Structural Mechanisms Of PD-L1 And CTLA-4 Antibodies Mediated Tumor Immunotherapy

In recent years,antibody drugs that target immuno-checkpoints have achieved great success in the treatment of tumors.Several PD-1,PD-L1 and CTLA-4 antibodies have been approved by FDA for clinical treatment of tumors.However,the structural mechanism of these antibody drugs is not yet clear and some problems such as drug resistance,immune side reactions occured during use.Therefore,we conducted a series of studies on the structure and function of PD-L1 and CTLA-4 antibodies,providing ideas for subsequent antibody optimization and design small molecule drugs.For study the structure and function of PD-L1 antibodies,we first screened the nanobody KN035 targeting PD-L1 by immunizing the camel.The antibody which does not cross-binding with other molecules on the surface of T cells,has high recognition specificity and stable physical and chemical properties.Functional experiments show that KN035 can activate T cells to secrete IFN-γ in vitro and with the ability to inhibit tumor cell proliferation in vivo.To clarify the mechanism of KN035,we solved the crystal structure of the PD-L1/KN035 complex.The structure showed that KN035 mainly used its longer CDR3 region to recognize the front beta-sheet surface of PD-L1 with 7 hydrogen bonds and 2 ionic bonds.Through site-directed mutagenesis and determination of dissociation constant,we identified I54,Y56,E58,Q66,R113 and M115 as hot spot amino acids on the surface of PD-L1.To further compare the difference between KN035 and clinically used antibody drugs,we determinated the crystal structure of Atezolizumab/PD-L1 complexe.This structure shows that Atezolizumab binds PD-L1 mainly through the 3 CDR regions of the heavy chain and LCDR3.The CDR regions of this antibody are rich in aromatic amino acids that form a number of intermolecular π-π and cation-π interactions with PD-L1.By stacking the PD-1/PD-L1,KN035/PD-L1 and Atezolizumab/PD-L1 complex structures,we found that PD-1,KN035 and Atezolizumab bind to similar region of the front beta-sheet surface of PD-L1.For another important immune checkpoint molecule,CTLA-4,we screened nanobody KN044 through a similar technical route.KN044 can not only activate T cells to secrete IFN-γ in vitro but also clear tumor cells in vivo.By solving the crystal structure of the KN044/CTLA-4 complex,we found that all three CDR regions of KN044 were involved in the binding of CTLA-4.As a comparison,we also determinated the crystal structures of Ipilimumab/CTLA-4 complexe.Comparing B7-1/CTLA-4,KN044/CTLA-4 and Ipilimumab/CTLA-4 complex structures,we found that B7-1,KN044 and Ipilimumab bind to the front beta-sheet surface of CTLA-4 IgV domain,indicating that KN044 and Ipilimumab can competitively bind CTLA-4 with B7.At the same time,we identified K95,I93,V46 and L106 on the surface of CTLA-4 as important amino acid residues through site-directed mutagenesis and measurement of dissociation constant.Finally,according to the binding characteristics of KN035 and KN044,a novel bispecific Nanobody that binds both PD-L1 and CTLA-4 was designed by grafting the CDR3 region of KN035 and the CDR1 and CDR2 regions of KN044.The bispecific nanobody has certain binding ability to both PD-L1 and CTLA-4.In summary,We have clarified the molecular mechanisms by which these antibody drugs exert therapeutic effects and identified the key amino acids on the surface of PD-L1 and CTLA-4 molecules,which laid an important structural basis for the development of the next generation of tumor immunological drugs.