CN109929036A - A kind of antibody screening method that epitope is special and the antibody screened - Google Patents

Abstract

An epitope-specific antibody screening method to solve the problem of low screening efficiency of functional antibodies with therapeutic effects in the antibody library screening technology, including the steps of using functional epitope peptide screening and live cell screening; also provides the method using this method A fully human anti-PD-1 monoclonal antibody obtained by screening the phage display antibody library, targeting specific functional epitopes, can effectively block the binding of PD-1 to its ligand, and has high affinity and low EC50. It can produce therapeutic effects on animal models at low doses. The invention greatly improves the screening efficiency of functional antibodies, and provides a potential safer and more economical immunotherapy drug for patients with malignant tumors and immune diseases.

Description

Epitope-specific antibody screening method and screened antibody

technical field

The invention belongs to the field of biomedicine, and particularly relates to an epitope-specific antibody screening method and the screened antibody.

Background technique

The "immune escape" of cancer cells is considered to be the main mechanism of tumorigenesis, progression and drug resistance. Tumors can protect themselves from immune system clearance by directly or indirectly suppressing T-cell signaling. Tumor immune checkpoint therapy (immune chenkpoint therapy) is a type of treatment that modulates T cell activity through a series of pathways such as co-suppression or co-stimulatory signaling to improve anti-tumor immune responses.

PD-1 (Programmed Cell Death Protein 1, full English name Programmed Cell Death Protein 1, also known as CD279) is a cell surface receptor expressed on activated T cells and pre-B cells (pro-B cells). It is an immune checkpoint that negatively regulates the immune system and promotes self-tolerance by inhibiting T cell inflammatory activity. More specifically, PD-1 promotes apoptosis of antigen-specific T cells in lymph nodes and reduces regulatory T cells. The dual mechanism of apoptosis of cells (Treg, also called suppressor T cells) prevents the occurrence of autoimmunity.

PD-1 is encoded by the PDCD1 gene, with a full length of 288 amino acids, of which the first 20 amino acids at the N-terminal are signal peptides, which are excised after maturation, the 21-170 amino acids are the extracellular segment, and the 171-191 amino acids are transmembrane region, amino acids 192-288 are intracellular tails.

PD-1 can bind to 2 ligands - B7 family members PD-L1 and PD-L2. In mice, PD-L1 is widely expressed on heart, lung, thymus, spleen, kidney and other organs, as well as almost all mouse tumor cell lines, and is highly expressed on a variety of human tumor cells; The expression of L2 is more limited, mainly in dendritic cells and a few tumor cell lines. Therefore, PD-1 and its ligand PD-L1 have become targets for anti-tumor immunotherapy.

At present, two anti-PD-1 targeted therapy drugs have been approved for marketing, namely Opdivo (generic name Nivolumab) and Keytruda (generic name Pembrolizumab). Opdivo has been approved for indications including unresectable or metastatic melanoma and Metastatic squamous non-small cell lung cancer, Keytruda is approved for unresectable or metastatic melanoma as well as non-small cell lung cancer, head and neck squamous cell carcinoma, classic Hodgkin lymphoma, urothelial carcinoma, Microsatellite hyperinstability carcinoma, etc.

The objective response rate (ORR) of Opdivo in a clinical trial of 120 patients with unresectable or metastatic melanoma was 32% (38/120), including 4 Complete remission (CR) and 34 partial remission (PR) patients; in another clinical trial of 117 patients with metastatic squamous non-small cell lung cancer, the objective response rate was 15% (17/117). In a randomized, controlled, open-label, multicenter clinical trial of Keytruda in 834 patients with unresectable or metastatic melanoma, the ORR of the two experimental groups (receiving different doses of Keytruda) was 33% (every three weeks). 10 mg/kg) and 34% (10 mg/kg every two weeks), the CR rates were 6% and 5%, the PR rates were 27% and 29%, respectively, while the control group (anti-CTLA-4 monotherapy) anti-ipilimumab treatment) had an ORR of 12%; in another randomized controlled, open-label, multicenter clinical trial of 305 patients with metastatic non-small cell lung cancer, the experimental arm (Keytruda treatment) had an ORR of 45% (including CR4 %, PR41%), while the control group (chemotherapy) ORR was 28% (including CR1%, PR27%). It can be seen from the above studies that although the anti-PD-1 therapy of Opdivo and Keytruda has significantly improved the ORR compared with chemotherapy and anti-CTLA-4 therapy, only about one-third of patients still benefit. , more than half of patients do not achieve remission.

