Anti-pd-l1 nanobody and preparation method and application thereof

Abstract

The application discloses an anti-PD-L1 nanobody and a preparation method and application thereof. The anti-PD-L1 nanobody is prepared through screening, has high affinity and specificity, can efficiently target PD-L1, has a simple structure, is easy to prepare, and can further prepare a PD-L1 binding molecule and an immunoconjugate, and has a wide application prospect in the fields of immunodetection and tumor treatment.

Description

[0001] This application claims priority to patent application number 202310427416.0 (the earlier application was filed on April 20, 2023, and is entitled "An anti-PD-L1 nanobody and its preparation method and application"). Technical Field

[0002] This invention belongs to the field of biotechnology and relates to an anti-PD-L1 nanobody, its preparation method, and its application. Background Technology

[0003] Programmed cell death 1 ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a human protein encoded by the CD274 gene. It is a type I transmembrane protein with a molecular weight of 40 kDa and belongs to the B7 family.

[0004] Studies have found that PD-L1 protein is mainly expressed in antigen-presenting cells (APCs), activated T cells, B cells, macrophages, placental trophoblast cells, cardiac endothelial cells, and thymic cortical epithelial cells. PD-L1 expression can be detected in various human tumor tissues, and the expression level is higher than in normal tissues. After the T cell surface receptor PD-1 binds to its ligand PD-L1, it recruits protein tyrosine phosphatase-1 (SHP-1) and SHP-2, generating a blocking signal that inhibits the phosphorylation of downstream effector PI3K / Akt pathways and the activation of mTOR and ERK2, while promoting CD4+ activation. + FoxP3 - T cells convert CD4 + FoxP3 + Treg differentiation. In short, increased PD-L1 expression on the surface of tumor cells in the tumor microenvironment, when combined with PD-1 on the surface of activated T cells, transmits negative regulatory signals, leading to apoptosis or immunodeficiency of tumor antigen-specific T cells, thereby suppressing the immune response. Currently, it has been found that blocking the PD-1 / PD-L1 signaling pathway with antibodies can restore the killing ability of T cells against tumor cells, helping cancer patients clear cancer cells with their own immunity. Antibodies targeting the PD-1 / PD-L1 signaling pathway have broad application prospects in the field of tumor treatment.

[0005] Nanobodies were first reported by Belgian scientists in the journal Nature in 1993. A naturally occurring antibody lacking the light chain exists in the peripheral blood of alpacas. This antibody contains only a single heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. The VHH structure, cloned and expressed independently, exhibits structural stability and antigen-binding activity comparable to the original heavy chain antibody, and is currently the smallest known unit capable of binding to target antigens. VHH crystals are 2.5 nm in size, 4 nm in length, and have a molecular weight of only 15 kDa, hence the name nanobody (Nb). Nanobodies offer advantages such as small molecular weight, easy penetration into dense tissues and even the blood-brain barrier; high affinity, strong specificity, good solubility and stability; weak immunogenicity in humans and good biocompatibility; high expression in prokaryotic or eukaryotic systems, making them easy to produce; and simple Nb structure, making them easy to engineer.

[0006] In conclusion, the development of novel nanobodies targeting PD-L1 is of great significance to the field of tumor therapy. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides an anti-PD-L1 nanobody, its preparation method, and its applications. This invention screens and prepares anti-PD-L1 nanobodies with high affinity, which have broad application prospects in fields such as immune detection and tumor treatment.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides an anti-PD-L1 nanobody, wherein the complementarity-determining region of the VHH chain of the anti-PD-L1 nanobody is selected from any one of the following groups:

[0010] (1) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively;

[0011] (2) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively;

[0012] (3) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively;

[0013] (4) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0014] In this invention, anti-PD-L1 nanobodies are screened. These anti-PD-L1 nanobodies only include the heavy chain variable region, which has high affinity and specificity, can efficiently target PD-L1, have a simple structure, are easy to prepare, and have broad application prospects in fields such as immune detection and tumor treatment.

[0015] SEQ ID NO:1: GFTVSNSAVS.

[0016] SEQ ID NO:2: LIDSNRNTV.

[0017] SEQ ID NO:3: KGIWGSTL.

[0018] SEQ ID NO:4: GSIFRITVA.

[0019] SEQ ID NO:5:VITDNGSTD.

[0020] SEQ ID NO:6:SRARPVRWT.

[0021] SEQ ID NO:7: GFTFSTSFVS.

[0022] SEQ ID NO:8: RISPGGASTS.

[0023] SEQ ID NO:9: REIYSGRFSGFDY.

[0024] SEQ ID NO:10: GIISDINMVA.

[0025] SEQ ID NO:11: KIFRGGSTY.

[0026] SEQ ID NO: 12: ARIQIRADDYSDY.

[0027] Understandably, on the other hand, this invention claims protection for the heavy chain variable region VHH of the nanobody, wherein the VHH complementarity-determining region is selected from any one of the following groups:

[0028] (1) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively;

[0029] (2) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively;

[0030] (3) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively;

[0031] (4) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0032] It is understood that any derivative sequence that retains the ability to bind to PD-L1, based on any of the above-mentioned amino acid sequences, by selectively adding, deleting, modifying and / or substituting at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid, is within the scope of protection of this invention.

[0033] In one specific embodiment of the present invention, the derived sequence, which has been modified by adding, deleting, altering and / or substituting at least one amino acid and is capable of retaining the ability to specifically bind to PD-L1, is an amino acid sequence with homology or sequence identity of at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0034] Preferably, the amino acid sequence of the VHH chain of the anti-PD-L1 nanobody is selected from any one of the sequences shown in SEQ ID NO:13-SEQ ID NO:16 or sequences with at least 80% homology thereto, including but not limited to amino acid sequences with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0035] SEQ ID NO:13:

[0036] QVQLQESGGGLVQPGGSLTLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNVKNTVYLQMNSLKPEDTAVYYCAKGIWGSTLTGQGTQVTVSS.

[0037] SEQ ID NO:14:

[0038] QVQLQESGGGLVQPGGSLRLSCAGSGSIFRITPVAWYRQAPEKQRELVAVITDNGSTDYGDFVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNSRARPVRWTWGQGTQVTVSS.

[0039] SEQ ID NO:15:

[0040] QVQLQESGGGLVQPGGSLRLSCAASGFTFSTSFVSWVRQAPGKGLEWVSRISPGGASTSYADSVKGRFTISRDNAENTLYLQMNSLKPEDTALYYCAREIYSGRFSGFDYWGQGTQVTVSS.

[0041] SEQ ID NO:16:

[0042] QVQLQESGGGLVQPGGSLRLSCAASGIISDINMVAWYRQAPGKQRELVAKIFRG GSTYYTDFVKGRFTISRENAKNTVYLQMNSLKPDDTAVYYCNARIQIRADRDYSDYW GQGTQVTVSS.

[0043] In a second aspect, the present invention provides a PD-L1 binding molecule, wherein the PD-L1 binding molecule contains the anti-PD-L1 nanobody described in the first aspect or the heavy chain variable region VHH.

[0044] In this invention, a PD-L1 heavy chain single-domain antibody (nanobody) with high affinity and specificity is screened and prepared. Based on this, various different PD-L1 binding molecules can be prepared, such as those linked with optional tag sequences for co-expression and / or purification; or they can be humanized to obtain humanized antibodies; or they can be used to prepare multispecific antibodies, including bivalent antibodies and / or multivalent antibodies, wherein the multispecific antibodies contain one or more of the anti-PD-L1 nanobodies and / or VHH domains described in the first aspect, and may also contain antibodies targeting any other antigen.

[0045] In one specific embodiment, the tag sequence includes any one or a combination of at least two of the following: Fc tag, HA tag, GGGS sequence, FLAG tag, Myc tag, or 6His tag.

[0046] In another specific embodiment, the PD-L1 binding molecule is a monomer, dimer, or polymer.

[0047] In a third aspect, the present invention provides a humanized antibody obtained from the nanobody described in the first aspect.

[0048] In another preferred embodiment, the humanized antibody has an amino acid sequence shown in any one of SEQ ID NO:23-42.

[0049] In another preferred embodiment, the humanized antibody has an amino acid sequence shown in any one of SEQ ID NO: 30, 33, 36, 40, 41.

[0050] In another preferred embodiment, the humanized antibody has the amino acid sequence shown in SEQ ID NO:30 or 40.

[0051] In a fourth aspect, the present invention provides a chimeric antigen receptor (CAR) containing an extracellular domain, the extracellular domain comprising an anti-PD-L1 nanobody as described in the first aspect of the present invention, an anti-PD-L1 binding molecule as described in the second aspect of the present invention, or a humanized antibody as described in the third aspect of the present invention.

[0052] In another preferred embodiment, the extracellular domain further includes a signal peptide.

[0053] In another preferred embodiment, the extracellular domain also includes other exogenous proteins.

[0054] In another preferred embodiment, the CAR has the structure shown in Formula Ia:

[0055] L-Nb-H-TM-C-CD3ζ(Ia)

[0056] In the formula,

[0057] L represents the absence of a signal peptide sequence;

[0058] Nb is a specific binding domain;

[0059] H represents the area with no hinge or no connection.

[0060] TM represents a transmembrane domain;

[0061] C is the co-stimulation signal structure domain;

[0062] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers);

[0063] The "-" indicates a linking peptide or peptide bond.

[0064] In another preferred embodiment, the L is selected from the signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or combinations thereof.

[0065] In another preferred embodiment, the Nb targets PD-L1.

[0066] In another preferred embodiment, the Nb is an anti-PD-L1 nanobody.

[0067] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0068] In another preferred embodiment, H is the human IgG1 Fc hinge region.

[0069] In another preferred embodiment, the TM is selected from the transmembrane regions of the following histones: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants / modified forms thereof, or combinations thereof.

[0070] In another preferred embodiment, C is selected from the co-stimulatory domains of the following histones: 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 mutants / modified forms thereof, or combinations thereof.

[0071] In another preferred embodiment, the CAR includes the intracellular domain of a cytokine.

[0072] In another preferred embodiment, the cytokines include: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), growth factors, chemokines, or combinations thereof; preferably, the cytokines are interleukins.

[0073] In another preferred embodiment, the CAR includes the intracellular domain of interleukin.

[0074] In another preferred embodiment, the interleukin is selected from the group consisting of IL-12, IL-2, IL-15, IL-21, or combinations thereof; preferably IL-12.

[0075] In a fifth aspect of the invention, a fusion protein is provided, the fusion protein having:

[0076] (Z1) First protein, the first protein comprising: an anti-PL-L1 nanobody as described in the first aspect of the present invention, an anti-PD-L1 binding molecule as described in the second aspect of the present invention, or a humanized antibody or its active fragment as described in the third aspect of the present invention.

[0077] (Z2) A second protein, which includes an Fc fragment or cytokine; and

[0078] (Z3) An optional linker located between the first protein and the second protein.

[0079] In another preferred embodiment, the cytokines include: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), growth factors, chemokines, or combinations thereof.

[0080] In another preferred embodiment, the second protein is interleukin.

[0081] In another preferred embodiment, the interleukin is selected from the group consisting of IL-12, IL-2, IL-15, IL-21, or combinations thereof.

[0082] In another preferred embodiment, the interleukin is IL-12.

[0083] In another preferred embodiment, the Fc fragment is the Fc fragment of human IgG.

