0125023
`
`pyCEAtrp207-l* which contained heavy chain as insoluble bodies, were
`1 ysed and centrifuged as above; the pe 11 et was resuspended in the
`same buffer, sonicated and re-centrifuged. This pellet was washed
`. once with buffer, then suspended in 6M guanidine HCl, O.lM Tris HC1,
`pH 8, lnf'i EOTA, 20 mg/ml sodium sulfite and 10 mg/ml sodium
`tetrathionate and allowed to react at 25• for about 16 hrs. The
`r.eaction mixture w~s diaJyze~ against BM urea, O.lM Tris HCl, pH 8,
`and stored at 4•, to give a 3 mg/ml solution of y-Sso3•
`
`650 ul of cell lysate from cells of various!_. coli strains
`producing various lgG chdins, was added to 500 mg urea. To this was
`added a-mercaptoethanol to 2011-1, Tris-HCl, pH 8.5 to SOnM and EDTA
`to lmM, and in some experiments, y-SS03 was added to 0.1 mg/ml.
`After standing at 25° for 30-90 mins., the reaction mixtures were
`dialyzed at 4• against a buffer composed of O.lM sodium glycinate,
`pH"· 10·.a, O.SM urea, lOnM glycine ethyl ester, 5mM reduced
`glutathione, O.lnt-1 oxidized glutatnione. This buffer was prepared
`from r,2-saturated water and tne dialysis was performed in a capped
`Wheaton bottle._ After lfr48 hours, dialysis bags were transferred
`to 4° phosphate buffered saline containing lrrf'i PMSF and dialysis
`continued another lfr24 hrs.· Dialysates were assayed by ELISA as
`described in paragraph E.2 for ability to bind CEA. The results
`below show the values obtained oy comparison with the standard curve
`in x ng/ml anti-CEA. Also shown are the reconstitution efficiencies
`ca,ku,lated,. f;r:om» the EL1'SA, r.e-sponses.,. minus the background" {108~
`ng/ml) of cells producing K chain only, and from estimates of the
`levels of y and K cnains in the reaction mixtures.
`ng/ml
`anti-CEA
`
`Percent
`recombination
`
`!_. coli W3110 producing IFN-aA {control)
`f. coli (W3110/pKCEAtrp207-l*)
`f. coli (W3110/pKCEAtrp207-l*), plus y-SS03
`f. coli (W3110/pKCEAtrp207-l*6, pyCEAint2)
`Hybridoma anti-CEA K-SS0 3 and y-SS03
`
`0
`108
`848
`1580
`540
`
`0.33
`0.76
`0.40
`
`5
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`10
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`15
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`25°
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`
`E.4 Preparation of Chimeric Antibody
`Figures 11 and 12 show the construction of an expression vector
`for a chimeric heavy (garrrna) cha in which comprises the murine anti
`CEA variable region and human y-2 constant region.
`
`A ONA sequence encoding the human gamma-2 heavy chain is
`prepared as fol 1 ows:
`the cDNA 1 ibrary obtained by standard
`'
`techniques from a human multiple myeloma cell line is probed with
`5 1 GGGCACTCGACACAA 3' to obtain the plasmid containing the cDNA
`insert for human gamma-2 cha in (Takahashi, et tl·, Cel 1, 29: 671
`(1982), incorporated herein b~ reference), and analyzed to verify
`its identity with the known sequence in human ganrna-2 {Ellison, J.~
`et tl·, Proc. Natl. Acad. Sci. (USA}, 79: 1984 (1982) incorporated
`herein by reference).
`
`As shown. in Figure 11. two fragments are obtained from this
`cloned human gamma 2 plasmid (py2). The first fragment is formed by
`digestion with Pvull followed by digestion with Ava III, and
`purification of the smaller DNA fragment, which contains a portion
`-
`of the constant region, using 6 percent rAGE. The second fragment·
`is obtained by digesting the py2 with any restriction enzyme which
`cleaves in the 3 1 untranslated region of y2, as deduced from the
`nucleotide sequence, filling in with Klenow and dNTPs, cleaving with
`Ava III, and isolating the smaller fragment using 6 percent PAGE.
`lT.b~, cnoi ce .. o:f. a two step, two fragment composj tton to. supp.ly.. the.
`~vull-3' untranslated fragment provides a.cleaner path to product
`due to the proximity of the AvalII site to the 3 terminal end thus
`avoiding additi~nal restriction sites in the gene sequence matching
`the 3• untranslated region site.J· pyCEA207-l* is digested with EcoR
`1. treated with Klenow and dNTPs to fill in the cohesive end, and
`digested with Pvu II, the large vector fragment containing promoter
`isolated by 6 perc~nt PAGE.
`
`rhe. location an.d- DNA sequence· surr-oundi'ng the- Pvult site in· the
`
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`

`

`0125023
`
`-50-
`
`mouse garmna-1 gene are.identical to the location and DNA sequence
`surrounding the Pvull site in the human garmna-2 gene.
`
`The plasmid resulting from a three way ligation of the foregoing
`fragments, pChiml, contains, under the influence of trp promoter,
`the variable and part of the· constant region of· murine anti--CEA
`gamma 1 chain, and a portion of the galtlila 2 human chain. pChiml
`will, in fact, express a chimeric heavy chain. when transfonned into
`f· coli, but one wherein the change from mouse to human does not
`take place at the variable to constant junction.
`
`Figure·· 12 shows modification of pChiml to construct pChim2 so
`that the resulting protein from expression will contain variable
`region from murine anti CEA antibody and constant region from the
`numan y-2 chain. First, a fragment is prepared from pChiml by
`treating with Nco I, blunt ending with Klenow and dNTPs~ cleaving
`with Pvu II, and.isolating the large vector fragment which is almost
`the complete pla~mid except for short segment in the constant coding·
`region for mouse anti CEA. A second fragment is prepared from the
`previously described py2 by treating with Pvu II, followed by
`treating with any restriction enzyme which cleaves in the variable
`region, blunt ending with Klenow and dNTPs and isolating the short
`fragment which comprises the junction between variable and constant
`regions of this chain.
`
`Ligation of the foregoing two fragments produces an intennediate
`plasmid which is correct except for an extraneous DNA fragment which
`contains a small portion of the constant region of the murine anti
`CEA antigen, and a small portion of the variable region of the human
`gamma chain. This repair can be made by excising the Xba I to Pvu
`II fragment and cloning into M13 phage as described by Messing
`et_!]_., Nucleic Acids Res. 9-: 309 C19.81), followed by in vitro site
`directed deletion mutagenesis as described by Adelman, et tl·, DNA
`2, 183 (1983) which is incorporated herein by reference. The
`
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`

