WO2023011654A1 - 抗pd-1纳米抗体及其应用 - Google Patents

抗pd-1纳米抗体及其应用 Download PDF

Info

Publication number
WO2023011654A1
WO2023011654A1 PCT/CN2022/110672 CN2022110672W WO2023011654A1 WO 2023011654 A1 WO2023011654 A1 WO 2023011654A1 CN 2022110672 W CN2022110672 W CN 2022110672W WO 2023011654 A1 WO2023011654 A1 WO 2023011654A1
Authority
WO
WIPO (PCT)
Prior art keywords
nanobody
antibody
cancer
segment
fusion protein
Prior art date
Application number
PCT/CN2022/110672
Other languages
English (en)
French (fr)
Inventor
屈向东
潘琴
都业杰
金后聪
Original Assignee
启愈生物技术(上海)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 启愈生物技术(上海)有限公司 filed Critical 启愈生物技术(上海)有限公司
Publication of WO2023011654A1 publication Critical patent/WO2023011654A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention relates to the field of biotechnology, in particular to anti-PD-1 nanobody and its application.
  • the immune system can recognize tumor antigens and eliminate tumor cells.
  • the activation of oncogenes causes tumor cells to change themselves and the tumor microenvironment, which disrupts the balance between the immune system and tumor cells.
  • the tumor microenvironment can also suppress the immune system by releasing immunosuppressive factors, such as IL-10, TGF- ⁇ , etc.
  • Immunosuppressive proteins (such as PD-1, PD-L1) are also highly expressed on the surface of tumor cells.
  • T cell apoptosis When effector T cells bind to tumor cells, PD-L1 interacts with PD-1 and induces T cell apoptosis.
  • PD-L1 interacts with PD-1 and induces T cell apoptosis.
  • tumors grow rapidly and metastasize. If the host's immune system is artificially activated and redirected to tumor cells, tumor tissue can theoretically be eliminated, and the theory of immunotherapy has been widely proven in clinical treatment.
  • PD-1 programmed death receptor 1
  • CD279 cluster of differentiation 279
  • PD-1 CD279
  • the human PD-1 coding gene PDCD1 is located at 2q37.3, with a full length of 2097bp, consisting of 6 exons, and the translation product is a PD-1 precursor consisting of 288 amino acids. Protein, the mature protein is obtained after cutting the signal peptide composed of the first 20 amino acids.
  • PD-1 includes an extracellular immunoglobulin variable region IgV domain, a hydrophobic transmembrane domain and an intracellular domain.
  • the N-terminal ITIM motif of the intracellular tail domain contains two phosphorylation sites, and the C-terminal has one ITSM motif.
  • PD-1 is a membrane protein that belongs to the CD28 immunoglobulin superfamily and is mainly expressed on the surface of activated T cells. In addition, it is also expressed in low abundance in thymus CD4-CD8-T cells, activated NK cells and monocytes .
  • PD-1 has two ligands, PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC) of the B7 protein family, and the amino acid sequences of PD-L1 and PD-L2 are 40% identical . The difference between the two is mainly in the different expression patterns.
  • PD-L1 is constitutively low-expressed in APCs, non-hematopoietic cells (such as vascular endothelial cells, islet cells) and immune privileged parts (such as placenta, testis and eyes), and inflammatory cytokines such as Type I and type II interferon, TNF- ⁇ and VEGF can all induce the expression of PD-L1.
  • PD-L2 is only expressed in activated macrophages and dendritic cells.
  • the ITSM motif of PD-1 will be tyrosine phosphorylated, which will lead to the dephosphorylation of downstream protein kinases Syk and PI3K, and inhibit the downstream AKT, ERK and other pathways. Activation, ultimately inhibiting the transcription and translation of genes and cytokines required for T cell activation, negatively regulating T cell activity.
  • Tumor cells In tumor cells, tumor cells and the tumor microenvironment negatively regulate T cell activity and inhibit immune response by up-regulating the expression of PD-L1 and binding to PD-1 on the surface of tumor-specific CD8+ T cells.
  • Tumor cells can up-regulate the expression of PD-L1 through the following four ways: 1. Amplification of the gene encoding PD-L1 (9p24.1); 2. Activation of EGFR, MAPK, PI3K-Akt signaling pathways, HIF-1 transcription factors, etc. Up-regulate the expression of PD-L1 from the transcriptional level; 3. Induction of Epstein-Barr virus (Epstein-Barr virus-positive gastric cancer and nasopharyngeal carcinoma show high expression of PD-L1); 4.
  • the stimulation of inflammatory factors such as interferon- ⁇ can also induce the expression of PD-L1 and PD-L2.
  • Inflammatory factors can induce other cells in the tumor microenvironment, including macrophages, dendritic cells, and stromal cells to express PD-L1 and PD-L2, while tumor-infiltrating T cells that can recognize tumor antigens can secrete interferon- ⁇ , thereby Inducing the upregulation of PD-L1 expression, this process is called "adaptive immune resistance", and tumor cells can achieve self-protection through this mechanism.
  • PD-L1 and PD-L2 have been found in various solid tumors and hematologic malignancies.
  • PD-Ls have been found in various solid tumors and hematologic malignancies.
  • the purpose of the present invention is to provide anti-PD-1 nanobody and its application.
  • the present invention provides an anti-PD-1 nanobody, which includes: VHH chain, the complementarity determining region of the VHH chain includes CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is as SEQ ID NO: 7 As shown, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 9 or SEQ ID NO: 10.
  • the amino acid sequence of the VHH chain includes: selected from any one of SEQ ID NO: 5, 6 or SEQ ID NO: 12-18, or at least 85%, 90%, 95%, Sequences of 97%, or 99% identity.
  • the present invention also provides a fusion protein, which includes: the anti-PD-1 nanobody.
  • the fusion protein further includes: an Fc segment; the Fc segment is fused to the N-terminal or C-terminal of the anti-PD-1 Nanobody.
  • the fusion protein also includes: other functional fragments except the anti-PD-1 Nanobody and the Fc segment; the Fc segment is fused with the anti-PD-1 Nanobody, and/or, The Fc fragment is fused with the other functional fragments.
  • the Fc segment is selected from human IgG1, IgG2, IgG3, or IgG4 or variants thereof; preferably, the Fc segment is selected from a variant of human IgG1; preferably, the Fc domain Choose to eliminate the immune effector function, preferably including any of the following mutations, the following mutations are counted according to EU:
  • the present invention also provides an anti-PD-1 antibody molecule, which includes: one or more of the anti-PD-1 nanobody; or includes: the fusion protein; the anti-PD-1 antibody molecule is monovalent Antibodies, bivalent or multivalent antibodies, bispecific antibodies, or multispecific antibodies.
  • the present invention also provides a chimeric antigen receptor, comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular domain, the extracellular antigen binding domain comprising the anti-PD-1 nanobody.
  • the present invention also provides a nucleic acid molecule encoding: the anti-PD-1 nanobody, or the fusion protein.
  • the present invention also provides a vector comprising the nucleic acid molecule.
  • the present invention also provides a host cell transformed with the vector.
  • the present invention also provides an antibody-drug conjugate comprising the anti-PD-1 nanobody and a drug coupled to the anti-PD-1 nanobody.
  • the present invention also provides a pharmaceutical composition, which contains: the anti-PD-1 nanobody, or the fusion protein, and one or more pharmaceutically acceptable carriers, diluents or excipients agent.
  • the present invention also provides the use of the anti-PD-1 nanobody in the preparation of medicines for inhibiting or treating diseases.
  • the present invention also provides the use of the anti-PD-1 nanobody for preventing or treating diseases by stimulating immune function, such as preparing a vaccine for prevention or treatment.
  • the disease is a PD-1-mediated disease.
  • the disease is cancer; preferably, the cancer includes breast cancer, lung cancer, gastric cancer, colon cancer, kidney cancer, melanoma, bladder cancer, head and neck cancer, lymphoma, skin malignancy, or non- Small Cell Lung Cancer.
  • the present invention has the following beneficial effects:
  • the antibody sequence of the present invention is novel, and it is expected to be used as a therapeutic antibody for the treatment of various malignant tumors with high expression of PD-1.
  • the PD-1 nanobody of the present invention has strong specificity and high affinity; in addition, the VHH chain of the nanobody can be combined with various molecular chains to form different antibody molecular forms, such as combining to form a dual-target or multi-target antibody. specific antibody.
  • Figure 1 is a graph showing the results of ELISA detection of PD-1 nanobody binding to human PD-1 protein.
  • Figure 2 is a graph showing the results of ELISA detection of the binding activity of PD-1 antibody blocking PD-L1/PD-1.
  • Figure 3 is a graph showing the results of LISA detection of PD-1 nanobody binding to cynomolgus monkey PD-1 protein.
  • Figure 4 is a graph showing the binding results of the humanized PD-1 nanobody Fc fusion protein to human PD-1 protein by ELISA.
  • Fig. 5 is a diagram showing the results of ELISA detection of humanized PD-1 Nanobody Fc fusion protein blocking the binding of human PD-L1 protein to PD-1 protein.
  • Figure 6 is a graph showing the binding results of the humanized PD-1 nanobody Fc fusion protein to human PD-1 protein by ELISA.
  • Fig. 7 is a diagram showing the results of ELISA detection of humanized PD-1 nanobody Fc fusion protein blocking the binding of human PD-L1 protein to PD-1 protein.
  • Figure 8 and Figure 9 are the detection results of IL-2 secretion in the supernatant of the mixed lymphocyte reaction for 48 hours.
  • Fig. 10 and Fig. 11 are diagrams showing the detection results of IFN- ⁇ secretion in the supernatant of the mixed lymphocyte reaction for 120 hours.
  • Fig. 12 is a graph showing the detection results of anti-PD-1 antibody on IL-2 secretion of SEB-stimulated PBMC proliferation for 48 hours.
  • Fig. 13 is a graph showing the results of the drug efficacy test of the antibody against the mouse colon cancer CDX model MC38-hPDL1-mClaudin18.2.
  • Fig. 14 is a graph of tumor growth curves of each group after grouping.
  • Fig. 15 is a graph showing the weight of mouse tumor D17 in each group added with each antibody of the present invention and a control antibody.
  • Fig. 16 is a graph of body weight of mice in each group.
  • Antibody, Ab refers to an immunoglobulin molecule (immunoglobulin, Ig) that contains at least one antigen-binding site and is capable of specifically binding to an antigen.
  • Antigen is a substance that can induce an immune response in the body and specifically binds to an antibody.
  • the binding of antibody and antigen is mediated by the interaction formed between them, including hydrogen bond, van der Waals force, ionic bond and hydrophobic bond.
  • the region on the surface of an antigen that binds to an antibody is called an "antigenic determinant" or "epitope".
  • each antigen has multiple determinants.
  • antibody referred to in the present invention is understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, antibodies comprising at least two different antigen-binding domains Multispecific antibodies (eg, bispecific antibodies), as well as single domain antibodies or nanobodies.
  • Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies of other origin.
  • the antibodies of the present invention can be derived from any animal, including but not limited to immunoglobulin molecules of humans, non-human primates, mice, rats, cows, horses, chickens, camels, and alpacas.
  • Antibodies may contain additional alterations, such as unnatural amino acids, Fc effector function mutations, and glycosylation site mutations. Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of antibodies, and immunoglobulin molecules comprising any other modification to the antigen recognition site, so long as these antibodies exhibit the desired biological activity.
  • the basic structure of a conventional antibody is a Y-shaped monomer connected by two identical heavy chains (heavy chain, H) and two identical light chains (light chain, L) through disulfide bonds.
  • Each chain is composed of 2 to 5 structural domains (also known as functional regions) of about 110 amino acids with similar sequences but different functions.
  • the amino acid sequence of the light chain and the heavy chain near the N-terminus of the antibody molecule changes greatly, and the formed domain is called the variable region (variable region, V region); the relatively constant region near the C-terminal amino acid sequence is called the constant region (constant region) , area C).
  • VH and VL The V regions of the heavy chain and light chain are called VH and VL, respectively, and each of VH and VL has three regions whose amino acid composition and sequence are highly variable, called hypervariable region (HVR); the formation of this region is related to the expression of antigen
  • HVR hypervariable region
  • the complementary spatial conformation is also called complementarity determining region (CDR).
  • the three CDRs of VH can be represented by VHCDR1, VHCDR2, and VHCDR3, respectively, and the three CDRs of VL can be represented by VLCDR1, VLCDR2, and VLCDR3, respectively.
  • a total of 6 CDRs of VH and VL together form the antigen-binding site.
  • the diversity of amino acids in the CDR region is the molecular basis for the specific binding of antibodies to a large number of different antigens.
  • the composition and sequence of amino acids outside the CDRs in the V region have relatively little change, which is called the framework region or framework region (FR).
  • FR framework region
  • VH and VL each have four framework regions, denoted by FR1, FR2, FR3, and FR4, respectively.
  • Each VH and VL consists of three CDRs and four FRs, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant regions of the heavy and light chains are called CH and CL, respectively.
  • the heavy chain constant regions of IgG, IgA, and IgD have three domains, CH1, CH2, and CH3, and the heavy chain constant regions of IgM and IgE have four domains, CH1, CH2, CH3, and CH4.
  • the hinge region between CH1 and CH2 is rich in proline, so it is easy to stretch and bend, and can change the distance between the two arms of the Y shape, which is conducive to the simultaneous binding of the two arms to the epitope.
  • Antigen-binding fragments of conventional antibodies refer to Fab fragments, F(ab')2 fragments, Fv fragments, ScFv fragments, etc. that have antigen-binding activity.
  • Fab fragment fragment of antigen binding, Fab refers to an antibody fragment composed of VL, VH, CL and CH1 domains, which binds to a single antigenic epitope (monovalent).
  • papain hydrolyzes IgG to form two identical Fab segments and one Fc segment
  • pepsin hydrolyzes IgG to form one F(ab')2 segment and several polypeptide fragments (pFc').
  • Single-chain variable fragment scFv single chain antibody fragment
  • scFv single chain antibody fragment
  • HCAb heavy chain antibody
  • CH1 immunoglobulin G
  • Fab antibody antigen binding fragment
  • VHH variable domain of the heavy chain of heavy-chain antibody
  • a nanobody nanobody containing only the VHH domain
  • Nanobody nanobody
  • Singledomain antibody singledomain antibody
  • Nanobodies are easily engineered to form multivalent forms. Due to its small molecular weight, nanobodies are encoded by a single gene, which is easy to carry out genetic engineering, and can polymerize multiple nanobodies through short linking sequences, and even connect and combine with Fab fragments, Fv fragments, ScFv fragments, etc. of conventional antibodies. Formation of multivalent or multispecific antibody structures. Bivalent or multivalent antibodies can recognize the same epitope, but with higher affinity than monovalent antigens. Bispecific or multispecific antibodies can bind to different targets, or different binding regions on the same target, and have stronger antigen recognition ability than monovalent antibodies.
  • Nanobodies are easy to form new fusion molecules with other structures (such as BSA, IgG-Fc, etc.).
  • the nanobody is directionally combined with its target antigen, and the part fused with the nanobody can perform the corresponding function, so it can be used in combination with other drugs, or used in diagnosis and as an experimental research tool in various fields .
  • Nanobody screening can be divided into alpaca immunization, lymphocyte extraction, nanobody library construction, phage library construction, specific phage screening, E. coli expression, antibody purification and other steps.
  • fused refers to linking components directly by peptide bonds or via linker fragments, or by intermolecular interactions. Within a single peptide chain, fusion refers to direct linking by peptide bonds or linking via linking fragments.
  • Fc refers to a crystallizable fragment (fragment crystallizable), which has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cell surface Fc receptors (FcR).
  • the Fc fragment comprises the constant region polypeptides of the antibody except the heavy chain constant region CH1. Fc fragments bind to cells with corresponding Fc receptors on their surface and produce different biological effects.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the Fab segment of the antibody binds to the antigenic epitope of the virus-infected cell or tumor cell, and its Fc segment interacts with the killer cell (NK FcR binding on the surface of cells, macrophages, etc.) mediates killer cells to directly kill target cells.
  • ADCP is antibody-dependent cellular phagocytosis (antibody-dependent cellular phagocytosis).
  • the mechanism of ADCP is that the target cells acted by antibodies activate the Fc ⁇ R mechanism on the surface of macrophages, induce phagocytosis, internalize target cells and acidify and degrade phagosomes.
  • the heavy chain antibody HCAb
  • the Fc segment of the heavy chain antibody only has CH2 and CH3 domains.
  • the Fc segment of the antibody can optionally eliminate immune effector functions, including but not limited to combinations of the following mutations (according to EU counts):
  • the number and position of the CDR amino acid residues of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules.
  • Humanized antibody refers to an antibody obtained by grafting the CDR sequence of a non-human antibody into the framework of a human antibody variable region. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. In order to avoid the decreased activity caused by the decreased immunogenicity, minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain the activity.
  • Sequence identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides, and is the degree to which two polynucleotides or two polypeptides have the same bases or amino acids.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operably linked.
  • expression vectors useful in recombinant DNA techniques are usually in the form of plasmids.
  • Chimeric Antigen Receptor T-Cell refers to T cells that can recognize specific target antigens in a non-restricted manner by MHC after genetic modification, and continue to activate and expand.
  • Chimeric antigen receptor is the core component of CAR-T, which consists of three main structures: extracellular antigen-binding domain, transmembrane domain and intracellular domain.
  • the structure of chimeric antigen receptor (CAR) contains signal peptide, extracellular antigen binding domain, hinge region, transmembrane region, intracellular co-stimulatory and intracellular signaling domain sequentially from N-terminus to C-terminus.
  • Antibodies or antigen-binding fragments of antibodies can be used as extracellular antigen-binding domains, which are important components of CAR.
  • Nanobodies have unique properties such as low molecular weight, good water solubility, and strong stability, and have recently been used to develop CAR-T and target delivery.
  • Antibody-Drug Conjugates refer to binding proteins linked to one or more chemical drugs (optionally therapeutic or cytotoxic agents).
  • Antibody-drug conjugates can be obtained by linking cytotoxic small molecules (cytotoxin) and antibodies through permanent or unstable chemical linkers.
  • Fully human single-domain antibody-drug conjugate (UdADC), which conjugates human nanobody and drug, has been confirmed in tumor organoids, tumor microspheres and mouse tumor models. This new type of drug has significant advantages over traditional antibody-drug conjugates.
  • PD-1-His recombinant protein (protein number QPP11) was purchased from Yiqiao Sino (Beijing) Biotechnology Co., Ltd., product number: 10377-H08H.
  • PD-1-hFc recombinant protein protein number QP11308
  • PD-1 extracellular region sequence selected from UNIPROT, amino acids 24-170 of sequence number Q15116 fused with FC of human IgG1, the sequence is shown below.
  • Load the eukaryotic expression vector pTargeT construct the expression clone, stably transfect the expressed protein with CHOS cells, and purify the obtained protein.
  • PD-L1-hFc recombinant protein protein number QP004
  • PD-L1 extracellular region sequence selected from UNIPROT, amino acids 1-238 of sequence number Q9NZQ7 fused with FC of human IgG1, the sequence is shown below.
  • Load the eukaryotic expression vector pQD construct the expression clone, use 293E cells to transiently express the protein, and purify the obtained protein.
  • the control antibody is the anti-PD-1 antibody Nivolumab (5C4), which is obtained by fusion of the following two sequences, loaded into the eukaryotic expression vector pQD, and an expression clone is constructed, and the protein is transiently expressed by 293E cells, and the protein QP32103211 is obtained by purification.
  • 5C4 anti-PD-1 antibody Nivolumab
  • Example 2 Alpaca immunization and construction of phage immune library
  • Alpaca immunization Using PD-1 extracellular region recombinant protein (Shenzhou Yiqiao, 10377-H08H)) as an antigen to immunize a healthy alpaca (vicugna pacos, alpaca, lama pacos), using Freund's complete adjuvant CFA for the first time , immunized with 0.3 mg of antigenic protein; from the second to the fifth times, the incomplete Freund's adjuvant IFA was used to emulsify and mix with the antigen, and then injected subcutaneously at multiple points to immunize with 0.2 mg of antigenic protein.
  • PBMC peripheral blood mononuclear cells
  • RT-PCR reverse transcribe into cDNA by RT-PCR
  • design primers for PCR amplification of VHH genes design primers for PCR amplification of VHH genes
  • an immune library Peripheral blood lymphocytes were separated, and 50 mL of peripheral blood was collected.
  • PBMCs were separated according to the instructions of the lymphocyte separation medium, and total RNA of PBMCs was extracted with TRIzol reagent. Reverse transcription, see III First-Strand Synthesis System for RT-PCR instructions, a total of 8 ⁇ g RNA transcribed.
  • VHH fragments of about 750 bp were recovered from rubber tapping for the second round of nested PCR.
  • the vector for constructing the phage library is pComb3XSS.
  • the pComb3XSS phagemid vector is digested with SfiI
  • pComb3XSS is digested into two large fragments of 1672bp (SS stuffer) and 3301bp (target fragment of the vector).
  • the vector has His tag and HA tag for convenience Purification and detection. Ligation of the vector and the target fragment
  • the vector and the target fragment were respectively digested with sfiI, digested at 50°C overnight, and then the target fragment was recovered by tapping the gel.
  • a total of 10 electroporations were performed. Immediately after electroporation, 1 mL of 2YT medium was added for recovery. A total of 100 ml of recovery products were recovered at 37°C and 180 rpm for 45 minutes. 100 ⁇ L was taken to measure the library capacity, and the rest was centrifuged, and 5 mL of 2YT was added to resuspend and coated A total of 8 pieces are placed on a 200mm flat plate. On the second day, there were 73 clones in 10 -5 , so the library capacity was 7.3 ⁇ 10 8 (73*100*10 5 ).
  • the first round (1st) of panning In the immunotube, coat the antigen, 5ng/ul, 1ml, overnight at 4°C.
  • Blocking Block the immunotube with 2% MPBS at 37°C for 1 hour.
  • Subtraction Add 1800ul 2% MPBS, then add 200ul input phage, add antigen for subtraction at a final concentration of 50ng/ul, rotate at room temperature for 1hr.
  • Binding Transfer the supernatant to an immunotube coated with antigen, and rotate at room temperature for 1 hr. wash: Wash the immunotube 8 times with 1xPBST, and 4 times with 1xPBS. Elution: 800ul 100mM TEA, 10min at room temperature.
  • Neutralization Transfer the eluate to a 1.5ml EP tube, add 400ul 1M pH7.4 Tris.
  • Infection: add the eluted phage after neutralization to 10ml TG1 with OD600 0.5. 37°C for 4min. Measure the titer, and spread the rest of the bacterial solution on a large plate. Incubate overnight at 37°C.
  • Packaging 2nd input phage 2xTY medium scraped bacteria, inoculated into 50ml 2xTY medium + amp + 1% glucose to OD ⁇ 0.1, 37 degrees 200rpm, grow ⁇ 1 hour 20mins, to OD ⁇ 0.4-0.6. Add 500ul M13KO7, centrifuge at 37°C for 40mins to remove the supernatant, and resuspend the pellet in 100ml 2xTY medium+amp+kana. 30 degrees 200rpm overnight. 2ND input phage precipitation: Centrifuge overnight at 4200rpm for 15min. Take 40ml of supernatant, add 10ml PEG/NaCl, mix well, and place on ice for 20 minutes. Centrifuge, 4200rpm, 15min.
  • the second round (2 nd ) of panning In an immunotube, coat the antigen at 2ng/ul, 1ml, overnight at 4°C.
  • Blocking Block the immunotube with 2% MPBS at 37°C for 1 hour.
  • Subtraction Add 1800ul 2% MPBS, then add 200ul input phage, add antigen for subtraction at a final concentration of 50ng/ul, rotate at room temperature for 1hr.
  • Binding Transfer the supernatant to an immunotube coated with antigen, and rotate at room temperature for 1 hr. wash: Wash the immunotube 8 times with 1xPBST, and 4 times with 1xPBS. Elution: 800ul 100mM TEA, 10min at room temperature.
  • Neutralization transfer the eluate to a 1.5ml EP tube, add 400ul 1M pH7.4 Tris.
  • Packaging 2nd output phage 2xTY medium scraped bacteria, inoculated into 50ml 2xTY medium + amp + 1% glucose to OD ⁇ 0.1, 37 degrees 200rpm, grow ⁇ 1hour 20mins, to OD ⁇ 0.4-0.6. Add 500ul M13KO7, centrifuge at 37°C for 40mins to remove the supernatant, and resuspend the pellet in 100ml 2xTY medium+amp+kana. 30 degrees 200rpm overnight. 2ND output phage precipitation: Centrifuge overnight at 4200rpm for 15min. Take 40ml of supernatant, add 10ml PEG/NaCl, mix well, and place on ice for 20 minutes. Centrifuge, 4200rpm, 15min.
  • Immune library screening and identification After 2 rounds of panning, single clones were picked for phage ELISA, and positive clones binding to PD-1 were screened. Simultaneously screen clones that are positive for binding to PD-1 and positive for blocking PD-1 and PD-L1 binding. The positive clones were sequenced to obtain the VHH sequence of the antibody PD-1 antibody.
  • the ELISA screening method is as follows:
  • Packaging phage and soluble expression pick clones: P1-P4 total 4 96-well plates. Single clones were inoculated in 180ul 2YT+amp and cultured at 37°C for 2 hours.
  • Packaging phage Add 100ul 2YT+M13K07 to 30ul, let stand at 37°C for 1 hour, add 50ul 2YT+amp+kan, shake overnight at 30°C. The supernatant was collected by centrifugation for ELISA.
  • Soluble expression 120ul bacterial liquid, after 3 hours of shaking culture, add 60ul 2YT, add 1M IPTG to the final concentration of 1mM. Shake overnight at 30°C. The supernatant was collected by centrifugation for ELISA.
  • Phage ELISA coated with QP1138, negative control human IgG (protein number QP11851186), PDL1.mFc (manufactured by us) 2ng/ul, 60ul/well 4 plates each, coated overnight at 4 degrees, washed 3 times with PBS. 5% milk 200ul/well was blocked at room temperature for 1h.
  • PDL1.mFc add 1ng/ul QP1138, 60ul/well, room temperature for 1 hour, add 20ul phage supernatant and 40ul 2% MPBS, mix well, room temperature for 1 hour. Wash the plate 3 times with PBST, add 60ul anti-M13HRP (Shenzhou, 11973-MM05T-200), room temperature for 1 hour.
  • Blocking phage ELISA operating method coating protein QP004 (PD-L1-hFC) 4ng/ul 60ul/well, add QPP11 (PD1.His, Yiqiao Shenzhou) 1ng/ul, 60ul/well, 60ul MPBS+20ul phage supernatant , add 60ul anti-M13HRP (Shenzhou, 11973-MM05T-200) to incubate for 1 hour, wash the plate 5 times with PBST, develop color with TMB, stop with H 2 SO 4 .
  • Soluble blocking ELISA operating method coat protein QP004 (PD-L1-hFC) 2ng/ul 60ul/well, overnight at 4, wash the plate 3 times with PBS, block with 5% milk 200ul/well for 1h. Wash the plate 3 times with PBS. 30ul biontin-QP1138 0.1ng/ul+30ul expression supernatant, incubate for 1 hour, wash the plate 3 times with PBST, add Strep-HRP 1:5000, incubate 60ul/well for 1 hour, wash the plate 5 times with PBST, TMB color development, H2SO4 terminated .
  • the present invention provides two obtained unique nanobodies, numbered QP3120 and QP3126 respectively, and the amino acid sequences are shown in SEQ ID NO:5 and 6.
  • CDR1, CDR2, and CDR3 of the Nanobody sequence are shown in bold and underlined, and the sequence numbers are SEQ ID NO: 7, 8, and 9, respectively.
  • CDR1, CDR2, and CDR3 of the Nanobody sequence are shown in bold and underlined, and the sequence numbers are SEQ ID NO: 7, 8, and 10, respectively.
  • Example 4 Nanobody construction of FC fusion protein, cloning, expression, and purification of protein
  • Cloning design and construction The C-terminal of the nanobody VHH of the anti-PD-1 antibody was fused with human IgG1FC to construct the nanobody FC fusion protein antiPD-1VHH-FC.
  • a eukaryotic expression plasmid was constructed to transiently express the protein in HEK293 cells, and the protein was purified by protein A affinity chromatography and other methods.
  • the protein numbers of the two PD-1VHH-FC fusion proteins continued to use QP3120 and QP3126.
  • the human IgG1 Fc segment sequence is shown in SEQ ID NO: 11.
