Proc. Natl. Acad. Sci. USA
`Vol. 81, pp. 6851-6855, November 1984
`Immunology
`
`Chimeric human antibody molecules: Mouse antigen-binding
`domains with human constant region domains
`(transfect.lon/protoplast fusion/calcium phosphate transrection/intronk controlling tkments/transfectoma)
`
`
`SHERIE L. MORRISON*, M. JACQUELINE JOHNSONt, LEONARD A. HERZENBERGt, AND VERNON T. Q1:t:
`Coluf!lbi� University.
`_ollege of Physicians and Surgeons.
`
`for Cancer Research. •Depanment of Microbiology and the Cancer Center. Institute
`
`
`New York, NY 10032: tDepanment of Genetics, Stanford University School of Medicine.
`
`Stanford, CA 94305; and *Becton-Dickinson
`Monoclonal Center. 2375 Garcia Avenue , Mountain View, CA 94043
`
`_C
`
`Contributed by Leonard A. Heri.enberg, August I, 1984
`
`ABSTRACT We have created mouse-human antibody
`molecules of defined antigen-binding specificity by taking the
`variable region genes of a mouse antibody-producing myeloma
`ceU line with known antigen-binding specificity and joining
`them to human immunoglobulin constant region genes using
`recombinant DNA techniques. Chimeric genes were construct­
`ed that utilized the rearranged and expressed antigen-binding
`variable region cxons from the myeloma cell line S107, which
`produces an lgA (K) anti-phosphocholine antibody. The
`heavy chain variable region exon was joined to human lgG 1 or
`IgG2 heavy chain constant region genes, and the light chain
`variable region exon from the same myeloma was joined to the
`human " light chain gene. These genes we.re translected into
`mouse myeloma ceU lines, generating translormed ceUs that
`produce chimeric mouse-human lgG (K) or lgG (K) anti-pbos·
`phocholine antibodies. The transformed ceU lines remained tu­
`morigenic in mice and the chimeric molecules were present in
`the ascltic nuids and sera of tumor-bearing mice.
`
`The capability to transfer immunoglobulin genes into lym­
`phoid cells where they produce protein in quantities suffi­
`cient for structural studies (1-3) provides us with the oppor­
`tunity to generate and characterize novel immunoglobulin
`molecules. Cloned variable (V) region genes from mouse or
`rat hybridoma cell lines can be ligated to human constant (C)
`region genes and we would expect that these chimeric genes
`can be transfected into mouse myeloma cells, which then
`will produce novel human antibody molecules. We would
`thus produce antibodies that are largely human but which
`have antigen-binding specificities generated in mice. The ad­
`ditional potential for in vitro manipulation and alteration of
`both the antigen-binding site and the structures correlated
`with biological effector functions of these antibody mole­
`cules using recombinant DNA techniques would introduce a
`powerful approach for further understanding antibody struc­
`ture, function, and immunogenetics.
`As we show here, both chimeric mouse heavy chain V re­
`gion exon (VH}-human heavy chain C region genes and chi­
`meric mouse light chain V region exon (V,.}-human K light
`chain gene constructs are expressed when transfected into
`mouse myeloma cell lines. When both chimeric heavy and
`light chain genes are transfected into the same myeloma cell,
`an intact tetrameric (H2L2) chimeric antibody is produced.
`In this study we used VH and V,. exons from the mouse phos­
`phocholine (PCho)-binding antibody-producing S107 myelo­
`ma cell line (4, 5). Chimeric mouse-human anti-PCho anti­
`bodies were produced in culture by appropriate transfected
`cell lines or by "transfectomas" obtained when such cell
`lines are injected into mice.
`
`MATERIALS AND METHODS
`Chimeric Genes. The cloned S107 VH and S107 v .. genes
`were gifts from Matthew Scharff (Albert Einstein College of
`Medicine, Bronx, NY). The S107 VH gene was spliced to
`human lgGl and lgG2 C region genes by using Sal I linkers
`as shown in Fig. lA. Both constructs were inserted into the
`vector pSV2�H-gpt (1, 6). The S107 V,. gene was spliced to
`the human K gene at a unique HindUI site located in the large
`intron between the K light chain joining and C (J,. and C,.)
`region exons as shown in Fig. lB. This chimeric light chain
`gene construct was inserted into both PSV2�H-gpt and
`pSV2-neo plasmid vectors (7).
