Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential

Abstract

Background Nanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II. Methods and results Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdc em26 Il2rg em26 Nju (NCG) mice transplanted with CEM cells without any obvious decrease in body weight. Further studies on NCG mice model with patient-derived T-ALL cells, dhuVHH6-PE38 treatment, significantly prolonged mice survival with ~40% survival improvement. However, it was also noticed that although dhuVHH6-PE-LR showed strong antitumor effect in vitro, its in vivo antitumor efficacy was disappointing. Conclusion We have successfully constructed a targeted CD7 molecule-modified nanobody (CD7 molecule-improved nanobody) immunotoxin dhuVHH6-PE38 and demonstrated its potential for treating CD7-positive malignant tumors, especially T-cell acute lymphoblastic leukemia.

131I-labeled Anti-HER2 Camelid sdAb as a Theranostic Tool in Cancer Treatment

“…Consequently, α-particle therapy might be more suited in a micrometastatic setting. In line with antibody-drug conjugates, sdAbs have been successfully conjugated with cytotoxic payloads like DM1 or PE38-toxin, showing efficient tumor growth control without systemic toxicity (43–45). However, selecting highly internalizing sdAbs will be mandatory to induce significant cytotoxic effects.…”

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

“…A similar fusion construct of a monomeric CD38-specific nanobody with PE38 resulted in highly selective cytotoxicity against multiple myeloma cell lines and patient-derived multiple myeloma cells ( 23 ). And CD7-specific nanobodies fused to PE38 showed cytotoxic efficacy in CD7-expressing T-cell acute lymphoblastic leukemia in vitro and in a preclinical mouse model in vivo ( 91 , 92 ). Genetic fusion of an anti-EGFR nanobody to cucurmosin, a pumpkin toxin from the family of type 1 ribosome inactivating proteins induced cell death of EGFR-expressing cells lines in vitro ( 93 ).…”

Immunotoxins: Targeted Toxin Delivery for Cancer Therapy

“…5 Nanobody-based immunotoxins on several targets including CD7, CD38, and VEGFR2 were constructed and tumor-inhibitory effects were evaluated. [114][115][116]…”