WO2023072213A1 - 一种pd-l1结合分子及其应用 - Google Patents

一种pd-l1结合分子及其应用 Download PDF

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WO2023072213A1
WO2023072213A1 PCT/CN2022/128027 CN2022128027W WO2023072213A1 WO 2023072213 A1 WO2023072213 A1 WO 2023072213A1 CN 2022128027 W CN2022128027 W CN 2022128027W WO 2023072213 A1 WO2023072213 A1 WO 2023072213A1
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seq
binding molecule
antibody
cdr3
binding
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闫加庆
李艳敏
张丽丽
霍永庭
路力生
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广东菲鹏制药股份有限公司
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    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • G01—MEASURING; TESTING
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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
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    • 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®
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

Definitions

  • the invention relates to the technical field of monoclonal antibodies, in particular to a PD-L1 binding molecule and its application.
  • PD-L1 Programmed cell death 1 ligand 1
  • surface antigen differentiation cluster 274 cluster of differentiation 274, CD274
  • B7-H1 The third member, B7-H1 was discovered for the first time.
  • PD-L1 protein is widely expressed in activated T, B cells and macrophages.
  • the interaction between PD-L1 and the PD-1 protein on T cells can inhibit the activation of T cells, cause the apoptosis of T cells, and play a negative regulatory role in the immune response.
  • tumor cells and tumor-associated antigen presenting cells highly express PD-L1
  • tumor-infiltrating lymphocytes highly express PD-1 under long-term stimulation of tumor antigens.
  • the combination of PD-L1 and PD-1 can induce T cell apoptosis, incapacity, and exhaustion, thereby inhibiting the activation, proliferation, and anti-tumor function of tumor antigen-specific CD8+ T cells, and achieving tumor immune escape.
  • Nanobodies are currently the smallest antibody molecules, and their molecular weight is 1/10 of that of ordinary antibodies. In addition to the antigen reactivity of monoclonal antibodies, nanobodies also have some unique functional characteristics, such as small molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetrability, and strong targeting , Humanization is simple, and the preparation cost is low, which almost perfectly overcomes the defects of traditional antibody such as long development cycle, low stability, and harsh storage conditions.
  • Nanobodies binding to PD-L1 have been disclosed in the art, there is still a need in the art for Nanobodies with better binding affinity and specificity.
  • One of the objectives of the present invention is to provide an antibody that can specifically bind to PD-L1 and its application.
  • the present application solves the technical problems in this field through the following technical solutions.
  • a PD-L1 binding molecule comprising at least one immunoglobulin single variable domain, said at least one immunoglobulin single variable domain comprising any one of the following (i) to (vi) CDR1, CDR2, and CDR3:
  • the PD-L1 binding molecule as described in item 2 the amino acid sequence of the VHH is as SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 9 shown in any one.
  • PD-L1 binding molecule according to any one of items 1 to 3, said PD-L1 binding molecule further comprising an immunoglobulin Fc region;
  • the C-terminus of the immunoglobulin single variable domain is linked to the N-terminus of the immunoglobulin Fc region.
  • the PD-L1 binding molecule as described in item 4 the amino acid sequence of the Fc region of the immunoglobulin is shown in the 120th-346th amino acid of SEQ ID NO:44.
  • a host cell comprising the expression vector of item 8 or the polynucleotide of item 7 integrated in its genome.
  • a method for preparing the PD-L1 binding molecule described in any one of items 1 to 6, comprising culturing the host cell described in item 9 under conditions that allow the production of the PD-L1 binding molecule and recovering and isolating the obtained The PD-L1 binding molecule.
  • a phage displaying Nanobodies wherein the PD-L1 binding molecule according to any one of items 1-6 is displayed on the surface of the phage.
  • a kit for detecting PD-L1, comprising the PD-L1 binding molecule described in any one of items 1 to 6 or the phage displaying Nanobody described in item 11.
  • kit according to item 13 further comprising a solid-phase carrier, and the PD-L1 binding molecule or the phage displaying the nanobody is immobilized on the solid-phase carrier.
  • kit as described in item 13 which also includes a detectable label that can be linked to the PD-L1 binding molecule; and/or a PD-L1 standard or a PD-L1 conjugate standard and/or a substrate corresponding to a detectable label; and/or an ELISA reagent;
  • said detectable label is attached to said PD-L1 binding molecule or present in a kit separately.
  • the phage of the antibody is used as the detection antibody of PD-L1
  • the presence of PD-L1 in the sample to be tested is detected by enzyme-linked immunosorbent assay.
  • the sample to be tested is coated on a solid phase carrier, and the PD-L1 binding molecule carrying or not carrying a detectable marker or the phage displaying the Nanobody is used as a detection antibody to detect the presence of PD-L1 .
  • An immunoconjugate comprising a therapeutic agent and a PD-L1 binding molecule as described in any one of items 1 to 6 conjugated to the therapeutic agent;
  • the therapeutic agent comprises a toxin, radioisotope, drug or cytotoxic agent.
  • a bispecific or multispecific antibody comprising the PD-L1 binding molecule described in any one of items 1 to 6, and another or several other antibodies functionally linked to the PD-L1 binding molecule.
  • Antibodies or antibody fragments with antigen-binding properties comprising the PD-L1 binding molecule described in any one of items 1 to 6, and another or several other antibodies functionally linked to the PD-L1 binding molecule.
  • a pharmaceutical composition comprising the PD-L1 binding molecule described in any one of items 1-6; preferably, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, carrier or diluent.
  • the PD-L1 binding molecule as described in any one of items 1 to 6, the immunoconjugate as described in item 17, the bispecific or multispecific antibody as described in item 18, and the pharmaceutical composition as described in item 19 Use in the preparation of medicines for treating PD-L1-mediated diseases;
  • the PD-L1-mediated disease is preferably cancer, more preferably a cancer with high expression of PD-L1.
  • the cancers include but not limited to lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, Head and neck cancer is preferably breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, and melanoma.
  • a method for diagnosing, treating, preventing or alleviating a disease, disease or condition related to PD-L1 in a subject which comprises administering a therapeutically effective amount of any one of items 1 to 6 to the above subject.
  • FIG. 2 Electrophoresis diagram of the second round of nested PCR in Example 2.
  • Figure 3 Diagram of flow cytometry detection results in Example 5.
  • a shows the flow cytometry detection results of R1014, R1015, R1016, R1017, R1018, R1019, R1020;
  • b shows the flow cytometry detection results of R1021, R1022, R1023, R1024, R1025, R1026, R1027 Figure;
  • c shows the results of flow cytometry detection of R1028;
  • d shows the results of flow cytometry detection of R1148, R1149, R1150.
  • Figure 4a Binding curve of fusion protein R1015 in Example 6.
  • Figure 4b Binding curve of fusion protein R1016 in Example 6.
  • Figure 4c Binding curve of fusion protein R1019 in Example 6.
  • Figure 4d Binding curve of fusion protein R1148 in Example 6.
  • Figure 4e The binding curve of the fusion protein R1149 in Example 6.
  • Figure 4f The binding curve of the fusion protein R1150 in Example 6.
  • Figure 4g Binding curve of fusion protein R0999 in Example 6.
  • Figure 4h The binding curve of the fusion protein R0516 in Example 6.
  • Figure 4i Binding curve of fusion protein R0323 in Example 6.
  • Figure 5 shows the binding curve and IC50 value of fusion protein blocking membrane expressed human PD-L1 protein and free human PD-1 using detection method (A) in Example 7.
  • a shows the binding curve and IC50 value of R1148, R1149, R1150 blocking membrane expressed human PD-L1 protein and free human PD-1
  • b shows R1014, R1015, R1016, R1017, R1018, R1019, R1027 blocking Binding curves and IC50 values of membrane-expressed human PD-L1 protein and free human PD-1.
  • Figure 6 shows the binding curve and IC50 value of fusion protein blocking membrane expressed human PD-L1 protein and free human PD-1 using detection method (B) in Example 7.
  • a shows the binding curve and IC50 value of R1148, R1149, R1150 blocking membrane expressed human PD-L1 protein and free human PD-1
  • b shows R1014, R1015, R1016, R1017, R1018, R1019, R1027 blocking Binding curves and IC50 values of membrane-expressed human PD-L1 protein and free human PD-1.
  • Figure 7 shows the binding curve and IC50 value of fusion protein blocking membrane expressed human PD-L1 protein and free human CDSO using detection method (A) in Example 9.
  • Figure 8 shows the binding curve and IC50 value of fusion protein blocking membrane expressed human PD-L1 protein and free human CDSO using detection method (B) in Example 9.
  • Nanobody refers to a heavy chain antibody lacking a light chain (e.g. derived from camels), a single domain antibody obtained by cloning its variable region (VHH), which is a minimally functional antigen-binding antibody. Fragment, the relative molecular mass (Mr) is only about 15000. Nanobodies have the characteristics of small molecular weight, strong stability, good solubility, easy expression, and low immunogenicity. VHH usually contains three hypervariable regions, called “complementarity determining regions (CDRs)", and these three CDRs are CDR1, CDR2, and CDR3, respectively.
  • CDRs complementarity determining regions
  • immunoglobulin Fc region refers to the crystallizable fragment of an immunoglobulin antibody following papain digestion.
