0022-1767/88/141 1 l—4053SO2.00/0
`Tar. JOURNAL or IMMUNOLOGY
`Copyright © 1988 by The American Association of Immunologists
`
`Vol. 14]. 4053-4060, No. ll. December 1. 1988
`Printed in U. S.A.
`
`CLONING AND HIGH LEVEL EXPRESSION OF A CHIMERIC ANTIBODY WITH
`SPECIFICITY FOR HUMAN CARCINOEMBRYONIC ANTIGEN
`
`CATHERINE B. BEIDLER, JAMES R. LUDWIG, JOSE CARDENAS, JULIA PHELPS,
`CAROL G. PAPWORTH, EVAN MELCHER, MICHAEL SIERZEGA. LAURA J. MYERS.
`BARBARA W. UNGER, MARY FISHER, GARY S. DAVID, AND M. JACQUELINE JOHNSON‘
`
`From the Department oflmmunobiology. Hybritech. 1710. San Diego. CA 92121
`
`A mouse/human chimeric antibody has been con-
`structed by using variable light and variable heavy
`regions from a murine hybridoma specific for hu-
`man carcinoembryonic antigen (CEA) (CEM231.6.7).
`These V regions were combined with x and 7-1 con-
`stant region genes cloned from human lymphocytes.
`The chimeric constructs were sequentially electro-
`porated into murine non-lg-producing myeloma
`(P3.653) and hybridoma (SP2/0) cell. Significant dif-
`ferences were seen in expression levels between the
`two cell types. High levels of expression (24 to 32
`ug/ml/106 cells) were seen with several of the anti-
`CEA SP2/0 transfectomas but not with the P3.653
`cells. The SP2/0 transfectoma lines were adapted to
`serum-free. chemically defined media and grown in
`large scale fermentation cultures where they con-
`tinued to secrete high levels of antibody. The chi-
`meric antibodies remain reactive against human
`CEA with affinity constants comparable to that of
`the parental hybridoma antibody. High level expres-
`sion will make practical the production of chimeric
`antibodies for in vivo therapeutic and diagnostic
`purposes.
`
`The therapeutic and in vivo diagnostic potential of mAb
`has been recognized for some time. Applications involv-
`ing naked antibody. immunotoxin or immunodrug con-
`jugates, and radiolabeled mAb have all been attempted
`(1-5). Murine mAb, however, have limited utility in re-
`peat dose protocols in that they usually elicit an immune
`response in humans (6-8). An obvious alternative would
`be to produce human mAb to reduce the immune re-
`sponse. In the few cases where human hybridomas have
`been produced they have usually proven to be unstable
`and to secrete lg at low levels (9). Furthermore, clinically
`useful human mAb against tumor-associated Ag have yet
`to be demonstrated (10). Another possible means of di-
`minishing the immune response is by decreasing the
`percentage of the antibody which is recognized by the
`human immune system as foreign. Several groups have
`achieved this by constructing chimeric antibodies con-
`taining human constant regions combined with murine
`V regions (11-13). Recently this technology has been
`
`Received for publication May 16. 1988.
`Accepted for publication September 8. 1988.
`The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`advertisement in accordance with 18 U.S.C. Section 1734 solely to indi-
`cate this fact.
`' Address correspondence and requests for reprints to M. J. Johnson,
`Hybritech lnc.. 11085 Torreyanna Road. San Diego. CA 92121.
`
`applied to mAb of potential clinical value (14-17). Our
`group has been working with several of these antibodies,
`including CEM23l.6.7. an antibody to CEA2 which was
`developed at Hybritech Inc. We have succeeded in pro-
`ducing a chimeric form of this antibody which can be
`expressed in mammalian cells. In addition, we have de-
`veloped several anti-CEA transfectoma lines which se-
`crete chimeric antibody in the range of 24 to 32 ug/ml/
`106 cells. After adaptation to serum-free chemically de-
`fined media and growth in large scale fermenters, these
`cells were able to secrete lg at levels of approximately
`100 pg/ml. Such expression levels have important impli-
`cations in producing chimeric antibodies for clinical use.
`Levels of expression under 50 pg/ml make large scale
`purification of antibody difficult and costly, as clinical
`protocols call for single doses up to l g (7, l8—22).
`
`MATERIALS AND METHODS
`
`Isolation of human K and 7-1 constant region genes. DNA was
`isolated from PBL of individuals homozygous for the two common
`Gm haplotypes, azg andfbn (23). Ten micrograms of azg haplotype
`DNA was digested with the restriction enzymes Hindlll and BamHI
`according to the supplier’s conditions (BRL, Gaithersburg. MD). The
`digested DNA was separated by electrophoresis in 0.6% LMT agarose
`gel [FMC Corp. Rockland. Maine) in 40 mM Tris-acetate and 2 mM
`EDTA (pH 8.0) and the 6 to 7.5-kb size DNA was excised from the
`gel. This DNA was directly ligated into a pUC 18 cloning vector.
