`Vol. 84, pp. 3439-3443, May 1987
`Medical Sciences
`
`Chimeric mouse-human lgG 1 antibody that can mediate lysis of
`cancer cells
`
`/antibody-dependent cellular
`
`
`/tumor antigen/complement-dependent cytolysis
`
`(lmmuooglobulln domain cDNA/DNA transrectioo
`cytololdclty)
`ALVIN Y. Lm*t, RANDY R. ROBINSON*, KARL ERIK HELLSTR6M:t§, E. DAVID MURRAY, JR.*11,
`c. PAUL CHANG*, AND lNGEGERD HELLSTR6M:tll
`•International Genetic Engineering, Inc., 1545 17th Street, Santa Monica, CA 90404; *Oncogen, 3005 First Avenue, Seattle, WA 98121; and Depanments of
`WA 98195
`fPathology and llMicrobiology, University of Washington Medical School, Seattle,
`Communicated by Paul D. Boyer, January 28, 1987 (received for review December 22, 1986)
`A chimeric mouse-human antibody has been
`ABSTRACT
`created that recognizes an antigen found on the surface of cells
`from many carcinomas. ImmunogJobulin constant (C) domains
`of the mouse monoclonal antibody L6, CY2a and C,., were
`substituted by the human Cy1 and C,. by recombining cDNA
`modules encoding variable or C domains. The cDNA constructs
`were transfected into lymphoid cells for antibody production.
`The chimeric antibody and mouse L6 antibody bound to
`carcinoma cells with equal aftlnJty and mediated complement·
`dependent cytolysis. In the presence of human effector cells, the
`chimeric antibody gave antibody-dependent cellular cytotox
`iclty at 100 times lower concentration than that needed for the
`mouse L6 antibody. The chimeric antibody, but not the mouse
`L6 antibody, ls effective against a melanoma line expressing
`small amounts of the L6 antigen. The ftndings point to the
`usefulness of the chimeric antibody approach for obtaining
`agents with strong antitumor activity for possible therapeutic
`use in man.
`
`In this study we have generated a mouse-human chimeric
`L6 antibody in which the mouse constant domains C.yia and
`C" are substituted by the human Cy1 and C". First, the cDNAs
`encoding the immunoglobulin genes were isolated. Next,
`restriction enzyme recognition sites were created in the
`cDNA sequences at the V /C junction (where V stands for
`variable) (9) by in vitro mutagenesis using oligodeoxyribo
`nucleotides (10). The chimeric cDNAs thus constructed were
`then introduced into lymphoid cells by DNA transfection.
`The chimeric antibody isolated from the transfectants was
`compared with the mouse L6 for effector functions.
`
`MATERIALS AND METHODS
`
`DNA Transfection of Mouse Sp2/0 Lymphoid Cells. Expres·
`sion plasmid pING2114 (50 µ,g), linearized at a unique site
`(Aat II) in the nonessential bacterial region (see Fig. 3A), was
`transfected into 107 mouse Sp2/0 cells (CRL 1581, ATCC) by
`electroporation (11, 12). Transfonnants were selected in
`Dulbecco's modified Eagle's medium (DMEM) supplement
`ed with 10% (vol/vol) fetal bovine serum (HyClone, Logan,
`UT) and G418 at 0.8 mg/ml (GIBCO). The transfection
`frequency was between 10-5 and 10-•. Human antibody in
`the medium was detected by ELISA (13).
`Isolation of Chimeric Antibody. Antibody-producing cells
`were grown to a density of 106 cells per ml and then shifted
`to serum-free DMEM 24 hr before harvest. Antibody secret·
`ed by the cells was concentrated by ultrafiltration, then
`chromatographed on a DEAE·cellulose column equilibrated
`in 40 mM NaCl/10 mM sodium phosphate, pH 8.0. The
`antibody in the flow-through was further purified to apparent
`homogeneity on protein A-Sepharose (14). For production of
`ascites fluid, 11>6 cells were iajected into pristane-primed
`BALB/c mice. The chimeric antibody was purified by anti
`human lgG-Sepharose chromatography (14).
