CA2450289A1 - Method of producing an antibody to epidermal growth factor receptor - Google Patents

Abstract

The present invention is directed to a method of producing an antibody Epidermal Growth Factor Receptor (EGFR). The method includes producing transformed cells that express EGFR antibodies, Culturing the transformed cells, harvesting the transformed cells to collect the EGFR antibodies, and purifying the EGFR
antibodies.

Description

METHOD OF PRODUCING AN AN~3UDY TI? RP1~ERMAL
CROWT~I FAC~'QR RECEPTOR
F>F~ OF TI31~ lNVIrN'MON
[01~ The present invention relates to a method of producing an antibody specific for Epidermal Growth Factor Receptor.
BACKGROUND of THE INVENTION
[0~] Augiogenesis, which refers to the formation of capillaries from pre-existing vessels in the embryo and adult urgattism, is known to he a key element in tumor grovuth, survival and metastasis, Growth factors and their receptors, insludiu$ epidermal growth factor (EGF~ and transforming growth factors (TGF°cx), which activate BGFR, are thought to play a mle in tumor as<giogetresis. Binding of these growth factors m their cell surface receptors induces receptt~r activatioua, which itdtiates and madihes signal transduction pathways and leads to cell proliferation and differentiation.
[Q3J EGFIt is a 170 kh membrane-spanning glycoprotein with ari extracellular ligand biadzng domain, a transmembrane region and a cytaplasmic protein tyrosine kinase domain. See, e.g., l3aselga et al.~ Epidermal Growtkt Factor Rcceptar:
Potential Target far Anti-tumor Agents, The Crater for Biomedical Continuing Education (240Q).
8indiug of specii;c ligrands, such as BCiF and TNF-a, to EFGR results in EGFR
autophosphorylaxion, activation of the receptor's cytopiasmic tyrosine kinase domain, and initiation of multiple signal transduction pathways that regulate tumor growth and survival. The EGFR
pathway also influences ~oduction, of various other angi4garic factors, such as VEGF slid basis ftbrablastfc growth factor (bFC)i;), in tumors.

Docket No. I 1245/49502 j(l4] Previous studies directed to biocki~g ~GFR have demo~rated that such a blockade can inhibit tumor growth. Yario~us different inhibitors of EGL'R have been utilized; for example, I~GFR-specii3c srrtall ntolecuies and rnanc~clonal antibodies have been developed, inclus#ing tfe monaclau~al antibody cetuxiniab, which is currently iu clinical trials.
[05j Current methods of producing EGFR antibodies, however, have not resulted in a slgtti~cant Yield ofthe antibodies. Accordingly, there is an unmet reed in the art for a method of gmducing a high yield of LGFR antilaodies.
S~R'Y 4~ INVENTTON
jQ6J The present invention is directed to a method of producing antibodies to ~GFR.
The method includes groducing trarlsfornned cells that express the HGFR
amibodies, Culturing the transformed cells, t~a,rvesting the transformed cells to collect the 13GFR
antibodies, and purifying tlxe ~GFxt. antibodies. In particular, the method involves selecting a transformant with D1~A, that encodes an EGFR antibody, cultivating the transforma~nt in an inocul~tm cultivation medium to create an inoculuaa, scaling..ug the inoculum in scale-up medium, stirring the inoculurn in a production medium to produce and accumulate LGF'R antibodies ia1 a culture, harvesting the BGFIZ antibodies from the culturr~, and purifying the fiGPR amibodics.
»R»F D&SCRIPTIQN OF TI~ 1~RAWI1,TGS
j47] Figure 'i is cpNA sequence of Heavy chain [081 Figure 2 cDNA light chain nrYO~ e~sa~as ~.mx 2 Docket Na. 11~4SI49S02 [09j Figure 3 is the amino acid sequenca of the heavy chain with the signal sequence italicized, the CDlts underlined, and the constant region bolded. The beginning of the constant region is indicated by (-).
[10j Ffgure 4 is the amino said of the light chain with the signal sequence italicized, tha Cl~Rs underlined, and thg constant region bolded. The beginning oPthe constant region is indicated by (-~.
DETA1~.FD DESCIt~T~ON ~F'rI1WFON
[1lj 'The present invention relates to a method of producing antibodies to EGFR. The antibodies of the present ixmention can be monoclonal or polyclonal antibodies ox any other suitable type of an antibody, such as a fragment or a dorivative of an arttiba~iy, a single chain antibody (scFv) or a synthetic liamologue of the antibody, provided that the antibody has the same binding characteristics as, or that have binding characteristics comparable tp, those of the whole antibody. As used herein, unless otherwise indicated or clemr from the conrext, antibody domains, regions and fragmepts are accorded standard detlniaons as are well lanown in the art. Sere, e.g., Abbas et al., rCellular acrd Molecular Imnamology, W.l~. Sounders Cauipapy,1"hitadelphia; PA (1991).
[12j Cleaving a whale antibody can produce antibody fragments, or by expressing DNA
that encodes the fragment. Fraguaents of antibodies can be prepared by methods described by I,.attioyi et al., J. Immunal Jkferhods, 5f: 23S-213 (1983) and by'Parham, J. ~lmmunol 131: zg95-29Q2 ( 1983). Such fragments can contain one or both Fob fragments or tire F(ab')z ftagtaenr. Such fragments can ale contaip single-chain fira.~nant variable region antibodies, i.e, scFv, dibodies, or other antibody fragments. ~'referably the antibody fragtttents contain all six completnentarity-determining regions of the whole antibody, Nro~r e~se4s_a.ooC 3 l3ocket hTo. 1124S149S03 althaugh fragmenu containing fewer dtan all of such regions. such as three, front or five ~C~Rs, can also be functicmai, x'h~e ann'b4dy fragment can also be conjugated to a carrier molecule, Same suitable carrier molecules include keyhole limpet hemocyanin and bovine serum albumen. Conjugation can be carried ant by sx~ethads known in the arc.
[13j Antibodies of the present invention also include those for which binding characteristics have been improved by direct mutation, methods of a~inlty maturation, phage display, or chain shuffling. Afi-utity and speclficlty can he tnodii:ied or improved by mutating CI)Rs and screening for antigen binding sites having the desired characteristics (see, e.~, Yang et al., J. Mol. Bio., 254: 392..43 (1995)). CI~R.s ire mutated in a variety of ways. One way is to randomize individual residues or combinatiprts of residues so that in a population of otherwise identical antigen binding sites, ail twenty amino acids are #bund at particular poxitions. Alternatively, mutations are induced aver a raage of CDR
residues by error proae PCR methods (see, e.g., Hawkiits et al., J. Mot.
$ip..226: 889-896 (I992)). Phage display vectors containing heavy and light chain variable region genes are propagated in rnt~tator strains of ~ coli (see, e.g., f.ow et al. J, Mol.
Bio., 250: 3S9-368 ( 1996)). These methods of rnutagenesis are illustrative of the many mehods !mown to one of skill in the art.
~I~1] The antibodies ofttte present inven'Aon can also be bispecific andlor multivalent.
A variety of chemical arid recombinant methods have been developed for the production of bispecific andlor multivalent antibody fragments. For a review, see FIQlliger and Winter, Crrrr: spin Biatechrrnd. 4: 44b~449 (1993); Carter et al., J.
Ifemarorhrrapy 4:~463-47t1 (I99S); Pliickthun and Peek, Immunoteotarrolagy 3, 83-i~5 (1999).
l3ispecifieity and/or bivalency has bean accomplished by f~sizag two scFv molecules via flexible linkers, ieucirio zipper uaotifs, CHC~,-heteradimerizatlon, a~ by association of scFv molecules to NY01 84~~$.9-~

