Abstract
Heterobifunctional crosslinking reagents are small molecular weight chemicals containing two different reactive groups that have become important tools in generating conjugates of two different biomolecules, such as two proteins. The resulting bioconjugates are hybrid molecules or proteins, a new category of biomolecules that exhibit the combined functions of the two parent biomolecules. An important category of hybrid proteins are conjugates of antibodies with other effector molecules, such as drugs or toxins. These antibody conjugates or immunoconjugates have a variety of the applications in medicine, with particular emphasis on the treatment of cancer. The most commonly used heterobifunctional crosslinking reagents for the synthesis of antibody conjugates contain an N-hydroxysuccinimide ester moiety, which allows derivatization of amino groups in proteins. The chemical modification of a functionally important amino group in the antigen-binding region of an antibody causes impairment or loss of the antigen binding function, resulting in a defective antibody conjugate that lacks one of its component functions. Furthermore, even if the chemical derivatization does not affect the antigen binding function, the subsequent coupling of an effector protein at or near the antigen-binding region can also cause the loss of the antigen binding function for steric reasons. In this chapter, heterobifunctional crosslinking reagents are described that allow the generation of antibody conjugates where the effector proteins are coupled to the antibody carbohydrate moieties. Because antibody carbohydrate moieties are distal from the antigen-binding region, the use of carbohydrate-directed heterobifunctional crosslinking reagents, such as S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), prevents inactivation of the antigen-binding function. The synthesis of two carbohydrate-directed heterobifunctional crosslinking reagents is described. Coupling protocols for the preparation of antibody conjugates with effector proteins of different sizes using carbohydrate-directed heterobifunctional crosslinking reagents are also provided.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Notes
- 1.
*
N-(É›-maleimidocaproic acid) hydrazide (EMCH) and N-(Îş-maleimidoundecanoic acid) hydrazide (KMUH) are carbohydrate-directed heterobifunctional crosslinking reagents where the hydrazide group and maleimide group are separated by 5 (EMCH) or 10 (KMUH) methylene groups, respectively.
References
Vogel, C.-W. (ed.) (1987) Immunoconjugates. Antibody Conjugates in Radioimaging and Therapy of Cancer. Oxford University Press, New York, NY.
Rodwell, J. D. (ed.) (1988) Antibody-Mediated Delivery Systems. Marcel Dekker, New York, NY.
Vogel, C.-W. and Bredehorst, R. (1997) Immunoconjugates, in Encyclopedia of Human Biology, Vol. 4, 2nd Ed. (Dulbecco, R. L., ed.), Academic Press, San Diego, CA, pp. 112.1–112.15.
Kreitman, R. J. (1999) Immunotoxins in cancer therapy. Curr. Opin. Immunol. 11, 570–578.
Trail, P. A., King, H. D., and Dubowchik, G. M. (2003) Monoclonal antibody drug immunoconjugates for targeted treatment of cancer. Cancer Immunol. Immunother. 52, 328–337.
Wong, S. S. (ed.) (1991) Chemistry of Protein Conjugation and Cross-Linking. CRC Press, Boca Raton, FL.
Hermanson, G. T. (ed.) (1996) Bioconjugate Techniques. Academic Press, San Diego, CA.
Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-protein conjugation. Biochem. J. 173, 723–737.
Zara, J., Wood, R., Boon, P., Kim, C.-H., Pomato, N., Bredehorst, R., et al. (1991) A carbohydrate-directed heterobifunctional cross-linking reagent for the synthesis of immunoconjugates. Anal. Biochem. 194, 156–162.
Zara, J., Pomato, N., McCabe, R. P., Bredehorst, R., and Vogel, C.-W. (1995) Cobra venom factor immunoconjugates: Effects of carbohydrate-directed versus amino group-directed conjugation. Bioconjug. Chem. 6, 367–372.
Ottenheijm, H. C. J., Liskamp, R. M. J., van Nispen, S. P. J. M., Boots, H. A., and Tijhuis, M. W. (1981) Total synthesis of the antibiotic sparsomycin, a modified uracil amino acid monoxodithioacetal. J. Organic Chem. 46, 3273–3283.
Peters, L. C., Brandhorst, J. S., and Hanna, M. G., Jr. (1979) Preparation of immunotherapeutic autologous tumor cell vaccines from solid tumors. Cancer Res. 39, 1353–1360.
Haspel, M. V., McCabe, R. P., Pomato, N., Janesch, J. J., Knowlton, J. V., Peters, L. C., et al. (1985) Generation of tumor cell-reactive human monoclonal antibodies using peripheral blood lymphocytes from actively immunized colorectal cancer patients. Cancer Res. 45, 3951–3960.
McCabe, R. P., Peters, L. C., Haspel, M. V., Pomato, N., Carrasquillo, J. A., and Hanna, M. G., Jr. (1988) Preclinical studies on the pharmacokinetic properties of human monoclonal antibodies to colorectal cancer and their use for detection of tumors. Cancer Res. 48, 4348–4353.
Vogel, C.-W. and Müller-Eberhard, H. J. (1984) Cobra venom factor: Improved method for purification and biochemical characterization. J. Immunol. Methods 73, 203–220.
Roberts, W. K. and Selitrennikoff, C. P. (1986) Isolation and partial characterization of two antifungal proteins from barley. Biochim. Biophys. Acta 880, 161–170.
Zara, J. J., Wood, R. D., Bredehorst, R., and Vogel, C.-W. (1992) S-(2-Thiopyridyl)-L-cysteine, a heterobifunctional crosslinking reagent. US Patent no. 5,157,123.
Vogel, C.-W. and Müller-Eberhard, H. J. (1981) Induction of immune cytolysis: Tumor-cell killing by complement is initiated by covalent complex of monoclonal antibody and stable C3/C5 convertase. Proc. Natl. Acad. Sci. USA 78, 7707–7711.
Petrella, E. C., Wilkie, S. D., Smith, C. A., Morgan, A. C., Jr., and Vogel, C.-W. (1987) Antibody conjugates with cobra venom factor. Synthesis and biochemical characterization. J. Immunol. Methods 104, 159–172.
Vogel, C.-W. (1988) Synthesis of antibody conjugates with cobra venom factor using heterobifunctional crosslinking reagents, in Antibody-Mediated Delivery Systems (Rodwell, J. D., ed.), Marcel Dekker, New York, NY, pp. 191–224.
Lee, D. S. C. and Griffiths, B. W. (1984) Comparative studies on Iodo-bead and Chloramine-T methods for radioiodination of human α-fetoprotein. J. Immuol. Methods 74, 181–189.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.
Vogel, C.-W. (1987) Antibody conjugates without inherent toxicity: The targeting of cobra venom factor and other biological response modifiers, in Immunoconjugates. Antibody Conjugates in Radioimaging and Therapy of Cancer (Vogel, C.-W., ed.), Oxford University Press, New York, NY, pp. 170–188.
Vogel, C.-W. (1988) Antibody conjugates with cobra venom factor as selective agents for tumor cell killing, in Cytolytic Lymphocytes and Complement: Effectors of the Immune System, Vol. 2 (Podack, E. R., ed.), CRC Press, Boca Raton, FL, pp. 135–151.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Miochem. 72, 248–254.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Vogel, CW. (2004). Preparation of Immunoconjugates Using Antibody Oligosaccharide Moieties. In: Niemeyer, C.M. (eds) Bioconjugation Protocols. Methods in Molecular Biology™, vol 283. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-813-7:087
Download citation
DOI: https://doi.org/10.1385/1-59259-813-7:087
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-098-4
Online ISBN: 978-1-59259-813-7
eBook Packages: Springer Protocols