`
`34
`33
`maots selected by ampicillin or tetracycline resistance where
`also coataios tbe SV40 viral origin of rcpticatioa. Fiers et al.,
`appropriate. Plasmids from the traosformaots arc prepared,
`Nature, 273:113 (1978); Mulligan and Berg, Science, 209:
`1422-1427 (1980); Pavlakis et al., Proc. Na1l Acad. Sci.
`analyzed by restriction endonuclease digestion, and/or
`sequenced is by the rnetbod or Messing Cl al, Nudeic Adds
`USA, 78: 7398-7402 (1981). The immediate early promoter
`of lbe human cytomegalovirus is coaveniently obtained as a s Res., 9: 309 (1981) or by tbe method of Maxam et al.,
`Methods in Enzymology 65: 499 (1980).
`Hindlll E restriction fragment. Greenaway el al., Gene, 18:
`355-360 (1982). A system for expressing DNA io mamma-
`Particularly useful in tbe practice of Ibis invention are
`Liao hosts using the bovine papilloma virus as a vector is
`expression vectors that provide for the transient expression
`disclosed in U.S. Pat. No. 4,419,446. A modification of tbis
`i.o mammalian cells of DNA encoding Lbe target polypeptide.
`ibcd · us p l N 4 601 978 "- lso 10 In general, transient expression in.volves the use of an
`· d
`1
`0
`expression vector tba.t is able to replicate efficiently in a bost
`• ~ a
`sys em IS c.scn
`· a
`•
`•
`·
`·
`ID
`Gray et al., Nature, 29: 503-508 (1982) on expressing
`cell, sucb tbat the bost cell accumulates many copies of tbe
`cDNAeacoding immune interferon in monkey cells;• Reyes
`expression vector and, in turn, syalbesizes higb levels of a
`et al., Nature, 291: 598-601 (1982) on expression of bumao
`desired polypeptide encoded by the expression vector. Trao-
`1}-i.oterferoa cDNA in mouse cells under the coatrol of a
`sicat expression systems, comprising a suitable expression
`thymidine kinase promoter from herpes simplex virus, is vector aad a bost cell, allow for lbe convenient positive
`Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79:
`ideatification of polypeptides encoded by cloned DNAs, 3S
`5166-5170 (1982) oa expression of the human interferon ~l
`well as for the rapid screening of sucll polypeptides for
`gene in culiurcd mouse and rabbit cells, and Gorman ct al.,
`desired biological or physiological properties. Thus, trao-
`Proc. Natl. Aced. Sci. USA, 79: 6777-6781 (1982) oo
`sient expression systems are particularly useful in tbe invea-
`expression of bacterial CAT sequences io CV-1 monkey 20 lion for purposes of identifying aaalogs and variants of the
`target polypeptide that bave target polypeptide-like activity.
`kidney cells, chicken embryo fibroblasts, Cbinese hamster
`ovary cells, Hel.a cells, and mouse NJH-3TI cells using tbe
`Otber methods, vectors, and bost cells suitable [or adap-
`Rous sarcoma virus long tenninal repeat as a promoter.
`tatioa to the synthesis of the target polypeptide i.o recombi-
`(e) Eobaocer Element Compoocnt
`nant vertebrate cell culture arc described in Gclbing et al.,
`Transcription of DNA encoding the target polypeptide of 2S Nature, 293: 620-625 ( L981 ]; Mantci el al., Nature, 28 l :
`this invention by higher eukaryotes is often increased by
`4()-46 (1979]; Levinson et al..; EP ll 7,060; and EP 117,058.
`inserting an enhancer sequeoce ioto tbe vector. Enhancers
`A particularly useful plasmid for mammalian cell culture
`arc cis-acting elements of DNA, usually about from 10-300
`expression of lbe target polypeptide is pRK.5 (EP pub. no.
`bp, that act on a promoter to increase its transcription.
`307,247) or pSVl6B.
`Enhancers are relatively orientation and position indcpen- 30 Selection aod Transformation of Hos1 Cells
`denl having been found 5' (Laimios et al., Proc. Nad. Acad.
`Suitable host cells for cloning or expresfilng Lbe vector.;
`Sci. USA, 78: 993 (1981D and 3' (Lusky et al., MoL Cell Bio.
