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.

  • Article
  • Published:

Small antibody mimetics comprising two complementarity-determining regions and a framework region for tumor targeting

Nature Biotechnology volume 25, pages 921–929 (2007)Cite this article

Abstract

Here we show that fusion of two complementarity-determining regions (CDRs), VHCDR1 and VLCDR3, through a cognate framework region (VHFR2) yields mimetics that retain the antigen recognition of their parent molecules, but have a superior capacity to penetrate tumors. The antigen-recognition abilities of these ∼3 kDa mimetics surpass those of comparable fragments lacking the framework region. In vivo activities of the mimetics suggests that the structural orientation of their CDRs approximates the conformation of the CDRs in the complex of the parent antibody with antigen. We linked the antibody mimetics to the bacterial toxin colicin Ia to create fusion proteins called “pheromonicins,” which enable targeted inhibition of tumor growth. In mice bearing human malignant tumors, pheromonicins directed against tumor-specific surface markers show greater capacity to target and penetrate tumors than their parent antibodies. Rational recombination of selected VH/VL binding sites and their framework regions might provide useful targeting moieties for cytotoxic cancer therapies.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure and activity of mimetics and fusion molecules.
Figure 2: In vitro killing activity of pheromonicins and competition ability of synthetic VHCDR1-VHFR2-VLCDR3 mimetics.
Figure 3: In vivo effects of PMC-EBV against solid tumors.
Figure 4: Fluorescence images of tumors in BALB/c mice treated with FITC-labeled PMC-EBV, HB-168 IgG, PMC-SA, PMC-LC or HB-8627 IgM.
Figure 5: Fluorescence images of tumors in SCID mice treated with FITC-labeled synthetic VHCDR1-VHFR2-VLCDR3 mimetic, PMC-EBV or parent HB-168 IgG.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Hoogenboom, H.R. Selecting and screening recombinant antibody libraries. Nat. Biotechnol. 23, 1105–1116 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Borg, N.A. et al. The CDR3 regions of an immunodominant T cell receptor dictate the 'energetic landscape' of peptide-MHC recognition. Nat. Immunol. 6, 171–180 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Laune, D. et al. Systematic exploration of the antigen binding activity of synthetic peptides isolated from the variable regions of immunoglobulins. J. Biol. Chem. 272, 30937–30944 (1997).

    Article  CAS  PubMed  Google Scholar 

  4. Ewert, S., Huber, T., Honegger, A. & Plückthun, A. Biophysical properties of human antibody variable domains. J. Mol. Biol. 325, 531–553 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Holliger, P. & Hudson, P. Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23, 1126–1136 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Binz, H.K., Amstutz, P. & Plückthun, A. Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23, 1257–1267 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Binz, H.K. et al. Engineered proteins as specific binding reagents. Curr. Opin. Biotechnol. 16, 459–469 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Heap, C.J. et al. Analysis of a 17-amino acid residue, virus-neutralizing microantibody. J. Gen. Virol. 86, 1791–1800 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Casset, F. et al. A peptide mimetic of an anti-CD4 monoclonal antibody by rational design. Biochem. Biophys. Res. Commun. 307, 198–205 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Qin, W. et al. Fusion protein of CDR mimetic peptide with Fc inhibit TNF-α induced cytotoxicity. Mol. Immunol. 43, 660–666 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Souriau, C. et al. New binding specificities derived from Min-23, a small cysteine-stabilized peptide scaffold. Biochemistry 44, 7143–7155 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Aburatani, T., Ueda, H. & Nagamune, T. Importance of a CDR H3 basal residue in VH/VL interaction of human antibodies. J. Biochem. 132, 775–785 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. De Genst, E. et al. Chemical basis for the affinity maturation of a camel single domain antibody. J. Biol. Chem. 279, 53593–53601 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Rothlisberger, D., Honegger, A. & Plückthun, A. Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. J. Mol. Biol. 347, 773–789 (2005).

    Article  PubMed  Google Scholar 

  15. Hoffman, G.J. et al. Monocolonal antibody against a 250,000-dalton glycoprotein of Epstein-Barr virus identifies a membrane antigen and a neutralizing antigen. Proc. Natl. Acad. Sci. USA 77, 2979–2983 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hellstrom, I. et al. Monoclonal mouse antibodies raised against human lung carcinoma. Cancer Res. 46, 3917–3923 (1986).

    CAS  PubMed  Google Scholar 

  17. Gong, M. & Kieff, E. Intracellular trafficking of two major Epstein-Barr virus glycoproteins, gp350/220 and gp110. J. Virol. 64, 1507–1516 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Thorley-Lawson, D.A. & Gross, A. Persistence of the Epstein-Barr virus and the origins of associated lymphomas. N. Engl. J. Med. 350, 1328–1337 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Thorley-Lawson, D.A. & Geilinger, K. Monoclonal antibodies against the major glycoprotein (gp350/220) of Epstein-Barr virus neutralize infectivity. Proc. Natl. Acad. Sci. USA 77, 5307–5311 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cramer, W.A. et al. Structure-function of the channel-forming colicins. Annu. Rev. Biophys. Biomol. Struct. 24, 611–641 (1995).