Docket Na. 1124~1495~2 form bivalent monuspecific diabadies and related srcttctures. The addition of multimerixation sequences at the carboxy o: amino terminus of the sclw v or Fob fxagmeuts has achieved multivalesicyp by using, for example, p33, streptavidin, and helix;um-helix motifs. For example, by dimerixatian via the helix-turn-helix motif of an scFv &isian protein of Vita form (scFvl)-hinge-helbt tin» helix-(scFv2), a tctravalenc bispecific miaiantibody is produced having tw4 sct=v binding sites for each of two target arnagens.
Improved avidity can also been obtained by providing three functional antigen binding sites. .Far example, scFv matecules with sitartenad linkers connecting the VH
and t~i.
donnains associate to form a triabody (Koxtt et ah Pmtein Bng 1 Q:423-433 ( 1997)).
[ISj Production of 1gG-type bispeoific antibodies, which t°esecnble 1gG
antibodies in that they possess a more or less coutpletelgG constant domain structure, has been achieved by chemical cross-linking of two different IgC3 molecules or by co-expression of two antibodies from the same cell. ate strategy developed to overcome umvanted pairings between 'two different sess of IgG heavy and light chains ccr-expressed in transfected cells is tnodiftcation of the Crt3 domains oftwo heavy chains to reduce homodimerixation between like antibody heavy chains. Merchant et al., .Nat_ ,~ioxechru~logy 16: 677-b$1 (1998j. In that method, light chain mispairing was eliminated by requiring the use of identical light chains far each binding site of those bispeci&c antibodies.
[lbj In same cases, it is desirable to maintain functional ar structural aspeots other than antigEn specificity. Far example, bath complement-mediated ~cy~axlcity (CMCj and antibody-dependent cell-medfated cytatoxicity {ApCCj, which require the presence and function of Fc region heavy chain cattstattt domairxs, are lost in most bispeci$c antibodies.
Caloma and Morris created a hamagesreaus population of bivalent BsAb molecules with NYOT 6AbB96 1.,DOC 5 pocket No. 11245/49502 an Fc domain by fusing a scFv to the C-terminus of a complete heavy chaen. Co-enpression of the fusion with ari antibody light chain resulted in the production of a hornoge~eous population ofbivaleut, bispecifc molecules drat bind to one antigen at one and arid to a second antigen at the other end (Coloiua and Morrison, Naa.
~'avtechnology 15,159-lb3 (199?)). however, this molecule had $ reduced ability to activate complement and was incapable of effecting ClvtC. Furthermore, the CH3 domain bouad to high ai~nity Fc rece~ (FcYRI) with r~iCed affinity. ?hu et al., PCTIUS01/16929., have described the replaccrxient of Ig variable domains with singly chain Fvs in order to produce tctram.Gric Y~-lilee pmtaips tttrtt (1) are bispecifc and biva[ern,

(2) are s4bstantlally homogeneous with no canstcaittts regarding selection of antigen-binding sites,