  • 293E cells were maintained at a culture density of 0.2-3 ⁇ 10 6 /ml, cultured in the maintenance phase medium (GIBCO Freestyle 293 expression medium), and the cells to be transfected were centrifuged to change the medium one day before transfection to adjust the cells The density is 0.5-0.8 ⁇ 10 6 /ml. On the day of transfection, the density of 293E cells was 1-1.5 ⁇ 10 6 /ml.
  • the mixture of plasmid and PEI was slowly added to the 293E cells, placed in 8% CO 2 , 120 rpm, and cultured in a shaker at 37°C. On the fifth day of transfection, the cell supernatant was collected by centrifuging at 4700 rpm for 20 min in a horizontal centrifuge.
  • Protein A affinity chromatography purification pass the column with the equilibrium solution, at least 3CV, the actual volume is 20ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, and the flow rate is 1ml/min; pass the supernatant of the culture solution after centrifugation through the column , load 40ml of sample, flow rate 0.33ml/min; pass the column with equilibrium solution, at least 3CV, actual volume 20ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the balance solution, flow rate 0.33ml/min; For the column, when the UV280 rises to 15mAU, the elution peak (PAC-EP) is collected, and when the UV280 drops to 15mAU, the collection is stopped, and the flow rate is 1ml/min. After the sample is collected, adjust the PAC-EP to neutral with pH adjusting solution.
  • PAC-EP elution peak
  • Example 5 ELISA detection of PD-1 nanobody Fc fusion protein binding to human PD-1 protein
  • coated plate coated with QP1138 (PD-1–hFC) 1 ⁇ g/ml, 60 ⁇ l/well, overnight at 4°C, PBS*3; blocking: 5% milk/PBS, 200 ⁇ l/well, incubated at 25°C for 1 hour;
  • Antigen Incubate PD-1 Nanobody PD-1VHH-FC QP3120 and QP3126 respectively, 5-fold dilution starting from 25 ⁇ g/ml, 8 gradients, 60 ⁇ l/well, 25°C, 1h, PBST*5;
  • Secondary antibody anti-Fab-HRP , 1:8000 dilution, 60 ⁇ l/well, 25°C, 1h, PBST*5; color development: TMB 100 ⁇ l/well, 5-10min, 2M H 2 SO 4 to stop the reaction, read at 450nm.
  • the results are shown in Figure 1, the PD-1 nanobody Fc fusion proteins all bind to human PD-1 protein.
  • Example 6 ELISA detection of PD-1 nanobody Fc fusion protein blocking human PD-L1 and PD-1 protein binding
  • Coating protein QP1138 (PD1-FC) 2 ⁇ g/ml 50 ⁇ l/well, overnight at 4°C. Wash 3 times with PBS. Blocking: 3% BSA 250 ⁇ l/well, incubate at room temperature for 1h. Prepare 2 ⁇ g/ml PDL1-mouse FC and different concentrations of antibodies respectively, mix in equal volumes, and incubate at room temperature for 1 h. Wash 3 times with PBST and 3 times with PBS. Incubate with secondary antibody: HRP-mouse IgG (1:5000) 50 ⁇ l/well, wash 6 times with PBST and 3 times with PBS. Color development: TMB 100 ⁇ l/well, color development 10min. 2M H 2 SO 4 50 ⁇ l/well to stop. The results are shown in Figure 2, the PD-1 nanobody Fc fusion protein can block the binding of human PD-L1 and PD-1 protein.
  • Example 7 ELISA detection of PD-1 nanobody Fc fusion protein binding to cynomolgus monkey PD-1 protein
  • Coat rabbit His antibody (GenScript, A00174) 2 ⁇ g/ml 50 ⁇ l/well, overnight at 4°C. Wash 3 times with PBS. Blocking: 3% BSA 250 ⁇ l/well, incubate at room temperature for 1h. Incubate monkey PD1-his (sinobio 90251-C08H) at 1 ⁇ g/ml, 50 ⁇ l/well, at 25°C for 1 hour, and wash 3 times with PBS. Incubate with an initial antibody concentration of 10 ⁇ g/ml and dilute 8 gradients. 50 ⁇ l/well, 1h at 25°C, washed 6 times with PBS.
  • Example 8 Nanobody humanized design, cloning, expression, and protein purification
  • Nanobody humanization design By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy and light chain variable region germline genes with high homology of QP3120 and QP3126 were used as templates, and sheep The CDRs of the camel nanobodies were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then select some important amino acid residues for back mutation combination. The amino acid residues are identified and annotated by the Kabat numbering system.
  • Primers were designed for PCR to build the VH gene fragments of each humanized antibody, and then homologously recombined with the expression vector pQD with signal peptide and constant region gene (FC) fragments to construct the antibody full-length expression vector VH-FC-pQD.
  • FC constant region gene
  • VH contains the gene fragments required for recombination and the expression vector pQD (with signal peptide and constant region gene (FC) fragment) recovered by BsmBI enzyme digestion is added to DH5a competent cells at a ratio of 3:1, 0 ° C ice bath for 30 min, 42 ° C hot Hit for 90s, add 5 times the volume of LB medium, incubate at 37°C for 45min, spread on LB-Amp plate, culture at 37°C overnight, pick single clones and send them for sequencing to obtain the target clones.
  • pQD with signal peptide and constant region gene (FC) fragment
  • Cloning construction The humanized design clone above was transformed into a PD-L1-FC fusion protein whose C-terminus is human IgG1FC.
  • the reconstituted plasmid was expressed in HEK293 cells, purified by protein A affinity chromatography, and a total of 7 humanized PD-1VHH-FC fusion proteins QP629-QP635 were obtained, the sequences of which were shown in SEQ ID NO: 12-18.
  • the back end of the antibody is connected to the FC segment of human IgG1, and the sequence is shown in SEQ ID NO: 11.
  • 293E cells were maintained at a culture density of 0.2-3 ⁇ 10 6 /ml, cultured in the maintenance phase medium (GIBCO Freestyle 293 expression medium), and the cells to be transfected were centrifuged to change the medium one day before transfection to adjust the cells The density is 0.5-0.8 ⁇ 10 6 /ml. On the day of transfection, the density of 293E cells was 1-1.5 ⁇ 10 6 /ml.
  • the mixture of plasmid and PEI was slowly added to the 293E cells, placed in 8% CO 2 , 120 rpm, and cultured in a shaker at 37°C. On the fifth day of transfection, the cell supernatant was collected by centrifuging at 4700 rpm for 20 min in a horizontal centrifuge.
  • Protein A affinity chromatography purification pass the column with the equilibrium solution, at least 3CV, the actual volume is 20ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, and the flow rate is 1ml/min; pass the supernatant of the culture solution after centrifugation through the column , load 40ml of sample, flow rate 0.33ml/min; pass the column with equilibrium solution, at least 3CV, actual volume 20ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the balance solution, flow rate 0.33ml/min;
  • the column starts to collect the elution peak (PAC-EP) when UV280 rises to 15mAU, stops collecting when UV280 drops to 15mAU, and the flow rate is 1ml/min. After the sample is collected, adjust the PAC-EP to neutral with pH adjusting solution.
  • PAC-EP elution peak
  • Example 9 ELISA detection of humanized PD-1 nanobody Fc fusion protein binding to human PD-1 protein
  • Example 10 ELISA detection of humanized PD-1 nanobody Fc fusion protein blocking human PD-L1 and PD-1 protein white combination
  • Coating protein QP1138 (PD1-FC) 2 ⁇ g/ml 50 ⁇ l/well, overnight at 4°C. Wash 3 times with PBS. Blocking: 3% BSA 250 ⁇ l/well, incubate at room temperature for 1h. Prepare 2 ⁇ g/ml PDL1-mouse FC, 15 ⁇ g/ml of humanized nanobodies such as QP509 with different concentrations, and 30 ⁇ g/ml of positive control, dilute 1:3, mix with equal volume, and incubate at room temperature for 1 h. Wash 3 times with PBST and 3 times with PBS.
  • the first form was the fusion of anti-PD-1 VHH in the FC segment of human IgG1 to eliminate FC ⁇ R function (EU Counting L234A/L235A) such as the N-terminal sequence of SEQ ID NO: 19;
  • the second form is the fusion of anti-PD-1 VHH to the C-terminus of human IgG1 FC segment mutation to eliminate FC ⁇ R function (EU counting L234A/L235A), protein number and The serial number is shown in Table 4.
  • Example 11 ELISA detection of humanized PD-1 nanobody Fc fusion protein binding to human PD-1 protein
  • Example 12 ELISA detection of humanized PD-1 nanobody Fc fusion protein blocking human PD-L1 and PD-1 protein white combination
  • Coating protein QP1138 (PD1-FC) 2 ⁇ g/ml 50 ⁇ l/well, overnight at 4°C. Wash 3 times with PBS. Blocking: 3% BSA 250 ⁇ l/well, incubate at room temperature for 1h. Prepare 2 ⁇ g/ml PDL1-mouse FC, 15 ⁇ g/ml of humanized nanobodies such as QP509 with different concentrations, and 30 ⁇ g/ml of positive control, dilute 1:3, mix with equal volume, and incubate at room temperature for 1 h. Wash 3 times with PBST and 3 times with PBS.
  • Example 13 Humanized Nanobody Fc Fusion Protein Promotes T Cell Proliferation in Mixed Lymphocyte Reaction
  • Mixed lymphocyte reaction refers to the co-culture of human T cells and allogeneic dendritic cells. Lymphocytes are stimulated by alloantigens to activate and proliferate, and produce a variety of cytokines.
  • Anti-PD-1 antibodies are antibodies Concentration-dependent By blocking the immunosuppressive signal of PD-1/PD-L1 binding, stimulate T cell proliferation to release cytokines such as IL-2/IFN- ⁇ , etc. The release of IL-2/IFN- ⁇ was detected by ELISA, and the biological activity of anti-PD-1 antibody in stimulating T cells to proliferate in vitro was studied.
  • Monocytes in PBMC were isolated, and rhGM-CSF and rhIL-4 were added to induce DC (inducible dendritic cells); CD4+T cells in another donor PBMC were isolated.
  • the results are shown in Figure 8, Figure 9, Figure 10, and Figure 11: the humanized PD-1 Nanobody fused to the N-terminal and C-terminal of Fc can stimulate T cell proliferation in mixed lymphocyte reaction (MLR), enhance Production of IL-2 and IFN- ⁇ .
  • MLR mixed lymphocyte reaction
  • the various forms of humanized PD-1 nanobody fused to the N-terminus and C-terminus of Fc, QP916, QP3517, QP3519, QP917, QP918, QP919, QP3518 can all be effective in mixed lymphocyte reaction (MLR). Stimulates T cell proliferation and enhances IL-2 and IFN- ⁇ production, superior to the control Novilumab analog (QP32103211).
  • MLR mixed lymphocyte reaction
  • Example 14 PD-1 nanobody stimulates the biological activity of human PBMC proliferation in vitro
  • PBMC Human peripheral blood mononuclear cells
  • PBMC Human peripheral blood mononuclear cells
  • PD-L1 antibody can enhance T cell proliferation and release cytokines such as IL-2/IFN- ⁇ by blocking the immunosuppressive signal of PD-1/PD-L1 binding.
  • the release of IL-2/IFN- ⁇ was detected by ELISA, and the biological activity of Q-1801 in PBMC in vitro proliferation assay was further studied.
  • PBMC cells in 96-well plates prepare different concentrations of SEB and add them to PBMC cell wells, then add anti-PD-1 antibody, mix gently, and culture for 2-5 days.
  • ELISA was used to detect the secretion of IL-2 in the cell culture supernatant
  • ELISA was used to detect the secretion of IFN- ⁇ in the cell culture supernatant.
  • the results shown in Figure 12 show that QP3517, QP918, QP3519, and QP919 can significantly enhance the activation and proliferation of PBMC and enhance the production of IL-2 in SEB-stimulated PBMC in vitro proliferation experiments, which are not inferior to the control antibody novilumab.
  • Humanized PD-1 Nanobody fused to the N-terminal and C-terminal of Fc can stimulate T cell proliferation and enhance IL-2 production in SEB-stimulated PBMC.
  • Example 15 Anti-human PD1 antibody inhibits tumor growth in animal drug effects in vivo
  • mice Collect cultured MC38-hPDL1 cells by centrifugation, disperse the cells with 1 ⁇ PBS, and prepare a cell suspension with a cell density of 5 ⁇ 10 6 cells/ml.
  • 0.1ml of cell suspension was subcutaneously inoculated into the right flank of C57BL/6-hPD1 mice to establish the MC38-hPDL1 tumor-bearing mouse model.
  • the mice were randomly divided into groups of 7 mice. All animals were weighed and tumor volumes were measured with calipers. According to the tumor volume, random grouping method was used for grouping to ensure that the tumor volumes of different groups were similar.
  • the day of grouping was D0, and the administration began on that day. The detailed administration method, dosage and route of administration are shown in the table below.
  • the dosing volume was 10 ⁇ L/g.
  • tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the long diameter and b represents the short diameter).
  • a represents the long diameter and b represents the short diameter.
  • T/C (%) (T-T0)/(C-C0) ⁇ 100% (T0 and C0 are the tumor volumes of the treatment group and the control group when grouping respectively, and T and C are the treatment group after administration respectively group and control group at a specific time point)
  • the average tumor volume of mice in the Vehicle control group was 705.60 ⁇ 105.85mm3 on the 17th day after administration.
  • the mean tumor volumes of the nanobody molecule QP918 (5mg/kg), QP918 (10mg/kg), QP3517 (5mg/kg) and QP3517 (10mg/kg) groups were 23.24 ⁇ 16.14mm3, 26.28 ⁇ 13.27mm3, 14.63 ⁇ 13.32mm3, and 11.59 ⁇ 8.42mm3, TGI were 107.95%, 107.41%, 109.29% and 109.68%, respectively, and the mean tumor volume of the control molecule QP32103211 (10mg/kg) was 47.99 ⁇ 8.42mm on the 17th day after administration 27.75mm3, TGI is 104.01% (see table below).
  • both the control molecule QP32103211 and the two nanobody molecules significantly inhibited the growth of the tumor (p ⁇ 0.001), and QP918 and QP3517 performed slightly better than the trend of the control molecule QP32103211.
  • the body weight of the mice did not decrease significantly during the administration process, indicating that the antibody molecules had no obvious toxic and side effects on the mice.
  • the results of the analysis of tumor weight were similar to those of tumor volume.
  • the average tumor weight of mice in the Vehicle control group was 741.07 ⁇ 318.89mg on the 17th day after administration, and the average tumor weight in the antibody molecule QP918-5mpk, QP918-10mpk, QP3517-5mpk and QP3517-10mpk treatment groups was 17 days after administration.
  • the weights were 9.73 ⁇ 16.38mg, 23.37 ⁇ 38.99mg, 9.76 ⁇ 22.33mg and 11.66 ⁇ 27.04mg respectively; the average tumor weight of the control molecule QP32103211-10mpk treatment group was 35.57 ⁇ 57.34mg on day 17 after administration. (see table below)
  • the screened antibody molecule has a better ability to inhibit tumors in vivo than the control molecule QP32103211.
  • FIGS. 13 to 16 The experimental results are shown in FIGS. 13 to 16 , among which, the results of the drug efficacy test of the antibody against the mouse colon cancer CDX model MC38-hPDL1-mClaudin18.2 are shown in FIG. 13 .
  • Fig. 14 is the tumor growth curve of each group after grouping.
  • Fig. 15 is the weight of tumor D17 of mice in each group added with each antibody of the present invention and control antibody.
  • Figure 16 is the body weight curves of mice in each group. It can be seen from the experimental results that the PD-1 nanobody QP918 and QP3517 of the present invention have a better effect of inhibiting tumor growth than the control antibody Novilumab analog (QP32103211) in the animal drug effect in vivo.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Plant Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供抗PD-1纳米抗体及其应用。抗PD-1纳米抗体包括VHH链。通过筛选获得了纳米抗体序列，并提供人源化的抗体，以及含有该纳米抗体序列的Fc融合蛋白。所述抗PD-1纳米抗体有望作为治疗性抗体用于各种PD-1高表达的恶性肿瘤的治疗。

Description

抗PD-1纳米抗体及其应用 技术领域
本发明涉及生物技术领域，具体涉及抗PD-1纳米抗体及其应用。
背景技术
在经典免疫监视理论中，免疫***可以识别肿瘤抗原并将肿瘤细胞消除。致癌基因的激活导致肿瘤细胞改变自身及肿瘤微环境，使得免疫***和肿瘤细胞间的平衡被打破。当免疫***和肿瘤细胞进入逃逸阶段，肿瘤细胞的恶性程度会提高，而肿瘤细胞丢失MHC分子使其避免被免疫细胞识别和消除。肿瘤微环境也可以通过释放免疫抑制因子抑制免疫***，如IL-10，TGF-β等。肿瘤细胞表面也会高表达免疫抑制蛋白(如PD-1，PD-L1)，当效应T细胞与肿瘤细胞结合时，PD-L1与PD-1相互作用并诱导T细胞发生凋亡，这是肿瘤对免疫***产生耐受的主要原因之一，肿瘤迅速生长，发生转移。如果人为激活宿主的免疫***并将其重定向到肿瘤细胞，从理论上来讲肿瘤组织就能够被清除，而免疫治疗的理论已经在临床治疗中得到广泛证明。
PD-1(程序性死亡受体1)，也称为CD279(分化簇279)，是一种重要的免疫抑制分子。PD-1(CD279)最早于1992年被报道，人PD-1编码基因PDCD1位于2q37.3，全长2097bp，由6个外显子组成，翻译产物为288个氨基酸组成的PD-1前体蛋白，剪切前20个氨基酸组成的信号肽后得到成熟蛋白质。PD-1包括胞外免疫球蛋白可变区IgV结构域，疏水跨膜结构域和胞内结构域，胞内尾部结构域N端ITIM基序包含2个磷酸化位点，C端则是一个ITSM基序。PD-1是膜蛋白，属于CD28免疫球蛋白超家族，主要表达在激活后的T细胞表面，此外还在胸腺的CD4-CD8-T细胞、活化的NK细胞和单核细胞有低丰度表达。PD-1有2个配体，分别是B7蛋白家族的PD-L1(CD274,B7-H1)和PD-L2(CD273,B7-DC)，PD-L1和PD-L2氨基酸序列有40％相同。两者区别主要在于表达模式不同，PD-L1组成性的低表达于APCs、非造血细胞(如血管内皮细胞、胰岛细胞)和免疫豁免部位(如胎盘、睾丸和眼睛)，炎性细胞因子如I型和II型干扰素、TNF-α和VEGF等均可以诱导PD-L1的表达。PD-L2则只在被激活的巨噬细胞和树突细胞中有表达。PD-1与PD-L1在激活的T细胞结合后，PD-1的ITSM基序发生酪氨酸磷酸化，进而导致下游 蛋白激酶Syk和PI3K的去磷酸化，抑制下游AKT、ERK等通路的活化，最终抑制T细胞活化所需基因及细胞因子的转录和翻译，发挥负向调控T细胞活性的作用。
在肿瘤细胞中，肿瘤细胞及肿瘤微环境通过上调PD-L1表达并与肿瘤特异的CD8+T细胞表面的PD-1结合，负调控T细胞活性，抑制免疫反应。肿瘤细胞可以通过以下4种途径上调PD-L1表达：1.编码PD-L1的基因扩增(9p24.1)；2.EGFR、MAPK、PI3K-Akt信号通路激活，HIF-1转录因子等可以从转录水平上调PD-L1的表达；3.EB病毒的诱导(EB病毒阳性的胃癌和鼻咽癌表现为PD-L1高表达)；4.表观遗传学的调控。在肿瘤微环境中，interferon-γ等炎症因子的刺激同样可以诱导PD-L1和PD-L2的表达。炎症因子可以诱导肿瘤微环境中其他细胞，包括巨噬细胞、树突状细胞和基质细胞表达PD-L1和PD-L2，而能够识别肿瘤抗原的肿瘤浸润性T细胞能够分泌interferon-γ，进而诱导PD-L1表达上调，这一过程被称为“适应性免疫抵抗”，肿瘤细胞通过这一机制可以实现自我保护。有越来越多的证据表明肿瘤利用PD-1依赖的免疫抑制免疫逃避。在各种实体瘤和血液***恶性肿瘤种均已经发现PD-L1和PD-L2的高表达。此外，PD-Ls的表达与肿瘤细胞的不良预后之间具有很强相关性，已证明的包括食道癌、胃癌、肾癌、卵巢癌、膀胱癌、胰腺癌和黑色素瘤等。
发明内容
本发明的目的是提供抗PD-1纳米抗体及其应用。
为了达到上述目的，本发明提供一种抗PD-1纳米抗体，其包括：VHH链，所述VHH链的互补决定区包括CDR1、CDR2和CDR3，所述CDR1的氨基酸序列如SEQ ID NO：7所示，所述CDR2的氨基酸序列如SEQ ID NO：8所示，所述CDR3的氨基酸序列如SEQ ID NO：9或SEQ ID NO：10所示。
可选的，所述VHH链的氨基酸序列包括：选自SEQ ID NO:5、6或SEQ ID NO:12-18中任意一项所示，或与其具有至少85％、90％、95％、97％、或99％同一性的序列。
本发明还提供了一种融合蛋白，其包括：所述的抗PD-1纳米抗体。
可选的，所述的融合蛋白还包括：Fc段；所述Fc段与所述抗PD-1纳米抗体的N端或C端融合。
可选的，所述融合蛋白还包括：除所述抗PD-1纳米抗体和所述Fc段以外的其他功能片段；所述Fc段与所述抗PD-1纳米抗体融合，和/或，所述Fc段与所述其他功能片段融合。
可选的，所述的Fc段选自人源IgG1、IgG2、IgG3、或IgG4或其变体；优选的，所述Fc段选自人源IgG1的变体；优选的，所述Fc结构域选择消除免疫效应功能，优选包含以下任一突变方式，以下突变为根据EU计数：
Figure PCTCN2022110672-appb-000001
本发明还提供了一种抗PD-1抗体分子，其包括：一个或多个所述的抗PD-1纳米抗体；或包括：所述的融合蛋白；所述抗PD-1抗体分子为单价抗体、双价或多价抗体、双特异抗体、或多特异抗体。
本发明还提供了一种嵌合抗原受体，包括胞外抗原结合域、跨膜结构域和胞内结构域，所述胞外抗原结合域包括所述的抗PD-1纳米抗体。
本发明还提供了一种核酸分子，其编码：所述的抗PD-1纳米抗体，或，所述的融合蛋白。
本发明还提供了一种载体，其包含所述的核酸分子。
本发明还提供了一种宿主细胞，其采用所述的载体转化得到。
本发明还提供了一种抗体药物偶联物，其包含所述的抗PD-1纳米抗体，以及偶联于 所述抗PD-1纳米抗体的药物。
本发明还提供了一种药物组合物，其含有：所述的抗PD-1纳米抗体，或，所述的融合蛋白，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明还提供了所述的抗PD-1纳米抗体在制备用于抑制或治疗疾病的药物中的用途。
本发明还提供了所述的抗PD-1纳米抗体在用于通过刺激免疫功能达到预防或治疗疾病的效果的用途，如用于制备预防或治疗用的疫苗。
可选的，所述的疾病为PD-1介导的疾病。
可选的，所述的疾病为癌症；优选的，所述的癌症包括乳腺癌、肺癌、胃癌、肠癌、肾癌、黑色素瘤、膀胱癌、头颈癌、淋巴瘤、皮肤恶性肿瘤、或非小细胞肺癌。
相对于现有技术，本发明具有以下有益效果：
1、本发明的抗体序列新颖，有望作为治疗性抗体用于各种PD-1高表达的恶性肿瘤的治疗。
2、本发明的PD-1纳米抗体特异性强，亲和力高；此外，纳米抗体的VHH链可与各种分子链组合形成不同的抗体分子形式，例如组合形成针对双靶点或多靶点的特异性抗体。
附图说明
图1为ELISA检测PD-1纳米抗体结合人PD-1蛋白结果图。
图2为ELISA检测PD-1抗体阻断PD-L1/PD-1的结合活性结果图。
图3为LISA检测PD-1纳米抗体结合食蟹猴PD-1蛋白结果图。
图4为ELISA检测人源化PD-1纳米抗体Fc融合蛋白对人PD-1蛋白结合结果图。
图5为ELISA检测人源化PD-1纳米抗体Fc融合蛋白阻断人PD-L1蛋白与PD-1蛋白的结合结果图。
图6为ELISA检测人源化PD-1纳米抗体Fc融合蛋白对人PD-1蛋白结合结果图。
图7为ELISA检测人源化PD-1纳米抗体Fc融合蛋白阻断人PD-L1蛋白与PD-1蛋白的结合结果图。
图8、图9为混合淋巴细胞反应48小时上清中IL-2分泌的检测结果图。
图10、图11为混合淋巴细胞反应120小时上清中IFN-γ分泌的检测结果图。
图12为抗PD-1抗体对SEB刺激PBMC增殖48小时IL-2分泌的检测结果图。
图13为抗体对小鼠结肠癌CDX模型MC38-hPDL1-mClaudin18.2的药效试验的结果图。
图14为各个组在分组后的肿瘤生长曲线图。
图15为加入本发明各抗体及对照抗体各组小鼠肿瘤D17重量图。
图16为各组小鼠的体重曲线图。
具体实施方式
以下结合附图和实施例对本发明的技术方案做进一步的说明。
本实验中未注明具体条件的实验方法，通常是按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
术语：
“抗体”(antibody，Ab)指包含至少一个抗原结合位点并能特异性结合抗原的免疫球蛋白分子(immunoglobulin，Ig)。
“抗原”是在机体内能诱发免疫应答且与抗体特异性结合的物质。抗体与抗原的结合依靠二者间形成的相互作用来介导，包括氢键、范德华力、离子键以及疏水键。抗原表面与抗体结合的区域为“抗原决定簇”或“表位”，一般来说，每个抗原有多个决定簇。
本发明所提及的术语“抗体”以其最广泛的含义理解，并包含单克隆抗体(包括全长单克隆抗体)、多克隆抗体、抗体片段、包含至少两个不同的抗原结合结构域的多特异性抗体(例如，双特异性抗体)，以及单域抗体或纳米抗体。抗体还包括鼠源抗体、人源化抗体、嵌合抗体、人抗体以及其它来源的抗体。本发明的抗体可以来源于任何动物，包括但不限于人、非人灵长类动物、小鼠、大鼠、牛、马、鸡、骆驼、羊驼的免疫球蛋白分子等。抗体可以含有另外的改变，如非天然氨基酸，Fc效应子功能突变和糖基化位点突变。抗体还包括翻译后修饰的抗体、包含抗体的抗原决定簇的融合蛋白，以及包含对抗原识别位点的任何其它修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。
常规抗体的基本结构是由两条完全相同的重链(heavy chain，H)和两条完全相同的轻链(light chain，L)通过二硫键连接的呈Y形的单体。每条链分别由2～5个约含110个氨基酸，序列相似但功能不同的结构域(又称功能区)组成。抗体分子中轻链和重链靠近N端的氨基酸序列变化较大，形成的结构域称为可变区(variable region，V区)；靠近C端的氨基酸序列相对恒定的区域称为恒定区(constant region，C区)。重链和轻链的V区分别称为VH和VL，VH和VL各有3个区域的氨基酸组成和排列顺序高度可变，称 为高变区(hypervariable region，HVR)；该区域形成与抗原表位互补的空间构象，又被称为互补决定区(complementarity determining region，CDR)。VH的3个CDR可分别用VHCDR1、VHCDR2、VHCDR3表示，VL的3个CDR分别用VLCDR1、VLCDR2、VLCDR3表示。VH和VL共6个CDR共同组成抗原结合部位(antigen-binding site)。CDR区氨基酸的多样性是抗体与数量庞大的不同抗原特异性结合的分子基础。V区中CDR之外的氨基酸组成和排列顺序相对变化不大，称为骨架区或框架区(framework region，FR)。VH和VL各有4个骨架区，分别用FR1、FR2、FR3、FR4表示。每个VH和VL有三个CDR和四个FR组成，从氨基端到羧基端排列顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的恒定区分别称为CH和CL。IgG、IgA、IgD的重链恒定区有CH1、CH2、CH3三个结构域，IgM、IgE的重链恒定区有CH1、CH2、CH3、CH4四个结构域。CH1和CH2之间为铰链区(hinge region)，含有丰富的脯氨酸，因此易伸展弯曲，能改变Y形两个臂之间的距离，有利于两臂同时结合抗原表位。
