Development of novel PDL1 single domain antibodies

Abstract

Nanobody against PDL1 and its use. Nanobody against PDL1 and its sequence are provided. Polynucleotides encoding the nanobody, corresponding expression vectors and host cells capable of expressing the nanobody, as well as a method for producing the nanobody. The nanobody can specifically bind to PDL1 and has high affinity; has good PD-1-PDL1 blocking activity; the nanobody only blocks the PD-1 and PDL1 pathways and does not affect the PD-1 and PD-L2 pathways, thereby avoiding side effects such as interstitial pneumonia and improving safety; the nanobody can more comprehensively activate the immune system to kill tumors, and patients who are ineffective against PD-1 or resistant to PD-1 drugs can also achieve therapeutic effects; the PDL1 nanobody not only inhibits the PD-1-PDL1 pathway, but also blocks the co-inhibitory function of B7.1 and PDL1, which is favorable to the comprehensive activation of T cell function and cytokine production, thereby achieving better immune effects.

Description

[Technical field]

[0001] Technical Field The present invention belongs to the biomedical or biopharmaceutical technical field and specifically relates to a single domain antibody specifically targeting PDL1 and uses thereof. [Background technology]

[0002] Background technology PD-1 / PDL1 immunotherapy is one of the most successful tumor immunotherapies, which has revolutionized tumor treatment and led the transformation of cancer treatment. Immune checkpoint inhibitors are a type of drug that fully utilizes the body's own immune system to fight cancer, and they have the potential to kill cancer cells by blocking the PD-1 / PDL1 signaling pathway, which has the potential to treat many types of tumors, effectively improving the survival of patients with advanced tumors and becoming a "miracle" drug for tumor patients.

[0003] At present, several PD-1 antibodies and PD-1 inhibitors have been marketed at home and abroad, which are similar to PD-1 antibodies, and can block the immune suppression mediated by the binding of PDL1 to PD-1 on the surface of T cells, and reactivate T cells to recognize and kill tumor cells, thereby suppressing tumor growth. Unlike PD-1 monoclonal antibodies, PD-1 antibodies bind to PDL1 expressed on tumor cells or tumor-infiltrating immune cells, whereas PD-1 monoclonal antibodies bind to PD-1.

[0004] However, current PD-1 antibodies have the following limitations: (1) in addition to blocking the PD-1 and PDL1 pathways, PD-1 antibodies may affect the PD-1 and PD-L2 pathways, leading to side effects such as interstitial pneumonia; (2) some patients may be ineffective in treatment with PD-1 monoclonal antibodies or may be resistant to PD-1 drugs, and alternative drugs are needed for such patients; (3) the efficacy rate and safety of PD-1 antibodies still need to be improved.

[0005] Therefore, there is a strong need in this field to develop novel drugs that inhibit the PD-1 / PDL1 signal pathway, which have better therapeutic effects, a wider range of application, and better safety. Summary of the Invention

[0006] Summary of the Invention It is an object of the present invention to provide nanobodies against PDL1 and uses thereof. In a first aspect of the present invention, there is provided a Nanobody against PDL1. In another preferred embodiment, said Nanobody against PDL1 is capable of specifically binding to PDL1.

[0007] In another preferred embodiment, the complementarity determining regions (CDRs) of said Nanobody against PDL1 are one or more selected from the following group: (1) CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9; (2) CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11, and CDR3 represented by SEQ ID NO: 12; (3) CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 13, and CDR3 represented by SEQ ID NO: 14; (4) CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 13, and CDR3 represented by SEQ ID NO: 15; (5) CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 16, and CDR3 represented by SEQ ID NO: 17; (6) CDR1 represented by sequence number 18, CDR2 represented by sequence number 19, and CDR3 represented by sequence number 20.

[0008] In another preferred embodiment, any one of the above amino acid sequences further includes a derived sequence, optionally having at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid addition, deletion, modification and / or substitution, and capable of maintaining the ability to specifically bind to PDL1.

[0009] In another preferred embodiment, the derived sequence, which has undergone the addition, deletion, modification and / or substitution of at least one amino acid and is capable of retaining the ability to specifically bind to PDL1, has an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or sequence identity.

[0010] In another preferred embodiment, said CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain. In another preferred embodiment, said Nanobody against PDL1 further comprises a framework region FR. In another preferred embodiment, the framework region FR is derived from the amino acid sequence shown in SEQ ID NO: 1-6.

[0011] In another preferred embodiment, the framework region FR is one or more selected from the following group: (1) FR1 represented by SEQ ID NO: 21, FR2 represented by SEQ ID NO: 22, FR3 represented by SEQ ID NO: 23, and FR4 represented by SEQ ID NO: 24; (2) FR1 represented by SEQ ID NO: 25, FR2 represented by SEQ ID NO: 26, FR3 represented by SEQ ID NO: 27, and FR4 represented by SEQ ID NO: 24; (3) FR1 represented by SEQ ID NO: 28, FR2 represented by SEQ ID NO: 29, FR3 represented by SEQ ID NO: 30, and FR4 represented by SEQ ID NO: 24; (4) FR1 represented by SEQ ID NO: 31, FR2 represented by SEQ ID NO: 29, FR3 represented by SEQ ID NO: 32, and FR4 represented by SEQ ID NO: 24; (5) FR1 represented by SEQ ID NO: 33, FR2 represented by SEQ ID NO: 22, FR3 represented by SEQ ID NO: 34, and FR4 represented by SEQ ID NO: 24; (6) FR1 represented by SEQ ID NO: 35, FR2 represented by SEQ ID NO: 36, FR3 represented by SEQ ID NO: 37, and FR4 represented by SEQ ID NO: 24.

[0012] In another preferred embodiment, the amino acid sequence of the VHH chain of said Nanobody against PDL1 is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a combination thereof.

[0013] In another preferred embodiment, said Nanobodies against PDL1 include humanized antibodies, camelid-derived antibodies and chimeric antibodies. In another preferred embodiment, said nanobody against PDL1 is a llama derived antibody.

[0014] In a second aspect of the invention there is provided an antibody against PDL1, the antibody comprising one or more VHH chains of a Nanobody against PDL1 according to the first aspect of the invention. In another preferred embodiment, the amino acid sequence of the VHH chain of said Nanobody against PDL1 is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a combination thereof.

[0015] In another preferred embodiment, the antibody against PDL1 may be a monomeric, bivalent and / or multivalent antibody. In another preferred embodiment, the antibody against PDL1 is a bivalent antibody.

[0016] In a third aspect of the invention, there is provided a chimeric antigen receptor CAR containing an extracellular domain comprising a nanobody against PDL1 as described in the first aspect of the invention, or an antibody against PDL1 as described in the second aspect of the invention.

[0017] In another preferred embodiment, the extracellular domain further comprises a signal peptide. In another preferred embodiment, the extracellular domain further comprises another exogenous protein. In another preferred embodiment, the antibody comprising the extracellular domain has an amino acid sequence as set forth in SEQ ID NO: 1-6.

[0018] In another preferred embodiment, the antibody comprising the extracellular domain has an amino acid sequence that has ≧85% identity, preferably ≧90%, more preferably ≧95% identity with SEQ ID NO:7-17, or has 1, 2 or 3 amino acid differences compared to SEQ ID NO:1-6. In another preferred embodiment, the CAR has the structure shown in formula Ia: L1-Nb-H-TM-C-CD3ζ (Ia) (In the formula, L is either absent or a signal peptide sequence. Nb is the specific binding domain. H is absent or is the hinge region. TM is the transmembrane domain. C is the costimulatory signal domain. CD3ζ is an intracellular signaling sequence derived from CD3ζ (including wild-type or mutant / modified forms thereof). The "-" is a connecting peptide or a peptide bond.

[0019] In another preferred embodiment, each of said L's is a signal peptide of a protein selected from the group consisting of CD8, GM-CSF, CD4, CD28, CD137, or a mutant / modified version thereof, or a combination thereof. In another preferred embodiment, the Nb targets PDL1. In another preferred embodiment, the Nb is a PDL1 nanobody.

[0020] In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, IgG, or a combination thereof. In another preferred embodiment, the H is a human IgG1 Fc hinge region.

[0021] In another preferred embodiment, the TM is a transmembrane domain of a protein selected from the group consisting of CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutant / modified versions thereof, or combinations thereof.

[0022] In another preferred embodiment, C is a costimulatory domain of a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutant / modified forms thereof, or combinations thereof.

[0023] In a fourth aspect of the present invention there is provided a recombinant protein comprising: (i) a nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention; and (ii) An optional tag sequence to aid in expression and / or purification. In another preferred embodiment, the tag sequence comprises an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

[0024] In another preferred embodiment, the recombinant protein specifically binds to PDL1. In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein. In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

[0025] In another preferred embodiment, the recombinant protein specifically binds to PDL1. In another preferred embodiment, the tag sequence is an Fc tag.

[0026] In a fifth aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of a nanobody against PDL1 as defined in the first aspect of the invention, or an antibody against PDL1 as defined in the second aspect of the invention, or a chimeric antigen receptor as defined in the third aspect of the invention, or a recombinant protein as defined in the fourth aspect of the invention.

[0027] In another preferred embodiment, the polynucleotide encodes a protein selected from the group consisting of a nanobody against PDL1 according to the first aspect of the invention or an antibody against PDL1 according to the second aspect of the invention.

[0028] In another preferred embodiment, the present invention relates to a nucleic acid molecule encoding a Nanobody against PDL1 according to the invention. The nucleic acid according to the invention may be RNA, DNA or cDNA. In a sixth aspect of the invention there is provided an expression vector containing a polynucleotide according to the fifth aspect of the invention.

[0029] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or combinations thereof.

[0030] In another preferred embodiment, the expression vector is selected from the group consisting of pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, and the like. In another preferred embodiment, the expression vector is a pcDNA3.4-hIgG1-Fc2 vector. In another preferred embodiment, the expression vector further comprises a member selected from the group consisting of a promoter, a transcription enhancer element WPRE, a long terminal repeat sequence LTR, and the like.

[0031] In a seventh aspect of the invention there is provided a host cell which contains an expression vector according to the sixth aspect of the invention or has incorporated into its genome a polynucleotide according to the fifth aspect of the invention.

[0032] In another preferred embodiment, the host cell comprises a prokaryotic or eukaryotic cell. In another preferred embodiment, the host cell is selected from the group consisting of E. coli, yeast cells, and mammalian cells. In another preferred embodiment, the host cell is a 293F cell.

