WO2020001526A1 - Pd-l1结合多肽及其用途 - Google Patents

Pd-l1结合多肽及其用途 Download PDF

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WO2020001526A1
WO2020001526A1 PCT/CN2019/093225 CN2019093225W WO2020001526A1 WO 2020001526 A1 WO2020001526 A1 WO 2020001526A1 CN 2019093225 W CN2019093225 W CN 2019093225W WO 2020001526 A1 WO2020001526 A1 WO 2020001526A1
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binding polypeptide
amino acid
cancer
seq
acid sequence
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French (fr)
Inventor
须涛
孙艳
齐浩
杜海菁
胡婷婷
杨艳玲
李芳芳
赵奇
秦颂兵
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Suzhou Smartnuclide Biopharmaceutical Co Ltd
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Suzhou Smartnuclide Biopharmaceutical Co Ltd
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Priority to EP19824565.6A priority Critical patent/EP3816186A4/en
Priority to CN201980044384.8A priority patent/CN112351998B/zh
Priority to JP2021522125A priority patent/JP7332194B2/ja
Publication of WO2020001526A1 publication Critical patent/WO2020001526A1/zh
Priority to US17/137,188 priority patent/US12378317B2/en
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    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • 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
    • A61K51/1027—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 against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K51/1045—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 against animal or human tumor cells or tumor cell 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
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    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the invention relates to the field of biomedicine.
  • the present invention relates to a specific PD-L1 binding polypeptide and uses thereof, particularly to use in detecting and / or diagnosing PD-L1 related diseases such as cancer.
  • PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA.
  • Two cell-surface glycoprotein ligands of PD-1, PD-L1 and PD-L2 have been identified and they have been shown to down-regulate T cell activation and cytokine secretion after binding to PD-1 (Freeman et al.
  • PD-L1 B7-H1
  • PD-L2 B7-DC
  • B7-H1 and PD-L2 B7-DC are both B7 homologues that can bind PD-1 but not other members of the CD28 family (Blank et al. 2004).
  • PD-L1 expression has been found in several mouse and human cancers, including human lung, ovarian, colon, melanoma, and various myeloma (Iwai et al.
  • ECT Emission computed tomography
  • SPECT Single-Photon Emission Computed Tomography
  • PET Positron Emission Tomography
  • a diagnostic agent that can be used to detect tumor cells expressing PD-L1, and in particular a PD-L1 antibody-based diagnostic agent suitable for ECT detection.
  • the present invention combines traditional PET technology with antibodies and similar molecules, and uses antibodies to identify target cells, thereby providing a snapshot of all tumors throughout the body.
  • the present invention relates to the use of PD-L1 heavy chain single domain antibody (VHH) or derivative molecules as a tool for imaging agents to identify cells of interest.
  • VHH heavy chain single domain antibody
  • the invention provides a programmed death ligand 1 (PD-L1) binding polypeptide, which is capable of specifically binding PD-L1 and comprising at least one immunoglobulin single variable domain, wherein An immunoglobulin single variable domain contains:
  • CDR1 which comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having one or more amino acid residue substitutions, deletions or additions to SEQ ID NO: 5,
  • CDR2 which comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having one or more amino acid residue substitutions, deletions or additions to SEQ ID NO: 6, for example, the amino acid sequence shown in SEQ ID NO: 13, with
  • CDR3 which comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having one or more amino acid residue substitutions, deletions or additions to SEQ ID NO: 7.
  • the at least one immunoglobulin single variable domain comprises:
  • CDR2 which comprises the amino acid sequence shown in SEQ ID NO: 13, and
  • CDR3 which contains the amino acid sequence shown in SEQ ID NO: 7.
  • the immunoglobulin single variable domain comprises at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 80% of the amino acid sequence of SEQ ID NO: 1. Amino acid sequence with 99% sequence identity.
  • the immunoglobulin single variable domain comprises only one lysine residue.
  • the immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-2, 8-12, and 18-22.
  • the immunoglobulin single variable domain is VHH.
  • the PD-L1 binding polypeptide does not block the binding of PD-1 to PD-L1.
  • the invention provides a nucleic acid molecule encoding a PD-L1 binding polypeptide of the invention.
  • the invention provides an expression vector comprising a nucleic acid molecule of the invention operably linked to an expression control element.
  • the invention provides a host cell comprising a nucleic acid molecule of the invention or transformed with an expression vector of the invention and capable of expressing the PD-L1 binding polypeptide.
  • the invention provides a method for producing a PD-L1 binding polypeptide of the invention, comprising:
  • step b) optionally further purifying and / or modifying the PD-L1 binding polypeptide obtained from step b).
  • the present invention provides a conjugated molecule comprising the PD-L1 binding polypeptide of the present invention and at least one detectable label conjugated to the PD-L1 binding polypeptide.
  • the detectable label is selected from a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme.
  • the detectable label is selected from 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb, 83 Sr or other ⁇ -, ⁇ -, or positron emitters, for example, the detectable label is 68 Ga or 125 I.
  • the PD-L1 binding polypeptide is conjugated to the detectable label via a chelator.
  • the chelator is selected from NOTA, DOTA, TETA or NETA.
  • the detectable label is 68 Ga and the chelator is NOTA.
  • the PD-L1 binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12 or 22.
  • the invention provides a method for detecting the presence of PD-L1 and / or the expression level of PD-L1 in a biological sample, comprising:
  • the difference in complex formation between the biological sample and the control sample indicates the presence of PD-L1 and / or the expression level of PD-L1 in the sample.
  • the present invention provides a diagnostic agent for detecting and / or diagnosing a PD-L1-related disease such as cancer, which comprises the PD-L1 binding polypeptide of the present invention and / or the conjugate molecule of the present invention, and any Selected physiologically acceptable carrier.
  • the diagnostic agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.
  • the present invention provides the use of a PD-L1 binding polypeptide of the present invention and / or a conjugate molecule of the present invention in the preparation of a diagnostic agent for detecting and / or diagnosing a PD-L1 related disease such as cancer.
  • the diagnostic agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.
  • the invention provides a method for detecting and / or diagnosing a PD-L1-related disease, such as cancer, in a subject, comprising administering to the subject a PD-L1 binding polypeptide of the invention and / or a conjugate of the invention Molecular and / or diagnostic agents of the invention.
  • the method further comprises the step of imaging the subject, such as ECT imaging.
  • the ECT imaging is SPECT imaging.
  • the ECT imaging is PET imaging.
  • the cancer highly expresses PD-L1
  • the cancer is selected from lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, bladder cancer, breast cancer, liver cancer, lymph Tumor, malignant hematological disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer, osteosarcoma.
  • the invention provides a kit comprising a PD-L1 binding polypeptide of the invention and / or a conjugated molecule of the invention and / or a diagnostic agent of the invention.
  • FIG. 1 shows a biochemical analysis of the heavy chain single domain antibody 109, where FIGS. 1A and 1B are SEC-HPLC analysis of 109-chis and 109, respectively, and FIG. 1C is a SDS-Page analysis of purified proteins of 109-chis and 109.
  • Figure 2 shows the binding curve of 109-chis and PD-L1.
  • FIG. 3 shows the in vitro activity of the heavy chain single domain antibody of PD-L1, wherein FIG. 3A is a competitive ELISA curve of 109-chis, and FIG. 3B is a flow cytometric detection of the binding effect of 109-chis on the cell surface PD-L1.
  • Figure 3C is the immunohistochemical results of 109-chis animal tumor sections
  • Figure 4 shows the labeling of single-domain antibodies with positron nuclides, of which Figure 4A is a flowchart of single-domain antibodies conjugated with a chelator and radionuclide 68 Ga, and Figure 4B is a mass spectrometric analysis of proteins 109 and 109-NOTA. 4C is the mass spectrum analysis of 67 Ga-NOTA-109, and Fig. 4D is the binding curve and EC50 value of 67 Ga-NOTA-109-chis to PD-L1.
  • Figure 5 shows the identification and analysis of mutant 109-K64Q-RDNSE-cHis.
  • Figure 5A shows the analysis of mutant 109-R73N & K75E-cHis and 109-K86R & P87A-cHis ELISA activity.
  • Figure 5B shows the mutant antibody 109-RDNSE-cHis And 109-K64Q-cHis ELISA activity analysis
  • Figure 5C is a mass spectrometric analysis of the NOTA conjugate of the mutant.
  • FIG 6 shows in vitro positron labeled single domain antibody 68 Ga-NOTA-109 analysis, wherein FIG 6A is a 68 Ga-NOTA-109-chis of ITLC analysis, FIG. 6B is a 68 Ga-NOTA-109-chis of SEC-HPLC and in vitro stability, C is the result of cell endocytosis experiment of 68 Ga-NOTA-109-chis.
  • FIG 7 shows in vitro profile 68 Ga-NOTA-109, wherein FIG 7A is a body 68 Ga-NOTA-109-chis each tissue distribution, FIG. 7B is a distribution in vivo target of the present ratio of 68 Ga-NOTA-109-chis of 7C is in vivo PET imaging 68 Ga-NOTA-109-chis FIG 7D is a biological profile body 68 Ga-NOTA-109-chis of.
  • Figure 8 shows the SPECT results of the 125I- labeled single domain antibody 109.
  • Figure 9 shows the in vitro stability of 68 Ga-NOTA-109 in PBS and serum (FBS).
  • Figure 10 shows the in vitro temperature stability of 68 Ga-NOTA-109 in PBS.
  • Figure 11 shows the in vivo stability of 68 Ga-NOTA-109.
  • Figure 12 shows immunological activity detection of 68 Ga-NOTA-109.
  • Figure 13 shows PET imaging of 68 Ga-NOTA-109 in different xenografts in the same mouse.
  • A Still image of 68 Ga-NOTA-109 at 1 hour after injection;
  • B ROI quantitative analysis of PET image of 68 Ga-NOTA-109 in tumors.
  • Figure 14 shows autoradiography and immunohistochemical analysis of 68 Ga-NOTA-109 PET imaging of different xenografts in the same mouse.
  • A PD-L1 immunohistochemical staining of tumors.
  • B A375 (i), A375 / A375-HPD-L1 (ii), A375-HPD-L1 (iii) 68GA-NOTA-109 autoradiography analysis (top) and autoradiography quantitative analysis (bottom) .
  • antibody or “immunoglobulin” used interchangeably herein, whether referring to heavy chain antibodies or conventional 4-chain antibodies, are used as general terms to include full-length antibodies, their individual And all its parts, domains or fragments (including but not limited to antigen binding domains or fragments, such as VHH domain or VH / VL domain, respectively).
  • sequence as used herein (eg, in terms of “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.) is generally understood To include both related amino acid sequences and nucleic acid or nucleotide sequences encoding said sequences, unless a more limited explanation is required herein.
  • domain refers to a folded protein structure capable of maintaining its tertiary structure independently of the rest of the protein.
  • a domain is responsible for the individual functional properties of a protein, and in many cases can be added, removed, or transferred to other proteins without losing the rest of the protein and / or the function of the domain.
  • immunoglobulin domain refers to a spherical region of an antibody chain, such as a chain of a conventional 4-chain antibody or a heavy chain antibody, or a polypeptide consisting essentially of such a spherical region.
  • the immunoglobulin domain is characterized in that it maintains the immunoglobulin folding characteristics of the antibody molecule, which consists of 2 sandwiches of about 7 antiparallel ⁇ -sheet strands arranged in two beta sheets optionally stabilized by conservative disulfide bonds .
  • immunoglobulin variable domain refers to the term "framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3” “Or” FR3 “and four" framework regions “of” framework region 4 "or” FR4 ", wherein the framework regions are referred to in the art and hereinafter as” complementarity determining region 1 " Or the three “complementarity determining regions” or “CDRs” of “CDR1", “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer antibody specificity to an antigen by having an antigen-binding site.
  • immunoglobulin single variable domain refers to an immunoglobulin variable domain capable of specifically binding an epitope without pairing with other immunoglobulin variable domains.
  • An example of an immunoglobulin single variable domain in the meaning of the present invention is a "domain antibody”, such as immunoglobulin single variable domains VH and VL (VH domain and VL domain).
  • Another example of a single variable domain of an immunoglobulin is the "VHH domain” (or simply "VHH") of the camelid family as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHH, V H H domains, VHH VHH antibodies and antibody fragments are known as “heavy chain antibody” (i.e., “antibody devoid of light chains”) antigen Binding immunoglobulin variable domains (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R .: “Naturally occurring antibodies devoid of light chains”; Nature 363,446- 448 (1993)).
  • VHH domain is used to associate the variable domain with the heavy chain variable domain (which is referred to herein as the "VH domain") present in conventional 4-chain antibodies and the conventional 4-chain antibodies Distinguish between light chain variable domains (which are referred to herein as "VL domains").
  • the VHH domain specifically binds epitopes without the need for additional antigen binding domains (this is in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the lower epitope is recognized by the VL domain together with the VH domain).
  • the VHH domain is a small stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • FR1 contains amino acid residues at positions 1-30
  • CDR1 contains amino acid residues at positions 31-35
  • FR2 contains amino acids at positions 36-49
  • CDR2 contains positions 50-65
  • FR3 contains amino acid residues at positions 66-94
  • CDR3 contains amino acid residues at positions 95-102
  • FR4 contains amino acid residues at positions 103-113.
  • the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., according to One or more positions of the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering).
  • the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • Camelidae-derived VHH domains can be obtained by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a human conventional 4-chain antibody.
  • Humanization also referred to herein as “sequence optimization", in addition to humanization, “sequence optimization” may also encompass other modifications to the sequence, such as removal, by one or more mutations that provide improved properties of VHH Potential post-translational modification sites).
  • a humanized VHH domain may contain one or more fully human framework region sequences.
  • epitopes or the interchangeable term “antigenic determinant” refers to any epitope on an antigen to which the paratope of an antibody binds.
  • Antigenic determinants usually contain chemically active surface groups of molecules, such as amino acid or sugar side chains, and often have specific three-dimensional structural characteristics and specific charge characteristics.
  • epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which can be "linear "Epitope or” conformational "epitope. See, for example, Epitope Mapping Protocols Methods Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • a linear epitope the points of all interactions between a protein and an interacting molecule (such as an antibody) exist linearly along the primary amino acid sequence of the protein.
  • an interacting molecule such as an antibody
  • points of interaction exist across protein amino acid residues that are separated from each other.
  • Epitope for a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, for example, Epitope Mapping Protocols Methods Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • linear epitopes can be determined, for example, by simultaneously synthesizing a large number of peptides on a solid support, where these peptides correspond to portions of a protein molecule, and allowing these peptides to interact with the support while still attached to the support. Antibody response.
  • These techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci.
  • conformational epitopes can be identified by determining the spatial configuration of amino acids, such as by, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (ibid.).
  • Antibodies can be screened competitively for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete with antigen binding. High-throughput methods based on their cross-competition to obtain antibodies that bind to the same epitope are described in International Patent Application WO03 / 48731. Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present invention for binding to the same epitope on PD-L1.
  • the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (such as an immunoglobulin single variable domain of the invention) molecule can bind.
  • the specificity of an antigen-binding molecule can be determined based on its affinity and / or affinity.
  • the affinity expressed by the dissociation equilibrium constant (KD) of an antigen and an antigen-binding protein is a measure of the strength of the binding between an epitope and an antigen-binding site on the antigen-binding protein: the smaller the KD value, the greater the The stronger the binding strength (or the affinity can also be expressed as the association constant (KA), which is 1 / KD).
  • affinity can be determined in a known manner.
