anti-PD-1 monoclonal antibody and obtaining method thereofTechnical Field
The invention relates to the technical field of antibody engineering, in particular to a fully human anti-PD-1 monoclonal antibody, and an obtaining method and application thereof.
Background
Immunomodulation plays an extremely important role in the immune response of the body, while activation of immunocompetent cells is critical to the regulation of the overall immune response. Studies have shown that T cell activation and proliferation are dependent on dual signaling pathways. The concept of "co-stimulatory signals" was based on the two-signal model of T cell activation in 1970 by Bretscher and Cohn, i.e., T cell activation requires not only the presentation of MHC-antigen peptide complexes by APCs to antigen-specific T cells to provide a first signal, but also the participation of multiple co-stimulatory molecules in providing a secondary, ancillary signal (co-stimulatory signal); with the progress of research, the costimulatory signal gradually becomes a hot spot field of immunology; these synergistic signaling molecules include mainly two superfamily of CD28/B7 and TNFR/TNF. PD-1/PD-L1 is a member of the CD28/B7 superfamily and can mediate negative co-stimulation signals. The PD-1/PD-L1 signal channel can effectively inhibit T, B cell functions, inhibit T cell proliferation, reduce secretion of cytokines IL-2, IL-10 and IFN-gamma, play an important role in immune regulation and have important significance in research of diseases such as tumor immunity, autoimmunity, transplantation immunity, asthma, virus infection and the like.
PD-1 belongs to immunoglobulin superfamily type I transmembrane protein, and has a molecular weight of about 50-55 KD. Originally obtained by subtractive hybridization techniques in apoptotic T-cell hybridomas, and were designated programmed cell death factor 1 (programed celldeath1) because of their association with apoptosis. The gene encoding PD-1 is PD-CD1, maps to human chromosome 2q37.3, and has 23% homology with CTLA4 gene. PD-1 is composed of an intracellular region, a transmembrane region and an extracellular region, wherein the extracellular region comprises an immunoglobulin variable region IgV-like domain; two tyrosine residues at the N end of the intracellular region and other amino acid residues jointly form an Immunoreceptor Tyrosine Inhibition Motif (ITIM), and the ITIM plays a role in antagonizing an antigen receptor stimulation signal through the phosphorylation of tyrosine, so that a negative regulation function is played in the immune response process; the PD-1 molecule can be inducibly expressed on the surfaces of activated T cells, B cells, NK cells, monocytes and dendritic cells, and can be combined with ligands PD-L1 and PD-L2 to generate inhibition effect on the activation of lymphocytes, thereby inhibiting the immune response of immune cells.
PD-L1(CD274 or B7H1) and PD-L2(CD273 or B7DC) are two ligands of PD1, both of which are located on human chromosome 9p24.2 and start in the same direction with an interval of about 42 kb;
they belong to the B7 family and thus, like other members, both PD-L1 and PD-L2 are structurally composed of an IgV-like domain, an IgC-like domain, a transmembrane region and a short, conserved cytoplasmic tail; the cytoplasmic tail of PD-L1 is more conserved between species than PD-L2. PD-L1 is inducibly expressed on activated T cells, B cells, dendritic cells, monocytes and various types of tumor cells (e.g., lung, liver, breast, ovarian, kidney, head and neck, esophagus, skin, squamous cell carcinoma, etc.); PD-L2 is expressed primarily on activated macrophages, dendritic cells and individual tumor cells (e.g., hodgkin's lymphoma). The interaction between PD-L1 on the surface of the tumor and PD-1 can cause the apoptosis of tumor antigen specific T cells, so that the tumor cells escape from the immune monitoring of the organism. The anti-PD-1 monoclonal antibody can promote the proliferation of tumor antigen specific T cells by blocking a PD-1/PD-L1 signal path, plays a role in killing tumor cells, can effectively improve the immunotherapy effect, and has the potential of treating various tumors.
At present, monoclonal antibody medicines aiming at PD-1 or PD-L1 (shown in Table 1) are carried out by a plurality of large international pharmaceutical companies, wherein a PD-1 inhibitor Opdvio (Nivolumab) of Baishishishishiqianbao is approved to be marketed in Japan within 7 months of 2014; PD-1 inhibitor of saxadong was approved by FDA for marketing in 9 months 2014; the first indication for both drugs was melanoma. With the advance of clinical items of various companies, indications have been expanded to the fields of lung cancer, breast cancer, hematological cancer, and the like.
TABLE 1 anti-PD-1 antibodies currently undergoing clinical trials
At present, the fully human antibody is the main direction for the development of therapeutic antibodies, and the emergence of antibody library technology provides a good technical platform for the preparation and screening of human antibodies. The antibody library technology bypasses the hybridoma process necessary in the previous monoclonal antibody development process, and can obtain various antibody genes and antibody molecular fragments even without an immunization process. Phage antibody libraries were the earliest and most widely used antibody libraries at present.
