Screening and Activity of Human ScFv Antibody Targeting the Extracellular Region of Fibroblast Growth Factor R3
Fibroblast growth factor (FGF) is the largest family of growth and differentiation polypeptide factors in mesodermal and epithelial cells. Fibroblast growth factor plays an important role in many biological processes, such as embryonic development, wound healing, hematopoiesis and angiogenesis. In addition, studies have shown that fibroblast growth factor can increase the infiltration of many kinds of tumor cells, such as prostate, bladder, kidney, breast, pancreas and so on.
At present, more than 20 kinds of fibroblast growth factors have been found, which have different effects on different types of cells. However, only five kinds of fibroblast growth factor receptors (fibroblast growth factor receptors) were found. At the protein level, these receptors have 55% to 72% homology. The structure of FGFR includes an extracellular ligand binding region, a transmembrane region and an intracellular kinase region. Ligand binding regions contain three distinct immunoglobulin-like domains (called immunoglobulin I, II and III). Different splicing effects of fibroblast growth factor R1-3 mRNA form two subtypes of alpha and beta. Fibroblast growth factor R3 has two different mutants, IIIb and IIIc. The two variants have different affinity activities: IIIC is more widely distributed and can bind to various kinds of fibroblast growth factors (FGF1, FGF2, FGF4, and FGF9); IIIb preferentially binds to fibroblast growth factor 1, which can bind to fibroblast growth factor 8 and fibroblast growth factor 9 at a lower level. In the presence of heparin, the binding of fibroblast growth factors and fibroblast growth factors (FGFRs) induces receptor dimerization, which results in the phosphorylation of intracellular kinase region and the activation of downstream signal cascades. After ligand receptor binding, fibroblast growth factor (FGFs) initiates various signal transduction pathways: elevated intracellular calcium level, induction of mitogen-activated protein kinase and protein kinase C pathway, activation of adenylate cyclase and induction of proto-oncogenes c-myc and c-fos.
It has been found that special mutations of fibroblast growth factor R3 lead to activation of tyrosine kinase activity, leading to some syndromes related to skeletal development, multiple myeloma, neck tumors and bladder tumors. Recent studies have found that the signal of fibroblast growth factor receptor (FGFR) is essential for the survival of prostate cancer cells in vitro. Recently, FGFR3 has been used as a potential therapeutic target for multiple myeloma. Although there is evidence that the mutation of activated fibroblast growth factor R3 exists in cancer tissues, little is known about the expression of fibroblast growth factor R3 in cancer tissues. Recently, after gene expression analysis using gene chip technology, it was found that fibroblast growth factor R3 was overexpressed in bladder cancer samples compared with normal tissues. The level of gene expression was further confirmed at protein level by Western blot and immunohistochemical analysis. In fact, the overproduction of this protein seems to be more likely to occur in transitional tumors than in gene mutations. All these data suggest that fibroblast growth factor R3 may be a very attractive therapeutic target for urological tumors. Bladder cancer is one of the second most common malignant tumors in the reproductive and urinary system. About 40% - 50% of bladder tumors show mutations in the FGFR3 gene; epidermal tumors are more likely to occur (80%) than invasive tumors.
With the increasing interest of fibroblast growth factor R3 as a therapeutic target for different tumors and the discovery of its overexpression in transitional cell tumors, we have begun to develop human antibodies for treatment using phage display technology. Phage antibody display is currently the best way to develop human antibodies for research, clinical and therapeutic purposes. However, for antibody development, the FGFR3 molecule is very difficult to understand, because the homology of mouse and human fibroblast growth factor R3 is very high (92%). Only recently, it has been reported that a Fab fragment targeting a subtype of FGFR3 has been developed by using a commercialized Fab library with a very large storage capacity (2.1*1010). In our experiment, we used two open scFv antibody libraries Tomlinson_I+J(MRC Geneservices,Cambridge,United_Kingdom). The storage capacity of both libraries is about 1.4*108. Compared with IgG and Fabs, scFvs have better invasiveness, faster clearance and better specificity. In this report, we have screened some specific human scFv antibodies against subtype III C of FGFR3a. These antibodies have been demonstrated by FACS to react with bladder cancer cell line RT112 and inhibit cell proliferation, which has potential for further treatment.