`Functional Tests of the Chimeric L6 Antibody. The follow
`ing tests were included: (l) measurement of antibody binding
`to target cells, either positive or negative for reactivity with
`the mouse L6; (ii) competitive inhibition of binding of L6 to
`these cells; (iii) assays for CDC and ADCC. The binding tests
`were performed using a Coulter model EPIC-C cell sorter (8).
`The assays for CDC and ADCC were carried out on 51Cr
`labeled target cells (2, 3) that were exposed to antibodies and
`human serum or peripheral blood leukocytes over a 4-hr
`period.
`
`The presence of tumor-associated antigens at the cell surface
`is a characteristic of many cancers. Since these antigens are
`either absent or found in much lower amounts in nonnal cells,
`it should be possible to use antibodies for targeting of tumors.
`A sizeable collection of relatively tumor-specific monoclonal
`antibodies (mAb) of mouse origin is available (1). Some of
`these mAb possess tumoricidal activity in the presence of
`human effector cells [antibody-dependent cellular cytotox
`icity (ADCC)] or serum [complement-dependent cytotoxicity
`(CDC)] (2, 3). It has been shown (4) that partial tumor
`regression can be achieved when mAb possessing such
`functional activity are given to patients. One complication
`preventing repeated use of mouse mAb in man is that they are
`immunogenic. Furthennore, mouse mAb may interact less
`efficiently with human effector cells to mediate tumor de
`struction.
`A method made possible by recombinant DNA technology
`was chosen to generate chimeric mouse-human antibodies. It
`entails the replacement of the mouse constant (C) domain
`regions with the corresponding human equivalents (5-7). In
`principle, antibody molecules obtained by this approach
`should retain their specificity for antigen and thus their
`usefulness for targeting, be much less immunogenic to man,
`and perhaps have increased antitumor activity.
`The mouse mAb L6 [IgG2a(K)] binds to a carbohydrate
`antigen found at the surface of cells from human carcinomas
`of the lung, breast, colon, and ovary (8). L6can mediate CDC
`with human complement or ADCC with human effector cells
`(2). mAb L6 may thus be of use for tumor targeting, either in
`its native fonn or after conjugation of anticancer agents.
`The publication costs of this article were defrayed in part by paae charge
`be hereby marked '' advertiument' •
`must therefore
`payment. This article
`
`in accordance with 18 U.S.C. §1734 solely to indicate
`
`this fact.
`
`3439
`
`J ,joining;
`Abbreviations: V, variable;
`
`mAb, monoclonal
`C, constant;
`cytolysis; ADCC, an
`
`antibody(ies); CDC, complement-dependent
`SV40, simian virus 40; H,
`cellular cytotoxicity;
`tibody-dependent
`heavy.
`tro whom reprint requests should be addressed.
`University of Cali
`
`11Present address: Department of Biochemistry,
`fornia, Riverside, CA 92521.
`
` 1 of 5
`
`BI Exhibit 1032
`
`
`
`3440 Medical Sciences: Liu et al.
`
`Proc. Natl. Acad. Sci. USA 84 (1987)
`
`met asp tro leu
`C23AGTTTGTCTTAAGGCACCACTGAGCCCAAGTCTTAGACATCATG GAT TGG CTG
`All I
`11Al31 dol.
