WO2018024237A1 - 抗pd-l1纳米抗体及其应用 - Google Patents

抗pd-l1纳米抗体及其应用 Download PDF

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WO2018024237A1
WO2018024237A1 PCT/CN2017/095884 CN2017095884W WO2018024237A1 WO 2018024237 A1 WO2018024237 A1 WO 2018024237A1 CN 2017095884 W CN2017095884 W CN 2017095884W WO 2018024237 A1 WO2018024237 A1 WO 2018024237A1
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nanobody
seq
antibody
cells
protein
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PCT/CN2017/095884
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French (fr)
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沈晓宁
缪小牛
刘晓林
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信达生物制药(苏州)有限公司
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Priority to KR1020187016857A priority Critical patent/KR102037016B1/ko
Priority to ES17836418T priority patent/ES2899036T3/es
Priority to EP17836418.8A priority patent/EP3369745B1/en
Priority to JP2018527234A priority patent/JP6670935B2/ja
Priority to CA3007021A priority patent/CA3007021C/en
Priority to AU2017305366A priority patent/AU2017305366B2/en
Priority to US16/068,815 priority patent/US11274153B2/en
Priority to BR112018068998-0A priority patent/BR112018068998A2/pt
Publication of WO2018024237A1 publication Critical patent/WO2018024237A1/zh

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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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • 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
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to the field of biomedical or biopharmaceutical technology, and more particularly to Nanobodies against PD-L1 and their coding sequences and uses.
  • PD-L1 Programmed death 1 ligand 1
  • CD274 is a member of the B7 family and is a ligand for PD-1.
  • PD-L1 is a type I transmembrane protein with a total of 290 amino acids, including one IgV-like region, one IgC-like region, one transmembrane hydrophobic region, and one intracellular region consisting of 30 amino acids.
  • PD-L1 has a negative regulatory effect on immune responses.
  • PD-L1 is also expressed in other tissues such as thymus, heart, placenta, etc. Endothelial cells, as well as various non-lymphoids such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, head and neck cancer.
  • PD-L1 has a broad spectrum of regulation of autoreactive T, B cells and immune tolerance, and plays a role in peripheral tissue T and B cell responses. The high expression of PD-L1 on tumor cells is associated with poor prognosis in cancer patients.
  • PD-1 Programmed death-1
  • CD279 is a member of the CD28 family. Its cytoplasmic region contains two tyrosine residues, one located near the N-terminus. In the immunoreceptor tyrosine-based inhibitory motif (ITIM), one near the C-terminus is located in the immunoreceptor tyrosine-based switch motif (ITSM).
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITMS immunoreceptor tyrosine-based switch motif
  • PD-1 is mainly expressed on the surface of activated T lymphocytes, B lymphocytes and macrophages. Under normal circumstances, PD-1 can inhibit the function of T lymphocytes and promote the function of Treg, thereby inhibiting the autoimmune response and preventing the occurrence of autoimmune diseases.
  • PD-L1 expressed by tumor cells combined with PD-1 can promote tumor immune escape by inhibiting lymphocytes.
  • the combination of PD-L1 and PD-1 can lead to a variety of biological changes, leading to immune regulation, such as inhibition of lymphocyte proliferation and activation, inhibition of CD4+ T cell differentiation into Th1 and Th17 cells, inhibition of inflammatory cytokine release Wait.
  • Nanobodies are currently the smallest antibody molecules with a molecular weight of 1/10 of that of a normal antibody.
  • nano-antibodies possess some unique functional properties, such as small molecular mass, strong stability, good solubility, easy expression, weak immunogenicity, strong penetrability and strong targeting. It is simple in humanization, low in preparation cost, etc. It almost completely overcomes the defects of long development cycle, low stability and harsh storage conditions of traditional antibodies.
  • a CDR region of a complementarity determining region of a VHH chain of an anti-PD-L1 Nanobody wherein the complementarity determining region CDR of the VHH chain is represented by CDR1, SEQ ID NO of SEQ ID NO: : CDR2 shown in 6 and CDR3 shown in SEQ ID NO: 7.
  • said CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • a VHH chain of an anti-PD-L1 Nanobody comprising a framework region FR and a complementarity determining region CDR according to the first aspect of the invention, wherein the framework region FR is
  • VHH chain of the anti-PD-L1 Nanobody is as shown in SEQ ID NO.: 8 or 14.
  • an anti-PD-L1 Nanobody which is a Nanobody directed against a PD-L1 epitope and which has the amino acid set forth in SEQ ID NO.: 8 or SEQ ID NO.: Sequence of VHH chains.
  • the present invention provides a polynucleotide encoding a protein selected from the group consisting of the CDR regions of the anti-PD-L1 Nanobody VHH chain of the first aspect of the invention, the present invention The VHH chain of the anti-PD-L1 Nanobody described in the above aspect, or the anti-PD-L1 Nanobody of the third aspect of the invention.
  • the polynucleotide has a nucleotide sequence as shown in SEQ ID NO.: 9 or 15.
  • the polynucleotide comprises DNA or RNA.
  • an expression vector comprising the polynucleotide of the fourth aspect of the invention.
  • the invention provides a host cell comprising the expression vector of the fifth aspect of the invention, or the polynucleotide of the fourth aspect of the invention integrated in the genome.
  • the host cell comprises a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of E. coli, yeast cells.
  • the anti-PD-L1 Nanobody has the amino acid sequence set forth in SEQ ID NO.: 8 or 14.
  • an immunoconjugate comprising:
  • a coupling moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  • the coupling moiety is a drug or a toxin.
  • the coupled moiety is a detectable label.
  • the conjugate is selected from the group consisting of: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or is capable of producing a detectable agent
  • Product enzymes radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, pre- A drug activating enzyme (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate comprises: a multivalent (e.g., bivalent) VHH chain of the anti-PD-L1 Nanobody of the second aspect of the invention, as described in the third aspect of the invention Anti-PD-L1 Nanobody.
  • a multivalent (e.g., bivalent) VHH chain of the anti-PD-L1 Nanobody of the second aspect of the invention as described in the third aspect of the invention Anti-PD-L1 Nanobody.
  • the multivalent protein refers to a VHH chain comprising a plurality of repeating anti-PD-L1 Nanobodies according to the second aspect of the present invention in the amino acid sequence of the immunoconjugate.
  • the anti-PD-L1 Nanobody of the third aspect of the invention refers to a VHH chain comprising a plurality of repeating anti-PD-L1 Nanobodies according to the second aspect of the present invention in the amino acid sequence of the immunoconjugate.
  • the ninth aspect of the invention provides the use of the anti-PD-L1 Nanobody of the third aspect of the invention for preparing (a) an agent for detecting a PD-L1 molecule; (b) a medicament for treating a tumor .
  • the detection comprises flow detection, cellular immunofluorescence detection.
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is in the form of an injection.
  • the pharmaceutical composition is for use in the preparation of a medicament for treating a tumor, the tumor being selected from the group consisting of The following groups: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, or bladder tumor.
  • the use is non-diagnostic and non-therapeutic.
  • a recombinant protein comprising:
  • the tag sequence includes a 6His tag and an HA tag.
  • the recombinant protein specifically binds to the PD-L1 protein.
  • a thirteenth aspect of the invention provides the use of the VHH chain according to the second aspect of the invention, the Nanobody of the third aspect of the invention, or the immunoconjugate of the eighth aspect of the invention, Used to prepare medicaments, reagents, test plates or kits;
  • the reagent, the detection plate or the kit is used for: detecting the PD-L1 protein in the sample;
  • the agent is for treating or preventing a tumor expressing a PD-L1 protein (ie, PD-L1 positive).
  • the tumor comprises: gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, or adrenal gland Tumor.
  • a fourteenth aspect of the present invention a method for detecting a PD-L1 protein in a sample, the method comprising the steps of:
  • a method for treating a disease comprising administering the nanobody of the third aspect of the invention or the immunoconjugate of the eighth aspect of the invention to a subject in need thereof.
  • the subject comprises a mammal, such as a human.