The phage display technology is a technology firstly established by Smith, which inserts a gene coding a foreign protein or polypeptide into a phage capsid protein gene, and leads the foreign protein or polypeptide and the phage capsid protein to be fused and expressed on the surface of a phage. The phage antibody library is obtained by expressing antibodies with different specificities or functional fragments thereof (Fab, Fv, ScFv) on the surface of phage by using the above principle, and then screening by using antigen. The phage antibody library is divided into immune library and non-immune library according to the source of antibody gene, and the non-immune library comprises natural library, semi-synthetic library and fully-synthetic library. Screening of phage antibody libraries mimics the process of antibody affinity maturation, typically by coating the antigen on a solid phase medium, adding the phage antibody library to be screened, and performing several rounds of "adsorption-washing-elution-amplification" (i.e., panning) until high affinity specific antibodies are screened.
Disclosure of Invention
The invention provides an anti-PD-1 monoclonal antibody; the invention screens the anti-PD-1 monoclonal antibody from the total synthetic antibody library, and then constructs a method of synthesizing a phage antibody light chain library with small capacity through computer aided design analysis, and constructs a library for the mutation of the complementarity determining regions CDR1, 2 and 3 of the light chain of the anti-PD-1 monoclonal antibody DFPD1-1 obtained by primary screening; after screening, the monoclonal antibodies DFPD1-3 and DFPD1-7 with higher affinity are selected, and then heavy chain CDR1, 2 and 3 region mutation libraries are screened, and finally the high-affinity anti-PD-1 monoclonal antibody is screened.
In order to achieve the above object, the process for obtaining an anti-PD-1 monoclonal antibody of the present invention comprises:
(1) and performing biopanning on the anti-PD-1 single-chain antibody, and obtaining an antibody sequence DFPD1-1 with higher affinity from a totally synthesized ScFv phage library through enrichment screening of a three-wheel antibody library, wherein the heavy chain is DFPD1-H1(SEQ NO.1), and the light chain is DFPD1-L1(SEQ NO. 5).
(2) Constructing a light chain complementarity determining region CDR1, 2 and 3 mutation library by computer tertiary structure simulation based on DFPD1-1, and carrying out biopanning and screening and identification on the antibody library to obtain antibody sequences DFPD1-2, DFPD1-3, DFPD1-4, DFPD1-5, DFPD1-6 and DFPD1-7 of 6 different light chains, wherein the corresponding light chain sequences are respectively DFPD1-L2(SEQ NO.6), DFPD-L3(SEQ NO.7), DFPD1-L4(SEQ NO.8), DFPD1-L5(SEQ NO.9), DFPD1-L6(SEQ NO.10) and DFPD1-L7(SEQ NO. 11); the 7 single-chain antibodies were compared for affinity at the phage level.
(3) Two clones DFPD1-3 and DFPD1-7 with high affinity are selected, heavy chain complementarity determining region CDR1, 2 and 3 libraries are constructed, and the libraries are subjected to biopanning and screening of positive clones, so that five different single-chain antibody sequences DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13 are obtained. Wherein the light chain variable region sequences of DFPD1-9, DFPD1-11 and DFPD1-12 are DFPD1-L3, and the light chain variable region sequences of DFPD1-10 and DFPD1-13 are DFPD 1-L7; the heavy chain variable region sequences of DFPD1-9 and DFPD1-10 are DFPD1-H2(SEQ NO.2), the heavy chain variable regions of DFPD1-11 and DFPD1-13 are DFPD1-H3(SEQ NO.3), and the heavy chain variable regions of DFPD1-12 are DFPD1-H4(SEQ NO. 4). The single-chain antibodies were subjected to affinity comparison at the phage level.
(4) Cloning the monoclonal heavy chain variable region gene, the light chain variable gene and the light and heavy chain constant region gene in the step (3) to a eukaryotic expression vector, transfecting host cells, obtaining the full antibody of the monoclonal antibody, and then comparing the affinity with other biological functions.
An anti-PD-1 monoclonal antibody obtained by the above method, comprising: a light chain and a heavy chain; the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are denoted LCDR1, LCDR2 and LCDR3, respectively; LCDR1 includes any one of RASQNIHSYLD, RASQNVSNWLD, RASQSIHNYLD, RASQDINNWLDRASQDVRTYLD, RASQGINSWLD or RASQSVSNYLD; LCDR2 includes any one of EASTRAS, DASNRAT, NASTRAT, DASTLAT, gastrt, or DASTRAT; LCDR3 includes either QQALKLPIT, QQSRHIPLT, QQELHLPLT, QQNVNLPLT, QQDIDLPLT, QQSYRLPLT or QQNMQLPLT.
Wherein, the light chain variable region amino acid sequence comprises any one of SEQ NO.5, SEQ NO.6, SEQ NO.7, SEQ NO.8, SEQ NO.9, SEQ NO.10 or SEQ NO. 11.
