WO2018233574A1 - 一种抗pd-l1人源化纳米抗体及其应用 - Google Patents

一种抗pd-l1人源化纳米抗体及其应用 Download PDF

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WO2018233574A1
WO2018233574A1 PCT/CN2018/091639 CN2018091639W WO2018233574A1 WO 2018233574 A1 WO2018233574 A1 WO 2018233574A1 CN 2018091639 W CN2018091639 W CN 2018091639W WO 2018233574 A1 WO2018233574 A1 WO 2018233574A1
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nanobody
seq
antibody
protein
cells
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French (fr)
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安康
安文琪
范蓓
马小伟
潘若文
张宝献
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华兰生物工程股份有限公司
华兰基因工程有限公司
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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

Definitions

  • the present invention relates to the field of biomedical or biopharmaceutical technology, and more particularly to anti-PD-L1 Nanobodies and their uses.
  • T cells are the basis of anti-tumor immunity, and activation and proliferation of T cells requires a dual signal. That is, in addition to the first signal generated by the combination of the T cell receptor (TCR) and the MHC-antigen peptide complex expressed on the surface of antigen-presenting cells (APCs); a plurality of co-stimulatory molecules are required to participate in the second signal provided ( Co-stimulatory signal).
  • TCR T cell receptor
  • APCs antigen-presenting cells
  • T cells are activated by the first signal provided by the TCR and the synergistic stimulation signal, and are activated by the activation threshold, generating effects and memory cells, and exerting an immune defense function, and the activation process causes a
  • a second signal provided by a costimulatory molecule it will result in unresponsive or specific immune tolerance of T cells, and even induces cells to enter apoptosis.
  • the costimulatory signal is overrepresented, it may lead to abnormal activation of immune cells, leading to various autoimmune diseases. Therefore, the positive and negative costimulatory signals obtained by immune cells need to reach a relative balance so that the immune response can be properly turned on and properly terminated, playing an important role in the body's resistance to foreign antigen invasion and prevention of autoimmune diseases. .
  • costimulatory molecules have become one of the new hot spots in immunology research.
  • the molecules that mediate the costimulatory signals mainly include the tumor necrosis factor/tumor necrosis factor receptor (TNF/TNFR) superfamily and the immunoglobulin superfamily, such as CD28/B7. These costimulatory molecules conduct signals in a manner that interacts with the ligand.
  • TNF/TNFR tumor necrosis factor/tumor necrosis factor receptor
  • CD28/B7 immunoglobulin superfamily
  • Programmed death factor 1 / programmed death factor 1 ligand 1 as a new member of the CD28/B7 costimulatory molecule superfamily, can mediate negative co-stimulatory signals, can effectively inhibit T, B cell function and proliferation, while reducing the secretion of cytokines IL-2, IL-10 and IFN- ⁇ , which are involved in the study of tumor immunity, transplant immunity, viral infection, autoimmune and other diseases. Significance.
  • PD-1 is a member of the immunoglobulin B7-CD28 family. It consists of an extracellular segment, a hydrophobic transmembrane region, and an intracellular segment. Its intracellular domain contains an immunoreceptor tyrosine-inhibiting motif (immunoreceptor tyrosine-based). Inhibitor motif, ITIM), immunoreceptor tyrosine-based switch motif (ITSM). Among them, the activation of ITSM is closely related to the effector T cell response activity.
  • PD-1 can be expressed on activated CD4 + T cells, CD8 + T cells, B cells, natural killer T cells, monocytes and dendritic cells. In addition, PD-1 is also expressed in regulatory T cells (Treg) and promotes proliferation of Treg cells and suppresses immune responses.
  • Treg regulatory T cells
  • Programmed death 1 ligand 1 (PD-L1), also known as CD274, is a member of the B7 family and is a ligand for PD-1.
  • PD-L1 is a type I transmembrane protein with a total of 290 amino acids and a molecular weight of approximately 30-35 KD.
  • Human PD-L1 molecules can be constitutively expressed in non-lymphoid tissues such as placenta, heart, liver, lung, kidney, skeletal muscle and some hematopoietic cells, and also moderately expressed in lymphoid tissues such as thymus, lymph nodes and spleen.
  • PD-L1 is also expressed in cancer tissues such as lung cancer, liver cancer, breast cancer, ovarian cancer, etc., and cancer tissues can also up-regulate their expression after induction.
  • cancer tissues such as lung cancer, liver cancer, breast cancer, ovarian cancer, etc.
  • cancer tissues can also up-regulate their expression after induction.
  • activation of the PD-1/PD-L1 signaling pathway minimizes the damage of the immune response to surrounding tissues and prevents autoimmune diseases.
  • activation of the PD-1/PD-L1 signaling pathway alters the local microenvironment of the tumor, resulting in a decrease in T cell immune effects, thereby mediating tumor immune escape and promoting tumor growth.
  • the expression of PD-L1 on tumors is associated with decreased survival of esophageal cancer, pancreatic cancer and other types of cancer, highlighting this pathway as a new promising target for tumor immunotherapy and has been confirmed by numerous experiments.
  • Nanobodies a novel small molecule antibody fragment, were cloned from the heavy chain variable region (VHH) of the camelid natural heavy chain antibody.
  • VHH heavy chain variable region
  • Nano-antibodies have excellent biological properties, molecular weight 12-15KD, which is one-tenth of intact antibodies. It overcomes the drawbacks of large molecular weight of natural antibodies and has complete antigen-binding sites with good tissue penetration. Sex, high specificity and good water solubility. Due to its special structural properties, it combines the advantages of traditional antibodies and small molecule drugs, and almost completely overcomes the shortcomings of traditional antibody development, low stability, and harsh storage conditions, and has gradually become a new generation of antibody therapy. Strength, showing broad application prospects in immunodiagnosis and treatment
  • the present invention provides a Nanobody specific for PD-L1 and is capable of effectively blocking the binding of PD-L1 to PD-1.
  • a VHH chain of an anti-PD-L1 Nanobody comprising a complementarity determining region CDR comprising the CDR1 set forth in SEQ ID NO.: CDR2 set forth in SEQ ID NO.: 6 and CDR3 set forth in SEQ ID NO.: 7 (or consisting of said CDR1, CDR2 and CDR3).
  • the PD-L1 is human PD-L1.
  • the amino acid sequence of any one of the above amino acid sequences further comprises at least one of adding, deleting, modifying and/or substituting, such as 1-3, preferably 1-2, more preferably 1) amino acid and retains a high affinity binding to PD-L1, blocking the binding of PD-L1 to PD-1.
  • the VHH chain further comprises a framework region FR, the CDR1, CDR2 and CDR3 being separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the frame area FR is
  • the framework region FR contains the FR1, FR2, FR3 and FR4 (SEQ ID NO.: 1-4 or SEQ ID NO.: 10-13).
  • amino acid sequence of the VHH chain of the anti-PD-L1 Nanobody is as shown in SEQ ID NO.: 8 or 14.
  • a heavy chain variable region of an anti-human PD-L1 antibody comprising three complementarity determining regions CDR1, CDR2, and CDR3, and the three CDRs comprising SEQ ID NO. CDR1 shown in 5, CDR2 shown in SEQ ID NO.: 6, CDR3 shown in SEQ ID NO.: 7.