`0125023
`
`-51-
`
`Xba I-Pvu II fragment thus modified is ligated back into the
`intermediate plasmid to form pChim2. This plasmid then is capable
`of expressing in a suitable host a cleanly constructed murine
`variable/human constant chimeric heavy chain.
`
`ln. an analogous fashfon,. but using mRNA templates. for cDNA
`construction for human kappa rather than y chain, the expression
`plasmid for chimeric light chain is prepared.
`
`The foregoing two plasmids are then double transformed into
`E.coli W3110, the cells grown and the chains reconstituted as set
`forth in paragraph E.1-E.3 supra.
`
`E.5 Preparation of Altered Murine Anti-CEA Antibody
`
`E.5.1 Construction of Plasmid Vectors for Direct Expression of
`Altered Murine Anti-CEA Heavy Chain Gene
`The cysteine residues, and the resultant disulfide bonds in the
`region of amino acids 216-230 in the constant region of murine
`anti-CEA heavy chain are suspected to be important for complement
`fixation (Klein, et!!•, Proc. Natl. Acad. Sci., (USA), 78: 524
`(1981)) but not for. the antigen binding property of the resulting
`antibody. To decrease the probability of incorrect disulfide bond
`fo·rmation· during re'c·on·stftlifion· accordl'ng to .the process of the
`invention herein, the nucleotides encoding the amino acid residues
`226-232 which includes codons for three cysteines, are deleted as
`follows:
`
`A "deleter" deoxyoligonucelotide. 5' CTAACACCATGTCAGGGT is used
`to delete the relevant portions of the gene from pyCEAtrp207-l* by
`the procedure of Wallace. et!!_ •• Science. 209: 1396 (i980) or of
`Adelman,- et &·, ~ 2, 183 (1983). Briefly. the "deleter"
`deoxyoligonucelotide is annealed with denatured pyCEAtrp207-l* DNA,
`
`5
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`

`0125023
`
`-52-
`
`and primer repair synthesis carried out in vitro, followed by
`screening by hybridization of presumptive deletion clones with P32
`labelled deleter sequence.
`
`5
`
`10
`
`E.5.2
`Production of Cysteine Deficient Altered Antibody
`The plasmid prepared in E.5 .1 is transformed into an £· co~ i
`strain previously transformed with pKCEAtrp207-l* as described
`above. The cel 1 s are grown, extracted for recombinant antibody
`chains, and the altered antibody reconstituted as described in
`E.1.10.
`
`E.6 Preparation of Fab
`
`E.6.1 Construction of a Plasmid Vector for Direct Expression
`of Murine Anti-CEA Gamma 1 Fab FraOTnent Gene
`pyCEAFabtro207-l*
`Figure 13 presents the construction of pyCEAFabtrp207-l*. 5 µg
`of pBR322 was digested with Hind III, the cohesive ends made flush
`by treating with Klenow and dNTPs; digested with Pst I, and treated
`with BAP. The large vector fragment, fragment I, was recovered
`using 6 percent PAGE followed by electroelution.
`
`5 µg of pyCEAtrp207-l* was digested with both BamH I and Pst I
`and)" the- -1szo bp, ONA fragment (fragment I.I) contain.ing the trp
`promoter and the gene sequence encoding the variable region
`continuing into constant region and further into the anti-CEA ganma
`1 chain hinge region, was isolated and purified after
`electrophoresis.
`
`Express ion of the anti-CEA ganma l chain Fab fragment rather
`than complete heavy chain requires that a termination codon be
`constructed at the appropriate location in the gene. For this, the
`2 60 bp Nco I - Nde l DNA fragment from 20 µg of the py298 was
`isolated and purified. A 13 nucleotide DNA priner, the complement
`
`15
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`