An anti-PD-1 monoclonal antibody obtained by the above method, comprising: a light chain and a heavy chain; the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are denoted HCDR1, HCDR2 and HCDR3, respectively; HCDR1 comprises SNNGMH or SNYGMH; HCDR2 comprises either VIWYDGSKK, VIWYDSSRK or VIWYDSTKK; HCDR3 comprises TAVYYCATNNDYW or TAVYYCATNTDYW.
Wherein, the amino acid sequence of the heavy chain variable region comprises any one of SEQ NO.1, SEQ NO.2, SEQ NO.3 or SEQ NO. 4.
The invention also provides an antibody, polypeptide or protein containing the light chain or the heavy chain.
The invention also provides an antibody containing the light chain or the heavy chain, wherein the antibody can block the combination of PD-1 and a ligand PD-L1 thereof and inhibit the biological activity of PD-1.
The invention also provides a polynucleotide sequence or a combination comprising the light chain or the heavy chain.
Wherein, the invention also provides a recombinant DNA expression vector containing the polynucleotide sequence or the combination; the DNA sequence of the vector comprises an amino acid sequence encoding the variable and constant regions of the heavy chain or the variable and constant regions of the light chain of the anti-PD 1 antibody.
The invention also provides a host cell for transfecting the recombinant DNA expression vector, wherein the host cell comprises prokaryotic cells such as escherichia coli and the like, yeast or mammalian cells.
Preferably, the host cell comprises a HEK293E cell, a CHO cell or a NS0 cell.
The invention also provides an antibody containing the light chain or the heavy chain, which is used for full-antibody, single-chain antibody, single-domain antibody, bispecific antibody, antibody drug conjugate or chimeric antigen receptor T cell immunotherapy.
Wherein, the invention also provides a monoclonal antibody, an artificial vector, a medicament or a pharmaceutical composition containing the light chain or the heavy chain
The invention also provides a detection reagent or a kit containing the light chain or the heavy chain.
Wherein the anti-PD-1 monoclonal antibody comprises a full-length antibody and a fragment of the anti-PD-1 monoclonal antibody, the fragment including but not limited to Fab, Fab ', F (ab')2Fv, or ScFv.
Wherein the full length antibody is fully human.
Wherein the heavy chain constant region of the anti-PD-1 monoclonal antibody comprises IgG1, IgG2, IgG3, and IgG 4; the light chain constant region comprises C κ or Cλ。
Preferably: the heavy chain constant region is IgG 4.
Preferably, the light chain constant region is ck.
Wherein, the CDR is a complementary-determining region (complementary-determining region); the ScFv is a single-chain antibody (single-chain-chainfragmentvariable); the ADCs are antibody-drug conjugates (antibody-drugs); the CAR-T is a chimeric antigen receptor T cell immunotherapy (Chimeric antigen receptor T-Cell immunotherapy); the HEK293E cells were human embryonic kidney293E cells (humanembryonicedney 293 Ecell); the CHO cell is a Chinese hamster ovary cell (Chinese hamster ovary); NS0 cells were mouse NS0 thymoma cells.
Compared with the prior art, the invention has the beneficial effects that:
the monoclonal antibody provided by the invention can prevent or treat diseases by eliminating and inhibiting the activity of PD-1, wherein the diseases are selected from cancers, infectious diseases or immune system diseases. The cancer includes, but is not limited to, lung cancer, kidney cancer, melanoma, breast cancer, liver cancer, head and neck cancer, skin cancer, squamous cell carcinoma, ovarian cancer, bone cancer, colorectal cancer, bladder cancer, stomach cancer, pancreatic cancer, prostate cancer, hodgkin lymphoma, follicular lymphoma, chronic or acute leukemia, solid tumor. The infectious diseases include, but are not limited to, HIV virus infection, hepatitis virus (type a, B, and C) infection, herpes virus infection, influenza virus infection. The immune system diseases include but are not limited to lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, myasthenia gravis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune hepatitis, scleroderma, polyarteritis nodosa, Wegener's granulomatosis.
