Engases-Catalyzed Glycoengineering Of Glycoproteins And Novel Antibody Drug Discovery

As a significant structure of living organisms,glycans are widely involved in various biological functions,of which protein glycosylation is an important glycan form and more than 50%mammalian proteins and over 70%protein-drugs are carrying glycosylation,participating in various biological functions,such as protein folding,protein transport,cell adhesion and immune response.The glycosylation of monoclonal antibodies,as the most important posttranslational modification of mAbs,is closely associated with its druggability,including the efficacy,in vivo metabolism,stability and immunogenicity.However,owing to the wide varieties and complexities,the precise control of protein glycosylation is still an arduous task full of challenge.This paper made a deep research in developing and improving ENGase-catalyzed protein glycoengineering technology,by which we remodeled the antibody glycans to give glycoengineered m Abs and glyco-site specific antibody drug conjuates（gsADCs）as well as selective cell surface N-glycan editing and labeling.We prepared the antibodies with optimized glycoforms rapidly,which showed enhanced ADCC and activity against cancer cells,in a manner of“one-pot glycoengineering”strategy.Meanwhile,we successfully synthesized a pool of site-specific ADCs through introducing non-natural glycan structures onto Fc glycosylation site with following drug conjugation.The glyco-site specific ADCs overcome the disadvantages of traditional random conjugated ADCs in heterogeneous conjugation site and numbers,bad quality control and poor reproducibility in pharmacodynamics and pharmacokinetics.Furtherly,we utilizied the method of ENGase-catalyzed protein glycoengineering technology to establish a novel cell surface glycan editing technology,realizing the selective editing and labeling of cell surface glycan subtype,providing a new chemical and biological method in cell surface glycan remodeling and glycan structure-function relationship studies.A diversified preparation technology of glycan substrates,improved high-efficient antibody glycoengineering technology and the application of such technologies in designing novel glycoengineered therapeutic antibodies was conducted and discussed respectively in the first part of the dissertation.Monoclonal antibody,mAb,which plays an important role in the treatment of tumors,inflammation and autoimmunity,now becomes a research focus in therapeutic biological drug discovery,especially the development of high-efficient and low-toxic therapeutic antibodies based on new technologies.Among them,design and preparation of glycoengineered antibodies,by controlling antibody Fc-glycans to improve and optimize its tumor-killing effect and druggability,provides a new avenue in therapeutic mAb drug discovery.Conventional antibody expression systems inevitably produce the mAbs with heterogeneous glycoforms.Even worse,regulations of the antibody glycosylation by reforming the glycosyltransferases in mammalian cell expression systems always involve various functional enzymes,and tedious work in optimizing expression systems when regarding different glycan structures and diverse antibodies.These limitations are not conductive to the design and screening of glycan structures of novel antibody drugs.Based on previous researches,we developed and optimized ENGases-catalyzed antibody glycoengineering technology.Taking advantage of the hydrolytic activity of wild-type endoglycosidase and the glycosynthase activity of its mutants,together with the synthetic diverse defined glycan substrates,we synthesized a variety of glycoengineered mAbs for screening.Firstly,we extracted and purified the sialylglycopeptide（SGP）from egg yolk as homogeneous glycan substrates precursor in developing a semi-synthesis strategy of glycan oxazoline,in which the chemical and enzymatic methods was combined for fast derivation of SGP,and 4 pure native glycans with defined structures and the successive oxazolines were synthesized for preparing glycoengineered antibodies.Then,wild-type Endo-S and fucosidase were used to remove the heterogeneous glycans and fucose on Herceptin,and the synthetic glycan oxazoline substrates were retransglycosylated to the deglycosylated antibody in the presence of Endo-S D233Q.We also developed a“one-pot”antibody glycoengineering strategy and prepared 4 glycoengineered antibodies.The establishment of“one-pot”glycoengineering greatly reduced the process of glycoengineered antibody preparation,improved the preparation efficiency and provided an efficient technology basis for the screening of the diversified glycoengineered antibodies and the further optimization of their druggability.The second part is the research and discovery of glycosite-specific antibody drug conjugates.Antibody drug conjugates,a complex that the cytotoxic drugs were covalently conjugated to a high tumor specificity antibody by a linker,reduces drug cytotoxicity,improves the targeting delivery,and enhances the therapeutic efficiency.The conventional method for preparing ADCs was mainly by introducing drugs to the side chains of lysine or cystine randomly.The conjugation sites and the number of drugs is highly heterogeneous in traditional ADCs,resulting in a series of problems of preparation technology,quality control and PK/PD optimization.Developing site-specific and quantitative conjugation method is the key point of novel ADC researches.Based on“one-pot”ENGase-catalyzed glycoengineering technology,we introduced reactive groups（e.g.azide or alkyne）to the non-natural glycan moiety when modifying the antibody Fc-glycans.Then we conjugated the synthetic drugs by“click chemistry”to realize the site-specific