`Escherichia coli M15 cells were transformed and selected on X—gal,
`isopropyl
`thiogalactoside. ampicillin plates. Transformants were
`screened by using a human 7-2 probe (gift of T. Honjo, Osaka
`University]. A clone containing a 7.5-kb insert corresponding to the
`human 7-1 gene was identified by restriction digestion with Sau3A
`and Mbol
`(24). The fragment was subsequently subcloned into
`pBR322 and designated pHGlZ.
`Human DNA of theflan haplotype was partially digested with Mbol
`according to the supplier's conditions (BRL) and DNA in the M, range
`of 12 to 24 kb was isolated and cloned into EMBL—3 phage arms
`obtained from Stratagene. Inc. (San Diego. CA] by using methods
`described by Davis et al. (25). The resulting library was screened
`with a human x probe (gift of P. Hieter. Johns Hopkins University)
`to identify a recombinant phage which was designated OHKFI. DNA
`from this phage clone was digested with restriction enzymes Hindlll
`and Bamlil (BRL) and a 5.2-kb fragment was isolated on DEAE 81
`paper after electrophoresis. The isolated fragment was ligated into
`Hindlll/Barnlil digested pUC 18. After transformation. ampicillin-
`resistant colonies were screened to identify the human K-fragment
`which was subsequently subcloned into pBR322 and designated
`pHFKl.
`Isoiation of murine CEM23l.6. 7 V“ and V,_ genesfrorn EMBL—3
`libraries. The expressed 1 and x genes from the murine hybridoma
`CEM23l.6.7 were cloned by Mbol partial digestion of the genomic
`DNA followed by ligation of the 12- to 20-kb size selected DNA into
`EMBL—3 phage arms by using protocols provided by Stratagene. Inc.
`Recombinant phage containing lg genes were identified by screening
`with murine probes representing the 7-1, Jh3-4. x and J1. region
`sequences. The 7 and x constant region oligonucleotide probes were
`
`2 Abbreviations used in this paper: CEA, carcinoembryonic Ag: V“ and
`VL. variable heavy H and L chain region; DTT. dithiothreitol.
`4053
`
`Genzyme Ex. 1040, pg 925
`
`Genzyme Ex. 1040, pg 925
`
`

`
`4054
`
`CHIMERIC ANTIBODY WITH SPECIFICITY FOR HUMAN CEA
`
`derived from sequences in the NIH Data Bank (Accession #J00453.
`J00545) and synthesized by using standard techniques (Molecular
`Biosystems Inc., San Diego, CA]. The Jh3-4 region plasmid was a
`gift from Dr. P. Tucker (University of Texas Health Science Center.
`Dallas. TX]; the Jk sequence containing plasmid was a gift from Dr.
`M. Shulman (University of Toronto, Toronto, Canada).
`The rearranged Ig clones were identified by duplicate hybridiza-
`tions to either the 7-1 and Jh probes for the H chain gene for the K
`and J1. probes for the L chain gene.
`Construction of chimeric Ig gene vectors. The murine K and 7
`genes were first isolated from the recombinant EMBL-3 phage and
`subcloned into Bluescript M13SK* (Stratagene) for restriction map-
`ping. After this. the V,_ exon was isolated as a 3.8-kb Hindlll frag-
`ment and subcloned in pHKF1 to form a chimeric L chain gene
`plasmid——pHKCE-10. The V” exon was subcloned into pHG1Z by
`cutting at the upstream Clal site in the Bluescribe vector, followed
`by a HindIII partial digest and isolation of a 5.6-kb Clal-Hindlll
`fragment. The resulting H chain chimeric plasmid was designated
`pHGCE-30.
`Construction of chimeric Ig expression vectors. In order to insert
`the chimeric lg genes into expression vectors, a Clal linker was
`inserted at the EcoRI site in both pSV2gpt and pSV2neo: insertion
`of the Clal linker does not destroy the EcoRI site. The resulting
`expression vectors pSV2gpt-Cla and pSV2neo-Cla were then digested
`with Clal and BamHI as was the DNA from the pHKCE-10 and
`pHGCE-30 chimeric plasmids. Fragments of 9 and 12.7 kb were
`isolated from the L and H chain chimeric vectors, respectively, and
`ligated into the two expression vectors. E. coli HB10l competent
`cells (BRL) were used in the transformation. DNA from ampicillin
`resistant plasmids was analyzed by restriction digest to identify the
`four expression plasmids: pGCEMK. pGCEMG1 (pSV2gpt based).
`pNCEMK. and pNCEMG1 (pSV2neo based).