Drawings
FIG. 1, plasmid map of pScFvDisb-s;
FIG. 2 shows an electrophoretogram of a heavy chain and a linker region amplified by using DFPD1-1 as a template in the construction of a light chain mutation library;
FIG. 3 is an electrophoresis diagram of a light chain library gene amplified using a synthesized light chain mutant library as a template in the construction of the light chain mutant library;
FIG. 4, electrophoretogram of VLCDR123M-DFPD1-1 mutant library obtained by amplification in light chain mutant library construction;
FIG. 5 is an electrophoretogram of the cleavage product of plasmid pScFvDisb-s by double cleavage with NcoI-HF and NotI in the light chain mutation library construction;
FIG. 6, monoclonal phageELISA, identifies the relative affinities of phage-Abs in light chain mutation libraries;
FIG. 7, gradient dilution phageELISA to identify the relative affinities of phage-Abs in light chain mutation libraries;
FIG. 8 is an electrophoretogram of heavy chain library genes amplified using a synthesized heavy chain mutant library as a template in the construction of a heavy chain mutant library;
FIG. 9 is an electrophoretogram of light chain and linker regions amplified by using DFPD1-3 and DFPD1-7 plasmids as templates in the construction of heavy chain mutation library;
FIG. 10, electrophoretogram of VHCDR123M-DFPD1-3 mutant library obtained by amplification in heavy chain mutant library construction;
FIG. 11, electrophoretogram of VHCDR123M-DFPD1-7 mutant library obtained by amplification in heavy chain mutant library construction;
FIG. 12, monoclonal phageELISA to identify the relative affinities of phage-Abs in heavy chain mutation libraries;
FIG. 13, gradient dilution phageELISA to identify the relative affinities of phage-Abs in heavy chain mutation libraries;
FIG. 14, map of pTSE plasmid vector;
FIG. 15, whole antibody binding assay with PD-1 at molecular level;
FIG. 16, competitive inhibition assay of whole antibody with PD-L1;
FIG. 17, PD-1 binding assay of whole antibodies to cell surface;
Detailed Description
The detailed implementation method of the invention refers to the examples, experimental methods and reagents described in the examples, and all the experimental methods and reagents are conventional unless otherwise specified. The following examples are intended only to illustrate and explain the invention and are not intended to limit the invention in any way.
The invention provides a monoclonal antibody specifically binding to PD-1, wherein the heavy chain variable region sequence comprises SEQ NO.1, 2, 3 and 4, and the light chain variable region sequence comprises SEQ NO.5, 6, 7, 8, 9, 10 and 11.
Preferably, the heavy chain variable region sequence of the monoclonal antibody specifically binding to PD-1 comprises seq id No.2, 3, 4 and the light chain variable region sequence is selected from seq id No.7, 11.
The amino acid sequences of the light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 of the antibody light chain or functional fragment thereof are selected from one of the following sets of amino acid sequences (as shown in table 2) by light chain phage library screening.
TABLE 2 amino acid sequences of the respective CDR regions of the light chain
| NO | LCDR1 | LCDR2 | LCDR3 |
| A | RASQNIHSYLD | EASTRAS | QQALKLPIT |
| B | RASQNVSNWLD | DASNRAT | QQSRHIPLT |
| C | RASQSIHNYLD | NASTRAT | QQELHLPLT |
| D | RASQDINNWLD | DASTLAT | QQNVNLPLT |
| E | RASQDVRTYLD | GASTRAT | QQDIDLPLT |
| F | RASQGINSWLD | DASTRAT | QQSYRLPLT |
| G | RASQSVSNYLD | DASTRAT | QQNMQLPLT |
The complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain of the antibody or functional fragment thereof are represented by HCDR1, HCDR2 and HCDR3, respectively, as screened by heavy chain phage library: HCDR1 is SNNGMH or SNYGMH; HCDR2 is either VIWYDGSKK, VIWYDSSRK or VIWYDSTKK; HCDR3 is TAVYYCATNNDYW or TAVYYCATNTDYW.
Preferably, the monoclonal antibody that specifically binds PD-1 comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 sequences, as screened by a heavy chain phage library. Wherein the sequence of the heavy chain variable region HCDR1 is selected from SNNGMH and SNYGMH amino acids; the light chain variable region LCDR1 sequence is an amino acid selected from RASQSIHNYLD, RASQSVSNYLD; the heavy chain variable region HCDR2 sequence is an amino acid selected from VIWYDGSKK, VIWYDSSRK; the light chain variable region LCDR2 sequence is an amino acid selected from NASTRAT, DASTRAT; the heavy chain variable region HCDR3 sequence is an amino acid selected from TAVYYCATNNDYW, TAVYYCATNTDYW; the light chain variable region LCDR3 sequence is selected from QQELHLPLT, QQNMQLPLT amino acids.
The invention relates to a method for obtaining a specific antibody by utilizing a fully synthetic ScFv single-chain phage antibody library, wherein the fully humanized monoclonal antibody specifically combined with PD-1 is obtained by utilizing phage antibody library technology screening, and the method comprises the following steps:
(1) and performing biological panning on the anti-PD-1 single-chain antibody, and obtaining an antibody sequence DFPD1-1 with higher affinity through three rounds of enrichment screening of an antibody library.
(2) Based on DFPD1-1, constructing light chain CDR1, 2 and 3 mutation libraries through computer aided design, performing biopanning on the antibody libraries and screening and identifying positive clones to obtain antibody sequences DFPD1-2, DFPD1-3, DFPD1-4, DFPD1-5, DFPD1-6 and DFPD1-7 of 6 different light chains. The 7 single-chain antibodies were compared for affinity at the phage level.
