Cancer Immunology, Immunotherapy

, Volume 53, Issue 4, pp 345–357 | Cite as

A soluble single-chain T-cell receptor IL-2 fusion protein retains MHC-restricted peptide specificity and IL-2 bioactivity

  • Kimberlyn F. Card
  • Shari A. Price-Schiavi
  • Bai Liu
  • Elizabeth Thomson
  • Esperanza Nieves
  • Heather Belmont
  • Janette Builes
  • Jin-an Jiao
  • Javier Hernandez
  • Jon Weidanz
  • Linda Sherman
  • John L. Francis
  • Ali Amirkhosravi
  • Hing C. Wong
Original Article


Antibody-based targeted immunotherapy has shown promise as an approach to treat cancer. However, many known tumor-associated antigens are not expressed as integral membrane proteins and cannot be utilized as targets for antibody-based therapeutics. In order to expand the limited target range of antibodies, we have constructed a soluble single-chain T-cell receptor (TCR) fusion protein designated 264scTCR/IL-2. This fusion protein is comprised of a three-domain HLA-A2-restricted TCR specific for a peptide epitope of the human p53 tumor suppressor protein, which is overexpressed in a broad range of human malignancies. The 264scTCR/IL-2 fusion protein has been expressed at high levels in mammalian cells, and milligram quantities have been purified. MHC-restricted antigen-specific binding properties are maintained in the single-chain, three-domain TCR portion of the fusion protein, and the IL-2 portion retains bioactivity similar to that of free recombinant IL-2. Moreover, this fusion protein is capable of conjugating target and effector cells, remains intact in the blood and substantially increases the half life of the IL-2 portion of the molecule. Finally, the 264scTCR/IL-2 fusion protein can be used to stain tumor cells and is capable of reducing lung metastases in an experimental model of metastasis. Thus, TCR-based fusion proteins may provide a novel class of targeted immunotherapeutics for cancer.


T cell receptors Tumor immunity Molecular biology Cytokines 



This work was supported in part by grant no. 1R43CA88615-01 from the National Institutes of Health.