`GTCGACTCT---------------------------�
`S•l I
`le•ct•t peptide
`IFA1
`trp asn leu leu phe leu met ala ala ala gln ser ala gln ala gln
`TGG AAC TTG CTA TTC CTG ATG GCA GCT GCC CAA AGT GCC CAA GCA CAG
`
`ile gln leu val gln s�r gty p�o gtu lgu lys lys p�o gty g!u thr
`ATC CAG TTG GTG CAG TCT GGA CCT GAG CTG AAG AAG CCT GGA GAG ACA
`1c_::� tyr gly
`val lys ile ser cys lys ala ser gly tyr thr phe t�
`GTC AAG ATC TCC TGC AAG GCT TCT GGG TAT ACC TTC ACA AJ\C TAT GGA
`89111
`COR 1,FR2
`FR21
`met asn trp val lys gln ala pro gly lys gly leu lys trp met gly
`ATG AAC TGG GTG AAG CAG GCT CCA GGA AAG GGT TTA AAG TGG ATG GGC
`Ahallt
`COR2
`trp ile asn thr tyr thr gly gln pro thr tyr ala aso asp ohe lys
`TGG ATA AAC ACC TAC ACT GGA CAG CCA ACA TAT GCT GAT GAC TTC AAG
`Nde t
`COR2,FR3
`gly aro phe ala ohe ser leu glu thr ser ala tyr thr ala tyr leu
`GGA CGG TTT GCC TTC TCT TTG GAA ACC TCT GCC TAC ACT GCC TAT TTG
`
`R;
`
`gln ile asn asn leu lys asn glu asp met ala thr tyr ohe cys ala
`CAG ATC AAC AAC CTC AAA AAT GAG GAC ATG GCT ACA TAT TTC TGT GCA
`FR3,COR3
`COR3,FR4
`arg phe ser tyr gly asn ser arg tyr ser asp tyr tro gly qln gly
`AGA TTT AGC TAT GGT AAC TCA CGT TAC TCT GAC TAC TGG GGC CAA GGC
`01p2.2 -------JH2·------
`thr thr leu thr val ser ser ala lys thr thr ala pro ser
`ACC ACT CTC ACA GTC TCC TCA GCC AAA ACA ACA GCC CCA TCG:----
`Gc---AG-G---MJH2Apol
`----------
`
`8
`
`L6 V.-;
`
`leader peptide
`met asp phe gln val gln ile phe ser phe leu leu
`C9CCCCAAGACAAAATG GAT TTT CAA GTG CAG ATT TTC AGC TTC CTG CTA
`-GTc·---------- s·s111
`!FRI
`ile ser ala ser val ile met ser arg gly gln ile val leu ser qln
`ATC AGT GCT TCA GTC ATA ATG TCC AGA GGA CAA ATT GTT CTC TCC CAG
`
`ser pro ala ile leu ser ala ser pro gly glu lys v�l t�r lgu thr
`TCT CCA GCA ATC CTG TCT GCA TCT CCA GGG GAG AAG GTC ACA TTG ACT
`
`p
`F,111,COJ!I 9
`o
`0
`0 CQ.R1JFR,i
`0
`0
`0
`
`c9s afg a!a ser ser ser val ser phe met a�n tfp tyr qln q!n lys
`TGC AGG Gee AGC TCA AGT GTA AGT TTC ATG AAC TGG TAC CAG CAG AAG
`Kpnl
`o
`FR2,COR2
`pro gly ser ser pro lys pro trp ile tyr ala thr ser asn leu ala
`F10. 1. Nucleotide sequences and predicted
`
`CCA GGA TCC TCC CCC AAA CCC TGG ATT TAT GCC ACA TCC AAT TTG GCT
`amino acid sequences of the L6 V H (A) and V. (B).
`BomHI
`The framework (FR) and complementarity deter
`
`COR 21FR3
`mining region (CDR) segments arc indicated. The
`
`ser glu phe pro gly arg phe ser gly glu trp ser gly thr ser tyr
`diversity (D) segment in VH is underlined.
`TCT GAG TTC CCT GGT CGC TTC AGT GGC GAG TGG TCT GGG ACC TCT TAC
`Circles
`
`above the amino acid residues indicate that these
`residues matched to those obtained from peptide
`ser leu ala ile ser arg val glu ara gtu a�o ala ata t�r tyr tyr
`
`sequencing. The V H sequence is present in plasmid
`pH3-6a. The C23 at the 5' end was removed by
`TCT CTC GCA ATC AGC AGA GTG GAG GCT GAA GAT GCT GCC ACT TAT TAC
`J.s
`
`
`BAL-31 nuclease digestion. The resultant DNA
`F.(t3,C0!13 p
`0
`0 COR3,FR4
`0
`0
`0
`0
`sequence at the 5' end is shown below the first line.
