WO2020248926A1 - 抗PDL1和TGFβ的双功能融合蛋白及其用途 - Google Patents

抗PDL1和TGFβ的双功能融合蛋白及其用途 Download PDF

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WO2020248926A1
WO2020248926A1 PCT/CN2020/094855 CN2020094855W WO2020248926A1 WO 2020248926 A1 WO2020248926 A1 WO 2020248926A1 CN 2020094855 W CN2020094855 W CN 2020094855W WO 2020248926 A1 WO2020248926 A1 WO 2020248926A1
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antibody
antigen
binding fragment
seq
fusion protein
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PCT/CN2020/094855
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English (en)
French (fr)
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宋德勇
徐洪光
韩镇
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山东博安生物技术有限公司
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Priority to JP2021560523A priority Critical patent/JP7297090B2/ja
Priority to EP20822451.9A priority patent/EP3929215A4/en
Priority to BR112021024820A priority patent/BR112021024820A2/pt
Priority to KR1020217040151A priority patent/KR20220007118A/ko
Priority to CN202080000954.6A priority patent/CN114206926B/zh
Priority to AU2020290119A priority patent/AU2020290119A1/en
Priority to CA3135988A priority patent/CA3135988A1/en
Priority to US17/601,891 priority patent/US20220213195A1/en
Publication of WO2020248926A1 publication Critical patent/WO2020248926A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C07K16/2827—Immunoglobulins [IGs], 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 B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • the invention relates to the field of biomedicine or biopharmaceutical technology. Specifically, the present invention relates to anti-PDL1 antibodies, anti-PDL1 and TGF ⁇ bifunctional fusion proteins and variants and antigen-binding fragments thereof, as well as methods for their preparation and methods and uses for the treatment of cancer.
  • PD1 programmed cell death-1
  • PDL1 programmed cell death-1 ligand 1
  • PD1 is expressed on the surface of activated T cells and NK cells, and tumor cells pass a large number of Express PDL1, inhibit the maturation and proliferation of T cells and NK cells after binding to PD1, and escape immune surveillance (Iwai et al., PNAS99: 12293-7, 2002) (Ohigashi et al., Clin Cancer Res 11: 2947-53 ,2005). Therefore, blocking the interaction between the two can reactivate T cells, recognize and kill tumor cells, and benefit patients.
  • TGF ⁇ can inhibit the growth of early tumors, but as the tumor continues to progress, tumor cells become less sensitive to TGF ⁇ . At this time, TGF ⁇ promotes tumor growth by promoting epithelial-stromal metastasis and suppressing the immune system (Bierie et ah , Nat Rev Cancer. 2006; 6:506-20).
  • TGF ⁇ RII The extracellular domain of TGF ⁇ RII is only 136 amino acid residues long and can bind to TGF ⁇ 1 (Lin et al, J Biol Chem. 1995; 270: 2747-54). Soluble TGF ⁇ RII-Fc has been tested as an anticancer agent, and it has been shown to inhibit the growth of malignant mesothelioma in a mouse model (Suzuki et al, Clin Cancer Res. 2004; 10: 5907-18) (Suzuki et al., Clin Cancer) Res. 2004; 10:5907-18).
  • Merck's M7824 bifunctional fusion protein (Chinese application number CN201580007865.3) is an anti-PDL1 antibody Avelumab and TGF ⁇ RII connected through (G 4 S) 4 G. It has completed some phase I and phase II clinical trials and has been used in a variety of solid tumors. Performs well in the medium (MSB0011359C).
  • VL light chain variable region
  • VH heavy chain variable region
  • LCDR light chain complementarity determining region
  • HCDR heavy chain complementarity determining region
  • LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 The various embodiments of HCDR3 and HCDR3 can be implemented individually or in any combination.
  • the present invention relates to an antibody or its antigen binding fragment or bifunctional fusion protein and its preparation method and application.
  • the present invention relates to antibodies or antigen-binding fragments thereof, comprising three heavy chain complementarity determining regions, wherein the HCDR1 amino acid sequence is shown in SEQ ID NO: 8 and the HCDR2 amino acid sequence is shown in SEQ ID NO: 9
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 10.
  • the antibody or antigen-binding fragment thereof further comprises three light chain complementarity determining regions, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 7 is shown.
  • the antibody or antigen-binding fragment thereof provided by the present invention includes the heavy chain variable region shown in SEQ ID NO: 2; preferably, it also contains the light chain variable region shown in SEQ ID NO:1.
  • the present invention relates to an antibody or an antigen-binding fragment thereof, comprising three light chain complementarity determining regions, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 11, and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 6
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 12; and/or three heavy chain complementarity determining regions, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 13, and the amino acid sequence of HCDR2 is shown in SEQ ID NO: 14, and HCDR3
  • the amino acid sequence is shown in SEQ ID NO: 15.
  • the present invention relates to an antibody or antigen-binding fragment thereof comprising the light chain variable region of the amino acid sequence shown in SEQ ID NO: 3, and/or the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 4.
  • sequence of the light chain constant region of the antibody or antigen-binding fragment thereof in any of the foregoing aspects is SEQ ID NO: 16.
  • sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof in any of the foregoing aspects is SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof provided by the present invention preferably comprises the light chain variable region of the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: The light chain constant region of the amino acid sequence shown in: 16 and the heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof provided by the present invention preferably comprises the light chain variable region of the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: The light chain constant region of the amino acid sequence shown in 16 and the heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 17.
  • the present invention relates to the antibody or antigen-binding fragment thereof according to any one of the foregoing aspects, including monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2 , Fv, scFv or dsFv fragments, etc.
  • any of the aforementioned antibodies or antigen-binding fragments thereof bind to PDL1.
  • the present invention relates to a bifunctional fusion protein, which comprises the antibody or antigen-binding fragment thereof according to any one of the foregoing aspects, and a TGF ⁇ RII fragment.
  • the TGF ⁇ RII fragment comprises the extracellular domain of TGF ⁇ RII.
  • the antibody or antigen-binding fragment thereof in any one of the foregoing aspects is connected to the TGF ⁇ RII fragment through a linker.
  • the TGF ⁇ RII fragment is connected to the antibody or antigen-binding fragment thereof.
  • the C-terminus of the heavy chain constant region of the fragment is preferably connected by a (G 4 S) 4 G (ie GGGGSGGGGSGGGGSGGGGSG) linker.
  • the sequence of the heavy chain constant region of the bifunctional fusion protein is SEQ ID NO: 22, preferably Specifically, the sequence of the TGF ⁇ RII fragment of the bifunctional fusion protein is SEQ ID NO: 23; specifically, the structure of the bifunctional fusion protein is: an antibody or an antigen-binding fragment thereof-(G 4 S) 4 G-TGF ⁇ RII fragment.
  • the sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 1
  • the sequence of the heavy chain variable region is SEQ ID NO: 2
  • the sequence of the constant region of the light chain is SEQ ID NO: 16
  • the sequence of the constant region of the heavy chain is SEQ ID NO: 22
  • the sequence of the TGF ⁇ RII fragment is SEQ ID NO: 23; specifically The sequence where the heavy chain constant region and the TGF ⁇ RII fragment are connected via (G 4 S) 4 G is SEQ ID NO: 18.
  • the sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 3
  • the sequence of the heavy chain variable region is SEQ ID NO: 4
  • the sequence of the constant region of the light chain is SEQ ID NO: 16
  • the sequence of the constant region of the heavy chain is SEQ ID NO: 22
  • the sequence of the TGF ⁇ RII fragment is SEQ ID NO: 23; specifically The sequence where the heavy chain constant region and the TGF ⁇ RII fragment are connected via (G 4 S) 4 G is SEQ ID NO: 18.
  • any one of the above-mentioned bifunctional fusion proteins binds PDL1 and TGF ⁇ .
