0022- 1767/86/137?» 106630200/O
`THE JOURNAL or IMMUNOLOGY
`Copyright © 1986 by The American Association of Immunologists
`
`Vol. 137. l066~1074. No. 3. August 1, 1986
`Printed in U.S.A.
`
`A GENETICALLY ENGINEERED MURINE/HUMAN CHIMERIC ANTIBODY
`RETAINS SPECIFICITY FOR HUMAN TUMOR-ASSOCIATED ANTIGEN
`
`BARBARA G. SAHAGAN, HAIMANTI DORAI. JO SALTZGABER-MULLER. FRANCES TONEGUZZO.
`CATHY A. GUINDON. SARAH P. LILLY, KEVIN W. MCDONALD. DAVID V. MORRISSEY,
`BARRY A. STONE. GARY L. DAVIS, PHYLLIS K. MCINTOSH, AND GORDON P. MOORE
`
`From New Technology Research. E. I. DuPont de Nemours and Co.. 331 Treble Cove Road. N. Billerica. MA 01862
`
`Chimeric immunoglobulin genes were constructed
`by fusing murine variable region exons to human
`constant region exons. The ultimate goal was to
`produce an antibody capable of escaping surveil-
`lance by the human immune system while retaining
`the tumor specificity of a murine monoclonal. The
`murine variable regions were isolated from the func-
`tionally expressed x and 7 1 immunoglobulin genes
`of the murine hybridoma cell line B6.2, the secreted
`monoclonal antibody of which reacts with a surface
`antigen from human breast, lung, and colon carci-
`nomas. The x and -y 1 chain fusion genes were co-
`introduced into non-antibody producing murine
`myeloma cells by electroporation. Transfectants
`that produced murine/human chimeric antibody
`were obtained at high frequency as indicated by
`immunoblots probed with an antisera specific for
`human immunoglobulin. Enzyme-linked immu-
`noabsorbent assay analysis demonstrated that this
`chimeric antibody was secreted from the myeloma
`cells and retained the ability to bind selectively to
`membrane prepared from human tumor cells. The
`chimeric immunoglobulin was also shown by indi-
`rect fluorescence microscopy to bind to intact hu-
`man carcinoma cells with specificity expected of
`B6.2. The ability of chimeric antibody to recognize
`human tumor-associated antigen makes feasible a
`novel approach to cancer immunotherapy.
`
`For a number of years. researchers have attempted to
`use tumor-specific antibodies in the diagnosis and treat-
`ment of cancer (1). These efforts have been intensified
`by development of the technology to produce monoclonal
`antibodies of defined antigen specificity (2). Tumor-spe-
`cific monoclonal antibodies can be used in a number of
`
`ways. For example. they can be radioactively labeled, and
`then used to image tumors in whole body scans (3). In
`some cases, monoclonal antibodies alone can inhibit the
`
`growth of tumor cells (4). whereas in other systems in-
`hibition of tumor growth can be achieved by complexing
`the antibody with various toxic substances (5). Although
`such treatments have encountered numerous difficulties,
`some recent successes have been reported (6), and im-
`munotherapy may be of great clinical value in the future.
`
`Received for publication April 22. 1986.
`Accepted for publication May 6. 1986.
`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.
`
`A major limitation in the clinical use of murine-derived
`monoclonal antibodies is the immune response elicited
`against foreign protein. which may render the antibody
`ineffective and also may harm the patient (1). Although
`treatment with human monoclonals is under investiga-
`tion. human hybridoma cell lines are largely unavailable,
`and where they do exist, are usually unstable and pro-
`duce low amounts of immunoglobulin (lg) (7).
`The potential to engineer antibodies of improved utility
`and lacking unwanted side effects exists through the
`modification of Ig genes by using recombinant DNA tech-
`niques. One approach to circumvent the antigenicity of a
`murine monoclonal antibody in humans is to construct
`molecules that incorporate the specificity of the mono-
`clonal into a human antibody. This can be accomplished
`by fusion of the murine variable (V) region exons with
`the human K or -y constant (C)‘ region exon(s). The seg-
`mented structure of the Ig heavy and light chain genes
`and the conservation of this structure among mammals
`allows the construction of the appropriate murine/hu-
`man fusions. Because most lg antigenicity resides in the
`C domain, creation of murine V/human C “chimeric" lg
`should result in an antibody that has the specificity of a
`murine monoclonal, yet fails to elicit a human immune
`response (8). Furthermore. such chimeric proteins may
`interact more effectively with the human cellular im-
`mune system by virtue of their human C domain and
`thus provide more beneficial therapy than would the
`corresponding murine antibody.
`Murine/human chimeric antibodies in which the mu-
`rine V region specifies binding of a non-biological hapten
`have been described by others (9). Here we report the
`extension of this technology by construction of a chimera
`with specificity for an antigen associated with certain
`human carcinomas. The murine monoclonal from which
`
`the V exons were derived was isolated by Colcher et al.
