Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Immunotherapy

Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

Abstract

WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2)-restricted peptide derived from Wilms tumor protein 1 (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single-chain variable fragment (scFv) antibody specific for the T-cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant (KD)=3 nm) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD=2 pm) binding to the T-cell epitope and to tumor targets and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor-expressing human T- or NK-92-MI-transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T-cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high-affinity TCR-like antibodies could be rapidly explored for potential clinical development.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

References

  1. Doubrovina E, Carpenter T, Pankov D, Selvakumar A, Hasan A, O'Reilly RJ . Mapping of novel peptides of WT-1 and presenting HLA alleles that induce epitope-specific HLA-restricted T cells with cytotoxic activity against WT-1(+) leukemias. Blood 2012; 120: 1633–1646.

    Article  CAS  Google Scholar 

  2. Santomasso BD, Roberts WK, Thomas A, Williams T, Blachere NE, Dudley ME et al. A T-cell receptor associated with naturally occurring human tumor immunity. Proc Natl Acad Sci USA 2007; 104: 19073–19078.

    Article  CAS  Google Scholar 

  3. Keilholz U, Letsch A, Busse A, Asemissen AM, Bauer S, Blau IW et al. A clinical and immunologic phase 2 trial of Wilms tumor gene product 1 (WT1) peptide vaccination in patients with AML and MDS. Blood 2009; 113: 6541–6548.

    Article  CAS  Google Scholar 

  4. Yee C . Adoptive T cell therapy: Addressing challenges in cancer immunotherapy. J Transl Med 2005; 3: 17.

    Article  Google Scholar 

  5. Dahan R, Reiter Y . T-cell-receptor-like antibodies - generation, function and applications. Expert Rev Mol Med 2012; 14: e6.

    Article  Google Scholar 

  6. Cohen CJ, Denkberg G, Lev A, Epel M, Reiter Y . Recombinant antibodies with MHC-restricted, peptide-specific, T-cell receptor-like specificity: new tools to study antigen presentation and TCR-peptide-MHC interactions. J Mol Recognit 2003; 16: 324–332.

    Article  CAS  Google Scholar 

  7. Chames P, Hufton SE, Coulie PG, Uchanska-Ziegler B, Hoogenboom HR . Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library. Proc Natl Acad Sci USA 2000; 97: 7969–7974.

    Article  CAS  Google Scholar 

  8. Lev A, Noy R, Oved K, Novak H, Segal D, Walden P et al. Tumor-specific Ab-mediated targeting of MHC-peptide complexes induces regression of human tumor xenografts in vivo. Proc Natl Acad Sci USA 2004; 101: 9051–9056.

    Article  CAS  Google Scholar 

  9. Stewart-Jones G, Wadle A, Hombach A, Shenderov E, Held G, Fischer E et al. Rational development of high-affinity T-cell receptor-like antibodies. Proc Natl Acad Sci USA 2009; 106: 5784–5788.

    Article  CAS  Google Scholar 

  10. Holler PD, Chlewicki LK, Kranz DM . TCRs with high affinity for foreign pMHC show self-reactivity. Nat Immunol 2003; 4: 55–62.

    Article  CAS  Google Scholar 

  11. Denkberg G, Reiter Y . Recombinant antibodies with T-cell receptor-like specificity: novel tools to study MHC class I presentation. Autoimmun Rev 2006; 5: 252–257.

    Article  CAS  Google Scholar 

  12. Sastry KS, Too CT, Kaur K, Gehring AJ, Low L, Javiad A et al. Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody. J Virol 2011; 85: 1935–1942.

    Article  CAS  Google Scholar 

  13. Sergeeva A, Alatrash G, He H, Ruisaard K, Lu S, Wygant J et al. An anti-PR1/HLA-A2 T-cell receptor-like antibody mediates complement-dependent cytotoxicity against acute myeloid leukemia progenitor cells. Blood 2011; 117: 4262–4272.

    Article  CAS  Google Scholar 

  14. Verma B, Neethling FA, Caseltine S, Fabrizio G, Largo S, Duty JA et al. TCR mimic monoclonal antibody targets a specific peptide/HLA class I complex and significantly impedes tumor growth in vivo using breast cancer models. J Immunol 2010; 184: 2156–2165.

    Article  CAS  Google Scholar 

  15. Willemsen RA, Debets R, Hart E, Hoogenboom HR, Bolhuis RL, Chames P . A phage display selected fab fragment with MHC class I-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes. Gene Ther 2001; 8: 1601–1608.

    Article  CAS  Google Scholar 

  16. Dao T, Yan S, Veomett N, Pankov D, Zhou L, Korontsvit T et al. Targeting the intracellular WT1 oncogene product with a therapeutic human antibody. Sci Transl Med 2013; 5: 176ra133.

    Article  Google Scholar 

  17. Tassev DV, Cheng M, Cheung NK . Retargeting NK92 cells using an HLA-A2-restricted, EBNA3C-specific chimeric antigen receptor. Cancer Gene Ther 2012; 19: 84–100.