The efficacy of anti-PD-1 antibody in the treatment of malignant tumors is mainly related to its blocking effect on the binding of PD-1/PD-L1. The binding epitope and its polymorphism of anti-PD-1 antibody on PD-1 molecule, as well as binding affinity (generally expressed by equilibrium dissociation constant KD value), specificity and other factors will affect anti-PD-1 antibody Blocking effect on PD-1/PD-L1 binding.

Phage display antibody library is a technology for high-throughput screening of antibodies. Currently, the extracellular region of recombinantly expressed target molecule is often used as target antigen to screen antibody library in the industry. The extracellular region of these target molecules has only one or several functional epitopes, usually composed of 5-15 amino acids, which are responsible for ligand binding, so only antibodies directed against specific functional epitopes have drug potential. The recombinantly expressed extracellular region fragments used for antibody library screening have a large number of dominant antigenic epitopes in addition to individual functional domains that exert biological functions. Therefore, most of the antibodies obtained by screening only have binding activity, but do not block their The neutralizing activity of biological functions has no drug potential, and a large number of functional experiments and epitope identification work are required to select functional antibodies with therapeutic effects. There are also cases of using synthetic epitope peptides to screen antibody libraries. However, due to the possible differences between synthetic epitope peptides and antigenic epitopes in natural conformation, antibodies that can bind to epitope peptides may not necessarily bind to antigenic epitopes on the surface of living cells. combine. In conclusion, the existing antibody library screening technology has a low hit rate of functional antibodies specific to epitopes with drug potential, which seriously affects the efficiency of high-throughput screening and increases the cost of later clone identification.

SUMMARY OF THE INVENTION

The present invention provides a method for efficiently screening an antibody library, which can solve the problem that the existing antibody library screening technology has a low hit rate of functional antibodies specific to epitopes with drug-forming potential. The present invention also provides an anti-PD-1 monoclonal antibody obtained by this screening method, which has different binding epitopes from Nivolumab and Pembrolizumab, and has high affinity and good specificity, which can solve the problem of some patients with existing anti-PD -1 The problem of ineffective treatment.

The technical scheme of the present invention is as follows:

A screening method for epitope-specific functional antibodies, comprising the following two screening steps:

Step 1: For the antigenic epitope with biological function, synthesize the epitope peptide and couple it to the solid-phase medium, and perform the first round of screening from the phage display antibody library to obtain the phage that specifically binds to the epitope peptide and expand it. increase;

Step 2: Obtain or construct a cell line that highly expresses the target antigen on the cell surface, and use live cells to further screen the phage that binds to the cell surface antigen from the phage obtained in the first round of screening to prepare functional antibodies.

In order to enhance the specificity of the screening, step 1 and step 2 can be screened in sequence for 2-5 rounds. For the technical flow chart of antibody screening according to this technical scheme, please refer to Figure 1.

In step 1, the amino acid sequence of the antigenic epitope with biological function can be obtained according to methods such as protein crystallization, fragment deletion/point mutation affinity analysis for ligands/receptors, software prediction, etc. The antigenic epitope peptide is composed of A cyclic peptide consisting of 10 to 15 amino acids, the amino acid composition of the cyclic peptide meets the following conditions:

(1) There is only one cysteine in the cyclic peptide, and the cyclic peptide is covalently coupled to the solid medium through the sulfhydryl group of the cysteine;

(2) There is a linking peptide consisting of 2 or 3 amino acids on both sides of the cysteine in the cyclic peptide, and the amino acids constituting the linking peptide can only be glycine or proline;

(3) Cyclic peptides contain an epitope consisting of 5-8 amino acids.

Preferably, the linking peptide connected to the amino terminus of cysteine in the cyclic peptide is composed entirely of glycine, and the linking peptide linked to the carboxyl terminus of cysteine contains 1 to 2 prolines.

Specifically, when used to screen monoclonal antibodies against the functional epitope composed of amino acids 75-82 of human PD-1 (the amino acid sequence is QTDKLAAF), the epitope peptide used in step 1 is cyclic and It conforms to the sequence recorded in SEQ ID NO: 13; the cell line used in step 2 is a cell line that highly expresses human PD-1 on the cell surface, preferably, the cell line is an expression of exogenous human PD transformed by gene recombination technology -1 gene cell line, and the cell line does not express PD-1 in the natural state (ie, before genetic modification), preferably, the expression density of human PD-1 on the cell surface is 10,000 cells/cell to 500,000 cells/cell.