`(10) by injection into mice of a membrane-enriched ex-
`tract from metastatic cells of a human breast carcinoma.
`This monoclonal. B6.2. has been shown to bind to the
`surface of various human carcinoma cells but not to
`
`sarcomas, melanomas. hematopoietic tumors. or most
`normal human tissues (1 1). We describe the isolation of
`genes coding for the B6.2 antibody, their DNA sequence,
`construction of murine/human gene fusions. and their
`expression in murine myeloma cells. The demonstration
`that the resultant chimeric antibody retains specificity
`
`‘Abbreviations used in this paper: C. constant: Eco—gpt. E. coli Xan-
`thine—guanine phosphoribosyl transferase gene; neo. Tn5 neomycin re-
`slstance gene: Ag8. P3X63.653-Ag8: Sp2/O. Sp2/O-Ag14; NS~l. P3/NS1/
`l—Ag4-1.
`1066
`
`Genzyme Ex. 1041, pg 933
`
`Genzyme Ex. 1041, pg 933
`
`

`
`MURINE/HUMAN ANTI-TUMOR CHIMERIC ANTIBODY
`
`1067
`
`for human tumor-associated antigen and intact human
`carcinoma cells raises the possibility that this approach
`may be generally applicable to the construction of agents
`useful in human immunotherapy.
`
`MATERIALS AND METHODS
`
`DNA isolation and analysis. High m.w. DNA was isolated from
`myeloma cells as described (12). Ten micrograms of each DNA were
`digested with the specified restriction enzyme (2 U enzyme/pg DNA)
`according to the supplier’s conditions (New England Biolabs, Beverly,
`MA). DNA was separated by electrophoresis through 0.8% agarose
`slab gels in 40 mM Tris-acetate and 2 mM EDTA (pH 8.0) and was
`transferred to nitrocellulose (13). Probes used in the analysis of
`genomic DNA were derived from cloned lg genes generously supplied
`by Dr. P. Leder (Harvard Medical School, Boston, MA). The probe
`used to detect the light chain gene was a 2.3 kb Hind Ill/Bam I-I1
`DNA fragment containing the murine Cx exon (see Fig. 1A, bottom).
`The heavy chain probe was a 1.4 kb Pst 1 fragment derived from a
`portion of the murine JH-Cu intron (see Fig. 1B, bottom). After
`restriction enzyme digestion, probes were purified by agarose gel
`electrophoresis, were electroeluted, were labeled with [”P]-dCTP by
`nick-translation (New England Nuclear, Boston, MA), and were hy-
`bridized to filters (12).
`Construction and screening (J >\ phage libraries. The genes
`coding for the heavy and light chains of B6.2 were isolated from
`clone libraries constructed in bacteriophage A (14). The libraries
`were screened by plaque hybridization (15) with nick-translated DNA
`fragments or end-labeled oligonucleotides (12, 16). To clone the gene
`coding for the heavy chain, a total Eco R1 digest of B6.2 DNA was
`fractionated in a Bull's-Eye gel apparatus (Hoeffer Scientific, San
`Francisco, CA). and fractions containing the desired DNA fragment
`were cloned into >\gtWES-)\B (17). For the light chain. a 5 to 15 kb
`fraction of B6.2 DNA from a total Bam H1 digest was cloned into A
`Charon 30 (18).
`Nucleotide sequencing Of B6.2 Ig genes. The nucleotide sequence
`of the B6.2 Vx exon was determined by primer extension of mRNA
`(19). Total RNA was isolated by guanidinium/phenol extraction, and
`poly A* mRNA was selected by oligo[dT)-cellulose chromatography
`(12). A 16 nucleotide DNA primer corresponding to the complement
`of the 5’ end of the murine Cu domain (20) was synthesized on a
`model 380A Advanced Biosystem DNA synthesizer. By using the [”P)
`end labeled oligonucleotide as primer and reverse transcriptase (Mo-
`lecular Genetics Resources, Tampa, FL). fully extended cDNA was
`synthesized for sequencing by the method of Maxam and Gilbert
`(21). Two other primers from different regions of the gene (see Fig.
`3) were also used for extension, and the overlapping sequences were
`compared for confirmation. The identity of the B6.2 K-containing
`genomic clone was verified by direct plasmid sequencing (22) of the
`first hypervariable region of the V domain. The B6.2 heavy chain V
`region was subcloned into M13mp8 and —mp9 (23), followed by
`sequence determination by using the method of Sanger (24).
