Cancer Immunology, Immunotherapy

, Volume 53, Issue 3, pp 217–226

Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors

  • Winfried Wels
  • Markus Biburger
  • Tina Müller
  • Benjamin Dälken
  • Ulrike Giesübel
  • Torsten Tonn
  • Christoph Uherek


Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells’ potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.


Targeted therapy Single chain Fv antibody fragment Immunotoxin Pseudomonal exotoxin A Cyotoxic T lymphocytes Natural killer cells Chimeric antigen receptor Epidermal growth factor receptor ErbB2/HER2 


  1. 1.
    Altenschmidt U, Kahl R, Moritz D, Schnierle BS, Gerstmayer B, Wels W, Groner B (1996) Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes. Clin Cancer Res 2:1001–1008PubMedGoogle Scholar
  2. 2.
    Altenschmidt U, Klundt E, Groner B (1997) Adoptive transfer of in vitro-targeted, activated T lymphocytes results in total tumor regression. J Immunol 159:5509–5515PubMedGoogle Scholar
  3. 3.
    Altenschmidt U, Schmidt M, Groner B, Wels W (1997) Targeted therapy of schwannoma cells in immunocompetent rats with an erbB2-specific antibody-toxin. Int J Cancer 73:117–124CrossRefPubMedGoogle Scholar
  4. 4.
    Alvarez-Vallina L, Hawkins RE (1996) Antigen-specific targeting of CD28-mediated T cell co-stimulation using chimeric single-chain antibody variable fragment-CD28 receptors. Eur J Immunol 26:2304–2309PubMedGoogle Scholar
  5. 5.
    Azemar M, Djahansouzi S, Jäger E, Solbach C, Schmidt M, Maurer AB, Mross K, Unger C, von Minckwitz G, Dall P, Groner B, Wels W (2003). Regression of cutaneous tumor lesions in patients intratumorally injected with a recombinant single-chain antibody-toxin targeted to ErbB2/HER2. Breast Cancer Research and Treatment 82:155–164Google Scholar
  6. 6.
    Azemar M, Schmidt M, Arlt F, Kennel P, Brandt B, Papadimitriou A, Groner B, Wels W (2000) Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapid tumor regression in vivo. Int J Cancer 86:269–275Google Scholar
  7. 7.
    Benhar I, Azriel R, Nahary L, Shaky S, Berdichevsky Y, Tamarkin A, Wels W (2000) Highly efficient selection of phage antibodies mediated by display of antigen as lpp-OmpA’ fusions on live bacteria. J Mol Biol 301:893–904CrossRefPubMedGoogle Scholar
  8. 8.
    Bonini C, Ferrari G, Verzeletti S, Servida P, Zappone E, Ruggieri L, Ponzoni M, Rossini S, Mavilio F, Traversari C, Bordignon C (1997) HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia [see comments]. Science 276:1719–1724CrossRefPubMedGoogle Scholar
  9. 9.
    Brentjens RJ, Latouche JB, Santos E, Marti F, Gong MC, Lyddane C, King PD, Larson S, Weiss M, Riviere I, Sadelain M (2003) Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. Nat Med 9:279–286CrossRefPubMedGoogle Scholar
  10. 10.
    Brocker T, Karjalainen K (1995) Signals through T cell receptor-zeta chain alone are insufficient to prime resting T lymphocytes. J Exp Med 181:1653–1659PubMedGoogle Scholar
  11. 11.
    Bukowski JF, Warner JF, Dennert G, Welsh RM (1985) Adoptive transfer studies demonstrating the antiviral effect of natural killer cells in vivo. J Exp Med 161:40–52PubMedGoogle Scholar
  12. 12.
    Chaudhary VK, Queen C, Junghans RP, Waldmann TA, FitzGerald DJ, Pastan I (1989) A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature 339:394–397PubMedGoogle Scholar
  13. 13.
    Darcy PK, Haynes NM, Snook MB, Trapani JA, Cerruti L, Jane SM, Smyth MJ (2000) Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL. J Immunol 164:3705–3712PubMedGoogle Scholar
  14. 14.