`0125023'
`
`-53-
`
`of which encodes the last 3 C-terminal amino acids of the fab gene
`and 2 oases of the 3 needed for the stop codon, was synthesized by
`the phosphotriester method (supra). The probe hybridizes to·
`nucleotides 754 to 767 (figure 4) which has the following sequence:
`AspCysGlyStop
`5 1 GGGATTGTGGTTG
`3,1
`
`The third base of the stop codon is provided by the terminal
`nucleotide of the filled-in Hind III site from pBR322 cleavage
`described above. 500 ng of this primer was used in a primer repair
`reaction by phosphorylation at the 5' end in a reaction with 10 ·
`units T4 DNA kinase containing 0.5 nivt ATP in 20 µl, and mixing with
`-200 ng of the Nco 1-Nde I DNA fragment. The mixture was heat
`denatured for 3 minutes at 95° and quenched in dry-ice ethanol. The
`denatured DNA solu.tion ·was made 60rrM NaCl, 7mM MgC1 2, 7 rrt-1 Tris
`HCl
`(pH 7 .4), 12 mM in each dNTP and 12 units DNA Polymerase I-Lar9e
`Fragment was added. After 2 hours incubation at 37°C, this primer
`repair reaction was phenol/CHC13 extracted, ethanol precipitated,
`digested with BamH I and the reaction electrophoresed through a 6
`percent polyacrylamide gel. -song of the 181 bp blunt end to BamH
`I ONA fragment, fragment III> was isolated and purified.
`
`-100 ng of fragment I, -100 ng each of fragments II and III were·
`1Tg~a·ted o·verY1fght and' 1:r·arisfofmed~ fo'to E. coli Kl2 strain 2~94.
`- --.·
`Plasmid DNA from several tetracycline resistant transformants wa~ · ·
`analyzed for the proper construction and the nucleotide sequence
`through the repair blunt end filled-in Hind III -junction was
`determined for verification of the TGA stop codon.
`
`Production of Fab Protein
`E.6.2
`· The plasmid prepared in E.6~1 is transformed into an f. coli
`strain previously transformed with pKCEAtrp207-1* as described
`above.
`·the cells are grown> extracted for recombinant antibody
`chains and the Fab protein reconstituted as described in E.1.10.
`
`5
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`..
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`-54-
`
`0125023'
`
`5
`
`The appended c·laims set. out the principal areas for
`which a monopoly is presently claimed.
`In addition, the
`following preferred features should be noted:
`the antibody of claim 3 which is directed against CEA;
`th~ antibody of claim 3 wherein the heavy chain is of
`· the gamma family;
`the antibody of claim 3 wherein the light chain is of
`the kappa family;
`the composition of matter of claim 8 which is
`io mammalian;
`the composition of matter of claim 8 which is
`immunoreactive against CEA;
`the sequence of claim 9 which is a mammalian heavy
`chain;
`the sequence of claim 9 which is anti-CEA heavy chain;
`the sequence of claim 10 which is a mammalian light
`chain;
`the sequence of claim 10 which is anti-CEA light
`chain;
`the recombinant host cells of claim 16 which are
`microbial host cells;
`the method of claim 17 wherein the vector of b) and
`the vector of d) are transformed into the same host cell
`culture, and
`the sequence of a) and the sequence of c) are inserted
`into the same replicable expression vector;
`tJ1_e metJ1qd of c),aim 1 7 whe:r:.e.in t.Qe DN~ s~eq:u.ene,e of. a.t
`encodes mammalian heavy chain, and the DNA sequence of c)
`encodes mammalian light chain; and wherein both DNA
`fragments encode amino acid sequences of the same mammalian
`antibody;
`the method of claim 17 wherein the DNA fragment of a)
`encodes a chimeric hybrid heavy chain and the DNA sequence
`of c) encodes a chimeric light chain; and
`the method of any one of claims 17 to 19 wherein said
`vectors, a:i=-e t:v-ansformed., i 0nto the same hos't ce·ll culture·.
`
`15
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`

`-55-
`
`0125023
`
`CLAIMS
`
`1.
`
`An immunoglobulin produced by r~combinant host cells.
`
`5
`
`An immunoglobulin substanti~l1y fr~e of other proteins
`2.
`with which it is normally associate~ in· vertebrate cells.··.
`
`10
`
`The· immunog_lobulin o.f claim l or 2 which is a
`3 .• ,
`mammalian antibody, in that the amino acid sequences of all
`four chains are homologous to the sequences in the
`corresponding chain~ in an antibo~y derived from a
`mammalian species.
`
`The immunoglobulin of claim 1 or 2. which is a hybrid
`4.
`15 antibody, a composite non-specific immunoglobulin, a
`chimeric antibody, or an altered-antibody.
`
`A chimeric antibody of clai~~-wh~rei~ the constant
`5.
`regions of all four chains are homologous ~o the
`. ..
`.
`20 corresponding constant regions of ~n~ntibcidy of a first
`mammalian species, and the amino a~i9 sequence of the
`variable regions of all four chains are homologous to the·
`variable regions in an antibody derived from a second,
`different, mammalian species.
`
`25
`
`A- compos.i-tion, of matter- consisting~ essentially of a-
`6:-
`univalent antibody.
`
`A composition of matter consisting essentially of Fab
`7.
`30 protein.
`
`A composition of matter of claiin. 6 or claim 7 which
`8.
`is produced by recombinant host c~).ls.
`
`35_ 9.
`A sequence of amino·acids produced by recombinant
`host cells corresponding to immunoglobulin heavy chain.
`
`. ... -'
`
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`Page 4008
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`

`

`-56-
`
`0125023
`
`A sequence of amino acids producea by recombinant host
`10.
`cells corresponding to immunoglobulin_ light chain.
`
`A sequence of claim 9 or claim 10 which is a chimeric
`11.
`heavy chain or light chain, respectively.
`
`5
`
`10
`
`A sequence of claim 11 wherein that portion of the
`12.
`sequence which corresponds to the constant region is
`homologous to corresponding sequence of an antibody derived
`from humans, and the amino acid sequence of the variable
`region is homologous to the corresponding amino acid
`sequence of an antibody derived from non-human mammalian
`species.
`
`A DNA sequence which encodes for the immunoglobulin
`15 13.
`of claim 1 or 2, the composition of matter of claim 6 or
`the amino acid sequence Qf claim 9 or claim 10.
`
`A replicable expression vector capable of expressing
`14.
`in a suitable host cell the DNA sequence of claim 13.
`
`20
`
`An expression plasmid which comprises the DNA sequence
`15.
`of claim 14 operably linked to a promoter compatible with a
`suitable host cell.
`
`25
`
`Recombinant host cells or host cell cultures
`16.
`transfcfr'rned wit:h fne· vector of claim 14 or 15.
`
`A method for preparing immunoglobulins in recombinant
`17.
`30 host cells which method comprises
`a) preparing a DNA sequence encoding heavy chain,
`b) inserting the sequence of a} into a replicable
`expression vector operably linked to a- suitable promoter,
`c) preparing a DNA sequence encoding light chain,
`d) inserting the sequence of c} into a replicable
`expresion vector operably linked to a suitable promoter,
`
`35
`
`BIOEPIS EX. 1002
`Page 4009
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`