(3) Two clones DFPD1-3 and DFPD1-7 with high affinity are selected, heavy chain CDR1, 2 and 3 libraries are constructed, biopanning and positive clone screening are carried out on the libraries, and five single-chain antibodies with different sequences, namely DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13, are obtained. The single-chain antibodies were subjected to affinity comparison at the phage level.
(4) Cloning the monoclonal heavy chain variable region gene, the light chain variable gene and the light and heavy chain constant region gene in the step (3) to a eukaryotic expression vector, transfecting host cells to obtain the full antibody of the monoclonal antibody, and then comparing the affinity with other biological functions.
The specific embodiment is as follows:
the invention is described in detail below with reference to the figures and examples.
Example 1 biopanning of anti-PD-1 Single chain antibodies
A series of gene cloning methods are adopted to modify a pCom3 vector (purchased from China plasmid vector strain cell strain gene collection center) so as to be used for constructing and expressing a phage single-chain antibody library. The transformed vector is named pScFvDisb-s, the plasmid map of the transformed vector is shown in figure 1, and a fully synthetic phage antibody library is constructed on the basis of the vector.
The immune tube was coated with PD-1-His as antigen in an amount of 5. mu.g/500. mu.l/tube and was coated overnight at 4 ℃. Then 4% skimmed milk powder/PBST is used for respectively sealing the immune tube and the fully synthetic phage antibody library, and the immune tube and the fully synthetic phage antibody library are sealed for 1h at room temperature. Adding the sealed phage antibody library into an immune tube for antigen-antibody binding, wherein the phage input amount is about 109-1012And reacting at room temperature for 1 h. PBST-PBS washed away unbound phage, 0.1MPH2.2 Glycine-HCl elution, 1.5MPH8.8 Tris-HCl neutralization elution of phage antibody solution to pH 7.0.
And infecting 10ml of TG1 bacterial solution growing to the logarithmic phase with the neutralized phage, standing in an incubator at 37 ℃ for 30min, taking out part of the bacterial solution, performing gradient dilution, and coating on a 2YTAG plate for calculating the output of the phage. The remaining bacterial solution was centrifuged and the supernatant discarded, and the pellet was resuspended in a small volume of medium, aspirated and plated on a 2YTAG large plate in preparation for the next round of screening.
Scraping the infected and plated thallus from a large plate, inoculating the thallus to a 2YTAG liquid culture medium, shaking to logarithmic phase, adding M13 helper phage for superinfection, culturing at 28 ℃ overnight to amplify the phage, and depositing and purifying the phage by PEG/NaCl for the next round of screening. Three rounds of phage library enrichment screening were performed.
Example 2 screening of anti-PD-1 phage Single-chain antibody Positive clones
After three rounds of screening, well-separated monoclonal colonies were picked, inoculated into a 96-well deep-well plate containing 2YTAG liquid medium, cultured at 37 ℃ and 220rpm until its logarithmic phase, and added at about 10/well10The helper phage M13KO7 of (1), was statically infected at 37 ℃ for 30 min. 4000rpm, centrifugation for 15min, discarding supernatant, bacterial body with 2YTAK heavy suspension precipitation, 28 degrees C, 220rpm culture overnight. And (4) centrifuging at 4000rpm and 4 ℃ for 15min, and sucking amplified phage supernatant for ELISA identification. Screening to obtain single-chain antibody DFPD1-1 with high affinity, wherein the heavy chain variable region is named as DFPD1-H1The amino acid sequence is shown in SEQ NO. 1; the light chain variable region is named as DFPD1-L1, and the amino acid sequence is shown in SEQ NO. 5.
Example 3 in vitro affinity maturation of the selected anti-PD-1 Single-chain antibody DFPD1-1
Construction of the CDR1, 2, 3 mutant library of the light chain of DFPD1-1
Primers PVLF1 and PVLR1 were designed to PCR amplify light chain library genes (FIG. 3) using the synthesized light chain mutant library (SEQ NO.12) as a template; primers PVHF1 and PVHR1 were designed to amplify the heavy chain and linker regions using DFPD1-1 plasmid as a template (FIG. 2). Reaction conditions are as follows: 30s at 95 ℃,1 cycle; 95 ℃ for 15s, 60 ℃ for 10s, 72 ℃ for 30s,3 cycles; storing at 95 deg.C for 15s, 72 deg.C for 40s,25cycles,72 deg.C for 5min, and 4 deg.C. And recovering the PCR target fragment by using the universal recovery kit for the Tiangen.
