Preparation Of Anti-CD133 Nanobodies And In Vitro Assessment Of The Cytolytic Peptides Anti-tumoral Activity

Currently the general opinion on the cause of refractoriness and resistence to chemotherapy of cancer diseases is attributed to a unique cancer cell population, cancer stem cells(CSC) — rare cells with indefinite potential for self-renewal that drive tumorigenesis. Accordingly, the effective approach to increase the treatment of cancer is to identify and understand the role of valuable CSC biomarkers and develop novel CSC-targeting therapy. CD133, also known as Prominin-1, a pentaspan transmembrane glycoprotein overexpressed in various solid tumours, is a commonly used biomarker to identify and isolation of CSC population from tumor mass. At present, the widely employed anti-CD133 mAb, such as AC133, 293C3 and AC141 were mainly used to distinguish CD133+ CSC populations. Nevertheless, owing to nearly no effect on growth of this cell groups, their further application in cancer therapy was limited. Therefore, it is necessary to develop novel anti-CD133 antibody with tumor and CSC kill activities. However, the lacking of information about the role of CD133 in the development of CSC hindered the design and production of therapeutic antibody targeting CD133. Considering, the feasible way to develop CSCtargeting therapy is conjugating cytotoxicity reagents with antibody(antibody-drug conjugate, ADC) to gain effective treatment. One of method is to conjugate cell killing peptides, such as E.coli Colicin Ia, Rana peptides Brevinin-2R, pseudomonas exotoxin A-PE38 and Lys-Asp-Glu-Leu(KDEL) peptide etc. The other promising conjugate is cytotoxic drug, monomethyl auristatin F, which are drawing wide concern. There are evidences that these cytotoxicity reagents conjugated with antibody could inhibit CSC maker expression of and suppress the tumor cell growth.Nanobodies(Nbs) are camel derived single domain heavy chain antibody fragments which naturaly devoid of light chain. It is the available smallest antibody fragment with intact activity of antigen binding. Owing to single domain property of Nbs, there are several advantages over heterotetrameric conventional antibody. Such as high soluble in water, low cost in production and binding inaccessible epitopes. The most important property is Nbs can easily conjugated with cytotoxicity reagents, and nearly lost no antigen-binding activity. These features make Nbs a promising candidate for production of ADC.The purpose of this work is to prepare camel derived nanobodies which specifically bind the CD133 and analyze its antigen binding property.The gene segment coding of CD133 extracellular region were PCR amplified and ligated into pET28 a plasmid and construction of a prokaryotic expression vector. The recombinant CD133 protein was expressed by IPTG induction and purified with Ni-affinity chromatography. Construct a naive single-domain antibody library of Xinjiang Camelus Bactrianus.By three round Affinity screening, we could aquire VHHs. The Enzyme-linked immunosorbent(ELISA) and Western blot were applied to assay the the specific binding to CD133 protein.At the same time, One camel and two New Zealand rabbits were immunized to aquire high titer of poly antibodies. ELISA and Western blot were applied to assay polyclonal antibody. We also applied CCK-8 to test the inhibition abilities of Colicin Ia and Brevinin-2R towards three cancer cell ines(B16 cells,Cavo-3 cells,Skov-3 cells).The ELISA results revealed that the titer of anti-CD133 antibody raised from camel reached about 1:106 after the 5th immunization, and the titer of anti-CD133 antibody raised from rabbit reached about 1:5×105 after the 4th immunization.The antiCD133 antisera could bind his-CD133 specifically in Western blot. We have successfully constructed The na?ve single-domain antibody library of Xinjiang Camelus Bactrianus that it contained 1.4×109 cfu individual transformants. And the titer of VHH phage rescuring titer was up to 1.7×1014 pfu/mL. After three round enrichment screening,we aquire 56 positive clones.Through test the sequences of genes by company,we got three expected sequence and one VHH-13 protein. The Enzyme-linked immunosorbent(ELISA) and Western blot reveled that VHH-13 protein specific binding to CD133 protein.The outcome of CCK-8 revealed that Colicin Ia could not inhibit the growth of Cavo-3 cells、Skov-3 cells and B16 cells. Brevinin-2R could inhibit the growth of Cavo-3 cells and B16 cells lines.It will offer experimental basis for toxin molecules coupled to nanobody.