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


RESULTS
Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed inEscherichia coliand 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-Prkdcem26Il2rgem26Nju (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.


CONCLUSIONS
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.
The Cyrus Tang Hematology Center; Collaborative Innovation Center of Hematology, Soochow University; Suzhou Cancer Immunotherapy and Diagnosis Engineering Center, Suzhou.
Yuan
Y
Yu
The Cyrus Tang Hematology Center; Collaborative Innovation Center of Hematology, Soochow University; Suzhou Cancer Immunotherapy and Diagnosis Engineering Center, Suzhou.
Jialu
J
Li
Central Laboratory, Department of Hematology, The Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing.
Xuejun
X
Zhu
Collaborative Innovation Center of Hematology, Soochow University; Department of Hematology, The First Affiliated Hospital of Soochow University, Suzhou.
Xiaowen
X
Tang
Department of Hematology-Oncology, The First People's Hospital of Hefei, Hefei, People's Republic of China.
Yangyi
Y
Bao
Department of Hematology-Oncology, The First People's Hospital of Hefei, Hefei, People's Republic of China.
Xiang
X
Sun
The Cyrus Tang Hematology Center; Collaborative Innovation Center of Hematology, Soochow University.
Yuhui
Y
Huang
Central Laboratory, Department of Hematology, The Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing.
Fang
F
Tian
The Cyrus Tang Hematology Center; Collaborative Innovation Center of Hematology, Soochow University.
Xiaomei
X
Liu
The Cyrus Tang Hematology Center; Collaborative Innovation Center of Hematology, Soochow University; Suzhou Cancer Immunotherapy and Diagnosis Engineering Center, Suzhou.
Lin
L
Yang
D036002:ADP Ribose Transferases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism; D002460:Cell Line; D004705:Endocytosis; D005098:Exotoxins / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000627:therapeutic use*; D016688:Mice, Inbred NOD; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy*; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use; D061905:Single-Domain Antibodies / Q000627:therapeutic use*; D013778:Tetrazolium Salts / Q000378:metabolism; D037521:Virulence Factors / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays; D055815:Young Adult; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D000941:Antigens; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D013778:Tetrazolium Salts; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C476329:2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt
CD7; Pseudomonas exotoxin A; T-cell acute lymphoblastic leukemia; humanized nanobody; patient-derived xenograft model; recombinant immunotoxins