WO2016197497A1 - 一种抗pd-1的单克隆抗体及其获得方法 - Google Patents

一种抗pd-1的单克隆抗体及其获得方法 Download PDF

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WO2016197497A1
WO2016197497A1 PCT/CN2015/091842 CN2015091842W WO2016197497A1 WO 2016197497 A1 WO2016197497 A1 WO 2016197497A1 CN 2015091842 W CN2015091842 W CN 2015091842W WO 2016197497 A1 WO2016197497 A1 WO 2016197497A1
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antibody
dfpd1
seq
heavy chain
monoclonal antibody
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French (fr)
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周海平
李晓敏
周俊杰
裴爽
昝琰璐
白义
白先宏
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Beijing Jingyitaixiang Technology Development Co Ltd
Beijing Dongfang Biotech Co Ltd
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Beijing Jingyitaixiang Technology Development Co Ltd
Beijing Dongfang Biotech Co Ltd
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Priority to NZ728874A priority patent/NZ728874A/en
Priority to CA2972833A priority patent/CA2972833C/en
Priority to BR112017010744-9A priority patent/BR112017010744B1/pt
Priority to JP2017510906A priority patent/JP6588534B2/ja
Priority to EP20191281.3A priority patent/EP3770177A1/en
Priority to EA201792584A priority patent/EA036779B1/ru
Priority to US15/501,812 priority patent/US20170240644A1/en
Priority to AU2015398193A priority patent/AU2015398193B2/en
Priority to MX2017008662A priority patent/MX372821B/es
Priority to KR1020177004050A priority patent/KR101876689B1/ko
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    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the invention relates to the technical field of antibody engineering, in particular to a monoclonal antibody against PD-1 which is fully human, and a method and application thereof.
  • Immunoregulation plays an extremely important role in the body's immune response, and activation of immune-active cells is critical to the regulation of the overall immune response. Studies have shown that T cell activation and proliferation are dependent on a dual signaling pathway. The concept of "co-stimulatory signal" was proposed by Bretscher and Cohn in the 1970 T-cell activation dual-signal model, that is, T cell activation requires not only the presentation of MHC-antigen peptide complex by APC to antigen-specific T cells.
  • a variety of co-stimulatory molecules are required to participate in the provision of the auxiliary second signal (co-stimulatory signal); as the research progresses, the synergistic stimulation signal gradually becomes a hotspot of immunology; these synergistic signal molecules mainly include Two superfamilies of CD28/B7 and TNFR/TNF.
  • PD-1/PD-L1 a member of the CD28/B7 superfamily, mediates negative costimulatory signals.
  • PD-1/PD-L1 signaling pathway can effectively inhibit the function of T and B cells, inhibit the proliferation of T cells, and reduce the secretion of cytokines IL-2, IL-10 and IFN- ⁇ , which plays an important role in immune regulation. Its role, and in the study of tumor immunity, autoimmunity, transplant immunity, asthma, viral infections and other diseases have important significance.
  • PD-1 belongs to the immunoglobulin superfamily type I transmembrane protein with a molecular weight of approximately 50-55 KD. Originally obtained by subtractive hybridization in apoptotic T cell hybridoma, it was named Programmed Cell Death 1 because it is involved in apoptosis.
  • the coding gene of PD-1 is PD-CD1, which is located on 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, and its extracellular region contains an immunoglobulin variable region IgV-like domain; two tyrosine residues at the N-terminus of the intracellular region and other amino acids The residues together constitute an immunoreceptor tyrosine inhibition motif (ITIM), which exerts a function of antagonizing the antigen receptor stimulation signal by phosphorylation of tyrosine, thereby exerting a negative regulatory function in the immune response process; PD -1 molecule can be induced in the surface of activated T cells, B cells, NK cells, monocytes and dendritic cells, and combined with its ligands PD-L1, PD-L2 to activate lymphocytes Inhibition, thereby suppressing the immune response of immune cells.
  • ITIM immunoreceptor tyrosine inhibition motif
  • 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 composed of an IgV-like domain, an IgC-like domain, a transmembrane region, and a short and conserved cytoplasmic tail in the protein structure; Compared with PD-L2, the cytoplasmic tail of PD-L1 is more conserved among different species.
  • PD-L1 is activated T cells, B cells, dendritic cells, monocytes and various types of tumor cells (such as lung cancer, liver cancer, breast cancer, ovarian cancer, kidney cancer, head and neck cancer, esophageal cancer, skin cancer). Inducible expression on squamous cell carcinoma, etc.; PD-L2 is mainly expressed on activated macrophages, dendritic cells and individual tumor cells (such as Hodgkin's lymphoma). Tumor surface The interaction of PD-L1 with PD-1 can lead to tumor antigen-specific T cell apoptosis, allowing tumor cells to escape from offline immune surveillance.
  • Anti-PD-1 monoclonal antibody promotes tumor antigen-specific T cell proliferation by blocking PD-1/PD-L1 signaling pathway, plays a role in killing tumor cells, can effectively improve the effect of immunotherapy, and has the potential to treat various types of tumors. .
  • human-derived antibodies are the main direction of therapeutic antibody development, 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 development of the monoclonal antibody in the past, and even obtains various antibody genes and antibody molecular fragments without the need of an immunological process.
  • the phage antibody library is the earliest and currently the most widely used antibody library.
  • the phage display technology is a technique first established by Smith to insert a gene encoding a foreign protein or polypeptide into a phage coat protein gene, and to express a foreign protein or polypeptide to a phage surface by fusion with a phage coat protein.
  • the phage antibody library utilizes the above principle to express different specific antibodies or functional fragments thereof (Fab, Fv, ScFv) on the surface of phage, and then screened with antigen.
  • the phage antibody library is divided into an immunological library and a non-immune library according to the source of the antibody gene, and the non-immune library includes a natural library, a semi-synthetic library, and a fully synthetic library.
  • Screening of phage antibody libraries mimics the process of antibody affinity maturation, usually by coating the antigen on a solid phase medium, adding the phage antibody library to be screened, and through several rounds of "adsorption-wash-elution-amplification” processes (ie Panning) until screening for high affinity specific antibodies.
  • the present invention provides a monoclonal antibody against PD-1; the present invention screens a monoclonal antibody against PD-1 from a fully synthetic antibody library, and then constructs a small-capacity synthetic phage antibody light chain library by computer-aided design analysis.
  • the method is to establish a mutation in the CDR1, 2, and 3 regions of the complementarity determining region of the anti-PD-1 monoclonal antibody DFPD1-1 light chain obtained by the primary screening; after screening, select the higher affinity monoclonal antibodies DFPD1-3 and DFPD1. -7, and then the heavy-chain CDR1, 2, and 3 region mutations were screened, and finally a high-affinity monoclonal antibody against PD-1 was screened.
  • the process for obtaining a monoclonal antibody against PD-1 of the present invention includes:
  • DFPD1-1 Biopanning of anti-PD-1 single-chain antibody, a high affinity antibody sequence DFPD1-1 was obtained from the fully synthetic ScFv phage library by enrichment screening of three rounds of antibody library.
  • the strand is DFPD1-H1 (SEQ NO. 1) and the light chain is DFPD1-L1 (SEQ NO. 5).
  • the antibody sequences of six different light chains are DFPD1-2, DFPD1-3, DFPD1-4, DFPD1-5, DFPD1-6, DFPD1-7, and their corresponding light chain sequences are 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), DFPD1-L7 (SEQ NO. 11);
  • the above seven single-chain antibodies were compared for affinity at the phage level.
  • DFPD1-3 and DFPD1-7 Two clones with high affinity, DFPD1-3 and DFPD1-7, were selected, and the CDR1, 2, and 3 libraries of the heavy chain complementarity determining region were constructed and the library was screened for biopanning and positive clones.
  • the light chain variable region sequences of DFPD1-9, DFPD1-11 and DFPD1-12 are DFPD1-L3, while the light chain variable region sequences of DFPD1-10 and DFPD1-13 are DFPD1-L7; DFPD1-9 and DFPD1-
  • the heavy chain variable region sequence of 10 is the heavy chain variable region of DFPD1-H2 (SEQ NO. 2), DFPD1-11 and DFPD1-13 Is DFPD1-H3 (SEQ NO. 3), and the heavy chain variable region of DFPD1-12 is DFPD1-H4 (SEQ NO. 4). Affinity comparisons of the above single chain antibodies were performed at the phage level.
