Abstract
Nanobodies (Nbs) are the smallest antigen-binding, single domain fragments derived from heavy-chain-only antibodies from Camelidae. Among the several advantages over conventional monoclonal antibodies, their small size (12–15 kDa) allows them to extravasate rapidly, to show improved tissue penetration, and to clear rapidly from blood, which are important characteristics for cancer imaging and targeted radiotherapy. Herein, we identified Nbs against CD33, a marker for acute myeloid leukemia (AML). A total of 12 Nbs were generated against recombinant CD33 protein, out of which six bound natively CD33 protein, expressed on the surface of acute myeloid leukemia THP-1 cells. The equilibrium dissociation constants (KD) of these six Nbs and CD33 range from 4 to 270 nM, and their melting temperature (Tm) varies between 52.67 and 67.80 °C. None of these Nbs showed leukemogenicity activity in vitro. The selected six candidates were radiolabeled with 99mTc, and their biodistribution was evaluated in THP-1-tumor-bearing mice. The imaging results demonstrated the fast tumor-targeting capacity of the Nbs in vivo. Among the anti-CD33 Nbs, Nb_7 showed the highest tumor uptake (2.53 ± 0.69 % injected activity per gram (IA/g), with low background signal, except in the kidneys and bladder. Overall, Nb_7 exhibits the best characteristics to be used as an anti-CD33 targeting vehicle for future diagnostic or therapeutic applications.
Highlights
Acute myeloid leukemia is an aggressive clonal malignancy of myeloid progenitors characterized by clinical and biological heterogeneity [1]
A total of 12 Nbs were generated against recombinant CD33 protein, out of which six bound natively CD33 protein, expressed on the surface of acute myeloid leukemia THP-1 cells
After four rounds of phage-display selection on recombinant CD33 coated on microtiter plates, the periplasmic extracts of 78 individual clones were confirmed to bind the recombinant human CD33 ectodomain in an ELISA
Summary
Acute myeloid leukemia is an aggressive clonal malignancy of myeloid progenitors characterized by clinical and biological heterogeneity [1]. It is the most common leukemia in adults, accounting for 30% of all leukemias, with over 400,000 cases worldwide, in 2018 [2,3]. The current standard of care for medically fit patients consists of intensive chemotherapy, followed by allogeneic hematopoietic transplantation. Despite these aggressive treatments, there is high risk of relapse, with cure rates between 35% and 40% for younger, fitter patients, while the prognosis is worse for patients above 65 years of age, with a five-year survival below 5% [6,7]. The risk of relapse remains high for multifactorial reasons, mainly due to the LSC plasticity that resists the different selection pressures from chemotherapeutics [8,9]
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