常规抗体的“抗原结合片段”指具有抗原结合活性的Fab片段、F(ab’)2片段、Fv片段、ScFv片段等。“Fab片段”(fragment of antigen binding,Fab)，意指由VL、VH、CL和CH1结构域组成的抗体片段，与单个抗原表位结合(单价)。本领域技术人员可知，木瓜蛋白酶水解IgG形成2个相同的Fab段和1个Fc段；胃蛋白酶水解IgG形成1个F(ab’)2段和若干多肽碎片(pFc’)。若F(ab’)2重链间二硫键断裂，可形成2个Fab’片段，后者可进一步被酶解成Fv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区。单链可变片段scFv(single chain antibody fragment)，或称单链抗体，由抗体重链可变区和轻链可变区通过连接片段(linker)连接而成。
1993年，Hamers实验室发现，骆驼血清中除了常规的四联抗体外，还有大量类似免疫球蛋白G(immunoglobulins，IgG)的分子。这类分子称为重链抗体(HCAb)，天然缺失传统抗体轻链和重链恒定区CH1，但仍具备对抗原的强结合力。Hamers实验室还分析鉴别了骆驼血清中重链抗体的结构和序列，发现重链抗体的抗原结合区仅由可变区片段构成，其相当于传统抗体抗原结合片段(Fab)的功能等同物。因此，人们把重链抗体的抗原识别区片段称为VHH(variable domain of theheavy chain of heavy-chain antibody，VHH)，并在此基础上开发出只含有VHH结构域的纳米抗体(nanobody)。纳米抗体(nanobody)又称单域抗体(singledomain antibody，sdAb)。
纳米抗体容易改造、形成多价形态。纳米抗体由于分子量小，是单一基因编码，容易进行基因工程改造，并且可以通过短小的连接序列聚合多个纳米抗体，甚至可以与常规抗 体的Fab片段、Fv片段、ScFv片段等进行连接及组合，形成多价或者是多特异的抗体结构。双价或者多价的抗体能识别同一种表位，但是比单价的抗原亲和力更高。双特异性或多特异性抗体，可以结合不同靶点，或者是同样靶点上的不同结合区域，比单价抗体具有更强的抗原识别能力。
纳米抗体容易和其他结构(如BSA、IgG-Fc等)形成新的融合分子。在新的融合分子中，纳米抗体与其靶抗原定向结合，与纳米抗体融合的部分就能发挥相应的功能，因此可以与其他药物联用，或者是应用于诊断和充当多种领域的实验研究工具。纳米抗体筛选可分为羊驼免疫、淋巴细胞提取、纳米抗体文库构建、噬菌体库构建、特异性噬菌体筛选、大肠杆菌表达、抗体纯化等步骤。
术语“融合”指由肽键直接连接或借助连接片段连接组分，或通过分子间相互作用进行融合。在单条肽链中，融合是指由肽键直接连接或借助连接片段连接。
术语“Fc”、“Fc段”或“Fc片段”是指可结晶片段(fragment crystallizable)，无抗原结合活性，是抗体与效应分子或细胞表面Fc受体(FcR)相互作用的部位。Fc片段包含抗体除重链恒定区CH1之外的恒定区多肽。Fc片段与表面具有相应Fc受体的细胞结合，产生不同的生物学作用。在ADCC效应中(抗体依赖的细胞介导的细胞毒性作用，antibody-dependent cell-mediated cytotoxicity)，抗体的Fab段结合病毒感染的细胞或肿瘤细胞的抗原表位，其Fc段与杀伤细胞(NK细胞、巨噬细胞等)表面的FcR结合，介导杀伤细胞直接杀伤靶细胞。ADCP为抗体依赖性细胞吞噬作用(antibody-dependent cellular phagocytosis)，ADCP的机制是抗体作用的靶细胞激活巨噬细胞表面上的FcγR机制，诱导吞噬，使靶细胞内化和被吞噬体酸化降解。由于重链抗体(HCAb)天然缺失传统抗体轻链和重链恒定区CH1，因此重链抗体的Fc段仅具有CH2和CH3结构域。
本领域技术人员可知，Fc变体或突变具有多种形式，在不同的实施例中，本领域技术人员可选择不同的Fc变体。抗体的Fc段可选择消除免疫效应功能，包含但不限于以下突变的组合(根据EU计数)：
Figure PCTCN2022110672-appb-000002
本发明所述抗体或抗原结合片段的CDR氨基酸残基在数量和位置上符合已知的Kabat编号规则。
“人源化抗体”是指将非人源抗体的CDR序列移植到人的抗体可变区框架得到的抗体。此类框架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。为避免免疫原性下降的同时引起的活性下降，可对人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
“序列同一性”指两个多核苷酸序列之间或两个多肽之间的序列相似性，是两个多核苷酸或两个多肽之间具有相同碱基或氨基酸的程度。
术语“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，其中附加的DNA区段可以连接到病毒基因组中。某些载体能够在引入它 们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。此外，某些载体能够指导与它们可操作地连接的基因的表达。通常，在重组DNA技术中有用的表达载体通常呈质粒的形式。
嵌合抗原受体T细胞(Chimeric Antigen Receptor T-Cell，CAR-T)是指经基因修饰后，能以MHC非限制性方式识别特定目的抗原，并且持续活化扩增的T细胞。嵌合抗原受体(CAR)是CAR-T的核心部件，由三个主要的结构组成：胞外抗原结合域、跨膜结构域和胞内结构域。嵌合抗原受体(CAR)的结构从N端到C端，依次含有信号肽、胞外抗原结合域、铰链区、跨膜区、胞内共刺激和胞内信号域。抗体或抗体的抗原结合片段可用作胞外抗原结合域，是CAR中重要的组成部分。纳米抗体具有独特的性质例如分子量少，水溶性好，稳定性强等，近来已被用于开发为CAR-T并靶向递送。
Antibody-Drug Conjugates(ADCs，抗体药物偶联物)，是指与一个或多个化学药物(任选地可以是治疗剂或细胞毒性剂)连接的结合蛋白。可通过永久的或者不稳定的化学连接体(linker)将细胞毒小分子(cytotoxin)和抗体连接，获得抗体药物偶联物。将人源纳米抗体与药物偶联的全人源纳米抗体偶联药物(Fully human single-domain antibody-drug conjugate，UdADC)，在肿瘤类器官、肿瘤微球及小鼠肿瘤模型中均证实了该类新型药物相比传统抗体偶联药物具有显著优势。
实施例1：抗原及对照抗体准备
PD-1-His重组蛋白(蛋白编号QPP11)，购买自义翘神州(北京)生物技术有限公司，货号：10377-H08H。
PD-1-hFc重组蛋白(蛋白编号QP1138)，公司内部生产，PD-1胞外区序列(选自UNIPROT，序列号Q15116第24-170位氨基酸)融合人IgG1的FC，序列如下所示。装入真核表达载体pTargeT，构建表达克隆，用CHOS细胞稳定转染表达蛋白，纯化获得蛋白。
SEQ ID NO：1 QP1138
Figure PCTCN2022110672-appb-000003
Figure PCTCN2022110672-appb-000004
PD-L1-hFc重组蛋白(蛋白编号QP004)，公司内部生产，PD-L1胞外区序列(选自UNIPROT，序列号Q9NZQ7第1-238位氨基酸)融合人IgG1的FC，序列如下所示。装入真核表达载体pQD，构建表达克隆，用293E细胞瞬转表达蛋白，纯化获得蛋白。
SEQ ID NO：2 QP004
Figure PCTCN2022110672-appb-000005
对照抗体为抗PD-1抗体Nivolumab(5C4)，由以下两条序列融合得到，装入真核表达载体pQD，构建表达克隆，用293E细胞瞬转表达蛋白，纯化获得蛋白QP32103211。
SEQ ID NO：3>QD3210：
Figure PCTCN2022110672-appb-000006
SEQ ID NO：4>QD3211：
Figure PCTCN2022110672-appb-000007
Figure PCTCN2022110672-appb-000008
实施例2：羊驼免疫以及构建噬菌体免疫库
羊驼免疫：用PD-1胞外区重组蛋白(义翘神州，10377-H08H))作为抗原，免疫一只健康羊驼(vicugna pacos,alpaca,lama pacos)，首次使用弗式完全佐剂CFA，免疫0.3mg抗原蛋白；第2到第5次均使用弗式不完全佐剂IFA乳化与抗原混合后皮下多点注射，免疫0.2mg抗原蛋白。
建库：取50ml外周血分离外周血单个核细胞(PBMC)，提取PBMC总RNA，RT-PCR反转录成cDNA，设计引物PCR扩增VHH基因，建免疫库。外周血淋巴细胞分离，采集50mL外周血，按照淋巴细胞分离液使用说明分离PBMC，用TRIzol试剂提取PBMC总RNA。反转录，参见
Figure PCTCN2022110672-appb-000009
III First-Strand Synthesis System for RT-PCR说明书，共转录8μg RNA。巢式PCR第一轮，割胶回收750bp左右的VHH片段进行巢式PCR第二轮。构建噬菌体文库载体为pComb3XSS，pComb3XSS噬菌粒载体通过SfiI单酶切，将pComb3XSS酶切为1672bp(SS stuffer)与3301bp(载体目的片段)两个大片段，该载体带有His tag与HA tag方便纯化与检测。载体与目的片段连接将载体与目的片段分别用sfiI进行酶切，50℃过夜酶切，然后割胶回收目的片段。连接摩尔比例为Vector：VHH＝1:3。电转化共进行了10次电转化，电转化后立即加入1mL 2YT培养基复苏，共计100ml复苏产物，37℃，180rpm复苏45min，取100μL测定库容量，其余离心，加入5mL 2YT重悬，涂布于200mm的平板上共8块。第二天10 -5共有73个克隆，因此库容量为7.3×10 8(73*100*10 5)。
实施例3：纳米抗体免疫库筛选鉴定
用PD1胞外区重组蛋白筛选2轮。
第一轮(1st)淘筛：在免疫管中，包被抗原，5ng/ul，1ml，4℃过夜。封闭：2％MPBS封闭免疫管，37℃，1小时。消减：加入1800ul 2％MPBS，再加入200ul input phage，加入终浓度50ng/ul消减用抗原，室温旋转1hr。结合：将上清转移至包被抗原的免疫管中，室温旋转1hr。wash：1xPBST洗免疫管8遍，1xPBS洗4遍。洗脱：800ul 100mM TEA，室温10min。中和：转移洗脱液至1.5ml EP管中，加入400ul 1M pH7.4Tris.侵染：中和后的洗脱phage，加入到10ml OD600＝0.5的TG1中。37℃4min。测滴度，其余菌液涂大平板。37℃，培养过夜。
包装2nd input phage：2xTY培养基刮菌，菌接种到50ml 2xTY培养基+amp+1％glucose至OD～0.1,37度200rpm，生长～1hour 20mins,至OD～0.4-0.6。加入500ul  M13KO7，37度感染40mins离心去上清，沉淀重悬100ml 2xTY培养基+amp+kana。30度200rpm过夜。2ND input phage沉淀：过夜菌液离心，4200rpm，15min。取上清40ml，加入10ml PEG/NaCl，混匀后，冰上放置20分钟。离心，4200rpm，15min。弃上清。瞬时离心，吸弃上清。加入1ml 1xPBS重悬沉淀。13000rpm，离心10min。取上清转移至新的1.5EP管中。用于接下来的淘洗，或者加入0.5ml 50％甘油-80℃冻存。