  • the monoclonal antibody against PD-1 obtained by the above method comprises: a light chain and a heavy chain; the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are represented by LCDR1, LCDR2 and LCDR3, respectively;
  • LCDR1 comprises RASQNIHSYLD, RASQNVSNWLD, Any of RASQSIHNYLD, RASQDINNWLD RASQDVRTYLD, RASQGINSWLD or RASQSVSNYLD;
  • LCDR2 includes any of EASTRAS, DASNRAT, NASTRAT, DASTLAT, GASTRAT or DASTRAT;
  • LCDR3 includes QQALKLPIT, QQSRHIPLT, QQELHLPLT, QQNVNLPLT, QQDIDLPLT, QQSYRLPLT or QQNMQLPLT Any one.
  • 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.kind.
  • the monoclonal antibody against PD-1 obtained by the above method comprises: a light chain and a heavy chain; the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are represented by HCDR1, HCDR2 and HCDR3, respectively; HCDR1 comprises SNNGMH or SNYGMH; HCDR2 comprises any of VIWYDGSKK, VIWYDSSRK or VIWYDSTKK; HCDR3 comprises TAVYYCATNNDYW or TAVYYCATNTDYW.
  • heavy chain variable region amino acid sequence comprises any one of SEQ NO. 1, SEQ NO. 2, SEQ NO. 3 or SEQ NO.
  • the present invention also provides an antibody, polypeptide or protein comprising the above light chain or the above heavy chain.
  • the present invention also provides an antibody comprising the above light chain or the above heavy chain, which is capable of blocking the binding of PD-1 to its ligand PD-L1 and inhibiting the biological activity of PD-1.
  • the present invention also provides a polynucleotide sequence or combination comprising the above light chain or the above heavy chain.
  • the present invention also provides a recombinant DNA expression vector comprising the above polynucleotide sequence or combination; the DNA sequence of the vector comprises a heavy chain variable region encoding an anti-PD1 antibody and a constant region or a light chain variable region And the amino acid sequence of the constant region sequence.
  • the present invention also provides a host cell transfected with the above recombinant DNA expression vector, the host cell comprising a prokaryotic cell such as Escherichia coli, a yeast or a mammalian cell.
  • a prokaryotic cell such as Escherichia coli, a yeast or a mammalian cell.
  • the host cell comprises HEK293E cells, CHO cells or NSO cells.
  • the present invention also provides an antibody comprising the above light chain or the above heavy chain for use in a total antibody, a single chain antibody, a single domain antibody, a bispecific antibody, an antibody drug conjugate or a chimeric antigen receptor T cell Immunotherapy.
  • the present invention also provides a monoclonal antibody, an artificial vector, a drug or a drug containing the above light chain or the above heavy chain Pharmaceutical composition
  • the present invention also provides a detection reagent or kit comprising the above light chain or the above heavy chain.
  • the monoclonal antibody against PD-1 comprises a full length antibody and a fragment of an anti-PD-1 monoclonal antibody, including but not limited to Fab, Fab', F(ab') 2 , Fv or ScFv.
  • anti-PD-1 heavy chain constant region comprising a monoclonal antibody IgG1, IgG2, IgG3 and lgG4; comprising a light chain constant region C ⁇ or C ⁇ .
  • the constant region is IgG4.
  • the light chain constant region is C ⁇ .
  • the CDR is a complementarity-determining region
  • the ScFv is a single-chain fragment variable
  • the ADCs are antibody-drug conjugates
  • -T is Chimeric Antigen Receptor T-Cell Immunotherapy
  • the HEK293E cells are human embryonic kidney 293E cells
  • CHO cells are Chinese hamster ovary cells (Chinese hamster ovary cells)
  • NS0 cells are mouse NSO thymoma cells.
  • the invention has the following beneficial effects:
  • the monoclonal antibody provided by the present invention can prevent or treat a disease by eliminating or inhibiting PD-1 activity, wherein the disease is selected from a cancer, an infectious disease, or an immune system disease.
  • 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's 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, type B, and type C) infection, herpes virus infection, and influenza virus infection.
  • the immune system diseases include, without limitation, lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, myasthenia gravis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune hepatitis, scleroderma, nodularity Arteritis, Wegener granulomatosis.
  • FIG. 1 Electrophoresis map of a light chain library gene amplified by a synthetic light chain mutant library as a template in the construction of a light chain mutation library;
  • Figure 8 Electropherogram of a heavy chain library gene amplified by a synthetic heavy chain mutant library as a template in the construction of a heavy chain mutant library
  • Figure 14 Figure of the pTSE plasmid vector
  • Figure 17 PD-1 binding assay of whole antibody to cell surface
  • the present invention provides a monoclonal antibody that specifically binds to PD-1, the heavy chain variable region sequence comprising SEQ NO. 1, 2, 3, 4, and the light chain variable region sequence comprising SEQ NO. 6, 6, 8, 9, 10, 11.
  • the heavy chain variable region sequence of the monoclonal antibody that specifically binds to PD-1 comprises SEQ NO. 2, 3, 4, and the light chain variable region sequence is selected from the group consisting of SEQ NO.
  • amino acid sequences of the light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 of the antibody light chain or a functional fragment thereof are selected from the group consisting of the following respective amino acid sequences (as shown in Table 1) by light chain phage library screening.
  • the CDR1, CDR2 and CDR3 of the antibody heavy chain or its functional fragment heavy chain are represented by HCDR1, HCDR2 and HCDR3, respectively, by the heavy chain phage library: HCDR1 is SNNGMH or SNYGMH; HCDR2 is VIWYDGSKK, VIWYDSSRK or VIWYDSTKK Any of them; HCDR3 is TAVYYCATNNDYW or TAVYYCATNTDYW.
  • the monoclonal antibody that specifically binds to PD-1 comprises a heavy chain variable region of the HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences by screening of a heavy chain phage library.
  • the heavy chain variable region HCDR1 sequence is an amino acid selected from the group consisting of SNNGMH, SNYGMH;
  • the light chain variable region LCDR1 sequence is an amino acid selected from the group consisting of RASQSIHNYLD, RASQSVSNYLD;
  • the heavy chain variable region HCDR2 sequence is selected from An amino acid of 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 the group consisting of TAVYYCATNNDYW, TAVYYCATNTDYW; and the light chain variable region LCDR3
  • the sequence is an amino acid selected from the group consisting of QQELHLPLT and QQNMQLPLT.
  • the invention utilizes a method for obtaining a specific antibody by using a fully synthetic ScFv single-chain phage antibody library, which is obtained by screening a monoclonal antibody specific for PD-1 by using a phage antibody library technology, and the steps thereof are as follows:
  • the vector pCom3 vector (purchased from the Chinese plasmid vector strain cell strain gene collection) was modified by a series of gene cloning methods for use in the construction and expression of a phage single-chain antibody library.
  • the transformed vector was named pScFvDisb-s, and its plasmid map is shown in Figure 1. Based on this vector, a fully synthetic phage antibody library was constructed.
  • the tube was immunized with PD-1-His as an antigen, and the antigen coating amount was 5 ug/500 ul/tube, and coated at 4 ° C overnight.
  • the immunotubes and the fully synthetic phage antibody library were blocked with 4% skim milk powder/PBST, respectively, and blocked at room temperature for 1 h.
  • the blocked phage antibody library was added to the immunotube for antigen-antibody binding, and the phage input amount was about 10 9 -10 12 , and the reaction was carried out at room temperature for 1 h.
  • the above-mentioned neutralized phage was infected with 10 ml of TG1 bacterial solution grown to log phase, and allowed to stand in a 37 ° C incubator for 30 min, and some of the bacterial liquid was taken out for gradient dilution and applied to 2YTAG plates for calculation of phage yield. .
  • the remaining bacterial liquid was centrifuged to discard the supernatant, and the bacterial pellet was resuspended in a small amount of medium, and then aspirated and applied to a 2YTAG large plate to prepare for the next round of screening.
  • the above-mentioned infected plate was scraped from the large plate, and the cells were inoculated to 2YTAG liquid medium. After the logarithmic phase, the M13 helper phage super infection was added, and the phage was expanded overnight at 28 ° C, and the PEG/NaCl was purified by sedimentation. Phage were used for the next round of screening. A total of three rounds of phage library enrichment screening were performed.