  • a framework region FR of an anti-PD-L1 Nanobody VHH chain is provided, wherein the framework region FR of the VHH chain is represented by FR1, SEQ ID NO: 2 of SEQ ID NO: FR2, FR3 shown in SEQ ID NO: 3, and FR4 shown in SEQ ID NO: 4.
  • FIG. 1 is a SDS-PAGE diagram of antigen protein and Nanobody purification.
  • A is a nucleic acid molecule standard
  • B is a purified hPD-L1 (ECD)-Fc protein
  • C is purified by TEV enzymatic digestion to remove Fc-tag protein.
  • hPD-L1 (ECD) protein is a purified PD-L1Nb-Fc protein
  • E is a biotinylated PD-1-Fc protein, all of which are expressed by HEK293F cells.
  • Fig. 2 is a storage capacity detection map of the constructed library.
  • the constructed library was diluted by gradient plating, and the number of clones was determined by taking 1/5 gradient dilution of 104 times, 105 times, and 106 times, and the number of clones was determined to determine the library size.
  • Figure 3 is a plot of the insertion rate of the constructed library, which is the result of the insertion rate of the constructed nanobody library.
  • the DNA bands of the gel pores from left to right are: the first is the DNA molecular marker, and the other is the detection insertion.
  • the PCR product of the fragment has a PCR product band of about 500 bp; after detection, the insertion rate of the library reached 95.8%.
  • Figure 4 shows the enrichment process of PD-L1 Nanobody. After the first round of panning, there was no enrichment. The second round of panning showed 4 times enrichment, and the third round of panning showed 210 times enrichment.
  • Figure 5 is a purification diagram of a strain of PD-L1 Nanobody expressed by Escherichia coli, and the Nanobody corresponding to the amino acid sequence of SEQ ID NO. 8 is purified by nickel column resin gel affinity chromatography, and the PD-L1 Nanobody is Electropherogram of SDS-PAGE. The results showed that the PD-L1 Nanobody could achieve a purity of more than 90% after the purification process.
  • Figure 6 is a blockade effect of FACS detection of PD-L1 Nanobodies, and HEK293F cells transiently expressing human full-length PD-L1 protein were co-reacted with each group of Nanobodies and biotinylated hPD-1-Fc protein.
  • Figure 7 is a purification diagram of eukaryotic expression humanized PD-L1 Nanobody, and four humanized PD-L1 Nanobodies are expressed by HEK293F cells, wherein A is a protein molecular standard, and B is an amino acid of SEQ ID NO.
  • A is a protein molecular standard
  • B is an amino acid of SEQ ID NO.
  • the humanized PD-L1Nb protein encoded by the sequence, the antibody expressed contains an Fc-tag protein, and the protein purity is over 90%.
  • Figure 8 is a blockade effect of FACS detection of humanized PD-L1 Nanobody, co-reacting with EBC-1 cells naturally expressing PD-L1 protein with humanized Nanobody and biotinylated hPD-1-Fc protein, It can be seen that the binding rate of PD-1-Fc-biotin to EBC-1 cells in the blank group and the negative control group was above 90%, and PD-1-Fc was added after the addition of PD-L1 Nanobody and humanized Nanobody. - The binding rate of biotin to EBC-1 cells was only below 10%, indicating that the added Nanobody can significantly block the interaction of PD-1 with PD-L1.
  • Figure 9 shows the results of affinity determination of PD-L1 Nanobody.
  • the affinity of PD-L1 Nanobody was determined by BiaCore T200. It can be seen that the affinity of humanized pre-humanized pre-humanized antibody was 2.34 ⁇ 10 -9 M, after humanization. The affinity of the Nanobody was 2.26 x 10 -9 M. Humanization did not affect the affinity of the antibody.
  • Figure 10 is the specific result of ELISA for detection of PD-L1 Nanobody, showing PD-L1 nano before and after humanization.
  • the rice antibody only interacts with human-derived and monkey-derived PD-L1, and does not interact with the murine source and other members of the PD-L1 family.
  • the two Nanobodies have better specificity.
  • Figure 11 shows the MOA assay for the inhibition of PD-1/PD-L1 interaction by Nanobodies, in which the Nanobody before humanization is substantially more active than the positive control antibody, and the Nanobody of the present invention after humanization The activity was comparable to the positive control group.
  • Figure 12 shows that both the Nanobody of the present invention and the humanized engineered antibody can effectively activate T cells, and the activation effect is similar to that of the positive control antibody.
  • Figure 13 shows the mode of administration in the study of the nanobody antibody tumor suppressor activity of the present invention.
  • Figure 14 shows that mice immunized with humanized Nb-Fc showed a good control of tumor volume relative to the control group, and there was no significant increase, indicating that humanized Nb-Fc has significant tumor suppression. effect.
  • Figure 15 shows that the humanized Nb-Fc of the invention has superior solubility to the control antibody.
  • Figure 16 shows that there is no significant change in the purity of humanized Nb-Fc.
  • Figure 17 shows that there is no significant change in the binding of humanized Nb-Fc and CHO-PDL1 cells.
  • the inventors have successfully obtained a class of anti-PD-L1 Nanobodies through extensive screening through extensive and in-depth research.
  • the experimental results show that the PD-L1 Nanobodies obtained by the present invention can effectively block PD-L1 and PD-1.
  • the interaction between the two, surprisingly, the PD-L1 Nanobody after humanization of the present invention is more effective in blocking the binding of PD-L1 to PD-1, and is identified by BiaCore T200, after humanization.
  • the PD-L1 Nanobody has high affinity, good stability, and significant tumor inhibition. The present invention has been completed on this basis.
  • the present invention immunizes a camel using a human PD-L1 antigen protein to obtain a high-quality immuno Nanobody gene library.
  • the PD-L1 protein molecule is then coupled to the ELISA plate to display the correct spatial structure of the PD-L1 protein.
  • the antigen in this form is screened for the immuno Nanobody gene library using the phage display technology (Camel Heavy Chain Antibody Phage Display Gene Bank) Thereby, a Nano-antibody gene specific for PD-L1 was obtained. This gene was further transferred to Escherichia coli to obtain a highly specific Nanobody strain which was highly expressed in Escherichia coli.
  • Nanobody of the invention As used herein, the terms " Nanobody of the invention”, “anti-PD-L1 Nanobody of the invention”, “PD-L1 Nanobody of the invention” are used interchangeably and refer to both specific recognition and binding to PD-L1 ( Nanobodies including human PD-L1). Particularly preferred is the amino acid sequence of the VHH chain as shown in SEQ ID NO.: 8 or 14.
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; a constant region of the light chain and The first constant region of the heavy chain is opposite, and the variable region of the light chain is opposite the variable region of the heavy chain.
  • VL variable region
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • single domain antibody VHH
  • nanobody a single domain antibody consisting of only one heavy chain variable region.
  • VHH single domain antibody
  • CH1 light chain and heavy chain constant region 1
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules that are combined with the antibodies or fragments thereof of the invention to form Conjugate.
  • the present invention also encompasses cell surface markers or antigens that bind to the anti-PD-L1 protein antibody or fragment thereof.
  • variable region are used interchangeably with “complementarity determining region (CDR).
  • the heavy chain variable region of the antibody comprises three complementarity determining regions, CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.
  • the terms "antibody of the invention”, “protein of the invention”, or “polypeptide of the invention” are used interchangeably and refer to a polypeptide which specifically binds to a PD-L1 protein, such as a protein having a heavy chain variable region. Or a polypeptide. They may or may not contain an initial methionine.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the invention encompasses any protein or protein conjugate having a heavy chain comprising a variable region and a fusion expression product (ie, an immunoconjugate and a fusion expression product), so long as the variable region is linked to the heavy chain of an antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain,
  • the variable region (CDR) is divided into four framework regions (FR), and the amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction.
  • These CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen.
  • the invention includes those molecules having an antibody heavy chain variable region with a CDR, as long as the CDRs thereof have 90% or more (preferably 95% or more, optimally 98% or more) homology to the CDRs identified herein. Sex.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of an antibody of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed by the 6
  • the antibody of the present invention refers to a polypeptide comprising the above CDR regions having PD-L1 protein binding activity.
