Logo of brjcancerBJC HomepageBJC Advance online publicationBJC Current IssueSubmitting an article to BJCWeb feeds
Br J Cancer. 2000 Jul; 83(2): 252–260.
Published online 2000 Jun 15. doi:  10.1054/bjoc.2000.1226
PMCID: PMC2363493

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning


In various clinical studies, Hodgkin’s patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the non-human therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics. © 2000 Cancer Research Campaign

Keywords: CD30, phage display, chain shuffling, human antibody, guided selection

Full Text

The Full Text of this article is available as a PDF (153K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Barth S, Huhn M, Matthey B, Klimka A, Galinski EA, Engert A. Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions. Appl Environ Microbiol. 2000 Apr;66(4):1572–1579. [PMC free article] [PubMed]
  • Beiboer SH, Reurs A, Roovers RC, Arends JW, Whitelegg NR, Rees AR, Hoogenboom HR. Guided selection of a pan carcinoma specific antibody reveals similar binding characteristics yet structural divergence between the original murine antibody and its human equivalent. J Mol Biol. 2000 Feb 25;296(3):833–849. [PubMed]
  • Bosslet K, Mennel HD, Rodden F, Bauer BL, Wagner F, Altmannsberger A, Sedlacek HH, Wiegandt H. Monoclonal antibodies against epitopes on ganglioside GD2 and its lactones. Markers for gliomas and neuroblastomas. Cancer Immunol Immunother. 1989;29(3):171–178. [PubMed]
  • Brüggemann M, Neuberger MS. Strategies for expressing human antibody repertoires in transgenic mice. Immunol Today. 1996 Aug;17(8):391–397. [PubMed]
  • Chothia C, Lesk AM, Tramontano A, Levitt M, Smith-Gill SJ, Air G, Sheriff S, Padlan EA, Davies D, Tulip WR, et al. Conformations of immunoglobulin hypervariable regions. Nature. 1989 Dec 21;342(6252):877–883. [PubMed]
  • Chothia C, Lesk AM, Gherardi E, Tomlinson IM, Walter G, Marks JD, Llewelyn MB, Winter G. Structural repertoire of the human VH segments. J Mol Biol. 1992 Oct 5;227(3):799–817. [PubMed]
  • Diehl V, Kirchner HH, Schaadt M, Fonatsch C, Stein H, Gerdes J, Boie C. Hodgkin's disease: establishment and characterization of four in vitro cell lies. J Cancer Res Clin Oncol. 1981;101(1):111–124. [PubMed]
  • Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. [PMC free article] [PubMed]
  • Figini M, Marks JD, Winter G, Griffiths AD. In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation. J Mol Biol. 1994 May 27;239(1):68–78. [PubMed]
  • Figini M, Obici L, Mezzanzanica D, Griffiths A, Colnaghi MI, Winter G, Canevari S. Panning phage antibody libraries on cells: isolation of human Fab fragments against ovarian carcinoma using guided selection. Cancer Res. 1998 Mar 1;58(5):991–996. [PubMed]
  • Gruss HJ, Dower SK. The TNF ligand superfamily and its relevance for human diseases. Cytokines Mol Ther. 1995 Jun;1(2):75–105. [PubMed]
  • Hoogenboom HR. Designing and optimizing library selection strategies for generating high-affinity antibodies. Trends Biotechnol. 1997 Feb;15(2):62–70. [PubMed]
  • Hoogenboom HR, de Bruïne AP, Hufton SE, Hoet RM, Arends JW, Roovers RC. Antibody phage display technology and its applications. Immunotechnology. 1998 Jun;4(1):1–20. [PubMed]
  • Horn-Lohrens O, Tiemann M, Lange H, Kobarg J, Hafner M, Hansen H, Sterry W, Parwaresch RM, Lemke H. Shedding of the soluble form of CD30 from the Hodgkin-analogous cell line L540 is strongly inhibited by a new CD30-specific antibody (Ki-4). Int J Cancer. 1995 Feb 8;60(4):539–544. [PubMed]
  • Huston JS, McCartney J, Tai MS, Mottola-Hartshorn C, Jin D, Warren F, Keck P, Oppermann H. Medical applications of single-chain antibodies. Int Rev Immunol. 1993;10(2-3):195–217. [PubMed]
  • Jespers LS, Roberts A, Mahler SM, Winter G, Hoogenboom HR. Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. Biotechnology (N Y) 1994 Sep;12(9):899–903. [PubMed]