  • the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc region of IgM and IgE is present in each polypeptide chain Contains three heavy chain constant domains (CH domains 2-4).
  • the "detection antibody” refers to an antibody specifically anti-PD-L1, with a detectable label.
  • the "detectable marker” refers to a marker located on the detection antibody and used to determine the presence or absence and the amount of PD-L1 in the sample to be detected. Such as: enzymes, fluorescent labels, nuclides, quantum dots, colloidal gold, etc.
  • the marker is selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, ⁇ -D-galactosidase, urease, catalase, or glucoamylase.
  • the "substrate corresponding to the detectable label” refers to a color that can be catalyzed by the label of the detection antibody to display the recognition signal of the binding of the detection antibody to PD-L1. Described substrate is such as: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS for horseradish peroxidase; p-nitrophenyl phosphate, p-NPP); and so on.
  • OPD o-phenylenediamine
  • TMB tetramethylbenzidine
  • ABTS horseradish peroxidase
  • p-nitrophenyl phosphate p-NPP
  • amino acid refers to the twenty common naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C ); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine ( Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S ), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn;
  • the invention provides a nanobody, and the nanobody is selected from a camel-derived natural single-domain heavy chain antibody library.
  • a Nanobody of the invention can specifically bind to PD-L1 with high affinity.
  • a Nanobody of the invention has a heavy chain variable region (VHH) as set forth in SEQ ID NO: 1-19.
  • a Nanobody of the invention has a heavy chain variable region (VHH) as set forth in SEQ ID NO: 7, 3, 6, 2, 8 and 9.
  • the amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 29, 30 and 31, respectively (based on IMGT numbering).
  • sequence of the heavy chain variable region of the Nanobody is SEQ ID NO: 3
  • amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 23, 24 and 25, respectively (based on IMGT numbering).
  • sequence of the heavy chain variable region of the Nanobody is SEQ ID NO: 6
  • amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 26, 27 and 28, respectively (based on IMGT numbering).
  • sequence of the heavy chain variable region of the Nanobody is SEQ ID NO: 2
  • amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 20, 21 and 22, respectively (based on IMGT numbering).
  • the amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 32, 33 and 34, respectively (based on IMGT numbering).
  • the amino acid sequences of its CDR1, CDR2 and CDR3 are SEQ ID NO: 35, 36 and 37, respectively (based on IMGT numbering).
  • the numbering method of the antibody sequence is not limited to the IMGT method, and other methods can also be used to number the antibody sequence, such as Kabat, Chothia, Martin, AHo and other methods.
  • the CDR sequences of antibodies may differ where different numbering schemes are used. Unless otherwise specified, the antibody numbers in this application are numbered using the IMGT method.
  • the present invention uses the IMGT numbering system to mark the CDR region, and the CDR region marked by other methods also belongs to the protection scope of the present invention.
  • the invention also includes variants, derivatives and analogs of said Nanobodies.
  • variants derivatives and analogs of said Nanobodies.
  • variants derivatives and analogs of said Nanobodies.
  • analogs refer to polypeptides that substantially retain the same biological function or activity of the Nanobodies of the invention.
  • polypeptide variants, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues
  • the base may or may not be encoded by the genetic code, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a propolypeptide sequence, or a fusion polypeptide).
  • Nanobody of the present invention can be added to the amino-terminal or carboxyl-terminal of the Nanobody.
  • these added amino acid sequences are favorable for expression (such as signal peptide), favorable for purification (such as 6XHis sequence), or other sequences that can promote the activity, expression amount or stability of the Nanobody.
  • the invention also includes DNA molecules encoding the Nanobodies of the invention or variants, derivatives thereof.
  • the DNA molecules can be all artificially synthesized, or can be obtained by PCR amplification.
  • the coding sequence of the Nanobody of the present invention can be modified, for example, using codons preferred by the host cell to eliminate sequences that are not conducive to gene transcription and translation.
  • the DNA sequence encoding the Nanobody of the present invention or its variants and derivatives After obtaining the DNA sequence encoding the Nanobody of the present invention or its variants and derivatives, it is cloned into a suitable expression vector, and then transformed into a suitable host cell. Finally, the transformed host cells are cultured, and the novel nanobody of the present invention is obtained by separation and purification.
  • said Nanobody is fused to an immunoglobulin Fc region.
  • the C-terminus of the Nanobody is fused to the N-terminus of the Fc region of an immunoglobulin.
  • the immunoglobulin Fc region is from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE.
  • amino acid sequence of the Fc region of the immunoglobulin is shown in amino acid 120 to amino acid 346 of SEQ ID NO:44.
  • the present invention provides an isolated polynucleotide encoding the aforementioned Nanobody.
  • polynucleotide is generally RNA or DNA, polynucleotides may be single-stranded or double-stranded, but are preferably double-stranded DNA. Unless otherwise stated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as expressly indicated the sequence of.
  • the present invention provides an expression vector, which comprises the aforementioned polynucleotide.
  • expression vector includes plasmids, cloning vectors, viral vectors, and the like.
  • Various vectors known in the art can be used. For example, a commercially available vector is selected, and then the nucleotide sequence encoding the Nanobody of the present invention is operably linked to the expression control sequence to form an expression vector.
  • the present invention provides a host cell comprising the aforementioned expression vector or the aforementioned polynucleotide integrated in its genome.
  • the term "host cell” includes both prokaryotic and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Host cells for expressing Nanobodies include Escherichia coli, yeast cells, insect cells, COS cells, CHO cells, and the like. Preferably, the host cell is a eukaryotic cell, more preferably a CHO cell.
  • the cell After the transformed host cell is obtained, the cell can be cultured under conditions suitable for expressing the Nanobody of the present invention, thereby expressing the Nanobody; and then the expressed Nanobody can be isolated.
  • the present invention provides a phage displaying Nanobody, and the surface of the phage displays the aforementioned Nanobody.
  • Phage display technology is used in the construction of the nanobody library, and the DNA sequence of the foreign protein or polypeptide is inserted into the appropriate position of the structural gene of the phage coat protein, so that the foreign gene is expressed with the expression of the coat protein, and at the same time, the foreign protein is Phage reassembly and display on the phage surface.
  • the present invention provides a kit for detecting PD-L1, and the kit can be used for the detection of PD-L1.
  • the kit contains: the nanobody of the present invention or a phage (phagemid) displaying the nanobody.
  • the sample to be tested can be coated on a solid phase carrier, and the Nanobody of the present invention can be used as a detection antibody for detection.
  • the Nanobody can be linked to a detectable label, or can be linked to a Another antibody (anti-antibody) that detects the marker binds, so as to know the presence of PD-L1 in the sample to be tested. It should be understood that after obtaining the Nanobody of the present invention, various methods known in the art can be adopted to detect PD-L1, and these methods are all included in the present invention.
  • the detection antibody used in the kit of the present invention After the detection antibody used in the kit of the present invention is determined, various markers that are routinely used in the art for detection in combination with the detection antibody can be used as detectable markers.
  • the present invention has no particular limitation on the marker used, as long as it can bind to the detection antibody and can accurately indicate the presence or absence and the amount of PD-L1 in the sample to be detected after appropriate treatment are available.
  • the marker can be selected from (but not limited to): horseradish peroxidase, alkaline phosphatase, glucose oxidase, ⁇ -D-galactosidase, urease, catalase, or Glucoamylase.
  • the detection antibody is labeled with horseradish peroxidase (HRP).
  • Antibody labeling methods are well known in the art, for example, the simple sodium periodate method or the glutaraldehyde two-step method for HRP-labeling antibodies.
  • Described substrate is for example: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS for horseradish peroxidase; p-nitrophenyl phosphate, p-NPP).
  • OPD o-phenylenediamine
  • TMB tetramethylbenzidine
  • ABTS horseradish peroxidase
  • p-NPP p-nitrophenyl phosphate
  • the quality control product can be, for example, a PD-L1 standard product.
  • multiple standards containing known concentrations of PD-L1 can be set during the detection process.
  • a conventional method can be used for the setting method of the standard. Using the standard, the standard curve is set as follows: use the OD value detection result of the standard as the vertical axis (Y axis), and the standard concentration as the horizontal axis (X axis) to draw the quantitative standard curve of the PD-L1 kit. Therefore, according to the OD value obtained by testing the sample to be tested, the concentration of PD-L1 in the sample to be tested can be calculated using the standard curve.
  • the kit preferably also contains other auxiliary reagents, and the auxiliary reagents are some reagents routinely used in enzyme-linked immunoassays.
  • the properties and methods of their formulation are well known to those skilled in the art.
  • the reagents include: chromogen, washing solution, stop solution, and sensitizing diluent.
  • the coating antibody is coated on a solid phase carrier.
  • the present invention has no particular limitation on the solid phase carrier used, as long as it can be coupled (linked) with the coated antibody.
  • the solid phase carrier is selected from: microtiter plates (also called multi-well plates, such as 96-well plates) or microspheres.
  • the solid phase carrier used is a microtiter plate (elisa plate), and the microtiter plate is a polystyrene plate with a size of 12 ⁇ 8 detachable strips.
  • the nanobody used in the kit of the present invention has extremely excellent binding properties (high specificity) to PD-L1. According to the above method, as long as an antigen control of known concentration is set, a concentration standard curve is made, and the PD-L1 content in the sample to be tested can be obtained by comparing the concentration standard curve.