(3) Comprise ~c coustaat domains and retain associated functions, and (4) can be groducsd in mammalian or other cells without ftuther processing. By a sinular method, bispeci~c manovalent Fob-like proteins can be produced.
[17j Preferably, the antibodies of the subject invention are monoclonal antibodies. The antibodies of the present invemion are also preferably chimeric antibodies having a variable ragian of an antibody of one species, for example, a mouse, and a constant region of as antibody of a different species, for example; a human. Alternatively, the antibodies of the present invention can be humanized amibodies having hypervariable or ootnpleme~ntarity-determining regions (CDRs) of an antibody fmrn one species, for example, a mouse, arid framework variable regions and a constant i~gion of a human antibody. Also alternatively, the antibodies of the prescnt invention can be human antibodies having bath a constant region and a variable region of a human antibody.
[I~] IrJ one embodiment oftha present invention, tha 1~GFR antibody is a fully human, monoclonal antibody specific for EGFR, such as, for example, A:~X-EGF
(Ahgenix, Inc).
NYU18358~1.D~JC

locket Na. 1124S1495a2 A13~C-EFC'.r binds E .rarFR with high specificity, blocking binding of EC>;Rto both its ligands, EGJ~ and TNF-alpha. The sequence and characterization of A)3X~~GF is disclosed in U.S. Patent Na, b,~35,883 at col. 2l;, lice 62 through col. 29, line 36 and in FICi. 29-34, which is iticorpArated by reference herein. .fee r~dso Yang et al., Cririccrd Rev.
~ncallHer~tnd., 3$ (1): 7-23, 2001, which is also incorporated by reference herein, [19] !n a preferred embodiment, the EC',rFR antibody is a humanized monoclonal antibody specific for EGFR with corctplementarity determining regions as disclosed in TJ.S. Patent No. 4,93,533 to lVlendelsohn et al (AT~C ~$50b, i~fiS07, I-~8508 and Hfi8509), which is incorporated by reference herein.
[20] In a more preferred embcxlimeut, the EGFR antibody is a chimeric antibody, such as, for example, cetuximab, which specifically binds EGFR and blocks binding of a ligatJd, Such as E~'xF or T NF-a, 'co EGF.R.. 'This blockage results in inhibition of tumor growth, which includes inhibition of tumor ixwasian, metastasis, cell repair, and angiogenesis, by interfering with the effects of >rGFR activation. In addition, or alternatively, cetuximab may pmtnote internalization mf the rccepeor-antibody complex, preventing further stimulation ofthe receptor by its li~;and ar any other mechanism.
Further eharacterizat~att ttf cetuxima~b is disclosed in ~T.S. Application Nos. 081973,Od5 to Goldstein et al., arid ~91b35,974 to Teufel; WO 99db~023 to Waksal et al., and WO
QQI$9459 to Waksal, all of which are inct~rporated by reference herein.
[~L] Natwititstanding the exact nature or characteristics of an EG1 R
antibody, the mefiltod of producing an EGFR antibody according to the present invention generally includes the steps of producing trat~.sfotmed cells that e~.prass EGFR
antibodies (the transforming step), culturing the transformed cells (the culturing step), harvesting the IVYQ9 B45B46_i.GOC

Docket hlo. 11~4~f49502 transformed cells to collect the EGFR antibodies (the harvesting step), and purifying the EGFR antibodies (the purifying step).
[~Z~ With respect to the transforming step, a D1'~lA encoding an EGrFR
antibody Is isolated and inserted into a replicable vector for further cloning or for expression. The DNA encoding the EGFR antibody caa be generated by methods known in the art, including, but are not limited to, production in hybridoma sells. These methods are described in various publications, including the itnutunological method described by Kohler and Milstein, Nature256: 4~5-499 (lpl~) and Campbeh in °'Monoctanal Antibody Technology, The Production and Characterization of Rodent and Human Hybridamas" in Surdon et al., Eds., J:.aboratary Techniques iu Hiochemistry and Molecular Biology, Vohune 13, Elsevier Science Publishers, Amsterdam (1985); as well as by the recombinant DNA tnethads described by Fuse et al. in 5cierrc~ Z4G: 1275-1281 (1989).
[Z3] Methods for incorporating the DNA into a vector are well known in the art and include direct cloning, site specific recotn6inatian using recombinases, homologous recotnbiliation, and other suitable Methods of constructing a recombinant vector.See ~erterally, Sandbrook et al. Molecular Clanir~g: A laboratory .Mameal 3"a edition, Cold Spring Harbor Press (1989).
[24] Vectors useful in the present invention are also well laiown in the art and include ftu example, bacterial or viral veewrs. Suitable bacterial vectors include plasmids such as pBR322-based plasmids, 8luescript, pSI~.F, and p&T23», arid bacteriaphagesy c.g., latubda and M13 based vectors. Suitable viral vectors include r8troviral vectors, adeaoviral vectors, adeno-associated viral vectors, hetpesvira! vectors, SV40 viral vectors, po3yoma virus vectors, papilloma vixus vectors, picnovirus vectors, vaccinia virus vectors, or other NYAi &4584CZ,? DOC