  • an anti-PD-L1 Nanobody which is a Nanobody directed against a PD-L1 epitope and which has the amino acid set forth in SEQ ID NO.: 8 or SEQ ID NO.: Sequence of VHH chains.
  • the anti-PD-L1 Nanobody is immobilized (or supported) on a solid support or a semi-solid support.
  • the present invention provides a polynucleotide encoding a protein selected from the group consisting of the VHH chain of the anti-PD-L1 Nanobody of the first aspect of the invention or the second aspect of the invention Said anti-PD-L1 Nanobody.
  • the polynucleotide has a nucleotide sequence as shown in SEQ ID NO.: 9 or 15.
  • the polynucleotide comprises DNA or RNA.
  • an expression vector comprising the polynucleotide of the third aspect of the invention is provided.
  • the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or a combination thereof.
  • the expression vector comprises a viral vector, such as a lentivirus, an adenovirus, an AAV virus, a retrovirus, or a combination thereof.
  • a host cell comprising the expression vector of the fourth aspect of the invention, or a polynucleotide thereof according to the third aspect of the invention, is integrated into the host cell.
  • the host cell comprises a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of E. coli, yeast cells.
  • a method for producing an anti-PD-L1 Nanobody comprising the steps of:
  • the method further comprises the step of: (c) further purifying and/or modifying the PD-L1 Nanobody of step (b).
  • the anti-PD-L1 Nanobody has the amino acid sequence set forth in SEQ ID NO.: 8 or 14.
  • a polypeptide (preferably a recombinant polypeptide) comprising:
  • an optional modification label selected from the group consisting of: a chemical label or a biomarker, optionally bound or coupled to the polypeptide.
  • polypeptide is a fusion polypeptide.
  • the chemical label is an isotope, an immunotoxin, and/or a chemical.
  • the biomarker is biotin, avidin or an enzyme label.
  • the fusion is immobilized (or supported) on a solid support or a semi-solid support.
  • an immunoconjugate comprising:
  • a coupled moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the coupling moiety is a drug or a toxin.
  • the coupled moiety is a detectable label.
  • the conjugate is selected from the group consisting of: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or is capable of producing a detectable agent
  • Product enzymes radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, pre- A drug activating enzyme (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate comprises: a multivalent (e.g., bivalent) VHH chain of the anti-PD-L1 Nanobody of the first aspect of the invention, as described in the second aspect of the invention Anti-PD-L1 Nanobody.
  • a multivalent (e.g., bivalent) VHH chain of the anti-PD-L1 Nanobody of the first aspect of the invention as described in the second aspect of the invention Anti-PD-L1 Nanobody.
  • the multivalent protein refers to a VHH chain comprising the plurality of repeating anti-PD-L1 Nanobodies according to the first aspect of the present invention in the amino acid sequence of the immunoconjugate.
  • the anti-PD-L1 Nanobody of the second aspect of the invention refers to a VHH chain comprising the plurality of repeating anti-PD-L1 Nanobodies according to the first aspect of the present invention in the amino acid sequence of the immunoconjugate.
  • the immunoconjugate is immobilized (or supported) on a solid support or a semi-solid support.
  • the anti-PD-L1 Nanobody of the second aspect of the invention for the preparation of (a) an agent for detecting a PD-L1 molecule; (b) for blocking PD- a formulation in which L1 binds to PD-1; (c) a drug for treating a tumor.
  • the detection comprises flow detection, cellular immunofluorescence detection.
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is in the form of an injection.
  • the pharmaceutical composition is used for preparing a medicament for treating a tumor, the tumor being selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, Colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, or bladder tumor.
  • the use is non-diagnostic and non-therapeutic.
  • a recombinant protein comprising:
  • the tag sequence comprises a 6His tag and an HA tag.
  • the recombinant protein specifically binds to the PD-L1 protein.
  • a thirteenth aspect of the invention provides the use of the VHH chain according to the first aspect of the invention, the Nanobody of the second aspect of the invention, or the immunoconjugate of the eighth aspect of the invention, Used to prepare medicaments, reagents, test plates or kits;
  • the reagent, the detection plate or the kit is used for: detecting the PD-L1 protein in the sample;
  • the agent is for treating or preventing a tumor expressing PD-L1 (ie, PD-L1 positive).
  • the tumor comprises: melanoma, gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer , or adrenal tumors.
  • a fourteenth aspect of the present invention a method for detecting a PD-L1 protein in a sample, the method comprising the steps of:
  • a method for treating a disease comprising administering the nanobody of the second aspect of the invention or the immunoconjugate of the eighth aspect of the invention to a subject in need thereof.
  • the subject comprises a mammal, such as a human.
  • a framework region FR of a VHH chain of the anti-PD-L1 Nanobody wherein the framework region FR of the VHH chain is represented by FR1 of SEQ ID NO.: 1, SEQ ID NO.: FR2 shown in 2, FR3 shown in SEQ ID NO.: 3, and FR4 shown in SEQ ID NO.: 4.
  • a kit comprising: (a) the nanobody according to the second aspect of the present invention, the fusion of the seventh aspect of the present invention, and the present invention The immunoconjugate of the eighth aspect, the recombinant protein of the twelfth aspect of the invention; (b) a container.
  • a CAR-T cell comprising a chimeric antigen receptor CAR, the antigen binding domain of the CAR having the VHH chain of the first aspect of the invention is provided Or the Nanobody of the second aspect of the invention.
  • Figure 1 shows the absorbance values of nine PD-L1 Nanobody positive clones screened by phage display technology.
  • FIG. 2 is a preliminary analysis of the blocking effect of PD-L1 Nanobodies by FACS. The results indicate that the Nanobody Nb43 specific to PD-L1 of the present invention has a good blocking effect on the binding of PD-L1 to PD-1.
  • Figure 3 is a FACS detection of humanized Nanobody (Nb43) and humanized PD-L1 Nanobody Fc fusion protein (MY1909) blocking PD-L1/PD-1 binding.
  • FIG. 4 shows that the humanized PD-L1 Nanobody Fc fusion protein (MY1909) stimulates T cell activation.
  • Figure 5 shows the detection of the inhibitory activity of the humanized PD-L1 Nanobody Fc fusion protein (MY1909) on tumor growth.
  • Figure 6 shows the detection of the inhibitory activity of MY1909 on tumor growth.
  • the inventors have successfully obtained a class of anti-PD-L1 Nanobodies through extensive and intensive research through extensive screening.
  • the experimental results show that a PD-L1 Nanobody obtained by the present invention can effectively block the interaction between PD-L1 and PD-1, and the PD-L1 Nanobody which is humanized by the present invention can also be effective. Block PD-L1 binding to PD-1.
  • the present invention has been completed on this basis.
  • the present invention utilizes a human PD-L1 extracellular domain antigen protein to immunize a camel to obtain a high quality nanobody phage display library.
  • the PD-L1 protein molecule is then coupled to the ELISA plate to display the correct spatial structure of the PD-L1 protein.
  • the antigen in this form is screened for the immuno Nanobody gene library using the phage display technology (Camel Heavy Chain Antibody Phage Display Gene Bank) Thereby, a Nano-antibody gene specific for PD-L1 was obtained. This gene was further transferred to Escherichia coli to obtain a highly specific Nanobody strain which was highly expressed in Escherichia coli.