  • the present invention provides an immunoconjugate comprising a therapeutic agent and the aforementioned Nanobody conjugated to the therapeutic agent;
  • the therapeutic agent comprises a toxin, radioisotope, drug or cytotoxic agent.
  • immunoconjugate is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.
  • the present invention provides a bispecific or multispecific antibody comprising the aforementioned Nanobody, and an antibody or antibody fragment functionally linked to the Nanobody having another or several other antigen-binding properties.
  • bispecific or multispecific antibody refers to a molecule that can bind to two or more different epitopes of the same antigen or that can bind to two or more different antigens.
  • the present invention provides a pharmaceutical composition, which includes a PD-L1 binding molecule (such as the aforementioned nanobody, immunoconjugate, bispecific or multispecific antibody);
  • a PD-L1 binding molecule such as the aforementioned nanobody, immunoconjugate, bispecific or multispecific antibody
  • the pharmaceutical composition includes a pharmaceutically acceptable excipient, carrier or diluent.
  • the term "pharmaceutical composition” is in a form that permits the biological activity of the active ingredients to be effective, and does not contain additional ingredients that would be unacceptably toxic to the subject to which the composition will be administered.
  • the pharmaceutical composition also includes a pharmaceutically acceptable excipient, carrier or diluent, specifically, any and all solvents, dispersion media, coatings, antibacterial agents that are physiologically compatible. Agents and antifungal agents, isotonic and absorption delaying agents, etc., are used to prolong the shelf life or potency of antibodies.
  • the present invention has creatively developed PD-L1 binding molecules with high affinity and strong specificity to PD-L1, such as PD-L1 Nanobody, which can block the binding of human PD-L1 protein to free human PD-1 and free human CD80 , the nanobody and the phage displaying the nanobody can be used to prepare a kit for detecting PD-L1, and the PD-L1 nanobody and its immunoconjugate, bispecific or multispecific antibody, and pharmaceutical composition are prepared Promising applications in drugs for the treatment of PD-L1-mediated diseases.
  • PD-L1 Nanobody which can block the binding of human PD-L1 protein to free human PD-1 and free human CD80
  • the nanobody and the phage displaying the nanobody can be used to prepare a kit for detecting PD-L1, and the PD-L1 nanobody and its immunoconjugate, bispecific or multispecific antibody, and pharmaceutical composition are prepared Promising applications in drugs for the treatment of PD-L1-mediated diseases.
  • Embodiment 1 the preparation of material
  • the genes encoding human PD-L1 (NP_054862.1), monkey PD-L1 (XP_015292694.1), mouse PD-L1 (NP_068693.1), human PD-1 (NP_054862.1) and human CD80 (NP_005182.1)
  • the target gene was cloned into plasmids containing His, human Fc or mouse Fc, and then transfected into HEK293 cells, a transient expression system, for transient expression to obtain hPD-L1-His (R0201 for short), cynoPD-L1-His, mPD - L1-His, hPD-L1-hFc, hPD-1-hFc (abbreviated as R0479), hPD-1-mFc (abbreviated as R0323) and hCD80-hFc secreted protein.
  • the target genes encoding human PD-L1 (NP_054862.1), monkey PD-L1 (XP_015292694.1) and mouse PD-L1 (NP_068693.1) were cloned into the above plasmids containing His, human Fc or mouse Fc, and then Transfected into CHO cells for membrane protein expression, and then obtained cell lines overexpressing PD-L1 membrane protein, namely CHO-hPD-L1, CHO-cynoPD-L1 and CHO-mPD-L1.
  • the construction of the phage library displaying nanobodies was completed by Chengdu Apak Biotechnology Co., Ltd.
  • 50ml of alpaca peripheral blood was extracted, and lymphocytes were separated by lymphocyte separation medium.
  • RNA was extracted using RNA extraction reagent Trizol (purchased from Invitrogen), and the integrity of RNA was detected by 1% agarose electrophoresis, see Figure 1 for details.
  • Alpaca total cDNA was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).
  • Nanobody genes were performed by 2 rounds of nested PCR.
  • the first round of nested PCR system is shown in Table 1:
  • the NPR-19001 primer sequence is: 5'-CTTGGTGGTCCTGGCTGC-3' (SEQ ID NO: 45);
  • the NPR-19002 primer sequence is: 5'-GTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO: 46);
  • Reaction program 98°C, 52min; 98°C for 10s, 55°C for 30s, 72°C for 45s, a total of 25 cycles; 72°C, 7min; after the reaction, gel electrophoresis and gel tapping to recover the target VHH fragment of about 700bp.
  • the second round of nested PCR used conventional methods to design primers and reaction procedures to amplify the target gene fragments, and performed 2% agarose electrophoresis.
  • the vector and the target fragment (i.e. the VHH fragment after the second round of nested PCR amplification and purification) were respectively digested with SfiI, digested overnight at 50°C and recovered.
  • the vector carrying the target gene fragment was transformed into Escherichia coli competent E.coli TG1 cells by electroporation, and the calculated bacterial library capacity was 2.55 ⁇ 10 9 cfu.
  • helper phage M13KO7 was added according to the MOI of 20 times to continue the culture, and the standard purification method of PEG-NaCl was used to purify twice to construct the phage library.
  • the titer of the identified phage library was 1.41 ⁇ 10 13 cfu/mL.
  • the ELISA method was used to screen positive clones that combined with human PD-L1, and the nucleotide sequence was sequenced. The nucleotide sequences were translated into amino acid sequences by software and then compared, and classified into 19 categories according to CDR3 differences and amino acid differences in the phylogenetic tree. The best-performing sequences of each category are shown in Table 2 below.
  • the screening method is as follows: First, the hPD-L1-His and mPD-L1-hFc antigens were diluted to 2 ⁇ g/mL with 0.05M carbonate buffer (pH 9.6), coated at 100 ⁇ L/well overnight at 4°C; Wash 3 times with PBST, add 300 ⁇ L 5% skim milk to each well, block at 37°C for 1 hour; wash 3 times with PBST, add 100 ⁇ L/well of phages that have undergone three rounds of panning, incubate for 45 minutes at 37°C; wash 5 times with PBST , respectively added horseradish peroxidase-labeled goat anti-Alpaca secondary antibody (diluted 1:1W with PBS), 100 ⁇ L/well, incubated at 37°C for 45min; washed the plate 5 times with BST. Add 100 ⁇ L of TMB chromogenic solution to each well for color development, and react at 37°C for 5 min; add 50 ⁇ L of stop solution to each well to
  • Embodiment 4 Preparation of fusion protein
  • the target gene sequences encoding nanobodies were cloned into plasmids containing mouse IgG1 Fc, and then transfected into transient expression system HEK293 cells for transient expression to obtain corresponding secreted proteins (ie, fusion proteins, C-terminal of VHH and Fc
  • secreted proteins ie, fusion proteins, C-terminal of VHH and Fc
  • the abbreviations of each fusion protein are R1014, R1015, R1016, R1017, R1018, R1019, R1148, R1149, R1150, R1020, R1021, R1022, R1023, R1024, R1025, R1026, R1027, R1028 and R1029 .
  • the fusion protein contains two identical peptide chains, the VHH sequence of each fusion protein is shown in the above table 3, and the full-length amino acid sequence of one of the chains of the partial fusion protein is shown in any of SEQ ID NO: 38-43 in the following table 5 amino acid sequence.
  • the amino acid sequence of one CH2-CH3 of the immunoglobulin Fc region is shown in the 120th-346th amino acid of SEQ ID NO: 44 (Table 5 italics).
  • Example 5 Detection of binding activity of fusion protein to membrane-expressed human PD-L1 protein
  • FCM flow cytometry
  • fusion protein to be detected and the standard antibody at an appropriate dilution factor to 2E5/well CHO-hPD-L1 cells, incubate at 2-8°C for 30 minutes, wash once with 200 ⁇ l/well 1 ⁇ PBS, and add 1:1 to 100 ⁇ l/well 500 diluted E-anti-hIgG fluorescent secondary antibody (diluted with 1 ⁇ PBS containing 3% BSA), incubated at 2-8°C for 30min, washed once with 200 ⁇ l/well 1 ⁇ PBS, then washed with 100 ⁇ l/well 1 ⁇ PBS Resuspended for flow cytometry detection.
  • E-anti-hIgG fluorescent secondary antibody diluted with 1 ⁇ PBS containing 3% BSA
  • Embodiment 6 Detection of fusion protein affinity
  • Biomembrane interferometry was used to detect the positively binding fusion proteins (R1015, R1016, R1019, R1148, R1149, R1150) obtained in Example 5 and the reference antibodies obtained in Example 1 (referred to as R0999 and R0516), human PD- 1 protein (referred to as R0323) protein and human PD-L1 protein (referred to as R0201) affinity.
  • the detection method is as follows: 5 ⁇ g/ml of R0201 is solidified on the Anti-Penta-HIS (HIS1K) (18-5120, Fortebio) probe of the molecular interaction analyzer ForteBIO (Octet OK e ), and the probe is balanced for 90 seconds, respectively, with The analyte diluted at an appropriate concentration binds for 180s, then dissociates for 300s. After the probe is regenerated and neutralized in 10mM glycine, follow this method for the next cycle of equilibrium, binding, dissociation and regeneration neutralization. All cycle speeds were set at 1000 rpm and the experimental temperature was 30°C.
  • Example 7 Fusion proteins block the binding of membrane-expressed human PD-L1 protein and free human PD-1
  • fusion proteins obtained in Example 4 (R1015, R1016, R1019, R1148, R1149, R1150) and the R0999, R0516 obtained in Example 1, blocking cell CHO-hPD-L1 and free human were detected by flow cytometry (FCM). Binding of PD-1 protein. Detection methods (A) and (B) are as follows:
  • the detection method (A) is as follows:
  • the detection method (B) is as follows:
  • the positive binding fusion proteins (R1148, R1149 and R1150) obtained in Example 7 and the binding regions of the reference antibodies obtained in Example 1 (referred to as R0999 and R0516) and human PD-L1 protein (referred to as R0201) were grouped by competition ELISA .
  • the detection method is as follows:
  • FCM Flow cytometry
  • the detection method (A) is as follows:
  • fusion protein to be detected and the standard antibody at an appropriate dilution factor to 2E5/well CHO-hPD-L1 cells, incubate on ice in the dark for 30 minutes, then add hCD80 protein to a 96-well V-shaped plate (according to 50 ⁇ L/well), and store on ice.
  • the detection method (B) is as follows:
  • hCD80 protein to 2E5/well CHO-hPD-L1 cells, incubate on ice in the dark for 30 minutes, then add the fusion protein to be detected and the standard antibody at an appropriate dilution to a 96-well V-plate (according to 50 ⁇ L/well), ice Incubate in the dark for 30 minutes; centrifuge (300g/5min), discard the supernatant, add 200 ⁇ L FCM buffer to wash once, centrifuge (300g/5min), discard the supernatant, add 1:500 diluted PE-anti- hIgG fluorescent secondary antibody, incubated on ice in the dark for 30 minutes; after centrifugation and washing, add 100 ⁇ L 1 ⁇ PBS to resuspend, and detect on the machine.
  • a phage-displayed nanobody library was constructed, and positive clones that bind to human PD-L1 were screened out by ELISA method to obtain PD-L1-binding molecules that specifically bind to PD-L1, such as FBP002-1128, FBP002-1136, FBP002-1136, FBP002-1369, FBP002-1389, FBP002-1471, FBP002-2002, FBP002-2056, FBP002-2058, FBP002-1191, FBP002-1217, FBP002-1118, F BP002-1134, FBP002- 1153, FBP002-1184, FBP002-1224, FBP002-1249, FBP002-1075, FBP002-1095, R1015, R1016, R1019, R1148, R1149, R1150, R1014, R1017, R1018, R1020, R 1021, R1022, R1023, R1024, R1025,

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Abstract

一种PD-L1结合分子及其应用，涉及单克隆抗体技术领域。提供了所述PD-L1结合分子的制备方法、其用途和包含其的试剂盒、免疫缀合物和药物组合物。

Description

一种PD-L1结合分子及其应用
优先权声明
本申请要求申请号为202111274164.X，申请日为2021年10月29日，发明名称为一种PD-L1结合分子及其应用的中国发明专利申请的优先权，其全部内容通过引用并入本文中。
技术领域
本发明涉及单克隆抗体技术领域，具体来说是一种PD-L1结合分子及其应用。
背景技术
细胞程序性死亡-配体1(Programmed cell death 1 ligand 1，PD-L1)也称为表面抗原分化簇274(cluster of differentiation 274，CD274)，是由华裔学者陈列平教授在1999年作为B7家族的第3个成员B7-H1首次发现。PD-L1蛋白广泛表达于活化T、B细胞和巨噬细胞。PD-L1与T细胞上的PD-1蛋白相互作用会抑制T细胞的活化，引起T细胞的凋亡，在免疫应答中起负性调控作用。在肿瘤微环境中，肿瘤细胞和肿瘤相关抗原呈递细胞(antigen presenting cells，APCs)高表达PD-L1，肿瘤浸润性淋巴细胞在肿瘤抗原长期刺激下高表达PD-1。PD-L1与PD-1结合后可诱导T细胞凋亡、失能、耗竭，进而抑制肿瘤抗原特异性CD8+T细胞的激活、增殖和抗肿瘤功能，实现肿瘤免疫逃逸。
单克隆抗体在癌症的检测及生物靶向治疗方面成功的应用，引起了肿瘤治疗的变革。然而，传统的单抗(150kD)分子质量过大，难穿透组织，造成肿瘤区域的有效浓度较低，治疗效果不充分；传统的抗体具有很高的免疫原性，而改造的抗体很难达到原来的亲和力。此外，完全人源化的传统抗体开发周期长，生产成本高，稳定性不够等诸多因素限制其在临床中的应用及普及。
纳米抗体是目前最小的抗体分子，其分子量是普通抗体的1/10。纳米抗体除具备单克隆抗体的抗原反应性外，还拥有一些独特的功能特性，如分子质量小，稳定性强、可溶性好、易表达、免疫原性弱、穿透性强、靶向性强、人源化简单，制备成本低廉等，几乎完美克服了传统抗体开发周期长，稳定性较低，保存条件苛刻等缺陷。
尽管本领域中公开了一些结合PD-L1的纳米抗体，但是本领域仍然对具有较优的结合亲和力和特异性的纳米抗体存在需求。
发明内容
本发明的目的之一在于提供一种可以特异性结合PD-L1的抗体及其应用。
具体而言，本申请通过以下技术方案解决了本领域的技术问题。
1.一种PD-L1结合分子，其包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含选自以下(i)至(vi)任一项的CDR1、CDR2和CDR3：
(i)如SEQ ID NO：29所示的CDR1、如SEQ ID NO：30所示的CDR2、和如SEQ ID NO：31所示的CDR3；
(ii)如SEQ ID NO：23所示的CDR1、如SEQ ID NO：24所示的CDR2、和如SEQ ID NO：25所示的CDR3；
(iii)如SEQ ID NO：26所示的CDR1、如SEQ ID NO：27所示的CDR2、和如SEQ ID NO：28所示的CDR3；
(iv)如SEQ ID NO：20所示的CDR1、如SEQ ID NO：21所示的CDR2、和如SEQ ID NO：22所示的CDR3；
(v)如SEQ ID NO：32所示的CDR1、如SEQ ID NO：33所示的CDR2、和如SEQ ID NO：34所示的CDR3；或
(vi)如SEQ ID NO：35所示的CDR1、如SEQ ID NO：36所示的CDR2、和如SEQ ID NO：37所示的CDR3。
2.如项目1所述PD-L1结合分子，所述免疫球蛋白单一可变结构域为VHH。
3.如项目2所述PD-L1结合分子，所述VHH的氨基酸序列如SEQ ID NO：7、SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：2、SEQ ID NO：8或SEQ ID NO：9任一所示。
4.如项目1～3任一所述PD-L1结合分子，所述PD-L1结合分子还包含免疫球蛋白Fc区；
任选地，所述免疫球蛋白单一可变结构域的C端与免疫球蛋白Fc区的N端连接。