`herein are the prokaryote, yeast, or bigber eukaryote cells
`3: 1108 (19&3D lo lbe lranscription unil, within an iotron
`described above. Suitable prokaryotes include eubacteria,
`(Banerji el al., Cell, 33: 729 [1983D as well as wilhio tbe
`such as Gram-negative or Gram-positive organisms, for
`coding sequence itself (Osborne et al., Mol. Cell Bio., 4: JS example, E.coli, Bacilli such asB. subtilis, Pscudomooas
`species such as P. aerugirwsa, Salmonella typhi.murium, or
`1293 (1984D. Many enhancer sequences arc now known
`from mammalian genes (globin, elastase, albumin,
`Serratiamareescans.OnepreferredE.coliclooinghostis£.
`coli 294 (ATCC 31,446), although other strains such as £.
`o.-Cctoprotein and insulin). TypicaUy, however, one will use
`an enhancer from a eukaryotic cell virus. Examples iodude
`coli B, E. coli Xt776 (ATCC 31,537), and£. coli W3110
`the SV40 enhancer on tbe late side of the replication origin 40 (ATCC 27,325) arc suitable. Tbcsc examples are illustrative
`(bp 100-270), ibe cytomegalovirus early promoter eabaacer,
`rather than limiting. Preferably tbe bosl cell should secrete
`tbe polyoma enhancer oa tbe late side of tbe replication
`minimal amounts of proteolytic enzymes. Alternatively, in
`origio, and adenovirus enhancers. See also Yaniv, Nature,
`vitro methods of cloning, e.g. PCR or otbcr nucleic acid
`297: 17-18 (1982) on enhancing elemeats for activation of
`polymerase reactions, are suitable.
`eukaryotic promoters. 1be enhancer may be spliced into the 45
`In addition to prokaryotes, eukaryotic microbes sucb as
`vector at a position S' or 3' lo the target polypeptide DNA,
`filamentous fungi or yeast are suitable hosts for target
`but is preferably located at a site 5' Crom tbe promoter.
`polypeptide-encoding vectors. Saccharomycescerevisiae, or
`common baker's yeast, is the most commooly llScd among
`(f) TranscTiption Termination Component
`Expression vectors used in eukaryotic bosl cells (yeast,
`lower eukaryotic host microorganisms. However, a number
`fungi, insect, plant. animal, buman, or nucleated cells from so of other genera, species, and strains are commonly available
`aod useful herein, sucb as Schiwsaccharomyces pombe
`otber multicellular organisms) will also contain sequences
`(Beach and Nurse, Nature, 290: 140 (1981); EP 139,383
`necessary for tbe termination of transcription and for stabi-
`lizi.og the mRNA Sucb sequences ace commonly available
`published May 2, 198S), Kluyvcromyces hosts (U.S. Pat.
`No. 4,943,529) such as, e.g., K lactis [Louveacourt et al, J.
`from tbe 5' and, occasionally 3' untranslated regions of
`eukaryotic or viral DNAs or cDNAs. These regions contain ss Bacterial., 737 (1983)), JC fragilis, K. bulgaricus, JC
`thennotolerans, and K. marxiaruis, yarrowia (EP 402,226),
`nucleotide segments uanscnbed as poJyadcnylated frag-
`meats i.o tbe untranslated portion of the mRNA encoding the
`Pichia pasroris (EP L83,070; Sreekrisbm et al., J. Basic
`target polypeptide. The 3' untranslated regions also include Microbial., 28: 265-278 (1988)], Candida, Tn'choderma
`reesia [EP 244,2341), Neurospora crassa (Case el al., Proc.
`transcription termination sites.
`Construction of suilable vC(;lors containing one or more of 60 Natl. A cad. Sci. USA, 76: 5259-5263 (1979)), and tilameo-
`the above listed components tbe desired coding and control
`tous fungi such as, e.g, Ncurospora, PeniciUium, TolyPOCla-
`sequences employs standard ligation techniques. Isolated
`dium (WO 91/00357 published Jao.10, 1991], and Aspcrgil-
`lus bosts sucb as A. nidulans [Ballance et al., Biocliem.
`plasmids or DNA fragments arc cleaved, tailored, and reli-
`gated ill. the form desired to generate the plasmids required.
`Biophys. Res. Commun. 112: 284-289 (1983); Tilbum et al.,
`For analysis to confirm correct sequences in plasmids 65 Gene, 26: 205-221 (1983); Yelton et al., Proc. Natl. Acad.
`Sci. USt\, 81: 1470-1474 {1984)] and A. niger[Kelly and
`constructed, the ligation mixtures are used to transform£.
