Immunoconjugate preparation

TPT is a small hapten with an MW of 421D, and to stimulate an immune response, TPT was coupled to keyhole limpet hemocyanin via the Mannich reaction. The resultant immunoconjugate preparation was found to contain 90 + 42 molecules of TPT per molecule of KLH. 

In a somewhat similar scheme, Noguchi et al. (1992) prepared a carboxylate spacer arm by reacting 6-bromohexanoic acid with a dextran polymer. The carboxylate then was aminated with ethylene diamine to form an amine-terminal spacer (Figure 25.15). This dextran derivative finally was reacted with N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Chapter 5, Section 1.1) to create the desired sulfhydryl-reactive polymer (Section 2.4, this chapter). The SPDP-activated polymer then could be used to prepare an immunoconjugate composed of an antibody against human colon cancer conjugated with the drug mitomycin-C. 

Foglesong, P.D., Winkler, M.A., Price, J.O., Marshall, G.D., Reagh, S.H., Bush, D.A., Hixson, K.S., and West, W.H. (1989) Preparation and analysis of bifunctional immunoconjugates containing monoclonal antibodies OKT3 and BABR1. Cancer Immunol. Immunother. 30(3), 177-184. 

Noguchi, A., Takahashi, T., Yamaguchi, T., Kitamura, K., Takakura, Y., Hashida, M., and Sezaki, H. (1992) Preparation and properties of the immunoconjugate composed of anti-human colon cancer monoclonal antibody and mitomycin C—Dextran conjugate. Bioconjugate Chem. 3, 132-137. 

Wawrzynczak, E. J and Thorpe, P. E. (1987) Methods for preparing immunotoxms effect of the linkage on activity and stability, in Immunoconjugates Antibody Conjugates in Radioimaging and Therapy of Cancer (Vogel, C.-W., ed.), Oxford University Press, New York and Oxford, pp. 28-55. 

Barth et alJ30 have reported the preparation of boronated starburst dendrimer— monoclonal antibodies immunoconjugates as a potential delivery system for boron neutron capture therapy. 31 32 Starburst dendrimers have also been employed as linker molecules for the covalent connection of synthetic porphyrins to antibodies J33 ... 

Ogden JR, Leung K, Runda SA, Telander MW, Avner BP, Liao SK, Thurman GB, Oldham RK (1989) Immunoconjugates of doxorubicin and murine antihuman breast carcinoma monoclonal antibodies prepared via an N-hydroxysuccinimide active ester intermediate of cis-aconityl-doxorubicin preparation and in vitro cytotoxicity. Mol Biother 1 170-174... 

For the production of pAbs the purity of the hapten used to prepare an immunoconjugate should be as high as possible. After synthesis of haptenic substances, closely related substances may be present in small quantities, leading to the production of nonspecific Abs and, consequently, to unwanted cross-reactivities of the antisera. This is not a problem with mAb, because a single cell line producing only one kind of Ab with the desired properties can be selected. 

Scheme 8 Preparation of the Tn-clustered immunoconjugates 36, 37, and 39. (a) NaOH, MeOH, 95% (b) 35, NHS, EDC, DMF, DIEA or HOAt, HATU, DMF, collidine, 35-40% (c) H2N(CH2)3NHBoc, IIDQ, CH2CI2 (d) TFA, CH2CI2 (e) AcS(CH2)C02Pfp (SAMA-OPfp), DIEA, CH2CI2, 81% (f) NaOMe, MeOH (degassed), 85%.

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