Albumin Fusion Protein-based Nano-adaptor For Antibodies Delivery

Immune checkpoint inhibitors have been approved for the treatment of various types of tumors,and obtained phased achievements.Continuous and in-depth clinical research shows that the therapeutic effect of immune checkpoint blockade therapy in different patients are quite different,and the clinical response rate is generally low.Combined immune checkpoint immunotherapy or bispecific/multi-specific antibody can improve the therapeutic effect on tumors effectively.In the previous research of our group,two kinds of antibodies are immobilized on the surface of nanoparticles to achieve the function of bispecific antibodies.On this basis,this research proposed the nanoparticle assembled from albumin fusion protein and hydrophobic polymer polylactic acid（PLA）,which can be used for delivering antibodies efficiently.The fusion protein is composed by Fcγreceptor I（FcγRI）and serum albumin（SA）,in which FcγRI can specifically bind to the Fc domain of antibody,and SA can bind to hydrophobic PLA.The nanoparticle,named nano-adaptor,can bind various antibodies efficiently and conveniently for antibodies delivery that to enhance the efficacy of immunotherapy.This dissertation is mainly described in three parts as below:1.The recombinant strain Mut⁺GS115-p PICZαA-m FcγRI-MSA has been constructed by pichia pastoris protein expression system,which can express and secrete m FcγRI-MSA fusion protein efficiently.The m FcγRI-MSA fusion protein with a yield of more than 300 mg/L and a purity of more than 98%was purified from the culture supernatant,and it has the complete protein structure.We confirmed that it has similar affinity with the recombinant truncated m FcγRI,and it can bind with therapeutic antibodies with specificity and high efficiency.The affinity constant of m FcγRI-MSA is 2.92×10^-8 M.It has similar affinity with different therapeutic antibodies of the same subtype.2.The fusion protein nano-adaptor NP_{m FcγRI-MSA}has been successfully constructed with m FcγRI-MSA and PLA.At the mass ratio of m FcγRI-MSA and PLA_130K was 5:1,we have obtained stable nanoparticles which can bind antibodies efficiency.The optimal ratio of fusion protein nano-adaptor and monoclonal antibodies was 10:1,and the fusion protein nano-adaptor has the ability to bind antibodies efficiently.Finally,it is confirmed that the universal antibody delivery platform NP_{m FcγRI-MSA} has good stability before and after binding antibodies.The fusion protein nano-adaptor may realize the universality of antibody delivery platform for screening antibody combinations targeting different tumor diseases.3.The fusion protein nano-adaptor bindingαPD-1,αPD-L1 andαNKG2A called trispecific-like antibody(NP_{m FcγRI-MSA@3Ab})has been constructed.At the cellular level,we have demonstrated that trispecific-like antibody can bind to tumor cells and immune cells specifically,and enhancing the interaction between effector cells and target cells,which can strengthen the killing ability of immune cells to tumor cells through immune checkpoint blockade effect.Studies at the animal level have shown that the trispecific-like antibody can enhance enrichment of antibodies in tumor tissue.The three-specific nano-adaptor has showed significant tumor inhibition effect in some mouse tumor models,such as 4T1 breast cancer and B16-F10melanoma,and has no obvious toxic and side effects.Taking advantage of nanoparticles,we design the nano-adaptor based on albumin fusion protein to achieve the combination of multiple antibodies.It has increased the enrichment of antibodies within tumor.The nano-adaptor shows potential to solve the limitations of antibody monotherapy,which can enhance the response rate of antibody treatment.As a universal antibody delivery platform,NP_{m FcγRI-MSA} has good biodegradability and safety,which is expected to accelerate the development and clinical transformation of antibody combination delivery.