    Eshhar Z (1997) Tumor-specific T-bodies: towards clinical application. Cancer Immunol Immunother 45:131–136CrossRefPubMedGoogle Scholar
  15. 15.
    Finney HM, Lawson AD, Bebbington CR, Weir AN (1998) Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product. J Immunol 161:2791–2797PubMedGoogle Scholar
  16. 16.
    Fitzer-Attas CJ, Schindler DG, Waks T, Eshhar Z (1998) Harnessing Syk family tyrosine kinases as signaling domains for chimeric single chain of the variable domain receptors: optimal design for T cell activation. J Immunol 160:145–154PubMedGoogle Scholar
  17. 17.
    Gong JH, Maki G, Klingemann HG (1994) Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 8:652–658PubMedGoogle Scholar
  18. 18.
    Gross G, Waks T, Eshhar Z (1989) Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A 86:10024–10028PubMedGoogle Scholar
  19. 19.
    Hombach A, Heuser C, Sircar R, Tillmann T, Diehl V, Pohl C, Abken H (1998) An anti-CD30 chimeric receptor that mediates CD3-zeta-independent T-cell activation against Hodgkin’s lymphoma cells in the presence of soluble CD30. Cancer Res 58:1116–1119PubMedGoogle Scholar
  20. 20.
    Hoogenboom HR, de Bruine AP, Hufton SE, Hoet RM, Arends JW, Roovers RC (1998) Antibody phage display technology and its applications. Immunotechnology 4:1–20PubMedGoogle Scholar
  21. 21.
    Jensen M, Tan G, Forman S, Wu AM, Raubitschek A (1998) CD20 is a molecular target for scFvFc:zeta receptor redirected T cells: implications for cellular immunotherapy of CD20+ malignancy. Biol Blood Marrow Transplant 4:75–83PubMedGoogle Scholar
  22. 22.
    Kane LP, Lin J, Weiss A (2000) Signal transduction by the TCR for antigen. Curr Opin Immunol 12:242–249Google Scholar
  23. 23.
    Klapper LN, Kirschbaum MH, Sela M, Yarden Y (2000) Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors. Adv Cancer Res 77:25–79PubMedGoogle Scholar
  24. 24.
    Kreitman RJ (2003) Recombinant toxins for the treatment of cancer. Curr Opin Mol Ther 5:44–51PubMedGoogle Scholar
  25. 25.
    Kreitman RJ, Wilson WH, Bergeron K, Raggio M, Stetler-Stevenson M, FitzGerald DJ, Pastan I (2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N Engl J Med 345:241–247Google Scholar
  26. 26.
    Kreitman RJ, Wilson WH, White JD, Stetler-Stevenson M, Jaffe ES, Giardina S, Waldmann TA, Pastan I (2000) Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies. J Clin Oncol 18:1622–1636PubMedGoogle Scholar
  27. 27.
    Maurer-Gebhard M, Schmidt M, Azemar M, Altenschmidt U, Stocklin E, Wels W, Groner B (1998) Systemic treatment with a recombinant erbB-2 receptor-specific tumor toxin efficiently reduces pulmonary metastases in mice injected with genetically modified carcinoma cells. Cancer Res 58:2661–2666PubMedGoogle Scholar
  28. 28.
    McGuinness RP, Ge Y, Patel SD, Kashmiri SV, Lee HS, Hand PH, Schlom J, Finer MH, McArthur JG (1999) Anti-tumor activity of human T cells expressing the CC49-zeta chimeric immune receptor. Hum Gene Ther 10:165–173Google Scholar
  29. 29.
    Mendelsohn J, Baselga J (2000) The EGF receptor family as targets for cancer therapy. Oncogene 19:6550–6565CrossRefPubMedGoogle Scholar
  30. 30.
    Mezzanzanica D, Canevari S, Mazzoni A, Figini M, Colnaghi MI, Waks T, Schindler DG, Eshhar Z (1998) Transfer of chimeric receptor gene made of variable regions of tumor-specific antibody confers anticarbohydrate specificity on T cells. Cancer Gene Ther 5:401–407PubMedGoogle Scholar
  31. 31.
    Moritz D, Groner B (1995) A spacer region between the single chain antibody- and the CD3 zeta-chain domain of chimeric T cell receptor components is required for efficient ligand binding and signaling activity. Gene Ther 2:539–546.PubMedGoogle Scholar