  • Nanobody of the invention As used herein, the terms " Nanobody of the invention”, “anti-PD-L1 Nanobody of the invention”, “PD-L1 Nanobody of the invention” are used interchangeably and refer to both specific recognition and binding to PD-L1 ( Nanobodies including human PD-L1). Particularly preferred is the amino acid sequence of the VHH chain as shown in SEQ ID NO.: 8 or 14.
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • single domain antibody As used herein, the terms “single domain antibody”, “nanobody” have the same meaning and refer to the variable region of a cloned antibody heavy chain, constructing a single domain antibody consisting of only one heavy chain variable region, which is fully functional. The smallest antigen-binding fragment. Typically, the antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • CH1 light chain and heavy chain constant region 1
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules that are combined with the antibodies or fragments thereof of the invention to form Conjugate.
  • the present invention also encompasses cell surface markers or antigens that bind to the anti-PD-L1 protein antibody or fragment thereof.
  • variable region are used interchangeably with “complementarity determining region (CDR).
  • the heavy chain variable region of the antibody comprises three complementarity determining regions, CDR1, CDR2, and CDR3.
  • the complementarity determining region CDR comprises CDR1 set forth in SEQ ID NO.: 5, CDR2 set forth in SEQ ID NO.: 6, and CDR3 set forth in SEQ ID NO.: 7 (or Consists of the CDR1, CDR2 and CDR3).
  • RFTASMG SEQ ID NO: 5
  • AADDDYYAFLSRGARDFRY (SEQ ID NO: 7)
  • the heavy chain variable region of the antibody further comprises a framework region FR, the CDR1, CDR2 and CDR3 being separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the frame area FR is
  • NYADSVRGRFTISKDNAKNTLYLQINSLKPEDTAVYYC (SEQ ID NO: 3)
  • the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.
  • the terms "antibody of the invention”, “protein of the invention”, or “polypeptide of the invention” are used interchangeably and refer to a polypeptide which specifically binds to a PD-L1 protein, such as a protein having a heavy chain variable region. Or a polypeptide. They may or may not contain an initial methionine.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the invention encompasses any protein or protein conjugate having a heavy chain comprising a variable region and a fusion expression product (ie, an immunoconjugate and a fusion expression product), so long as the variable region is linked to the heavy chain of an antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are divided into four framework regions (FR), four FR amino acid sequences. Relatively conservative, not directly involved in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen.
  • the invention includes those molecules having an antibody heavy chain variable region with a CDR, as long as the CDRs thereof have 90% or more (preferably 95% or more, optimally 98% or more) homology to the CDRs identified herein. Sex.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of an antibody of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed by the 6
  • the antibody of the present invention refers to a polypeptide comprising the above CDR regions having PD-L1 protein binding activity.
  • the term also encompasses variant forms of a polypeptide comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include, but are not limited to, one or more (typically 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained using an antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof.
  • the invention also includes fragments of the Nanobodies of the invention.
  • the fragment will have at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
  • “conservative variant of the antibody of the present invention” means having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3, compared to the amino acid sequence of the antibody of the present invention. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the present invention also provides a polynucleotide molecule encoding the above antibody or a fragment thereof or a fusion protein thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides which hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • One possible method is to synthesize related sequences by artificial synthesis, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecule (nucleic acid, protein, etc.) to which the present invention relates includes biomolecules existing in an isolated form.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention may be used alone or in combination or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or capable of producing detectable products. Enzyme.
  • Therapeutic agents that can be bound or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides; 2. biotoxic; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; Particles; 6. liposomes; 7. nanomagnetic particles; 8. drug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (eg , cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for binding to a PD-L1 protein molecule, and thus can be used for treating a tumor.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-described Nanobody of the present invention (or a conjugate thereof) and pharmaceutically An acceptable carrier or excipient.
  • a safe and effective amount e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram body weight to about 50 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the Nanobody is provided with a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label or a fluorescent label.
  • the colloidal gold label can be carried out by methods known to those skilled in the art.
  • the anti-PD-L1 Nanobody is labeled with colloidal gold to obtain a colloidal gold-labeled Nanobody.
  • the anti-PD-L1 Nanobody of the present invention has good specificity and high potency.
  • the invention also relates to methods of detecting PD-L1 protein.
  • the method steps are substantially as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of PD-L1 protein in the dissolved sample.
  • the sample to be used is not particularly limited, and a representative example is a cell-containing sample present in the cell preservation solution.
  • the present invention also provides a kit comprising the antibody (or a fragment thereof) or a test plate of the present invention.
  • the kit further comprises a container, instructions for use, a buffer, and the like.
  • the present invention also provides a detection kit for detecting PD-L1 levels, the kit comprising an antibody recognizing a PD-L1 protein, a lysis medium for dissolving a sample, and a detection of a desired universal reagent and a buffer, such as various Buffer, detection mark, substrate detection, etc.
  • the test kit can be an in vitro diagnostic device.
  • the Nanobody of the present invention has a wide range of biological application value and clinical application value, and its application relates to various fields such as diagnosis and treatment of diseases related to PD-L1, basic medical research, and biological research.
  • a preferred application is for clinical diagnosis and targeted therapy for PD-L1.
  • Nanobody of the present invention is highly specific for a human PD-L1 protein having the correct spatial structure.
  • the nanobody of the present invention has a strong affinity.
  • the plasmid was mixed with the transfection reagent PEI 1:3 and allowed to stand for 10 min, then added to HEK293F cells, and cultured for 5-6 days at 37 ° C in a 6% CO 2 shaker incubator;
  • the eluted protein was ultrafiltered into PBS, the yield was measured, and the sample was subjected to SDS-PAGE to detect the purity of the antigen, and the remaining proteins were stored in a refrigerator at -80 ° C;
  • the antigen with a purity greater than 90% is selected for subsequent camel immunization.
  • the ligation product was transformed into electroporation competent cell TG1, and the PD-L1 nanobody library was constructed and the storage capacity was determined.
  • the storage capacity was 1.0 ⁇ 10 9 CFU;
  • the ProtSA server is used to calculate the solvent contactability of the residue, that is, the ratio of the folded state of the residue to the solvent contactable area of the unfolded state is greater than 40%.
  • the residue is a residue that is exposed to the solvent;
  • Example 6 Eukaryotic expression and purification of humanized PD-L1 blocking Nanobody
  • Example 7 Detection of blocking function of humanized PD-L1 Nanobody by flow cytometry
  • Example 8 Activation of humanized blocking PD-L1 Nanobody Fc fusion protein in CD4+ T cells in dendritic cell-T cell mixed lymphocyte reaction
  • the obtained dendritic cells were resuspended in RPMI complete medium, 2 ⁇ 10 5 /mL. Then, 50 ⁇ L per well was added to a 96-well U-shaped bottom plate (Costar: 3799), and cultured in an incubator;
  • Example 9 Inhibitory activity of humanized blocking PD-L1 Nanobody Fc fusion protein on tumor growth
  • NOD/SCID non-obese diabetic/severe combined immunodeficient mice. This was accomplished by subcutaneous transplantation of NOD/SCID mice expressing the human PD-L1 melanoma cell line A375 and human peripheral blood mononuclear cells PBMC. The detailed steps are as follows:
  • A375 and PBMC are mixed before injection and injected subcutaneously;
  • Fc fusion protein (MY1909) was administered intraperitoneally for the first time 24 hours after tumor inoculation, and then administered once a week for administration. The dose is 2mg/kg;
  • Example 10 Inhibitory activity of MY1909 on tumor growth
  • the in vivo activity of MY1909 was studied using a subcutaneously transplanted tumor model of C57BL/6 mice.