  • the present invention relates to a nucleic acid that encodes the antibody or antigen-binding fragment or bifunctional fusion protein of any one of the foregoing aspects.
  • the present invention relates to a vector comprising the nucleic acid described in the previous aspect, or it can express the antibody or antigen-binding fragment or bifunctional fusion protein of any one of the foregoing aspects.
  • the vector can be a viral vector; preferably, the viral vector includes, but is not limited to, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector; preferably, the vector can be a non-viral vector Vector;
  • the vector can be a mammalian expression vector; preferably, the expression vector can be a bacterial expression vector; preferably, the expression vector can be a fungal expression vector.
  • the present invention relates to a cell that can express the antibody or antigen-binding fragment or bifunctional fusion protein of any one of the foregoing aspects.
  • the cell is a bacterial cell; preferably, the bacterial cell is an E. coli cell, etc.; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast
  • the cells are Pichia pastoris cells, etc.; preferably, the cells are mammalian cells; preferably, the mammalian cells are Chinese hamster ovary cells (CHO), human embryonic kidney cells (293), B cells, T cells, DC cells or NK cells, etc.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, bifunctional fusion protein, nucleic acid, carrier or cell according to any one of the foregoing aspects.
  • the pharmaceutical composition also Contains pharmaceutically acceptable excipients, preferably, the pharmaceutically acceptable excipients include one or more of the following: pharmaceutically acceptable solvents, dispersants, additives, plasticizers, etc. .
  • the pharmaceutical composition may also contain other therapeutic agents.
  • other therapeutic agents include chemotherapeutic agents, immunotherapeutic agents, or hormone therapy agents. The combined administration of the antibody or antigen-binding fragment and other therapeutic agents can enhance the therapeutic effect.
  • the "enhancement of the therapeutic effect” refers to enhancing the therapeutic effect of other therapeutic agents or therapies.
  • the antibody or antigen-binding fragment provided by the present invention can be administered alone or in combination with other therapeutic agents or therapies.
  • other therapeutic agents or therapies include chemotherapeutic agents, immunotherapeutic agents, hormone therapy agents, radiation therapy, surgical treatment.
  • kits comprising the antibody or antigen-binding fragment thereof of the present invention, or a bifunctional fusion protein, or a nucleic acid encoding the antibody or antigen-binding fragment or bifunctional fusion protein.
  • the present invention relates to the use of the antibody or antigen-binding fragment, bifunctional fusion protein, nucleic acid, carrier or cell of any one of the foregoing aspects in the preparation of drugs for treating or preventing diseases.
  • the present invention relates to the use of the antibody or antigen-binding fragment, bifunctional fusion protein or nucleic acid of any one of the foregoing aspects in the preparation of a diagnostic or detection kit.
  • a method for treating or preventing diseases comprising administering the antibody or antigen-binding fragment, bifunctional fusion protein, nucleic acid, carrier, cell or pharmaceutical composition of any aspect of the present invention to a recipient in need Examiner.
  • a method of diagnosis or detection comprising administering the antibody or antigen-binding fragment, bifunctional fusion protein, nucleic acid or kit of any aspect of the present invention to a subject or sample in need.
  • the method is a method of diagnosing or detecting a disease.
  • the present invention relates to the use of the antibody or antigen-binding fragment, bifunctional fusion protein, nucleic acid, carrier, cell or pharmaceutical composition of any one of the foregoing aspects for the treatment or prevention of diseases.
  • the present invention relates to the use of the antibody or antigen-binding fragment, bifunctional fusion protein, nucleic acid or kit of any one of the foregoing aspects for detection or diagnosis.
  • the use is for diagnosing or detecting diseases.
  • the disease is cancer.
  • the cancer includes gastric cancer, esophageal cancer, head and neck cancer, bladder cancer, cervical cancer, sarcoma, cell tumor, lung cancer, colon cancer, ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, liver cancer, melanin Tumors, breast cancer, myeloma, glioma, leukemia, lymphoma, etc.
  • the present invention relates to a method for preparing the antibody or antigen-binding fragment or bifunctional fusion protein of any one of the foregoing aspects, comprising transfecting cells with the above-mentioned vector, and expressing the antibody or the antibody or its An antigen-binding fragment or a bifunctional fusion protein; or including the expression of the antibody or an antigen-binding fragment or a bifunctional fusion protein with the aforementioned cells.
  • the antibody or antigen-binding fragment or bifunctional fusion protein provided by the present invention has one or more of the following advantages: enhanced TGF ⁇ 1 binding activity, enhanced PDL1 affinity, enhanced ability to block the binding of PDL1 and PD1, and enhanced blocking of TGF ⁇ 1 Functional activity, enhanced ability to promote T cells to secrete IFN- ⁇ , better immune regulation effect, and better tumor suppression effect.
  • Figure 1 shows the serum titers of BoAn-hMab1 mice after five immunizations (2500-fold dilution).
  • Figure 2 shows the binding of the bifunctional fusion protein to the TGF ⁇ 1 protein.
  • Figure 3 shows that the bifunctional fusion protein promotes the secretion of IFN- ⁇ from CD4+T.
  • Figure 4 shows the blocking effect of the bifunctional fusion protein on TGF ⁇ 1 function.
  • Figure 5A shows the body weight of the MC38-hPD-L1 tumor model mouse
  • Figure 5B shows the tumor volume of the MC38-hPD-L1 tumor model mouse
  • Figure 5C shows the tumor weight of the MC38-hPD-L1 tumor model mouse.
  • PDL1 protein (Sinobiological, catalog number: 10084-H08H); PD1 protein (Sinobiological, catalog number: 10377-H08H); TGF ⁇ 1 (Sinobiological, catalog number: 10804-HNAC); NaHCO 3 (Sinobiological, 10018960); TMB (Beijing Meikewande, catalog number: 1001); STREP/HRP (R&D, catalog number: 890803); HBS-EP+1 ⁇ buffer (GE, catalog number: BR-1008-26); microtiter plate (Suzhou Beaver, catalog number: 40301); biosensor chip (GE, catalog number: BR100530); 96-well round bottom plate (Corning, catalog number: 3799); anti-human IgG Fc amino coupling kit (GE, cat#BR -1008-39);
  • the serum titer (2500-fold dilution) detected by ELISA is shown in Figure 1.
  • PL1Q15, PL1Q16, PL1Q18, PL1Q19, PL1Q21 mice are Freund's adjuvant immunized mice
  • PL1Q24 mice are gold adjuvant immunized mice.
  • spleens Six mice with a higher serum titer in 1.1 were sacrificed, the spleens were dissected, and the spleens were ground and broken with a syringe rubber stopper and filtered with a strainer (FALCON, catalog number: 352350). The filtered spleen cells were frozen for later use. After RNA is extracted, cDNA is obtained by reverse transcription. The establishment of the phage library is carried out according to the method described in Carlos F. Barbas III, Phage display: A laboratory manual, and the variable regions of the heavy and light chains are obtained from the cDNA by PCR. , And then the variable regions of the heavy and light chains were obtained by overlapping extension PCR to obtain single-chain Fv (scFv).
  • scFv single-chain Fv
  • the scFv was digested with SfiI enzyme (NEB, catalog number: #R0123L) and then passed through T4DNA ligase (Yiqiao Shenzhou). ) Was ligated with plasmid pCOMB3x (China Plasmid Vector Strain Cell Line Gene Collection Center, BIOVECTOR510837), and then the ligated product was electrotransfected into E. coli TG1 competent cells (Lucigen, catalog number: A96595-2), and the transfected TG1 After culturing on a shaker at 37°C and 220 rpm, phage infection was added, and then the culture supernatant was recovered and purified to obtain a phage library.