[0084] In a sixth aspect, the present invention provides a nucleic acid molecule containing the nucleic acid encoding the anti-PD-L1 nanobody or the heavy chain variable region VHH described in the first aspect, the anti-PD-L1 binding molecule described in the second aspect, the humanized antibody or its active fragment described in the third aspect, the chimeric antigen receptor described in the fourth aspect, or the fusion protein described in the fifth aspect.

[0085] In one specific embodiment, the nucleic acid molecule may encode the anti-PD-L1 nanobody, the heavy chain variable region VHH, the PD-L1 binding molecule, the humanized antibody, the chimeric antigen receptor, or the fusion protein, and may be RNA, DNA, or cDNA.

[0086] In a seventh aspect, the present invention provides a recombinant expression vector containing the nucleic acid molecule described in the sixth aspect.

[0087] In one specific embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, or other gene transfer systems, or a combination of at least two of these. Preferably, the expression vector includes viral vectors, such as lentiviruses, adenoviruses, AAV viruses, or retroviruses, or a combination of at least two of these.

[0088] In another specific embodiment, the expression vector is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, or pComb3XSS, etc.

[0089] In another specific embodiment, the expression vector further includes a selection from the group consisting of promoters, transcriptional enhancement elements (WPREs), or long terminal repeat sequences (LTRs).

[0090] Eighthly, the present invention provides a recombinant cell containing the nucleic acid molecule described in the sixth aspect or the recombinant expression vector described in the seventh aspect.

[0091] Preferably, the nucleic acid molecule is integrated into the genome of the recombinant cell.

[0092] Preferably, the starting cells of the recombinant cells include prokaryotic cells or eukaryotic cells, and are further selected from Escherichia coli, yeast cells or mammalian cells.

[0093] In a ninth aspect, the present invention provides a method for preparing the anti-PD-L1 nanobody described in the first aspect, the method comprising:

[0094] The gene encoding the anti-PD-L1 nanobody described in the first aspect is inserted into an expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell for culture, and the anti-PD-L1 nanobody is isolated from the culture.

[0095] In a tenth aspect, the present invention provides the use of the anti-PD-L1 nanobody described in the first aspect, the anti-PD-L1 binding molecule described in the second aspect, the humanized antibody or its active fragment described in the third aspect, the chimeric antigen receptor described in the fourth aspect, or the fusion protein described in the fifth aspect in the targeted binding of PD-L1.

[0096] In this invention, anti-PD-L1 nanobodies with high specificity and affinity are screened and prepared, which have broad application prospects, such as in vitro immunoassay for non-disease diagnosis and / or treatment purposes, and research on the basic behavior of PD-L1; they can also be used to prepare PD-L1 binding agents, including: drugs for the prevention and / or treatment of PD-L1 high-expression diseases; and / or reagents for detecting PD-L1 high-expression diseases.

[0097] In the eleventh aspect, the present invention provides the use of the anti-PD-L1 nanobody described in the first aspect, the anti-PD-L1 binding molecule described in the second aspect, the humanized antibody or its active fragment described in the third aspect, the chimeric antigen receptor described in the fourth aspect, or the fusion protein described in the fifth aspect, the nucleic acid molecule described in the sixth aspect, the recombinant vector described in the seventh aspect, or the recombinant cell described in the eighth aspect in the preparation of a drug for targeted treatment of PD-L1-related diseases.

[0098] Preferably, the PD-L1-related disease is a disease with high PD-L1 expression.

[0099] Preferably, the PD-L1 hyperexpression disease is a tumor that expresses PD-L1.

[0100] Preferably, the tumors expressing PD-L1 include, but are not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, ovarian cancer, esophageal cancer, bladder (urothelial) cancer, melanoma, Merkel cell carcinoma, etc.

[0101] On the twelfth page, the present invention provides an immunoconjugate comprising the anti-PD-L1 nanobody described in the first aspect, the anti-PD-L1 binding molecule described in the second aspect, the humanized antibody or its active fragment described in the third aspect, or the fusion protein and the conjugation portion described in the fifth aspect, wherein the conjugation portion is selected from any one or a combination of at least two of proteins, small molecule compounds, fluorescein, radioisotopes, contrast agents or fatty acids.

[0102] Preferably, the anti-PD-L1 nanobody is coupled to the conjugated portion via chemical bonds or linkers.

[0103] In one specific embodiment, the radioactive isotope includes:

[0104] (i) a diagnostic isotope selected from the group consisting of any one or at least two of the following: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, or Re-188; and / or

[0105] (ii) A therapeutic isotope selected from the group consisting of any one or a combination of at least two of the following: 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, or Yb-177.

[0106] In a thirteenth aspect, the present invention provides a pharmaceutical composition comprising any one or a combination of at least two of the following: the anti-PD-L1 nanobody described in the first aspect, the anti-PD-L1 binding molecule described in the second aspect, the humanized antibody or its active fragment described in the third aspect, the chimeric antigen receptor described in the fourth aspect, the fusion protein described in the fifth aspect, the nucleic acid molecule described in the sixth aspect, the recombinant vector described in the seventh aspect, the recombinant cell described in the eighth aspect, or the immunoconjugate described in the twelfth aspect.

[0107] In a fourteenth aspect, an engineered immune cell is provided, the engineered immune cell containing an expression vector as described in the seventh aspect of the present invention or having an exogenous nucleic acid molecule as described in the sixth aspect of the present invention integrated into its genome, or expressing a chimeric antigen receptor as described in the fourth aspect of the present invention.

[0108] In another preferred embodiment, the engineered immune cells are selected from the group consisting of:

[0109] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0110] (ii) Chimeric antigen receptor γδ T cells (CAR-T cells);

[0111] (iii) Chimeric antigen receptor NKT cells (CAR-NKT cells);

[0112] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0113] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or combinations thereof.

[0114] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0115] In a fifteenth aspect, a kit is provided, the kit comprising:

[0116] (1) A first container containing, in the first aspect of the present invention, an anti-PD-L1 nanobody as described in the first aspect of the present invention, an anti-PD-L1 binding molecule as described in the second aspect of the present invention, a humanized antibody or an active fragment thereof as described in the third aspect of the present invention, a chimeric antigen receptor as described in the fourth aspect of the present invention, an immunoconjugate as described in the thirteenth aspect of the present invention, recombinant cells or combinations thereof as described in the eighth aspect of the present invention; and / or

[0117] (2) A second container, wherein the second container contains a secondary antibody against the contents of the first container;

[0118] or,

[0119] The kit contains a detection plate, which includes a substrate (support plate) and a test strip. The test strip contains an anti-PD-L1 nanobody as described in the first aspect of the present invention, an anti-PD-L1 binding molecule as described in the second aspect, a humanized antibody or its active fragment as described in the third aspect, a chimeric antigen receptor as described in the fourth aspect, an immunoconjugate as described in the thirteenth aspect of the present invention, or a combination thereof.

[0120] In another preferred embodiment, the kit also includes an instruction manual, according to which the kit is used for non-invasive detection of PD-L1 expression in a test subject.

[0121] In another preferred embodiment, the kit is used for the detection of PD-L1 overexpression diseases.

[0122] In another preferred embodiment, the PD-L1 high expression disease includes, but is not limited to: solid tumors, hematologic malignancies, immune diseases, or combinations thereof.

[0123] In another preferred embodiment, the diseases with high PD-L1 expression include, but are not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, ovarian cancer, esophageal cancer, bladder (urothelial) cancer, melanoma, Merkel cell carcinoma, etc., or combinations thereof.

[0124] In a sixteenth aspect of the invention, a method for preventing and / or treating PD-L1-related diseases is provided, the method comprising: administering to a desired subject an anti-PD-L1 nanobody as described in the first aspect of the invention, an anti-PD-L1 binding molecule as described in the second aspect, a humanized antibody or an active fragment thereof as described in the third aspect, a chimeric antigen receptor as described in the fourth aspect, a fusion protein as described in the fifth aspect, an immunoconjugate as described in the thirteenth aspect, a pharmaceutical composition as described in the twelfth aspect of the invention, engineered immune cells as described in the fourteenth aspect of the invention, or a combination thereof.

[0125] In another preferred embodiment, the PD-L1-related disease is a disease with high PD-L1 expression.

[0126] In another preferred embodiment, the object includes mammals, such as humans.

[0127] In another preferred embodiment, the PD-L1 high expression disease includes, but is not limited to: solid tumors, hematologic malignancies, immune diseases, or combinations thereof.

[0128] In another preferred embodiment, the diseases with high PD-L1 expression include, but are not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, ovarian cancer, esophageal cancer, bladder (urothelial) cancer, melanoma, Merkel cell carcinoma, etc., or combinations thereof.

[0129] In another preferred embodiment, the method may be used in combination with other treatment methods.

[0130] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy, etc.

[0131] In a seventeenth aspect of the present invention, a diagnostic method for anti-PD-L1 related diseases is provided, comprising the steps of:

[0132] (i) Obtaining a sample from a diagnostic subject and contacting the sample with an anti-PD-L1 nanobody as described in the first aspect of the invention, an anti-PD-L1 binding molecule as described in the second aspect, a humanized antibody or its active fragment as described in the third aspect, an immunoconjugate as described in the tenth aspect of the invention, or a combination thereof; and

[0133] (ii) Detect whether an antigen-antibody complex is formed, wherein the formation of a complex indicates that the subject is a confirmed patient with PD-L1 hyperexpression disease.

[0134] In another preferred embodiment, the PD-L1-related disease is a disease with high PD-L1 expression.

[0135] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues or organs.

[0136] In another preferred embodiment, the PD-L1 high expression disease includes, but is not limited to: solid tumors, hematologic malignancies, immune diseases, or combinations thereof.

[0137] In another preferred embodiment, the diseases with high PD-L1 expression include, but are not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, ovarian cancer, esophageal cancer, bladder (urothelial) cancer, melanoma, Merkel cell carcinoma, etc., or combinations thereof.

[0138] In this invention, a PD-L1 heavy chain single-domain antibody (nanobody) with high affinity and specificity is screened and prepared. Based on this, chemical markers, biological markers, small molecule compounds, and proteins can be further linked to prepare immunoconjugates for use in the fields of immunodetection and therapy. For example, the chemical markers can be isotopes, immunotoxins, and / or chemical drugs; the biological markers can be biotin, avidin, or enzyme-labeled substances; the small molecule compounds include, but are not limited to, drugs or toxins with clear or potential therapeutic or adjuvant treatment for tumors or autoimmune diseases; the contrast agents include gold nanoparticles / nanorobars or magnetic nanoparticles; and the proteins include, but are not limited to, antibodies Fc, HRP, antibodies, enzymes, cytokines, viral capsid proteins, VLPs, and other bioactive proteins or peptides.

[0139] Preferably, the protein includes IL-12 protein. Attached Figure Description

[0140] Figure 1 Image showing the SDS-PAGE results for verifying the purity of purified PD-L1;

[0141] Figure 2 The image shows the results of SDS-PAGE identification of the purified nanobodies.

[0142] Figure 3 This is a graph showing the results of the identification of recombinant proteins from the antigen.

[0143] Figure 4 The image shows the half-image of the ELISA results for detecting the binding of candidate PD-L1 nanobodies to human PD-L1 protein.

[0144] Figure 5 Figure 2 / 2 shows the results of ELISA detection of the binding of candidate PD-L1 nanobody to human PD-L1 protein;

[0145] Figure 6 The graph shows the results of determining the affinity of nanobodies for PD-L1 at a single concentration using the SPR method.

[0146] Figure 7 Figure showing the results of FACS detection of the binding activity of PDL1 nanobody with PDL1 expressed in Raji cells;

[0147] Figure 8 A graph showing the blocking effect of PD-L1 nanobody on PD-1 / PD-L1 interaction as detected by ELISA;

[0148] Figure 9 Figure showing the results of identifying the blocking activity of Raji cell surface nanobodies;

[0149] Figure 10Figure showing the results of SDS-PAGE identification of the purity of anti-PDL1-Fc nanobody protein;

[0150] Figure 11 The diagram shows the binding results of the NbsPD-L1-Fc fusion protein to human PD-L1 protein.

[0151] Figure 12 The figure shows the results of ELISA detection of the binding ability of Nbs-PD-L1-Fc fusion protein to PD-L1 protein;

[0152] Figure 13 The graph shows the affinity results of the NbsPD-L1-Fc fusion protein determined by the SPR method.

[0153] Figure 14 Figure 1 shows the results of FACS assay for detecting the binding activity of NbsPDL1-1-Fc with Raji-PDL1 / Raji cells.

[0154] Figure 15 This is a diagram showing the specificity of PD-L1 nanobody binding to PDL1 protein.

[0155] Figure 16A The identification results of humanized single-domain antibodies of PDL1-1 are shown in the figure (PDL1-1v4, PDL1-1v7, PDL1-1v9, PDL1-1v1 and PDL1-1v2);

[0156] Figure 16B The identification results of PDL1-1 humanized single-domain antibodies are shown in the figure (PDL1-1v3, PDL1-1v10, PDL1-1v8, ​​PDL1-1v5, PDL1-1v6, PDL1-1v10, PDL1-1v13, PDL1-1v19 and PDL1-1v17);

[0157] Figure 16C The identification results of PDL1-1 humanized single-domain antibodies are shown in the figure (PDL1-1v11, PDL1-1v14, PDL1-1v16, PDL1-1v15, PDL1-1v12 and PDL1-1v18).

[0158] Figure 17 The graph shows the results of the PDL1-1 humanized single-domain antibody binding affinity assay.

[0159] Figure 18 The graph shows the results of the PDL1-1 humanized single-domain antibody binding affinity assay.

[0160] Figure 19 The graph shows the results of the PDL1-1 humanized single-domain antibody binding affinity assay.

[0161] Figure 20The graph shows the results of the PDL1-1 humanized single-domain antibody binding affinity assay.

[0162] Figure 21 The image shows the results of the detection of blocking activity of the PDL1-1 humanized single-domain antibody.

[0163] Figure 22 This is a graph showing the results of the blocking activity assay of the PDL1-1 humanized single-domain antibody at the cellular level.

[0164] Figure 23 The image shows the activation detection results of PBMCs by PDL1 single-domain antibody and humanized antibody;

[0165] Figure 24 The image shows the activation detection results of PBMCs by PDL1 single-domain antibody and humanized antibody;

[0166] Figure 25 The graph shows the detection results of the killing effect of the PDL1-1 humanized heavy chain single-domain antibody on target cells.

[0167] Figure 26 A diagram simulating the expression pattern of a fusion protein;

[0168] Figure 27 This is a gel electrophoresis analysis diagram of the fusion protein;

[0169] Figure 28 The graph shows the results of PD-L1 binding activity assays for fusion proteins 1-4.

[0170] Figure 29 The graph shows the results of PD-L1 binding activity assay for fusion protein 5-fusion 9.

[0171] Figure 30 Sensing images of fusion proteins 1-4 and their corresponding fitting curves;

[0172] Figure 31 The sensor images and corresponding fitting curves of fusion proteins 5-9 are shown.

[0173] Figure 32 The fusion proteins 4, 7, and 8 enhance the killing effect of effector cells on target cells. Detailed Implementation

[0174] Through extensive and in-depth research and screening, the inventors unexpectedly obtained, for the first time, an anti-PD-L1 nanobody with high affinity and high specificity. A fusion protein was prepared using this anti-PD-L1 nanobody and IL-12 protein. This fusion protein can both target tumor lesions and release IL-12 cytokines to stimulate immune cells to exert their immune-killing function, thus providing a better diagnostic tool for the treatment of PD-L1-related diseases, such as tumors. This invention was completed based on this.

[0175] the term

[0176] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0177] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0178] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0179] In specific embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the nanobody of the present invention, as well as the amino acid sequence of the VHH chain, are shown in the table below:

[0180]

[0181] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. 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 and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0182] As used herein, the terms "single-domain antibody," "VHH," "nanobody," and "single-domain antibody (sdAb, or nanobody)" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain.

[0183] In this invention, the terms "nanobody of the present invention," "antibody of the present invention," "protein of the present invention," or "peptide of the present invention" are used interchangeably and all refer to peptides that specifically bind to PD-L1, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0184] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called backbone regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally β-sheeted and linked by three CDRs forming a linking loop, and in some cases, partially β-sheeted structures. The CDRs in each chain are tightly packed together by 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)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0185] As used herein, the terms "hypervariant region," "highly variable region," "complementarity determining region," and "complementarity determining region (CDR)" are used interchangeably. In a preferred embodiment of the invention, the heavy chain variable region of the nanobody or antibody includes three complementarity determining regions, CDR1, CDR2, and CDR3. In another preferred embodiment of the invention, the heavy chain of the nanobody or antibody includes a heavy chain variable region and a heavy chain constant region.

[0186] The antigen-binding properties of an antibody can typically be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This region is divided into four backbone regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of similar antibodies can be compared to determine which amino acids constitute the FR or CDR regions.

[0187] The heavy chain variable region of the nanobodies or antibodies of the present invention is critical because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0188] In specific embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the nanobody of the present invention, as well as the amino acid sequence of the VHH chain, are shown in the table below:

[0189]

[0190] As those skilled in the art will recognize, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the described CD38-targeting nanobodies or fragments thereof.

[0191] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0192] As used herein, the term "humanized antibody" has the meaning conventionally understood by those skilled in the art, referring to antibodies obtained by humanizing alpaca-derived antibodies, thereby reducing the immunogenicity of alpaca-derived antibodies. Based on the anti-PD-L1 nanobody of the present invention, those skilled in the art know how to humanize it to obtain a humanized antibody. In specific embodiments, the humanized antibody of the present invention has the amino acid sequence shown in any one of SEQ ID NO: 23-42; preferably any one of SEQ ID NO: 30, 33, 36, 40, 41; more preferably the amino acid sequence shown in SEQ ID NO: 30 or 40.

[0193] To improve the affinity of humanized antibodies, those skilled in the art can perform reversion mutations on humanized antibodies to obtain humanized antibodies with enhanced affinity.

[0194] Based on the teachings of this invention, those skilled in the art will also know how to obtain the active fragments of the antibodies of this invention, i.e., antibody fragments having the same or similar immune activity as the antibodies of this invention. Therefore, this invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. For example, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0195] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0196] The term "antibody of the present invention" includes not only polypeptides containing the aforementioned CDR region that have PD-L1 protein binding activity, but also variants of polypeptides containing the aforementioned CDR region that have the same function as the antibody of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibody of the present invention.

[0197] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0198] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0199] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to the table below.

[0200] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0201] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0202] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0203] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0204] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0205] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0206] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0207] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0208] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0209] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0210] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0211] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0212] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, 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.

[0213] The nanobodies, antibodies, or humanized antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified portions, or any combination of the above substances.

[0214] Detectable markers 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 capable of producing detectable products.

[0215] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0216] PD-L1 related diseases

[0217] Based on the teachings of this invention, those skilled in the art will know that the nanobodies, their active fragments, or derivatives of this invention can be used to treat PD-L1-related diseases.

[0218] The PD-L1-related diseases are diseases with high PD-L1 expression. In a specific implementation, the PD-L1-high expression diseases are tumors that express PD-L1; including but not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, ovarian cancer, esophageal cancer, bladder (urothelial) cancer, melanoma, Merkel cell carcinoma, etc.

[0219] Reagent test kit

[0220] The present invention also provides a kit containing the anti-PD-L1 nanobody, antibody, or humanized antibody (or fragment thereof) or detection plate of the present invention. In a preferred embodiment, the kit further includes a container, instructions for use, buffer, etc.

[0221] This invention also provides a detection kit for detecting PD-L1 protein levels. The kit includes the antibody of this invention that recognizes PD-L1 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0222] Pharmaceutical Composition

[0223] Based on the antibody of the present invention, the present invention also provides a pharmaceutical composition comprising, as an active ingredient, the anti-PD-L1 nanobody of the present invention, an anti-PD-L1 binding molecule, a humanized antibody, a chimeric antigen receptor, a fusion protein, a recombinant protein, a host cell, an engineered immune cell, an immunoconjugate, or a combination thereof. The pharmaceutical composition of the present invention may also optionally contain a pharmaceutically acceptable carrier.

[0224] These substances are typically formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH usually around 5-8, preferably around 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0225] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0226] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals, wherein this safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0227] application

[0228] As described above, the nanobodies, their active fragments, or derivatives of the present invention have broad biological and clinical application value, and their applications involve multiple fields such as the diagnosis and treatment of diseases related to PD-L1 protein, basic medical research, and biological research. A preferred application is for the clinical diagnosis, prevention, and treatment of PD-L1 protein.

[0229] The present invention also provides a method for stimulating T-cell-mediated immune responses targeting mammalian tumor cell populations or tissues, comprising the following steps: administering the CAR-T cells of the present invention to a mammal.

[0230] In one embodiment, the present invention includes a type of cell therapy in which patient-associated T cells (or allogeneic donor cells) are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This approach results in an extremely low probability of graft-versus-host disease, and the antigen is recognized by T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-lasting, durable antibodies that lead to sustained tumor control.

[0231] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and persist for months to years. Furthermore, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-T cells can induce a specific immune response against tumor cells that highly express antigens recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention elicit a specific immune response against tumor cells that highly express PD-L1. In specific embodiments, the PD-L1-highly expressing tumors include, but are not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, ovarian cancer, esophageal cancer, bladder (urothelial) cancer, melanoma, Merkel cell carcinoma, etc.

[0232] Typically, activated and expanded cells, as described herein, can be used to treat and prevent diseases such as tumors. Therefore, this invention provides a method for treating cancer comprising administering a therapeutically effective amount of the CAR-T cells of this invention to a subject in need of treatment.

[0233] The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may include target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0234] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the treatment (or prevention) of a disease. The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.

[0235] When referring to "immunologically effective dose," "antitumor effective dose," "tumor-inhibitory effective dose," or "therapeutic dose," the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. Pharmaceutical compositions including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 7 Administered at a dose of cells per kg body weight (inclusive of all integer values ​​within the range). The T-cell composition may also be administered multiple times at these doses. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a medical professional by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0236] The composition can be administered in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient via intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered via intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0237] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded immune cells of the present invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0238] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 5 One to 1×10 10 The modified T cells of this invention are administered to a patient, for example, via intravenous infusion.

[0239] Main advantages of the invention

[0240] 1. The anti-PD-L1 nanobody of the present invention has high affinity and high specificity, thereby enabling it to efficiently target PD-L1;

[0241] 2. The anti-PD-L1 nanobody of the present invention has a simple structure and is easy to prepare, thus having broad application prospects in the fields of immune detection and tumor treatment.

[0242] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0243] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0244] In specific embodiments of the present invention, the abbreviations and key terms are defined as follows.

[0245]

[0246]

[0247] Example 1

[0248] This embodiment screens for heavy chain single-domain antibodies against PD-L1.

[0249] 1. Preparation of immune antigens

[0250] The extracellular domain of the antigen PD-L1 (1-238) was synthesized using a gene, and a six-histidine tag was added to the C-terminus. The resulting protein was subcloned into the eukaryotic expression vector pcDNA3.1 to construct an expression vector for the recombinant PD-L1 protein (PD-L1-His). The amino acid sequence is shown in SEQ ID NO. 17. The constructed PD-L1-His protein was then extracted as a plasmid, sequenced correctly, and transfected into FreeStyle DNA. TM 293-F cells were cultured in shake flasks at 37℃, 120 rpm, and 5% CO2 for 3.5 days. The expression supernatant was collected by centrifugation, filtered through a 0.45 μm filter membrane, and subjected to HisTrap. TM FF was used for affinity purification, and the sample was collected after elution with 500mM imidazole. The sample was then concentrated using an ultrafiltration concentrator with buffer replacement. SDS-PAGE was used to identify protein purity. Results are as follows: Figure 1 As shown, the positions of the antigen proteins in the electrophoresis were as expected, the bands were clear, and the purity was high. No broken or degraded bands were observed in the PD-L1 protein.

[0251] Lanes 1 and 2 contain non-reduced PD-L1 protein, lanes 4 and 5 contain reduced PD-L1 protein, and lanes 3 and 6 contain protein molecular weight standards. The protein concentration was quantified using a spectrophotometer at 2 mg / mL.

[0252] SEQ ID NO.17:

[0253] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYG GADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER.

[0254] 2. Building a library

[0255] A healthy alpaca (1.5 years old, male, weighing approximately 160 catties, provided by Qingdao Kangda Biotechnology Co., Ltd.) was selected for immunization. Multiple subcutaneous injections were administered to the neck and shoulder. After 5 immunizations, 100 mL of peripheral blood lymphocytes were extracted from the alpaca. The PBMCs were isolated using Alpaca Peripheral Blood Lymphocyte Separation Solution KIT from Tianjin Haoyang Biotechnology Co., Ltd. RNA was extracted using the Novozymes RNA Keeper Tissue Atabilizer extraction kit. The extracted RNA was reverse transcribed into cDNA using the HiScript III 1st StrandcDNA Synthesis Kit (Novozymes, R312-01). Nested PCR was used to amplify the nucleic acid fragments of the variable region of the heavy chain antibody.

[0256] First round of PCR:

[0257] Upstream primer F1: GTCCTGGCTGCTCTTCTACAAGG (SEQ ID NO.43);

[0258] Downstream primer R1: GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO.44);

[0259] Fab of heavy chain antibody was obtained by PCR amplification using cDNA as a template. The reaction procedure is as follows:

[0260]

[0261] Electrophoresis was performed using a 2% agarose gel. Bands of approximately 700 bp were cut, and the target gene was recovered using a gel extraction kit (Novizan) according to the instructions.

[0262] Second round of PCR:

[0263] Using the first-round PCR product as a template, nanobodies were obtained by PCR amplification again. The reaction system and reaction procedure are shown in the table.

[0264] Upstream primer F2: atggcccaggtgcagctgcagGAGTCTGGRGGAGG (SEQ ID NO.45);

[0265] Downstream primer R2: gtggtgtgaggagacGGTGACCTGGGT (SEQ ID NO.46);

[0266] The reaction system is as follows:

[0267] Components volume First round PCR products 1μL Upstream primer F2 (10 μM) 1μL Downstream primer R2 (10 μM) 1μL <![CDATA[2×PCR Bestaq TM Master Mix]]> 25μL <![CDATA[ddH2O]]> Make up to 50 μL

[0268] The PCR reaction procedure is as follows:

[0269]

[0270]

[0271] The target heavy chain single-domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pMES4 using restriction endonucleases (PstⅠ and BsteⅡ, purchased from NEB). Fresh competent cells TG1 were prepared, and the product was electroporated into competent cells TG1. The cells were then plated using a serial dilution method, and the library size was calculated to be 10⁻¹⁰. 11 Twenty clones were randomly selected for colony PCR and sequencing verification. The results showed that the insertion rate was 100%, and the heavy chain single-domain antibody phage display library against PD-L1 was successfully constructed.

[0272] 3. Screening for single-domain antibodies targeting the PD-L1 heavy chain.

[0273] PD-L1-his fusion protein (SEQ ID NO.18) at 10 μg / mL was coated onto immunotubes and incubated overnight at 4°C. The next day, the immunotubes were blocked with 1 mL of PBS (PBS containing 2% skim milk powder) at 37°C for 1 h, and simultaneously, 100 μL of phage library (from the heavy chain single-domain antibody phage display library constructed above) was blocked. The blocked phage antibody library was added to the immunotubes and incubated overnight at 4°C. The liquid in the immunotubes was discarded, and the tubes were washed with PBS, PBST, and PBS+NaCl, with the washing intensity increasing in each round. After three rounds of washing, the liquid in the immunotubes was discarded, and 1 mL of 0.2 mol / L glycine-hydrochloric acid (pH 2.2) was added for elution, followed by neutralization to pH with 1 mol / L Tris. 7.4; Logarithmically growing *E. coli* TG1 was added to the immunofluorescence tube for infection. The eluted bacterial solution was resuspended and evenly spread on 2YT-AG (A: ampicillin sodium, G: glucose) plates. After incubation at 37°C overnight, all colonies were collected. An appropriate amount of bacterial solution was added to 100 mL of 2YT-AG liquid medium and cultured until the absorbance (A) at 600 nm was 0.7. Helper phage M13KO7 was added at an infection coefficient (MOI) of 50:1. The culture was incubated at 25°C for 30 min, then at 37°C and 150 rpm for 1 h. IPTG was added to a final concentration of 0.15 mmol / L, and the culture was incubated at 30°C and 200 rpm for 10 h. The phage antibody was recovered by PEG8000 precipitation, and a certain amount was used for the next round of screening. This process was repeated for 3 rounds of screening. Finally, positive clones were enriched, achieving the goal of using phage display technology to screen for PD-L1 specific antibodies.

[0274] SEQ ID NO.18:

[0275] MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGAD YKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERHHHHHH.

[0276] 4. Screen for positive clones using phage enzyme-linked immunosorbent assay (ELISA).

[0277] Select the selected single colonies and incubate them overnight at 37°C and 220 rpm in 96-well plates containing 2×YT-AG medium until saturation; then transfer the saturated bacterial culture to OD200. 600 The concentration was approximately 0.5. Helper phage M13KO7 was added at an infection coefficient (MOI) of 50:1. After standing at 25°C for 30 min, the mixture was incubated at 37°C and 150 rpm for 1 h. Kanamycin was added to a final concentration of 20 μg / mL, and IPTG to a final concentration of 0.15 mM. Induction was performed at 30°C and 200 rpm for 12 h. The supernatant was collected by centrifugation as phage antibody. The antibody was coated with the target antigen (PDL1-his) and control antigen (BSA) and incubated overnight at 4°C. The phage antibody bound at 37°C for 1 h, followed by washing. Anti-M13 antibody bound at 37°C for 45 min, followed by washing. TMB single-component chromogenic solution was added, and the absorption peak was measured at 450 nm. If the OD value of the sample well was greater than 3 times the OD value of the control well, it was identified as a positive monoclonal colony and sent for sequencing. Variable region genes of 11 candidate antibodies were obtained.

[0278] Example 2

[0279] This embodiment provides a preliminary evaluation and identification of heavy chain single-domain antibodies against PD-L1.

[0280] 1. Expression and purification of heavy chain single-domain antibodies in Escherichia coli

[0281] Plasmids were extracted from the 11 single-domain antibodies (numbered PDL1-1, PDL1-3, PDL1-4, PDL1-6, PDL1-8, PDL1-13, PDL1-14, PDL1-16, PDL1-19, PDL1-20, and PDL1-21) obtained from sequencing analysis in Example 1, and then transformed into BL21(DE3) competent cells by heat shock. 1 mL of positive clone bacterial culture was added to 100 mL of LB liquid medium (containing 100 μg / mL ampicillin) in a clean bench and cultured at 37°C with shaking until OD. 600 =Approximately 0.8, add IPTG to a final concentration of 1 mM, and incubate overnight at 30°C with shaking. The next day, centrifuge at 8000 rpm for 10 min, collect the bacterial cells, resuspend the precipitate in 1.5 mL of pre-chilled TES buffer, and stir on ice for 30 min. Add 3.0 mL of TES / 4 (TES diluted 4-fold with pure water), and continue stirring on ice for 30 min. Centrifuge at 9000 rpm at 4°C for 10 min, collect the supernatant (periplasmic protein extract), and perform SDS-PAGE electrophoresis analysis. Based on the protein purification system, HisTrap was used. TM FF was used for affinity purification, and the sample was collected after elution with 300mM imidazole buffer. Antibody concentration was quantified using a spectrophotometer, and antibody purity was identified by SDS-PAGE. Results are shown below. Figure 2As shown, the extracted proteins were positioned as expected by electrophoresis, with clear bands and high purity. No broken or degraded bands were observed in any of the PD-L1 proteins. In the table, 12 represents the protein molecular weight standard, and 1-11 represent 11 purified nanobody protein samples: PDL1-1, PDL1-3, PDL1-4, PDL1-6, PDL1-8, PDL1-13, PDL1-14, PDL1-16, PDL1-19, PDL1-20, and PDL1-21.

[0282] 2. Construction of recombinant antigen protein

[0283] An expression vector for the recombinant protein PDL1 and human Fc (PDL1-Fc) was constructed. The PDL1 and human Fc genes were cloned into the pcDNA3.1 vector. After correct sequencing, the vector was transfected into healthy FreeStyle™ 293-F cells and cultured in shake flasks at 37℃, 120 rpm, and 5% CO2 for 3.5 days. The supernatant was collected, purified using a Protein A column, eluted with citrate-sodium citrate buffer, and concentrated using ultrafiltration. Protein purity was identified by SDS-PAGE. The results are shown below. Figure 3 As shown, lane 1: non-reduced SDS-PAGE of PDL1-Fc protein; lane 2: protein molecular weight standard; lane 3: reduced SDS-PAGE of PDL1-Fc protein. This indicates that the electrophoretic positions of the recombinant antigen protein are as expected, the bands are clear, and the purity is high. No broken or degraded bands were observed in the PDL1-Fc recombinant protein. Protein concentration was quantified using a spectrophotometer; the protein concentration was 2 mg / mL.

[0284] 3. ELISA detection of the binding of candidate PD-L1 nanobodies to human PD-L1 protein.

[0285] 200 ng / well of human PDL1-Fc fusion protein (SEQ ID NO.19) was coated onto plates and incubated overnight at 4°C. The obtained heavy chain single-domain antibody was then serially diluted 3-fold from 10 μg / mL in 12 steps, blocked at 37°C for 1 h. 100 μL of the blocked whole antibody was added to each well, and the plates were incubated at 37°C for 1 h. After washing, 100 μL / well of HRP-labeled anti-his tag antibody (His Tag Antibody HRP, Sino Biological) was added to each well, and the plates were incubated at 37°C for 45 min. After washing, 100 μL / well of TMB substrate chromogenic solution was added to each well, and the plates were incubated at 25°C for 10 min. The reaction was terminated with 100 μL / well of 1M H2SO4. The absorbance at 450 nm was measured using a microplate reader. Figure 4 and Figure 5 Data processing and parameter fitting were performed using Graphd Prism Software 5.0 to calculate EC.50 The results are shown in Tables 1 and 2. Among the 11 nanobodies screened, 4 prokaryotic expression nanobodies, PDL1-1, PDL1-3, PDL1-13, and PDL1-14, were bound to the PDL1-Fc fusion protein. The corresponding amino acid sequences are shown in SEQ ID NO:13-SEQ ID NO:16, respectively.