An antibody screened by the above method can specifically bind to amino acids 75-82 from the N-terminus of the human PD-1 molecule (the amino acid sequence is QTDKLAAF), and the complementarity determining regions CDR1, CDR2, CDR3 has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 respectively, and the complementarity determining regions CDR1, CDR2, CDR3 of the light chain have SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 5, respectively The amino acid sequence of ID NO: 6.

Preferably, the heavy chain variable region has the amino acid sequence of SEQ ID NO: 7, and the light chain variable region has the amino acid sequence of SEQ ID NO: 8.

Preferably, the heavy chain is of IgG4 subtype and has the amino acid sequence of SEQ ID NO: 9, and the light chain has the amino acid sequence of SEQ ID NO: 10.

Preferably, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO: 11, and the light chain is encoded by the nucleotide sequence of SEQ ID NO: 12.

The antibody is secreted and expressed by Chinese hamster ovary cells (CHO cells).

Competitive inhibition assays (Example 6) and point mutation assays (Example 7) showed that the antibody has different binding epitopes to Nivolumab and Pembrolizumab, and can also bind to the D85G point mutation PD-1 that cannot bind to Pembrolizumab.

The antibody has a lower IC50 value than Nivolumab and Pembrolizumab (Example 5), can enhance the response of human T lymphocytes to SEB stimulation (Example 8), and can more effectively block PD-1 and PD at low doses. -L1 binding and at lower doses have therapeutic effect on malignancies (Example 9).

The antibody can be used alone or in combination with other drugs and excipients to prepare a drug for treating malignant tumors and a drug for treating diseases of the immune system.

The beneficial effects of the present invention include that, using the antibody library screening method of the present invention, the hit rate of functional antibodies specific to epitopes can be increased to more than 30%, and the number and cost of clone identification can be greatly reduced. The anti-PD-1 monoclonal antibody of the present invention has different human PD-1 binding epitopes from Nivolumab and Pembrolizumab, can also bind to the point mutation PD-1 that cannot be combined with Pembrolizumab, and has high affinity with PD-1. The low IC50 value is more effective in blocking the combination of PD-1 and PD-L1 at low doses, and has a therapeutic effect on malignant tumors at lower doses, while the application of low-dose antibodies can reduce the effect of drugs on the body. stimulus, lower drug costs and prices have positive implications.

Description of drawings

Figure 1 Technical roadmap for epitope-specific functional antibody screening

Figure 2 The results of the competitive inhibition experiment of the fully human anti-PD-1 monoclonal antibody 5L12 on PD-1/PD-L1 binding in Example 5

Figure 3 The results of the competition inhibition experiment of the binding of the fully human anti-PD-1 monoclonal antibody 5L12 to PD-1 by other anti-PD-1 antibodies in Example 6

Figure 4 Example 7 ELISA test results of the binding ability of fully human anti-PD-1 monoclonal antibody 5L12 to D85G mutant PD-1

Figure 5 The test results of the fully human anti-PD-1 monoclonal antibody 5L12 in Example 8 to enhance the ability of human T lymphocytes to respond to SEB stimulation

Figure 6 The results of the high-dose group of the tumor inhibition experiment of the fully human anti-PD-1 monoclonal antibody 5L12 in the animal model of Example 9

Fig. 7 The results of the low-dose group of the tumor inhibition experiment of the fully human anti-PD-1 monoclonal antibody 5L12 in the animal model of Example 9.

Detailed ways

Example 1. Synthesis of PD-1 epitope peptide and screening of phage display antibody library

(1) Epitope peptide synthesis and magnetic bead coupling

According to the sequence of SEQ ID NO: 13, the cyclic epitope peptide of human PD-1 was chemically synthesized (entrusted to Sangon Bioengineering (Shanghai) Co., Ltd.), the epitope peptide includes the amino acid QTDKLAAF at positions 75-82 of human PD-1 , and the cysteine M for magnetic bead coupling and the linking peptides on both sides consisting of 3 amino acids, with a molecular weight of 1475 Da. The synthesized epitope peptide was dissolved in PBS (phosphate buffered saline, pH 7.2, containing 20 mM sodium phosphate, 150 mM sodium chloride) at 10 mg/mL for use;

Wash 2 mL of amino-derived magnetic beads (Thermofisher Scientific, product number 21352, the weight of each mL of magnetic beads is 2.5 g, containing about 12 μmol of amino groups) with 2 mL of PBS, wash three times, and magnetically separate the magnetic beads;

Dissolve Sulfo-SMCC (Thermofisher Scientific, Product No. 22122) in PBS at a concentration of 4.8 mg/mL, mix 200 μL with the above magnetic beads, and react at 4°C for 2 hours;

Separate the magnetic beads and wash them with PBS. Divide the magnetic beads into two parts. One part is resuspended in 1 mL of PBST containing 1% BSA (PBS containing 0.1% Tween 20), which is recorded as magnetic beads A; the other part is added to 5 mL The cyclic epitope peptides were reacted at 4°C for 2 hours, the magnetic beads were separated and washed with PBST, and resuspended in 1 mL of PBST containing 1% BSA, which was denoted as magnetic beads B.