The primers are shown below:
PVLF1：5’-GATATCCAGATGACCCAGAGC-3’
PVLR1：5’-CTAAGCGGCCGCTTTGATCTCCACTTTGGTGC-3’
PVHF1：5’-CATACCATGGCCCAGGTGCAGCTGGTGGAGTCTG-3’
PVHR1：5’-GCTCTGGGTCATCTGGATATCGGATCCACCACC-3’
and carrying out overlapPCR amplification on the two parts of PCR reaction products to obtain the light chain mutation library gene of DFPD 1-1. Reaction conditions are as follows: 30s at 95 ℃,1 cycle; 15s at 95 ℃, 30s at 72 ℃,4 cycles; (addition of primers PVHF1 and PVLR1),95 ℃ 15s, 72 ℃ 40s,25 cycles; preserving at 72 deg.C for 5min and 4 deg.C. The fragments of interest were recovered from the Tiangen Universal recovery kit and the corresponding PCR product was named VLCDR123M-DFPD1-1 (FIG. 4).
The plasmid pScFvDisb-s was double digested with NcoI-HF and NotI, the digested product was electrophoresed through 0.8% agarose gel (FIG. 5), and the gel was excised and recovered; the VLCDR123M-DFPD1-1PCR product was double digested with NcoI-HF and NotI, respectively. And recovering the PCR enzyme digestion product by using a universal recovery kit. The recovered PCR fragment was mixed with pscfvdib-s in a molar ratio of 4: ligation was performed at 16 ℃ for 4h with T4DNA ligase at a ratio of 1. The ligation products were electroporated into TG1 competence. The cells were cultured for 1 hour at 37 ℃ in SOC medium for recovery. Taking the bacteria liquid according to the proportion, coating a flat plate, and calculating the storage capacity. The rest bacterial liquid is centrifuged at 4000rpm for 15min at room temperature. The supernatant was discarded, the pellet was spread on a 2YTAG large plate, and cultured overnight at 37 ℃ in an inverted state.
Construction of an antibody library volume of about 108The accuracy of sequence analysis of 20 clones randomly picked from the antibody library is 95%, and the capacity of the antibody library far exceeds the diversity of the antibody library.
3.2. Biopanning of phage antibody libraries and screening for positive clones
Screening is carried out according to the method of example 1, clone sequencing with higher affinity is obtained, 6 different single-chain antibody sequences are obtained, and are respectively named as DFPD1-2, DFPD1-3, DFPD1-4, DFPD1-5, DFPD1-6 and DFPD1-7, the corresponding light chain variable regions are named as DFPD1-L2, DFPD1-L3, DFPD1-L4, DFPD1-L5, DFPD1-L6 and DFPD1-L7, and the corresponding amino acid sequences are respectively shown in SEQ NO.6, SEQ NO.7, SEQ NO SEQ NO.8, SEQ NO.9, SEQ NO.10 and SEQ NO. 11. The relative affinities of the monoclonal phageELISA for identifying the phage-Abs are shown in FIG. 3.
3.3. Identification of affinity of anti-PD 1 single-chain antibody by gradient dilution phageELISA
Clones obtained in this example 2 were subjected to monoclonal phage display and purification, and a phage gradient dilution ELISA experiment was performed to identify the affinity of the phage-Abs.
PD1-His was coated with carbonate buffer pH9.6 and was coated overnight at 4 ℃. PBST was washed three times and blocked at 4% mil-PBST 37 ℃ for 1 h. The purified phase was diluted three times with 4% mil-PBST, 100. mu.l of the diluted sample was added to each well and allowed to stand at room temperature for 1 h. The ELISA plate was washed with PBST, and the anti-M13-HRP monoclonal antibody diluted with 4% skim milk powder was added to the ELISA plate and left at room temperature for 1 h. The TMB color development kit develops color at room temperature for 5 min. With 2MH2SO4The color development was stopped, 50. mu.l/well. Enzyme labelThe optical density value is measured by a single wavelength of 450 nm. The results show that several different selected phage antibodies can bind to PD-1, the affinities of DFPD1-3 and DFPD1-7 are obviously higher than those of other clones (FIG. 4), and DFPD1-3 and DFPD1-7 are selected for further experiments.
Example 4 in vitro affinity maturation of selected anti-PD-1 Single-chain antibodies DFPD1-3, DFPD1-7
4.1 construction of DFPD1-3 and DFPD1-7 heavy chain CDR1, 2, 3 mutant libraries
Primers PVHF2 and PVHR2 were designed to PCR amplify the heavy chain pool gene using the synthetic heavy chain mutant pool (SEQ NO.13) as template (FIG. 8); primers PVLF2 and PVLR2 were designed to amplify light chain and linker regions respectively using DFPD1-3 and DFPD1-7 plasmids as templates (FIG. 9), wherein the left side is DFPD1-3 and the right side is DFPD 1-7. Reaction conditions are as follows: 30s at 95 ℃,1 cycle; 95 ℃ for 15s, 60 ℃ for 10s, 72 ℃ for 30s,3 cycles; 95 ℃ for 15s, 72 ℃ for 40s,25 cycles; preserving at 72 deg.C for 5min and 4 deg.C. And recovering the PCR target fragment by using the universal recovery kit for the Tiangen.
