Anti-human pd1 fully human antibody and use thereof

Abstract

The application discloses an anti-human PD1 full human antibody and application thereof, and relates to the field of antibodies, and specifically discloses an anti-human PD1 full human antibody, which comprises three heavy chain variable regions HCDR and three light chain variable regions LCDR; the three heavy chain variable regions comprise: HCDR1 is an amino acid sequence as shown in SEQ ID NO. 4, HCDR2 is an amino acid sequence as shown in SEQ ID NO. 5, and HCDR3 is an amino acid sequence as shown in SEQ ID NO. 6; and the three light chain variable regions comprise: LCDR1 is an amino acid sequence as shown in SEQ ID NO. 1, LCDR2 is an amino acid sequence as shown in SEQ ID NO. 2, and LCDR3 is an amino acid sequence as shown in SEQ ID NO. 3. The application has reduced immunogenicity, and affinity maturation and activity are greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an anti-human PD1 fully human antibody and application thereof. BACKGROUND

[0002] The formation of tumor growth microenvironment not only depends on some cytokines secreted by itself, but also can escape immune surveillance and regulation through certain signal pathways, so as to make the body immune tolerant to tumor cells, and further lead to rapid progression of tumor.

[0003] PD1 is a type I transmembrane glycoprotein of immunoglobulin superfamily, a member of CD28 superfamily and a therapeutic antibody, which is an immunosuppressive molecule. It was first cloned from the hybridoma 2B4.11 of apoptotic mouse T cells and discovered by Professor Yasutomi in Kyoto University in 1992. PD1 protein is expressed in activated T cells, B cells and the like. Compared with other members of the CD28 superfamily, PD1 has more extensive expression, which may indicate that its role in immune response is more complex than other members of the CD28 superfamily. The most remarkable feature of PD1 molecule is that the tail of the cytoplasmic region contains two tyrosine residues, which are involved in the formation of immunoreceptor tyrosine-based inhibitory motifs (ITIM) and immunoreceptor tyrosine-based switch motifs (ITSM) domains. ITIM can restore the phosphorylation of the cytoplasmic segment and play an antagonistic role in the function of antigen receptors, and the tyrosine residues on ITSM are essential for the negative regulation of PD1.

[0004] Previous PD1 antibodies (including drugs such as keytruda which have been marketed) are mainly hybridoma humanized antibodies, and the activity is low, and there is basically no fully human PD1 antibody. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide an anti-human PD1 fully human antibody.

[0006] To achieve the purpose of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides an anti-human PD1 fully human antibody, characterized in that it comprises three heavy chain variable regions HCDRs and three light chain variable regions LCDRs; the three heavy chain variable regions comprise: HCDR1 is the amino acid sequence shown in SEQ ID NO. 3, HCDR2 is the amino acid sequence shown in SEQ ID NO. 4, and HCDR3 is the amino acid sequence shown in SEQ ID NO. 5; the three light chain variable regions LCDRs are as follows: LCDR1 is the amino acid sequence shown in SEQ ID NO. 1, the amino acid sequence of LCDR2 is RAS, and LCDR3 is the amino acid sequence shown in SEQ ID NO. 2.

[0008] In a second aspect, the present application provides a nucleic acid molecule encoding the anti-human PD1 fully human antibody of the first aspect.

[0009] In a third aspect, the present application provides an expression vector, characterized in that the expression vector contains the nucleic acid molecule of the second aspect.

[0010] In a fourth aspect, the present application provides a host cell, characterized in that the host cell contains at least one copy of the expression vector of the third aspect.

[0011] In a fifth aspect, the present application provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the anti-human PD1 fully human antibody of the first aspect, and a pharmaceutically acceptable carrier and / or diluent.

[0012] In a sixth aspect, the present application provides the use of any one or at least two of the anti-human PD1 fully human antibody of the first aspect, the host cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for preventing, alleviating, improving or inhibiting a disease or disorder.

[0013] Preferably, the disease or disorder is selected from the group consisting of cancer and / or autoimmune disease.

[0014] The positive progress effect of the present application is that the present application reduces immunogenicity while affinity maturation, and the activity is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure is a schematic diagram of the panning output set for the His tag antigen detection of the present application.

[0016] Figure 2 The figure is a schematic diagram of the supernatant ranking of the candidate molecules of the present application.