(2) Screening of phage-displayed antibody libraries

Collect 20 mL of blood from 50 healthy volunteers, mix them, and follow the method of "Construction and Diversity Analysis of Natural Human IgG Fab Phage Antibody Library" (Chinese Journal of Immunology, 2010, 26 (11): 1007-1010) Construction of phage-displayed antibody libraries.

Mix the magnetic beads A prepared in step (1) with 1 mL of the phage-displayed antibody library (5×10^12 PFU) diluted with PBST containing 1% BSA, rotate and mix at room temperature for 1 hour, and remove the magnetic beads A by magnetic force. Then add the magnetic beads B prepared in step (1), rotate and mix at room temperature for 1 hour, magnetically separate the magnetic beads B, wash the magnetic beads B with PBST three times, add 0.2 mol/LGlycine-HCl (pH 2.2) solution for mild The phages on the magnetic beads B were washed and collected by shaking for 20 min, and then neutralized with 1mol/LTris-HCl (pH 9.1). The obtained phages were infected with E. coli and amplified to become the secondary library and stored spare.

Example 2. Secondary screening of functional antibodies in live cells with high PD-1 expression

(1) Construction of cell lines that highly express PD-1 on the cell surface

The full-length PD-1 coding gene (PDCD1 gene, containing the N-terminal signal peptide coding sequence, see NCBI Reference Sequence: NM_005018.2 for the specific sequence) was cloned, inserted into the pcDNA3.1 expression vector, and amplified in E. coli 293T cells were transfected (ATCC number CRL-3216), and high-expressing cell lines were screened according to the literature "Flow cytometric detection and quantitation of the epidermal growth factor receptor incomparison to Scatchard analysis in human bladder carcinoma cell lines. Cytometry. 1994 Sep 1;17( 1): 75-83" and "Antibody-dependent cellularcytotoxicity mediated by cetuximab against lung cancer cell lines. ClinCancer Res. 2007 Mar 1; 13(5): 1552-61 "Determination of the surface of each cell line by flow cytometry The PD-1 expression density of 2H-3 was selected from the cell line numbered 2H-3 with the highest expression density. It was determined that the average expression density of PD-1 on the surface of cell line 2H-3 was 12,000 cells/cell. Serum medium (Thermofisher Scientific, Cat. No. 11686029) was used for suspension culture, and the number of cells was expanded to more than 5×10^6.

(2) Live cell screening of anti-PD-1 antibodies

The blank 293T cells grown in logarithmic phase were resuspended in 1 mL DMEM medium at a density of 1×10^6 cells/mL, and the phage screened in Example 1 (2) was added (centrifuged and resuspended in 1 mL DMEM cultured). After mixing, incubate at 37°C for 1 hour, remove cells by low-speed centrifugation, and repeat the process once to remove ineffective antibodies bound to 293T cells and reduce the background value of screening; then add to the centrifuged supernatant 5 × 10^5 2H-3 cells with high PD-1 expression prepared in Example 2(1) were incubated at 37°C for 1 hour after mixing, and the cells were harvested by low-speed centrifugation. pH 2.5) Wash the phage from the cells, and then neutralize it with 0.5 times the volume of Tris-HCl (pH 7.4) to infect E. coli with the obtained phage for amplification.

According to the screening methods of Example 1 (2) and Example 2 (2), two rounds of screening were performed on the phage obtained above in turn, and the final obtained phage was monocloned and amplified, and 60 clones were selected by ELISA method. Identification of the affinity and binding epitope of phage to PD-1 (refer to the Phage-ELISA identification method of "Journal of Immunology, Vol. 28, No. 6: 489-496, June 2012"), using recombinantly expressed human PD-1 extracellular segment full-length protein (amino acids 20-170) and chemically synthesized human PD-1 amino acids 75-82 (sequence QTDKLAAF) were coated on 96-well plates, and the chemically synthesized sequences were LDSPDR, Short peptides such as LAPKAQ and RSQPGQ were used as negative controls to coat 96-well plates, then phage and HRP-labeled anti-phage antibodies were added, and HRP substrate was added to detect the binding situation; flow cytometry was used to identify the effect of phage on live cells 2H-3 Affinity of surface PD-1; 22 clones that were both positive for binding to PD-1 and bound to a specific epitope of PD-1 (amino acids 75-82 QTDKLAAF) were identified by ELISA and flow cytometry, and the positive rate was 36.67%.

