Skip to main content
SpringerLink
Log in

Generation of Bispecific Antibodies by Chemical Conjugation

  • Chapter
  • First Online:
Bispecific Antibodies

Abstract

Bispecifc antibodies (bsAbs) are emerging as a promising new class of biotherapeutics. Although Ig domain fusion by DNA engineering is the prevalent methodology for producing bsAbs, a significant number of studies are being performed with chemically crosslinked Abs. By using different starting material and various conjugation strategies, bispecific fragments, full-length Abs, or combinations thereof can be generated. Two types or crosslinkers are used for the generation of bsAbs: hetero- or homobifunctional. Here we compare the pros and cons of the two types and we describe the use of the crosslinkers succinimidyl -3(2-pyridylthiol)propionate (SPDP), Traut’s reagent,sulpho-[succinimidyl-4-(N-maleimidomethyl)-4-cyclohexane-1-carboxylate] (SMCC), o-phenylenedimaleimide (o-PDM). We also review the clinical studies carried out with some of the products generated by these methodologies. The main hurdle for the transfer of chemically conjugated bsAbs to clinical applications has been the low yield and heterogeneity of the products. In this chapter we describe the generation of bisFabs, crosslinked Fabs with engineered Cys residues, to improve the efficiency and quality of bsAbs. We also discuss the use of non-natural amino acids along with click chemistry reactions and other approaches for the addition of new functionalities into bsAbs. These new technologies may help to improve both the yield and the homogeneity of bsAbs as well as their pharmacokinetics properties, paving the way for future clinical applications.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Brennan M, Davison PF, Paulus H (1985) Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. Science 229(4708):81–83

    Article  PubMed  CAS  Google Scholar 

  • Brocchini S, Godwin A, Balan S, Choi JW, Zloh M, Shaunak S (2008) Disulfide bridge based PEGylation of proteins. Adv Drug Deliv Rev 60(1):3–12

    Article  PubMed  CAS  Google Scholar 

  • Bundy BC, Swartz JR (2010) Site-specific incorporation of p-propargyloxyphenylalanine in a cell-free environment for direct protein-protein click conjugation. Bioconjug Chem 21(2):255–263

    Article  PubMed  CAS  Google Scholar 

  • Carrico IS, Carlson BL, Bertozzi CR (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3(6):321–322

    Article  PubMed  CAS  Google Scholar 

  • Chatal JF, Campion L, Kraeber-Bodere F, Bardet S, Vuillez JP, Charbonnel B, Rohmer V, Chang CH, Sharkey RM, Goldenberg DM, Barbet J (2006) Survival improvement in patients with medullary thyroid carcinoma who undergo pretargeted anti-carcinoembryonic-antigen radioimmunotherapy: a collaborative study with the French Endocrine Tumor Group. J Clin Oncol 24(11):1705–1711

    Article  PubMed  CAS  Google Scholar 

  • de Graaf AJ, Kooijman M, Hennink WE, Mastrobattista E (2009) Nonnatural amino acids for site-specific protein conjugation. Bioconjug Chem 20(7):1281–1295

    Article  PubMed  Google Scholar 

  • Deiters A, Cropp TA, Summerer D, Mukherji M, Schultz PG (2004) Site-specific PEGylation of proteins containing unnatural amino acids. Bioorg Med Chem Lett 14(23):5743–5745

    Article  PubMed  CAS  Google Scholar 

  • Doppalapudi VR, Tryder N, Li L, Aja T, Griffith D, Liao FF, Roxas G, Ramprasad MP, Bradshaw C, Barbas CF 3rd (2007) Chemically programmed antibodies: endothelin receptor targeting CovX-Bodies. Bioorg Med Chem Lett 17(2):501–506

    Article  PubMed  CAS  Google Scholar 

  • Fury MG, Lipton A, Smith KM, Winston CB, Pfister DG (2008) A phase-I trial of the epidermal growth factor receptor directed bispecific antibody MDX-447 without and with recombinant human granulocyte-colony stimulating factor in patients with advanced solid tumors. Cancer Immunol Immunother 57(2):155–163

    Article  PubMed  CAS  Google Scholar 

  • Gavrilyuk JI, Wuellner U, Salahuddin S, Goswami RK, Sinha SC, Barbas CF 3rd (2009) An efficient chemical approach to bispecific antibodies and antibodies of high valency. Bioorg Med Chem Lett 19(14):3716–3720

    Article  PubMed  CAS  Google Scholar 

  • Glennie MJ, McBride HM, Worth AT, Stevenson GT (1987) Preparation and performance of bispecific F(ab' gamma)2 antibody containing thioether-linked Fab' gamma fragments. J Immunol 139(7):2367–2375

    PubMed  CAS  Google Scholar 

  • Gu J, Ghayur T (2010) Rationale and development of multispecifc antibody drugs. Expert reviews of clinical pharmacology 3(4):491–508

    Article  CAS  Google Scholar 

  • Hollander N (2009) Bispecific antibodies for cancer therapy. Immunotherapy 1(2):211–222