Docket loo. 1 i245h19502 suitable vectors. pNA expression icy a suitable vector can he canirohed by inducible or uninducibk regulatory sequences. Generally, a vector useful in the presem invention can, therefore, also include any or all aftile following: signal peptide, a leader seduence, one or mare marker genes, a promoter, and a transcription termination sequence.
[25j Ottce a suitable expression vector according to the present invention is identified, the expression vector is inuoduced into a host cell. Any suitable method of introducing the dxprcssion vector into a bast cell can be employed, including calcimn phosphate precipitation, nuclear Injection, and elecuoporatian, for example. The Bast cells of the present invention can Include prnkaryot3c and eukaryotic organisms, such as, far example, tnatnmaiian cells. Preferably, the host cells are mammalian cells such as, for example, SP2I0 cells, NSO cells, CUS-7 cells, Chinese hamster ovary (CHO) ceps, and cells lines of lyutphaid origin, such as lymphoma, tnyeloma, or hyhridama cells, for example.
Outer eukaryotlc host, such as yeasts and plants, can alternatively be used.
[2G~ For those EGFR antibodies of the present invention that captain bath a light and heavy chain, these chtiins can be transformed into separate cell cultures, either ofthe same.
or of differing specks. Alternatively, the light and heavy chains can be ca-transfbtmed into a single cell culture by using separate vectors or a single expression vector thn contains the coding genes for bath the Light and heavy chain.
[Z'] poring the culturing step, the txaasfarmed cells are cultured by preparing and cultivating au inoculum (the cultivatitra phase), scaling up the itiocuium in a series of biareactors (the scale-up phase), and prattucing aria accumumiu~
E~ii~R.~u~it..~.s-t~o~~..__ ......_ the inocuium (the production phase).
JVYOt B~i5B4B ~.pOC 9 Docket No. 11245189502 [2$1 In the cultivation phase, the transformed cells fratn the transforming step are recovered into an inoeulum cultivation medium to create an inoculum. 'fhe transformed bast cells are cultured by methods known in the art' in a liquid medium containing assimilable sources s~f carbon (carbohydrates such as glucose or lactase), nitrogen (amino acids, peptides, proxsins or their degradation products such as peptones, aumouium salts or the like), and inorganic salts (sulFates, phosphates andlor carbonates of sodium, potassium, magnesium autd calcium). The inoaulum cultivation medium preferably includes a conventional nutrient medium such as Dttlbecco's Madi~ed Eagle's Ma~1iunct (L~h~IVL) (Sigma), Ham°s 7F10 (sigma), Minimal ~SSesatisl Medium (1VIEM7 ($;gma7, RPM'1-1 G44 (Sigma) or NCTC-135, [~9j Any ofthese media can be supplemented as necessary with amino acids (glutamine), hprmones ar other growth factors (insulin, tratisferrin. or epidermal growth factor), vitamins, salts (zinc sulfate, sodium chloride, phosphate), buffers, nucleotides, antibiotics, ionic surfactants, and glucose or an equivalent energy source.
The medium can fuxther contain trace elements that are growth psomating substances, such as iron chelates (e.g., chelate B, Invitrogen Corp,, Carlsbad , CA), aad manganese.
During the cultivation phases, culture coz<ditions, such as temperature, pH, and the like, are monitored to ensues rapid cell growth.
[30) Furring the scale-up phase, the inaculum is scaled-up in scale-up medium through sequential steps of cultivation. Such steps can be performed in any suitable container, including cell culture flasks. stir bottles, roller bottles, rotary bioreactors, and spinner flasks. The seal~up medium also iuclt~des a conventional nutrient medium and can include amino acids supplied by hydralysates (e.g., HySoy, Guest lnternatianal, Chic$go, !x.), harnlanes or other growth factors, vitamins, salts, bu~'ers, nucleotides, antibiotics, NY07 fi45846_i.L~ l~

Docket No. 11245l495Q2 ionic surfactants, iron chelates, and glucose or an equivalent energy source.
X7uring the scale-up phase in biareactars, the pal, o~cygen saturation, and waste products of the inoeuluan are monitored.
x"31] During the production phase, the cells are transferred to a stir tack or airlift biareactar and fed with a complex growth medium eontainang sugars, amine acids, salts, trace elements and growth factors, which are combined in such quantities so as to maintain the pal, osmolality, and other essential parameters of the growth medium for consistent, robust, rapid cell growth. The use of osmaprotect$nt compounds, such as betaine or proline, fnr example, can be used to pmrect cells from osmotic stress while enhancing antibody pradttctivity. The temperature, dissolved oxygen, pig pressure, gas flaw rate, and stir rate ors also controlled during the production phase. During tha production phase, the cells develop within themselves the EGFR antibodies or secrete the EGFR
antibodies into the stuxounding medium as a by-product of growth. Those cells that develop ECFR.
antibodies within their structures can lae chemically or mechanically fragmented in order to harvest the F.FGR antibodies. Mare complex cells such as mammalian cells can produce sugar-modified cellulax products and secrete the EGFR antibodies into the cell culture medium for isolation.
[~Z] During the harvesting step, the BG1,R antibodies arc removed from the cell cuiiure by any means ktiawn in the art. Far example, when the ECiFR antibodies are produced inizacellularly by the transformed cells, centrifugation or ultrafxitration can he used to remove fife host cells or lysed calls. Where the EGFR antibodies are secreted into the medium, tha antibodies can be removed from the mixture of compounds fed to the cells and from the by-products of the cells themselves by using eammeraially available protein nrYOf Bs5H4s_r.pcac 11 Iaocket No. 1124S/49502 concentration filters, such as, for example, Amieon or Millipore ~ellicon ulua~Itration units.
[33j Ihiring the purifying step, the EGFIt antibodies are suaiected to oua or more purification steps, including various chromatography methods. samples of such purification procedures include anion exchange chromatography and canon exchange chrontatagaphy, as well as various fdliration methods, such as tangential #1ow filtration using P'ellicon~ membranes (Millipore, Bitlerica, MA), for example, nanoftltration using DYSO falters (Pall Corporation, fast Hills, NS~, for example, reduce potential viral contamination and appropriate sire dead end filtration (such as 0.45um and 0.2pm filters), fractionation on a hydrophobic interaction chromatography (e.g. on phenyl sepharose), ethanol precipitation, isoelectric focusing, Reverse Phase HPLC, chmmatography on silica, chromatography on HEPARTN SBPHAR081=,~' further ion exchange chromatography, chromatofocusing, SDS PAGE, ammonium sulfate precipitation, hydroxylapatite chromatography, gel tleerraphoresis, dialysis, and affunity chromatography (e.g., using protein A, protein G, an antibody, a specific substrate, ligand or antigen as the capture reagent).
[3A] The antibodies of the present invention rail also be modified or derivatized.
lrxampies of such modifxeation include post translation modifications, such as glycosylation (both O-Iinlu~d and N-linlted), acetylation, phosphorylation, ubiquitinacion, and the like. These modi~tcatiotts can be carried out in viva using the host cell machinery or in vitro following isalatiQn of the antibody fraut the host celh [3~] It is understood that the 8 ,l"rFlt antibodies of the inventio~x can be mixed with a pharmaceutically acceptable carrier, or diluted by a carrier, andlor enclosed within a NY01 645848 9.~OC 1~