`

`-57-
`
`0125023
`
`e) transforming host cell ciulture wiih the vector of
`b) and host cell culture with the vector of d},
`f) recoveririg light chain and heavy chain from cell
`culture,
`g} reconstituting light and heavy chain,
`wherein steps f) and g) may be performed either
`sequentially in either order, or simul·taneously.
`
`5
`
`15
`
`A method for preparing Fab protein in recombinant
`18.
`10 host cells which method comprises
`a) preparing a DNA sequence encoding the Fab region
`of heavy chain,
`b} inserting the sequence of a) into a replicable
`expression vector operably linked to a suitable promoter,
`c) preparing a DNA sequence encoding light chain,
`d) inserting the sequence of c) into a replicable
`expression vector operably linked to a suitable promoter,
`e) transforming host cell culture with the vector of
`b) and host cell culture with the vector of d},
`f) recovering light chain and Fab protein of heavy
`chain from cell culture,
`g) reconstituting light and heavy Fab region chains;
`wherein steps f) and g) may either be performed
`sequentially in either order or simultaneously.
`
`20
`
`25
`
`A method for preparing univalent antibody in
`19.
`recombinant hos.t cells which method comprises_
`a} preparing a DNA sequence encoding heavy chain,
`b) inserting the sequence of a) into a replicable
`30 expression vector operably linked to a suitable promoter,
`c) preparing a DNA sequence encoding light chain,
`d) .inserting the sequence of c) into a replicable
`expression vector operably linked to a suitable promoter,
`e) preparing a DNA sequence encoding the Fe portion
`of heavy chain,
`
`35
`
`.
`
`. .. ··.~·
`:• .·.
`.....
`··,·· ..
`
`BIOEPIS EX. 1002
`Page 4010
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`

`

`-58-
`
`0125023
`
`f) inserting the sequence of e) into a replicable
`expression vector operably linked to a suitable promoter,
`g) transforming host cell culture with the vector of
`b~ host cell cultur~ with the vectbr of d), and host ce11·
`culture with the vector off),
`h) recovering light chain, hea_vy chain, and Fe portion
`of heavy chain from cell culture,
`i} reconstituting light chain, heavy chain, and Fe
`portion of heavy ch~in,
`wherein steps h) and i) may be performed
`sequentially in either order or simultaneously.
`
`5
`
`10
`
`15
`
`A method for preparing heavy chain or light chain
`20.
`which method comprises
`a) preparing a DNA sequence encoding heavy or light
`chain,
`b) inserting said sequence into a replicable
`expression vector operably linked to a suitable promoter,
`c) transforming host cell culture with the vector of
`20 b), and
`d} recovering heavy or light chain from cell culture.
`
`25
`
`A method for preparing Fab region of heavy chain as a
`21.
`polypeptide which method comprises
`a) preparing a DNA sequence encoding Fab region of
`heavy chain,
`bs).
`i,nser~i-ng, sa--1-d" sequence into a rep-1-icable
`expression vector operably linked to a suitable promoter,
`d) transforming host cell culture with the vector of
`
`30 b),
`
`d) recovering Fab region of heavy chain from cell
`culture.
`
`BIOEPIS EX. 1002
`Page 4011
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`

`

`0125023
`
`Fe fragment
`
`Fig. 1.
`
`BIOEPIS EX. 1002
`Page 4012
`
`

`

`1
`
`101
`
`201
`
`301
`
`401
`
`haeII I
`hae I
`hph I
`t.thl 11
`tth 111
`.
`GTTGCTGTGG TTGTCTGGTG TTGAAGGAGA CATTGTGATG ACCCAGTCTC ACAAATTCAT GTCCACATCA GTAGGAGACA GGGTCAGCAT CACCTGCAAG
`CAACGACACC AACAGACCAC AACTTCCTCT GTAACACTAC TGGGTCAGAG TGTTTAAGTA CAGGTGTAGT CATCCTC·TGT CCCAGTCGTA GTGGACGTTC
`·
`•
`·
`·
`sfaNI
`·
`
`scrF I
`ncf I
`scrFI
`scrFI
`fnu4HI
`bbv
`fok I
`hfnfI
`hpa II
`ecoRI I
`ecoRII
`GCCAGTCAGG ATGTGGGTGC TGCTATAGCC TGGTATCAAC AGAAACCAGG ACAATCTCCT AAACTACTGA TTTACTG~GC ATCCACCCGG CACACTGGAG
`CGGTCAGTCC TACACCCACG ACGATATCGG ACCATAGTTG TCTTTGGTCC TGTTAGAGGA TTTGATGACT AAATGACCCG TAGGTGGGCC GTGTGACCTC
`.
`fok I
`~ sfaNI
`
`xhotl
`sau3A
`sau3A
`hfncII
`.
`.
`hphI
`dpil
`dpnl
`TCCCTGATCG CTTCACAGGC AGTGGATCTG GGACAGATTT CACTCTCACC ATTAGCAATG TGCAGTCTGA TGACTTGGCA GATTATTTCT GTCAACAATA
`AGGGACTAGC GAAGTGTCCG TCACCT~GAC CCTGTCTAAA GTGAGAGTGG TAATCGTTAC ACGTCAGACT ACTGAACCGT CTAATAAAGA CAGTTGTTAT
`
`,t:2
`;6
`
`sau96
`lpaI
`fnu4HI
`mboII
`bbv
`aluI sfaNI
`mn1I
`. avail aluI
`hfncII
`TAGCGGGTAT CCTCTCACGT TCGGTGClGG GACCAAGCTG GAGCTGAAAC GGGCTGATGC TGCACCAACT GTATCCATCT TCCCACCATC CAGTGAGCAG
`ATCGCCCATA GGAGAGTGCA AGCCACGACC CTGGTTCGAC CTCGACTTTG CCCGACTACG ACGTGGTTGA CATAGGTAGA AGGGTGGTAG GTCACTCGTC
`fokl
`
`mnll
`ddeI
`mnlI
`acyI
`mbo1I
`xmnI
`TTAACATCTG GAGGTGCCTC AGTCGTGTGt TTCTTGAACA ACTTCTACCC CAAAGACATC AATGTCAAGT GGAAGATTGA TGGCAGTGAA CGACAAAATG
`AATTGTAGAC CTCCACGGAG TCAGCAC'ACG AAGAACTTGT TGAAGATGGG GTTTCTGTAG TTACAGTTCA CCTTCTAACT ACCGTCACTT GCTGTTTTAC
`
`Fig.2A~
`
`0
`..l
`·N·
`u,
`0
`N
`(,,)
`
`BIOEPIS EX. 1002
`Page 4013
`
`

