Nanobody 2F8-based PNA-mediated pre-targeting for the treatment of multiple myeloma

Our project aims to use Nb2F8, a CD38-binding single domain antigen-binding fragment (sdAb), as a theragnostic tool for the diagnosis and treatment of multiple myeloma (MM). This nanobody can be radiolabeled with different radionuclides for diagnostic and therapeutic use. Despite its suitable properties, the high renal reuptake remains a major limitation for therapeutic applications. To bypass this drawback, a pre-targeting system, based on 2 complementary peptide nucleic acids (PNA) sequences HP1 and HP2 has been developed. A pentapeptide motif specifically recognizable by the enzyme Sortase A was introduced in the Nb2F8 sequence which allowed the conjugation to HP1. We assessed the binding affinities of this 2F8-HP1 conjugate by biolayer interferometry (BLI) and by flow cytometry using CD38+RPMI-8226 cells. We found that this Nb2F8-HP1 conjugate retained the excelling binding properties and could still be recognized by HP2.To evaluate the feasibility and specificity of the pre-targeting approach, an in vitro assay was designed and included (1) RPMI-8226 cells incubated with the primary targeting agent 2F8-HP1 and then 68Ga-DOTA-HP2, (2) RPMI-8226 cells incubated with nonconjugated Nb2F8 before the addition of 2F8-HP1, (3) RPMI-8226 cells incubated with an excess of cold HP2 prior to incubation with the radiolabeled form and (4) RPMI-8226 cells incubated with radiolabeled HP2 alone. The retained radioactivity measured demonstrated that the pre-targeting system allowed to bring the radionuclide to myeloma cells. Moreover, it shown that the hybridization between the complementary nucleotides is necessary to realize the pre-targeting and that there are no non-specific interactions of RPMI-8226 cells with HP2. Considering these promising results, the pre-targeting system can be suitable for in vivo applications. The biodistribution of 2F8-HP1 and the specificity of the probes will be evaluated in mice model xenografts. If the selective and specific binding of this pre-targeting system would be confirmed also in vivo, the Nb2F8 could assume a new significant role for further purposes as theragnostic tool.