`Construction of chimeric lg genes. Chimeric Ig genes were con-
`structed by ligation of cloned murine V exons with human x or 7, C
`regions. The human CK (25) and C7, exons were kindly provided by
`Dr. P. Leder. The human CK exon, located on a 954 base Eco Rl-
`Hhal fragment, was inserted between the Eco R1 and Bam H1 sites
`of pSV2gpt (26) (see Fig. 4A). The human C71 exons are contained
`on a 6.5 kb Hind III-‘Bam H1 fragment inserted between the Eco RI
`and Bam H1 sites of pSV2neo (27) (see Fig. 4B). The resulting
`plasmids are 5.5 and 11.5 kb, respectively. The murine Vx region is
`a 4.8 kb Eco Rl—Xmnl fragment; the Xmnl site used is within the x
`gene intron (Fig. 2A). Through linker conversion (12), this fragment
`was inserted into the unique Eco R1 site of the vector. The chimeric
`light chain plasmid, pSV2gpt/B6.2V._huCx, is 10.2 kb and carries
`the E. coli xanthine-guanine phosphoribosyl transferase gene (Eco-
`gpt) (see Fig. 4A). The murine VH region is within the cloned 5.0 Eco
`RI fragment (see Fig. 2B). Ligation of this exon into the Eco RI site
`of
`the heavy chain vector yielded the plasmid pSV2neo/
`B6.2VHhuC7,, which is 16.5 kb and carries the Tn5 neomycin re-
`sistance gene (neo) (see Fig. 4B). The transcriptional orientation of
`the chimeric genes is opposite to that of the marker genes.
`Cell lines. The P3X63.653-Ag8 (Ag8: ATCC CRL 1580) and Sp2/
`0-Ag14 (Sp2/0; ATCC CRL 1581) cell lines used for transfection are
`non-lg secreting murine myelomas. The myeloma J558L (28). kindly
`provided by Dr. A. Hayday (Yale University, New Haven, CT), syn-
`thesizes only A light chain. For immunofluorescence analysis. two
`lines of human lung carcinoma cells were used. These were A549.El,
`a subline of A549 (ATCC CCLI85) and 9812 (generously made avail-
`able by the late Dr. J. Fogh, Sloan-Kettering Cancer Institute, New
`York). All lines were grown in Dulbecco’s modified Eagle‘s medium
`supplemented with 10 to 20% fetal calf serum (both from M. A.
`
`Bioproducts, Walkersville. MD).
`Introduction of DNA into myeloma cells by electroporation. DNA
`was introduced into murine myeloma cells by electroporation as
`described (29, 30). Approximately 107 cells were subjected to an
`electric field of 3.8 kV/cm in 0.5 ml of phosphate-buffered saline
`(PBS) at 4°C with 20 pg each of pSV2gpt/B6.2VLhuCx linearized
`within the pBR322 sequence and pSV2neo/B6.2 VHhuC'y1, linearized
`at the Xho I site (Fig. 4). After incubation at 37°C for 48 h, the
`transfected cells were switched to Dulbecco’s medium containing
`mycophenolic acid (GIBCO, Gaithersburg, MD; 1.0 g/ml for Sp2/O
`and Ag8 and 6.0 ;g/ml for J558L), 250 ug/ml xanthine and 15 ;g/
`ml hypoxanthine for selection of plasmid-containing cells.
`Immunoblot analysis. Cell lines were analyzed for synthesis of Ig
`by lysis of 1 X 107 cells in 180 ill of 1% Triton X-100, 0.5% deoxy-
`cholate, 0.1% sodium dodecyl sulfate (SDS), 0.01 M Na phosphate
`(pH 7.5), 0.1 M NaCl, 0.001 M EDTA, and 0.001 M pheny1methyl-
`sulfonyl fluoride. Lysates were centrifuged for 5 min in an Eppendorf
`centrifuge to remove nuclei and cell debris, and protein concentra-
`tion of the supernatant was determined by using a Protein Assay Kit
`(Biorad, Richmond, CA). Fifty microliters of protein from each su-
`pernatant were denatured by adjusting the concentrations to 50 mM
`Tris-Cl (pH 6.8), 2% SDS, 2 mM EDTA, 10% glycerol. and 5% (3-
`mercaptoethanol, and boiling for 2 min. Electrophoresis was on a
`12% SDS-polyacrylamide gel with appropriate protein standards
`(BRL, Gaithersburg, MD). Protein was transferred to nitrocellulose
`by using a Bio-Rad Trans-Blot Cell apparatus at 75 mA for 4 h (31).
`Immunologic detection of heavy and light chain Igs was carried out
`by using the avidin-biotin system (32). To reduce background, filters
`were treated with Blotto (5% non-fat dry milk) (33) for 60 min.
`Biotinylated goat anti-human IgG (7 specific) and goat anti-human x
`or horse anti-murine IgG (Vector Laboratory, Burlingame, CA) were
`diluted in Blotto and were incubated for 3 hr with the filters. After
`three washes with 10 mM Tris-Cl (pH 7.5), 0.9% NaCl. and 0.05%
`Tween 20 (34), the filters were treated with horseradish peroxidase
`avidin D (Vector Laboratory) for 1 hr in Blotto. The visible signal
`was produced by incubation in 1 mg/ml diaminobenzidine, 1 mg/ml
`imidazole. and 1 ul/ml 30% H202 at room temperature.