[0286] SEQ ID NO.19:

[0287] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMIS YGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPP NEREPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0288] Table 1

[0289] PDL1-1 PDL1-3 PDL1-4 PDL1-6 PDL1-8 EC50 (ug / ml) 0.008252 0.01278 ～0.001032 1.821 9.787

[0290] Table 2

[0291] PDL1-13 PDL1-14 PDL1-16 PDL1-19 PDL1-20 PDL1-21 EC50 (ug / ml) 0.06611 0.1235 0.002282 ～4.598 2.518 12.26

[0292] Example 3

[0293] In this embodiment, the SPR method (i.e., BIcore) was used to identify the binding ability of the PD-L1 single-domain antibody protein to PD-L1.

[0294] A single-concentration affinity screening test was used, in which all nanobodies were tested at the same single concentration, and the binding activity differences between the nanobodies were detected by comparing them in parallel. Capture antibodies were conjugated to the surface of a CM5 chip using a Human Antibody Capture Kit. The kinetic parameters of antibody-antigen interactions were determined using a multi-cycle kinetics method. Purified PDL1-his was diluted to 1 μg / mL with HBS-EP buffer and captured and immobilized on the chip surface at 25℃ and 5 μL / min for 1 min, with a capture response value of approximately 400 RU. Nanobodies PDL1-1, PDL1-3, PDL1-13, and PDL1-14 were diluted to 1.5 μg / mL with HBS-EP buffer as the mobile phase. The test conditions were 25℃, 30 μL / min, binding for 60 s, dissociation for 120 s, and regeneration under 10 mM glycine hydrochloride buffer (pH 2.5) at 30 μL / min for 90 s. The results were analyzed as follows: Figure 6 As shown in Table 3, antibody affinity is an important indicator for evaluating antibody molecules, and surface plasmon resonance (SPR) technology is the recognized gold standard for detecting antibody affinity. We used the BIAcore T100 system and employed a single-concentration affinity screening method. The binding activity parameters for PDL1-1, PDL1-3, and PDL1-13 were 0.5, 0.6, and 0.2, respectively, while PDL1-14 showed no activity.

[0295] Table 3

[0296]

[0297]

[0298] Example 4

[0299] In this embodiment, the FACS method was used to detect the binding activity between the nanobody and Raji-PDL1 / Raji cells.

[0300] Raji cells (Beijing Kangyuan Bochuang, KC-1886) carrying the full-length human PD-L1 gene plasmid were purchased and cultured to the logarithmic growth phase. Cells were resuspended in FACS buffer and the cell density was adjusted to 3E6 / mL, with 100 μL seeded per well. The heavy chain single-domain antibody obtained in Example 2 (positive control group: KN035 from KNJ) was then diluted 5-fold with FACS buffer to 30 μg / mL, and the cells were resuspended in 100 μL / well. The cells were incubated at 4°C for 60 min, washed three times with PBS, and then the secondary antibody (THE) was added. TMHis Tag Antibody [FITC], mAb, and Mouse were used to resuspend cells. Cells were incubated at 4°C for 60 min, washed three times with PBS, and resuspended in 100-200 μL of PBS per well. MFI was detected using Beckman / CytoFLEX. Data processing and parameter fitting were performed using Graphd Prism Software 5.0, and EC50 values ​​were calculated. Results are shown below. Figure 7 As shown in Table 4, PDL1-1 and PDL1-3 exhibit high binding activity to PDL1 protein expressed in Raji cells and show typical dose-dependent characteristics.

[0301] Table 4

[0302] PDL1-1 PDL1-3 <![CDATA[EC 50 (ug / ml)]]> 0.0606 0.07298

[0303] Example 5

[0304] In this embodiment, a competitive ELISA was used to detect the blocking effect of the PD-L1 heavy chain single-domain antibody on the interaction between PD-1 and PD-L1.

[0305] PDL1-his protein was diluted to 2 μg / mL with PBS, and 100 μL / well was added to a 96-well ELISA plate. The plate was incubated overnight at 4°C. The heavy chain single-domain antibody obtained in Example 2 was serially diluted 13 times from 200 μg / mL with 2.5% PBST milk, with a final concentration of 50 μg / mL for each well containing recombinant PD1-Fc (SEQ ID NO. 20). (The blank group contained no antibody or protein, only an equal volume of buffer.) The plates were blocked at 37°C for 1 h, and then incubated at 37°C for 1 h. After washing, anti-IgG antibody (purchased from Zhongshan Jinqiao) was added, and the plates were incubated at 37°C for 45 min. After washing, TMB substrate chromogenic solution was added to 100 μL / well, and the plates were incubated at 25°C for 10 min. The incubation was terminated with 100 μL / well of 1M H2SO4. The absorbance was measured at 450 nm using a microplate reader. Data processing and parameter fitting were performed using Graphd Prism Software 5.0, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The result is as follows Figure 8 As shown in Table 5, PDL1-1 and PDL1-3 can block the binding of PDL1-his protein to PD1-Fc within a certain concentration range, and this effect is dose-dependent. The IC50 values ​​for PDL1-1 and PDL1-3 are also shown. 50 The values ​​were 2.323 μg / mL and 1.312 μg / mL, respectively, indicating similar blocking activities.

[0306] Table 5

[0307] PDL1-1 PDL1-3 <![CDATA[IC 50 (ug / ml)]]> 2.323 1.312

[0308] SEQ ID NO.20:

[0309] PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0310] Example 6

[0311] This embodiment uses the FACS method to detect the blocking activity of the antibody at the cellular level (cell-based RBA).

[0312] Raji cells carrying the full-length human PD-L1 gene plasmid were cultured to the logarithmic growth phase. Cells were resuspended in FACS buffer and the cell density was adjusted to 3E6 / ml, with 100 μL seeded per well. 50 μL / well of PD1-Fc and 50 μL / well of the test antibody were added to resuspend the cells, and the cells were incubated at 4°C for 60 min. The cells were washed three times with 200 μL of PBS, centrifuged at 300g / 5 min, and the supernatant was discarded. Secondary antibody SA-488 was diluted with FACS buffer, and cells were resuspended at 100 μL / well, incubated at 4°C for 60 min. 200 μL / well of PBS was added to wash three times, centrifuged at 300g / 5 min, and the supernatant was discarded. Cells were resuspended in 100 μL / well of PBS and analyzed. Results are as follows: Figure 9 As shown in Table 6, PDL1-1 and PDL1-3 can block the binding of PDL1 protein to PD1-Fc on the surface of Raji cells within a certain concentration range, and this effect is dose-dependent. The IC50 values ​​for PDL1-1 and PDL1-3 are... 50 The values ​​were 0.06263 ug / ml and 0.04058 ug / ml, respectively, indicating similar blocking activities.

[0313] Table 6

[0314] PDL1-1 PDL1-3 IC50 (ug / ml) 0.06263 0.04058

[0315] Example 7

[0316] This embodiment analyzes the specificity of PD-L1 nanobody binding to PDL1-Fc protein.

[0317] Different proteins (200 ng / well) were coated with antigens HpaA, UreB, HtrA, HER2, BCMA, PD1, IL-12, IL-18, IL-15, Pertuzuman, HAS, BSA, and PDL1. All proteins were sourced from our own laboratory. Coating was performed overnight at 4°C. Antibodies (PDL1-1 0.2 μg, PDL1-3 0.2 μg, PDL1-13 0.2 μg, PDL1-14 0.2 μg) were added. Detection was performed in replicates at 37°C for 1 h. After washing, HRP-labeled anti-his-tag antibody was added at 100 μL / well to the ELISA plate. The plate was incubated at 37°C for 45 min. After washing, TMB substrate chromogenic solution was added at 100 μL / well, and the reaction was terminated with 100 μL / well of 1M H2SO4. Absorbance was measured at 450 nm using a microplate reader. Data processing was performed using Graphd Prism Software 5.0. Figure 15 Specificity experiments showed that PDL1-1, PDL1-3, PDL1-13, and PDL1-14 specifically bind to human PDL1 and do not bind to other unrelated proteins.

[0318] Example 8

[0319] This embodiment utilizes mammalian cells to prepare the NbsPDL1-Fc fusion protein.

[0320] Clones identified as positive single-chain antibodies (PDL1-1, PDL1-3, and PDL1-13) were sequenced. The sequencing results were analyzed, and the positive single-chain variable region gene was amplified using primers HF (ccttaagggcgtgcagtgccaggtgcagctgcaggagtc; SEQ ID NO.47) and HR (gatttgggctcgctagctgaggagacggtgacctggg; SEQ ID NO.48). The amplification reaction system is shown in Table 7, and the reaction procedure is shown in Table 8. The positive single-chain antibody gene was cloned into a pcDNA3.1(+) vector containing human Fc (SEQ ID NO.21) via homologous recombination. The reaction system is shown in Table 9, and the reaction conditions were 37℃ for 30 min.

[0321] SEQ ID NO.21:

[0322] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0323] Table 7

[0324] Phagocytic template 0.5μL HF primers (10 μM) 1μL HR primers (10 μM) 1μL 2×Phanta Max Master Mix 15μL Sterilized deionized water 12.5μL sum 30μL

[0325] Table 8

[0326]

[0327] Table 9

[0328] Vector pABG1-Fc (0.02 pmol) 0.4μL PD-L1 target fragment (0.06 pmol) 0.3~3μL Exnase II 1μL 5×CE II buffer 2μL Sterilized deionized water 3.6–6.3 μL sum 10μL

[0329] Plasmids were extracted using an endotoxin-free plasmid extraction reagent, quantified spectrophotometer-based, and sequenced. FreeStyle™ 293-F cells were collected by centrifugation one day before transfection, and the cell density was adjusted to 1×10⁻⁶ cells / year. 6 Transfection ratio: cells / mL (cells:medium = 20:1); Polyethylenimine Lineae (PEI) 2 μL / mL; Plasmid 1 μg / mL; 40 μg of plasmid was dissolved in 2 mL of Opti-MEM medium, gently mixed, then 80 μL of PEI transfection reagent was added, vortexed for 10 s to mix thoroughly, and incubated at 25°C for 13 min. 2 mL of the mixture was added to 40 mL of healthy FreeStyle™ 293-F cells, gently mixed, and cultured in a shaker at 37°C, 120 rpm, and 5% CO2 for 4 days. The supernatant was collected for SDS-PAGE electrophoresis analysis. Protein A affinity purification was performed using a protein purification system, and the sample was eluted with 0.1 M citrate-sodium citrate buffer. Antibody concentration was quantified using a spectrophotometer, and antibody purity was identified by SDS-PAGE. Figure 10As shown, the power source positions of both reduced and non-reduced NbPDL1-Fc fusion proteins were as expected, with clear bands and high purity. No broken or degraded bands were observed in any of the NbPDL1-Fc fusion proteins. Lanes 1 and 2 contain the non-reduced NbPDL1-1-Fc fusion protein, lane 3 contains the protein molecular weight standard, and lanes 4 and 5 contain the reduced NbPDL1-1-Fc and NbPDL1-3-Fc fusion proteins.

[0330] Example 9

[0331] This embodiment detects the binding of the candidate NbsPD-L1-Fc fusion protein to human PD-L1 protein.

[0332] Human PDL1-his protein was used to coat plates with 200 ng / well and incubated overnight at 4°C. Then, the heavy chain single-domain antibody obtained in Example 7 (positive control group: KN035 containing Fc (SEQ ID NO. 22) from KNJ Biopharma) was serially diluted 3-fold from 10 μg / mL in 12 gradients, blocked at 37°C for 1 h. 100 μL of the blocked whole antibody was added to each well, and the plates were incubated at 37°C for 1 h. After washing, HRP-labeled anti-his-tag antibody was added to each well at 100 μL / well, and the plates were incubated at 37°C for 45 min. After washing, TMB substrate chromogenic solution was added to each well, and the plates were incubated at 25°C for 10 min. The reaction was terminated with 100 μL / well of 1M H2SO4. The absorbance was measured at 450 nm using a microplate reader. Data processing and parameter fitting were performed using Graphd Prism Software 5.0, and EC50 was calculated. 50 The values ​​and results are shown in Table 10 and Figure 11 As shown, the NbPDL1-1-Fc fusion protein, NbPDL1-3-Fc fusion protein, and NbPDL1-13-Fc fusion protein exhibit better binding activity and typical dose-dependent characteristics compared to the control antibody containing FcKN035.

[0333] Table 10

[0334] NbPDL1-1-Fc NbPDL1-3-Fc NbPDL1-13-Fc KN035-Fc <![CDATA[EC 50 (ug / ml)]]> 0.01237 0.01106 0.01212 0.04186

[0335] SEQ ID NO.22:

[0336] QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPT CTLVTSSGAFQYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAGIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0337] Example 10

[0338] This embodiment examines the binding ability of the candidate NbPD-L1-Fc fusion protein to the PD-L1 (Cynomolgus) protein.

[0339] Using Cynomolgus PDL1-his protein-coated plates (100 ng / well), incubated overnight at 4°C. The coating solution was discarded, and 2.5% PBS (milk containing 2.5% skim milk powder) was added to each well (120 μL). The heavy chain single-domain recombinant antibody obtained in Example 7 (positive control: KN035 from Corninger) was serially diluted 3-fold from 50 μg / mL in 2.5% PBST for 12 consecutive times. The plates were blocked at 37°C for 1 hour. The blocking solution was discarded, and 100 μL of the blocked whole antibody was added to each well. The plates were incubated at 37°C for 1 hour, washed 6 times with PBST, and 100 μL of HRP-labeled goat anti-human IgG antibody was diluted 1:3000 with 2.5% PBST milk and added to each well. The plates were incubated at 37°C for 45 minutes, washed 6 times with PBST, and the washing solution was discarded. TMB substrate chromogenic solution was added to each well, and the plates were incubated at 25°C for 10 minutes. The solution was terminated with 100 μL of 1MH2SO4 per well. The absorbance was measured at 450 nm using a microplate reader. The results are as follows: Figure 12 As shown in Table 11, NbPDL1-1-Fc and NbPDL1-3-Fc can effectively bind to Cynomolgus PDL1 protein in a dose-dependent manner.

[0340] Table 11

[0341] NbPDL1-1-Fc NbPDL1-3-Fc NbPDL1-13-Fc NbPDL1-14-Fc <![CDATA[EC 50 (ug / ml)]]> 0.01625 0.009659 5.099 31.88

[0342] Example 11

[0343] In this embodiment, the SPR method (i.e., BIcore) was used to identify the binding ability of the PD-L1 single-domain antibody Fc fusion protein to PD-L1.

[0344] A single-concentration affinity screening test was used, in which all nanobodies were tested at the same single concentration, and the binding activity differences between the nanobodies were detected by comparing them in parallel. The capture antibody was coupled to the surface of a CM5 chip using a Human Antibody Capture Kit, and the kinetic parameters of the antibody-antigen interaction were determined using a multi-cycle kinetics method. The purified PDL1-his was diluted to 1 μg / mL with HBS-EP buffer and captured and immobilized on the chip surface at 25℃ and a flow rate of 5 μl / min for 1 min, with a capture response value of approximately 400 RU. The nanobodies PDL1-1-Fc, PDL1-3-Fc, PDL1-13-Fc, and PDL1-14-Fc were diluted to 1.5 μg / mL with HBS-EP buffer as the mobile phase. The test conditions were 25℃, 30 μl / min, binding for 60 s, dissociation for 120 s, and regeneration conditions were 10 mM glycine hydrochloride buffer at pH 2.5, 30 μl / min for 90 s. The results were then analyzed.

[0345] As shown in Table 12 and Figure 13 As shown, NbPDL1-1-Fc, NbPDL1-3-Fc, and NbPDL1-13-Fc can effectively bind PD-L1, with binding activity parameters of 8.2, 12.7, and 2.4, respectively, while NbPDL1-14 has no binding activity.

[0346] Table 12

[0347] sample Binding value (60s) RU Dissociation value (120s) RU Dissociation percentage % Combined with activity evaluation parameters NbPDL1-1-Fc 83.1 -8.4 10.1 8.2 NbPDL1-3-Fc 105.7 -8.8 8.3 12.7 NbPDL1-13-Fc 112.3 -51.8 46.1 2.4

[0348] Example 12

[0349] In this embodiment, the FACS method was used to detect the binding activity of the NbPDL1-1-Fc fusion protein and Raji-PDL1 / Raji cells.

[0350] Raji cells (Beijing Kangyuan Bochuang, KC-1886) carrying the full-length human PDL1 gene plasmid were purchased and cultured to the logarithmic growth phase. Cells were resuspended in FACS buffer and the cell density was adjusted to 3E6 / mL, with 100 μL seeded per well. The heavy chain single-domain antibody obtained in Example 7 (positive control group: KN035 containing Fc from KN035) was then diluted 5-fold with FACS buffer to 30 μg / mL, and cells were resuspended in 100 μL / well. Cells were incubated at 4°C for 60 min. Cells were washed three times with PBS, and the secondary antibody (THE) was added. TM His Tag Antibody [FITC], mAb, and Mouse were used to resuspend cells, and incubated at 4°C for 60 min. Cells were washed three times with PBS, and resuspended in 100 μL PBS per well. MFI was detected using Beckman Coulter / CytoFLEX. Data processing and parameter fitting were performed using Graphd Prism Software 5.0, and EC50 values ​​were calculated. The results are shown in Table 13. Figure 14 As shown, the NbPDL1-1-Fc fusion protein, the NbPDL1-3-Fc fusion protein, and the Fc-containing KN035 have comparable affinity.

[0351] Table 13

[0352] KN035-Fc NbPDL1-1-Fc NbPDL1-3-Fc <![CDATA[EC 50 (ug / ml)]]> 0.1392 0.2041 0.1968

[0353] Example 13

[0354] This embodiment involves the humanization of a PDL1-1 single-domain antibody.

[0355] Humanization was performed using a protein surface amino acid humanization method. The fully human sequence was aligned to IMGT using a reference homologous sequence, and protein surface amino acid humanization was carried out according to design principles. Site-directed mutations were performed on the amino acids to complete the antibody humanization.

[0356] The antibody PDL1-1 was humanized to obtain 20 humanized antibody strains.

[0357] Humanized sequence

[0358] PDL1-1v1

[0359] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS (SEQ ID NO. 23).

[0360] PDL1-1v2

[0361] QVQLVESGGGLVKPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.24)。

[0362] PDL1-1v3

[0363] QVQLLESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.25)。

[0364] PDL1-1v4

[0365] EVQLQESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.26)。

[0366] PDL1-1v5

[0367] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNVKNTVYLQMNSLRAEDTAVYYCAKGWGSTLWGQGTTVTVSS(SEQ ID NO.27)。

[0368] PDL1-1v6

[0369] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNSKNTVYLQMNSLKAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.28)。

[0370] PDL1-1v7

[0371] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.29)。

[0372] PDL1-1v8

[0373] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKGIWGSTLTGQGTTVTVSS(SEQ ID NO.30)。

[0374] PDL1-1v9

[0375] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTQVTVSS(SEQ ID NO.31)。

[0376] PDL1-1v10-

[0377] EVQLLESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIASNRNTVYAQSVRGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.32)。

[0378] PDL1-1v11

[0379] QVQLQESGGGLVKPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.33)。

[0380] PDL1-1v12

[0381] QVQLVESGGGLVQPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.34)。

[0382] PDL1-1v13

[0383] QVQLVESGGGLVKPGGSLTLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.35)。

[0384] PDL1-1v14

[0385] QVQLVESGGGLVKPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNVKNSVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.36)。

[0386] PDL1-1v15

[0387] QVQLVESGGGLVKPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.37)。

[0388] PDL1-1v16

[0389] QVQLVESGGGLVKPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNAKNSVYLQMNSLKAEDTAVYYCAKGIWGSTLWGQGTTVTVSS(SEQ ID NO.38)。

[0390] PDL1-1v17

[0391] QVQLVESGGGLVKPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNAKNSVYLQMNSLRPEDTAVYYCAKGIWGSTLWGQGTTVTVSS (SEQ ID NO. 39).

[0392] PDL1-1v18

[0393] QVQLVESGGGLVKPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAKGIWGSTLTGQGTTVTVSS (SEQ ID NO. 40).

[0394] PDL1-1v19

[0395] QVQLVESGGGLVKPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIDSNRNTVYAQSVRGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTQVTVSS (SEQ ID NO. 41).

[0396] PDL1-1v20

[0397] QVQLVESGGGLVKPGGSLRLSCAASGFTVSNSAVSWARQAPGKGLEWVSLIASNRNTVYAQSVRGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAKGIWGSTLWGQGTTVT VSS (SEQ ID NO. 42).

[0398] Example 14

[0399] This embodiment identifies the function of the PD-L1-1 humanized single-domain antibody.

[0400] 14.1 Preparation of PDL1-1 humanized single-domain antibody protein from mammalian cells

[0401] The humanized single-domain antibody sequence gene of PDL1-1 was synthesized into the vector pcDNA3.1. FreeStyle™ 293-F cells were transfected, and the cell density was adjusted to 1×10⁻⁶ cells / year. 6Cells / mL, transfection ratio (cells:medium = 20:1); Polyethylenimine Lineae (PEI) 2 μL / mL; plasmid 1 μg / mL; then add 80 μL of PEI transfection reagent, incubate at 25℃ for 10 min; add to FreeStyle™ 293-F cells, and culture in shake flasks at 37℃, 120 rpm, 5% CO2 level for 3.5 days. Based on the protein purification system, affinity purification was performed using HisTrap™ FF, and samples were collected after elution with 300 mM imidazole buffer. The supernatant was collected for SDS-PAGE electrophoresis analysis.

[0402] The results are as follows Figures 16A-16C As shown, non-reduced SDS-PAGE gel electrophoresis was used to identify the humanized single-domain antibody against PDL1-1. Figure 16A 1: PDL1-1v4, 2: PDL1-1v7, 3: PDL1-1v9, 4: PDL1-1v1, 5: PDL1-1v2, 6: Protein molecular weight standard; Figure 16B 1: PDL1-1v3, 2: PDL1-1v10, 3: PDL1-1v8, ​​4: PDL1-1v5, 5: PDL1-1v6, 6: Protein molecular weight standard, 7: PDL1-1v20, 8: PDL1-1v13, 9: PDL1-1v19, 10: PDL1-1v17; Figure 16C 1: PDL1-1v11, 2: PDL1-1v14, 3: PDL1-1v16, 4: PDL1-1v15, 5: PDL1-1v12, 6: PDL1-1v18, 7: Protein molecular weight standard. The results showed that the humanized single-domain antibody protein electrophoresis positions were as expected, the bands were clear, and the purity was high; no broken or degraded bands were observed.

