Chemical modifications of the biotin carrier in pretargeted avidin-biotin radionuclide therapy may be of a paramount importance for tuning the amount of the radioactivity delivered to the cancer cells by the labelled biotins. We report here the synthesis of a collection of new synthetic DOTA-constructs bearing two (+)-biotin molecules (bis-biotins), designed for the creation of multimeric Av units (tetramers) bonded to the antibody. All the syntheses were carried out following the solid phase strategy, and growing up the molecules on a Rink Amide resin. The biotin heads are connected through spacers containing PEG or non-PEG residues. Molecular modelling calculations suggested that the Av cross-linking ability of the bis-biotins depends mainly on the spacers’ length, the best results being expected for arms affording distances in the range of 10-25 Å between the biotin carboxylate atoms, in the fully extended conformation. SEC-HPLC MALLS analysis of the products of our Av/bis-biotins reaction mixtures confirmed this hypothesis. The bis-biotin 16, where the non-PEG linker ensured a distance between the biotin moieties of 26.7 Å, gave about 50% of Av oligomers while the shorter analogue 18 (19.5 Å) afforded 100% of a Av polymer containing about 21 protein units. Remarkably, both the bis-biotins i.e. 16 and 18 were well soluble in aqueous solutions and showed an excellent stability against the action of peptidases.