed 'canonical structures*. Further work by.Cha^a,.et;kL·· (Nature. 1989 Dec 21-28:342(6252):877-83) and

<img file="IN313DEN2012A_D0174.tif" />

antibodies.

Some of thc antibodiea described above were analyzed to determine fee canonical class for each of fee antibody's complementarity determining regions (CDRs). As is known, canonical l

Classes have only been assigned for CDR1 and CDR2 of the antibody heavy chain, along with CDR1, CDR2 and CDR3 of the antibody light chain. The table below (41.1) summarizes the results of the analysis. The Canonical Class data is in the form of 'HCDR1-HCDR2-LCDR1LCDR2-LCDR3, wherein “HCDR refers to fee heavy chain CDR and “LCDR refers to the light chain CDR. Thus, for example, a canonical class of 1-3-2-1-5 refers to an antibody that has a HCDR1 that falls into canonical class 1, alJCDR2 that fells into canonical class 3, a LCDR) that fells into canonical class 2,« LCDR2 that falls into canonical class I, and a LCDR3 feat feUs into • .· ? ,»!«- ^v·» - ’ · · »· · | _f canonical class 5.

<img file="IN313DEN2012A_D0175.tif" />

21Γ

318

333

342

150

173

21Γ 1-3-4-1-1

318 1-3-4-1-1

333 1-3-4-1-1

342 1-3-4-1-1

3-1-4-1-1

150 3-Y-4-1-1

173 3-Y-4-1-1 /oy . &lt;';·Λ W ^:·

Each CDR (except for H3) Wai lSMgned to a canonical structureif it satisfice the length requirement and matches the key residues defined in the canonical class. The anrino adds defined for each antibody can be found, for example, in foe articles by Chothia, ct al. referred to above.

. . . Eordvafcnts

The foregoing description and Examples detail certain preferred embodiments Ofthe invention and describes foe best mode contemplated by the inventors, ft will be appreciated, however, that ho matter how'detailed the foregoing may appear in text, the invention may be practised in many ways and the invention should be construed in accordance with the appended claims and any e^uvaleots thercof >