`Nucleotide sequencing of CEM231.6.7 V region genes. The nu-
`cleotide sequences of the VL and VH exons were determined by using
`primer extension of ssDNA. Initially, murine sequences derived from
`the NIH Data Base corresponding to the Jm and Jks sequences were
`used as primers. Protocols followed were those recommended by the
`suppliers of the sequencing kits (Stratagene. Inc. and Sequenase
`from U. S. Biochemicals, Cleveland. Ohio). Additional primers needed
`to complete the sequencing of the V“ and V._ exons were synthesized
`on a model 8700 Biosearch DNA Synthesizer (New Brunswick Corp..
`Edison. NJ).
`Transfection of DNA into murine cells by electroporation. DNA
`was introduced into the myeloma line 133.653 and hybridoma line
`SP2/O-Agl4 by electroporation (26. 28). Chimeric Ig K plasmid DNA
`was linearized with Pvul in a nonessential part of the vector and 20
`pg was added to 107 cells in 0.3 ml HBSS [GIBCO. Gaithersburg, MD)
`with 6 mM dextrose. 0.25 M HEPES. The mixture was kept at 4°C
`for 10 min, then electoporated by using conditions of three 100-[.l.S
`pulses of 300 V each. Cells were incubated at 37°C, 5% CO2 for 72
`h then plated at 105 cells/well in media containing mycophenolic
`acid (GIBCO) 1 ltg/ml. 250 pg/ml xanthine, and 150 pg/ml hypoxan-
`thine for selection of cells containing gpt-resistant plasmids or 1
`mg/ml G418 (GIBCO) for cells containing neo-resistant plasmids.
`Cells were first identified which expressed the chimeric K protein.
`these were then pooled and the CEM chimeric 7 gene was introduced
`by electroporation. Conditions used for transfection of the 7 chimeric
`plasmid were either three pulses of 100 [LS at 300 V or one pulse of
`5 ms at 250 V.
`Cell culture. Hybridoma and transfectoma clones were cultured
`at 5% CO2. 37°C in a modified Dulbecco‘s modified Eagle‘s medium
`F12 (Irvine Scientific. Irvine, CA) containing 5% FCS (29). Transfec-
`tomas secreting chimeric antibody were adapted to serum-free con-
`ditions by direct transfer into modified Dulbecco‘s modified Eagle’s
`medium without serum. Large scale culture was accomplished by
`maintaining logarithmic growth in serum—free medium through 100-
`, 250-. 500-. and 1000—m1 spinner flasks and into 10- and 90-liter
`fermenters (Marubishi. Japan).
`ELISA assay of chimeric CEM K expression. Drug-resistant cells
`were analyzed for K expression in 96-well plates (Titertek. McLean.
`VA) by using standard ELISA techniques (30) with goat anti-human
`K and alkaline phosphatase goat anti-human K-chain antibody re-
`agents (Tago #4106 and #4296, Burlingame, CA). Plates were coated
`with 50 pl of goat anti-human K chain at 5 /.g/ml in 10 mM phosphate
`(pH 7 to 8), and dried overnight at 37°C. Plates were washed with
`PBS + 0.1% Tween-20.
`then with H20. Fifty microliters of cell
`supernatant were added to each well and incubated for 2 h at room
`temperature. After washing, 100 pl of a goat anti-human K chain
`alkaline phosphatase conjugate diluted 1/100 in RPMI. 10% horse
`serum. and 3% goat serum were added to each well. Plates were
`incubated for 1 h. at 20°C on a rotator and then washed as described
`above. p-Nitrophenylphosphate substrate was added at 150 pl/well
`and incubated on a rotator at 37°C for 45 min. The reaction was
`
`quenched with the addition of 50 pl/well of 500 mM EDTA. and the
`OD were read at 405 nm by using the Bio-Tek EL310 microplate
`autoreader.
`ELISA qf assembled chimeric antibody expression. Detection of
`assembled antibodies was carried out by coating the microtiter plate
`wells with goat anti-human IgG antibody reagent (Tago 33100) at 5
`ug/ml in 10 mM phosphate pH 7 to 8. Plates were dried overnight at
`37°C. then washed with PBS and 0.1% Tween-20. then H20. Fifty
`microliters of the cell supernatant were added to each well and the
`experiments were continued as described above in the chimeric CEM
`K expression ELISA assays. Purified protein from a human hybrid-
`oma (YCA343. developed at Hybritech Inc.) was used as a positive
`control. Because this is a 7-2/It protein, the conjugated antibody
`reagent used was an alkaline phosphatase-conjugated goat anti-
`human >\-chain antibody (Tago #4108).
