Abstract
We have devised a strategy based on polymerase chain reaction (PCR) for the rapid cloning of functional antibody genes as single-chain immunotoxins. RNA from a hybridoma producing an antibody (OVB3) that reacts with ovarian cancer cells was used as a template to make the first strand of a cDNA. Then a second strand was synthesized and amplified by using two sets of DNA primers that (i) hybridized to the ends of the light- and heavy-chain variable regions, (ii) encoded a linker peptide, and (iii) contained appropriate restriction enzyme sites for cloning. After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into an Escherichia coli expression vector containing a portion of the Pseudomonas exotoxin gene. Clones encoding recombinant single-chain immunotoxins were expressed in E. coli and the protein product was assessed for its ability to bind to or kill cells bearing the OVB3 antigen. By using this approach it should be possible to rapidly clone the functional variable region sequences of many different antibodies from hybridoma RNA.
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- Better M., Chang C. P., Robinson R. R., Horwitz A. H. Escherichia coli secretion of an active chimeric antibody fragment. Science. 1988 May 20;240(4855):1041–1043. doi: 10.1126/science.3285471. [DOI] [PubMed] [Google Scholar]
- Bird R. E., Hardman K. D., Jacobson J. W., Johnson S., Kaufman B. M., Lee S. M., Lee T., Pope S. H., Riordan G. S., Whitlow M. Single-chain antigen-binding proteins. Science. 1988 Oct 21;242(4877):423–426. doi: 10.1126/science.3140379. [DOI] [PubMed] [Google Scholar]
- Chaudhary V. K., Mizukami T., Fuerst T. R., FitzGerald D. J., Moss B., Pastan I., Berger E. A. Selective killing of HIV-infected cells by recombinant human CD4-Pseudomonas exotoxin hybrid protein. Nature. 1988 Sep 22;335(6188):369–372. doi: 10.1038/335369a0. [DOI] [PubMed] [Google Scholar]
- Chaudhary V. K., Queen C., Junghans R. P., Waldmann T. A., FitzGerald D. J., Pastan I. A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature. 1989 Jun 1;339(6223):394–397. doi: 10.1038/339394a0. [DOI] [PubMed] [Google Scholar]
- Chiang Y. L., Sheng-Dong R., Brow M. A., Larrick J. W. Direct cDNA cloning of the rearranged immunoglobulin variable region. Biotechniques. 1989 Apr;7(4):360–366. [PubMed] [Google Scholar]
- Huston J. S., Levinson D., Mudgett-Hunter M., Tai M. S., Novotný J., Margolies M. N., Ridge R. J., Bruccoleri R. E., Haber E., Crea R. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879–5883. doi: 10.1073/pnas.85.16.5879. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jinno Y., Ogata M., Chaudhary V. K., Willingham M. C., Adhya S., FitzGerald D., Pastan I. Domain II mutants of Pseudomonas exotoxin deficient in translocation. J Biol Chem. 1989 Sep 25;264(27):15953–15959. [PubMed] [Google Scholar]
- Jones P. T., Dear P. H., Foote J., Neuberger M. S., Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. 1986 May 29-Jun 4Nature. 321(6069):522–525. doi: 10.1038/321522a0. [DOI] [PubMed] [Google Scholar]
- Kondo T., FitzGerald D., Chaudhary V. K., Adhya S., Pastan I. Activity of immunotoxins constructed with modified Pseudomonas exotoxin A lacking the cell recognition domain. J Biol Chem. 1988 Jul 5;263(19):9470–9475. [PubMed] [Google Scholar]
- Orlandi R., Güssow D. H., Jones P. T., Winter G. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 May;86(10):3833–3837. doi: 10.1073/pnas.86.10.3833. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pastan I., FitzGerald D. Pseudomonas exotoxin: chimeric toxins. J Biol Chem. 1989 Sep 15;264(26):15157–15160. [PubMed] [Google Scholar]
- Pastan I., Willingham M. C., FitzGerald D. J. Immunotoxins. Cell. 1986 Dec 5;47(5):641–648. doi: 10.1016/0092-8674(86)90506-4. [DOI] [PubMed] [Google Scholar]
- Pirker R., FitzGerald D. J., Hamilton T. C., Ozols R. F., Willingham M. C., Pastan I. Anti-transferrin receptor antibody linked to Pseudomonas exotoxin as a model immunotoxin in human ovarian carcinoma cell lines. Cancer Res. 1985 Feb;45(2):751–757. [PubMed] [Google Scholar]
- Riechmann L., Clark M., Waldmann H., Winter G. Reshaping human antibodies for therapy. Nature. 1988 Mar 24;332(6162):323–327. doi: 10.1038/332323a0. [DOI] [PubMed] [Google Scholar]
- Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
- Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
- Sastry L., Alting-Mees M., Huse W. D., Short J. M., Sorge J. A., Hay B. N., Janda K. D., Benkovic S. J., Lerner R. A. Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5728–5732. doi: 10.1073/pnas.86.15.5728. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scharf S. J., Horn G. T., Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. doi: 10.1126/science.3461561. [DOI] [PubMed] [Google Scholar]
- Siegall C. B., Chaudhary V. K., FitzGerald D. J., Pastan I. Cytotoxic activity of an interleukin 6-Pseudomonas exotoxin fusion protein on human myeloma cells. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9738–9742. doi: 10.1073/pnas.85.24.9738. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siegall C. B., Xu Y. H., Chaudhary V. K., Adhya S., Fitzgerald D., Pastan I. Cytotoxic activities of a fusion protein comprised of TGF alpha and Pseudomonas exotoxin. FASEB J. 1989 Dec;3(14):2647–2652. doi: 10.1096/fasebj.3.14.2556314. [DOI] [PubMed] [Google Scholar]
- Skerra A., Plückthun A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 1988 May 20;240(4855):1038–1041. doi: 10.1126/science.3285470. [DOI] [PubMed] [Google Scholar]
- Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
- Verhoeyen M., Milstein C., Winter G. Reshaping human antibodies: grafting an antilysozyme activity. Science. 1988 Mar 25;239(4847):1534–1536. doi: 10.1126/science.2451287. [DOI] [PubMed] [Google Scholar]
- Vitetta E. S., Fulton R. J., May R. D., Till M., Uhr J. W. Redesigning nature's poisons to create anti-tumor reagents. Science. 1987 Nov 20;238(4830):1098–1104. doi: 10.1126/science.3317828. [DOI] [PubMed] [Google Scholar]
- Willingham M. C., FitzGerald D. J., Pastan I. Pseudomonas exotoxin coupled to a monoclonal antibody against ovarian cancer inhibits the growth of human ovarian cancer cells in a mouse model. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2474–2478. doi: 10.1073/pnas.84.8.2474. [DOI] [PMC free article] [PubMed] [Google Scholar]