Selection Novel Anti-human PD-1 Antibody From The Targeted Epitope Mammalian Cell Displayed Antibody Library

Antibody drugs play important roles in biological pharmaceutical industries, and they have been broadly used from the initial anti-transplant rejection to the therapies of cancer, autoimmune diseases, infections, cardiovascular/cerebrovascular diseases, ophthalmic diseases and bio-security prevention and control. The development of therapeutic antibodies experienced four stages: polyclonal antiserum, murine monoclonal antibodies, humanized antibodies and fully human antibodies. Humanized and fully human antibodies have become the trends of antibody drugs. Among the fully human antibody preparation techniques, such as, human-human hybridoma technology, transgenic mice, antibody library technology and EB virus transformed human B lymphocytes, the antibody library technology is one of the most commonly used one.Phage antibody library technology is the most widely used technology of antibody development at present. Adalimumab(Humira) is the first approved human monoclonal antibody drugs screened from this technology platform, and it has been the world’s best-selling drug for several years(2012-2015).However, compared with widely used mammalian cell expression system for preparing the antibody on the large scale, the prokaryotic expression system used by phage lacks the processes of precise protein folding and modification; the antibodies screened from it would not have the conformational and post-translational modifications of the natural antibodies. Therefore, the use of mammalian expression system to display the antibodies with natural conformations and post translational modifications will have great practical value and prospect. However, due to the transfection efficiencies, culture system and other limitations, the capacity of the mammalian cell antibody library is small(about 106), which limits its wide application. But in recent years, computer-aided molecular design has been an promising technology to optimize antibody candidates. Through targeting specific epitopes, computer aided molecular design helps to improve the proportion of functional antibodies and the affinities of them to a certain extent. Therefore, the combination of mammalian cells display system and computer aided molecular design is helpful to obtain antibody library with high quality within limited storage capacity and will be helpful to improve antibody library quality and screening efficiency.Immunotherapy of cancer have shown good treatment effects after experiencing a long period of exploration and major setbacks, and they have been expected to change the strategies of currentcancer treatments. The successful uses of the monoclonal antibodies targeting the “immune-checkpoint molecules” such as CTLA-4 and PD-1/PD-L1 or antibody structure-based CART technique, which lay the foundations of antibody in the field of cancer immunotherapy. Programmed death 1 receptor(PD-1) is mainly expressed in activated T cells and B cells, and it negatively regulates the immune response(also known as an "immune checkpoint molecule"): it binds to the PD-L1 and PD-L2 ligands on the surface of NK cells and other cells, thus activates the negative regulatory signals and leads to the suppression of the hyperactivation of the immune cells.Studies have shown that PD-L1 are highly expressed on the surface of many kinds of tumor cells and the immune tolerance is formed through the PD-1/PD-L1 signaling pathway, which contributed tumor evading the body’s immune responses. Specifically blocking PD-1/PD-L1 signaling pathway can effectively activate the immune system and kill the tumor cells. Therefore, PD-1/PD-L1 has become a hot target for tumor immunotherapy. The foreign pharmaceutical giants have launched a fierce competition for the development of PD-1 monoclonal antibody drug. Among them, Nivolumab developed by BMS has been approved by FDA for the clinical treatments of malignant melanoma, squamous non-small cell lung cancer, renal cell carcinoma. In addition to the use of a single drug, the clinical trials which evaluate the synergistic use of Nivolumab and other targeted drug are undergoing and good progress has been made. However, there are currently only two domestic companies which get clinical approval for the PD-1 antibody. Therefore, it is of great theoretical and practical significance to screen the novel PD-1 antibodies with independent intellectual property rights.In this paper, we construct the mammalian cell antibody displaying system and get the PD-1 targeted antibody library through computer-aided design. Candidate antibodies are screened through high throughput flow cytometry; they are further evaluated in in vitro and in vivo models for the efficacies. The results are as follows:1.Construction and validation of mammalian cell display systemTo realize the basic principles for antibody library selection, such as antibody displaying on mammalian cell surface, expressing single antibody gene in single cells, we constructed a vector(name as “PFRT/KIg G1 Fab HTM”)for expressing Fab domain on the mammalian cell membrane based on sitespecific integration system FRT-Flp-In?. At the same time, we have constructed the full-length antibody expression vector PRT/KIg G1, which could be used for antibody candidate expression screened from the antibody library. Flow cytometry and confocal laser scanning results showed that PFRT/KIg G1 Fab HTM was able to display Fab antibody on the surface of mammalian cell membranes. The transient expressions of several antibodies, such as anti-Ebola virus antibody, anti-West Nile virus antibody, anti-PD-1antibodies and anti-CD3 antibody, show that each antibody in the full-length antibody expression vector PRT/KIg G1 could obtain the expression levels to 100μg/m L. The cells that stably expressed the anti-PD-1 antibody and anti-CD3 antibody sequences were constructed, and the expression level of the antibody was up to 1.2g/L after 14 days fed-batch cultivation. These results suggest that the PRT/KIg G1 vector is a universal and efficient expression vector for the expression of full-length antibody. We further use red and green fluorescence protein genes to verify whether single cell could express single antibody gene in the FRT-Flp-In system.The results showed that single fluorescent protein gene are expressed in one signal cell in the stably transfected FRT-Flp-In? system, which indicate that the FRT-Flp-In? site-specific integration system can achieve the aim that “one gene one cell”. We expressed the Fab structure of PD-1 antibody MIL75 onto the CHO cell surface by using the mammalian surface display system and flow cytometry results showed that PD-1-FITC bind to the cells in a dose dependent way, which suggests that FACS could be used to achieve high throughput screening in this displaying system.2. Construction and screening of PD-1 targeted antibody libraryBased on molecular modeling and the docking the spatial structure of PD-1 and PD-L1 complexes were built. The key amino acid residues of PD-L1 in recognizing PD-1 molecules were identified according to the interaction between PD-L1 and PD-1. Using the subgroup of light and heavy chain variable region of Nivolumab antibody sequence(Ig KV3-11*01, Ig HV3-33*01) as the template. The accessible surface area of CDR involving interaction with antigen was replaced with similar amino acid mutations. The targeted antibody library was constructed resembles the recognition mode of PD-1/PDL1. Through mammalian cells platform, anti-PD-1 targeted antibody library was constructed, Results from transformant count and the second generation high-throughput sequencing showed that the antibody library covers the most theoretical diversity of the light chain and heavy chain sequence(sequences diversity of measured vs sequences diversity of theoretical design, light chain 96/96; heavy chain 7321/8640). 3 rounds of FACS are further used for the screening and enrichment of constructed cell library. The positive clones were obtained and sequenced. After sequence alignment and analysis, 120 antibody sequences were divided into 10 groups according to sequence homology and backbone carbon atom RMS displacement and 13 representative sequences were selected from them. 13 representative full-length antibodies were expressed and the expression results show that the 13 m Abs had high expression levels(15.2 μg/m L ~ 74.5 μg/m L). among these, the expressions of FV63(59 μg/m L) and FV78(74.5 μg/m L) were significantly higher than that of the control antibody MIL75(28.7 μg/m L). 5antibodies of higher affinity were further obtained from the specific binding affinity assays(1-4×10-10M). we evaluated the stabilities of space structure, the molecular space of PD-1 epitope and other parameters through bioinformatics, structural biology and computer aided molecular simulation technology. Eventually, two strains with higher functional activity, FV74 and FV78 were identified. results from Preliminary functional evaluation show that the expression levels of FV78 is much higher than the marketed antibody-MIL75.3.Evaluation of biological activity of new antibody FV78 targeting PD-1We further evaluated the functions of the FV78 antibody in vivo and in vitro. The results showed that the FV78 specifically recognized human PD-1, and did not interact with other members of the CD28 family(CD28, CTLA4 and BTLA and ICOS); competitive ELISA results suggest that FV78 can effectively block the interaction between PD-1 and PD-L1/PD-L2, and the EC 50 is 9.9 ug / ml. FV78 antibody could effectively activated T cells to release IFN gamma in PBMC or DC-CD4+T cells co-culture system. The graft versus host disease(GVHD) model was constructed using PBMC Intravenous infused into deficiency mice NPG for evaluating the immune-activity of the anti PD-1 antibody. The results showed that the antibody FV78 can effectively activate the immune system, aggravate the reaction in GVHD, and accelerate the death of mice(median survival time(MST), FV78 vs N.S=41 days vs 36 days, P < 0.05). Compared with the positive control group MIL75, antibody FV78 has a little superiority(MST, FV78 vs MIL75=41 days vs 39 days, P = 0.08). In vivo and in vitro results indicated that the new antibody FV78 was not weaker than the positive antibody MIL75.