`Transfection. Protoplast fusion and calcium phosphate
`precipitation techniques (1, 8, 9) were used to transfect these
`chimeric immunoglobulin genes into the J558L myeloma cell
`line (a >. light chain-producing mouse myeloma cell line) and
`an immunoglobulin nonproducing derivative of the P3 my­
`eloma cell line. Mycophenolic acid (GIBCO) was used for
`selection of cells transfected with pSV2�-gpt vectors as de­
`scribed (1, 3). G418 (GIBCO) at 1.0 mg/ml was used for se­
`lection of cells transfected ,with pSV2-neo vectors (7).
`Light and heavy chain chimeric immunoglobulin genes
`were transfected sequentially by protoplast fusion using
`G418 selection for the chimeric light chain gene vector and
`mycophenolic acid for the chimeric heavy chain gene vector.
`The protoplast fusion transfection procedure used was as de­
`scribed (1).
`Transfection using the calcium phosphate precipitation
`procedure was done by transfecting a mixture of 40 µg each
`of the chimeric light and chimeric heavy chain pSV2�H-gpt
`vectors into 5 x 106 cells. Mycophenolic acid was used to
`select for transformed cell lines as described (1).
`Antigen Binding. PCho binding of antibody secreted into
`the culture supernates of transfected cell lines was analyzed
`by using a solid-phase radioimmunoassay described previ­
`ously (10). PCho-keyhole limpet hemocyanin antigen was
`bound to 96-well polyvinyl plates; bindint of chimeric anti­
`PCho antibodies was detected by using 1 I-labeled protein
`A or 1251-labeled-anti-human lgG. PCho-binding antibodies
`in biosynthetically labeled culture supernates and cell ly­
`sates of transfected cell lines also were analyzed by binding
`the biosynthetically labeled antibody to PCho-coupled Seph­
`arose 48 (Pharmacia) and then eluting the bound antibody
`with PCho-hapten. The bound and eluted antibody was ex­
`amined by NaDodS04/polyacrylamide gel electrophoresis
`(NaDodS04/PAGE). Biosynthetic-labeling procedures were
`as described (11).
`ldlotope Analysis. Three hybridoma anti-idiotope antibod­
`ies, also kindly provided by Matthew Scharff and Angela
`
`The publication costs of this anicle were defrayed in part by page charge
`
`
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`Abbreviations: V, variable; C, constant; J,joining; "'heavy chain;
`L• light chain; PCho, phosphoeholine; kb, kilobase(s); NEPHGE.
`nonequilibrium pH gradient electrophoresis.
`
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`6852
`
`Immunology: Morrison et al.
`
`Proc. Natl. Acad. Sci. USA 81 (1984)
`
`A
`
`bJD.HI
`
`(Seel) Sel I (Hind Ill)
`
`pSV26H-S 107 HuG 1
`
`t
`
`t
`
`Mouse VDJ
`
`Human constant region
`
`pBR322 orl
`
`SV40 ori
`
`B
`
`bDJ.HI t Mouse VJ
`
`pSV26H-S107 Huk
`
`Human kappa
`
`bm.HI
`
`t
`
`pBR322 orl
`
`SV40 ori
`
`Eco-gpt or
`Eco-neo
`
`F1G. 1. Schematic diagrams (not drawn to scale) of the chimeric mouse-human heavy chain gene vector (A) and the chimeric light chain
`gene vectors (B). DNA fragment sizes are as follows: HindlII-BamHI human IgGl (HuGl) or lgG2 (HuG2) heavy chain gene, 7 kilobases (kb);
`mouse Sl07 VH gene, 4.5 kb; HindIIl-BamHl human c. gene (HuK), 10 kb; and mouse Sl07 v. gene, 3.5 kb. The Hindlll sites in the human
`IgGl and IgG2 heavy chain genes were ligated to the Sac I site of the mouse Sl07 VH gene with Sal I linkers.
`
`Guisti (Albert Einstein College of Medicine), were usea to
`analyze the Vw-VL (Lis light chain) domain structure of the
`chimeric human anti-PCho antibodies. These antibodies
`(TC102.l.2, Tl39.2, and Tl56.l.l), recognizing three inde­
`pendent idiotopes (12), were used to immunoprecipitate bio­
`synthetically labeled material eluted with PCho from the
`PCho-Sepharose 4B matrix. Immunoprecipitates were ana­
`lyzed by NaDodS04/PAGE.