  • Affinity is a measure of the strength of binding between an antigen-binding molecule (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing it) and a relevant antigen. Affinity is related to the affinity between the antigen-binding sites on its antigen-binding molecule and the number of related binding sites present on the antigen-binding molecule.
  • PD-L1 binding polypeptide means any polypeptide capable of specifically binding PD-L1, such as a single domain antibody of the present invention that specifically binds PD-L1.
  • PD-L1 binding polypeptide or may refer to a monovalent polypeptide that binds PD-L1 (ie, a polypeptide that binds to one epitope of PD-L1), and a bivalent or multivalent binding polypeptide (that is, a binding polypeptide that binds more than one epitope) ).
  • the "PD-L1 binding polypeptide” of the present invention may comprise at least one immunoglobulin single variable domain such as VHH that binds PD-L1.
  • the PD-L1 binding polypeptide of the invention will be at a preferred range of 10 -7 to 10 -11 mol / L (M), more preferably 10 -8 to 10 -11 mol / L, as measured in a Biacore or KinExA assay, and even more preferably from 10 -9 to 10 -11 M, even more preferably 10 -10 to 10 -11 M or less, a dissociation constant (KD), and / or at least 10 7 M -1, preferably at least 10 8 M - 1.
  • An association constant (KA) of at least 10 9 M -1 , more preferably at least 10 10 M -1 , such as at least 10 11 M -1 binds an antigen to be bound (ie PD-L1).
  • Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • the specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays described herein (e.g., Radioimmunoassay (RIA), Enzyme Immunoassay (EIA) and Sandwich Competitive Assay.
  • SPR surface plasmon resonance
  • RIA Radioimmunoassay
  • EIA Enzyme Immunoassay
  • amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as is known and agreed in the art.
  • amino acid difference refers to the insertion, deletion or substitution of a specified number of amino acid residues at a certain position of a reference sequence compared to another sequence.
  • substitution will preferably be a conservative amino acid substitution, which means that the amino acid residue is replaced with another amino acid residue having a similar chemical structure, and it has an effect on the function, activity or other biological properties of the polypeptide Less or substantially no impact.
  • conservative amino acid substitutions are preferably one amino acid in the following group (i)-(v) is replaced by another amino acid residue in the same group: (i) smaller Aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) Polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) Polar positively charged residues: His, Arg, and Lys; (iv) Larger aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys; and (v) Aromatic residues: Phe, Tyr, and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala Or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr Substitution; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
  • sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences.
  • sequence similarity indicates the percentage of amino acids that are the same or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is usually measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.
  • sequence identity For the determination of sequence identity, see, for example, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York , 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis Analysis Molecular Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • a polypeptide or nucleic acid molecule is considered “substantially isolated” when another protein / polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) is separated.
  • a polypeptide or nucleic acid molecule is considered to be “substantially isolated” when it has been purified at least 2 times, in particular at least 10 times, more particularly at least 100 times and up to 1000 times or more.
  • the "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by suitable techniques, such as suitable chromatography techniques, such as polyacrylamide gel electrophoresis.
  • subject means a mammal, especially a primate, and especially a human.
  • the invention provides a programmed death ligand 1 (PD-L1) binding polypeptide, which is capable of specifically binding PD-L1 and comprising at least one immunoglobulin single variable domain, wherein the at least An immunoglobulin single variable domain contains:
  • CDR1 which comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having one or more amino acid residue substitutions, deletions or additions to SEQ ID NO: 5,
  • CDR2 which comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having one or more amino acid residue substitutions, deletions or additions to SEQ ID NO: 6, for example, the amino acid sequence shown in SEQ ID NO: 13, with
  • CDR3 which comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having one or more amino acid residue substitutions, deletions or additions to SEQ ID NO: 7.
  • the at least one immunoglobulin single variable domain comprises:
  • CDR2 which comprises the amino acid sequence shown in SEQ ID NO: 13, and
  • CDR3 which contains the amino acid sequence shown in SEQ ID NO: 7.
  • the immunoglobulin single variable domain comprises at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 80% of the amino acid sequence of SEQ ID NO: 1. Amino acid sequence with 99% sequence identity.
  • the immunoglobulin single variable domain is derived from SEQ ID NO: 1.
  • the polypeptide of SEQ ID NO: 1 contains a total of 3 lysine groups with free amino groups (located at positions 64, 75, and 86 of SEQ ID NO: 1). These groups can be used to chelate Conjugation of mixtures such as NOTA.
  • substitutions in these lysine groups can be performed.
  • the immunoglobulin single variable domain comprises only one lysine residue.
  • the immunoglobulin single variable domain comprises a K64Q substitution relative to SEQ ID NO: 1.
  • the immunoglobulin single variable domain comprises K86R and P87A substitutions relative to SEQ ID NO: 1.
  • the immunoglobulin single variable domain comprises H71R, R73N, A74S, and K75E substitutions relative to SEQ ID NO: 1.
  • the immunoglobulin single variable domain comprises R73N and K75E substitutions relative to SEQ ID NO: 1.
  • the immunoglobulin single variable domain comprises K64Q, H71R, R73N, A74S, and K75E substitutions relative to SEQ ID NO: 1.
  • the protein or nucleic acid may consist of the sequence, or it may have additional amino acids or cores at one or both ends of the protein or nucleic acid Glycylic acid, but still has the activity according to the invention.
  • the PD-L1 binding polypeptide of the present invention may further include a tag suitable for expression and / or purification, including but not limited to a His tag and the like.
  • the immunoglobulin single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-2, 8-12, and 18-22.
  • the immunoglobulin single variable domain is VHH.
  • the PD-L1 binding polypeptide does not block the binding of PD-1 to PD-L1. Not blocking the binding of PD-1 to PD-L1 can also reduce potential side effects by avoiding affecting the normal function of PD-L1.
  • the epitope to which the PD-L1 binding polypeptide of the present invention binds to PD-L1 is different from the PD-1 and PD-L1 binding sites in the prior art, so that the PD-L1 binding polypeptide of the present invention does not affect other target resistances.
  • the PD-L1 binding polypeptide of the invention does not block the interaction of a therapeutic antibody against PD-L1 with PD-L1. This makes it possible to monitor the cancer using the PD-L1 binding polypeptide of the present invention or a derivative conjugated molecule thereof while administering a therapeutic antibody against PD-L1 for cancer treatment.
  • the invention in another aspect, relates to a nucleic acid molecule encoding a PD-L1 binding polypeptide of the invention.
  • the nucleic acid of the invention may be RNA, DNA or cDNA.
  • a person skilled in the art can select a nucleic acid molecule encoding the PD-L1 binding polypeptide of the present invention according to need or conventional means.
  • the nucleic acids of the invention may also be in the form of a vector, which may be present in the vector and / or may be part of a vector such as a plasmid, a cosmid or YAC.
  • the vector may be, in particular, an expression vector, that is, a vector that provides expression of the PD-L1 binding polypeptide in vitro and / or in vivo (ie, in a suitable host cell, host organism, and / or expression system).
  • the expression vector typically contains at least one nucleic acid of the invention that is operably linked to one or more suitable expression regulatory elements (e.g., promoter, enhancer, terminator, etc.). Selection of the elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the PD-L1 binding polypeptide of the present invention, such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.
  • the nucleic acids of the invention can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on information about the amino acid sequence of the polypeptides of the invention given herein, and / or can be obtained from a suitable natural Sources are separated.
  • the invention relates to a host cell that expresses or is capable of expressing one or more PD-L1 binding polypeptides of the invention and / or a nucleic acid or vector of the invention.
  • Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include Gram-negative bacteria strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacteria strains (e.g., Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains) cells.
  • Gram-negative bacteria strains e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacteria strains e.g., Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains
  • Suitable fungal cells include cells of species Trichoderma, Neurospora, and Aspergillus; or include Saccharomyces (e.g. Saccharomycescercerevisiae), schizont Schizosaccharomyces (e.g. Schizosaccharomyces pombe), Pichia (e.g. Pichia pastoris and Pichia methanolica) and Han Cells of the species Hansenula.
  • Saccharomyces e.g. Saccharomycescercerevisiae
  • schizont Schizosaccharomyces e.g. Schizosaccharomyces pombe
  • Pichia e.g. Pichia pastoris and Pichia methanolica
  • Han Cells of the species Hansenula e.g. Pichia pastoris and Pichia methanolica
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • the present invention can also use amphibious cells, insect cells, plant cells, and any other cells in the art for expressing heterologous proteins.
  • the invention also provides a method for producing a PD-L1 binding polypeptide of the invention, which method generally comprises the following steps:
  • the PD-L1 binding polypeptide of the invention can be produced in a cell as described above in an intracellular manner (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cell and optionally further purified; or it can be a cell Produced exogenously (eg, in a medium in which the host cells are cultured), followed by isolation from the medium and optionally further purification.
  • the PD-L1 binding polypeptide of the present invention can also be obtained by other methods for producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.
  • the present invention provides a conjugated molecule comprising the PD-L1 binding polypeptide of the present invention and at least one detectable label conjugated to the PD-L1 binding polypeptide.
  • the detectable label includes, but is not limited to, a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme.
  • Fluorescent agents that can be used for conjugation include, but are not limited to, isothionine fluorescein, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, and fluorescent amines; chemiluminescent agents that can be used for conjugation Includes, but is not limited to, luminol, isoluminol, aromatic acridinium esters, imidazole, acridine salts, and oxalates; bioluminescent agents that can be used for conjugation include, but are not limited to, luciferin, luciferase, and jellyfish luminescence protein.
  • Paramagnetic ions that can be used for conjugation include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), diamond (II), nickel (II), copper (II), neodymium (III) ), Gadolinium (III), gadolinium (III), gadolinium (III), vanadium (II), gadolinium (III), gadolinium (III), gadolinium (III), and gadolinium (III), or radiopaque materials, Such as dam, diatrizoate, ethyl iodine oil, gallium citrate, iodocaric acid, iodoin acid, iodadamine, cholic acid, iodosinic acid, iodocolamide, iohexol, iopanol, iodine Panic acid, Iopexic acid, Iodixifaic acid, Iodosuccinic acid
  • the detectable label is a radionuclide.
  • Radionuclides that can be used for conjugation are, for example, radionuclides with an energy between 20-4000KeV, including but not limited to 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52m Mn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb, 83 Sr or other ⁇ -, ⁇ -, or positron emitters.
  • the detectable label is 68 Ga or 125 I.
  • the PD-L1 binding polypeptide can be conjugated to the detectable label via a chelator.
  • the PD-L1 binding polypeptide of the present invention can be reacted with a reagent having a long tail, and the long tail is attached with a plurality of integrators for binding ions. group.
  • Such tails can be, for example, polylysine, polysaccharides, or other polymers with derivatized or derivatizable chains with pendant groups that can bind chelating groups such as ethylenediaminetetraacetic acid (EDTA) , Diethylene triamine pentaacetic acid (DTPA), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid), NOTA, TETA, NETA, porphyrin , Polyamines, crown ethers, thiosemicarbazone, polyoximes, and similar groups known to be useful for this purpose.
  • Chelating agents can be attached to antibodies using standard chemical methods.
  • a detectable label is conjugated to a PD-L1 binding polypeptide of the invention via a chelator.
  • the chelating agents used include, but are not limited to, NOTA, DOTA, TETA or NETA.
  • the detectable label is 68 Ga and the chelator is NOTA.
  • the PD-L1 binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12 or 22.
  • the present invention provides a method for preparing a radionuclide such as 68 Ga-labeled conjugated molecules of the present invention, comprising 1) conjugating the PD-L1 binding polypeptide of the present invention with a chelating agent to generate said Conjugate of PD-L1 binding polypeptide with a chelator; and 2) contacting the product of step 1) with a radionuclide such as 68 Ga, whereby the radionuclide such as 68 Ga is labeled by the chelation of the chelator PD-L1 binds a polypeptide.
  • the chelator is NOTA
  • the PD- is generated by reacting the PD-L1 binding polypeptide with p-SCN-Bn-NOTA or p-NH2-Bn-NOTA.
  • L1 binds a conjugate of a polypeptide to NOTA.
  • the invention provides a method for preparing a 125 I-labeled conjugated molecule of the invention, comprising 1) reacting a PD-L1 binding polypeptide of the invention with 125 I in the presence of chloramine T; and 2 ) Optionally, the reaction is terminated with sodium metabisulfite.
  • the invention provides a method for detecting the presence of PD-L1 and / or the expression level of PD-L1 in a biological sample, comprising:
  • the difference in complex formation between the biological sample and the control sample indicates the presence of PD-L1 and / or the expression level of PD-L1 in the sample.
  • the biological sample is an ex vivo sample.
  • the invention provides a composition comprising a PD-L1 binding polypeptide of the invention and / or a conjugate molecule of the invention, and an optional physiologically acceptable carrier.
  • the composition can be used as a diagnostic agent, for example, a diagnostic agent for detecting and / or diagnosing PD-L1-related diseases.
  • the present invention provides a diagnostic agent for detecting and / or diagnosing a PD-L1-related disease such as cancer, comprising the PD-L1 binding polypeptide of the present invention and / or the conjugate molecule of the present invention, and any Selected physiologically acceptable carrier.
  • the diagnostic agent is a contrast agent.
  • the PD-L1 binding polypeptide of the present invention and / or the conjugated molecule of the present invention are particularly suitable for in vivo imaging, such as Emission Computed Tomography (ECT).
  • ECT Emission Computed Tomography
  • the PD-L1 binding polypeptide of the present invention and / or the conjugated molecule of the present invention can be applied to single-photon emission computed tomography (SPECT) and positron emission tomography (SPECT) according to different labels.
  • SPECT single-photon emission computed tomography
  • SPECT positron emission tomography
  • Positron, Tomography, PET In tumor diagnosis, high-resolution tumor imaging can be provided and quantitative analysis can be performed through images.
  • the SPECT imaging may further include SPECT / CT imaging
  • the PET imaging may further include PET / CT imaging, which may provide a better imaging effect.
  • the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.
  • the present invention provides the use of a PD-L1 binding polypeptide of the present invention and / or a conjugate molecule of the present invention in the preparation of a diagnostic agent for detecting and / or diagnosing a PD-L1 related disease such as cancer.
  • the diagnostic agent is a contrast agent.
  • the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent
  • the invention provides a method for detecting and / or diagnosing a PD-L1-related disease, such as cancer, in a subject, comprising administering to the subject a PD-L1 binding polypeptide of the invention and / or a conjugate of the invention Molecular and / or diagnostic agents of the invention.
  • the method further comprises the step of imaging the subject, such as ECT imaging.
  • the ECT imaging is SPECT imaging.
  • the ECT imaging is PET imaging. Imaging techniques and devices for scanning by SPECT or PET are well known in the art and any such known ECT imaging technique and device can be used.
  • Diseases that can be detected and / or diagnosed by the PD-L1 binding polypeptide of the present invention and / or the conjugated molecule of the present invention and / or the diagnostic agent of the present invention include those that abnormally increase PD-L1 expression in cells, tissues or organs Diseases, such as infectious diseases, cancer, etc.
  • Preferred cancers that can be detected and / or diagnosed using the PD-L1 binding polypeptide of the present invention have high expression of PD-L1.
  • Non-limiting examples include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (such as metastatic malignant melanin Tumors), bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor and osteosarcoma.
  • Examples of other cancers that can be detected and / or diagnosed by the methods of the present invention include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or eye melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer , Endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra Cancer, penile cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, ureter Cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pit
  • kits comprising a PD-L1 binding polypeptide of the invention and / or a conjugated molecule of the invention and / or a diagnostic agent of the invention.
  • the kit is used to perform the method of the invention.
  • Kits generally include a label indicating the intended use of the contents of the kit.