`Hynes, EMDO J., 4: 475-479 (1985)].
`coli K.12 strain 294 (ATCC 31,446) and successful trans for-
`
`921 of 1033
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`10
`
`35
`Suitable host cells for the expression of glycosylated
`target polypeptide are derived from multicellular organisms.
`Such host cells are capable o[ complel( processing aad
`glycosylatioo activities. Jo principle, any higher eukaryotic
`cell culture is workable, whether from vertebrate or i.over- 5
`tebrate culture. El(amples of invertebrate cells include plant
`aod insect cells. Numerous baculoviral strains and variants
`and corresponding permissive insect host cells from hosts
`such as Spodoptera frugiperda (caterpillar), Aedes aegypri
`(mosquito), Aedes albopictus (mosquito), Drosophila me/a-
`rwgaster (fruitfiy). and Bombyx. mori host cells have been
`identified. See, e.g., Luckow el al., Bio/Technologvy 6:
`47-55 (1988); Miller ct al., in Generic Engineering Setlow,
`J. IC. ct a., eds., Vol. 8 (J>lenum Publishing, 1986}, pp.
`277-279; and Maeda et al., Nature, 315: 592-594 (1985). A
`variety of such viral strains are publicly available, e.g., the 15
`L-1 variant of Autographa califomica N"PV and the Bm-5
`strain of Bombyx mon· NPV, and sucb viruses may be used
`as the virus herein according to the present invention,
`particularly for transfection of Spodoptera frugiperda cells.
`Plan• cell curures of cotton, corn, potato, soybean, petunia, 20
`lomalo, a.ad tobacco ca.a be utilized as hosts. Typically, plant
`cells arc transfected by incubation with certain strains of the
`bacteriumAgrobacteriwn twnefaciens, which bas been pre(cid:173)
`viously manipulated to contain the target polypeptide DNA.
`During incubation of the plant cell cuhure with A . is
`tumefaciens, the DNA encoding target polypeptide is 1taos(cid:173)
`ferred to the plant cell host such that it is transfected, and
`will, under appropriate conditions, express the targel
`polypeptide DNA. ln addition, regulatory and signal
`sequences compatible with plant C:ells are available, sucb as 30
`the oopaLiae synthase promoter aod polyadeoylatioo signal
`sequences. Depickcr et al.,J . Mo/. Appl. Gen., 1: 561 (1982).
`lo addition, DNA segments isolated from lhe upstream
`region of the T-DNA 780 gene are capable of activatiag or
`increasing lranscription levels of plaot-expressible genes in 35
`recombinant DNA-oootaining pJaot tissue. See EP 321,196
`published Jun. 21, 1989.
`However, interest bas bceo greatest in vertebrate cells,
`and propagation of vertebrate cells in culture (tissue culture)
`bas become a routine procedure in recent years [T&SSue 40
`Culture, Academic Press, Kruse and Patterson, editors
`(1973)]. Examples of useful mammalian host cell lines are
`monkey kidney CVl line transformed by SV40 (COS-7,
`ATCC CRL 1651); human embryonic kidney lioe (293 or
`293 cells subcloned for growth io suspension cultuJe, Gra- 45
`ham el al., J. Gen Vuvl, 36: 59 (1977]); baby hamster
`kidney cells (BHK, ATCC CCL 10); Chiocse hamster ovary
`cells/-DHFR (CHO, Urlaub and Chasin, Proc. Nari. Acad.
`Sci. USA, 77: 4216 (1980D; mouse sertoli cells (TM4,
`Malher,Biol. Reprod., 23; 243-251 [1980D; monkey kidney so
`cells (CV! ATCC CCL 70); African green monkey kidney
`cells (VERO· 76. PJCC CRL-1587); human cervical carci(cid:173)
`noma cells (HELA, ATCC CCL 2); canine kidney cells
`(MOCK, A'.I'CC .CCL 34); buffalo rat liver cells (BRL 3A.
`ATCC CRL 1442); human lung cells (Wl38, ATCC CCL 55
`75); human liver oclls (Hep 02, HS 8065); mouse mammary
`tumor (MMT 060562, ATCC CCL51); TRI cells (Malber et
`al., Annals N.Y. Acad. Sci., 38.3: 44-68 [1982D; MRC 5
`cells; FS4 cells; and a human hepatoma cell line (Hep G2).
`Preferred host cells arc human embryonic kidney 293 and 60
`Chinese hamster ovary cells.