`Quantitation of assembled chimeric antibodies. The quantita-
`tion of intact antibody was accomplished essentially as described in
`the assembled lg ELISA. however. we were able to use purified
`chimeric CEM antibody (7/K) instead of the human 7/A antibody as
`calibrator. Standard curves were generated by plating serial dilu-
`tions of a known concentration of the chimeric CEM antibody in a
`range of 5 to 180 ng/ml. The optical densities were read at 405 nm
`and each sample was quantitated against the appropriate standard
`curve.
`
`Protein characterization of chimeric CEM antibodies. For analy-
`sis of chimeric antibody proteins, the xCEM449. 18 and xCEM449.08
`transfectomas clones. the SP2/0 recipient and the CEM231 parental
`cell lines were each plated in serum free media at a density of 2 x
`105/ml. After 3 days in culture, cells were pelleted and the super-
`natant fractions were concentrated 7- to 10-fold in an Amicon
`Stirred Cell apparatus with a YM 30 filter. The concentrated super-
`natants from these cell lines as well as purified antibody from a
`human hybridoma (YCA343) and a purified human K Bence Jones
`protein (Kallestad, Austin. TX] were analyzed by modified Laemmli
`10% SDS-PAGE reducing and nonreducing gels (31. 32) as well as
`by western blotting of PAGE gels (25). Approximately 2.5 pg of Ig
`protein was analyzed per sample. Reduced samples included 0.1 M
`171"!‘ in the SDS sample buffer. All samples were boiled for 5 min
`before loading. Low m.w. protein standards (Pharmacia. Piscataway,
`NJ) were routinely chromatographed on the gels. Protein gels were
`stained in methanolzacetic acid: H20 [4:1:6) with Coomassie blue for
`30 min. then destained for approximately 2 h before photographing.
`For Western blot analysis. protein was transferred to nitrocellulose
`(Schleicher & Schuell, Keene. NH) by using a Hoeffer Trans-Blot
`apparatus at 100 mA for 2 h. Detection of human and murine H and
`L chain Ig proteins was carried out with ‘Q51-labeled anti-human or
`anti-murine lg antibodies (Tago #4106, 3100. 4100 and Fisher
`BioTech *1 137-21, Pittsburgh, PA). The nitrocellulose filters were
`incubated overnight with the iodinated antibody probes in Blotto (5%
`w/v nonfat dry milk in PBS), then washed in PBS. 0.1% Tween for
`1 h at room temperature. After this, the filters were exposed over-
`night using Kodak X—AR film at —80°C.
`Affinity constant determinations of chimeric CEM antibody.
`Assays were performed to determine the K. of the chimeric CEM
`antibodies (xCEM449.l8 and xCEM449.08) for the CEA Ag. Affinity
`was determined by Scatchard analysis (26) of standard solid phase
`radioimmunoassay data (27). The following reagents were added to
`wells of a microtiter plate and incubated overnight at room temper-
`ature with mixing: 20 pl of Sepharose bound goat anti-human IgG.
`25 M1 concentrated culture media containing the chimeric antibody.
`50 [[1 of ‘Z51-CEA (purified) and either 25 ill of unlabeled competitor
`CEA or cell media. Competition assays were generated by the addi-
`tion of unlabeled CEA. Immune complexes bound to the anti-human
`IgG Sepharose were collected onto glass fiber filters and counted in
`a gamma counter. A Scatchard plot of bound/free vs bound antigen
`permitted calculation of the affinity constant as defined by the
`negative slope of the line.
`
`RESULTS
`
`Isolation and sequencing Qf the CEM231.6.7 genes.
`Phage clones were isolated which hybridized to the mouse
`7-1 and Jh probes or K and J, probes to identify the H
`and L chain CEM lg genes, respectively. Restriction map-
`ping of the DNA from isolated phage enabled us to sub-
`clone smaller regions representing the V” and V1, exons
`into plasmid vectors.
`The V“ exon and major H chain intron were isolated on
`a 5.6-kb Sstl fragment and subcloned into the Bluescript
`M13SK-vector (Stratagene, Inc.). This plasmid was des-
`
`Genzyme Ex. 1040, pg 926
`
`Genzyme Ex. 1040, pg 926
`
`

`
`CHIMERIC ANTIBODY WITH SPECIFICITY FOR HUMAN CEA
`
`4055
`
`ignated pMHCE-30. The VL and K gene sequences were
`isolated on a 10-kb BamHI fragment and subcloned into
`pBR322. Detailed restriction maps are shown in Figure
`1. From these maps we found that the CEM V] gene had
`rearranged to the Jks sequence, although we were unable
`to determine whether the Vh gene was rearranged to the
`J,,, or J,,2 exon.
`The hybridoma variable regions were sequenced using
`both the Stratagene and Sequenase systems following
`the protocols recommended by the supplier. The genomic
`DNA sequences representing the cloned heavy and light
`chain CEM23l.6.7 genes are shown in Figure 2. The
`absence of several restriction sites discovered during
`mapping of the heavy chain gene was clarified during
`sequencing as having resulted from a deletion which
`includes the entire Jhg region of the gene. This deletion
`is indicated in Figure 2.