  • the well-separated monoclonal colonies were picked and inoculated into 96-well deep-well plates supplemented with 2YTAG liquid medium, and cultured at 37 ° C, 220 rpm to their logarithmic growth phase, adding about 10 10 per well.
  • the phage M13KO7 was infected at 37 ° C for 30 min. After centrifugation at 4000 rpm for 15 min, the supernatant was discarded, and the cells were resuspended in 2YTAK and cultured overnight at 28 ° C, 220 rpm. After centrifugation at 4000 rpm for 15 min at 4 ° C, the amplified phage supernatant was aspirated for ELISA.
  • the single-chain antibody DFPD1-1 with higher affinity was screened, and its heavy chain variable region was named DFPD1-H1, its amino acid sequence is shown in SEQ NO.1; its light chain variable region is named 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
  • the primers PVLF1 and PVLR1 were designed to synthesize the light chain library gene using the synthetic light chain mutation library (SEQ NO. 12) as a template (Fig. 3); the primers PVHF1 and PVHR1 were designed to amplify the heavy chain with the DFPD1-1 plasmid as a template. And the linker area ( Figure 2). Reaction conditions: 95 ° C for 30 s, 1 cycle; 95 ° C for 15 s, 60 ° C for 10 s, 72 ° C for 30 s, 3 cycles; 95 ° C for 15 s, 72 ° C for 40 s, 25 cycles, 72 ° C for 5 min, 4 ° C storage. The PCR target fragment was recovered by the Tiangen Universal Recovery Kit.
  • the primers are as follows:
  • the two-part PCR reaction product was subjected to Overlap PCR amplification to obtain a light chain mutant library gene of DFPD1-1.
  • Reaction conditions 95 ° C for 30 s, 1 cycle; 95 ° C for 15 s, 72 ° C for 30 s, 4 cycles; (add primers PVHF1 and PVLR2), 95 ° C for 15 s, 72 ° C for 40 s, 25 cycles; 72 ° C for 5 min, stored at 4 ° C.
  • the PCR target fragment was recovered by the Tiangen Universal Recovery Kit, and the corresponding PCR product was named VLCDR123M-DFPD1-1 (Fig. 4).
  • the plasmid pScFvDisb-s was digested with NcoI-HF and NotI, and the digested product was subjected to 0.8% agarose gel electrophoresis (Fig. 5), and the gel was recovered.
  • the VLCDR123M-DFPD1-1 PCR product was separately treated with NcoI-HF and NotI. Perform double digestion.
  • the PCR-cut products were recovered using a universal recovery kit.
  • the recovered PCR fragment was ligated with pScFvDisb-s in a ratio of 4:1 by T4 DNA ligase at 16 ° C for 4 h.
  • the ligation product was electroporated into a TG1 competent state.
  • the culture was incubated for 1 h at 37 ° C using SOC medium.
  • the bacteria solution was taken in proportion and plated to calculate the library capacity.
  • the remaining bacterial solution was centrifuged at 4000 rpm for 15 min at room temperature. The supernatant was discarded, and the pellet was plated on a 2YTAG large plate and cultured at 37 ° C overnight.
  • the library capacity of the constructed antibody library was about 10 8 , and 20 clones were randomly picked from the above antibody library for sequence analysis with a correct rate of 95%.
  • the antibody library storage capacity far exceeded the diversity of the antibody library.
  • DFPD1-7 the corresponding light chain variable regions are named DFPD1-L2, DFPD1-L3, DFPD1-L4, DFPD1-L5, DFPD1-L6, DFPD1-L7, and their corresponding amino acid sequences are shown in SEQ NO. SEQ NO. 7, SEQ NO. 8, SEQ NO. 9, SEQ NO. 10, and SEQ NO.
  • the relative affinities of phage-Abs identified by monoclonal phage ELISA are shown in Figure 3.
  • the clone obtained in this Example 2 was subjected to display and purification of a monoclonal phage, and a phage gradient dilution ELISA assay was performed to identify the affinity of the phage-Abs.
  • PD1-His was coated with carbonate buffer pH 9.6 and coated overnight at 4 °C.
  • the cells were washed three times with PBST and blocked with 4% milk-PBST at 37 ° C for 1 h.
  • the purified phage was diluted three times with 4% milk-PBST, and 100 ul of the diluted sample was added to each well, and allowed to stand at room temperature for 1 hour.
  • 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 allowed to stand at room temperature for 1 h.
  • the TMB color development kit was developed in color and developed at room temperature for 5 min.
  • the color development was stopped with 2M H 2 SO 4 , 50 ⁇ l/well.
  • the optical density value was measured by a microplate reader at a single wavelength of 450 nm.
  • the results showed that several different phage antibodies were able to bind to PD-1, and the affinity of DFPD1-3 and DFPD1-7 was significantly higher than that of other clones (Fig. 4).
  • DFPD1-3 and DFPD1-7 were selected.
  • Example 4 in vitro affinity maturation of the single-chain antibody DFPD1-3, DFPD1-7, which was screened against PD-1
  • the primers PVHF2 and PVHR2 were designed to synthesize the heavy chain library gene using the synthetic heavy chain mutation library (SEQ NO. 13) as a template (Fig. 8).
  • the primers PVLF2 and PVLR2 were designed with DFPD1-3 and DFPD1-7 as templates. Amplify its light chain and linker region (Fig. 9), with DFPD1-3 on the left and DFPD1-7 on the right. Reaction conditions: 95 ° C for 30 s, 1 cycle; 95 ° C for 15 s, 60 ° C for 10 s, 72 ° C for 30 s, 3 cycles; 95 ° C for 15 s, 72 ° C for 40 s, 25 cycles; 72 ° C for 5 min, stored at 4 ° C.
  • the PCR target fragment was recovered by the Tiangen Universal Recovery Kit.
  • the primers are as follows:
  • the two-part PCR reaction product was subjected to Overlap PCR amplification to obtain heavy chain mutant library genes of DFPD1-3 and DFPD1-7.
  • Reaction conditions 95 ° C for 30 s, 1 cycle; 95 ° C for 15 s, 72 ° C for 30 s, 4 cycles; (add primers PVHF 2 and PVLR 2), 95 ° C for 15 s, 72 ° C for 40 s, 25 cycles; 72 ° C for 5 min, stored at 4 ° C.
  • the PCR target fragment was recovered by 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 digested with NcoI-HF and NotI, and the digested product was subjected to 0.8% agarose gel electrophoresis (Fig. 5), and the gel was recovered; NcoI-HF and NotI were used to respectively VHCDR123M-DFPD1-3 and The VHCDR123M-DFPD1-7 PCR product was double digested.
  • the PCR-cut products were recovered using the Tiangen Universal Recovery Kit.
  • the recovered PCR fragment was ligated with pScFvDisb-s in a ratio of 4:1 by T4 DNA ligase at 16 ° C for 4 h.
  • the ligation product was electroporated into a TG1 competent state.
  • the culture was incubated for 1 h at 37 ° C using SOC medium.
  • the bacteria solution was taken in proportion and plated to calculate the library capacity.
  • the remaining bacterial solution was centrifuged at 4000 rpm for 15 min at room temperature. The supernatant was discarded, and the pellet was plated on a 2YTAG large plate and cultured at 37 ° C overnight.
  • Each antibody library has a library capacity of approximately 10 7 , and the antibody library capacity far exceeds the diversity of the antibody library. Twenty clones were randomly picked from the above antibody library for sequence analysis, and the correct rate was 90%.
  • the two antibody libraries constructed above were subjected to phage display, purification and precipitation. Single-chain antibodies against PD1 were then panned from the pool.
  • the biopanning method of the phage antibody library is the same as in the case 1.
  • the screening method for single-chain antibody-positive clones resistant to PD-1 was the same as in Example 2. It was found that a total of five different anti-PD-1 antibody sequences were screened and named as DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, DFPD1-13.
  • the light chain variable region sequence of DFPD1-9, DFPD1-11 and DFPD1-12 is DFPD1-L3, and the light chain variable region sequences of DFPD1-10 and DFPD1-13 are DFPD1-L7; DFPD1-9 and DFPD1-
  • the heavy chain variable region sequence of 10 is DFPD1-H2
  • the heavy chain variable region of DFPD1-11 and DFPD1-13 is DFPD1-H3
  • the heavy chain variable region of DFPD1-12 is DFPD1-H4.
  • the relative affinities of phage-Abs by monoclonal phage ELISA are shown in Figure 12.
  • the clone obtained in the present Example 4.2 was subjected to display and purification of a monoclonal phage, and the affinity of the phage-abs was identified by a phage gradient dilution ELISA test in the same manner as in Example 3, 3.1.
  • the results showed that several different phage antibodies were able to bind to PD1, and the affinity was not very different (Fig. 13), among which DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12, DFPD1-13 were slightly better. These single-chain antibodies were selected for subsequent testing.
  • the heavy chain VH and light chain VK genes of the above antibodies were separately cloned into the vector pTSE (Fig. 14) containing the heavy and light chain constant region genes, encoding the human constant region ⁇ 4 (see SEQ NO. 14) and the kappa chain (see The pTSE vector of SEQ NO. 15) (pTSE vector structure is shown in Figure 14, and the preparation process is described in paragraph [0019] on page 3 of the specification of CN103525868A).
  • HEK293E cells were transiently transfected for full antibody expression. Whole antibody protein was obtained using an AKTA instrument protein A affinity column purification.
  • the affinity of the whole antibody was determined by the capture method.
  • Anti-human IgG was coupled to the surface of the CM5 chip, and DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13 were diluted, respectively, to ensure that approximately 300 RU of antibody was captured by anti-human IgG.
  • PD-1 was set to a series of concentration gradients (1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.9063 nM, 1.9531 nM, 0.9766 nM) through the surface of the stationary phase to determine the affinity of the antibody. .
  • concentration gradients 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.9063 nM, 1.9531
  • PD-1-His was coated with a pH 9.6 carbonate buffer, 60 ng/well/100 ⁇ l, and coated overnight at 4 °C. Wash five times with 300 ⁇ l/well PBST, and then add 1% BSA-PBS for 2 h at 37 °C. Different dilutions of total anti-DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13 were added. The highest concentration of the five whole antibodies was 16 ug/ml, and 11 gradients were used to make 11 gradients. The last well was used as a negative control—that is, only the dilution PBS was added and incubated at 37 ° C for 1 h.
  • the cells were washed five times with 300 ⁇ l/well PBST, and then incubated with 1% BSA-PBS 1:40000 diluted Anti-Human Fc-HRP secondary antibody for 1 h at 37 °C.
  • the TMB color development kit was developed in color, 100 ⁇ l/well, and developed at room temperature for 8 min, and then stopped color development with 2 M H 2 SO 4 , 50 ⁇ l/well. 450 nm / 630 nm reading.
  • the experimental results are shown in Figure 15. All antibodies bind well to the PD-1 molecule.
  • PDL1-Fc was coated with carbonate at pH 9.6 and coated overnight at 4 °C. Wash PBST five times and block with 1% BSA-PBS for 2 h at 37 °C.
  • the four whole antibodies of DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13 were diluted with 4 ⁇ g/ml of PD1-His, and the molar ratio of whole antibody to PD-1 started from 10:1. Five-fold gradient dilutions, 9 dilutions per sample, and 1 h incubation at 37 °C.
  • the PBST was washed five times, and HRP-labeled mouse anti-His antibody diluted with 1% BSA-PBS was added and incubated at 37 ° C for 1 h.
  • the TMB color development kit was developed in color, 100 ⁇ l/well, and developed at room temperature for 8 min. Color development was stopped with 10% H 2 SO 4 , 50 ⁇ l/well. 450 nm / 630 nm reading.
  • DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 and DFPD1-13 all inhibited the binding of PD-1 to PD-L1 (see Fig. 16 for the results).
  • the overexpressed CHO stable cell line of PD-1 was first constructed and named PD1-CHO. After coating 96-well plates with gelatin, PD1-CHO cells were trypsinized and then stopped, resuspended by centrifugation, diluted to 2 ⁇ 10 5 cells/ml, and 100 ⁇ l per well was plated in 96-well plates for 12 wells ⁇ 6 rows. That is, 2 ⁇ 10 4 cells/well, 5% CO 2 , and cultured at 37 ° C overnight. The medium was discarded the next day, washed once with 350 ul of pre-cooled PBS, 2% freshly prepared PFA was fixed for 5 min, and washed twice with PBS.
  • the diluted anti-PD-1 total anti-antibody was added to the cell plate, and the dilution was PBS containing 0.5% BSA.
  • the sample concentration was from 100 ug/ml, diluted 8 times, a total of 12 dilutions, and incubated for 30 min at room temperature. The supernatant was then discarded, washed 3 times with 350 ul PBS, and a 1:5000 dilution of horseradish peroxidase-labeled goat anti-human secondary antibody was added and incubated for 15 min at room temperature.

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Abstract

本发明属于抗体工程技术领域，提供了一种全人源的抗PD-1的单克隆抗体、其制备方法及用途。从全合成抗体库中筛选得到抗PD-1的单克隆抗体，然后通过亲和力成熟技术依次对所得抗体的轻链CDR1-3区和重链CDR1-3区突变建库，筛选获得高亲和力的抗PD-1的抗体。所述抗体可用于肿瘤、炎症、自身免疫性疾病的治疗。

Description

一种抗PD-1的单克隆抗体及其获得方法 技术领域
本发明涉及抗体工程技术领域，具体涉及全人源的抗PD-1的单克隆抗体、及其获得方法与应用。
背景技术
免疫调节在机体免疫应答过程中起着极其重要的作用，而免疫活性细胞的活化对整个免疫应答的调节极其关键。研究表明，T细胞活化和增殖依赖于双重信号途径。“协同刺激信号”的概念是1970年Bretscher和Cohn在T细胞活化双信号模型的基础上提出来的，即T细胞的活化不仅需要通过APC递呈MHC-抗原肽复合物给抗原特异性T细胞来提供第一信号外，还需要多种协同刺激分子参与提供辅助的第二信号(协同刺激信号)；随着研究的深入，协同刺激信号逐渐成为免疫学的热点领域；这些协同信号分子主要包括CD28/B7和TNFR/TNF两大超家族。PD-1/PD-L1作为CD28/B7超家族的成员，能介导负性协同刺激信号。PD-1/PD-L1信号通路能有效抑制T、B细胞功能，使T细胞增殖受抑，同时减少细胞因子IL-2、IL-10和IFN-γ的分泌，在免疫调节中发挥着重要的作用，并在肿瘤免疫、自身免疫、移植免疫、哮喘、病毒感染等疾病的研究中有着重要的意义。
PD-1属于免疫球蛋白超家族I型跨膜蛋白，分子量约为50-55KD。最初是在凋亡的T细胞杂交瘤中通过消减杂交技术得到的，因其与细胞凋亡有关故命名为程序性细胞死亡因子1(Programmed Cell Death 1)。PD-1的编码基因是PD-CD1，定位于人染色体2q37.3，和CTLA4基因具有23％的同源性。PD-1由胞内区、跨膜区和胞外区构成，其胞外区包含一个免疫球蛋白可变区IgV样结构域；其胞内区N端的两个酪氨酸残基与其他氨基酸残基共同组成了一个免疫受体酪氨酸抑制基序(ITIM)，ITIM通过酪氨酸的磷酸化发挥拮抗抗原受体刺激信号的功能，从而在免疫应答过程中发挥负性调控功能；PD-1分子可在活化的T细胞、B细胞、NK细胞、单核细胞和树突状细胞表面诱导性地表达，并与其配体PD-L1、PD-L2相结合而对淋巴细胞的活化产生抑制作用，从而抑制免疫细胞的免疫应答反应。
PD-L1(CD274或B7H1)和PD-L2(CD273或B7DC)是PD1的两个配体，这两个配体基因均定位于人染色体9p24.2，并起始于同一方向，间隔42kb左右；它们同属B7家族，因而同其他成员一样，在蛋白结构上PD-L1和PD-L2均由IgV样结构域、IgC样结构域、跨膜区和短而保守的胞质区尾巴组成；与PD-L2相比，PD-L1的胞质尾巴在不同种属间更为保守。PD-L1在活化的T细胞、B细胞、树突状细胞、单核细胞和多种类型的肿瘤细胞(如肺癌、肝癌、乳腺癌、卵巢癌、肾癌、头颈癌、食道癌、皮肤癌、鳞状细胞癌等)上诱导性表达；PD-L2主要表达于活化的巨噬细胞、树突细胞和个别肿瘤细胞(如霍奇金淋巴瘤)上。肿瘤表面的 PD-L1与PD-1相互作用，可导致肿瘤抗原特异性T细胞凋亡，使肿瘤细胞逃脱机体免疫监控。抗PD-1的单抗通过阻断PD-1/PD-L1信号通路促进肿瘤抗原特异性T细胞增殖，发挥杀伤肿瘤细胞的作用，能有效提高免疫治疗效果，具有治疗多种类型肿瘤的潜力。
目前多家大型国际制药公司都在进行针对PD-1或PD-L1的单抗药物(如表1所示)，其中百事美施贵宝的PD-1抑制剂Opdvio(Nivolumab)于2014年7月获日本批准上市；默沙东的PD-1抑制剂于2014年9月获FDA批准上市；这两个药物的首个适应症均为黑色素瘤。随着各公司临床项目的推进，适应症已扩展到肺癌、乳腺癌、血液癌症等领域。
表1、当前正在开展临床实验的抗PD-1抗体
Figure PCTCN2015091842-appb-000001
目前全人源性抗体是治疗性抗体发展的主要方向，抗体库技术的出现为人源抗体的制备筛选提供了良好的技术平台。抗体库技术绕过了以往单抗研制过程中必须的杂交瘤过程，甚至不需要经过免疫过程即可获得各种抗体基因及抗体分子片段。噬菌体抗体库是最早出现也是目前应用最广泛的抗体库。
噬菌体展示技术是由Smith首先建立的一种将编码外源蛋白或多肽的基因插入噬菌体衣壳蛋白基因，使外源蛋白或多肽与噬菌体衣壳蛋白融合表达于噬菌体表面的技术。噬菌体抗体库就是利用了上述原理将不同特异性的抗体或其功能性片段(Fab、Fv、ScFv)表达在噬菌体表面，再用抗原进行筛选。噬菌体抗体库根据抗体基因的来源分为免疫库和非免疫库，非免疫库又包括天然库、半合成库和全合成库。噬菌体抗体库的筛选模拟了抗体亲和力成熟的过程，通常将抗原包被在固相介质上，加入待筛选的噬菌体抗体库，通过数轮“吸附-洗涤-洗脱-扩增”的过程(即淘选)直至筛选到高亲和力特异的抗体。
发明内容
本发明提供了一种抗PD-1的单克隆抗体；本发明从全合成抗体库中筛选出抗PD-1的单克隆抗体，然后通过计算机辅助设计分析，构建小容量合成噬菌体抗体轻链库的方法，对初筛获得的抗PD-1单克隆抗体DFPD1-1轻链的互补决定区CDR1、2、3区突变建库；筛选后，选取亲和力较高的单克隆抗体DFPD1-3和DFPD1-7，再对其重链CDR1、2、3区突变建库进行筛选，最终筛选到了高亲和力的抗PD-1的单克隆抗体。
为实现上述目的，本发明一种抗PD-1的单克隆抗体的获得过程包括：
(1)、抗PD-1单链抗体的生物淘选，通过三轮抗体库的富集筛选，从全合成的ScFv噬菌体库中获得了一种亲和力较高的抗体序列DFPD1-1，其重链为DFPD1-H1(SEQ NO.1)，轻链为DFPD1-L1(SEQ NO.5)。
(2)、以DFPD1-1为基础，通过计算机三级结构模拟，构建轻链互补决定区CDR1、2、3突变库并对该抗体库进行生物淘选和阳性克隆的筛选及鉴定，获得了6种不同轻链的抗体序列DFPD1-2、DFPD1-3、DFPD1-4、DFPD1-5、DFPD1-6、DFPD1-7，他们对应的轻链序列分别为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)、DFPD1-L7(SEQ NO.11)；将上述7种单链抗体在噬菌体水平进行亲和力比较。
(3)、选出两株亲和力较高的克隆DFPD1-3和DFPD1-7，构建重链互补决定区CDR1、2、3库并对该库进行生物淘选和阳性克隆的筛选，获得了五种不同的单链抗体序列DFPD1-9，DFPD1-10、DFPD1-11、DFPD1-12、DFPD1-13。其中DFPD1-9、DFPD1-11和DFPD1-12的轻链可变区序列是DFPD1-L3，而DFPD1-10和DFPD1-13的轻链可变区序列是DFPD1-L7；DFPD1-9和DFPD1-10的重链可变区序列是DFPD1-H2(SEQ NO.2)，DFPD1-11和DFPD1-13的重链可变区 是DFPD1-H3(SEQ NO.3)，DFPD1-12的重链可变区是DFPD1-H4(SEQ NO.4)。将上述单链抗体在噬菌体水平上进行亲和力比较。
(4)、将(3)中所述单克隆重链可变区基因和轻链可变基因及轻重链恒定区基因克隆到真核表达载体，转染宿主细胞，获得单克隆抗体的全抗再进行亲和力和其他生物学功能比较。
通过上述方法获得的抗PD-1的单克隆抗体，包括：轻链和重链；所述轻链的互补决定区CDR1、CDR2和CDR3分别用LCDR1、LCDR2和LCDR3表示；LCDR1包含RASQNIHSYLD、RASQNVSNWLD、RASQSIHNYLD、RASQDINNWLD RASQDVRTYLD、RASQGINSWLD或RASQSVSNYLD中的任一种；LCDR2包含EASTRAS、DASNRAT、NASTRAT、DASTLAT、GASTRAT或DASTRAT中的任一种；LCDR3包含QQALKLPIT、QQSRHIPLT、QQELHLPLT、QQNVNLPLT、QQDIDLPLT、QQSYRLPLT或QQNMQLPLT中的任一种。
其中，所述轻链可变区氨基酸序列包含SEQ NO.5、SEQ NO.6、SEQ NO.7、SEQ NO.8、SEQ NO.9、SEQ NO.10或SEQ NO.11中的任一种。
通过上述方法获得的抗PD-1的单克隆抗体，包括：轻链和重链；所述重链的互补决定区CDR1，CDR2和CDR3分别用HCDR1、HCDR2和HCDR3表示；HCDR1包含SNNGMH或SNYGMH；HCDR2包含VIWYDGSKK、VIWYDSSRK或VIWYDSTKK中的任一种；HCDR3包含TAVYYCATNNDYW或TAVYYCATNTDYW。
其中，所述重链可变区氨基酸序列包含SEQ NO.1、SEQ NO.2、SEQ NO.3或SEQ NO.4中的任一种。
其中，本发明还提供了一种包含上述轻链或上述重链的抗体、多肽或蛋白质。
其中，本发明还提供了一种包含上述轻链或上述重链的抗体，所述抗体能够阻断PD-1与其配体PD-L1的结合，抑制PD-1的生物学活性。
其中，本发明还提供了一种包含上述轻链或上述重链的多核苷酸序列或组合。
其中，本发明还提供了一种包含上述多核苷酸序列或组合的重组DNA表达载体；所述载体的DNA序列中包含编码抗PD1抗体的重链可变区及恒定区或轻链可变区及恒定区序列的氨基酸序列。
其中，本发明还提供了一种转染上述重组DNA表达载体的宿主细胞，所述宿主细胞包含大肠杆菌等原核细胞、酵母或哺乳动物细胞。
优选地，所述宿主细胞包含HEK293E细胞、CHO细胞或NS0细胞。
其中，本发明还提供了一种含有上述轻链或上述重链的抗体，用于全抗、单链抗体、单域抗体、双特异抗体、抗体药物偶联物或嵌合抗原受体T细胞免疫疗法。
其中，本发明还提供了一种含有上述轻链或上述重链的单克隆抗体、人工载体、药物或 药物组合物
其中，本发明还提供了一种含有上述轻链或上述重链的检测试剂或试剂盒。
其中，所述抗PD-1的单克隆抗体包含全长抗体和抗PD-1单克隆抗体的片段，所述片段包含但不限于Fab、Fab’、F(ab’)2、Fv或ScFv。
其中，所述全长抗体是全人源的。
其中，所述抗PD-1的单克隆抗体的重链恒定区包含IgG1、IgG2、IgG3和IgG4；所述轻链恒定区包含Cκ或Cλ。
优选地：所述恒定区为IgG4。
优选地，所述轻链恒定区为Cκ。
其中，所述CDR为互补决定区(complementarity-determining region)；所述ScFv为单链抗体(single-chain fragment variable)；所述ADCs为抗体药物偶联物(antibody-drug conjugates)；所述CAR-T为嵌合抗原受体T细胞免疫疗法(Chimeric Antigen Receptor T-Cell Immunotherapy)；所述HEK293E细胞为人胚肾293E细胞(human embryonic kidney293E cell)；CHO细胞为中国仓鼠卵巢细胞(chinese hamster ovary cell)；NS0细胞为小鼠NS0胸腺瘤细胞。
本发明相对于现有技术而言，具备的有益效果是：
本发明提供的单克隆抗体可以通过消除、抑制PD-1活性来预防或治疗疾病，其中所述疾病选自癌症、感染性疾病或免疫系统疾病。所述癌症包括但不限于肺癌、肾癌、黑色素瘤、乳腺癌、肝癌、头颈癌、皮肤癌、鳞状细胞癌、卵巢癌、骨癌、结直肠癌、膀胱癌、胃癌、胰腺癌、前列腺癌、霍奇金淋巴瘤、滤泡性淋巴瘤、慢性或急性白血病、实体瘤。所述感染性疾病包括不限于HIV病毒感染、肝炎病毒(A型、B型和C型)感染、疱疹病毒感染、流感病毒感染。所述免疫系统疾病包括不限于红斑狼疮、类风湿关节炎、强直性脊柱炎、重症肌无力、多发性硬化症、自身免疫性溶血性贫血、自身免疫性肝炎、硬皮病、结节性多动脉炎、Wegener肉芽肿病。
说明书附图
图1、pScFvDisb-s质粒图谱；
图2、轻链突变库构建中以DFPD1-1为模板扩增的重链及linker区电泳图；
图3、轻链突变库构建中以合成的轻链突变库为模板扩增的轻链库基因电泳图；
图4、轻链突变库构建中通过扩增获得的VLCDR123M-DFPD1-1突变库的电泳图；
图5、轻链突变库构建中通过NcoI-HF和NotI对质粒pScFvDisb-s进行双酶切的酶切产物的电泳图；
图6、单克隆phage ELISA鉴定phage-Abs的相对亲和力；
图7、梯度稀释phage ELISA鉴定phage-Abs的相对亲和力；
图8、重链突变库构建中以合成的重链突变库为模板扩增的重链库基因的电泳图；
图9、重链突变库构建中以DFPD1-3和DFPD1-7质粒为模板扩增的轻链及linker区的电泳图；
图10、重链突变库构建中通过扩增获得的VHCDR123M-DFPD1-3突变库的电泳图；
图11、重链突变库构建中通过扩增获得的VHCDR123M-DFPD1-7突变库的电泳图；
图12、单克隆phage ELISA鉴定phage-Abs的相对亲和力；
图13、梯度稀释phage ELISA鉴定phage-Abs的相对亲和力；
图14、pTSE质粒载体图；
图15、全抗体与PD-1在分子水平上结合试验；
图16、全抗体与PD-L1的竞争抑制试验；
图17、全抗体与细胞表面的PD-1结合试验；
具体实施方式
本发明详细的实施方法参见实施例，实施例中所述的实验方法和试剂，若无特殊说明均为常规实验方法和试剂。以下实施例仅用于说明和解释本发明，而不是以任何方式限制本发明。
本发明提供了一种特异性结合PD-1的单克隆抗体，所述重链可变区序列包含SEQ NO.1、2、3、4,所述轻链可变区序列包含SEQ NO.5、6、7、8、9、10、11。
优选地，所述特异性结合PD-1的单克隆抗体的重链可变区序列包含SEQ NO.2、3、4，所述轻链可变区序列选自SEQ NO.7、11。
通过轻链噬菌体库筛选，所述抗体轻链或其功能性片段的轻链互补决定区LCDR1、LCDR2和LCDR3的氨基酸序列选自以下各氨基酸序列中的一组(如表1所示)。
表1、轻链各个CDR区的氨基酸序列
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
通过重链噬菌体库筛选，所述抗体重链或其功能性片段重链的互补决定区CDR1，CDR2和CDR3分别用HCDR1、HCDR2和HCDR3表示：HCDR1为SNNGMH或SNYGMH；HCDR2为VIWYDGSKK、VIWYDSSRK或VIWYDSTKK中的任一种；HCDR3为TAVYYCATNNDYW或TAVYYCATNTDYW。
优选地，通过重链噬菌体库筛选，所述特异性结合PD-1的单克隆抗体包含HCDR1、HCDR2和HCDR3序列的重链可变区及含LCDR1、LCDR2和LCDR3序列的轻链可变区。其中,所述重链可变区HCDR1序列为选自SNNGMH、SNYGMH的氨基酸；所述轻链可变区LCDR1序列为选自RASQSIHNYLD、RASQSVSNYLD的氨基酸；所述重链可变区HCDR2序列为选自VIWYDGSKK、VIWYDSSRK的氨基酸；所述轻链可变区LCDR2序列为选自NASTRAT、DASTRAT的氨基酸；所述重链可变区HCDR3序列为选自TAVYYCATNNDYW、TAVYYCATNTDYW的氨基酸；所述轻链可变区LCDR3序列为选自QQELHLPLT、QQNMQLPLT的氨基酸。
本发明利用全合成ScFv单链噬菌抗体库获得特异性抗体的方法，所述全人源的特异性结合PD-1的单克隆抗体是利用噬菌体抗体库技术筛选而得，其步骤在于：
(1)、抗PD-1单链抗体的生物淘选，通过三轮抗体库的富集筛选，获得了一种亲和力较高的抗体序列DFPD1-1。
(2)、以DFPD1-1为基础，通过计算机辅助设计，构建轻链CDR1、2、3突变库并对该抗体库进行生物淘选和阳性克隆的筛选及鉴定，获得了6种不同轻链的抗体序列DFPD1-2、DFPD1-3、DFPD1-4、DFPD1-5、DFPD1-6、DFPD1-7。将上述7种单链抗体在噬菌体水平进行亲和力比较。
(3)、选出两株亲和力较高的克隆DFPD1-3和DFPD1-7，构建重链CDR1、2、3库并对该库进行生物淘选和阳性克隆的筛选，获得了五种不同序列的单链抗体DFPD1-9，DFPD1-10，DFPD1-11，DFPD1-12，DFPD1-13。将上述单链抗体在噬菌体水平上进行亲和力比较。
(4)、将(3)中所述单克隆重链可变区基因和轻链可变基因及轻重链恒定区基因克隆到真核表达载体，转染宿主细胞，获得单克隆抗体的全抗，再进行亲和力和其他生物学功能比较。
具体实施例：
以下结合附图和实施例详述本发明。
实施例1、抗PD-1单链抗体的生物淘选
采用一系列基因克隆的方法对载体pCom3载体(购自中国质粒载体菌株细胞株基因保藏中心)进行改造，使之用于噬菌体单链抗体库的构建和表达。改造后的载体命名为pScFvDisb-s，其质粒图谱如图1所示，并以此载体为基础，构建全合成噬菌体抗体库。
以PD-1-His为抗原包被免疫管，抗原包被量为5ug/500ul/管，4℃包被过夜。再用4％脱脂奶粉/PBST分别封闭免疫管和全合成噬菌体抗体库，室温封闭1h。封闭后的噬菌体抗体 库加入免疫管中进行抗原抗体结合，噬菌体投入量约为109-1012个，室温反应1h。PBST-PBS洗去未结合的噬菌体，0.1M PH2.2的Glycine-HCl洗脱，用1.5M PH8.8的Tris-HCl中和洗脱下来的噬菌体抗体溶液至PH7.0左右。
将上述中和后的噬菌体感染10ml生长至对数期的TG1菌液，37℃培养箱中静置30min，取出部分菌液进行梯度稀释，涂布于2YTAG平板上，用于计算噬菌体产出量。剩余的菌液离心弃上清，将菌体沉淀重悬于少量培养基，吸出后涂布于2YTAG大平板，为下一轮筛选做准备。
将上述感染后涂板的菌体从大平板上刮下，接菌至2YTAG液体培养基，摇至对数期后加入M13辅助噬菌体超感染，28℃培养过夜扩增噬菌体，PEG/NaCl沉降纯化噬菌体用于下一轮筛选。共进行三轮噬菌体库富集筛选。
实施例2、抗PD-1噬菌体单链抗体阳性克隆的筛选
经过三轮筛选后，挑取分隔良好的单克隆菌落，接种于加有2YTAG液体培养基的96孔深孔板，37℃，220rpm培养至其对数生长期，每孔加入约1010的辅助噬菌体M13KO7，37℃静止感染30min。4000rpm，离心15min，弃去上清，菌体用2YTAK重悬沉淀，28℃，220rpm培养过夜。4000rpm，4℃离心15min，吸取扩增后的噬菌体上清进行ELISA鉴定。筛选得到亲和力较高的单链抗体DFPD1-1，其重链可变区命名为DFPD1-H1,其氨基酸序列见SEQ NO.1；其轻链可变区命名为DFPD1-L1,氨基酸序列见SEQ NO.5。
实施例3、对筛选出的抗PD-1单链抗体DFPD1-1进行体外亲和力成熟
3.1.DFPD1-1轻链CDR1、2、3突变库的构建
设计引物PVLF1和PVLR1以合成的轻链突变库(SEQ NO.12)为模板，PCR扩增轻链库基因(图3)；设计引物PVHF1和PVHR1以DFPD1-1质粒为模板扩增其重链及linker区(图2)。反应条件：95℃30s，1cycle；95℃15s，60℃10s，72℃30s,3cycles；95℃15s，72℃40s,25cycles,72℃5min，4℃保存。天根通用回收试剂盒回收PCR目的片段。
引物如下所示：
PVLF1：
Figure PCTCN2015091842-appb-000002
PVLR1：
Figure PCTCN2015091842-appb-000003
PVHF1：
Figure PCTCN2015091842-appb-000004
PVHR1：
Figure PCTCN2015091842-appb-000005
将上述两部分PCR反应产物进行Overlap PCR扩增获得DFPD1-1的轻链突变库基因。反应条件：95℃30s，1cycle；95℃15s，72℃30s,4cycle；(加入引物PVHF1和PVLR2),95℃15s，72℃40s,25cycles；72℃5min，4℃保存。天根通用回收试剂盒回收PCR目的片段，对应的PCR产物命名为VLCDR123M-DFPD1-1(图4)。
用NcoI-HF和NotI对质粒pScFvDisb-s进行双酶切，酶切产物经0.8％琼脂糖凝胶电泳(图5)，切胶回收；用NcoI-HF和NotI分别对VLCDR123M-DFPD1-1PCR产物进行双酶切。PCR酶切产物采用通用回收试剂盒进行回收。回收后的PCR片段与pScFvDisb-s按摩尔比4：1比例经T4DNA连接酶16℃连接4h。将连接产物电击法转化至TG1感受态中。使用SOC培养基37℃培养1h复苏。按比例取菌液，涂平板，计算库容量。其余菌液4000rpm，室温下离心15min。弃上清，沉淀涂布于2YTAG大平板上，37℃倒置培养过夜。
构建抗体库库容大约为108，从上述抗体库中分别随机挑取20个克隆进行序列分析正确率95％，抗体库库容远远超过抗体库的多样性。
3.2.噬菌体抗体库的生物淘选和阳性克隆的筛选
在按照实施例1的方法进行筛选，得到亲和力较高的克隆测序，共得到6种不同的单链抗体序列，分别命名为DFPD1-2、DFPD1-3、DFPD1-4、DFPD1-5、DFPD1-6、DFPD1-7，对应的轻链可变区命名为DFPD1-L2、DFPD1-L3、DFPD1-L4、DFPD1-L5、DFPD1-L6、DFPD1-L7，它们对应的氨基酸序列分别见SEQ NO.6、SEQ NO.7、SEQ NO.8、SEQ NO.9、SEQ NO.10和SEQ NO.11。单克隆phage ELISA鉴定phage-Abs的相对亲和力如图3所示。
3.3.梯度稀释phage ELISA鉴定抗PD1单链抗体的亲和力
将本实施例2中获得的克隆进行单克隆phage的展示和纯化，进行phage梯度稀释ELISA实验鉴定phage-Abs的亲和力。
用pH9.6的碳酸盐缓冲液包被PD1-His，4℃包被过夜。PBST洗涤三次，4％milk-PBST 37℃封闭1h。将纯化后的phage用4％milk-PBST三倍稀释，每孔加入100ul稀释后的样品，室温静置1h。用PBST洗涤ELISA板，将4％脱脂奶粉稀释后的anti-M13-HRP单克隆抗体加入ELISA板中，室温放置1h。TMB显色试剂盒显色，室温显色5min。用2M H2SO4终止显色，50μl/孔。酶标仪450nm单波长测定光密度值。结果显示筛选出的几株不同的噬菌体抗体均能与PD-1进行结合，而且DFPD1-3，DFPD1-7的亲和力明显高于其它克隆(图4)，选取DFPD1-3和DFPD1-7进行下一步试验。
实施例4、对筛选出的抗PD-1的单链抗体DFPD1-3，DFPD1-7再次进行体外亲和力成熟
4.1DFPD1-3和DFPD1-7重链CDR1、2、3突变库的构建
设计引物PVHF2和PVHR2以合成的重链突变库(SEQ NO.13)为模板，PCR扩增重链库基因(图8)；设计引物PVLF2和PVLR2以DFPD1-3和DFPD1-7质粒为模板分别扩增其轻链及linker区(图9)，其中，左侧为DFPD1-3，右侧为DFPD1-7。反应条件：95℃30s，1cycle；95℃15s，60℃10s，72℃30s,3cycles；95℃15s，72℃40s,25cycles；72℃5min，4℃保存。天根通用回收试剂盒回收PCR目的片段。
引物如下所示：
PVHF2：
Figure PCTCN2015091842-appb-000006
PVHR2：
Figure PCTCN2015091842-appb-000007
PVLF2：
Figure PCTCN2015091842-appb-000008
PVLR2：
Figure PCTCN2015091842-appb-000009
将上述两部分PCR反应产物进行Overlap PCR扩增获得DFPD1-3和DFPD1-7的重链突变库基因。反应条件：95℃30s，1cycle；95℃15s，72℃30s,4cycles；(加入引物PVHF2和PVLR2)，95℃15s，72℃40s,25cycles；72℃5min，4℃保存。天根通用回收试剂盒回收PCR目的片段，将对应的产物命名为VHCDR123M-DFPD1-3(图10)和VHCDR123M-DFPD1-7(图11)。
用NcoI-HF和NotI对质粒pScFvDisb-s进行双酶切，酶切产物经0.8％琼脂糖凝胶电泳(图5)，切胶回收；用NcoI-HF和NotI分别对VHCDR123M-DFPD1-3和VHCDR123M-DFPD1-7PCR产物进行双酶切。PCR酶切产物采用天根通用回收试剂盒进行回收。回收后的PCR片段与pScFvDisb-s按摩尔比4：1比例经T4DNA连接酶16℃连接4h。将连接产物电击法转化至TG1感受态中。使用SOC培养基37℃培养1h复苏。按比例取菌液，涂平板，计算库容量。其余菌液4000rpm，室温下离心15min。弃上清，沉淀涂布于2YTAG大平板上，37℃倒置培养过夜。
构建了2个不同的抗体库。每个抗体库库容大约为107，抗体库库容远远超过抗体库的多样性。从上述抗体库中分别随机挑取20个克隆进行序列分析，正确率为90％。
4.2噬菌体抗体库的生物淘选和阳性克隆的筛选
将上述构建的两种抗体库，进行phage展示，纯化和沉淀。然后从该库中淘选抗PD1的单链抗体。噬菌体抗体库的生物淘选方法同实施案例1。抗PD-1的单链抗体阳性克隆的筛选的方法同实施案例2。结果发现共筛选到5种不同的抗PD-1抗体序列，分别命名为DFPD1-9，DFPD1-10，DFPD1-11，DFPD1-12，DFPD1-13。其中DFPD1-9，DFPD1-11和DFPD1-12的轻链可变区序列是DFPD1-L3，而DFPD1-10和DFPD1-13的轻链可变区序列是DFPD1-L7；DFPD1-9和DFPD1-10的重链可变区序列是DFPD1-H2，DFPD1-11和DFPD1-13的重链可变区是DFPD1-H3，DFPD1-12的重链可变区是DFPD1-H4。单克隆phage ELISA鉴定phage-Abs的相对亲和力如图12所示。
4.3.梯度稀释phage ELISA鉴定抗PD1单链抗体的亲和力
将本实施例4.2中获得的克隆进行单克隆phage的展示和纯化，进行phage梯度稀释ELISA实验鉴定phage-abs的亲和力，方法同实施例3中3.1。结果显示筛选出的几株不同的噬菌体抗体均能与PD1进行结合，亲和力相差不是很大(图13)，其中DFPD1-9，DFPD1-10，DFPD1-11，DFPD1-12，DFPD1-13略好，选择这几种单链抗体进行后面试验。
实施例5、抗PD-1全抗体DFPD1-9、DFPD1-10、DFPD1-11、DFPD1-12、DFPD1-13亲和力 鉴定
5.1抗PD-1全抗体的制备
将上述抗体的重链VH和轻链VK基因分别克隆至装有重链和轻链恒定区基因的载体pTSE(图14)，编码人恒定区γ4(见SEQ NO.14)和κ链(见SEQ NO.15)的pTSE载体中(pTSE载体结构如图14所示，制备过程参见CN103525868A说明书第3页第[0019]段)。瞬时转染HEK293E细胞，进行全抗体表达。使用AKTA仪器protein A亲和柱纯化获得全抗体蛋白。
5.2BIAcore X100测定全抗体的亲和力
采用捕获法测定全抗体的亲和力。将抗人IgG偶联到CM5芯片表面，分别稀释DFPD1-9，DFPD1-10，DFPD1-11，DFPD1-12和DFPD1-13保证大约300RU左右的抗体被抗人IgG捕获。将PD-1设置一系列的浓度梯度(1000nM，500nM，250nM，125nM，62.5nM，31.25nM，15.625nM，7.8125nM，3.9063nM,1.9531nM，0.9766nM)流经固定相表面，测定抗体的亲和力。结果发现筛出的抗体的亲和力相差不是太大(表3)。
表3、抗PD1全抗体亲和力常数测定数值
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全抗体与PD-1的结合实验
用pH9.6的碳酸盐缓冲液包被PD-1-His，60ng/孔/100μl，4℃过夜包被。用300μl/孔PBST洗五次，再加入1％BSA-PBS在37℃封闭2h。加入不同稀释度的全抗DFPD1-9，DFPD1-10，DFPD1-11，DFPD1-12和DFPD1-13。五种全抗体最高浓度是16ug/ml，4倍稀释做11个梯度，最后一个孔作为阴性对照——即只加稀释液PBS，37℃孵育1h。用300μl/孔PBST洗五次，再加入用1％BSA-PBS 1:40000稀释的Anti-Human Fc-HRP二抗37℃孵育1h。TMB显色试剂盒显色，100μl/孔，室温显色8min，然后用2M H2SO4终止显色，50μl/孔。450nm/630nm读数。实验结果如图15所示，所有抗体均能很好的与PD-1分子结合。
5.4全抗体竞争抑制PD-L1与PD-1结合
用pH9.6的碳酸盐包被PDL1-Fc，4℃包被过夜。PBST洗涤五次，1％BSA-PBS 37℃封闭2h。用4μg/ml的PD1-His分别稀释DFPD1-9，DFPD1-10，DFPD1-11，DFPD1-12和DFPD1-13这五种全抗体，全抗体与PD-1的摩尔比从10：1开始，五倍梯度稀释，每个样品做9个稀 释度，37℃孵育1h。PBST洗五次，加入用1％BSA-PBS稀释的HRP标记的小鼠抗His抗体，37℃孵育1h。TMB显色试剂盒显色，100μl/孔，室温显色8min。用10％H2SO4终止显色，50μl/孔。450nm/630nm读数。结果如图16所示，DFPD1-9，DFPD1-10，DFPD1-11，DFPD1-12和DFPD1-13均能抑制PD-1与PD-L1的结合(结果见图16)。
实例6、抗PD-1抗体与细胞表面PD-1结合试验
先构建过表达的PD-1的CHO稳定细胞系，将它命名为PD1-CHO。用明胶包被96孔板后，PD1-CHO细胞胰酶消化后中止，离心重悬，稀释至2×105细胞/ml，每孔100ul铺于96孔板中，共12孔×6排，即2×104细胞/孔，5％CO2，37℃培养过夜。第二天弃去培养基，用350ul预冷的PBS洗一遍，2％新鲜配制的PFA固定5min，PBS洗2遍。
细胞板中加入倍比稀释好的抗PD-1全抗，稀释液为含0.5％BSA的PBS。样品浓度从100ug/ml起，8倍稀释，共12稀释度，室温孵育30min。然后弃去上清，350ulPBS洗3遍后，加入1:5000稀释的辣根过氧化物酶标记羊抗人二抗,室温孵育15min。
再用350ulPBS洗3遍，每孔加入100ulTMB显色液，室温显色15-30min。每孔加入50ul2M H2SO4中止显色，酶标仪450nm读数。用Graphpad prism软件处理结果，计算结合常数(见图17)。
对于本领域的普通技术人员而言，具体实施例只是对本发明进行了示例性描述，显然本发明具体实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种非实质性的改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围之内。

Claims (12)

  1. 一种抗PD-1的单克隆抗体，其特征在于；包括：轻链和重链；所述轻链的互补决定区CDR1、CDR2和CDR3分别用LCDR1、LCDR2和LCDR3表示；LCDR1包含RASQNIHSYLD、RASQNVSNWLD、RASQSIHNYLD、RASQDINNWLD RASQDVRTYLD、RASQGINSWLD或RASQSVSNYLD中的任一种；LCDR2包含EASTRAS、DASNRAT、NASTRAT、DASTLAT、GASTRAT或DASTRAT中的任一种；LCDR3包含QQALKLPIT、QQSRHIPLT、QQELHLPLT、QQNVNLPLT、QQDIDLPLT、QQSYRLPLT或QQNMQLPLT中的任一种。
  2. 根据权利要求1所述的抗PD-1的单克隆抗体，其特征在于；所述轻链可变区氨基酸序列包含SEQ NO.5、SEQ NO.6、SEQ NO.7、SEQ NO.8、SEQ NO.9、SEQ NO.10或SEQ NO.11中的任一种。
  3. 一种抗PD-1的单克隆抗体，其特征在于；包括：轻链和重链；所述重链的互补决定区CDR1，CDR2和CDR3分别用HCDR1、HCDR2和HCDR3表示；HCDR1包含SNNGMH或SNYGMH；HCDR2包含VIWYDGSKK、VIWYDSSRK或VIWYDSTKK中的任一种；HCDR3包含TAVYYCATNNDYW或TAVYYCATNTDYW。
  4. 根据权利要求3所述的抗PD-1的单克隆抗体，其特征在于；所述重链可变区氨基酸序列包含SEQ NO.1、SEQ NO.2、SEQ NO.3或SEQ NO.4中的任一种。
  5. 一种抗体、多肽或蛋白，其特征在于；所述抗体、多肽或蛋白包含权利要求1-4任一所述的轻链或重链。
  6. 一种多核苷酸序列或组合，其特征在于；所述多核苷酸序列或组合包含权利要求1-4任一所述的轻链或重链。
  7. 一种重组DNA表达载体，其特征在于；所述重组DNA表达载体包含权利要求6所述的多核苷酸序列或组合。
  8. 一种宿主细胞，其特征在于；所述宿主细胞在转染所述重组DNA表达载体时用到，所述宿主细胞包含原核细胞、酵母或哺乳动物细胞。
  9. 根据权利要求1-4任一所述的抗PD-1的单克隆抗体，其特征在于；所述抗体为全人源抗体；所述抗体的重链恒定区包含IgG1、IgG2、IgG3或IgG4；所述抗体的轻链恒定区包含Cκ或Cλ。
  10. 一种全抗、单链抗体、单域抗体、双特异抗体、抗体药物偶联物或嵌合抗原受体T细胞免疫疗法，包含权利要求1-4任一所述的轻链或重链。
  11. 一种单克隆抗体、人工载体、药物或药物组合物，其特征在于；所述单克隆抗体、人工载体、药物或药物组合物包含权利要求1-4任一所述的轻链或重链。
  12. 一种检测试剂或试剂盒，其特征在于；所述检测试剂或试剂盒包含权利要求1-4任一所述的轻链或重链。
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