`Host cells are lraasfected and preferably transformed with
`the above-described expression or cloning vectors of Ibis
`iovent.ioo aod cultured in oonveotional nutrient media modi(cid:173)
`fied a.s appropriate for inducing promoters, selecting 65
`lransfonnants, or amplifying tbe genes encoding the desired
`sequences.
`
`US 6,407,213 Bl
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`36
`Transfectioo refers to the ta.king up of an exprc.ssion
`vector by a host cell wbetber or not any coding sequenel.'S
`are in fact expre~d. Numerous melh6ds of transfectioo are
`known to the ordinarihy skilled artisan, for example, CaP04
`and electropora1ion. Successful transfection is generally
`recognized when any indication of the operation of this
`vector occurs within the host cell.
`Transformation means introducing DNA into an organism
`so tbal the DNA is replicable, either as an extracbromosomal
`element or by chromosomal integraot. Depending o.n the
`host cell used, transformation is done using standard tech(cid:173)
`niques appropriate to such cells. The calcium treatment
`employing calcium chloride, as described in section 1.82 of
`Sambrook et al.. supra, is generally used for prokaryotes or
`other cells that coniain substantial cell-wall barriera. Infec(cid:173)
`tion with Agrobacrerium tumefaciens is used for lra.nsfor(cid:173)
`matioa of cerlain plant cells, as described by Shaw ct al.,
`Gene, 23: 315 (1983) and WO 89/05859 published Jun. 29,
`1989. For mammalian cells witbo\ll such cell walls, tbe
`calcium phosphate precipitation method descnbed in sec(cid:173)
`tions 16.30-16.37 of Sambrook el al., supra, is preferred.
`General aspects of mammalian cell host system transforma(cid:173)
`tions have been described by Axel io U.S. PaL No. 4,399,216
`issued Aug. 16, 1983. Traosformations into yeast are typi(cid:173)
`cally carried out according to the method of Van Soliogen et
`al.,J. Bact., 130: 946 (1977) and Hsiao et a)., Proc. Natl.
`Acad. Sci. (USA), 76: 3829 (1979). However, other methods
`for introducing DNA illto cells such as by nuclear i.t!jection,
`electroporation, or protoplasl fusion may also be used.
`Culturina the Host Cells
`Prokaryotic cells used to produce the target polypeptide of
`lhis invention are cultured in suitable media as described
`generally in Sambrook et al., supra.
`The mammalian bosl cells used to produce the target
`polypeptide of this invention may be cultured in a variety of
`media. Commercially available media such as Ham's FlO
`(Sigma), Minimal Essential Medium QMEM], Sigma),
`RPMI-1640 (Sigma), and Oulbecco's Modified Eagle's
`Medium ([DMEM], Sigma) are suitable for culturing the
`host cells. In addition. any of the media described in Ham
`aod Wallace, Meth. Enz., 58: 44 (1979), Barnes and Sato,
`Anal. Biodiem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704;
`4,657,866; 4,927.762; or 4,.560,655; WO 90/03430; WO
`87/00195; U.S . Pal. No. Re. 30.985, may be used a.sculrure
`media for the host cello;. Any of these media may be
`supplemented as necessary with hormones and/or other
`growth (aclors (such as insulin, lransferrin, or epidennal
`growth factor), salts (sucb as sodium chloride, calcium,
`magnesium, and phosphate), buffers (such as HEPES),
`nucleosides (such as adenosine and Lbymidioe), antibiotics
`(such as GeotamycinTM drug), trace elements (defined as
`inorganic compounds usually present at final concentrations
`in the micromolar range), and glucose or ao equivalent
`energy source. Any olbor necessary supplements may also
`be included al appropriate concenlralions lbat would be
`known to those skilled in tbe art. The culLure conditions,
`sucb as temperah1re, pH, and the like, are those previously
`used with the host cell selected for expression, and will be
`apparent to the ordinarily skilled artisan.
`The host cells referred to in this disdosure encompass
`cells io in vitro culture a.swell as cells tbat are within a bast
`animal.
`Il is further envisiooed that the target polypeptides of this
`invention may be produced by homologous recombination, ,
`or with recombinant production methods utilizing control
`clemeats introduced into cells already conlaining DNA
`encoding the target polypeptide curreoUy io use in the field.
