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Engineering THIOMABs for Site-Specific Conjugation of Thiol-Reactive Linkers

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1045))

Abstract

Antibody conjugates are used in many therapeutic and research applications and are generated by chemically linking a cysteine or lysine residue to potent chemotherapeutic drugs or other functional groups through a flexible linker. Recently, we have engineered THIOMABs (antibodies with engineered reactive cysteine residues) for site-specific conjugation and showed that these antibody conjugates display homogeneous labeling with optimal in vitro and in vivo characteristics. Here, we describe protocols for engineering, selection, and site-specific conjugation of THIOMABs with thiol-reactive linkers.

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References

  1. Reichert RM (2012) Marketed therapeutic antibodies compendium. MAbs 4:413–415

    PubMed  Google Scholar 

  2. Reichert JM, Dhimolea E (2012) The future of antibodies as cancer drugs. Drug Discov Today

    Google Scholar 

  3. Beck A, Haeuw JF, Wurch T, Goetsch L, Bailly C, Corvaia N (2011) The next generation of antibody–drug conjugates comes of age. Discov Med 10:329–339

    Google Scholar 

  4. Carter PJ, Senter PD (2008) Antibody–drug conjugates for cancer therapy. Cancer J 14:154–169

    Article  PubMed  CAS  Google Scholar 

  5. Polakis P (2005) Arming antibodies for cancer therapy. Curr Opin Pharmacol 5:382–387

    Article  PubMed  CAS  Google Scholar 

  6. Doronina SO, Toki BE, Torgov MY, Mendelsohn BA, Cerveny CG, Chace DF, DeBlanc RL, Gearing RP, Bovee TD, Siegall CB, Francisco JA, Wahl AF, Meyer DL, Senter PD (2003) Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat Biotechnol 21:778–784

    Article  PubMed  CAS  Google Scholar 

  7. Fossella F, McCann J, Tolcher A, Xie H, Hwang LL, Carr C, Berg K, Fram R (2005) Phase II trial of BB-10901 (huN901-DM1) given weekly for four consecutive weeks every 6 weeks in patients with relapsed SCLC and CD56-positive small cell carcinoma. J Clin Oncol 23:660S

    Google Scholar 

  8. Francisco JA, Cerveny CG, Meyer DL, Mixan BJ, Klussman K, Chace DF, Rejniak SX, Gordon KA, DeBlanc R, Toki BE, Law CL, Doronina SO, Siegall CB, Senter PD, Wahl AF (2003) cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood 102:1458–1465

    Article  PubMed  CAS  Google Scholar 

  9. Helft PR, Schilsky RL, Hoke FJ, Williams D, Kindler HL, Sprague E, DeWitte M, Martino HK, Erickson J, Pandite L, Russo M, Lambert JM, Howard M, Ratain MJ (2004) A phase I study of cantuzumab mertansine administered as a single intravenous infusion once weekly in patients with advanced solid tumors. Clin Cancer Res 10:4363–4368

    Article  PubMed  CAS  Google Scholar 

  10. Henry MD, Wen SH, Silva MD, Chandra S, Milton M, Worland PJ (2004) A prostate-specific membrane antigen-targeted monoclonal antibody-chemotherapeutic conjugate designed for the treatment of prostate cancer. Cancer Res 64:7995–8001

    Article  PubMed  CAS  Google Scholar 

  11. Liu CN, Tadayoni BM, Bourret LA, Mattocks KM, Derr SM, Widdison WC, Kedersha NL, Ariniello PD, Goldmacher VS, Lambert JM, Blattler WA, Chari RVJ (1996) Eradication of large colon tumor xenografts by targeted delivery of maytansinoids. Proc Natl Acad Sci U S A 93:8618–8623

    Article  PubMed  CAS  Google Scholar 

  12. Naito K, Takeshita A, Shigeno K, Nakamura S, Fujisawa S, Shinjo K, Yoshida H, Ohnishi K, Mori M, Terakawa S, Ohno R (2000) Calicheamicin-conjugated humanized anti-CD33 monoclonal antibody (gemtuzumab zogamicin, CMA-676) shows cytocidal effect on CD33-positive leukemia cell lines, but is inactive on P-glycoprotein-expressing sublines. Leukemia 14:1436–1443