Example 3. Construction of a fully human anti-PD-1 monoclonal antibody expression system

(1) Identification of heavy and light chain variable regions

From the 22 phage-positive clones finally obtained in Example 2, 5 clones with higher affinity identified by ELISA were selected for sequencing, and 1 clone numbered 5L12 was selected for further development. The sequencing results show that the heavy chain variable region of 5L12 has the amino acid sequence of SEQ ID NO: 7, and the light chain variable region has the amino acid sequence of SEQ ID NO: 8. Further antibody sequence analysis shows that the complementarity determining regions CDR1, CDR2 and CDR3 respectively have the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain respectively have SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 5 and SEQ ID NO: 3. The amino acid sequence of ID NO: 6.

(2) Construction and transfection of expression vector

Using the light and heavy chain variable region gene sequence of 5L12 as a template, the heavy chain gene (IgG4 type) and light chain gene of a fully human antibody are synthesized by overlapping PCR, wherein the coding sequence of the heavy chain is the nucleotide of SEQ ID NO: 11 sequence, the coding sequence of the signal peptide at the 5' end of the heavy chain is the nucleotide sequence of SEQ ID NO: 14, and the coding sequence of the light chain is the nucleotide sequence of SEQ ID NO: 12. The coding sequences of the heavy and light chains were inserted into the pcDNA3.1(+) vector (Invitrogen) to construct the expression vector pcDNA3.1-anti-PD-1 for the fully human anti-PD-1 monoclonal antibody, and the Lipofectamine2000Reagent kit was used to construct the expression vector pcDNA3.1-anti-PD-1. CHO-K1 cells (ATCC number CRL-9618) were transfected, positive clones were screened with G418 and Zeocin, and cell culture supernatants were taken to screen high-expressing cell lines by ELISA. The specific experimental process of transfection and screening was in accordance with the patent CN201010125241 (Grant Bulletin No. CN102167742B) for the example.

(3) Small amount of expression and purification

The screened high-expressing cell lines were expanded with CHO cell serum-free medium, and the fully human antibody 5L12 was isolated and purified with Protein A affinity column (product of GE). The purified antibody was dialyzed with PBS, and finally absorbed by UV light. Legal Quantity.

Example 4. Determination of Affinity of Fully Human Anti-PD-1 Monoclonal Antibody 5L12

The affinity of the fully human anti-PD-1 monoclonal antibody 5L12 prepared in Example 3 to human PD-1 was determined by surface plasmon resonance (SPR). The BIACORE3000 system was used to couple the sensor chip with purified 5L12 antibody, and operate according to the operating instructions of the system to detect the full-length protein of the extracellular segment of PD-1 recombinantly expressed by 5L12 and CHO cells (NCBI reference sequence NM_005018.2). 20-170 amino acids) and chemically synthesized PD-1 amino acids 75-82 (sequence QTDKLAAF) and short peptides with sequences LDSPDR, LAPKAQ, RSQPGQ, the test results are shown in Table 1, 5L12 extracellular PD-1 affinity The affinity of the full-length segment and the epitope peptide QTDKLAAF is 10 ^-11 M.

Table 1 The results of determining the affinity of fully human anti-PD-1 monoclonal antibody 5L12 with different peptides by SPR method

Example 5. Competitive inhibition experiment of fully human anti-PD-1 monoclonal antibody 5L12 on PD-1/PD-L1 binding

The protein to be labeled PD-L1 (the extracellular segment of human PD-L1 recombinantly expressed in CHO cells) was diluted with 0.025M carbonate buffer pH9.5 to a concentration of 1% (mass volume ratio) and put into a dialysis bag. FITC was prepared into a 0.1 mg/ml solution with the same buffer and placed in a small beaker, the dialysis bag was immersed in the FITC solution, and stirred for 24 h at 4°C in the dark. Take out the labeling solution in the dialysis bag, pass through the column with Sephadex G-50, remove free fluorescein, and collect the fluorescent antibody FITC-PD-L1 for use.