`
`922 of 1033
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`BI Exhibit 1002
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`US 6,407,213 Bl
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`37
`For example, a powe rful promoter/cnbancer element, a
`sup17rcssor, or an exogenous transcription modulatory ele·
`men! is inserted in the genome of the in~ended bost cell in
`proximity and orientation sufficient to influence the tran(cid:173)
`scription of ONA encoding the desired target polypeptide.
`The control element does not encode the target polypep1ide
`of this foveation, but the DNA is present in the bost cell
`genome. One next screens for cells making the target
`polypeptide of this invention, or increased or decreased
`levels of expression, as desiredl.
`Detecting Gene Amplification/Expression
`Gene amplification and/or expression may be measured in
`a sample directly, for example, by conventional Southern
`blotting, nonbero blotting to quantitate the transcription of
`mRNA(lbomas,Proc. NatlAcad. Sci. USA, 77: 5201-5205 JS
`(1980]), dot blotting (DNA analysis). or i n situ
`hybridization, using an appropriately labeled probe, based
`on the sequences provided beccin. Various labels may be
`employed, most commonly radioisotopes, particularly 32P.
`However, other techniques may also be employed, such as 1.0
`using biotin-modified nucleotides for introduction into a
`polynucleotide. The biotin then serves as the site for binding
`to avidio or antibodies, which may be labeled with a wide
`variety of labels, such as radionuclides, fluorescers,
`enzymes, or the like. Alternatively, antibodies may be 2S
`employed that can recognize specific duplexes, including
`DNA !luplexes, RNA duplexes, and DNA-RNA hybrid
`duplexes or DNA-protein duplexes. The antibodies 'in tum
`may be labeled and the assay may be carried out where lbe
`duplex is bound to a surface, so that upon the formation of 30
`duplex o n the surface, the presence of antibody bound to the
`duplex can be detected.
`Gene expression, alternatively, may be measured by
`immunological roethods, such as imrnunoh'istochemical
`staining of tissue sections and assay of cell culture or body 35
`fiuids, to quaotitate directly tbe expression of gene product.
`With lmmunohistochemical staining techniques, a cell
`sample is prepared, typically by dehydration and fixation,
`followed by reaction with labeled antibodies specific for the
`gene p(Qduct coupled, where the labels arc usually visually 40
`detectable, such as enzymatic labels, fluorescent labels,
`luminesa:nt labels, and the like. A particularly sensitive
`staining technique suitable for use in the present invention is
`described by Hsu el aL, Am. J. Clin. Path., 75: 734-738
`(1980)
`Antibodies useful for immuoohistochemica1 staining and/
`or assay of sample fluids may be either monoclonal or
`polyclonal, and may be prepared in .any mammal.
`Conveniently, tbe antibodies may be prepared against a
`native target polypeptide or against a syuthetic peptide based so
`on the DNA sequences provided herein as described further
`in Section 4 below.
`Purification of the Target Polypeptide
`The target polypeptide preferably is recovered from the
`culture medium as a secreted polypeptide, although it also ss
`may be recovered from host cell lysates wb~n direcUy
`expressed without a secretory signal.
`When the target polypeptide is expressed io a recorobi·
`nan! cell other tba.n one of human origin, tbe target polypep(cid:173)
`tide is completely free of proteins or polypeptides ofbuman 60
`origin. However, it is necessary to purify the target polypep(cid:173)
`tide from recombinant cell proteins or polypeptides to obtain
`preparations I.hat are substantially homogeneous as 10 the
`target polypeptide. As a first step, the culture medium or
`lysate is centrifuged to remove particulate cell debris, The 6S
`membrane and soluble protein fractions are then separated.
`The target polypeptide may then be purified from the soluble
`
`38
`protein fraction and from the membrane fraction of the
`culture lysate, depending on whether the target polypeptide
`is membrane bound. The following procedures are exem(cid:173)
`plary of suitable purification procedures: fractionation oo
`immuaoaffinity or ion-exchange cohunns; ethanol precipi(cid:173)
`tation; reverse phase HPLC; chromatography on silica or on
`a cation exchange resio such as DEAE; chromatofocusing;
`SOS-PAGE; ammonium sulfate precipitation; gel filtration
`using, foe example, Sepbadex G-75; and protein A
`10 Sepharose columns to remove contaroioan~ such as lgG.