  • SfiI enzyme NEB, catalog number: #R0123L
  • T4DNA ligase Yamaqiao Shenzhou
  • Magnetic bead screening Biotinylate PDL1-mFc protein (Acrobiosystems, catalog number: PD1-H52A3) (the molar ratio of protein to biotin is 1:2): Change the PDL1-mFc protein to 0.1M pH8.0NaHCO 3 In the medium, a precision balance weighs an appropriate amount of biotin (Thermo, 21335) and dissolves it in ultrapure water, immediately adds an appropriate amount of dissolved biotin to the PDL1-mFc protein solution, and incubates on a rotating mixer in the dark for 40 minutes. After the labeling is completed Change the PDL1-mFc protein solution to PBS.
  • Phage clones express scFv in Escherichia coli, and then detect the blocking of PDL1/PD1 binding by scFv by ELISA, and clones that retain the blocking activity for subsequent molecular construction.
  • ELISA detection of scFv blocking the binding of PD1/PDL1 Dilute PD1 protein to 0.5 ⁇ g/mL with pH9.6CBS, coat the ELISA plate, 100 ⁇ L/well, incubate at 4°C overnight; wash the plate with 3% skimmed milk powder at 37°C Closed for 1h. After washing the plate, add 50 ⁇ L of scFv periplasm to each well.
  • biotin-labeled PDL1-Fc protein final concentration 0.2 ⁇ g/mL
  • STREP/HRP diluted with PBST
  • 100 ⁇ L/well 100 ⁇ L/well
  • incubate at 37°C for 1h.
  • the magnetic beads screened clones BA533, BA603, BT613, BA623, BA649, BA669, BA466 and the plate screened clone BA705 were sent to Invitrogen Biotechnology Co., Ltd. for sequencing.
  • the amino acid sequences of each cloned light chain variable region and heavy chain variable region are listed in Table 1.
  • nucleotide sequence encoding VH into the vector pCDNA3.4 (Life Technology) with the nucleotide sequence encoding SEQ ID NO: 18, wherein SEQ ID NO: 18 contains The heavy chain constant region sequence and TGF ⁇ RII sequence of the IgG1 antibody linked by (G 4 S) 4 G, the nucleotide sequence encoding VL is inserted into the amino acid sequence encoding the antibody light chain constant region (SEQ ID NO: 16) Nucleotide sequence vector pCDNA3.4 (Life Technology), then transfected HEK293 cells, and cultured in a shaker at 37°C ⁇ 8%CO 2 ⁇ 125rpm for 6-7 days,
  • the amino acid sequence of the variable region of Avelumab of Merck was determined by IMGT database (the sequence of the variable region of the heavy chain is SEQ ID NO: 19, and the sequence of the variable region of the light chain is SEQ ID NO: 20), and the entire gene is synthesized and inserted into the vector pCDNA3 .4, expressed by HEK293 cells, and the purified bifunctional fusion protein was named PLTGB-M7824-IgG1 (heavy chain variable region sequence is SEQ ID NO: 19, light chain variable region sequence is SEQ ID NO: 20, light The sequence of the chain constant region is SEQ ID NO: 21, and the sequence of the heavy chain constant region and TGF ⁇ RII is SEQ ID NO: 18).
  • TGF ⁇ 1 Dilute TGF ⁇ 1 with pH9.6CBS to different concentrations (0.2 ⁇ g/mL, 0.05 ⁇ g/mL, 0.0125 ⁇ g/mL), respectively coat the ELISA plate, 100 ⁇ L/well, incubate at 4°C overnight; wash the plate with 3% skimmed milk powder Block at 37°C for 1h; after washing the plate, add 100 ⁇ L of candidate bifunctional fusion protein (1 ⁇ g/mL) diluted in PBST (PBS+0.05% Tween20) to each well, incubate at 37°C for 1h; then add goat anti-human IgG/HRP and incubate at 37°C 1h; After washing the plate, add TMB for color development, add 50 ⁇ L 2M H 2 SO 4 to each well for 10 minutes to stop color development, and read OD450 on the microplate reader.
  • Table 3 and Figure 2 The results are shown in Table 3 and Figure 2.
  • the OD values of the candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1, PLTGBQ21-BA669-IgG1 at each concentration are higher than those of the control group PLTGB-M7824-IgG1, indicating that the candidate bifunctional fusion protein has a better effect on TGF ⁇ 1 Binding capacity.
  • Purify by size exclusion chromatography (Shanghai Bogelong, catalog number: AG319109): equilibrate the column with 1.5CV buffer, load the sample, the sample volume is not more than 3% CV, continue to rinse with buffer, and collect after peaking Objective Bifunctional fusion protein.
  • IFN- ⁇ secretion values of candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1 and PLTGBQ21-BA669-IgG1 at the same concentration were higher than those of the control group PLTGB-M7824-IgG1.
  • the candidate bifunctional fusion protein PLTGBQ19-BA533-IgG1, PLTGBQ21-BA669-IgG1 may have better immune regulation and anti-tumor properties than the control group PLTGB-M7824-IgG1.
  • the antibody binding kinetics was detected by BIAcore8K instrument.
  • the anti-human IgG antibody was coupled to the CM5 biosensor chip by the anti-human IgG Fc amino coupling kit to obtain about 1000 RU (response unit). Dilute PDL1 with HBS-EP+1 ⁇ buffer to 50nM, and then dilute it twice, for a total of 5 concentrations (50nM, 25nM, 12.5nM, 6.25nM, 3.125nM), and set a blank control.
  • the determination steps and conditions are as follows: bifunctional fusion protein injection 2 ⁇ g/mL, injection time 70s, flow rate 5 ⁇ L/min, stable for 5s; PDL1 protein binding and dissociation: binding 60s, flow rate 30 ⁇ L/min, dissociation 450s; regeneration: Regenerate with 3M MgCl 2 buffer solution for 30 seconds and start 3 times.
  • a one-to-one Languir model (BIAcore Evaluation Software version 3.2) was used to calculate the binding constant (ka) and dissociation constant (kd), and the equilibrium dissociation constant KD was calculated from kd/ka.
  • the affinity data of each bifunctional fusion protein is shown in Table 5.
  • MV-1-Lu cells were resuspended on the basis and added to a white 96-well plate (Corning, catalog number: 3917), 50 ⁇ L/well, 2000 cells/well. Dilute the bifunctional fusion protein to 333.3 ng/mL with complete medium, and then sequentially dilute it by 3 times to a total of 6 concentrations, and add it to the cell wells at 25 ⁇ L/well.
  • TGF ⁇ 1 Dilute TGF ⁇ 1 to 2ng/mL with complete medium and add it to cell wells at 25 ⁇ L/well.
  • the final concentration of the bifunctional fusion protein 83.3ng/mL, 27.8ng/mL, 9.26ng/mL, 3.09ng/mL, 1.03ng/mL, 0.34ng/mL.
  • the CellTiter-Glo kit Promega, catalog number: G7571 was used to detect the fluorescence value in the 96-well plate (the fluorescence value can represent the number of cells).
  • the candidate bifunctional fusion proteins PLTGBQ19-BA533-IgG1 and PLTGBQ21-BA669-IgG1 have better activity to block the function of TGF ⁇ 1 than the control group PLTGB-M7824-IgG1.
  • the candidate bifunctional fusion protein PLTGBQ19-BA533-IgG1, PLTGBQ21-BA669-IgG1 can better block the TGF pathway than the control group PLTGB-M7824-IgG1, and have better immune regulation and anti-tumor properties.
  • the IgG1 monoclonal antibody corresponding to the bifunctional fusion protein was constructed.
  • the monoclonal antibody corresponding to PLTGBQ19-BA533-IgG1 is BA533-IgG1 (the heavy chain variable region sequence is SEQ ID NO: 2, and the light chain variable region sequence is SEQ ID NO:1
  • the light chain constant region sequence is SEQ ID NO: 16, the heavy chain constant region sequence is SEQ ID NO: 17);
  • the corresponding monoclonal antibody of PLTGBQ21-BA669-IgG1 is BA669-IgG1 (the heavy chain variable region sequence is SEQ ID NO: 4
  • the light chain variable region sequence is SEQ ID NO: 3, the light chain constant region sequence is SEQ ID NO: 16, and the heavy chain constant region sequence is SEQ ID NO: 17);
  • PLTGB-M7824-IgG1 corresponding monoclonal antibody is Avelumab ( The heavy chain variable region sequence is SEQ ID NO: 19, the light chain variable region sequence is SEQ ID
  • Mouse colon cancer MC38-hPD-L1 cells were purchased from Beijing Biocytogen Biotechnology Co., Ltd.
  • the culture conditions were DMEM (Gibco, catalog number: 11965-092) medium plus 10% FBS (Gibco, catalog number: 10099-141C), 1% P/S (Gibco, catalog number: 15070-063), 37°C , 5% CO 2 incubator culture. Passage 2-3 times a week.
  • Collect MC38-hPD-L1 cells in good condition resuspend them in PBS and adjust the cell concentration to 5 ⁇ 10 6 /ml, and inoculate 0.1 ml of cell suspension subcutaneously on the dorsal side of the forelimb of each mouse. Randomization was started when the average tumor volume reached 87mm 3 . There are 7 groups, 6 animals in each group. The administration was started on the day of grouping. The anti-PD-L1 monoclonal antibody was diluted with PBS. Two doses of each antibody: 3mg/kg and 10mg/kg, intraperitoneal injection, once every 2 days, The solvent control group was given the same volume of PBS as a control.
  • the candidate antibody has better anti-tumor effect than the control antibody, and high doses also have comparable anti-tumor effect.
  • group End point tumor weight (g) Vehicle(Q2D,i.p.) 2.12 ⁇ 0.24 BA533-IgG1 (3mg/kg, Q2D, i.p.) 0.92 ⁇ 0.26 BA669-IgG1 (3mg/kg, Q2D, i.p.) 0.77 ⁇ 0.13 Avelumab (3mg/kg, Q2D, i.p.) 1.22 ⁇ 0.33 BA533-IgG1 (10mg/kg, Q2D, i.p.) 0.46 ⁇ 0.12 BA669-IgG1 (10mg/kg, Q2D, i.p.) 0.39 ⁇ 0.08 Avelumab (10mg/kg, Q2D, i.p.) 0.42 ⁇ 0.09

Abstract

提供了抗PDL1抗体及其抗原结合片段，还提供了抗PDL1和TGFβ的双功能融合蛋白及其制备方法和用途。该抗体或抗原结合片段或双功能融合蛋白具有以下功能中的一种或多种优势：增强的TGFβ1结合活性、增强的PDL1亲和力、增强的阻断PDL1和PD1结合的能力、增强的阻断TGFβ1功能活性、增强的促进T细胞分泌IFN-γ的能力、更好的免疫调节效果、更好的肿瘤抑瘤效果。

Description

抗PDL1和TGFβ的双功能融合蛋白及其用途 技术领域
本发明涉及生物医学或生物制药技术领域。具体而言，本发明涉及抗PDL1的抗体、抗PDL1和TGFβ的双功能融合蛋白及其变体和抗原结合片段，以及其制备方法和用于治疗癌症的方法和用途。
背景技术
PD1(程序化细胞死亡-1)和PDL1(程序化细胞死亡-1配体1)是一类重要的免疫检查点蛋白，其中PD1表达于激活的T细胞和NK细胞表面上，肿瘤细胞通过大量表达PDL1，同PD1结合之后抑制T细胞和NK细胞的成熟和增殖，进而逃离免疫监视(Iwai et al.,PNAS99:12293-7,2002)(Ohigashi et al.,Clin Cancer Res 11:2947-53,2005)。因而通过阻断两者相互作用可以重新激活T细胞，识别并杀灭肿瘤细胞，使患者获益。
TGFβ可以抑制早期肿瘤的生长，但随着肿瘤的持续进展，肿瘤细胞对TGFβ变得不再敏感，这时TGFβ通过促进上皮-间质转移和抑制免疫***，转而促进肿瘤生长(Bierie et ah,Nat Rev Cancer.2006；6:506-20)。
TGFβRII的胞外结构域仅长136个氨基酸残基，可结合TGFβ1(Lin et al,J Biol Chem.1995；270:2747-54)。可溶TGFβRII-Fc经过作为抗癌剂的实验，并且显示其在鼠模型中可抑制恶性间皮瘤生长(Suzuki et al,Clin Cancer Res.2004；10:5907-18)(Suzuki等，Clin Cancer Res.2004；10:5907-18)。
Merck公司的M7824双功能融合蛋白(中国申请号CN201580007865.3)为抗PDL1抗体Avelumab与TGFβRII通过(G 4S) 4G连接，其已完成部分I期及II期临床试验，在多种实体瘤中表现良好(MSB0011359C)。
然而，面对患者对于疾病治疗的药物需求，尤其是抗体药物的需求，临床上仍然亟待提供一种结合活性更高、药效更优的抗PDL1的抗体或抗PDL1和TGFβ的双功能融合蛋白。
发明内容
本发明全文中关于VL(轻链可变区)、VH(重链可变区)、LCDR(轻链互补决定区)、HCDR(重链互补决定区)、LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3的各个实施方式可以各自单独实施，也可以任意组合实施。
本发明涉及一种抗体或其抗原结合片段或双功能融合蛋白及其制备方法和用途。
在本发明的一方面，本发明涉及抗体或其抗原结合片段，包含3个重链互补决定区，其中HCDR1氨基酸序列如SEQ ID NO:8所示，HCDR2氨基酸序列如SEQ ID NO:9所示，HCDR3氨基酸序列如SEQ ID NO:10所示。进一步地，所述抗体或其抗原结合片段还包含3个轻链互补决定区，其中LCDR1氨基酸序列如SEQ ID NO:5所示，LCDR2氨基酸序列如SEQ ID NO:6所示，LCDR3氨基酸序列如SEQ ID NO:7所示。
在本发明的一方面，本发明提供的抗体或其抗原结合片段包含SEQ ID NO:2所示的重链可变区；优选还含有SEQ ID NO:1所示的轻链可变区。
在本发明的一方面，本发明涉及抗体或其抗原结合片段，包含3个轻链互补决定区，其中LCDR1氨基酸序列如SEQ ID NO:11所示，LCDR2氨基酸序列如SEQ ID NO:6所示， LCDR3氨基酸序列如SEQ ID NO:12所示；和/或3个重链互补决定区，其中HCDR1氨基酸序列如SEQ ID NO:13所示，HCDR2氨基酸序列如SEQ ID NO:14所示，HCDR3氨基酸序列如SEQ ID NO:15所示。
在另一方面，本发明涉及抗体或其抗原结合片段包含SEQ ID NO:3所示氨基酸序列的轻链可变区，和/或SEQ ID NO:4所示氨基酸序列的重链可变区。
在另一方面，前述任一方面所述抗体或其抗原结合片段的轻链恒定区的序列为SEQ ID NO:16。
在另一方面，前述任一方面所述抗体或其抗原结合片段的重链恒定区的序列为SEQ ID NO:17。
具体地，本发明提供的抗体或其抗原结合片段优选包含SEQ ID NO:1所示氨基酸序列的轻链可变区，SEQ ID NO:2所示氨基酸序列的重链可变区，SEQ ID NO:16所示氨基酸序列的轻链恒定区和SEQ ID NO:17所示氨基酸序列的重链恒定区。或者，本发明提供的抗体或其抗原结合片段优选包含SEQ ID NO:3所示氨基酸序列的轻链可变区，SEQ ID NO:4所示氨基酸序列的重链可变区，SEQ ID NO:16所示氨基酸序列的轻链恒定区和SEQ ID NO:17所示氨基酸序列的重链恒定区。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段包括单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体、Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段等。
在另一方面，上述任意一种抗体或其抗原结合片段结合PDL1。
在另一方面，本发明涉及一种双功能融合蛋白，其包括前述任一方面所述的抗体或其抗原结合片段，以及TGFβRII片段，优选地，所述TGFβRII片段包含TGFβRII胞外域。
在另一方面，所述双功能融合蛋白中，前述任一方面所述抗体或其抗原结合片段与TGFβRII片段之间通过接头连接，优选地，所述TGFβRII片段连接在所述抗体或其抗原结合片段的重链恒定区的C端，优选通过(G 4S) 4G(即GGGGSGGGGSGGGGSGGGGSG)接头连接，优选地，所述双功能融合蛋白的重链恒定区的序列为SEQ ID NO:22，优选地，所述双功能融合蛋白的TGFβRII片段的序列为SEQ ID NO:23；具体地，所述双功能融合蛋白结构为：抗体或其抗原结合片段-(G 4S) 4G-TGFβRII片段。
在另一方面，所述双功能融合蛋白中，所述抗体或其抗原结合片段的轻链可变区的序列为SEQ ID NO:1，重链可变区的序列为SEQ ID NO:2，优选地，所述双功能融合蛋白中，轻链恒定区的序列为SEQ ID NO:16，重链恒定区的序列为SEQ ID NO:22，TGFβRII片段的序列为SEQ ID NO:23；具体地，重链恒定区与TGFβRII片段通过(G 4S) 4G连接的序列为SEQ ID NO:18。
在另一方面，所述双功能融合蛋白中，所述抗体或其抗原结合片段的轻链可变区的序列为SEQ ID NO:3，重链可变区的序列为SEQ ID NO:4，优选地，所述双功能融合蛋白中，轻链恒定区的序列为SEQ ID NO:16，重链恒定区的序列为SEQ ID NO:22，TGFβRII片段的序列为SEQ ID NO:23；具体地，重链恒定区与TGFβRII片段通过(G 4S) 4G连接的序列为SEQ ID NO:18。
在另一方面，上述任意一种双功能融合蛋白结合PDL1和TGFβ。
在另一方面，本发明涉及一种核酸，其编码前述任一方面所述的抗体或其抗原结合片段或双功能融合蛋白。
在另一方面，本发明涉及一种载体，其包含前一方面所述的核酸，或其可表达前述任 一方面所述的抗体或其抗原结合片段或双功能融合蛋白。优选地，所述载体可以是病毒载体；优选地，所述病毒载体包含但不限于慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体等；优选地，所述载体可以是非病毒载体；优选地，所述载体可以是哺乳细胞表达载体；优选地，所述表达载体可以是细菌表达载体；优选地，所述表达载体可以是真菌表达载体。
在另一方面，本发明涉及一种细胞，其可表达前述任一方面所述的抗体或其抗原结合片段或双功能融合蛋白的细胞。优选地，所述细胞为细菌细胞；优选地，所述细菌细胞为大肠杆菌细胞等；优选地，所述细胞为真菌细胞；优选地，所述真菌细胞为酵母细胞；优选地，所述酵母细胞为毕赤酵母细胞等；优选地，所述细胞为哺乳动物细胞；优选地，所述哺乳动物细胞为中国仓鼠卵巢细胞(CHO)、人胚胎肾细胞(293)、B细胞、T细胞、DC细胞或NK细胞等。
在另一方面，本发明涉及一种药物组合物，其包含前述任一方面所述的抗体或其抗原结合片段、双功能融合蛋白、核酸、载体或细胞，优选地，所述药物组合物还包含药学上可接受的赋形剂，优选地，所述药学上可接受的赋形剂包括以下中的一种或多种：药学上可接受的溶剂、分散剂、附加剂、塑形剂等。
在另一方面，所述药物组合物还可以包含其他治疗剂。在一些实施方式中，其他治疗剂包括化疗剂、免疫治疗剂或激素治疗剂。所述抗体或抗原结合片段与其他治疗剂的联合施用能够增强治疗效果。
在另一方面，所述“增强治疗效果”是指增强其他治疗剂或疗法的治疗效果。本发明提供的所述抗体或抗原结合片段可以单独施用，也可以与其他治疗剂或疗法联合施用。在一些实施方式中，其他治疗剂或疗法包括化疗剂、免疫治疗剂、激素治疗剂、放射治疗、手术治疗。
在另一方面，提供一种试剂盒，其包含本发明所述的抗体或其抗原结合片段，或包含双功能融合蛋白，或包含编码抗体或其抗原结合片段或双功能融合蛋白的核酸。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、双功能融合蛋白、核酸、载体或细胞在制备治疗或预防疾病的药物中的应用。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、双功能融合蛋白或核酸在制备诊断或检测试剂盒中的应用。
在另一方面，提供一种治疗或预防疾病的方法，包括将本发明的任一方面所述抗体或抗原结合片段、双功能融合蛋白、核酸、载体、细胞或药物组合物给予有需要的受试者。
在另一方面，提供一种诊断或检测的方法，包括将本发明的任一方面所述抗体或抗原结合片段、双功能融合蛋白、核酸或试剂盒给予有需要的受试者或样本。优选地，所述方法是诊断或检测疾病的方法。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、双功能融合蛋白、核酸、载体、细胞或药物组合物用于治疗或预防疾病的用途。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、双功能融合蛋白、核酸或试剂盒用于检测或诊断的用途。优选地，所述用途是诊断或检测疾病的用途。
在另一方面，所述疾病是癌症。
在另一方面，所述癌症包括胃癌、食道癌、头颈癌、膀胱癌、***、肉瘤、细胞瘤、肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤等。
在另一方面，本发明涉及制备前述任一方面所述的抗体或其抗原结合片段或双功能融合蛋白的方法，包括使用上述载体转染细胞，并由转染的细胞表达所述抗体或其抗原结合片段或双功能融合蛋白；或者包括用上述细胞表达所述抗体或其抗原结合片段或双功能融合蛋白。