The genetically recombinant 293T cells expressing human PD-1 on the surface were resuspended in PBS solution of pH 7.2 at a density of 1×10 ⁶ cells/ml; the fixed subsaturated concentration of fluorescently labeled protein FITC-PD-L1 and The unlabeled purified antibody 5L12, Nivolumab (Opdivo), Pembrolizumab (Keytruda), and human IgG (control) were mixed with 4× serial dilutions, added to the cell suspension, incubated at 4 °C for 60 min, and the cells were harvested by centrifugation. After washing twice with FCS-PBS buffer, the fluorescence intensity of cells was detected by flow cytometry and analyzed by Cellquest software. Three replicate tubes were set for each concentration of the competing antibody, and the IC50 value of the median inhibitory concentration was calculated, and the maximum fluorescence intensity represented the average fluorescence intensity obtained without the competing antibody.

The experimental results are shown in Figure 2, indicating that the antibody 5L12 can completely block the binding of the fluorescently labeled antibody FITC-PD-L1 to 293T cells expressing PD-1 on the surface, and its IC50 value is about 30 ng/mL, which is 1 of Nivolumab and Pembrolizumab respectively. /20, 1/2, indicating that 5L12 can more effectively block the binding of PD-1 to its ligand, and the minimum dose that can exert the effect is lower.

Example 6. Competitive inhibition experiment of other anti-PD-1 antibodies on the binding of fully human anti-PD-1 monoclonal antibody 5L12 to PD-1

5L12 was labeled with FITC by the method of Example 5, and FITC-5L12 fluorescent antibody was prepared for use.

The genetically reconstituted 293T cells expressing human PD-1 on the surface were resuspended in PBS solution of pH 7.2 at a density of 1×10 ⁶ cells/ml; the fixed subsaturated concentration of fluorescently labeled protein FITC-5L12 was respectively and 10 × Serial dilutions of unlabeled antibodies Nivolumab (Opdivo), Pembrolizumab (Keytruda), and human IgG (control) were mixed, added to the cell suspension, incubated at 4 °C for 60 min, centrifuged to harvest the cells, and washed with 1% FCS-PBS buffer After washing twice, the fluorescence intensity of cells was detected by flow cytometer and analyzed by Cellquest software. Three replicate tubes were set for each concentration of the competing antibody, and the maximum fluorescence intensity represents the average fluorescence intensity obtained without the competing antibody.

The experimental results are shown in Figure 3. The results show that Pembrolizumab has a competitive inhibitory effect on the binding of 5L12 to human PD-1, while Nivolumab has no obvious competitive inhibitory effect on 5L12, which indicates that the binding epitope of 5L12 on human PD-1 is similar to that of Nivolumab ( The main binding epitope is 25LDSPDR30, reference: An unexpected N-terminal loop in PD-1 dominates binding by nivolumab. Nature Communications, 2017, 8: 14369), but different from Pembrolizumab (the main binding epitope is Pro83～Gly90, reference : High-resolution crystal structure of the therapeutic antibody pembrolizumab bound to the human PD-1. Scientific Reports. 2016, 6, Article number: 35297) close, or the binding of pembrolizumab to PD-1 sterically hinders the binding of 5L12 to PD-1 combine.

Example 7. The ability of fully human anti-PD-1 monoclonal antibody 5L12 to bind to D85G mutant PD-1

The 85th aspartic acid of human PD-1 was mutated to glycine (D85G mutation) by genetic recombination, and the genes encoding the extracellular segment of human PD-1 and the extracellular segment of human PD-1 (D85G) were inserted into pcDNA3. 1 vector and transfected into CHO cells for transient expression and purification.

The purified human PD-1 extracellular segment and human PD-1 extracellular segment (D85G) protein at a concentration of 1 mg/mL were coated at 100 μL/well in a 96-well plate, and 5L12 was prepared from 1 mg/mL. 10× serial dilution, 100 μL/well was added to 96-well plate, and then HRP-labeled rabbit anti-human IgG was added for color reaction, and the data was read with a microplate reader. The ELISA results are shown in Figure 4. The results show that 5L12 can bind to both human PD-1 extracellular segment and human PD-1 extracellular segment (D85G), while Pembrolizumab is reported to be unable to bind to D85G-mutated PD-1 (Reference: Structural basis for blocking PD-1-mediated immune suppression by therapeutic antibody pembrolizumab. Cell Research 2017.27:147-150.), indicating that 5L12 and Pembrolizumab have different binding sites.