`Target polypeptide variants in which residues have been
`d~lttcd, inserted or substituted are recovered in the same
`fashion, taking account of any substantial changes in prop(cid:173)
`erties occasioned by I be variation. For example, preparation
`of a target polypeptide fusion with another proteia or
`polypeptide, e.g. a bacterial or viral antigen, facilitates
`purification; an immuooa.ffinity column containing antibody
`to the antigen (or containing antigen, where the target
`polypeptide is an antibody) can be used to adsorb the fusion_
`lmmunoaffinity columns such as a rabbit polyclonal anti·
`target polypeptide column can be employed to absorb the
`target polypeptide variant by binding it to at least one
`remaining immune epitope. A protease inhibitor such as
`phenyl methyl sulfonyl fiuoride (PMSF) also may be useful
`to inhibit proteolytic degradation during purification., and
`antibiotics may be included to prevent the growth of adven-
`titious contaminants. One skilled in the art will appreciate
`that purification methods suitable for native target polypep(cid:173)
`tide may require modification to account for changes in the
`character of tbe target polypeptide or its variants upon
`expression ia recombinant cell culture.
`Covalent Modifications o[ Target Polypeptides
`Covalent modifications of target polypeptides are
`included within the scope of tills invention. One type of
`covalent modification included within the scope of this
`invention is a target polypeptide fragment. Target polypep-
`tide fragments having up to about 40 amino acid residues
`may be convenienlly prepared by chemical syntbesis, or by
`enzymatic or chemical cleavage of the full-length target
`polypeptide or varia;nt target polypeptide. Other types of
`covalent modifications of the target polypeptide or frag.
`meats thereof arc introduced into the molecule by reacting
`specific am.ioo acid residues of the target polypeptide or
`fragments thereof with an organic derivatizing agent that is
`45 capable of reacting with selected side chains or the N- or
`C-terminal residues.
`Cysteinyl residues most commonly are reacted witb
`a-baloacetates {and correspooding amines), sucb as cbloro·
`acetic acid or cbloroacetamide, to give carboxymetl\yl or
`carboxyamidometbyl derivatives. Cysteioyl residues also
`are derivatized by reaction with bromotrifluoroacetone,
`a-bromo-~ ·(5-imidozoyl)propionic acid, cbloroacetyl
`phosphate, N-alkylmaleimidcs,3-oitro-2-pyridyl disulfide,
`methy12-pyridyldisulfide, p-cbloromercuribenzoate,
`2-chloromercuri-4-aitropbenol, or chloro-7-nitrobenzo-2·
`oxa-l,3-diazolc.
`Histidy) residues are derivatizcd by reaction with dielh·
`ylpyrocarbooate at pH 55-7.0 becau.se th.is agent is rela(cid:173)
`livcly specific for the bistidyl side chain . Para·
`bromopheoacyl bromide also is useful; the reaction is
`preferably performed ia 0.1M sodium cacodylate at pH 6.0.
`Lysinyl and amino terminal residues are reacted with
`succinic or other cart:>oxylic acid anhydrides. Derivatization
`with these agents has the effect of reversing the charge of the
`lysinyl residues. Other suitable reagents for derivatizing
`a-amioo-cootaioing residues include imidoesters such as
`methyl picolinimidate; pyridoxal phosphate; pyridoxal;
`
`923 of 1033
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`39
`cbloroborobydride; trinitrobenzenesulfonic acid;
`0-metbylisourea; 2,4-pentaaedione; and transaminase(cid:173)
`catalyzed reaction with glyoxylate.
`Arginyl residues are modified by reaction with one or
`several conventional reagents, among them phenylglyoxal, 5
`2,3-hutanedione, 1,2-cyclohexanedione, and ninbydrin.
`Derivatization of arginine residues requires that the reaction
`be performed in alkaline conditions because of the high pK,,
`of the guaoidine functional group. Furthermore, these
`reagents may react with the groups of Lysine as well as the 10
`arginine epsilon-amino group.
`The specific modification of tyrosyl residues may be
`made, witb particular interest in so introducing spectral
`labels into tyrosyl residues by reaction with aromatic dia(cid:173)
`zoo.ium compounds or tetranitrometbaoc. Most commonly, is
`N-acetylirnidizole and tetranitromethane arc used to fonn
`0-acetyl tyrosyl species and 3-nitro derivatives, respec(cid:173)
`tively. Tyrosyl residues arc iodinated using 1251 or 131
`( to
`prepare labeled proteins for use in radioimmunoassay, the
`chloramine T method described above being suitable.