Docket No. 112451~4~502 carrier, which can, for example, be irv the form of a capsule, sachet, paper or ether container. When the Barrier serves as a diluent, it cart be a solid, semi-solid, or liquid material, which acts as a vehicle, excipient ar medium 'for the active ingredient. Suitable pharmaceutically acceptable carriers inclpde, for example, one or more ofwater, saline, pho~h~e buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers can farther comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the lsinding proteins. The compositions of the injection can, as is well known is the are, be ~urraul~sted so ass to prQVide quick, sustained or delayed release of the active ingredient.
~3~j The BGFR antibodies of this invention can also he in a variety of fornxs.
These include, for example, solid, semi-solid and liquid dosage forms, such as tal7lets, pills, powdars, liquid solutions, dispersions or suspensions, Iipasomes, suppositories, injectable and infusible solutions. Thus, the cotnpasition lae in the form of tablets, lozenges, sachets.
cachets, elixirs, suspensions, aerosols (as a solid or in a liquid medium), ointments containing far example up to 10'/o by weight of the active compound. soft and hard gelatin capsules, suppositories, injection solutions, suspensions, sterile packaged powders andas a topical patch. The preferred form depends on the intended mode of administration and therapeutic application.
fiXANIPLBS
>?xample 1: Froduei~t~~ Transformed Cells that Express EGFR Antibodies [37] The myeloma cell lice SP~!IO-Agi4 (ATCG CItL-1581), which is a line that was farmed by fusing 13A1»Elc spleen Bells (&am mouse immunized with sheep RB~s) with Hraa sa5as~.~.voC 13 rocket No. 11z451A~9so2 the P3~.G3Ag8 mygloma (see Shulznan et al., Nature 27fi: 269-270 {1978)) was transfatmed to excptess EGfR antibodies. The cell line was expanded in tissue culture flasks {1L) and total cell R1~TA was prepared by lysing washed cells in gaunidine isothiacyauate containing 2-tnercaptoethanal (25 mL), shearing the solution in a~ Bounce hamogani~er to degrade cell DNA, and layering the preparation on a CsCI
cushion {10 m>:,). After centrifugatiau (24,040 rpm, tG hrs), the RNA pellet was resuspended in Tl:
buffer and precipitated with ethanol. The poly A (t) rnRbTA. fraction was isolated by binding to and elution from align dT cellulose.
~3$j A cflN'A library was prepared using the poly A(+) mRNA as template and oligo dt as primer. The second strand was synthesized by nick translatiau using ltNase H and DNA polymerase 1, The double stranded DNA was passed through a G 75 Sepharose calumu {2 ml-.) to remove oligo dT and small products and then ilgated to a polylinker with tile sequence: 5'-AATTCTCGAh'a'~'CTAGA-3' encoding an 1=caR.X four base sticky end for ligation to the cloning vector, and the restriction sites far :XhoI
and Xbai far subsequent manipulations of the eDNAs. The ligaied cDNA was then axe-selected to enrich for fuh length by electrophoresis on a S% polyacrylamide gel. The appropriate size ficactir~ns {~-15EI0 by for H chain and -900 6p for L chaia cDNA) were elecu~oeluted fronn gel slices and ligated to EcaRI-digested lambda gtt 0 phage DNA.
[39] Libraries were generated by packag~g the ligation products in vitro and plating the recarnbinant phage on lawns o~~ coti strain C600 HFL. Phage containing H and L
cDNAs were identified by phage filter lifts tfiat were hybridized with radialabeled aligonucleatides specific for the mouse kappa and gatntna constant regions.
nrrar sasaa~ i.aQC 14 digesrion and agarase gel electrophoresis. Isolates with the longest cDNA
inserts were sulxlosled in a plasmid vector and analyzed by DNA sequencing.
[41.J itt order to idenxify the correct L chain aDNA, a sample ofmouse I;GFR
xrttibody was sequenced by automated Bdman degradation after first separating H and L
chains by reducing SDS gel electmpharesis and blotting to membranes. 'Ihe sequence obtained for the L chain matched one of the cpNAs.
~4~] The V regions were adapted for expression by ligating the body of each to a synthetic DNA duplex encoding the sequence between the closest unique restriction site to the V/C jutlction and the exact boundary of the V region. To this was ligated a second short iutrott sequence, which when joined restores a functional splice donpr site to the V
region- At the end afthe itttron for the L chain is a BamHI site and at the end ofthe H
chain intron is a I~indlIl site. The adapted L chain V region was then isolated as a Xbal-Bam~iI fragment (the Xbai site was in the original linker used for cDNA
cIorying) while the adapted H fihain V region was isolated as a Xhol-Hind3ll f~nent.
[43] The ~sxpression vector, containing human kappa and human gamma 1 constant regions, was digested with Xbax and BamH.i and used far the insertion of the adapted light chain variable region. The resulting plasmid was then digested with XhoI and HindIII and used far the insertion of the adapped Fi chain V region. 'final vector for expression of the E~'x>;1~ antibody was identified by restriction analyses. Set forth in Figure 1 is the nucleotide sequence of the heavy chain cI~NA and in figure 2 is the nucleotide sequence of the light chain cl~lNA.
NYor 645aas_7.AOC 15 I~cket lrFo. 11245149502 [d4] The final vecear was introduced into hybridoma sp210 Agl4 cells by protoplast fusion. 'rhe bacteria harboring the vector were Brawn to an optical density of U.S at 600 nm at which time chlc~ramphenicol was added to arrest growth and amplify the vector copy number. The following day the bacteria were treated with lyst~xyme to remove the cell wall and the resulting protoplasts were fused to the hybridotna cells with polyethylezxe ~lycoI (1500 mI_). Aver the fusion, the cells were grown in antibiotics to kill any surviving bacteria and were plated in 96-well microtiter plates. The selection medium [containing methotrexaze (NTTX) at 0.1 pMj was added aflea 2~-48 hr to allow tunly the trarisfected cells to gtow, by vlmte of thCir expression of the marker gene (dihydrafolaxe reductase) present in the expression plasmid.
[45a After two weeps, several MT~~ resistant clot~s were obtained that were then tested for antibody expression. Culture supematauts were added to wells coated with an anti human Ig (Fc-specific) antibody as the capture reagent. 1'he detectiaet system was an HRp-coxtjugated goat anti-human l~appa antibody. Ttte tn~jority of clones were found for be secreting humatl antibody determinants and the three highest producers were adapted to grow at 1 and then 5 pM MTX. The lines were subcloned by limiting dilutiazt and the productivity of the subclanes was tested by seeding cells at 2 x ltd cells per mL in growth medium and measuring the accumulated antibody ou. day ~. The cell lines were subclones again arid they all produced between 110 and 13~ mglL of antibody in the 7-day production assay.
NY07 645~48_1.DOC 16 Docket No. I 1245/9502 Example ~: Pren~in~ and C~!_tiya~Tnoculitm ~4b] Transformed sells from >rxample 1 were recovered inter an itsoculum cultivation indium drat included the companems listed is Table i (referred to herein as "inoculum Cultivation Medium A:') In~rcdieat Araaant Dulbecco's Modified Eagle's Medium (DMFM) 90°/n N~Teas~ Ion Glutamine ~i mM