`Construction of the chimeric H and L chain expres-
`sion vectors. The CEM231 V“ gene was isolated on a 5.6-
`kb fragment which resulted from a Clal complete, Hindlll
`partial restriction digest. This fragment was then sub-
`cloned into a pBR322 based vector—pHGlZ. which con-
`tained our previously cloned human ‘y-1 gene as described
`in Materials and Methods. A 3.8-kb Hindlll fragment
`representing the VL leader was fused to the human x gene
`in the pBR322 based vector——pHKFl.
`The chimeric lg H and L chain gene fragments were
`then subcloned into the eukaryotic expression vectors
`pSV2gpt and pSV2neo as shown in Figure 3. A and B.
`The L chain expression vector, designated pGCEMK, con-
`tains the gpt gene and the H chain chimeric expression
`vector, pNCEMG1 contains the neo gene. The alternate
`expression vectors pNCEMK and pGCEMGl. were also
`constructed in the same manner but are not shown.
`
`Introduction of chimeric genes into cells. Twenty mi-
`crograms of the pGCEMK vector was linearized at the
`Puul site and introduced into both SP2/O and P3.653
`cells by electroporation (28, 33, 34). Recipient cells were
`selected in mycophenolic acids, with observed transfec-
`tion efficiencies of approximately 1 X 10'“. After 2 wk,
`supernatant fractions were assayed for “human" K-chain
`expression and cells from the four highest scoring super-
`natants of SP2/O-xCEM x and P3.653-xCEM K were
`pooled. respectively. These cells were then expanded in
`number and used as recipients for the introduction of the
`chimeric 7-chain gene. Twenty micrograms of the plas-
`mid pNCEMGl were linearized with Pvul, then electro-
`porated into the K expressing SP2/O and P3.653 cells.
`Cells were maintained in mycophenolic acid for 72 h
`post-transfection and then plated in media containing
`mycophenolic acid and G418.
`
`ELISA analysis qf xCEM transfectomas. Supernatant
`fractions from cells resistant to both drugs were assayed
`for the presence of assembled antibodies using standard
`ELISA procedures (see Materials and Methods). The ini-
`tial screening data, shown in Figure 4 represent 214
`supernatant samples from a total of four electroporation
`experiments.
`The efficiencies of obtaining drug resistant colonies
`following transfection differed somewhat between the
`four experiments; in the first transfection 34% of the
`wells plated were resistant to the antibiotics, whereas in
`the second, third and fourth transfections, the percent-
`ages were 68%, 57 %. and 63%, respectively. No consist-
`ent differences in transfection efficiencies was seen be-
`tween the two different cell lines. The SP2/O transfec-
`tions when assayed however, produced more colonies
`secreting chimeric lg as compared to the P3.653 cells.
`The transfection experiments using P3.653 cells pro-
`duced only one colony which secreted Ig in a range >O.4
`ELISA OD units while the SP2/0 transfections produced
`43 colonies with OD readings >0.4. The range of OD
`readings from 0.2 to 0.5 seen with many of the P3.653
`colonies assayed was consistent with earlier expression
`data observed with an anti-TNP producing transfectoma.
`In these transfectomas, similar OD readings corre-
`sponded to a range of 1 to 5 pg/ml/10“ cells (our unpub-
`lished results).
`The four transfections also differed in the parameters
`used for introduction of the second chimeric gene, the 7-
`chain. In experiments 1 and 3 conditions used were three
`100-ps pulses of 300 V each, whereas in experiments 2
`and 4 one 5—ms pulse of 250 V was used. As already
`discussed, transfection efficiencies did not vary greatly
`among three of the four experiments, however more lg
`expressing colonies were seen in experiments where the
`latter conditions were used to introduce the 7-chain gene.
`Another parameter distinguishing the four transfec-
`tions was the association of drug resistance genes with a
`particular Ig gene. The two P3.653 transfections which
`gave rise to lg expressing cells were carried out by using
`the pNCEMK vector followed by the pGCEMG1 vector.
`The inverse combination of these vectors (i.e., pNCEMG1
`and pGCEMK) did not give rise to lg-expressing colonies.
`Different results were seen with the SP2/O cells; here the
`combination of pGCEMK followed by pNCEMG1 yielded
`lg-expressing colonies. Again the inverse vector combi-
`nation was attempted, but drug-resistant colonies did not
`express chimeric lg at detectable levels. Because of the
`small sample set represented here, it is difficult to draw
`conclusions as to which paired set of vectors might ex-
`press the most lg protein.