[0017] Figure 3 The figure is a schematic diagram of the SDS-PAGE protein purity identification of the present application.

[0018] Figure 4 Typical SEC-HPLC profile for screening molecules of the present application.

[0019] Figure 5 ELISA assay for antibody binding activity to antigen of the present application.

[0020] Figure 6 FACS assay for antibody binding activity to cells of the present application.

[0021] Figure 7 FACS assay for antibody competition activity of the present application.

[0022] Figure 8 Results of chemiluminescence experiment of the present application. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application.

[0024] The preparation method of the anti-human PD1 fully human antibody of the present application comprises the following steps: constructing a gene library of human antibodies; 15 mL of Ficoll-Paque density gradient separation liquid (purchased from GE Company, catalog number: 17144003S) is slowly added into a 50 mL centrifuge tube. The centrifuge tube is tilted and 15 mL of collected normal human blood is slowly added along the tube wall to maintain a clear separation interface between the Ficoll-Paque density gradient separation liquid and the normal human blood. The 50 mL centrifuge tube containing the blood and the separation liquid is centrifuged at about 15°C for 20 min, wherein the centrifuge is set to 400g, and the acceleration is 3 and the deceleration is 0. After centrifugation, the whole liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, the middle layer is Ficoll-Paque liquid, and there is a narrow band of white cloud layer mainly composed of PBMC at the junction of the upper and middle layers, i.e. the PBMC cell layer. The plasma mixture in the upper layer is carefully removed with a sterile Pasteur pipette, and then a new sterile Pasteur pipette is used to collect the PBMC to obtain separated PBMC. The separated PBMC is first washed with PBS twice, then centrifuged at 1500 rpm for 10 min at 4°C, and finally resuspended with 1.5 mL of PBS and counted by a cell counter (CountStar, CountStar Altair). The total RNA is extracted from the separated PBMC cells by a conventional method. The extracted total RNA is reverse transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa Company, catalog number: 6210A). Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers are designed at the front end of the V region and the rear end of the first constant region of the heavy chain and light chain, respectively, and the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody are obtained after PCR. After recovering the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody, a fragment containing the light and heavy chain variable regions of the antibody is amplified by fusion PCR method, and then the PCR product and the phage display vector are subjected to enzyme digestion, recovery and ligation. The ligation product is recovered by a recovery kit (Omega, catalog number: D6492-02). Finally, the transformed Escherichia coli SS320 (Lucigen, MC1061 F) is transformed into the competent Escherichia coli SS320 (Lucigen, MC1061 F) by an electrotransformation instrument (Bio-Rad, MicroPulser), and the transformed Escherichia coli SS320 bacterial liquid is coated on a 2-YT solid plate (the solid plate is prepared by 1.5% tryptone, 1% yeast extract, 0.5% NaCl, 1.5% agar, and the mass volume g / mL is prepared) with ampicillin resistance.

[0025] The present application uses biotinylated PD-1-Fc fusion protein to screen the full human antibody phage display library to obtain the Fab antibody fragment of anti-PD-1 which is displayed on the phage and specifically binds to human PD-1. The specific screening method used in the present application is as follows: the magnetic bead method is used to screen the antibody library, before each round of screening of the full human antibody phage display library using biotinylated PD-1-Fc fusion protein, the phage binding to Fc is removed by screening the antibody library using CD40-Fc-biotin, and then the phage monoclonal specifically binding to human PD-1 protein is screened using biotin-labeled PD-1-Fc fusion protein. After washing to remove the nonspecifically adsorbed phage, the obtained phage is amplified using E. coli SS320 cells and then used for the next round of screening. In each round of screening, the ratio of input phage to output phage is calculated and used as an index of enrichment of PD-1 specific phage.

[0026] The enrichment of each library to PD-1 is also confirmed by phage library ELISA, in which the phage binding to PD-1 is highly enriched after three rounds of screening.

[0027] The screened phage Fab is tested for binding to PD-1-Fc-biotin and IL23-Fc-biotin. The phage clone showing affinity to only PD-1-Fc-biotin is selected, DNA is extracted and sequenced.

[0028] A phage clone B10 containing a unique Fab sequence is obtained, which encodes the amino acid sequence as shown below.