    Article  CAS  PubMed  Google Scholar 

  21. Qiu, X-Q., Jakes, K.S., Kienker, P.K., Finkelstein, A. & Slatin, S.L. Major transmembrane movement associated with colicin Ia channel gating. J. Gen. Physiol. 107, 313–328 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Kienker, P.K., Qiu, X-Q., Slatin, S.L., Finkelstein, A. & Jakes, K.S. Transmembrane insertion of the colicin Ia hydrophobic hairpin. J. Membr. Biol. 157, 27–37 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Jakes, K.S., Kienker, P.K. & Finkelstein, A. Channel-forming colicins: translocation (and other deviant behaviour) associated with colicin Ia channel gating. Q. Rev. Biophys. 32, 189–205 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Qiu, X.Q. et al. An engineered multidomain bactericidal peptide as a model for targeted antibiotics against specific bacteria. Nat. Biotechnol. 21, 1480–1485 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Qiu, X.Q., Zhang, J., Wang, H. & Wu, G.Y. A novel engineered peptide, a narrow-spectrum antibiotic, is effective against vancomycin-resistant Enterococcus faecalis. Antimicrob. Agents Chemother. 49, 1184–1189 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Jakes, K.S., Abrams, C.K., Finkelstein, A. & Slatin, S.L. Alteration of the pH-dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis. J. Biol. Chem. 265, 6984–6991 (1990).

    CAS  PubMed  Google Scholar 

  27. Gao, X. et al. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat. Biotechnol. 22, 969–976 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Yu, Y.A. et al. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins. Nat. Biotechnol. 22, 313–320 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Midelfort, K.S. et al. Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody. J. Mol. Biol. 343, 685–701 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Carter, P.J. Potent antibody therapeutics by design. Nat. Rev. Immunol. 6, 343–357 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Adams, G.P. et al. High affinity restricts the localization and tumor penetration of single-chain Fv antibody molecules. Cancer Res. 61, 4750–4755 (2001).

    CAS  PubMed  Google Scholar 

  32. Fujimori, K., Covell, D.G., Fletcher, J.E. & Weinstein, J.N. Modeling analysis of the global and microscopic distribution of immunoglobulin G, F(ab′)2, and Fab in tumors. Cancer Res. 49, 5656–5663 (1989).

    CAS  PubMed  Google Scholar 

  33. Mattes, M.J. et al. Cell surface antigens of human ovarian and endometrial carcinoma defined by mouse monoclonal antibodies. Proc. Natl. Acad. Sci. USA 81, 568–572 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bang, S., Nagata, S., Onda, M., Kreitman, R.J. & Pastan, I. HA22(R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity. Clin. Cancer Res. 11, 1545–1550 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Vallera, D.A. et al. Molecular modification of a recombinant, bivalent anti-human CD3 immunotoxin (Bic30 results in reduced in vivo toxicity in mice. Leuk. Res. 29, 331–341 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Krauss, J. et al. Targeting malignant B-cell lymphoma with a humanized anti-CD22 scFv-angiogenin immunoenzyme. Br. J. Haematol. 128, 602–609 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Qiu, X.Q., Jakes, K.S., Finkelstein, A. & Slatin, S.L. Site-specific biotinylation of colicin Ia: A probe for protein conformation in the membrane. J. Biol. Chem. 269, 7483–7488 (1994).

    CAS  PubMed  Google Scholar 

  38. Harlow, E. Lane, D. (eds.) Immunohistology. in Using Antibodies, A Laboratory Manual. Chapter 6. Cold Spring Harbor Laboratory Press (1998) (Science Publication, Beijing, 93–129 (2002)).

    Google Scholar 

Download references

Acknowledgements

This work was supported by National Science Foundation of China grants 30430220, 30271557, 30571942 and 30672137 and Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education to X.-Q.Q. and H.W. We would like to acknowledge the help and scientific critique of P. Kienker, H. Li, J.C. Hou and C.A. Deng during the preparation of this manuscript. We would also like to acknowledge the help of J. Zhang, P. Dai, Z.P. Zhen, Y.C. Huang, F.L. Cai, S.Y. Qiu and X.F. Lu in DNA scanning, histology and fusion peptide purification.

Author information

Authors and Affiliations

  1. Key Laboratory of Transplant Immunology, Ministry of Health, State Key Laboratory of Biotherapy, No. 37 Wai Nan Guo-xue-Xiang, Chengdu, 610041, P.R. of China

    Xiao-Qing Qiu & Shi-Tao Yue

  2. Department of Lab Medicine, Division of Clinical Immunology, West China Hospital, No. 37 Wai Nan Guo-xue-Xiang, Chengdu, 610041, P.R. of China

    Bei Cai & Lan-Lan Wang

  3. Laboratory of Genetics, West China Second University Hospital, West China School of Medicine, Sichuan University, No. 37 Wai Nan Guo-xue-Xiang, Chengdu, 610041, P.R. of China

    He Wang

Authors
  1. Xiao-Qing Qiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. He Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bei Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lan-Lan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shi-Tao Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X-Q.Q. and H.W. prepared mimetics and fusion molecules, measured in vitro and in vivo killing activity and did immunolabeling/fluorescent/pathology assays; B.C. and L.-L.W. did the cytometry assays; S.-T.Y. carried out SDS-PAGE and sizing chromatography.

Corresponding author

Correspondence to Xiao-Qing Qiu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 2383 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Qiu, XQ., Wang, H., Cai, B. et al. Small antibody mimetics comprising two complementarity-determining regions and a framework region for tumor targeting. Nat Biotechnol 25, 921–929 (2007). https://doi.org/10.1038/nbt1320

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1320

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing