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WO2023116781A1 - 一种新型pd1单域抗体的开发 - Google Patents

一种新型pd1单域抗体的开发 Download PDF

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WO2023116781A1
WO2023116781A1 PCT/CN2022/140768 CN2022140768W WO2023116781A1 WO 2023116781 A1 WO2023116781 A1 WO 2023116781A1 CN 2022140768 W CN2022140768 W CN 2022140768W WO 2023116781 A1 WO2023116781 A1 WO 2023116781A1
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seq
single domain
antibody
domain antibody
present
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PCT/CN2022/140768
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English (en)
French (fr)
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杨林
游凤涛
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博生吉医药科技(苏州)有限公司
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Definitions

  • the invention relates to the field of biomedicine.
  • the present invention relates to anti-PD1 single domain antibodies.
  • the specific immune response of T cells is stimulated by the first stimulating signal formed by the combination of T cell receptors and antigen peptide MHC complexes; at the same time, the intensity and magnitude of the immune response mediated by T cells is determined by immune checkpoint proteins (including co-repressors and co-stimulatory molecules).
  • immune checkpoint proteins including co-repressors and co-stimulatory molecules.
  • immune checkpoint molecules include: cytotoxic lymphocyte antigen 4 (Cytotoxic lymphocyte antigen-4, CTLA-4), programmed death Protein 1 (Programmed death-1, PD-1), T cell immunoglobulin and mucin domain 3 (TIM-3), T cell immunoglobulin and ITIM structure (T cell Ig and ITIM domain, TIGIT), etc.
  • immune checkpoints Under normal physiological conditions, the main function of immune checkpoints is to maintain self-tolerance and protect normal tissues from damage by the immune system.
  • lymphocytes In the tumor microenvironment, due to long-term exposure to tumor antigens, lymphocytes will abnormally express certain co-suppressor factors, resulting in dysfunction of tumor-specific T cells.
  • a large number of clinical data show that antibodies against co-inhibitory signals can enhance tumor-specific T cell responses. Therefore, these immune checkpoint proteins have become potential targets for anti-tumor immunotherapy, and CAR-T cells can also further improve the therapeutic effect on solid tumors by co-expressing PD1 antibodies.
  • the purpose of the present invention is to provide a single domain antibody against PD-1 and its application.
  • an anti-PD-1 single domain antibody characterized in that, the complementarity determining region CDR of the VHH chain of the anti-PD-1 single domain antibody is one selected from the following group or Various:
  • the anti-PD-1 single domain antibody can specifically bind to PD-1.
  • the anti-PD-1 single domain antibody can block the binding of PD-1 and PD-L1.
  • any amino acid sequence in the amino acid sequence further includes at least one (such as 1-3, preferably 1-2, more Preferably 1) amino acid derivative sequence that can retain the ability to specifically bind to PD-1.
  • the anti-PD-1 single domain antibody further includes a framework region FR.
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the framework regions FR1, FR2, FR3 and FR4 have a sequence derived from SEQ ID NO: 1, 2, 3 or 4.
  • amino acid sequence of the VHH chain of the anti-PD-1 single domain antibody is selected from the sequences shown in SEQ ID NO: 1, 2, 3 or 4.
  • the anti-PD-1 single domain antibody includes monomer, bivalent antibody (bivalent antibody), and/or multivalent antibody.
  • the multivalent body comprises more than 2 VHH chains of the single domain antibody according to the first aspect of the present invention, preferably 2, 3 or 4.
  • the anti-PD-1 single domain antibody is a bivalent body.
  • the anti-PD-1 single domain antibody has a structure as shown in Formula I from the N-terminal to the C-terminal direction:
  • L is the connecting peptide
  • Each of P1 and P2 is a VHH chain.
  • said P1 and P2 each independently comprise a sequence as shown in SEQ ID NO: 1, 2, 3 or 4.
  • P1 has the amino acid sequence shown in SEQ ID NO:3
  • P2 has the amino acid sequence shown in SEQ ID NO:2.
  • P1 has the amino acid sequence shown in SEQ ID NO:3
  • P2 has the amino acid sequence shown in SEQ ID NO:1.
  • P1 has the amino acid sequence shown in SEQ ID NO:2
  • P2 has the amino acid sequence shown in SEQ ID NO:1.
  • the anti-PD-1 single domain antibody includes humanized antibody, camelid antibody and chimeric antibody.
  • an anti-PD-1 antibody comprising one or more VHH chains of the anti-PD1 single domain antibody according to the first aspect of the present invention.
  • amino acid sequence of the VHH chain of the anti-PD-1 single domain antibody is selected from the sequences shown in SEQ ID NO: 1, 2, 3 or 4.
  • the anti-PD-1 antibody includes monomer, bivalent antibody (bivalent antibody), and/or multivalent antibody.
  • a polynucleotide encoding a protein selected from the group consisting of: the anti-PD-1 single domain antibody as described in the first aspect of the present invention, or the anti-PD-1 single domain antibody as described in the present invention The antibody of the second aspect.
  • the present invention relates to a nucleic acid molecule encoding the anti-PD-1 single domain antibody of the present invention.
  • a nucleic acid of the invention may be RNA, DNA or cDNA.
  • an expression vector containing the polynucleotide according to the third aspect of the present invention is provided.
  • the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or combinations thereof.
  • the expression vector is pcDNA3.4-hIgG1-Fc2 plasmid.
  • a host cell contains the expression vector according to the fourth aspect of the present invention, or the polynucleotide according to the third aspect of the present invention is integrated in its genome.
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.
  • the host cells are 293F cells.
  • a method for producing an anti-PD-1 single domain antibody comprising the steps of:
  • step (c) Optionally, purifying and/or modifying the anti-PD-1 single domain antibody obtained in step (b).
  • an immunoconjugate which comprises:
  • a coupling moiety selected from the group consisting of detectable labels, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.
  • the radionuclides include:
  • isotopes for diagnosis are selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
  • the isotope for treatment is selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb- 177, or combinations thereof.
  • the coupling moiety is a detectable label.
  • the coupling moiety is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer X-ray tomography) contrast agents, or capable of producing Detectable products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles , prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), or nanoparticles in any form.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • the immunoconjugate comprises: a multivalent (eg bivalent) VHH chain of the anti-PD-1 single domain antibody according to the first aspect of the present invention.
  • the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repeated VHH chains of the anti-PD-1 single domain antibody according to the first aspect of the present invention.
  • the anti-PD-1 single domain antibody as described in the first aspect of the present invention the anti-PD-1 antibody as described in the second aspect of the present invention, or the ninth aspect of the present invention
  • the use of the immunoconjugate is characterized in that it is used to prepare:
  • the PD-1 signaling pathway-related diseases include cancer or autoimmune diseases.
  • the cancer or tumor is selected from the group consisting of blood tumors, lymphomas, solid tumors, or combinations thereof.
  • the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse massive B cell lymphoma (DLBCL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse massive B cell lymphoma
  • the lymphoma is selected from the group consisting of Hodgkin's lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukocyte (CLL) , small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), and other complex B-cell non-Hodgkin lymphomas.
  • HL Hodgkin's lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • CLL chronic lymphocytic leukocyte
  • SLL small lymphocytic lymphoma
  • MZL marginal zone lymphoma
  • MCL mantle cell lymphoma
  • BL Burkitt lymphoma
  • the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer , ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or its combination.
  • gastric cancer peritoneal metastasis of gastric cancer
  • liver cancer liver cancer
  • kidney tumor lung cancer
  • small intestine cancer bone cancer
  • prostate cancer colorectal cancer
  • breast cancer colorectal cancer
  • cervical cancer cervical cancer
  • ovarian cancer lymphoma
  • nasopharyngeal cancer adrenal tumor
  • bladder tumor bladder tumor
  • NSCLC non-small cell lung cancer
  • glioma endometri
  • the reagent shown is a diagnostic reagent, preferably, the diagnostic reagent is a detection chip or a detection plate.
  • the diagnostic reagent is used for detecting PD-1 or its fragments in a sample.
  • a pharmaceutical composition which contains:
  • a recombinant protein is provided, and the recombinant protein has:
  • the tag sequence includes Fc tag, HA tag and His tag.
  • the recombinant protein specifically binds to PD-1.
  • kits which contains the anti-PD-1 single domain antibody as described in the first aspect of the present invention, the anti-PD-1 antibody as described in the second aspect of the present invention. 1, or the immunoconjugate as described in the seventh aspect of the present invention.
  • a method for preventing and/or treating diseases related to PD-1 signaling pathway comprising, administering the anti-PD-1 as described in the first aspect of the present invention to a subject in need.
  • a single domain antibody, anti-PD-1 antibody according to the second aspect of the present invention, or immunoconjugate according to the seventh aspect of the present invention comprising, administering the anti-PD-1 as described in the first aspect of the present invention to a subject in need.
  • the subject includes mammals, such as humans.
  • the PD-1 signaling pathway-related diseases include cancer or autoimmune diseases.
  • a method for in vitro detection of PD-1 or a fragment thereof in a sample comprising the steps of:
  • the serotype of PD-1 includes: PD-11, PD-12, PD-15, PD-16, PD-17, PD-18, PD-19.
  • the serotypes of PD-1 include: PD-15, PD-18, and PD-19.
  • the detection includes diagnostic or non-diagnostic.
  • a method for diagnosing diseases related to PD-1 signaling pathway comprising the steps of:
  • a method for preparing a recombinant polypeptide is provided, and the recombinant polypeptide is the anti-PD-1 single domain antibody described in the first aspect of the present invention, or the anti-PD-1 single domain antibody described in the second aspect of the present invention.
  • Anti-PD-1 antibody the method comprises:
  • Figure 1 shows the results of detecting PD1-Fc protein expression by SDS-PAGE.
  • Lane 1 represents the size of PD1-Fc protein, and lane M represents protein Mraker.
  • Figure 2 shows the results of flow cytometry detection of the binding of PD1-Fc protein to the PDL1 overexpression cell line CHO.
  • Figure 3 shows the results of the immune titer ELISA detection of the immune serum.
  • Figure 4 shows the alignment results of the phage display library sequences.
  • Figure 5 shows the results of antigen solid-phase panning, and a total of 4 different antibody sequences were screened.
  • Figure 6 shows the results of screening antibodies by cross-selection of cells and solid-phase panning.
  • Figure 7 shows the binding of the screened PD1 single domain antibody to the PD1 overexpression cell line CHO cells.
  • Figure 8 shows the functional verification of PD1 single domain antibody (monovalent) blocking activity.
  • Nivolumab is the positive control PD1 antibody
  • LAB190417-2-B1, LAB190417-2-C10, LAB190417-2-F3, and LAB190417-3-H6 are four cloned PD1 antibodies screened by flow cytometry.
  • Figure 9 shows the functional verification of PD1 single domain antibody (bivalent) blocking activity.
  • Nivolumab is the positive control PD1 antibody
  • 2-C10-(G4S)3-2-F3 is the positive control PD1 antibody
  • 2-C10-(G4S)3-2-B1 is the three constructed Bivalent PD1 antibody.
  • the present invention uses the PD1-Fc antigen protein to immunize camels, uses phage display technology to screen the immune single domain antibody gene library (phage display library), and performs panning and identification, thereby obtaining the single domain antibody gene against PD-1 .
  • Relevant experimental results show that the anti-PD-1 single domain antibody obtained in the present invention can effectively bind to PD-1, and has excellent blocking activity on the binding of PD-1/PD-L1. The present invention has been accomplished on this basis.
  • the terms “antibody of the invention”, “antibody of the invention”, “single domain antibody against PD-1 of the invention”, “single domain antibody against PD-1 of the invention”, “anti-PD-1 "Single domain antibody against PD-1” and “single domain antibody against PD-1” have the same meaning and can be used interchangeably, both refer to antibodies that specifically recognize and bind to PD-1.
  • antibody or "immunoglobulin” is a heterotetrameric protein of about 150,000 Daltons with identical structural features, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain .
  • VL variable region
  • Specific amino acid residues form the interface between the variable domains of the light and heavy chains.
  • single domain antibody As used herein, the terms “single domain antibody”, “VHH”, “nanobody”, “single domain antibody” (single domain antibody, sdAb, or nanobody nanobody) have the same meaning and are used interchangeably, Refers to cloning the variable region of the heavy chain of an antibody to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, after obtaining the antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.
  • VHH single domain antibody
  • variable means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable domains are called the framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each contain four FR regions in a roughly b-sheet configuration connected by three CDRs that form connecting loops, which in some cases may form partial b-sheet structures.
  • the CDRs in each chain are in close proximity through the FR regions 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, for example involved in the antibody-dependent cytotoxicity of the antibody.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokine), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention to form of conjugates.
  • the present invention also includes cell surface markers or antigens that bind to the nanobody against PD-1 or a fragment thereof.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to a polypeptide that specifically binds to a PD-1 protein, such as a protein with a heavy chain variable region or peptides. They may or may not contain starting methionine.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the present invention includes any protein or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) having a heavy chain containing a variable region, as long as the variable region is compatible with the heavy chain of the antibody of the present invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • the terms “specifically bind”, “selectively bind”, “selectively bind” and “specifically bind” refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of about less than 10 "7M , eg, about less than 10 "8M , 10 "9M , or 10 "10M or less.
  • KD affinity
  • the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR), which is separated into four framework regions (FR), four FR amino acids
  • CDR variable region
  • FR framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a ring structure, and the ⁇ sheets formed by the FRs in between are close to each other in the spatial structure.
  • the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody.
  • Which amino acids constitute FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • 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. Therefore, the present invention includes those molecules having antibody heavy chain variable regions with CDRs, as long as the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein sex.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention.
  • the polypeptide fragments, 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 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 in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • the antibody of the present invention refers to a polypeptide that has PD-1 protein binding activity and includes the above CDR region.
  • the term also includes variant forms of polypeptides comprising the above CDR regions that have the same function as the antibodies of the present invention. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with the DNA encoding the antibody of the present invention under high or low stringency conditions
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof.
  • the invention also includes fragments of the antibodies of the invention.
  • the fragment has 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 an antibody of the invention.
  • “conservative variants of the antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acid sequences compared with the amino acid sequence of the antibody of the present invention.
  • An amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • a polynucleotide encoding a mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 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 its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.
  • biomolecules nucleic acid, protein, etc.
  • the biomolecules involved in the present invention include biomolecules in an isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • the host cell may 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, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after exponential growth and treated with CaCl2 using procedures well known in the art. Another way is to use MgCl2. Transformation can also be performed by electroporation, if desired.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional media according to the host cells used.
  • the culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
  • the recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, 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 can be used alone, or combined or conjugated 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 (computed tomography) contrast agents, or substances capable of producing a detectable product. enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biological toxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), etc.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • PD-1 and "programmed death receptor 1" both refer to the PD-1 immune checkpoint receptor, which can limit the activity of T cells and limit autoimmunity during the inflammatory response of peripheral tissues.
  • PD-1 is the main mechanism of immune resistance in the tumor microenvironment. T cells induce the expression of PD-1 after activation. When bound to a ligand of PD-1, the phosphorylase SHP2 involved in T cell activation is inhibited. At the same time, because PD-1 binding inhibits the TCR stop signal, this signaling pathway can change the connection time between T cell-APC or T cell-target cell.
  • PD-1 is highly expressed in Treg cells, and Treg cells will enhance their own proliferation in the presence of PD-1 ligands. Because many tumors highly express infiltrating Treg cells, which may further suppress the immune response of effector cells, blocking the PD-1 signaling pathway will enhance the anti-tumor immune response by reducing and/or inhibiting the activity of intratumoral Treg cells.
  • PD-1 has two ligands PD-L1 and PD-L2.
  • PD-1 is expressed on the majority of tumor infiltrating lymphocytes (TILs) derived from a variety of tumor types.
  • TILs tumor infiltrating lymphocytes
  • CD4+ TILs with high expression of PD-1 are mainly concentrated in CD4+ Treg cells in tumor tissues.
  • CD8+ TILs with high expression of PD-1 showed an incompetent or exhausted state, and by comparing PD-1-TILs in melanoma, PD-1+ TILs secreted fewer cytokines.
  • PD-1 ligands are also highly expressed on the surface of tumor cells in many tumors.
  • PD-L1 the ligand of PD-1
  • T cells the high expression of PD-L1 in tumor cells in mice can inhibit the anti-tumor immune response mediated by T cells.
  • IHC immunohistochemistry
  • flow cytometry it is concluded that a variety of human tumors constitutively express PD-1 ligands at a certain level.
  • PD-1 ligands will largely determine whether immunotherapeutic strategies to block this pathway are feasible, since the main role of PD-1 ligands in cancer is thought to be immunosuppressive in the tumor microenvironment, and PD-1 1 combined with its ligands, PD-L1 and PD-L2, can only inhibit the function of lymphocytes.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, 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 about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody (or its conjugate) of the present invention and a pharmaceutically acceptable acceptable carrier or excipient.
  • a pharmaceutically acceptable acceptable carrier or excipient include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the invention can also be used
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the present invention provides an anti-PD-1 single domain antibody, which can specifically bind to PD-1.
  • the complementarity determining region CDR of the VHH chain of the anti-PD-1 single domain antibody of the present invention is one or more selected from the following group:
  • the anti-PD-1 single domain antibody includes one or more VHH chains having the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4.
  • the anti-PD-1 single domain antibody includes monomer, bivalent body (bivalent antibody), tetravalent body (tetravalent antibody), and/or multivalent body (multivalent antibody).
  • the multivalent antibody comprises two or more of the above-mentioned VHH chains or the above-mentioned single domain antibody, preferably two, three or four.
  • the anti-PD-1 single domain antibody is a bivalent body.
  • bivalent body (bivalent antibody) of the present invention has a structure as shown in Formula I:
  • said P1 and P2 each independently comprise a sequence as shown in SEQ ID NO: 1, 2, 3 or 4.
  • P1 has the amino acid sequence shown in SEQ ID NO:3
  • P2 has the amino acid sequence shown in SEQ ID NO:2.
  • P1 has the amino acid sequence shown in SEQ ID NO:3
  • P2 has the amino acid sequence shown in SEQ ID NO:1.
  • P1 has the amino acid sequence shown in SEQ ID NO:2
  • P2 has the amino acid sequence shown in SEQ ID NO:1.
  • the present invention also relates to methods for detecting PD-1.
  • the steps of the method are roughly as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of PD-1 in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or its fragment) or detection plate of the present invention.
  • the kit further includes a container, instructions for use, buffer and the like.
  • the present invention also provides a detection kit for detecting the level of PD-1, which includes an antibody that recognizes PD-1, a lysis medium for dissolving samples, general reagents and buffers required for detection, such as various buffers solution, detection label, detection substrate, etc.
  • the test kit may be an in vitro diagnostic device.
  • the antibody of the present invention has a wide range of biological and clinical application values, and its application involves the diagnosis and treatment of diseases related to the PD-1 signaling pathway, basic medical research, biological research and other fields.
  • a preferred application is for clinical diagnosis, prevention and treatment of diseases related to PD-1 signaling pathway.
  • the antibody of the present invention can specifically bind to PD-1, and has a high neutralizing activity against PD-1;
  • the antibody of the present invention can efficiently block PD-1/PD-L1 binding
  • the PD1 nanobody of the present invention has the advantages of small molecular weight, fast tissue penetration, high solubility and stability, high antigen binding specificity, weak immunogenicity, etc., and is easier for CAR-T cells to co-express PD1 antibody. Can be used in cancer treatment.
  • Agar (Sigma, CAT#A1296); Peptone (Sigma, CAT#93926); Yeast Extract (OXOID, CAT#: LP0021); Sodium Chloride (Aladdin, CAT#: C111533); Potassium Chloride (A Latin, CAT#:P112133); Magnesium Sulfate (Sinopharm, CAT#:10013018); Magnesium Chloride (Sinopharm, CAT#:10012818); Glucose (Sanko, CAT#:GT1991); SfiI (NEB, CAT#:R0123L); T4 DNA ligase (TaKaRa, CAT#:2011A); PrimeScript TM II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#:6210B); NuHi power mix (Xinhai Biology, CAT#: NH9303); 3M sodium acetate (pH5.2- 6)
  • the constructed PD1-Fc protein expression vector was subjected to large-scale plasmid extraction, and after transiently transfecting 293 cells, cultured continuously for 8 days, collected the supernatant of the medium by centrifugation, filtered through a 0.45 ⁇ m filter membrane, and transferred the filtrate to sterile centrifugation tube, purify the antibody using a Protein A column.
  • peripheral blood 100ml of peripheral blood was collected, and PBMC were sorted using lymphocyte separation medium.
  • the PCR product was analyzed by electrophoresis using 1% agarose, and fragments with a molecular weight of about 750 bp were separated.
  • the PCR product was recovered using a gel recovery kit, and the concentration was determined with NanoDrop.
  • the PCR product was analyzed by electrophoresis using 1% agarose, and fragments with a molecular weight of about 750 bp were separated.
  • the PCR product was recovered using a gel recovery kit, and the concentration was determined with NanoDrop.
  • M13KO7 volume 10 ⁇ volume ⁇ OD600 ⁇ 5 ⁇ 108/M13KO7 titer
  • Titer calculation select a plate with the number of spots between 30-300, take the average value of two plates, multiply the number of spots by the dilution factor and multiply by 100 to get the titer.
  • the solid-phase panning steps of the phage display library are as follows:
  • Titer calculation select a plate with the number of spots between 30-300, take the average value of two plates, multiply the number of spots by the dilution factor and multiply by the elution volume.
  • the cell-based panning steps of the phage display library are as follows:
  • a recombinant cell line overexpressing the target protein incubate the phage library with empty cells and cells overexpressing the target protein in sequence, and after several times of washing to remove non-specifically bound phage, use glycine or TEA to bind to the cell surface
  • the recombinant phages were eluted and amplified; after 3-4 rounds of panning, single clones were selected for ELISA detection.
  • M13KO7 volume 10 ⁇ volume ⁇ OD600 ⁇ 5 ⁇ 108/M13KO7 titer
  • VHH antibody sequences obtained from the analysis were gene synthesized and subcloned in tandem with human IgG1Fc into the expression vector pcDNA3.4-hIgG1-Fc2. After the vector was verified to be correct by sequencing, the endotoxin-free plasmid was prepared using the Qiagen plasmid extraction kit.
  • the three candidate antibodies were randomly combined to construct a bivalent antibody, and the expression form of the bivalent antibody was 2-C10-(G4S) 3 -2-F3, 2-C10-(G4S) 3 -2-B1, 2-F3-( G4S) 3 -2-B1 were synthesized separately and subcloned in tandem with human IgG1Fc into the expression vector pcDNA3.4-hIgG1-Fc2. After the vector was verified to be correct by sequencing, the endotoxin-free plasmid was prepared using the Qiagen plasmid extraction kit.
  • LVTransm transfection reagent and the antibody expression vector pcDNA3.4-hIgG1-Fc2 Take out the LVTransm transfection reagent and the antibody expression vector pcDNA3.4-hIgG1-Fc2 from the refrigerator, thaw at room temperature, and mix completely by blowing up and down with a pipette gun. Remove the PBS buffer and warm to room temperature. Take 500 ⁇ L LVTransm to one well of a 24-well plate, add 4 ⁇ g pcDNA3.4-hIgG1-Fc2, pipette up and down to mix well, add 12 ⁇ L LVTransm, immediately use a pipette to mix up and down, and let stand at room temperature for 10 minutes . The mixture here is called DNA/LVTransm complex.
  • the medium supernatant was collected by centrifugation, filtered with a 0.45 ⁇ m filter membrane, and the filtrate was transferred to a sterile centrifuge tube for subsequent flow cytometry and ELISA detection.
  • CHO-K1 and CHO-K1-PD1 cell lines from liquid nitrogen, and adjust the cell state to logarithmic growth phase.
  • the two types of cells were divided into several parts, and the number of cells in each part was 5*10 ⁇ 5 cells.
  • the culture supernatant was collected by centrifugation, filtered through a 0.45 ⁇ m filter membrane, and the filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a Protein A column.
  • the final concentrations are 30 ⁇ g/mL, 10 ⁇ g/mL, 3.333 ⁇ g/mL mL, 1.111 ⁇ g/mL, 0.3704 ⁇ g/mL, 0.1235 ⁇ g/mL, 0.04115 ⁇ g/mL, 0.01372 ⁇ g/mL, 0.004572 ⁇ g/mL, and then added to the corresponding wells. After co-cultivation for 18 hours, 25 ⁇ L of One-Glo reagent was added to each well, and the luciferase activity in the wells was detected using a Tecan M1000pro microplate reader.
  • the PD1-Fc recombinant protein was immobilized on the CM5 chip using 10mM Acetate buffer, and the prepared single domain antibody was used as the mobile phase to detect the binding ability of the candidate single domain antibody to the target protein PD1.
  • the PD1-Fc antigen was prepared using the above-mentioned experimental method 1, and the expression of PD1-Fc protein was detected by SDS-PAGE. The result is shown in Figure 1.
  • the secondary antibody is APC-anti human Fc, according to the results of flow cytometry ( Figure 2), PD1-Fc can bind to PDL1, has activity, and can be used for alpaca immunization and antibody production filter.
  • PBMC isolation and amplification of VHH fragments were performed using Experimental Methods 4 and 5 above. Specifically, collect peripheral blood from the immunized American llama in Example 1.2, provide total RNA, reverse-transcribe it into cDNA, use single-domain antibody amplification primers to perform two rounds of PCR, and agarose gel electrophoresis PCR product results . PCR bands of about 1000 bp and 750 bp were obtained in the first round of PCR, and the 750 bp fragment recovered from the gel was used as a template for the second round of PCR. A band of about 450bp was obtained in the second round of PCR, and a VHH fragment was obtained.
  • the construction of the phage display library and the diversity analysis were carried out using the above-mentioned experimental method 6. Specifically, the VHH fragment obtained in Example 1.3 was digested with SfiI and subcloned into the phage display vector pComF, and electrotransformed into SS320 Escherichia coli competent cells to construct a single domain antibody phage display library. From them, 20 single clones were randomly selected for sequencing, and the diversity of the constructed phage display library was analyzed.
  • the solid-phase panning of the phage display library was performed using the above-mentioned experimental method 7. Specifically, PD1-Fc and Fc recombinant proteins were coated respectively, and the constructed phage display library was screened and enriched four times to enrich positive clones.
  • the second and third rounds of solid-phase panning were selected for Phage ELISA experiments; they were coated with PD1-Fc antigen protein and detected by Phage Elisa, and the control groups were directly blocked well plates and Fc-coated wells. Select clones with an S/N ratio above 5 and no binding to Fc for sequencing, and analyze sequenced antibodies.
  • the underlined part of the sequence is the CDR region.
  • Phage display library cells and solid-phase panning were performed using the experimental method 8 described above. Specifically, using CHO and CHO-PD1 cells and PD1-Fc antigen, the constructed phage display library was screened and enriched five times to enrich positive clones.
  • PD1-Fc and Fc antigen proteins were respectively coated, and Phage Elisa was used for detection, and the control group was a directly closed well plate.
  • the antigen ELISA test results were compared with the test results of the control group, and the clone with a larger ratio was selected for sequencing analysis and antibody sequence analysis.
  • vector construction and expression detection were performed on the four VHH antibody sequences screened and sequenced in Example 1.6.
  • the CMV promoter, signal peptide, and human IgG1 Fc tag were added to the N-terminal and C-terminal of the candidate antibody sequence by Overlap PCR, and the purified PCR product was transiently transfected into 293 cells, and the antibody was expressed for flow cytometric detection.
  • Results The results of FACS detection are shown in Figure 7.
  • the 3-H6, 2-C10, 2-B1 and 2-F3 clones obtained by panning the phage display library were all PD1-specific binding antibodies.
  • the antibody was expressed and purified for subsequent verification of blocking function.
  • the PD1-PDL1 blocking activity was detected using the Jurkat-PD1-NFAT-Luc reporter gene cell line and aAPCCHO-PDL1 cells.
  • the Nivolumab positive control can block the interaction of PD1/PDL1, with an EC50 of 0.6374 ⁇ g/mL.
  • PD1 single domain antibodies LAB190417-2-B1, LAB190417-2-C10 and LAB190417-2-F3 can all block the interaction of PD1/PDL1.
  • the three candidate antibodies were randomly combined to construct a bivalent antibody, and the expression form of the bivalent antibody was 2-C10-(G4S)3-2-F3, 2-C10-(G4S)3-2-B1, 2-F3-( G4S) 3-2-B1, after antibody purification, use Jurkat-PD1-NFAT-Luc reporter gene cell line and aAPCCHO-PDL1 cells to detect PD1-PDL1 blocking activity.
  • the Nivolumab positive control can block the interaction of PD1/PDL1, with an EC50 of 0.4887 ⁇ g/mL. All the bivalent PD1 antibodies to be tested can block the PD1/PDL1 interaction, among which 2-C10-(G4S)3-2-B1 has the highest blocking activity.
  • the PD1-Fc recombinant protein was immobilized on the CM5 chip using 10mM Acetate buffer, and the prepared bivalent single domain antibody was used as the mobile phase to detect the binding ability of the candidate single domain antibody to the target protein PD1.

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Abstract

本发明提供了抗PD-1单域抗体。具体地，本发明通过实验筛选获得了多个抗PD-1的单域抗体，并构建了重组的二价抗PD-1抗体。本发明获得的抗PD-1的单域抗体能够高效地与PD-1结合，并能够高效阻断与PD-1/PD-L1的结合，具备在相关疾病治疗中的应用前景。

Description

一种新型PD1单域抗体的开发 技术领域
本发明涉及生物医药领域。具体地说，本发明涉及抗PD1单域抗体。
背景技术
T细胞的特异性免疫应答是由T细胞受体与抗原肽MHC复合物结合后形成的第一刺激信号所激发的；同时T细胞介导的免疫应答的强度和幅度则是被免疫检验点蛋白(包括共抑制因子和共刺激分子)所调节。经过20多年的研究，免疫检验点的分子被发现在调节T细胞免疫应答中发挥了关键作用，这些分子包括：细胞毒性淋巴细胞抗原4(Cytotoxic lymphocyte antigen-4,CTLA-4)，程序性死亡蛋白1(Programmed death-1,PD-1)，T细胞免疫球蛋白和粘蛋白结构-3(T cell immunoglobulin and mucin domain 3,TIM-3)，T细胞免疫球蛋白和ITIM结构(T cell Ig and ITIM domain,TIGIT)等。
正常生理状况下，免疫检验点的主要功能是维持自身耐受和保护正常组织免受免疫系统损伤。在肿瘤微环境中，由于长时间接触肿瘤抗原，淋巴细胞会异常表达某些共抑制因子，从而造成肿瘤特异性T细胞发生功能紊乱。大量的临床数据显示针对共抑制信号的抗体能够提高肿瘤特异性T细胞的应答。因此，这些免疫检验点蛋白成为了抗肿瘤免疫治疗的潜在靶点，而且，CAR-T细胞也可以通过共表达PD1抗体来进一步改善对实体肿瘤的治疗疗效。
然而，目前的抗体的性能尚难以令人满意。因此本领域需要开发新的、性能优异针对上述靶点的抗体药物，以满足临床上的需求。
目前大部分抗体还是以单克隆抗体来源的，阻断活性有限，而且由于单克隆抗体的分子量较大，不易于CAR-T细胞同时进行PD1抗体的共表达。
因此，本领域有需要开发一种新型的PD1纳米抗体。
发明内容
本发明的目的在于提供一种针对PD-1的单域抗体及其应用。
在本发明的第一方面，提供了一种抗PD-1单域抗体，其特征在于，所述抗PD-1单域抗体的VHH链的互补决定区CDR为选自下组的一种或多种：
(1)SEQ ID NO:5所示的CDR1、SEQ ID NO:9所示的CDR2、和SEQ ID NO:10所示的CDR3；
(2)SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:8所示的CDR3；
(3)SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:7所示的CDR3；和
(4)SEQ ID NO:11所示的CDR1、SEQ ID NO:12所示的CDR2、和SEQ ID NO:13所示的CDR3。
在另一优选例中，所述的抗PD-1单域抗体能够与PD-1特异性结合。
在另一优选例中，所述的抗PD-1单域抗体能够阻断PD-1和PD-L1的结合。
在另一优选例中，所述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与PD-1特异性结合能力的衍生序列。
在另一优选例中，所述抗PD-1单域抗体还包括框架区FR。
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。
在另一优选例中，所述的框架区FR1、FR2、FR3和FR4具有衍生自SEQ ID NO:1、2、3或4的序列。
在另一优选例中，所述的抗PD-1单域抗体的VHH链的氨基酸序列选自SEQ ID NO:1、2、3或4所示的序列。
在另一优选例中，所述的抗PD-1的单域抗体包括单体、二价体(二价抗体)、和/或多价抗体。
在另一优选例中，所述的多价体包含2个以上的本发明第一方面所述的单域抗体的VHH链，优选为2个、3个或4个。
在另一优选例中，所述的抗PD-1的单域抗体为二价体。
在另一优选例中，所述抗PD-1的单域抗体从N端至C端方向具有如式I所示的结构：
P1-L-P2       (I)
其中，
“-”为肽键；
L为连接肽，
P1、P2各自为VHH链。
在另一优选例中，所述的L的序列为(G4S)n，其中，n为正整数(例如1、2、3、4、5或6)，优选地，n＝3。
在另一优选例中，所述P1、P2各自独立地包含如SEQ ID NO:1、2、3或4所示的序列。
在另一优选例中，P1具有SEQ ID NO:3所示的氨基酸序列，P2具有SEQ ID NO:2所示的氨基酸序列。
在另一优选例中，P1具有SEQ ID NO:3所示的氨基酸序列，P2具有SEQ ID NO:1所示的氨基酸序列。
在另一优选例中，P1具有SEQ ID NO:2所示的氨基酸序列，P2具有SEQ ID NO:1所示的氨基酸序列。
在另一优选例中，所述的抗PD-1单域抗体包括人源化抗体、骆驼源抗体、嵌合抗体。
在本发明的第二方面，提供了一种抗PD-1的抗体，所述抗体包括一个或多个如本发明第一方面所述的抗PD1的单域抗体的VHH链。
在另一优选例中，所述的抗PD-1单域抗体的VHH链的氨基酸序列选自SEQ ID NO:1、2、3或4所示的序列。
在另一优选例中，所述的抗PD-1的抗体包括单体、二价体(二价抗体)、和/或多价抗体。
在本发明的第三方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：如本发明第一方面所述的抗PD-1单域抗体、或如本发明第二方面所述的抗体。
在另一优选例中，本发明涉及编码本发明的抗PD-1单域抗体的核酸分子。本发明的核酸可为RNA、DNA或cDNA。
在本发明的第四方面，提供了一种表达载体，所述表达载体含有如本发明第三方面所述的多核苷酸。
在另一优选例中，所述的表达载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其他基因转移系统、或其组合。
在另一优选例中，所述的表达载体为pcDNA3.4-hIgG1-Fc2质粒。
在本发明的第五方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第四方面所述的表达载体，或其基因组中整合有如本发明第三方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞。
在另一优选例中，所述的宿主细胞为293F细胞。
在本发明的第六方面，提供了一种产生抗PD-1单域抗体的方法，包括步骤：
(a)在适合产生单域抗体的条件下，培养如本发明第五方面所述的宿主细胞，从而获得含所述抗PD-1单域抗体的培养物；
(b)从所述培养物中分离和/或回收所述的抗PD-1单域抗体；和
(c)任选地，对步骤(b)获得的抗PD-1单域抗体进行纯化和/或修饰。
在本发明的第七方面，提供了一种免疫偶联物，所述免疫偶联物含有：
(a)如本发明第一方面所述的抗PD-1单域抗体、或如本发明第二方面所述的针对PD-1的抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP，或其组合。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188，或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133、Yb-169、Yb-177，或其组合。
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，所述偶联部分选自下组：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))或任何形式的纳米颗粒。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第一方面所述的抗PD-1单域抗体的VHH链。
在另一优选例中，所述多价是指，在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第一方面所述的抗PD-1单域抗体的VHH链。
在本发明的第八方面，提供了如本发明第一方面所述的抗PD-1单域抗体，如本发明第二方面所述的抗PD-1的抗体，或如本发明第九方面所述的免疫偶联物的用途，其特征在于，用于制备：
(1)预防和/或治疗PD-1信号通路相关的疾病的药物；
(2)检测PD-1信号的试剂。
在另一优选例中，所述的PD-1信号通路相关疾病包括癌症或自身免疫性疾病。
在另一优选例中，所述癌症或肿瘤选自下组：血液肿瘤、淋巴瘤、实体瘤、或其组合。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、或其组合。
在另一优选例中，所述淋巴瘤选自下组：霍奇金淋巴瘤(HL)，弥漫大B细胞淋巴瘤(DLBCL)、滤泡淋巴瘤(FL)、慢性淋巴细胞白细胞(CLL)、小淋巴细胞淋巴瘤(SLL)、边缘区淋巴瘤(MZL)、套细胞淋巴瘤(MCL)、伯基特淋巴瘤(BL)以及其他复杂B细胞非霍奇金淋巴瘤。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、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、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、睾丸癌、结直肠癌、尿路肿瘤、甲状腺癌、或其组合。
在另一优选例中，所示试剂为诊断试剂，较佳地，所述的诊断试剂为检测片或检测板。
在另一优选例中，所述诊断试剂用于：检测样品中的PD-1或其片段。
在本发明的第九方面，提供了一种药物组合物，所述药物组合物含有：
(i)如本发明第一方面所述的抗PD-1单域抗体、如本发明第二方面所述的抗PD-1的抗体，或如本发明第七方面所述的免疫偶联物；和
(ii)药学上可接受的载体。
在本发明的第十方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的抗PD-1单域抗体、或如本发明第二方面所述的抗PD-1的抗体；和
(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Fc标签、HA标签和His标签。
在另一优选例中，所述的重组蛋白特异性结合于PD-1。
在本发明的第十一方面，提供了一种试剂盒，所述试剂盒含有如本发明第一方面所述的抗PD-1单域抗体、如本发明第二方面所述的抗PD-1的抗体，或如本发明第七方面所述的免疫偶联物。
在本发明的第十二方面，提供了一种预防和/或治疗PD-1信号通路相关疾病的方法，所述方法包括，给需要的对象施用如本发明第一方面所述的抗PD-1单域抗体、如本发明第二方面所述的抗PD-1的抗体，或如本发明第七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。
在另一优选例中，所述的PD-1信号通路相关疾病包括癌症或自身免疫性疾病。
在本发明的第十三方面，提供了一种体外检测样品中PD-1或其片段的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第一方面所述的抗PD-1单域抗体、如本发明第二方面所述的抗PD-1的抗体，或如本发明第七方面所述的免疫偶联物接触；
(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-1或其片段。
在另一优选例中，所述PD-1的血清型包括：PD-11、PD-12、PD-15、PD-16、PD-17、PD-18、PD-19。
在另一优选例中，所述的PD-1的血清型包括：PD-15、PD-18、PD-19。
在另一优选例中，所述的检测包括诊断性的或非诊断性的。
在本发明的第十四方面，提供了一种PD-1信号通路相关疾病的诊断方法，包括步骤：
(i)从诊断对象获取一样品，将所述的样品与如本发明第一方面所述的抗PD-1单域抗体、如本发明第二方面所述的抗PD-1的抗体、或如本发明第七方面所述的免疫偶联物接触；和
(ii)检测是否形成抗原-抗体复合物，其中形成复合物就表示所述的对象患有PD-1信号通路相关疾病。
在本发明的第十五方面，提供了一种重组多肽的制备方法，所述的重组多肽是如本发明第一方面所述的抗PD-1单域抗体、如本发明第二方面所述的抗PD-1的抗体，所述的方法包括：
(a)在适合表达的条件下，培养如本发明第五方面所述的宿主细胞；和
(b)从培养物中分离出所述的重组多肽。
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
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。
图1显示了SDS-PAGE检测PD1-Fc蛋白表达的结果。泳道1代表PD1-Fc蛋白的大小，泳道M代表蛋白Mraker。
图2显示了细胞流式检测PD1-Fc蛋白与PDL1过表达细胞株CHO的结合情况的结果。
图3显示了免疫血清的免疫效价ELISA检测结果。
图4显示了噬菌体展示库序列的比对结果。
图5显示了抗原固相淘选结果，共筛选到4条不同的抗体序列。
图6显示了细胞和固相淘选交叉进行筛选抗体的结果。
图7显示了筛选的PD1单域抗体与PD1过表达细胞株CHO细胞的结合情况。
图8显示了PD1单域抗体(单价)阻断活性功能验证。Nivolumab为阳性对照PD1抗体，LAB190417-2-B1，LAB190417-2-C10，LAB190417-2-F3，LAB190417-3-H6为流式筛选的4个克隆的PD1抗体。
图9显示了PD1单域抗体(双价)阻断活性功能验证。Nivolumab为阳性对照PD1抗体，2-C10-(G4S)3-2-F3，2-C10-(G4S)3-2-B1，2-F3-(G4S)3-2-B1为构建的3个双价的PD1抗体。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，成功获得多个抗PD-1的单域抗体。具体地，本发明利用PD1-Fc抗原蛋白免疫骆驼，利用噬菌体展示技术筛选免疫单域抗体基因库(噬菌体展示库)，并进行淘选和鉴定，从而获得了针对PD-1的单域抗体基因。相关实验结果表明，本发明获得的抗PD-1的单域抗体能够有效地与PD-1结合，并且对PD-1/PD-L1的结合具有优异的阻断活性。在此基础上完成了本发明。
术语
如本文所用，术语“本发明抗体”、“本发明的抗体”、“本发明的抗PD-1的单域抗体”、“本发明抗PD-1的单域抗体”、“抗PD-1的单域抗体”、“抗PD-1的单域抗体”具有相同的含义，可互换使用，均指特异性识别和结合于PD-1的抗体。
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)”、“单域抗体”(single domain antibody,sdAb，或纳米抗体nanobody)具有相同的含义并可互换使用，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b-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b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针对PD-1的纳米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-1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。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于10 -8M、10 -9M或l0 -10M或更小的亲和力(KD)结合。
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-1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A进行氨基酸替换而产生。
表A
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))等。
PD-1
如本文所用，术语“PD-1”、“程序性死亡受体1”均指PD-1免疫检验点受体，其在外周组织炎症应答的时候能够限制T细胞的活性，以及限制自身免疫。 PD-1是肿瘤微环境中产生免疫抵抗的主要机制。T细胞在活化后诱导PD-1的表达。当与PD-1的一个配体结合后，参与T细胞激活的磷酸化酶SHP2被抑制。同时，因为PD-1结合抑制了TCR停止信号，这条信号通路能够改变T细胞—APC或T细胞—靶细胞之间的连接时间。PD-1高表达于Treg细胞，Treg细胞在PD-1配体存在的情况下会增强自身的增殖。因为许多肿瘤高表达浸润的Treg细胞，可能会进一步抑制效应细胞的免疫应答，所以阻断PD-1信号通路会通过减少并/或抑制瘤内Treg细胞的活性从而增强抗肿瘤免疫应答。
PD-1有两个配体PD-L1和PD-L2。
PD-1表达在来源多种肿瘤类型的大部分肿瘤浸润淋巴细胞(TILs)中。高表达PD-1的CD4+TIL，主要集中在肿瘤组织中CD4+Treg细胞。高表达PD-1的CD8+TILs表现出无能或疲惫的状态，通过比较黑色素瘤内PD-1-TILs，PD-1+TILs分泌更少的细胞因子。就像PD-1高表达于许多肿瘤的TILs，PD-1配体也高表达于多种肿瘤的肿瘤细胞表面。在实体瘤细胞表面主要表达PD-1的配体PD-L1，促使小鼠肿瘤细胞高表达PD-L1能够抑制T细胞介导的抗肿瘤免疫应答。的确，这些发现为阻断PD-1信号通路增强在肿瘤微环境中抗肿瘤效应细胞的功能提供了基础。基于免疫组化(IHC)和流式细胞仪的分析得出多种人类肿瘤在一定水平组成型高表达PD-1配体。PD-1配体的表达模式将主要决定阻断这条通路的免疫治疗策略是否可行，因为PD-1配体在癌症中的主要作用被认为是在肿瘤微环境中发挥免疫抑制，而且PD-1与它的配体，PD-L1和PD-L2，结合后只能抑制淋巴细胞的功能。
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)：腹膜内、静脉内、或局部给药。
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。
抗PD-1的单域抗体
本发明提供了抗PD-1的单域抗体，其能够特异性结合于PD-1。
本发明的抗PD-1单域抗体的VHH链的互补决定区CDR为选自下组的一种或多种：
(1)SEQ ID NO:5所示的CDR1、SEQ ID NO:9所示的CDR2、和SEQ ID NO:10所示的CDR3；
(2)SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:8所示的CDR3；
(3)SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:7所示的CDR3；和
(4)SEQ ID NO:11所示的CDR1、SEQ ID NO:12所示的CDR2、和SEQ ID NO:13所示的CDR3。
在本发明的一个优选例中，所述所述抗PD-1的单域抗体包括一条或多条具有如SEQ ID NO:1、2、3或4所示氨基酸序列的VHH链。
在本发明中，所述抗PD-1的单域抗体包括单体、二价体(二价抗体)、四价体(四价抗体)、和/或多价体(多价抗体)。所述多价抗体包含2个以上的上述VHH链或上述单域抗体，优选为2个、3个或4个。
在本发明的一个优选例中，所述所述抗PD-1的单域抗体为二价体。
本发明的二价体(二价抗体)具有如式I所示的结构：
P1-L-P2       (I)
其中，
“-”为肽键；L为连接肽，P1、P2VHH链。
在本发明的一个优选例中，所述的L的序列为(G4S)n，其中，n为正整数(例如1、2、3、4、5或6)，优选地，n＝3。
在另一优选例中，所述P1、P2各自独立地包含如SEQ ID NO:1、2、3或4所示的序列。
在另一优选例中，P1具有SEQ ID NO:3所示的氨基酸序列，P2具有SEQ ID NO:2所示的氨基酸序列。
在另一优选例中，P1具有SEQ ID NO:3所示的氨基酸序列，P2具有SEQ ID NO:1所示的氨基酸序列。
在另一优选例中，P1具有SEQ ID NO:2所示的氨基酸序列，P2具有SEQ ID NO:1所示的氨基酸序列。
检测方法
本发明还涉及检测PD-1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-1的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测PD-1水平的检测试剂盒，该试剂盒包括识别PD-1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-1信号通路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-1信号通路相关疾病的临床诊断、预防和治疗。
本发明的主要优点包括：
1)本发明抗体能够特异性结合PD-1，且对PD-1中和活性较高；
2)本发明抗体能够高效地阻断PD-1/PD-L1结合；
3)本发明的PD1纳米抗体具有分子量小，组织穿透快，溶解度和稳定性高，抗原结合特异性高，免疫原性弱等优点，更易于CAR-T细胞对PD1抗体的共表达。可用于癌症治疗。
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。
实验试剂、耗材及设备：
试剂：琼脂(Sigma,CAT#A1296)；蛋白胨(Sigma,CAT#93926)；酵母提取物(OXOID,CAT#:LP0021)；氯化钠(阿拉丁,CAT#:C111533)；氯化钾(阿拉丁,CAT#:P112133)；硫酸镁(国药,CAT#:10013018)；氯化镁(国药,CAT#:10012818)；葡萄糖(生工,CAT#:GT1991)；SfiI(NEB,CAT#:R0123L)；T4 DNA ligase(TaKaRa,CAT#:2011A)；PrimeScript TM II 1st Strand cDNA Synthesis Kit(TaKaRa,CAT#:6210B)；NuHi power mix(新海生物,CAT#:NH9303)；3M醋酸钠(pH5.2-6)(Sigma,CAT#:126-96-5)；DNA片段回收试剂盒(TakaRa,CAT#:9761)；胶回收试剂盒(Qiagen,CAT#:28706)；天根质粒大抽试剂盒(天根,CAT#:DP117)；PmeI限制性内切酶(NEB,CAT#:R0560L)；HRP-M13(Sino Biolo,CAT#:11973-MM05)；PE-anti-Human IgG(eBioscience,Cat#:12-4998-82)；Rabbit anti-Llama IgG(H+L)Secondary Antibody[HRP](Novus，CAT#NBP1-75095)；SS320感受态(iCarTab)；pComF噬菌体展示载体(iCarTab)。
耗材；50mL Falcon离心管(Corning,CAT#352070)；电击杯(Bio-Rad 0.2cm)；RNase free 1.5ml EP管(QSP,CAT#:509-GRD-Q)；200μLRNase free PCR管(Axygen,PCR-02D-C)；T125摇瓶(Corning,CAT#431143)。
设备；电转仪(Eppendorf Multiporator)；离心机(湘仪H1650R)；恒温培养箱(上海精宏DNP-9052)；恒温震荡培养箱(朗越DZ-85A)；超净工作台(苏净安泰SW-CJ-1FD)；PCR仪(Applide Biosystems ABI2720)；生物安全柜(海尔，HR40-IIA2)；流式细胞仪(Thermo Attune Nxt flow cytometer)。
实验方法
1.PD1-Fc抗原制备
1)通过基因合成PD1胞外段(25AA-167AA)序列，在C端添加human IgG1Fc标签，亚克隆至真核表达载体中，构建抗原表达载体。
2)将构建好的PD1-Fc蛋白表达载体进行质粒大抽，瞬时转染293细胞后，连续培养8天，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，使用Protein A柱子纯化抗体。
2.抗原免疫美洲无峰驼
采用上述制备的抗原免疫1只羊驼，皮下多点免疫5次。免疫流程如下表1所示。
表1羊驼免疫方案
Figure PCTCN2022140768-appb-000001
Figure PCTCN2022140768-appb-000002
3.免疫效价的检测
1)采集5ml外周血，将收集有血液样本的离心管置于37℃培养箱内放置1小时；然后将血液样本转移至4℃过夜；
2)将血清转移至一个新的无菌离心管中，5000rpm离心20min；采用ELISA检测免疫效价。
4.PBMC分离
采集100ml外周血，使用淋巴细胞分离液分选PBMC。
提取RNA，使用PrimeScript TM II 1st Strand cDNA Synthesis Kit进行反转录，制备cDNA。方法如下：
在200μLPCR中配制表2的反应混合液MIX1：
表2
Figure PCTCN2022140768-appb-000003
65℃保温5min后，冰上迅速冷却；
在上述PCR管中配制表3的反应液：
表3
Figure PCTCN2022140768-appb-000004
吹打混匀后，分装80μL/管，置于PCR仪中42℃1小时，70℃热失活15分钟，最后将cDNA样品置于冰上或-20℃长期保存。
5.VHH片段的扩增
如表4所示配置第一轮PCR反应体系(50μL/管)。
表4
Figure PCTCN2022140768-appb-000005
配置好PCR反应体系后，按照如下表5程序设置PCR仪：
表5
Figure PCTCN2022140768-appb-000006
使用1％的琼脂糖进行电泳分析PCR产物，分离分子量大小为750bp左右的片段。使用胶回收试剂盒回收PCR产物，并用NanoDrop测定浓度。
按表6配置二轮PCR反应体系(50μL/管)
表6
Figure PCTCN2022140768-appb-000007
配置好PCR反应体系后，按照表7的程序设置PCR仪：
表7
Figure PCTCN2022140768-appb-000008
使用1％的琼脂糖进行电泳分析PCR产物，分离分子量大小为750bp左右的片段。使用胶回收试剂盒回收PCR产物，并用NanoDrop测定浓度。
6.噬菌体展示库的构建
噬菌体展示载体的构建:
1)使用SfiI分别酶切pCom F载体和上述获得的VHH PCR胶回收产物，50℃酶切过夜。
2)使用1％的琼脂糖凝胶分离pCom F载体片段，切取5000bp的载体片段进行胶回收；同时使用DNA片段回收试剂盒纯化PCR酶切产物，并用NanoDrop测定浓度。
3)酶切好的pCom F载体和VHH片段使用T4ligase连接，16℃连接过夜。
噬菌体连接产物电转化大肠杆菌：
1)准备电转杯、连接产物和电转感受态置于冰上预冷；
2)取预冷的建库连接产物加入到电转感受态中，置于冰上1min，向每个电转杯中加入70μL DNA/感受态混合物，将电转杯放于冰上；
3)按照2500V，5ms进行电转；
4)电击结束后，立刻加入平衡至室温的SOC培养基重悬菌体，37℃摇床培养1小时。
5)取菌液15mL直接进行噬菌体拯救，其余5mL电转产物加入等体积的50％甘油，均匀混合后保存在-80℃。
6)另外取菌液20μL，加入980μL 2YT培养基进行稀释后，取100μL稀释后产物，加入900μL 2YT培养基进行第二步稀释，取50μL均匀涂布在含有氨苄青霉素的LB平板上，37℃培养过夜。
7)第二天，取出平板，计算每个连接能够产生的克隆数，计算库容。
8)同时挑取平板上的单克隆20个到含氨苄青霉素的2YT培养基中，37℃震荡培养约6-8小时，送菌液测序(测序通用引物M13R)，计算库的多样性。
7.噬菌体展示库的淘选
噬菌体展示库的复苏和拯救及噬菌体沉淀步骤如下：
1)将电转后的转化产物用2YT稀释调整至OD600为0.2左右，加入终浓度为100ug/mL的氨苄青霉素置于恒温摇床中，37℃，225rpm培养，至OD600为0.5时停止；
2)投入M13KO7，摇匀后37℃静置30min后，37℃，225rpm培养1h。
3)M13KO7体积＝10×体积×OD600×5×108/M13KO7滴度
4)将菌液6000rpm离心10min后用2YT-AK培养基重悬，25℃，200rpm过夜培养；
5)将菌液10000rpm离心15min；
6)弃去沉淀，将上清转至新的离心管，管内加入1/5菌液体积的PEG/NaCl， 混匀后放置4℃，静置2h。
7)将沉淀的噬菌体上清，10000rpm，4℃，离心30min；弃去上清，每个50ml离心管的沉淀(噬菌体)用1ml无菌PBS重悬；
8)将重悬的噬菌体转移至1.5mLEP管中，置于离心机中，12000g，4℃，离心5min；
9)将上清转移至新的1.5mlEP管，每管加入250μl的PEG/NaCl，混匀后4℃静置10min,
10)12000g离心10min，弃上清，加入1mlPBS重悬，
11)12000g离心5min，弃沉淀，将上清转移至新的1.5mlEP管，
12)12000g离心5mi，将上清转移至新的1.5mlEP管，得到噬菌体原始库。
13)取10μl沉淀投入90μl 2YT培养基，记做10 -1，依次往后10倍稀释至10 -9，取10 -7、10 -8、10 -9三个梯度20μl稀释好的样品投入200μl事先准备好的OD600为0.5的ER2738，混匀后放于37℃水浴锅，静置10min，每108μl涂1块LB-AMP固体平板，37℃过夜，第二天数斑以确定Titer。
14)Titer计算：选取斑数在30-300之间的平板，两块平板取平均值，斑数量乘以稀释倍数再乘以100得到titer。
8.噬菌体展示库的淘选(panning)
噬菌体展示库的固相panning步骤如下：
使用ELISA板，包被目的蛋白，经历数次洗脱步骤后，使用TEA将结合在固定抗原上的重组噬菌体洗脱下来，并进行扩增；经历3-4轮淘选后，挑选单克隆进行测序。
1)用PBS稀释抗原至50μg/ml，150μl/well，共包被3well，置于4℃包被过夜；
2)吸去target蛋白，用3％MPBS室温封闭1h；
3)将lib phage或上轮扩增的沉淀用450μl 3％MPBS稀释，将6×10 11Pfu的phage(每孔2×10 11Pfu)投入到对照蛋白包被孔中，每孔投入150μl的稀释好的phage，室温孵育1h；
4)吸去target蛋白的MPBS，并将对照孔中的phage转至包被target蛋白的孔中，室温孵育1-1.5h；
5)将phage吸去，用0.05％PBST洗涤8-10次，每次2-3min，再用PBS洗涤4-5次，每次2-3min，同时用PBS洗涤事先封闭的1.5mlEP管；
6)用1×TEA洗脱6～8min，每孔200μl，收集洗脱下来的产物至预先封闭过的EP管中，每孔再加入100μl Tris-HCl中和；
7)取10μl output产物投入90μl 2YT培养基，记做100，依次往后10倍稀 释至10 -2，取10 1、10 0、10 -1、10 -2四个梯度各20μl稀释好的样品投入200μl事先准备好的OD600为0.5的ER2738(10 1是直接将未稀释的产物20μl加入ER2738)，混匀后放于37℃水浴锅，静置10min，每108μl涂1块LB-AMP固体平板，37℃过夜，第二天数斑以确定Titer。
8)Titer计算：选取斑数在30-300之间的平板，两块平板取平均值，用斑数量乘以稀释倍数再乘以洗脱体积。
噬菌体展示库的cell-based panning步骤如下：
使用过表达靶蛋白的重组细胞株，将噬菌体库依次与空细胞和过表达靶蛋白的细胞进行孵育后，经数次洗涤洗去非特异性结合的噬菌体后，使用甘氨酸或TEA将结合在细胞表面上的重组噬菌体洗脱下来，并进行扩增；经历3-4轮淘选后，挑选单克隆进行ELISA检测。
1)提前一天用1％PBSA封闭1.5ml EP管，4℃过夜；
2)Target cell和control cell各取1x107，用PBS洗涤三次，用10mL1％PBSA重悬，放置于脱色摇床室温低速封闭1h；