本发明提供的抗体或抗原结合片段或双功能融合蛋白具有以下一种或多种优势：增强的TGFβ1结合活性、增强的PDL1亲和力、增强的阻断PDL1和PD1结合的能力、增强的阻断TGFβ1功能活性、增强的促进T细胞分泌IFN-γ的能力、更好的免疫调节效果、更好的肿瘤抑瘤效果。
附图说明
图1示出了BoAn-hMab1小鼠五免后血清滴度(2500倍稀释)。
图2示出了双功能融合蛋白与TGFβ1蛋白结合。
图3示出了双功能融合蛋白促进CD4+T分泌IFN-γ。
图4示出了双功能融合蛋白对TGFβ1功能的阻断效应。
图5A示出了MC38-hPD-L1肿瘤模型小鼠体重；图5B示出了MC38-hPD-L1肿瘤模型小鼠肿瘤体积；图5C示出了MC38-hPD-L1肿瘤模型小鼠瘤重。
具体实施方式
下面结合具体实施例，进一步阐述本发明。所描述的实施例是本发明一部分实施例，而不是全部的实施例。应理解，举出以下实施例是为了向本发明所属技术领域的一般专业人员就如何利用本发明之方法和组合物提供一个完整的公开和说明，并非用于限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
材料和试剂：PDL1蛋白(Sinobiological，目录号：10084-H08H)；PD1蛋白(Sinobiological,目录号：10377-H08H)；TGFβ1(Sinobiological,目录号：10804-HNAC)；NaHCO 3(国药，10018960)；TMB(北京梅科万德，目录号：1001)；STREP/HRP(R&D，目录号：890803)；HBS-EP+1×缓冲液(GE，目录号：BR-1008-26)；酶标板(苏州海狸，目录号：40301)；生物传感器芯片(GE，目录号：BR100530)；96孔圆底板(Corning，目录号：3799)；抗人IgG Fc氨基偶联试剂盒(GE，cat#BR-1008-39)；
实施例1.抗PDL1和TGFβ的双功能融合蛋白的制备
1.1 PDL1蛋白免疫
将PDL1蛋白与弗氏完全佐剂(Sigma，目录号：F5881-10ML)、弗氏不完全佐剂(Sigma，目录号：F5506-10ML)或金佐剂(Sigma，目录号：T2684-1mL)乳化后免疫博安生物全人抗体转基因小鼠BoAn-hMab1(按照中国专利CN103571872B记载的方法制备)。本次共免疫11只小鼠，包括6只雄性和5只雌性，鼠龄为6月-12月，共免疫5次，选取血清滴度较高的6只小鼠用上述PDL1蛋白进行加强免疫。采用ELISA检测的血清滴度(2500倍稀释)见图1。其中PL1Q15、PL1Q16、PL1Q18、PL1Q19、PL1Q21小鼠为弗氏佐剂免疫小鼠，PL1Q24小鼠为金佐剂免疫小鼠。
1.2噬菌体库的建立
处死1.1中血清滴度较高的6只小鼠，解剖取出脾脏，把脾脏用注射器胶塞研磨破碎 并用滤网(FALCON，目录号：352350)过滤，把滤过的脾细胞冷冻备用。提取RNA后反转录获得cDNA，噬菌体库的建立步骤参照Carlos F.Barbas III，Phage display：A laboratory manual中记载的方法进行，用PCR的方法从cDNA中获得重链和轻链的可变区，再将重链和轻链的可变区通过重叠延伸PCR的方法获得单链Fv(scFv)，scFv经SfiI酶(NEB，目录号：#R0123L)酶切后通过T4DNA连接酶(义翘神州)与质粒pCOMB3x(中国质粒载体菌株细胞株基因保藏中心，BIOVECTOR510837)连接，然后将连接产物电转染至大肠杆菌TG1感受态细胞(Lucigen，目录号：A96595-2)中，转染后的TG1经37℃220rpm摇床培养后加入噬菌体侵染，然后回收培养物上清浓缩纯化获得噬菌体库。1.3噬菌体库的筛选
平板筛选：CBS缓冲液的制备：称取Na 2CO 3(国药，10019260)1.59g、NaHCO 3 2.93g加蒸馏水至1L配制成CBS缓冲液。用CBS缓冲液稀释PDL1蛋白至10μg/mL，按100μL/孔加入到筛选平板(Costar，42592)中，每个库用8个孔，4℃放置过夜；第二天弃掉孔板中蛋白溶液，用2％BSA(Solarbio，A8010)封闭平板1h，加入噬菌体库(2x10 12/孔)样品37℃孵育2h，用PBST(PBS+0.05％Tween20)洗涤4-10次后，用洗脱缓冲液(向500ml超纯水中加入4.2ml浓盐酸(科密欧)，用甘氨酸粉末(Biotopped，BG0617-500)调节pH至2.2)洗脱PDL1特异结合的噬菌体。
磁珠筛选：将PDL1-mFc蛋白(Acrobiosystems，目录号：PD1-H52A3)进行生物素化(蛋白与生物素摩尔比为1:2)：将PDL1-mFc蛋白换液至0.1M pH8.0NaHCO 3中，精密天平称取适量生物素(Thermo，21335)用超纯水溶解，立即向PDL1-mFc蛋白溶液中加入适量溶解后的生物素，在旋转混合仪上避光旋转孵育40min，标记完成后将PDL1-mFc蛋白溶液换液至PBS中。将生物素化的PDL1-mFc蛋白(3μg/库)与磁珠(Invitrogen Dynabeads M-280 Streptavidin，目录号：00355871)(10μL/库)室温结合1h，经2％BSA封闭后与噬菌体库室温孵育2h，洗涤4-10次后用洗脱缓冲液(pH 2.2)洗脱PDL1特异结合的噬菌体。
噬菌体克隆通过大肠杆菌表达scFv，然后通过ELISA检测scFv对PDL1/PD1结合的阻断，保留阻断活性的克隆进行后续分子构建。
ELISA检测scFv阻断PD1/PDL1的结合：用pH9.6CBS稀释PD1蛋白至0.5μg/mL，包被酶标板，100μL/孔，4℃孵育过夜；洗板后用3％脱脂奶粉于37℃封闭1h。洗板后每孔加入scFv周质50μL。然后加入生物素标记的PDL1-Fc蛋白(终浓度0.2μg/mL)，50μL/孔，37℃孵育1h；洗板后加入PBST稀释的STREP/HRP，100μL/孔，37℃孵育1h。洗板后每孔加入100μL TMB显色，10min后每孔加入50μL 2M H 2SO 4终止显色，用酶标仪读取OD450。
1.4双功能融合蛋白分子的构建与生产
将磁珠筛选的克隆BA533、BA603、BT613、BA623、BA649、BA669、BA466和平板筛选的克隆BA705送至Invitrogen生物技术有限公司测序。每个克隆的轻链可变区和重链可变区的氨基酸序列记载于表1中。
表1候选克隆的轻链可变区和重链可变区的氨基酸序列(CDR以下划线标出)
Figure PCTCN2020094855-appb-000001
Figure PCTCN2020094855-appb-000002
Figure PCTCN2020094855-appb-000003
通过可变区基因扩增(2*Phanta Max Master Mix，厂家：Vazyme，目录号：P515-AA)、信号肽与可变区重叠延伸、同源重组(ClonExpressⅡOne Step Cloning Kit，厂家：Vazyme，目录号：C112-01)等方法，最终把编码VH的核苷酸序列片段***带有编码SEQ ID NO:18的核苷酸序列的载体pCDNA3.4(Life Technology)，其中SEQ ID NO:18包含由(G 4S) 4G连接的IgG1抗体的重链恒定区序列及TGFβRII序列，把编码VL的核苷酸序列片段***带有编码抗体轻链恒定区氨基酸序列(SEQ ID NO:16)的核苷酸序列的载体pCDNA3.4(Life Technology)，然后转染HEK293细胞，并在37℃\8％CO 2\125rpm摇床中培养6-7天，最后培养上清通过Protein A填料纯化获得表2中的双功能融合蛋白克隆用于后续检测。
表2抗PDL1-TGFβ的双功能融合蛋白克隆
PLTGBQ19-BA533-IgG1 PLTGBQ24-BA603-IgG1 PLTGBQ16-BA466-IgG1
PLTGBQ21-BA649-IgG1 PLTGBH03-BT613-IgG1 PLTGBQ24-BA705-IgG1
PLTGBQ21-BA669-IgG1 PLTGBQ21-BA623-IgG1 /
对照双功能融合蛋白的生产：Merck公司的M7824双功能融合蛋白为抗PDL1抗体Avelumab与TGFβRII通过(G 4S) 4G连接，其已完成部分I期及II期临床试验，在多种实体瘤中表现良好。通过IMGT数据库确定Merck公司的Avelumab的可变区氨基酸序列(重链可变区序列为SEQ ID NO:19，轻链可变区序列为SEQ ID NO:20)，全基因合成后***到载体pCDNA3.4，通过HEK293细胞进行表达，纯化的双功能融合蛋白命名为PLTGB-M7824-IgG1(重链可变区序列为SEQ ID NO:19，轻链可变区序列为SEQ ID NO:20，轻链恒定区序列为SEQ ID NO:21，重链恒定区及TGFβRII序列为SEQ ID NO:18)。
1.5 ELISA检测双功能融合蛋白对PDL1-PD1相互结合的阻断
用pH9.6CBS稀释PD1蛋白至0.5μg/mL，包被酶标板，100μL/孔，4℃孵育过夜；洗板后用3％脱脂奶粉37℃封闭1h；洗板后分别加入PBST(PBS+0.05％Tween20)稀释不同浓度(0.5μg/mL、0.25μg/mL、0.125μg/mL、0.0625μg/mL、0.03125μg/mL、0.015625μg/mL)双功能融合蛋白，50μL/孔，然后加入标记有生物素的PDL1-Fc蛋白(终浓度0.2μg/mL)，50μL/孔，37℃孵育1h；洗板后加入PBST稀释的STREP/HRP，100μL/孔，37℃孵育1h。洗板后加入TMB显色，10min后每孔加入50μL 2M H 2SO 4终止显色，酶标仪上读取OD450。结果表明，本发明的候选双功能融合蛋白(PLTGBQ16-BA466-IgG1、PLTGBQ19-BA533-IgG1、PLTGBQ24-BA603-IgG1、PLTGBQ21-BA623-IgG1、PLTGBQ21-BA649-IgG1、PLTGBQ21-BA669-IgG1、PLTGBQ24-BA705-IgG1、PLTGBH03-BT613-IgG1)均能有效阻断PD1与PDL1的结合。
1.6 ELISA检测双功能融合蛋白与TGFβ1蛋白结合
用pH9.6CBS稀释TGFβ1至不同浓度(0.2μg/mL、0.05μg/mL、0.0125μg/mL)，分别包被酶标板，100μL/孔，4℃孵育过夜；洗板后用3％脱脂奶粉37℃封闭1h；洗板后每孔加入100μL PBST(PBS+0.05％Tween20)稀释的候选双功能融合蛋白(1μg/mL)，37℃孵育1h；然后加入山羊抗人IgG/HRP，37℃孵育1h；洗板后加入TMB显色，10min每孔加入50μL 2M H 2SO 4后终止显色，酶标仪上读取OD450。结果见表3和图2。
候选双功能融合蛋白PLTGBQ19-BA533-IgG1、PLTGBQ21-BA669-IgG1在各个浓度上的OD值均高于对照组PLTGB-M7824-IgG1，说明候选双功能融合蛋白有更好的对TGFβ1有更好的结合能力。
进而预示候选双功能融合蛋白PLTGBQ19-BA533-IgG1、PLTGBQ21-BA669-IgG1相较对照组PLTGB-M7824-IgG1能更好的阻断TGF通路，临床上有更好的免疫调节和抗肿瘤特性。
表3 ELISA检测双功能融合蛋白与TGFβ1蛋白结合数据(对应图2)
Figure PCTCN2020094855-appb-000004
实施例2双功能融合蛋白的表征
2.1双功能融合蛋白的生产
用ExpiCHO(Thermo，目录号:A29129)表达***转染并表达双功能融合蛋白，用蛋白A(GE，Mabselect SuRe)柱纯化表达上清：细胞培养液以4500g离心后用0.45μm滤膜过滤，上样后并平衡后用0.1M pH3.2甘氨酸缓冲液洗脱目的双功能融合蛋白，并用pH8.01M Tris进行中和。用分子排阻色谱(上海博格隆，目录号：AG319109)进行精纯：用1.5CV缓冲液平衡柱子，样品上样，样品体积不大于3％CV，用缓冲液继续冲洗，出峰后收集目的双功能融合蛋白。
2.2混合淋巴反应/MLR检测双功能融合蛋白活性
把RPMI 1640(Gibco，目录号：11875-093)、FBS(Gibco，目录号：10091-148)、HEPES(Solarbio，目录号：H1090)按照90:10:1混合以配制完全培养基，分别用完全培养基重悬DC细胞(ALLCELLS，目录号：PB-DC001F-0.3M)和CD4+T细胞(ALLCELLS，目录号：PB009-2F-C-3M)，按1:10细胞比例加入到96孔圆底板中，最终体积150μL/孔。用完全培养基把双功能融合蛋白稀释2个浓度：4μg/mL、0.4μg/mL，分别加入细胞孔中，50μL/孔，最终体积200μL。培养5天后，用IFN-γ试剂盒(cisbio，目录号：62HIFNGPEG)检测培养上清。检测结果见表4和图3。
候选双功能融合蛋白PLTGBQ19-BA533-IgG1、PLTGBQ21-BA669-IgG1在相同浓度上的IFN-γ分泌值均高于对照组PLTGB-M7824-IgG1。
进而预示候选双功能融合蛋白PLTGBQ19-BA533-IgG1、PLTGBQ21-BA669-IgG1相较对照组PLTGB-M7824-IgG1可能有更好的免疫调节和抗肿瘤特性。
表4混合淋巴反应/MLR检测双功能融合蛋白活性数据(对应图3)
Figure PCTCN2020094855-appb-000005
Figure PCTCN2020094855-appb-000006
2.3 SPR检测双功能融合蛋白与PDL1蛋白的亲和力
抗体结合动力学使用BIAcore8K仪器检测。通过抗人IgG Fc氨基偶联试剂盒将抗人IgG抗体偶联到CM5生物传感器芯片上以获得约1000RU(应答单位)。把PDL1用HBS-EP+1×缓冲液稀释至50nM，然后2倍稀释，共5个浓度(50nM、25nM、12.5nM、6.25nM、3.125nM)，并设置空白对照。测定步骤及条件如下：双功能融合蛋白进样2μg/mL，进样时间70s，流速5μL/min，稳定5s；PDL1蛋白结合与解离：结合60s，流速30μL/min，解离450s；再生：用3M MgCl 2缓冲液再生30s，启动3次。使用一对一Languir模型(BIAcore Evaluation Software version 3.2)计算结合常数(ka)和解离常数(kd)，由kd/ka计算得到平衡解离常数KD。各双功能融合蛋白亲和力数据见表5。
结果表明，本发明的候选双功能融合蛋白PLTGBQ19-BA533-IgG1和PLTGBQ21-BA669-IgG1与对照组PLTGB-M7824-IgG1有基本相当的PDL1亲和力。其中PLTGBQ19-BA533-IgG1的PDL1亲和力优于对照组。
表5双功能融合蛋白与PDL1蛋白亲和力
名称 ka(1/Ms) kd(1/s) KD(M)
PLTGBQ16-BA466-IgG1 1.14E+06 2.32E-03 2.03E-09
PLTGBQ19-BA533-IgG1 1.50E+06 7.95E-04 5.29E-10
PLTGBQ24-BA603-IgG1 1.70E+06 1.64E-03 9.63E-10
PLTGBQ21-BA623-IgG1 1.34E+06 1.79E-03 1.34E-09
PLTGBQ21-BA649-IgG1 1.85E+06 1.67E-03 9.04E-10
PLTGBQ21-BA669-IgG1 1.78E+06 1.36E-03 7.64E-10
PLTGBQ24-BA705-IgG1 1.45E+06 8.37E-04 5.77E-10
PLTGBH03-BT613-IgG1 1.39E+06 1.01E-03 7.30E-10
PLTGB-M7824-IgG1 4.98E+05 3.00E-04 6.03E-10
2.4用MV-1-Lu细胞检测双功能融合蛋白对TGFβ1功能的阻断效应
把EMEM(ATCC，目录号：30-2003)、FBS(Gibco，目录号：10099-141)、HEPES(Solarbio，目录号：H1090)按照90:10:1混合以配制完全培养基，用完全培养基重悬MV-1-Lu细胞，加入到白色96孔板中(Corning，目录号：3917)，50μL/孔，2000细胞/孔。用完全培养基稀释双功能融合蛋白至333.3 ng/mL，然后依次3倍稀释，共6个浓度，加入到细胞孔中，25μL/孔。用完全培养基把TGFβ1稀释至2ng/mL，加入到细胞孔中，25μL/孔。双功能融合蛋白终浓度：83.3ng/mL、27.8ng/mL、9.26ng/mL、3.09ng/mL、1.03ng/mL、0.34ng/mL。培养4天后用CellTiter-Glo试剂盒(Promega，目录号：G7571) 检测96孔板中的荧光值(该荧光值可代表细胞数)，检测结果见图4。
可以看出，候选双功能融合蛋白PLTGBQ19-BA533-IgG1、PLTGBQ21-BA669-IgG1相对于对照组PLTGB-M7824-IgG1，有更好的阻断TGFβ1功能的活性。
进而预示候选双功能融合蛋白PLTGBQ19-BA533-IgG1、PLTGBQ21-BA669-IgG1相较对照组PLTGB-M7824-IgG1能更好的阻断TGF通路，有更好的免疫调节和抗肿瘤特性。
实施例3抗PD-L1单抗在小鼠MC38-hPDL1结肠癌模型中的药效测试
构建双功能融合蛋白对应的IgG1单抗，PLTGBQ19-BA533-IgG1对应单抗为BA533-IgG1(重链可变区序列为SEQ ID NO:2，轻链可变区序列为SEQ ID NO:1，轻链恒定区序列为SEQ ID NO:16，重链恒定区序列为SEQ ID NO:17)；PLTGBQ21-BA669-IgG1对应单抗为BA669-IgG1(重链可变区序列为SEQ ID NO:4，轻链可变区序列为SEQ ID NO:3，轻链恒定区序列为SEQ ID NO:16，重链恒定区序列为SEQ ID NO:17)；PLTGB-M7824-IgG1对应单抗为Avelumab(重链可变区序列为SEQ ID NO:19，轻链可变区序列为SEQ ID NO:20，轻链恒定区序列为SEQ ID NO:21，重链恒定区序列为SEQ ID NO:17)。
小鼠结肠癌MC38-hPD-L1细胞购买自北京百奥赛图基因生物技术有限公司。培养条件为DMEM(Gibco,目录号：11965-092)培养基中加10％FBS(Gibco,目录号：10099-141C)、1％P/S(Gibco,目录号：15070-063)，37℃、5％CO 2培养箱培养。一周传代2-3次。B-hPD-L1人源化小鼠，雌性，6-8周龄，购买自百奥赛图江苏基因生物技术有限公司。
收集状态良好的MC38-hPD-L1细胞，PBS重悬混匀，调整细胞浓度至5×10 6/ml，将0.1ml细胞悬液接种于每只小鼠的前肢背侧皮下。当肿瘤平均体积达到87mm 3时开始随机分组。共7组，每组6只，分组当天开始给药，用PBS稀释抗PD-L1单抗，每种抗体两个剂量：3mg/kg、10mg/kg，腹腔注射，每2天给药一次，溶剂对照组给予相同体积的PBS作为对照。每周测量小鼠称重、肿瘤体积2-3次，肿瘤体积(mm 3)＝0.5×长径×短径 2。当对照组肿瘤体积达到2000mm 3时，对小鼠实行安乐死，剥取肿瘤进行称重。结果见图5A、图5B、图5C及表6、表7。
如图5A所示，小鼠体重稳定增长，无不良反应出现。
如图5B和7C所示，BA533-IgG1、BA669-IgG1、Avelumab给药剂量为10mg/kg时，均可显著抑制小鼠MC38-hPD-L1肿瘤的生长，相对肿瘤抑制率TGI(％)≥80％，三者之间没有统计学差异。BA533-IgG1、BA669-IgG1、Avelumab给药剂量为3mg/kg时，BA533-IgG1和BA669-IgG1的肿瘤抑制率优于Avelumab。
上述结果说明，低剂量下，候选抗体较对照抗体有更好的抑瘤效果，高剂量也具有相当的抑瘤效果。
表6 MC38-hPD-L1肿瘤模型小鼠肿瘤体积数据(平均值±SEM)(对应图5B)
组 重点肿瘤体积(mm 3)
Vehicle(Q2D,i.p.) 1972±224
BA533-IgG1(3mg/kg,Q2D,i.p.) 780±185
BA669-IgG1(3mg/kg,Q2D,i.p.) 679±113
Avelumab(3mg/kg,Q2D,i.p.) 969±208
BA533-IgG1(10mg/kg,Q2D,i.p.) 465±131
BA669-IgG1(10mg/kg,Q2D,i.p.) 392±83
Avelumab(10mg/kg,Q2D,i.p.) 408±90
表7 MC38-hPD-L1肿瘤模型瘤重数据(平均值±SEM)(对应图5C)
组 终点瘤重(g)
Vehicle(Q2D,i.p.) 2.12±0.24
BA533-IgG1(3mg/kg,Q2D,i.p.) 0.92±0.26
BA669-IgG1(3mg/kg,Q2D,i.p.) 0.77±0.13
Avelumab(3mg/kg,Q2D,i.p.) 1.22±0.33
BA533-IgG1(10mg/kg,Q2D,i.p.) 0.46±0.12
BA669-IgG1(10mg/kg,Q2D,i.p.) 0.39±0.08
Avelumab(10mg/kg,Q2D,i.p.) 0.42±0.09

Claims (10)

  1. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包含3个重链互补决定区，其中，HCDR1氨基酸序列如SEQ ID NO:8所示，HCDR2氨基酸序列如SEQ ID NO:9所示，HCDR3氨基酸序列如SEQ ID NO:10所示。
  2. 根据权利要求1所述的抗体或其抗原结合片段，还包含3个轻链互补决定区，其中，LCDR1氨基酸序列如SEQ ID NO:5所示，LCDR2氨基酸序列如SEQ ID NO:6所示，LCDR3氨基酸序列如SEQ ID NO:7所示。
  3. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包含SEQ ID NO:2所示的重链可变区；优选还含有SEQ ID NO:1所示的轻链可变区。
  4. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，所述抗原结合片段包括单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体、Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段。
  5. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段结合PDL1。
  6. 一种双功能融合蛋白，所述双功能融合蛋白包括根据权利要求1-5中任一项所述的抗体或其抗原结合片段，以及TGFβRII片段，所述抗体或其抗原结合片段与所述TGFβRII片段通过接头连接。
  7. 一种核酸或细胞，所述核酸编码根据权利要求1-5任一项所述的抗体或其抗原结合片段，或根据权利要求6所述的双功能融合蛋白，所述细胞表达根据权利要求1-5任一项所述的抗体或其抗原结合片段，或根据权利要求6所述的双功能融合蛋白。
  8. 一种药物组合物，其包含根据权利要求1-5中任一项所述的抗体或其抗原结合片段，或根据权利要求6所述的双功能融合蛋白，或根据权利要求7所述的核酸或细胞。
  9. 一种试剂盒，其包含根据权利要求1-5中任一项所述的抗体或其抗原结合片段，或根据权利要求6所述的双功能融合蛋白，或根据权利要求7所述的核酸。
  10. 根据权利要求1-5中任一项所述的抗体或其抗原结合片段，或根据权利要求6所述的双功能融合蛋白，或权利要求7所述的核酸用于治疗、预防、检测或诊断疾病的用途，优选地，所述疾病是癌症；更优选为胃癌、食道癌、头颈癌、膀胱癌、***、肉瘤、细胞瘤、肺癌、结肠癌、卵巢癌、肾脏癌、结肠直肠癌、胰脏癌、肝癌、黑色素瘤、乳腺癌、骨髓瘤、神经胶质瘤、白血病和淋巴瘤。
PCT/CN2020/094855 2019-06-10 2020-06-08 抗PDL1和TGFβ的双功能融合蛋白及其用途 WO2020248926A1 (zh)

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