This paper presents the generation and evaluation of a novel potential drug delivery platform for biologics, based on recombinant spider silk. Targeting CD40 for activation of antigen presenting cells, in order to overcome tumor induced T cell tolerance, have shown promising results in cell and animal models. However, further trials have gained limited results due to severe side reactions. To overcome this, we have investigated a strategy for a localized CD40 activation. A CD40 agonist based on a single chain variable fragment (scFv<inf>CD40</inf>) was enzymatically coupled to silk structures, that were then used to stimulate cells in vitro. A reporter cell line responsive to CD40 agonists was used to evaluate the bioactivity of the developed scFv<inf>CD40</inf>-silk, and to optimize the method. Once the bioactivity was confirmed, human primary B cells derived from healthy donors were stimulated with the scFv<inf>CD40</inf>-silk construct. The resulting B cell response was characterized both by upregulated surface expression of the activation marker CD86 (3 fold), suggesting an improved antigen-presenting capacity, and by B cell proliferation (4 fold) generating an expanded B cell population. The detected upregulation of the costimulatory molecule CD86 on the B cells implies a potential of the functionalized silk to steer the tumor-specific T cell response from tolerance to immune activation, including the onset of appropriate effector functions. Finally, we investigated the usability of the novel silk format microspheres for CD40-mediated cell activation in vitro. Here, we were able to demonstrate that scFv<inf>CD40</inf>-coupled silk microspheres gave a pronounced activation of the CD40-expressing reporter cell line, supporting the suitability of silk microspheres for the delivery of biologics with immune modulatory purposes.