  • the term also encompasses variant forms of a polypeptide comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include, but are not limited to, one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained using an antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof.
  • the invention also includes fragments of the Nanobodies of the invention.
  • the fragment will have at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
  • “conservative variant of the antibody of the present invention” means having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3, compared to the amino acid sequence of the antibody of the present invention. Amino acid A similar or similar amino acid is replaced to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the present invention also provides a polynucleotide molecule encoding the above antibody or a fragment thereof or a fusion protein thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • One possible method is to synthesize related sequences by artificial synthesis, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecule (nucleic acid, protein, etc.) to which the present invention relates includes biomolecules existing in an isolated form.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention may be used alone or in combination or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or capable of producing detectable agents The enzyme of the product.
  • Therapeutic agents that can be bound or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides; 2. biotoxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; Particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents ( For example, cisplatin) or any form of nanoparticles, and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for binding to a PD-L1 protein molecule, and thus can be used for treating a tumor.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-described Nanobody of the present invention (or a conjugate thereof) and pharmaceutically An acceptable carrier or excipient.
  • a safe and effective amount e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram body weight to about 50 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the Nanobody is provided with a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label or a fluorescent label.
  • the colloidal gold label can be carried out by methods known to those skilled in the art.
  • the anti-PD-L1 Nanobody is labeled with colloidal gold to obtain a colloidal gold-labeled Nanobody.
  • the anti-PD-L1 Nanobody of the present invention has good specificity and high potency.
  • the invention also relates to methods of detecting PD-L1 protein.
  • the method steps are substantially as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of PD-L1 protein in the dissolved sample.
  • the sample to be used is not particularly limited, and a representative example is a cell-containing sample present in the cell preservation solution.
  • the present invention also provides a kit comprising the antibody (or a fragment thereof) or a test plate of the present invention.
  • the kit further comprises a container, instructions for use, a buffer, and the like.
  • the present invention also provides a detection kit for detecting PD-L1 levels, the kit comprising an antibody recognizing a PD-L1 protein, a lysis medium for dissolving a sample, and a detection of a desired universal reagent and a buffer, such as various Buffer, detection mark, substrate detection, etc.
  • the test kit can be an in vitro diagnostic device.
  • the Nanobody of the present invention has a wide range of biological application value and clinical application value, and its application relates to various fields such as diagnosis and treatment of diseases related to PD-L1, basic medical research, and biological research.
  • a preferred application is for clinical diagnosis and targeted therapy for PD-L1.
  • Nanobody of the present invention is highly specific for a human PD-L1 protein having the correct spatial structure.
  • the nanobody of the present invention has a strong affinity.
  • the plasmid was mixed with the transfection reagent PEI 1:3 and allowed to stand for 20 min, then added to HEK293F cells, and cultured for 5-6 days at 37 ° C in a 6% CO 2 shaker incubator;
  • hPD-L1(ECD)-Fc protein was digested, and 1 mg of hPD-L1(ECD)-Fc protein was digested with 0.1 mg of TEV, and digested at 4 °C for 16 hours to flow the protein solution through the nickel.
  • the column was passed through a Protein A column, and the effluent was collected and sampled for SDS-PAGE detection (the results are shown in Figure 1C).
  • the ligation product was transformed into electroporation competent cell TG1, and the PD-L1 nanobody library was constructed and the storage capacity was determined.
  • the storage capacity was 1.3 ⁇ 10 9 CF ⁇ (the results are shown in Fig. 2).
  • a crude antibody was obtained by an infiltration method, and the antibody was transferred to an antigen-coated ELISA plate and allowed to stand at room temperature for 1 hour.
  • Example 4 Nanobody expression and purification in host strain Escherichia coli:
  • the plasmid of the clone obtained by the previous sequencing analysis was electrotransformed into Escherichia coli WK6, and coated on LA+Glucose, a culture plate containing ampicillin and glucose, and cultured at 37 ° C overnight;
  • the nano-antibody with a purity of more than 90% was prepared by nickel column ion affinity chromatography, and the purification result is shown in FIG. 5 .
  • HPD-1-Fc-Biotin protein Preparation of hPD-1-Fc-Biotin protein (HPD-1-Fc preparation method is the same as in Example 1, SDS-PAGE detection results are shown in Figure 1E), and the method of protein biotin refers to the biotin reagent specification;
  • the ProtSA server is used to calculate the solvent contactability of the residue, that is, the ratio of the folded state of the residue to the solvent contactable area of the unfolded state is greater than 40%.
  • the residue is a residue that is exposed to the solvent;
  • CHOK1-PDL1 cells were plated: CHI TD-PDL1 was passaged 1-2 days before. The culture supernatant was discarded and washed with PBS. Add appropriate amount of trypsin to digest at 37 ° C / 5% CO 2 for 3-5 min. Add 4 times trypsin volume to the medium, transfer the cells to a 50 ml centrifuge tube and count. Take the required volume of cells, 230g, and centrifuge for 10min. The medium was added and the cells were resuspended to 4 x 10 5 cells/mL. The cells were added to a 96-well white cell culture plate at 100 ⁇ l/well. The side holes were added with PBS, 200 ⁇ l/well. The cells were cultured overnight in a 37 ° C / 5% CO 2 incubator.
  • Bio-GloTM buffer was melted in advance, added to Bio-GloTM substrate, and mixed. After 6 hours, Bio-GloTM Reagent was added at 80 ⁇ l/well. Leave at room temperature for 5-10 min. reading.
  • Example 7 Expression and purification of humanized PD-L1 Nanobody in eukaryotic cell HEK293
  • the humanized and humanized PD-L1Nb sequence was synthesized into pFUSE-IgG1 vector (purchased from Invivogen), and pFUSE-IgG1-Nb (humanized) plasmid was extracted with Omega plasmid extract kit. ;
  • the plasmid was mixed with the transfection reagent PEI 1:3 and allowed to stand for 20 min, then added to HEK293F cells, and cultured in a 6% CO 2 shaker incubator at 37 ° C for 5-6 days;
  • Example 8 Detection of blocking function of humanized PD-L1 Nanobody by flow cytometry
  • Chip regeneration Regeneration was carried out by rinsing with 10 mM Glycine for the next antibody assay.
  • Example 10 Identification of the specificity of purified Nanobodies by enzyme-linked immunosorbent assay (ELISA):
  • the Nanobody before and after humanization can interact with PD-L1 of human and monkey sources, but not with the PD-L1 of mouse source.
  • the two antibodies have better species specificity; and the Nanobodies before and after humanization do not interact with the PD-L1 family members, and have good family specificity.
  • PBMC separation Take 50 ml of fresh blood from the donor, add 2.5 times PBS, gently add to 1 pipe of FiColl (Thermo), 4 tubes, 400 g, centrifuge for 30 min, stop at 0 deceleration. The middle white strip was pipetted into PBS (Gibco) and washed twice with PBS.
  • DC cell isolation 5 ml of T cell culture medium was added to the isolated PBMC cells, cultured at 37 ° C, 6% CO 2 , adherent culture for 2 hr, and the suspension cell suspension was aspirated for CD4+ cell separation, and the remaining cells were added with 3 ml of DC medium. After 2 days of culture, 3 ml of DC medium was added, and then cultured for 5 days, then rTNFa (R&D Systems) (1000 U/ml), IL-1b (R&D Systems) (5 ng/ml), IL-6 (R&D Systems) (10 ng) were added. /ml) and 1 ⁇ M PGE2 (Tocris) were cultured for 2 days as DC cells for lymphocyte mixed reaction (MLR).
  • MLR lymphocyte mixed reaction
  • CD4+ cell isolation PBMC was statically cultured for 2 hr, the suspended cell liquid was aspirated into a 15 ml centrifuge tube, centrifuged at 200 g for 10 min, and 500 ⁇ l of the separation solution, 100 ⁇ l of AB type serum, 100 ⁇ l of purified antibody were resuspended in the pellet, and incubated at 4 ° C for 20 min. Wash once with the separation solution, add 500 ⁇ l Bead Buffer for 15 min, remove the Bead in the magnetic field, wash the T cell medium once, resuspend in 8 ml medium, and incubate at 37 ° C, 6% CO 2 . (Operation in accordance with the 'Human CD4+T cell Enrichment Kit' (19052, Stemcell) instructions).
