Preparation And Functional Study Of A New Human Antibody Targeting AXL

The TAM receptor tyrosine kinase（RTK）family（Tyro3,AXL and Mer）is a class of proteins that have been researched in recent years.The TAM receptor family of proteins has a micro amount of expression in normal tissues and cells;it is an important factor in maintaining homeostasis and innate immune responses.There have many results shown that AXL proteins in the TAM family are highly expressed on the surface of a variety of primary and metastatic tumor cells,it is considered to be a potential tumor target.The AXL molecule contains three phosphokinase sites(Y⁷⁷⁹,Y⁸2121 and Y⁸⁶⁶),which are key sites for the transmission of various signaling pathways.The ligands of the TAM receptor family are mainly growth arrest specific gene 6（Gas6）and protein S1（PROS1）.Gas6 and PROS1 are structurally similar,both containing sex hormone-binding globulin（SHBG）andγ-carboxyglutamate（Gla）domains to mediate their activity.As the sole ligand of AXL,Gas6 binds to AXL in a paracrine or autocrine manner,and its SHBG domain binds to the Ig-like domain of the extracellular domain of AXL,promoting the dimerization of the phosphorylation tail of AXL to transmit downstream signals.It has been shown that the normal expression of the AXL-Gas6signaling pathway regulates vascular permeability in the liver.AXL protein has been researched in promoting tumor cell migration,invasion,tumor metastasis and tumor cell drug resistance formation.In the past decade,it was found that AXL-Gas6 signal way promotes tumor development in tumor microenvironment.There have several therapeutic drugs targeting AXL,including small molecule inhibitors,anti-AXL antibodies and AXL decoy protein.There are several AXL small molecule inhibitors have entered clinical trials,like the small molecule drug R428（BGB324）has achieved significant inhibit effects in high-invasive breast cancer cell growth and metastasis,liver cancer metastasis and glioma cell metastasis.However,there is still a preclinical study on targeting AXL therapeutic antibodies.In this study,the bio-informatics method was used to analyze the AXL-Gas6interaction pattern and key amino acid residue sites,combined with molecular biology and cell biology methods to verify;through the screening of the natural phage antibody library to obtain a human antibody targeting AXL;then the phage library-derived AXL antibody as parent antibody to construct an affinity matured antibody library for screening,and an affinity matured AXL antibody which specifically blocked AXL-Gas6interaction was obtained finally.The main research contents of thesis include:1.Analytical confirmation of AXL-Gas6 interaction sites;2.Screening of high affinity antibodies of targeting AXL;3.Evaluate the functional activity of novel targeting AXL antibodies.The research results obtained are as follows:1.Analytical confirmation of AXL-Gas6 interaction mode and siteAs a key ligand for activating downstream signaling of AXL,Gas6 binds to the AXL protein on the surface of tumor cells in an autocrine or paracrine manner,activating the activity of AXL intracellular phosphorylation tail to regulate the development of tumor cells.In this part,it was confirmed that AXL-Gas6 was combined in the main mode and the secondary mode by analyzing the pattern of AXL-Gas6 interaction,and the key amino acid sites involved in the main mode of interaction of AXL-Gas6 were analyzed.According to the analysis results,construct five AXL Fc fusion proteins:AXL extracellular domain（AXL-ECD）wild type(WT:G³²,D⁸⁷,V⁹²,G¹²⁷),high affinity mutant(M1/M2:AXL-ECD-M1(G³²S,D⁸⁷G,V⁹²A,G¹²⁷R),AXL-ECD-M2(G³²A,D⁸⁷A,V⁹²A,G¹²⁷A)and low affinity mutant proteins(M3/M4:AXL-ECD-M3(E⁵⁶R,T⁷⁷R),AXL-ECD-M4(E⁵⁹R,T⁷⁷R)).The results showed that the five fusion proteins of AXL-ECD were specifically recognized by anti-AXL;the affinity with Gas6 protein was significantly different,and the high affinity mutant AXL-ECD-M1/M2 binding with Gas6 is higher than AXL-ECD-WT,while the affinity of low affinity mutant AXL-ECD-M3/M4 is much lower than AXL-ECD-WT.The high affinity mutant of AXL-ECD-M1/M2 can specifically capture free Gas6 protein to inhibit tumor cell migration and the up-regulation of p-AXL expression by inducing Gas6;low affinity AXL-ECD-M3/M4 lacks blocking activity.The above results indicate that the three amino acid positions E⁵⁶,E⁵99 and T⁷77 are key amino acid sites involved in the AXL-Gas6 interaction mode,and as key functional epitopes in the function of AXL-Gas6 signal way.2.Screening of high affinity antibodies of targeting AXL,and evaluate the functional activity of antibody（1）Screening of novel targeting AXL high-affinity human antibodiesA novel AXL antibody was obtained by three rounds of affinity panning of the native phage antibody library,named as anti-AXL-64 with a KD value of 4.42*10^-9M.Preliminary validation of the phage-derived AXL antibody anti-AXL-64 revealed that it can binding with human and murine AXL proteins in a dose-dependent manner,blocking AXL-Gas6 interaction but not directly recognizing key epitopes of AXL-Gas6interaction.Based on the above results,bio-informatics method was used to affinity-mature the anti-AXL-64 to construct an affinity matured antibody library.The antibody library was stably transfected into Flp-In?-CHO cells,screened by mammalian cell display technology,the cells expressing high affinity antibodies were enriched by three-rounds flow sorting,and the antibody sequence of the third round of cells was amplified and sequenced.Selected 150 clones for sequencing analysis randomly and finally selected 18 representative antibodies for identification.After full-length expression of 18 antibody clones,the supernatant expression level and the binding ability with human AXL protein were detected.The blocking activity of the AXL-Gas6 interaction initially obtained three candidate antibodies,named DAXL-11,DAXL-32 and DAXL-88.These three strains of antibodies were purified and further screened.From the results,the recognition epitope of AXL protein which DAXL-32recognized was drifted,so it lost the activity of blocking AXL-Gas6 binding,and could not recognize the AXL protein on the surface of tumor cells;DAXL-11 had no higher affinity with human AXL protein compared with the parent antibody,the ability to block AXL-Gas6 interaction and recognize AXL on tumor cell surface was reduced;DAXL-88 affinity increased(3.702×10^-10M),and the ability to recognize AXL protein on the surface of tumor cells was not changed,the recognition epitope did not shift significantly,and it is effectively blocked the AXL-Gas6 interaction.Therefore,the antibody DAXL-88 was finally selected for evaluation of in vitro and in vivo functional activity.（2）Evaluate the functional activity of DAXL-88The novel AXL antibody DAXL-88 obtained by screening was evaluated for in vitro and in vivo functional activity.Flow cytometry results have confirmed that DAXL-88 can effectively recognize AXL protein on the surface of tumor cells A549and SKOV3 cells.Competition ELISA results suggest that DAXL-88 can block the interaction of AXL-Gas6 effectively.Cell migration assay showed that the antibody DAXL-88 blocked the migration of A549 and SKOV3 cells induced by free Gas6effectively;similar result were verified in cell invasion experiments.Western blot results indicated that the antibody DAXL-88 inhibit the expression of p-AXL and its downstream signaling molecules p-Akt and p-Erk,which were up-regulated by free Gas6 in a concentration-dependent manner;when the final concentration of antibody DAXL-88 was 1μg/mL,the inhibitory effects on p-AXL,p-Akt and p-Erk were sustained.The mouse tumor-bearing model was established by using SKOV3 cells,and the anti-tumor activity of the antibody DAXL-88 was evaluated.The single-dose experiment showed that DAXL-88（10 mg/kg）treatment group had no significant inhibitory effect on tumor size compared with saline group（Control group）.HE staining analysis showed that DAXL-88 treatment group promoted tumor cells necrosis,and AXL staining in the tumor tissue of the DAXL-88 treatment group showed that the expression of AXL protein was significantly decreased（P=0.0193）.This finding provides an experimental basis for the use of the antibody DAXL-88 as an adjunct.