  1. 1.
    Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM (1996) Phenotypic analysis of antigen-specific T lymphocytes. Science 274:94PubMedGoogle Scholar
  2. 2.
    Anderson KS, Alexander J, Wei M, Cresswell P (1993) Intracellular transport of class I MHC molecules in antigen processing mutant cell lines. J Immunol 151:3407PubMedGoogle Scholar
  3. 3.
    Bauer RJ, Dedrick RL, White ML, Murray MJ, Garovoy MR (1999) Population pharmacokinetics and pharmacodynamics of the anti-CD11a antibody hu1124 in human subjects with psoriasis. J Pharmacokinet Biopharm 27:397CrossRefPubMedGoogle Scholar
  4. 4.
    Becker JC, Varki N, Gillies SD, Furukawa K, Reisfeld RA (1996) Long-lived and transferable tumor immunity in mice after targeted interleukin-2 therapy. J Clin Invest 98:2801PubMedGoogle Scholar
  5. 5.
    Chung S, Wucherpfennig KW, Friedman SM, Hafler DA, Strominger JL (1994) Functional three-domain single-chain T-cell receptors. Proc Natl Acad Sci USA 91:12654PubMedGoogle Scholar
  6. 6.
    Donohue JH, Rosenberg SA (1983) The fate of interleukin-2 after in vivo administration. J Immunol 130:2203PubMedGoogle Scholar
  7. 7.
    Dummer R, Gore ME, Hancock BW, Guillou PJ, Grobben HC, Becker JC, Oskam R, Dieleman JP, Burg G (1995) A multicenter phase II clinical trial using dacarbazine and continuous infusion interleukin-2 for metastatic melanoma. Clinical data and immunomonitoring. Cancer 75:1038PubMedGoogle Scholar
  8. 8.
    Engel I, Ottenhoff TH, Klausner RD (1992) High-efficiency expression and solubilization of functional T cell antigen receptor heterodimers. Science 256:1318PubMedGoogle Scholar
  9. 9.
    Gregoire C, Rebai N, Schweisguth F, Necker A, Mazza G, Auphan N, Millward A, Schmitt-Verhulst AM, Malissen B (1991) Engineered secreted T-cell receptor alpha beta heterodimers. Proc Natl Acad Sci USA 88:8077PubMedGoogle Scholar
  10. 10.
    Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA (1982) Lymphokine-activated killer cell phenomenon. Lysis of natural killer- resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med 155:1823PubMedGoogle Scholar
  11. 11.
    Grussenmeyer T, Scheidtmann KH, Hutchinson MA, Eckhart W, Walter G (1985) Complexes of polyoma virus medium T antigen and cellular proteins. Proc Natl Acad Sci USA 82:7952PubMedGoogle Scholar
  12. 12.
    Hank JA, Albertini MR, Schiller J, Sondel PM (1993) Activation of multiple effector mechanisms to enhance tumor immunotherapy. J Immunother 14:329PubMedGoogle Scholar
  13. 13.
    Hank JA, Robinson RR, Surfus J, Mueller BM, Reisfeld RA, Cheung NK, Sondel PM (1990a) Augmentation of antibody dependent cell mediated cytotoxicity following in vivo therapy with recombinant interleukin 2. Cancer Res 50:5234PubMedGoogle Scholar
  14. 14.
    Hank JA, Sosman JA, Kohler PC, Bechhofer R, Storer B, Sondel PM (1990b) Depressed in vitro T cell responses concomitant with augmented interleukin-2 responses by lymphocytes from cancer patients following in vivo treatment with interleukin-2. J Biol Response Mod 9:5PubMedGoogle Scholar
  15. 15.
    Hank JA, Surfus J, Gan J, Chew TL, Hong R, Tans K, Reisfeld R, Seeger RC, Reynolds CP, Bauer M, et al (1994) Treatment of neuroblastoma patients with antiganglioside GD2 antibody plus interleukin-2 induces antibody-dependent cellular cytotoxicity against neuroblastoma detected in vitro. J Immunother 15:29PubMedGoogle Scholar
  16. 16.
    Harvill ET, Morrison SL (1995) An IgG3-IL2 fusion protein activates complement, binds Fc gamma RI, generates LAK activity and shows enhanced binding to the high affinity IL-2R. Immunotechnology 1:95CrossRefPubMedGoogle Scholar
  17. 17.
    Harvill ET, Fleming JM, Morrison SL (1996) In vivo properties of an IgG3-IL-2 fusion protein. A general strategy for immune potentiation. J Immunol 157:3165PubMedGoogle Scholar
  18. 18.
    Hilyard KL, Reyburn H, Chung S, Bell JI, Strominger JL (1994) Binding of soluble natural ligands to a soluble human T-cell receptor fragment produced in Escherichia coli. Proc Natl Acad Sci USA 91:9057PubMedGoogle Scholar
  19. 19.
    Hinds PW, Finlay CA, Quartin RS, Baker SJ, Fearon ER., Vogelstein B, Levine AJ (1990) Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the “hot spot” mutant phenotypes. Cell Growth Differ 1:571PubMedGoogle Scholar
  20. 20.
    Hurford RK Jr, Dranoff G, Mulligan RC, Tepper RI (1995) Gene therapy of metastatic cancer by in vivo retroviral gene targeting. Nat Genet 10:430PubMedGoogle Scholar
  21. 21.
    Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R, et al (1988) 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 USA 85:5879PubMedGoogle Scholar
  22. 22.
    Iggo R, Gatter K, Bartek J, Lane D, Harris AL (1990) Increased expression of mutant forms of p53 oncogene in primary lung cancer. Lancet 335:675PubMedGoogle Scholar
  23. 23.
    Kendra K, Gan J, Ricci M, Surfus J, Shaker A, Super M, Frost JD, Rakhmilevich A, Hank JA, Gillies SD, Sondel PM (1999) Pharmacokinetics and stability of the ch14.18-interleukin-2 fusion protein in mice. Cancer Immunol Immunother 48:219CrossRefPubMedGoogle Scholar
  24. 24.
    Klausner RD, Lippincott-Schwartz J, Bonifacino JS (1990) The T cell antigen receptor: insights into organelle biology. Annu Rev Cell Biol 6:403PubMedGoogle Scholar
  25. 25.
    Lewis LD, Cole BF, Wallace PK, Fisher JL, Waugh M, Guyre PM, Fanger MW, Curnow RT, Kaufman PA, Ernstoff MS (2001) Pharmacokinetic-pharmacodynamic relationships of the bispecific antibody MDX-H210 when administered in combination with interferon gamma: a multiple-dose phase-I study in patients with advanced cancer which overexpresses HER-2/neu. J Immunol Methods 248:149PubMedGoogle Scholar
  26. 26.
    Lin AY, Devaux B, Green A, Sagerstrom C, Elliott JF, Davis MM (1990) Expression of T cell antigen receptor heterodimers in a lipid-linked form. Science 249:677PubMedGoogle Scholar
  27. 27.
    Lode HN, Xiang R, Varki NM, Dolman CS, Gillies SD, Reisfeld RA (1997) Targeted interleukin-2 therapy for spontaneous neuroblastoma metastases to bone marrow. J Natl Cancer Inst 89:1586CrossRefPubMedGoogle Scholar
  28. 28.
    Lode HN, Xiang R, Dreier T, Varki NM, Gillies SD, Reisfeld RA (1998) Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy. Blood 91:1706PubMedGoogle Scholar
  29. 29.
    Lustgarten J, Marks J, Sherman LA (1999) Redirecting effector T cells through their IL-2 receptors. J Immunol 162:359PubMedGoogle Scholar
  30. 30.
    McLaughlin R, O’Hanlon D, McHale T, Connolly CE, Given HF (2001) Prognostic implications of p53 and bcl-2 expression in 108 women with stage two breast cancer. Ir J Med Sci 170:11PubMedGoogle Scholar
  31. 31.
    Motzer RJ, Rakhit A, Schwartz LH, Olencki T, Malone TM, Sandstrom K, Nadeau R, Parmar H, Bukowski R (1998) Phase I trial of subcutaneous recombinant human interleukin-12 in patients with advanced renal cell carcinoma. Clin Cancer Res 4:1183PubMedGoogle Scholar
  32. 32.
    Motzer RJ, Rakhit A, Ginsberg M, Rittweger K, Vuky J, Yu R, Fettner S, Hooftman L (2001) Phase I trial of 40-kd branched pegylated interferon alfa-2a for patients with advanced renal cell carcinoma. J Clin Oncol 19:1312PubMedGoogle Scholar
  33. 33.
    Nastala CL, Edington HD, McKinney TG, Tahara H, Nalesnik MA, Brunda MJ, Gately MK, Wolf SF, Schreiber RD, Storkus WJ, et al (1994) Recombinant IL-12 administration induces tumor regression in association with IFN-gamma production. J Immunol 153:1697PubMedGoogle Scholar
  34. 34.
    Pardoll DM (1995) Paracrine cytokine adjuvants in cancer immunotherapy. Annu Rev Immunol 13:399PubMedGoogle Scholar
  35. 35.
    Peng LS, Penichet ML, Morrison SL (1999) A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity. J Immunol 163:250PubMedGoogle Scholar
  36. 36.
    Penichet ML, Harvill ET, Morrison SL (1997) Antibody-IL-2 fusion proteins: a novel strategy for immune protection. Hum Antibodies 8:106PubMedGoogle Scholar
  37. 37.
    Posey JA, Raspet R, Verma U, Deo YM, Keller T, Marshall JL, Hodgson J, Mazumder A, Hawkins MJ (1999) A pilot trial of GM-CSF and MDX-H210 in patients with erbB-2-positive advanced malignancies. J Immunother 22:371PubMedGoogle Scholar
  38. 38.
    Pullarkat V, Deo Y, Link J, Spears L, Marty V, Curnow R, Groshen S, Gee C, Weber JS (1999) A phase I study of a HER2/neu bispecific antibody with granulocyte-colony-stimulating factor in patients with metastatic breast cancer that overexpresses HER2/neu. Cancer Immunol Immunother 48:9CrossRefPubMedGoogle Scholar
  39. 39.
    Reddy KR, Wright TL, Pockros PJ, Shiffman M, Everson G, Reindollar R, Fried MW, Purdum PP 3rd, Jensen D, Smith C, et al (2001) Efficacy and safety of pegylated (40-kd) interferon alpha-2a compared with interferon alpha-2a in noncirrhotic patients with chronic hepatitis C. Hepatology 33:433CrossRefPubMedGoogle Scholar
  40. 40.
    Rosenberg SA, Spiess PJ, Schwarz S (1983) In vivo administration of interleukin-2 enhances specific alloimmune responses. Transplantation 35:631PubMedGoogle Scholar
  41. 41.