  • a test model was constructed by subcutaneously inoculation of C57BL/6 mice colon cancer cell line MC38 (MC38-hPD-L1) expressing human PD-L1, and the inhibitory activity of MY1909 on its tumor was evaluated. The detailed steps are as follows:
  • MC38-hPD-L1 tumor cells (1 ⁇ 10 6 /cell) were inoculated subcutaneously in the right side of C57BL/6 mice, and the cells were cultured in 10% heat-inactivated fetal bovine serum (ExCell Biology) and 50 ⁇ g/mL. In the DMEM medium of hygromycin B, 1 ⁇ 10 6 cells were resuspended in 100 ⁇ L of PBS). When the average tumor-bearing tumor volume of the mice reached about 64 mm 3 , 24 tumor-bearing mice were randomly divided into 3 experimental groups, 6 in each group, and the grouping day was defined as day 0.
  • mice Three groups of mice were given isotype control (Isotype control) hIgG1 (group 01) as negative control, and Analogue to Atezolizumab (Roche) (group 02) as positive control and MY1909 (group 03) on day 0.
  • the products were diluted to 0.5 mg/mL with PBS, administered at a dose of 5 mg/kg, intraperitoneally, twice a week, for a total of 3 weeks.
  • Ti and Ci were the average tumor volume of Day i days in the treatment group and the control group, respectively.
  • TGI Tumor growth inhibition
  • Ti and Ci were the average tumor volume of Day i days in the treatment group and the control group, respectively;
  • T0 and C0 are the average tumor volume of Day 0 days in the treatment group and the control group, respectively.
  • the tumor growth inhibition rate (TGI%) of the MY1909 monotherapy group was 58.19% compared with the Isotype hIgG1 control group, with a statistically significant anti-MC38-hPD-L1 tumor.
  • the role of growth P ⁇ 0.05.
  • 5 tumors completely resolved, and there was no evidence of tumor growth at the end of the experiment (day 34). This indicates that MY1909 can significantly inhibit tumor growth at a dose of 5 mg/kg.

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Abstract

提供了一种抗PD-L1人源化纳米抗体及其应用。具体地，提供了一种抗蛋白程序性死亡配体1(PD-L1)的纳米抗体。该抗PD-L1纳米抗体能有效阻断PD-L1与程序性死亡分子1(PD-1)的结合，因此可成功阻断PD-L1对表达PD-1的T细胞的抑制作用。该PD-L1纳米抗体可应用于PD-L1相关疾病的预防、诊断、治疗。进一步还提供了人源化的PD-L1纳米抗体序列，人源化后的纳米抗体仍具有阻断PD-L1与PD-1结合的功能，并具有高亲和力及高特异性，可作为肿瘤免疫靶点抗体药物。

Description

一种抗PD-L1人源化纳米抗体及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及抗PD-L1纳米抗体及其应用。
背景技术
细胞介导的细胞免疫应答是抗肿瘤免疫的基础，T细胞的活化和增殖需要双重信号。即除了需要T细胞受体(TCR)与表达在抗原递呈细胞(APCs)表面的MHC-抗原肽复合物结合产生的第一信号外；还需要多种协同刺激分子参与提供的第二信号(协同刺激信号)。
免疫应答初始阶段，T细胞在TCR提供的第一信号及协同刺激信号的共同作用下，达到其激活阈而被激活，产生效应及记忆性细胞，发挥免疫防御功能，而该激活过程又引起一系列协同刺激抑制性分子的上调表达，以维持其处于持续的激活状态或抑制其过度活化增殖。如果缺少协同刺激分子提供的第二信号，将会导致T细胞的无反应或特异性免疫耐受，甚至诱导细胞进入凋亡。相反，若协同刺激信号反映过度，则可能导致免疫细胞异常激活，从而引发各种自身免疫性疾病。因此，免疫细胞所获得的正性和负性协同刺激信号需要到达相对平衡以使免疫应答适时开启又能恰当终止，在机体抵抗外来抗原入侵和防止自身免疫性疾病的发生中起着重要的作用。
随着广泛而深入的研究，协同刺激分子已成为免疫学研究新的热点之一。介导协同刺激信号的分子主要包括肿瘤坏死因子/肿瘤坏死因子受体(TNF/TNFR)超家族和免疫球蛋白超家族，如CD28/B7等两大超家族。这些共刺激分子以手提配体相互作用的方式传导信号。
程序性死亡因子1/程序性死亡因子1配体1(PD-1/PD-L1)作为CD28/B7协同刺激分子超家族的新成员，可以介导负性协同刺激信号，能有效抑制T、B细胞功能和增殖，同时减少细胞因子IL-2、IL-10和IFN-γ的分泌，该抑制途径参与的免疫调节在肿瘤免疫、移植免疫、病毒感染、自身免疫等疾病的研究中都具有重要意义。
PD-1是免疫球蛋白B7-CD28家族成员之一，由胞外段、疏水性跨膜区、胞内段组成，其胞内段含有免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif,ITIM)、免疫受体酪氨酸转换基序(immunoreceptor tyrosine-based switch motif,ITSM)。其中，ITSM的激活与效应性T细胞应答活性密切相关。PD-1可表达于活化的CD4 +T细胞、CD8 +T细胞、B细胞、自然杀伤T细胞、单核细胞和树突状细胞上。另外，PD-1也表达于调节性T细胞(regulatory T cell,Treg)，并能促进Treg细胞的增殖，抑制免疫应答。
程序性死亡因子1配体1(programmed death 1ligand 1，PD-L1)又称CD274，为B7家族成员，是PD-1的配体。PD-L1属于I型跨膜蛋白，共290个氨基酸，分子量约为30-35KD。人PD-L1分子可组成性表达于胎盘、心、肝、肺、肾、骨骼肌和部分造血细胞等非淋巴组织，在胸腺、淋巴结和脾脏等淋巴组织中也有适度表达。此外，PD-L1在肺癌、肝癌、乳腺癌、卵巢癌等癌组织细胞上也具有表达，且癌组织经过诱导亦可上调其表达。在健康的机体中，PD-1/PD-L1信号通路的激活可最大程度减少免疫反应对周围组织的损伤，避免发生自身免疫疾病。然而，PD-1/PD-L1信号通路激活改变肿瘤局部微环境使得T细胞免疫效应降低，从而介导肿瘤免疫逃逸，促进肿瘤生长。PD-L1在肿瘤上的表达与食管癌、胰腺癌和其它类型的癌症的生存率下降相关，突出了该通路可以作为新的有前途的肿瘤免疫治疗靶点，并以得到诸多实验证实。