  • MLR experiment Mix mature DC cells with CD4+ cells, volume 200 ⁇ l per well, 10000 DC cells, 100000 CD4+ cells, add antibodies, DC, T cells, MLR as negative control, DC+T cells+ Anti-CD3/CD28 magnetic beads were used as positive controls for 5 days in mixed culture.
  • the cisbio kit Human IL2 Kit 1000 Test, Human IFN gamma 1000 test was used to detect IL2 and IFN-gamma concentrations.
  • the Nanobody of the present invention stimulated the donor to produce higher cytokines than the positive control, and after humanization, the cytokine produced was comparable to the positive control. Therefore, both the Nanobody of the present invention and the humanized antibody can effectively activate T cells, and the activation effect is similar to that of the positive control antibody.
  • MC38 cells (MC38-PDL1) expressing human PD-L1 (Nanjing Yinhe Co., Ltd.) were used in PD-L1 transfection.
  • the gene mouse measures the anti-tumor effect of humanized Nb-Fc.
  • the MC38-PDL1 tumor-bearing mouse model was first established by subcutaneous vaccination. After tumor formation, different antibodies (see Example 5) and different doses were administered. The tumor volume and body weight of each group were monitored during the administration. The frequency of administration was 2 times/week, the monitoring frequency was 2 times/week, and the monitoring was continued for 5 weeks.
  • the dosage and manner of administration were as shown in Table 5 and FIG.
  • MC38/PD-L1 cell suspension preparation MC38 cells were dispersed in PBS (1 ⁇ ), and the cell density was 1 ⁇ 10 7 cells/ml to prepare MC38 cell suspension;
  • mice 25 C57Bl/6 background PD-L1 mice were shaved on the right side, and subcutaneously injected with MC38/PD-L1 cells 1 ⁇ 10 6 / 0.1 ml / piece. 6 days after tumor cell inoculation the tumor volume of each mouse is detected, selected tumor volume in the range of 25 mice 87.4mm 3 ⁇ 228.4mm 3 by tumor volume average packet.
  • mice inoculated with humanized Nb-Fc had a good control of the tumor volume relative to the control group, and there was no significant increase in the case.
  • the sourced Nb-Fc has a significant tumor suppressive effect.
  • Ll, 73.3 ⁇ l, 80 ⁇ l, 86.7 ⁇ l, 93.3 ⁇ l, 100 ⁇ l, 106.7 ⁇ l, IBI301 Buffer was made up to a total volume of 200 ⁇ l.
  • the PEG concentration gradients were 4%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, respectively.
  • the long-term thermal stability of the antibody was evaluated by detecting changes in the purity and biological activity of the antibody (see Example 5) at 40 ° C for 30 days.
  • the purity of the antibody of interest after standing at 40 ° C for 0, 14 and 30 days was measured by the method of SEC.
  • the purity of the humanized Nb-Fc was not significantly changed.
  • the FACS method was used to detect the binding of accelerated stability test samples and CHO-PDL1 cells. The steps are as follows:

Abstract

一种针对人程序性死亡因子PD-L1的纳米抗体，该抗体具有阻断PD-L1与受体PD-1结合的功能。公开了该纳米抗体及编码该纳米抗体的基因序列，相应的表达载体和能够表达该纳米抗体的宿主细胞，以及该纳米抗体的生产方法。同时还公开了人源化的PD-L1纳米抗体序列，人源化后的纳米抗体仍然具有阻断PD-L1与PD-1结合的功能、较高的亲和力及较好的特异性。

Description

抗PD-L1纳米抗体及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及针对PD-L1的纳米抗体及其编码序列和用途。
背景技术
程序性死亡因子1配体1(programmed death 1ligand 1，PD-L1)又称CD274，为B7家族成员，是PD-1的配体。PD-L1属于I型跨膜蛋白，共290个氨基酸，包含1个IgV样区、1个IgC样区、1个跨膜疏水区和1个由30个氨基酸组成的胞内区。
与其他B7家族分子不同的是，PD-L1具有负向调节免疫应答的作用。研究发现，PD-L1主要表达于活化的T细胞、B细胞、巨噬细胞和树突状细胞等，除淋巴细胞外，PD-L1也表达于其他多种组织如胸腺、心脏、胎盘等的内皮细胞，以及各类非淋巴系如黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌、头颈癌等。PD-L1在调节自身反应性T、B细胞和免疫耐受方面具有一定广泛性，并且在外周组织T和B细胞应答起作用。PD-L1在肿瘤细胞上的高表达与癌症患者的不良预后相关。
与PD-L1相结合的程序性死亡因子1(programmed death-1，PD-1)又称CD279，是CD28家族成员，其胞质区含有2个酪氨酸残基，靠近N端的1个位于免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif，ITIM)中，靠近C端的1个位于免疫受体酪氨酸转化基序(immunoreceptor tyrosine-based switch motif，ITSM)中。PD-1主要表达在活化的T淋巴细胞、B淋巴细胞和巨噬细胞表面。在正常情况下，PD-1能够抑制T淋巴细胞的功能，促进Treg的功能，从而抑制自身免疫应答，防止自身免疫性疾病的发生。但在肿瘤的发生中，肿瘤细胞表达的PD-L1与PD-1结合后却能通过对淋巴细胞的抑制性作用促进肿瘤的免疫逃逸。PD-L1与PD-1的结合可导致多种生物学变化，引起免疫调控，如能够抑制淋巴细胞的增殖和活化、抑制CD4+T细胞向Th1和Th17细胞分化、抑制炎性细胞因子的释放等。