`Carboxyl side groups (aspartyl or glulamyl) are selec(cid:173)
`tively modified by reaction with earbodiimides (R1
`N=O=N-R1), where R and R' are different alkyl groups,
`such as l-cyclobexyl-3-(2-morpbolinyl-4-ethyl) carbodiim(cid:173)
`ide or l-elhyl-3-(4-a?.onia-4,4-dimelhylpentyl) carbodiim- 25
`ide. Fucthermore, aspartyl and glutamyl residues are con(cid:173)
`verted to asparaginyl and glutaminyl residues by reaction
`with ammonium ions.
`Derivatization with bif-uactional agents is useful for
`crosslinkiag target polypeptide to a water-insoluble support 30
`matrix or surface for use in the method for purifying
`anti-target polypeptide antibodies, and vice versa. Com(cid:173)
`monly used crosslinking agents include, e.g., 1.1-bis
`( diazoace ty 1)-2-pheny le thane, gluta ra ldehyde ,
`N-bydrox.ysuccinimide esters, for example, esters with 35
`4-azidosalicylic acid, bomobifunctionaJ imidoesters, includ(cid:173)
`ing disuccioimidyl esters such as 3,3'-ditbiobis
`(succinimidylpropionate), and bifunctional maleimides such
`as bis-N-maleimido-1,8-octane. Derivatizing agents such as
`melhyl-3-[(p-azidopbenyl)dilhio ]propioimidate yield photo- 40
`activatable intermediates that are capable of forming
`crosslinks in the presence of light. Alternatively, reactive
`water-insoluble malrices such as cyanogen bromide(cid:173)
`activated carbohydrates and tbe reactive substrates
`described in U.S. PaL Nos. 3,969,287; 3,691,016; 4,195, 45
`128; 4,247,642; 4,229,537; and 4,330,440 arc employed for
`protein immobilization.
`Glutaminyl and asparaginyl residues are [requeotly dea(cid:173)
`midated to tbe corresponding glutamyl and aspartyl
`residue$, resp«tively. Alternativciy, these residues are dea- so
`midated under mildly acidic conditions. Either form of these
`residues falls within tbe scope of this invention.
`Other modificafion include hydroxylation of prolinc and
`lysine, pbophorylation of hydroxyl groups of seryl or threo(cid:173)
`nyl resides, melhylation of the o.-amino groups of lysine, 55
`arginine, and histidine side chains, (T. E. Creighton, Protein:
`Structure and Molecular Properties, W. H. Freeman & Co.,
`Saa Francisco, pp. 79~6 (1983}), acetylation of the
`N-terminal amine, and amidaatioon of any C-terminal car(cid:173)
`boxyl group.
`Another type of covalent modification of the target
`polypeptide included witbin the ~pe of this invention
`comprises altering the native gluoosylatuion pattern of the
`polypeptide. By aliering is meant deleting one or more
`carbohydrate moieties fou nd in the native target 6S
`polypeptide, and/or adding one or more glyoosylation sites
`that arc not present i.u the native target polypeptide.
`
`40
`Gylcosylatioo of polypeptides is typically either N·linked
`or 0-linked refers to the auacbment of the carbonhydrate
`moiety to tbe side chain o[ an asparaginc reisdue. The
`tri-peplide sequences asparagine-X-resine and asparagine(cid:173)
`X-tbreooine, where Xis any aminoe acid except proline, are
`the recoginition sequences for enzymatic at!achmen1 of the
`carbohydrate moiety to the asparagioe side chain. Thus, tbe
`presence of either of these tri-peptide sequences in a
`polypeptide creates a potential glycosylatioo site. 0-1.inked
`glycosylatioo refers to the attachment of one of the sugars
`N-acetylgaclosamine, galactosc, o r xylosc, to a
`bydroxyamino acid, most oommonly serine or threonine,
`allbougb 5-bydroxyproline or 5-hydroxylysine may also be
`used.
`Addition of glycosylation sites to the target polypeptide is
`conveniently accomplished by altering the amino acid
`sequence such that it contains one or more of bte above(cid:173)
`dcscribed tri-peptide sequences (for N-linked glycosylation
`sites). The alteration may also be made by the addition of,
`20 or subslirution by, ooe or more serine or tbeonille resides to
`the native target polypeptide sequence (for 0-linked glyco(cid:173)
`sylatloo sices). For case, the target polypeptide amino acid
`sequences is preferably altered through changes at the DNA
`level, particularly by mutating the DNA encoding the target
`polypeptide at preselected bases such that condon.s are
`generated 1hat will translate into the desired amino acids.