`Enzyme-linked immunoabsorbent assay (ELISA) analysis. Mi-
`crotiter plates were coated with 20 pg per well of partially purified
`membrane from the human colorectal carcinoma cell line LSl74T
`(ATCC CLl88). Membranes were prepared by a modification of the
`method of Colcher et al. (35). A 10 g pellet of cells was suspended in
`100 ml of 10 mM Tris-Cl (pH 7.2) and 0.2 mM CaCl2 and was
`sonicated 6 X 10-sec at setting 4 of a Branson sonifier. The lysate
`was pressure homogenized in a Parr cell disruption bomb for 5 min
`at 1000 lb/in“, and was then centrifuged at 1000 X G for 5 min. The
`resultant supernatant was resonicated for 2 min at 10-sec intervals
`at setting 6. The lysate was centrifuged at 10.000 X G for 10 min.
`and the protein concentration was determined by the Lowry test.
`For negative controls. membrane was prepared from a cell line to
`which B6.2 is known not to bind (A375, ATCC CRL1619. a human
`myeloma). This material was prepared and was fixed to microtiter
`plates exactly as was the membrane of LS1 74T cells described above.
`Growth medium from transfectomas or their parental lines was
`collected, was centrifuged, and was serially diluted with Dulbecco’s
`buffered saline containing 1% bovine serum albumin (BSA). Fifty
`microliters of each dilution were incubated per well for 1 hr at 37°C.
`An equal volume of horseradish peroxidase-conjugated goat anti-
`human IgG (Fc fragment) serum (The Jackson Laboratory Immuno-
`research. Avondale, PA) or goat anti-murine IgG (Kirkegaard and
`Perry Laboratories lnc., Gaithersburg, MD) was added, and the in-
`cubation was continued for another 1 hr. Plates were then washed
`four times with 10 mM Tris-Cl (pH 8.0) and 0.05% Tween 20. Fifty
`microliters of a mixture containing 0.2% o-phenylenediamine,
`0.015% H202, 17 mM citric acid, 65 mM Na phosphate (pH 6.3), and
`0.01% methiolate were added per well to produce a color change.
`The reaction was stopped after 2 to 3 min with 4.5 M H2804. and
`the OD was measured at 490 nm in an MR60O Dynatech Products
`microplate reader. To determine the concentration of antibody se-
`creted by transfectomas. ELISA were performed by using microtiter
`plates coated with 500 ng/well of goat F(ab’)2 anti-human lgG (Tago
`Inc., Burlingame. CA), and purified human IgG (Cappel Worthington,
`Malvern, PA) was used to generate a standard curve.
`immunofluorescence. Human lung carcinoma cells grown to 85%
`confluence were washed in cold PBS, and were then incubated for 1
`hr at 4°C in 100 pl PBS containing 5 pg/ml of chimeric or murine
`B6.2 purified as described (manuscript in preparation). After wash-
`ing, the cells were incubated in PBS for 1 hr at 4°C with a 1/100
`dilution (~80 ug/ml final concentration) of fluorescein-conjugated
`antibody specific for either murine or human IgG. These were rho-
`damine-conjugated sheep anti-human IgG or fluorescein-conjugated
`sheep anti-murine IgG (Cappell-Worthington, Cooper Biomedical,
`Malvern, PA). The cells were washed in PBS containing 0.1% BSA,
`
`Genzyme Ex. 1041, pg 934
`
`Genzyme Ex. 1041, pg 934
`
`

`
`1068
`
`MURINE/HUMAN ANTI—TUMOR CI-IIMERIC ANTIBODY
`
`A
`
`>~ W is
`~s‘°&°<:.?"
`
`B
`
`“‘ £3
`ca‘!
`<2> $’ -3:
`
`.
`
`fl
`
`J6_
`||_2-
`3.5-
`6.4-
`5?—
`
`8.0-
`5.0-
`
`Q. -
`
`'
`
`is
`
`LVJ
`
`C
`
`e
`Qgbe
`
`x
`Q3;
`LVDJ
`
`Q5:
`
`Figure 1. Southern analysis of B6.2. NS-1. and BALE/c murine liver
`DNA. DNA was digested with the indicated restriction enzyme and was
`iwbridized with human C region probes as described in Materials and
`Methods. Autoradiograms of Ram 1-11-digested DNA probed with rnurlne
`Cr [panel A] and Eco R1-digested DNA analyzed with a murine heavy
`chain intron probe [panel B]. The diagrams at the bottom of panels A
`and B indicate the probes that were used for Southern analysis. The m.w.
`lkb] of |g—(:ontalnIng fragments were determined by comparison with Hind
`I[|-digested phage A DNA.
`
`were fixed with 1% formaldehyde in PBS. and then were photo-
`graphed by epiflunrescence.
`
`RESULTS
`
`isolation of the B6.2 lg genes. To identify the 1g genes
`and assess the configuration of restriction sites around
`the heavy and light Chain V exons. high m.w. B6.2 DNA
`was digested with various restriction enzymes and was
`analyzed by the method of Southern [13] (Fig. 1). Light
`chain genes were probed with a 2.3 kb Hind III;’Bam H1
`fragment containing the murine Cx region [Fig. IA). Bam
`H1-digested DNA from B6.2 and P3,/NS]/1-Agni-1 [NS-1.