  • the term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • the PDL1-Fc fusion protein for immunization was expressed from CHO cells and purified by Protein A affinity chromatography. A Xinjiang Bactrian camel was selected for immunization. After 4 times of immunization, 100ml peripheral blood lymphocytes of camel were extracted and total RNA was extracted using RNA extraction kit provided by QIAGEN. Super-Script III FIRST STRANDSUPERMIX kit (Thermo Fisher Scientific) was used to reverse the extracted RNA according to the instructions. Recorded as cDNA. Amplification of a nucleic acid fragment encoding the variable region of a heavy chain antibody using nested PCR:
  • Upstream primer GTCCTGGCTGCTCTTCTACAAGGC (SEQ ID NO: 14);
  • Downstream primer GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 15).
  • Upstream primer GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 16);
  • Downstream primer GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT (SEQ ID NO: 17).
  • the target heavy chain single domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pCDisplay-3 (Creative Biolabs, Cat: VPT4023) using restriction enzymes PstI and NotI.
  • the product was then electrotransformed into E. coli electrotransformed competent cells TG1 to construct a heavy chain single domain antibody phage display library against PD-L1 and verify the library. By gradient dilution plating, the storage capacity was calculated to be 1.33 ⁇ 10 8 .
  • 24 clones were randomly selected for colony PCR. The results show that the insertion rate has reached 100%.
  • the obtained PD-L1 binding-positive phages were infected with blank E. coli and plated. Subsequently, 96 single colonies were selected for cultivation, and phages were produced and purified. The plate was coated with a PDL1-Fc fusion protein at 4 ° C overnight, and the obtained sample phage (control group was a blank phage) was added and reacted at room temperature for 1 hour. After washing, a primary anti-mouse anti-HA tag antibody (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) was added and reacted at room temperature for 1 hour.
  • a primary anti-mouse anti-HA tag antibody purchased from Beijing Kangwei Century Biotechnology Co., Ltd.
  • a secondary antibody goat anti-mouse alkaline phosphatase-labeled antibody (purchased from Amyjet Technology Co., Ltd.) was added and reacted at room temperature for 1 hour. After washing, an alkaline phosphatase coloring solution was added, and the absorption value was read at a wavelength of 405 nm. When the OD value of the sample well is greater than 3 times the OD value of the control well, it is judged as a positive clone well.
  • the bacteria of the positive cloning wells were transferred to an LB liquid containing 100 ⁇ g / mL ampicillin and cultured in order to extract a plasmid and perform sequencing.
  • the amino acid sequence of the single-domain antibody is shown in Table 1, where 109-chis is a recombinant protein with a His tag at the c-terminus.
  • the CDR sequences are shown in boxes.
  • nucleotide sequences encoding the single domain antibodies of Table 1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4.
  • the CDR sequences of the 109 antibody are CDR1: GFSLDDSDMG (SEQ ID NO: 5); CDR2: IASDRSTYYTPSVKG (SEQ ID NO: 6); and CDR3: APRLAYTTAMTCEGDFAY (SEQ ID ID NO: 7).
  • Primers are designed based on the nucleotide sequence of the single-domain antibody. PCR is performed using the gene-synthesized DNA as a template, and then cloned into pCDNA4 (Invitrogen, Cat V86220) vector. The gene sequence is determined to determine the target cloned gene. The correctness of the sequence.
  • the recombinantly constructed single domain antibody fusion protein particles were transfected into HEK293 cells for antibody expression.
  • the recombinant expression plasmid was diluted with Freestyle293 medium (Thermo Fisher Scientific, Cat. No. 12338018) and the PEI (polyethyleneimine) solution required for transformation was added.
  • the plasmid / PEI mixture was separately added to the HEK293 cell suspension and placed at 37 ° C. % CO 2 , cultured at 90 rpm.
  • EX293 medium Sigma, batch number 14571C
  • 109-chis single domain antibody was purified by Ni + resin gel affinity chromatography to obtain a purified antibody with a purity of more than 95% by SDS-PAGE electrophoresis; 109 single domain antibody First, one-step enrichment was performed by affinity chromatography, and then purified by ion chromatography to obtain 109 purified antibodies with SDS-PAGE electrophoresis purity of 95% or more (as shown in Figures 1A-C).
  • the PDL1-Fc fusion protein was obtained by transient expression of HEK293 and purification by nickel column affinity chromatography.
  • the obtained PDL1-Fc fusion protein was coated at 0.5 ⁇ g / well at 4 ° C. overnight, and the plate was then added with a serial dilution series of the obtained PD-L1 single domain antibody protein, and reacted at room temperature for 1 hour.
  • an anti-his-horseradish peroxidase (HRP) -labeled antibody purchased from Abcam, article number ab1187) was added and reacted at room temperature for 1 hour.
  • a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • Example 3 In vitro activity analysis of PD-L1 antibody protein
  • PDL1-muFc fusion protein (using murine Fc) and PD1-Fc fusion protein were cloned into pCDNA4 vector (Invitrogen, Cat V86220) and expressed by HEK293 cells.
  • PDL1-muFc fusion protein 0.5 ⁇ g / well overnight at 4 ° C, then add 10 ⁇ g PD1-Fc to each well (the blank group does not add any antibodies or proteins, only equal volume buffer solution) and the initial dilution concentration is 3 ⁇ g / L Heavy chain single domain antibody 109-chis (control group was buffered), 2-fold dilution, 12 gradients, and reacted for 1 hour at room temperature.
  • anti-his-HRP purchased from Abcam
  • a color developing solution was added, and an absorption value was read at a wavelength of 405 nm.
  • the binding showed an S-curve binding trend. It is considered that the antibody has no blocking effect.
  • PDL1-muFc fusion protein 0.5 ⁇ g / well overnight at 4 ° C, then add 10 ⁇ g PD1-Fc to each well (the blank group does not add any antibodies or proteins, only equal volume buffer solution) and the initial dilution concentration is 3 ⁇ g / L Heavy chain single domain antibody 109-chis (control group was buffered), 2-fold dilution, 12 gradients, and reacted for 1 hour at room temperature. Then, anti-Fc-HRP (purchased from Abcam) was added, and the reaction was performed at room temperature for 1 hour. Then, a color developing solution was added, and an absorption value was read at a wavelength of 405 nm.
  • A375 cells (A375-PDL1 cells) stably expressing human PD-L1 protein on the membrane were obtained by stably transfecting human melanoma A375 cells with human PD-L1 full-length protein gene.
  • the tumorigenic cell line MCF-7 which does not express PD-L1, was used as a negative control.
  • the MCF-7 cell line was obtained from the American Type Culture Collection (ATCC) under the accession number ATCC HTB-22 and cultured as recommended. Cells were cultured to> 90% confluence before use.
  • Anti-PD-L1 single domain antibody 109 was used to perform flow cytometric analysis on these several cell lines for quantitative analysis of indirect immunofluorescence staining. Determine the number of receptors for each cell surface.
  • Cells were then washed with 1 ml of ice-cold flow cytobuffer (PBS, containing 2% bovine serum albumin), centrifuged at 300 ⁇ g for 5 minutes, and resuspended in 0.5 ⁇ L buffer. Add 100 ⁇ L of a 1:50 dilution of the secondary antibody-PE conjugate with PBS and incubate on ice for 45 minutes in the dark. The cells were then washed twice with 1 mL of ice-cold buffer, centrifuged at 300 ⁇ g for 5 minutes, and resuspended in 500 ⁇ L of buffer.
  • PBS ice-cold flow cytobuffer
  • Flow cytometry was performed on a Beckman Coulter Cytomics FC500MPL. For each tube, a minimum of 5 ⁇ 10 4 events were collected. All analyses were single color and PE was detected in FL1. Forward scattering (FS) and side scattering (SS) data demonstrate that all cell populations are closely clustered.
  • FS Forward scattering
  • SS side scattering
  • the experimental temperature of Biacore was 25 ° C, and the injection flow rate was 50 ⁇ l / min.
  • the analyte is diluted with a buffer HBS-EP + buffer to a certain concentration, which causes it to flow through the blank reference channel and the activation channel in sequence.
  • the signal value generated on the blank reference channel reflects the non-specific adsorption of the analyte on the chip. In some cases, the signal value generated on the activation channel reflects the specific binding between the analyte and the ligand.
  • the analyte was injected for 300s, and then HBS-EP + buffer was injected for 180s to dissociate the analyte from the ligand.
  • the 109 antibody was diluted to 7 gradients of 200ng / ml, 100ng / ml, 50ng / ml, 25ng / ml, 12.5ng / ml, 6.25ng / ml, 3.125ng / ml. Repeated injection and dissociation of analytes of different concentrations.
  • Immunohistochemical sections were prepared according to conventional methods well known to those skilled in the art and stained with anti-his-horseradish peroxidase (HRP) labeled antibodies.
  • HRP anti-his-horseradish peroxidase
  • the single domain antibody of the present invention is labeled with a chelating agent p-SCN-Bn-NOTA and a radionuclide 68 Ga according to the flowchart shown in FIG. 4A.
  • Single-domain antibody 109-chis (3 mg) was dissolved in 0.05 M sodium carbonate buffer at pH 8.7, and p-SCN-Bn-NOTA (Macrocyclics, Cat. No. B-605) was added in a 10-fold molar excess. The mixture was stirred and protected from light at room temperature for at least 18 hours.
  • the conjugated product was purified using a PD-10 column (GE) to remove excess p-SCN-Bn-NOTA, and the single domain antibody was concentrated to ⁇ 1 mg / mL by ultrafiltration.
  • SEC-HPL analysis was performed using 0.1M phosphate buffer at pH 7.0 as the mobile phase.
  • the amount of chelated NOTA was determined by a Thermo Scientific LTQ Orbitrap XL liquid chromatography mass spectrometer.
  • the single domain antibody concentration was determined by the BCA protein concentration determination kit. Chromatographic conditions: Instrument Waters ACQUITY UPLC, using ACQUITY BEH C18 1.7 ⁇ m (2.1 ⁇ 100mm) chromatographic column, the temperature of the autosampler is set to 4 ° C, the flow rate is 0.25mL / min, the temperature of the column is 50 ° C, the temperature of the 4th column is used for gradient elution, and the mobile phase is 0.1 % Formic acid water (A)-0.1% formic acid acetonitrile (B). The molecular weight characteristics were calculated using Thermo Scientific Protein Deconvolution 4.0. The result of mass analysis of the obtained single domain antibody 109-chis-NOTA is shown in FIG. 4B.
  • Figure 4B shows the molecular weight of 109-chis protein is 13603Da
  • the right side shows that after p-SCN-Bn-NOTA is conjugated with NOTA, each molecule of NOTA is conjugated with the corresponding molecular weight.
  • the increase is 450, which is 14053 Da
  • 2 molecules of NOTA are conjugated
  • the corresponding molecular weight is increased by 900 Da, which is 14503 Da.
  • the mass spectrometry results of 109-chis-NOTA showed that after the conjugation reaction of 109-chis, the product was a mixture of 1 molecule and 2 molecules of NOTA respectively.
  • GaCl 3 was dissolved in 0.05M HCL, 1/2 volume of 0.2M sodium acetate solution was added, and 1/4 volume of 0.1M sodium acetate buffer containing 109-chis-NOTA pH 5.3 was added. The pH of the reaction system was 4.5. Between ⁇ 4.7, react at room temperature for 10 minutes, and remove unreacted ion gallium through a PD-10 column. Thermo Scientific LTQ Orbitrap XL LC-MS / MS was used to determine the amount of chelated 67 Ga. The single domain antibody concentration was determined by the BCA protein concentration determination kit. Chromatographic conditions: Instrument Waters ACQUITY UPLC, using ACQUITY BEH C18 1.7 ⁇ m (2.1 ⁇ 100mm) chromatographic column.
  • the temperature of the autosampler is set to 4 ° C. At a flow rate of 0.25mL / min, the column temperature is 50 ° C. 4 ⁇ L is injected for gradient elution. The mobile phase is 0.1% formic acid. Water (A)-0.1% formic acid acetonitrile (B). The obtained mass analysis results of 69 and 71 Ga-NOTA-109-chis are shown in Fig. 4C.
  • the PDL1-Fc fusion protein was obtained by transient expression of HEK293 and purification by nickel column affinity chromatography.
  • the resulting PDL1-Fc fusion protein was coated at 0.5 ⁇ g / well at 4 ° C overnight, and then a 109-chis PD-L1 single domain antibody protein or a PD-L1 single domain antibody protein chelate product obtained in 4.2 was added to the dilution series at room temperature. The reaction was continued for 1 hour. After washing, an anti-his horseradish peroxidase-labeled antibody was added and reacted at room temperature for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • the application software SotfMaxPro v5.4 was used for data processing and mapping analysis. After four-parameter fitting, the antibody-PD-L1 binding curve and EC50 value shown in Figure 4D were obtained to reflect the antibody 109-chis or its labeled form to PD-L1. Affinity.
  • the 109 antibody protein structure contains a total of 3 lysine groups with free amino groups, which can be used for subsequent labeling.
  • the lysine on the surface of the antibody was mutated, and to maintain affinity, lysine adjacent amino acids were also carried Mutation, a total of four mutants shown below were obtained: 109-R73N & K75E-cHis, 109-K86R & P87A-cHis, 109-RDNSE-cHis, 109-K64Q-cHis, as shown in Table 3, and examined the antibodies and PD after mutation -L1 binding ability is affected.
  • the K64Q mutation is located in CDR2, which results in a mutated CDR2, which contains the amino acid sequence IASDRSTYYTPSVQG (SEQ ID NO: 13).
  • the PDL1-Fc fusion protein was obtained by transient expression of HEK293 and purification by nickel column affinity chromatography.
  • the obtained PDL1-Fc fusion protein was coated at 0.5 ⁇ g / well at 4 ° C. overnight, and the plate was then added with a serial dilution series of the obtained PD-L1 single domain antibody protein, and reacted at room temperature for 1 hour. After washing, an anti-his horseradish peroxidase-labeled antibody was added and reacted at room temperature for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 405 nm.
  • Application software SotfMaxPro v5.4 was used for data processing and mapping analysis. Through four-parameter fitting, the antibody-PD-L1 binding curve and EC50 value were obtained to reflect the affinity of the antibody to PD-L1.
  • the mutation of the amino acid on the surface of the antibody does not affect the binding performance of the antigen antibody.
  • the labeling efficiency of the chelating agent was further analyzed, and the mutant antibody was subjected to NOTA conjugation for mass spectrometric analysis and identification using the method described in 4.1.
  • the mutant antibody 109-K64Q-RDNSE-cHis was subjected to NOT conjugation and identified by mass spectrometry using the method described in 4.1. Mass spectrometry results showed that the mutated single-domain antibody 109-K64Q-RDNSE-cHis reacted with the chelating agent p-SCN-Bn-NOTA, and the product was a pure final product conjugated to only one molecule of NOTA.
  • ITLC SA is pre-cut to 1 cm x 12 cm strips and the ends of the strips are marked 1 cm from each other with a pencil.
  • RCP radiochemical purity
  • SEC-HPLC chromatographic conditions were: SEC-HPLC was performed on an Agilent 1100 series HPLC equipped with a Tokousv3000SL (4.6 ⁇ 250mm) column and a Raytest GABI radioactive detector. SEC analysis was performed using 0.1M phosphate buffer at pH 7.0 as the mobile phase.
  • FIG. 6A A representative SEC-HPLC chromatogram of a PD-10 column purified radiolabeled single domain antibody is shown in Figure 6A.