Bovine Insulin 7.5 mglL

l3ovinc Trausferrin 7.5 mglr., 8ovlne Serum Albumen (BSA) 1.0 giL

Ethanolarnine 30 uM

Selenium 40 nM

Mercaptoetlzanol 30 ltM

Oxaloacetate 150 m~lL.

Irxample 3: Pre~'~~; and Cultfvat~l~-an lnoculum [4'7j Transformed ceps from Example 1 were recovered into an ipoeulum cultivation medium that included the components listed in Table 2 (referred xo herein as "xaoculum Cultivation Medium »"). Inoculum Cultivation Medium B differed from Inoculum Cultivation Medium A irt that bovine insulin was replaced with reccorubinant human insulin and bovine transferrin was replaced ~w~h an inorganic ixon chelator.
Ira addition, the concentration of amino acids, salts, and vitamins in DMi;M and NCTG.135 and xbe NYOf &f5R4~9_DOG

Docket No. 112~51~9502 concentration of glutamine ware approximately doubled to that present in Inacuium Cultivadort Medium A. Further, au inorganic salt, such as zinc sulFate, apd an ianie surfactant, such as platonic Fb8 were added to the inaculum cultivation medium.
TABt~~ 2 Ingrediet Atnn~unt ~MM~M 9o~ro N~x~-1

4~lutamine S mM

Human Recombinant Insulin 20.0 mg/L

Inorganic chclate (inorganic?.S mglL
iron chelator) BSA 1.0 g!L

lrtbanolatnine 30 pM

Oxaloacetate 1 SO
mglL

Selenium ~0 nlV1 Mereapmetltanol 30 pM

zinc aulfate 1 pM

Plurania F68 I glL

Frxaunple 4: ~- ~~~ulum [4~~ Inoculum created from &xarnple 2 was scaled up izx scale-up medium thraugb sequential steps of cultivation in toll culture flasks, roller brntles, &nd spinner flasks. The scale-up medium izscluded the components listed in Table 3 (referred to herein as "Scalc-LTp Medium A").
NYOf 645948 f.pQC ~$

is l~o. ~ mas~a9so2 ~'A3 Iagredient Amoant ~oculum Cultivation 1.83 Medium A gII.

Sodium 131carbonate 3.55 g/l.

Plurouie fi68 1 gIL

Metltatrexate 5 uM

HySoy, OF (Quest); 3 .25 gIL

~xcyte VILE 5.Q
mLIL

{a9~] The Iuoculum was again scate~d up in a second scalo-up medium thsaugh sequential steps of cultivation in cell culture flasks, roller bottles, and spinner flasks. The scale-up medium included the components listed in Table 4 (referred to herein as "Scale-LTp Medium B"). Scale-Up Medium F3 died from Scale~Up Medium A in that Inoculum Cultivation Medium B was used instead oflnaeulum Cultivation Medium A. In additiati, chelate 8 obtained fraxn Invitmgen was added and phuanic F68 was eliminated.

rngredicut Amouat lnoculurn Gultivatian18.67 Medium ~ AIL

Sodiu~t Hicarbonate 3.55 gli.