  • Kabat EA, Wu TT. Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. J Immunol. 1991 Sep 1;147(5):1709–1719. [PubMed]
  • Khazaeli MB, Conry RM, LoBuglio AF. Human immune response to monoclonal antibodies. J Immunother Emphasis Tumor Immunol. 1994 Jan;15(1):42–52. [PubMed]
  • Klimka A, Barth S, Matthey B, Roovers RC, Lemke H, Hansen H, Arends JW, Diehl V, Hoogenboom HR, Engert A. An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETA') is a potent immunotoxin against a Hodgkin-derived cell line. Br J Cancer. 1999 Jun;80(8):1214–1222. [PMC free article] [PubMed]
  • Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. [PubMed]
  • Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 1991 Dec 5;222(3):581–597. [PubMed]
  • McCafferty J, Fitzgerald KJ, Earnshaw J, Chiswell DJ, Link J, Smith R, Kenten J. Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display. Appl Biochem Biotechnol. 1994 May-Jun;47(2-3):157–173. [PubMed]
  • McCall AM, Amoroso AR, Sautès C, Marks JD, Weiner LM. Characterization of anti-mouse Fc gamma RII single-chain Fv fragments derived from human phage display libraries. Immunotechnology. 1998 Jun;4(1):71–87. [PubMed]
  • Meredith RF, Khazaeli MB, Grizzle WE, Orr RA, Plott G, Urist MM, Liu T, Russell CD, Wheeler RH, Schlom J, et al. Direct localization comparison of murine and chimeric B72.3 antibodies in patients with colon cancer. Hum Antibodies Hybridomas. 1993 Oct;4(4):190–197. [PubMed]
  • Newton DL, Xue Y, Olson KA, Fett JW, Rybak SM. Angiogenin single-chain immunofusions: influence of peptide linkers and spacers between fusion protein domains. Biochemistry. 1996 Jan 16;35(2):545–553. [PubMed]
  • Persic L, Horn IR, Rybak S, Cattaneo A, Hoogenboom HR, Bradbury A. Single-chain variable fragments selected on the 57-76 p21Ras neutralising epitope from phage antibody libraries recognise the parental protein. FEBS Lett. 1999 Jan 25;443(2):112–116. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Schnell R, Linnartz C, Katouzi AA, Schön G, Bohlen H, Horn-Lohrens O, Parwaresch RM, Lange H, Diehl V, Lemke H, et al. Development of new ricin A-chain immunotoxins with potent anti-tumor effects against human Hodgkin cells in vitro and disseminated Hodgkin tumors in SCID mice using high-affinity monoclonal antibodies directed against the CD30 antigen. Int J Cancer. 1995 Oct 9;63(2):238–244. [PubMed]
  • Schwab U, Stein H, Gerdes J, Lemke H, Kirchner H, Schaadt M, Diehl V. Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin's disease and a subset of normal lymphoid cells. Nature. 1982 Sep 2;299(5878):65–67. [PubMed]
  • Schwarting R, Gerdes J, Dürkop H, Falini B, Pileri S, Stein H. BER-H2: a new anti-Ki-1 (CD30) monoclonal antibody directed at a formol-resistant epitope. Blood. 1989 Oct;74(5):1678–1689. [PubMed]
  • Shawler DL, Bartholomew RM, Smith LM, Dillman RO. Human immune response to multiple injections of murine monoclonal IgG. J Immunol. 1985 Aug;135(2):1530–1535. [PubMed]
  • Smith TJ. Purification of mouse antibodies and Fab fragments. Methods Cell Biol. 1993;37:75–93. [PubMed]
  • Tai MS, McCartney JE, Adams GP, Jin D, Hudziak RM, Oppermann H, Laminet AA, Bookman MA, Wolf EJ, Liu S, et al. Targeting c-erbB-2 expressing tumors using single-chain Fv monomers and dimers. Cancer Res. 1995 Dec 1;55(23 Suppl):5983s–5989s. [PubMed]
  • Watzka H, Pfizenmaier K, Moosmayer D. Guided selection of antibody fragments specific for human interferon gamma receptor 1 from a human VH- and VL-gene repertoire. Immunotechnology. 1998 Jan;3(4):279–291. [PubMed]
  • Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR. Making antibodies by phage display technology. Annu Rev Immunol. 1994;12:433–455. [PubMed]

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...