`C9S gln g!n trp asn ser asn pro leu thr phe qly ala qly thr lys
`
`An Apa I site was introduced by the oligonucleo
`TGC CAG CAG TGG AAT AGT AAC CCA CTC ACr, TTC GGT GCT GGG ACC AAG
`tide primer MJH2Apal. The V. sequence is pres
`ent in plasmid pL3-12a. The C13 at the 5' end was
`
`removed by oligonucleotide-mediated mutagcne
`leu glu leu lys
`
`sis. A Hindlll site was introduced by the oligonu
`CTG GAG CTG AAA-----------------------
`
`cleotide primer JKHindlll.
`
` 2 of 5
`
`BI Exhibit 1032
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`
`3441
`
`Proc. Natl. Acad. Sci. USA 84 ( 1987)
`placed upstream of the SV40 promoter (9). The selectable
`marker is the Tn.5 neo gene that confers resistance to the drug
`G418.
`The heavy-chain plasmid pING2111 was constructed by
`first joining the mouse VH cDNA module in a Sal I-Apa I
`DNA fragment with the human C,,1 cDNA module in an Apa
`I-BamHl DNA fragment. The ligated fragments were then
`inserted into plNG2012E cleaved by Sal I-BamHI. The
`light-chain plasmid pING2119 was constructed by joining the
`mouse V,. cDNA module in a Sal I-HindlII DNA fragment
`with the human C,. cDNA module in a Hindill-BamHl DNA
`fragment. The same vector fragment was used (Fig. 3A). In
`both plasmids the cDNA gene is placed 11 nucleotide
`residues downstream of the SV40 19S 3'-splice acceptor (9).
`The cDNA ends in a segment approximately A10G20, where
`it is joined to the SV 40 transcription-termination/polyaden
`ylylation sequences. Fig. 3B shows the incident nucleotide
`sequence changes made at the V /Cjunction as a result of the
`gene construction.
`A two-gene plasmid, pING2114, was constructed from
`pING2111 and plNG2119 in which the light- and heavy-chai?
`gene transcription units are in tandem (Fig. 3A). By usi� this
`plasmid, we introduced an equal ratio of heavy- and light
`chain genes into recipient cells. Unexpectedly, we observed
`that there was a consistently higher expression of heavy than
`of light chain in all transfected cell lines examined (data not
`shown). The two transcription units differ in that the light
`chain gene is about 700 base pairs shorter than the heavy
`chain gene, and the C,. gene segment has a higher A+T
`content. This imbalance was reduced by introducing more
`light-chain gene c.opies carried on a second plasmid with a
`different selectable marker [pING212la, an Eco-gpt (22)
`version of plNG2119).
`Two initial Sp2/0 transformants, D7 and 3E3, obtained by
`transfection with plNG2114 were cultured for the isolation of
`chimer:ic antibody. D7 secretes 10% of the antibody produced
`by 3E3-K (17 µ.g/liter) and 'Y (77 µ.g/liter) chains for D7
`compared to K (100 µ.g/liter) and 'Y (700 µ.g/liter) chains for
`3E3.
`Binding Characteristics or Chimeric Versus Mouse L6 An
`tibody. Table 1 shows that the chimeric L6 antibody binds to
`cells from a human colon carcinoma (line C-3347) that
`express 5 x l� molecules per cell of the antigen defined by
`the mouse L6 mAb (8). In a competition assay, 50% inhibition
`of binding was achieved by the same amount of the chimeric
`and mouse L6 (Fig. 4). Cells from a T-cell line, HSB-2, did
`not bind either mouse L6 or the chimeric antibody. Data on
`the melanoma line M-2669, clone 13 (3), are also included in
`Table l, since this line, which expresses a low level of the
`L6-defined antigen, was used for the functional studies (see
`below).
`Chimeric L6 Antibody Mediates CDC and ADCC. Fig. 5
`shows that both the chimeric and mouse L6 antibodies lysed
`tumor cells in the presence of human complement. The
`experiment further showed that the chimeric L6 gave higher
`CDC at all dilutions of the complement.
`
`Medical Sciences: Liu et al.