Example 8. Test of fully human anti-PD-1 monoclonal antibody 5L12 enhancing the ability of human T lymphocytes to respond to SEB stimulation

Peripheral blood from healthy volunteers was diluted 1:10 into cell culture medium and whole blood was plated (150 μL per well) into 96-well plates with 10× serial dilutions of fully human anti-PD-1 monoclonal Antibody 5L12 was pre-incubated for 30-60 minutes, and then 1 μg/mL of staphylococcal enterotoxin B (SEB) was added for 2-4 days. The supernatant was collected and the IL-2 content was measured by ELISA. The results are shown in Figure 5, indicating that 5L12 can increase IL-2 production in whole blood cells stimulated by SEB, and the level of IL-2 was correlated with the supplemental dose of 5L12.

Example 9. Tumor inhibition experiment of fully human anti-PD-1 monoclonal antibody 5L12 in animal model

1. Construction of human PBMC immune reconstitution mice

Human PBMCs (peripheral blood mononuclear cells) from healthy donors were isolated using human lymphocyte separation medium, and resuspended in RPMI-1640 medium at 8 × 10 ⁷ /mL for later use; use serum mouse IgG<5μg/mL Adult NOD/SCID mice were injected with 2×10 ⁷ PBMC cells through the tail vein, and 250 μL of peripheral blood was collected from the retro-orbital venous plexus of mice on days 0, 14, 28, 42, and 56, respectively. PE-labeled mouse antibody human CD3 was used. Monoclonal antibody and FITC-labeled mouse anti-human CD19 monoclonal antibody were combined with flow cytometry to detect the content of CD3+ T cells and CD19+ B cells, and the content of human IgG in mouse serum was determined by ELISA to determine the effect of immune reconstitution.

2. Construction of mouse tumor model and treatment with anti-human PD-1 antibody 5L12

Human non-small cell lung cancer cell line HCC827 (high PD-L1 expression) was subcutaneously implanted into human PBMC immunoreconstructed mice at a dose of 1×10 ⁶ cells/mouse, and anti-PD was administered after the tumor grew to 250 mm ² after implantation. -1 treatment. The mice were divided into high-dose group and low-dose group. The high-dose group was given 3 mg/kg of 5L12 (dissolved in PBS at a concentration of 1 mg/mL), Nivolumab, and Pembrolizumab, administered once every 10 days. Four times of administration, the tumor size was measured with a caliper every 1 week; the administration dose of the low-dose group was adjusted to 0.5 mg/kg, and other test methods remained unchanged; and PBS was used as a control.

The experimental results are shown in Figures 6 and 7, indicating that 5L12 can significantly inhibit tumor growth at low doses, and the effective dose is significantly lower than that of Nivolumab and Pembrolizumab.

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Trp Met Leu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Tyr Pro Ser Thr Gly Tyr Thr Glu Tyr Ser Glu Asn Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 10

<211> 219

<212> PRT

<213> artificial sequence()

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Ser Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 11

<211> 1332

<212> DNA

<213> artificial sequence()

<400> 11

gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ccggcggctc cctgcggctg 60

tcctgcgccg cctccggcta caccttcacc tcctactgga tgctgtgggt gcggcaggcc 120

cccggcaagg gcctggagtg ggtgtcctac atctacccct ccaccggcta caccgagtac 180

tccgagaact tcaaggaccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc ccggggcgtg 300

cggtactact tcgactactg gggccagggc accctggtga ccgtgtcctc cgcctccacc 360

aagggcccct ccgtgttccc cctggccccc tgctcccggt ccacctccga gtccaccgcc 420

gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgtc ctggaactcc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtcctc cggcctgtac 540

tccctgtcct ccgtggtgac cgtgccctcc tcctccctgg gcaccaagac ctacacctgc 600

aacgtggacc acaagccctc caacaccaag gtggacaagc gggtggagtc caagtacggc 660

cccccctgcc ccccctgccc cgcccccgag ttcctgggcg gcccctccgt gttcctgttc 720

ccccccaagc ccaaggacac cctgatgatc tcccggaccc ccgaggtgac ctgcgtggtg 780

gtggacgtgt cccaggagga ccccgaggtg cagttcaact ggtacgtgga cggcgtggag 840

gtgcacaacg ccaagaccaa gccccgggag gagcagttca actccaccta ccgggtggtg 900

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 960

tccaacaagg gcctgccctc ctccatcgag aagaccatct ccaaggccaa gggccagccc 1020

cgggagcccc aggtgtacac cctgcccccc tcccaggagg agatgaccaa gaaccaggtg 1080

tccctgacct gcctggtgaa gggcttctac ccctccgaca tcgccgtgga gtgggagtcc 1140

aacggccagc ccgagaacaa ctacaagacc accccccccg tgctggactc cgacggctcc 1200

ttcttcctgt actcccggct gaccgtggac aagtcccggt ggcaggaggg caacgtgttc 1260

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320

tccctgggca ag 1332

<210> 12

<211> 657

<212> DNA

<213> artificial sequence()