`Tbe DNA mulation(s) may be made using melbods
`described above under tbe beading of "Amino Acid
`Sequence Variants of Target Polypeptide".
`Another means of increasing tbe number of carbohydrate
`moieties on lhc target polypeptide is by chemical or enzy(cid:173)
`matic coupling glycosides to Lhe polypeptides. These pro·
`ccdures are advantageous in that they do not require pro(cid:173)
`duction of the polypeptide in a host cell that bas
`glycosylation capabilities for N- or 0-linkcd glycosylation.
`Depending on the couple mode used, the suga~s) may be
`attached to (a) arginine and histidine, (b} fxcc carboxyl
`groups, (c) free sulfbydryl groups such as those of cysteine,
`(d) free hydroxyl groups such as those of serine, threonine,
`or hydroxyproline, (e) aromatic residues such as those of
`phenylalanine, tyrosine, or lryptopban, or (f) the amide
`group of glut.amine. These methods are described in WO
`87/05330 published Sep. 11, 1987, and in Aplin and Wriston
`(CRC Cric. Rev. Biochem., pp. 259-306 [1981].
`Removal of carbohydrate moieties present on the native
`tar.get polypeptide may be accomplished chemically or enzy(cid:173)
`matically. Chemical deglycosylation requires exposure of
`tbe polypeptide to the compound trilluorometbanesulfonic
`acid, or an equivalent compound. This treatmepl results in
`the cleavage of most or all sugars except the linking sugar
`(N-acetylglucosamine or N -acetylgalactosamine), while
`leaving tbe polypeptide intact. Chemical deglycosylation is
`described by Hakimuddin et al. (Arch. Bioch2m. Biophys.,
`259:52 [1987D and by Edge et al. (Anal. Biochem., 118:131
`[198ID. Enzymatic cleavage of carbohydrate moieties oo
`polypeptides can be achieved by tbe use of a variety of codo-
`and exo-glycosidases as described by Thotakura et al (Meth.
`Enzymol. 138:350 [1987D.
`Glycosylalion al potential glycosylation sites may be
`60 prevented by the use of the compound tunicamycin as
`described by Duskin el al. (J. Biol. ChenL, 257:3105
`[1982]}. Tunicarnycin blocks the formation of protein-N(cid:173)
`glycoside linkages.
`Another type of covalent modification or the target
`polypeptide comprises linking tbe target polypeptide to
`various noopro1cinaceous polymers, e.g. polyethylene
`glycol, polypropylcnc glycol or polyoicyalkylenes, in tbe
`
`924 of 1033
`
`BI Exhibit 1002
`
`
`
`US 6,407,213 Bl
`
`41
`maooer sci forth io U.S. PaL Nos. 4,640,835; 4 ,496,689;
`4,301,144; 4,670,417; 4,791,192 or 4,179,337.
`The target polypeptide also may be entrapped )11 micro(cid:173)
`capsuJes prepared, for example, by coacervation techniques
`or by ioterfacial polymerizatioo (for example, bydroxym- s
`cthylcellulose or gela1in-microcapsules and poly(cid:173)
`[metbylmetbacylatc ]microcapsules, respectively), in colloi-
`dal drug deliverysystems (for example, liposomcs, albumin
`D;Jicrospbercs, microcmulsioos, naoo-particles and
`oaoocapsulcs), or in macroemulsions. Such techniques ue IO
`dl:>closed in Reminaton's Plwrm{)r;e,.iicq{ Scien&~. l61b
`edition, Osol, A., Ed., (1980).
`Target polypeptide preparations are also useful in gener·
`ating antibodies, for screening for binding partoer:s, as
`standards in assays for the target polypeptide (e.g. by LS
`labeling the target polypeptide for use as a standard in a
`radioimmunoassay, enzyme-linked immunoassay, or
`radioreceptor assay), in affinity purification techniques, and
`in competitive-type receptor binding assays when labeled
`with radioiodine, enzymes, fiuoropbores, spin labels, and the 20
`like:.
`Si'nce it is often difficult to predict in advance tbe char(cid:173)
`acteristics of a variant target polypeptide, it will be appre(cid:173)
`ciated tbat some screening of the recovered variant will be
`needed to select the optimal variant. For example, a change 25
`in the immunological character of tho target polypeptide
`molecule, such as affinity for a given antigen or antibody, is
`measured by a competitive-type immunoassay. The varianl
`is assayed for cbaoges in lbe suppression or enba