`Chain Composition. Monoclonal anti-hu­
`Immunoglobulin
`man lgG and anti-human K antibodies (Becton-Dickinson)
`were used to immunoprecipitate biosynthetically labeled chi­
`meric human anti-PCho antibodies for analyses using two­
`dimensional nonequilibrium pH gradient PAGE (NEPH'.GE)
`(11, 13). PCho-coupled Sepharose 4B also was used for af­
`finity purification.
`Tunicamycin
`lmmunoglobulln Heavy Chain Glyoosylation.
`
`(Calbiochem-Behring) was used to inhibit asparagine-linked
`glycosylation of biosynthetically labeled antibody from
`mouse cell lines producing mouse-human chimeric immuno­
`globulins (11). PCho-binding antibody from tunicamycin­
`treated cells was analyzed by NaDodS04/PAGE. Proce-
`
`dures for tunicamycin treatment were as described (11).
`Chimeric Mouse-Human Antibody Production in Mice.
`Transfected J558L cells producing chimeric mouse-human
`antibody were injected intraperitoneally into BALB/c mice
`(106 cells per mouse). Sera and ascitic fluids from tumor­
`bearing mice were analyzed for human anti-PCho antibody
`by a solid-phase radioimmunoassay (10) and by immunoelec­
`trophoresis using a polyclonal anti-human lgG antiserum.
`
`RESULTS
`We obtained expression of chimeric mouse V region-human
`C region genes in transfected J558L and the immunoglobulin
`nonproducing P3 myeloma cell lines. When both light chain
`and heavy chain chimeric genes (see Fig. 1) were expressed
`in the same cell, tetrameric (H2L2) antigen-binding antibod­
`ies were obtained. Biosynthetically labeled antibody mole­
`cules secreted by J�58L cells expressing chimeric genes
`were bound and hapten-eluted from PCho-Sepharose 4B.
`Autoradiograms of two-dimensional NEPHGE analyses
`(Fig. 2) show immunoglobulin polypeptide chains of the ex-
`
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`Immunology: Morrison et al
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`Proc. Natl Acad. Sci. USA 81 (1984) 6853
`
`pected charge and relative molecular mass. Identical two­
`dimensional gel analysis results were obtained with immuno­
`precipitates by using monoclonal anti-human K and IgG anti­
`bodies. Similar results were obtained when the chimeric
`IgG2 (K) antibodies produced in the transfected nonproduc­
`ing P3 cell line were analyzed. Since the recipient P3 cell line
`does not produce endogenous immunoglobulin polypeptide
`chains, only the chimeric mouse-human heavy and light
`chains were seen (data not shown). PCho binding by the chi­
`meric antibody produced in the J558L cell line required the
`specific association of both the V H and V L domains of the
`Sl07 myeloma protein connected to human C region poly­
`peptides. Antibody secreted by transfected J558L cells ex­
`pressing only the chimeric heavy chain and the endogenous
`J558L light chain did not bind PCho (data not shown).
`Further verification of the appropriate polypeptide folding
`of the mouse V H and V L domains attached to human C re­
`gion polypeptide chains was done by analyses detecting the
`presence of idiotopes known to occur on the parental Sl07
`PCho-binding antibody molecule. Three monoclQnal anti­
`idiotope antibodies-two recognizing idiotopes requiring the
`association of light and heavy V region domains (TC102.l.2
`and Tl39.2) and the third (Tl56.l.l) recognizing an epitope
`preseqt on the heavy chain V region domain (12)-were
`found to react with the mouse-human chimeric anti-PCho
`antibodies (see Fig. 3). This is good evidence that the mouse
`Sl07 antigen-binding domains have folded into their intend­
`ed structures.
`The conformation of IgG heavy chain C regions depends
`
`Basic
`
`A
`
`HuG2-
`
`B
`
`HuGl-
`
`•
`
`t
`
`•
`
`t
`
`HuK
`
`Acidic
`
`-67
`
`-45
`
`-30
`
`-67
`
`-45
`
`-30
`
`•
`
`t
`
`..
`
`t
`
`J558>..