The primers are shown below:
PVHF2：‘5CATACCATGGCCCAGGTGCAGCTGGTGGAGTCTG3’
PVHR2：‘5TGAGGAGACGGTGACCAGGGTGCCCTG3’
PVLF2：‘5CTGGTCACCGTCTCCTCAGGTGGTGGTGGTAGC3’
PVLR2：‘5CTAAGCGGCCGCTTTGATCTCCACTTTGGTGC3’
the two-part PCR reaction product was amplified by overlapPCR to obtain heavy chain mutant library genes of DFPD1-3 and DFPD 1-7. Reaction conditions are as follows: 30s at 95 ℃,1 cycle; 15s at 95 ℃, 30s at 72 ℃,4 cycles; (addition of primers PVHF2 and PVLR2), 95 ℃ 15s, 72 ℃ 40s,25 cycles; preserving at 72 deg.C for 5min and 4 deg.C. The fragments of interest from PCR were recovered using the Tiangen Universal recovery kit and the corresponding products were named VHCDR123M-DFPD1-3 (FIG. 10) and VHCDR123M-DFPD1-7 (FIG. 11).
The plasmid pScFvDisb-s was double digested with NcoI-HF and NotI, the digested product was electrophoresed through 0.8% agarose gel (FIG. 5), and the gel was excised and recovered; the PCR products of VHCDR123M-DFPD1-3 and VHCDR123M-DFPD1-7 were double digested with NcoI-HF and NotI, respectively. And recovering the PCR enzyme digestion product by adopting a universal recovery kit for radix asparagi. The recovered PCR fragment was mixed with pscfvdib-s in a molar ratio of 4: ligation was performed at 16 ℃ for 4h with T4DNA ligase at a ratio of 1. The ligation products were electroporated into TG1 competence. The cells were cultured for 1 hour at 37 ℃ in SOC medium for recovery. Taking the bacteria liquid according to the proportion, coating a flat plate, and calculating the storage capacity. The rest bacterial liquid is centrifuged at 4000rpm for 15min at room temperature. The supernatant was discarded, the pellet was spread on a 2YTAG large plate, and cultured overnight at 37 ℃ in an inverted state.
2 different antibody libraries were constructed. Each antibody library volume was approximately 107The repertoire of antibodies far exceeds the diversity of antibody repertoires. 20 clones were randomly picked from the antibody library and subjected to sequence analysis, and the accuracy was 90%.
4.2 biopanning of phage antibody libraries and screening for positive clones
The two antibody libraries constructed above were subjected to phase display, purification and precipitation. Single chain antibodies against PD1 were then panned from the pool. The biopanning procedure for phage antibody libraries was the same as in example 1. The method for screening the anti-PD-1 single-chain antibody positive clone was the same as in example 2. As a result, 5 different anti-PD-1 antibody sequences were found to be co-screened, and were designated DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, and DFPD1-13, respectively. Wherein the light chain variable region sequences of DFPD1-9, DFPD1-11 and DFPD1-12 are DFPD1-L3, and the light chain variable region sequences of DFPD1-10 and DFPD1-13 are DFPD 1-L7; the heavy chain variable region sequences of DFPD1-9 and DFPD1-10 are DFPD1-H2, the heavy chain variable regions of DFPD1-11 and DFPD1-13 are DFPD1-H3, and the heavy chain variable region of DFPD1-12 is DFPD 1-H4. The relative affinities of the monoclonal phageELISA for identifying phage-Abs are shown in FIG. 12.
4.3. Identification of affinity of anti-PD 1 single-chain antibody by gradient dilution phageELISA
Clones obtained in example 4.2 were subjected to monoclonal phase display and purification, and a phase gradient dilution ELISA assay was performed to identify the affinity of phase-abs, as in 3.1 of example 3. The results showed that several different phage antibodies were selected to bind to PD1 with a slightly different affinity (FIG. 13), with DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, and DFPD1-13, which were selected for later testing.
Example 5 anti-PD-1 Whole antibodies DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, DFPD1-13 affinity identification
5.1 preparation of anti-PD-1 Total antibody
The heavy chain VH and light chain VK genes of the above antibody were cloned into the vector pTSE (FIG. 14) containing heavy chain and light chain constant region genes, and the pTSE vector encoding human constant region gamma 4 (see SEQ NO.14) and kappa chain (see SEQ NO.15), respectively (the structure of pTSE vector is shown in FIG. 14, and the preparation process is described in page 3 [0019] of the CN103525868A instruction). HEK293E cells were transiently transfected for whole antibody expression. The whole antibody protein was obtained by purification using AKTA instruments proteinA affinity column.