`

`sau3A
`fnu4HI
`dpnI
`alul
`mnll hfncII
`hgal
`bbv
`bell
`GCGTCCTGAA CAGTTGGACT GATCAGGACA GCAAAGACAG CACCTACAGC ATGAGCAGCA CCCTCACGTT GACCAAGGAC GAGTATGAAC GACATAACAG
`~GCAGGACTT GTCAACCTGA CTAGT~CTGT CGTTTCTGTC GTGGATGTCG TACTCGTCGT GGGAGTGCAA CTGGTTCCTG CTCATACTTG CTGTATTGTC
`
`sau96
`hga I
`mnl I
`haelII
`ddeI
`.
`alul
`avall
`acyI
`hphl
`hael
`CTATACCTGT GAGGCCACTC ACAAGACATC AACTTCACCC ATTGTCAAGA GCTTCAACAG GAATGAGTGT TAGAGACAAA GGTCCTGAGA CGCCACCACC
`GATATGGACA CTCCGGTGAG TGTTCT,GTAG TTGAAGTGGG TAACAGTTCT CGAAGTTGTC CTTACTCACA ATCTCTGTTT CCAGGACTCT GCGGTGGTGG
`
`mnll
`mn1I
`hgfA
`.
`mn1I
`ddeI
`mboII
`aluI
`aluI
`mn1I
`AGCTCCCCAG CTCCATCCTA TCTTCCCTTC TAAGGTCTTG .GAGGCTTCCC CACAAGCGAC CTACCACTGT TGCGGTGCTC CAAACCTCCT CCCCACCTCC
`TCGAGGGGTC GAGGTAGGAT AGAAGGGAAG ATTCCAGAAC CTCCGAAGGG GTGTTCGCTG GATGGTGACA ACGCCACGAG GTTTGGAGGA GGGGTGGAGG
`fok I
`.
`·
`
`~
`..3
`
`mn1I
`hfnfI
`xmnl
`mn1I mn1I
`TTCTCCTCCT CCTCCCTTTC CTTGGCTTTT ATCATGCTAA TATTTGCAGA AAATATTCAA TAAAGTGAGT CTTTGCACTT GA
`AAGAGGAGGA GGAGGGAAAG GAACCGAAAA TAGTACGATT ATAAACGTCT TTTATAAGTT ATTTCACTCA GAAACGTGAA CT
`
`501
`
`601
`
`701
`
`801
`
`nucleotfdes: 882
`
`Fig. 28.
`
`0 ....
`N
`u,
`0
`N
`ca:,
`
`BIOEPIS EX. 1002
`Page 4014
`
`

`

`20
`10
`1
`~9
`leu leu trp leu ser gly val glu gly asp fle val met thr gln ser hfs lys phe met ser thr ser val gly asp arg val ser
`UUG CUG UGG UUG UCU GGU GUU GAA G~A GAC AUU GUG AUG ACC CAG UCU CAC AAA UUC AUG UCC ACA UCA GUA GGA GAC AGG GUC AGC
`
`G
`
`50
`40
`30
`fle thr cys lys ala ser gln asp val gly ala ala fle ala trp tyr gln gln lys pro gly gln ser pro lys leu leu ile tyr trp
`AUC ACC UGC AAG GCC AGU CAG GAU GUG GGU GCU GCU AUA GCC UGG UAU CAA CAG AAA CCA GGA CAA UCU CCU AAA CUA CUG AUU UAC UGG
`
`80
`. 60
`70
`ala ser thr arg hfs thr gly val pro a~p arg phe thr gly ser gly ser gly thr asp phe thr leu thr fle ser asn val gln ser
`GCA ucc ACC CGG CAC ACU GGA GUC CCU GAU CGC uuc ACA GGC AGU'GGA ucu GGG ACA GAU uuc ACU cue ACC AUU AGC AAU GUG CAG ucu
`.
`90
`100
`110
`asp asp leu ala asp tyr phe cys gln gln tyr ser gly tyr pro leu thr phe gly ala gly thr lys leu glu leu lys arg ala asp
`GAU GAC UUG GCA GAU UAU UUC UGU CAA C~~ UAU AGC GGG UAU CCU CUC ACG UUC GGU GCU GGG ACC AAG CUG GAG CUG AAA CGG GCU GAU
`
`130
`120
`140
`ala ala pro thr val ser fle phe pro p~o ser ser glu gln leu thr ser gly gly ala ser val val cys phe leu asn asn phe tyr
`GCU GCA CCA ACU GUA UCC AUC UUC CCA CCA UCC AGU GAG CAG UUA ACA UCU GGA GGU GCC UCA GUC GUG UGC UUC UUG AAC AAC UUC UAC
`
`1~
`160
`170
`pro lys asp fle asn val lys trp lys fle asp gly ser glu arg gln asn gly val leu asn ser trp thr asp gln asp ser lys asp
`CCC AAA GAC AUC AAU GUC AAG UGG AAG AUU GAU GGC AGU GAA CGA CAA AAU GGC GUC CUG AAC AGU UGG ACU GAU CAG GAC AGC AAA GAC
`
`200
`190
`180
`ser thr tyr ser met ser ser thr leu thr, leu thr lys asp glu tyr glu arg hfs asn ser tyr thr cys glu ala thr hfs lys thr
`AGC ACC UAC AGC AUG AGC AGC ACC CUC ACG UUG ACC AAG GAC GAG UAU GAA CGA CAU AAC AGC UAU ACC UGU GAG GCC ACU CAC AAG ACA
`
`210
`214
`ser thr ser pro fle val lys ser phe as~ arg asn glu cys AM
`UCA ACU UCA CCC AUU GUC AAG AGC UUC AAG AGG AAU GAG UGU UAG AGACAAAGGUCCUGAGACGCCACCACCAGCUCCCCAGCUCCAUCCUAUCUUCCCUUCUAA
`
`GGUCUUGGAGGCUUCCCCACAAGCGACCUACCACUGUUGCGGUGCUCCAAACCUCCUCCCCACCUCCUUCUCCUCCUCCUCCCUUUCCUUGGCUUUUAUCAUGCUAAUAUUUGCAGAAAA
`UAUUCAAUAAAGUGAGUCUUUGCACUUGA
`
`~l
`
`~ .......
`3
`
`0 ....
`"' (JI
`
`0
`t\')
`c..,
`
`BIOEPIS EX. 1002
`Page 4015
`
`

`

`1
`
`101
`
`201
`
`301
`
`401
`
`ddel
`sau96
`avail mntI·
`s fa NI
`aha I I I
`M nfl
`al ul
`GAGTCAGCAC TGAACACGGA CCCCTC·ACGA TGAACTTCGG GCTCAGCTTG ATTTACCTTG TCCTTGTTTT AAAAGTTGTC CAGTGTGAAG TGATGCTGGT
`OTCAGTCGTG ACTTGTGCCT GGGGAGTGCT ACTTGAAGCC CGAGTCGAAC TAAATGGAAC AGGAACAAAA TTTTCAACAG GTCACACTTC ACTACGACCA
`
`I
`
`sau96
`
`scrFI
`fnu4HI
`' mn1I
`h 1 n f I
`h 1 n fl
`h 1n f I
`b b v m n 1I
`e.c o R 11 a v a I I
`GGAGTCTGGG GGAGTCTTAA TGGAGCCTGG AGGGTCCCTG AAACTCTCCT GTGCAGCCTC TGGATTCACT TTCAGTAGAT ATGCCATGTC TTGGGTTCGC
`CCTCAGACCC CCTCAGAATT ACCTCGGACC TCCCAGGGAC TJTGAGAGGA CACGTCGGAG ACCTAAGTGA AAGTCATCTA TACGGTACAG AACCCAAGCG
`hphl
`mnll
`hpaII
`hinfl
`mboII
`hfnfI
`CAGACTCCGG AGAAGAGGCT GGAGTGGGTC GCAACCATTA GTAGTGGTGG TAGTTCACAC CTTCCATCCA GACAGTGTGA AGGGCGATTC ACCATCTCCA
`GTCTGAGGCC TCTTCTCCGA CCTCACICAG CGTTGGTAAT CATCACCACC ATCAAGTGTG GAAGGTAGGT CTGTCACACT TCCCGCTAAG TGGTAGAGGT
`,
`fok I
`
`mnl I
`mntI
`.
`mn1I
`haeIIi
`ddel
`ddel
`rsaI
`GAGACAATGC CAAGAACACC CTGTACCTGC AAATGAGCAG TCTGAGGTCT GAGGACACGG CCATGTATTA CTGTGCAAGA CCCCCTCTTA TTTCGTTAGT
`CTCTGTTACG GTTCTTGTGG GACATGGACG TTTACTCGTC AGACTCCAGA CTCCTGTGCC GGTACATAAT GA~ACGTTCT GGGGGAGAAT AAAGCAATCA
`
`xhoII
`scrFI
`sau96
`sau3A
`ecoRII
`mnlI
`mnll
`haeJII
`dpnI
`ddeI
`ddeI hphI
`A~CGGACTAT GCTATGGACT ACTGGGGTCA AGGAACCTCA GTCACCGTCT CCTCAGCCAA AACGACACCC CCATCTGTCT ATCCACTGGC CCCTGGATCT
`TCGCCTGATA CGATACCTGA TGACCCCAGT TCCTTGGAGT CAGTGGCAGA GGAGTCGGTT TTGCTGTGGG GGTAGACAGA TAGGTGACCG GGGACCTAGA
`
`-
`
`(/\
`
`:E
`
`Fig.4A.
`
`/
`
`0 .....
`I\)/
`.·~
`/<II
`0
`I\)
`(,i)
`
`BIOEPIS EX. 1002
`Page 4016
`
`

