Immuno-targeting the multifunctional CD38 using nanobody

Li, Ting; Qi, Shile; Unger, Mandy; Hou, Yun Nan; Deng, Qi; Liu, Jun; Lam, Connie Mo Ching; Wang, Xianwang; Du, Xin; Zhang, Peng; Koch-Nolte, Friedrich; Hao, Quan; Zhang, Hongmin; Lee, Hon Cheung; Zhao, Yong

doi:10.1038/srep27055

Cited by 61 publications

(79 citation statements)

References 59 publications

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“…As shown in Fig. 6A (IP:Nb375), both CD38 and CIB1 could be pulled down in the wild-type LP-1 cells by the immobilized CD38 nanobody, Nb-375, which binds to an epitope overlapping with Nb-1053 (19). Background contamination by endogenous CIB1 during IP was assessed using CD38-KO cells (compare Fig.…”

Section: Visualization Of Intracellular Interaction Of Cd38 With Cib1mentioning

confidence: 94%

“…S1). Crystallography identifies a 3D array of 22 or 23 different residues comprising each epitope (19), making the combination unique to the protein (CD38) that possesses both. The nanobodies were fused with the N-terminal (LucN) or C-terminal (LucC) fragment of Gaussia princeps luciferase, respectively.…”

Section: Significancementioning

confidence: 99%

“…With the verification of the DepID method, it was then applied to two multiple myeloma cell lines derived from patients to determine if type III CD38 is naturally expressed in cells. Both LP-1 and OPM2 endogenously express high levels of CD38 (19). Cell lines, based on LP-1 and OPM2, stably expressing LucN-1053 and LucC-551 were generated.…”

Section: Significancementioning

confidence: 99%

“…Type III CD38 in the ER should have its catalytic-terminal domain facing the cytosol. We targeted two separate epitopes on the C-domain simultaneously using two different nanobodies (Nb-1053 and Nb-551) that we have previously generated (19) (Fig. S1).…”

Section: Significancementioning

confidence: 99%

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Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels

Liu

Zhao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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CD38 catalyzes the synthesis of the Ca 2+ messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca 2+ stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells. Because type III CD38 is topologically amenable to cytosolic regulation, we used yeast-two-hybrid screening to identify cytosolic Ca 2+ and integrinbinding protein 1 (CIB1), as its interacting partner. The results from immunoprecipitation, ELISA, and bimolecular fluorescence complementation confirmed that CIB1 binds specifically to the catalytic domain of CD38, in vivo and in vitro. Mutational studies established that the N terminus of CIB1 is the interacting domain. Using shRNA to knock down and Cas9/guide RNA to knock out CIB1, a direct correlation between the cellular cADPR and CIB1 levels was demonstrated. The results indicate that the type III CD38 is functionally active in producing cellular cADPR and that the activity is specifically modulated through interaction with cytosolic CIB1.CD38 | cyclic ADP-ribose | membrane topology | calcium signaling | CIB1 C yclic ADP-ribose (cADPR) is a second-messenger molecule regulating the endoplasmic Ca 2+ stores in a wide range of cells spanning three biological kingdoms (1-4). Equally widespread is the enzyme activity that cyclizes nicotinamide-adenine dinucleotide (NAD) to produce cADPR (5). The first fully characterized enzyme was the ADP-ribosyl cyclase, a small soluble protein in Aplysia (6). It was thus surprising that the soluble cyclase shows sequence homology with the carboxyl (C-) domain of a mammalian membrane protein, CD38, a surface antigen first identified in lymphocytes (7). Subsequent work shows that CD38 is indeed a novel enzyme catalyzing not only the synthesis of cADPR but its hydrolysis to ADP-ribose as well (8,9). Gene ablation studies establish that CD38 is the dominant enzyme in mammalian tissues responsible for metabolizing cADPR and is important in regulating diverse physiological functions, ranging from neutrophil chemotaxis to oxytocin release and insulin secretion (10-12).It is generally believed that CD38 is a type II protein with a single transmembrane segment and a short amino(N)-tail of 21 residues extending into the cytosol, whereas the major portion of the molecule, its catalytic C-domain, is facing outside (13). This membrane topology seems paradoxical to CD38 functioning as a signaling enzyme producing an intracellular messenger. A vesicular mechanism has been proposed, positing that CD38 can be endocytosed, together with transport proteins, into endolysosomes. The transporters can then...

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Section: Visualization Of Intracellular Interaction Of Cd38 With Cib1mentioning

confidence: 94%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 2 more Smart Citations

Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels

Liu

Zhao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Due to their small molecular weight, 13 rapid tissue penetration, 14 high solubility and stability, 15 high specificity of antigen binding, and ability for large amounts to be synthesized, 16 they are often used in cancer treatment. Including the structural recombinant immunotoxins, 17 the therapeutic fusion proteins, they are are formed from the fusion of the antibody part that can specifically bind to tumor cell surface antigens and the part with cytotoxic activity. 18 In our previous study, we fused an anti-CD7 nanobody that we screened with PE38, a 38 kDa-molecular weight truncated Pseudomonas exotoxin A (PE), to construct the univalent and divalent recombinant immunotoxins VHH6-PE38 and dVHH6-PE38.…”

Section: Introductionmentioning

confidence: 99%

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

Yuan

Zhu

et al. 2017

IJN

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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.

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Enzymatic and Endogenous Synthesis of Nad(p)‐derived Calcium‐mobilizing Messengers

Lee¹,

Zhao²

2022

Nucleic Acids in Medicinal Chemistry and Chemical Biology

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Immuno-targeting the multifunctional CD38 using nanobody

Cited by 61 publications

References 59 publications

Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels

Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels

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

Enzymatic and Endogenous Synthesis of Nad(p)‐derived Calcium‐mobilizing Messengers

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