The Study Of A Novel Nanobody-based Bispecific T-cell Engager In Treating PD-L1 Overexpression Melanoma

Purpose:In the tumor microenviroment,programmed death ligand-1（PD-L1）caused immune escape by interacting the corresponding receptor PD-1 on T cells,which resulted T cell defunction and exhaustion.Immune checkpoint blockade（ICB）therapy brought a breakthrough in the treatment of various malignant tumors,which blocked PD-1/PD-L1 interaction and reversed immunosuppressive microenvironment.Especially for advanced and unresectable melanoma,melanoma is refractory to most standard systemic treatments.The current ICB antibodies still faced the shortage of low patients’responsibility and economic burden.Hence,this study designed a PD-L1×CD3 bispecific nanobody aimed at blocking PD-L1 and activating CD3 on T cells,as a novel bispecific T-cell engager in PD-L1 overexpression melanoma.Method:（1）By utilizing an artificial intelligence（AI）tool called Alpha-fold2,the binding region between Bi TE PD-L1×CD3 and CD3 domain and the epitope was predicted.From a structural view,this illustrated the accurate binding sites of Bi TE PD-L1×CD3 and CD3.（2）Bi TE PD-L1×CD3,anti-PD-L1 Nb and anti-CD3 Nb were expressed by E.coli.Then they were purified and characterized.（3）The PD-1/PD-L1 blockade effect of Bi TE PD-L1×CD3 was quantified by the PD-1/PD-L1 Blockade Bioassay.The evaluation of activation on T cells was achieved by detecting two cytokines TNF-αand IFN-γin the supernatant.A PD-L1stable expression melanoma cell line A375^PDL1was constructed to explore the relationship between PD-L1 expression level and cytotoxic effect and therapeutic efficiency.（4）A humanized melanoma mouse model was constructed to explore the in vivo therapeutic effect of Bi TE PD-L1×CD3.The immunofluorescence result of tumor tissue sections illustrated that Bi TE PD-L1×CD3 could activate T cells inside the tumor.The H&E result of five organs proved the in vivo biosafety of Bi TE PD-L1×CD3.Results:（1）The result of homology modelling and molecular docking showed that Asp31in the CDR1 region and Lys104 in the F-G loop region formed a stable salt bridge.Trp52A in CDR2 formed a hydrogen bond with Tyr99 in CD3-ε.And the CDR3region Trp100 formed a hydrogen bond with Gly54 of the B-C loop.According to the predicted binding sites,the binding mode of Bi TE PD-L1×CD3 to CD3εshared the same binding region as the reported UCHT1-sc Fv antibody,indicating its similar activation on CD3.（2）Bi TE PD-L1×CD3,anti-PD-L1 Nb and anti-CD3 Nb were expressed successfully by the strain BL21（DE3）and the vector p Cold II via low temperature induction.Three nanobodies were purified by affinity chromatography and characterized by SDS-PAGE and Western Blot.（3）The result of PD-1/PD-L1 Blockade Bioassay showed that the IC₅₀value of Bi TE PD-L1×CD3 Nb was 4.208g/m L lower than Nivolumab（1.212g/m L）and the cetuximab as the negative control showed no effect.A PD-L1 overexpression melanoma cell line A375^PD-L1was constructed successfully.The cytotoxic effect of Bi TE PD-L1×CD3 on A375^PD-L1was 1.2-2 fold higher than A375^WTin a dose-dependent and PD-L1 expression level-dependent manner.（4）After the treatment in the humanized melanoma mouse model,the inhibition tumor growth curve and the tumor tissue sections from three experimental groups showed Bi TE PD-L1×CD3 had a better therapeutic effect than Durvalumab（the positive control）.Meanwhile,the expression level of anti-Human CD69 on the tumor tissue sections was compared and found that all three experimental groups showed different expression levels.The CD69 expression showed a higher level in Bi TE PD-L1×CD3 group,which illustrated the activation of intratumoral T cells.By comparison of mice weight and appearance and H&E result of different organs,the result confoirmed Bi TE PD-L1×CD3 had a very good in vivo biosafety.Conclusion:This study constructed a PD-L1×CD3 nanobody as a novel Bi TE aimed at blocking PD-L1 and activating T cells for PD-L1 overexpression melanoma.The result of homology modeling and molecular docking based on Alpha-fold2 predicted that the epitope of Bi TE PD-L1×CD3 similar to the reported UCHT1-sc Fv antibody.This study identified that Bi TE PD-L1×CD3 could activate T cell and disrupt PD-1/PD-L1 engagement both in vitro and in vivo.Furthermore,the cytotoxic activity of Bi TE PD-L1×CD3 on A375 was related to PD-L1 expression level amd administration dose.In melanoma mice models,the treatment result showed that Bi TE PD-L1×CD3 had a better therapeutic effect than Durvalumab（the positive control）.,which indicated that Bi TE PD-L1×CD3 was superior to anti-PD-L1antibody.Bi TE PD-L1×CD3 also showed a very good in vivo biosafety.