`
`the fusion partner used to generate B6.2) yielded multi-
`ple. diffuse bands of hybridization the average sizes of
`which were 11.0. 8.5. 6.4. and 5.7 kb. Liver DNA showed
`
`hybridization to a single 16 kb Bam H1 fragment. The
`diffuse bands found in 136.2 and NS-1 DNA and their
`
`varying intensities indicated the presence of multiple
`gene copies. This result. which has also been observed
`for NS-1 and its fusion progeny by others [36]. suggested
`that the x gene had been amplified. To clone the B6.2 Vx
`exon. complete Barn H1 digests were ligated into the it
`vector Charon 30 [17]. When this library was screened
`with the murinc x-containing probe. multiple clones were
`isolated that were shown after additional analysis to
`contain the C exon but to lack the B6.2 Vx exon.
`
`To identify the functionally rearranged VK exon from
`among the it clones containing amplified x genes. at B6.2
`VK exon-specific probe was synthesized. To generate the
`necessary information. the sequence of mRNA coding for
`the B6.2 Vx exon was determined by primer extension of
`an oligonucleotidc complementary to the 5’ end of the Ca
`exon [25]. The probe. a 34-nucleotide oligomcr corre-
`sponding to the derived sequence of the third hypervari-
`able region (nucleotide 34'? to 381: see Fig. 3A). was used
`to rescrcen it clones isolated by hybridization with the K-
`containing DNA fragment [see above and Fig. IA). Of 45
`«containing clones tested. two were found to hybridize
`with the oligomer and to contain the functional Vx exon.
`The B6.2 light chain gene is contained on a Barn H1
`fragment of approximately 9 kb as shown in Figure 2A;
`in the genome. this fragment is within the broad band of
`hybridization with average size of 8.5 kb [Fig IA).
`Heavy chain genes were identified by Southern analy-
`sis by using as probe a 1.4 kb Pst I fragment from a
`portion of the murinc JH—C,u intron (Fig. 15‘. bottom). As
`expected. a unique heavy chain-containing fragment was
`seen in B6.2 DNA that was not found in NS-1 or liver
`
`DNA: this corresponds to the rearranged form of the gene
`(Fig. 1B. top]. DNA from this B6.2-specific 5 kb Eco R1
`fragment was cloned into )tgtWES—i\B {I6}. and the re-
`sultant phage library was screened with the heavy chain
`probe {see above]. A partial restriction map of the isolated
`clone. which contains the rearranged B6.2 heavy chain
`V region and its enhancer. is shown in Figure 2B.
`Nucleotide sequences (J the B6.2 V exons. As de-
`scribed above. the sequence of the B6.2 light chain mRNA
`was determined by primer extension (Fig. 3A]. To confirm
`that this mRNA in fact codes for the B6.2 light chain
`protein. the sequence of the 20 N-terminal amino acids
`of secreted :4 chain was determined by Edman degrada-
`
`Ii-c
`35,
`R
`Q,
`1:?
`~
`I-‘tgu re 2. Restriction enzyme cleavage
`6;
`‘lg Q,‘
`{tag
`3..
`g 32-"
`Q
`;.
`gt
`sites in B6.2 V rcgiomcontaining clones.
`~
`an
`d‘
`an
`-6°
`4-
`:5:
`-R‘
`at‘
`is
`in panel A. a 9 kh Bam H1 fragment
`cioncd in it Charon 30 that contains the
`B6.2 VJ5 region. the K specific enhancer.
`1.. N5
`E,
`C,
`and the murine Cu exon is shown. This
`insert corresponds to one of the x-contain-
`lng fragments in the diffuse band of hy-
`bridization of Figure la the average size
`of which is 8.5 kb. Panel B shows a 5 kb
`Eco RI
`fragment cloned in itgtWES—»\B
`that contains the B6.2 V11}, exon and the
`,|«l'SpE'("lflC enhancer sequence. This insert
`corresponds to the 5 kb genomic fragment
`shown in Figure 1B.
`
`A.
`
`B
`
`'
`
`‘
`
`H
`
`as \ .,‘
`“gas ,3?