[0029] The amino acid sequence encoding the light chain variable region of B10 Fab is as follows:

[0030] EIVLTQSPATLSLSPGERATLSCRASQSVQSYLAWYQQKPGQAPRLLIYRASRRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQYNEFSLTFGQGTKLEIK

[0031] The amino acid sequence encoding the heavy chain variable region of B10 Fab is as follows:

[0032] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSAISGGGRYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVRFGESWFSYWGQGTTVTVSS

[0033] The anti-human PD1 full human antibody includes three heavy chain variable regions HCDR and three light chain variable regions LCDR; the three heavy chain variable regions include: HCDR1 is the amino acid sequence shown in SEQ ID NO. 3, HCDR2 is the amino acid sequence shown in SEQ ID NO. 4, and HCDR3 is the amino acid sequence shown in SEQ ID NO. 5; the three light chain variable regions include: LCDR1 is the amino acid sequence shown in SEQ ID NO. 1, LCDR2 is the amino acid sequence shown in RAS, and LCDR3 is the amino acid sequence shown in SEQ ID NO. 2. The specific sequence information is as follows: SEQ ID NO. 1: SQSVQSY. SEQ ID NO. 2: LQYNEFSLT. SEQ ID NO. 3: GFTFSSYD. SEQ ID NO. 4: ISGGGRYT. SEQ ID NO. 5: AVRFGESWFSY.

[0034] The nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region of the phage B10 clone are respectively connected into the pTT5 vector, and expressed and purified in the form of complete antibody in expi293F cells (Thermo Fisher). The Fab candidate molecules are subjected to specific affinity detection by the ELISA method.

[0035] Construction of affinity matured antibody library

[0036] The amino acid sequence of the antibody Fab is provided, and a phage display vector is constructed into a plasmid, and the expression activity is verified. The plasmid is used as a template to perform multi-point saturation mutation, and then the antibody gene is recombined by the method of in vitro ligation, and finally the recombined antibody Fab gene sequence is inserted into the pAM vector, and then a mutated antibody library is obtained. Sequencing and sequence analysis verification, as shown in Table 1, four affinity matured antibody libraries (AM570, AM571, AM572, AM573) are constructed, the fragment size of the amplified gene is consistent with the designed size. The actual library capacity of the electric transformation is greater than the theoretical library capacity. Compared with the parent amino acid, the mutation amino acid distribution of each library is relatively uniform, which meets the requirements.

[0037] Table 1 Library sequencing result statistics table

[0038]

[0039] Screening of antibody affinity matured antibody library

[0040] Using an immunograft screening method, mutants with significantly enhanced affinity compared to the parent antibody were selected from four qualified antibody mutant libraries. This process included the following four parts: 1.1 Phage titer determination in the initial screening: Phages were selected using trypsin elution. Statistical analysis of the input and output of each screening round showed varying degrees of enrichment across different screening methods. 1.2 Enrichment detection of the selected collection library: The selected collection was prepared into a supernatant, such as... Figure 1 As shown, the binding activity with the PD-1-His antigen was detected by ELISA, and the antibody concentration in the supernatant was then determined using a standard IgG Mix. The correlation between titer and signal was calculated using software to determine the enrichment status. 1.3, Sequencing Data Analysis Results: Candidate clones from the initial monoclonal screening underwent sequencing analysis to remove repetitive and problematic sequences. Four representative sequences were selected. (See figure...) Figure 2 As shown, the four positive clones were sorted by affinity. 1.4, Clone Sequencing and Construction: Based on the sorting and sequence comparison, and after communicating with the clones, sequences 1 and 26 were selected as templates, and four additional mutants were designed, resulting in a total of seven clones.

[0041] Candidate molecule detection specifically includes the following six parts:

[0042] 2.1 SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis): Based on the selected candidate molecule sequences, the molecules were constructed and expressed in the ExpiCHOs expression system. After cell expansion and cis-transformation, the cell supernatant was collected, and antibody purification was performed according to standard protein purification procedures. Figure 3 As shown in the figure, the experimental results indicate that the purified antibody has a purity greater than 95%.