`

`ncol
`
`xhoII
`scrFI
`sfaNl
`fokl
`sau3A
`scrF·I
`scrFI
`hphl ecoRI I
`frj.u4HI
`dpnl
`bamHI
`ecoRJI
`ecoRII
`bstEII
`bbv
`ddel
`GtTGCCCAAA CTAACTCCAT GGTGACCCTG GGATGCCTGG TCAAGGGCTA TTTCCCTGAG CCAGTGACAG TGACCTGGAA CTCTGGATCC CTGTCCAGCG
`C~AtGGGTTT GATTGAGGTA CCACTG~GAC CCTACGGACC AGTTCCCGAT AAAGGGACTC GGTCACTGTC ACTGGA~tTT GAGACCTAGG GACAGGTCGC
`sau96
`fnu4HI
`mn1I
`bbv ddeI
`pvuII
`hphI
`pstl:
`aluI
`haeIII
`ddeI
`mnll
`mnlI
`hgfA
`alul
`GTGTGCACAC CTTCCCAGCT GTCCTGQAGT CTGACCTCTA CACTCTGAGC AGCTCAGTGA CTGTCCCCTC CAGCCCTCGG CCCAGCGAGA ccGrCACCTG
`CACACGTGTG GAAGGGTCGA CAGGAC~TCA GACTGGAGAT GTGAGACTCG TCGAGTCACT GACAGGGGAG GTCGGGAGCC GGGTCGCTCT GGCAGTGGAC
`scrFI
`ha ell I
`ncfl
`· fnu4HI
`scrFI
`rsal
`ecoRII
`bg1I
`hpaII
`bbv
`ndel
`CAACGTTGCC CACCCGGCCA GCAGCAQCAA GGTGGACAAG AAAATTGTGC CCAGGGATTG TGGTTGTAAG CCTTGCATAT GTACAGTCCC AGAAGTATCA
`GTJGCAACGG GTGGGCCGGT CGTCGTGQTT CCACCTGTTC TTTTAACACG GGTCCCTAAC ACCAACATTC GGAACGTATA CATGTCAGGG TCTTCATAGT
`
`sau3A
`mstII
`dpnl mn1I
`hfnfI
`hphI
`fokl
`accI
`mboII mboII
`avaI
`fokl hg1A
`ddeI
`TCTGTCTTCA TCTTCCCCCC AAAGCCC~AG GATGTGCTCA CCATTACTCT GACTCCTAAG GTCACGTGTG TTGTGGTAGA CATCAGCAAG GATGATCCCG
`AGACAGAAGT AGAAGGGGGG TTTCGG~TTC CTACACGAGT GGTAATGAGA CTGAGGATTC CAGTGCACAC AACACCATCT GTAGTCGTTC CTACTAGGGC
`
`smal
`scrFI
`scrFI
`ncfl
`ncfl
`hpaII
`mn1I
`pvuII
`sau96
`ddeI
`avaI mnll
`aluI hgal
`hgfA
`alul
`a,aII
`AGGTCCAGTT CAGCTGGTTT GTAGATGATG TGGAGGTGCA CACAGCTCAG ACGCAACCCC GGGAGGAGCA
`TCCAGGTCAA GTCGACCAAA CATCTACTAC ACCTCCACGT GTGTCGAGTC TGCGTTGGGG CCCTCCTCGT
`
`ddeI
`GTTCAACAGC ACTTTCCGCT CAGTCAGTGA
`CAAGTTGTCG TGAAAGGCGA GTCAGTCACT
`
`501
`
`601
`
`701
`
`801
`
`901
`
`Fig. 48.
`
`"
`
`0-·
`
`-1'
`
`0 ....
`N
`U'I
`0
`N
`(A)
`
`BIOEPIS EX. 1002
`Page 4017
`
`

`

`1001
`
`1101
`
`1201
`
`1301
`
`1401
`
`1501
`
`fnu4HI
`bbv
`scrFI
`taql
`hfncII
`alul
`ecoRII
`ACTTCCCATC ATGCACCAGG ACTGGCTCAA TGGCAAGGAG TTCAAATGCA GGGTCAACAG TGCAGCTTTC CCTGCCCCCA TCGAGAAAAC CATCTCCAAA
`TGAAGGGTAG TACGTGGTCC TGACCGAGTT ACCGTTCCTC AAGTTTACGT CCCAGTTGTC ACGTCGAAAG GGACGGGGGT AGCTCTTTTG GTAGAGGTTT
`
`tr
`
`,
`
`haelII
`hael
`ball
`mnll
`rsal
`ACCAAAGGCA GACCGAAGGC TCCACAGGTG TACACCATTC CACCTCCCAA GGAGCAGATG GCCAAGGATA AAGTCAGTCT GACCTGCATG ATAACAGACT
`TGGTTTCCGT CTGGCTTCCG AGGTGTCCAC ATGTGGTAAG GTGGAGGGTT CCTCGTCTAC CGGTTCCTAT TTCAGTCAGA CTGGACGTAC TATTGTCTGA
`
`fnu4HI .
`ddel
`bbv ·
`mboll mboII
`TCTTCCCTGA AGACATTACT GTGGAGTGGC AGTGGAATGG GCAGCCAGCG GAGAACTACA AGAACACTCA GCCCATCATG AACACGAATG GCTCTTACTT
`AGAAGGGACT TCTGTAATGA CACCTCACCG TCACCTTACC CGTCGGTCGC CTCTTGATGT TCTTGTGAGT CGGGTAGTAC TTGTGCTTAC CGAGAATGAA
`sau96
`mnl I
`mboII
`mnll
`mboII
`alul
`acct
`ddel
`hphI
`haelll
`CGTCTACAGC AAGCTCAATG TGCAGAAGAG CAACTGGGAG GCAGGAAATA·'CTTTCACCTG CTCTGTGTTA CATGAGGGCC TGCACAACCA CCATACTGAG
`GCAGATGTCG TTCGAGTTAC ACGTCTTCTC GTTGACCCTC CGTCCTTTAT GAAAGTGGAC GAGACACAAT GTAcrqccGG ACGTGTTGGT GGTATGACTC
`scrFI
`sau3A
`sau96
`avail
`mn1I
`mnll
`hfnfl
`· mn1I
`·
`mn1I
`ecoRII
`dpnI
`~AGAGCCTCT CCCACTCTCC TGGTAAATGA TCCCAGTGTC CTTGGAGCCC TCTGGTCCTA CAGGACTCTG ACACCTACCT CCACCCCTCC CTGTATAAAT
`TTCTCGGAGA GGGTGAGAGG ACCATTTACT AGGGTCACAG GAACCTCGGG AGACCAGGAT GTCCTGAGAC TGTGGATGGA GGTGGGGAGG GACATATTTA
`AAAGCACCCA GCACTGCCTT GGGAAAAA
`TTTCGTGGGT CGTGACGGAA CCCTTTTT
`
`Fiff 4C.
`
`-J
`
`-.:6"
`
`0
`.A
`I\)
`01
`0
`I\)
`c:.,
`
`BIOEPIS EX. 1002
`Page 4018
`
`