`
`Ban
`
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`
`Genzyme Ex. 1040, pg 927
`
`Figure 1. Partial restriction maps of
`CEM23l .6.7 V" and VL chain genes. A
`BamHl fragment containing the L chain
`gene was subcloned out of the original
`phage vector into pBR322 to form pMLCE—
`10. An Sstl fragment containing the H V“
`chain region was similarly subcloned into
`Bluescribe (M13 SK‘) to form pMl-{CE-30.
`The murine enhancers are denoted by E.
`
`Genzyme Ex. 1040, pg 927
`
`

`
`4056
`
`CHIMERIC ANTIBODY WITH SPECIFICITY FOR HUMAN CEA
`
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`Tannecacrsnrrrncrrsseaiccnnccssrncncrssesreccrsnrcecrrencasscnsrssarcrsssecnsnr1rcncrcrenccerraccnar
`.
`_
`.
`_
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`_
`_
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`— — — - —» — — — —— —
`- — - — — — — —
`— — — »
`— —_—¢—~—— »—.-.—»—,
`-A—.._.—~ ,__..-,-_ ___-.___-__‘__,____ -___,
`atI1:5Ten:rennrsnnccsrnssrrssccnrorsnecrcnssaacrnaccansrsrccsrceccrasacccrsrcrnaasrsnsnsrssrnnrsarrn
`LgsfilaLPulleTgrLeuHla9EFHsnfiPg19rThrEIgValPrDHspHrgPheThrB]9§ePGlHSEFGIgThrRspPheThrLauThrIleThrfi5n
`
`200
`
`300
`
`500
`
`:n.Jnr ~~«>
`IFrem9unrk 4 —~—>
`lED9 3 —-—>
`:1 Q
`:JK5:
`1
`BTGCRHYETGHHGHEETGEERGRTTRTTTCTBTETGERRCRTTBBRHYTRTCEGCYCRCGTTCGGTBCTGGERCCRHGCTBGRGCTERnnCGT:RE?2ER
`—--—.————.-——-_—-——»—-——.———->—-__,—___.—---.—-__.---.V,-—-._-_»_ . _ . _ _ _ --_ _ _ _ , .. --_ . c _ _ _ , __
`‘
`A-~—+
`‘
`cncsrtannerreresnccsrcranrnansncnoncsrrsInnccrrnarnsocsnsIscnnsccncsncccrearrcsaccrcsncrrrscnrrcarer
`UslGlnSwrGluHspLauRlaR9pTgrPh=CgsLauGInHi3Trp9snTgrPraLauThrPh9G15fllaGl3ThrLgsL$uGluLauLgsHrg
`
`600
`
`of
`gequence
`Figure2. Nucleotide
`CEM231.6.7 genomic variable regions.
`Sequences were determined as described
`in the materials and methods. A, L chain
`
`-—«—, ——~-»—-—-. ~-— 618
`C"'"3'C‘”C‘"”"
`GRHRHGRBTHGHHHRRRH
`
`variable region; B. H chain variable re- B
`I5Ignal Peptide Intron --->
`--->
`{Signal Pap:.aa - Exam 1
`'
`gion. A 375-bp deletion that spans the
`entire JH2 region resulted in the loss of
`GarcnscflcraancacncnccacTcnccérssncrECasscTcnnrIrasrrrrccrrsrccrrarrrrsnnaocrnnr1rsrnefisnreasrricracc
`-—--,4-——»
`¢
`_
`+
`_
`,
`V
`,_
`_
`9
`.
`—- — - — ———+——-— -——— ~«—— ——-»+-—-— -——— -——— —--— -—-— A———«~—-- ———A+--~— ~-—-»~-— ———~+
`Pstl, Hlndlll. and Bglll restriction sites.
`ETHBTCBTGRDTTGTBYETEGTBRBTSGTRCCTEHGETEEEHGTTRRRYCRRRRGBRQCRGEHHTRRRRTTTYECRTTHRRERTCTCYRETCRRHBRCBG
`HctRsDSFrHr9LEuRsnLSuVa|Ph¢LeuUe|Lou|loL¢uLgs
`
`IS|gnal nap.
`IExon 2 ~——>
`rotisIsrecccnassnnarasanacnrrsrrrarrrcniravirravitrstrssrancasrrircieaccaocnrrcrersiiiscassrsiccnors
`._-,, _..._. K V _ _
`. -__.-_-- _,__,_-, _,_-+t,__ _....___,
`r—7-.,—<._—-—-.
