Preparation And Analysis Of Nanobody Against Immune Checkpoint LAG-3

ObjectiveThe nanobody phage library after LAG-3 immunization was constructed by phage technology.The gene sequence of anti-LAG-3 nanobody was screened from the library.The specific anti-LAG-3 nanobody was obtained by expression and purification,and the property of the nanobody was characterized.MethodsThe nanobody DNA fragments were obtained by two rounds of nested PCR using total c DNA from peripheral blood lymphocytes of immunized alpaca as a template.DNA fragments and phage vector fragments were digested with restriction enzymes and T4 ligase was used to construct recombinant phage plasmid.The plasmid was transformed into XL1-Blue competent cells by electroporation.After selection on antibiotic plates,we obtained the original immune library of nanobody.The quality of the phage library was analyzed by single colony PCR and gene sequencing.The original phage library was screened by four rounds of "adsorption-eluting-enrichment" using the solid-phase antigen screening method.Monoclonal phages were randomly selected from the screened library and some phages displaying specific anti-LAG-3 nanobody were screened by phage ELISA.The nanobody sequence was obtained by gene sequencing,and then the prokaryotic expression system was constructed by PCR and homologous recombination.Nanobody expression was induced using IPTG.After extraction of periplasmic expressed proteins,the proteins were purified using the Prism A column.SDS-PAGE was used to verify the purification results.Western blot and ELISA were used to verify the specific affinity of the nanobody to LAG-3 molecules The actual molecular weight and amino acid coverage of the nanobodies were verified by HPLC-MS and the affinity and dissociation ability of anti-LAG-3 nanobodies were determined by surface plasmon resonance.The inhibitory effect of anti-LAG-3 nanobody on the binding of LAG-3 to its ligand FGL-1 was verified by competitive ELISA.Results1.We successfully obtained the DNA fragments of the nanobody and constructed the nanobody phage library of the LAG-3 immunized.The library capacity is 6.54 ×108CFU/ml,and the positive fragment insertion rate is 90.90%.The sequencing results of the monoclonal clones selected from the library showed that they were all nanobody gene sequences and the sequence diversity was good.2.After four rounds of solid-phase antigen screening,the phage library with anti-LAG-3 nanobody was 133-fold enriched.A total of 26 phages displaying high-affinity anti-LAG-3 nanobody were obtained by phage ELISA.The nanobody gene sequences were classified into three types.3.The expression strain(pET-22b(+)-BL21-VHH)was constructed,and the expression and purification process of nanobody were improved.Three kinds of anti-LAG-3 nanobodies(VHH-L1-3,VHH-L3-2,VHH-L13-2)with a purity of about 90% were obtained.The specificity and affinity of anti-LAG-3 nanobody to LAG-3 were good.Among them,the affinity of VHH-L13-2 nanobody was relatively high,the molecular weight detected by HPLC-MS was consistent with the theoretical value,and the amino acid coverage was 100%.SPR results showed that the affinity of VHH-L13-2 nanobody with LAG-3 molecule reached the nanomolar level,with KD value of 3.971 x 10-9M.The binding of LAG-3 to its major ligand FGL-1 was inhibited by the VHH-L13-2 nanobody.ConclusionSuccessfully constructed a phage library of LAG-3 immunized nanobody by phage display technology,and screened three genotype-specific anti-LAG-3 nanobodies.Successfully obtained highly purified anti-LAG-3 nanobody by prokaryotic expression system,and the specific affinity was up to nanomolar level.The binding of LAG-3 to its major ligand FGL-1 was inhibited by the anti-LAG-3 nanobody.