Antibody-recruiting Strategy To Reconstitute The Fc Functions Of Nanobody

Nanobodies exhibit many advantages,such as high specificity and affinity,good stability,low immunogenicity and easy production.Consequently,nanobody-based therapeutic strategies and drugs have been widely developed.However,nanobodies lack the Fc region of a conventional antibody,which possesses many functions,e.g.,antibody-dependent cell-mediated cytotoxicity?ADCC?and complement-dependent cytotoxicity?CDC?,essential for effective immunotherapy.Additionally,the molecular weight of nanobodies is only one-tenth of that of conventional antibodies,which leads to the short serum half-life and seriously affect the stable anti-tumor performance of nanobodies.To address these deficiencies,firstly,the extracellular secretory expression level of transpeptidase Sortase A?Srt A,EC number:3.4.22.70?in Escherichia coli?E.coli?was efficiently enhanced by optimizing a series of conditions.Then,a series of nanobodies with antibody-recruiting ability were constructed via Srt A-mediated ligation?SML?/fusion expression,and the reconstituted Fc functions of these engineered nanobodies were subsequently confirmed.The main results are as follows:?1?Efficient secretory expression of Srt A in E.coli was achieved.Firstly,Pel B was screened out to mediate extracellular secretion of Srt A.With a subsequent codon optimization,the extracellular Srt A could reach 11.7 U/m L in shake flask after 36 h.Secondly,five recombinant E.coli harboring different molecular chaperones expression plasmids were constructed after codon optimization,among which the strain co-expressing Gro ES/Gro EL/Tig showed the highest Srt A secretion enhancement.The extracellular Srt A could rise to 34.0 U/m L in shake flask after 36 h.Finally,after codon optimization,glycine was screened out as the best additive to boost the secretion of Srt A.Under the optimal glycine supplementation strategy?termed two-stage glycine supplementation strategy:0.5%and 1.0%glycine were added at 0and 6 h post-induction,respectively?,the extracellular Srt A could rise to 100.4 U/m L in 7-L bioreactor after 48 h,which presents the highest level of extracellular Srt A ever reported.?2?The Fc functions of nanobodies were reconstituted via site-specific C-terminal 2,4-dinitrophenyl?DNP?modification.Firstly,four 7D12-DNP conjugates D1,D2,D3 and D4 were successfully constructed by using SML.Secondly,the in vitro results showed that all four conjugates could specifically recruit anti-DNP antibodies on EGFR⁺cancer cells,and kill them via ADCC and CDC.The results also indicated that D2?with PEG₃ as the linker?had the highest capacity to induce cell death,the cell lysis of A431 cells via ADCC and CDC after treated with100 n M D2 were 28.2%and 31.1%,respectively.Thirdly,in vivo pharmacokinetic assays showed that the half-lives of conjugates could be significantly prolonged in the presence of anti-DNP antibodies,the half-life of D2 in OVA-DNP pre-immunized mice could even reach31.6 h,which is 174.6-fold higher than that of 7D12.Finally,the A431 xenograft mice model was constructed to evaluate the in vivo antitumor ability of D2.The results showed the tumor inhibition rate in D2 group could reach 65.9%,which is 3-fold higher than that in 7D12 group.?3?The Fc functions of nanobodies were reconstituted via non-specificity-recruiting domain or multi-recruiting domain.Firstly,two non-specificity-recruiting nanobodies 7D12-ZZ and ZZ-7D12 were constructed.Secondly,the in vitro results showed that both of them could recruit antibodies on EGFR⁺cancer cells,and induced significant cell death.Thirdly,in vivo pharmacokinetic assays showed that the half-lives of 7D12-ZZ and ZZ-7D12 were 25.9and 29.5 h,respectively,significantly higher than that of 7D12.Fourthly,the triple-negative breast cancer xenograft mice model was constructed.The results showed that the tumor weight in 7D12-ZZ and ZZ-7D12 were 10.9%and 5.3%of that in 7D12 group,indicating that both7D12-ZZ and ZZ-7D12 possess high anti-tumor activities.Finally,two multi-recruiting nanobodies 7ZD and Z7D were constructed via SML,the flow cytometry and in vitro cytotoxicity showed that 7ZD and Z7D could recruit higher level of antibodies and induce stronger cytotoxicity than 7D12-ZZ and ZZ-7D12 in the presence of anti-DNP antibodies.?4?The Fc functions of bispecific nanobodies?bs Nbs?were reconstituted via site-specific C-terminal Rhamnose?Rha?modification.Firstly,two bs Nbs 7D12-C7b and C7b-7D12 were constructed through fusion expression,and 7D12-C7b was selected due to the higher affinity in flow cytometry evaluation.Secondly,three bs Nb-Rha conjugates R1,R2 and R3 were constructed by using SML.Thirdly,the in vitro results showed that these three conjugates could specifically recruit anti-Rha antibodies on EGFR⁺/HER2⁺cancer cells,and kill them via ADCC/CDC.The results also indicated that R2?with PEG₃ as the linker?had the highest capacity to induce cell death.A431,SKBR3 and A431/HER2 cells treated with 50 n M R2 could cause 26.5%,16.3%and 26.4%cell lysis via ADCC,and,30.1%,23.4%and 33.4%cell lysis via CDC,respectively.