第二轮(2 nd)淘筛：在免疫管中，包被抗原，2ng/ul，1ml，4℃过夜。封闭：2％MPBS封闭免疫管，37℃，1小时。消减：加入1800ul 2％MPBS，再加入200ul input phage，加入终浓度50ng/ul消减用抗原，室温旋转1hr。结合：将上清转移至包被抗原的免疫管中，室温旋转1hr。wash：1xPBST洗免疫管8遍，1xPBS洗4遍。洗脱：800ul 100mM TEA，室温10min。中和：转移洗脱液至1.5ml EP管中，加入400ul 1M pH7.4Tris.侵染：中和后的洗脱phage，加入到10ml OD600＝0.5的TG1中。37℃4min。测滴度，其余菌液涂大平板。37℃，培养过夜。
包装2nd output phage：2xTY培养基刮菌，菌接种到50ml 2xTY培养基+amp+1％glucose至OD～0.1,37度200rpm，生长～1hour 20mins,至OD～0.4-0.6。加入500ul M13KO7，37度感染40mins离心去上清，沉淀重悬100ml 2xTY培养基+amp+kana。30度200rpm过夜。2ND output phage沉淀：过夜菌液离心，4200rpm，15min。取上清40ml，加入10ml PEG/NaCl，混匀后，冰上放置20分钟。离心，4200rpm，15min。弃上清。瞬时离心，吸弃上清。加入1ml 1xPBS重悬沉淀。13000rpm，离心10min。取上清转移至新的1.5EP管中。用于接下来的淘洗，或者加入0.5ml 50％甘油-80℃冻存。
淘选策略及结果如下所示：
表1淘选策略
  1 st 2 nd–QP1138 2 nd–QPP11
antigen QP1138 5ng/ul QP1138 2ng/ul QPP11 2ng/ul
表2淘选结果
  1 st 2 nd–QP1138 2 nd–QPP11
NB089 1E6 1.5E8 2.3E6
免疫库筛选鉴定：经过2轮淘筛，挑取单克隆进行噬菌体ELISA，筛选结合PD-1的阳性克隆。同时筛选结合PD-1阳性及阻断PD-1及PD-L1结合阳性的克隆。将阳性克隆测序，获得抗体PD-1抗体的VHH序列。ELISA筛选方法如下所示：
包装phage和可溶性表达：挑选克隆：P1-P4总共4块96孔板。单克隆接种于180ul 2YT+amp中，37℃培养2小时。包装phage：30ul加入100ul 2YT+M13K07，37℃静置1小时，加入50ul 2YT+amp+kan，30℃震荡过夜。离心取上清用于ELISA。可溶性表达：120ul菌液，经过3小时震荡培养后，加入60ul 2YT,加入1M IPTG至终浓度1mM。30℃震荡过夜。离心取上清用于ELISA。
phage ELISA：包被QP1138，阴性对照human IgG(蛋白编号QP11851186)，PDL1.mFc(自己生产)2ng/ul，60ul/well各4块板，4度包被过夜，PBS洗3遍。5％milk 200ul/孔室温封闭1h。在PDL1.mFc中，加入1ng/ul QP1138,60ul/孔，室温1小时，加入20ul phage上清和40ul 2％MPBS，混匀，室温1小时。PBST洗板3次，加入60ul anti-M13HRP(义翘神州，11973-MM05T-200)，室温1小时。PBST洗板5次，TMB显色100ul/孔，室温10分钟，2M H2SO4 100ul/孔终止反应。QP1138结合阳性，QP11851186不结合的克隆为PD-1特异性结合克隆，送测序。
blocking phage ELISA操作方法：包被蛋白QP004(PD-L1-hFC)4ng/ul 60ul/孔，加入QPP11(PD1.His，义翘神州)1ng/ul,60ul/孔，60ul MPBS+20ul phage上清，加入60ul anti-M13HRP(义翘神州，11973-MM05T-200)孵育1小时，PBST洗板5次，TMB显色，H 2SO 4终止。
可溶性阻断ELISA操作方法：包被蛋白QP004(PD-L1-hFC)2ng/ul 60ul/孔，4度过夜，PBS洗板3次，5％milk 200ul/孔封闭1h。PBS洗板3次。30ul biontin-QP1138 0.1ng/ul+30ul表达上清，孵育1小时，PBST洗板3次，加入Strep-HRP 1:5000，60ul/孔孵育1小时，PBST洗板5次后，TMB显色，H 2SO 4终止。
选择结合PD-1阳性及同时能阻断PD-1与PD-L1结合的克隆。将阳性克隆测序，获得抗PD-1抗体的VHH序列。本发明提供2个获得的独特的纳米抗体，编号分别QP3120和QP3126，氨基酸序列如SEQ ID NO：5、6所示。
SEQ ID NO：5 QP3120
Figure PCTCN2022110672-appb-000010
Figure PCTCN2022110672-appb-000011
其中，加粗并下划线所示为纳米抗体序列的CDR1、CDR2、CDR3，序列编号分别为SEQ ID NO：7、8、9。
SEQ ID NO：6 QP3126
Figure PCTCN2022110672-appb-000012
Figure PCTCN2022110672-appb-000013
其中，加粗并下划线所示为纳米抗体序列的CDR1、CDR2、CDR3，序列编号分别为SEQ ID NO：7、8、10。
由以上序列可知，QP3120和QP3126的CDR区仅CDR3具有一个氨基酸的差异，因此QP3120和QP3126序列高度相似。
实施例4：纳米抗体构建FC融合蛋白，克隆、表达、纯化蛋白
克隆设计构建：将抗PD-1抗体的纳米抗体VHH的C端融合人IgG1FC，构建纳米抗体FC融合蛋白antiPD-1VHH-FC。构建真核表达质粒在HEK293细胞中瞬转表达蛋白，通过protein A亲和层析等方法纯化蛋白，2个PD-1VHH-FC融合蛋白的蛋白编号继续采用QP3120、QP3126。人IgG1Fc段序列如SEQ ID NO：11所示。
蛋白表达：293E细胞培养密度维持在0.2-3×10 6/ml之间，维护阶段培养基(GIBCO Freestyle 293表达培养基)进行培养，转染前一天将待转染细胞离心换液，调整细胞密度为0.5-0.8×10 6/ml。转染当天，293E细胞密度为1-1.5×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为100μg/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO 2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
Protein A亲和层析纯化：用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，当UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
实施例5：ELISA检测PD-1纳米抗体Fc融合蛋白对人PD-1蛋白结合
实验步骤：包板：包被QP1138(PD-1–hFC)1μg/ml，60μl/孔，4℃过夜，PBS*3；封闭：5％milk/PBS，200μl/孔，25℃，孵育1h；抗原：分别孵育PD-1纳米抗体PD-1VHH-FC QP3120和QP3126，25μg/ml起5倍稀释，8个梯度，60μl/孔，25℃，1h，PBST*5；二抗：抗Fab-HRP，1：8000稀释，60μl/孔，25℃，1h，PBST*5；显色：TMB 100μl/孔，5-10min，2M的H 2SO 4终止反应，450nm读数。结果如图1所示，PD-1纳米抗体Fc融合蛋白均结合人PD-1蛋白。
实施例6：ELISA检测PD-1纳米抗体Fc融合蛋白阻断人PD-L1和PD-1蛋白结合
包被蛋白QP1138(PD1-FC)2μg/ml 50μl/孔，4℃过夜。PBS洗3次。封闭：3％BSA250μl/孔，室温孵育1h。分别配制2μg/ml PDL1-小鼠FC和不同浓度抗体，等体积混匀，室温孵育1h。PBST洗3次，PBS洗3次。孵育二抗：HRP-小鼠IgG(1：5000)50μl/孔，PBST洗6次，PBS洗3次。显色：TMB 100μl/孔，显色10min。2M H 2SO 4 50μl/孔终止。结果如图2所示，PD-1纳米抗体Fc融合蛋白能阻断人PD-L1和PD-1蛋白结合。
实施例7：ELISA检测PD-1纳米抗体Fc融合蛋白对食蟹猴PD-1蛋白结合
包被兔His抗体(金斯瑞，A00174)2μg/ml 50μl/孔，4℃过夜。PBS洗3次。封闭：3％BSA 250μl/孔，室温孵育1h。孵育猴PD1-his(sinobio 90251-C08H)1μg/ml，50μl/孔，25℃1h，PBS洗3遍。孵育抗体起始浓度10μg/ml，稀释8个梯度。50μl/孔，25℃1h，PBS洗6遍。孵育二抗HRP-抗人FC，1:5000倍稀释，60μl/孔，25℃1h，PBST洗6遍。TMB显色，H 2SO 4终止反应。酶标仪设置450nm读数。结果如图3所示，PD-1纳米抗体Fc融合蛋白均结合食蟹猴PD-1蛋白。
实施例8：纳米抗体人源化设计，克隆、表达、纯化蛋白
纳米抗体人源化设计：通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，将QP3120、QP3126同源性高的重轻链可变区种系基因作为模板，将羊驼纳米抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。其中氨基酸残基由Kabat编号***确定并注释。设计引物PCR搭建各人源化抗体VH基因片段，再与带信号肽及恒定区基因(FC)片段的表达载体pQD同源重组，构建抗体全长表达载体VH-FC-pQD。利用在线软件DNAWorks(v3.2.4)(http://helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp信号肽+VH+30bp FC-3’。按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。带信号肽及恒定区基因(FC)片段的表达载体pQD的构建及酶切，利用限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建带信号肽及恒定区基因(FC)片段的表达载体pQD。BsmBI酶切载体，切胶回收备用。重组构建表达载体VH-FC-pQD。VH含重组所需基因片段与BsmBI酶切回收表达载体pQD(带信号肽及恒定区基因(FC)片段)按3：1比例分别加入DH5a感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
各克隆人源化设计轻重链可变区序列及蛋白表达编号如下表3所示，其中所有抗体在其C端融合人IgG1-FC恒定区。
表3 QP3120、QP3126人源化设计
Figure PCTCN2022110672-appb-000014
克隆构建：将上面人源化设计克隆转化为C端为人IgG1FC的PD-L1-FC融合蛋白。重新构建的质粒在HEK293细胞中进行表达，通过protein A亲和层析纯化，一共获得7个人源化PD-1VHH-FC融合蛋白QP629-QP635，序列为SEQ ID NO：12-18所示的纳米抗体后端连接人IgG1FC段，序列如SEQ ID NO：11所示。
蛋白表达：293E细胞培养密度维持在0.2-3×10 6/ml之间，维护阶段培养基(GIBCO Freestyle 293表达培养基)进行培养，转染前一天将待转染细胞离心换液，调整细胞密度为0.5-0.8×10 6/ml。转染当天，293E细胞密度为1-1.5×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为100μg/100ml细胞，使用PEI和质粒的质量比为2：1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO 2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