<400> 12

gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga ccgggtgacc 60

atcacctgca agtcctccca gtccctgctg gactcctcca cccggaagaa ctacctggcc 120

tggtaccagc agaagcccgg caaggccccc aagctgctga tctactgggc ctccacccgg 180

gagtccggcg tgccctcccg gttctccggc tccggctccg gcaccgactt caccctgacc 240

atctcctccc tgcagcccga ggacttcgcc acctactact gcaagcagtc ctacaacctg 300

tggaccttcg gccagggcac caaggtggag atcaagcgga ccgtggccgc cccctccgtg 360

ttcatcttcc ccccctccga cgagcagctg aagtccggca ccgcctccgt ggtgtgcctg 420

ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480

tccggcaact cccaggagtc cgtgaccgag caggactcca aggactccac ctactccctg 540

tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgag 600

gtgacccacc agggcctgtc ctcccccgtg accaagtcct tcaaccgggg cgagtgc 657

<210> 13

<211> 15

<212> PRT

<213> artificial sequence()

<400> 13

Cys Pro Pro Ala Gln Thr Asp Lys Leu Ala Ala Phe Ala Ala Ala

1 5 10 15

<210> 14

<211> 66

<212> DNA

<213> artificial sequence()

<400> 14

atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatatcc 60

agagga 66

Claims (10)

1. a kind of screening technique for the functional antibodies that epitope is special, characterized in that including following two screening step:

Step 1: synthetic antigen epitope peptide is simultaneously coupled on solid-phase media, and first run screening is carried out from phage displaying antibody library, It obtains and the bacteriophage of epitope peptide specific binding and amplification；

Step 2: obtaining or building is in the cell strain of cell surface height expression purpose antigen, is obtained with living cells from first run screening Bacteriophage in further bacteriophage of the screening in conjunction with cell surface antigen.

2. according to the method described in claim 1, it is characterized in that, epitope peptide described in step 1 is by 10 to 15 amino The cyclic peptide of acid composition, the Amino acid profile of cyclic peptide meet the following conditions:

Have in cyclic peptide and only 1 cysteine, cyclic peptide pass through on the sulfydryl covalent coupling to solid-phase media of the cysteine；

Respectively there are one section of link peptide by 2 or 3 Amino acid profiles in the two sides of cysteine in cyclic peptide, constitute the amino of link peptide Acid can only be glycine or proline；

The epitope being made of containing one section 5-8 amino acid in cyclic peptide.

3. according to the method described in claim 2, it is characterized in that, one section of link peptide being connected in cyclic peptide with cysteine aminoterminal It is made of completely glycine, 1 to 2 proline is contained in one section of link peptide being connected with cysteine c-terminus.

4. a kind of screening technique of the special anti-human PD-1 functional antibodies of epitope, characterized in that using described in claim 1 Method, and epitope peptide used in step 1 is the cyclic peptide with amino acid sequence documented by SEQ ID NO:13, Cell strain used in step 2 is the cell strain of cell surface height expression PD-1, and PD-1 is in the expression quantity of cell surface 10,000/cell is to 500,000/cell.

5. a kind of antibody screened using method described in claim 1, can specificity combination people's PD-1 molecule it is extracellular Section, characterized in that complementary determining region CDR1, CDR2, CDR3 of heavy chain be respectively provided with SEQ ID NO:1, SEQ ID NO:2, The amino acid sequence of SEQ ID NO:3, complementary determining region CDR1, CDR2, CDR3 of light chain be respectively provided with SEQ ID NO:4, The amino acid sequence of SEQ ID NO:5, SEQ ID NO:6.

6. antibody according to claim 5, characterized in that heavy chain variable region has the amino acid sequence of SEQ ID NO:7 Column, light chain variable region have the amino acid sequence of SEQ ID NO:8.

7. antibody according to claim 5, characterized in that heavy chain has the amino acid sequence of SEQ ID NO:9, light chain Amino acid sequence with SEQ ID NO:10.

8. antibody according to claim 5, characterized in that heavy chain is nucleotide sequence coded by SEQ ID NO:11's, gently Chain is nucleotide sequence coded by SEQ ID NO:12's.

9. a kind of composition contains monoclonal antibody as claimed in claim 6 and pharmaceutically acceptable carrier.

10. antibody described in claim 6-9 is in preparing treating malignant tumor drug and disease of immune system therapeutic agent Purposes.