[0043] 2.2 SEC verification: such as Figure 4 As shown, the chromatographic column was an XBridge BEH200A, SEC 3.5 µm, 7.8 × 300 mm; the mobile phase was 0.15 M PB, pH 7.4; the flow rate was 0.5 mL / min; the sample loading volume was 20 μL; and the detector parameters were: detection wavelength 280 nm, bandwidth 16 nm, reference wavelength 360 nm, bandwidth 100 nm, peak width (response time) > 0.1 min (2 s); slit width 4 nm; and negative absorbance baseline 100 mAU. After the chromatographic system stabilized, the sample was centrifuged at 12000 rpm at 4℃, and the sample solution was accurately measured and injected into the liquid chromatograph. The purity of the sample was calculated using the peak area normalization method, as shown in Table 2.

[0044] Table 2 Statistical Table of Full-Length Antibody SEC Results

[0045]

[0046] 2.3 Affinity kinetics assay: Open the GATOR instrument and related software, select the Kinetics experiment mode. The affinity of two antibodies (AE-01 and AE-23) was improved by more than two times, as shown in Table 3.

[0047] Table 3 Summary of antibody affinity kinetics

[0048]

[0049] 2.4 ELISA method for detecting antibody binding activity to PD-1-his: By ELISA method, the test plate was coated with 1.0 μg / ml of PD-1-his, a sample concentration gradient was set, and the binding activity OD value was measured to detect the binding affinity of the antibody to the target antigen. As shown in Table 4, the data showed that the series of antibodies had strong binding to the antigen, the curve had clear upper and lower platforms, the window was large, and the activity was good. Figure 5

[0050] 2.5 FACS method for detecting antibody binding activity to CHO-hPD-1 cells: By FACS method, CHO-hPD-1 cells were evenly spread in a 96-well cell plate, all sample concentration gradients were set, and the cells were combined, then 3 μg / ml of Alex488 labeled goat anti-mouse secondary antibody / Alex488 labeled goat anti-human secondary antibody was added to each well, and the binding activity OD value was measured after binding to detect the binding strength of the series of antibodies to CHO-hPD1 cells. As shown in Table 5, the data showed that the series of antibodies all had binding to CHO-hPD1 cells, and the AE-01 antibody had the strongest binding. Figure 6

[0051] 2.6 FACS method for detecting the competition activity of the series of antibodies to PD-L1-mFc: By FACS method, CHO-hPD-1 cells were evenly spread in a 96-well cell plate, a constant concentration of PD-L1-mFc and a concentration gradient of the sample to be tested were set, and the cells were combined, then 3 μg / ml of Alex488 labeled goat anti-mouse / Alex488 labeled goat anti-human secondary antibody was added to each well, and the competition activity OD value was measured after binding to detect the competition strength of the series of antibodies to PD-L1-mFc. As shown in Table 6, the data showed that the competition strength of the series of antibodies was relatively strong. Figure 7

[0052] Luciferase experiment

[0053] ​​​The cells CD3L-PD-L1-CHO, huPD-1-NF-AT Jurkat were mixed at a total volume of 50 μl / well in a 96-well plate, wherein the final density of the cells CD3L-PD-L1-CHO was 2E4 (cell / well) and the final density of the cells huPD-1-NF-AT Jurkat was 1E5 (cell / well). After the test antibody was diluted to a certain concentration, it was added to the 96-well cell plate and incubated with the cells. After the incubation, the 96-well cell culture plate was placed in a fluorescence chemiluminescence analyzer to read the chemiluminescence value. The data were analyzed by four-parameter curve fitting using Graphpad Prism software to obtain the EC50 value. As shown in the data Figure 8 The data show that the curve of the humanized antibody has a clear upper and lower platform, a large window, and good activity.

[0054] In addition, it should be understood that although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description of the specification is only for the sake of clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A fully human antibody against human PD1, characterized in that, It includes three heavy chain variable regions (HCDRs) and three light chain variable regions (LCDRs); the three heavy chain variable regions (HCDRs) are as follows: HCDR1 is the amino acid sequence shown in SEQ ID NO.3, HCDR2 is the amino acid sequence shown in SEQ ID NO.4, and HCDR3 is the amino acid sequence shown in SEQ ID NO.5; the three light chain variable regions (LCDRs) are as follows: LCDR1 is the amino acid sequence shown in SEQ ID NO.1, LCDR2 is RAS, and LCDR3 is the amino acid sequence shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fully human anti-human PD1 antibody as described in claim 1.

3. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains at least one copy of the expression vector of claim 3.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the fully human anti-PD1 antibody of claim 1, and a pharmaceutically acceptable carrier and / or diluent.

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