3)向control cell中投入1x1011的噬菌体，孵育1h，target cell继续封闭；
4)将孵育结束的细胞置于离心机中，1000g离心5min，弃去target cell上清(1％PBSA)，并将control cell的上清小心吸出，加入到target cell管中，重悬后，置于摇床，低速孵育1h；
5)将target cell置于离心机中，1000g离心5min；(同时用PBS洗涤三次昨天封闭的1.5ml EP管)，弃去target cell上清，加入4ml PBS重悬，分装至封闭的1.5ml EP管，用PBS离心洗涤5次，再将细胞转移至另外四个EP管，继续洗涤5次，然后将细胞转移至同一个EP管中；
6)用200μl PBS重悬细胞，然后加入200μl 2xTEA迅速吹吸，直至溶液不再粘稠，再加入200μl Tris-HCl中和，即得到产物；
7)测定Output库的titer，同固相panning方案。
9.Phage ELISA
1)分装2YT-Amp培养基至96孔深孔板，每孔500μl，挑取output平板上的单克隆，37℃，225rpm培养至OD600直至0.5；最后两孔H11和H12不挑克隆，只放培养基，作为空白对照；
2)同时用CBS包被抗原至ELISA板，浓度1μg/ml，100μl/well，37℃，包被2h；
3)另取一块96孔深孔板，分装2YT-A培养基，每孔500μl，用排枪依次吸取OD600为0.5的菌液10μl至新分装的96孔板中，置于37℃，225rpm培养过 夜，此为送样测序菌液；
4)向OD600为0.5的菌液中，加入M13KO7，混匀后放于37℃，静置15min；
5)M13KO7体积＝10×体积×OD600×5×108/M13KO7滴度
6)将侵染结束的菌液置于摇床上，37℃，225rpm培养45min；
7)将菌液置于离心机中，4000rpm离心10min，弃去上清，用2YT-AK培养基重悬，每孔800μl，重置于摇床中，30℃，210rpm过夜培养。
8)同时ELISA板甩掉抗原，用PBST洗液洗三遍后，用3％MPBS封闭250μl/well，4℃过夜；并额外封闭一块空白板，作为BLANK；
9)第二天，将96孔深孔板置于离心机中，4000rpm离心10min；将ELISA板中的牛奶弃去，用200μL PBST洗涤4次；在每孔先加入50μl PBST，再一一对应加入50μL离心后的噬菌体上清，置4℃孵育1h；弃去上清，并用PBST洗涤5次；用PBST稀释HRP-Anti M13二抗，每孔100μl，4℃孵育45min后，洗去二抗，PBST洗5次，TMB常温显色10min，盐酸终止，读数，选取S/N比大的值的克隆，用保种的菌液送测。
10.VHH真核表达载体的构建
根据噬菌体单克隆Elisa检测结果，挑选阳性克隆进行测序，获得VHH抗体序列。将分析获得的VHH抗体序列分别进行基因合成，与human IgG1Fc串联亚克隆至表达载体pcDNA3.4-hIgG1-Fc2中。载体经测序验证无误后，使用Qiagen质粒大抽试剂盒制备去内毒素质粒备用。
11.二价抗体表达载体的构建
将候选的3个抗体随机组合构建二价抗体，二价抗体表达形式为2-C10-(G4S) 3-2-F3，2-C10-(G4S) 3-2-B1，2-F3-(G4S) 3-2-B1分别进行基因合成，与human IgG1Fc串联亚克隆至表达载体pcDNA3.4-hIgG1-Fc2中。载体经测序验证无误后，使用Qiagen质粒大抽试剂盒制备去内毒素质粒备用。
12.单域抗体的表达
从冰箱中取出LVTransm转染试剂及抗体表达载体pcDNA3.4-hIgG1-Fc2，室温解冻后，用移液枪上下吹打完全混匀。取出PBS缓冲液，温热至室温。取500μLPBS至24孔板的一个孔，加入4μg pcDNA3.4-hIgG1-Fc2，移液枪上下吹打充分混匀后，加入12μL LVTransm，立即用移液器上下吹打混匀，室温下静置10分钟。此处的混合物称为DNA/LVTransm复合物。
将上述532μL DNA/LVTransm复合物加入到1.5mL 293F细胞中，轻轻晃动充分混匀。将细胞置于37℃、5％CO 2培养箱，130RPM培养6～8小时后，加入 1.5mL新鲜的293培养基，将细胞重新放回培养箱中继续培养。
连续培养3天后，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，进行后续的流式和ELISA检测。
13.流式检测重组抗体与靶蛋白的结合
从液氮中复苏CHO-K1和CHO-K1-PD1细胞株，调整细胞状态至对数生长期。将两种细胞分别分为若干份，每份细胞的数量为5*10^5个细胞。将表达的抗体分别孵育靶细胞，充分混匀后，室温孵育1小时。800xg室温离心5分钟，去掉含有抗体的上清，使用PBS洗涤细胞3次。加入1μL PE标记的Anti-human IgG，充分混匀后，室温避光孵育30分钟。800xg室温离心5分钟，去掉含有二抗的上清，使用PBS洗涤细胞3次。使用500uL PBS重悬细胞，进行流式分析。
14.单域抗体的表达纯化
从冰箱中取出LVTransm转染试剂及单链抗体表达载体，室温解冻后，用移液枪上下吹打完全混匀。取出PBS或HBSS缓冲液，温热至室温。取2mL PBS至6孔板的一个孔，分别加入130μg pcDNA3.4-hIgG1-Fc2，移液枪上下吹打充分混匀后，加入400μL LVTransm，立即用移液器上下吹打混匀，室温下静置10分钟。
将上述DNA/LVTransm复合物加入到50mL 293F细胞中，轻轻晃动充分混匀。将细胞置于37℃、5％CO2培养箱，130RPM培养6～8小时后，加入50mL新鲜的293培养基，将细胞重新放回培养箱中继续培养。
连续培养7天后，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，使用Protein A柱子纯化抗体。
15.PD1-PDL1阻断活性检测
复苏Jurkat-PD1-NFAT-Luc报告基因细胞株及aAPCCHO-PDL1细胞，连续继代培养至对数生长期，在96孔板中，每孔接种2x10^4个效应细胞Jurkat-PD1-NFAT-Luc，按照1:1加入靶细胞aAPCCHO-PDL1。在对应的孔中加入梯度稀释的检测抗体(阳性抗体：Nivolumab；待检测抗体)，按照3倍梯度稀释抗体，连续稀释9个梯度，终浓度依次为30μg/mL，10μg/mL，3.333μg/mL，1.111μg/mL，0.3704μg/mL，0.1235μg/mL，0.04115μg/mL，0.01372μg/mL，0.004572μg/mL，然后加入对应孔中。共培养18h后，每孔加入25μL One-Glo试剂，使用Tecan M1000pro酶标仪检测孔内的荧光素酶活性数值。
16.重组抗体亲和力检测
将PD1-Fc重组蛋白使用10mM Acetate缓冲液固定在CM5芯片上，分别以制备的单域抗体作为流动相，检测候选单域抗体与靶蛋白PD1的结合能力。
实施例1 PD1单域抗体的制备及纯化
1.1 PD1-Fc重组蛋白活性检测
使用上述的实验方法1制备PD1-Fc抗原，SDS-PAGE检测PD1-Fc蛋白的表达。结果如图1所示。
使用CHO-PDL1细胞与PD1-Fc进行孵育，二抗为APC-anti human Fc，根据流式检测结果(图2)，PD1-Fc可以结合PDL1，具有活性，可以用于羊驼免疫和抗体的筛选。
1.2免疫效价ELISA检测
使用上述的实验方法2免疫美洲无峰驼。并使用实验方法3进行免疫效价的检测。具体地，从免疫的羊驼分离血清，按照图3所示的稀释梯度进行有限稀释，与PD1-His(义翘神州，Cat#10377-H08H)抗原预包被的96孔板进行ELISA实验检测。
结果：如图3所示，免疫效价ELISA检测结果显示PD1免疫效价较高，进行一次冲击免疫，10天后采集150mL外周血进行噬菌体展示库的构建。
1.3 VHH片段的扩增
使用上述的实验方法4和5进行PBMC分离和VHH片段的扩增。具体地，从实施例1.2中经过免疫的美洲无峰驼采集外周血，提供总的RNA，逆转录为cDNA后，使用单域抗体扩增引物进行两轮PCR，琼脂糖凝胶电泳PCR产物结果。第一轮PCR分别获得1000bp和750bp左右的PCR条带，胶回收750bp的片段作为二轮PCR的模板。第二轮PCR获得450bp左右的条带，获得VHH片段。
1.4噬菌体展示库多样性分析
使用上述的实验方法6进行噬菌体展示库的构建和多样性分析。具体地，将实施例1.3中获得的VHH片段使用SfiI酶切后亚克隆至噬菌体展示载体pComF中，电转化SS320大肠杆菌感受态细胞，构建单域抗体噬菌体展示库。从其中随机挑选20个单克隆进行测序，分析构建噬菌体展示库的多样性。
结果：如图4所示。测序结果比对显示噬菌体展示库空载率及抗体重复率不高于10％。
1.5噬菌体展示库固相淘选及产物的Phage ELISA鉴定
使用上述的实验方法7进行噬菌体展示库的固相淘选。具体地，分别包被PD1-Fc和Fc重组蛋白，将构建的噬菌体展示库进行四次筛选富集，富集阳性克隆。
1.6噬菌体展示库固相淘选产物phage ELISA
从实施例1.5中富集的噬菌体阳性克隆中，挑选噬菌体单克隆，使用上述的实验方法8进行Phage ELISA鉴定。
具体地，选取固相淘选第2轮和第3轮output进行Phage ELISA实验；分别包被PD1-Fc抗原蛋白，Phage Elisa检测，对照组为直接封闭的孔板和Fc包被的孔。选择S/N比值在5以上，且与Fc不结合的克隆进行测序，分析序列抗体。
结果：如图5所示，抗原固相淘选，共获得4条不同的抗体序列，其VHH链氨基酸序列及CDR氨基酸序列如表8和表9所示，用于后续构建真核表达载体，进行瞬时转染表达和流式分析。
由于采用固相筛选，获得的大部分为Fc阳性的克隆，后续安排细胞和固相淘选交叉进行排除Fc抗体的干扰。
表8本发明单域抗体VHH链序列
Figure PCTCN2022140768-appb-000009
Figure PCTCN2022140768-appb-000010
其中，序列下划线标记处为CDR区。
表9各抗体VHH链CDR区序列
Figure PCTCN2022140768-appb-000011
1.7噬菌体展示库细胞和固相淘选交叉进行
使用上述的实验方法8交叉进行噬菌体展示库细胞和固相淘选。具体地，使用CHO和CHO-PD1细胞以及PD1-Fc抗原，将构建的噬菌体展示库进行5次筛选富集，富集阳性克隆。
1.8噬菌体展示库固相和细胞交叉淘选产物Phage ELISA
从实施例1.7中富集的噬菌体阳性克隆中，挑选噬菌体单克隆，使用上述的实验方法9进行Phage ELISA鉴定。
具体地，分别包被PD1-Fc和Fc抗原蛋白，Phage Elisa检测，对照组为直接封闭的孔板。将抗原ELISA检测结果和对照组的检测结果进行比值，挑选比值较大的克隆进行测序分析，分析抗体序列。