  • the retention time of the radiolabeled single domain antibody is practically unchanged from the retention time of the corresponding unlabeled single domain antibody (except for the time difference due to the physical separation of the UV and gamma detectors; the data is not shown ).
  • the radiolabeled product 68 Ga-NOTA-109 was labeled and placed in PBS and serum (FBS), stored at room temperature for 0, 2, 4 h, and analyzed by HPLC to observe its stability in PBS and FBS.
  • the results are shown in Figures 9A and B. The results showed that the prepared radiolabeled products were stored in PBS and serum at room temperature for 4 hours, and there was no difference between the peak shape and radiolabeled, and the radiochemical purity was> 98%, and no obvious shedding was observed.
  • the radiolabeled product 68 Ga-NOTA-109 was labeled and placed in PBS, and stored at 55-75 ° C for 4 hours ( Figure 10), and its in vitro temperature stability was measured.
  • the results showed that the prepared radiopharmaceutical was left at 55 ° C for 4 h in PBS, the peak shape was not different from that after radiolabeling, the radiochemical purity was> 98%, and no obvious shedding was observed.
  • PBS at 65 and 75 ° C for 4 h, there was a degradation peak after the main peak, and the amount of degradation increased with increasing temperature.
  • Cells were plated in 6-well plates, 8 ⁇ 10 5 cells per well, 3 mL medium / well, 37 ° C, 5% CO2 overnight culture; cells were removed, and incubated at 4 ° C for 30min; all 6-well plates were removed, and the medium was changed to Medium containing 10 ⁇ ci ( ⁇ 50nM) of hot-labeled protein, incubate at 4 ° C for 1 h; replace 3 of the medium with 25 ⁇ M of unlabeled protein as a non-specific adsorption control sample; aspirate the supernatant containing unlabeled protein.
  • Protein-binding medium wash the cells twice with 2 mL of pre-chilled PBS containing 1% BSA; replace the medium with a growth medium, and incubate at 37 ° C and 5% CO2 for 1 h and 2 h; discard the culture after incubation at different times Base supernatant, add 2 mL of pH 3.0 containing 20 mM sodium acetate PBS, incubate for 15 min at 4 ° C, add 2 mL of pH 3.0 containing 20 mM sodium acetate PBS again, the supernatant of the two parts is mixed, which is the cpm value of the cell surface bound part ; The remaining cell pellet was resuspended in PBS containing 0.5% SDS, this part is the cell endocytosis part; the gamma-counter detected the cpm value of the cell binding and endocytosis part, and carried out attenuation correction.
  • the results are shown in Figure 6C, indicating that the 68 Ga intracellular endocytos
  • mice Healthy mice were selected. Each mouse was injected with 0.1 mL of 68 Ga-NOTA-109 with a radiochemical purity of 98% through the tail vein. Urine was collected 2 hours after the injection, and analyzed by HPLC to determine its stability in vivo.
  • Example 8 Determination of 68 Ga-NOTA-109 immune activity
  • the immunological activity of 68 Ga-NOTA-109 was determined as follows:
  • the gamma counter measures the cpm value of the cell suspension and the washed supernatant, and corrects the decay.
  • Example 8 68 Ga-NOTA-109 in vivo distribution
  • ⁇ 10 ⁇ g of radiolabeled single domain antibody (-100 ⁇ Ci / 10 ⁇ g) was administered to mice via tail vein injection. Mice were placed in filter-lined cages until euthanized. Five mice were euthanized at each time point, the tissue of interest was dissected, and counted on a gamma counter. Collect data for blood, kidney, liver, spleen, lung, heart, intestine, stomach, muscle, skin, brain, bone and tumor. Take the injected dose as the total injected dose. For each organ, the% injected dose was determined based on the total injected dose, and the organs were weighed to determine the% injected dose per gram (% ID / g).
  • Figure 7B shows the results of tumor tissue and blood uptake ratios and tumor tissue and contralateral normal muscle uptake ratios for these experiments.
  • 109 single domain antibody showed good tumor uptake in A375-PDL1 tumors expressing the target, with a maximum value of about 5% of the injected dose per gram of tissue 30 minutes after the injection (PI), and at 4 hours PI, the peak tumor: muscle The ratio is still greater than 6.
  • the uptake of tumor tissue in mice is higher than that of the normal muscle on the opposite side, and the uptake in bone after 4h is low, indicating that the marker is stable in the body without 68 Ga removal. This marker may become a good tumor Developer.
  • mice were anesthetized with isoflurane and placed on a PET bed, and ⁇ 10 ⁇ g of a radiolabeled single domain antibody ( ⁇ 100 ⁇ Ci / 10 ⁇ g) was administered to the mice via tail vein injection.
  • a continuous PET scan was performed at 120 minutes to analyze the radiation absorption of tumors, muscles and kidneys at multiple time points, as shown in the upper and middle graphs of Figure 7C.
  • Image reconstruction was performed using a two-dimensional ordered subset expectation maximization algorithm.
  • the blocking experiment was given the therapeutic drug KN035 (blocking the binding of PD1 and PDL1) 24 hours in advance.
  • the mice were anesthetized with isoflurane and placed on a PET bed.
  • ⁇ 10 ⁇ g of radiolabeled single domain antibody ( ⁇ 100 ⁇ Ci / 10 ⁇ g) were administered to mice via tail vein injection.
  • a continuous PET scan was performed at 120 minutes to analyze the radiation absorption of tumors, muscles and kidneys at multiple time points, as shown in the lower graph of Figure 7C. Biodistribution was analyzed using the MIM software activity time curve, as shown in Figure 7D.
  • A375-PD-L1 xenograft tumors were clearly visible from 5 to 120 minutes after injection of the PET tracer 68 Ga-NOTA-109, while MCF-7 xenografts with PD-L1 negative expression were injected. No uptake was seen after 120 min.
  • the A375-PD-L1 tumor has good contrast compared to the contralateral side.
  • 68 Ga-NOTA-109 has significant concentration in the kidneys of model mice, which indicates that it is mainly metabolized by the kidneys. 68 Ga-NOTA-109 has a short half-life and will not cause damage to model rats.
  • the tumor uptake value of 68 Ga-NOTA-109 reached a maximum of about 5-6% ID / g. With the change of time, the intake value did not decrease significantly. 120 minutes after injection, the tumor pair 68 Ga-NOTA-109 The uptake value can still be maintained at about ⁇ 4% ID / g.
  • Significant uptake of 68 Ga-NOTA-109 by the kidneys confirms that the PET tracer is primarily metabolized by the kidneys. In addition, as shown in FIG.
  • the tumor has a significantly higher uptake of 68 Ga-NOTA-109 than normal tissues such as muscles, and the tumor-to-muscle uptake ratios (T / NT) are all greater than 5, which is beneficial for obtaining high-resolution PET images of tumors.
  • T / NT tumor-to-muscle uptake ratios
  • the left hind leg, right hind leg, and right front leg of the same mouse were subcutaneously inoculated with different cancer cells A375-HPD-L1 and A375, respectively.
  • the tumor size was about 250-350 mm 3 .
  • radiolabeled single domain antibody (-100 ⁇ Ci / 10 ⁇ g) was administered to mice via tail vein injection.
  • a static PET scan was performed 1 h after dosing, as shown in Figure 13A.
  • Image reconstruction was performed using a two-dimensional ordered subset expectation maximization algorithm.
  • the radioactivity (MBq / mL) was calculated by the region of interest (ROI) method, and the obtained value was divided by the injection dose to obtain the PET tracer uptake value (% ID / g) of each tissue (assuming a tissue density of 1g / ml) ).
  • the calculation results are shown in Figure 13B.
  • Example 9 125 I-labeled single domain antibody 109 and its imaging
  • the A375-PDL1 tumor model was inoculated according to the method of 8.1.
  • the mice were anesthetized with isoflurane and placed on a SPECT bed.
  • ⁇ 10 ⁇ g of radiolabeled single domain antibody ( ⁇ 100 ⁇ Ci / 10 ⁇ g) were injected into the tail vein of the mice.
  • SPECT scans were performed at 1h, 2h, and 4h to analyze the radioactive absorption of tumors, tissues, and organs at multiple time points, as shown in Figure 8.