Protein A亲和层析纯化：用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，在UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速为1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
实施例9：ELISA检测人源化PD-1纳米抗体Fc融合蛋白对人PD-1蛋白结合
包板anti-his抗体1ug/ml，60ul/孔，4℃overnight，PBST洗2遍。封闭5％skim milk，200ul/孔，室温1h，PBST洗2遍。孵育抗原PD1-his，1ug/ml，60ul/孔，室温1h， PBST洗5遍。孵育抗体133.3nM起，4倍稀释，最后一个100倍稀释，60ul/孔，室温1h，PBST洗5遍。孵育二抗anti-hFc HRP，1：5000，60ul/孔，室温1h，PBST洗5遍。TMB显色10min。450nm读数。
结果如图4所示，ELISA检测人源化PD-1纳米抗体Fc融合蛋白对人PD-1蛋白结合，选择结合且EC50值低的分子进一步验证。
实施例10：ELISA检测人源化PD-1纳米抗体Fc融合蛋白阻断人PD-L1和PD-1蛋 白结合
包被蛋白QP1138(PD1-FC)2μg/ml 50μl/孔，4℃过夜。PBS洗3次。封闭：3％BSA250μl/孔，室温孵育1h。分别配制2μg/ml PDL1-小鼠FC和不同浓度QP509等人源化纳米抗体15μg/ml，阳性对照30μg/ml，1：3稀释，等体积混匀，室温孵育1h。PBST洗3次，PBS洗3次。孵育二抗：HRP-小鼠IgG(1：5000)50μl/孔，PBST洗6次，PBS洗3次。显色：TMB 100μl/孔，显色10min。2M H 2SO 4 50μl/孔终止。
结果如图5所示，ELISA检测人源化PD-1纳米抗体Fc融合蛋白能阻断人PD-L1蛋白与PD-1蛋白的结合。
为了评估抗PD-1纳米抗体功能活性，设计了人源化纳米抗体Fc融合蛋白的两种形式，具体地，第一种形式为将抗PD-1VHH融合在人IgG1FC段突变消除FCγR功能(EU计数L234A/L235A)如序列SEQ ID NO：19组成的N端；第二种形式为将抗PD-1VHH融合在人IgG1FC段突变消除FCγR功能(EU计数L234A/L235A)的C端，蛋白编号及序列号如表4所示。
表4人源化纳米抗体Fc融合蛋白的两种形式设计
Figure PCTCN2022110672-appb-000015
实施例11：ELISA检测人源化PD-1纳米抗体Fc融合蛋白对人PD-1蛋白结合
包板anti-his抗体1ug/ml，60ul/孔，4℃overnight，PBST洗2遍。封闭5％skim milk，200ul/孔，室温1h，PBST洗2遍。孵育抗原PD1-his，1ug/ml，60ul/孔，室温1h，PBST洗5遍。孵育抗体133.3nM起，4倍稀释，最后一个100倍稀释，60ul/孔，室温1h，PBST洗5遍。孵育二抗anti-hFc HRP，1：5000，60ul/孔，室温1h，PBST洗5遍。TMB显色10min。450nm读数。结果如图6所示，人源化PD-1纳米抗体融合在Fc的N端及C端均结合人PD-1蛋白。
实施例12：ELISA检测人源化PD-1纳米抗体Fc融合蛋白阻断人PD-L1和PD-1蛋 白结合
包被蛋白QP1138(PD1-FC)2μg/ml 50μl/孔，4℃过夜。PBS洗3次。封闭：3％BSA250μl/孔，室温孵育1h。分别配制2μg/ml PDL1-小鼠FC和不同浓度QP509等人源化纳米抗体15μg/ml，阳性对照30μg/ml，1：3稀释，等体积混匀，室温孵育1h。PBST洗3次，PBS洗3次。孵育二抗：HRP-小鼠IgG(1：5000)50μl/孔，PBST洗6次，PBS洗3次。显色：TMB 100μl/孔，显色10min。2M H 2SO 4 50μl/孔终止。
结果如图7所示，人源化PD-1纳米抗体融合在Fc的N端及C端均能阻断人PD-L1蛋白与PD-1蛋白的结合。
实施例13：人源化纳米抗体Fc融合蛋白在混合淋巴细胞反应中促进T细胞增殖
混合淋巴细胞反应是指将人T细胞和同种异体树突细胞混合共培养，淋巴细胞接受同种异型抗原的刺激而发生活化、增殖，产生种类众多的细胞因子，抗PD-1抗体呈抗体浓度依赖通过阻断PD-1/PD-L1结合的免疫抑制信号，刺激T细胞增殖释放细胞因子如IL-2/IFN-γ等。通过ELISA检测IL-2/IFN-γ释放量，研究抗PD-1抗体在混合淋巴细胞反应中刺激T细胞体外增殖生物活性。
分离PBMC中的单核细胞，加入rhGM-CSF和rhIL-4，诱导为DC(诱导性树突状细胞)；分离另一个donor PBMC中的CD4+T细胞。将DC细胞与T细胞按1:10比例混合，加入不同浓度的抗PD-1抗体，混合培养2-5天，检测培养上清中IL-2和IFN-γ的表达。结果如图8，图9，图10，图11所示：人源化PD-1纳米抗体融合在Fc的N端及C端在混合淋巴细胞反应(MLR)中均能刺激T细胞增殖，增强IL-2和IFN-γ的产生。
综上所述，人源化PD-1纳米抗体融合在Fc的N端及C端的各种形式，QP916,QP3517,QP3519,QP917,QP918,QP919,QP3518在混合淋巴细胞反应(MLR)中均能刺激T细胞增殖，增强IL-2和IFN-γ的产生，优于对照Novilumab类似物(QP32103211)。
实施例14：PD-1纳米抗体刺激人PBMC体外增殖生物活性
人外周血单个核细胞(PBMC)由多种白细胞组成，主要包括单核细胞，B细胞，T细胞，NK细胞，树突状细胞和巨噬细胞等。在体外加入超抗原SEB刺激PBMC，通过其中的APC细胞的递呈和激活，淋巴细胞活化增殖，产生种类众多的细胞因子。PD-L1抗体通过阻断PD-1/PD-L1结合的免疫抑制信号，增强T细胞增殖释放细胞因子如IL-2/IFN-γ等。通过ELISA检测IL-2/IFN-γ释放量，进一步研究Q-1801在PBMC体外增殖实验中的生物活性。将PBMC细胞接种于96孔板中，配制不同浓度SEB加入PBMC细胞孔中，再加入抗PD-1抗体，轻轻混匀，培养2-5天。ELISA检测细胞培养上清中IL-2分泌量，ELISA检测细胞培养上清中IFN-γ分泌量。结果如图12显示QP3517,QP918,QP3519,QP919在SEB刺激的PBMC体外增殖实验中能显著增强PBMC的激活和增殖，增强IL-2的产生，不劣于对照抗体novilumab。人源化PD-1纳米抗体融合在Fc的N端及C端在SEB刺激PBMC中均能刺激T细胞增殖，增强IL-2的产生。
实施例15：抗人PD1抗体在体内动物药效中抑制肿瘤生长
实验目的：通过CDX动物药效试验验证抗人PD1抗体抑制肿瘤生长
实验步骤：离心收集培养的MC38-hPDL1细胞，用1×PBS分散细胞，制备成细胞密度为5×10 6个/ml细胞悬液。取0.1ml细胞悬液皮下接种至C57BL/6-hPD1小鼠右侧肋部皮下来建立MC38-hPDL1荷瘤小鼠模型。当C57BL/6-hPD1小鼠平均肿瘤体积到达约73mm 3时，将小鼠随机分组，每组7只。称量所有动物的体重，并用游标卡尺测量肿瘤体积。根据肿瘤体积，采用随机分组方法进行分组，保证不同组别间的肿瘤体积相似。分组当天为D0，并于当天开始给药。详细的给药方法、给药剂量和给药途径见下表。
表5：动物药效试验的给药参数
Figure PCTCN2022110672-appb-000016
给药体积为10μL/g。
开始给药后，每周测量三次小鼠体重和肿瘤体积。肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。当最后一次给药后三天终止实验，处死小鼠，取瘤称重、拍照。
选用以下分析方法进行数据分析：
肿瘤增殖率，T/C(％)＝(T-T0)/(C-C0)×100％(T0和C0分别为分组时治疗组和对照组瘤体积，T和C分别为给药后治疗组和对照组在某一特定时间点的瘤体积)
肿瘤抑制率，TGI(％)，计算公式为：TGI％＝(1-T/C)×100％
实验结果：
Vehicle对照组小鼠在给药后第17天平均肿瘤体积为705.60±105.85mm3。纳米抗体分子QP918(5mg/kg)、QP918(10mg/kg)、QP3517(5mg/kg)和QP3517(10mg/kg)组在给药后第17天平均肿瘤体积分别为23.24±16.14mm3、26.28±13.27mm3、14.63±13.32mm3、和11.59±8.42mm3，TGI分别为107.95％、107.41％、109.29％和109.68％，对照分子QP32103211(10mg/kg)在给药后第17天平均肿瘤体积为47.99±27.75mm3，TGI为104.01％(见下表)。和Vehicle组相比，对照分子QP32103211和两个纳米抗体分子均显著抑制了肿瘤的生长(p<0.001)，且QP918和QP3517表现略优于对照分子QP32103211的趋势。而且小鼠体重在给药过程中没有发生明显下降，说明抗体分子对小鼠没有明显毒副作用。
表6 PD-1抗体抑制肿瘤生长的肿瘤体积分析结果
Figure PCTCN2022110672-appb-000017
Figure PCTCN2022110672-appb-000018
肿瘤重量的分析结果与肿瘤体积的结果相似。Vehicle对照组小鼠在给药后第17天平均肿瘤重量为741.07±318.89mg，抗体分子QP918-5mpk、QP918-10mpk、QP3517-5mpk和QP3517-10mpk治疗组在结束给药后第17天平均肿瘤重量分别为9.73±16.38mg、23.37±38.99mg、9.76±22.33mg和11.66±27.04mg；对照分子QP32103211-10mpk治疗组在给药后第17天平均肿瘤重量为35.57±57.34mg。(见下表)
表7 PD-1抗体抑制肿瘤生长的肿瘤重量分析结果
Figure PCTCN2022110672-appb-000019
因此通过CDX动物药效试验，我们发现筛选出的抗体分子具有比对照分子QP32103211更优的体内抑制肿瘤能力。
实验结果如图13至图16所示，其中，抗体对小鼠结肠癌CDX模型MC38-hPDL1-mClaudin18.2的药效试验的结果如图13所示。图14是各个组在分组后的肿瘤生长曲线。图15是加入本发明各抗体及对照抗体各组小鼠肿瘤D17重量。图16是各组小鼠的体重曲线。从实验结果中可见，本发明的PD-1纳米抗体QP918,QP3517比对照抗体Novilumab类似物(QP32103211)在体内动物药效中表现出更好的抑制肿瘤生长的效果。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。
Figure PCTCN2022110672-appb-000020
Figure PCTCN2022110672-appb-000021
Figure PCTCN2022110672-appb-000022
Figure PCTCN2022110672-appb-000023
Figure PCTCN2022110672-appb-000024
Figure PCTCN2022110672-appb-000025
Figure PCTCN2022110672-appb-000026

