Preparation Of E23sFv-humanized Antibody And Its Application In HER2 Targeted Tumor Killing

As a well-documented anti-tumor target,therapeutic strategies targeting HER2(p185^{erb B2/neu})typically involve monoclonal antibodies and single-chain antibody?scFv?-conjugated effectors.The monoclonal antibodies have been widely used in clinic such as Herceptin,which are usually humanized out of parental murine monoclonal antibodies with little immunogenicity-related side effect after long-term use.The scFv-based conjugation is still under preclinical investigation with much more complicated manipulation,among which humanization of murine scFv is a prerequisite for clinical use.In this study,we chose the murine scFv e23 sFv for humanization due to its well-defined HER2 binding ability in our previously released serial reports.The present study aimed at strategy improvement of e23 sFv humanization in order to balance efficiency and diversity,and validation of humanized scFv-based antitumor therapeutics.For humanization strategy,two improvements were made in this study.F irstly,based on traditional engraftment of murine complementarity-determining region?CDR?into human framework?FR?,we extended scope for human FR templates.We selected e23sFv-homologous FR sequences derived from both individual human antibodies and consensus human antibody subclasses,such that comparison of the two origins of FRs could provide structure-function clues.Secondly,in the case of FR engraftment with consensus human antibody subclasses,it is necessary to mutate key FR residues back to the mouse ones in order to maintain the conformation of humanized antibody and avoid loss of affinity.Such mutagenesis usually involves site-directed mutation with antibody structure-based rational design and random mutation with improved binding diversity but much more labor consumption.In this study,we tried to combine site-directed mutation with random mutation.Thirteen FR residuals of consensus human antibody subclasses were chosen as point mutation sites,and in order to enhance diversity of mutants,non-mutated loci of these 13 residues were included as well,followed by random combination of 13 loci with each locus-derived mutant and control,which gave rise to 213 of phage-displayed mutated scFvs.Four humanized scFvs with high affinity were identified after four rounds of affinity panning and phage ELISA screening.As a result,we obtained two candidates,SGF1 and SGF2,which were engrafted to human antibody individual sequences,and four candidates,P1h2,P1h3,P2h2 and P2h5,which were engrafted to human antibody consensus sequence.The structures of e23 sFv,SGF1 and P1h2 were modeled based on sequence homology and each scFv was docked to extra cellular domain?ECD?of HER2.The recognition epitopes of humanized scFvs were predicted to be located in domain IV of HER2 ECD.The main chain carbon atomic conformation of SGF1 is closer to that of e23 sFv in comparison with P1h2.However,P1h2-HER2 interaction was more intensive than SGF1-HER2 interaction.All the humanized scFvs were prokaryotically expressed in inclusion body.The purified and refolded proteins were functionally evaluated as follows.1)Affinity: The affinity of humanized scFvs to human recombinant HER2?hrHER2?was observed by surface plasmon resonance?SPR?.Affinities of P1h2,P1h3 and SGF1 were about 10 times higher than that of e23 sFv.However,affinities of P2h2 and P2h5 were similar to e23 sFv.Cell ELISA verified binding of humanized scFvs to cellular HER2 at the cell surface level,and the results were consistent with SPR.2)Epitopes: Competitive flow cytometry was performed with fluorescein labeled humanized scFvs,and the results showed that humanized scFvs recognize the identical HER2 epitope with e23 sFv.3)Internalization: Confocal microscopy assay showed that P1h2,P1h3 and SGF1 could specifically bind to and enter HER2 positive cells,whereas P2h2,P2h5 and SGF2 could not be internalized under the same conditions.4)Immunogenicity: Cytokine production of PBMCs stimulated with humanized scFvs were detected by ELISPOT and antibody conjugated beads.Production of IFN-? and TNF-? by PBMCs stimulated with humanized scFvs was reduced to 1/20 level of e23 sFv stimulation.It is noteworthy that SGF1,derived from individual human antibody sequence,led to much higher level of cytokines than the ones derived from human consensus sequences,suggesting the relatively stronger immunogenicity of humanized scFvs with individual human sequence origin.In summary,P1h2 and P1h3 were selected for further functional tests due to their low immunogenicity,high affinity and internalization activity.For anti-tumor therapy,we explored the use of P1h2 and P1h3 in both indirect killing via complement-dependent cytotoxicity?CDC?and direct killing via immunoproapoptotic strategy we established before.1)C DC strategy: Human IgG1 Fc was fused to the C terminus of humanized scFvs to generate a series of scFv-Fc fusion proteins.The fusion proteins were eukaryotically expressed and purified by protein A affinity chromatography.Flow cytometry and in vitro CDC assays showed that Fc fusion proteins could bind specifically to HER2-positive tumor cells and cause cell death.The killing capacities of P1h2-Fc and P1h3-Fc were higher than that of e23sFv-Fc in HER2 highly expressing breast cancer cells and HER2 moderate ly expressing lung cancer cells.2)Immunoproapoptotic strategy: Humanized scFvs were used to construct scFv-Fdt-tBid.The fusion proteins were prokaryotically expressed in soluble fractions and purified by Ni-NTA affinity chromatography.Flow cytometry and in vitro cyotoxicity assay showed that P1h2-Fdt-tBid and P1h3-Fdt-tBid specifically inhibited proliferation of HER2-positive tumor cells.In HER2 highly expressing breast cancer cells and HER2 moderately expressing lung cancer cells,P1h2-Fdt-tBid and P1h3-Fdt-tBid showed anti-tumor function comparable to e23sFv-Fdt-tBid.In vivo anti-tumor activities of P1h2-Fdt-tBid and P1h3-Fdt-tBid were confirmed using three tumor models of NOD-SCID mice?orthotopic tumors of breast cancer,orthotopic tumors of gastric cancer,subcutaneous transplants of lung cancer?.Of note,the in vivo anti-tumor effects of P1h2-Fdt-tBid and P1h3-Fdt-t Bid were stronger than that of e23sFv-Fdt-tBid in breast cancer and gastric cancer models,whereas in lung cancer model,there was no difference in anti-tumor effects between humanized scFv-based proapoptotic molecules and e23sFv-Fdt-tBid.In conclusion,the present study optimized the CDR transplantation strategy for antibody humanization,and obtained e23sFv-humanized P1h2 and P1h3 with reduced immunogenicity,improved affinity and internalization activity.The application of the two humanized scFvs in anti-tumor therapy involving CDC strategy and immunoproapoptotic strategy was validated in vitro and in vivo.This study offers a paradigm for humanizing scFv-based therapeutics with regard to FR manipulation strategy and safety-efficacy evaluation.