  • A375-PD-L1 xenografts were clearly visible. As can be seen from the figure, the A375-PD-L1 tumor has good contrast compared with the contralateral side. Similar to 68 Ga-NOTA-109 in model mice, 125 I-109 has significant concentration in the kidney, which indicates that it is mainly metabolized by the kidney.

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Abstract

提供了一种特异性结合PD-L1的单域抗体多肽及其用途，特别是在检测和/或诊断PD-L1相关疾病例如癌症中的用途。

Description

PD-L1结合多肽及其用途 技术领域
本发明涉及生物医药领域。具体而言，本发明涉及一种特异性PD-L1结合多肽及其用途，特别是在检测和/或诊断PD-L1相关疾病例如癌症中的用途。
背景技术
程序性死亡-1(PD-1)是CD28受体家族的成员，该家族包括CD28、CTLA-4、ICOS、PD-1和BTLA。该家族的最初成员CD28和ICOS通过添加单克隆抗体后增强T细胞增殖的功能而发现(Hutloff等(1999)，Nature 397:263-266；Hansen等(1980)，Immunogenics 10:247-260)。已经鉴定了PD-1的两种细胞表面糖蛋白配体，PD-L1和PD-L2，已经表明它们在与PD-1结合后下调T细胞活化和细胞因子分泌(Freeman等(2000)，J Exp Med 192:1027-34；Latchman等(2001)，Nat Immunol 2:261-8；Cater等(2002)，Eur J Immunol 32:634-43；Ohigashi等(2005)，Clin Cancer Res 11:2947-53)。PD-L1(B7-H1)和PD-L2(B7-DC)都是可与PD-1结合但是不与其他CD28家族成员结合的B7同源物(Blank等2004)。PD-L1的表达已经在几种鼠和人类癌症中发现，包括人肺癌、卵巢癌、结肠癌、黑色素瘤和各种骨髓瘤(Iwai等(2002)，PNAS 99:12293-7；Ohigashi等(2005)，Clin Cancer Res 11:2947-53)。已有的结果显示，肿瘤细胞高表达的PD-L1通过增加T细胞的凋亡从而在肿瘤的免疫逃逸中起着重要的作用。检测患者中的PD-L1的表达可用于肿瘤的诊断，或者为肿瘤的抗-PD1或抗PD-L1免疫治疗提供临床诊断依据。然而，先前报道的针对PD-L1的抗体迄今并没有成功地用来有效地早期检测和/或诊断癌症。
发射型计算机断层成像术(Emission Computed Tomography，ECT)已用于肿瘤的诊断。ECT包括单光子发射计算机断层成像术(Single-Photon Emission Computed Tomography，SPECT)和正电子发射断层成像术(Positron Emission Tomography，PET)，其提供了高分辨率的肿瘤成像并且可通过图像进行定量分析。
本领域需要能够用于检测表达PD-L1的肿瘤细胞的诊断剂，特别是适 合于ECT检测的基于PD-L1抗体的诊断剂。
发明概述
本发明将传统PET技术与抗体和类似分子结合起来，使用抗体来识别目标细胞，从而提供全身所有肿瘤的快照。本发明涉及使用PD-L1重链单域抗体(VHH)或衍生分子作为成像剂的工具，来识别感兴趣的细胞。
在一方面，本发明提供一种程序性死亡配体1(PD-L1)结合多肽，其特征在于能够特异性结合PD-L1且包含至少一个免疫球蛋白单一可变结构域，其中所述至少一个免疫球蛋白单一可变结构域包含：
CDR1，其包含SEQ ID NO:5所示氨基酸序列或相对于SEQ ID NO:5具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列，
CDR2，其包含SEQ ID NO:6所示氨基酸序列或相对于SEQ ID NO:6具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列，例如包含SEQ ID NO:13所示氨基酸序列，和
CDR3，其包含SEQ ID NO:7所示氨基酸序列或相对于SEQ ID NO:7具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列。
在一些具体实施方案中，所述至少一个免疫球蛋白单一可变结构域包含：
CDR1，其包含SEQ ID NO:5所示氨基酸序列，
CDR2，其包含SEQ ID NO:13所示氨基酸序列，和
CDR3，其包含SEQ ID NO:7所示氨基酸序列。
在一些实施方案中，其中所述免疫球蛋白单一可变结构域包含与SEQ ID NO：1的氨基酸序列具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％序列相同性的氨基酸序列。
在一些实施方案中，所述免疫球蛋白单一可变结构域只包含一个赖氨酸残基。
在一些实施方案中，所述免疫球蛋白单一可变结构域包含选自SEQ ID NO：1-2、8-12和18-22的氨基酸序列。
在一些实施方案中，所述免疫球蛋白单一可变结构域是VHH。
在一些实施方案中，所述PD-L1结合多肽不阻断PD-1与PD-L1的结合。
在另一方面，本发明提供一种核酸分子，其编码本发明的PD-L1结合多肽。
在另一方面，本发明提供一种表达载体，其包含与表达调控元件可操作地连接的本发明的核酸分子。
在另一方面，本发明提供一种宿主细胞，其包含本发明的核酸分子或以本发明的表达载体转化，并能够表达所述PD-L1结合多肽。
在另一方面，本发明提供一种产生本发明的PD-L1结合多肽的方法，包括：
a)在允许所述PD-L1结合多肽表达的条件下培养本发明的宿主细胞；
b)从得自步骤a)的培养物回收由所述宿主细胞表达的PD-L1结合多肽；及
c)任选进一步纯化和/或修饰得自步骤b)的PD-L1结合多肽。
在另一方面，本发明提供一种缀合分子，其包含本发明的PD-L1结合多肽，以及与所述PD-L1结合多肽缀合的至少一种可检测标记。
在一些实施方案中，其中所述可检测标记选自放射性核素、荧光剂、化学发光剂、生物发光剂、顺磁离子和酶。
在一些实施方案中，其中所述可检测标记选自 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 64Cu、 67Cu、 67Ga、 68Ga、 68Ge、 86Y、 90Y、 89Zr、 94mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 55Co、 72As、 75Br、 76Br、 82mRb、 83Sr或其它γ-、β-、或正电子发射体，例如，所述可检测标记为 68Ga或 125I。
在一些实施方案中，其中所述PD-L1结合多肽通过螯合剂与所述可检测标记缀合。
在一些实施方案中，其中所述螯合剂选自NOTA、DOTA、TETA或者NETA。
在一些实施方案中，其中所述可检测标记是 68Ga且所述螯合剂为NOTA。
在一些实施方案中，其中所述PD-L1结合多肽包含SEQ ID NO:12或22所示氨基酸序列。
在另一方面，本发明提供一种检测生物学样品中PD-L1的存在和/或PD-L1的表达水平的方法，包括：
a)在本发明的PD-L1结合多肽和/或本发明的缀合分子与PD-L1之间能够形成复合物的条件下，使所述生物学样品和对照样品接触本发明的PD-L1结合多肽和/或本发明的缀合分子；
b)检测复合物的形成，
其中所述生物学样品与对照样品之间复合物形成的差异指示样品中PD-L1的存在和/或PD-L1的表达水平。
在另一方面，本发明提供一种用于检测和/或诊断PD-L1相关疾病如癌症的诊断剂，其包含本发明的PD-L1结合多肽和/或本发明的缀合分子，以及任选的生理学上可接受的载体。在一些实施方案中，所述诊断剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
在另一方面，本发明提供了本发明的PD-L1结合多肽和/或本发明的缀合分子在制备用于检测和/或诊断PD-L1相关疾病如癌症的诊断剂中的用途。在一些实施方案中，所述诊断剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
在另一方面，本发明提供一种在对象中检测和/或诊断PD-L1相关疾病如癌症的方法，包括给所述对象施用本发明的PD-L1结合多肽和/或本发明的缀合分子和/或本发明的诊断剂。
在一些实施方案中，所述方法还包括对所述对象进行成像例如ECT成像的步骤。在一些实施方案中，所述ECT成像是SPECT成像。在一些实施方案中，所述ECT成像是PET成像。
在本发明各个方面的一些实施方案中，其中所述癌症高表达PD-L1，例如所述癌症选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体癌、骨肉瘤。
在另一方面，本发明提供一种试剂盒，其包含本发明的PD-L1结合多肽和/或本发明的缀合分子和/或本发明的诊断剂。
附图说明
图1示出重链单域抗体109的生物化学分析，其中图1A与图1B分别为109-chis和109的SEC-HPLC分析，图1C为109-chis和109的纯化蛋白SDS-Page分析。
图2示出109-chis与PD-L1的结合曲线图谱。
图3示出PD-L1的重链单域抗体的体外活性，其中图3A为109-chis的竞争ELISA曲线图谱，图3B为流式细胞仪检测109-chis对细胞表面PD-L1结合效果，图3C为109-chis动物肿瘤切片的免疫组化结果
图4示出用正电子核素标记单域抗体，其中图4A为用螯合剂和放射性核素 68Ga缀合单域抗体的流程图，图4B为蛋白109和109-NOTA的质谱分析，图4C为 67Ga-NOTA-109的质谱分析，图4D为 67Ga-NOTA-109-chis对PD-L1的结合曲线及EC50值。
图5示出对突变体109-K64Q-RDNSE-cHis的鉴定与分析，其中图5A为突变体109-R73N&K75E-cHis和109-K86R&P87A-cHis ELISA活性分析，图5B为突变抗体109-RDNSE-cHis和109-K64Q-cHis ELISA活性分析，图5C为突变体的NOTA缀合物的质谱分析。
图6示出正电子核素标记单域抗体 68Ga-NOTA-109的体外分析，其中图6A为 68Ga-NOTA-109-chis的ITLC分析，图6B为 68Ga-NOTA-109-chis的SEC-HPLC及体外稳定性，C为 68Ga-NOTA-109-chis的细胞内吞实验结果。
图7示出 68Ga-NOTA-109的体外分布，其中图7A为 68Ga-NOTA-109-chis的体内各组织分布，图7B为 68Ga-NOTA-109-chis的体内分布靶本比，图7C为 68Ga-NOTA-109-chis的体内PET成像，图7D为 68Ga-NOTA-109-chis的体内生物分布曲线。
图8示出 125I标记单域抗体109的SPECT结果。
图9示出 68Ga-NOTA-109分别在PBS和血清(FBS)中的体外稳定性。
图10示出 68Ga-NOTA-109在PBS中的体外温度稳定性。
图11示出 68Ga-NOTA-109的体内稳定性。
图12示出 68Ga-NOTA-109的免疫活性检测。
图13示出 68Ga-NOTA-109在同一小鼠体内不同移植瘤的PET成像。A：注射后1小时， 68Ga-NOTA-109的静态图像；B：根据PET图像的ROI定量分析， 68Ga-NOTA-109在肿瘤中的生物分布。
图14示出同一小鼠体内不同移植瘤用 68Ga-NOTA-109进行PET成像后的放射自显影及免疫组化分析。A：肿瘤的PD-L1免疫组织化学染色。B：A375(i)、A375/A375-HPD-L1(ii)、A375-HPD-L1(iii)肿瘤中68GA-NOTA-109 的放射自显影分析(上)及放射自显影定量分析(下)。
发明详述
定义
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook等人,“Molecular Cloning:A Laboratory Manual”(第2版),第1-3卷,Cold Spring Harbor Laboratory Press(1989)；Lewin,“Genes IV”，Oxford University Press,New York,(1990)；及Roitt等人,“Immunology”(第2版),Gower Medical Publishing,London,New York(1989)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
除非另有说明，否则可互换使用的术语“抗体”或“免疫球蛋白”在本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用，术语(多肽或蛋白的)“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个的功能性质，且在许多情况下可添加、移除或转移至其他蛋白而不损失蛋白的其余部分和/或结构域的功能。
如本文所用的术语“免疫球蛋白结构域”是指抗体链(例如常规4链抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征，其由排列在两个β折叠中任选由保守二硫键稳定的约7个反平行β折叠股的2层夹层组成。
如本文所用的术语“免疫球蛋白可变结构域”是指基本上由本领域及 下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
如本文所用的术语“免疫球蛋白单一可变结构域”是指能够在不与其他免疫球蛋白可变结构域配对的情况下特异性结合抗原表位的免疫球蛋白可变结构域。本发明含义中的免疫球蛋白单一可变结构域的一个实例为“结构域抗体”，例如免疫球蛋白单一可变结构域VH及VL(VH结构域及VL结构域)。免疫球蛋白单一可变结构域的另一实例为如下文定义的骆驼科的“VHH结构域”(或简称为“VHH”)。“VHH结构域”，亦称为重链单域抗体、VHH、V HH结构域、VHH抗体片段和VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.:“Naturally occurring antibodies devoid of light chains”；Nature 363,446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规4链抗体中的重链可变结构域(其在本文中称为“VH结构域”)以及存在于常规4链抗体中的轻链可变结构域(其在本文中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
在本发明的上下文中，术语“重链单域抗体”、“VHH结构域”、“VHH”、“V HH结构域”、“VHH抗体片段”、“VHH抗体”以及
Figure PCTCN2019093225-appb-000001
及“
Figure PCTCN2019093225-appb-000002
结构域”(“Nanobody”为Ablynx N.V.公司，Ghent，Belgium的商标)可互换使用。
例如Riechmann及Muyldermans,J.Immunol.Methods 231,25-38(1999)的图2中所示，对于骆驼科的VHH结构域所应用的氨基酸残基，根据Kabat等人给出的VH结构域的一般编号法来编号(“Sequence of proteins of  immunological interest”,US Public Health Services,NIH Bethesda,MD,公开案第91号)。根据该编号法，FR1包含在位置1-30处的氨基酸残基，CDR1包含在位置31-35处的氨基酸残基，FR2包含在位置36-49处的氨基酸，CDR2包含在位置50-65处的氨基酸残基，FR3包含在位置66-94处的氨基酸残基，CDR3包含在位置95-102处的氨基酸残基，且FR4包含在位置103-113处的氨基酸残基。
然而应注意，如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。
本领域中已知对VH结构域的氨基酸残基进行编号的替代方法，所述替代方法还可以类似地应用于VHH结构域。然而，除非另有说明，否则在本说明书、权利要求书及附图中，将遵循如上所述的根据Kabat且适于VHH结构域的编号。VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本文所述的目的。
获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden et al.,Journal of Immunological Methods,240(2000)185–195；Li et al.,J Biol Chem.,287(2012)13713–13721；Deffar et al.,African Journal of Biotechnology Vol.8(12),pp.2645-2652,17June,2009和WO94/04678。
源自骆驼科的VHH结构域可通过以人常规4链抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而经“人源化”(本文中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其他修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列。
此外，本领域技术人员还将了解，有可能将一个或多个上述CDR“移植”于其他“支架”(包括但不限于人支架或非免疫球蛋白支架)上。适于所 述CDR移植的支架及技术在本领域中是已知的。
如本文所用，术语“表位”或可互换使用的术语“抗原决定簇”指抗体的互补位所结合的抗原上的任何抗原决定簇。抗原决定簇通常包含分子的化学活性表面基团，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性”表位或“构象”表位。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