纳米抗体作为一种新型的小分子抗体片段，由驼类天然的重链抗体重链可变区(VHH)克隆获得。纳米抗体具有优良的生物学特性，分子量12-15KD，是完整抗体的十分之一，在克服了天然抗体分子量大的弊端的同时又拥有完整的抗原结合位点，具有很好的组织穿透性，特异性高，水溶性好。因其特殊的结构性质，兼具了传统抗体与小分子药物的优势，几乎完美克服了传统抗体的开发周期长，稳定性较低，保存条件苛刻等缺陷，逐渐成为新一代抗体治疗中的新兴力量，在免疫诊断和治疗中显示出广阔的应用前景
本研究将重点开发与PD-L1高亲和力结合，且可以阻断PD-L1/PD-1结合的纳米抗体，尤其是人源化之后的PD-L1纳米抗体仍然保持较高的阻断活性。
发明内容
本发明提供了一种特异性针对PD-L1的纳米抗体，并能够有效阻断PD-L1与PD-1的结合。
在本发明的第一方面，提供了一种抗PD-L1纳米抗体的VHH链，所述VHH链包括互补决定区CDR，所述的互补决定区CDR包括SEQ ID NO.:5所示的CDR1，SEQ ID NO.:6所示的CDR2，和SEQ ID NO.:7所示的CDR3(或由所述CDR1、CDR2和CDR3构成)。
在另一优选例中，所述PD-L1为人PD-L1。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与PD-L1高亲和力结合、阻断PD-L1与PD-1结合的衍生序列。
在另一优选例中，所述VHH链还包括框架区FR，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述的框架区FR由
(a)SEQ ID NO.:1所示的FR1，SEQ ID NO.:2所示的FR2，SEQ ID NO.:3所 示的FR3，SEQ ID NO.:4所示的FR4组成；或
(b)SEQ ID NO.:10所示的FR1，SEQ ID NO.:11所示的FR2，SEQ ID NO.:12所示的FR3，SEQ ID NO.:13所示的FR4组成。
在另一优选例中，所述的框架区FR含有所述的FR1、FR2、FR3和FR4(SEQ ID NO.:1-4或SEQ ID NO.:10-13)。
在另一优选例中，所述的抗PD-L1纳米抗体的VHH链的氨基酸序列如SEQ ID NO.:8或14所示。
此外，还提供一种抗人PD-L1抗体的重链可变区，所述的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3，并且3个CDR包括SEQ ID NO.:5所示的CDR1，SEQ ID NO.:6所示的CDR2，SEQ ID NO.:7所示的CDR3。
本发明第二方面，提供了一种抗PD-L1纳米抗体，它是针对PD-L1表位的纳米抗体，并且具有如SEQ ID NO.:8或SEQ ID NO.:14中所示的氨基酸序列的VHH链。
在另一优选例中，所述抗PD-L1纳米抗体固定(或负载于)在固体载体或半固体载体。
本发明第三方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：本发明第一方面所述的抗PD-L1纳米抗体的VHH链或本发明第二方面所述的抗PD-L1纳米抗体。
在另一优选例中，所述多核苷酸具有如SEQ ID NO.:9或15所示的核苷酸序列。
在另一优选例中，所述的多核苷酸包括DNA或RNA。
本发明第四方面，提供了一种表达载体，所述表达载体含有本发明第三方面所述的多核苷酸。
在另一优选例中，所述的表达载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其他基因转移系统、或其组合。优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
本发明第五方面，提供了一种宿主细胞，所述宿主细胞含有本发明第四方面所述的表达载体，或其基因组中整合有本发明第三方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞。
本发明第六方面，提供了一种产生抗PD-L1纳米抗体的方法，包括步骤：
(a)在适合产生纳米抗体的条件下，培养本发明第五方面所述的宿主细胞，从而获得含所述抗PD-L1纳米抗体的培养物；以及
(b)从所述培养物中分离或回收所述的抗PD-L1纳米抗体。
在另一优选例中，所述方法还包括步骤：(c)近一步纯化和/或修饰得步骤(b)的PD-L1纳米抗体。
在另一优选例中，所述的抗PD-L1纳米抗体具有如SEQ ID NO.:8或14所示的氨基酸序列。
本发明第七方面，提供了一种多肽(优选重组多肽)，所述多肽含有：
(a)如本发明第一方面所述的抗PD-L1纳米抗体的VHH链、或如本发明第二方面所述的抗PD-L1纳米抗体；
(b)任选的、与所述多肽结合或偶联的选自下组的修饰标记物：化学标记物或生物标记物。
在另一优选例中，所述的多肽为融合多肽。
在另一优选例中，所述化学标记为同位素、免疫毒素和/或化学药物。
在另一优选例中，所述生物标记为生物素、亲和素或酶标记。
在另一优选例中，所述融合物固定(或负载于)在固体载体或半固体载体。
本发明第八方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第一方面所述的抗PD-L1纳米抗体的VHH链、或如本发明第二方面所述的抗PD-L1纳米抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第一方面所述的抗PD-L1纳米抗体的VHH链、如本发明第二方面所述的抗PD-L1纳米抗体。
在另一优选例中，所述多价是指，在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第一方面所述的抗PD-L1纳米抗体的VHH链、本发明第二方面所述的抗PD-L1纳米抗体。
在另一优选例中，所述免疫偶联物固定(或负载于)在固体载体或半固体载体。
本发明第九方面，提供了本发明第二方面所述的抗PD-L1纳米抗体的用途，用于制备(a)用于检测PD-L1分子的试剂；(b)用于阻断PD-L1与PD-1结合的制剂；(c)用于治疗肿瘤的药物。
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测。
本发明第十方面，提供了一种药物组合物，含有：
(i)本发明第一方面抗PD-L1纳米抗体的VHH链、或如本发明第二方面所述 的抗PD-L1纳米抗体、或本发明第八方面所述的免疫偶联物；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备治疗肿瘤的药物，所述的肿瘤选自下组：胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。
本发明第十一方面，提供了本发明第二方面所述抗PD-L1纳米抗体的一种或多种的用途：
(i)用于检测人PD-L1分子；
(ii)用于流式检测；
(iii)用于细胞免疫荧光检测；
(iv)用于治疗肿瘤；
(v)用于肿瘤诊断。
在另一优选例中，所述用途为非诊断的和非治疗的。
本发明第十二方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区VHH的序列或如本发明第二方面所述的纳米抗体的序列；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签和HA标签。
在另一优选例中，所述的重组蛋白特异性结合于PD-L1蛋白。
本发明第十三方面，提供了如本发明第一方面所述的VHH链、如本发明第二方面所述的纳米抗体、或本发明第八方面所述的免疫偶联物的用途，它们被用于制备药剂、试剂、检测板或试剂盒；
其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1蛋白；