`
`RESULTS
`
`Isolation or Mouse cDNA. A cDNA library was generated
`from the L6 hybridoma cells by priming poly(Ar RNA with
`oligo(dT) as described (9, 15). The probes used to screen the
`library were a J,.5 ollgonucleotide, d(GGTCCCAGCAC
`CGAACG), for the light chain and a JH2 oligonucleotide,
`d(TGGCTGAGGAGACTGTGAGAG) for the heavy chain
`(where J stands for joining and H stands for heavy). Two
`methods (16, 17) were used to determine that the L6 K mRNA
`contains J,.5 sequences and that the L6 ')'2a mRNA contains
`JH2 sequences.
`Preparation or Mouse V-Region cDNA Modules. Restriction
`enzyme sites were engineered into the immunoglobulin
`cDNA around the V /C border for recombining mouse V
`regions to human C modules. The oligonucleotide MJH2Apal
`�(ATGGGCCCTTTGTGCTGGCTGAGGAGACTGT)
`(with the restnct1on enzyme site underlined)) was used for
`mutagenesis of the VH cDNA; and the oligonucleotide
`JKHindIII [d(CTCAAGCTIGGTCCC)) for that of the V K
`cDNA. Restriction sites on the 5' side of the ATG codon were
`also created. The oligonucleotide d(GAAAATCCATTI
`TGTCGACGGG) was used to generate a Sal I site eight
`residues on the 5' side of the V,. ATG codon. By cleaving with
`Sal I the ollgo[d(GC)) segmerit on the 5' side of the cDNA
`insert was removed. To remove the oligo[d(GC)) segment on
`the 5' side of the VH cDNA, the nuclease BAL-31 (18) was
`used. The digested products were inserted into the vector
`M13mpl9 (19) in such a way that the M13 Sal I site became
`a convenient site on the 5' side of the ATG codon. The DNA
`sequences of V H and V,. are shown in Fig. 1.
`Human C-Regioo cDNA Modules. Human C-region cDNAs
`were isolated from libraries generated from the mRNA from
`two human lymphoblastoid cell lines, GM1500 and GM2146
`(Human Genetic Mutant Cell Repository). The human C,.1
`module has been described (9). The cDNA clone pGMH6
`contains an Apa I site 16 nucleotide residues on the 3' side of
`the V /C border (Fig. 2).
`The human C,. module is a composite of two K cDNAs
`isolated from the GM1500 and GM2146 libraries. In plasmid
`pGML60, the 3'-untranslated region was derived from the K
`mRNA of GM2146 while the coding region was from that of
`GM1500. The JKHindIII oligonucleotide described above
`was used to engineer a Hindlll recognition site at a position
`of the human J,. segment analogous to that in the mouse J"
`segment.
`Chimeric L6 Heavy- and Light-Chain Exp�ion PlasmJds.
`The cDNA constructs were inserted into the vector se
`quences of plasmid plNG2012E (9). Directionality of inser
`tion was achieved by using a Sal I-BamHI bracket.
`pING2012E contains regulatory sequences derived from
`plasmid pLl (20) that furnished the early promoter and splice
`donor-acceptor of simian virus 40 (SV40); and from plasmid
`pSV2neo (21) that furnished the transcription termination/
`polyadenylylation signals of SV40. We added the mouse
`immunoglobulin heavy-chain gene transcription enhancer,
`
`pGMH6 human Cyl module
`--GI GTC ACC GTC TCT TCAI GCC TCC ACC AAG GGC Cf A TCG G------
`Apa I
`BSI E 11
`pGML60 human CK module
`c. clone does not contain a convenient
`-��- JK4--------------�
`recombination site; and an oligonucle
`otide containing a Hindlll site was used
`--����������������-
`' Sou3AI
`to introduce this site into the cDNA.
`-���1- C -T-
`The C-domain cDNA modules are
`Hind Ill
`pGMH6 and pGML60.
`
`Ftc;. 2. Human C-domain cDNA
`gene modules. The relevant sequences
`at the V /C junction of human Cy1 and
`c. cDNA clones are shown. The C�1
`clone contains a BstEil and an Apa I
`site that can be used to recombine with
`different V-domain cDNA genes. The
`
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`3442
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`Medical Sciences: Liu et al.