5.如项目4所述PD-L1结合分子，所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO：44的第120位-第346位氨基酸所示。
6.如项目1～5任一所述PD-L1结合分子，所述PD-L1结合分子包括如SEQ ID NO：38-43任一所示氨基酸序列。
7.一种分离的多核苷酸，其编码项目1～6任一项所述的PD-L1结合分子。
8.一种表达载体，其包含项目7所述的多核苷酸。
9.一种宿主细胞，所述宿主细胞包含项目8所述的表达载体或其基因组中整合有项目7的多核苷酸。
10.一种制备项目1～6任一项所述PD-L1结合分子的方法，其包括在允许产生所述PD-L1结合分子的条件下培养项目9所述的宿主细胞并回收和分离所述PD-L1结合分子。
11.一种展示纳米抗体的噬菌体，所述噬菌体表面展示有项目1～6任一所述的PD-L1结合分子。
12.一种用于检测PD-L1的试剂盒，其包括项目1～6任一所述的PD-L1结合分子或项目11所述的展示纳米抗体的噬菌体。
13.如项目12所述试剂盒，所述试剂盒中还包括固相载体，所述的PD-L1结合分子或展示纳米抗体的噬菌体被固定于固相载体中。
14.如项目13所述试剂盒，所述试剂盒中还包括能与所述的PD-L1结合分子连接的可检测标记物；和/或PD-L1标准品或PD-L1偶联物标准品；和/或与可检测标记物相对应的底物；和/或酶联免疫反应试剂；
优选地，所述的可检测标记物被连接于所述的PD-L1结合分子或分离地存在于试剂盒中。
15.一种用于治疗目的或非治疗目的的检测待测样品中PD-L1的存在情况的方法，以项目1～6任一所述的PD-L1结合分子或项目11所述的展示纳米抗体的噬菌体作为PD-L1的检测抗体，通过酶联免疫吸附测定法来检测待测样品中PD-L1的存在情况。
16.如项目15所述的方法，其具体包括以下步骤：
将待测样品包被于固相载体上，以携带或不携带可检测标记物的所述的PD-L1结合分子或所述的展示纳米抗体的噬菌体作为检测抗体，检测PD-L1的存在情况。
17.一种免疫缀合物，其包括治疗剂和与所述治疗剂缀合的如项目1～6任一所述的PD-L1结合分子；
优选地，所述治疗剂包括毒素、放射性同位素、药物或细胞毒剂。
18.一种双特异性或多特异性抗体，其包括项目1～6任一所述的PD-L1结合分子，以及与所述PD-L1结合分子功能性连接的具有另一种或另几种抗原结合特性的抗体或抗体片段。
19.一种药物组合物，其包括项目1～6任一所述的PD-L1结合分子；优选地，所述药物组合物包括可药用赋形剂、载体或稀释剂。
20.如项目1～6任一所述PD-L1结合分子、如项目17所述免疫缀合物、如项目18所述双特异性或多特异性抗体、以及如项目19所述药物组合物在制备用于治疗PD-L1介导的疾病的药物中的用途；
所述PD-L1介导的疾病优选为癌症，更优选为高表达PD-L1的癌症。所述的癌症包括但不限于肺癌、肝癌、卵巢癌、***、皮肤癌、膀肮癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌、头颈癌，优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤。
21.一种在受试者中诊断、治疗、预防或减轻与PD-L1相关的疾病、病症或状况的方法，其包括向上述受试者施用治疗有效量的如项目1～6任一所述PD-L1结合分子和/或如项目19所述药物组合物。
附图说明
为了更清楚地说明本发明实施方式的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的 某些实施方式，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1.实施例2中提取的总RNA的电泳图。
图2.实施例2中第2轮巢式PCR电泳图。
图3.实施例5中流式细胞术检测结果图。其中，a显示了R1014、R1015、R1016、R1017、R1018、R1019、R1020的流式细胞术检测结果图；b显示了R1021、R1022、R1023、R1024、R1025、R1026、R1027的流式细胞术检测结果图；c显示了R1028的流式细胞术检测结果图；d显示R1148、R1149、R1150的流式细胞术检测结果图。
[根据细则91更正 08.12.2022]　
图4a.实施例6中融合蛋白R1015的结合曲线图。
图4b.实施例6中融合蛋白R1016的结合曲线图。
图4c.实施例6中融合蛋白R1019的结合曲线图。
图4d.实施例6中融合蛋白R1148的结合曲线图。
图4e.实施例6中融合蛋白R1149的结合曲线图。
图4f.实施例6中融合蛋白R1150的结合曲线图。
图4g.实施例6中融合蛋白R0999的结合曲线图。
图4h.实施例6中融合蛋白R0516的结合曲线图。
图4i.实施例6中融合蛋白R0323的结合曲线图。
图5.显示了实施例7中使用检测方法(A)，融合蛋白阻断膜表达人PD-L1蛋白和游离人PD-1的结合曲线和IC50值。其中，a显示了R1148、R1149、R1150阻断膜表达人PD-L1蛋白和游离人PD-1的结合曲线和IC50值；b显示了R1014、R1015、R1016、R1017、R1018、R1019、R1027阻断膜表达人PD-L1蛋白和游离人PD-1的结合曲线和IC50值。
图6.显示了实施例7中使用检测方法(B)，融合蛋白阻断膜表达人PD-L1蛋白和游离人PD-1的结合曲线和IC50值。其中，a显示了R1148、R1149、R1150阻断膜表达人PD-L1蛋白和游离人PD-1的结合曲线和IC50值；b显示了R1014、R1015、R1016、R1017、R1018、R1019、R1027阻断膜表达人PD-L1蛋白和游离人PD-1的结合曲线和IC50值。
图7.显示了实施例9中使用检测方法(A)，融合蛋白阻断膜表达人PD-L1蛋白和游离人CDS0的结合曲线和IC50值。
图8.显示了实施例9中使用检测方法(B)，融合蛋白阻断膜表达人PD-L1蛋白和游离人CDS0的结合曲线和IC50值。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。
如本文所用，所述的“纳米抗体”是指缺失轻链的重链抗体(如：来源于骆驼体内)，克隆其可变区(VHH)得到的单域抗体，是最小的功能性抗原结合片段，相对分子质量(Mr)仅约为15000。纳米抗体具有分子质量小、稳定性强、可溶性好、易表达，免疫原性低等特点。VHH通常含有三个高变区，称为“互补决定区(CDR)”，这三个CDR分别为CDR1、CDR2、CDR3。
如本文所用，“免疫球蛋白Fc区”或“Fc区”是指在木瓜蛋白酶消化后免疫球蛋白抗体的可结晶片段。在IgG，IgA和IgD抗体同种型中，Fc区由来自抗体两条重链的CH2结构域和CH3结构域的两个相同的蛋白片段构成；IgM和IgE的Fc区在每个多肽链中包含三个重链恒定结构域(CH结构域2-4)。
如本文所用，所述的“检测抗体”是指特异性地抗PD-L1的抗体，具有可检测标记物。
如本文所用，所述的“可检测标记物”是指位于检测抗体上的，用于确定待检测样品中PD-L1的存在与否以及存在的量的标志物。如：酶、荧光标记、核素、量子点、胶体金等。优选的，所述的标记物选自：辣根过氧化物酶(HRP)、碱性磷酸酯酶(AP)、葡萄糖氧化酶、β-D-半乳糖苷酶、脲酶、过氧化氢酶、或葡萄糖淀粉酶。
如本文所用，所述的“与可检测标记物相对应的底物”是指可被检测抗体的标记物所催化显色，用于显示检测抗体与PD-L1发生结合的识别信号。所述的底物比如：用于辣根过氧化物酶的邻苯二胺(OPD)、四甲基联苯胺(TMB)、ABTS；用于碱性磷酸酯酶的对硝基苯磷酸酯(p-nitrophenyl phosphate，p-NPP)；等等。
如本文所用，术语“氨基酸(amino acid)”是指二十个常见的天然存在的氨基酸。天然存在的氨基酸包括丙氨酸(Ala；A)、精氨酸(Arg；R)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、半胱氨酸(Cys；C)；谷氨酸(Glu； E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)；组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、甲硫氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)、丝胺酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。
1、抗PD-L1纳米抗体
本发明提供了一种纳米抗体，所述的纳米抗体筛选自驼源性天然单域重链抗体库。
本发明所述的纳米抗体，能高亲和力特异性与PD-L1结合。在一个实施方案中，本发明的纳米抗体具有SEQ ID NO：1-19所示的重链可变区(VHH)。在优选的实施方案中，本发明的纳米抗体具有SEQ ID NO：7、3、6、2、8和9所示的重链可变区(VHH)。在所述纳米抗体的重链可变区的序列为SEQ ID NO：7的情况下，其CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO：29、30和31(基于IMGT编号方法)。在所述纳米抗体的重链可变区的序列为SEQ ID NO：3的情况下，其CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO：23、24和25(基于IMGT编号方法)。在所述纳米抗体的重链可变区的序列为SEQ ID NO：6的情况下，其CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO：26、27和28(基于IMGT编号方法)。在所述纳米抗体的重链可变区的序列为SEQ ID NO：2的情况下，其CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO：20、21和22(基于IMGT编号方法)。在所述纳米抗体的重链可变区的序列为SEQ ID NO：8的情况下，其CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO：32、33和34(基于IMGT编号方法)。在所述纳米抗体的重链可变区的序列为SEQ ID NO：9的情况下，其CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO：35、36和37(基于IMGT编号方法)。需要指出的是，抗体序列的编号方法不限于IMGT方法，也可以使用其他方法对抗体序列进行编号，例如Kabat，Chothia，Martin，AHo等方法。在使用不同编号方法的情况下，抗体的CDR序列可能不同。在没有特别指出的请下，本申请中抗体的编号为使用IMGT方法进行编号。