    Article  CAS  Google Scholar 

  18. Oren R, Hod-Marco M, Haus-Cohen M, Thomas S, Blat D, Duvshani N et al. Functional comparison of engineered T cells carrying a native TCR versus TCR-like antibody-based chimeric antigen receptors indicates affinity/avidity thresholds. J Immunol 2014; 193: 5733–5743.

    Article  CAS  Google Scholar 

  19. Renshaw J, Orr RM, Walton MI, Te Poele R, Williams RD, Wancewicz EV et al. Disruption of WT1 gene expression and exon 5 splicing following cytotoxic drug treatment: antisense down-regulation of exon 5 alters target gene expression and inhibits cell survival. Mol Cancer Ther 2004; 3: 1467–1484.

    CAS  PubMed  Google Scholar 

  20. Yang L, Han Y, Suarez Saiz F, Minden MD . A tumor suppressor and oncogene: the WT1 story. Leukemia 2007; 21: 868–876.

    Article  CAS  Google Scholar 

  21. Sugiyama H . WT1 (Wilms' tumor gene 1): biology and cancer immunotherapy. Jpn J Clin Oncol 2010; 40: 377–387.

    Article  Google Scholar 

  22. O'Reilly RJ, Dao T, Koehne G, Scheinberg D, Doubrovina E . Adoptive transfer of unselected or leukemia-reactive T-cells in the treatment of relapse following allogeneic hematopoietic cell transplantation. Semin Immunol 2010; 22: 162–172.

    Article  CAS  Google Scholar 

  23. Rezvani K, Brenchley JM, Price DA, Kilical Y, Gostick E, Sewell AK et al. T-cell responses directed against multiple HLA-A*0201-restricted epitopes derived from Wilms' tumor 1 protein in patients with leukemia and healthy donors: identification, quantification, and characterization. Clin Cancer Res 2005; 11: 8799–8807.

    Article  CAS  Google Scholar 

  24. Kohrt HE, Muller A, Baker J, Goldstein MJ, Newell E, Dutt S et al. Donor immunization with WT1 peptide augments antileukemic activity after MHC-matched bone marrow transplantation. Blood 2011; 118: 5319–5329.

    Article  CAS  Google Scholar 

  25. Borbulevych OY, Do P, Baker BM . Structures of native and affinity-enhanced WT1 epitopes bound to HLA-A*0201: implications for WT1-based cancer therapeutics. Mol Immunol 2010; 47: 2519–2524.

    Article  CAS  Google Scholar 

  26. Mailander V, Scheibenbogen C, Thiel E, Letsch A, Blau IW, Keilholz U . Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity. Leukemia 2004; 18: 165–166.

    Article  CAS  Google Scholar 

  27. de Wildt RM, Mundy CR, Gorick BD, Tomlinson IM . Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat Biotechnol 2000; 18: 989–994.

    Article  CAS  Google Scholar 

  28. Zhao Q, Zhu Z, Dimitrov DS . Yeast display of engineered antibody domains. Methods Mol Biol 2012; 899: 73–84.

    Article  CAS  Google Scholar 

  29. Zhao Q, Chan YW, Lee SS, Cheung WT . One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector. Protein Expr Purif 2009; 68: 190–195.

    Article  CAS  Google Scholar 

  30. Chen W, Feng Y, Zhao Q, Zhu Z, Dimitrov DS . Human monoclonal antibodies targeting nonoverlapping epitopes on insulin-like growth factor II as a novel type of candidate cancer therapeutics. Mol Cancer Ther 2012; 11: 1400–1410.

    Article  CAS  Google Scholar 

  31. Cheung NK, Guo H, Hu J, Tassev DV, Cheung IY . Humanizing murine IgG3 anti-GD2 antibody m3F8 substantially improves antibody-dependent cell-mediated cytotoxicity while retaining targeting in vivo. Oncoimmunology 2012; 1: 477–486.

    Article  Google Scholar 

  32. Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH, Geiger TL et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 2004; 18: 676–684.

    Article  CAS  Google Scholar 

  33. Wittrup KD, Thurber GM, Schmidt MM, Rhoden JJ . Practical theoretic guidance for the design of tumor-targeting agents. Methods Enzymol 2012; 503: 255–268.

    Article  CAS  Google Scholar 

  34. Dimitrov DS, Marks JD . Therapeutic antibodies: current state and future trends—is a paradigm change coming soon? Methods Mol Biol 2009; 525: 1–27, xiii.

    Article  CAS  Google Scholar 

  35. Zhao Q, Feng Y, Zhu Z, Dimitrov DS . Human monoclonal antibody fragments binding to insulin-like growth factors I and II with picomolar affinity. Mol Cancer Ther 2011; 10: 1677–1685.

