Research Of Transplanted Skeleton Of CDR3 Based On The Camel Single Domain Antibody Framework Regions

Biopharmaceutical therapy which based on the monoclonal antibody has become an important means to deal with the threat to human life, especially for cancer and other diseases. The limited ability of organization(such as a solid tumor) penetrating and the high costs of production or storage have promoted the researchers to generate the smaller sized genetically engineered antibodies, such as miniaturization(Fab, scFv and nanobody) or the new structural scaffold antibody mimetics or analogs.Nanobody are derived from the variable domain of the heavy-chain antibodies(HcAbs) that occur naturally in the serum of Camelidae. The variable domain of the heavy chain from HcAbs is fully functional and currently the smallest naturally derived antigen-binding fragment. Owing to single domain property of Nbs,there are several advantages over heterotetrameric mAb. Such as relatively non-immunogenic, high ability of organization penetrating and binding inaccessible epitopes, strong folding ability, high stability under extreme conditions, low costs,samll size, good soluablity, high stability, the VHH antibody holds great promise in immunological modulation and therapy application. In addition, the structural properties of nanobody that its antigen binding site consites only of single domain make nanobodies to be a promising candidates of next generation therapeutic molecules.NBL42 is an antibody which has only a CDR3 and two framework regions on both sides of the CDR3. This antibody has both a higher specific binding activity and a significant inhibitory activity to lysozyme. From the analysis of the sequence of NBL42, we come to know that it has a longer CDR3(17 amino acids). Besides, it also has the difference of three amino acid residues of the FR3 sequences of VHH with all the known FR3. One amino acid residue in FR4 is different from the amino acid residue at the sama position of the others in majority of VHH antibodies. Therefore, itis envisaged that this antibody framework region whether can be a generic transplantation skeleton for the grafting of CDR3.The purpose of this study is mainly in three aspects: Firstly, the analysis of the relationship between the structure of NBL42 and its antigen binding activity.Secondly, it is verified that whether this framework regions can be used as a universal framework for CDR3-transplanted peptide antibody. Finally, the generation of anti-CD47 antibody peptide and its activity analysis. The aim of this research is generating engineered antibody, developing a universa structure for the grafting of these small molecule antibody fragments which are based on the development of camel single-domain antibody fragments. Thus can provide the theoretical basis for designing novel antibodies in the future.There are three main part research contents in the thesis:1. The analysis of the relationship between the structure of NBL42 and its antigen binding activity.Firstly, the NBL42 protein was prepared by DNA recombinant techniques. The sequence of NBL42 which without leading NBL42 region and the hinge region was recovered by PCR cloning from a plasmid containing full NBL42-LH sequence. The gene fragment in pET30 a was expressed, the expressed protein in E. coli was purified with nickel column purification and identified by ELISA. The NBL42 Nanobodies which is removal of the leader and hinge region still shows higher capacity of lysozyme binding. The affinity constant, measured accurately by non-competitive ELISA, was found to be 1.2 × 106 L/mol, in terms of Kd to be 8.33 × 10-7 mol/L. The IC50 value was measured to be 14.01 ?g/mL, the inhibitory effect of NBL42 was showed good, and a high inhibitory activity was also showed. NBL42 Nanobodies retained 64.60% of its maximal antigen-binding activity after 0.5h of incubation at95 ?. These results indicated that the VHH antibody which was derived from the CDR3 retained the good thermal stability and high affinity.Finally, the antigen binding activity of nanobodies expressed in different parts in E. coli was compared in ELISA. The soluble NBL42 showed a better activity to bind lysozyme than the refolding form of insoluble protein.The refolding form of insoluble Lys3 still has a good antigen-binding activity the same as the periplasmic Lys3.2. The analysis of whether this NBL42 framework region can be used as a universal framework for CDR3-transplanted peptide antibody based on its binding activity of the antigen-antibody.In order to determine the feasibility of NBL42 to be the transplant skeleton for CDR3 peptide antibodies, following studies was made:Firstly, the FR3 and FR4 region of NBL42 was used as a receptor, and the CDR3 region of VHHA4 was used as a donor. NBL42-A4CDR3 recombinant antibody fragment of DNA was constructed which then connected onto the pET32 a expression vector for protein expression and purification, The binding properties of lysozyme and alliinase was detected by ELISA. The results showed that NBL42-A4CDR3 was successfully built into pET32 a, was successfully expressed to be NBL42-A4CDR3 recombinant antibodies in the soluble form. ELISA indicates that grafted antibody NBL42-A4CDR3 having similar binding activity with VHHA4. The length of the CDR3 of these two antibodies are close, but there is no significant sequence similarity of their amino acids. VHHA4 was expressed as inclusion originally, but won soluble expression when CDR3 transplanted into the framework of NBL42.Secondly, cAb-Lys3(combined with lysozyme, having 19 amino acids in CDR3)and murine monoclonal antibody B6H12(binding CD47 integrin-associated protein)were chosen as transplant donor, even though the specificity, affinity, CDR3 lengths and the expression levels of these two antigens were different. And NBL42(lysozyme binding) which only containing FR3-FR4 and CDR3 region was chosed as the receptor for these CDR3 sequences from various sources.NBL42-Lys3H3 and NBL42-B6H12H3 were prepared by total gene synthesis.The gene fragment in pET22b(+) was expressed and purified. These two CDR3 peptide antibodies were expressed in the periplasm. Western Blotting results showed that the two peptide antibodies were expressed at 16 kD and 28 kD respectively.ELISA showed that these two CDR3-grafting peptide antibody do not have the ability to bind their antigen lysozyme.3. The generation and activity analysis of anti-CD47 antibody peptide.This thesis attempts to transforme the structure of antibody and generate the framework of smaller sized genetically engineered antibodies which can bind CD47.There are three following strategies to design anti-CD47 antibody peptide:Firstly, the complete framework region of cAbBCII10(combined within the?-lactamase) can accept most of the three CDRs of camel VHH antibody subfamily II for grafting. The complete framework region of cAbBCII10 had a potential to be a universal backbone for CDR grafting. Secondly, Qiu used VHFR2 to combain the antibody VHCDR1 with VLCDR3. SDS-PAGE showed that anti-CD47 antibody peptide cAbCII10-B6H12H3, B6H12/H1-FR2-L3 was expressed and purified successfully. ELISA indicated that cAbBCII10-B6H12H3 or B6H12/H1-FR2-L3 did not bind CD47 specifically.This study analyzed the possibility of the framework region of NBL42 to be a universal generic skeleton for CDR3-grafting systematically. This study has achieved partial success that a CDR3 of a camel VHH antibody has been transplanted onto the framework of NBL42, and it was proved to be feasible that NBL42-A4CDR3 has obtained the antigen-specific activity of donor antibody. The experiments showed NBL42 having a potential to be the skeleton for CDR-grafting preliminary. However,the use of three antibodies of other sources have not been successful, because the constructed peptide antibody did not show the expected antigen-binding activity. It may be related to the source of CDR3 of grafted antibody or the composition of aminoacids of the CDR3 closely. The role of the sequence of CDR3 in transplanted antibodies need further analysis, to provide a theoretical basis for clarifying the relationship between the activity and the structure of the antibody...