and quantitative conjugation of drug payloads onto Fc-glycans,offering an approach in designing ADCs with highly homogeneous glycan structures,conjugation sites and the number of drugs.From the structural characteristics of the terminal sialic acid and/or galactose on SGP,we introduced aldehyde group into glycan structures by NaIO₄ selective oxidation and/or galactose oxidase incubation.The formed aldehyde group was occupied by the following functionalization with azide or alkyne tag via oxime forming reaction or reductive amination reaction to give non-natural glycan substrates respectively.Enzyme-catalyzed antibody glycoengineering technology can specificity transfer these glycan substrates to the Asn297 on Fc domain,by which 1 Pertuzumab and 1novel mAb,2 Rituximabs and 7 Herceptins with different functional groups were well prepared and characterized.After that,we designed and synthesized a series of different drug-linkers,which were further applied to the site-specific conjugation of the synthetic glycoengineered antibodies,giving 25 site-specific ADCs with specific structures,linking methods and numbers,including the design of antibodies with different targets,dual drug strategy carrying two different drugs,and different drug releasing strategies.The anti-tumor activities of these gsADCs showed that the cleavable linker,like Val-Cit,can promote the release of drugs,with IC₅₀ reached to a level of 0.1nM.In summary,this part provided a new idea for site-specific ADC preparation,explored the structure function relationship and the design strategy of the glycosite-specific ADC,and provided a research basis for the glycosite-specific ADC.The third part of the dissertation introduced the establishment of a novel cell surface glycan editing and labeling strategy.We applied the ENGase-catalyzed protein glycoengineering technology to cell surface glycan remodeling to selectively edit the glycan subtypes and realize their fluorescence labeling.The glycosylation of cell surface protein is highly related to the cell-cell interaction,receptor binding,immunoregulation,tumor differentiation and escape,invasion of bacteria and virus etc.The method of cell surface glycan labeling based on chemical biology is an important tool for the function study and visualization of glycoprotein and its glycosylation.However,due to the variety and complexity of glycosylation,the selective labeling of precise glycoforms is still a challenge needed to be overcome in this field.Herein,to selectively hydrolyze the N-glycan of the cell surface glycoprotein,we used the specific substrate recognition of ENGases and its mutant enzymes to realize the transglycosylation of synthetic glycans to certain types of glycan acceptors,with precise glycans assembling onto the cell surface glycoproteins.Furthermore,we established the selectively labeling of the cell surface glycan using azide-modified non-natural glycan substrates.We edited and labeled the surface glycans of 5 different cell lines with core-fucosylation by Endo-F3 and its mutant,and verified the specific fucose-targeting labeling by FRET.The ENGase-catalyzed cell surface glycan labeling strategy firstly supplied a tool for selectively labeling of specific glycan subtypes,realizing the selectively labeling of N-glycan.In another hand,this method could remodel the heterogeneous glycans on cell surface to homogeneous glycoforms,providing a novel and powerful tool for investigating the effect of the precise glycoforms on cell surface glycoproteins and their functions,and promoting the development of the chemical glycobiology researches and the biological function investigation of protein glycosylation.The fourth part of the dissertation is the design and synthesis of FRET fluorescence probe based on the glycan structures and its applications in the detection of endo-glycosidase or bacteria.Endoglycosidases are mainly derived from bacteria while different bacteria secrete endoglycosidases with different activities towards specific glycan substrates.Therefore,with the substrate specificity of different ENGases,we designed relevant fluorescence probes to realize the selective detection of the ENGases and bacteria.Forster Resonance Energy Transfer,FRET,is an energy transfer phenomenon occurs when the distance of two energy overlapping fluorescence molecules is short than 10 nm.Herein,based on the structure of SGP,we labeled Cy3/Cy5 fluorescence probes to the opposite site of the oligosaccharide chains repectively and the FRET was successfully detected,which faded after Endo-M incubation,giving a kit for Endo-M and relevant bacteria assay.Furtherly,we designed and synthesized a FRET probe based on the antibody glycosylation in detecting Endo-S or similar enzymes.This kind of fluorescence probe has a wide application in the discovery,identification and activity detection of endoglycosidases,showing a promising future.The last part made a summary of overall researches.This part summarizes the work in whole doctoral research brifly,and prospected the systematic study or methods that are worth being carried out later.In a summary,this dissertation studied the endoglycosidases in applying to glycoengineered antibodies and novel glycosite-specific antibody drug conjugates preparation,and cell surface specific glycan labeling.“One-pot”synthesis of glycan oxazoline,“one-pot”antibody glycoengineering,the preparation and evaluation of glycosite-specific ADCs and ENGase-catalyzed cell surface glycan labeling were successfully established and developed,providing a good foundation for the further application of endoglycosidase and the researches of glycosite-specific ADCs,also providing a theoretical guidance and practical experience toward endoglycosidase applications.