Bispecific antibodies are capable of binding to two different antigens or to two distinct epitopes on the same antigen, the latter called biparatopic antibodies. Their dual specificity enables completely new therapeutic strategies and applications which were not achievable with the classical monoclonal antibody format. In this work, we constructed and comparatively characterized new antibody-based formats with multiple specificities, based on Dual-Variable-Domain Immunoglobulins, Strand Exchange Engineered Domains, and the IgG-scFv format, for two different innovative applications. The first part of the thesis work was dedicated to the development of novel biparatopic antibodies against granulocyte-macrophage colony stimulating factor and the investigation of their efficiency on the monomeric soluble target. The aim of the second part of the thesis was T cell immunomodulation through receptor co-ligation. Bispecific antibodies binding the T cell receptor as well as the inhibitory receptor PD-1 have been generated and characterized in vitro in relevant bioassays.