[0033] In an eighth aspect of the invention there is provided an engineered immune cell which contains an expression vector according to the sixth aspect of the invention or has an exogenous polynucleotide according to the fifth aspect of the invention integrated into its chromosome.

[0034] In another preferred embodiment, the engineered immune cells are selected from the group consisting of: (i) Chimeric antigen receptor αβ T cells (CAR-T cells); (ii) chimeric antigen receptor γδ T cells (CAR-T cells); (iii) chimeric antigen receptor NKT cells (CAR-NKT cells); (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0035] In another preferred embodiment, the engineered immune cells comprise autologous or allogeneic αβ T cells, γδ T cells, NK T cells, NK cells, or a combination thereof. In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0036] In a ninth aspect of the present invention there is provided a method for producing a Nanobody against PDL1 comprising the steps of: (a) obtaining a culture comprising a Nanobody against PDL1 by culturing a host cell according to the seventh aspect of the invention under conditions suitable for the production of a Nanobody; (b) isolating and / or recovering said Nanobody against PDL1 from said culture; and (c) Optionally, purifying and / or modifying the Nanobody against PDL1 obtained from step (b).

[0037] In a tenth aspect of the invention there is provided a method of producing an engineered immune cell according to the eighth aspect of the invention, the method comprising the step of obtaining said engineered immune cell by transducing into an immune cell a polynucleotide according to the fifth aspect of the invention or an expression vector according to the sixth aspect of the invention. In another preferred embodiment, the method further comprises the step of performing functional and efficacy detection on the resulting engineered immune cells.

[0038] In an eleventh aspect of the present invention there is provided an immunoconjugate comprising: (a) a Nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a recombinant protein according to the fourth aspect of the invention; and (b) a conjugate moiety selected from the group consisting of a detectable marker, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a magnetic nanoparticle, a viral coat protein or VLP, or a combination thereof.

[0039] In another preferred embodiment, said moiety (a) is a Nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention. In another preferred embodiment, the (a) moiety and the conjugated moiety are conjugated via a chemical bond or a linker.

[0040] In another preferred embodiment, the radionuclide comprises: (i) a diagnostic isotope 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 a combination thereof; and / or (ii) A therapeutic isotope 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 a combination thereof.

[0041] In another preferred embodiment, the conjugated moiety is a drug or a toxin. In another preferred embodiment, the drug is a drug for targeting and treating a disease with high PDL1 expression. In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like. In another preferred embodiment, the drug is a cytotoxic drug.

[0042] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0043] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, and typical cytotoxic drugs include, for example, Auristatin, Camptothecin, Duocarmycin, Etoposide, Maytansine and Maytansinoids (e.g., DM1 and DM4), Taxanes, Benzodiazepines or Benzodiazepine containing drugs (e.g., Pyrrolo[1,4]benzodiazepines (PBDs), Indolinobenzodiazepines, and Oxazolidinobenzodiazepines), Vinca alkaloids, or combinations thereof.

[0044] In another preferred embodiment, the toxin is auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatins, duocarmycins, dolastatins, adriamycin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, The toxin is selected from the group consisting of vincristine, vinblastine, colchicine, dihydroxyanthracenedione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-octastasis, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0045] In another preferred embodiment, the conjugated moiety is a detectable marker. In another preferred embodiment, the conjugated moiety is selected from the group consisting of a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme generating a detectable product, a radionuclide, a biotoxin, a cytokine (such as IL-2), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a magnetic nanoparticle, a prodrug activating enzyme (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or a nanoparticle of any type.

[0046] In another preferred embodiment, said immune complex comprises multivalent (eg bivalent) VHH chains of a Nanobody against PDL1 according to the first aspect of the invention. In another preferred embodiment, said multivalent means that the amino acid sequence of said immune complex comprises a plurality of repeated similar or different VHH chains of a Nanobody against PDL1 according to the first aspect of the invention.

[0047] In a twelfth aspect of the present invention there is provided the use of an active ingredient selected from the group consisting of a nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, or an engineered immune cell according to the eighth aspect of the invention, or an immune complex according to the eleventh aspect of the invention, or a combination thereof, for the preparation of: (a) a drug for preventing and / or treating a disease with high PDL1 expression; (b) Reagents for detecting diseases with high PDL1 expression.

[0048] In another preferred embodiment, the reagent is a diagnostic reagent, preferably, the diagnostic reagent is a detection sheet or a detection plate. In another preferred embodiment, the diagnostic reagent is for detecting PDL1 protein or a fragment thereof in a sample. In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.

[0049] In a thirteenth aspect of the present invention, there is provided a method for detecting PDL1 protein or a fragment thereof in a sample in vitro, the method comprising the steps of: (1) contacting said sample in vitro with a nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, or an engineered immune cell according to the eighth aspect of the invention, or an immune complex according to the eleventh aspect of the invention, or a combination thereof; (2) detecting whether an antigen-antibody complex is formed, where the formation of the complex indicates the presence of a PDL1 protein or a fragment thereof in the sample. In another preferred embodiment, the detection includes diagnostic or non-diagnostic detection.

[0050] In a fourteenth aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) as an active ingredient, a nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, or an engineered immune cell according to the eighth aspect of the invention, or an immune complex according to the eleventh aspect of the invention, or a combination thereof; and (ii) A pharma- ceutically acceptable carrier, diluent or excipient.

[0051] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of an injection and a freeze-dried preparation.

[0052] In another preferred embodiment, the pharmaceutical composition comprises 0.01-99.99% of the nanobody against PDL1 described in the first aspect of the present invention, or the antibody against PDL1 described in the second aspect of the present invention, or the chimeric antigen receptor described in the third aspect of the present invention, or the recombinant protein described in the fourth aspect of the present invention, or the immune complex described in the eleventh aspect of the present invention, or a combination thereof, and 0.01-99.99% of a pharmaceutical carrier, the percentages being mass percentages in the pharmaceutical composition.

[0053] In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1×10 3 -1×10 8 cells / mL, preferably 1 x 10 4 -1×10 7 cells / mL.

[0054] In a fifteenth aspect of the present invention, there is provided a kit comprising: (1) a first container containing a nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, or an engineered immune cell according to the eighth aspect of the invention, or an immune complex according to the eleventh aspect of the invention, or a combination thereof; and / or (2) a second container containing a second antibody against the contents of the first container; or, The kit comprises a base sheet (support plate) and a detection plate comprising an antibody against PDL1 described in the first aspect of the invention, or an antibody against PDL1 described in the second aspect of the invention, or a recombinant protein described in the third aspect of the invention, or an immune complex described in the eighth aspect of the invention, or a combination thereof.

[0055] In another preferred embodiment, the kit further includes instructions, and the kit is used to non-invasively detect the expression of PDL1 in a subject, as described in the specification.

[0056] In another preferred embodiment, the detection kit is used for detecting a disease in which PDL1 is highly expressed. In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.

[0057] In a sixteenth aspect of the present invention, there is provided a method for preventing and / or treating a disease with high PDL1 expression, comprising the step of administering to a subject in need thereof a nanobody against PDL1 as described in the first aspect of the present invention, or an antibody against PDL1 as described in the second aspect of the present invention, or a chimeric antigen receptor as described in the third aspect of the present invention, or a recombinant protein as described in the fourth aspect of the present invention, or an engineered immune cell as described in the eighth aspect of the present invention, or an immune complex as described in the eleventh aspect of the present invention, or a pharmaceutical composition as described in the fourteenth aspect of the present invention, or a combination thereof.

[0058] In another preferred embodiment, the subject comprises a mammal, such as a human. In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like. In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from the subject (autologous cells).

[0059] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from a healthy individual (allogeneic cells). In another preferred embodiment, the method may be used in combination with other therapeutic methods. In another preferred embodiment, the other therapeutic methods include chemotherapy, radiation therapy, targeted therapy, and the like.

[0060] In a seventeenth aspect of the present invention, there is provided a diagnostic method for a disease with high PDL1 expression, comprising the steps of: (i) obtaining a sample from a subject to be diagnosed and contacting said sample with a nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, or an engineered immune cell according to the eighth aspect of the invention, or an immune complex according to the eleventh aspect of the invention, or a combination thereof; and (ii) detecting whether an antigen-antibody complex is formed, where the formation of the complex indicates that the subject is a confirmed patient of a disease with high PDL1 expression.

[0061] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from another tissue organ. In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.

[0062] In an eighteenth aspect of the present invention there is provided a method for producing a recombinant polypeptide, said recombinant polypeptide being a Nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, comprising the steps of: (a) culturing a host cell according to the fifth aspect of the invention under conditions suitable for expression; and (b) isolating the recombinant polypeptide from the culture. [Brief description of the drawings]

[0063] Description of the drawings [Figure 1] FIG. 1 shows the results of identification of PDL1-Fc protein by SDS-PAGE. [Diagram 2] FIG. 2 shows the results of ELISA detection of immune sera.

[0064] [Diagram 3] Figure 3 shows the results of detection of PCR products by agarose gel electrophoresis, where Figure 3A shows two PCR bands of approximately 1000 bp and 750 bp obtained in the first PCR. The 750 bp fragment was recovered from the gel and used as a template for the second PCR. Figure 3B shows a band of approximately 450 bp obtained in the second PCR, which was identified by agarose gel electrophoresis and proved to be a VHH fragment. [Figure 4] FIG. 4 shows the results of a sequence alignment of the phage display library.

[0065] [Diagram 5] Figures 5-9 show the results of phage ELISA of the solid-phase panning screening products of the phage display library, where red represents clones that simultaneously bind to both the PDL1 antigen and Fc, blue represents clones that bind only to the PDL1 target antigen and do not bind to Fc, and green represents clones that bind only to the PDL1 target antigen, do not bind to Fc, and have relatively strong binding to the PDL1 target antigen. [Figure 6] Figures 5-9 show the results of phage ELISA of the solid-phase panning screening products of the phage display library, where red represents clones that simultaneously bind to both the PDL1 antigen and Fc, blue represents clones that bind only to the PDL1 target antigen and do not bind to Fc, and green represents clones that bind only to the PDL1 target antigen, do not bind to Fc, and have relatively strong binding to the PDL1 target antigen.

[0066] [Figure 7] Figures 5-9 show the results of phage ELISA of the solid-phase panning screening products of the phage display library, where red represents clones that simultaneously bind to both the PDL1 antigen and Fc, blue represents clones that bind only to the PDL1 target antigen and do not bind to Fc, and green represents clones that bind only to the PDL1 target antigen, do not bind to Fc, and have relatively strong binding to the PDL1 target antigen. [Figure 8] Figures 5-9 show the results of phage ELISA of the solid-phase panning screening products of the phage display library, where red represents clones that simultaneously bind to both the PDL1 antigen and Fc, blue represents clones that bind only to the PDL1 target antigen and do not bind to Fc, and green represents clones that bind only to the PDL1 target antigen, do not bind to Fc, and have relatively strong binding to the PDL1 target antigen. [Figure 9] Figures 5-9 show the results of phage ELISA of the solid-phase panning screening products of the phage display library, where red represents clones that simultaneously bind to both the PDL1 antigen and Fc, blue represents clones that bind only to the PDL1 target antigen and do not bind to Fc, and green represents clones that bind only to the PDL1 target antigen, do not bind to Fc, and have relatively strong binding to the PDL1 target antigen.

[0067] [Figure 10] 10 and 11 show the results of flow cytometry analysis of the overlap products detected by transient transfection. [Figure 11] 10 and 11 show the results of flow cytometry analysis of the overlap products detected by transient transfection.

[0068] [Figure 12] Figures 12 to 14 show the binding of purified antibodies to the recombinant cell line CHO-K1 / PDL1 overexpressing PDL1, as detected by flow cytometry. [Figure 13] Figures 12 to 14 show the binding of purified antibodies to the recombinant cell line CHO-K1 / PDL1 overexpressing PDL1, as detected by flow cytometry. [Figure 14] Figures 12 to 14 show the binding of purified antibodies to the recombinant cell line CHO-K1 / PDL1 overexpressing PDL1, as detected by flow cytometry.

[0069] [Figure 15]FIG. 15 shows the results of detecting the PD-1-PDL1 blocking activity of monovalent antibodies. [Figure 16] FIG. 16 shows the results of detecting the PD-1-PDL1 blocking activity of bivalent antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Specific embodiments The present inventors have conducted extensive and in-depth research and conducted a large amount of screening, and for the first time have developed several nanoantibodies against PDL1 and bivalent antibodies constructed therewith. Specifically, in the present invention, llamas were immunized with human-derived PDL1 protein to obtain a gene library of high-quality immune nanoantibodies. Then, PDL1 protein molecules were coupled to a microplate, and using the principle of specific antigen-antibody binding, this form of antigen was used to screen the immune nanoantibody gene library (llama heavy chain antibody phage display gene library) by phage display technology to obtain genes of PDL1-specific nanoantibodies. In addition, from the relevant experimental results, the nanoantibodies against PDL1 obtained in the present invention and bivalent antibodies constructed therewith can effectively bind to PDL1 protein, and at the same time have blocking function and can block the interaction with its ligand PD-1. Based on this, the present invention has been completed. The nanoantibodies targeting PDL1 developed in the present invention are useful as a new therapeutic means for targeting and treating diseases such as gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, and lymphoma.

[0071] term As used herein, the terms "antibody of the present invention", "antibody of the present invention", "nanobody against PDL1 of the present invention", "nanobody against PDL1 of the present invention", "nanobody against PDL1", and "nanobody against PDL1" have the same meaning and can be used interchangeably, and all refer to an antibody that specifically recognizes and binds to PDL1 protein (including human PDL1 protein).

[0072] The antibody numbers and corresponding SEQ ID NOs of the Nanobodies of the invention are given in Table 1 below. [Table 1] Note: Each number in the table represents a sequence number, i.e., "1" represents "SEQ ID NO: 1," and the sequence numbers of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4 shown in the table are the numbers of the amino acid sequences.

[0073] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons with similar structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains depends on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by multiple constant regions. Each light chain has a variable region (VL) at one end followed by a constant region at the other end. The constant region of the light chain faces the first constant region of the heavy chain, and the variable region of the light chain faces the variable region of the heavy chain. Special amino acid residues form an interface between the light and heavy chain variable regions.

[0074] As used herein, the terms "single domain antibody", "VHH", "nanobody", "single domain antibody (adAb, or nanobody)" have the same meaning and can be used interchangeably and refer to the variable region of the heavy chain of a cloned antibody, and a single domain antibody (VHH) consisting of only one heavy chain variable region is constructed and is the smallest fully functional antigen-binding fragment. Typically, an antibody lacking the natural light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the heavy chain of the antibody is further cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0075] As used herein, the term "variable" refers to the fact that certain portions of the variable regions of antibodies differ in sequence, which constitutes the binding and specificity of each particular antibody to its particular antigen. However, variability is not uniformly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. In the variable regions, the relatively conserved portions are called framework regions (FRs). Natural heavy and light chain variable regions each contain four FR regions that are essentially in a β-sheet structure, connected by three CDRs that form connecting loops, and sometimes in a partial β-sheet structure. The CDRs in each chain are in close contact with each other 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 region is not directly involved in binding of an antibody to an antigen, but exhibits different effector functions, such as participating in antibody-dependent cellular toxicity of the antibody.

[0076] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates of the antibodies or fragments thereof of the present invention conjugated to drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules.Furthermore, the present invention includes cell surface markers or antigens conjugated to the nanobody or fragments thereof against PDL1.

[0077] As used herein, the terms "heavy chain variable region" and "VH" can be used interchangeably. As used herein, the terms "variable region" and "Complementarity determining region (CDR)" can be used interchangeably.

[0078] In one preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions: CDR1, CDR2, and CDR3. In one preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region.

[0079] In the present invention, the terms "antibody of the present invention", "protein of the present invention" or "polypeptide of the present invention" can be used interchangeably and all refer to a polypeptide that specifically binds to the PDL1 protein, such as a protein or polypeptide having a heavy chain variable region, which may or may not contain an initial methionine.

[0080] The invention also provides other proteins or fusion expression products of the antibodies of the invention, specifically, the invention includes any protein or protein complex and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region similar to or having at least 90%, preferably at least 95%, homology with the heavy chain variable region of an antibody of the invention.

[0081] In general, the antigen-binding properties of an antibody are characterized by three specific regions located in the heavy chain variable region, called the variable region (CDR), which are divided into four framework regions (FR), the amino acid sequences of which are relatively conserved and not directly involved in binding reactions. These CDRs form a ring structure, in which the FRs form a β-sheet that brings them into spatial proximity, and the CDRs in the heavy chain and the corresponding CDRs in the light chain constitute the antigen-binding site of the antibody. Comparison of the amino acid sequences of similar antibodies can identify which amino acids constitute the FR or CDR region.

[0082] The heavy chain variable region of the antibody of the present invention is of particular interest because at least a portion of it is involved in antigen binding. Therefore, molecules having a heavy chain variable region of a CDR-containing monoclonal antibody are included in the present invention as long as the CDRs have 90% or more (preferably 95% or more, most preferably 98% or more) homology with the CDRs identified herein.

[0083] The present invention includes not only complete antibodies, but also immunologically active antibody fragments or fusion proteins consisting of antibodies and other sequences, and therefore further includes fragments, derivatives and analogs of said antibodies.

[0084] As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that maintains essentially the same biological function or activity as an antibody of the invention. A fragment, derivative, or analog of a polypeptide of the invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted, whether or not such substituted amino acid residues are encoded by the genetic code, or (ii) a polypeptide in which one or more amino acid residues have been substituted, or (iii) a polypeptide in which the mature polypeptide has been fused to another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iiiV) a polypeptide in which an additional amino acid sequence has been fused to the polypeptide (e.g., a leader sequence, a secretory sequence, a sequence for purifying the polypeptide, a protein precursor sequence, or a fusion protein formed with a 6His tag). Based on the disclosure of the present specification, these fragments, derivatives, and analogs are within the scope of what is known to those of skill in the art.

[0085] The antibody of the present invention refers to a polypeptide comprising the above CDR region, which has an activity of binding to PDL1 protein. The term further includes mutated forms of the polypeptide comprising the above CDR region, which has the same function as the antibody of the present invention. These mutated forms include, but are not limited to, deletion, insertion and / or substitution of one or more amino acids (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and addition of one or more amino acids (usually 20 or less, preferably 10 or less, more preferably 5 or less) to the C-terminus and / or N-terminus. For example, in the art, substitution with an amino acid having a close or similar function usually does not change the function of the protein. Furthermore, addition of one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. The term further includes active fragments and active derivatives of the antibody of the present invention.

[0086] Variants of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency, and polypeptides or proteins obtainable with antisera against the antibody of the present invention.

[0087] The present invention also provides fusion proteins comprising other polypeptides, such as antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention further includes fragments of the antibodies of the present invention. Typically, the fragments comprise at least about 50 contiguous amino acids of an antibody of the present invention, 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.

[0088] In the present invention, a "conservative variant of the antibody of the present invention" refers to a polypeptide in which, compared to the amino acid sequence of the antibody of the present invention, 10 or less, preferably 8 or less, more preferably 5 or less, and most preferably 3 or less amino acids have been substituted with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution as shown in Table 2. [Table 2]

[0089] Furthermore, the present invention provides a polynucleotide molecule encoding the above-mentioned antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

[0090] A polynucleotide encoding a mature polypeptide of the present invention includes a coding sequence encoding only the mature polypeptide, a coding sequence for the mature polypeptide and various additional coding sequences, a coding sequence for the mature polypeptide (and any additional coding sequences) and non-coding sequences.

[0091] The term "polynucleotide encoding a polypeptide" may be a polynucleotide that encodes the polypeptide, or it may further comprise additional coding and / or non-coding sequences.

[0092] The present invention further relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% homology between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to (1) low ionic strength and high temperature, such as 0.2xSSC, 0.1% SDS, hybridization and elution at 60°C, or (2) the inclusion of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll at 42°C, or (3) hybridization only when the homology between the two sequences is at least 90%, preferably 95%. And the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0093] The full length nucleotide sequence of the antibody of the present invention or a fragment thereof can usually be obtained by PCR amplification, recombinant techniques or artificial synthesis. Applicable methods, especially when the fragment length is short, are to synthesize the relevant sequence by artificial synthesis. Usually, multiple small fragments are first synthesized and then concatenated to obtain a long fragment of the sequence. The coding sequence of the heavy chain can also be fused together with an expression tag (e.g., 6His) to form a fusion protein.

[0094] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant techniques, typically by cloning the sequence into a vector, introducing it into a cell, and isolating the relevant sequence from the host cell which has been grown in the usual manner. Biological molecules (nucleic acids, proteins, etc.) according to the present invention include biological molecules that exist in isolated form.

[0095] At present, it is already possible to obtain the DNA sequence encoding the protein of the present invention (or a fragment or derivative thereof) entirely by chemical synthesis. Furthermore, this DNA sequence may be introduced into various known DNA molecules (or vectors, etc.) or cells well known in the art. Mutations may also be introduced into the protein sequence of the present invention by chemical synthesis.

[0096] The present invention further relates to vectors containing the appropriate DNA sequences as described above and appropriate promoter or control sequences, which can be used to transform an appropriate host cell so as to express the protein.

[0097] 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, typically including bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, insect cells such as fruit fly S2 or Sf9, and animal cells such as CHO, COS7, and 293 cells.

[0098] Transformation of host cells by DNA recombination may be performed by conventional techniques familiar to those skilled in the art. When the host is a prokaryotic cell, such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the process used being well known in the art. Another method uses MgCl2. Optionally, transformation may be by electroporation. When the host is a eukaryotic organism, conventional mechanical methods such as calcium phosphate precipitation, microinjection, electroporation, and DNA transfection methods such as lipofection are used.

[0099] The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, a conventional medium may be selected as the medium for culture. The culture is performed under conditions suitable for the growth of the host cells. When the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cells are further cultured.

[0100] The recombinant polypeptide in the above method can be expressed intracellularly or in the cell membrane, or can be secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various isolation methods based on its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out), centrifugation, osmotic shock, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques, as well as combinations of these methods.

[0101] The antibodies of the present invention may be used alone, or may be conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modulating moiety, or any combination thereof.

[0102] Detectable markers used for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes that produce a detectable product. Therapeutic agents that can be conjugated or complexed with the antibodies of the present invention include, but are not limited to, 1. radionuclides, 2. biotoxins, 3. cytokines, such as IL-2, 4. gold nanoparticles / nanorods, 5. viral particles, 6. liposomes, 7. magnetic nanoparticles, 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.

[0103] PDL1 protein Programmed cell death 1 ligand 1 (PDL1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a human protein encoded by the CD274 gene. PDL1 is a type I transmembrane protein with a size of 40 kDa, which has been implicated in the suppression of the immune system in some special cases (e.g., pregnancy, tissue transplantation, autoimmune diseases, and certain diseases such as hepatitis). Normally, the immune system responds to foreign antigens that accumulate in lymph nodes or spleen, promoting the development of antigen-specific cytotoxic T cells (proliferation of CD8+ T cells). Meanwhile, programmed cell death receptor 1 (PD-1) can transmit an inhibitory signal when bound to PDL1, reducing the proliferation of CD8+ T cells in lymph nodes, and PD-1 can suppress the recruitment of antigen-specific T cells in lymph nodes by regulating the Bcl-2 gene.

[0104] PD-1 / PDL1 The binding of PDL1 and PD-1 acts as an inhibitory signal that mediates T cell activation, suppresses the killing function of T cells, and plays a negative regulatory role in the body's immune response.

[0105] Drug Composition The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or fusion protein and a pharma- ceutically acceptable carrier. Usually, these substances are formulated in a non-toxic, inert, pharma- ceutically acceptable aqueous carrier, and the pH value is usually about 5-8, preferably about 6-8, depending on the nature of the substance to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by any conventional route, including, but not limited to, intraperitoneal, intravenous, or topical administration.

[0106] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned antibody (or its complex) of the present invention and a pharma- ceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof. The drug formulation corresponds to the administration form. The pharmaceutical composition of the present invention may be an injection, which can be prepared by a conventional method, for example, in physiological saline or an aqueous solution containing glucose and other auxiliary agents. The pharmaceutical composition is prepared under sterile conditions in the case of an injection or solution. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight daily. The polypeptide of the present invention can also be used in combination with other therapeutic agents.

[0107] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, which is usually at least about 10 μg / kg body weight, and in most cases is less than about 50 mg / kg body weight, and preferably the dosage is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage should further take into account factors such as the mode of administration and the health condition of the patient, all of which are within the skill of a skilled physician.

[0108] Nanobody against PDL1 In the present invention, said Nanobodies against PDL1 include monomers, dimers (bivalent antibodies), tetramers (tetravalent antibodies) and / or multimers (multivalent antibodies). In one preferred embodiment of the present invention, said Nanobody against PDL1 comprises a VHH chain having one or more of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.

[0109] Labeled antibodies In one preferred embodiment of the present invention, the antibody has a detectable marker, more preferably the marker is selected from the group consisting of an isotope, a colloidal gold marker, a colored marker or a fluorescent marker.

[0110] Colloidal gold markers may be performed by methods known to those skilled in the art. In one preferred embodiment of the present invention, an antibody against the PDL1 protein is labeled with colloidal gold to obtain an antibody labeled with colloidal gold. The nanobodies against PDL1 of the present invention are capable of effectively binding to the PDL1 protein.

[0111] Detection Method The present invention also relates to a method for detecting PDL1 protein or fragments thereof, which essentially comprises the steps of obtaining a cell and / or tissue sample, lysing the sample in a medium, and detecting the level of PDL1 protein in the lysed sample. In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells present in a cell preservation solution.

[0112] kit The present invention also provides a kit containing the antibody (or fragment thereof) or cassette of the present invention, and in a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer solutions, etc.

[0113] The present invention also provides a detection kit for detecting the level of PDL1 protein, which includes an antibody that recognizes the PDL1 protein, a decomposition medium for dissolving the sample, and general-purpose reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit may be an in vitro diagnostic device.

[0114] application As described above, the antibody of the present invention has broad biological and clinical application value, and its applications cover many fields such as diagnosis and treatment of diseases related to PDL1 protein, basic medical research, biological research, etc. One preferred application is for clinical diagnosis, prevention and treatment of PDL1 protein.

[0115] The present invention also provides a method of stimulating an immune response by T cells targeting a mammalian tumor cell population or tissue, comprising administering to a mammal a CAR-T cell of the present invention.

[0116] In one embodiment, the invention involves cell therapy in which a patient's autologous T cells (or allogeneic donor) are isolated, activated, and genetically modified to generate CAR-T cells, which are then infused back into the patient. This approach has a very low incidence of graft-versus-host reactions, and antigens are recognized by T cells without MHC restriction. Also, a single CAR-T can treat all cancers expressing that antigen. Unlike antibody therapy, CAR-T cells can replicate in the body, resulting in long-term tumor suppression durability.

[0117] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo proliferation and persist for months to years. CAR-mediated immune responses can also be part of an adoptive immunotherapy process, in which the CAR-T cells can induce a specific immune response against tumor cells that highly express the antigen recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention can induce a specific immune response against tumor cells that highly express PDL1.

[0118] Treatable cancers include non-vascularized or essentially non-vascularized tumors, and vascularized tumors. The types of cancers treated with the CAR of the present invention include, but are not limited to, gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, etc.

[0119] Generally, as described herein, the activated and expanded cells can be used to treat and prevent diseases such as tumors. Thus, the present invention provides a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-T cells of the present invention.

[0120] The CAR-T cells of the present invention can be administered alone or in combination with diluents and / or other components, such as IL-2, IL-17, or other cytokines or cell populations, as a pharmaceutical composition. Briefly, a pharmaceutical composition of the present invention comprises a target cell population as described herein, optionally in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients.

[0121] The pharmaceutical composition of the present invention can be administered in a form suitable for the disease to be treated (or prevented). The amount and frequency of administration will depend on factors such as the patient's illness and the type and severity of the patient's illness, or will be determined by clinical trials.

[0122] When referring to an "immunologically effective amount," "antitumor effective amount," "tumor suppression effective amount," or "therapeutic amount," the exact amount of the composition of the present invention to be administered will be determined by a physician (taking into account the age, weight, tumor size, degree of infection or metastasis, and individual differences in the disease of the patient (subject). The pharmaceutical composition comprising T cells described herein may be administered in an amount of up to 10 4 -10 9 Dosage of 10 cells / kg body weight, preferably 10 5 -10 7The T cells may be administered in doses of 100 cells / kg body weight (including all integer values ​​within the range). The T cell compositions may be administered at these doses several times. The cells may be administered by injection techniques known in immunotherapy (e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment plan for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient's disease status and adjusting the treatment accordingly.

[0123] The compositions may be administered to a subject by any convenient means, including aerosol, injection, oral, infusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the invention are preferably administered by intravenous injection. The T cell compositions may be injected directly into a tumor, lymph node or site of infection.

[0124] In one embodiment of the invention, cells activated and expanded by the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are used to administer to a patient in conjunction with (e.g., before, simultaneously, or after) any number of relevant therapeutic procedures, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir and interleukin-2, azacitidine (known as ARA-C), or treatment with natalizumab for MS patients or efalizumab for psoriasis patients, or other treatments for PML patients. In a further embodiment, the T cells of the invention can be combined with chemotherapy, radiation, immunosuppressive agents such as cyclosporine A, azathioprine, methotrexate, mycophenolate mofetil, or FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell compositions of the invention are administered to a patient in conjunction with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), and cyclophosphamide. For example, in one embodiment, a subject may undergo high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, after transplant, the subject receives an infusion of expanded immune cells of the invention. In another embodiment, the expanded cells are administered pre- or post-surgery.

[0125] The dosage of the above treatment administered to a patient will vary depending on the exact nature of the disease being treated and the recipient being treated. The dosage rate administered to a human can be implemented according to accepted practices in the art. Typically, a single treatment or course of treatment will be administered in the range of 1×10 5 pieces~1×10 10 The modified T cells of the present invention can be administered to a patient, for example, by means of intravenous infusion.

[0126] Reagents, materials and equipment Main Reagents Agar (Sigma, CAT# A1296), peptone (Sigma, CAT#93926), yeast extract (OXOID, CAT#: LP0021), sodium chloride (Aladdin, CAT#: C111533), potassium chloride (Aladdin, CAT#: P112133), magnesium sulfate (Kokuyo Pharmaceutical, CAT#: 10013018), magnesium chloride (Kokuyo Pharmaceutical, CAT#: 10012818), glucose (Seiko, CAT#: GT1991), SfiI (NEB, CAT#: R0123L), T4 DNA ligase (TaKaRa, CAT#: 2011A), PrimeScript (商標)II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#: 6210B), NuHi power mix (Shinkai Seibutsu, CAT#: NH9303), 3 M sodium acetate (pH 5.2-6) (Sigma, CAT#: 126-96-5), DNA fragment recovery kit (TakaRa, CAT#: 9761), gel recovery kit (Qiagen, CAT#: 28706), Amane Plasmid Maxi Kit (Amane, CAT#: DP117), 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 sensitive cells (iCarTab), pComF phage display vector (iCarTab), NHS-biotin (APExBIO, CAT#: A8002), HRP-streptavidin (Boster, CAT#: BA1088), streptavidin magnetic beads (NEB, CAT#:S14205), antibody affinity detection buffer: HBS-EP+ 10X (GE, Cat# BR100669), amino coupling kit (GE, Cat#BR100050), 10 mM glycine 2.5 (GE, Cat#BR100356), S series CM5 chip (GE, Cat#29149603), PBS (Gbico, CAT# 14190-250), DMEM (Gbico, CAT# 41965-062), RPMI 1640 (Gbico, CAT# 61870044), FBS (Gbico, CAT# 10099-141), genomic DNA purification kit (Lifetech, CAT# K0512), polybrene (Sigma, CAT# 107689-10G), LVtransm transfection reagent (iCarTab, Cat# LVTran100), lymphocyte isolation fluid (Stem Cell, CAT# 18051).

[0127] Main materials 50 mL Falcon centrifuge tubes (Corning, CAT# 352070), electroporation cups (Bio-Rad 0.2 cm), RNase free 1.5 mL EP tubes (QSP, CAT#: 509-GRD-Q), 200 μL RNase free PCR tubes (Axygen, PCR-02D-C), T125 shake flasks (Corning, CAT# 431143), 15 mL Falcon centrifuge tubes (Corning, CAT# 430052), 6-well plates (Corning, CAT# 3516), 96-well plates (Corning, CAT# 3365).

[0128] Main Facilities Electroporation device (Eppendorf Multiporator), centrifuge (Xiangyi H1650R), constant temperature incubator (Shanghai Jinghong DNP-9052), constant temperature shaking culture incubator (Langyue DZ-85A), clean bench (SUZHOU ANTAI AIR TECH CO.,LTD., SW-CJ-1FD), PCR device (Applied Biosystems ABI2720), safety cabinet (Haier, HR40-IIA2), flow cytometer (Thermo Attune Nxt flow cytometer), Thermo 3111 CO2 incubator, BiaCore T200.

[0129] Amino acid sequence SEQ ID NO:1 (Nanoantibody 17-A06) MAQVKLEESGGGLVQAGGSLRLSCVASGRSFITYAVGWFRQAPGKEREFVASINWSGAMTYYTDSVNGRFAISRDNAKNTVYLQMNNLKLEDTAVYYCASTISAVTPTNGYQNWGQGTQVTVSS SEQ ID NO:2 (Nanobody 17-C12) MAQVKLEESGGGLVQPGGSLTLSCAASGRTFGFYGWFRQAPGKEREFVAGITWGGSVTSYADSVKGRFTISRDNAKNTVYLQMNSLKPENTAVYYCARSPRVTTTPREFDVWGQGTQVTVSS SEQ ID NO:3 (Nanobody 17-A01) MAAVQLVDSGGGLVQPGGSLRLSCAASGRTFGFYGWFRQAPGKEREFVAAITWGGSAISYEDSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCARSTRVTTNPREYDYWGQGTQVTVSS SEQ ID NO:4 (Nanobody 17-G06) MAAVQLVESGGGLVQPGGSLRLSCAASGRTFGFYGWFRQAPGKEREFVAAITWGGSAISYDDSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCARSMRVTTNPREYDYWGQGTQVTVSS SEQ ID NO:5 (Nanobody 23-G3) MAAVQLVESGGGLVQAGGSLRLSCVASGRSFITYAVGWFRQAPGKEREFVASVNWSGAMTYYADAVNGRFTISRDNAKNTVYLQMNNLKLEDTAVYYCAATISAVTPTNGYQNWGQGTQVTVSS

[0130] SEQ ID NO:6 (Nanobody 29-H9) RLQLVESGGGLVQAGGSLRLSCAASGFTFSTYAMSWYRGVPEKERELVAFISSDGGGTTYRDSVKGRFTISRDNGKNTVYLQMNSLKPEDTGVYYCARGLAQIWGQGTQVTVSS SEQ ID NO:7 (CDR1 of nanobody 17-A06, 23-G3) GRSFITYA SEQ ID NO:8 (CDR2 of nanobody 17-A06) INWSGAMT SEQ ID NO:9 (CDR3 of nanobody 17-A06) ASTISAVTPTNGYQN SEQ ID NO: 10 (CDR1 of nanobodies 17-C12, 17-A01, 17-G06) GRTFGF

[0131] SEQ ID NO: 11 (CDR2 of Nanobody 17-C12) ITWGGSVT SEQ ID NO: 12 (CDR3 of Nanobody 17-C12) ARSPRVTTTPREFDV SEQ ID NO: 13 (CDR2 of nanobodies 17-A01, 17-G06) ITWGGSAI SEQ ID NO: 14 (CDR3 of nanobody 17-A01) ARSTRVTTNPREYDY SEQ ID NO: 15 (CDR3 of Nanobody 17-G06) ARSMRVTTNPREYDY

[0132] SEQ ID NO: 16 (CDR2 of nanobody 23-G3) VNWSGAMT SEQ ID NO: 17 (CDR3 of Nanobody 23-G3) AATISAVTPTNGYQN SEQ ID NO: 18 (CDR1 of nanobody 29-H9) GFTFSTYA SEQ ID NO: 19 (CDR2 of nanobody 29-H9) ISSDGGGT SEQ ID NO: 20 (CDR3 of nanobody 29-H9) ARGLAQI

[0133] SEQ ID NO: 21 (FR1 of nanobody 17-A06) MAQVKLEESGGGLVQAGGSLRLSCVAS SEQ ID NO: 22 (FR2 of nanobody 17-A06, 23-G3) VGWFRQAPGKEREFVAS SEQ ID NO: 23 (FR3 of nanobody 17-A06) YYTDSVNGRFAISRDNAKNTVYLQMNNLKLEDTAVYYC SEQ ID NO: 24 (FR4 of nanoantibodies 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, 29-H9) WGQGTQVTVSS SEQ ID NO: 25 (FR1 of nanobody 17-C12) MAQVKLEESGGGLVQPGGSLTLSCAAS

[0134] SEQ ID NO: 26 (FR2 of nanobody 17-C12) YGWFRQAPGKEREFVAG SEQ ID NO: 27 (FR3 of nanobody 17-C12) SYADSVKGRFTISRDNAKNTVYLQMNSLKPENTAVYYC SEQ ID NO: 28 (FR1 of nanobody 17-A01) MAAVQLVDSGGGLVQPGGSLRLSCAAS SEQ ID NO: 29 (FR2 of nanobodies 17-A01, 17-G06) YGWFRQAPGKEREFVAA SEQ ID NO: 30 (FR3 of nanobody 17-A01) SYEDSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC

[0135] SEQ ID NO: 31 (FR1 of Nanobody 17-G06) MAAVQLVESGGGLVQPGGSLRLSCAAS SEQ ID NO: 32 (FR3 of nanobody 17-G06) SYDDSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC SEQ ID NO: 33 (FR1 of nanobody 23-G3) MAAVQLVESGGGLVQAGGSLRLSCVAS SEQ ID NO: 34 (FR3 of nanobody 23-G3) YYADAVNGRFTISRDNAKNTVYLQMNNLKLEDTAVYYC SEQ ID NO: 35 (FR1 of nanobody 29-H9) RLQLVESGGGLVQAGGSLRLSCAAS

[0136] SEQ ID NO: 36 (FR2 of nanobody 29-H9) MSWYRGVPEKERELVAF SEQ ID NO: 37 (FR3 of nanobody 29-H9) TYRDSVKGRFTISRDNGKNTVYLQMNSLKPEDTGVYYC

[0137] The main advantages of the present invention are: 1. The PDL1 nanobody of the present invention can bind specifically to PDL1 with high affinity. 2. The PDL1 nanobody of the present invention has good PD-1-PDL1 blocking activity and high neutralizing activity against PDL1. 3. In the present invention, a monovalent PDL1 nanobody and a bivalent PDL1 nanobody are simultaneously constructed, both of which have obvious blocking activity. 4. The PDL1 nanobody of the present invention only blocks the PD-1 and PDL1 pathways, and does not affect the PD-1 and PD-L2 pathways, thereby avoiding side effects such as interstitial pneumonia and improving safety. 5. The PDL1 nanobody of the present invention can activate the immune system more comprehensively to kill tumors, and patients with PD-1 ineffectiveness or PD-1 drug resistance can obviously benefit and achieve therapeutic effects, thereby broadening the range of patients.

[0138] 6. The PDL1 nanobody of the present invention not only inhibits the PD-1-PDL1 pathway, but also blocks the co-inhibitory function of B7.1 and PDL1, which is beneficial to the overall activation of T cell function and cytokine production, thus achieving better immune efficacy. 7. The PDL1 nanobody of the present invention is cheaper and easier to mass produce than polyclonal antibodies, lowering production costs and thereby reducing the price of drugs produced therefrom. 8. The PDL1 nanobody of the present invention has a wide range of application, is stable over a wide temperature range, and can exert its function even at high temperatures. Unlike conventional antibody fragments, the unfolding of nanobodies at high temperatures has been proven to be fully reversible. Nanobodies are also stable at extreme pH values ​​and can survive in gastric juices. 9. In terms of structure, nanobodies have a hydrophilic aspect, so compared to conventional antibodies, the PDL1 nanobodies of the present invention do not suffer from the solubility and aggregation problems associated with conventional antibodies.

[0139] The present invention will be further described below with reference to specific examples. It is understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. Experimental methods for which no specific conditions are given in the following examples are generally performed according to standard conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommended conditions. Unless otherwise stated, percentages and parts are by weight. EXAMPLES

[0140] Example 1. Antigen production method 1. The sequence of the PDL1 extracellular domain (19AA-238AA) was prepared by gene synthesis, and an IgG1 Fc tag was added to the C-terminus. The sequence was then subcloned into a eukaryotic expression vector to construct an antigen PDL1-Fc protein expression vector. 2. Plasmid was extracted from the constructed PDL1-Fc protein expression vector, and then transiently transfected into 293 cells. The cells were then cultured for 8 days, centrifuged to collect the culture supernatant, which was then filtered through a 0.45 μm filter membrane and the filtrate was transferred to a sterile centrifuge tube and purified using a protein A column to obtain PDL1-Fc protein. 3. PDL1-Fc protein was identified by SDS-PAGE detection.

[0141] As shown in Figure 1, SDS-PAGE detection showed that the actual SDS-PAGE molecular weight of PDL1-Fc was approximately 65 kD, which was consistent with the electrophoretic detection results of ACRO's PDL1-Fc product, and the band purity of the PDL1-Fc of the present invention was high, indicating that the expression and purification effects were good. The purified PDL1-Fc protein is an antigen, which is also called an immunogen or an immunogen.

[0142] Example 2. Immunization of llamas with antigens One llama was immunized subcutaneously at multiple sites with the PDL1-Fc protein antigen produced above, and the three immunization series are shown in Table 3. [Table 3]

[0143] Example 3. Detection of immune titers 1.5 mL of peripheral blood was collected after the third immunization in Example 1, and the centrifuge tube in which the blood sample was collected was placed in an incubator at 37° C. for 1 hour, and then the blood sample was transferred to 4° C. and placed overnight. 2. The serum was transferred to a new sterile centrifuge tube and centrifuged at 5000 rpm for 20 min, and then anti-camel IgG(H+L) secondary antibody and anti-camel VHH secondary antibody were selected using a 96-well plate pre-coated with cleaved Fc antigen from PDL1, and ELISA experiments were performed to detect the immune titer.

[0144] As shown in the ELISA detection results in Figure 2, the immune sera were positive and had higher titers when detected with anti-camel IgG (H+L) secondary antibody compared with the non-immune (negative) sera.

[0145] The above results showed that the immune serum specifically bound to PDL1, and the OD450 value changed gradiently with the dilution gradient of the serum, indicating that the immunization was successful. 100 mL of peripheral blood was collected and prepared for library construction.

[0146] Example 4. Isolation of PBMCs and obtaining VHH antibody fragments 1.150 mL of peripheral blood prepared in Example 1 was collected, and PBMCs were separated using lymphocyte separation solution.

[0147] 2. Extract RNA and PrimeScript (商標) The cDNA was prepared by reverse transcription using the II 1st Strand cDNA Synthesis Kit. (1) Reaction mixture Mix 1 in Table 4 was prepared in a 200 μL PCR tube. [Table 4] (2) After incubation at 65°C for 5 minutes, the mixture was quickly cooled on ice. (3) The reaction mixture shown in Table 5 was prepared in the above PCR tube. [Table 5] (4) After mixing evenly by blowing, the mixture was divided into 80 μL / tube and placed in a PCR machine at 42°C for 1 hour, then heat inactivated at 70°C for 15 minutes. Finally, the cDNA samples were stored on ice or at -20°C for long periods.

[0148] 3. Amplification of VHH fragments (1) The first PCR reaction system (50 μL / tube) shown in Table 6 was prepared. [Table 6] After preparing the PCR reaction system, the PCR machine was set up with the program in Table 7. [Table 7] (2) Agarose gel electrophoresis of PCR products: The PCR products were analyzed by electrophoresis on a 1% agarose gel, and a single step with a molecular weight of about 750 bp was separated. The PCR products were recovered using a gel recovery kit, and the concentration was measured using a NanoDrop. (3) Prepare the second PCR reaction system (50 μL / tube) shown in Table 8. [Table 8] After preparing the PCR reaction system, the PCR machine was set up with the program in Table 9. [Table 9] (4) Agarose gel electrophoresis analysis of the second PCR products: The PCR products were analyzed by electrophoresis on a 1% agarose gel to separate the VHH fragments with a molecular weight of about 400 bp. The VHH PCR products were collected using a gel collection kit and the concentration was measured using a NanoDrop.

[0149] The results of agarose gel electrophoresis of the PCR products are shown in Figure 3. Figure 3A shows two PCR bands of about 1000 bp and 750 bp obtained in the first PCR, and the 750 bp fragment was recovered from the gel and used as a template for the second PCR, and Figure 3B shows a band of about 450 bp obtained in the second PCR, which was identified by agarose gel electrophoresis and proved to be a VHH fragment.

[0150] The VHH fragments were enzymatically digested with SfiI and then subcloned into the phage display vector pComF, and the ligation products were electrotransformed into SS320 E. coli responsive cells to construct a single domain antibody phage display library.

[0151] Example 5. Construction of a phage display library 1. Construction of phage display vector: (1) The pComF vector and the VHH PCR gel recovered product obtained in Example 4 above were each enzymatically digested with SfiI and incubated at 50° C. overnight. (2) The pCom F vector fragment was separated on a 1% agarose gel, and the 5000 bp vector fragment was excised and gel-collected. At the same time, the PCR enzyme cleavage product was purified using a DNA fragment collection kit, and the concentration was measured using a NanoDrop. (3) The enzymatically cleaved pCom F vector and the VHH fragment were ligated by T4 ligase and incubated at 16°C overnight.

[0152] 2. Electrotransformation of E. coli with the phage ligation products: (1) An electroporation cup, ligation products and electrotransformation-competent cells were prepared and pre-cooled on ice. (2) Take the pre-chilled library construction ligation products into electrotransformation-competent cells and place on ice for 1 min, then place 70 μL of DNA / competent cells mixture into each electroporation cup and place the electroporation cup on ice. (3) Electrical transformation was performed at 2500 V for 5 ms. (4) After the electric shock, the cells were immediately resuspended in SOC medium equilibrated to room temperature and cultured in a shaker at 37°C for 1 hour.

[0153] (5) 15 mL of the bacterial liquid was removed and used for phage rescue. The remaining 5 mL of the electrotransformation product was added to an equal volume of 50% glycerol, mixed uniformly, and stored at -80°C. (6) Separately, 20 μL of the bacterial liquid was taken and diluted with 980 μL of 2YT medium, and then 100 μL of the diluted product was taken and diluted a second time with 900 μL of 2YT medium. 50 μL was then evenly spread onto an ampicillin-containing LB plate and cultured overnight at 37°C. (7) The next day, the plates were removed, the number of clones obtained from each ligation was calculated, and the library volume was calculated. (8) At the same time, 20 single clones from the plate were inoculated into ampicillin-containing 2YT medium and cultured with shaking at 37°C for approximately 6-8 hours. The bacterial liquid was then sequenced (using the common sequencing primer M13R) to calculate the diversity of the library.

[0154] As shown in Figure 4 , the sequence alignment results of the phage display library showed that the blank rate and antibody overlap rate of the phage display library were good, and the capacity of the E. coli library was 6.53E8.

[0155] Example 6. Renaturation and rescue of phage display libraries and phage precipitation 1. The product after electrical transformation in Example 5 was diluted with 2YT to adjust the OD600 to about 0.2, ampicillin was added to a final concentration of 100 μg / mL, and the culture was placed in a thermostatic shaker at 37°C and 225 rpm. Culture was stopped when the OD600 reached 0.5. 2. M13KO7 was added and the mixture was shaken uniformly, then allowed to stand at 37°C for 30 minutes and further cultured at 37°C and 225 rpm for 1 hour. 3. M13KO7 volume = 10 x volume x OD600 x 5 x 10 8 / M13KO7 titer. 4. The bacterial solution was centrifuged at 6000 rpm for 10 min, then resuspended in 2YT-AK medium and cultured overnight at 25°C and 200 rpm. 5. The bacterial solution was centrifuged at 10,000 rpm for 15 minutes.

[0156] 6. The precipitate was discarded and the supernatant was transferred to a new centrifuge tube. PEG / NaCl (1 / 5 of the bacterial liquid volume) was added to the tube, mixed uniformly, and then allowed to stand at 4°C for 2 hours. 7. The precipitated phage supernatant was centrifuged at 10,000 rpm at 4° C. for 30 minutes, the supernatant was discarded, and the precipitate (phage) in each 50 mL centrifuge tube was resuspended in 1 mL of sterile PBS. 8. The resuspended phages were transferred to a 1.5 mL EP tube, placed in a centrifuge, and centrifuged at 12,000 g and 4° C. for 5 min. 9. The supernatant was transferred to a new 1.5 mL EP tube, 250 μL of PEG / NaCl was added to the tube, and after uniform mixing, the tube was allowed to stand at 4° C. for 10 minutes. 10. The mixture was centrifuged at 12,000 g for 10 minutes, the supernatant was discarded, and the mixture was resuspended in 1 mL of PBS.

[0157] 11. The mixture was centrifuged at 12,000 g for 5 minutes, the precipitate was discarded, and the supernatant was transferred to a new 1.5 mL EP tube. 12. The mixture was centrifuged at 12,000 g for 5 minutes, and the supernatant was transferred to a new 1.5 mL EP tube to obtain an initial phage library. 13. Take 10 μL of the precipitate and add it to 90 μL of 2YT medium. -1 Then, in order, -9 Dilute 10-fold to 10 -7 , 10 -8 , 10 -9 20 μL of the diluted sample from the three-stage gradient was added to 200 μL of previously prepared ER2738 with an OD600 of 0.5, mixed uniformly, placed in a water bath at 37°C, and left to stand for 10 min. Then, 108 μL of each was spread onto an LB-AMP solid plate and left at 37°C overnight. The next day, the colonies were counted to determine the titer. 14. Calculation of titer: Select plates with colony numbers between 30-300, take the average of the two plates, multiply the colony number by the dilution factor, and then multiply by 100 to obtain the titer. After renaturation, rescue and counting, the phage display library is ready for subsequent solution panning of the phage display library.

[0158] Example 7. Solution panning of phage display libraries Streptavidin magnetic beads were used to bind the pre-biotinylated target antigen, which was then incubated with the phage library to be screened, non-specifically bound phages were washed away, and recombinant phages bound to the target antigen were eluted with TEA and amplified. After 3-4 rounds of screening, single clones were selected for sequencing.

[0159] 1. Biotinylated target antigen was diluted with PBS to 50, 10, 5, and 5 μg / ml (different concentrations were used each time), mixed with streptavidin magnetic beads at 4°C, and incubated for 1 hour. Free target antigen was then removed by washing. 2. Approximately 6×10 11 Pfu 150 μL of library phages or previous amplification products were diluted to 600 μL and placed in ELISA wells pre-coated with Fc, and Fc removal was performed three times in total to remove Fc-bound phages. 3. The Fc-removed phages were mixed with streptavidin magnetic beads and incubated at 4°C for 1 h to remove streptavidin-bound phages. 4. The magnetic beads were discarded, and the phages were diluted to approximately 1 mL with 1% PBSA, mixed with the magnetic beads pre-bound with the target antigen in step 1, and incubated at 4° C. for 1 h.

[0160] 5. Aspirate the phages and wash the magnetic beads 3-5 times with PBS, 2-3 min each time. Simultaneously wash a 1.5 mL EP tube previously blocked with MPBS with PBS. 6. The phages bound to the magnetic beads were eluted with 600 μL of 1×TEA for 10 min, and the eluted product was transferred to a pre-blocked EP tube and neutralized with 300 μL of Tris-HCl. 7. Take 10 μL of the output product and add it to 90 μL of 2YT medium to make it 100, then add 10 -2 Dilute 10-fold to 10 1 , 100 , 10 -1 , 10 -2 20 μL of diluted sample from the four-step gradient was added to 200 μL of ER2738 (OD600 0.5) (10 1 20 μL of the undiluted product was added to the ER2738, mixed uniformly, and then placed in a water bath at 37°C for 10 min. 108 μL of the product was then spread onto an LB-AMP solid plate, incubated at 37°C overnight, and the colonies were counted the next day to determine the titer. 8. Calculation of titer: Select plates with colony counts between 30-300, take the average of the two plates, multiply the colony count by the dilution factor and then by the elution volume. The constructed phage display library was coated with Fc protein, depleted six times, and then incubated with streptavidin magnetic beads pre-bound with biotin-PDL1 to enrich for phages that specifically bound PDL1, and enriched four times. The input and output of each round were statistically calculated, and the enrichment factor of each round was calculated.

[0161] The calculation results shown in Table 10 indicate that the enrichment factor (input / output) showed clear enrichment of the phage library, indicating that a phage library that specifically binds to PDL1 was successfully constructed. [Table 10-1] [Table 10-2]

[0162] Example 8. Cell-based panning of phage display libraries Using a recombinant cell line overexpressing the target protein, the phage library was incubated with blank cells and cells overexpressing the target protein in sequence, and then non-specifically bound phages were removed by several washings. The recombinant phages bound to the cell surface were eluted with glycine or TEA and amplified. After 3-4 rounds of screening, single clones were selected for ELISA detection. 1. One day before, a 1.5 mL EP tube was blocked with 1% PBSA and left at 4°C overnight. 2. 1x10 target cells and control cells each 7 The cells were harvested, washed three times with PBS, resuspended in 10 mL of 1% PBSA, and blocked at low speed for 1 h at room temperature on a destaining shaker. 3. Control cells were incubated with 1 x 10 11 The phages were added and incubated for 1 h, and the target cells were then blocked.

[0163] 4. After incubation, place the cells in a centrifuge and centrifuge at 1000g for 5 min. Discard the target cell supernatant (1% PBSA), and carefully aspirate the control cell supernatant and place it in the target cell tube, resuspend, then place on a shaker and incubate at low speed for 1 h. 5. Place the target cells in a centrifuge and centrifuge at 1000g for 5 min (at the same time, wash the 1.5 mL EP tubes blocked one day earlier with PBS three times), discard the target cell supernatant, add 4 mL of PBS to resuspend, place into separate blocked 1.5 mL EP tubes, centrifuge with PBS and wash five times, then transfer the cells to another four EP tubes, wash five times, and then transfer the cells to the same EP tubes. 6. After resuspending the cells in 200 μL of PBS, add 200 μL of 2×TEA and quickly blow and suck until the solution becomes non-viscous. Then add 200 μL of Tris-HCl to neutralize the solution and obtain the product. 7. Measure the titer of the outlibrary, similar to steps 7 and 8 in the solution panning scheme of Example 7.

[0164] The phage library screening can be performed by selecting the method in Example 7 or Example 8 depending on the experimental conditions, but in the present invention, the method in Example 7 is preferably used.

[0165] Example 9. Phage ELISA The 1.2YT-Amp medium was divided into 96-well deep well plates, and 500 μL / well was used to pick single clones from the output plate and cultured at 37°C, 225 rpm until the OD600 reached 0.5. The last two wells, H11 and H12, were used as blank controls, containing only medium and no clones. 2. At the same time, antigen was coated onto ELISA plates in CBS at a concentration of 1 μg / mL, 100 μL / well, and coated at 37℃ for 2h. 3. Separately, take one 96-well deep well plate, add 500 μL of 2YT-A medium to each well, and use a multichannel pipette to suck up 10 μL of bacterial solution with an OD600 of 0.5 into a new 96-well plate. Culture overnight at 37°C and 225 rpm to use as the sample bacterial solution for sequencing. 4. M13KO7 was added to a bacterial solution with an OD600 of 0.5, mixed uniformly, and then allowed to stand at 37°C for 15 minutes. 5. M13KO7 volume = 10 x volume x OD600 x 5 x 10 8 / M13KO7 titer.

[0166] 6. After the invasion, the bacterial solution was placed on a shaker and cultured at 37°C and 225 rpm for 45 min. 7. The bacterial solution was placed in a centrifuge and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded and the cells were resuspended in 2YT-AK medium at 800 μL / well. The cells were placed in a shaker and cultured overnight at 30° C. and 210 rpm. 8. At the same time, the antigen was discarded from the ELISA plate, and the plate was washed three times with PBST washing solution, and then blocked with 3% MPBS at 250 μL / well and left overnight at 4° C. Then, an extra blank plate was blocked to serve as a blank. 9. The next day, the 96-well deep well plate was placed in a centrifuge and centrifuged at 4000 rpm for 10 min. The milk in the ELISA plate was discarded and washed 4 times with 200 μL of PBST. 50 μL of PBST was added to each well first, and then 50 μL of the phage supernatant after centrifugation was added correspondingly, and incubated at 4°C for 1 h. The supernatant was discarded and washed 5 times with PBST. HRP-anti-M13 secondary antibody was diluted with PBST, incubated at 4°C for 45 min with 100 μL / well, and then the secondary antibody was washed and removed, washed 5 times with PBST, colored with TMB at room temperature for 10 min, stopped with hydrochloric acid, and the value was read, and the clone with the highest S / N ratio was selected, and the preserved bacterial liquid was used for measurement.

[0167] The second and third output products in Example 7 were selected for phage ELISA detection. They were coated with streptavidin, biotin-PDL1-Fc and Fc protein, respectively, and the recombinant phage supernatant was taken for ELISA detection. The control group was a well plate that was simply blocked.

[0168] As shown in Figure 5-9, the ELISA detection results of the second and third output products, in which the red marks represent clones that simultaneously bind to both PDL1 antigen and Fc, the blue marks represent clones that only bind to PDL1 target antigen and do not bind to Fc, and the green marks represent clones that only bind to PDL1 target antigen and do not bind to Fc, and have relatively strong binding to PDL1 target antigen (i.e., clones to be subjected to sequencing). Among them, clones with an S / N ratio of 4 or more and that do not bind to Fc were selected and sequenced to analyze the antibody sequences.

[0169] Upon analysis, solution panning with streptavidin magnetic beads and biotinylated antigen yielded a total of six different antibody sequences, namely 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, and 29-H9 of the present invention.

[0170] The bacterial suspension of the target clone was taken and amplified by PCR, a signal peptide was added to the N-terminus of the VHH, and IgG1-Fc was added to the C-terminus. The PCR product was transiently transfected into HEK293 cells, and the expressed antibody supernatant was taken for FACS detection.

[0171] Example 10. Amplification, transient transfection and detection of overlapping PCR products 1. First PCR: Amplification of CMV, VHH and Fc (1) A PCR reaction system [50 μL / reaction] as shown in Table 11 was prepared (multiple CMV and Fc fragments may be produced) (CMV fragment amplification primers: CMV-F and PCom-R1; VHH fragment amplification primers: PCom-F1 and PCom-R2; Fc fragment amplification primers: PCom-F2 and PGK-R). [Table 11] Here, the PCR reaction program is as follows: 95℃ 10 minutes 95℃ 15 seconds 56℃ 30 seconds 25 cycles 68℃ 60 seconds 68℃ 10 minutes (2) 50 μL of the PCR product was taken, 1 / 10 volume of 10x buffer solution was added, and analyzed by electrophoresis on a 1% agarose gel. The bands of CMV and Fc were approximately 750 bp in size, and the band of VHH was approximately 560 bp in size. (3) The target band was excised from the gel, and the PCR product was purified and its concentration was measured using a NanoDrop (if the concentration was too high, it was diluted before subsequent reactions).

[0172] 2. Second PCR: Linking CMV, VHH and Fc by overlap expression PCR (1) A PCR reaction system as shown in Table 12 was prepared. [Table 12] Here, the PCR reaction program is as follows: 95℃ 10 minutes 95℃ 15 seconds 60℃ 30sec 15 cycles 68℃ 120 seconds

[0173] (2) After the PCR reaction program was completed, 2 μL each of primers CMV-F and PGK-R was added, and the following PCR reaction program was performed. The program for the following PCR reaction is as follows: 95℃ 15 seconds 60℃ 30 seconds 20 cycles 68℃ 120 seconds 68℃ 10 minutes (3) The overlap PCR product was purified using TakaRa's DNA fragment recovery kit, and the concentration was measured using NanoDrop. At least 10 μg of PCR product was required for the subsequent cell transfection verification. (4) The transfection process is similar to that of eukaryotic expression vectors.

[0174] Example 11. Transient transfection and expression of single domain antibodies 1. Take out the LVTransm transfection reagent and antibody expression vector pcDNA3.4-hIgG1-Fc2 or overlap PCR product from the refrigerator, thaw at room temperature, and mix thoroughly and homogenously by pipetting up and down. Take out the PBS buffer and warm it to room temperature. Put 500 μL of PBS into one well of a 24-well plate, add 4 μg of pcDNA3.4-hIgG1-Fc2, mix thoroughly and homogenously by pipetting up and down, then add 12 μL of LVTransm, immediately mix thoroughly and homogenously by pipetting up and down, and leave at room temperature for 10 minutes. This mixture is called the DNA / LVTransm complex.

[0175] 2. Add 532 μL of the DNA / LVTransm complex to 1.5 mL of 293F cells and gently shake to mix thoroughly and homogenously. Place the cells in a 37℃, 5% CO2 incubator and culture at 130 RPM for 6-8 hours. Then add 1.5 mL of fresh 293 medium and return the cells to the incubator for further culture. 3. After 3 consecutive days of culture, the culture supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube for subsequent flow cytometry and ELISA detection.

[0176] Example 12. Construction of VHH eukaryotic expression vectors According to the results of ELISA detection of the phage single clones in Example 9, positive clones were selected and sequenced to obtain the sequences of VHH antibodies. The sequences of the obtained VHH antibodies were analyzed and gene synthesis was carried out for each, which was then linked in series with human IgG1 Fc and subcloned into the expression vector pcDNA3.4-hIgG1-Fc2. After the vector was verified to be correct by sequencing, the endotoxin-removing plasmid was prepared by Qiagen Plasmid Maxi Kit for use.

[0177] Example 13. Detection of binding of recombinant antibodies to target proteins by flow cytometry 1. CHO-K1 and CHO-K1-PDL1 cell lines were regenerated from liquid nitrogen and the cells were adjusted to the logarithmic growth phase. 2. Divide each of the two types of cells into several groups, and the number of cells is 5 x 10 5 It was individual cells at a time. 3. The expressed antibodies were incubated with the target cells CHO-K1 and CHO-K1-PDL1, respectively, thoroughly mixed to homogeneity, and then incubated at room temperature for 1 hour. The cells were centrifuged at 4,800×g at room temperature for 5 minutes, the antibody-containing supernatant was discarded, and the cells were washed three times with PBS.

[0178] 5.1 μL of PE- or Alexa Fluor 488-labeled anti-human IgG was added, thoroughly mixed uniformly, and then incubated at room temperature for 30 minutes away from light. The cells were centrifuged at 6,800×g at room temperature for 5 minutes, the supernatant containing the secondary antibody was discarded, and the cells were washed three times with PBS. 7. The cells were resuspended in 500 μL of PBS and analyzed by flow cytometry.

[0179] A CMV promoter, a signal peptide, and a human IgG1 Fc tag were added to the N-terminus and C-terminus of the candidate antibody sequence by overlap PCR as described in Example 10, and the PCR products were purified and transiently transfected into 293 cells as described in Example 11, and the expressed antibodies were detected by flow cytometry.

[0180] As shown in Figures 10 and 11, the results of detection by FACS showed that 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, and 29-H9 were able to specifically bind to the CHO-K1 / PDL1 recombinant cell line, but did not or barely bound to CHO-K1. The sequence was synthesized and inserted into the expression vector pcDNA3.4-hIgG1-Fc2 for expression and purification of candidate antibodies.

[0181] Example 14. Antibody expression and purification 1. Take out the LVTransm transfection reagent and single-chain antibody expression vector from the refrigerator, thaw at room temperature, and then mix thoroughly and homogenously by blowing up and down with a pipette. Take out the PBS or HBSS buffer and warm it to room temperature. Put 2 mL of PBS into one well of a 6-well plate, add 130 μg of pcDNA3.4-hIgG1-Fc2, and mix thoroughly and homogenously by blowing up and down with a pipette. Then add 400 μL of LVTransm, immediately blow up and down with a pipette to mix thoroughly and homogenously, and leave it at room temperature for 10 minutes. 2. The above DNA / LVTransm complex was added to 50 mL of 293F cells and gently shaken to mix thoroughly and homogenously. The cells were placed in a 37℃, 5% CO2 incubator and cultured at 130 RPM for 6-8 hours, after which 50 mL of fresh 293 medium was added and the cells were returned to the incubator for further culture.

[0182] 3. After 7 days of continuous culture, the culture supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane, the filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a protein A column. Expression vectors for candidate PDL1 antibodies (i.e., 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, 29-H9) were transiently transfected into 293F cells, transfection supernatants were collected, and candidate antibodies were purified with Protein A. Candidate antibodies were diluted 5-fold starting from 10 μg / mL in a 4-step gradient, and the binding of the purified antibodies to the recombinant cell line CHO-K1 / PDL1, which overexpresses PDL1, was detected using a flow cytometer. The flow cytometry results shown in Figures 12-14 indicate that the binding of the candidate antibodies to the overexpressing cell lines changed with decreasing concentration of the candidate antibodies, and it can be seen that the purified 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, and 29-H9 antibodies can all specifically bind to PDL1-overexpressing cell lines.

[0183] Example 15. Detection of PD-1-PDL1 blocking activity Jurkat-PD-1-NFAT-Luc reporter gene cell line and aAPC CHO-PDL1 cells were regenerated and serially subcultured to logarithmic growth phase and cultured in 96-well plates at 2 × 10 4 Inoculate effector cells Jurkat-PD-1-NFAT-Luc at 1:1 with target cells aAPCCHO-PDL1 was added. Gradient-diluted detection antibody (positive antibody: Nivolumab; test antibody) was added to the corresponding wells, and the antibody was diluted in a 3-fold gradient, and then serially diluted in a 9-step gradient, with final concentrations of 30 μg / mL, 10 μg / mL, 3.333 μg / mL, 1.111 μg / mL, 0.3704 μg / mL, 0.1235 μg / mL, 0.04115 μg / mL, 0.01372 μg / mL, and 0.004572 μg / mL, respectively, and then added to the corresponding wells. After 18 h of co-culture, 25 μL of One-Glo reagent was added to each well, and the luciferase activity in the wells was detected using a Tecan M1000pro microplate reader.

[0184] As shown in Figure 15, the positive control nivolumab was able to block the PD-1 / PDL1 interaction, with an EC50 of 0.3217 μg / mL. Among the tested antibodies, the PDL1 single domain antibodies 17-A06, 23-G3, and 29-H9 were all able to block the PD-1 / PDL1 interaction, but the remaining three posterior antibodies did not have blocking function. However, the EC50 of the PDL1 candidate antibodies 17-A06, 23-G3, and 29-H9 was higher than that of the positive control antibody nivolumab, which was presumed to be because the single domain antibodies were small and the space occupied by the epitope was limited. The three antibodies with blocking function were randomly combined to construct a bivalent antibody and further detected the blocking activity.

[0185] The three candidate antibodies were randomly combined to construct bivalent antibodies, and the expression forms of the bivalent antibodies were 17-A06-G4S-23-G3, 17-A06-G4S-29-H9, and 23-G3-G4S-29-H9. After purification, the antibodies were expressed in Jurkat-PD-1-NFAT-Luc reporter gene cell line and aAPC The PD-1-PDL1 blocking activity of bivalent antibodies was detected in CHO-PDL1 cells.

[0186] As shown in Figure 16, the positive controls Nivolumab (PD-1 antibody, NIV20) and Atezolizumab (PDL1 antibody, Atezolizumab) were able to block the PD-1 / PDL1 interaction with comparable EC50. All of the bivalent PDL1 antibodies tested were able to block the PD-1 / PDL1 interaction, and the blocking ability of the bivalent antibodies was consistent with the positive controls.

[0187] Example 16. Detection of the affinity of recombinant antibodies PDL1 candidate antibodies (Fc tag) were conjugated to a Protein A probe at 5 μg / mL, and gradient-diluted PDL1-His was used as the mobile phase to give final concentrations of 2.6 μg / mL, 1.3 μg / mL, 0.65 μg / mL, 0.325 μg / mL, and 0.1625 μg / mL, respectively, and affinity detection was performed with OCTET R2.

[0188] Affinity detection was performed on the three bivalent PDL1 antibodies using OCTET R2, and the results of the affinity detection are shown in Table 13. [Table 13] The results showed that all three bivalent antibodies had high affinity, with the highest affinity being 4.198 × 10 -9 M, 2.729×10 -9 M, 1.915×10 -9 It was shown that M.

[0189] All documents related to the present invention are incorporated herein by reference as if each document were incorporated individually. After reading the above content of the present invention, it will be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalent forms thereof are within the scope of the claims of the present invention.

Claims

1. A Nanobody against PDL1, characterized in that the complementarity determining regions CDRs of the VHH chain of said Nanobody against PDL1 are one or more selected from the group consisting of: (1) CDR1 as set forth in SEQ ID NO: 7, CDR2 as set forth in SEQ ID NO: 8, and CDR3 as set forth in SEQ ID NO: 9; (2) CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11, and CDR3 represented by SEQ ID NO: 12; (3) CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 13, and CDR3 represented by SEQ ID NO: 14; (4) CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 13, and CDR3 represented by SEQ ID NO: 15; (5) CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 16, and CDR3 represented by SEQ ID NO: 17; (6) CDR1 represented by SEQ ID NO: 18, CDR2 represented by SEQ ID NO: 19, and CDR3 represented by SEQ ID NO:

20.

2. The Nanobody against PDL1 according to claim 1, characterized in that the VHH chain of the Nanobody against PDL1 further comprises a framework region FR, said framework region FR being one or more selected from the group consisting of: (1) FR1 represented by SEQ ID NO: 21, FR2 represented by SEQ ID NO: 22, FR3 represented by SEQ ID NO: 23, and FR4 represented by SEQ ID NO: 24; (2) FR1 represented by SEQ ID NO: 25, FR2 represented by SEQ ID NO: 26, FR3 represented by SEQ ID NO: 27, and FR4 represented by SEQ ID NO: 24; (3) FR1 represented by SEQ ID NO: 28, FR2 represented by SEQ ID NO: 29, FR3 represented by SEQ ID NO: 30, and FR4 represented by SEQ ID NO: 24; (4) FR1 represented by SEQ ID NO: 31, FR2 represented by SEQ ID NO: 29, FR3 represented by SEQ ID NO: 32, and FR4 represented by SEQ ID NO: 24; (5) FR1 represented by SEQ ID NO: 33, FR2 represented by SEQ ID NO: 22, FR3 represented by SEQ ID NO: 34, and FR4 represented by SEQ ID NO: 24; (6) FR1 represented by SEQ ID NO: 35, FR2 represented by SEQ ID NO: 36, FR3 represented by SEQ ID NO: 37, and FR4 represented by SEQ ID NO:

24.

3. The nanobody against PDL1 according to claim 1, characterized in that the amino acid sequence of the VHH chain of the nanobody against PDL1 is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a combination thereof.

4. An antibody against PDL1, characterized in that it comprises one or more nanobodies against PDL1 according to claim 3.

5. The antibody against PDL1 according to claim 4, characterized in that the antibody comprises a monomeric, bivalent and / or multivalent antibody.

6. A polynucleotide encoding a protein selected from the group consisting of the nanobody against PDL1 according to claim 1 or the antibody against PDL1 according to claim 4.

7. An expression vector comprising the polynucleotide according to claim 6.

8. A host cell comprising the expression vector according to claim 7 or having the polynucleotide according to claim 6 integrated into its genome.

9. 1. A method for producing a Nanobody against PDL1, characterized in that the method comprises the steps of: (a) obtaining a culture comprising a Nanobody against PDL1 by culturing a host cell according to claim 8 under conditions suitable for the production of the Nanobody; (b) isolating and / or recovering said Nanobody against PDL1 from said culture; and (c) Optionally, the Nanobody against PDL1 obtained in step (b) is purified and / or modified.

10. An immunoconjugate characterized in that it comprises: (a) a nanobody against PDL1 according to claim 1 or an antibody against PDL1 according to claim 4; and (b) a conjugate moiety selected from the group consisting of a detectable marker, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a magnetic nanoparticle, a viral coat protein or a VLP, or a combination thereof.

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