可使用本领域中熟知的许多表位定位技术鉴别给定抗原的表位。参见例如Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。举例而言，线性表位可通过例如以下方法来确定：在固体支持物上同时合成大量肽，其中这些肽对应于蛋白质分子的各部分，且使这些肽在仍然与支持物连接的情况下与抗体反应。这些技术在本领域中为已知的且描述于例如美国专利第4,708,871号；Geysen等人(1984)Proc.Natl.Acad.Sci.USA 81:3998-4002；Geysen等人(1986)Molec.Immunol.23:709-715中。类似地，构象表位可通过诸如通过例如x射线结晶学及2维核磁共振确定氨基酸的空间构形加以鉴别。参见例如Epitope Mapping Protocols(同上)。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利申请WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本发明的抗体分子竞争结合PD-L1上的相同表位的抗体及其抗原结合片段。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本发明的免疫球蛋白单一可变结构域)分子可结合的不同类型抗原或表位的数目。可基于抗原结合分子的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合分 子之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合分子(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合分子上的抗原结合位点之间的亲和力，以及存在于抗原结合分子上的相关结合位点的数目。
如本文所用，术语“PD-L1结合多肽”意指任何能够特异性结合PD-L1的多肽，例如本发明的特异性结合PD-L1的单域抗体。
“PD-L1结合多肽”或者可以指结合PD-L1的单价多肽(即与PD-L1的一个表位结合的多肽)，以及二价或多价结合多肽(即结合一个以上表位的结合多肽)。本发明的“PD-L1结合多肽”可以包含至少一个结合PD-L1的免疫球蛋白单一可变结构域如VHH。
通常，本发明的PD-L1结合多肽将以如于Biacore或KinExA测定中测量的优选10 -7至10 -11摩尔/升(M)、更优选10 -8至10 -11摩尔/升、甚至更优选10 -9至10 -11M、甚至更优选10 -10至10 -11M或更低的解离常数(KD)，和/或以至少10 7M -1、优选至少10 8M -1、更优选至少10 9M -1，更优选至少10 10M -1、例如至少10 11M -1的缔合常数(KA)结合所要结合的抗原(即PD-L1)。任何大于10 -4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如表面等离子体共振术(SPR)测定、Scatchard测定和/或本文所述的竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定。
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。在比较两个氨基酸序列时，术语“氨基酸差异”是指与另一序列相比，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在取代的情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电 残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
两个多肽序列之间的“序列相同性”指示序列之间相同氨基酸的百分比。“序列相似性”指示相同或代表保守氨基酸取代的氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987和Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991。
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“基本上分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“基本上分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“基本上分离的”多肽或核酸分子优选基本上为均质的。
如本文所用的术语“对象”意指哺乳动物，尤其灵长类动物，尤其是人。
本发明的PD-L1结合多肽
在一方面，本发明提供一种程序性死亡配体1(PD-L1)结合多肽，其特征在于能够特异性结合PD-L1且包含至少一个免疫球蛋白单一可变结构域，其中所述至少一个免疫球蛋白单一可变结构域包含：
CDR1，其包含SEQ ID NO:5所示氨基酸序列或相对于SEQ ID NO:5具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列，
CDR2，其包含SEQ ID NO:6所示氨基酸序列或相对于SEQ ID NO:6具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列，例如包含SEQ ID NO:13所示氨基酸序列，和
CDR3，其包含SEQ ID NO:7所示氨基酸序列或相对于SEQ ID NO:7具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列。
在一些具体实施方案中，所述至少一个免疫球蛋白单一可变结构域包含：
CDR1，其包含SEQ ID NO:5所示氨基酸序列，
CDR2，其包含SEQ ID NO:13所示氨基酸序列，和
CDR3，其包含SEQ ID NO:7所示氨基酸序列。
在一些实施方案中，其中所述免疫球蛋白单一可变结构域包含与SEQ ID NO：1的氨基酸序列具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％序列相同性的氨基酸序列。
在一些实施方案中，所述免疫球蛋白单一可变结构域衍生自SEQ ID NO:1。SEQ ID NO:1的多肽一共含有3个带有游离氨基的赖氨酸基团(分别位于SEQ ID NO：1的第64位、第75位和第86位)，这些基团可用于与螯合剂例如NOTA的缀合。为了优化结合多肽与螯合剂例如NOTA的缀合物的纯度，保证每mol多肽能且只能缀合1mol螯合物，可以对这些赖氨酸基团中的进行取代。例如，在一些优选实施方案中，所述免疫球蛋白单一可变结构域只包含一个赖氨酸残基。
此外，在对赖氨酸残基进行取代的同时，为了保留合适的结合能力，可以对赖氨酸残基的邻近残基进行突变。例如，在一些实施方案中，所述免疫球蛋白单一可变结构域相对于SEQ ID NO:1包含K64Q取代。在一些实施方案中，所述免疫球蛋白单一可变结构域相对于SEQ ID NO:1 包含K86R和P87A取代。在一些实施方案中，所述免疫球蛋白单一可变结构域相对于SEQ ID NO:1包含H71R、R73N、A74S和K75E取代。在一些实施方案中，所述免疫球蛋白单一可变结构域相对于SEQ ID NO:1包含R73N和K75E取代。在一些实施方案中，所述免疫球蛋白单一可变结构域相对于SEQ ID NO:1包含K64Q、H71R、R73N、A74S和K75E取代。本申请实验表明，此类突变并不破坏对PD-L1的结合亲和力，即使其可能位于CDR内(例如K64Q)。本文中的氨基酸位置编号均参考SEQ ID NO:1。
“包含”一词在本文中用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有本发明所述的活性。例如，本发明的PD-L1结合多肽还可以包含适合于表达和/或纯化的标签，包括但不限于His标签等。
在一些具体实施方案中，所述免疫球蛋白单一可变结构域包含选自SEQ ID NO：1-2、8-12和18-22的氨基酸序列。
在一些实施方案中，所述免疫球蛋白单一可变结构域是VHH。
在一些实施方案中，所述PD-L1结合多肽不阻断PD-1与PD-L1的结合。不阻断PD-1与PD-L1的结合也可以通过避免影响PD-L1正常功能而减少潜在的副作用。此外，本发明的PD-L1结合多肽与PD-L1结合的表位与现有技术中PD-1与PD-L1结合的位点不同，从而本发明的PD-L1结合多肽可不影响其他针对阻断PD-1与PD-L1的相互作用而设计的抗体例如治疗性抗体的效力。因此，在一些实施方案中，本发明的PD-L1结合多肽不阻断针对PD-L1的治疗性抗体与PD-L1的相互作用。这使得可以在施用针对PD-L1的治疗性抗体进行癌症治疗的同时，使用本发明的PD-L1结合多肽或其衍生的缀合分子对癌症进行监测。
核酸、载体、宿主细胞
在另一方面，本发明涉及编码本发明的PD-L1结合多肽的核酸分子。本发明的核酸可为RNA、DNA或cDNA。本领域技术人员可根据需要或常规手段选择编码本发明的PD-L1结合多肽的核酸分子。
本发明的核酸也可呈载体形式，可存在于载体中和/或可为载体的一 部分，该载体例如质粒、粘端质粒或YAC。载体可尤其为表达载体，即可提供PD-L1结合多肽体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本发明的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本发明的PD-L1结合多肽的表达有用或必需的调控元件及其他元件的具体实例，例如启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本发明的核酸可基于关于本文给出的本发明的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
在另一方面中，本发明涉及表达或能够表达一种或多种本发明的PD-L1结合多肽和/或含有本发明的核酸或载体的宿主细胞。本发明的优选宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
适合的细菌细胞包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
适合的真菌细胞包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
适合的哺乳动物细胞包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本发明也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本发明还提供产生本发明的PD-L1结合多肽的方法，所述方法通常包含以下步骤：
-在允许表达本发明的PD-L1结合多肽的条件下培养本发明的宿主细胞；及
-从培养物回收由所述宿主细胞表达的PD-L1结合多肽；及
-任选进一步纯化和/或修饰本发明的PD-L1结合多肽。
本发明的PD-L1结合多肽可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本发明的PD-L1结合多肽的方法中的蛋白分离及纯化技术为本领域技术人员所公知。
然而，本发明的PD-L1结合多肽也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
缀合分子
在另一方面，本发明提供一种缀合分子，其包含本发明的PD-L1结合多肽，以及与所述PD-L1结合多肽缀合的至少一种可检测标记。
所述可检测标记包括但不限于放射性核素、荧光剂、化学发光剂、生物发光剂、顺磁离子和酶。
可用于缀合的荧光剂包括但不限于异硫氨酸荧光素、罗丹明、藻红蛋白、藻蓝蛋白、别藻蓝蛋白、邻苯二醛和荧光胺；可用于缀合的化学发光剂包括但不限于鲁米诺、异鲁米诺、芳族吖啶酯、咪唑、吖啶盐和草酸酯；可用于缀合的生物发光剂包括但不限于荧光素、荧光素酶和水母发光蛋白。可用于缀合的顺磁离子包括但不限于铬(III)、锰(II)、铁(III)、铁(II)、钻(II)、镍(II)、铜(II)、钕(III)、钐(III)、镱(III)、钆(III)、钒(II)、铽(III)、镝(III)、钬(III)和铒(III)，或者是不透辐射的材料，如坝、泛影酸盐、乙碘油、柠檬酸镓、碘卡酸、碘因他酸、碘达胺、胆影酸、碘沙酸、碘古酰胺、碘海醇、碘帕醇、碘番酸、碘普西酸、碘西法酸、碘丝酸、碘砜葡胺、碘酞硫、碘替酸、碘他拉酸、碘曲西酸、碘克沙酸、羟泛影酸、碘泊酸盐、葡甲胺、甲泛葡胶、甲泛影盐、丙碘酣和氧化亚铊。 可用于缀合的酶包括但不限于辣根过氧化物酶等。
优选地，所述可检测标记是放射性核素。可用于缀合的放射性核素例如是能量在20-4000KeV之间的放射性核素，包括但不限于 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 64Cu、 67Cu、 67Ga、 68Ga、 68Ge、 86Y、 90Y、 89Zr、 94mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 55Co、 72As、 75Br、 76Br、 82mRb、 83Sr或其它γ-、β-、或正电子发射体。在一些实施方案中，所述可检测标记为 68Ga或 125I。
将可检测标记与多肽缀合的方法是本领域技术人员熟知的。例如，在一些实施方案中，所述PD-L1结合多肽可以通过螯合剂与所述可检测标记缀合。
为了用放射性核素如 68Ga标记本发明的PD-L1结合多肽，可以使本发明的PD-L1结合多肽和具有长尾的试剂反应，该长尾附着有多个用于结合离子的整合基团。这样的尾可以是例如聚赖氨酸、多聚糖或其它具有侧基的衍生的或可衍生的链的聚合物，该侧基可结合螯合基团，例如乙二胺四乙酸(EDTA)、二乙烯三胺五乙酸(DTPA)、DOTA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四羧酸)、NOTA、TETA、NETA、卟啉、聚胺、冠状醚、双缩氨硫脲、聚肟以及己知可用于此目的类似基团。可以使用标准的化学方法将螯合剂连接到抗体上。在一些实施方案中，可检测标记通过螯合剂与本发明的PD-L1结合多肽缀合。所用的螯合剂包括但不限于NOTA、DOTA、TETA或者NETA等。
在一些具体实施方案中，其中所述可检测标记是 68Ga且所述螯合剂为NOTA。在一些实施方案中，其中所述PD-L1结合多肽包含SEQ ID NO:12或22所示氨基酸序列。
在另一方面，本发明提供一种制备本发明的放射性核素如 68Ga标记的缀合分子的方法，其包含1)使本发明的PD-L1结合多肽与螯合剂缀合以生成所述PD-L1结合多肽与螯合剂的缀合物；和2)使步骤1)的产物与放射性核素如 68Ga接触，由此放射性核素如 68Ga通过螯合剂的螯合作用标记本发明的PD-L1结合多肽。在一些实施方案中，所述螯合剂是NOTA，且步骤1)中通过使所述PD-L1结合多肽与p-SCN-Bn-NOTA或p-NH2-Bn-NOTA反应而生成所述PD-L1结合多肽与NOTA的缀合物。
在另一方面，本发明提供一种制备本发明的 125I标记的缀合分子的方法，其包含1)使本发明的PD-L1结合多肽在氯胺T存在下与 125I反应；和2)任选地，用偏重亚硫酸钠终止反应。
检测/诊断用途
在另一方面，本发明提供一种检测生物学样品中PD-L1的存在和/或PD-L1的表达水平的方法，包括：
a)在本发明的PD-L1结合多肽和/或本发明的缀合分子与PD-L1之间能够形成复合物的条件下，使所述生物学样品和对照样品接触本发明的PD-L1结合多肽和/或本发明的缀合分子；
b)检测复合物的形成，
其中所述生物学样品与对照样品之间复合物形成的差异指示样品中PD-L1的存在和/或PD-L1的表达水平。在一些实施方案中，所述生物学样品是离体样品。
在另一方面，本发明提供一种组合物，其包含本发明的PD-L1结合多肽和/或本发明的缀合分子，以及任选的生理学上可接受的载体。所述组合物可以作为诊断剂，例如用于检测和/或诊断PD-L1相关疾病的诊断剂。
在另一方面，本发明提供一种用于检测和/或诊断PD-L1相关疾病例如癌症的诊断剂，其包含本发明的PD-L1结合多肽和/或本发明的缀合分子，以及任选的生理学上可接受的载体。在一些实施方案中，所述诊断剂是造影剂。
本发明的PD-L1结合多肽和/或本发明的缀合分子特别适合于体内成像，例如适用于发射型计算机断层成像术(Emission Computed Tomography，ECT)。例如，本发明的PD-L1结合多肽和/或本发明的缀合分子根据标记的不同可应用于单光子发射计算机断层成像术(Single-Photon Emission Computed Tomography，SPECT)和正电子发射断层成像术(Positron Emission Tomography，PET)。在肿瘤诊断中可提供高分辨率的肿瘤成像并且可通过图像进行定量分析。所述SPECT成像还可以包括SPECT/CT成像，且所述PET成像还可以包括PET/CT成像，其可以提供更优的成像效果。
因此，在一些实施方案中，所述造影剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
在另一方面，本发明提供了本发明的PD-L1结合多肽和/或本发明的缀合分子在制备用于检测和/或诊断PD-L1相关疾病例如癌症的诊断剂中的用途。在一些实施方案中，所述诊断剂是造影剂。在一些实施方案中，所述造影剂是ECT造影剂，例如SPECT造影剂或PET造影剂
在另一方面，本发明提供一种在对象中检测和/或诊断PD-L1相关疾病例如癌症的方法，包括给所述对象施用本发明的PD-L1结合多肽和/或本发明的缀合分子和/或本发明的诊断剂。
在一些实施方案中，所述方法还包括对所述对象进行成像例如ECT成像的步骤。在一些实施方案中，所述ECT成像是SPECT成像。在一些实施方案中，所述ECT成像是PET成像。用于通过SPECT或PET扫描的成像技术和装置是本领域公知的且可使用任何此类己知的ECT成像技术和装置。
可通过本发明的PD-L1结合多肽和/或本发明的缀合分子和/或本发明的诊断剂检测和/或诊断的疾病包括使细胞、组织或器官中PD-L1表达异常升高的疾病，例如感染性疾病、癌症等。
使用本发明的PD-L1结合多肽可以检测和/或诊断的优选的癌症高表达PD-L1，非限制性的例子包括肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤(例如转移的恶性黑色素瘤)、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤。可以用本发明的方法检测和/或诊断的其他癌症的例子包括：骨癌、胰腺癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病，包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、输尿管癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。本发明也可用于检测和/或诊断转移性癌，特别是表达PD-L1的转移性癌(Iwai等(2005)Int Immunol 17：133-144)。
在另一方面，本发明提供一种试剂盒，其包含本发明的PD-L1结合多肽和/或本发明的缀合分子和/或本发明的诊断剂。所述试剂盒用于实施本发明的方法。试剂盒一般包括表明试剂盒内容物的预期用途的标签。术语标签包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
实施例
