A Humanized Nanobody Targeting The Ligand-binding Site Of Transforming Growth Factor Receptor-βⅡ(TβRⅡ)

Purpose and significanceTissue fibrosis and severe scar formation are common adverse events in the process of wound healing.If the event occurs in the main organs,it will be life-threatening,if it occurs in a large area on the body surface,it will affect the normal physiological function of the skin,and if it occurs on the face,it will seriously affect the patient’s self-confidence.Transforming growth factor-β1(TGF-β1)is one of the main members of the TGF-βsuperfamily and plays an important role in tissue fibrosis and scar formation.Blocking the TGF-β/Smad signal pathway activated by TGF-β1 has important clinical value in the prevention or treatment of tissue fibrosis and scar formation.TGF-βreceptor(TβR)is a member of serine/threonine kinase receptor family and is a single transmembrane protein.There are three subtypes of TβRI,TβRⅡ and TβRⅡI,among which TβRI and TβRⅡ are involved in the activation of TGF-β1 signal pathway.TGF-β1 first binds to TβRⅡ,the latter is phosphorylated in the intracellular domain and recruits TβRI to form heterodimers.The activated TβRI then activates the Smad signaling pathway and stimulates fibroblasts to differentiate into myofibroblasts,and secretes a large amount of type I and ⅡI collagen,which leads to tissue fibrosis and scar formation.Therefore,using antibody drugs to block the binding reaction between TGF-β1 and TβRⅡ may effectively block TGF-β1/Smad signal pathway and effectively block tissue fibrosis and scar formation.Nanobody,also known as single domain antibody,is a new type of small molecule antibody constructed from the variable region of alpaca single chain heavy chain antibody.It is about 15KDa in size,has good physical and chemical stability and tissue penetration ability,and can be produced efficiently and cheaply by microbial genetic engineering system,so it has a broad application prospect in the research and development of therapeutic antibodies.Screening the nanobodies targeting the ligand TGF-β1 located at the interface of TβRⅡmay reduce or block the signal transmitted by TGF-β1,and even become an effective strategy to inhibit scar formation.In this study,a kind of nanobody targeting TβRⅡligand binding domain was screened from humanized nanobody phage display library,an efficient preparation method was established,and its biological activity in vitro was studied preliminarily.It will lay a solid foundation for the further development of nanobody drugs against fibrosis and scar formation.Research contents and methods1.Design and synthesis of antigenic peptidesThe 3D structural model of the binding of TGF-β1 and TβRⅡwas analyzed by CN3D4.1 software,and the interface between them was found.The randomly curled peptide was selected as antigen peptide and synthesized by chemical synthesis.2.Phage display and screening of anti-TβRⅡnanobodies.The antigen peptides were coated in the aminoenzyme plate,and the humanized nanobody phage display library(3×10~9)from commercial sources was used for three rounds of washing.The positive clones were identified by ELISA,and the DNA sequences were analyzed after sequencing.3.Construction and preparation of recombinant engineering bacteria with nanobodiesThe target gene of nanobody was designed according to the preference codon of E.coli,and the His6 tag sequence was introduced into the 3’end.It was cloned into the NdeⅠand Hind ⅡI sites of the p ET21b expression vector according to the conventional method.The target gene was introduced into the host E.coli shuffle T7-B by thermal shock,so that the target gene could be efficiently expressed under the drive of T7 promoter.The expression of nanobody was analyzed by SDS-PAGE method,the expression product was identified by Western blot,and the high-level expression clone was selected as the reserve of engineering bacteria.Shake flask scale fermentation culture and IPTG induced expression.After ultrasonic fragmentation,the distribution of the target expression product in the supernatant and precipitation was analyzed by electrophoresis to determine whether the target protein expression product existed in the form of soluble protein or inclusion body protein.According to the physical and chemical properties of the target protein,the protein was purified by cation exchange(CM-Sepharose FF column)and nickel ion chelate chromatography(Ni-NTAcolumn).The buffer was replaced by molecular sieve