`
`ct Elf?‘
`,§,g?g¢°
`
`.H~§'-E,
`qf‘-*q§.'q‘°;§
`
`g .~ ct‘:
`39° q_"3‘¢9
`
`Genzyme Ex. 1041, pg 935
`
`Genzyme Ex. 1041, pg 935
`
`

`
`MURINE/HUMAN ANTI-TUMOR CHIMERIC ANTIBODY
`
`1069
`
`A
`
`54
`27
`CCT ATG CAT CA6 ATC AGC ATG GGC ATC AAG ATG GAG TCA CAG ACT CAG GTC TTT
`Ile
`Thr Gln Val Pha
`MET His Gln
`Ila Ser Mel Gly
`Lys Mel Glu Se! Gln
`BI
`I08
`GTA RAC ATG TTG CTG TGG TTG TCT GGT GTT GAT GGA'GAC ATT GTG ATG ACC CAG
`Val Yyr Mai Lou Lou Trp Lau Ser Gly Val Asp Gly
`§_sQ
`lI_e E] Q E! gr;
`I35
`I62
`TOT CAA AAA TTC ATG TCC ACA TCA GTG GGA GAC AGG GTC AGC GTC ACO TGG AAG
`5_-r%|_-y1mm1S_erm§e_rwGJ"£25Lqws_erwmQy_su_s
`
`P3
`I89
`2:5
`GCC AGT CAG AAT GTG GTC ACT AAT GTA GCC TGG TAT CAA CAG ACA CCA GGA CAA
`msgmmwmmmygflmmmmm may
`
`.....P}...3J.9
`243
`TCT cor AAA GCA CTG ATT TAO TGG GcA Tcc TAC CGG TAC AGT GGA GTC ccT GAT
`§°_'l’_'9l-_-x_!A|al.2.u ll:IEEAEEMAr9Tv'S°rG|yV=|PwAsn
`.~.~w.A.~.M.~...w.~.4
`297
`324
`CGC TTC TCA GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC AGC AAT GTG
`Arg Phe Se! Gly Set Gly S97 Gly Thr Asp Phe Thv Leu Th!
`150
`551’ 55“ V3‘
`351
`378
`CAG TCT GGA GAC TTG GCA GAG TAT TTC TGT CAG CAA TAT AAC AGC TAT OCT CTC
`Gln Ser Gly Asp Lou Ala Glu
`Tyr Phe Cy: Gln Gln Tyr Asn Sar Tyr Pro Leu
`P
`
`405
`'
`AC6 TTC GGT GGT (366 A60 AAG CTG GAG CTG AAA CGG
`Lys Lau Glu Lau Lys Arg
`Thy PM Gly Ala Gly Tnr
`
`B
`
`54
`27
`ATG GAC AGG CTT ACT TCT TCA TTC CTG CTG CTG ATT GTC OCT GCA T AT GTC TTG
`MET Asp Avg Lou Tm so: set Pho Lou Lou Lou
`III VII Pro Ala T Y’ Val Lou
`3|
`I08
`rec
`'CAA GTT ACT CTA AAA GAG TCT sec ccT GGG ATA TTG AAG ccc TCA CAG Acc
`£.LJl_5fl9§i!!(mTm
`ser<mmT_hrm|=x_s¢mS_°v§x£v2§x||_o
`I62
`[35
`cTc AGT CTG ACT TGT TcT Trc Tcr GGG TTT TcA ore AGC ACT 'rcT GGT ATG GGT
`L¢3_lJ§fl£T_l"§1:§.eLEh.q_5.flfl¥E5_°'L_9|iS£'T_?"§§!Q1.M_¢iG|Y
`216
`I59
`GTA GGC TGG ATT CGT CAG CCT TCA GGA AAG AGT CTG GAG TGG CTG GOA CAC ATT
`Val Gly Trp
`Ila Avg Gln Pro Se: Gly
`Lys Sor Lou Glu Tip Lou Ala His
`Ila
`243
`270
`TGG TGG AAT GAT GAG AGG TAC TAT AAC CCA TCC CTG AAG AAC CAG CTC ACA ATC
`Trp Trp Asn Asp Glu Arg Tyr Ty! Asn Pro Se! Leu Lys Asn Gln Lau Thr
`Ila
`Psi]
`297
`324
`me AAG GAT Acc rec AGA AAc CAG TAT ‘rec TCA AGA TCA GOA TGT GGA CAC TGC
`Se! Lys Asp Thr Ser Arg Asn Gln Tyr Se! Ser Arg Ser Pro Cys Gly His Cys
`35!
`378
`AEA TAC TGC CAC TTA CTA CTG TGC TCG TAT CCC CTA AGG GGG TAC TTT GAC TAO
`Arg Tyr Cys His Leu Lou Leu Cys Ser Tyr Pro Leu Avg Gly Tyr Phs Asp Tyr
`405
`TGG GGC CAA GGC ACC ACT CTC ACA GTC TCC TCA G
`Trp Gly Glrl Gly
`Thr Th: Lau Thv Val
`Ser Ser Val
`
`Figure 3. DNA sequences encoding the V regions of the B6.2 mono-
`clonal antibody. Panel A shows the sequences of the B6.2 VL gene. The
`sequence of x message was determined by primer extension of poly A*
`RNA. The positions of the three primers used (P1. P2. and P3) are indicated
`by wavy lines. P, was also used for direct sequencing of supercoiled DNA
`from a genomic clone of the B6.2 Vx gene (see text). Panel B shows the
`sequence of the B6.2 VH gene. The genomic B6.2 V" gene was sequenced
`from the indicated Pst I site and the Bam Hl site 5’ of the leader exon
`(Fig. 28]. The slash denotes the position of the first intron [sequence not
`shown]. For panels A and B, the arrow indicates the start of the mature
`x and 7 proteins. The underlined amino acids are those that were
`confirmed by direct Edman degradation, whereas the remainder were
`translated from the nucleic acid sequence.