下面将通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所描述的实施例范围中。
实施例1：针对PD-L1的重链单域抗体的筛选
1.1文库的构建
免疫用的PDL1-Fc融合蛋白由CHO细胞表达，经Protein A亲和层析纯化得到。选取一只新疆双峰驼进行免疫。4次免疫结束后，提取骆驼100ml外周血的淋巴细胞并使用QIAGEN公司提供的RNA提取试剂盒提取总RNA，使用Super-Script III FIRST STRANDSUPERMIX试剂盒(Thermo Fisher Scientific)按照说明书将提取的RNA反转录成cDNA。用巢式PCR扩增编码重链抗体的可变区的核酸片段：
第一轮PCR：
上游引物：GTCCTGGCTGCTCTTCTACAAGGC(SEQ ID NO:14)；
下游引物：GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO:15)。
第二轮PCR：
以第一轮PCR产物作模板，
上游引物：GATGTGCAGCTGCAGGAGTCTGGRGGAGG(SEQ ID NO:16)；
下游引物：GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO:17)。
回收目标重链单域抗体核酸片段，并使用限制性内切酶PstI及NotI将其克隆进入噬菌体展示用载体pCDisplay-3(Creative Biolabs，Cat：VPT4023)中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建针对PD-L1的重链单域抗体噬菌体展示文库并对文库进行检定。通过梯度稀释铺板， 计算库容的大小为1.33×10 8。为检测文库的插入率，随机选取24个克隆做菌落PCR。结果显示插入率已达到100％。
1.2针对PD-L1的重链单域抗体淘选
用PDL1-Fc融合蛋白10μg/孔包被平板，4℃放置过夜。第二天用1％脱脂奶室温封闭2小时后，加入100μl噬菌体(8×10 11tfu，来自1.1所构建的骆驼重链单域抗体噬菌展示文库)，在室温下作用1小时。之后用PBST(PBS中含有0.05％吐温20)洗5遍，以洗掉不结合的噬菌体。最后用三乙基胺(100mM)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，产生并纯化噬菌体用于下一轮的筛选。相同筛选过程重复3-4轮。由此，阳性的克隆被富集，达到了利用噬菌体展示技术筛取抗体库中PD-L1特异抗体的目的。
1.3用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆
3-4轮淘选后，获得的PD-L1结合阳性的噬菌体感染空白大肠杆菌并铺板。随后挑选96个单菌落分别培养，生产并纯化噬菌体。用PDL1-Fc融合蛋白包被平板4℃过夜，将获得的样品噬菌体(对照组为空白噬菌体)加入，室温下反应1小时。洗涤之后加入一抗小鼠抗-HA标签抗体(购自北京康为世纪生物科技有限公司)，室温反应1小时。洗涤之后加入二抗山羊抗-小鼠碱性磷酸酶标记抗体(购自艾美捷科技有限公司)，室温反应1小时。洗涤之后加入碱性磷酸酶显色液，405nm波长读取吸收值。当样品孔OD值大于对照孔OD值3倍以上时，判为阳性克隆孔。将阳性克隆孔的菌转移至含有100μg/mL氨苄霉素的LB液体中培养以便提取质粒并进行测序。
根据序列比对软件Vector NTI分析各个克隆的蛋白序列。把CDR1、CDR2、CDR3序列均相同的克隆视为同一抗体株，而CDR序列不同的克隆视为不同抗体株。最终获得命名为109的PD-L1重链单域抗体
实施例2：针对PD-L1的重链单域抗体的初步评价鉴定
2.1构建PD-L1单域抗体的基因克隆
单域抗体的氨基酸序列如表1所示，其中109-chis为c末端带His标签的重组蛋白。CDR序列用方框示出。
表1
Figure PCTCN2019093225-appb-000003
编码表1的单域抗体的核苷酸序列如SEQ ID NO:3和SEQ ID NO:4所示。
109单域抗体的核苷酸序列：
Figure PCTCN2019093225-appb-000004
109-chis的核苷酸序列：
Figure PCTCN2019093225-appb-000005
109抗体的CDR序列分别为CDR1：GFSLDDSDMG(SEQ ID NO:5)；CDR2：IASDRSTYYTPSVKG(SEQ ID NO:6)；和CDR3：APRLAYTTAMTCEGDFAY(SEQ ID NO:7)。
依据单域抗体的核苷酸序列设计引物，以全基因合成的DNA为模板进行PCR扩增，然后克隆至pCDNA4(Invitrogen，Cat V86220)载体中，经基因序列测定，以确定所获得目的克隆基因序列的正确性。
2.2用哺乳动物细胞制备PD-L1抗体蛋白
将上述重组构建的单域抗体融合蛋白质粒转染HEK293细胞进行抗体表达。将重组表达质粒用Freestyle293培养基(Thermo Fisher Scientific，货号12338018)稀释并加入转化所需PEI(聚乙烯亚胺)溶液，将质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，10％CO 2，90rpm中培养。四小时后再补加EX293培养基(Sigma，批号14571C)，2mM谷氨酰胺，135rpm培养。24小时后加3.8mM丙戊酸VPA(Sigma，批号P4543)。培养5～6天后，收集瞬时表达培养上清液，109-chis单域抗体通过Ni+树脂凝胶亲和层析法，纯化得到SDS-PAGE电泳纯度达95％以上的纯化抗体；109单域抗体首先通过亲和层析法进行一步富集，然后利用离子层析柱纯化得到SDS-PAGE电泳纯度达95％以上的109纯化抗体(如图1A-C所示)。
2.3检测PD-L1重链单域抗体对人PD-L1蛋白的特异性结合
PDL1-Fc融合蛋白由HEK293瞬时表达及镍柱亲和层析纯化获得。得到的PDL1-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后加入获得的PD-L1单域抗体蛋白的梯度稀释系列，室温下反应1小时。洗涤之后加入抗-his-辣根过氧化物酶(HRP)标记抗体(购自Abcam，货号ab1187)，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。应用软件SotfMaxPro v5.4进行数据处理和作图分析，通过四参数拟合，得到如图2所示的抗体对PD-L1结合曲线及EC50值(2.891ng/mL)，其反映出109抗体与PD-L1的亲和能力。
实施例3：PD-L1抗体蛋白体外活性分析
3.1竞争ELISA考察PD-L1重链单域抗体对PD-1与PD-L1相互作用的阻断效果
PDL1-muFc融合蛋白(使用鼠Fc)与PD1-Fc融合蛋白克隆至pCDNA4载体(Invitrogen，Cat V86220)由HEK293细胞表达获得。PDL1-muFc融合蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入10μg PD1-Fc(空白组不加入任何抗体或蛋白，只加入等体积缓冲液)以及起始稀释浓度为3μg/L的重链单域抗体109-chis(对照组为缓冲液)，2倍稀释，12个梯度，室温下反 应1小时。之后加入抗-his-HRP(购自Abcam公司)，室温反应1小时。之后加入显色液，405nm波长读取吸收值。在过量的PD-1存在条件下，随着重链单域抗体109-chis浓度的升高，结合呈现S曲线结合趋势。则认为抗体无阻断效果。
PDL1-muFc融合蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入10μg PD1-Fc(空白组不加入任何抗体或蛋白，只加入等体积缓冲液)以及起始稀释浓度为3μg/L的重链单域抗体109-chis(对照组为缓冲液)，2倍稀释，12个梯度，室温下反应1小时。之后加入抗-Fc-HRP(购自Abcam公司)，室温反应1小时。之后加入显色液，405nm波长读取吸收值。随着重链单域抗体109-chis浓度的升高，PD-1的结合呈现平稳结合趋势。则认为PD-1与PDL1结合不受抗体存在的影响。结果如图3A所示，109与PDL1结合的表位与PD-1的表位并不相同，从而PD-1蛋白的存在不会影响109蛋白与PD-L1蛋白的结合。
3.2通过流式细胞仪检测PD-L1重链单域抗体对细胞表面PD-L1结合效果
通过构建人PD-L1全长蛋白基因的人黑色素瘤A375细胞稳定转染细胞株，获得在膜上稳定表达人PD-L1蛋白的A375细胞(A375-PDL1细胞)。以不表达PD-L1的肿瘤发生细胞系MCF-7为阴性对照。MCF-7细胞系得自美国典型培养物保藏中心(ATCC)，保藏号为ATCC HTB-22，并且按照推荐培养。在使用前将细胞培养至>90％汇合。使用抗PD-L1单域抗体109对这几种细胞系进行流式细胞分析，用于定量分析间接免疫荧光染色。以确定对于每个细胞表面受体的数量。
使用细胞解离缓冲液(PBS+10mM EDTA)而不是胰蛋白酶，将粘着细胞从细胞培养皿上洗脱，以避免细胞表面受体的蛋白水解。将细胞在PBS中洗涤两次，并且在冰冷的缓冲液(PBS+0.5％BSA w/v)中再悬浮至5～10×10 6个细胞/ml的浓度。将100μL细胞的等分试样与5μg一抗混合，并且在冰上孵育45分钟。细胞随后用1ml冰冷的流式细胞缓冲液(PBS，含有2％牛血清白蛋白)洗涤，在300×g下离心5分钟，并且在0.5μL缓冲液再悬浮。加入100μL二抗-PE缀合物与PBS的1:50稀释液，并且在冰上避光孵育45分钟。细胞随后用1mL冰冷的缓冲液洗涤两次，在300×g下离心5分钟，并且在500μL的缓冲液中再次悬浮。
在Beckman Coulter Cytomics FC500MPL上进行流式细胞分析。对于每个管，收集最少5×10 4个事件。所有分析为单一颜色，在FL1中检测PE。向前散射(FS)和侧散射(SS)数据证明所有细胞群体紧密成群。
流式细胞术用于评价细胞的体外PDL1表达(如图3B所示)，其中A375-PDL1细胞显示最高水平的PDL1表达。MCF-7细胞显示几乎无PDL1表达。
3.3鉴定PD-L1单域抗体蛋白对PD-L1结合能力(Biacore)
Biacore的实验温度为25℃，进样流速为50μl/min。分析物用缓冲液HBS-EP+缓冲液稀释至某一浓度，使其依次流过空白参比通道和活化通道，空白参比通道上产生的信号值反映了分析物在芯片上产生的非特异性吸附情况，活化通道上产生的信号值则反映了分析物与配体间的特异性结合。分析物进样300s，随后HBS-EP+缓冲液进样180s，使分析物从配体上进行解离。109抗体稀释至200ng/ml、100ng/ml、50ng/ml、25ng/ml、12.5ng/ml、6.25ng/ml、3.125ng/ml 7个梯度。不同浓度的分析物重复进样、解离。
Biacore T200记录的数据采用Biacore T200 Evaluation Software进行分析。
测量的抗PD-L1抗体的结合亲和力结果见表2。其KD值在1.7nM。结果表明109蛋白对PD-L1靶标蛋白的亲和力显著。
表2. 109抗体对PD-L1的结合亲和力
Ka 2.534 X 10 5M -1s -1
Kd 4.382 X 10 -4s -1
KD 1.729 X 10 -9M
3.4免疫组化
用年龄在6～8周范围的雌性裸小鼠进行体内研究。在无特定病原体(SPF)环境中饲养裸鼠，可随意获取食物和水，标准12小时白天-夜晚照明循环。对于异种移植，在小鼠右前腿皮下植入100μl的细胞(A375-PDL1或MCF-7)/PBS。细胞接种密度约为5～6×10 6个细胞/小鼠。在异氟烷麻醉下实施植入。在这些条件下，在多于80％的注射的动物中，在3～4周后得到可用的肿瘤(A375-PDL1或MCF-7)(100-300μg)。通过解剖从小鼠收集肿瘤， 并且整个肿瘤用福尔马林进行固定，4℃保存至免疫组化处理。按照本领域技术人员熟知的常规方法制备免疫组化切片并用抗-his-辣根过氧化物酶(HRP)标记抗体染色。
图3C免疫组化的图片结果可以看出，利用109-chis作为一抗进行免疫结合，A375-PDL1肿瘤PD-L1表达为阳性，PD-L1主要表达于细胞的表面。而MCF-7肿瘤，在细胞表面PD-L1为阴性，这一结果和流式分析相一致。
实施例4：单域抗体核素标记
按照图4A所示的流程图用螯合剂p-SCN-Bn-NOTA和放射性核素 68Ga标记本发明的单域抗体。
4.1单域抗体与p-SCN-Bn-NOTA反应
单域抗体109-chis(3mg)溶解于pH8.7的0.05M碳酸钠缓冲液中，添加过量10倍摩尔比的p-SCN-Bn-NOTA(Macrocyclics，货号B-605)。将混合物避光搅拌室温至少反应18小时，缀合产物使用PD-10柱(GE)纯化去除过量的p-SCN-Bn-NOTA，超滤管将单域抗体浓缩至～1mg/mL。以pH 7.0的0.1M磷酸盐缓冲液作为流动相进行SEC-HPL分析。
通过Thermo Scientific LTQ Orbitrap XL型液相色谱质谱联用仪质谱测定螯合NOTA数量。通过BCA蛋白浓度测定试剂盒测定单域抗体浓度。色谱条件为：仪器Waters ACQUITY UPLC，使用ACQUITY
Figure PCTCN2019093225-appb-000006
BEH C18 1.7μm(2.1×100mm)色谱柱，自动进样器温度设为4℃，以0.25mL/min的流速，50℃的柱温，进样4柱温进行梯度洗脱，流动相为0.1％甲酸水(A)-0.1％甲酸乙腈(B)。并使用Thermo Scientific Protein Deconvolution 4.0计算分子量特征。所得的单域抗体109-chis-NOTA质谱分析结果如图4B所示。
从质谱结果可以看出，图4B左图示出109-chis蛋白的分子量为13603Da，右图示出p-SCN-Bn-NOTA在经过NOTA缀合后，每缀合一分子的NOTA，相应分子量增加为450，即14053Da，缀合2分子的NOTA，相应分子量增加900Da，即14503Da。109-chis-NOTA的质谱结果显示，109-chis经过缀合反应后，产物分别为缀合1分子和2分子NOTA的混合物。
4.2合成 69、71Ga-NOTA-109-chis
GaCl 3溶解于0.05M的HCL中，添加1/2体积0.2M醋酸钠溶液，并 加入1/4体积含有109-chis-NOTA pH为5.3的0.1M醋酸钠缓冲液，反应体系的pH在4.5～4.7之间，室温反应10分钟，经PD-10柱去除未反应的离子镓。Thermo Scientific LTQ Orbitrap XL型液相色谱质谱联用仪质谱测定螯合 67Ga数量。通过BCA蛋白浓度测定试剂盒测定单域抗体浓度。色谱条件为：仪器Waters ACQUITY UPLC，使用ACQUITY
Figure PCTCN2019093225-appb-000007
BEH C18 1.7μm(2.1×100mm)色谱柱，自动进样器温度设为4℃，以0.25mL/min的流速，50℃的柱温，进样4μL进行梯度洗脱，流动相为0.1％甲酸水(A)-0.1％甲酸乙腈(B)。所得的 69、71Ga-NOTA-109-chis质谱分析结果如图4C所示。
69、71Ga-109-chis-NOTA的质谱结果显示，产物分别为螯合1分子和2分子 69、71Ga的混合物。分子量为14120为1个单域抗体标记了1个NOTA和1个 69、71Ga，分子量为14637为1个单域抗体标记了2个NOTA和2个 69、71Ga，结果说明，在此条件下，NOTA缀合反应及Ga冷标记成功获得相应产物。
4.3  69、71Ga-NOTA-109-chis对PD-L1结合能力鉴定
PDL1-Fc融合蛋白由HEK293瞬时表达及镍柱亲和层析纯化获得。得到的PDL1-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后加入109-chis PD-L1单域抗体蛋白或4.2获得的PD-L1单域抗体蛋白螯合产物的梯度稀释系列，室温下反应1小时。洗涤之后加入抗-his辣根过氧化物酶标记抗体，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。应用软件SotfMaxPro v5.4进行数据处理和作图分析，通过四参数拟合，得到如图4D所示抗体对PD-L1结合曲线及EC50值以反映抗体109-chis或其标记形式对PD-L1的亲和能力。
结果见图4D，其中纵坐标为OD405，横坐标为PD-L1单域抗体蛋白浓度(单位ng/mL)；方形分别代表109-chis，菱形代表 69、71Ga-NOTA-109-chis。两种分子对PD-L1的亲和力相当。
4.4合成 68Ga-NOTA-109-chis
用0.05M的无菌HCL淋洗Eckert&Ziegler GalliaPharm锗68/镓68(Ge68/Ga 68)发生器制备 68Ga淋洗液，添加1/2体积0.2M醋酸钠溶液，并加入1/4体积含有109-chis-NOTA pH为5.3的0.1M醋酸钠缓冲液，反应体 系的pH在4.5～4.7之间，室温反应10分钟，经PD-10柱去除未反应的离子镓并同时将产物缓冲体系更换为生理盐水。利用剂量校正器分析 68Ga-NOTA-109-chis注射剂的总放射性浓度。
实施例5：109抗体结构突变
109抗体蛋白结构上一共含有3个带有游离氨基的赖氨酸基团，该基团可以用于后续标记。为了优化抗体标记螯合物的纯度，保证每mol抗体能且只能缀合1mol螯合物，对抗体表面的赖氨酸进行了突变，并且为了保持亲和力，对赖氨酸邻近氨基酸也进行了突变，共得到4种如下所示的突变体：109-R73N&K75E-cHis、109-K86R&P87A-cHis、109-RDNSE-cHis、109-K64Q-cHis，如表3所示，并考察突变后抗体与PD-L1结合能力是否受影响。
表3 109-chis突变体序列
Figure PCTCN2019093225-appb-000008
其中，K64Q突变位于CDR2中，由此导致突变的CDR2，其包含氨基酸序列IASDRSTYYTPSVQG(SEQ ID NO:13)。
5.1突变抗体对PD-L1结合能力鉴定(ELISA法)
PDL1-Fc融合蛋白由HEK293瞬时表达及镍柱亲和层析纯化获得。得到的PDL1-Fc融合蛋白0.5μg/孔4℃过夜包被平板，之后加入获得的PD-L1单域抗体蛋白的梯度稀释系列，室温下反应1小时。洗涤之后加入抗-his 辣根过氧化物酶标记抗体，室温反应1小时。洗涤之后加入显色液，405nm波长读取吸收值。应用软件SotfMaxPro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对PD-L1结合曲线及EC50值，以反映抗体对PD-L1的亲和能力。
结果如图5A-B所示，其中纵坐标为OD405，横坐标为PD-L1单域抗体蛋白浓度(单位ng/mL)；单个赖氨酸被突变的突变体的EC50与野生型109-chis的EC50相比有所下降，说明突变体的亲和力并未破坏。
5.2突变抗体NOTA标记分析