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。
然而，目前本领域尚缺乏令人满意的针对PD-L1的纳米抗体。因此，本领域迫切 需要开发新的有效针对PD-L1的特异性纳米抗体。
发明内容
本发明的目的就是提供一类有效针对PD-L1的特异性纳米抗体。
在本发明的第一方面，提供了一种种抗PD-L1纳米抗体VHH链的互补决定区CDR区，所述VHH链的互补决定区CDR由SEQ ID NO：5所示的CDR1、SEQ ID NO：6所示的CDR2，SEQ ID NO：7所示的CDR3组成。
在另一优选例中，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
本发明第二方面，提供了一种抗PD-L1纳米抗体的VHH链，所述VHH链包括框架区FR和本发明第一方面所述的互补决定区CDR，所述的框架区FR由
(a)SEQ ID NO：1所示的FR1，SEQ ID NO：2所示的FR2，SEQ ID NO：3所示的FR3，SEQ ID NO：4所示的FR4组成；或
(b)SEQ ID NO：10所示的FR1，SEQ ID NO：11所示的FR2，SEQ ID NO：12所示的FR3，SEQ ID NO：13所示的FR4组成。
在另一优选例中，所述的抗PD-L1纳米抗体的VHH链如SEQ ID NO.:8或14所示。
本发明第三方面，提供了一种抗PD-L1纳米抗体，它是针对PD-L1表位的纳米抗体，并且具有如SEQ ID NO.:8或SEQ ID NO.:14中所示的氨基酸序列的VHH链。
本发明第四方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：本发明第一方面所述的抗PD-L1纳米抗体VHH链的CDR区、本发明第二方面所述的抗PD-L1纳米抗体的VHH链、或本发明第三方面所述的抗PD-L1纳米抗体。
在另一优选例中，所述多核苷酸具有如SEQ ID NO.:9或15所示的核苷酸序列。
在另一优选例中，所述的多核苷酸包括DNA或RNA。
本发明第五方面，提供了一种表达载体，所述表达载体含有本发明第四方面所述的多核苷酸。
本发明第六方面，提供了一种宿主细胞，所述宿主细胞含有本发明第五方面所述的表达载体，或其基因组中整合有本发明第四方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞。
本发明七方面，提供了一种产生抗PD-L1纳米抗体的方法，包括步骤：
(a)在适合产生纳米抗体的条件下，培养本发明第六方面所述的宿主细胞，从而 获得含所述抗PD-L1纳米抗体的培养物；以及
(b)从所述培养物中分离或回收所述的抗PD-L1纳米抗体。
在另一优选例中，所述的抗PD-L1纳米抗体具有如SEQ ID NO.:8或14所示的氨基酸序列。
本发明第八方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第二方面所述的抗PD-L1纳米抗体的VHH链、或如本发明第三方面所述的抗PD-L1纳米抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第二方面所述的抗PD-L1纳米抗体的VHH链、如本发明第三方面所述的抗PD-L1纳米抗体。
在另一优选例中，所述多价是指，在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第二方面所述的抗PD-L1纳米抗体的VHH链、本发明第三方面所述的抗PD-L1纳米抗体。
本发明第九方面，提供了本发明第三方面所述的抗PD-L1纳米抗体的用途，用于制备(a)用于检测PD-L1分子的试剂；(b)用于治疗肿瘤的药物。
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测。
本发明第十方面，提供了一种药物组合物，含有：
(i)本发明第一方面抗PD-L1纳米抗体VHH链的互补决定区CDR、本发明第二方面所述的抗PD-L1纳米抗体的VHH链、或如本发明第三方面所述的抗PD-L1纳米抗体、或本发明第八方面所述的免疫偶联物；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备治疗肿瘤的药物，所述的肿瘤选自 下组：胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。
本发明第十一方面，提供了本发明第三方面所述抗PD-L1纳米抗体的一种或多种的用途：
(i)用于检测人PD-L1分子；
(ii)用于流式检测；
(iii)用于细胞免疫荧光检测；
(iv)用于治疗肿瘤；
(v)用于肿瘤诊断。
在另一优选例中，所述用途为非诊断的和非治疗的。
本发明第十二方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第二方面所述的重链可变区VHH的序列或如本发明第三方面所述的纳米抗体的序列；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签和HA标签
在另一优选例中，所述的重组蛋白特异性结合于PD-L1蛋白。
本发明第十三方面，提供了如本发明第二方面所述的VHH链、如本发明第三方面所述的纳米抗体、或本发明第八方面所述的免疫偶联物的用途，它们被用于制备药剂、试剂、检测板或试剂盒；
其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1蛋白；
其中，所述药剂用于治疗或预防表达PD-L1蛋白(即PD-L1阳性)的肿瘤。
在另一优选例中，所述肿瘤包括：胃癌、淋巴瘤、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、或肾上腺肿瘤。
本发明第十四方面，提供了一种检测样品中PD-L1蛋白的方法，所述方法包括步骤：
(1)将样品与本发明第三方面所述的纳米抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在PD-L1蛋白。
本发明第十五方面，提供了一种治疗疾病的方法，所述方法包括，给需要的对象施用本发明第三方面所述的纳米抗体或本发明第八方面所述的免疫偶联物。
在另一优选例中，所述的对象包括哺乳动物，如人。
本发明第十六方面，提供了一种抗PD-L1纳米抗体VHH链的框架区FR，所述的VHH链的框架区FR由SEQ ID NO：1所示的FR1，SEQ ID NO：2所示的FR2，SEQ ID NO：3所示的FR3，SEQ ID NO：4所示的FR4组成。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1是抗原蛋白及纳米抗体纯化SDS-PAGE图，图中A为核酸分子标准，B为纯化的hPD-L1(ECD)-Fc蛋白，C为用TEV酶酶切去除Fc标签蛋白后纯化的hPD-L1(ECD)蛋白，D为表达纯化的PD-L1Nb-Fc蛋白，E为生物素化的PD-1-Fc蛋白，以上蛋白均由HEK293F细胞表达。
图2是构建文库的库容检测图，构建好的文库被梯度稀释后涂板，图中显示取1/5梯度稀释104倍、105倍、106倍的克隆数目，计算单克隆数确定文库大小。
图3是构建文库的插入率检测图，是构建的纳米抗体文库的插入率检测结果，从左到右凝胶孔的DNA条带分别是：第一道为DNA分子标记，其余孔道为检测插入片段的PCR产物，PCR产物带约为500bp；经检测，该文库的插入率达到95.8％。
图4是PD-L1纳米抗体筛选富集过程，经第一轮淘选后未出现富集，第二轮淘选出现4倍富集，第三轮淘选出现210倍富集。
图5是1株由大肠杆菌表达PD-L1纳米抗体的纯化图，对应SEQ ID NO.8氨基酸序列的纳米抗体，是经镍柱树脂凝胶亲和层析纯化后，PD-L1纳米抗体的SDS-PAGE的电泳图。结果显示，PD-L1纳米抗体经过该纯化过程，其纯度可达到90％以上。
图6是FACS检测PD-L1纳米抗体的阻断效果，用瞬转表达人全长PD-L1蛋白的HEK293F细胞与各组纳米抗体和生物素化的hPD-1-Fc蛋白共反应。
图7是真核表达人源化PD-L1纳米抗体纯化图，由HEK293F细胞表达四种人源化的PD-L1纳米抗体，其中A为蛋白分子标准，B、为由SEQ ID NO.10氨基酸序列编码的humanized PD-L1Nb蛋白，表达的该抗体含有Fc标签蛋白，蛋白纯度达到90％以上。
图8是FACS检测人源化PD-L1纳米抗体的阻断效果，用天然表达PD-L1蛋白的EBC-1细胞与人源化纳米抗体和生物素化的hPD-1-Fc蛋白共反应，可见由空白组及阴性对照组中PD-1-Fc-生物素与EBC-1细胞的结合率在90％以上，而加入PD-L1纳米抗体和人源化纳米抗体后，PD-1-Fc-生物素与EBC-1细胞的结合率仅在10％以下，这表明加入的纳米抗体能够明显阻断PD-1与PD-L1的相互作用。