`
`a
`
`b
`
`c
`
`d
`
`H2L2- -----
`
`Fm. 3. Autoradiograph of nonreduced NaI>odSO./PAGE anal­
`yses of />Cho-binding material from a chimeric IgG2 (K) antibody­
`producing J558L cell line immunoprecipitated with Staphylococcus
`aureus protein A (lane a), monoclonal anti-human K antibody (lane
`b). and anti-idiotope antibodies TC102.l.2 and Tl56.l.l (lanes c and
`d).
`
`on the presence of the asparagine-linked carbohydrate
`moeity in the CH2 domain of the molecule (4). The loss of
`this carbohydrate chain affects profoundly the overall do­
`main structure of this part of the immunoglobulin molecule.
`Concomitant with this structural change, the catabolism rate
`of the molecule is increased and biological effector functions
`such as complement-fixation are lost (14). Glycosylation of
`the mouse-human chimeric antibodies in mouse myeloma
`cells was deduced by determining the molecular masses of
`antibodies synthesized in the presence and absence of tuni­
`camycin, an antibiotic inhibitor of asparagine-linked glyco­
`sylation. Fig. 4 is an autoradiogram of NaDodS04/PAGE
`analysis of glycosylated and nonglycosylated chimeric heavy
`and light chains produced in transfected mouse myeloma
`cells. Though we cannot be certain that in the absence of
`tunicamycin the appropriate asparagine residue in the CH2
`domain is glycosylated, clearly, the lower molecular mass of
`the heavy chain synthesized in the presence of tunicamycin
`is as expected if a single N-linked carbohydrate were absent
`
`a
`
`b
`
`-·-
`
`-67
`
`-45
`
`-30
`
`-20
`
`FIG. 2. Autoradiographs of two-dimensional NEPHGE gels of
`mouse-human chimeric IgG2 (K) anti-PC antibody (A) and mouse­
`human chimeric IgGl (K) anti-PC antibody (B). Antibodies synthe­
`sized by transfected J558L cells were affi nity purified with PCho­
`Sepharose 4B. The electrophoretic mobility of the J558L >.chain had
`been determined previously (1). Size markers are shown in k.Da.
`
`FIG. 4. Autoradiograph of reduced NaI>odSO./PAGE analyses
`of chimeric IgG2 (K) antibody synthesized in the presence (lane a)
`and absence (lane b) of tunicamycin. Size markers are shown in
`k.Da.
`
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`6854 Immunology:
`Morrison et al
`
`Proc. Natl Acad. Sci. USA 81 (1984)
`
`anti-human lgG
`
`the immunogenicity of antibodies used in vivo, relative to
`
`
`
`monoclonal mouse hybridoma antibodies currently being
`used (15). Further, since the same V region can be joined to
`
`
`
`any C region, it is possible to use the C region of an appropri­
`
`ate human heavy chain isotype that will exhibit desired bio­
`F10. S. lmmunoclcctrophoresis of ascitic fluid from a BALB/c
`
`
`
`logical effector functions.
`
`mouse bearing a J558L transfectoma producing chimeric IgG2 (ic)
`The use of mammalian lymphoid cells to produce these
`with a polyclonal anti-human lgG
`anti-PCho antibodies developed
`
`
`
`antibodies rather than a prokaryotic expression system as­
`antiserum.
`
`
`sures that any post-translational modifications of the anti­
`from the polypeptide chain. Note that the light chain band is
`
`
`
`
`body molecules as they are synthesized, processed, folded,
`
`
`
`not affected by tunicamycin treatment. From these data, we
`
`and assembled in the eukaryotic endoplasmic reticulum are
`
`
`
`conclude that the mouse myeloma cell appropriately glyco­
`
`
`carried out correctly. For example, the immunoglobulins en­
`sylates the human heavy chain.
`
`coded by the transfected mouse-human chimeric immuno­
`
`When transfected J558L cells producing the human IgG2
`
`
`globulin genes in both mouse recipient cells were glycosylat­
`
`(K) chimeric anti-PCho antibody were grown as an ascitic
`
`
`ed, bound antigen, and presumably would be treated as nor­
`
`
`tumor in BALB/c mice, analyses of sera and ascitic fluids
`
`
`mal immunoglobulins if injected into humans. This obviates
`
`showed the presence of significant quantities of anti-PCho­
`
`
`the need for any post-synthetic in vitro modification or as­
`
`
`binding antibodies by radioimmunoassay. Fig. 5 shows the
`
`
`
`sembly that is required with prokaryotic synthesis of immu­
`
`
`results of immunoelectrophoretic analysis of ascitic fluids
`
`
`
`
`noglobulin polypeptides to produce functional antibody mol­
`
`
`from these transfectoma-bearing mice. Polyclonal anti-hu­
`
`ecules (16). Furthermore, we have shown that transfectomas
`
`man antiserum (a gift of E. A. Kabat, Columbia University,
`
`produce useable amounts of human antibodies either in tis­
`
`New York) was used to demonstrate the presence of human
`sue culture or in mice.
