NB22

Hematologic malignancies, particularly of B-cell origin, represent approximately 6.5% of all cancers. While conventional therapies remain first-line treatments, targeted therapies such as immunotoxins (ITs) offer promising alternatives, especially in relapsed or refractory cases. ITs are fusion proteins combining a targeting ligand with a potent toxin. CD22, a B-cell surface marker with restricted expression and rapid internalization, is an attractive target for IT development.

We developed a recombinant immunotoxin (rIT) by fusing a camelid-derived anti-CD22 nanobody (Nb22, ~15 kDa, ~400 bp) to a truncated diphtheria toxin (DT386, ~43 kDa, ~1,158 bp) via a human IgA1 hinge linker (~32 amino acids). The resulting fusion protein (~62 kDa, ~1,700 bp) was expressed in E. coli WK6 and characterized by SDS-PAGE and Western blot. Binding affinity to CD22 was evaluated by ELISA and flow cytometry. Cytotoxicity was assessed using MTT assays on Raji and Daudi (CD22⁺) and MOLT-4 (CD22⁻) cells.

The rIT exhibited high binding affinity to CD22 (Kaff ≈ 9.4 nM). Cytotoxicity assays demonstrated selective killing of Raji cells at low concentrations (1.6 nM), while MOLT-4 cells remained unaffected. In contrast, DT alone displayed non-specific toxicity only at high doses.

The selective cytotoxicity of the rIT demonstrates the potential of nanobody-based immunotoxins to target CD22⁺ cells while sparing non-target cells, highlighting an advantage over conventional toxin approaches. These results support further in vivo evaluation and optimization of Nb22-DT386 as a targeted therapy for B-cell hematologic malignancies.

The developed rIT is a promising candidate for targeted therapy of CD22⁺ hematologic malignancies, offering high specificity, potent cytotoxicity, and potential for reduced off-target effects.

Epithelial cell adhesion molecule (EpCAM) plays an important role in tumorigenesis. Camelids produce functional antibodies composed of heavy chains only that bind to their antigens via a single domain variable fragment known as nanobody. Nanobodies show multiple advantages over traditional monoclonal antibodies. Isolation of functional anti-EpCAM nanobodies (Nbs) was the main aim of this study. An immune nanobody library containing 10⁸ members was constructed previously. Anti -EpCAM nanobodies were isolated from camel immune library using phage display. Four consecutive rounds of biopanning were performed on immobilized EpCAM. Four nanobodies (Nb4, Nb5, Nb22, and Nb23) with highest signal intensity in monoclonal phage ELISA were selected. Affinity of these selected nanobodies for EpCAM was in the nanomolar range. Selected nanobodies significantly inhibited proliferation of MCF-7 cells. The in vivo study revealed that a significant reduction in tumor size occurred when treated with nanobodies Nb4 and Nb5, after 14 days monitoring. Our data revealed that nanobodies Nb4 and Nb5 could be considered as attractive theranostic agents for EpCAM overexpressing cancers.