`

`1
`wlO
`met asn phe gly leu ser leu fle tyr leu val leu val leu lys val val gln cys glu
`GAGUCAGCACUGAACACGGACCCCUCAC~ AUG AAC UUC GGG CUC AGC UUG AUU UAC CUU GUC CUG GUU UUA AAA GUU GUC CAG UGU GAA
`
`30
`20
`10
`val met leu val glu ser gly gly val le~ met glu pro gly gly ser leu lys leu ser cys ala ala ser gly phe thr phe ser arg
`GUG AUG CUG GUG GAG ucu GGG GGA GUC UUA AUG GAG CCU GGA GGG ucc CUG AAA cue ucc UGU GCA GCC ucu GGA uuc ACU uuc AGU AGA
`40
`50
`60
`tyr ala.met ser trp val arg gln thr pro glu lys arg leu glu trp val ala thr fle ser ser gly gly ser ser hfs leu proser
`UAU GCC AU~ ucu UGG GUU CGC CAG ACU cc~ GAG AAG AGG CUG GAG UGG GUC GCA ACC AUU AGU AGU GGU GGU AGU UCA CAC cuu CCA ucc
`70
`80
`90
`arg gln cys glu gly arg phe thr fle se~ arg asp asn ala lys asn thr leu tyr leu gln met ser ser leu arg ser glu asp thr
`AGA CAG UGU GAA GGG CGA UUC ACC AUC UCC1 AGA GAC AAU GCC AAG AAC ACC CUG UAC CUG CAA AUG AGC AGU CUG AGG UCU GAG GAC ACG
`
`120
`100
`110
`ala met tyr tyr cys ala arg pro pro leu ile ser leu val ala asp tyr ala met asp tyr trp gly gln gly thr ser val thr val
`GCC AUG UAU UAC UGU GCA AGA CCC CCU CUU: AUU UCG UUA GUA GCG GAC UAU GCU AUG GAC UAC UGG GGU CA~ GGA ACC UCA GUC ACC GUC
`
`150
`140
`130
`ser ser ala lys thr thr pro proser val tyr pro leu ala pro gly ser ala ala gln thr asn ser met val thr leu gly cys leu
`UCC UCA GCC AAA ACG ACA CCC CCA UCU GUC' UAU CCA CUG GCC CCU GGA UCU GCU GCC CAA ACU AAC UCC AUG GUG ACC CUG GGA UGC CUG
`
`160
`180
`170
`o()
`val lys gly tyr phe pro glu pro val thr val thr trp asn ser gly ser leu ser.ser gly val hfs thr phe pro ala val leu gln
`---
`GUC AAG GGC UAU UUC CCU GAG CCA GUG ACA GUG ACC UGG AAC UCU GGA UCC CUG UCC AGC GGU GUG CAC ACC UUC CCA GCU GUC CUG CAG ~
`
`200
`190
`210
`ser asp leu tyr thr leu ser ser ser val thr val proser ser pro arg proser glu thr val thr cys asn val ala hfs pro ala
`UCU GAC CUC UAC ACU CUG AGC AGC UCA GUG ACU GUC CCC UCC AGC CCU CGG CCC AGC GAG ACC GUC ACC UGC AAC GUU GCC CAC CCG GCC
`
`240
`230
`220
`ser ser thr lys val asp lys lys fle val pro arg asp cys gly cys lys pro cys fle cys thr val pro glu val ser ser val phe
`AGC AGC ACC AAG·GUG GAC AAG AAA AUU GUG CCC AGG GAU UGU GGU UGU AAG CCU UGC AUA UGU ACA GUC CCA GAA GUA UCA UCU GUC UUC
`
`Fig. SA.
`
`C)
`
`....
`~
`CJ1
`0
`N
`
`"'
`
`BIOEPIS EX. 1002
`Page 4019
`
`

`

`270
`260
`250
`11e phe pro pro lys pro lys asp val leu thr 11e thr leu thr pro lys val thr cys val val val asp ile ser lys asp asp pro
`AUC uuc CCC CCA AAG CCC AAG GAU GUG cue ACC AUU ACU CUG ACU CCU AAG GUC ACG UGU GUU GUG GUA GAC AUC AGC AAG GAU GAU CCC
`
`300
`290
`280
`glu val gln phe ser trp phe val asp asp val glu val his thr ala gln thr gln pro arg glu glu,·gln phe asn ser thr phe arg
`GAG GUC CAG UUC AGC UGG UUU GUA GAU GAU GUG GAG GUG CAC ·ACA GCU CAG ACG CAA CCC CGG GAG GAG CAG UUC AAC AGC ACU UUC CGC
`
`330
`320
`310
`ser val ser glu leu pro fle met hfs gfn asp trp leu asn gly lys glu phe lys cys arg val as~ ser ala ala phe pro ala pro
`UCA GUC AGU GAA CUU CCC AUC AUG CAC CAG GAC UGG CUC AAU GGC AAG GAG UUC AAA UGC AGG GUC AAC AGU GCA GCU UUC CCU GCC CCC
`
`360
`350
`340
`11e glu 1Ys thr 11e ser lys thr lys gly arg pro lys ala ·pro gln val tyr thr fle pro pro pro lys glu gln·met ala lys asp
`AUC GA