结果：如图6所示，细胞和固相淘选交叉进行，排除了Fc抗体的干扰，筛选到特异性结合PD-1的抗体。本实验中抗体序列高度富集，获得1条抗体序列，同固相挑选获得的2-C10抗体序列。
1.9单域抗体真核表达结合检测
在本实施例中，对实施例1.6中筛选并测序的4种VHH抗体序列进行载体构 建和表达检测。在候选抗体序列的N端和C端分别通过Overlap PCR添加CMV启动子、信号肽及human IgG1 Fc标签，纯化PCR产物瞬时转染293细胞，表达抗体进行流式检测。
结果：FACS检测结果如图7所示，噬菌体展示库淘选获得的3-H6，2-C10，2-B1和2-F3克隆均为PD1特异性结合抗体。抗体进行表达纯化，用于后续阻断功能的验证。
实施例2抗体功能检测
2.1 PD1-PDL1阻断活性检测(单价抗体)
使用Jurkat-PD1-NFAT-Luc报告基因细胞株及aAPCCHO-PDL1细胞进行PD1-PDL1阻断活性检测。
结果：如图8所示，Nivolumab阳性对照可以阻断PD1/PDL1的相互作用，EC50为0.6374μg/mL。待检测抗体中PD1单域抗体LAB190417-2-B1，LAB190417-2-C10和LAB190417-2-F3均可以阻断PD1/PDL1的相互作用。
2.2 PD1-PDL1阻断活性检测(二价抗体)
为提高抗体的阻断活性，在本实施例中，将3个抗体随机组合构建二价抗体再进行阻断活性检测。
将候选的3个抗体随机组合构建二价抗体，二价抗体表达形式为2-C10-(G4S)3-2-F3，2-C10-(G4S)3-2-B1，2-F3-(G4S)3-2-B1，纯化抗体后，使用Jurkat-PD1-NFAT-Luc报告基因细胞株及aAPCCHO-PDL1细胞进行PD1-PDL1阻断活性检测。
结果：如图9所示，Nivolumab阳性对照可以阻断PD1/PDL1的相互作用，EC50为0.4887μg/mL。待检测的二价PD1抗体均可以阻断PD1/PDL1的相互作用，其中2-C10-(G4S)3-2-B1阻断活性最高。
本实施例证明，构建的二价抗体具有高效阻断PD1/PDL1相互作用的活性。
2.3重组抗体亲和力检测
将PD1-Fc重组蛋白使用10mM Acetate缓冲液固定在CM5芯片上，分别以制备的二价单域抗体作为流动相，检测候选单域抗体与靶蛋白PD1的结合能力。
结果：如表10所示。
表10重组抗体亲和力检测结果
  ka kd KD
Nivolumab 3.987×10 6M -1s -1 8.747×10 -5s -1 2.194×10 -11M
2-C10-(G4S) 3-2-B1 1.562×10 6M -1s -1 3.289×10 -5s -1 2.106×10 -11M
2-C10-(G4S) 3-2-F3 3.181×10 5M -1s -1 8.918×10 -5s -1 2.804×10 -10M
2-F3-(G4S)3-2-B1 4.183×10 5M -1s -1 1.180×10 -4s -1 2.822×10 -10M
本实施例证明，本发明的三种二价抗体对PD-1均具有高亲和力，其中2-C10-(G4S) 3-2-B1亲和力高于现有的阳性对照Nivolumab。
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 (15)

  1. 一种抗PD-1单域抗体，其特征在于，所述抗PD-1单域抗体的VHH链的互补决定区CDR为选自下组的一种或多种：
    (1)SEQ ID NO:5所示的CDR1、SEQ ID NO:9所示的CDR2、和SEQ ID NO:10所示的CDR3；
    (2)SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:8所示的CDR3；
    (3)SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:7所示的CDR3；和
    (4)SEQ ID NO:11所示的CDR1、SEQ ID NO:12所示的CDR2、和SEQ ID NO:13所示的CDR3。
  2. 如权利要求1所述的抗PD-1单域抗体，其特征在于，所述的抗PD-1单域抗体的VHH链的氨基酸序列选自SEQ ID NO:1、2、3或4所示的序列。
  3. 如权利要求1所述的抗PD-1单域抗体，其特征在于，所述的抗PD-1单域抗体为单体、二价体或多价体。
  4. 如权利要求1所述的抗PD-1单域抗体，其特征在于，所述的抗PD-1单域抗体为二价体，并且从N端至C端方向具有如式I所示的结构：
    P1-L-P2 (I)
    其中，
    “-”为肽键；
    L为连接肽，
    P1、P2各自为VHH链。
  5. 如权利要求4所述的抗PD-1单域抗体，其特征在于，所述的L的序列为(G4S)n，其中，n为1-6的正整数(例如1、2、3、4、5或6)，优选地，n＝3。
  6. 如权利要求4所述的抗PD-1单域抗体，其特征在于，
    所述P1具有SEQ ID NO:5所示的CDR1、SEQ ID NO:9所示的CDR2、和SEQ ID NO:10所示的CDR3，且所述P2具有SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:7所示的CDR3；或
    所述P1具有SEQ ID NO:5所示的CDR1、SEQ ID NO:9所示的CDR2、和SEQ ID NO:10所示的CDR3；且所述P2具有SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:8所示的CDR3；或
    所述P1具有SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:8所示的CDR3，且所述P2具有SEQ ID NO:5所示的CDR1、SEQ ID NO:6所示的CDR2、和SEQ ID NO:7所示的CDR3。
  7. 如权利要求4所述的抗PD-1单域抗体，其特征在于，
    P1具有SEQ ID NO:3所示的氨基酸序列，且P2具有SEQ ID NO:1所示的氨基酸序列；或
    P1具有SEQ ID NO:3所示的氨基酸序列，且P2具有SEQ ID NO:2所示的氨基酸序列；或
    P1具有SEQ ID NO:2所示的氨基酸序列，且P2具有SEQ ID NO:1所示的氨基酸序列。
  8. 一种抗PD-1的抗体，所述抗体包括一个或多个如权利要求1所述的抗PD1的单域抗体的VHH链。
  9. 一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：如权利要求1所述的抗PD-1单域抗体、或如权利要求8所述的抗体。
  10. 一种表达载体，所述表达载体含有如权利要求9所述的多核苷酸。
  11. 一种宿主细胞，所述宿主细胞含有如权利要求10所述的表达载体，或其基因组中整合有如权利要求9所述的多核苷酸。
  12. 一种免疫偶联物，所述免疫偶联物含有：
    (a)如权利要求1所述的抗PD-1单域抗体、或如权利要求8所述的抗PD-1的抗体；和
    (b)选自下组的偶联部分：可检测标记物、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP，或其组合。
  13. 一种药物组合物，所述药物组合物含有：
    (i)如权利要求1所述的抗PD-1单域抗体、如权利要求8所述的抗PD-1的抗体，或如权利要求12所述的免疫偶联物；和
    (ii)药学上可接受的载体。
  14. 一种产生抗PD-1单域抗体的方法，包括步骤：
    (a)在适合产生单域抗体的条件下，培养如权利要求11所述的宿主细胞，从而获得含所述抗PD-1单域抗体的培养物；
    (b)从所述培养物中分离和/或回收所述的抗PD-1单域抗体；和
    (c)任选地，对步骤(b)获得的抗PD-1单域抗体进行纯化和/或修饰。
  15. 一种预防和/或治疗PD-1信号通路相关疾病的方法，所述方法包括，给需要的对象施用如权利要求1所述的抗PD-1单域抗体、如权利要求8所述的抗PD-1的抗体，或如权利要求12所述的免疫偶联物。
PCT/CN2022/140768 2021-12-21 2022-12-21 一种新型pd1单域抗体的开发 WO2023116781A1 (zh)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107474135A (zh) * 2017-02-17 2017-12-15 广西医科大学 抗PD‑1的纳米抗体PD‑1/Nb20及其制备方法与应用
CN107814845A (zh) * 2016-09-14 2018-03-20 浙江特瑞思药业股份有限公司 新的抗pd‑1纳米抗体及其应用
CN108285485A (zh) * 2018-01-08 2018-07-17 乌鲁木齐恒康致远生物技术有限公司 一种抗pd-1的单域抗体及其应用
CN108299561A (zh) * 2018-01-02 2018-07-20 暨南大学 一种pd-1纳米抗体及其克隆表达方法与应用
CN110003336A (zh) * 2019-04-12 2019-07-12 深圳普瑞金生物药业有限公司 Pd-1单域抗体、核苷酸序列及试剂盒
CN112442122A (zh) * 2019-09-04 2021-03-05 上海洛启生物医药技术有限公司 阻断型pd-1纳米抗体及其编码序列和用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107814845A (zh) * 2016-09-14 2018-03-20 浙江特瑞思药业股份有限公司 新的抗pd‑1纳米抗体及其应用
CN107474135A (zh) * 2017-02-17 2017-12-15 广西医科大学 抗PD‑1的纳米抗体PD‑1/Nb20及其制备方法与应用
CN108299561A (zh) * 2018-01-02 2018-07-20 暨南大学 一种pd-1纳米抗体及其克隆表达方法与应用
CN108285485A (zh) * 2018-01-08 2018-07-17 乌鲁木齐恒康致远生物技术有限公司 一种抗pd-1的单域抗体及其应用
CN110003336A (zh) * 2019-04-12 2019-07-12 深圳普瑞金生物药业有限公司 Pd-1单域抗体、核苷酸序列及试剂盒
CN112442122A (zh) * 2019-09-04 2021-03-05 上海洛启生物医药技术有限公司 阻断型pd-1纳米抗体及其编码序列和用途

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