Chelate B (Ynviuogen)2.Q
mLl~.

Met~otrexarte S 1tM

FIySay,'CTF Guest); 125 gll.

F.~ccyte VLE 5.0 mL2 nrvA~ sa~e~as_~.anc 19 Docket Nn. I 1245I49~oa lxaraple 6: .Production and Accumulation of EGFR Antibodies [Stlj InocuIum from ~ple 4 was transferrred to a 1,200 L stir tank. Production medium included the components listed iu Table 5 (referred to herein as "Production Ntedium A").
'PA~I,.E 5 Yngredyeat Arttpuat Inoculum Cultwation ~ 4.$3 Medium A gIL

Sodium Bioarl~~ate 3.~5 gIL

H3'~aYa ~ (Quest); 1.~5 gIL

Methotrexate 5 ulvl Excyte YLE 5.0 tnLlL

Hydrocortisone D.5 ~,M

[SI] Insulin, glutarnine, and Excyte were added to the sdr fault about three days (Day 3) after the inoculutm was tranferred to the stir tank. Excyte was added after four days (flay A~) and then aver five days (Day 5) after the inaculutta was taanferred.
Glutamine control after twa feeds (greater than 220 mg/i..); glucose control aRer two feed (grearer than 2.0 g/i.). The ply pf the contents of the stir tank was controlled at betwcen b.9 and 7.I at Day 4, or l 6-30 hours aver the glucose, glutarrritle and ExCyte wart added to the stir tank.
[5~j Temperature, dissolved axygen, pH, pressure and gas Ilow rate during the production phase were controlled.
nrrov e~eas~~.rxac 20 l7rocket No. 11245i495oz Example ?: Froductiort and Accumula~t'on of BGFR Antibodies [53j The inoculum from Example 5 was transferred to a 12,000 L stir tank.
Production differed from Example 6 in that Inaculum Cultivation Medium E was used instead of Inoaulum Cultivation Medium A. ~ addition, chelate H was added. The production medium included the components listed in Table 6 (referred w hercln as "Production Medium B").

Ingredient Amount lZxoculum Cultivation 1V(edium B 18.6? gII.
Sodium Piicarbonate3.55 gIL

Inot~ic Iron Chelate~.0 mLllh.

~x~y, ~' (~ue~t~ Las get.

Merhotrexat~ s ,~M

~y~e vlrl; 5.o mLra.

Hydrocortisone o.5 ~M

[S4] Twenty millimoles of,glueose, ~0 tnM of glutamiue, and 13 mLlI. ofExCyte were added to the stir tanl~ approximately 48 hours after the inoculum was transferred to the stir tank. At this time, under preferred conditions, there were greater than ?.0 x 105 viable cellslml. in the stir tai. '~veltty-dues point ~tve grams pert liter of ~IySoy was addtd ax Day 4 or Day S or when the cell concentration was a 2 x ilk' viable cellslmL.
The pH of the conc~eenrs of the stir tank was controlled at 6.~ at Day 4, as I ~-30 hours after the glucose, glatamiue and ~cCyte were added to the stir tank.
NY01 845846_'l.DOC 21 pa~i~t I~o. llz4sea~soz ~~SJ Alternatively, 20 millimales of glucose, 20 mM of glutamine, 15 mIJL of ExCyte and 90 mM Betaine were added to the stlr tank apprazcimately 48 hours after the inoculum was transferred to the stir tank. At this time, underpreferred conditions, there were greater than 7.0 x 105 viable cellslmL in the stir tank. Twenty~hree paint five grams per liter ofHySoy was added at lay ~ ar Day S or when the cell concentration wasp 2 x 106 viable cellslmL. The pH of the caatenxs of the stir tank was con'Arolled at 6.9 at Day ~l, or 16-3D hours after the glucose, glutamine and ExCyte were added to the stir tank.
[SGj Temperature, dissolved oxygen, pH, pzcssure and gas flow rate were controlled.
Example $: ~Iarves~in~Transfar~ted Cel)s to Called ~e EGFR. Anribadies [59j Cells were removed by cell clarification using depth filtration to obtain a culture broth. The cell broth was then concentrated via tangential flow filtration (TFF) using polyethexsulfane membranes. 'xhe harvested culture broth was them filtered agaiast a filter having a pons size of 0.2 mlcrans.
[SSj hxaxngle 9: Purifvina the EGFR Antibodies Produced by the Transformed Cells [39j The EGPIt antibodies of the harvested culture were purified using a sequence of affinity and ion exchange chromatography. In the affinity chramaiagraphy step, concentrated cell-free conditioned media is purif ed aver POROS~ A50 r~ambinant protein A matrix. The concentrated conditioned media was either loaded an an equilibrated Protein A matrix at a pH of 9.(14 and washed with equilibration buffer (10 rnM sodium phosphate buyer, p~ g.0) to remove unbound impurities ar the cell harvest supernatant was loaded an an equilibrated Protein A matrix at a pH of approximately 7.2 and washed with equilibration buffer {10 mM sodium phasphate,145 mM sodium chloride furor sasaas_r.t~oc 22 Docket No. 11245/49502 buffer, pH 7.2). The bound antibodies were eluted fmm the column of the Protein A
matrix using 7S mM acetic acid.
jb4] Following elution from the column, a low ptl treatment was performed to achieve significant inactivation in virus contaxaiaatian. Using 1.0 M acetic acid, the pH was lowered to x.00-3.50 and held for a minimum of b0 minutes. Using 1.Q M Tris base, the pH was then raised to 7.50-8.50.
jbl] Further purification was accomplished by concentration attd diafil#ratian against 10 mM sodium phosphate, pH 6.0, via TFF using polyetherSUlfone membranes. The BG>;R.
antibodies were rhea processed by anion exchange chmtaatograpliy over Q
Sepharose Fasi Flow matrix. The unbound product was eluted using 10 znM sodium phctphate, pH
6.0 buffer. The eluted antibodies were collected as a single fraction. Next, a DV50 virus teduction titration step was performed to remove a significant amount of virus.
[62] The antibodies were then concentrated and diafilured agair~ 10 mM sodium phosphate, 14S mM sodium chloride, pFI 7.24 via TFF using polyethersulfone membrane.
j63] The purified EGFIt antibodies were then filtered against a filter having a pore size of 0.2 microns. Set forth is the amino acid sequence afthe heavy chain in Figure 3 and the light chain is Figure 4. The signal seqiaenoes era italicized, the CDlts underlined, and the constant region bolded, with the beginning indicated by ( ). The antibodies were then be formulated in phpsphatc buffered saline with no stabilizers.
[b4] The foregoing description and examples have been set &trth merely to illusuate the invention and are not intended to be limiting. Since modif catic~as of the disclosed embodiments incorporating doe spirit and substance of the iaxvention may occur w persons skilled in the art, the invention should be construed to include everything within the scope NYP7 ~4& i.POC 2~