    Rosenberg SA, Lotze MT, Muul LM, Chang AE, Avis FP, Leitman S, Linehan WM, Robertson CN, Lee RE, Rubin JT, et al (1987) A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med 316:889PubMedGoogle Scholar
  42. 42.
    Rosenberg SA, Lotze MT, Yang JC, Aebersold PM, Linehan WM, Seipp CA, White DE (1989) Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg 210:474PubMedGoogle Scholar
  43. 43.
    Rosenberg SA, Yang JC, White DE, Steinberg SM (1998) Durability of complete responses in patients with metastatic cancer treated with high-dose interleukin-2: identification of the antigens mediating response. Ann Surg 228:307CrossRefPubMedGoogle Scholar
  44. 44.
    Royal RE, Steinberg SM, Krouse RS, Heywood G, White DE, Hwu P, Marincola FM, Parkinson DR, Schwartzentruber DJ, Topalian SL, et al. (1996) Correlates of Response to IL-2 Therapy in Patients Treated for Metastatic Renal Cancer and Melanoma. Cancer J Sci Am 2:91PubMedGoogle Scholar
  45. 45.
    Sherman LA, Hesse SV, Irwin MJ, La Face D, Peterson P (1992) Selecting T cell receptors with high affinity for self-MHC by decreasing the contribution of CD8. Science 258:815PubMedGoogle Scholar
  46. 46.
    Sondel PM, Kohler PC, Hank JA, Moore KH, Rosenthal NS, Sosman JA, Bechhofer R, Storer B (1988) Clinical and immunological effects of recombinant interleukin 2 given by repetitive weekly cycles to patients with cancer. Cancer Res 48:2561PubMedGoogle Scholar
  47. 47.
    Sosman JA, Hank JA, Moore KH, Borchert A, Schell K, Kohler PC, Goldstein D, Bechhofer R, Storer B, Albertini MR, et al. (1991) Prolonged interleukin-2 (IL-2) treatment can augment immune activation without enhancing antitumor activity in renal cell carcinoma. Cancer Invest 9:35PubMedGoogle Scholar
  48. 48.
    Temmim L, Baker H, Sinowatz F (2001) Immunohistochemical detection of p53 protein expression in breast cancer in young Kuwaiti women. Anticancer Res 21:743PubMedGoogle Scholar
  49. 49.
    Theobald M, Biggs J, Dittmer D, Levine AJ, Sherman LA (1995) Targeting p53 as a general tumor antigen. Proc Natl Acad Sci USA 92:11993PubMedGoogle Scholar
  50. 50.
    Theobald M, Biggs J, Hernandez J, Lustgarten J, Labadie C, Sherman LA (1997) Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes. J Exp Med 185:833PubMedGoogle Scholar
  51. 51.
    Thor Straten P, Guldberg P, Schrama D, Andersen MA, Moerch U, Seremet T, Siedel C, Reisfeld RA, Becker JC (2001) In situ cytokine therapy: redistribution of clonally expanded T cells. Eur J Immunol 31:250CrossRefPubMedGoogle Scholar
  52. 52.
    Tsung K, Meko JB, Peplinski GR, Tsung YL, Norton JA (1997) IL-12 induces T helper 1-directed antitumor response. J Immunol 158:3359PubMedGoogle Scholar
  53. 53.
    van Golen KL, Risin S, Staroselsky A, Berger D, Tainsky MA, Pathak S, Price JE (1996) Predominance of the metastatic phenotype in hybrids formed by fusion of mouse and human melanoma clones. Clin Exp Metastasis 14:95PubMedGoogle Scholar
  54. 54.
    Weber S, Traunecker A, Oliveri F, Gerhard W, Karjalainen K (1992) Specific low-affinity recognition of major histocompatibility complex plus peptide by soluble T-cell receptor. Nature 356:793CrossRefPubMedGoogle Scholar
  55. 55.
    Weil-Hillman G, Voss SD, Fisch P, Schell K, Hank JA, Sosman JA, Sugamura K, Sondel PM (1990) Natural killer cells activated by interleukin 2 treatment in vivo respond to interleukin 2 primarily through the p75 receptor and maintain the p55 (TAC) negative phenotype. Cancer Res 50:2683PubMedGoogle Scholar
  56. 56.
    Wiebke EA, Rosenberg SA, Lotze MT (1988) Acute immunologic effects of interleukin-2 therapy in cancer patients: decreased delayed type hypersensitivity response and decreased proliferative response to soluble antigens. J Clin Oncol 6:1440PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Kimberlyn F. Card
    • 1
  • Shari A. Price-Schiavi
    • 1
  • Bai Liu
    • 1
  • Elizabeth Thomson
    • 1
  • Esperanza Nieves
    • 2
  • Heather Belmont
    • 1
  • Janette Builes
    • 1
  • Jin-an Jiao
    • 6
  • Javier Hernandez
    • 3
  • Jon Weidanz
    • 4
  • Linda Sherman
    • 3
  • John L. Francis
    • 5
  • Ali Amirkhosravi
    • 5
  • Hing C. Wong
    • 1
  1. 1.Altor BioScience CorporationMiramarUSA
  2. 2.Dade-Behring CorporationNewarkUSA
  3. 3.The Scripps Research InstituteLa JollaUSA
  4. 4.Department of Pharmaceutical Sciences, School of PharmacyTexas Tech UniversityAmarilloUSA
  5. 5.Florida Hospital Cancer InstituteOrlandoUSA
  6. 6.Hematech LLCSioux FallsUSA

Personalised recommendations