    Article  PubMed  CAS  Google Scholar 

  • Jewett JC, Bertozzi CR (2010) Cu-free click cycloaddition reactions in chemical biology. Chem Soc Rev 39(4):1272–1279

    Article  PubMed  CAS  Google Scholar 

  • Junutula JR, Bhakta S, Raab H, Ervin KE, Eigenbrot C, Vandlen R, Scheller RH, Lowman HB (2008a) Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs. J Immunol Methods 332(1–2):41–52

    Article  PubMed  CAS  Google Scholar 

  • Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, Lu Y, Meng YG, Ng C, Yang J, Lee CC, Duenas E, Gorrell J, Katta V, Kim A, McDorman K, Flagella K, Venook R, Ross S, Spencer SD, Lee Wong W, Lowman HB, Vandlen R, Sliwkowski MX, Scheller RH, Polakis P, Mallet W (2008b) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26(8):925–932

    Article  PubMed  CAS  Google Scholar 

  • Karpovsky B, Titus JA, Stephany DA, Segal DM (1984) Production of target-specific effector cells using hetero-cross-linked aggregates containing anti-target cell and anti-Fc gamma receptor antibodies. J Exp Med 160(6):1686–1701

    Article  PubMed  CAS  Google Scholar 

  • Khaw BA, Rammohan R, Abu-Taha A (2005) Bispecific enzyme-linked signal-enhanced immunoassay with subattomole sensitivity. Assay Drug Dev Technol 3(3):319–327

    Article  PubMed  CAS  Google Scholar 

  • Khaw BA, Tekabe Y, Johnson LL (2006) Imaging experimental atherosclerotic lesions in ApoE knockout mice: enhanced targeting with Z2D3-anti-DTPA bispecific antibody and 99mTc-labeled negatively charged polymers. J Nucl Med 47(5):868–876

    PubMed  CAS  Google Scholar 

  • Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256(5517):495–497

    Article  PubMed  Google Scholar 

  • Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed Engl 40(11):2004–2021

    Article  PubMed  CAS  Google Scholar 

  • Kraeber-Bodere F, Bardet S, Hoefnagel CA, Vieira MR, Vuillez JP, Murat A, Ferreira TC, Bardies M, Ferrer L, Resche I, Gautherot E, Rouvier E, Barbet J, Chatal JF (1999) Radioimmunotherapy in medullary thyroid cancer using bispecific antibody and iodine 131-labeled bivalent hapten: preliminary results of a phase I/II clinical trial. Clin Cancer Res 5(Suppl 10):3190s–3198s

    PubMed  CAS  Google Scholar 

  • Kraeber-Bodere F, Rousseau C, Bodet-Milin C, Ferrer L, Faivre-Chauvet A, Campion L, Vuillez JP, Devillers A, Chang CH, Goldenberg DM, Chatal JF, Barbet J (2006) Targeting, toxicity, and efficacy of 2-step, pretargeted radioimmunotherapy using a chimeric bispecific antibody and 131I-labeled bivalent hapten in a phase I optimization clinical trial. J Nucl Med 47(2):247–255

    PubMed  CAS  Google Scholar 

  • Kraeber-Bodere F, Salaun PY, Oudoux A, Goldenberg DM, Chatal JF, Barbet J (2010) Pretargeted radioimmunotherapy in rapidly progressing, metastatic, medullary thyroid cancer. Cancer 116(4 Suppl):1118–1125

    Article  PubMed  CAS  Google Scholar 

  • Langer HF, von der Ruhr JW, Daub K, Schoenberger T, Stellos K, May AE, Schnell H, Gauss A, Hafner R, Lang P, Schumm M, Buhring HJ, Klingel K, Conrad S, Schaller M, van Zandvoort M, Jung G, Dimmeler S, Skutella T, Gawaz M (2010) Capture of endothelial progenitor cells by a bispecific protein/monoclonal antibody molecule induces reendothelialization of vascular lesions. J Mol Med 88(7):687–699

    Article  PubMed  CAS  Google Scholar 

  • Lee RJ, Fang Q, Davol PA, Gu Y, Sievers RE, Grabert RC, Gall JM, Tsang E, Yee MS, Fok H, Huang NF, Padbury JF, Larrick JW, Lum LG (2007) Antibody targeting of stem cells to infarcted myocardium. Stem Cells 25(3):712–717

    Article  PubMed  CAS  Google Scholar 

  • Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444

    Article  PubMed  CAS  Google Scholar 

  • Lum LG, Rathore R, Dizon D, Wang D, Al-Kadhimi Z, Uberti J, Ratanathahathorn V (2007) Induction of immune response and improved survival after infusions of T-cells armed with anti-CD3 x anti-Her2/Neu bispecifc antibody in stage IV breast cancer patients (PhaseI). Blood 110:2747

    Google Scholar 

  • Maullu C, Raimondo D, Caboi F, Giorgetti A, Sergi M, Valentini M, Tonon G, Tramontano A (2009) Site-directed enzymatic PEGylation of the human granulocyte colony-stimulating factor. FEBS J 276(22):6741–6750