Docket No. i 12~SIA9502 of the inventio~l thereof. The disclosures of aII citations in the specification are expressly incorporated herein by reference. Furthermore, laboratory protocols applicable to all production methods of the present invention that are described in Sambras~k et al, (2QQ0) Molecular Clotiirt~: A Lalaaratory Manual, f~ ed., bald Spring lE~arbor Laboratory Press, which are incorporated by reference herein.
IYYQ7 ~f584B_?.DOS

Claims (14)

What is claimed is:

1. A method of producing an antibody specific for epidermal growth factor receptor (EGFR) comprising:
producing transformed cells that express an EGFR antibody;
culturing the transformed calls;
harvesting the transformed cells to collect the EGFR antibody; and purifyiug the EGFR antibody.

2. The method of claim 1, wherein culturing the transformed cells comprises:
selecting a transformant having DNA encoding the EGFR antibody;
cultivating the transformant in inoculum cultivation medium to create an inoculum;
scaling-up the inoculum in scale-up medium; and stirring the inoculum in a production medium to produce and accumulate the EGFR antibody in a culture.

3. The method of claim 1 or 2, wherein the inoculum cultivation medium comprises 90% Duibecco's Modified Eagle's Medium (DMEM) and 10% NCTC-135.

4. The method of any one of claims 1-3, wherein the inoculum cultivation medium comprises 4 mM glutamine; 7.5 mg/L bovine insulin; 7.5 mg/L bovine transferrin; 1.4 g/L bovine serum albumen (BSA); 30 µM ethanolamine; 40 nM
selenium;
34 µM mercaptoethanol; and150 mg/L oxaloacetate.

3. The method of any one of claims 1-3, wherein the inoculum cultivation medium comprises 8 mM glutamine; 24.4 mg/L human recombinant insulin; 7.5 mg/L
inorganic iron chelator; 1.0 g/L bovine serum albumen (BSA); 30 µM
ethanolamine; 40 nM selenium; 30 µM mercaptoethanol; and 150 mg/L oxaloacetate.

6. The method of any one of claims 1-5, wherein the inoculum cultivation medium further comprises 1 µM zinc sulfate and 1 g/L pluronic F68.

7. The method of any one of claims 1-6, wherein the scale-up medium comprises 14.83 g/L inoculum cultivation medium; 3.55 g/L sodium bicarbonate;
1 g/L
pluronic F68; 5 µM methotrexate; 1.25 g/L HySoy, UF; and 5.0 mL/L Excyte VLE.

8. The method of any one of claims 1-6, wherein the scale-up medium comprises 14.67 g/L inoculum cultivation medium; 3.55 g/L sodium bicarbonate;
2.0 mL/L chelate B; 5 µM methotrexate; 1.25 g/L HySoy, UF; 5.0 mL/L Excyte VLE.

9. The method of any one of claims 1-8, wherein the production medium comprises 14.83 g/L inoculum cultivation medium; 3.55 g/L sodium bicarbonate;
1.25 g/L
HySoy, UF; 5 µM methotrexate; 5.0 mL/L Excyte VLE; and 0.5 µM
hydrocortisone.

10. The method of any one of claims 1-8, wherein the production medium comprises 14.67 g/L inoculum cultivation medium; 3.55 g/L sodium bicarbonate;
2.0 mL/L inorganic iron chelate; 1.25 g/L HySoy, UF; 5 µM methotrexate; 5.0 mL/L Excyte VLE; and 0.5 µM hydrocorrisone.

11. The method of any one of claims 1-10, wherein the EGFR antibody has an amino acid sequence of Figure 3 and/or Figure 4.

12. The method of any one of claims 2-10, wherein the DNA encoding the EGFR antibody has a nucleotide sequence of Figure 1 and/or Figure 2.

13. The use of the antibody specific for epidermal growth factor receptor for the treatment of EGFR-mediated diseases.

14. The use according to claim 13, wherein the antibody is produced according to the method defined by claim 1.