`
`tion (37). It was identical to that predicted by the nucleic
`acid sequence. To insure that the isolated clone is tran-
`scribed into the sequenced mRNA. the chimeric lg gene
`(shown in Fig. 4A) was partially sequenced (to base 145)
`by direct primer extension from supercoiled plasmid DNA
`(22). The determined DNA sequence was identical to that
`predicted by the data shown in Figure 3A.
`The DNA sequence of the B6.2 heavy chain V region
`(Fig. 3B) was determined by subcloning the V region into
`the bacteriophage vector Ml3mp8 and —mp9 (23) and
`sequencing by the method of Sanger (24). That this se-
`quence codes for the B6.2 heavy chain protein was veri-
`fied by sequencing the 34 N-terminal amino acids. As in
`
`the case for the light chain. the amino acid sequence of
`the heavy chain V region was identical to that predicted.
`The data shown in Figure 3 are discussed below.
`Fusion of murine V with human C exons and intro-
`duction into murine myeloma cells. The B6.2 heavy and
`light chain V regions were inserted into the unique clon-
`ing sites of plasmids containing the human x or 71 C
`exons (Fig. 4). These vectors, adapted from pSV2gpt (26),
`include transcriptional control signals from the SV40
`immediate early genes located 5’ of either the Eco-gpt
`(26) or neo genes (27). Expression of these markers allows
`selection of the vectors in medium containing mycophen-
`olic acid or the drug G-418, respectively. Upstream Ig
`control sequences and the cell type-specific enhancers
`are provided by the inserted murine DNA, whereas ter-
`mination and polyadenylation signals derive from the
`human C region.
`The plasmids containing Chimeric lg genes, pSV2gpt/
`B6.2VLhuC:< and pSV2neo/B6.2VHhuC,1 (Fig. 4), were co-
`introduced into myeloma cells by electroporation (29, 30).
`This technique has several advantages including a high
`frequency of co-transfection (30). The recipient cells Sp2/
`0, Ag8, or J558L were selected in medium containing
`mycophenolic acid or G-418. The fraction of cells that
`grew varied with the cell type and the selection; typically,
`0.1 to 1.0 X lO“‘ transformants [also termed “transfec-
`tomas”) (9) were observed. Although only one of the two
`marker genes was actually selected, the incidence of co-
`transfection was high.
`immunoblotting,
`Analysis
`(J
`transfectomas by
`ELISA, and immunofluorescent cell staining. The pro-
`duction of lg by transfectomas containing chimeric genes
`was assessed by immunoblotting of protein in cellular
`extracts. The filter shown in Figure 5A was probed with
`a mixture of antisera capable of recognizing both human
`7 and x chains. Transfectomas from all three cell types,
`which express both light and heavy chains, were clearly
`identified (lanes 5, 7, and 9). The size of heavy chain lg
`synthesized by the transfectomas was similar to human
`7 chains (lanes 1 and 10) and larger than that of B6.2.
`The K protein made by the transfectomas was slightly
`smaller than human x, but was larger than that of B6.2.
`The transfectomas shown in Figure 5A are among the
`better producers; however. a wide range of expression
`levels was observed in the large number of transfectomas
`analyzed. N0 lg was observed in extracts of recipient cells
`that had not been transfected with the chimeric genes
`(lanes 2. 6, and 8), although the production of A light
`chain by J558L was confirmed using an anti—A sera (data
`not shown). Some transfectomas were generated into
`which either the chimeric heavy or light chain gene. but
`not both. had been introduced. As shown in lanes 3 and
`
`4, these cells were perfectly capable of expressing light
`or heavy chain alone. These results in transfectomas are
`not consistent with reports from other laboratories of
`cellular toxicity of free heavy chain (38); however. this
`might be explained by the relatively low level of Ig expres-
`sion exhibited by the transfectomas (see below).
`Extracts of some of the same transfectomas shown in
`
`Figure 5A were probed with anti—murine lgG (heavy and
`light chain specific) (Fig. 5B). This antisera produced a
`strong reaction with the B6.2 cellular extract (lane 1), but
`as expected it failed to detect lg from the chimeric trans-
`fectomas (lanes 2 to 5).
`
`Genzyme Ex. 1041, pg 936
`
`Genzyme Ex. 1041, pg 936
`
`

`
`1070
`
`MURINE/HUMAN ANTI-TUMOR CHIMERIC ANTIBODY
`
`Figure 4. Structures of plasmids con-
`taining the B62 chimeric lg genes. Panel
`A. pSV2gpt/B€~.2V,_huCx.
`and panel B.
`pSV2neo,fB6.2-
`\-",.huC'y;.
`
`
`
`Bqi II
`
`IIIOI
`
`B.
`A.