`
`A
`
`Proc. Natl. Acad. Sci. USA 84 (1987)
`Table 1. Binding of chimeric L6 and mouse L6 antibodies to cell
`lines used as targets for functional assays
`
`Antibody
`
`Antibody
`concentration,
`1-'g/ml
`
`Binding ratio*
`
`GAM
`
`GAH
`
`plNG2114
`11.5kb
`
`Human colon carcinoma line C-3347
`Mouse L6
`
`Chimeric L6 (ascites)
`
`Chimeric L6 (cell culture)
`
`30
`10
`3
`30
`10
`3
`30
`10
`3
`
`-CTC ACA GTC TCC T;� I �cc �Ge AC� MG GGC CCA T-
`Ap• t
`
`Mouse L6
`
`Human melanoma line M-2669 (clone 13)
`30
`10
`3
`30
`10
`3
`
`Chimeric L6 (cell culture)
`
`38
`49
`40
`2
`2
`1
`1
`1
`1
`
`7
`3
`1
`NT
`NT
`NT
`
`1
`1
`1
`
`4
`4
`3
`108
`84
`42
`105
`86
`
`44
`
`NT
`NT
`NT
`4
`2
`1
`
`1
`1
`1
`
`Human T-cell line HSB-2
`Mouse L6
`
`Chimeric L6 (ascites)
`
`Chimeric L6 (cell culture)
`
`10
`10
`10
`
`*The binding ratio is the number of times a test sample is brighter
`than a control sample when treated with GAM (fluorescein isothio
`cyanate-coojugated goat anti-mouse immunoglobulin) or with GAH
`(fluorescein isothiocyanate-conjugated goat anti-human immuno
`globulin). For example, a ratio of 1 means that the test sample is as
`bright as the control; a ratio of 2 means that the test sample is twice
`as bright as the control. NT, not tested.
`
`(data not shown). Efficacy of the chimeric L6 was further
`demonstrated by its ability to lyse M-2669 melanoma cells
`(35% at 10 µ.g/ml, 27% at 0.1 µ.g/ml); the mouse L6 had no
`effect on these cells (9% at 10 µ.g/ml, as compared to 10%
`lysis with lymphocytes alone, cf. Table 1).
`
`DISCUSSION
`
`mo hu
`-ACC NAG C� GAG 8TG AAA CGA ACT--
`Hind111
`FIG. 3.
`(A) Expression plasmids. plNG2111 is the heavy-chain
`and pING2119 is the light-chain expression plasmid. They were used
`to construct plNG2114, a two-gene plasmid. Solid circles, mouse
`heavy-chain immunoglobulin gene enhancer; small arrows, SV40
`early promoter; diamonds, bidirectional SV40 transcription
`tennination/polyadenylylation signals. (B) Nucleotide changes made
`
`introduced by oligonucleotide-mediated mutagenesis. They are silent
`
`in the V /C junction. Dotted residues in the V HC�1 junction were
`changes. Circled residues in the V .c. junction are residues contrib
`
`uted by the human cDNA module to the mouse V. gene.
`
`Fig. 6 shows ADCC tests with cells from two cell lines. At
`a ratio of 100:1 human peripheral blood leukocytes to the
`colon carcinoma line C-3347 cells, the chimeric L6 killed a
`greater fraction of the target cells (a maximum of 98% versus
`63%) and gave 50% ADCC at 100 times lower concentration
`than the mouse L6 (0.01 µ.g/ml versus 1 µ.g/ml, Fig. 6A).
`Significant ADCC of C-3347 cells (24% as compared to 3%
`lysis with lymphocytes alone) was observed down to a 3:1
`ratio of effector cells to target cells when the chimeric L6 (at
`2.5 µ.g/ml) was used (Fig. 6B). Cell killing was specific
`because ADCC was not observed with the following three
`cell lines lacking detectable L6 antigens: B-cell lines DHL-10
`(Fig. 6C) and T51 (data not shown) and the T-cell line HSB-2
`
`50% Inhibilion
`:
`Mouse L6= l.9JJg/ml
`Chimeric L6 =l.9µ9/ml
`
`100
`c 80
`0
`:-E 60 ..0 E
`� 40
`�20
`
`l
`10
`Inhibiting Antibody (µg/ml)
`FIG. 4. Comparison between chimeric and standard mouse L6 in
`
`antibody inhibition assays, performed by fluorescence-activated cell
`sorting. C-3347 cells were incubated with the blocking antibodies
`before fluorescein isothiocyanate-conjugated mouse L6 (3 µg/ml)
`was added.