如上所述，抗体表面氨基酸的突变，并未影响抗原抗体的结合性能。进一步对其螯合剂的标记效率进行分析，利用如4.1所述的方法对突变抗体进行NOTA缀合并进行质谱分析鉴定。
从图5C的质谱结果可以看出，对于K75E及K64Q突变抗体，NOTA缀合效率并未因为氨基酸的突变而发生变化，而K86R突变后，NOTA基本不能成功缀合至抗体。该结果说明，K86为较容易与NOTA缀合的氨基酸。所以进一步以RDNSE突变蛋白为基础，进行K64Q突变，形成109-K64Q-RDNSE-cHis：
Figure PCTCN2019093225-appb-000009
利用4.1所述的方法对突变抗体109-K64Q-RDNSE-cHis进行NOTA缀合并进行质谱分析鉴定。质谱结果表明，突变后的单域抗体109-K64Q-RDNSE-cHis与螯合剂p-SCN-Bn-NOTA反应后，产物为纯的只缀合1分子的NOTA的终产物。
实施例6：正电子核素标记单域抗体 68Ga-NOTA-109体外分析
6.1薄层色谱ITLC分析
ITLC SA预切至1cm x 12cm条和用铅笔标记条各端彼此距离1cm。在展开槽中倾倒醋酸铵1M:甲醇(1:1V/V)溶液至深度3至4mm，覆盖槽，和令其平衡。在离ITLC条底部1cm铅笔线处加一滴 68Ga-109注射液。将 ITLC条放置在展开槽中和令其展开从加样点距离10cm(即至顶部铅笔线标志)。用辐射测量的ITLC扫描仪扫描ITLC，通过在层析图上对峰积分计算放射化学纯度(RCP)，分析结果如图6A所示，表明正电子核素 68Ga成功标记单域抗体109。
6.2 SEC-HPLC及体外稳定性分析
SEC-HPLC色谱条件为：在配备Tokou sv3000SL(4.6×250mm)柱和Raytest GABI放射性检测器的Agilent 1100系列HPLC上进行SEC-HPLC。以pH 7.0的0.1M磷酸盐缓冲液作为流动相进行SEC分析。
PD-10柱纯化的放射性标记的单域抗体的代表性的SEC-HPLC色谱图示于图6A。在280nm UV色谱图中，放射性标记单域抗体的保留时间实际上与相应的未标记的单域抗体的保留时间基本无变化(除了由于UV和γ检测器的物理分离的时间差异；数据未显示)。
生理盐水中室温保存3h，SEC结果显示，未见明显降解或 68Ga脱落现象(图6B)，说明在此条件下，放射性标记抗体基本稳定。
将放射性标记产物 68Ga-NOTA-109标记后分别放置于PBS及血清(FBS)中，室温保存0、2、4h，用HPLC分析，观察其在PBS及FBS中的稳定性。结果见图9A和B。结果显示，制备的放射性标记产物在PBS及血清中，室温放置4h，峰形与放射性标记后并无差别，放射化学纯度>98％，未见明显脱落。
随后，将放射性标记产物 68Ga-NOTA-109标记后分别放置于PBS中，55～75℃保存4h(图10)，测定其体外温度稳定性。结果显示，制备的放射性药物在PBS中55℃放置4h，峰形与放射性标记后并无差别，放射化学纯度>98％，未见明显脱落。而在PBS中65、75℃放置4h，主峰后有降解峰，降解量随着温度升高而升高。
6.3细胞结合及内吞分析
细胞铺板于6孔板中，每孔8×10 5cells，3mL培养基/孔，37℃，5％CO2过夜培养；细胞取出，4℃孵育30min；取出所有6孔板，将培养基更换为含有10μci(～50nM)热标记蛋白的培养基，4℃继续孵育1h；将其中3孔培养基更换为含有25μM未标记蛋白的培养基，作为非特异性吸附的对照 样品；吸取上清中含有未结合蛋白的培养基，用2mL预冷的含1％BSA的PBS洗涤细胞2次；将培养基更换为生长培养基，37℃，5％CO2继续孵育1h和2h；孵育不同时间后弃去培养基上清，添加2mL pH 3.0含20mM乙酸钠的PBS，4℃孵育15min，再次添加2mL pH 3.0含20mM乙酸钠的PBS，这两部分的上清混合，即为细胞表面结合的部分的cpm值；余下的细胞沉淀用含有0.5％SDS的PBS重悬，此部分为细胞内吞部分；γ计数仪检测细胞结合及内吞部分cpm值，并进行衰减校正，结果如图6C所示，表明发生细胞内吞后 68Ga基本不脱落，放射性标记抗体结构稳定。
6.4体内稳定性分析
选择健康小鼠，每只小鼠经尾静脉注射0.1mL放射化学纯度为98％的 68Ga-NOTA-109，于注射后2h取其尿液，将其进行HPLC分析，测定其体内稳定性。
结果如图11所示，放射性药物在小鼠体内2h后，其尿液中的放射性核素，主峰形与放射性标记后产物并无差别，主峰后有降解峰。
实施例8： 68Ga-NOTA-109免疫活性测定
按以下方法进行 68Ga-NOTA-109的免疫活性测定：
(1)细胞消化铺板，37℃，5％CO 2过夜培养，然后4℃孵育30min；
(2)将细胞消化后，重悬于500μL PBS中(pH 7.4)，细胞密度为3×10 6个细胞/管；分别提前0.5h添加过量1000倍的未标记109单域抗体或阻断性抗体KN035(可阻断PD1和PDL1结合的治疗性抗体)。
(3)将1μL(5μci/0.2μg)放射性反应液加入到细胞中，室温分别反应0.5、1、2、4小时；
(4)细胞600g离心2分钟，将上清置于一个新的流式分选管中；细胞用预冷的500μL PBS重复洗涤2次，所有洗涤的上清加入到第一次离心的上清流式管中，而细胞重悬于500μL PBS中；
(5)γ计数仪检测细胞悬液及洗涤上清的cpm值，并做衰变矫正。
结果如图12所示，反应4h后 68Ga-NOTA-109的细胞摄取量可达7.17％，提前用阻断药物KN035预处理的细胞摄取量可达6.93％，这两者基本一致，而过量未标记的单域抗体109预处理的实验组可显著阻止 68Ga-NOTA-109de摄取，其在0.5～4h的细胞摄取量基本在1～2％之间。
实施例8： 68Ga-NOTA-109体内分布
8.1用于研究 68Ga-NOTA-109的动物模型。
用6～8周龄的雌性裸小鼠进行体内研究。在SPF环境中饲养裸鼠，可随意获取食物和水，标准12小时白天-夜晚照明循环。对于异种移植，在小鼠右前腿皮下植入100μl的细胞(A375-PDL1或MCF-7)/PBS。细胞接种密度约为5～6×10 6个细胞/小鼠。在异氟烷麻醉下实施植入。在这些条件下，在多于80％的注射的动物中，在3～4周后得到可用的肿瘤(100-300mm 3)(A375-PDL1或MCF-7)。
8.2  68Ga-NOTA-109体内分布
将～10μg放射性标记的单域抗体(～100μCi/10μg)尾静脉注射给予小鼠。将小鼠放置在滤纸衬里的笼子中，直至安乐死。在每个时间点使五只小鼠安乐死，将目的组织解剖，并且在γ计数器上计数。对血液、肾、肝、脾、肺、心、肠、胃、肌肉、皮肤、脑、骨及肿瘤收集数据。以注射剂量为总注射剂量。对于每一种器官，基于该总注射剂量确定％注射剂量，并且将器官称重，用于确定每克的％注射剂量(％ID/g)。
表4显示了在携带A375-PD-L1肿瘤的完好雄性小鼠中 68Ga-NOTA-109摄取的生物分布数据。其为在具有皮下肿瘤的完好雄性小鼠中(n＝5) 68Ga-NOTA-109摄取的离体生物分布数据。在静脉给药后1、2、4h采集数据。数据以平均％ID/g±标准偏差(S.D.)表示。所述比值的误差以标准偏差的几何平均数计算。如图7A所示的数据，其中误差条代表该组的标准偏差，显示出 68Ga-NOTA-109良好的肿瘤特异性。
表4  68Ga-NOTA-109体内分布数据
Figure PCTCN2019093225-appb-000010
Figure PCTCN2019093225-appb-000011
如图7B显示对于这些实验的肿瘤组织和血液摄取比例以及肿瘤组织和对侧正常肌肉摄取比例的结果。109单域抗体在表达靶标的A375-PDL1肿瘤中显示良好的肿瘤摄取，最大值为在注射后(PI)30分钟约5％的注射剂量/克组织，并且在PI 4h时，峰值肿瘤:肌肉比仍大于6。小鼠肿瘤组织的摄取与对侧正常肌肉相比表现为高摄取，4h后骨中的摄取低，说明标记物在体内稳定，无脱 68Ga的现象，此标记物可能成为一种良好的肿瘤显像剂。
8.3  68Ga-NOTA-109体内PET成像
将小鼠异氟烷麻醉后置于PET床，～10μg放射性标记的单域抗体(～100μCi/10μg)尾静脉注射给予小鼠。在120分钟进行连续PET扫描，分析多个时间点的肿瘤、肌肉及肾脏的放射性吸收情况，如图7C上图和中图所示。采用二维有序子集期望最大化算法进行图像重建。在肿瘤、肌肉、肝等器官用感兴趣区(ROI)法计算放射性活度(MBq/mL)，所得值除以注射剂量获得各组织对PET示踪剂摄取值(％ID/g)(假定组织密度为1g/ml)。计算结果分别如图7D所示。
阻断实验采用提前24h给予治疗型药物KN035(可阻断PD1和PDL1结合)，待治疗型药物分布完成后，将小鼠异氟烷麻醉后置于PET床，～10μg放射性标记的单域抗体(～100μCi/10μg)尾静脉注射给予小鼠。在120分钟进行连续PET扫描，分析多个时间点的肿瘤、肌肉及肾脏的放射性吸收情况，如图7C下图所示。生物分布使用MIM软件活动时间曲线分析，如图7D所示。
如图7C和7D结果显示，注射上述PET示踪剂 68Ga-NOTA-109后5min到120min，A375-PD-L1移植瘤均清晰可见，而PD-L1阴性表达的MCF-7移植瘤在注射后120min未见有摄取。从图7C可以看到，A375-PD-L1肿瘤与对侧相比具有良好的对比度。
68Ga-NOTA-109在模型鼠的肾中具有显著的浓聚，这表明其主要通过肾代谢。 68Ga-NOTA-109半衰期较短，不会对模型鼠造成伤害。
注射后10min，肿瘤对 68Ga-NOTA-109摄取值达最高约5-6％ID/g，随着时间的变化，摄取值降低并不明显，注射后120min，肿瘤对 68Ga-NOTA-109摄取值仍能维持在约～4％ID/g。肾对 68Ga-NOTA-109摄取值显著，证实所述PET示踪剂主要通过肾代谢。此外，如图7D所示，肿瘤较正常组织如肌肉对 68Ga-NOTA-109摄取显著，肿瘤与肌肉摄取比值(T/NT)均大于5，这有利于获得高清晰度的肿瘤PET图像，便于肿瘤的诊断和治疗。
8.4：同一小鼠 68Ga-NOTA-109体内PET成像分析PD-L1靶向能力
为了进一步证明 68Ga-NOTA-109的PD-L1靶向能力和消除个体差异，分别在同一小鼠的左后腿、右后腿和右前腿皮下同时接种不同癌细胞A375-HPD-L1、A375-HPD-L1/A375混合物(1/1，v/v)和A375。实验开始时，肿瘤大小约250～350mm 3。
将～10μg放射性标记的单域抗体(～100μCi/10μg)尾静脉注射给予小鼠。在给药后1h进行静态PET扫描，如图13A所示。
采用二维有序子集期望最大化算法进行图像重建。对肿瘤用感兴趣区(ROI)法计算放射性活度(MBq/mL)，所得值除以注射剂量获得各组织对PET示踪剂摄取值(％ID/g)(假定组织密度为1g/ml)。计算结果分别如图13B所示。
结果所示，注射上述PET示踪剂 68Ga-NOTA-109后60min，A375-PD-L1移植瘤均清晰可见，放射性摄取达4.94±0.46％id/g。A375-HPD-L1/A375混合物(1/1，v/v)接种的移植瘤表现出较为中间值的摄取，而PD-L1阴性表达的A375移植瘤在注射后60min未见有摄取。从ROI计算得知，同一动物，三个移植瘤由于表达量的不同，其％ID/g也表现出明显的差异。
PET显像完成后，将肿瘤从动物体内剥离，对动物进行放射自显影和免疫组化染色分析(图14)。免疫组化检测的PD-L1表达显示，A375-HPD-L1和A375-HPD-L1/A375肿瘤的PD-L1阳性区域分别为30％±6.36和15％±4.24，而A375肿瘤基本未表达。体外肿瘤放射自显影成像结果进一步证实了免疫组化检测的结果。
实施例9： 125I标记单域抗体109及其成像
以0.05mol/L，PH＝7.5的PBS为溶剂配制10mg/mL的氯胺T溶液和偏重亚硫酸钠溶液，将2uL蛋白稀释到50uL PB溶液中，取50uL溶液加入1.5mCi(10uL)的Na 125I混合，混合溶液中加入氯胺T溶液，迅速混匀，室温下反应40s，加入偏重亚硫酸钠溶液50uL混合，终止反应。毛细管吸一滴反应液滴在层析纸尾部，用PBS作为展开剂展开，展开到层析纸顶部时终止，通过TLC扫描仪对标记率进行鉴定。经PD-10柱去除未反应的 125I并同时将产物缓冲体系更换为生理盐水。
按照8.1的方法接种A375-PDL1肿瘤模型，将小鼠异氟烷麻醉后置于SPECT床，～10μg放射性标记的单域抗体(～100μCi/10μg)尾静脉注射给予小鼠。分别在1h、2h、4h进行SPECT扫描，分析多个时间点的肿瘤及组织器官的放射性吸收情况，如图8所示。
注射 125I-109后120min，A375-PD-L1移植瘤均清晰可见。从图可以看到，A375-PD-L1肿瘤与对侧相比具有良好的对比度。和 68Ga-NOTA-109在模型鼠中的表现类似， 125I-109在肾中具有显著的浓聚，这表明其主要通过肾代谢。

Claims (27)

  1. 一种程序性死亡配体1(PD-L1)结合多肽，其特征在于能够特异性结合PD-L1且包含至少一个免疫球蛋白单一可变结构域，其中所述至少一个免疫球蛋白单一可变结构域包含：
    CDR1，其包含SEQ ID NO:5所示氨基酸序列或相对于SEQ ID NO:5具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列，
    CDR2，其包含SEQ ID NO:6所示氨基酸序列或相对于SEQ ID NO:6具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列，例如包含SEQ ID NO:13所示氨基酸序列，和
    CDR3，其包含SEQ ID NO:7所示氨基酸序列或相对于SEQ ID NO:7具有1或多个氨基酸残基取代、缺失或添加的氨基酸序列。
  2. 权利要求1的PD-L1结合多肽，其中所述至少一个免疫球蛋白单一可变结构域包含：
    CDR1，其包含SEQ ID NO:5所示氨基酸序列，
    CDR2，其包含SEQ ID NO:13所示氨基酸序列，和
    CDR3，其包含SEQ ID NO:7所示氨基酸序列。
  3. 权利要求1或2的PD-L1结合多肽，其中所述免疫球蛋白单一可变结构域包含与SEQ ID NO：1的氨基酸序列具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％序列相同性的氨基酸序列。
  4. 权利要求1-3任一项的PD-L1结合多肽，其中所述免疫球蛋白单一可变结构域只包含一个赖氨酸残基。
  5. 权利要求1-3任一项的PD-L1结合多肽，其中所述免疫球蛋白单一可变结构域包含选自SEQ ID NO：1-2、8-12和18-22的氨基酸序列。
  6. 权利要求1-5任一项的PD-L1结合多肽，其中所述免疫球蛋白单一可变结构域是VHH。
  7. 权利要求1-6任一项的PD-L1结合多肽，其不阻断PD-1与PD-L1的结合。
  8. 核酸分子，其编码权利要求1-7中任一项的PD-L1结合多肽。
  9. 表达载体，其包含与表达调控元件可操作地连接的权利要求8的核酸分子。
  10. 宿主细胞，其包含权利要求8的核酸分子或以权利要求9的表达载体转化，并能够表达所述PD-L1结合多肽。
  11. 产生权利要求1-7中任一项的PD-L1结合多肽的方法，包括：
    a)在允许所述PD-L1结合多肽表达的条件下培养权利要求10的宿主细胞；
    b)从得自步骤a)的培养物回收由所述宿主细胞表达的PD-L1结合多肽；及
    c)任选进一步纯化和/或修饰得自步骤b)的PD-L1结合多肽。
  12. 一种缀合分子，其包含权利要求1-7任一项的PD-L1结合多肽，以及与所述PD-L1结合多肽缀合的至少一种可检测标记。
  13. 权利要求12的缀合分子，其中所述可检测标记选自放射性核素、荧光剂、化学发光剂、生物发光剂、顺磁离子和酶。
  14. 权利要求13的缀合分子，其中所述可检测标记选自 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 64Cu、 67Cu、 67Ga、 68Ga、 68Ge、 86Y、 90Y、 89Zr、 94mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 55Co、 72As、 75Br、 76Br、 82mRb、 83Sr或其它γ-、β-、或正电子发射体，例如，所述可检测标记为 68Ga或 125I。
  15. 权利要求12-14中任一项的缀合分子，其中所述PD-L1结合多肽通过螯合剂与所述可检测标记缀合。
  16. 权利要求15的缀合分子，其中所述螯合剂选自NOTA、DOTA、TETA或者NETA。
  17. 权利要求16的缀合分子，其中所述可检测标记是 68Ga且所述螯合剂为NOTA。
  18. 权利要求17的缀合分子，其中所述PD-L1结合多肽包含SEQ ID NO:12所示氨基酸序列。
  19. 检测生物学样品中PD-L1的存在和/或PD-L1的表达水平的方法，包括：
    a)在权利要求1-7任一项的PD-L1结合多肽和/或权利要求12-18中任一项的缀合分子与PD-L1之间能够形成复合物的条件下，使所述生物学样品和对照样品接触权利要求1-7任一项的PD-L1结合多肽和/或权利要求12-18中任一项的缀合分子；
    b)检测复合物的形成，
    其中所述生物学样品与对照样品之间复合物形成的差异指示样品中PD-L1的存在和/或PD-L1的表达水平。
  20. 一种用于检测和/或诊断PD-L1相关疾病例如癌症的诊断剂，其包含权利要求1-7任一项的PD-L1结合多肽和/或权利要求12-18中任一项的缀合分子，以及任选的生理学上可接受的载体。
  21. 权利要求20的诊断剂，其是ECT造影剂，例如SPECT造影剂或PET造影剂。
  22. 权利要求1-7任一项的PD-L1结合多肽和/或权利要求12-18中任一项的缀合分子在制备用于检测和/或诊断PD-L1相关疾病例如癌症的诊断剂中的用途。
  23. 权利要求22的用途，其中所述诊断剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
  24. 一种在对象中检测和/或诊断癌症的方法，包括给所述对象施用权利要求1-7任一项的PD-L1结合多肽和/或权利要求12-18任一项的缀合分子和/或权利要求20-21任一项的诊断剂。
  25. 权利要求24的方法，还包括对所述对象进行ECT成像，例如SPECT成像或PET成像的步骤。
  26. 权利要求20-21任一项的诊断剂、权利要求22-23任一项的用途、或权利要求24-25任一项的方法，其中所述癌症高表达PD-L1，例如所述癌症选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体癌、骨肉瘤。
  27. 一种试剂盒，其包含权利要求1-7任一项的PD-L1结合多肽和/或权利要求12-18任一项的缀合分子和/或权利要求20-21任一项的诊断剂。
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