  32. 32.
    Moritz D, Wels W, Mattern J, Groner B (1994) Cytotoxic T lymphocytes with a grafted recognition specificity for ERBB2-expressing tumor cells. Proc Natl Acad Sci U S A 91:4318–4322PubMedGoogle Scholar
  33. 33.
    Olayioye MA, Neve RM, Lane HA, Hynes NE (2000) The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 19:3159–3167PubMedGoogle Scholar
  34. 34.
    Pai-Scherf LH, Villa J, Pearson D, Watson T, Liu E, Willingham MC, Pastan I (1999) Hepatotoxicity in cancer patients receiving erb-38, a recombinant immunotoxin that targets the erbB2 receptor. Clin Cancer Res 5:2311–2315PubMedGoogle Scholar
  35. 35.
    Pluckthun A, Pack P (1997) New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology 3:83–105CrossRefPubMedGoogle Scholar
  36. 36.
    Reinhold U, Liu L, Ludtke-Handjery HC, Heuser C, Hombach A, Wang X, Tilgen W, Ferrone S, Abken H (1999) Specific lysis of melanoma cells by receptor grafted T cells is enhanced by anti-idiotypic monoclonal antibodies directed to the scFv domain of the receptor. J Invest Dermatol 112:744–750CrossRefPubMedGoogle Scholar
  37. 37.
    Reiter Y, Brinkmann U, Jung SH, Lee B, Kasprzyk PG, King CR, Pastan I (1994) Improved binding and antitumor activity of a recombinant anti-erbB2 immunotoxin by disulfide stabilization of the Fv fragment. J Biol Chem 269:18327–18331PubMedGoogle Scholar
  38. 38.
    Reiter Y, Pastan I (1996) Antibody engineering of recombinant Fv immunotoxins for improved targeting of cancer: disulfide-stabilized Fv immunotoxins. Clin Cancer Res 2:245–252PubMedGoogle Scholar
  39. 39.
    Roberts MR, Cooke KS, Tran AC, Smith KA, Lin WY, Wang M, Dull TJ, Farson D, Zsebo KM, Finer MH (1998) Antigen-specific cytolysis by neutrophils and NK cells expressing chimeric immune receptors bearing zeta or gamma signaling domains. J Immunol 161:375–384PubMedGoogle Scholar
  40. 40.
    Samel D, Muller D, Gerspach J, Assohou-Luty C, Saas G, Tiegs G, Pfizenmaier K, Wajant H (2003) Generation of a FasL based proapoptotic fusion protein devoid of systemic toxicity due to cell surface antigen-restricted activation. J Biol Chem (in press)Google Scholar
  41. 41.
    Schmidt M, Maurer-Gebhard M, Groner B, Kohler G, Brochmann-Santos G, Wels W (1999) Suppression of metastasis formation by a recombinant single chain antibody-toxin targeted to full-length and oncogenic variant EGF receptors. Oncogene 18:1711–1721Google Scholar
  42. 42.
    Schmidt M, McWatters A, White RA, Groner B, Wels W, Fan Z, Bast RC (2001) Synergistic interaction between an anti-p185HER-2 Pseudomonas exotoxin fusion protein [scFv(FRP5)-ETA] and ionizing radiation for inhibiting growth of ovarian cancer cells that overexpress HER-2. Gynecol Oncol 80:145–155CrossRefPubMedGoogle Scholar
  43. 43.
    Schmidt M, Reiser P, Hills D, Gullick WJ, Wels W (1998) Expression of an oncogenic mutant EGF receptor markedly increases the sensitivity of cells to an EGF-receptor-specific antibody-toxin. Int J Cancer 75:878–884CrossRefPubMedGoogle Scholar
  44. 44.
    Schmidt M, Vakalopoulou E, Schneider DW, Wels W (1997) Construction and functional characterization of scFv(14E1)-ETA—a novel, highly potent antibody-toxin specific for the EGF receptor. Br J Cancer 75:1575–1584PubMedGoogle Scholar
  45. 45.
    Schnierle BS, Stitz J, Bosch V, Nocken F, Merget-Millitzer H, Engelstadter M, Kurth R, Groner B, Cichutek K (1997) Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells. Proc Natl Acad Sci U S A 94:8640–8645CrossRefPubMedGoogle Scholar
  46. 46.