`
`100
`
`The mouse mAb L6 recognizes a carbohydrate antigen
`present in abundance in a variety of carcinomas. Normal
`tissues express only trace amounts of the antigen (8). Based
`on this specificity there is justification in considering L6 for
`cancer treatment with the mAb used either alone (2) or as a
`carrier of anticancer agents. However, the immunogenicity
`of mouse L6 mAb in man is a disadvantage for its sustained
`use in patients, and its functional activity (ADCC and CDC)
`may be insufficient to effect optimal tumor destruction at the
`100
`.z:-80
`·u
`")( 60
`
`C-3347 Cells
`
`0 0 ;;.. 40 (.)
`
`�
`
`(Chimeric LG
`
`lMouse L6
`
`1:8
`
`1:4
`1:2
`Undiluled
`Dilution of Human Serum
`F1G. 5. Titration of human serum (as a source of complement) in
`the presence of chimeric or mouse L6 at 2.5 1-'g/ml.
`
` 4 of 5
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`BI Exhibit 1032
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`
`Medical Sciences: Liu et al.
`
`A
`
`100
`80
`
`C·3347Cells
`E/T =100/1
`
`r Lymphocytes alone
`_______ _!.. _________ _
`o......,... __ � ........ ��......_��.._�__...__�_..�
`0.1
`I
`10
`0.01
`0.0001 0.001
`Antibody (µg/ml)
`
`B
`
`100
`
`C·3347 Cells
`
`3443
`
`Proc. Natl. Acad. Sci. USA 84 (1987)
`
`
`the functional attributes of chimeric L6, should make it a
`
`
`
`strong candidate for therapeutic trials. Some of the antibodies
`induced in man to mouse mAb were directed to idiotypic
`
`determinants (26, 27). It remains to be seen whether the
`of the chimeric L6 will
`
`intmunogenicity of those determinants
`be different from that of the mouse L6.
`of the cDNA approach lies in the ease with
`The advantage
`gene cDNAs can be isolated. The
`which immunoglobulin
`
`
`technology used for the present work should make it possible to
`
`convert many other mouse mAb to chimeric antibodies with
`improved antitumor activity via ADCC and CDC mechanisms.
`
`
`The chimeric antibodies will augment the relatively few human
`
`
`mAb currently used in the treatment of cancer (28).
`We thank Cathy Shapiro, Phil Mack, Phil Mixter, Pam Smith,
`Susan Azemove, Grethe Lovold, and Pat McGowan for excellent
`technical assistance. We also thank Randy Wall for discussion, and
`Randy Wall, Carol Hersh, Arup Sen, Gary Wilcox, Perry Fell, Jeff
`Ledbetter, Peter Linsley, and EriR Milner for useful comments on
`
`
`
`the manuscript. The work was supported by INGENE and
`ONCOGEN.
`
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`for Cancer Detection and Therapy, eds. Baldwin, R. S. & Byers,
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`& Hellstrom,
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`8. Hellstrom, I., Hom, D., Linsley, P., Brown, J.P., Brankovan, V.
`
`100
`80
`60
`40
`20
`
`Effector Cells per Torget Cell
`
`lOO
`c
`Z" 80
`·u
`
`DHL-10 Cells
`E/T=lOO/I
`')( 60 0 0 :;.. 40 u
`�
`� 20 �ouse LG {Chimeric LG
`o�� ======t======:=a..-
`0.2
`0.02
`2
`Antibody (,µg/ml)
`(A) 1Titration of chimeric and mouse L6 antibodies iii
`
`FIG. 6.
`ADCC assays with human peripheral blood leukocytes.
`E/T, ef
`fector-target cell ratio. Two preparations of chimeric L6 were used.
`Titration of L6 (chimeric
`
`K. E. (1986) Cancer Res. 46, 3917-3923.
`9. Liu, A. Y., M ack, P. W., Champion, C. I. & Robinson, R.R.,
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`Gene, in press.