`- - - - — — ——.~——— ——-—.——-_ —_7—
`—
`ncnnéacncssarrcerrrnrcrIrGrnncannraangrnaraaénTnnaacaneearrercaaaaancresrcsrnnsaaacnfiacerccncqssrcqc
`GlgUalSlfiC9s
`icon l
`--—:
`-~—>
`iFrgmeuark 1
`TGRTGTGCRGCTGGTGGRGTCTGGGGGRBECTTHETBCRGCETEBRGGGTCCCGGRRHCTCTECTGTECHGCCYCTGGHTYCHCTTTCHGTRHETTTGGR
`«SE?EeéééiééééEéEEFEQESEE&EE?EEE?q$?eFuEEs¥E6u3E.Efi?EEE$éEEEe?¥¥E3éF.éé£aEEaEé¥EEé£.¢éEE?$s§E¥ém3$.§?E£§¥8é.A?aEE?
`flspValGlnLeuValGlu5arGlgGlgGlgL¢uUd1GlnprufilgGlgSarHrgLg9L9uSsrC3aRIafilaSkrGlgPh¢ThrPh9SarRsnPheGlg
`i7Famauark 2 ~——>
`ECDQ 2 —-->
`E——>
`RTGERéYBBRTTCBTCRGGCTCCRGHGQQGEBHETBGRGT5éGTC5CfiYRERTTRBTEBTBBDHGTHBTHECHTETRETRTBERGQCRERBTGRHBBECC
`F5612+és§ES?§.§éE-aé?EEé§5é¥é¥E¥¥EEE?éAEEEéééééééé F.?é?s§»§i£s§EE$EEé?e§¥Eé?éé?$é.E.¥éé ?s§Eé¥e rE?é?E%iE.-J iééééé
`HatH|sTPpllafirgfilhfilaPro5luLg95l3Lau5luTrpUalHlaTgrllwSarGl3G|gS&rSarThrIlBT5rTgrRleH5pThrValLgsGlgRrg
`rramauork 3 -—->
`:con 3
`;,-.»
`GRTTCRCCRTCTCCRGRBHCHRTCECRBGHRCRCCCTCTTCCTBEHRHTBHCCRGYETRHGGTCTGRGGRCRCGGCCRTGTTTIRETGTGERRGRGHYTH
`_a-7 »—-~¢——~— ~-——+-—--,»AA-+-——v —~--4«—4 — - — « — — — - » — A »~¢———- ——~—+————,~———+———— «—~_._—»7 7»-».
`CTRRGTSDTRGRGETEYETBTTRBGBTCETTBTGGERBHRGGREBTTTRCTBBTCREHTTCCRGRCTEETGTECCBGTRCHRRRTGHCRCGTTLTEVRRT
`ph9ThrlXESErHrgHepH:nPruHr9fl:nThrLfiuPheL9uG|nHe£ThrSarLauHrgS:rGluR:pThrfilaMetPhgl3rCg$R|aHrqflspTgr
`1Frameuork 4 --—>
`:naJor
`Intron —
`:JH-\ 7-->
`:
`CTHCGCTRNCHHCTacrastncrYCGHTGTCYGGEGCGCRGGGRCCRCGGvcficcsrcfccYCHGGIHHGCTGGCTTITTTLTIicioracfitifcnrru
`——~4 —»—~._»—— —~——+—~—— -_——+«—»4 ——..-¢_-4- ——~_+——~_ —-««+»——— ——~—¢»—A- «-449 --— «—--o—~-— ~- -4
`GnvscsniTorTGRrGnccnrsanscrncnsnccccscsrcccTsGTsccn515scnsAsGasTcearrcsacceennnnaoanasacnrsioaonvnau
`TgrfilaR3nR$nTgrTrpT9rph=RapVn1YrpGIgHlaGlgThrTHrValThrUa|SerSer
`375 bp delstlun at this site
`
`IGHRRTGGGHHHRERTTTYCTéRGRTCTCCCCRTGTCHGGCCRTCCTGGCYGRTGCRGHCRGRCHTCCTCRGCYCCCRuHCIT
`..._ —_-.._,-~ —_-—.--_-__—,A.~.-_ »_--._—r- _—~—.-.—,
`.-/~._V,— 77*-.. _.
`. —.-.— ea:
`ncrrrncccrrITCiaaaasnsrernsnsessracesrcc5BTesGacesncTac5rcrorcrsinoenciconéesrcronn
`
`100
`
`200
`
`3°“
`
`‘O0
`
`son
`
`emu
`
`Quantitation of SP2/O xCEM transfectomas. The
`highest OD readings from the initial ELISA screen were
`used to identify those cells producing the most chimeric
`Ig protein. This method only provides semiquantitative
`data due to the variation in cell density and colony num-
`ber among the individual wells, therefore several of the
`transfectomas (nine SP2/O and one P3) were expanded
`for a more thorough quantitation. We were unable to
`maintain lg expression in the one P3 transfectoma which
`was selected, however, the nine SP2/0 transfectomas
`remained positive for lg secretion. The three best SP2.0
`transfectomas secreted chimeric antibody in the range of
`8 to 17 pg/ml/105 cells as shown in Table IA. The highest
`
`producing cell population xCEM449 was selected for sub-
`cloning and further characterization.