图9是PD-L1纳米抗体亲和力测定结果，用BiaCore T200测定PD-L1纳米抗体的亲和力，可见人源化前人源化前纳米抗体的亲和力为2.34×10-9M，人源化后的纳米抗体的亲和力为2.26×10-9M。人源化改造并未影响抗体的亲和力。
图10是ELISA检测PD-L1纳米抗体的特异性结果，可见人源化前后的PD-L1纳 米抗体只与人源及猴源的PD-L1相互作用，不与鼠源及PD-L1家族其他成员发生相互作用，两株纳米抗体具有较好的特异性。
图11显示了MOA法检测纳米抗体对PD-1/PD-L1相互作用的抑制作用，其中人源化前的纳米抗体基本都比阳性对照组抗体活性强，人源化后本发明纳米抗体的活性则与阳性对照组相当。
图12显示了本发明的纳米抗体以及人源化改造后的抗体均可有效激活T细胞，且激活效果和阳性对照组抗体相似。
图13显示了本发明纳米抗体肿瘤抑制活性研究中的给药方式。
图14显示了接种了人源化Nb-Fc的小鼠，其肿瘤体积相对于对照组得到了很好的控制，并没有出现显著增加的情况，说明人源化Nb-Fc有明显的肿瘤抑制作用。
图15显示了本发明人源化Nb-Fc具有和对照抗体更优的可溶性。
图16显示人源化Nb-Fc的纯度没有明显变化。
图17显示人源化Nb-Fc和CHO-PDL1细胞的结合没有明显变化。
具体实施方式
本发明人通过广泛而深入的研究，经过大量的筛选，成功获得一类抗PD-L1纳米抗体，实验结果表明，本发明获得的PD-L1纳米抗体能够有效阻断PD-L1与PD-1之间的相互作用，令人意外的是，经本发明人源化后的PD-L1纳米抗体更能够有效阻断PD-L1与PD-1的结合，且经BiaCore T200鉴定，人源化后的PD-L1纳米抗体亲和力高，稳定性好，且肿瘤抑制作用显著。在此基础上完成了本发明。
具体地，本发明利用人源的PD-L1抗原蛋白免疫骆驼，获得高质量的免疫纳米抗体基因文库。然后将PD-L1蛋白分子偶联在酶标板上，展示PD-L1蛋白的正确空间结构，以此形式的抗原利用噬菌体展示技术筛选免疫纳米抗体基因库(骆驼重链抗体噬菌体展示基因库)，从而获得了PD-L1特异性的纳米抗体基因。再将此基因转至大肠杆菌中，从而获得了能在大肠杆菌中高效表达的、且特异性高的纳米抗体株。
如本文所用，术语“本发明纳米抗体”、“本发明的抗PD-L1纳米抗体”、“本发明PD－L1纳米抗体”可互换使用，均指特异性识别和结合于PD-L1(包括人PD-L1)的纳米抗体。特别优选的是VHH链的氨基酸序列如SEQ ID NO.:8或14所示的纳米抗体。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与 重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体(VHH)”、“纳米抗体”(nanobody)具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的抗PD-L1蛋白抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合PD-L1蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为 可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有PD-L1蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含纳米抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明纳米抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相 似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测 产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合PD-L1蛋白分子，因而可用于治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的纳米抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
标记的纳米抗体
在本发明的一个优选例中，所述纳米抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，抗PD-L1的纳米抗体用胶体金标记，得到胶体金标记的纳米抗体。
本发明的抗PD-L1纳米抗体具有很好的特异性，很高的效价。
检测方法
本发明还涉及检测PD-L1蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中PD-L1蛋白的水平。本
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测PD-L1水平的检测试剂盒，该试剂盒包括识别PD-L1蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的纳米抗体有广泛生物应用价值和临床应用价值，其应用涉及到与PD-L1相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对PD-L1的临床诊断和靶向治疗。
本发明的主要优点包括：
(a)本发明纳米抗体高特异性针对人的具有正确空间结构的PD-L1蛋白。
(b)本发明纳米抗体的亲和力强。
(c)本发明纳米抗体的生产简便。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：人PD-L1蛋白的表达纯化
(1)将人PD-L1的核苷酸序列合成在pCDNA3.1(-)载体(购自Invivogen)上，然后将其胞外段序列亚克隆至pFUSE-IgG1载体(购自Invitrogen)上，其中在hPD-L1(ECD)的C端引入一个TEV酶切位点，便于制备无Fc标签的hPD-L1(ECD)蛋白；
(2)用Omega质粒大提试剂盒提取构建好的pFΜSE-IgG1-hPD-L1(ECD)质粒；
(3)培养HEK293F细胞至OD为2.0×106个/mL；
(4)将质粒与转染试剂PEI 1:3混合均匀后静置20min，然后加入到HEK293F细胞中，37℃，6％CO2摇床培养箱中培养5-6天；
(5)收集细胞上清，与Protein A珠子在室温下结合1小时；
(6)用磷酸盐缓冲液pH7.0洗涤珠子后，再用0.1M pH3.0Glycine洗脱蛋白；
(7)将洗脱的蛋白超滤至PBS中，测定产量后取样进行SDS-PAGE检测(检测结果如图1B所示)，其余蛋白保存于-80℃冰箱；
(8)将表达的hPD-L1(ECD)-Fc蛋白进行酶切，用0.1mg TEV酶酶切1mg hPD-L1(ECD)-Fc蛋白，4℃酶切16小时，将蛋白液流过镍柱，再流过Protein A柱子，收集流出液，取样进行SDS-PAGE检测(检测结果如图1C所示)。
实施例2：PD-L1纳米抗体文库的构建
(1)将1mg hPD-L1(ECD)-Fc抗原与弗氏佐剂等体积混合，免疫一只新疆双峰驼，每周一次，共免疫7次，刺激B细胞表达抗原特异性的纳米抗体；
(2)7次免疫结束后，提取100mL骆驼外周血淋巴细胞并提取总RNA；
(3)合成cDNA并利用套式PCR扩增VHH；
(4)利用限制性内切酶PstI及NotI酶切20μg pMECS噬菌体展示载体(购自Biovector)及10μg VHH并连接两个片段；
(5)将连接产物转化至电转感受态细胞TG1中，构建PD-L1纳米抗体文库并测定库容，库容大小为1.3×109CFΜ(结果如图2所示)。
与此同时，随机挑取24颗克隆进行菌落PCR检测，结果表明所建文库的插入率为100％，图3显示菌落PCR结果。
实施例3：PD-L1纳米抗体的筛选及鉴定
抗体筛选：
(1)将溶解在100mM NaHCO3、pH 8.2中的10μg hPD-L1(ECD)抗原偶联在NUNC酶标板上，4℃放置过夜；
(2)第二天加入100μL 0.1％BSA，室温封闭2h；
(3)2h后，加入100μL噬菌体(2×1011CFΜ免疫骆驼纳米抗体噬菌展示基因库)，室温作用1h；
(4)用0.05％PBS+Tween-20洗5遍，以洗掉非特异的噬菌体；
(5)用100mM三乙醇胺将与PD-L1特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1细胞，37℃培养1h，产生并纯化噬菌体用于下一轮的筛选，相同筛选过程重复3轮，其富集结果如图4所示。
用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆：
(1)从上述2-3轮筛选后含有噬菌体的细胞培养皿中，挑选96个单个菌落并接种 于含有100μg/mL的氨苄青霉素的TB培养基(1升TB培养基中含有2.3g KH2PO4，12.52g K2HPO4，12g蛋白胨，24g酵母提取物，4mL甘油)中，生长至对数期后，加终浓度1mM的IPTG，28℃培养过夜。
(2)利用渗透法获得粗提抗体，并将抗体转移到经抗原包被的ELISA板中，在室温下放置1小时。
(3)用PBST洗去未结合的抗体，加入抗鼠抗HA抗体(购自北京康为世纪生物科技有限公司)，在室温下放置1小时。