本发明采用IMGT编号***标示CDR区，其他方法标示的CDR区也属于本发明的保护范围。
本发明也包括所述的纳米抗体的变体、衍生物和类似物。如本文所用，术语“变体”、“衍生物”和“类似物”是指基本上保持本发明的纳米抗体相同的生物学功能或活性的多肽。本发明的多肽变体、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或多肽原序列，或融合多肽)。根据本文的定义，这些变体、衍生物和类似物属于本领域技术人员公知的范围。
此外，在所述的纳米抗体的氨基端或羧基端还可添加其它基本上不影响本发明所述的纳米抗体的活性、表达量和稳定性的氨基酸序列。
优选地，这些添加的氨基酸序列有利于表达(如信号肽)，有利于纯化(如6XHis序列)，或其它可促进所述的纳米抗体的活性、表达量或稳定性的序列。
本发明还包括编码本发明的纳米抗体或其变体、衍生物的DNA分子。所述的DNA分子可以全部人工合成，也可用PCR扩增的方法获得。
为了进一步提高宿主细胞的表达量，可以对本发明的纳米抗体的编码序列进行改造，例如采用宿主细胞偏好的密码子，消除不利于基因转录及翻译的序列。
在获得了编码本发明纳米抗体或其变体、衍生物的DNA序列之后，将其克隆入合适的表达载体，再转入合适的宿主细胞。最后，培养转化后的宿主细胞，通过分离纯化得到本发明的新的纳米抗体。
在一些具体的实施例中，所述纳米抗体与免疫球蛋白Fc区融合。
在一些具体的实施例中，所述纳米抗体的C端与免疫球蛋白Fc区的N端融合。
在一些具体的实施例中，所述免疫球蛋白Fc区来自于IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgD、IgM或者IgE。
在一些具体的实施例中，所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO：44的第120位-第346位氨基酸所示。
2、多核苷酸
本发明提供了一种分离的多核苷酸，所述多核苷酸编码前述的纳米抗体。
如本文所用，术语“多核苷酸”通常是RNA或DNA，多核苷酸可以是单链或双链的，但优选是双链DNA。除非另有说明，否则特定的多核苷酸序列还隐含地涵盖其保守修饰的变体(例如简并的密码子取代)、等位基因、直向同源物、SNP和互补序列以及明确指出的序列。
3、表达载体
本发明提供了一种表达载体，所述表达载体包含前述的多核苷酸。
如本文所用，术语“表达载体”包括质粒、克隆载体、病毒载体等。可选用本领域已知的各种载体。比如，选用市售的载体，然后将编码本发明纳米抗体的核苷酸序列可操作地连于表达调控序列，可以形成表达载体。
4、宿主细胞
本发明提供了一种宿主细胞，所述宿主细胞包含前述的表达载体或其基因组中整合有前述的多核苷酸。
如本文所用，术语“宿主细胞”包括原核细胞和真核细胞。常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。用于表达纳米抗体的宿主细胞包括大肠杆菌、酵母细胞、昆虫细胞、COS细胞、CHO细胞等。优选地，该宿主细胞是真核细胞，更优选地是CHO细胞。
在获得转化的宿主细胞后，可在适合表达本发明纳米抗体的条件下培养该细胞，从而表达出纳米抗体；然后再分离出表达的纳米抗体。
5、噬菌体
本发明提供了一种展示纳米抗体的噬菌体，所述噬菌体表面展示有前述的纳米抗体。
在纳米抗体库的构建时采用噬菌体展示技术，将外源蛋白或多肽的DNA序列***到噬菌体外壳蛋白结构基因的适当位置，使外源基因随外壳蛋白的表达而表达，同时，外源蛋白随噬菌体的重新组装而展示到噬菌体表面。
6、试剂盒
基于本发明获得的纳米抗体，本发明提供一种检测PD-L1的试剂盒，所述的试剂盒可用于PD-L1的检测。
所述的试剂盒含有：本发明所述的纳米抗体或展示纳米抗体的噬菌体(噬菌粒)。在一些具体的实施例中，可以将待测样品包被于固相载体上，以本发明的纳米抗体作为检测抗体进行检测，所述的纳米抗体可连接可检测标记物，或可以与连接可检测标记物的另一抗体(抗抗体)结合，从而获知待测样品中PD-L1的存在情况。应理解，在获得了本发明的纳米抗体后，可以采取多种本领域已知的方式来实施PD-L1的检测，这些方式均包含在本发明中。
在确定了本发明的试剂盒所采用的检测抗体后，可以采用本领域常规可用于与检测抗体结合来进行检测的各种标记物作为可检测标记物。本发明对所采用的标记物没有特别的限制，只要是能够与所述的检测抗体结合，且在适当处理后能够准确地指示待检测样品中PD-L1的存在与否以及存在量的标记物均是可用的。例如，所述的标记物可以选自(但不限于)：辣根过氧化物酶、碱性磷酸酯酶、葡萄糖氧化酶、β-D-半乳糖苷酶、脲酶、过氧化氢酶、或葡萄糖淀粉酶。例如，所述的检测抗体采用辣根过氧化物酶(HRP)标记。抗体标记的方法在本领域是公知的，例如用简易过碘酸钠法或者戊二醛二步法进行HRP标记抗体。
当采用如上所示的一些酶标记物时，还需要采用一些与相应的酶结合的底物，从而可通过显色等方式来指示标记物的存在情况或者存在量。所述的底物例如：用于辣根过氧化物酶的邻苯二胺(OPD)、四甲基联苯胺(TMB)、ABTS；用于碱性磷酸酯酶的对硝基苯磷酸酯(p-nitrophenyl phosphate，p-NPP)。
为了消除假阳性和假阴性，宜在检测过程中设置质控(对照)。所述的质控品可以是例如PD-L1标准品。此外，为了获得定量结果，可以在检测过程中设置含已知浓度的多个PD-L1的标准品。对于标准品的设置方法可采用常规的方法。利用所述的标准品，标准曲线如下设置：用标准品的OD值检测结果为纵坐标(Y轴)，标准品浓度为横坐标(X轴)绘制成PD-L1试剂盒的定量标准曲线。从而，根据待测样品检测获得的OD值，利用标准曲线可计算出待测样品中PD-L1的浓度。
此外，为了使本发明的试剂盒在检测时更方便，所述的试剂盒中优选的还包含其它一些辅助试剂，所述的辅助试剂是酶联免疫试验中常规使用 的一些试剂，这些试剂的特性以及它们的配制方法均是本领域技术人员所熟知的。所述的试剂包括：显色剂、洗涤液、终止液，增敏稀释液。
所述的包被抗体被包被在固相载体上。本发明对所采用的固相载体没有特别的限制，只要其能够与包被抗体相偶联(连接)即可。例如，所述的固相载体选自：微量滴定板(又称为多孔板，如96孔板)或微球。
在本发明的一个实例中，采用的固相载体是微量滴定板(酶标板)，所述的微量滴定板是一种聚苯乙烯板，规格是12×8可拆卸条板。
由于本发明的试剂盒采用的纳米抗体对PD-L1具有极其优异的结合特性(高特异性)。按照上述方法，只要设置已知浓度的抗原对照，制作浓度标准曲线，通过比照浓度标准曲线就可以得出待测样品中的PD-L1含量。
7、免疫缀合物
本发明提供了一种免疫缀合物，其包括治疗剂和与所述治疗剂缀合的前述的纳米抗体；
优选地，所述治疗剂包括毒素、放射性同位素、药物或细胞毒剂。
如本文所用，术语“免疫缀合物”是与一个或多个其它物质(包括但不限于细胞毒性剂或标记)缀合的抗体。
8、双特异性或多特异性抗体
本发明提供了一种双特异性或多特异性抗体，其包括前述的纳米抗体，以及与所述纳米抗体功能性连接的具有另一种或另几种抗原结合特性的抗体或抗体片段。
如本文所用，术语“双特异性或多特异性抗体”是指可以与同一抗原的两个或多个不同抗原表位或者可以与两个或多个不同抗原相结合的一种分子。
9、药物组合物
本发明提供了一种药物组合物，其包括PD-L1结合分子(如前述的纳米抗体、免疫缀合物、双特异性或多特异性抗体)；
优选地，所述药物组合物包括可药用赋形剂、载体或稀释剂。
如本文所用，术语“药物组合物”是以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。在一些实施方式中，所述药物组合物还包括药学上可接 受的赋形剂、载体或稀释剂，具体的，可以包括生理学上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等，用来延长抗体的保存限期或效力。
有益效果包括：
本发明创造性的开发出了与PD-L1具有高亲和力、特异性强的PD-L1结合分子，如PD-L1纳米抗体能够阻断人PD-L1蛋白和游离人PD-1、游离人CD80结合，该纳米抗体及展示纳米抗体的噬菌体能够用于制备检测PD-L1的试剂盒，且该PD-L1纳米抗体及其免疫缀合物、双特异性或多特异性抗体、药物组合物在制备用于治疗PD-L1介导的疾病的药物中的应用前景广泛。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1：材料的制备
将编码人PD-L1(NP_054862.1)、猴PD-L1(XP_015292694.1)、鼠PD-L1(NP_068693.1)、人PD-1(NP_054862.1)和人CD80(NP_005182.1)的目的基因分别克隆到含有His、人Fc或鼠Fc的质粒中，随后转染到瞬转表达体系HEK293细胞进行瞬时表达，进而获得hPD-L1-His(简称R0201)、cynoPD-L1-His、mPD-L1-His、hPD-L1-hFc、hPD-1-hFc(简称R0479)、hPD-1-mFc(简称R0323)和hCD80-hFc分泌蛋白。
将编码人PD-L1(NP_054862.1)、猴PD-L1(XP_015292694.1)和鼠PD-L1(NP_068693.1)的目的基因克隆到上述含有His、人Fc或鼠Fc的 质粒中，随后转染到CHO细胞进行膜蛋白表达，进而获得过表达PD-L1膜蛋白的细胞株，分别为CHO-hPD-L1、CHO-cynoPD-L1和CHO-mPD-L1。
根据申请号201510465481.8中56号纳米抗体的序列和Drug Bank网站查询Avelumab可变区序列构建鼠IgG1亚型的对标抗体，其简称分别为R0999和R0516。
实施例2：纳米抗体文库的构建
展示纳米抗体的噬菌体库构建由成都阿帕克生物科技有限公司完成。将1.5mg人PD-L1抗原-mFc(购自Sino Biologics)与弗氏佐剂等体积混合，采取背部皮内、皮下多点注射的方式免疫1只羊驼，每周一次，共免疫4次，刺激B细胞表达抗原特异性的纳米抗体。4次免疫结束后，提取50ml羊驼外周血，采用淋巴细胞分离液分离得到淋巴细胞。采用RNA提取试剂Trizol(购自Invitrogen)提取总RNA，并通过1％琼脂糖电泳检测RNA的完整性，详见图1。使用cDNA合成试剂盒(购自Invitrogen)反转录获得羊驼总cDNA。
通过2轮巢氏PCR进行扩增纳米抗体基因。第一轮巢氏PCR体系如下表1：
表1
Figure PCTCN2022128027-appb-000001
其中：
NPR-19001引物序列为：5’-CTTGGTGGTCCTGGCTGC-3’(SEQ ID NO：45)；
NPR-19002引物序列为：5’-GTACGTGCTGTTGAACTGTTCC-3’(SEQ ID NO：46)；
反应程序：98℃，52min；98℃10s，55℃30s，72℃45s，共25循环；72℃，7min；反应结束后，凝胶电泳，割胶回收700bp左右的目的VHH片段。
第二轮巢氏PCR采用常规方法设计引物和反应程序扩增目的基因片段，并进行2％琼脂糖电泳，结果如图2所示，并割胶回收500bp左右的VHH片段。
将载体与目的片段(即第2轮巢式PCR扩增和纯化后VHH片段)分别用SfiI进行酶切，50℃过夜酶切并回收。采用T4连接酶以Vector：VHH＝1∶3的连接摩尔比例进行4℃孵育16h的连接反应。将带有目的基因片段的载体采用电转化方式转化至大肠杆菌感受态E.coli TG1细胞，并计算细菌文库库容为2.55×10 9cfu。按照20倍的感染复数MOI加入辅助噬菌体M13KO7继续培养，采用PEG-NaCl标准纯化方法纯化2次，构建噬菌体文库。经鉴定噬菌体文库滴度为1.41×10 13cfu/mL。
实施例3：纳米抗体的噬菌体淘选
将hPD-L1-His抗原包被至酶标板，采用固相筛选的方案按6块96孔酶标板进行筛选和亲和富集，采用浓度为0.2M且pH＝2.2的Gly-HCl洗脱特异性结合的噬菌体，然后立即用Tris-HCl中和缓冲液中和，重复3轮淘选。采用ELISA方法筛选与人PD-L1结合的阳性克隆，对核苷酸序列进行测序。将核苷酸序列经软件翻译成氨基酸序列后对比，并按CDR3不同和进化树中氨基酸差异分为19类，每类最佳表现序列如下表2所示。
表2
Figure PCTCN2022128027-appb-000002
Figure PCTCN2022128027-appb-000003
19个纳米抗体的氨基酸序列如下表3所示。
表3
Figure PCTCN2022128027-appb-000004
Figure PCTCN2022128027-appb-000005
纳米抗体的CDR的氨基酸序列(基于IMGT编号方法)如下表4所示。
表4
Figure PCTCN2022128027-appb-000006
筛选方法如下：首先分别将hPD-L1-His和mPD-L1-hFc抗原用0.05M碳酸盐缓冲液(pH 9.6)稀释至2μg/mL，按100μL/well，4℃包被过夜；弃包被液，PBST洗涤3次，每孔加入300μL 5％脱脂牛奶，37℃封闭1h；PBST洗涤3次，加入100μL/孔的经3轮淘选的噬菌体，37℃，孵育45min；PBST洗涤5次，分别加入辣根过氧化物酶标记的羊抗Alpaca二抗(用PBS按1∶1W稀释)，100μL/孔，37℃孵育45min；BST洗板5次。每孔加入100μL TMB显色液显色，37℃反应5min；每孔加入50μL终止液终止反应，于450nm下测光密度。
实施例4：融合蛋白的制备
将编码纳米抗体的目的基因序列分别克隆到含有鼠IgG1 Fc的质粒中，随后转染到瞬转表达体系HEK293细胞进行瞬时表达，进而获得相应的分泌蛋白(即融合蛋白，VHH的C端与Fc区的N端连接)，各个融合蛋白简称分别为R1014、R1015、R1016、R1017、R1018、R1019、R1148、R1149、R1150、R1020、R1021、R1022、R1023、R1024、R1025、R1026、R1027、R1028和R1029。融合蛋白含有两条相同的肽链，各个融合蛋白的VHH序列如上述表3所示，部分融合蛋白的其中一条链的全长氨基酸序列如下表5中SEQ ID NO：38-43任一所示氨基酸序列。免疫球蛋白Fc区的其中一条CH2-CH3的氨基酸序列如SEQ ID NO：44的第120位-第346位氨基酸所示(表5斜体部分)。
Figure PCTCN2022128027-appb-000007
Figure PCTCN2022128027-appb-000008
表5
Figure PCTCN2022128027-appb-000009
Figure PCTCN2022128027-appb-000010
实施例5：融合蛋白与膜表达人PD-L1蛋白的结合活性检测
采用流式细胞仪(FCM)检测实施例4获得的融合蛋白以及实施例1获得的R0999、R0516、与细胞CHO-hPD-L1的结合情况。检测方法如下：
向2E5/孔CHO-hPD-L1细胞加入适当稀释倍数的待检测融合蛋白和对标抗体，2-8℃孵育30min后，用200μl/孔1×PBS洗涤一次后，按100μl/孔加入1∶500稀释的E-anti-hIgG荧光二抗(用含3％BSA的1×PBS稀释)，2-8℃孵育30min后，用200μl/孔1×PBS洗涤一次后，用100μl/孔1×PBS重悬，进行流式细胞术检测。
流式细胞术检测结果图3所示，结果显示：R1015、R1016、R1019、R1148、R1149、R1150的EC50分别为0.148nM、0.247nM、0.773nM、0.445nM、0.497nM、0.512nM，表明这6个融合蛋白与膜表达人PD-L1蛋白具有很好的结合活性。
实施例6：融合蛋白亲和力检测
采用生物膜干涉技术(BLI)检测实施例5获得的结合阳性的融合蛋白(R1015、R1016、R1019、R1148、R1149、R1150)以及实施例1获得对标抗体(简称R0999和R0516)、人PD-1蛋白(简称R0323)蛋白与人PD-L1蛋白(简称R0201)的亲和力。
检测方法如下：将5μg/ml的R0201固化到分子互相作用分析仪ForteBIO(Octet OK e)的Anti-Penta-HIS(HIS1K)(18-5120，Fortebio)探针上，平衡探针90s，分别与合适浓度稀释的待检测物结合180s，然后解离300s，探针于10mM甘氨酸中再生中和以后，再按照这个方法进行下个循环的平衡，结合，解离以及再生中和。所有循环转速都设定为1000rpm，实验温度为30℃。
检测结果如下表6所示，结合曲线如图4所示，结果显示：R1015、R1016、R1019、R1148、R1149、R1150与人PD-L1蛋白的亲和力高。
表6
蛋白简称 kon(1/Ms) koff(1/s) KD(M)
R1015 7.69E+05 8.85E-05 1.15E-10
R1016 8.44E+05 1.18E-04 1.40E-10
R1019 6.48E+05 ＜1.0E-07 ＜1.0E-12
R1148 8.26E+05 9.34E-05 1.13E-10
R1149 4.10E+05 7.54E-05 1.84E-10
R1150 3.94E+05 4.87E-05 1.24E-10
R0999 8.57E+05 2.20E-05 2.57E-11
R0516 4.61E+05 4.67E-05 1.01E-10
R0323 1.42E+05 2.12E-04 7.78E-08
实施例7：融合蛋白阻断膜表达人PD-L1蛋白和游离人PD-1的结合
采用流式细胞仪(FCM)检测实施例4获得的融合蛋白(R1015、R1016、R1019、R1148、R1149、R1150)以及实施例1获得的R0999、R0516、阻断细胞CHO-hPD-L1和游离人PD-1蛋白的结合情况。检测方法(A)和(B)分别如下：
检测方法(A)如下：
向2E5/孔CHO-hPD-L1细胞加入适当稀释倍数的待检测融合蛋白和对标抗体，冰上避光孵育30min，再加入hPD-1-hFc蛋白至96孔V型板(按照50μL/孔加入)，冰上避光孵育30min；离心(300g/5min)，弃上清，加入200μL FCM缓冲液洗涤一次，离心(300g/5min)，弃上清，按100μl/孔加入1∶500稀释的PE-anti-hIgG荧光二抗，冰上避光孵育30min；离心洗涤后，加入100μL 1×PBS重悬，上机检测。
检测结果如图5所示，结果显示：R1148、R1149和R1150的IC50分别为6.431nM、6.330nM和8.813nM，表明这3个融合蛋白能阻断膜表达人PD-L1蛋白和游离人PD-1的结合。
检测方法(B)如下：
向2E5/孔CHO-hPD-L1细胞加入hPD-1-hFc蛋白，冰上避光孵育30min，再加入适当稀释倍数的待检测融合蛋白和对标抗体至96孔V型板(按照50μL/孔加入)，冰上避光孵育30min；离心(300g/5min)，弃上清，加入200μL FCM缓冲液洗涤一次，离心(300g/5min)，弃上清，按100μl/孔加入1∶500稀释的PE-anti-hIgG荧光二抗，冰上避光孵育30min；离心洗涤后，加入100μL 1×PBS重悬，上机检测。