    Article  PubMed  CAS  Google Scholar 

  13. Phillips GDL, Li GM, Dugger DL, Crocker LM, Parsons KL, Mai E, Blattler WA, Lambert JM, Chari RVJ, Lutz RJ, Wong WLT, Jacobson FS, Koeppen H, Schwall RH, Kenkare-Mitra SR, Spencer SD, Sliwkowski MX (2008) Targeting HER2-positive breast cancer with trastuzumab–DM1, an antibody–cytotoxic drug conjugate. Cancer Res 68:9280–9290

    Article  Google Scholar 

  14. Polson AG, Calemine-Fenaux J, Chan P, Chang W, Christensen E, Clark S, de Sauvage FJ, Eaton D, Elkins K, Elliott JM, Frantz G, Fuji RN, Gray A, Harden K, Ingle GS, Kljavin NM, Koeppen H, Nelson C, Prabhu S, Raab H, Ross S, Stephan JP, Scales SJ, Spencer SD, Vandlen R, Wranik B, Yu SF, Zheng B, Ebens A (2009) Antibody–drug conjugates for the treatment of non-Hodgkin’s lymphoma: target and linker-drug selection. Cancer Res 69:2358–2364

    Article  PubMed  CAS  Google Scholar 

  15. Tse KF, Jeffers M, Pollack VA, McCabe DA, Shadish ML, Khramtsov NV, Hackett CS, Shenoy SG, Kuang B, Boldog FL, MacDougall JR, Rastelli L, Herrmann J, Gallo M, Gazit-Bornstein G, Senter PD, Meyer DL, Lichenstein HS, LaRochelle WJ (2006) CR011, a fully human monoclonal antibody–auristatin E conjugate, for the treatment of melanoma. Clin Cancer Res 12:1373–1382

    Article  PubMed  CAS  Google Scholar 

  16. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, Kissler KM, Bernhardt SX, Kopcha AK, Zabinski RF, Meyer DL, Francisco JA (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10:7063–7070

    Article  PubMed  CAS  Google Scholar 

  17. Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H, Bhakta S, Nguyen T, Dugger DL, Li G, Mai E, Lewis Phillips GD, Hiraragi H, Fuji RN, Tibbitts J, Vandlen R, Spencer SD, Scheller RH, Polakis P, Sliwkowski MX (2010) Engineered thio-trastuzumab–DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin Cancer Res 16:4769–4778

    Article  PubMed  CAS  Google Scholar 

  18. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu YM, Meng YG, Ng C, Yang JH, Lee CC, Duenas E, Gorrell J, Katta V, Kim A, McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Wong WL, Lowman HB, Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26:925–932

    Article  PubMed  CAS  Google Scholar 

  19. Wang L, Amphlett G, Blattler WA, Lambert JM, Zhang W (2005) Structural characterization of the maytansinoid–monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry. Protein Sci 14:2436–2446

    Article  PubMed  CAS  Google Scholar 

  20. Junutula JR, Bhakta S, Raab H, Ervin KE, Eigenbrot C, Vandlen R, Scheller RH, Lowman HB (2008) Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs. J Immunol Methods 332:41–52

    Article  PubMed  CAS  Google Scholar 

  21. Tinianow JN, Gill HS, Ogasawara A, Flores JE, Vanderbilt AN, Luis E, Vandlen R, Darwish M, Junutula JR, Williams SP, Marik J (2010) Site-specifically 89Zr-labeled monoclonal antibodies for ImmunoPET. Nucl Med Biol 37:289–297

    Article  PubMed  CAS  Google Scholar 

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Bhakta, S., Raab, H., Junutula, J.R. (2013). Engineering THIOMABs for Site-Specific Conjugation of Thiol-Reactive Linkers. In: Ducry, L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology, vol 1045. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-541-5_11

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  • DOI: https://doi.org/10.1007/978-1-62703-541-5_11

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-540-8

  • Online ISBN: 978-1-62703-541-5

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