`immunoglobulin.
`
`Based on our previous experience with
`
`It is now feasible to shuffle exons or carry out other kinds
`mouse hybridoma-antibody production in mice, we conclude
`
`
`
`of directed mutagenesis to explore the human antibody mol­
`
`
`that the amounts of immunoglobulin visualized by this im­
`
`
`
`ecules C region structures required for carrying out diverse
`
`munoelectrophoretic analysis are similar to the levels seen
`
`
`
`functions. It is conceivable that an altered antibody
`antibody
`
`with lower-producing mouse bybridomas in vivo.
`
`
`
`molecule more effective in specific functions than naturally
`
`
`Transfectomas producing chimeric heavy and light chains
`occurring antibody molecule can be created. In a similar
`
`were produced either by cotransfection of both genes using
`
`manner, it should be possible to modify V regions to alter
`
`
`
`calcium phosphate precipitation or by sequential protoplast
`
`
`
`their interactions with antigen. Even more exciting would be
`
`fusion using vectors with different drug markers. For proto­
`de novo.
`
`to construct antigen-binding sites
`
`plast fusion, the heavy chains were introduced first on a
`A limitation yet to be overcome is the low frequency of
`
`
`
`plasmid expressing the Eco-gpt gene. One heavy chain-pro­
`
`transformants that produce chimeric light chains. The chi­
`(see Fig. lB) has the mouse
`meric light chain gene construct
`
`fusion ducing cell line then was transfected by protoplast
`
`
`
`with a plasmid containing the light chain gene and the neo
`
`V,. gene coupled to the presumed human intronic controlling
`
`
`
`marker. Transformants resistant to both mycophenolic acid
`
`
`element (ICE) or "enhancer"-that is, this construct con­
`and G418 were assayed for their production of anti-PCho
`
`tains the human intron with a sequence homologous to the
`
`
`
`antibodies. Of 77 transformants analyzed, only 7 were posi­
`
`
`established mouse ICE sequence (17). In contrast, the heavy
`
`tive for antibody by radioimmunoassay and only 2 of these
`
`
`
`chain chimeric mouse-human polypeptide is expressed effi­
`
`
`ciently. In the latter construct (sec Fig. lA), the ICE se­
`
`
`
`were found to be producing significant quantities of K light
`
`
`
`chain. In cotransfection experiments usins the calcium phos­
`
`
`quence is mouse-derived. Thus, the lower expression fre­
`
`
`
`phate precipitation protocol, the same phenomenon was ob­
`
`
`quency of the chimeric light chain gene may reflect a species
`
`served. Analyses of a large number of independent transfor­
`
`
`
`preference for the effective interaction of the ICE sequence
`
`( <10%) of the transfected cell mants revealed that a minority
`
`
`
`with factors or elements controlling immunoglobulin expres­
`
`lines produced both chimeric heavy and light chain polypep­
`
`sion in mouse lymphoid cells. This suggestion is consistent
`
`tides. Since our own unpublished data using the calcium
`
`with our earlier finding that the mouse light chain gene, pre­
`
`
`sumably due to its ICE sequence, is not expressed efficiently
`
`
`
`phosphate precipitation procedure demonstrate that two in­
`
`in a rat myeloma cell line (1). Construction of chimeric genes
`
`
`tact mouse immunoglobulin genes cotransfected into the
`
`
`of using entire mouse intronic sequences and elimination
`same cell, using the same protocol as described here, coex­
`
`
`
`most human intronic sequences, to determine if these will be
`
`press both gene products in the majority of transformed cell
`
`
`expressed efficiently, await further investigation.
`
`lines, these results suggest that appropriate transcriptional
`
`In this work we have taken the initial steps toward con­
`
`
`or translational controlling clements are absent in the chi­
`
`
`
`structing and efficiently expressing unique imrnunoglobulin
`
`meric light chain gene construction or that required tran­
`
`
`molecules. We expect that this technique will permit an in­
`
`scriptional or translational factors are absent in the mouse
`
`
`creased understanding of the structural and dynamic require­
`
`
`cell Lines transfected here.
`
`
`
`ments of antibody functions. It also will facilitate develop­
`DISCUSSION
`
`
`ment of antibody molecules for immunodiagnosis and immu­
`The opportunity to use recombinant DNA techniques to con­
`
`
`
`notherapy.
`struct novel antibody genes and then to produce antibody
`
`
`
`molecules in mammalian lymphoid cells transfected with
`We thank Letitia Wims and Linda Nakamura Roark, who provid­
`
`these genes in appropriate vectors provides a new approach
`ed invaluable
`
`
`assistance with these experiments. We also thank
`
`
`to understanding the structure, function, and immune prop­
`Olivia Gagliani
`
`
`for her patience in preparing this manuscript. This
`
`erties of antibody molecules. The use of chimeric mouse­
`research was supported in part by Grants K04 AI00408, Al 19042,
`
`AI 08917, CA 16858, CA 22736, CA 13969, and CA G4681 from the
`
`human antibody gene constructs permits us to study "near­
`of Health, Grant IMS-360 from the American
`National Institutes
`
`
`human" antibodies with desired antigen-binding specific­
`
`
`Cancer Society. and Grant SPO 13712-02-00 from the Becton-Dick­
`
`
`ities. For clinical applications, human antibody molecules
`
`inson Monoclonal Center.
`
`
`constructed by using recombinant DNA techniques should
`
`
`complement human-human hybridoma antibodies as useful
`
`
`
`immunotherapeutic and diagnostic reagents. The use of hu­
`1. Oi, V. T .. Morrison, S. L., Herzenbcrg,
`L.A. & Berg, P.
`
`might decrease or eliminate man or near-human antibodies
`
`
`(1983) Proc. Natl. Acad. Sci. USA 80, 825-829.
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`Immunology: Morrison et al.
`
`Proc. Natl. Acad. Sci. USA 81 (1984) 6855
`
`2. Gillies, S. D., Morrison, S. L., Oi, V. T. & Tonegawa, S.
`(1983) Cell 33, 717-728.
`3. Ochi, A .. Hawley, R. G., Hawley, T., Shulman, M. J., Trau­
`necker, A .. Kohler, G. & Hozumi, N. (1983) Proc. Natl. Acad.
`Sci. USA 80, 6351-6355.
`4. Kwan, S. P., Rudikoff, S., Seidman, J. G., Leder, P. &
`Scharff, M. D. (1981) J. Exp. Med. 153, 1366-1370.
`5. Clarke, C., Berenson, J., Governman, J., Boyer, P. D.,
`Crews, S., Siu, G. & Calame, K. (1982) Nucleic Acids Res. 10,
`7731-7749.
`6. Mulligan, R. C. & Berg, P. (1980) Science 209, 1422-1427.
`7. Mulligan, R. C. & Berg, P. (1981) Proc. Natl. Acad. Sci. USA
`78, 2072-2076.
`8. Sandri-Goldin, R. M., Goldin, A. L., Levine, M. & Glorioso,
`J. C. (1981) Mo/. Cell. Biol. l, 743-752.
`9. Chu, G. & Sharp, P. A. (1981) Gene 13, 197-202.
`
`10. Oi, V. T. & Herzenberg, L.A. (1979) Mo/. Immuno/. 16,
`1005-1017.
`11. Oi, V. T., Bryan, V. M .. Herzenberg, L.A. & Herzenberg,
`L. A. (1980) J. Exp. Med. 151, 1260-1274.
`12. Desaymaid. C., Guist, A. M. & Sharff, M. D. (1984) Mo/. lm­
`munol., in press.
`13. O'Farrell, P. Z .. Goodman, H. M. & O'Farrell, P. H. (1977)
`Cell 12, 1133-1142.
`14. Nose, M. & Wigzell, H. (1983) Proc. Natl. Acad. Sci. USA 80,
`6632-6636.
`15. Miller, R. A. & Levy, R. (1981) Lancet i, 226-229.
`16. Boss, M.A., Kenton, J. H., Wood, C. R. & Emtage, J. S.
`(1984) Nucleic Acids Res. 12, 3791-3806.
`17. Morrison, S. L. & Oi, V. T. (1984) Annu. Rev. lmmunol. 2,
`239-256.
`
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