检测结果如图6所示，结果显示：R1148、R1149和R1150的IC50分别为6.072nM、10.64nM和13.98nM，表明这3个融合蛋白能阻断膜表达人PD-L1蛋白和游离人PD-1的结合。
实施例8：融合蛋白结合表位分析
采用竞争ELISA将实施例7获得的结合阳性的融合蛋白(R1148、R1149和R1150)以及实施例1获得对标抗体(简称R0999和R0516)与人PD-L1蛋白(简称R0201)的结合区域进行分组。检测方法如下：
将5μg/ml的R0201固化到分子互相作用分析仪ForteBIO(Octet OK e)的Anti-Penta-HIS(HIS1K)(18-5120，Fortebio)探针上，平衡探针90s，分别与150nM的第1抗体结合180s，然后与150nM的第1抗体结合180s，探针于10mM甘氨酸中再生中和以后，再按照这个方法进行下个循环的平衡，结合以及再生中和。
检测结果如下表7所示，结果表明：R1148、R0999和R0516表位一致；R1149和R1150表位一致，与R0999、R0516表位不一致。
表7
AB# R1148 R1149 R1150 R0999 R0516
R1148 3.07％ 107.45％ 110.88％ 0.06％ 1.30％
R1149 110.09％ 4.24％ 4.45％ 111.57％ 87.67％
R1150 109.96％ 4.48％ 4.45％ 113.42％ 88.32％
R0999 3.96％ 106.86％ 108.81％ 1.13％ 2.87％
R0516 14.32％ 110.05％ 115.77％ 9.08％ 7.79％
实施例9：融合蛋白阻断膜表达人PD-L1蛋白和游离人CD80的结合
采用流式细胞仪(FCM)检测融合蛋白(R1148、R1149和R1150)以及实施例1获得R0999、R0516、阻断细胞CHO-hPD-L1和游离人CD80蛋白的结合情况。检测方法(A)和(B)分别如下：
检测方法(A)如下：
向2E5/孔CHO-hPD-L1细胞加入适当稀释倍数的待检测融合蛋白和对标抗体，冰上避光孵育30min，再加入hCD80蛋白至96孔V型板(按照50μL/孔加入)，冰上避光孵育30min；离心(300g/5min)，弃上清，加入200μL FCM缓冲液洗涤一次，离心(300g/5min)，弃上清，按100μl/孔加入1∶500稀释的PE-anti-hIgG荧光二抗，冰上避光孵育30min；离心洗涤后，加入100μL 1×PBS重悬，上机检测。
检测结果如图7所示，结果显示：R1148、R1149和R1150能阻断膜表达人PD-L1蛋白和游离人CD80的结合，且阻断效果与对标抗体相当。
检测方法(B)如下：
向2E5/孔CHO-hPD-L1细胞加入hCD80蛋白，冰上避光孵育30min，再加入适当稀释倍数的待检测融合蛋白和对标抗体至96孔V型板(按照50μL/孔加入)，冰上避光孵育30min；离心(300g/5min)，弃上清，加入200μL FCM缓冲液洗涤一次，离心(300g/5min)，弃上清，按100μl/孔加入1∶500稀释的PE-anti-hIgG荧光二抗，冰上避光孵育30min；离心洗涤后，加入100μL 1×PBS重悬，上机检测。
检测结果如图8所示，结果显示：R1148、R1149和R1150能阻断膜表达人PD-L1蛋白和游离人CD80的结合，且阻断效果与对标抗体相当。
综上，本发明实施例构建了噬菌体展示的纳米抗体文库，采用ELISA方法筛选出与人PD-L1结合的阳性克隆，得到特异性结合PD-L1的PD-L1结合分子，如FBP002-1128、FBP002-1136、FBP002-1136、FBP002-1369、FBP002-1389、FBP002-1471、FBP002-2002、FBP002-2056、FBP002-2058、FBP002-1191、FBP002-1217、FBP002-1118、FBP002-1134、FBP002-1153、FBP002-1184、FBP002-1224、FBP002-1249、FBP002-1075、FBP002-1095、R1015、R1016、R1019、R1148、R1149、R1150、R1014、R1017、R1018、R1020、R1021、R1022、R1023、R1024、R1025、R1026、R1027、R1028、R1029，这些PD-L1结合分子能够阻断人PD-L1蛋白和游离人PD-1、游离人CD80的结合。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (20)

  1. 一种PD-L1结合分子，其特征在于，所述PD-L1结合分子包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含选自以下(i)至(vi)任一项的CDR1、CDR2和CDR3：
    (i)如SEQ ID NO：29所示的CDR1、如SEQ ID NO：30所示的CDR2、和如SEQ ID NO：31所示的CDR3；
    (ii)如SEQ ID NO：23所示的CDR1、如SEQ ID NO：24所示的CDR2、和如SEQ ID NO：25所示的CDR3；
    (iii)如SEQ ID NO：26所示的CDR1、如SEQ ID NO：27所示的CDR2、和如SEQ ID NO：28所示的CDR3；
    (iv)如SEQ ID NO：20所示的CDR1、如SEQ ID NO：21所示的CDR2、和如SEQ ID NO：22所示的CDR3；
    (v)如SEQ ID NO：32所示的CDR1、如SEQ ID NO：33所示的CDR2、和如SEQ ID NO：34所示的CDR3；或
    (vi)如SEQ ID NO：35所示的CDR1、如SEQ ID NO：36所示的CDR2、和如SEQ ID NO：37所示的CDR3。
  2. 如权利要求1所述PD-L1结合分子，其特征在于，所述免疫球蛋白单一可变结构域为VHH。
  3. 如权利要求2所述PD-L1结合分子，其特征在于，所述VHH的氨基酸序列如SEQ ID NO：7、SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO：2、SEQ ID NO：8或SEQ ID NO：9任一所示。
  4. 如权利要求1～3任一所述PD-L1结合分子，其特征在于，所述PD-L1结合分子还包含免疫球蛋白Fc区；
    任选地，所述免疫球蛋白单一可变结构域的C端与所述免疫球蛋白Fc区的N端连接。
  5. 如权利要求4所述PD-L1结合分子，其特征在于，所述免疫球蛋白Fc区的氨基酸序列如SEQ ID NO：44的第120位-第346位氨基酸所示。
  6. 如权利要求1～5任一所述PD-L1结合分子，其特征在于，所述PD-L1结合分子包括如SEQ ID NO：38-43任一所示氨基酸序列。
  7. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码权利要求1～6任一项所述的PD-L1结合分子。
  8. 一种表达载体，其特征在于，所述表达载体包含权利要求7所述的多核苷酸。
  9. 一种宿主细胞，其特征在于，所述宿主细胞包含权利要求8所述的表达载体或其基因组中整合有权利要求7的多核苷酸。
  10. 一种制备权利要求1～6任一项所述PD-L1结合分子的方法，其特征在于，其包括在允许产生所述PD-L1结合分子的条件下培养权利要求9所述的宿主细胞并回收和分离所述PD-L1结合分子。
  11. 一种噬菌体，其特征在于，所述噬菌体表面展示有权利要求1～6任一所述的PD-L1结合分子。
  12. 一种用于检测PD-L1的试剂盒，其特征在于，其包括权利要求1～6任一所述的PD-L1结合分子或权利要求11所述的噬菌体。
  13. 如权利要求12所述试剂盒，其特征在于，所述试剂盒中还包括固相载体，所述的PD-L1结合分子或噬菌体被固定于固相载体中。
  14. 如权利要求13所述试剂盒，其特征在于，所述试剂盒中还包括能与所述的PD-L1结合分子连接的可检测标记物；和/或PD-L1标准品或PD-L1偶联物标准品；和/或与可检测标记物相对应的底物；和/或酶联免疫反应试剂；
    优选地，所述的可检测标记物被连接于所述的PD-L1结合分子或分离地存在于试剂盒中。
  15. 一种检测待测样品中PD-L1的存在情况的方法，其特征在于，以权利要求1～6任一所述的PD-L1结合分子或权利要求11所述的噬菌体作为PD-L1的检测抗体，通过酶联免疫吸附测定法来检测待测样品中PD-L1的存在情况。
  16. 如权利要求15所述方法，其特征在于，其具体包括以下步骤：
    将待测样品包被于固相载体上，以携带或不携带可检测标记物的所述的PD-L1结合分子或所述的噬菌体作为检测抗体，检测PD-L1的存在情况。
  17. 一种免疫缀合物，其特征在于，其包括治疗剂和与所述治疗剂缀合的如权利要求1～6任一所述的PD-L1结合分子；
    优选地，所述治疗剂包括毒素、放射性同位素、药物或细胞毒剂。
  18. 一种双特异性或多特异性抗体，其特征在于，其包括权利要求1～6任一所述的PD-L1结合分子，以及与所述PD-L1结合分子功能性连接的具有另一种或另几种抗原结合特性的抗体或抗体片段。
  19. 一种药物组合物，其特征在于，其包括权利要求1～6任一所述的PD-L1结合分子；
    优选地，所述药物组合物包括可药用赋形剂、载体或稀释剂。
  20. 如权利要求1～6任一所述PD-L1结合分子、如权利要求17所述免疫缀合物、如权利要求18所述双特异性或多特异性抗体、以及如权利要求19所述药物组合物在制备用于治疗PD-L1介导的疾病的药物中的用途。
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