(4)用PBST洗去未结合的抗体，加入山羊抗小鼠碱性磷酸酶标记抗体，在室温下放置1小时。
(5)用PBST洗去未结合的抗体，加入碱性磷酸酶显色液，于ELISA仪上，在405nm波长，读取吸收值。
(6)当样品孔OD值大于对照孔OD值3倍以上时(Ratio+/-＞3)，判为阳性克隆孔。
(7)将阳性克隆孔的菌转摇在含有100μg/mL的LB液体中以便提取质粒并进行测序。
实施例4：纳米抗体在宿主菌大肠杆菌中表达、纯化：
(1)将前面测序分析所获克隆株的质粒电转化到大肠杆菌WK6中，并将其涂布在LA+Glucose即含有氨苄青霉素和葡萄糖的培养平板上，37℃培养过夜；
(2)挑选单个菌落接种在5mL含有氨苄青霉素的LB培养液中，37℃摇床培养过夜；
(3)接种1mL的过夜菌种至330mL TB培养液中，37℃摇床培养，培养到OD值达到0.6-1时，加入IPTG，28℃摇床培养过夜；
(4)离心，收菌；
(5)利用渗透法，获得抗体粗提液；
(6)经镍柱离子亲和层析制备纯度达90％以上的纳米抗体，纯化结果如图5所示。
实施例5：流式细胞术检测纳米抗体的阻断功能
(1)制备hPD-1-Fc-Biotin蛋白(hPD-1-Fc制备方法同实施例1，SDS-PAGE检测结果如图1E所示)，蛋白生物素的方法参照生物素试剂说明书；
(2)每个样品取1×106个瞬转表达人PD-L1全长蛋白的HEK293F细胞重悬于0.5％BSA-PBS缓冲液中，加入上述纯化的PD-L1纳米抗体10μg，同时设置阴性对照(hIgG1)和空白组(PBS)，所有样本加入5μg hPD-1-Fc-生物素，4℃孵育20min；
(3)PBS洗涤2次细胞，加入eBioscience的SA-PE，4℃孵育20min，PBS洗涤2次细胞后用流式细胞仪(BD FACS Calibur)检测检测结果如图6所示。
PD-L1纳米抗体的人源化改造
(1)首先以SEQ ID NO.:8所示的PD-L1纳米抗体序列为模板在结构数据库中同源结构的搜索，共搜寻到1306个结构，取其中E value＝0.0并且序列等同性≥70％的34个结构；
(2)对这34个结构进行结构比对，并依据晶体结构分辨率大小和构建的进化树，最终选取包括3dwt在内的9个蛋白，进行基于SEQ ID NO.:8所示的PD-L1纳米抗体序列的多模板同源模建，最终获得的10个结构，再依据打分函数的高低排序，选取molpdf最低的结构，继续下面的工作；
(3)对模建的最优结构，利用ProtSA服务器计算残基的溶剂可接触性，即残基的折叠态相对于去折叠态的溶剂可接触面积的比值为判据，取大于40％的残基为暴露于溶剂外的残基；
(4)对模建的最优结构和DP-47进行序列比对，替换相应的暴露于溶剂的残基。最终确定出一种人源化PD-L1纳米抗体，由SEQ ID NO.14所示的氨基酸序列编码。人源化前后抗体序列对应如下表2：
表2
Figure PCTCN2017095884-appb-000001
人源化前后抗体骨架区与DP-47骨架区的同源性比较如下表3：
表3
Figure PCTCN2017095884-appb-000002
实施例6：MOA法检测抗PD-L1纳米抗体的活性
本实验采用已经上市的两种市售可得的抗PD-L1抗体(Atezolizumab，ATE和Durvalumab，DUR)作为阳性对照抗体，使用MOA检测细胞株(Promega)，通过检测 荧光报告基因的表达反应出NFAT信号的激活情况，从而检测抗体(序列见实施例5)对PD-1/PD-L1结合的抑制作用。步骤如下：
(1)活性检测前一天铺CHOK1-PDL1细胞：CHOK1-PDL1前1-2天传代。弃培养上清，PBS洗涤。加入适量胰酶于37℃/5％CO2消化3-5min。加入4倍胰酶体积的培养基，转移细胞至50ml离心管并计数。取所需体积细胞，230g，离心10min。加入培养基，重悬细胞至4×105个细胞/mL。将细胞加入96孔白色细胞培养板，100μl/孔。边孔加入PBS，200μl/孔。细胞于37℃/5％CO2培养箱中培养过夜。
(2)处理Jurkat-PD1细胞：活性检测前两天进行细胞传代。计数后取所需体积细胞，170g，离心5min。用assay buffer重悬细胞至1.25×106个细胞/ml。
(3)加入样品和Jurkat-PD1细胞至检测板：弃95μl/孔CHOK1-PDL1细胞上清。加入40μl样品(杂交瘤上清或梯度稀释的杂交瘤上清纯化抗体)及阳性对照、阴性对照。加入40μl Jurkat-PD1细胞。于37℃/5％CO2培养箱中培养6小时。
(4)检测：提前将Bio-GloTM buffer融化，加入Bio-GloTM substrate，混匀。6小时后，加入Bio-GloTM Reagent，80μl/孔。室温放置5-10min。读数。
实验结果如表4及图11所示，在各浓度下，本发明人源化前的纳米抗体基本都比阳性对照组抗体活性强，人源化后本发明纳米抗体的活性则与阳性对照组相当。因此Nb-Fc和人源化Nb-Fc抗体均可以有效阻断PD1/PD-L1的相互作用。
表4
Figure PCTCN2017095884-appb-000003
实施例7：人源化PD-L1纳米抗体在真核细胞HEK293中的表达及纯化
(1)将人源化前及人源化后的PD-L1Nb序列合成至pFUSE-IgG1载体(购自Invivogen)上，用Omega质粒大提试剂盒提取pFUSE-IgG1-Nb(人源化)质粒；
(2)培养HEK293F细胞至OD为2.0×106个/mL；
(3)将质粒与转染试剂PEI 1:3混合均匀后静置20min，然后加入到HEK293F细胞中，37℃，6％CO2摇床培养箱中培养5-6天；
(4)收集细胞上清，与Protein A珠子在室温下结合1小时；
(5)用磷酸盐缓冲液pH7.0洗涤珠子后，再用0.1M pH3.0Glycine洗脱蛋白；
(6)将洗脱的蛋白超滤至PBS中，测定产量后取样进行SDS-PAGE检测(检测结果如图1D和图7所示)，其余蛋白保存于-80℃冰箱，图7可见纯化后的人源化纳米抗体纯度达到90％以上。
实施例8：流式细胞术检测人源化PD-L1纳米抗体的阻断功能
方法同实施例5：
(1)每个样品取2×105个天然表达PD-L1的人肺癌细胞系EBC-1重悬于0.5％BSA-PBS缓冲液中，加入10μg纯化的人源化PD-L1纳米抗体，同时设置阴性对照(hIgG1)和空白组(PBS)，所有样本加入5μg hPD-1-Fc-生物素，4℃孵育20min；
(2)PBS洗涤2次细胞，加入eBioscience的SA-PE，4℃孵育20min，PBS洗涤2次细胞后上机检测，检测结果如图8所示：由空白组及阴性对照组可以看出PD-1-Fc-生物素与EBC-1细胞的结合率在90％以上，而加入PD-L1纳米抗体和人源化纳米抗体后，PD-1-Fc-生物素与EBC-1细胞的结合率在10％以下，这表明加入的纳米抗体能够明显阻断PD-1与PD-L1的相互作用。
实施例9：纳米抗体亲和力测定
用BiaCore T200进行检测，(1)固定：利用羧基氨基反应将固定相抗原固定在CM-5传感芯片表面；
(2)结合：将抗体用HBS缓冲液稀释成适当浓度(五个浓度梯度)，观察抗原抗体结合过程；
(3)芯片再生：进行下一个抗体测定时用10mM Glycine冲洗进行再生。
(4)实验结果分析，测定结果如图9所示，人源化前纳米抗体的亲和力为2.34×10-9M，人源化后的纳米抗体的亲和力为2.26×10-9M。人源化改造并未影响抗体的亲和力。
实施例10：酶联免疫法(ELISA)鉴定纯化的纳米抗体的特异性：
(1)通过常规方法将人源化前及人源化后的纳米抗体进行生物素化；
(2)包被抗原蛋白PD-L1(人)、PD-L1(鼠)、PD-L1(猴子)、PD-L2(人)、B7H4(人)、B7H3(人)：每孔0.5μg(5μg/mL，100μL)，包被IgG1为对照，4℃过夜；
(3)第二天用PBST洗涤3次，加入200μL的1％BSA室温下封闭2小时；
(4)将每株生物素化的纳米抗体稀释至10μg/mL，分别取100μL与各孔孵育，室温下反应1小时；
(5)用PBST洗去未结合的抗体，加入100μL streptavidin-HRP(1:1000稀释)，在室温下放置1小时；
(6)加入显色液，于ELISA仪上，在450nm波长，读取吸收值。根据吸收值判断纳米抗体的特异性，检测结果如图10所示：人源化前后的纳米抗体均能够与人源和猴源的PD-L1相互作用，而不与鼠源的PD-L1相互作用，两株抗体具有较好的种属特异性；且人源化前后的纳米抗体均不与PD-L1家族成员相互作用，具有较好的家族特异性。
实施例11：混合淋巴细胞实验
本实验将抗体和体外培养的、来源于不同供体的成熟DC细胞及CD4+T细胞共同孵育，通过检测体系中IL2和IFN-γ的相对表达量，从而反应出不同抗体对T细胞的激活作用。步骤如下：
(1)PBMC分离：取捐赠者新鲜血液50ml，添加2.5倍PBS，轻轻加入到FiColl(Thermo)12.5ml分4管，400g，30min离心，0减速度停止。吸取中间白色条带至PBS(Gibco)中，PBS洗2次。