[0403] 14.2 ELISA assay to identify the binding affinity of the humanized single-domain antibody to PD-L1.

[0404] Human PDL1-Fc recombinant protein was coated onto plates at 200 ng / well and incubated overnight at 4°C. Subsequently, 20 humanized single-domain antibodies obtained in section 14.1 were serially diluted 3-fold from 10 μg / mL in 12 steps, blocked at 37°C for 1 h, and then bound at 37°C for 1 h. After washing, HRP-labeled anti-his tag antibody (His Tag Antibody HRP, Sino Biological) was added, and the mixture was incubated at 37°C for 45 min. After washing, TMB substrate chromogenic solution was added, and the reaction was terminated with 1 M H2SO4. The absorbance was measured at 450 nm using a microplate reader. Graphd Prism Software 5.0 was used for data processing and parameter fitting to calculate EC50. 50Values ​​(Tables 14-17).

[0405] Table 14

[0406] PDL1-1V3 PDL1-1V10 PDL1-1V8 PDL1-1V6 PDL1-1V1 <![CDATA[EC 50 (ug / ml)]]> 0.03357 ～109.7 0.01084 0.05046 0.02747

[0407] Table 15

[0408] PDL1-1V4 PDL1-1V5 PDL1-1V7 PDL1-1V9 PDL1-1V1 <![CDATA[EC 50 (ug / ml)]]> 3.997 0.04781 0.028 0.02596 0.02508

[0409] Table 16

[0410] PDL1-1V11 PDL1-1V14 PDL1-1V16 PDL1-1V15 PDL1-1V2 EC50 (ug / ml) 0.02 0.0342 0.06285 0.04318 0.04228

[0411] Table 17

[0412]

[0413]

[0414] The binding activity of 20 purified humanized single-domain antibodies against PDL1-Fc protein was analyzed by ELISA. The results showed that ( Figures 17-20 The humanized single-domain antibody PD-L1-1 has high binding activity to the PDL1-Fc protein and exhibits typical dose-dependent characteristics.

[0415] 14.3 The forteBIO™ system was used to determine changes in the affinity of each mutant for its parents.

[0416] forteBIO TM Octet QK e The real-time detection system for biomolecular interactions is an eight-channel detection platform based on biomembrane interferometry (BLI). We utilize the high-throughput capability of this platform to compare and analyze the kinetic characteristics of interactions between different mutant antibody molecules and different mutant antigens. The specific method is as follows:

[0417] 1) Dilute the PDL1-Fc parent and each mutant to 100 nM with 10 mM PBS + 0.05% Tween-20 buffer, and add 200 μL / well to a 96-well plate for detection.

[0418] 2) Execute the Loading>Baseline>Asscociation>Disassociation process, setting Asscociation to 2 min and Disassociation to 3 min. Both the Baseline and Disassociation processes are performed in 10 mM PBS + 0.05% Tween-20 buffer. The Loading process corresponds to a specific concentration of PDL1-Fc dilution, and the Asscociation process corresponds to a specific concentration of mutant dilution.

[0419] 3) Data Analysis: Fitting analysis results yielded the affinity constant K for different PDL1-1 mutants. D .

[0420] The results are shown in Table 18. KD: equilibrium disassociation constant.

[0421] Table 18

[0422] Sample ID <![CDATA[K D (M)]]> 1v1 1.313E-08 1v2 1.428E-08 1v3 1.85E-08 1v4 1.316E-06 1v5 1.22E-08 1v6 2.205E-08 1v7 1.09E-08 1v8 5.901E-09 1v9 1.210E-08 1v10 3.05E-08 1v11 5.86E-09 1v12 3.890E-07 1v13 1.136E-08 1v14 7.37E-09 1v15 1.08E-08 1v16 1.43E-08 1v17 1.017E-08 1v18 4.158E-09 1v19 9.859E-09 1v20 3.25E-08

[0423] The affinity constants of the PDL1-Fc and PDL1-1 mutant recombinant proteins were determined using the capture method. The results of the determined affinity constants are shown in Table 18. The affinity of mutant 1v8 to PDL1-Fc was 5.901 nM, mutant 1v11 to PDL1-Fc was 5.86 nM, mutant 1v14 to PDL1-Fc was 7.37 nM, mutant 1v18 to PDL1-Fc was 4.158 nM, and mutant 1v19 to PDL1-Fc was 9.859 nM, which were significantly higher than those of the other mutants.

[0424] 14.4 ELISA assay to identify the blocking effect of PD-L1-1 humanized heavy chain single-domain antibody on the interaction between PD-1 and PD-L1.

[0425] PDL1-his protein was diluted to 2 μg / mL with PBS and incubated overnight at 4°C. The humanized heavy chain single-domain antibody obtained in 14.1 was serially diluted 13 times from 200 μg / mL in 2.5% PBST milk, with a final concentration of 50 μg / mL for each gradient. (The blank group contained no antibody or protein, only an equal volume of buffer). Blocking was performed at 37°C for 1 h, followed by binding at 37°C for 1 h. After washing, anti-IgG antibody (purchased from Zhongshan Jinqiao) was added, and the mixture was incubated at 37°C for 45 min. After washing, TMB substrate chromogenic solution was added, and the mixture was incubated at 25°C for 10 min. The reaction was terminated with 100 μL / well of 1M H2SO4. The absorbance was measured at 450 nm using a microplate reader. Data processing and parameter fitting were performed using Graphd Prism Software 5.0, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0426] The results of the blocking activity test are as follows Figure 21 As shown in Table 19, the competitive ELISA results indicate that humanized antibodies PDL1-1v8 and PDL1-1v18 can block the binding of PDL1-his protein to PD1-Fc, exhibiting similar blocking activity to PDL1-1 and showing a dose-dependent relationship. The IC50 values ​​for PDL1-1v8 and PDL1-1v18 are shown in Table 19. 50 The values ​​were 5.576 μg / mL and 5.216 μg / mL, respectively.

[0427] Table 19

[0428] PDL1-1v8 PDL1-1v11 PDL1-1v14 PDL1-1v18 PDL1-1v19 PDL1-1 <![CDATA[IC 50 (μg / mL)]]> 5.576 31.13 37.06 5.216 18.8 5.805

[0429] 14.5 Detection of the blocking activity of PDL1-1 humanized heavy chain single-domain antibody at the cellular level using FACS method

[0430] Raji cells carrying the full-length human PD-L1 gene plasmid were cultured to the logarithmic growth phase. Cells were resuspended in FACS buffer and the cell density was adjusted to 3E6 / mL, with 100 μL seeded per well. Cells were resuspended again with 50 μL / well of PD1-Fc and 50 μL / well of the test antibody, and incubated at 4°C for 60 min. Cells were washed three times with 200 μL / well of PBS, centrifuged at 300g / 5 min, and the supernatant was discarded. Secondary antibody SA-488 was diluted with FACS buffer, and cells were resuspended at 100 μL / well, and incubated at 4°C for 60 min. Cells were washed three times with 200 μL / well of PBS, centrifuged at 300g / 5 min, and the supernatant was discarded. Cells were resuspended in 100 μL / well of PBS and analyzed.

[0431] The results are as follows Figure 22As shown in Table 20, the humanized antibodies PDL1-1v8 and PDL1-1v18 can block the binding of Raji cells to PD1-Fc, exhibiting similar blocking activity to PDL1-1 and showing a dose-dependent relationship. The IC50 values ​​for PDL1-1v8 and PDL1-1v18 are... 50 The values ​​were 0.458 μg / mL and 0.6938 μg / mL, respectively.

[0432] Table 20

[0433]

[0434] 14.6 Activation of PBMCs by PDL1 Single-Domain Antibodies and Humanized Antibodies

[0435] Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of two healthy donors using density gradient centrifugation in human peripheral blood lymphocyte separation tubes (Dayou). Anti-CD3 antibody was diluted 1 / 2000 and added to the cells. PDL1-1, 1v8, 1v18, and KN035 were added at concentrations of 1 μg, 0.1 μg, 0.01 μg, and 0 μg, respectively. 1E5 PBMCs were added to each well and cultured for 5 days. The level of IFN-γ in the supernatant was detected using an interferon-gamma assay kit.

[0436] The results are as follows Figures 23-24 As shown, the PDL1 single-domain antibody PDL1-1 and the humanized antibodies 1v8 and 1v18 can enhance the secretion of interferon-gamma in PBMCs, that is, the PDL1 single-domain antibody enhances the activation of PBMCs, and this activation is dose-dependent.

[0437] 14.7 PDL1-1 humanized heavy chain single-domain antibody enhances the killing effect of effector cells on target cells.

[0438] Resuscitate Raji-PDL1 3E5 / mL cells, 50 μL per well, add NK92 cells at an effector-to-target ratio of 1:10 (effector-to-target killing assay shown in figure), add antibody 10 μg / mL, incubate overnight for 20 h, mix well, add CCK8 (Abixin), incubate at 37℃ for 4 h, and detect OD. 450 wavelength.

[0439] The results are as follows Figure 25 The humanized heavy chain single-domain antibody 1v18 of PDL1-1 showed better killing effect on target cells than KN035. The killing effect of 1v18 was comparable to that of KN035, but both were better than the unhumanized antibody PDL1-1.

[0440] Example 15

[0441] In this embodiment, a PD-L1-1 heavy chain single-domain antibody was fused with a mouse IL-12.

[0442] 15.1 PDL1-1 heavy chain single-domain antibody fusion with different forms of murine IL-12 protein

[0443] Fusion proteins of PD-L1-1 heavy chain single-domain antibody and murine IL-12 in different forms were constructed. Murine P35 / P40 and PD-L1-1 were linked together using (4GS)3 to construct forms ①-⑥, as shown in Table 21. Plasmids were extracted and co-transfected with the expressed protein at a 1:1 ratio to simulate the fusion protein form. Figure 26 Fusion 1-Fusion 4 consist of a single PDL1-1 heavy chain single-domain antibody fused with murine IL-12, while Fusion 5-Fusion 9 consist of two PDL1-1 heavy chain single-domain antibodies fused with murine IL-12.

[0444] Table 21

[0445]

[0446] The fusion protein was identified using non-reduced SDS-PAGE gel electrophoresis, and the results are as follows: Figure 27 As shown, 1: Fusion 1, 2: Fusion 2, 3: Fusion 3, 4: Fusion 4, 5: Fusion 5, 6: Fusion 6, 7; Fusion 7, 8: Fusion 8, 9: Fusion 9, 10: Protein molecular weight standards. The results show that the electrophoretic positions of the fusion proteins are as expected, the bands are clear, the purity is high, and no broken or degraded bands were observed.

[0447] 15.2 The binding ability of PD-L1-1 heavy chain single-domain antibody fused with IL-12 protein to PD-L1

[0448] Human PDL1-Fc recombinant protein was coated onto plates at 200 ng / well and incubated overnight at 4°C. Subsequently, the PD-L1-1 humanized heavy chain single-domain antibody obtained in section 15.1 was fused with IL-12 protein at 50 μg / mL, serially diluted 3-fold in 12 gradients. The PDL1-1 heavy chain single-domain antibody was also serially diluted 3-fold in 10 μg / mL in 12 gradients. Blocking was performed at 37°C for 1 h, followed by binding at 37°C for 1 h. After washing, HRP-labeled anti-his tag antibody (His Tag Antibody HRP, Sino Biological) was added, and the mixture was incubated at 37°C for 45 min. After washing, TMB substrate chromogenic buffer was added, and the reaction was terminated with 1 M H2SO4. The microplate was measured at 450 nm using a microplate reader. nm The light absorption value at wavelength. Graphd Prism Software 5.0 is used for data processing and parameter fitting to calculate EC. 50 value.

[0449] The results are as follows Figures 28-29As shown in Tables 22 and 23, ELISA detection showed that the binding activity of fusion 2 and fusion 4 proteins was relatively low, the binding activity of fusion 7 and fusion 9 proteins was relatively high, and the binding activity of other fusion proteins was comparable.

[0450] Table 22

[0451]

[0452] 15.3 The forteBIO™ system was used to determine changes in the affinity of PD-L1-1 heavy chain single-domain antibody fusion with IL-12 protein and PD-L1.

[0453] forteBIO TM Octet QK e The real-time detection system for biomolecular interactions is an eight-channel detection platform based on biomembrane interferometry (BLI). We utilize the high-throughput capability of this platform to compare and analyze the kinetic characteristics of interactions between different mutant antibody molecules and different mutant antigens. The specific method is as follows:

[0454] 1) Dilute the PDL1-Fc parent and fusion protein to 100 nM with 10 mM PBS + 0.05% Tween-20 buffer, and add 200 μL / well to a 96-well plate for detection.

[0455] 2) Execute the Loading>Baseline>Asscociation>Disassociation process, setting Asscociation to 2 min and Disassociation to 3 min. Both the Baseline and Disassociation processes are performed in 10 mM PBS + 0.05% Tween-20 buffer. The Loading process corresponds to a specific concentration of PDL1-Fc dilution, and the Asscociation process corresponds to a specific concentration of mutant dilution.

[0456] 3) Data Analysis: Fitting analysis results yielded the equilibrium dissociation constant K for different fusion proteins. D Calculated as a ratio kdis / kon.

[0457] The results are as follows Figures 30-31 As shown in Tables 24 and 25, forteBIO level detection showed that fusion 9 had the lowest affinity for PDL1 protein (0.0589 nM) and dissociation rate (kdis), while fusions 5-8 were comparable. The equilibrium dissociation constants K for fusions 1 and 2 were also shown. D The equilibrium dissociation constant K of fusion 3, 4 and PDL1-1 is relatively high. D quite.

[0458] Table 24

[0459] KD(M) kon(1 / Ms) kdis(1 / s) A12 Fusion 1 1.35E-08 4.77E+05 6.46E-03 B12 Fusion 2 1.15E-08 6.39E+05 7.35E-03 C12 Fusion 3 4.05E-09 9.24E+05 3.75E-03 D12 Fusion 4 7.40E-09 6.47E+05 4.78E-03 E12 PDL1-1 3.39E-09 1.66E+06 5.63E-03

[0460] Table 25

[0461] KD(M) kon(1 / Ms) kdis(1 / s) A12 Fusion 5 2.39E-10 6.31E+05 1.51E-04 B12 Fusion 6 1.31E-10 7.13E+05 9.36E-05 C12 Fusion 7 2.13E-10 7.48E+05 1.60E-04 D12 Fusion 8 1.58E-10 9.25E+05 1.46E-04 E12 Fusion 9 5.89E-11 9.69E+05 5.71E-05 F12 PDL1-1 2.96E-09 1.80E+06 5.32E-03

[0462] 15.4 Activation effect of PD-L1-1 heavy chain single-domain antibody fused with IL-12 protein on NK-92 cells

[0463] 100 μL of PD-L1-1 heavy chain single-domain antibody fusion IL-12 protein and WHO IL-12 standard working solution were respectively added to NK-92 cells prepared in 96-well plates to achieve final titers of 500.00 U / mL, 166.67 U / mL, 55.56 U / mL, 18.52 U / mL, 6.18 U / mL, 2.06 U / mL, 0.685 U / mL, and 0.23 U / mL, respectively. Each concentration was tested in duplicate, and the final cell concentration was 1 × 10⁻⁶ cells / well. 5 Cells were stimulated at (24±2) h and centrifuged at 1000 rpm for 5 min in a 96-well plate. The plate was diluted 60-fold with 1X Dilution Buffer R from the ELISA Kit, and 5 μL of the supernatant was added to 295 μL of the standard dilution buffer. The plate was vortexed at high speed for 5 min. 50 μL of 1X Biotinylated antibody was added to each well, and the plate was vortexed for 30 s to mix. The plate was incubated at room temperature for 120 min. After washing, 100 μL of 1X Steptavidin-HRP was added, and the plate was incubated at room temperature for 20 min. After washing, 100 μL of TMB was added, and the plate was incubated at 25°C in the dark for 18 min. The reaction was stopped by adding 100 μL of stop solution. Cells were then analyzed using a SpectraMax M2 microplate reader at 450 nm. nm OD values ​​were calculated, and data were processed using GraphPad Prism 6 software. OD values ​​were plotted on the ordinate, and dilution factor on the x-axis. A four-parameter fit was used to obtain the activity curve, Top value, Bottom value, and R-value. 2 Value, R 2 Value ≥ 0.95.

[0464] Table 26

[0465]

[0466]

[0467] As shown in Table 26, under the same molar mass conditions, the activation effect of mouse IL12 on NK-92 cells was 1, the activation effect of the fusion protein on NK-92 cells decreased by 2.5 times to fusion 4, and the activation effect of other fusion proteins increased to varying degrees. The nanobody PDL1-1 in the fusion protein had no activation effect on NK92. Therefore, it can be seen that there is a synergistic effect between mouse IL12 and heavy chain single domain antibody PDL1-1 in the fusion protein, which promotes the activation of NK92.

[0468] 15.5 PD-L1-1 heavy chain single-domain antibody fusion with IL-12 protein enhances the killing effect of effector cells on target cells.

[0469] MDAMB231 3E5 / mL cells were seeded in 50 μL per well. NK cells were added at effector-to-target ratios of 1:2.5 and 1:5. Antibody was added at 5 μg / mL. The cells were cultured overnight for 20 h. After mixing, CCK8 (Abixin) was added, and the cells were incubated at 37°C for 4 h. OD was then measured. 450 Wavelength, calculate lethality.

[0470] The results are as follows Figure 32 The PD-L1-1 heavy chain single-domain antibody fusion with IL-12 protein fusion 4, fusion 7 and fusion 8 showed stronger killing effect on target cells than murine IL-12 and were also superior to the PD-L1-1 heavy chain single-domain antibody, demonstrating the synergistic effect of murine IL12 and heavy chain single-domain antibody PDL1-1 in the fusion protein.

[0471] In summary, the present invention has screened and prepared anti-PD-L1 nanobodies, which have high affinity and specificity, can efficiently target PD-L1, have a simple structure, are easy to prepare, and can be further used to prepare PD-L1 binding molecules and immunoconjugates, etc., and have broad application prospects in the fields of immune detection and tumor treatment.

[0472] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-PD-L1 nanobody, characterized in that, The complementarity-determining region of the anti-PD-L1 nanobody is selected from any one of the following groups: (1) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; (2) The amino acid sequences are CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

2. The anti-PD-L1 nanobody according to claim 1, characterized in that, The amino acid sequence of the anti-PD-L1 nanobody is selected from any one of the sequences shown in SEQ ID NO: 13 or 14 or sequences with at least 80% homology to them.

3. A humanized antibody or an active fragment thereof, wherein the amino acid sequence of the humanized antibody is shown in any one of SEQ ID NO: 23-42.

4. The humanized antibody or its active fragment as described in claim 3, characterized in that, The amino acid sequence of the humanized antibody is shown in any one of SEQ ID NO: 30, 33, 36, 40, 41.

5. The humanized antibody or its active fragment as described in claim 4, characterized in that, The amino acid sequence of the humanized antibody is shown in SEQ ID NO: 30 or 40.

6. A chimeric antigen receptor (CAR) comprising an extracellular domain, the extracellular domain comprising an anti-PD-L1 nanobody as described in claim 1 or 2, or a humanized antibody as described in any one of claims 3-5.

7. A fusion protein, said fusion protein having: (Z1) First protein, the first protein comprising: The anti-PL-L1 nanobody as described in claim 1 or 2, or the humanized antibody or its active fragment as described in any one of claims 3-5; (Z2) A second protein, which includes an Fc fragment or cytokine; and (Z3) An optional linker located between the first protein and the second protein.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains the nucleic acid encoding the anti-PD-L1 nanobody of claim 1 or 2, the humanized antibody or its active fragment of any one of claims 3-5, the chimeric antigen receptor of claim 6, or the fusion protein of claim 7.

9. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 8.

10. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecule of claim 8 or the recombinant expression vector of claim 9.

11. A method for preparing the anti-PD-L1 nanobody according to claim 1 or 2, characterized in that, The method includes: The gene encoding the anti-PD-L1 nanobody according to claim 1 or 2 is inserted into an expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell for culture, and the anti-PD-L1 nanobody is isolated from the culture.

12. The use of the anti-PD-L1 nanobody according to claim 1 or 2 in the targeted binding of PD-L1, wherein the use is for purposes other than disease diagnosis and / or treatment.

13. The use of the anti-PD-L1 nanobody of claim 1 or 2, the humanized antibody or its active fragment of any one of claims 3-5, the chimeric antigen receptor of claim 6, the fusion protein of claim 7, the nucleic acid molecule of claim 8, the recombinant expression vector of claim 9, or the recombinant cell of claim 10 in the preparation of a drug for targeted therapy of solid tumors.

14. The application as described in claim 13, characterized in that, The solid tumors include solid tumors that express PD-L1.

15. The application as described in claim 14, characterized in that, The solid tumors expressing PD-L1 include any one of gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, ovarian cancer, esophageal cancer, bladder cancer, melanoma, or Merkel cell carcinoma.

16. A coupling, characterized in that, The conjugate contains the anti-PD-L1 nanobody of claim 1 or 2, the humanized antibody or its active fragment of any one of claims 3-5, the chimeric antigen receptor of claim 6, the fusion protein of claim 7, and the conjugate portion. The coupling portion is selected from any one or a combination of at least two of fluorescein, radioisotope, or contrast agent.

17. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one or a combination of at least two of the following: the anti-PD-L1 nanobody of claim 1 or 2, the humanized antibody or its active fragment of any one of claims 3-5, the chimeric antigen receptor of claim 6, the fusion protein of claim 7, the nucleic acid molecule of claim 8, the recombinant expression vector of claim 9, the recombinant cell of claim 10, or the conjugate of claim 16.

18. An engineered immune cell, said engineered immune cell containing the recombinant expression vector of claim 9 or having an exogenous nucleic acid molecule of claim 8 integrated into its genome, or expressing the chimeric antigen receptor of claim 6.

19. A kit comprising: (1) A first container containing the anti-PD-L1 nanobody of claim 1 or 2, the humanized antibody or its active fragment of any one of claims 3-5, the fusion protein of claim 7, or the conjugate of claim 16, or a combination thereof; and / or (2) A second container, wherein the second container contains a secondary antibody against the contents of the first container; or, The kit contains a detection plate comprising a substrate and a test strip, wherein the test strip contains the anti-PD-L1 nanobody of claim 1 or 2, the humanized antibody or its active fragment of any one of claims 3-5, the fusion protein of claim 7, or the conjugate of claim 16, or a combination thereof.

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