    Sedlmayr P, Rabinowich H, Elder EM, Ernstoff MS, Kirkwood JM, Herberman RB, Whiteside TL (1991) Depressed ability of patients with melanoma or renal cell carcinoma to generate adherent lymphokine-activated killer cells. J Immunother 10:336–346PubMedGoogle Scholar
  47. 47.
    Smyth MJ, Godfrey DI, Trapani JA (2001) A fresh look at tumor immunosurveillance and immunotherapy. Nat Immunol 2:293–299PubMedGoogle Scholar
  48. 48.
    Spyridonidis A, Schmidt M, Bernhardt W, Papadimitriou A, Azemar M, Wels W, Groner B, Henschler R (1998) Purging of mammary carcinoma cells during ex vivo culture of CD34+ hematopoietic progenitor cells with recombinant immunotoxins. Blood 91:1820–1827PubMedGoogle Scholar
  49. 49.
    Todorovska A, Roovers RC, Dolezal O, Kortt AA, Hoogenboom HR, Hudson PJ (2001) Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J Immunol Methods 248:47–66CrossRefPubMedGoogle Scholar
  50. 50.
    Tonn T, Becker S, Esser R, Schwabe D, Seifried E (2001) Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92. J Hematother Stem Cell Res 10:535–544CrossRefPubMedGoogle Scholar
  51. 51.
    Uherek C, Groner B, Wels W (2001) Chimeric antigen receptors for the retargeting of cytotoxic effector cells. J Hematother Stem Cell Res 10:523–534CrossRefPubMedGoogle Scholar
  52. 52.
    Uherek C, Tonn T, Uherek B, Becker S, Schnierle B, Klingemann H-G, Wels W (2002) Retargeting of natural killer-cell cytolytic activity to ErbB2-expressing cancer cells results in efficient and selective tumor cell destruction. Blood 100:1265–1273PubMedGoogle Scholar
  53. 53.
    Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, Murphy M, Novotny WF, Burchmore M, Shak S, Stewart SJ, Press M (2002) Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20:719–726CrossRefPubMedGoogle Scholar
  54. 54.
    Wang L, Liu B, Schmidt M, Lu Y, Wels W, Fan Z (2001) Antitumor effect of an HER2-specific antibody-toxin fusion protein on human prostate cancer cells. Prostate 47:21–28CrossRefPubMedGoogle Scholar
  55. 55.
    Wedekind JE, Trame CB, Dorywalska M, Koehl P, Raschke TM, McKee M, FitzGerald D, Collier RJ, McKay DB (2001) Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity. J Mol Biol 314:823–837CrossRefPubMedGoogle Scholar
  56. 56.
    Weijtens ME, Hart EH, Bolhuis RL (2000) Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production. Gene Ther 7:35–42PubMedGoogle Scholar
  57. 57.
    Wels W, Beerli R, Hellmann P, Schmidt M, Marte BM, Kornilova ES, Hekele A, Mendelsohn J, Groner B, Hynes NE (1995) EGF receptor and p185erbB-2-specific single-chain antibody toxins differ in their cell-killing activity on tumor cells expressing both receptor proteins. Int J Cancer 60:137–144PubMedGoogle Scholar
  58. 58.
    Wels W, Harwerth IM, Mueller M, Groner B, Hynes NE (1992) Selective inhibition of tumor cell growth by a recombinant single-chain antibody-toxin specific for the erbB-2 receptor. Cancer Res 52:6310–6317PubMedGoogle Scholar
  59. 59.
    Wikstrand CJ, Hale LP, Batra SK, Hill ML, Humphrey PA, Kurpad SN, McLendon RE, Moscatello D, Pegram CN, Reist CJ, Traweek ST, Wong AJ, Zalutsky MR, Bigner DD (1995) Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res 55:3140–3148PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Winfried Wels
    • 1
  • Markus Biburger
    • 1
  • Tina Müller
    • 1
  • Benjamin Dälken
    • 1
  • Ulrike Giesübel
    • 1
  • Torsten Tonn
    • 2
  • Christoph Uherek
    • 1
  1. 1.Chemotherapeutisches Forschungsinstitut Georg-Speyer-HausFrankfurt am MainGermany
  2. 2.Institute for Transfusion Medicine and Immunohematology RCBDSFrankfurtGermany

Personalised recommendations