Biomolecular Engineering 16 (1999) 113–118 Development of new biotin/streptavidin reagents for pretargeting D. Scott Wilbur a, *, Pradip M. Pathare a , Donald K. Hamlin a , Patrick S. Stayton b , Richard To b , Lisa A. Klumb b , Kent R. Buhler c , Robert L. Vessella c a Departments of Radiation Oncology, Uniersity of Washington, Seattle, WA 98195, USA b Department of Bioengineering, Uniersity of Washington, Seattle, WA 98195, USA c Department of Urology, Uniersity of Washington, Seattle, WA 98195, USA Abstract The high affinity of biotin for streptavidin has made this pair of molecules very useful for in vivo applications. To optimize reagents for one potential in vivo application, antibody-based pretargeting of cancer, we have prepared a number of new biotin and streptavidin derivatives. The derivatives developed include new radiolabeled biotin reagents, new protein biotinylation reagents, and new biotin multimers for cross-linking and/or polymerization of streptavidin. We have also modified streptavidin by site-directed mutation and chemical modification to improve its in vivo characteristics, and have developed new reagents for cross-linking antibody fragments with streptavidin. A brief overview of these new reagents is provided. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Biotin; Streptavidin; Site-directed mutation; Cancer www.elsevier.com/locate/geneanabioeng 1. Introduction The very high binding affinities of (strept)avidin and biotin provides the basis for their use in many in vitro and in vivo applications [1,2]. One application, termed ‘pretargeting’ [3 – 6], is being investigated by several research groups to obtain in vivo targeting of radionu- clides for diagnosis and therapy of medical conditions such as cancer and inflammation. Application of pre- targeting in cancer is described by Axworthy et al. in the preceding paper. An important aspect of pretarget- ing is that it can be conducted through several different reagent combinations [7]. Although the number of steps and reagents used in pretargeting makes it more com- plex to conduct than administration of radionuclides directly attached to monoclonal antibodies (mAbs), its use allows one to circumvent some of the inherent problems associated with tumor targeting of mAbs. Importantly, the use of reagents that have varying in vivo properties permits altering the pharmacokinetics and distribution of the molecule carrying the radionu- clide. The mAbs, streptavidin (or avidin), and biotin used in pretargeting have very different in vivo proper- ties, and those properties can be altered by chemical modification. Thus, our studies in pretargeting have focused on the development of improved mAb, biotin and streptavidin reagents. A brief review of some of the new reagents that have been developed follows. 2. New biotin reagents 2.1. Radiolabeled biotin deriaties The majority of pretargeting studies involve delivery of a radionuclide on a biotin derivative as the final step. We have chosen to use radionuclides of iodine (I-125, I-131, I-123) in our developmental studies. Previous experience with radioiodinated conjugates [8] led us to use arylstannyl biotin derivatives as intermediates which can be readily radioiodinated as a last synthetic step, By using the iodobenzamide labeling method for biotin derivatives, they are not subject to in vivo deiod- ination [9]. It is important to consider factors that affect the design of new radioiodinated biotin deriva- tives. For example, water solubility was of concern in the design of non-ionic radioiodinated biotin deriva- tives, particularly since they would include the iodoben- zamide functionality, which has a very low aqueous * Corresponding author. 1389-0344/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII:S1050-3862(99)00044-3