    Article  CAS  Google Scholar 

  36. Li Y, Li H, Yang F, Smith-Gill SJ, Mariuzza RA . X-ray snapshots of the maturation of an antibody response to a protein antigen. Nat Struct Biol 2003; 10: 482–488.

    Article  CAS  Google Scholar 

  37. Mareeva T, Martinez-Hackert E, Sykulev Y . How a T cell receptor-like antibody recognizes major histocompatibility complex-bound peptide. J Biol Chem 2008; 283: 29053–29059.

    Article  CAS  Google Scholar 

  38. Epel M, Carmi I, Soueid-Baumgarten S, Oh SK, Bera T, Pastan I et al. Targeting TARP, a novel breast and prostate tumor-associated antigen, with T cell receptor-like human recombinant antibodies. Eur J Immunol 2008; 38: 1706–1720.

    Article  CAS  Google Scholar 

  39. Noy R, Eppel M, Haus-Cohen M, Klechevsky E, Mekler O, Michaeli Y et al. T-cell receptor-like antibodies: novel reagents for clinical cancer immunology and immunotherapy. Expert Rev Anticancer Ther 2005; 5: 523–536.

    Article  CAS  Google Scholar 

  40. Gerber JM, Qin L, Kowalski J, Smith BD, Griffin CA, Vala MS et al. Characterization of chronic myeloid leukemia stem cells. Am J Hematol 2011; 86: 31–37.

    Article  Google Scholar 

  41. Engberg J, Yenidunya AF, Clausen R, Jensen LB, Sorensen P, Kops P et al. Human recombinant Fab antibodies with T-cell receptor-like specificities generated from phage display libraries. Methods Mol Biol 2003; 207: 161–177.

    CAS  PubMed  Google Scholar 

  42. Gilham DE, Debets R, Pule M, Hawkins RE, Abken H . CAR-T cells and solid tumors: tuning T cells to challenge an inveterate foe. Trends Mol Med 2012; 18: 377–384.

    Article  CAS  Google Scholar 

  43. Ramos CA, Dotti G . Chimeric antigen receptor (CAR)-engineered lymphocytes for cancer therapy. Expert Opin Biol Ther 2011; 11: 855–873.

    Article  CAS  Google Scholar 

  44. Mardiros A, Dos Santos C, McDonald T, Brown CE, Wang X, Budde LE et al. T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and anti-tumor effects against human acute myeloid leukemia. Blood 2013; 122: 3138–3148.

    Article  CAS  Google Scholar 

  45. Cheung NK, Dyer MA . Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat Rev Cancer 2013; 13: 397–411.

    Article  CAS  Google Scholar 

  46. Esser R, Muller T, Stefes D, Kloess S, Seidel D, Gillies SD et al. NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin. J Cell Mol Med 2012; 16: 569–581.

    Article  CAS  Google Scholar 

  47. Kruschinski A, Moosmann A, Poschke I, Norell H, Chmielewski M, Seliger B et al. Engineering antigen-specific primary human NK cells against HER-2 positive carcinomas. Proc Natl Acad Sci USA 2008; 105: 17481–17486.

    Article  CAS  Google Scholar 

  48. Cho D, Shook DR, Shimasaki N, Chang YH, Fujisaki H, Campana D . Cytotoxicity of activated natural killer cells against pediatric solid tumors. Clin Cancer Res 2010; 16: 3901–3909.

    Article  CAS  Google Scholar 

  49. Shook DR, Campana D . Natural killer cell engineering for cellular therapy of cancer. Tissue Antigens 2011; 78: 409–415.

    Article  CAS  Google Scholar 

  50. Cheever MA, Allison JP, Ferris AS, Finn OJ, Hastings BM, Hecht TT et al. The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research. Clin Cancer Res 2009; 15: 5323–5337.

    Article  Google Scholar 

Download references

Acknowledgements

This study was supported in part by Katie Find a Cure Fund, Cycle for Survival and Robert Steel Foundation. Technical services provided by the MSKCC Small-Animal Imaging Core Facility were supported in part by NIH Cancer Center Support Grant No. 2 P30 CA008748-48. We thank Dr Mamoru Ito of Central Institute for Experimental Animals, Kawasaki, Japan for kindly providing the DKO mice for our studies. We are grateful to Dr Gloria Koo, Hospital for Special Surgery, New York, NY, USA for her expertise and advice in handling these DKO mice. We thank Dr Dario Campana and St Jude Children’s Research Hospital for sharing with us the vector for chimeric antigen receptor. We also thank Dr Jian Hu for technical assistance.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to N-K V Cheung.

Ethics declarations

Competing interests

QZ, MA, DVT, RJO and N-KVC were named as inventors in patents related to WT1 filed by Memorial Sloan Kettering Cancer Center for which a license has been obtained. The other authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Leukemia website

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhao, Q., Ahmed, M., Tassev, D. et al. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential. Leukemia 29, 2238–2247 (2015). https://doi.org/10.1038/leu.2015.125

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2015.125

This article is cited by

Search

Quick links