Materials and methods
Cell lines, proteins, antibodies:
RT112, HEK293; recombinant human fibroblast growth factor R3a (IIIc)/Fc, fibroblast growth factor R1a (IIIc)/Fc, fibroblast growth factor 9, fibroblast growth factor 1 and epidermal growth factor. Mouse anti-human FGFR3 monoclonal antibody, sheep anti-human IgG (Fc specificity), mouse anti-c-myc monoclonal antibody, tubulin inhibitor; HRP-anti-c-myc antibody, anti-6His antibody, anti-M13 antibody, HRP-anti-M13 monoclonal antibody, FITC-rabbit anti-mouse IgG, R-phycoerythrin sheep anti-mouse IgG, mouse IgG TrueBlot.
Cloning and Cell Transfection of Fibroblast Growth Factor R3_cDNA
The extracellular region of fibroblast growth factor R3 (FGFR3) and the Fc_C end of human IgG were fused and expressed. The expression vectors pcDNA3.1-fibroblast growth factor R3 (IIIc) WT-Fc and pcDNA3.1-fibroblast growth factor R3 (IIIc) S249C-Fc were constructed. Then, HEK293T cells were transfected. Western blotting and FACS were used to detect protein expression and activity.
Screening of anti-fibroblast growth factor R3 specific scFv antibodies from phage library
Human scFv phage library Tomlin-son_I+J, auxiliary phage KM13, E.coli_TG1 and HB2151. Two phage libraries were cultured separately, and then 1:1 mixed phages were used for screening. Phage library screening and scFv expression were performed as shown in Figure 1. In the first round of screening, microporous plates were coated with 1ug_FGFR3 or human IgG protein. The phages were incubated with human IgG for 1 hour to remove the phages that could react with Fc, and then incubated with the holes coated with fibroblast growth factor R3 for 2 hours. Finally, the microporous plate was washed 10 times with 0.1% PBST (20 times in the next round of screening) and treated with 100 UL trypsin to elute the bound bacteriophage. Phage obtained by elution was screened in another round according to the method described by Goletz et al.
Phage ELISA
Using 0.3ug of fibroblast growth factor R3, fibroblast growth factor R1 or human IgG protein to coat the microporous plate, wash, seal, and add different concentrations of screened and purified phage suspension. After incubation, HRP-anti-M13 antibody was added, substrate color was added, and the results were read at 450 nm.
Monoclonal Phage ELISA
After two or three rounds of screening, the harvested bacteriophages were cultured and cloned. Then the monoclones were picked up by QPix high throughput automated clone screening system (Molecular Devices) and cultured on 96 microporous plates containing 100ul_2TY medium at 37 degrees. The next day, the cultures were diluted by the same medium at 1:100 degrees, then cultured in 37 degrees shaking for 2 hours, and added 25ul containing 109 auxiliary bacteriophages. Body KM13 was cultured on 2TY medium for 1 hour. After centrifugation, the bacteria were suspended in 2TY medium and cultured overnight at 30 degrees. Finally, 50 UL supernatant was centrifuged and analyzed by monoclonal phage ELISA.
Expression, Purification and ELISA Detection of Soluble ScFv Antibody
The highly specific clones were induced by E. coli_HB2151, harvested by centrifugation, and purified by affinity chromatography. The purified components were analyzed by SDS-PAGE and Coomassie brilliant blue staining. The purified components were further separated from scFv protein monomers by molecular sieve chromatography (scFv is prone to dimer or polymer).
Surface plasmon resonance analysis
BiacoreX was used to analyze the binding kinetics of soluble scFv and FGFR3, and the Kd value of each purified scFv was calculated. The specific binding sites of each scFv were determined by competitive binding analysis. The same method was used to analyze whether scFvs and FGF9, FGF1 and EGF could compete to bind to FGFR3.
Flow cytometry and confocal microscopy analysis
The binding activity of soluble scFv to fibroblast growth factor R3 at cell level was analyzed by loss cytometry. The antibody binding activity of RT112 cells was further analyzed by confocal microscopy.
Cell Proliferation Analysis
RT-112 cells were treated with different concentrations of anti-fibroblast growth factor R3_scFvs antibodies (0.02-2umol/L). After 48 hours, the cells were stained with MTT, and then 570 nm reading was performed. Cell viability was calculated by the following formula: Abs-scFv treated cells/Abs-control cells.
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