`|23456T89|0 |2345
`
`200-
`
`97-
`
`68-
`
`--C
`43-
`
`)2.0
`
`i-8
`
`I-6
`
`I-4
`
`
`
`fa
`
`
`
`OpticalDensityat490nm 9_GG
`
`lmrnunoblni analysis of transfeetomas containing the 36.2
`Figure 5.
`chimeric immunoglobulin genes. in par1etA.a mixture of antI—human ‘y
`and x chain antibodies was used to screen cell lysates of transfectornas
`into which light [L]. heavy [H]. or light plus heavy [1.+H] chain chimeras
`had been introduced. Lanes 1' and 10 contain 100 ng of human IgG.
`whereas lanes contain cellular lysates of Ag8 [lane 2}. Ag8/L [lane 3].
`Ags/H (tone 4). Ag8xL+H (tone 5]. J558L [tone 5]. J558L/L+H [lane 7).
`Sp2/0 [lane 8]. and Sp2/'0/L+H {lane 9]. ln panel B. a mixture of anti-
`bodies directed against murine x and 7 chains was used to probe cellular
`iysates of 86.2 [icme l}. Ag8(io.-1e 2]. Ags/L+H [lane 3]. Spam (lane 4].
`and Sp2/0]l.+H (lane 5]. Arrows Indicate the position of human -3: and in
`chains: the m.w. of protein markers are noted In kilodaltons.
`
`0-6
`
`0-4
`
`0-2
`
`To demonstrate that transfectornas containing chi-
`meric B6.2 genes were producing "human" x and 7 chains,
`culture supernatants were assayed for the presence of
`secreted. functional. chimeric antibody by ELISA [36].
`Antigen recognized by Bt-3.2 in the form of a partially
`purified membrane preparation (35) from human colorec-
`tal carcinoma cells LS1 74T was immobilized on microti-
`
`ter plates. and then antigen-antibody complexes were
`detected with horseradish peroxidase-conjugated anti-
`sera specific for human IgG. As shown in Figure 6 and
`listed in Table I. 60 of the 109 transfectomas tested gave
`a positive signal in the ELISA. indicating both that intact
`chimeric antibody was secreted. and this protein retained
`the ability to bind to human tumor-associated antigen.
`
`Figure 6. The level of lg in supernatants of chimeric transfectomas
`measured by ELISA. Supernatants that yieicied OD of less than 0.] were
`designated '-‘. those between 0.1 and 0.2 were designated ':“. and those
`greater than 0.2 were designated "+” (see Table i]. Parental [i.e.. untrans-
`fected) lines are indicated by the symbol "X".
`
`The parental [i.e.. untransfected] cell lines gave negative
`results.
`
`Figure 7 shows a representative ELISA for an lg-pro-
`ducing transfectoma [S25Dl). S25D1 gave a positive sig-
`nal with anti-human serum. (which did not bind B6.2)
`but failed to react with anti-murine serum. As a control.
`S25Dl did not bind to plates coated with partially purified
`membrane from a human myeloma cell line. A375. which
`
`Genzyme Ex. 1041, pg 937
`
`Genzyme Ex. 1041, pg 937
`
`

`
`MURINE/HUMAN ANTI-TUMOR CHIMERIC ANTIBODY
`
`1071
`
`TABLE I
`
`Level Qf secretion of chimeric antibody by transfectomas
`Number
`I§:‘;?::£,, Weakly
`[_)
`Positive“
`it/-)
`
`Recipient
`Cell Type
`
`,1_ra::t.r::t::nas
`Tested
`
`:D:?t1::f,
`H)
`
`Percent
`Positive
`
`Ag8
`SP2/O
`J558L
`
`Totals
`
`53
`25
`31
`
`1 09
`
`1 2
`10
`1 8
`
`40
`
`8
`O
`1
`
`9
`
`33
`15
`l 2
`
`60
`
`62
`60
`39
`
`55
`
`“ Transfectomas were scored based on their OD in ELISA (Fig. 6).
`“Negative” is defined as OD of 0.0.0 to 0.1. “weakly positive” denotes 0.1
`to 0.2. and “positive” indicates >0.2.
`
`0-5
`
`0-4
`
`0-3
`
`0-2
`
`
`
`OpticalDensityat490nm
`
`transfectomas was tested by comparison with known
`amounts of human IgG in an ELISA assay (see Materials
`and Methods). The better producing lines secrete approx-
`imately 1 pg/ml of chimeric antibody per 10“ cells per 24
`hr. Transfectoma cells were introduced into immunosup-
`pressed mice, and the level of chimeric lg monitored in
`the resultant ascites fluid. The mice produce approxi-
`mately 0.3 mg of chimeric protein per ml of ascites. Thus
`although the transfectomas do not appear to produce as
`much lg as most murine hybridomas, a sufficient quan-
`tity is made for many desired applications; a similar level
`of Ig production by transfectomas was observed previ-
`ously (9). In data to be presented elsewhere, we show by
`electrophoresis of purified c