chromatography(Sephadex G25 colum).The concentration of the target protein was detected by BCA.4.Specific binding of nanobodies to fibroblast surface receptorsWith NIH-3T3 as target cells,5×10~4cells/well were inoculated in 6-well plate and cultured in DEME medium containing 10%fetal bovine serum for24h,followed by serum-free starvation for 24h.After being blocked with5%BSA solution and co-incubated with different concentrations of TβRⅡnanobody.FITC-labeled mouse anti-His6 antibody was used to detect nanobody,and the specific binding nanobody on cell surface was observed by fluorescence microscope.5.Inhibitory effect of nanobody on the proliferation of fibroblastsThe blocking effect of nanobody on TGF-β1 stimulation signal was investigated by observing the growth inhibition effect of target cells.Fibroblast NIH-3T3 as target cells were cultured in DEME medium containing10%fetal bovine serum for 24 hours,followed by serum-free starvation for 24h.The cells were incubated with TGF-β1 and different concentrations of TβRⅡnanobodies for 48 hours,and the cell growth inhibition rate was detected by MTT.6.Effect of nanobodies on collagen expression of fibroblasts.The blocking effect of nanobody on TGF-β1 and the potential of anti-fibrosis and scar formation were investigated by observing the effect of nanobody on the expression of type I and ⅡI collagen in fibroblasts.NIH-3T3of fibroblasts were co-incubated with TGF-β1 and different concentrations of TβRⅡnanobodies,and then analyzed by Western blotting.The monoclonal antibodies against typeⅠand typeⅢcollagen were used as primary antibodies.7.Effect of nanobody on the migration ability of fibroblastsThe process of wound healing was simulated by cell scratch method in vitro to observe the effect of nanobody on the migration ability of fibroblasts and the blocking effect on TGF-β1.Result1.Design of antigen peptide and screening of phage nanobodyA random curled peptide composed of 15 amino acid residues was selected from the ligand binding interface of TβRⅡas antigen peptide.32positive clones were obtained after phage display and three rounds of washing.All of them were identified as two different nanobodies by DNA sequence analysis,which were named TβRⅡNb17 and TβRⅡNb21,respectively.2.Construction,expression and purification of nanobodies engineering bacteriaThe engineering bacteria expressing TβRⅡNb17 and TβRⅡNb21 were successfully constructed by using p ET21b as expression vector and E.coli Shuffle T7-B as expression host,and the two kinds of nanobodies existed as soluble expression products.The purity of the expressed product was more than 98%after two-step purification by cation exchange and nickel ion chelate chromatography.3.Specific binding of nanobodies to fibroblast surface receptorsBoth TβRⅡNb17 and TβRⅡNb21 nanobodies can specifically bind to the surface of fibroblast NIH-3T3 in a dose-dependent manner.4.Effect of nanobody on the proliferation of fibroblastsIn the absence of TGF-β1 stimulation,TβRⅡNb17 and TβRⅡNb21could inhibit the growth of fibroblasts in a dose-dependent manner,but only TβRⅡNb17 could significantly inhibit the proliferation of fibroblasts induced by TGF-β1.5.Effect of nanobody on collagen expression of fibroblasts.Both TβRⅡNb17 and TβRⅡNb21 nanobodies can inhibit the expression of typeⅠand typeⅢcollagen in fibroblasts,and inhibit the effect of TGF-β1on collagen expression.6.Effect of nanobody on the migration ability of fibroblastsThe effects of two kinds of nanobodies on the migration of fibroblasts were significantly different.TβRⅡNb17 could inhibit the migration of fibroblasts and inhibit the stimulating effect of TGF-β1,while TβRⅡNb21could promote the migration of fibroblasts,and had little effect on the stimulating effect of TGF-β1.ConclusionIn this study,the antigenic peptide of TβRⅡligand binding site was successfully obtained based on the ligand-receptor interface targeting strategy,and two humanized anti-TβRⅡnanobodies were successfully screened from the phage display library of nanobodies.Both of them were highly soluble and expressed in E.coli system,and could specifically bind to TβRⅡon the surface of fibroblasts.As a result,the stimulating effect of TGF-β1 on the expression of typeⅠand typeⅢcollagen was blocked,but there was a difference in the effect on the proliferation and migration of target cells.This study laid a solid foundation for the development of anti-TβRⅡnanobodies for the prevention and treatment of fibrosis and scar hyperplasia.