Identification And Characterization Of Nanobodies Binding With Programmed Death 1

Tumor immune escape is a major obstacle to anti-tumor immunotherapy.In recent years,the molecular of immune checkpoints,programmed death 1（PD-1）and its ligands,PD-L1 and PD-L2,play a key role in suppressing tumor immune responses.PD-1 is mainly expressed on the surface of activated T lymphocytes and tumor-infiltrating lymphocytes.A variety of tumor cells up-regulated expression of PD-L1,in order to escape from the immune surveillance system.Blocking the interaction of PD-1 and its ligands has a significant clinical therapeutic effect.By the end of 2017,the number of PD-1/PD-L1 inhibitory antibody drugs that have been approved by the FDA,has reached 7,mainly for melanoma,non-small cell lung cancer and renal cell carcinoma and other type of tumors.Although the antibody treatment for PD-1:PD-L1 pathway has achieved good results,the unsatisfactory treatment results and high treatment costs are problems that we have not yet resolved.Therefore,it is necessary to develop a novel therapeutic agent against the immune checkpoint PD-1:PD-L1.At the same time,the Fc fragment of the antibody plays a different role in the anti-tumor effect of PD-1 and PD-L1 antibodies.Recent studies have found that the role of the PD-L1 antibody depends on the Fc-mediated ADCC,whereas the PD-1antibody does not act on its Fc,and the anti-tumor activity of the PD-1 antibody containing Fc is inhibited.This indicates that the currently applied PD-1 antibody containing Fc fragment is not the most ideal molecular form.Moreover,due to the poor tissue accessibility and poor ability to penetrate tumor tissues,the effect of therapeutic effects is affected.Therefore,it is necessary to develop novel antibody drugs against PD-1.Nanobodies are small-molecular,single-domain antibodies obtained by genetic engineering techniques from camel heavy-chain antibody variable region genes.It has special advantages such as strong tumor penetration ability,more accessible antigenic epitopes,lower production cost,and stronger stability.At the same time,the nanobody itself does not contain an Fc fragment,and these properties are very suitable for the development of novel therapeutic antibodies against PD-1.Based on this,the main purpose of the present study is to obtain a nanobody that binds to PD-1 by phage display technology,and to study its binding specificity and stability,thus providing an experimental basis for the further development of a therapeutically effective nanobody.The content of this study is divided into the following three parts:1.Construction of anti-PD-1 phage antibody display libraryRecombinant PD-1 protein was used to immunize a Xinjiang bactrian camel.Peripheral blood was collected after six immunizations to obtain lymphocytes,then total RNA in lymphocytes was extracted,and cDNA was obtained by reverse transcription and then amplified by two rounds of nested PCR using specific primers.A VHH fragment with a molecular weight of approximately 500 bp was obtained.The obtained VHH fragment was constructed on a phage vector,and then the ligated product was electrotransformed into the E.coli TG1 competent state.The library capacity was calculated the next day based on the number of transformants grown,and the PCR positive rate of the transformants randomly selected overnight was selected.Then sent to the sequencing company to identify the sequence diversity.The results showed that the VHH phage display library with a library size of2.6×10⁸ cfu/m L was successfully constructed with high diversity（up to 100%）.2.Screening and preparation of anti-PD-1 nanobodiesA solid-phase affinity screening technique was used to screen the PD-l phage antibody display library for three rounds of affinity and positive clones were identified using a soluble monoclonal ELISA.The 46 positive clones identified were sent to the company for sequencing.Based on the sequence analysis results after sequencing,three sequences with higher sequence enrichment were selected,named VHH-B7,VHH-H5 and VHH-H12,and then it was subcloned from the pMECS vector onto the pET22b vector to induce expression of the nanobody.A large amount of nanobody was purified by affinity chromatography.The results showed that three anti-PD-1 nanobodies B7,H5 and H12 were successfully prepared.And the three kinds of nanobodies have high soluble expression and reach to 50 mg/L.3.Preliminary analysis of antigen binding and stability of anti-PD-1 nanobodiesThe binding activity of 3 nanobodies and its antigen PD-1 protein was detected by ELISA.The data processing software Excel,Graphpad prism 5 were used to draw the curve,and the affinity constant K_A was calculated after the curve formula was simulated.The ELISA method was used to measure the activity of nanobodies after treatment with temperature,acid and protease,and then the thermal stability,acid stability and protease hydrolysis stability of the three nanobodies were evaluated.The results showed that the three selected nanobodies had binding activity to antigens with high affinity,and the affinity constants were:K_A=1.19×10¹¹ L/mol,K_A=1.63×10¹¹ L/mol and K_A=1.59×10¹¹ L/mol.In the determination of stability,the three nanobodies have high thermal stability,and can retain more than half of the activity in a water bath at 80°C for 2 hours;the binding activity after 2.5 hours at37°C in an acidic condition of pH2.0.Basically maintained at a normal level,strong acid resistance;After 1 h of pepsin hydrolysis treatment,the 3 nanobodies still have a certain degree of activity,with a certain ability to pepsin hydrolysis.In the hydrolysis experiment of pepsin,there was a buffer-free protease treatment group.The results showed that the activity of the 3 kinds of nanobodies was not significantly different from the untreated control group（P>0.05）,and the antigen-binding activity of the antibody was not substantially affected.In the determination of storage stability,the 3nanobodies still had higher antigen binding activity at 4°C after 60 days of dark treatment,and H5 antibody still had more than 50%antigen binding activity after 120days of treatment.This indicates that the 3 anti-PD-1 nanobodies screened in this study have high thermal stability and high research and application potential.In summary,the anti-PD-1 VHH phage antibody display library was successfully constructed and a high-affinity multi-strain anti-PD-1 nanobody was screened from the library,and preliminary identification of these nano-antibodies was performed.It lays the foundation for the subsequent screening of anti-PD-1 nanobodies and the development and preparation of new PD-1 targeted drugs.