    Article  PubMed  CAS  Google Scholar 

  • Miller WH, Brosnan MJ, Graham D, Nicol CG, Morecroft I, Channon KM, Danilov SM, Reynolds PN, Baker AH, Dominiczak AF (2005) Targeting endothelial cells with adenovirus expressing nitric oxide synthase prevents elevation of blood pressure in stroke-prone spontaneously hypertensive rats. Mol Ther 12(2):321–327

    Article  PubMed  CAS  Google Scholar 

  • Nisonoff A, Rivers MM (1961) Recombination of a mixture of univalent antibody fragments of different specificity. Arch Biochem Biophys 93:460–462

    Article  PubMed  CAS  Google Scholar 

  • Popkov M, Gonzalez B, Sinha SC, Barbas CF 3rd (2009) Instant immunity through chemically programmable vaccination and covalent self-assembly. Proc Natl Acad Sci U S A 106(11):4378–4383

    Article  PubMed  CAS  Google Scholar 

  • 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(1):9–21

    Article  PubMed  CAS  Google Scholar 

  • Rader C, Sinha SC, Popkov M, Lerner RA, Barbas CF 3rd (2003) Chemically programmed monoclonal antibodies for cancer therapy: adaptor immunotherapy based on a covalent antibody catalyst. Proc Natl Acad Sci U S A 100(9):5396–5400

    Article  PubMed  CAS  Google Scholar 

  • Raso V, Brown M, McGrath J (1997) Intracellular targeting with low pH-triggered bispecific antibodies. J Biol Chem 272(44):27623–27628

    Article  PubMed  CAS  Google Scholar 

  • Repp R, van Ojik HH, Valerius T, Groenewegen G, Wieland G, Oetzel C, Stockmeyer B, Becker W, Eisenhut M, Steininger H, Deo YM, Blijham GH, Kalden JR, van de Winkel JG, Gramatzki M (2003) Phase I clinical trial of the bispecific antibody MDX-H210 (anti-FcgammaRI x anti-HER-2/neu) in combination with Filgrastim (G-CSF) for treatment of advanced breast cancer. Br J Cancer 89(12):2234–2243

    Article  PubMed  CAS  Google Scholar 

  • Reusch U, Sundaram M, Davol PA, Olson SD, Davis JB, Demel K, Nissim J, Rathore R, Liu PY, Lum LG (2006) Anti-CD3 x anti-epidermal growth factor receptor (EGFR) bispecific antibody redirects T-cell cytolytic activity to EGFR-positive cancers in vitro and in an animal model. Clin Cancer Res 12(1):183–190

    Article  PubMed  CAS  Google Scholar 

  • Segal D. SD Bast B (1995) Production of Bispecific Antibodies. In: Current Protocols in Immunology, Supplement 14. John Wiley & Sons, New York

    Google Scholar 

  • Shalaby MR, Shepard HM, Presta L, Rodrigues ML, Beverley PC, Feldmann M, Carter P (1992) Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. J Exp Med 175(1):217–225

    Article  PubMed  CAS  Google Scholar 

  • Songsivilai S, Clissold PM, Lachmann PJ (1989) A novel strategy for producing chimeric bispecific antibodies by gene transfection. Biochem Biophys Res Commun 164(1):271–276

    Article  PubMed  CAS  Google Scholar 

  • Valone FH, Kaufman PA, Guyre PM, Lewis LD, Memoli V, Ernstoff MS, Wells W, Barth R, Deo Y, Fisher J et al (1995) Clinical trials of bispecific antibody MDX-210 in women with advanced breast or ovarian cancer that overexpresses HER-2/neu. J Hematother 4(5):471–475

    Article  PubMed  CAS  Google Scholar 

  • Vasu C, Gorla SR, Prabhakar BS, Holterman MJ (2003) Targeted engagement of CTLA-4 prevents autoimmune thyroiditis. Int Immunol 15(5):641–654

    Article  PubMed  CAS  Google Scholar 

  • Wu P, Shui W, Carlson BL, Hu N, Rabuka D, Lee J, Bertozzi CR (2009) Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag. Proc Natl Acad Sci U S A 106(9):3000–3005

    Article  PubMed  CAS  Google Scholar 

  • Wuellner U, Gavrilyuk JI, Barbas CF 3rd (2010) Expanding the concept of chemically programmable antibodies to RNA aptamers: chemically programmed biotherapeutics. Angew Chem Int Ed Engl 49(34):5934–5937

    PubMed  CAS  Google Scholar 

Download references

Acknowledgement

We thank Dr. Byoung-Chul Lee for critically reading the manuscript.

Author information

Authors and Affiliations

  1. Department of Protein Chemistry, Research and Early Development, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA

    Diego Ellerman & Justin M. Scheer

Authors
  1. Diego Ellerman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Justin M. Scheer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Justin M. Scheer .

Editor information

Editors and Affiliations

  1. Inst. Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, Stuttgart, 70569, Germany

    Roland E. Kontermann

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Ellerman, D., Scheer, J.M. (2011). Generation of Bispecific Antibodies by Chemical Conjugation. In: Kontermann, R. (eds) Bispecific Antibodies. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-20910-9_3

Download citation

Publish with us

Policies and ethics

Search

Navigation