5.2BIAcoreX100 determination of the affinity of the Total antibody
The affinity of the whole antibody was determined by a capture method. Coupling of anti-human IgG to the surface of CM5 chip, dilution of DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13, respectively, ensured that about 300RU of antibody was captured by anti-human IgG. PD-1 was run through the stationary phase surface with a series of concentration gradients (1000nM, 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.8125nM, 3.9063nM,1.9531nM, 0.9766nM) and the affinity of the antibody was determined. It was found that the affinity of the screened antibodies did not differ too much (Table 3).
TABLE 3 determination of affinity constant of anti-PD 1 Total antibody
| Sample | ka(1/Ms) | kd(1/s) | KD |
| DFPD1-9 | 1.626E+4 | 1.045E-4 | 6.429E-9 |
| DFPD1-10 | 3.285E+4 | 1.300E-4 | 3.957E-9 |
| DFPD1-11 | 9.357E+3 | 1.015E-4 | 1.085E-8 |
| DFPD1-12 | 1.327E+4 | 2.975E-4 | 2.242E-8 |
| DFPD1-13 | 1.811E+4 | 1.079E-4 | 9.504E-9 |
5.3 binding experiments of the Whole antibody to PD-1
By pCarbonate buffer of H9.6 was coated with PD-1-His at 60 ng/well/100. mu.l overnight at 4 ℃. Wash five times with 300. mu.l/well PBST, add 1% BSA-PBS and block for 2h at 37 ℃. Different dilutions of total anti-DFPD 1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13 were added. The maximum concentration of five whole antibodies was 16. mu.g/ml, 11 gradients were made at 4-fold dilution, and the last well served as a negative control-i.e.dilution PBS alone was added and incubated for 1h at 37 ℃. The membrane was washed five times with 300. mu.l/well PBST and incubated for 1h at 37 ℃ with Anti-HumanFc-HRP secondary antibody diluted with 1% BSA-PBS1: 40000. Developing with TMB color development kit at 100 μ l/well for 8min at room temperature, and then developing with 2MH2SO4The color development was stopped, 50. mu.l/well. 450nm/630nm reading. The results are shown in FIG. 15, where all antibodies bound well to PD-1 molecules.
5.4 full antibody Competition inhibits PD-L1 binding to PD-1
PDL1-Fc was coated with carbonate at pH9.6 and overnight at 4 ℃. PBST was washed five times and blocked with 1% BSA-PBS37 ℃ for 2 h. Five kinds of whole antibodies, DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13, were diluted with 4. mu.g/ml PD1-His, respectively, and the molar ratio of the whole antibody to PD-1 was from 10: 1 start, five-fold gradient dilution, 9 dilutions per sample, and incubation for 1h at 37 ℃. PBST was washed five times, added with 1% BSA-PBS diluted HRP labeled mouse anti His antibody, 37 degrees C were incubated for 1 h. The TMB color development kit develops color at 100 mul/hole for 8min at room temperature. With 10% H2SO4The color development was stopped, 50. mu.l/well. 450nm/630nm reading. As shown in FIG. 16, DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13 all inhibited the binding of PD-1 to PD-L1.
Example 6 anti-PD-1 antibody binding assay to cell surface PD-1
Firstly, an over-expressed PD-1 CHO stable cell line is constructed and named as PD1-CHO, a 96-well plate is coated by gelatin, the digestion of PD1-CHO cells is stopped after pancreatin, the cell line is centrifugally resuspended and diluted to 2 × 105Cells/ml, 100. mu.l per well, in 96-well plates, × 6 rows of 12 wells, 2 × 104Cells/well, 5% CO2, cultured overnight at 37 ℃. The following day the medium was discarded and washed with 350. mu.l of pre-cooled PBSOnce, 2% freshly prepared PFA was fixed for 5min and washed 2 times with PBS.
anti-PD-1 full antibody diluted by times is added into a cell plate, and the diluent is PBS containing 0.5% BSA. The sample concentration was 8-fold diluted from 100. mu.g/ml for a total of 12 dilutions, and incubated at room temperature for 30 min. Then, the supernatant was discarded, washed with 350. mu.l of PBS for 3 times, and then a 1:5000 dilution of horseradish peroxidase-labeled goat anti-human secondary antibody was added, followed by incubation at room temperature for 15 min.
Then washed 3 times with 350. mu.l PBS, 100. mu.l TMB color developing solution is added into each hole, and color development is carried out for 15-30min at room temperature. The color development was stopped by adding 50. mu.l of 2MH2SO4 to each well and reading was done with a microplate reader at 450 nm. The results were processed with Graphpadprism software and binding constants were calculated (see figure 17).
It is obvious to those skilled in the art that the present invention is not limited to the above embodiments, and it is within the scope of the present invention to adopt various insubstantial modifications of the method concept and technical scheme of the present invention, or to directly apply the concept and technical scheme of the present invention to other occasions without modification.