其中，所述药剂用于治疗或预防表达PD-L1(即PD-L1阳性)的肿瘤。
在另一优选例中，所述肿瘤包括：黑色素瘤、胃癌、淋巴瘤、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、或肾上腺肿瘤。
本发明第十四方面，提供了一种检测样品中PD-L1蛋白的方法，所述方法包括步骤：
(1)将样品与本发明第二方面所述的纳米抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在PD-L1蛋白。
本发明第十五方面，提供了一种治疗疾病的方法，所述方法包括，给需要的对象施用本发明第二方面所述的纳米抗体或本发明第八方面所述的免疫偶联物。
在另一优选例中，所述的对象包括哺乳动物，如人。
本发明第十六方面，提供了一种抗PD-L1纳米抗体VHH链的框架区FR，所述的VHH链的框架区FR由SEQ ID NO.:1所示的FR1，SEQ ID NO.:2所示的FR2，SEQ ID NO.:3所示的FR3，SEQ ID NO.:4所示的FR4组成。
本发明第十七方面，提供了一种试剂盒，所述试剂盒中包括：(a)如本发明第二方面所述的纳米抗体、本发明第七方面所述的融合物、本发明第八方面所述的免疫偶联物、本发明第十二方面所述的重组蛋白；(b)容器。
本发明第十八方面，提供了一种CAR-T细胞，所述CAR-T细胞表达嵌合抗原受体CAR，所述CAR的抗原结合结构域具有如本发明第一方面所述的VHH链、或本发明第二方面所述的纳米抗体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了利用噬菌体展示技术筛选出的9种PD-L1纳米抗体阳性克隆吸光值。
图2是FACS初步鉴定PD-L1纳米抗体的阻断效果。结果表明本发明的特异性针对PD-L1的纳米抗体Nb43对PD-L1与PD-1的结合具有很好的阻断效果。
图3是FACS检测人源化纳米抗体(Nb43)和人源化PD-L1纳米抗体Fc融合蛋白(MY1909)阻断PD-L1/PD-1的结合。
图4显示了人源化PD-L1纳米抗体Fc融合蛋白(MY1909)可刺激T细胞的活化。
图5显示了人源化PD-L1纳米抗体Fc融合蛋白(MY1909)对肿瘤生长的抑制活性检测。
图6显示了MY1909对肿瘤生长的抑制活性检测。
具体实施方式
本发明人通过广泛而深入的研究，经过大量的筛选，成功获得一类抗PD-L1纳米抗体。实验结果表明，本发明获得的一株PD-L1纳米抗体能有效阻断PD-L1与PD-1之间的相互作用，且经本发明人人源化后的PD-L1纳米抗体亦能有效阻断PD-L1与PD-1结合。在此基础上完成了本发明。
具体地，本发明利用人源的PD-L1胞外段抗原蛋白免疫骆驼，获得高质量的纳米抗体噬菌体展示文库。然后将PD-L1蛋白分子偶联在酶标板上，展示PD-L1蛋白的正确空间结构，以此形式的抗原利用噬菌体展示技术筛选免疫纳米抗体基因库(骆驼重链抗体噬菌体展示基因库)，从而获得了PD-L1特异性的纳米抗体基因。 再将此基因转至大肠杆菌中，从而获得了能在大肠杆菌中高效表达的、且特异性高的纳米抗体株。
如本文所用，术语“本发明纳米抗体”、“本发明的抗PD-L1纳米抗体”、“本发明PD-L1纳米抗体”可互换使用，均指特异性识别和结合于PD-L1(包括人PD-L1)的纳米抗体。特别优选的是VHH链的氨基酸序列如SEQ ID NO.:8或14所示的纳米抗体。
Figure PCTCN2018091639-appb-000001
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体”、“纳米抗体”具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的抗PD-L1蛋白抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“V H”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。
在另一优选例中，所述的互补决定区CDR包括SEQ ID NO.:5所示的CDR1，SEQ ID NO.:6所示的CDR2，和SEQ ID NO.:7所示的CDR3(或由所述CDR1、CDR2和CDR3构成)。
RFTASMG(SEQ ID NO:5)
VSGAAST(SEQ ID NO:6)
AADDDYYAFLSRGARDFRY(SEQ ID NO:7)
在另一优选例中，所述抗体的重链可变区还包括框架区FR，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述的框架区FR由
(a)SEQ ID NO.:1所示的FR1，SEQ ID NO.:2所示的FR2，SEQ ID NO.:3所示的FR3，SEQ ID NO.:4所示的FR4组成；或
(b)SEQ ID NO.:10所示的FR1，SEQ ID NO.:11所示的FR2，SEQ ID NO.:12所示的FR3，SEQ ID NO.:13所示的FR4组成。
QVQLQESGGGSVQAGGSLRLSCAAS(SEQ ID NO:1)
WFRQAPGKEREGIAT(SEQ ID NO:2)
NYADSVRGRFTISKDNAKNTLYLQINSLKPEDTAVYYC(SEQ ID NO:3)
WGQGTQVTVSS(SEQ ID NO:4)
QVQLQESGGGLVQPGGSLRLSCAAS(SEQ ID NO:10)
WFRQAPGKGLEGIAT(SEQ ID NO:11)
NYADSVKGRFTISKDNSKNTLYLQINSLRDEDTAVYYC(SEQ ID NO:12)
WGQGTLVTVSS(SEQ ID NO:13)
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合PD-L1蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定区域来描述，称为 可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有PD-L1蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含纳米抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明纳米抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被 性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明 所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离 心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；9.疗剂(例如，顺铂)或任何形式的纳米颗粒等。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合PD-L1蛋白分子，因而可用于治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的纳米抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
标记的纳米抗体
在本发明的一个优选例中，所述纳米抗体带有可检测标记物。更佳地，所述的 标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，抗PD-L1的纳米抗体用胶体金标记，得到胶体金标记的纳米抗体。
本发明的抗PD-L1纳米抗体具有很好的特异性，很高的效价。
检测方法
本发明还涉及检测PD-L1蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中PD-L1蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测PD-L1水平的检测试剂盒，该试剂盒包括识别PD-L1蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的纳米抗体有广泛生物应用价值和临床应用价值，其应用涉及到与PD-L1相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对PD-L1的临床诊断和靶向治疗。