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`14. Johnstone, A. & Thorpe, R. (1982) lmmunochemistry in Practice
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`G. M. & Robinson, R. R.
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`Biol. 6, 703-706.
`13. Mixter, P. F., Wu, S. V., Studnicka,
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`(B) Titration of human peripheral blood leukocyte effector cells
`(2.5 µ.g/ml). (C)
`mediating ADCC in the presence of antibodies
`and mo use) in ADCC assays on the DHL-10
`15. Gubler, U. & Hoffman, B. J. (1983) Gene 25, 263-269.
`F. C. (1982) J. Biol. Chem. 251,
`T-cell line. 1F5 is a mouse mAb that recognizes the DHL-10 cells.
`16. White, B. A. & Bancroft,
`8569-8572.
`concentrations that can be attained in tumors after intrave
`
`
`
`Biochem. 131, 141-145.
`nous administration.
`18. Legerski, R. ]., Hodnett,].
`It is estimated that a major immunogenic site resides in the
`
`Acids Res. S, 1445-1463.
`
`CH2 region (23) of the lgG molecule. As one approach to
`J. (1983) Methods Enzymo/. 101, 20-78.
`decrease this p'roblem, one could generate chimeric mouse
`21. Southern, P. J. & Berg, P. (1982) J. Mo/. Appl. Genet. 1, 327-341.
`
`human antibodies thereby replacing the immunogenic C do
`22. Mulligan, R. C. & Berg, P. (1981) Proc. Natl. Acad. Sci. USA 78,
`
`mains of the mouse immunoglobulins with those of human
`23. Novotn9, J., Handschumacher,
`
`immunoglobulins. We did this using cDNA rather than cloned
`M. & Haber, E. (1986) J. Mo/.
`genomic DNA (24, 25). We sh<:>w here that this is a useful
`24. Sun, L. K., Curtis,
`E., Ghrayeb, J.,
`In cell line 3E3 and
`P., Rakowicz-Szulczynska,
`
`approach for producing chimeric antibody.
`H. (1986) Hybridoma S
`Morrison, S. L., Chang, N. & Koprowski,
`
`close to 1 µ.g/ml of IgGl protein was detected.
`its subclones
`Suppl. l, Sl7-S20.
`The chimeric antibody was found to bind to tumor cells as
`25. Sahagan, B. G., Dorai, H., Saltzgaber-Muller, J., Toneguzzo, F.,
`
`
`
`Guindon, C. A., Lilly, S. P., McDonald, K. W., Morrissey, D. V.,
`
`well as the mouse L6 antibody. The chimeric antibody was
`P. K. & Moore, G. P. (1986)
`Stone, B. A., Davis, G. L., Mcintosh,
`much more efficient than L6 in ADCC assays, killing a
`J. lmmunol. 137, 1066-1074.
`
`
`26. Goodman, G. E., Beaumier, P. L., Hellstrom, I., Femyhough, B.
`
`
`greater fraction of target cells at a concentration lower by a
`K. E. (1985) J. Clin. Oncol. 3, 340-352.
`factor of 100. Furthermore, the chimeric L6 killed cells from
`
`E. & Scars,
`
`
`a melanoma line that was refractory to ADCC by the mouse
`H. F. (1984) Proc. Natl. Acad. Sci. USA 81, 216-219.
`L6. In patients
`28. Irie, R. F. & Morton, D. L. (1986) Proc. Natl. Acad. Sci USA 83,
`
`one may speculate that the chimeric L6 would
`
`
`remain longer in the circulation. This, in combination with
`
`17. Nobrega, F. G., Dieckmann, C. L. & Tzagolotf, A. (1983) Anal.
`
`L. & Gray, H. B., Jr. (1978) Nucleic
`
`19. Messing,
`20. Okayama, H. & Berg, P. (1983) Mo/. Cell. Biol. 3, 280-289.
`
`2072-2076.
`
`Biol. 189, 715-721.
`
`& Hellstrom,
`
`27. Koprowski, H., Herlyn, D., Lubeck, M., Defreitas,
`
`8694-8698.
`
` 5 of 5
`
`BI Exhibit 1032
`
`