(2)DC细胞分离：取分离的PBMC细胞添加5ml T细胞培养基，37℃、6％CO2、贴壁培养2hr，吸取悬浮细胞液做CD4+细胞分离，剩下细胞添加3ml DC培养基，培养2天后添加3ml DC培养基，再培养第5天，然后添加rTNFa(R&D Systems)(1000U/ml)，IL-1b(R&D Systems)(5ng/ml)，IL-6(R&D Systems)(10ng/ml)和1μM PGE2(Tocris)培养2天，作为淋巴细胞混合反应(MLR)的DC细胞。
(3)CD4+细胞分离：PBMC静置培养2hr，吸取悬浮的细胞液至15ml离心管中，200g离心10min，沉淀加入500μl分离液、100μl AB型血清、100μl纯化抗体重悬，4℃孵育20min，用分离液清洗一次，再加入500μl Bead Buffer孵育15min，磁场去除Bead，T细胞培养基洗一次，使用8ml培养基重悬，37℃、6％CO2培养。(按照‘Human CD4+T cell Enrichment Kit’(19052，Stemcell)说明书操作)。
(3)MLR实验：将成熟后的DC细胞与CD4+细胞混合，每孔体积200μl，DC细胞10000个，CD4+细胞100000个，加入抗体，DC、T细胞、MLR作为阴性对照，DC+T细胞+anti-CD3/CD28磁珠作为阳性对照，混合培养5天，cisbio试剂盒(Human IL2Kit 1000Test、Human IFN gamma 1000test)检测IL2、IFN-gamma浓度。
实验结果如图12所示，本发明的纳米抗体(序列见实施例5)刺激供体产生的细胞因子均高于阳性对照，而经人源化后，所产生的细胞因子与阳性对照相当，因此，本发明的纳米抗体以及人源化改造后的抗体均可有效激活T细胞，且激活效果和阳性对照组抗体相似。
实施例12抗PD-L1纳米抗体的肿瘤抑制活性研究
本实验采用表达人PD-L1的MC38细胞(MC38-PDL1)(南京银河公司)在PD-L1转 基因小鼠测定人源化Nb-Fc的抗肿瘤作用。首先采用皮下接种的方式建立MC38-PDL1荷瘤小鼠模型，成瘤后分组，给予不同抗体(序列见实施例5)和不同剂量的治疗，监测给药期间各组小鼠瘤体积和体重变化，给药频率为2次/周，监测频率均为2次/周，连续监测5周，给药剂量和方式如表5及图13所示。
表5
Figure PCTCN2017095884-appb-000004
步骤如下：
1)MC38/PD-L1细胞悬液制备：以PBS(1×)分散MC38细胞，细胞密度为1×107个/ml，制备成MC38细胞悬液；
2)接种：25只C57Bl/6背景的PD-L1小鼠右侧背部剃毛，皮下注射MC38/PD-L1细胞1×106个/0.1ml/只。肿瘤细胞接种6天后检测各只小鼠瘤体积，挑选出瘤体积在87.4mm3～228.4mm3范围内的25只小鼠按瘤体积平均分组。
3)给药：见图13。
4)检测：每次给药前和测定体重和肿瘤体积，记录重量。采用电子天平测定体重，每周2次。
5)肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W2/2。
实验结果如图14所示，随着时间的推移，接种了人源化Nb-Fc的小鼠，其肿瘤体积相对于对照组得到了很好的控制，并没有出现显著增加的情况，说明人源化Nb-Fc有明显的肿瘤抑制作用。
实施例13.PEG沉淀法检测抗体的可溶性
本实验利用PEG沉淀的方法，通过检测备选抗体(序列见实施例5)在不同浓度的PEG中的溶解情况，反应了抗体的可溶性。步骤如下：
1)浓缩抗体样品至5mg/ml。
2)在96孔细胞培养板中加样，每孔40μl抗体样品，终浓度1mg/ml。第1至12列分别加入30％PEG 26.7μl、40μl、46.7μl、53.3μl、60μl、66.7
μl、73.3μl、80μl、86.7μl、93.3μl、100μl、106.7μl，IBI301Buffer补足至总体积200μl。PEG浓度梯度分别为4％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％。
3)室温放置1hr，测OD500nm。
研究结果如表6及图15所示，人源化Nb-Fc均在8％(w/v)PEG时出现浑浊，而对照抗体(已经上市的药物Humira)在6％(w/v)PEG时出现浑浊，表明本发明人源化Nb-Fc具有比对照抗体更优的可溶性。
表6
Figure PCTCN2017095884-appb-000005
实施例14.加速稳定性实验测定
本实验通过检测抗体(序列见实施例5)在40℃放置30天之后的纯度及生物学活性的变化，从而评价该抗体的长期热稳定性。使用SEC的方法测定了目的抗体在40℃放置0、14及30天后的纯度，实验结果如表7及图16所示，人源化Nb-Fc的纯度没有明显变化。本实验利用FACS方法检测了加速稳定性实验样品和CHO-PDL1细胞的结合情况，步骤如下：
1)细胞准备：将CHO-PDL1细胞计数，并稀释至2×106个细胞/ml，向U型底96孔板中加入100μl/孔，第一列孔补加50μl；
2)检测步骤：第一孔加入抗体，终浓度200nM，混匀，吸取50μl至第二孔，依次类推，阴性对照为IgG Control。冰浴20min。加PBS 100μl/孔，400g 5min去除上清，PBS洗细胞1遍。加1:100稀释的Goat anti-human IgG-PE(eBioscience)100μl/孔，冰浴20min，400g离心5min去除上清，加PBS 100μl/孔清洗1次，用100μl PBS重悬，FACS检测。
实验结果如图17所示，人源化Nb-Fc和CHO-PDL1细胞的结合没有明显变化。研究结果表明，人源化Nb-Fc具有较好的热稳定性。
表7
Figure PCTCN2017095884-appb-000006
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种抗PD-L1纳米抗体VHH链的互补决定区CDR区，其特征在于，所述VHH链的互补决定区CDR由SEQ ID NO：5所示的CDR1、SEQ ID NO：6所示的CDR2，SEQ ID NO：7所示的CDR3组成。
  2. 一种抗PD-L1纳米抗体的VHH链，其特征在于，所述VHH链包括框架区FR和权利要求1所述的互补决定区CDR，所述的框架区FR由
    (a)SEQ ID NO：1所示的FR1，SEQ ID NO：2所示的FR2，SEQ ID NO：3所示的FR3，SEQ ID NO：4所示的FR4组成；或
    (b)SEQ ID NO：10所示的FR1，SEQ ID NO：11所示的FR2，SEQ ID NO：12所示的FR3，SEQ ID NO：13所示的FR4组成。
  3. 一种抗PD-L1纳米抗体，其特征在于，它是针对PD-L1表位的纳米抗体，并且具有如SEQ ID NO.:8或SEQ ID NO.:14中所示的氨基酸序列的VHH链。
  4. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的蛋白质：权利要求1所述的CDR区、权利要求2所述的抗PD-L1纳米抗体的VHH链、或权利要求3所述的抗PD-L1纳米抗体。
  5. 如权利要求4所述的多核苷酸，其特征在于，具有如SEQ ID NO.:9或15所示的核苷酸序列。
  6. 一种表达载体，其特征在于，所述表达载体含有权利要求4所述的多核苷酸。
  7. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求6所述的表达载体，或其基因组中整合有权利要求4所述的多核苷酸。
  8. 一种产生抗PD-L1纳米抗体的方法，其特征在于，包括步骤：
    (a)在适合产生纳米抗体的条件下，培养权利要求7所述的宿主细胞，从而获得含所述抗PD-L1纳米抗体的培养物；以及
    (b)从所述培养物中分离或回收所述的抗PD-L1纳米抗体。
  9. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求2所述的抗PD-L1纳米抗体的VHH链、或如权利要求3所述的抗PD-L1纳米抗体；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  10. 权利要求3所述的抗PD-L1纳米抗体的用途，其特征在于，用于制备(a)用于检测PD-L1分子的试剂；(b)用于治疗肿瘤的药物。
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US20190023793A1 (en) 2019-01-24
AU2017305366A1 (en) 2018-06-21
CN107686520B (zh) 2023-01-03
EP3369745A4 (en) 2018-12-19
CA3007021C (en) 2023-08-29
CN107686520A (zh) 2018-02-13
KR102037016B1 (ko) 2019-10-25
AU2017305366B2 (en) 2019-06-20
JP2019528033A (ja) 2019-10-10
EP3369745A1 (en) 2018-09-05

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