`Subcloning and quantitation of the SP2/O xCEM
`transfectomas. The SP2/O derived xCEM449 cells were
`subcloned and supernatant fractions were assayed to
`quantitate levels of secreted lg. The standards used for
`the unsubcloned and subcloned transfectomas differed
`
`in the two assays. For the unsubcloned supernatant frac-
`tions, an HPLC-purified human 7-1/)\ antibody was used
`as a calibrator for quantitation. Later we were able to use
`HPLC-purified xCEM449.08 (7-1/K) chimeric antibody as
`the standard for quantitation of the supernatant frac-
`tions from the subcloned transfectomas. The data show
`
`Genzyme Ex. 1040, pg 928
`
`Genzyme Ex. 1040, pg 928
`
`

`
`CHIMERIC ANTIBODY WITH SPECIFICITY FOR HUMAN CEA
`
`4057
`
`(3.0. 405mm
`
`-
`
`Eco Ela
`
`Hind
`
`Amp
`
`panazz Dr:
`
`Hind
`Xbn
`
`Hlfld Eco Ban Xba
`
`pN[:Em31
`f 17.5 Kb]
`
`E”
`
`Xbn
`
`Hind
`
`[II]! HJIDEIIIUJEU EDEIJHEHIEIEED [ml] DD ElEllIl|J11I[l]l]ED
`
`D
`
`Human Gcmmal
`Figure 3. Structures oi the chimeric expression vectors. A, The V._
`region was isolated as a Hindlll fragment and inserted in front of the
`human K constant region of pl-lFK—l. The chimeric gene was then trans-
`ferred as a Bamlil/Clal fragment into pSV2gpt (Cla) to form the expres-
`sion vector pGCEMK. B. The V“ chain V region was isolated as a ClaI/
`Hindlll fragment and inserted in front of the human 7-l region in pHG1Z.
`The chimeric gene was then transferred as a Clal/Baml-ll fragment into
`pSV2neo (Cla) to form the expression vector pNCEMG.
`
`that four subclones secreted Ig in the range of 10 to 32
`ug/ml/ 106 cells (Table 1B). The two best subclones,
`xCEM449. 18 producing chimeric lg protein at 24 pg/ml/
`106 cells and XCEM449.08 producing 32 pg/ml/106 cells.
`were adapted to serum—free media for large scale produc-
`tion. In 90—liter fermenter cultures, in the absence of
`antibiotics, both subclones secreted >100 pg/ml of chi-
`meric antibody under terminal culture conditions.
`Analysis of xCEM449 transfectoma derived antibod-
`ies. Supernatant fractions from the xCEM449.18 and
`xCEM449.08 subclones, the SP2/0 recipient cell line, and
`the CEM231.6.7 parental hybridoma were concentrated
`7- to 10-fold. These along with purified human Bence
`Jones K protein (Kallestad, Austin, TX) and a human
`hybridoma-derived antibody (IgGl, A] were further ana-
`
`Figure 4. ELISA results from initial screening of transfectomas. 1,
`P3.653 recipient cells. electroporation of pNCEMK by using three 100-ps
`pulses at 300 V, sequential electroporation with pGCEMGl by using same
`conditions; 2, P3.653 recipient cells. electroporation pf pNCEMK using
`three 100-}LS pulses at 300 V, sequential electroporation with pGCEMGl
`using conditions of one 5 ms pulse of 250 volts; 3. SP2/O recipient cells,
`electroporation of pGCEMK by using three 1 00-us pulses at 300 V followed
`by sequential electroporation with pNCEMGl using same conditions: 4,
`SP2/0 recipient cells. electroporation of pGCEMK by using three 100-us
`pulses at 300 V, sequential electroporation with PNCEMG1 by using
`conditions of one 5—ms pulse of 250 V.
`
`TABLE I
`Quantitation Qfanti—CEA chimeric IgG"
`
`Transfectomas
`A. Unsubcloned transfectomas
`xCEM439
`xCEM449
`XCEM4-77
`B. Subcloned transfectomas
`xCEM449.08
`xCEM449. l 3
`xCEM449. 18
`xCEM449.24
`
`(fig/isle/r1e5E3o:e1Is)
`8
`1 7
`1 l
`
`32
`1 7
`24
`10
`
`° The calibrator used to quantitate lg production from the unsubcloned
`transfectomas was an I-IPLC-purified human hybridoma lg (YCA343)
`which is a 7-1/)\ protein. The calibrator used to quantitate lg production
`from the subcloned transfectomas was HPLC—purified chimeric antibody
`from xCEM449.08. All quantitation results were standardized for amount
`of Ig produced/ml of culture fluid/ 105 cells.
`
`Genzyme Ex. 1040, pg 929
`
`