In vivo evaluation of mono- and bispecific biologics derived from single-chain antibodies from camelids (nanobodies) : a study on their ability to modulate biological activities or to induce cell depletion. ; Évaluation in vivo de molécules biologiques mono ou bi-spécifiques dérivées des anticorps monocaténaires de lama (nanobodies) : étude portant sur leurs capacités à moduler les activités biologiques ou à induire la déplétion cellulaire

Nanobodies are derived from the variable part of camelid heavy chain antibodies. Among their remarkable properties, the single-chain structure confers to nanobodies the ability to be easily reformatted. In addition, nanobodies can act as modulators of biological activities. In this work, we evaluated in vivo the capacity of recombinant adeno-associated vectors (rAAV)-encoded nanobody-based biologics first to modulate biological activities, and then to induce cell depletion. Taking advantage of the availability of several nanobody clones in the lab, in this study the targets were the purinergic receptor P2X7 and the ADP-ribosyltransferase ARTC2.2. In the first part, we demonstrated that activation of P2X7 by ATP and NAD+ is blocked by 13A7-IgG1LSF construct. The s+16a-dimHLE construct is a potent blocker of ARTC2.2-dependent activation of P2X7. Conversely, activation of P2X7 by ATP and NAD+ is potentiated by 14D5-dimHLE. 13A7-IgG1LSF and s+16a-dimHLE protect CD69+ resident T cells and NKT cells (that express high levels of P2X7) from NAD+-induced cell death. In contrast, NAD+-induced cell death is increased by 14D5-dimHLE. An injection of NAD+ in vivo leads to the depletion of CD4+CD25+ regulatory T cells, increased by 14D5-dimHLE, while 13A7-IgG1LSF protects T cells from NAD+-induced depletion. In the second part, we evaluated the ability of mono- or bispecific nanobody-based biologics to induce cell depletion. A single injection of rAAV encoding these constructs achieves long-lasting depletion of cells expressing the targets. In this model, the intensity of cell depletion mediated by anti-P2X7 7E2-IgG2a and anti-ARTC2.2 s-14-IgG2a constructs correlates with the antigen density on the cell surface. However, the epitope recognized and the nature of the target protein seem also to influence cell depletion. The bispecific S-14-7E2-IgG2a construct fails to achieve selective depletion of CD4+CD25+ regulatory T cells expressing high levels of P2X7 and ARTC2.2. The intensity of cell depletion is lower than that ...