Claims (17)

  1. 一种抗PD-1纳米抗体，其特征在于，包括：VHH链，所述VHH链的互补决定区包括CDR1、CDR2和CDR3，所述CDR1的氨基酸序列如SEQ ID NO：7所示，所述CDR2的氨基酸序列如SEQ ID NO：8所示，所述CDR3的氨基酸序列如SEQ ID NO：9或SEQ ID NO：10所示。
  2. 根据权利要求1所述的抗PD-1纳米抗体，其特征在于，所述VHH链的氨基酸序列包括：选自SEQ ID NO:5、6或SEQ ID NO:12-18中任意一项所示，或与其具有至少85％、90％、95％、97％、或99％同一性的序列。
  3. 一种融合蛋白，其特征在于，包括：权利要求1或2所述的抗PD-1纳米抗体。
  4. 根据权利要求3所述的融合蛋白，其特征在于，还包括：Fc段；所述Fc段与所述抗PD-1纳米抗体的N端或C端融合。
  5. 根据权利要求4所述的融合蛋白，其特征在于，所述融合蛋白还包括：除所述抗PD-1纳米抗体和所述Fc段以外的其他功能片段；所述Fc段与所述抗PD-1纳米抗体融合，和/或，所述Fc段与所述其他功能片段融合。
  6. 根据权利要求3所述的融合蛋白，其特征在于，还包括：Fc段；所述的Fc段选自人源IgG1、IgG2、IgG3、或IgG4或其变体；优选的，所述Fc段选自人源IgG1的变体；优选的，所述Fc结构域选择消除免疫效应功能，优选包含以下任一突变方式，以下突变为根据EU计数：
    Figure PCTCN2022110672-appb-100001
  7. 一种抗PD-1抗体分子，其特征在于，包括：一个或多个权利要求1或2所述的抗PD-1纳米抗体；所述抗PD-1抗体分子为单价抗体、双价或多价抗体、双特异抗体、或多特异抗体。
  8. 一种嵌合抗原受体，包括胞外抗原结合域、跨膜结构域和胞内结构域，其特征在于，所述胞外抗原结合域包括权利要求1或2所述的抗PD-1纳米抗体。
  9. 一种核酸分子，其特征在于，其编码：权利要求1或2所述的抗PD-1纳米抗体，或，权利要求3至6中任意一项所述的融合蛋白。
  10. 一种载体，其特征在于，包含权利要求9所述的核酸分子。
  11. 一种宿主细胞，其特征在于，其采用权利要求10所述的载体转化得到。
  12. 一种抗体药物偶联物，其特征在于，包含权利要求1或2所述的抗PD-1纳米抗体，以及偶联于所述抗PD-1纳米抗体的药物。
  13. 一种药物组合物，其特征在于，其含有：权利要求1或2所述的抗PD-1纳米抗体，或，权利要求3至6中任意一项所述的融合蛋白，以及一种或多种药学上可接受的载 体、稀释剂或赋形剂。
  14. 权利要求1或2所述的抗PD-1纳米抗体在制备用于抑制或治疗疾病的药物中的用途。
  15. 权利要求1或2所述的抗PD-1纳米抗体在用于通过刺激免疫功能达到预防或治疗疾病的效果的用途。
  16. 根据权利要求14所述的用途，其特征在于，所述的疾病为PD-1介导的疾病。
  17. 根据权利要求16所述的用途，其特征在于，所述的疾病为癌症；优选的，所述的癌症包括乳腺癌、肺癌、胃癌、肠癌、肾癌、黑色素瘤、膀胱癌、头颈癌、淋巴瘤、皮肤恶性肿瘤、或非小细胞肺癌。
PCT/CN2022/110672 2021-08-06 2022-08-05 抗pd-1纳米抗体及其应用 WO2023011654A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110903505 2021-08-06
CN202110903505.9 2021-08-06

Publications (1)

Publication Number Publication Date
WO2023011654A1 true WO2023011654A1 (zh) 2023-02-09

Family

ID=85155326

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/110672 WO2023011654A1 (zh) 2021-08-06 2022-08-05 抗pd-1纳米抗体及其应用

Country Status (2)

Country Link
CN (1) CN115991771A (zh)
WO (1) WO2023011654A1 (zh)

Citations (5)

* 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
WO2018113258A1 (zh) * 2016-12-22 2018-06-28 安源医药科技（上海）有限公司 抗pd-1抗体及其用途
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
US20190352402A1 (en) * 2018-05-17 2019-11-21 Nanjing Leads Biolabs Co., Ltd. Antibody binding pd-1 and use thereof
CN112142842A (zh) * 2019-06-27 2020-12-29 启愈生物技术(上海)有限公司 抗PD-L1纳米抗体及其Fc融合蛋白和应用

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
US20190322747A1 (en) * 2016-09-14 2019-10-24 Zhejiang Teruisi Pharmaceutical Inc. Anti-pd-1 nano-antibody and application thereof
WO2018113258A1 (zh) * 2016-12-22 2018-06-28 安源医药科技（上海）有限公司 抗pd-1抗体及其用途
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
US20190352402A1 (en) * 2018-05-17 2019-11-21 Nanjing Leads Biolabs Co., Ltd. Antibody binding pd-1 and use thereof
CN112142842A (zh) * 2019-06-27 2020-12-29 启愈生物技术(上海)有限公司 抗PD-L1纳米抗体及其Fc融合蛋白和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YU SHENG, XIONG GUI, ZHAO SHIMEI, TANG YANBO, TANG HUA, WANG KAILI, LIU HONGJING, LAN KE, BI XIONGJIE, DUAN SILIANG: "Nanobodies targeting immune checkpoint molecules for tumor immunotherapy and immunoimaging (Review)", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, SPANDIDOS PUBLICATIONS, GR, vol. 47, no. 2, GR , pages 444 - 454, XP055944687, ISSN: 1107-3756, DOI: 10.3892/ijmm.2020.4817 *

Also Published As

Publication number Publication date
CN115991771A (zh) 2023-04-21

Similar Documents

Publication Publication Date Title
WO2022105832A1 (zh) 抗pd-l1纳米抗体及三功能融合蛋白
TWI754800B (zh) 新型抗ox40/pd-l1雙特異性抗體分子、新型抗vegf/gitr雙特異性抗體分子及其用途
WO2017084495A1 (zh) Pd-l1抗体、其抗原结合片段及其医药用途
WO2019184909A1 (zh) 新型抗体分子、其制备方法及其用途
US10774144B2 (en) Human programmed cell death 1 receptor antibody, method of preparing same, and use thereof
WO2021218874A1 (zh) 一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用
WO2021170082A1 (zh) 抗cd47/抗pd-l1抗体及其应用
WO2021043261A1 (zh) 抗pd-1单域抗体、其衍生蛋白及其医药用途
US20240124563A1 (en) Anti-Human MSLN Antibody And Application Thereof
TWI806087B (zh) 免疫細胞銜接多特異性結合蛋白及其製備和應用
CN115109156A (zh) 一种靶向bcma的纳米抗体及其应用
WO2023125561A1 (zh) 靶向tigit的抗体和双特异性抗体及其应用
WO2019192493A1 (zh) 抗人lag-3单克隆抗体及其应用
WO2023273595A1 (zh) 一种结合trop2的抗体及靶向trop2和cd3的双特异性抗体及其制备方法与应用
TWI806088B (zh) 具有H2L2與HCAb結構的結合蛋白
WO2023011654A1 (zh) 抗pd-1纳米抗体及其应用
TW202246341A (zh) 針對NKp46的抗體及其應用
WO2022002006A1 (zh) Fab-HCAb结构的结合蛋白
WO2023231705A1 (zh) 靶向SIRPα和PD-L1的双特异性抗体或其抗原结合片段及应用
WO2022247826A1 (zh) 靶向pd-l1和cd73的特异性结合蛋白
WO2022063100A1 (zh) 抗tigit抗体及双抗体和它们的应用
WO2022207921A1 (en) Novel tnfr2 binding molecules
TW202305011A (zh) 靶向ｐｄ－１和／或ｏｘ４０的特異性結合蛋白
TW202305007A (zh) 靶向pd-l1和cd73的特異性結合蛋白
TW202304963A (zh) 標靶bcma的多特異性抗體

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22852365

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE