Screening And Immobilizing Of Anti-?2M Nanobody For Preparation Of High-Performance Immunosorbent

Blood purification is a technology based on membrane separation or adsorption separation to remove endogenous and exogenous toxins from the patients' blood.It plays a crucial role in the treatment of critical illnesses such as liver failure,renal failure,and sepsis.Compared with water-soluble small molecule toxins,the effective removal of medium and large M.W.protein toxins are currently a bottleneck in this field.The immune adsorption technology depended on immobilized antibodies can highly recognize and specially remove target protein toxins in complex blood systems.Thus,safety and efficiency are the features of the immunosorbent.In this thesis,a VHH-based immunosorbent was developed for the specific blood purification of?2-microglobulin(?2M),a key pathogenic factor leading to dialysis-related amyloidosis(DRA)in end-stage kidney disease(ESKD)patients.In order to improve the antigen-binding activity of immobilized VHHs,the difference of amino and sulfhydryl groups in VHHs from different species was analyzed for the species selection principles.As a result,an anti-?2M VHH phage display library was constructed for the screening of specific VHHs.The amino and sulfhydryl immobilization of the selected VHHs were systematically studied,and high-performance blood purification adsorbent was prepared by optimizing the immobilization method.(1)Diversities analysis of VHHs generated from different camelids species.Amino and sulfhydryl groups are the most commonly used functional groups in the immobilization of protein,and modifications of amino and sulfhydryl groups without interrupting the protein structure determine the immobilized protein activities.In order to interpret the structural characteristics of VHHs from different species,an amino acid sequence library of VHHs from four camelid species(C.bact,C.drom,L.glam,and V.paco)was established by database search.Specifically,the amounts and locations of amino groups and sulfhydryl groups in the sequence were investigated.First,the amino acid sequences are highly conservative among all four VHHs,whereas significant differences are displayed at the non-conservative amino acid sequence between VHHs derived from Camelini and Lamini.On the other hand,there are 4 lysine sites that are more than 80%conserved on VHHs:H43,H64,H72 and H83.At least 90%of VHHs contain more than 3 lysine residues.VHHs derived from C.drom have significantly more lysine residues in average or in the CDR region than those of the other three species.For the analyzed VHHs,all have a strict conservative disulfide bond between H22 and H92.46%of the Camelini VHHs contain a second disulfide bond located between H33 and CDR3 but only 26.4%V.paco VHHs and 6.6%of L.glam VHHs contain the second disulfide bonds,mainly located between H50 and CDR3.In addition,only 5.0%of L.glam VHHs contain free sulfhydryl groups.Based on the facts that most of the L.glam VHHs contain only one pair of disulfide bond,without free sulfhydryl groups,and fewer amino groups,it is predicted that a VHHs library originated from L.glam will be more conducive to obtain VHHs suitable for the site-directed modification in preparation of immunosorbent.(2)Construction and screening of the phage display VHHs library.Two L.glam were immunized by human ?2M to construct an M13 phage display VHHs library,with the capacity reached up to 4.3×107 cfu,the positive clone percentage of 16 random clones was 100%,and the diversity reached 100%.The titer of the phage display library was 2.6×1012 cfu/mL,which satisfied for the experiment.After three rounds of biopanning and sequencing,20 VHHs belong to 9 subfamilies were obtained.From each subfamily,one clone was selected and purified to measure its affinity to ?2M by surface plasmon resonance technology,and the highest affinity of VHH R15 is 16.8 nM.(3)The influence of amino modification on VHH structure and function.To explore whether the VHHs surface amino groups revealed a preference in reaction,and to realize the site-specific modification and immobilization based on amino groups,four VHHs with different amino groups distributions were selected as model proteins and investigated the effect of amino modification on the structure and function of VHHs.The results showed that in the amino modification,the ?-amino and ?-amino groups on VHHs showed diverse reaction efficiency.Among the conservative amino sites on VHHs,H0 and K83 displayed high activity reach up to 50%?90%,especially,the reaction efficiency of K43,K64,and K75 is lower than 20%.But even so,it is still difficult to achieve a single-point amino modification with high uniformity only by controlling the reaction conditions.In addition,the structure and function analysis of the modified products showed that the amino modification of VHHs reduced stability,induced the secondary structure change,and weakened the antigen binding ability.VHHs were affected in varying degrees with significant individual variation.(4)Synthesis and evaluation of blood purification immunosorbent.The effect of VHHs immobilization methods on the performance of the adsorbent was further studied,and the VHHs immobilized via C-terminal sulfhydryl group showed significantly better affinity and activity retention than that via random surface amino groups.With the similar VHHs density,immunosorbent prepared by using the sulfhydryl group has 25%higher ?2M binding capacity than that using the amino group.Based on the above work,the ?2M selective immunosorbent was prepared by C-terminal sulfhydryl group site-directed immobilization of VHHs.The thermodynamic and kinetic characteristics of the target molecule were further investigated,and the potential clinical effect of the blood purification material was evaluated by establishing an in vitro perfusion model.The results showed that the coupling efficiency of VHH was 51%,and the coupling density reached 15.3 mg/mL on the adsorbent,with 74%of VHH maintained antigen binding activity.The immunosorbent exhibits a high adsorption capacity of up to 7.8 mg/mL,which was 19 times of the reported.In the external perfusion experiments,one perfusion treatment using 150 mL of immunosorbent can reduce the ?2M concentration of ordinary patients to a healthy level(<10 ?g/mL).Besides,the adsorbent has no detectable nonspecific adsorption to plasma proteins and has good reusability.In summary,this study indicated that screening and preparing specific VHHs for the development of new blood purification adsorption materials is a feasible technical strategy.Compared with ordinary antibodies,immunosorbent using VHHs as affinity ligands maintained high affinity and high selectivity with its advantages,including small molecules size,stable structures,easy prokaryotic expression,and cost-effective,showed good application prospects.This thesis provides theoretical basis,design ideas and technical methods for extracorporeal blood purification of ?2M and other blood toxins.