本发明的主要优点包括：
(a)本发明纳米抗体高特异性针对人的具有正确空间结构的PD-L1蛋白。
(b)本发明纳米抗体的亲和力强。
(c)本发明纳米抗体的生产简便。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：人PD-L1蛋白的表达纯化
(1)将人PD-L1的核苷酸序列合成在pCDNA3.1(-)载体上，然后将其胞外段序 列亚克隆至pFΜSE-IgG1载体上；
(2)用Omega质粒大提试剂盒提取构建的pFΜSE-IgG1-hPD-L1(ECD)质粒；
(3)培养HEK293F细胞至OD为2.0×10 6个/mL；
(4)将质粒与转染试剂PEI 1:3混合均匀后静置10min，然后加入到HEK293F细胞中，37℃，6％CO 2摇床培养箱中培养5-6天；
(5)收集细胞上清，与Protein A珠子在室温下结合1h；
(6)用磷酸盐缓冲液pH 7.0洗涤珠子后，再用0.1M pH 3.0Glycine洗脱蛋白；
(7)将洗脱的蛋白超滤至PBS中，测定产量后取样进行SDS-PAGE对抗原纯度进行检测，其余蛋白保存于-80℃冰箱；
(8)选取纯度大于90％的抗原进行后续骆驼的免疫。
实施例2：PD-L1纳米抗体文库的构建
(1)将1mg hPD-L1(ECD)-Fc抗原与弗氏佐剂等体积混合，免疫一只新疆双峰驼，每周一次，共免疫3次，刺激B细胞表达抗原特异性的纳米抗体；
(2)3次免疫结束后，提取100mL骆驼外周血淋巴细胞并提取总RNA；
(3)合成cDNA并利用套式PCR扩增VHH；
(4)利用限制性内切酶Pst I及Not I酶切20μg pMECS噬菌体展示载体(Biovector供应)及10μg VHH并连接两个片段；
(5)将连接产物转化至电转感受态细胞TG1中，构建PD-L1纳米抗体文库并测定库容，库容大小为1.0×10 9CFU；
与此同时，随机挑取24颗克隆进行菌落PCR检测，结果表明所建文库的插入率为达95.8％。上述结果说明，获得了库容和插入率均合格的PD-L1纳米抗体噬菌体展示文库。
实施例3：PD-L1纳米抗体的筛选及鉴定
抗体筛选：
(1)将溶解在100mM NaHCO 3、pH 8.2中的10μg hPD-L1(ECD)-Fc抗原(10μg Fc in NaHCO 3作为对照)偶联在NUNC酶标板上，4℃放置过夜；
(2)第二天加入100μL 0.01％BSA，室温封闭0.5h；
(3)2h后，加入100μL噬菌体(2×10 11CFU免疫骆驼纳米抗体噬菌展示基因库)，室温作用0.5h；
(4)用0.05％PBS+Tween-20洗5遍，以洗掉非特异的噬菌体；
(5)用100mM三乙醇胺将与PD-L1特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1细胞，37℃培养1h，产生并纯化噬菌体用于下一轮的筛选，相同筛选过程重复3轮，富集倍数分别为无富集，11.4倍和270倍。
用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆：
(1)从上述3轮筛选后含有噬菌体的细胞培养皿中，挑选600个单个菌落并接种于含有100μg/mL的氨苄青霉素的TB培养基(1L TB培养基中含有2.3g KH 2PO 4，12.52g K 2HPO 4，12g蛋白胨，24g酵母提取物，4mL甘油)中，生长至对数期后，加终浓度1mM的IPTG，28℃培养过夜；
(2)利用渗透法获得粗提抗体，并将抗体转移到经抗原包被的ELISA板中，在室温下放置1h；
(3)用PBST洗去未结合的抗体，加入鼠抗HA抗体，购自北京康为世纪生物科技有限公司)，在室温下放置1h；
(4)用PBST洗去未结合的抗体，加入山羊抗小鼠碱性磷酸酶标记抗体，在室温下放置1h；
(5)用PBST洗去未结合的抗体，加入碱性磷酸酶显色液，于ELISA仪上，在405nm波长，读取吸收值；
(6)当样品孔OD值大于对照孔OD值3倍以上时(Ratio+/-＞3)，判为阳性克隆孔。结果表明600颗克隆共出现95颗阳性克隆；
(7)将95颗阳性克隆孔的菌转摇在含有2mL氨苄青霉素浓度为100μg/mL的LB液体中以便提取质粒并进行测序；
(8)对比分析95株克隆系列，最终获得9种CDR3区序列明显差异纳米抗体，编号为Nb1，Nb4，Nb16，Nb23，Nb36，Nb43，Nb96，Nb102，Nb156(图1显示了其ELISA吸光值)。
实施例4：流式细胞术初步鉴定纳米抗体的阻断功能
(1)制备hPD-1(ECD)-Biotin蛋白(制备方法同实施例1，SDS-PAGE纯度验证)，蛋白生物素化的方法参照生物素试剂说明书。
(2)稳定表达PD-L1的A375转基因细胞的构建。
(3)制备PD-L1纳米抗体TG1菌株粗提裂解液，制备方法同实施例3。
(4)每个样品取1×10 6个稳转PD-L1细胞重悬于0.5％BSA-PBS buffer中，加入上述粗提液50μL，同时设置阴性对照(hIgG1)和空白组(PBS)，每孔加入5μg hPD-1(ECD)-Fc-Biotin，4℃孵育20min。
(5)PBS洗涤2次细胞，加入eBioscience的SA-PE，4℃孵育20min，PBS洗涤2次细胞后用流式细胞仪(BD FACS Calibur)检测结果如图2所示。
(6)从筛选获得的9株CDR3区序列明显差异纳米抗体中，发现其中1株(Nb 43)具有明显的阻断效果。
实施例5：PD-L1阻断型纳米抗体的人源化改造
(1)首先以SEQ ID NO.:8所示的PD-L1纳米抗体序列为模板在结构数据库中同源结构的搜索，共搜寻到1500个结构，取其中E value＝0.0并且序列等同性≥70％ 的40个结构；
(2)对这40个结构进行结构比对，并依据晶体结构分辨率大小和构建的进化树，最终选取包括3dwt在内的7个蛋白，进行基于SEQ ID NO.:8所示的PD-L1纳米抗体序列的多模板同源模建，最终获得的10个结构，再依据打分函数的高低排序，选取molpdf最低的结构，继续下面的工作；
(3)对模建的最优结构，利用ProtSA服务器计算残基的溶剂可接触性，即残基的折叠态相对于去折叠态的溶剂可接触面积的比值为判据，取大于40％的残基为暴露于溶剂外的残基；
(4)对模建的最优结构和DP-47进行序列比对，替换相应的暴露于溶剂的残基。最终确定出一种人源化PD-L1纳米抗体，由SEQ ID NO.14所示的氨基酸序列编码。人源化前后抗体序列对应如下表2：
表2
Figure PCTCN2018091639-appb-000002
人源化前后抗体骨架区与DP-47骨架区的同源性比较如下表3：
表3
Figure PCTCN2018091639-appb-000003
实施例6：人源化PD-L1阻断型纳米抗体真核表达纯化
(1)将人源化后的PD-L1纳米抗体序列合成至pFΜSE-IgG1载体(购自Invivogen)，用Omega质粒大提试剂盒提取pFΜSE-IgG1-Nb(humanized)质粒；
(2)培养HEK293F细胞至OD为2.0×10 6个/mL；
(3)将质粒与转染试剂PEI按照1:3混合均匀后静置10min，然后加入到HEK293F细胞中，37℃，6％CO 2摇床培养箱中培养5-6天；
(4)收集细胞上清，与Protein A珠子在室温下结合1h；
(5)用磷酸盐缓冲液pH 7.0洗涤珠子后，再用0.1M pH 3.0Glycine洗脱蛋白；
(6)将洗脱的蛋白超滤至PBS中，测定产量后取样进行SDS-PAGE检测才纯度，其余蛋白保存于-80℃冰箱；
(7)结果显示成功表达纯化获得Nb43高纯度人源化纳米抗体(SEQ ID NO.:14)。
实施例7：流式细胞术检测人源化PD-L1纳米抗体的阻断功能
方法同实施例4：
(1)每个样品取1×10 6个稳定表达PD-L1的细胞株重悬于0.5％BSA-PBS buffer中，加入10μg纯化的人源化PD-L1纳米抗体，同时设置阴性对照(hIgG1)和空白组(PBS)，所有样本加入5μg hPD-1(ECD)-Fc-Biotin蛋白，4℃孵育20min；
(2)PBS洗涤2次细胞，加入eBioscience的SA-PE，4℃孵育20min，PBS洗涤2次细胞后上机检测，检测结果如图3所示，Nb43表现出很好的阻断活性。
实施例8：人源化阻断型PD-L1纳米抗体Fc融合蛋白在树突细胞-T细胞混合淋巴反应中对CD4+T细胞的激活作用测定
(1)利用人淋巴细胞分离液(天津潮洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞PBMC；
(2)用无血清的RPMI 1640培养基培养1-2h，除去未贴壁的细胞，将细胞培养于含10％FBS，10ng/mL GM CSF以及20ng/mL IL 4的RPMI中；
(3)培养5-6天，加入10ng/mL的TNF-α并孵育24h，获得成熟的树突细胞；
(4)获得的树突细胞重悬于RPMI完全培养基中，2×l0 5/mL。然后在96孔U形底板(Costar:3799)中每孔加入50μL，培养箱中培养；
(5)利用磁珠分离试剂盒(Mi 1tenyi Bio tee:130-096-533)按照说明书方法从另一个供体PBMC中分离CD4 +T细胞；
(6)l×l0 4个树突细胞和l×10 5个CD4 +T细胞混合，于RPMI完全培养基中重悬并加入96孔培养板，每孔加入50μL细胞混液：每孔加入100μL稀释于RPMI完全培养基中的人源化PD-L1纳米抗体(SEQ ID NO.:14)Fc融合蛋白(MY1909)，培养5-7天后去上清，利用IFN-γELISA检测试剂盒(ebioscience)检测上清中IFN-γ水平；
结果如图4所示，可见PD-L1纳米抗体Fc融合蛋白可增强混合淋巴细胞反应中CD4 +T细胞的γ干扰素分泌，即PD-L1阻断型纳米抗体Fc融合蛋白增强了T细胞的活化。
实施例9：人源化阻断型PD-L1纳米抗体Fc融合蛋白对肿瘤生长的抑制活性
采用免疫缺陷NOD/SCID(非肥胖型糖尿病/重症联合免疫缺陷)小鼠研究其体内活性。通过对NOD/SCID小鼠皮下移植表达人PD-L1的黑色素瘤细胞系A375和人的外周血单个核细胞PBMC实验来完成。详细步骤如下：
(1)A375和PBMC在注射前混合，皮下注射；
(2)人源化阻断型PD-L1纳米抗体(SEQ ID NO.:14)Fc融合蛋白(MY1909)在肿瘤接种后24h第一次腹腔注射给药，之后每周给药一次，给药剂量为2mg/kg；
(3)PBS作为阴性对照，罗氏公司的TECENTRIQ作为阳性对照(benchmark，BMK)。每个实验组11只小鼠。每周两次观察肿瘤的形成，并用游标卡尺测量肿瘤长径和短径，计算肿瘤体积，绘制肿瘤生长曲线图(图5)。结果显示融合蛋白(抗体)MY1909在2mg/kg剂量下就可以显著抑制肿瘤生长。
实施例10：MY1909对肿瘤生长的抑制活性
采用C57BL/6小鼠皮下移植肿瘤模型研究MY1909的体内活性。通过对C57BL/6小鼠皮下接种表达人PD-L1的结肠癌细胞系MC38(MC38-hPD-L1)来构建试验模型，评价MY1909对其肿瘤的抑制活性。详细步骤如下：
(1)在受试C57BL/6小鼠右侧皮下接种MC38-hPD-L1肿瘤细胞(1×10 6/只，细胞培养于含10％热灭活胎牛血清(ExCell Biology)和50μg/mL hygromycin B的DMEM培养基中，1×10 6细胞使用100μL PBS重悬)。当小鼠平均荷瘤肿瘤体积达到约64mm 3时，将24只荷瘤鼠随机分入3个实验组，每组6只，分组当天定义为第0天。
(2)3组小鼠于第0天分别给予同型对照(Isotype control)hIgG1(01组)作为阴性对照、Analogue to Atezolizumab(Roche)(02组)作为阳性对照、MY1909(03组)，供试品都使用PBS稀释至0.5mg/mL，给药剂量5mg/kg，腹腔注射，给药频率每周2次，共给药3周。
(3)在分组后，实验中每周两次测量动物的肿瘤体积。肿瘤体积通过卡尺测量，公式为TV＝0.5a×b2，其中a是肿瘤的长径，b是肿瘤的短径。绘制肿瘤生长曲线图(图6)，考察各组供试品对肿瘤生长的抑制作用(表4)。结果显示MY1909在5mg/kg剂量下就可以显著抑制肿瘤生长。
表4.MY1909在皮下MC38-hPD-L1肿瘤模型中的抗肿瘤效果
Figure PCTCN2018091639-appb-000004
Figure PCTCN2018091639-appb-000005
备注：a.平均值±SEM；
b.相对肿瘤增殖率T/C(％)，是抗肿瘤活性评价指标：公式：T/C％＝Ti/Ci*100％；
Ti和Ci分别为处理组和对照组Day i天的平均肿瘤体积
c.肿瘤生长抑制(Tumor growth inhibition,TGI)：公式：％TGI＝(1-(Ti-T0)/(Ci-C0))*100％；
Ti和Ci分别为处理组和对照组Day i天的平均肿瘤体积；
T0和C0分别为处理组和对照组Day 0天的平均肿瘤体积
d.与第1组相比较。数据在利用独立样本T检验分析之前进行了SQRT转换。第2组和第3组相比，P>0.05.
(4)在分组治疗后第20天，与Isotype hIgG1对照处理组相比，MY1909单药治疗组的肿瘤生长抑制率(TGI％)为58.19％，具有统计学显著的抗MC38-hPD-L1肿瘤生长的作用(P<0.05)。MY1909单药治疗组中，5个肿瘤完全消退，截止实验终点时(第34天)无肿瘤生长迹象。表明MY1909在5mg/kg剂量下就可以显著抑制肿瘤生长。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种抗PD-L1纳米抗体的VHH链，其特征在于，所述VHH链包括互补决定区CDR，所述的互补决定区CDR包括SEQ ID NO.:5所示的CDR1，SEQ ID NO.:6所示的CDR2，和SEQ ID NO.:7所示的CDR3。
  2. 一种抗PD-L1纳米抗体，其特征在于，它是针对PD-L1表位的纳米抗体，并且具有如SEQ ID NO.:8或SEQ ID NO.:14中所示的氨基酸序列的VHH链。
  3. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的蛋白质：权利要求1所述的抗PD-L1纳米抗体的VHH链或权利要求2所述的抗PD-L1纳米抗体。
  4. 一种表达载体，其特征在于，所述表达载体含有权利要求3所述的多核苷酸。
  5. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求4所述的表达载体，或其基因组中整合有权利要求3所述的多核苷酸。
  6. 一种产生抗PD-L1纳米抗体的方法，其特征在于，包括步骤：
    (a)在适合产生纳米抗体的条件下，培养权利要求5所述的宿主细胞，从而获得含所述抗PD-L1纳米抗体的培养物；以及
    (b)从所述培养物中分离或回收所述的抗PD-L1纳米抗体。
  7. 一种多肽，其特征在于，所述多肽含有：
    (a)如权利要求1所述的抗PD-L1纳米抗体的VHH链、或如权利要求2所述的抗PD-L1纳米抗体；
    (b)任选的、与所述多肽结合或偶联的选自下组的修饰标记物：化学标记物或生物标记物。
  8. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求1所述的抗PD-L1纳米抗体的VHH链、或如权利要求2所述的抗PD-L1纳米抗体；
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  9. 权利要求2所述的抗PD-L1纳米抗体的用途，其特征在于，用于制备(a)用于检测PD-L1分子的试剂；(b)用于阻断PD-L1与PD-1结合的制剂；(c)用于治疗肿瘤的药物。
  10. 一种药物组合物，其特征在于，含有：
    (i)如权利要求1所述的抗PD-L1纳米抗体的VHH链、或如权利要求2所述的抗PD-L1纳米抗体、或如权利要求8所述的免疫偶联物；以及
    (ii)药学上可接受的载体。
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