Anti-PD-L1 and PD-L2 antibodies and their derivatives and uses

Abstract

This invention provides PD-L1 nanobodies, PD-L2 nanobodies, and bispecific antibodies containing both PD-L1 and PD-L2 nanobodies. These bispecific antibodies can simultaneously block the PD-1 / PD-L1 and PD-1 / PD-L2 pathways. The bispecific antibodies of this invention can restore T cell activity, enhance immune responses, and more effectively improve the inhibitory effect on tumor occurrence and development.

Description

Technical Field

[0001] This invention relates to the fields of biomedical or biopharmaceutical technology, and more specifically to an anti-PD-L1 and anti-PD-L2 antibody, its derivatives, and their uses. Background Technology

[0002] Programmed death-1 (PD-1), also known as CD279, is a member of the CD28 family. Its cytoplasmic region contains two tyrosine residues: one near the N-terminus in the immunoreceptor tyrosine-based inhibitory motif (ITIM), and the other near the C-terminus in the immunoreceptor tyrosine-based switch motif (ITSM). PD-1 is primarily expressed on the surface of activated T lymphocytes, B lymphocytes, and macrophages. Under normal conditions, PD-1 can inhibit T lymphocyte function and promote Treg function, thereby suppressing autoimmune responses and preventing the occurrence of autoimmune diseases.

[0003] Programmed death 1 ligand 1 (PD-L1), also known as CD274, is a member of the B7 family and is the ligand for PD-1. PD-L1 is a type I transmembrane protein, consisting of 290 amino acids, including an IgV-like region, an IgC-like region, a transmembrane hydrophobic region, and an intracellular region composed of 30 amino acids.

[0004] Unlike other B7 family molecules, PD-L1 plays a negative role in regulating immune responses. Studies have found that PD-L1 is primarily expressed in activated T cells, B cells, macrophages, and dendritic cells. Besides lymphocytes, PD-L1 is also expressed in endothelial cells of various tissues such as the thymus, heart, and placenta, as well as in various non-lymphatic cell lines such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, and head and neck cancer. PD-L1 has a broad role in regulating autoreactive T and B cells and immune tolerance, and it plays a role in peripheral tissue T and B cell responses. High expression of PD-L1 on tumor cells is associated with poor prognosis in cancer patients.

[0005] However, in tumorigenesis, the binding of PD-L1 expressed by tumor cells to PD-1 can promote tumor immune escape through its inhibitory effect on lymphocytes. The binding of PD-L1 to PD-1 can lead to a variety of biological changes and induce immune regulation, such as inhibiting lymphocyte proliferation and activation, inhibiting the differentiation of CD4+ T cells into Th1 and Th17 cells, and inhibiting the release of inflammatory cytokines.

[0006] Programmed death 1 ligand 2 (PD-L2), also known as CD273 or B7-DC, is another important ligand for PD-1. The interaction between PD-1 and PD-L2 inhibits the activation and proliferation of CD4+ T cells, reduces the release of cytokines, and increases tumor immune escape. Simultaneously blocking the interaction between PD-1 and both PD-L1 and PD-L2 can effectively relieve the suppression of the immune system by PD-1 pathway activation, activate the body's own immune system, and kill tumors.

[0007] However, to date, there are no publicly available bispecific single-domain antibodies that simultaneously target PD-L1 / PD-L2. As an emerging force in next-generation antibody diagnostics and therapy, single-domain antibodies have the characteristics of high stability, good water solubility, simple humanization, high targeting, and strong penetration. They play an unimaginable role in immune experiments, diagnosis, and therapy.

[0008] Therefore, there is an urgent need in this field for a bispecific single-domain antibody that can simultaneously target PD-L1 and PD-L2. Summary of the Invention

[0009] The purpose of this invention is to provide a bispecific antibody that can simultaneously target PD-L1 and PD-L2.

[0010] In a first aspect of the present invention, an anti-PD-L2 nanobody is provided, wherein the complementarity-determining regions (CDRs) of the VHH chain of the PD-L2 nanobody are composed of the following:

[0011] The amino acid sequence is as shown in SEQ ID NO:57 for CDR1; the amino acid sequence is as shown in SEQ ID NO:58 for CDR2; and the amino acid sequence is as shown in SEQ ID NO:59 for CDR3; or

[0012] The amino acid sequence of CDR1 as shown in SEQ ID NO:60; the amino acid sequence of CDR2 as shown in SEQ ID NO:61; and the amino acid sequence of CDR3 as shown in SEQ ID NO:62;

[0013] Alternatively, the amino acid sequence of the VHH chain of the anti-PD-L2 nanobody is as shown in SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 17 or 18.

[0014] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0015] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L2 nanobody is as shown in SEQ ID NO:3 (i.e., D-Na-96), 16 (i.e., HZ-D-Na-96-1), 12 (i.e., D-Ye-29) or 15 (i.e., HZ-D-Ye-29-3).

[0016] In another preferred embodiment, the anti-PD-L2 nanobody is humanized, and the amino acid sequence of the VHH chain of the anti-PD-L2 nanobody is as shown in 16 (i.e., HZ-D-Na-96-1) or 15 (i.e., HZ-D-Ye-29-3).

[0017] In another preferred embodiment, the PD-L2 nanobody can block the interaction between PD-1 and PD-L2.

[0018] In a second aspect of the invention, an anti-PD-L1 nanobody is provided, wherein the complementarity-determining regions (CDRs) of the VHH chain of the PD-L1 nanobody are composed of the following:

[0019] The amino acid sequences are as shown in SEQ ID NO:63 for CDR1; as shown in SEQ ID NO:64 for CDR2; and as shown in SEQ ID NO:65 for CDR3.

[0020] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0021] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L1 nanobody is as shown in SEQ ID NO:19 (i.e., K-Yr-13&14-02), 20 (i.e., K-Yr-13&14-09), 21 (i.e., K-Yr-13&14-16), or 22 (i.e., HZ-K-Yr-13&14-02-3).

[0022] In another preferred embodiment, the anti-PD-L1 nanobody is humanized, and the amino acid sequence of the VHH chain of the anti-PD-L1 nanobody is shown in SEQ ID NO:22 (i.e., HZ-K-Yr-13&14-02-3).

[0023] In another preferred embodiment, the PD-L1 nanobody can block the interaction between PD-1 and PD-L1.

[0024] In a third aspect of the present invention, a bispecific antibody is provided, the bispecific antibody comprising: an anti-PD-L1 nanobody as described in the second aspect of the present invention and an anti-PD-L2 nanobody as described in the first aspect of the present invention.

[0025] In another preferred embodiment, the bispecific antibody comprises 1-3 anti-PD-L1 nanobodies, preferably 1 or 2 anti-PD-L1 nanobodies.

[0026] In another preferred embodiment, the bispecific antibody comprises 1-3 anti-PD-L2 nanobodies, preferably 1 or 2 anti-PD-L2 nanobodies.

[0027] In another preferred embodiment, the bispecific antibody further comprises an Fc segment.

[0028] In another preferred embodiment, the Fc segment of the bispecific antibody is selected from the group consisting of the human IgG domain, the CH1+CL1 domain, or a combination thereof.

[0029] In another preferred embodiment, the human IgG domain is a modified mutant IgG domain, preferably an LALA mutant IgG domain.

[0030] In another preferred embodiment, the bispecific antibody contains a polypeptide with the structure shown in Formula I or Formula II, or simultaneously contains a polypeptide with the structures shown in Formula III and Formula IV.

[0031] A-L1-Fc1-L2-B (Formula I)

[0032] A-L3-B-L4-Fc1 (Formula II)

[0033] A-L5-Fc2-L6-Fc1 (Formula III)

[0034] B-L7-Fc2 (Formula IV)

[0035] in,

[0036] A and B are each independently an anti-PD-L1 nanobody as described in the second aspect of the present invention or an anti-PD-L2 nanobody as described in the first aspect of the present invention, and A and B are different antibodies;

[0037] L1, L2, L3, and L4 are each independently peptide bond or linker element;

[0038] Fc1 and Fc2 are both Fc segments of the antibody; where Fc1 is the human IgG domain (preferably the LALA mutant IgG domain) and Fc2 is the CH1+CL domain.

[0039] "-" represents a peptide bond.

[0040] In another preferred embodiment, the bispecific antibody has a polypeptide sequence as shown in Formula III and Formula IV, and the polypeptides of Formula III and Formula IV form a heterodimer a through disulfide bonds.

[0041] In another preferred embodiment, the bispecific antibody has a polypeptide with the structure shown in Formula I, and the polypeptide forms a homodimer i through disulfide bond interactions between Fc1.

[0042] In another preferred embodiment, the bispecific antibody has a polypeptide with the structure shown in Formula II, and the polypeptide forms a homodimer ii through disulfide bond interactions between Fc1.

[0043] In another preferred embodiment, the bispecific antibody has a polypeptide sequence as shown in Formula III and Formula IV, and the polypeptides of Formula III and Formula IV form a heterodimer a through disulfide bond interaction between Fc2, and the heterodimer ii forms a homodimer iii through disulfide bond interaction between Fc1.

[0044] In another preferred embodiment, the amino acid sequence of the PD-L1 nanobody is as shown in SEQ ID NO: 19, 20, 21 or 22, preferably SEQ ID NO: 22.

[0045] In another preferred embodiment, the bispecific antibody further includes the VHH chain of other anti-PD-L2 nanobodies, the amino acid sequences of which are shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0046] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L2 nanobody is as shown in SEQ ID NO:3, 16, 12 or 15, preferably SEQ ID NO:16 or 15.

[0047] In another preferred embodiment, the sequence of the connector elements is (4GS)n, where n is a positive integer (e.g., 1, 2, 3, 4, 5 or 6), preferably n = 4.

[0048] In another preferred embodiment, the sequence of the connector element is as shown in SEQ ID NO: 27, or has ≥85% (preferably 90%, more preferably 95%) sequence identity with the sequence shown in SEQ ID NO: 27.

[0049] In another preferred embodiment, the amino acid sequence of the human IgG domain of the LALA mutant is as shown in SEQ ID NO: 28, or has ≥85% (preferably 90%, more preferably 95%) sequence identity with the sequence shown in SEQ ID NO: 28.

[0050] In another preferred embodiment, the amino acid sequence of the CH1 domain is as shown in SEQ ID NO: 29, or has ≥85% (preferably 90%, more preferably 95%) sequence identity with the sequence shown in SEQ ID NO: 29.

[0051] In another preferred embodiment, the amino acid sequence of the CL domain is as shown in SEQ ID NO: 30, or has ≥85% (preferably 90%, more preferably 95%) sequence identity with the sequence shown in SEQ ID NO: 30.

[0052] In another preferred embodiment, the amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 23 or 24.

[0053] In another preferred embodiment, the bispecific antibody contains polypeptides with structures as shown in Formula III and Formula IV, wherein the amino acid sequence of the polypeptide of Formula III is shown in SEQ ID NO: 25, and the amino acid sequence of the polypeptide of Formula IV is shown in SEQ ID NO: 26.

[0054] In a fourth aspect of the invention, a separated polynucleotide is provided, the polynucleotide encoding an anti-PD-L2 nanobody as described in the first aspect of the invention, an anti-PD-L1 nanobody as described in the second aspect of the invention, or a bispecific antibody as described in the third aspect of the invention.

[0055] In another preferred embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48.

[0056] In another preferred embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO:33 (i.e., D-Na-96), 46 (i.e., HZ-D-Na-96-1), 42 (i.e., D-Ye-29) or 45 (i.e., HZ-D-Ye-29); preferably SEQ ID NO:46 or 45.

[0057] In another preferred embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO:49 (i.e., K-Yr-13&14-02), 50 (i.e., K-Yr-13&14-09), 51 (i.e., K-Yr-13&14-16), or 52 (i.e., HZ-K-Yr-13&14-02-3); preferably SEQ ID NO:52.

[0058] In another preferred embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO:53 or 54.

[0059] In another preferred embodiment, the polynucleotide comprises a first polynucleotide with the sequence shown in SEQ ID NO:55 and a second polynucleotide with the sequence shown in SEQ ID NO:56.

[0060] In a fifth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the fourth aspect of the invention.

[0061] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof; preferably, the expression vector includes viral vectors such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0062] In a sixth aspect of the invention, a host cell is provided, the host cell containing a vector as described in the fifth aspect of the invention, or having a polynucleotide as described in the fourth aspect of the invention integrated into its genome;

[0063] Alternatively, the host cells express anti-PD-L2 nanobodies as described in the first aspect of the present invention, anti-PD-L1 nanobodies as described in the second aspect of the present invention, or bispecific antibodies as described in the third aspect of the present invention.

[0064] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0065] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0066] In a seventh aspect of the invention, a method for generating anti-PD-L1 nanobodies, anti-PD-L2 nanobodies, or bispecific antibodies is provided, comprising the steps of:

[0067] (a) Under suitable conditions, host cells as described in the sixth aspect of the present invention are cultured to obtain a culture containing the said anti-PD-L1 nanobody, anti-PD-L2 nanobody, or bispecific antibody; and

[0068] (b) The culture obtained in step (a) is purified and / or separated to obtain the anti-PD-L1 nanobody, anti-PD-L2 nanobody, or bispecific antibody.

[0069] In another preferred embodiment, the purification can be performed by purifying and separating the target antibody using a protein A affinity column.

[0070] In another preferred embodiment, the purity of the purified and separated target antibody is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.

[0071] In an eighth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0072] (a) an anti-PD-L2 nanobody as described in the first aspect of the present invention, an anti-PD-L1 nanobody as described in the second aspect of the present invention, or a bispecific antibody as described in the third aspect of the present invention; and

[0073] (b) The coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0074] In another preferred embodiment, the radionuclide includes:

[0075] (i) a diagnostic isotope selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and / or

[0076] (ii) Therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or combinations thereof.

[0077] In another preferred embodiment, the coupling portion is a drug or toxin.

[0078] In another preferred embodiment, the drug is a cytotoxic drug.

[0079] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0080] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and microtubule inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0081] In another preferred embodiment, the toxin is selected from the group consisting of:

[0082] Otostatins (e.g., otostatin E, otostatin F, MMAE, and MMAF), chlortetracycline, tebuconazole, pectin, pectin A-chain, cobustatin, docalimicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxetine dione, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, absinthecin, absinthecin A-chain, saccharin A-chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachiin, Sapaonaria (Officialis) inhibitors, glucocorticoids, or combinations thereof.

[0083] In another preferred embodiment, the coupling portion is a detectable marker.

[0084] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanoran, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (such as DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), and chemotherapeutic agents (such as cisplatin).

[0085] In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) anti-PD-L2 nanobody as described in the first aspect of the invention, an anti-PD-L1 nanobody as described in the second aspect of the invention, or a bispecific antibody as described in the third aspect of the invention.

[0086] In another preferred embodiment, the term "multivalent" refers to the inclusion of multiple repeating anti-PD-L2 nanobodies, anti-PD-L1 nanobodies, or bispecific antibodies in the amino acid sequence of the immunoconjugate as described in the first aspect of the present invention, the second aspect of the present invention, or the third aspect of the present invention.

[0087] In a ninth aspect of the present invention, the use of an anti-PD-L2 nanobody as described in the first aspect of the present invention, an anti-PD-L1 nanobody as described in the second aspect of the present invention, or a bispecific antibody as described in the third aspect of the present invention, or an immunoconjugate as described in the eighth aspect of the present invention, for the preparation of pharmaceuticals, reagents, detection plates, or kits; wherein the reagents, detection plates, or kits are used to: detect PD-L1 and / or PD-L2 in a sample; wherein the pharmaceuticals are used to treat or prevent tumors expressing PD-L1 (i.e., PD-L1 positive) or tumors expressing PD-L2.

[0088] In another preferred embodiment, the coupling portion of the immunoconjugate is a diagnostic isotope.

[0089] In another preferred embodiment, the reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0090] In another preferred embodiment, the reagent for detecting PD-L1 and / or PD-L2 in the sample is a contrast agent for (in vivo) detection of PD-L1 and / or PD-L2 molecules.

[0091] In another preferred embodiment, the detection is either in vivo or in vitro.

[0092] In another preferred embodiment, the detection includes flow cytometry and cell immunofluorescence detection.

[0093] In another preferred embodiment, the agent is used to block the interaction between PD-1 and PD-L1, and / or to block the interaction between PD-1 and PD-L2.

[0094] In another preferred embodiment, the tumors include, but are not limited to: acute myeloid leukemia, chronic myeloid leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colon cancer, stomach cancer, liver cancer, leukemia, kidney tumors, lung cancer, small intestine cancer, bone cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumors, and bladder tumors.

[0095] In a tenth aspect of the invention, a pharmaceutical composition is provided comprising: (i) an anti-PD-L2 nanobody as described in the first aspect of the invention, an anti-PD-L1 nanobody as described in the second aspect of the invention, or a bispecific antibody as described in the third aspect of the invention, or an immunoconjugate as described in the eighth aspect of the invention; and (ii) a pharmaceutically acceptable carrier.

[0096] In another preferred embodiment, the conjugation portion of the immunoconjugate is a drug, a toxin, and / or a therapeutic isotope.

[0097] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating tumors, such as cytotoxic drugs.

[0098] In another preferred embodiment, the other drugs for treating tumors include paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, and pembrolizumab.

[0099] In another preferred embodiment, the drug is used to block the interaction between PD-1 and PD-L1, and / or to block the interaction between PD-1 and PD-L2.

[0100] In another preferred embodiment, the pharmaceutical composition is used to block the PD-1 / PD-L1 and / or PD-1 / PD-L2 signaling pathways.

[0101] In another preferred embodiment, the pharmaceutical composition is used to treat tumors that express PD-L1 protein (i.e., PD-L1 positive) and / or express PD-L2 protein (i.e., PD-L2 positive).

[0102] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0103] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for the prevention and treatment of tumors.

[0104] In an eleventh aspect of the present invention, one or more uses of the anti-PD-L2 nanobody as described in the first aspect of the present invention, the anti-PD-L1 nanobody as described in the second aspect of the present invention, or the bispecific antibody as described in the third aspect of the present invention, selected from the group consisting of:

[0105] (i) for the detection of human PD-L1 and / or PD-L2 molecules; (ii) for flow cytometry detection; (iii) for cellular immunofluorescence detection; (iv) for tumor treatment; (v) for tumor diagnosis; (vi) for blocking the interaction between PD-1 and PD-L1; and / or (vii) for blocking the interaction between PD-L2 and PD-1.

[0106] In another preferred embodiment, the tumor is a tumor that expresses PD-L1 protein (i.e., PD-L1 positive) and / or expresses PD-L2 protein (i.e., PD-L2 positive).

[0107] In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

[0108] In another preferred embodiment, the antibody is an antibody against PD-L1 and / or PD-L2.

[0109] In a twelfth aspect of the invention, a recombinant protein is provided, the recombinant protein having: (i) an anti-PD-L2 nanobody as described in the first aspect of the invention, an anti-PD-L1 nanobody as described in the second aspect of the invention, or a bispecific antibody as described in the third aspect of the invention; and (ii) an optional tag sequence for co-expression and / or purification.

[0110] In another preferred embodiment, the tag sequence includes a 6His tag, an HA tag, and an Fc tag.

[0111] In another preferred embodiment, the recombinant protein specifically binds to PD-L1 and / or PD-L2.

[0112] In a thirteenth aspect of the present invention, a method for detecting PD-L1 and / or PD-L2 in a sample is provided, the method comprising the steps of: (1) contacting the sample with an anti-PD-L2 nanobody as described in the first aspect of the present invention, an anti-PD-L1 nanobody as described in the second aspect of the present invention, or a bispecific antibody as described in the third aspect of the present invention; and (2) detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of PD-L1 and / or PD-L2 in the sample.

[0113] In a fourteenth aspect of the present invention, a method for treating a disease is provided, the method comprising administering to a desired subject an anti-PD-L2 nanobody as described in a first aspect of the present invention, an anti-PD-L1 nanobody as described in a second aspect of the present invention, a bispecific antibody as described in a third aspect of the present invention, an immunoconjugate as described in an eighth aspect of the present invention, or a pharmaceutical composition as described in a tenth aspect of the present invention.

[0114] In another preferred embodiment, the object includes mammals, preferably humans.

[0115] In a fifteenth aspect of the invention, a PD-L1 and / or PD-L2 detection reagent is provided, the detection reagent comprising an immunoconjugate as described in an eighth aspect of the invention and a detection-acceptable carrier.

[0116] In another preferred embodiment, the coupling portion of the immunoconjugate is a diagnostic isotope.

[0117] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0118] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0119] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0120] In another preferred embodiment, the test reagent is in liquid or powder form (e.g., aqueous solution, injection, lyophilized powder, tablet, lozenge, inhaler).

[0121] In a sixteenth aspect of the invention, a kit for detecting PD-L1 and / or PD-L2 is provided, the kit containing an immunoconjugate as described in an eighth aspect of the invention or a detection reagent as described in a fifteenth aspect of the invention, and instructions for use.

[0122] In another preferred embodiment, the specification states that the kit is used for non-invasive detection of PD-L1 and / or PD-L2 expression in a test subject.

[0123] In another preferred embodiment, the kit is used for the detection of tumors expressing PD-L1 protein (i.e., PD-L1 positive) and / or expressing PD-L2 protein (i.e., PD-L2 positive).

[0124] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0125] Figure 1 The binding activity of the purified anti-PD-L2 antibody to CHO-hPD-L2 cells was demonstrated.

[0126] Figure 2 The purified anti-PD-L2 antibody was shown to block the binding of PD-L2 to PD-1.

[0127] Figure 3 The binding activity of the D-Na-96 humanized antibody to CHO-hPD-L2 cells was demonstrated.

[0128] Figure 4 The study demonstrated the blocking effect of the D-Na-96 humanized antibody on the binding of PD-L2 and PD-1.

[0129] Figure 5 The binding activity of the humanized anti-PD-L1 nanobody to CHO-hPD-L1 cells was demonstrated.

[0130] Figure 6 The schematic diagrams of the structures of three different forms of anti-PD-L1 / PD-L2 bispecific antibodies are shown.

[0131] Figure 7 shows the interaction between the anti-PD-L1 / PD-L2 bispecific antibody of the present invention and CHO-hPD-L1 cells ( Figure 7A ) or CHO-hPD-L2 cells ( Figure 7B () binding activity.

[0132] Figure 8 shows the binding of the anti-PD-L1 / PD-L2 bispecific antibody of the present invention to PD-L1 and PD-1. Figure 8A ) and the binding of PD-L2 and PD-1 ( Figure 8B The blocking effect of ).

[0133] Figure 9 The study demonstrated the blocking effect of the anti-PD-L1 / PD-L2 bispecific antibody on the PDL1 / PDL2 / PD1 / luc signaling pathway. Detailed Implementation

[0134] Through extensive and in-depth research and screening, the inventors have developed, for the first time, a bispecific antibody against PD-L1 / PD-L2, comprising an anti-PD-L1 single-domain antibody and an anti-PD-L2 single-domain antibody. Experiments show that the bispecific antibody of this invention exhibits good binding activity to both PD-L1 and PD-L2 molecules, while simultaneously blocking the interaction between PD-1 and PD-L1, as well as the interaction between PD-1 and PD-L2. Furthermore, it can simultaneously block the PD-L1 / PD-1 and PD-L2 / PD-1 signaling pathways in vitro, activating the expression of downstream reporter genes, thus demonstrating good anti-tumor activity. This invention was completed based on these findings.

[0135] the term

[0136] 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. Other definitions are set forth throughout the application.

[0137] Bispecific antibodies

[0138] As used herein, the terms “bispecific antibody of the present invention”, “bispecific antibody of the present invention”, and “anti-PD-L1 / PD-L2 bispecific antibody” have the same meaning and refer to bispecific antibodies that specifically recognize and bind to PD-L1 and PD-L2.

[0139] The present invention provides a bispecific antibody against PD-L1 / PD-L2, comprising: an anti-PD-L1 single-domain antibody and an anti-PD-L2 single-domain antibody.

[0140] Preferably, the bispecific antibody of the present invention contains a polypeptide with the structure shown in Formula I or Formula II, or contains polypeptides with the structures shown in both Formula III and Formula IV.

[0141] A-L1-Fc1-L2-B (Formula I)

[0142] A-L3-B-L4-Fc1 (Formula II)

[0143] A-L5-Fc2-L6-Fc1 (Formula III)

[0144] B-L7-Fc2 (Formula IV)

[0145] in,

[0146] A and B are each independently an anti-PD-L1 single-domain antibody or an anti-PD-L2 single-domain antibody, and A and B are different antibodies;

[0147] L1, L2, L3, and L4 are each independently peptide bond or linker element;

[0148] Fc1 and Fc2 are both Fc segments of the antibody; where Fc1 is the human IgG domain (preferably the LALA mutant IgG domain) and Fc2 is the CH1+CL domain.

[0149] "-" represents a peptide bond.

[0150] In one embodiment, the bispecific antibody has a polypeptide with the structure shown in Formula I, and the polypeptide forms a homodimer i through disulfide bond interactions between Fc1.

[0151] In one embodiment, the bispecific antibody has a polypeptide with the structure shown in Formula II, and the polypeptide forms a homodimer ii through disulfide bond interactions between Fc1.

[0152] In one embodiment, the bispecific antibody has a polypeptide sequence as shown in Formula III and Formula IV, and the polypeptides of Formula III and Formula IV form a heterodimer a through disulfide bond interaction between Fc2, and the heterodimer ii forms a homodimer iii through disulfide bond interaction between Fc1.

[0153] As used herein, the terms "single-domain antibody," "VHH nanobody," and "nanobody" have the same meaning: to clone the variable region of an antibody heavy chain to construct a nanobody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies that are naturally missing 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 nanobody (VHH) consisting of only one heavy chain variable region.

[0154] 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 framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally folded and linked by three CDRs forming a linking loop, and in some cases, partially folded structures. The CDRs in each chain are tightly packed together by the FRs 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.

[0155] As used herein, the term "frame region" (FR) refers to the amino acid sequence inserted between CDRs, specifically those portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. Each immunoglobulin light and heavy chain has four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can be represented as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0156] Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the frame regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0157] As used herein, the term "human frame region" is a frame region that is substantially identical (approximately 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the frame region of a naturally occurring human antibody.

[0158] As used herein, the term "affinity" is theoretically defined by the balanced association between the intact antibody and the antigen. The affinity of the bispecific antibody of this invention can be assessed or determined by the KD value (dissociation constant) (or other assays), such as bio-layer interferometry (BLI), using a FortebioRed96 instrument.

[0159] As used herein, the term "connector" refers to one or more amino acid residues inserted into the immunoglobulin domain that provide sufficient mobility for both the light and heavy chain domains to fold into an exchangeable dual variable region immunoglobulin.

[0160] 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 that bind to the described PD-L1 / PD-L2 bispecific antibodies or fragments thereof.

[0161] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0162] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0163] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0164] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to PD-L1 and / or PD-L2 proteins, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0165] 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.

[0166] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame 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 the 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 antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0167] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0168] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0169] 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.

[0170] The term "antibody" in this invention refers to a bispecific antibody having PD-L1 and / or PD-L2 protein binding activity. This term also includes variants of polypeptides containing the same CDR region and having the same function as the antibodies of this 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. This term also includes active fragments and active derivatives of the antibodies of this invention.

[0171] 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.

[0172] The present invention also provides other polypeptides, such as fusion proteins comprising a single-domain antibody or a fragment thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the single-domain antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, 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.

[0173] 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 Table A.

[0174] Table A

[0175] 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

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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 polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] The 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 parts, or any combination of the above substances.

[0189] 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.

[0190] Therapeutic agents that can bind to or conjugate with the antibodies of the present 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)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0191] Pharmaceutical Composition

[0192] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 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): intratumoral, intraperitoneal, intravenous, or local administration.

[0193] The pharmaceutical compositions of the present invention can be directly used to bind PD-L1 and / or PD-L2 protein molecules, and are therefore suitable for the treatment of tumors. Furthermore, other therapeutic agents can be used concurrently.

[0194] 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 above-described single-domain antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, 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 by conventional methods using 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 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0195] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically 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, the dose is between about 10 micrograms per kilogram of body weight and 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 a skilled physician's expertise.

[0196] Labeled antibodies

[0197] In a preferred embodiment of the invention, the antibody carries a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0198] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the PD-L1 / PD-L2 bispecific antibody can be labeled with colloidal gold to obtain a colloidal gold-labeled antibody.

[0199] Detection methods

[0200] The present invention also relates to a method for detecting PD-L1 and / or PD-L2 proteins. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the levels of PD-L1 and / or PD-L2 proteins in the dissolved samples.

[0201] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0202] Reagent test kit

[0203] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention or a detection plate. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0204] This invention also provides a detection kit for detecting PD-L1 and / or PD-L2 levels. The kit includes antibodies that recognize PD-L1 and / or PD-L2 proteins, 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.

[0205] application

[0206] As described above, the single-domain antibody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of diseases related to PD-L1 and / or PD-L2, basic medical research, and biological research. A preferred application is for clinical diagnosis and targeted therapy against PD-L1 and / or PD-L2, such as tumor treatment.

[0207] The main advantages of this invention include:

[0208] (1) The nanobody of the present invention is highly specific to human PD-L1 protein with the correct spatial structure.

[0209] (2) The nanobody of the present invention has high specificity against human PD-L2 protein with the correct spatial structure.

[0210] (3) The nanoantibodies of the present invention have strong affinity.

[0211] (4) The nanoantibodies of the present invention are easy to produce.

[0212] (5) The present invention can simultaneously block the interaction between PD-L1 / PD-1 and PD-L2 / PD-1, relieve immunosuppression, and activate the body's immune system to kill tumors.

[0213] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0214] Table B: Summary of the sequence of this invention

[0215]

[0216]

[0217]

[0218] Example 1: Anti-human PD-L2 nanobody

[0219] 1.1 Construction of Nanobody Libraries

[0220] Animal Immunization

[0221] Two llamas were immunized with an equal volume of 1 mg human PD-L2 antigen (purchased from AcroBiosystems) and Freund's adjuvant, once a week for a total of four immunizations, stimulating B cells to express antigen-specific nanobodies. After the four immunizations, 50 ml of peripheral blood was extracted from the llamas, and lymphocytes were separated using lymphocyte separation medium. Total RNA was extracted using Trizol RNA extraction reagent (purchased from Invitrogen). Total cDNA from the llamas was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

[0222] Nanobody gene amplification

[0223] In the first round of PCR, IgG2 and IgG3 sequences were amplified from cDNA:

[0224] Table 1. Primers for the first round of PCR

[0225] name Sequence (5' to 3') SEQ ID NO: upstream primer GTCCTGGCTGCTCTTCTACAAGG 66 Downstream primer GGTACGTGCTGTTGAACTGTTCC 67

[0226] The PCR products were subjected to agarose gel electrophoresis, and the fragment at 750 bp was recovered from the gel for the second round of VHH sequence amplification. The primers for the second round of PCR amplification are as follows:

[0227] Table 2. Primers for the second round of PCR

[0228] name Sequence (5' to 3') SEQ ID NO: upstream primer CTAGTGCGGCCGCcTGGAGACGGTGACCTGGGT 68 Downstream primer CGCGGATCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG 69

[0229] Using the second-round PCR product as a template, a third-round PCR was performed to add a homologous arm to the VHH gene. The primers for the third-round PCR amplification are as follows:

[0230] Table 3. Primers for the third round of PCR

[0231]

[0232] The target fragment was recovered using a PCR purification kit (purchased from QIAGEN).

[0233] Library Construction

[0234] The linearized yeast display vector and the third-round PCR product were mixed and electroporated into *Saccharomyces cerevisiae* (purchased from ATCC) to construct anti-PD-L2 nanobody libraries from two animals, and the library sizes were determined to be 4.47 × 10⁻⁶. 7 and 4.14×10 7 .

[0235] 1.2 Screening of PD-L2 nanobodies

[0236] Biotinylation labeling of human PD-L2 protein

[0237] Dissolve human PD-L2 protein (purchased from AcroBiosystems) in an appropriate volume of double-distilled water. Following the instructions of the Biotin-labeled reagent kit (purchased from Thermo), dissolve biotin and mix it with the protein solution. Incubate at 4°C for 2 hours. Remove excess biotin using a desalting column (purchased from Thermo). Desalting column pretreatment and sample collection were performed according to the product instructions.

[0238] MACS enrichment of yeast that specifically binds to PD-L2

[0239] The VHH library constructed in Example 1.2 was inoculated into SD-CAA amplification medium (1L SD-CAA amplification medium contains 6.7g YNB, 5g tyrosine, 13.62g Na2HPO4·12H2O, 7.44g NaH2PO4 and 2% glucose), with the number of yeast cells inoculated being >10 × library capacity (initial amplification concentration = 0.5 OD). 600 Incubate overnight at 30℃ and 225 rpm ( / ml). Take 10× library volume of yeast cells, centrifuge at 3000 rpm for 5 min (the following centrifugation operations are all the same) to remove the culture medium, resuspend the yeast cells in SD-CAA induction medium, and adjust the initial concentration to 0.5 OD. 600 / ml, induce overnight. Determine the library concentration after induction, take 10× library volume of yeast cells, centrifuge to remove the culture medium. Resuspend the yeast cells in 50ml washing buffer (PBS + 0.5% BSA + 2mM EDTA), centrifuge to remove the supernatant. Resuspend the yeast cells in 10ml washing buffer.

[0240] Add biotin-labeled PD-L2 protein (final concentration 100 mM), incubate at room temperature for 30 min, collect yeast cells by centrifugation, and wash the yeast three times with 50 ml washing buffer. Resuspend the yeast cells in 5 ml washing buffer, add 200 μl of SA magnetic beads (purchased from Miltenyi), and incubate inverted mode for 10 min. Wash the yeast and magnetic bead mixture three times with washing buffer, and add the mixture to an LS purification column (purchased from Miltenyi). Place the LS purification column on a magnetic rack and wash with washing buffer to remove non-specifically bound yeast cells. Remove the purification column from the magnetic rack and add washing buffer to elute the yeast. Centrifuge the eluted yeast and transfer it to 200 ml of SD-CAA amplification medium for amplification.

[0241] High-affinity yeast cells were obtained by flow cytometry cell sorting.

[0242] MACS-enriched yeast cells were inoculated into SD-CAA amplification medium with an initial amplification concentration of 0.5 OD. 600 / ml. Incubate overnight in shake flasks at 30℃ and 225rpm. Resuspend yeast cells in SD-CAA induction medium (1L SD-CAA induction medium contains 6.7g YNB, 5g tyrosine, 13.62g Na2HPO4·12H2O, 7.44g NaH2PO4, 2% galactose, 2% raffinose, and 0.1% glucose) at an initial concentration of 0.5 OD. 600 / ml, induce overnight. Add 1:200 diluted anti-c-Myc mouse antibody (Thermo) and 100 nM biotin-labeled PD-L2 antigen, and incubate at room temperature for 10 min. Wash yeast three times with PBS, add 1:500 diluted goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (Invitrogen) and streptavidin APC conjugate fluorescent antibody (Invitrogen), and incubate at 4°C in the dark for 15 min. Resuspend cells in 2 ml PBS, and sort using a BD FACSAriaII instrument to obtain yeast cells with high binding capacity to PD-L2 antigen.

[0243] Extraction of antibody genes from PD-L2 nanobody candidate molecules

[0244] Yeast culture with high binding capacity to PD-L2 antigen, enriched by MACS and FACS, was cultured overnight at 30°C and 225 rpm in SD-CAA amplification medium. Yeast plasmids were extracted according to the yeast plasmid extraction kit (purchased from Tiangen). The plasmids were electroporated into Top10 competent cells (purchased from Tiangen), plated on ampicillin-resistant plates, and cultured overnight at 37°C. Single clones were picked and sequenced to obtain the VHH gene sequence.

[0245] 1.3 Construction, Expression, and Purification of Heavy Chain Antibodies

[0246] Antibody gene constructed into pCDNA3.1 expression vector

[0247] The VHH gene sequence was ligated to the human IgG1 (LALA mutant) Fc segment and constructed using homologous recombinase in a linearized pCDNA3.1 vector (purchased from Vazyme) and EcoRI / Not I double-digested, following the product's instructions. The homologous recombination product was transformed into Top10 competent cells, plated on ampicillin-resistant plates, incubated overnight at 37°C, and single clones were picked for sequencing and plasmid extraction.

[0248] Cell transfection and protein purification

[0249] Using ExpiCHO TMThe expression system kit (Thermo) was used to transfect the extracted plasmid into Expi-CHO cells according to the product instructions. After 5 days of cell culture, the supernatant was collected and the target protein was purified using protein A magnetic beads (GenScript). The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1-4 times the volume of the magnetic beads) and added to the sample to be purified. The sample was incubated at room temperature for 1 hour with gentle shaking. The sample was placed on a magnetic rack (Beaver), and the supernatant was discarded. The magnetic beads were washed 3 times with binding buffer. Elution buffer (0.1M sodium citrate, pH 3.2) was added at 3-5 times the volume of the magnetic beads, and the sample was shaken at room temperature for 5-10 minutes. The sample was then placed back on the magnetic rack, and the elution buffer was collected and transferred to a collection tube containing neutralization buffer (1M Tris, pH 8.54). The sample was then mixed to obtain the target protein.

[0250] 1.4 Purification of anti-PD-L2 antibody and binding to human PD-L2

[0251] CHO cells overexpressing human PD-L2 (CHO-hPD-L2 cells) were generated by transfecting the pCHO1.0 vector (purchased from Invitrogen) containing cloned human PD-L2 cDNA (purchased from Sino Biological). The expanded-cultured CHO-hPD-L2 cells were then adjusted to a cell density of 2 × 10⁻⁶ cells. 6 Cells / ml, 100 μl / well added to a 96-well flow cytometry plate, centrifuged and ready for use. Purified PD-L2 antibody was diluted with PBS, starting at 1000 nM and 3-fold diluted to 12 spots. 100 μl / well of the diluted sample was added to each well of the 96-well flow cytometry plate containing cells, incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of goat F(ab')2 anti-human IgG-Fc(PE) (purchased from Abcam) diluted 100-fold with PBS was added, incubated at 4°C for 30 min, and washed twice with PBS. Cells were resuspended in PBS with 100 μl / well, and the cells were analyzed and the corresponding MFI was calculated using a CytoFlex (Bechman) flow cytometer.

[0252] In the determination experiment using the method described above, the experimental results are as follows: Figure 1 As shown, all purified samples and CHO-hPD-L2 cells of this invention exhibit binding activity.

[0253] 1.5 PD-L2 antibody affinity assay

[0254] ForteBio affinity assays were performed according to existing methods (Estep, P et al., Determination of antibody-antigen affinity and epitope binding based on high-throughput method. MAbs, 2013.5(2):p.270-8). In short, the sensor was equilibrated offline in analytical buffer for 30 min, then detected online for 60 s to establish a baseline. The purified antibody obtained as described above was then loaded online onto the AHQ sensor. The sensor was then incubated in 100 nM PD-L2 antigen for 5 min, followed by dissociation in PBS for 5 min. Kinetic analysis was performed using a 1:1 binding model.

[0255] Table 4. Affinity of candidate molecules

[0256] serial number KD(M) Kon(1 / Ms) Koff(1 / s) D-Na-58 4.86E-10 6.68E+05 3.25E-04 D-Na-64 5.66E-10 6.52E+05 3.69E-04 D-Na-67 3.58E-09 9.33E+04 3.34E-04 D-Na-78 5.95E-09 5.81E+04 3.45E-04 D-Na-80 3.40E-09 1.27E+05 4.33E-04 D-Na-87 6.55E-10 4.40E+05 2.88E-04 D-Na-89 3.31E-09 3.39E+05 1.12E-03 D-Na-90 3.79E-09 4.63E+05 1.76E-03 D-Na-96 1.27E-09 8.62E+05 1.10E-03 D-Ye-10 5.22E-09 2.03E+05 1.06E-03 D-Ye-22 4.51E-09 1.83E+05 8.25E-04 D-Ye-29 1.93E-09 1.95E+05 3.76E-04 D-Ye-31 5.04E-09 1.28E+05 6.44E-04 D-Ye-32 5.69E-09 2.56E+05 1.46E-03

[0257] 1.6 Purification of anti-PD-L2 antibody to block the binding of PD-L2 to PD-1

[0258] CHO cells overexpressing human PD-1 (CHO-hPD-1 cells) were generated by transfecting the pCHO1.0 vector (purchased from Invitrogen) containing cloned human PD-1 cDNA (purchased from Sino Biological). The expanded-cultured CHO-hPD-1 cells were then adjusted to a cell density of 2 × 10⁻⁶ cells. 6 Cells / ml, 100 μl / well added to a 96-well flow cytometry plate, centrifuged and ready for use. The purified mutant sample was diluted with PBS, starting at 1000 nM and 3-fold diluted 12 times. 60 μl / well of the diluted sample was added to a 96-well sample dilution plate, along with 60 μl / well of biotinylated human PD-L2 protein (purchased from AcroBiosystems), to a final concentration of 1 μg / ml. The plate was incubated with the purified sample at 4°C for 30 min. 100 μl / well of the co-incubated sample was added to the above-mentioned 96-well flow cytometry plate containing cells, incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of APC goat anti-mouse IgG (minimum x reactivity) antibody (purchased from Biolegend), diluted 100-fold with PBS, was added, incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of cells was resuspended in PBS, and the cells were analyzed and the corresponding MFI was calculated using a CytoFlex (Bechman) flow cytometer.

[0259] In the determination experiment using the method described above, the experimental results are as follows: Figure 2 As shown, all purified samples of this invention can block the binding of PD-L2 and PD-1.

[0260] 1.7 Humanization Construction of PD-L2 Antibody

[0261] To reduce the immunogenicity of monoclonal antibodies in humans, DNA-96 and D-Ye-29 antibodies were humanized. The humanization method employed the VHH universal humanized framework transplantation technique, and the mutation of certain amino acids in the antibody framework 2 was completed according to the method reported in the literature (Vincke, C., et al., Humanized camel single-domain antibodies and general strategies for identifying universal humanized nanobody scaffolds. J Biol Chem 284(5):3273-3284).

[0262] This study used IMGT (http: / / www.imgt.org) to assess the humanization level of DNA-96, D-Ye-29, and humanized sequences. The results are shown in Table 5. The humanization level of all humanized samples was higher than 80%, which meets the requirements for later drug development.

[0263] Table 5. Humanized DNA-96 / D-Ye-29 sequences and their homology with humans

[0264] serial number phylogenetic homology D-Na-96 IGHV3-48*03 69.40％ HZ-D-Na-96-1 IGHV3-48*03 84.70％ HZ-D-Na-96-2 IGHV3-48*03 83.70％ HZ-D-Na-96-3 IGHV3-48*03 80.60％ D-Ye-29 IGHV3-23*01 71.1％ HZ-D-Ye-29-3 IGHV3-23*01 80.40％

[0265] The protein construction, expression, and purification methods were the same as in Example 1.3. Protein purity was determined using HPLC. The HPLC method was as follows: mobile phase: 150 mM Na₂HPO₄·12H₂O, pH 7.0; chromatographic conditions: detection wavelength: 280 nm, column temperature: 25 °C, flow rate: 0.35 ml / min, detection time: 20 min; Zenix-C SEC-300 column (SEPAX 4.6 × 300 mm, 3 μm).

[0266] Table 6. Purity test results of D-Na-96 / D-Ye-29 humanized antibody

[0267] serial number Monomer percentage (%) D-Na-96 100 HZ-D-Na-96-1 96.53 HZ-D-Na-96-2 99.92 HZ-D-Na-96-3 98.84 D-Ye-29 100 HZ-D-Ye-29-3 98.80

[0268] 1.8 Binding of D-Na-96 humanized sample with human PD-L2

[0269] This experiment tested the binding activity of the purified humanized D-Na-96 sample with CHO-hPD-L2 cells. The experimental method was the same as in Example 1.4, and the results are as follows. Figure 3 As shown, the humanized D-Na-96 sample exhibited good binding activity with CHO-hPD-L2 cells, with levels comparable to those of D-Na-96.

[0270] 1.9 Determination of Affinity for Humanized D-Na-96 / D-Ye-29 Samples

[0271] This experiment tested the binding affinity of the purified D-Na-96 / D-Ye-29 humanized sample to human PD-L2. The experimental method was the same as in Example 1.5. The experimental results are shown in Table 7. The D-Na-96 / D-Ye-29 humanized sample has good binding activity to human PD-L2 protein.

[0272] Table 7. Affinity of D-Na-96 / D-Ye-29 humanized samples

[0273] serial number KD(M) kon(1 / Ms) kdis(1 / s) D-Na-96 2.50E-09 2.62E+05 6.55E-04 HZ-D-Na-96-1 1.68E-09 3.44E+05 5.77E-04 HZ-D-Na-96-2 1.58E-09 3.23E+05 5.12E-04 HZ-D-Na-96-3 1.99E-09 2.97E+05 5.90E-04 D-Ye-29 1.93E-09 1.95E+05 3.76E-04 HZ-D-Ye-29-3 4.49E-09 3.86E+05 1.73E-03

[0274] 1.10 D-Na-96 humanized sample blocks PD-L2 binding to PD-1

[0275] This experiment tested the ability of the purified humanized D-Na-96 sample to block the binding of PD-L2 and PD-1. The experimental method was the same as in Example 1.6, and the results are as follows. Figure 4 As shown, all humanized samples of this invention can block the binding of PD-L2 and PD-1, and the blocking level is comparable to that of D-Na-96.

[0276] Example 2: Development of anti-human PD-L1 nanobodies

[0277] 2.1 Construction of Nanobody Libraries

[0278] Animal Immunization

[0279] One mg of human PD-L1 antigen (purchased from AcroBiosystems) was mixed with an equal volume of Freund's adjuvant and used to immunize two alpacas (one Llama and one Alpaca) at weeks 1, 2, 3, 5, and 7, respectively, to stimulate B cells to express antigen-specific nanobodies. After five immunizations, 300 ml of peripheral blood was extracted from the alpacas, and lymphocytes were separated using lymphocyte separation medium. Total RNA was extracted using Trizol RNA extraction reagent (purchased from Invitrogen). Total cDNA from the alpacas was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

[0280] Other methods for constructing nanolibraries are the same as in Example 1.1.

[0281] 2.2 Screening of PD-L1 nanobodies and construction, expression, and purification of heavy chain antibodies

[0282] This study screened nanobody sequences that specifically bind to human PD-L1 from the yeast display library constructed in Example 2.1, using the same screening method as in Example 1.2. The VHH gene sequence was linked to the human IgG1 (LALA mutant) Fc segment and constructed into the eukaryotic expression vector pCDNA3.1. High-purity heavy chain antibody proteins were prepared using the ExpiCHO expression system and magnetic bead affinity purification system. The construction, expression, and purification methods for the heavy chain antibody pairs were the same as in Example 1.3.

[0283] 2.3 PD-L1 antibody affinity assay

[0284] In this study, the binding activity of the obtained anti-PD-L1 antibody to human PD-L1 protein was detected using a ForteBio instrument, and the detection method was the same as in Example 1.5. The detection results are shown in Table 8. All three candidate molecules obtained in this study showed good binding activity to human PD-L1 protein.

[0285] Table 8. Affinity of candidate molecules

[0286] serial number KD(M) Kon(1 / Ms) Koff(1 / s) K-Yr-13&14-02 9.60E-10 5.85E+05 5.61E-04 K-Yr-13&14-09 2.56E-09 5.35E+05 1.37E-03 K-Yr-13&14-16 1.10E-08 5.53E+05 6.06E-03 ATE 1.30E-09 4.64E+05 6.04E-04

[0287] 2.4 Humanization of PD-L1 Antibody

[0288] To reduce the immunogenicity of the monoclonal antibody in the human body, the K-Yr-13&14-02 antibody was humanized. The humanization method was the same as in Example 1.7.

[0289] This study used IMGT (http: / / www.imgt.org) to assess the humanization level of K-Yr-13&14-02 and the humanized sequence. The results are shown in Table 9. The humanization level was higher than 80%, which meets the requirements for later-stage drug development.

[0290] Table 9. Humanization sequence of K-Yr-13&14-02 and its homology with humans

[0291] serial number phylogenetic homology K-Yr-13&14-02 IGHV3-11*05 74.20％ HZ-K-Yr-13&14-02-3 IGHV3-11*05 80.40％

[0292] The protein construction, expression, purification, and HPLC purity detection methods were the same as in Example 1.3. The results are shown in Table 10. A humanized anti-PD-L1 heavy chain antibody protein with high purity was obtained after one-step purification.

[0293] Table 10. Purity test results of HZ-K-Yr-13&14-02-3

[0294] serial number Monomer percentage (%) HZ-K-Yr-13&14-02-3 97.57

[0295] 2.5 Humanized anti-PD-L1 nanobody binds to human PD-L1

[0296] This experiment tested the binding activity of the purified humanized sample HZ-K-Yr-13&14-02-3 with CHO-hPD-L1 cells. The experimental method was the same as in Example 1.4, and the results are as follows. Figure 5 As shown, HZ-K-Yr-13&14-02-3 exhibits excellent binding activity with CHO-hPD-L1 cells, with levels comparable to K-Yr-13&14-02 and the control antibody ATE.

[0297] 2.6 Affinity assay of humanized anti-PD-L1 nanobody

[0298] This experiment tested the binding affinity of purified HZ-K-Yr-13&14-02-3 to human PD-L1. The experimental method was the same as in Example 1.5. The experimental results are shown in Table 11. HZ-K-Yr-13&14-02-3 has good binding activity with human PD-L2 protein.

[0299] Table 11. Affinity of humanized samples

[0300] serial number KD(M) kon(1 / Ms) kdis(1 / s) HZ-K-Yr-13&14-02-3 2.23E-10 4.45E+05 9.92E-05 ATE 1.16E-09 4.37E+05 5.07E-04

[0301] Example 3 Anti-PD-L1 / PD-L2 Bispecific Antibody

[0302] 3.1 Molecular construction of anti-PD-L1 / PD-L2 bispecific antibody

[0303] This study constructed three different forms of anti-PD-L1 / PD-L2 bispecific antibodies, and their structural diagrams are shown below. Figure 6 As shown.

[0304] Bi-201 contains a single peptide chain with the amino acid sequence shown in SEQ ID NO:23, comprising the anti-PD-L1 nanobody HZ-K-Yr-13&14-02-3, wherein the C-terminus of the nanobody's amino acid sequence is directly linked to a human IgG1 (LALA mutant) domain. The anti-PD-L2 nanobody HZ-D-NA-96-1 is linked to the C-terminus of the Fc via a flexible peptide chain (GGGGSGGGGSGGGGSGGGGSG) (SEQ ID NO:27).

[0305] Bi-202 contains a single peptide chain with the amino acid sequence shown in SEQ ID NO:24. The C-terminus of the anti-PD-L1 nanobody HZ-K-13&14-02-03 and the anti-PD-L2 nanobody HZ-D-NA-96-1 are linked by a flexible peptide chain (GGGGSGGGGSGGGGSGGGGSG) (SEQ ID NO:27). The C-terminus of HZ-D-NA-96-1 is directly linked to the human IgG1 (LALA mutant) domain.

[0306] Bi-203-204 contains two peptide chains. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO:25, with the C-terminus of the anti-PD-L1 nanobody HZ-K-Yr-13&14-02-3 directly linked to the CH1 amino acid sequence shown in SEQ ID NO:29 derived from human IgG1; the human IgG1 (LALA mutant) Fc (SEQ ID NO:) domain is directly linked to the C-terminus of the CH1 region, thereby obtaining peptide chain #1. Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:26, which contains the amino acid sequence SEQ ID NO:16 of the anti-PD-L2 nanobody HZ-D-NA-96-01, with the C-terminus of the nanobody amino acid sequence directly linked to the human κ light chain constant region (CL) amino acid sequence SEQ ID NO:30, thereby obtaining peptide chain #2.

[0307] 3.2 Expression and purification of anti-PD-L1 / PD-L2 bispecific antibody

[0308] In this embodiment, the nucleotide sequences encoding the anti-PD-L1 / PD-L2 bispecific antibodies Bi-201, Bi-202, and Bi-203-204 constructed in Example 3.1 were ligated into the commercially available eukaryotic expression vector pCDNA3.1(+) via a multiple cloning site. High-purity heavy chain antibody proteins were prepared using the ExpiCHO expression system and a magnetic bead affinity purification system. Protein construction, expression, purification, and HPLC purity detection methods were the same as in Example 1.3. The results are shown in Table 12; high-purity bispecific antibody proteins were obtained after one-step purification.

[0309] Table 12. Purity test results of anti-PD-L1 / PD-L2 bispecific antibodies

[0310] serial number Monomer percentage (%) Bi-201 98.26 Bi-202 97.67 Bi-203-204 99.26

[0311] 3.3 Affinity assay for anti-PD-L1 / PD-L2 bispecific antibodies

[0312] In this study, the binding activity of the obtained anti-PD-L1 / PD-L2 bispecific antibodies to human PD-L1 protein or human PD-L2 was detected using a ForteBio instrument, and the detection method was the same as in Example 1.5. The detection results are shown in Tables 13 and 14. The three candidate molecules obtained in this study all showed good binding activity to human PD-L1 and human PD-L2 proteins.

[0313] Table 13. Affinity of candidate molecules to human PD-L1 protein

[0314] serial number KD(M) Kon(1 / Ms) Koff(1 / s) Bi-201 4.81E-10 3.34E+05 1.61E-04 Bi-202 1.66E-09 2.07E+05 3.43E-04 Bi-203-204 1.59E-09 2.54E+05 4.05E-04 HZ-K-Yr-13&14-02-3 1.38E-09 2.24E+05 3.09E-04 ATE 7.97E-09 2.69E+05 2.15E-03

[0315] Table 14. Affinity of candidate molecules to human PD-L2 protein

[0316] serial number KD(M) Kon(1 / Ms) Koff(1 / s) Bi-201 4.45E-10 5.25E+05 2.34E-04 Bi-202 1.20E-09 3.29E+05 3.95E-04 Bi-203-204 1.14E-09 3.72E+05 4.24E-04 HZ-D-NA-96-1 1.11E-09 3.60E+05 4.00E-04

[0317] 3.4 Binding of anti-PD-L1 / PD-L2 bispecific antibodies to human PD-L1 or human PD-L2 on the cell surface

[0318] This experiment tested the binding activity of the purified anti-PD-L1 / PD-L2 bispecific antibody to CHO-hPD-L1 cells or CHO-hPD-L2 cells. The experimental method was the same as in Example 1.4, and the results are as follows. Figure 7A and 7B As shown, Bi-201, Bi-202, and Bi-203-204 all exhibited good binding activity with both CHO-hPD-L1 and CHO-hPD-L2 cells.

[0319] 3.5 Anti-PD-L1 / PD-L2 bispecific antibodies block the binding of PD-L2 / PD-L1 to PD-1

[0320] The cell density of the expanded-cultured CHO-hPD-1 cells was adjusted to 2 × 10⁻⁶. 6Cells / ml, 100 μl / well added to a 96-well flow cytometry plate, centrifuged and ready for use. The purified mutant sample was diluted with PBS, starting at 1000 nM and 3-fold diluted 12 times. 60 μl / well of the diluted sample was added to a 96-well sample dilution plate, along with 60 μl / well of biotinylated human PD-L2 protein or biotinylated human PD-L1 protein (purchased from AcroBiosystems), to a final concentration of 1 μg / ml. The plate was incubated with the purified sample at 4°C for 30 min. 100 μl / well of the co-incubated sample was added to the 96-well flow cytometry plate containing cells, incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of APC goat anti-mouse IgG (minimum x reactivity) antibody (purchased from Biolegend), diluted 100-fold with PBS, was added, incubated at 4°C for 30 min, and washed twice with PBS. Cells were resuspended in PBS at 100 μl / well and analyzed and the corresponding MFI was calculated on a CytoFlex (Bechman) flow cytometer.

[0321] In the determination experiment using the method described above, the experimental results are as follows: Figure 8A and 8B As shown, all purified bispecific antibody samples of this invention can block the binding of PD-L2 and PD-L1 to PD-1.

[0322] 3.6 Experiments using anti-PD-L1 / PD-L2 bispecific antibodies to block the PDL1 / PDL2 / PD1 / luc signaling pathway

[0323] PD-L1 and PD-L2 can be co-expressed on tumor cells or immune cells. In this embodiment, the simultaneous blocking effect of purified antibodies Bi-201, Bi-202, and Bi-203-204 on the PD-L1 / PD-1 and PD-L2 pathways was detected by co-incubating CHO cells (CHO-K1-PD-L1 / PD-L2) that co-express human PD-L1 and human PD-L2 and Jurkat cells that overexpress human PD-1 and contain the NFAT-luciferase reporter gene (Jurkat-PD-1-NFAT). The specific method is as follows.

[0324] The density of CHO-K1-PD-L1 / PD-L2 cells was adjusted to 5 × 10⁶. 5 Cells / ml, 100 μl / well were seeded into 96-well white cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator. The purified antibody and control antibody were serially diluted with 1640 complete medium and set aside. Jurkat-PD-1-NFAT cells were adjusted to a cell density of 2.5 × 10⁻⁶ cells / well using 1640 complete medium. 5Cells / ml, ready for use. Remove the white plate, aspirate the culture supernatant, dilute the above sample to the corresponding concentration, add 40 μl / well to the white plate, and simultaneously add 40 μl / well of Jurkat-PD-1-NFAT effector cell suspension. Incubate at 37°C, 5% CO2 for 6 hours. Add Bio-Glo™ reagent (Promega) to each well and read the fluorescence signal value using a multi-mode microplate reader.

[0325] Experimental results are as follows Figure 9 As shown, the anti-PD-L1 / PD-L2 bispecific antibody of the present invention can simultaneously block the PD-L1 / PD-1 and PD-L2 / PD-1 signaling pathways in vitro, activating the expression of downstream reporter genes. In contrast, anti-PD-L1 or anti-PD-L2 monoclonal antibodies cannot completely block this pathway and activate the expression of downstream reporter genes.

[0326] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Promis Biotechnology (Zhuhai) Co., Ltd. <120> Anti-PD-L1 and PD-L2 antibodies and their derivatives and their uses <130> P2022-1637 <150> CN202010246560.0 <151> 2020-03-31 <160> 71 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial sequence <220> <223> D-Na-58 VHH chain amino acid sequence <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Leu 35 40 45 Val Thr Ile Ser Arg Ser Gly Ser Thr Thr Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Leu Gln Asp Glu Val Tyr Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 2 <211> 119 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D-Na-64 VHH chain <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Met 20 25 30 Thr Ile Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Gly Trp Ser Thr Ser Lys Thr Val Tyr Lys Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Lys Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 3 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of D-Na-96 VHH chain <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ala 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Met 20 25 30 Thr Ile Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Gly Trp Thr Ser Gly Leu Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Lys Thr Met Tyr 65 70 75 80 Leu His Met Asn Asn Leu Lys Pro Glu Asp Thr Ser Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 4 <211> 124 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D‑Na‑67 VHH chain <400> 4 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Leu Thr Phe Ile Asn 20 25 30<000109​​35 40 45 Val Ala Ala Ile Ser Arg Phe Gly Gly Thr Thr Ser Tyr Ala Asp Ser 50 55 60 Val Lys Asp Arg Phe Ser Ile Thr Arg Asp Val Ala Lys Asp Thr Val 65 70 75 80 Tyr Leu Gln Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Asn Ser Arg Ile Leu Ser Arg Thr Ala Ala Asp Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115, 120 <210> 5 <211> 125 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D-Na-78 VHH chain <400> 5 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Val Ile Ser Ile Asn Tyr 20 25 30 Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Arg Phe Gly Val Thr Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Ser Ile Gly Ile Thr Arg Asp Asn Ala Lys Asp Thr 65 70 75 80 Met Tyr Leu Gln Thr Lys Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala Asn Ser Arg Ile Leu Ser Arg Thr Ala Lys Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 6 <211> 122 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D-Na-80 VHH chain <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Val Ser Phe Ile Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Arg Phe Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Ser Ile Thr Thr Asp Thr Lys Asp Thr Val Tyr Leu 65 70 75 80 Gln Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Asn Ser Arg Ile Leu Ser Arg Thr Ala Lys Asp Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 7 <211> 123 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D‑Na‑87 VHH chain <400> 7 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Asp Ala Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Leu 35 40 45 Ser Arg Ile Tyr Ser His Gly Ser Lys Ile Ile Tyr Ala Gly Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Pro Asp Asn Ile Pro Thr Thr Gly Glu Lys Tyr Asp Phe 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 8 <211> 123 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D-Na-89 VHH chain <400> 8 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ile Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ala Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Ser Ile Thr Arg Asp Ser Ala Lys Asp Thr Val Tyr 65 70 75 80 Leu Gln Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Ser Arg Ile Leu Ser Arg Thr Ala Lys Asp Tyr Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Glu Val Thr Val Ser Ser 115 120 <210> 9 <211> 123 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D‑Na‑90 VHH chain <400> 9 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ile Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ala Gly Gly Thr Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Asp Arg Phe Ser Ile Thr Arg Asp Ser Ala Lys Asp Thr Val Tyr 65 70 75 80 Leu Gln Thr Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Ser Arg Ile Leu Ser Arg Thr Ala Lys Asp Tyr Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 10 <211> 119 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D-Ye-10 VHH chain <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ala 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Arg Thr Phe Ser Ser Met 20 25 30 Thr Ile Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Gly Trp Ser Thr Ser Lys Thr Val Tyr Lys Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Lys Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 11 <211> 116 <212> PRT <213> artificial sequence <220><00​​​​​​​​​​​​​​​​​​50 55 60 Phe Ala Cys Ser Arg Asp Asp Ser Thr Asn Thr Ile Phe Leu Gln Met 65 70 75 80 Asn Ser Leu Glu Leu Glu Asp Thr Ala Leu Tyr Leu Cys Ala Ala Ala 85 90 95 Asp Ser Ser Gly Asp Asp Phe Glu Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 12 <211> 123 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D‑Ye‑29 VHH chain <400> 12 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Glu Phe Ser Phe Ile 20 25 30 Pro Thr Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Asp Leu Val 35 40 45 Ala Val Phe Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asn Asn Thr Lys Asn Ile Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Gly Val Pro Gly Pro His Tyr Asn Gly Pro Thr Ser Gly Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 13 <211> 119 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of D‑Ye‑31 VHH chain <400> 13 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Gly Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Met 20 25 30 Thr Ile Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Gly Trp Ser Thr Ser Lys Thr Val Tyr Lys Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Thr Asp Asn Ala Lys Lys Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 14 <211> 112 <212> PRT <213> Artificial sequence <220> [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​85 90 95 Ala Ala Gly Leu Gly Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 <210> 15 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of HZ-D-Ye-29-3 VHH chain <400> 15 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Glu Phe Ser Phe Ile 20 25 30 Pro Thr Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Val Phe Thr Ser Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Gly Val Pro Gly Pro His Tyr Asn Gly Pro Thr Ser Gly Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 16 <211> 119 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of HZ-D-Na-96-1 VHH chain <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Met 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ser Gly Ile Gly Trp Thr Ser Gly Leu Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 17 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of HZ-D-Na-96-2 VHH chain <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Met 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ser Gly Ile Gly Trp Thr Ser Gly Leu Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 18 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of HZ-D-Na-96-3 VHH chain <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Met 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Gly Ile Gly Trp Thr Ser Gly Leu Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 19 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the K-Yr-13&14-02 VHH chain <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Thr Phe Ile Ile Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Asn Trp Ser Gly Ser Met Thr Asn Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Tyr Val Gly Ala Thr Ile Ser Thr Ala His Ser Arg Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 20 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the K-Yr-13&14-09 VHH chain <400> 20 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Ala Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Ile Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Asn Trp Ser Gly Ser Met Thr Asn Tyr Thr Asp Ser Val ; 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Tyr Val Gly Ala Thr Ile Ser Thr Ala His Ser Arg Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 21 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of K‑Yr‑13&14‑16 VHH chain <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Arg Thr Phe Ile Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ser Ile Asn Trp Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg His Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Leu Ser Ala Val Val Val Thr Gln Ile Leu Asp Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 124 <212> PRT <213> artificial sequence <220> <223> Amino acid sequence of HZ-K-Yr-13&14-02-3 VHH chain <400> 22 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Thr Phe Ile Ile Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Thr Ile Asn Trp Ser Gly Ser Met Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Tyr Val Gly Ala Thr Ile Ser Thr Ala His Ser Arg Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 490 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of Bi-201 <400> 23 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Thr Phe Ile Ile Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Thr Ile Asn Trp Ser Gly Ser Met Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Tyr Val Gly Ala Thr Ile Ser Thr Ala His Ser Arg Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His 115 120 125 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 130 135 140 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 145 150 155 160 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 165 170 175 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 180 185 190 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 195 200 205 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 210 215 220 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 225 230 235 240 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 245 250 255 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 260 265 270 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 275 280 285 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 290 295 300 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 305 310 315 320 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 325 330 335 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly 340 345 350 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 355 360 365 Gly Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 370 375 380 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe 385 390 395 400 Ser Ser Met Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu 405 410 415 Glu Phe Val Ser Gly Ile Gly Trp Thr Ser Gly Leu Thr Val Tyr Ala 420 425 430 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 435 440 445 Ser Met Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 450 455 460 Tyr Tyr Cys Ala Ala Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp 465 470 475 480 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 485 490 <210> 24 <211> 490 <212> PRT <213> artificial sequence <220> <223> Bi-202 amino acid sequence <400> 24 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Thr Phe Ile Ile Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Thr Ile Asn Trp Ser Gly Ser Met Thr Asn Tyr Ala Asp Ser Val 50 55 60 [[ID=*]]Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 7* 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Tyr Val Gly Ala Thr Ile Ser Thr Ala His Ser Arg Tyr Asp 100 105 110 It seems there is a small error in the original text where "7*" is likely a typo. I've translated it as "70" in the English version above. If this is not what you intended, please correct the original text and I'll be happy to re-translate.Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 145 150 155 160 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 165 170 175 Ser Met Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu 180 185 190 Phe Val Ser Gly Ile Gly Trp Thr Ser Gly Leu Thr Val Tyr Ala Asp 195 200 205 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 210 215 220 Met Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 225 230 235 240 Tyr Cys Ala Ala Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro 260 265 270 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 275 280 285 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 290 295 300 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 305 310 315 320 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 325 330 335 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 340 345 350 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 355 360 365 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 370 375 380 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 385 390 395 400 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 405 410 415 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 420 425 430 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 435 440 445 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 450 455 460 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 465 470 475 480 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 485 490 <210> 25 <211> 452 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of Bi-203 <400> 25 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Thr Phe Ile Ile Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Thr Ile Asn Trp Ser Gly Ser Met Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Tyr Val Gly Ala Thr Ile Ser Thr Ala His Ser Arg Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro 450 <210> 26 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Bi-204 Amino Acid Sequence <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Met 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ser Gly Ile Gly Trp Thr Ser Gly Leu Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Met Asn Arg Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Arg Thr Val Ala Ala Pro Ser Val Phe 115 120 125 Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 130 135 140 Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 145 150 155 160 Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 165 170 175 Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 180 185 190 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 195 200 205 Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 210 215 220 Glu Cys 225 <210> 27 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Linker sequence <400> 27 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly 20 <210> 28 <211> 225 <212> PRT <213> Artificial sequence <220> <223> Fc <400> 28 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro 225 <210> 29 <211> 103 <212> PRT <213> artificial sequence <220> <223> CH1 <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys 100 <210> 30 <211> 107 <212> PRT <213> artificial sequence <220> <223> CL <400> 3 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 31 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of D-Na-58 VHH chain <400> 31 caggtgcagc tcgtggagtc aggcggagga ttggtgcagg ctgggggctc tctgacactc 60 tcctgtacag cctctggacg caccttcagt agctatgcca tggcctggtt ccgccaggct 120 ccagggaagg agcgtgagtt attagtaact atcagcagga gtggtagtac cacttactat 180 cttgactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240 ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcaaaaagg 300 tctctccaag acgaagttta ctactggggc caggggaccc aggtcaccgt ctcctca 357 <210> 32 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide Sequence of D-Na-64 VHH Chain <400> 32 gaggtgcagc tcgtggagtc agggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgcgcag cctctggacg caccttcagt agcatgacca tcggctggtt ccgccaaact 120 ccagggaagg agcgtgagtt tgtagcaggt attgggtgga gtacgagtaa aacagtctac 180 aaagactccg tgaagggccg attcaccatt tccacagaca atgccaagaa aacggtgtat 240 ctgcaaatga acaacctgaa acctgaggac acggccgttt attactgtgc agcagattca 300 atgaatagag gacagtttga ctactggggc caggggaccc aggtcaccgt ctcctca 357 <210> 33 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of D-Na-96 VHH chain <400> 33 caggtgcagc tcgtggagtc cgggggagga ttggtgcagg ctggggcctc tctgagactc 60 tcctgcgcag cctctggacg caccttcagt agcatgacca tcggctggtt ccgccaaact 120 ccagggaagg agcgtgagtt tgtagcaggt attgggtgga cgagtggtct cacagtctat 180 gcagactccg tgaagggccg attcaccatt tccagagaca atgccgagaa aacgatgtat 240 ctgcacatga acaatctgaa acctgaggac acgtccgttt attactgtgc agcagattca 300 atgaacagag gacagtttga ctactggggc caggggaccc aggtcaccgt ctcctca 357 <210> 34 <211> 372 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of D-Na-67 VHH chain <400> 34 caggtgcagc tcgtggagtc tggtggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag cctctggtgg actcaccttc attaactatg ccatgggctg gttccgccag 120 gctccaggga aggagcgtga gtttgtagca gctattagtc ggtttggtgg cactacatcc 180 tacgcagact ccgtgaagga ccgattcagc atcactagag atgttgccaa agacacggtg 240 tatttgcaaa cgaacagcct gaaacctgag gacacggccg tttattactg tgcagcaaac 300 tcccgtattc tgagtcgaac cgctgcggat tacgattact ggggccaggg gacccaggtc 360 accgtctcct ca 372 <210> 35 <211> 375 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of D‑Na‑78 VHH chain <400> 35 caggtgcagc tcgtggagtc cggcggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgaag cctctggagt catctccatt aactatgcca tggcctggtt ccgccaggct 120 ccagggaagg agcgtgagtt tgtagctgct attagtcggt ttggtgtaac tacgagttac 180 gcagactccg tgaaggaccg attcagcatc ggcatcacta gagataatgc caaagacaca 240 atgtatctgc aaacgaagag cctgaaacct gaggacacgg ccgtttatta ttgtgcagca 300 aactcccgta ttctgagtcg aaccgctaag gattatgatt actggggcca ggggacccag 360 gtcaccgtct cctca 375 <210> 36 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide Sequence of D-Na-80 VHH Chain <400> 3 gaggtgcagc tcgtggagtc tggtggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag tctctggagt ctccttcatt aactatgcca tgggctggtt ccgccaggct 120 ccagggaagg agcgtgagtt tgtagcagct attagtcggt ttggtggcac tacatcctac 180 gcagactccg tgaaggaccg attcagcatc actacagata ccaaagacac ggtgtatctg 240 caaacgaaca gcctgaaacc tgaggacacg gccgtttatt attgtgcagc aaactcccgt 300 attctgagtc gaaccgctaa ggattatgat tactggggcc aggggaccca ggtcaccgtc 360 tcctca 366 <210> 37 <211> 369 <212> DNA <213> artificial sequence <220> <223> Nucleotide sequence of D‑Na‑87 VHH chain <400> 37 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctgggggttc tctgagactc 60 tcctgtgcag cctctggatt cgcttttgat gcctatgcca tgacctgggt ccgacaggct 120 ccagggaagg gactggagtt tttgtcccgt atttatagtc atggaagtaa gatcatctac 180 gcaggttccg tgaagggccg attcaccatt ttcagagaca acgccaagaa cacggtgtat 240 ctgcaaatga acagcctgaa acctgaggac acggccgtgt attactgtgc agctggtccg 300 gacaacatac cgactacagg tgaaaagtat gacttctggg gtcaggggac ccaggtcacc 360 gtctcctca 369 <210> 38 <211> 369 <212> DNA <213> artificial sequence <220> <223> Nucleotide sequence of D‑Na‑89 VHH chain <400> 38 caggtgcagc tcgtggagtc agggggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag cctctggact caccttcatt aactatgcca tgggctggtt ccgccaggct 120 ccagggaagg agcgtgagtt tgtagcagct attagttggg ctggtggcac tacatcctac 180 gcagactccg tgaaggaccg attcagcatc actagagact ctgccaaaga cacggtgtat 240 ctgcaaacga acagcctgaa acctgaggac acggccgttt attattgtgc agcaaactcc 300 cgtattctga gtcgaaccgc taaggattat gattactggg gccaggggac cgaggtcacc 360 gtctcctca 369 <210> 39 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide Sequence of D-Na-90 VHH Chain <400> 39 caggtgcagc tcgtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag cctctggact caccttcatt aactatgcca tgggctggtt ccgccaggct 120 ccagggaagg agcgtgagtt tgtagcagct attagttggg ctggtggcac tacatcctac 180 gcagactccg tgaaggaccg attcagcatc actagagact ctgccaaaga cacggtgtat 240 ctgcaaacga acagcctgaa acctgaggac acggccgttt attattgtgc agcaaactcc 300 cgtattctga gtcgaaccgc taaggattat gattactggg gccaggggac ccaggtcacc 360 gtctcctca 369 <210> 40 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of D‑Ye‑10 VHH chain <400> 40 caggtgcagc tgcaggagtc tgggggagga ttggtgcagg ctggggcctc tctgagactc 60 tcctgcgcat cctctggacg caccttcagt agcatgacca tcggctggtt ccgccaaact 120 ccagggaagg agcgtgagtt tgtagcaggt attgggtgga gtacgagtaa aacagtctac 180 aaagactccg tgaagggccg attcaccatt tccacagaca atgccaagaa aacggtgtat 240 ctgcaaatga acaacctgaa acctgaggac acggccgttt attactgtgc agaagattca 300 atgaatagag gacagtttga ctactggggc caggggaccc aggtcaccgt ctccagt 357 <210> 41 <211> 348 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of D-Ye-22 VHH chain <400> 41 caggtgcagc tgcaggagtc tggaggagga ttggtgcagg ctggggactc tctgagactc 60 tcctgtatag gttctagacg taccttcacc atggcctggt tccgccaggc tccagggaag 120 gagcgtgaat ttgtggcagg tatttggggt cctgcggatc acacaatcta tgcaaactcc 180 gtgaagggcc gattcgcctg ctccagagac gattccacga atacgatttt tttgcaaatg 240 aacagcctgg aacttgagga cacggccctt tatctctgtg cagcagcgga ctccagtggg 300 gatgactttg agtattgggg ccaggggacc caggtcaccg tctccagt 348 <210> 42 <211> 369 <212> DNA <213> Artificial sequence <220><0​​​​​​​​​​gactccgtga agggccgatt caccatctcc agaaacaaca ccaagaacat agtgtatctg 240 caaatgaaca gcctcaaacc tgaggacacg gccgtctatt actgtagtgg agtcccaggt 300 ccacactaca atggtccgac atcggggata aactactggg gccaggggac ccaggtcacc 360 gtctccagt 369 <210> 43 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of D‑Ye‑31 VHH chain <400> 43 caggtgcagc tgcaggagtc tggaggagga ttggtgcagg ctgggggctc tctgggactc 60 tcctgcgcag cctctggacg caccttcagt agcatgacca tcggctggtt ccgccaaact 120 ccagggaagg agcgtgagtt tgtagcaggt attgggtgga gtacgagtaa aacagtctac 180 aaagactccg tgaagggccg attcaccatt tccacagaca atgccaagaa aacggtgtat 240 ctgcaaatga acaacctgaa acctgaggac acggccgttt attactgtgc agcagattca 300 atgaatagag gacagtttga ctactggggc caggggaccc aggtcaccgt ctccagt 357 <210> 44 <211> 336 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of D‑Ye‑32 VHH chain <400> 44 caggtgcagc tgcaggagtc tggaggaggc ttggtgcagg ctggggggtc tctgagactc 60 tcctgtgcag cctctgggag cgtcggcagt atcaatgcca tggcctggtt ccgccaggcg 120 ccagggaagc agcgcgacgt ggtcgcacat atgccttctg gtggtagcac acactatgca 180 gactccgtga agggccgatt caccgtctcc agagacaacg ccaagaacac ggtgtatcta 240 caaatgaaca gcctgaaagc tgaggacacg gccgtctatt actgttgggc agctggtctg 300 gggtggggcc aggggaccca ggtcaccgtc tccagt 336 <210> 45 <211> 369 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of HZ‑D‑Ye‑29‑3 VHH chain <400> 45 gaggtgcagc tgctggagag cggaggcgga ctggtgcagc ccggaggatc tctgagactg 60 agctgtgctg ctagtgggag cgagttctct tttatcccca caggatggta cagacaggcc 120 cctggaaaac agagagagct ggtggctgtg tttacctctg gagggctgac caactacgcc 180 gactctgtga aagggaggtt tactatcagc agggacaaca gcaagaacac agtgtatctc 240 cagatgaaca gtctgagagc cgaagacacc gccgtctatt attgcagcgg cgtgccagga 300 cctcattaca acgggcctac cagcggcatc aactactggg gacagggcac ccaggtgaca 360 gtgtcatcc 369 <210> 46 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide Sequence of HZ-D-Na-96-1 VHH Chain <400> 46 gaggtgcagc tggtggagag cggaggagga ctggtgcagc ccggcggctc tctgagactg 60 agctgcgccg cctccggaag gacctttagc tccatgacca tcggctggtt cagacaagcc 120 cccggcaagg gactggagtt cgtgagcggc atcggctgga caagcggact gaccgtgtat 180 gccgacagcg tcaagggaag gttcaccatc tctagggaca acgccaagaa cagcatgtat 240 ctgcagatga actctctgag ggccgaggac accgccgtgt actactgcgc cgccgacagc 300 atgaacagag gccagttcga ctactggggc caaggcacac tggtgacagt gagcagc 357 <210> 47 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of HZ-D-Na-96-2 VHH chain <400> 47 gaggtgcagc tggtggaaag cggcggagga ctggtgcagc ccggcggatc tctgagactc 60 agctgtgctg ccagcggaag gaccttcagc agcatgacca tcggctggtt cagacaagcc 120 cccggcaagg gactggagtt cgtgagcggc atcggctgga caagcggact gaccgtgtat 180 gccgacagcg tcaagggaag gttcaccatc tctagggaca atgccaagaa ctctctgtat 240 ctgcagatga actctctgaa gcccgaggat accgccgtgt actactgcgc cgccgatagc 300 atgaataggg gccagttcga ctactggggc caaggcacac tggtgacagt gagcagc 357 <210> 48 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of HZ-D-Na-96-3 VHH chain <400> 48 gaggtgcagc tggtggaaag cggcggagga ctggtgcagc ccggcggatc tctgagactg 60 agctgcgccg ccagcggaag gaccttctcc agcatgacca tcggctggtt taggcaagcc 120 cccggcaagg agagagagtt cgtgagcggc atcggctgga caagcggact gaccgtgtac 180 gccgatagcg tgaagggaag gttcaccatc tctagggaca acgccaagaa ctccatgtat 240 ctgcagatga actctctgaa gcccgaggat accgccgtgt actactgcgc cgccgacagc 300 atgaacagag gccagttcga ctactggggc caaggcacac tggtgacagt gtccagc 357 <210> 49 <211> 372 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of K-Yr-13&14-02 VHH chain <400> 49 caagtgcagc tggtagagtc tgggggagga ttggtcaagg ctggggactc tctgagactc 60 tcctgtgcag cctctggaca caccttcatt atttatgcca ttggctggtt ccgccaggct 120 ccagggaagg agcgtgagtt tgtagcaact attaactgga gtggtagtat gacaaactat 180 acagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240 ctccaaatga acagcctgaa acctgacgac acggccattt attactgtgc ggcttacgtc 300 ggtgcgacta tttccaccgc ccattcccga tatgactact ggggccaggg aacccaggtc 360 accgtgtcct ca 372 <210> 50 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide Sequence of K‑Yr‑13&14‑09 VHH Chain <400> 50 caagtgcagc tggtggagtc tgggggagga ttggtcaagg ctggggactc tctgagactc 60 tcctgtgcag cctctggacg caccttcact atttatgcca ttggctggtt ccgccaggct 120 ccagggaagg agcgtgagtt tgtagcaact attaactgga gtggtagtat gacaaactat 180 acagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240 ctccaaatga acagcctgaa acctgacgac acggccattt attactgtgc ggcttacgtc 300 ggtgcgacta tttccaccgc ccattcccga tatgactact ggggccaggg gaccctggtc 360 actgtctccg ca 372 <210> 51[[ID=3​​​​<213> Artificial sequence <220> <223> Nucleotide sequence of K-Yr-13&14-16 VHH chain <400> 51 gaagtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag cctctacacg caccttcatt acctatgcca tgggctggtt ccgccaggct 120 ccagggaagg agcgtgagtt tgtagcatct attaactgga gtggtgctag cacatactat 180 gcagactccg tgaagggccg attcaccatc tccagacaca acgccaagaa cacggtgtat 24o ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagctttg 300 tcagcagtag tagttacaca gatcctagac tatgactact ggggccaggg gaccctggtc 360 accgtctcct ca 372 <210> 52 L<211> 372 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of HZ-K-Yr-13&14-02-3 VHH chain <400> 52 caagtgcagc tggtggagag cggaggagga ctggtgaagc ccggcggctc tctgagactg 60 agctgtgccg cctccggcca caccttcatc atctacgcca tcggctggtt taggcaagcc 120 It should be noted that there is a typo in the original text where "24o" should be "240" in line 14 of the translation. cccggcaaag gactggagtt cgtggccacc atcaactgga gcggcagcat gaccaactac 180 gccgactccg tgaagggaag gttcacaatc tctagggaca acgccaagaa caccgtgtat 240 ctgcagatga actctctgaa gcccgacgac accgccgtgt actactgcgc tgcctatgtg 300 ggcgccacca tcagcacagc ccacagcaga tacgactact ggggacaagg cacactggtg 360 accgtgagca gc 372 <210> 53 <211> 1470 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of Bi-201 <400> 53 caagtgcagc tggtggagag cggaggagga ctggtgaagc ccggcggctc tctgagactg 60 agctgtgccg cctccggcca caccttcatc atctacgcca tcggctggtt taggcaagcc 120 cccggcaaag gactggagtt cgtggccacc atcaactgga gcggcagcat gaccaactac 180 gccgactccg tgaagggaag gttcacaatc tctagggaca acgccaagaa caccgtgtat 240 ctgcagatga actctctgaa gcccgacgac accgccgtgt actactgcgc tgcctatgtg 300 ggcgccacca tcagcacagc ccacagcaga tacgactact ggggacaagg cacactggtg 360 accgtgagca gcgacaaaac ccatacatgt cctccttgcc ccgcccctga ggctctctgga 420 ggccccagcg tgttcctgtt tccccccaag cccaaagata ccctcatgat ctccaggacc 480 cccgaagtga cctgcgtcgt ggtcgacgtg agccacgagg accctgaagt caagttcaac 540 tggtacgtcg atggcgtgga ggtgcacaac gctaagacca aaccccggga agagcagtac 600 aattccacct acagggtggt gtccgtcctg acagtgctgc accaagactg gctgaatgga 660 aaggagtca agtgcaaagt gaggaataag gccctccctg ctcccattga gaagaccatt 720 tccaaggcca aaggccagcc tcgggaaccc caggtgtaca cactgccccc ttccagggag 780 gagatgacca agaaccaggt gagcctcacc tgcctggtga agggcttcta ccctagcgac 840 attgctgtgg agtgggagag caacggccag cccgaaaaca actataagac aacccctccc 900 gtgctggaca gcgacggctc cttctttctg tactccaagc tcaccgtgga caagtccagg 960 tggcaacagg gaaacgtgtt ctcctgctcc gtgatgcacg aggccctcca caaccactac 1020 acccagaaga gcctgagcct gtcccctggc ggtggcggcg gtagtggcgg aggcgggagt 1080 ggcggtggag ggtcaggtgg tggaggctcg ggtgaggtgc agctggtgga gagcggagga 1140 ggactggtgc agcccggcgg ctctctgaga ctgagctgcg ccgcctccgg aaggaccttt 1200 agctccatga ccatcggctg gttcagacaa gcccccggca agggactgga gttcgtgagc 1260 ggcatcggct ggacaagcgg actgaccgtg tatgccgaca gcgtcaaggg aaggttcacc 1320 atctctaggg acaacgccaa gaacagcatg tatctgcaga tgaactctct gagggccgag 1380 gacaccgccg tgtactactg cgccgccgac agcatgaaca gaggccagtt cgactactgg 1440 ggccaaggca cactggtgac agtgagcagc 1470 <210> 54 <211> 1470 <212> DNA <213> Artificial Sequence <220> <223> Bi-202 Nucleotide Sequence <400> 54 caagtgcagc tggtggagag cggaggagga ctggtgaagc ccggcggctc tctgagactg 60 agctgtgccg cctccggcca caccttcatc atctacgcca tcggctggtt taggcaagcc 120 cccggcaaag gactggagtt cgtggccacc atcaactgga gcggcagcat gaccaactac 180 gccgactccg tgaagggaag gttcacaatc tctagggaca acgccaagaa caccgtgtat 240 ctgcagatga actctctgaa gcccgacgac accgccgtgt actactgcgc tgcctatgtg 300 ggcgccacca tcagcacagc ccacagcaga tacgactact ggggacaagg cacactggtg 360 accgtgagca gcggcggtgg cggcggtagt ggcggaggcg ggagtggcgg tggagggtca 420 ggtggtggag gctcgggtga ggtgcagctg gtggagagcg gaggaggact ggtgcagccc 480 ggcggctctc tgagactgag ctgcgccgcc tccggaagga cctttagctc catgaccatc 540 ggctggttca gacaagcccc cggcaaggga ctggagttcg tgagcggcat cggctggaca 600 agcggactga ccgtgtatgc cgacagcgtc aagggaaggt tcaccatctc tagggacaac 660 gccaagaaca gcatgtatct gcagatgaac tctctgaggg ccgaggacac cgccgtgtac 720 tactgcgccg ccgacagcat gaacagaggc cagttcgact actggggcca aggcacactg 780 gtgacagtga gcagcgacaa aacccataca tgtcctcctt gccccgcccc tgaggctgct 840 ggaggcccca gcgtgttcct gtttcccccc aagcccaaag ataccctcat gatctccagg 900 acccccgaag tgacctgcgt cgtggtcgac gtgagccacg aggaccctga agtcaagttc 960 aactggtacg tcgatggcgt ggaggtgcac aacgctaaga ccaaaccccg ggaagagcag 1020 tacaattcca cctacagggt ggtgtccgtc ctgacagtgc tgcaccaaga ctggctgaat 1080 ggaaaggagt acaagtgcaa agtgagcaat aaggccctcc ctgctcccat tgagaagacc 1140 atttccaagg ccaaaggcca gcctcgggaa ccccaggtgt acacactgcc cccttccagg 1200 gaggagatga ccaagaacca ggtgagcctc acctgcctgg tgaagggctt ctaccctagc 1260 gacattgctg tggagtggga gagcaacggc cagcccgaaa acaactataa gacaacccct 1320 cccgtgctgg acagcgacgg ctccttcttt ctgtactcca agctcaccgt ggacaagtcc 1380 aggtggcaac agggaaacgt gttctcctgc tccgtgatgc acgaggccct ccacaaccac 1440 tacacccaga agagcctgag cctgtcccct 1470 <210> 55 <211> 1356 <212> DNA <213> Artificial Sequence <220> <223> Bi-203 nucleotide sequence <400> 55 caagtgcagc tggtggagag cggaggagga ctggtgaagc ccggcggctc tctgagactg 60 agctgtgccg cctccggcca caccttcatc atctacgcca tcggctggtt taggcaagcc 120 cccggcaaag gactggagtt cgtggccacc atcaactgga gcggcagcat gaccaactac 180 gccgactccg tgaagggaag gttcacaatc tctagggaca acgccaagaa caccgtgtat 240 ctgcagatga actctctgaa gcccgacgac accgccgtgt actactgcgc tgcctatgtg 300 ggcgccacca tcagcacagc ccacagcaga tacgactact ggggacaagg cacactggtg 360 [[ID=十七]]accgtgagca gcgccagcac aaagggccct tccgtcttcc ctctggcccc ctccagcaag 420 tccacaagcg gaggaaccgc tgccctcggc tgcctcgtga aagactattt ccctgagccc 480 gtgacagtga gctggaatag cggcgctctc acctccggag tccacacctt ccccgctgtg 540 ctgcagagca gcggactgta tagcctgagc tccgtggtga ccgtgcctag ctcctccctc 600 ggcacccaga cctacatttg caatgtgaac cacaagccta gcaacaccaa ggtggacaag 660 It should be noted that there seems to be an incorrect label "十七" in the original text which has been retained as it is in the translation for the purpose of following the rules. You may want to check and correct the original text if necessary.aaggtggagc ccaagagctg cgacaaaacc catacatgtc ctccttgccc cgcccctgag 720 gctgctggag gccccagcgt gttcctgttt ccccccaagc ccaaagatac cctcatgatc 780 tccaggaccc ccgaagtgac ctgcgtcgtg gtcgacgtga gccacgagga ccctgaagtc 840 aagttcaact ggtacgtcga tggcgtggag gtgcacaacg ctaagaccaa accccgggaa 900 gagcagtaca attccaccta cagggtggtg tccgtcctga cagtgctgca ccaagactgg 960 ctgaatggaa aggagtacaa gtgcaaagtg agcaataagg ccctccctgc tcccattgag 1020 aagaccattt ccaaggccaa aggccagcct cgggaacccc aggtgtacac actgccccct 1080 tccagggagg agatgaccaa gaaccaggtg agcctcacct gcctggtgaa gggcttctac 1140 cctagcgaca ttgctgtgga gtgggagagc aacggccagc ccgaaaacaa ctataagaca 1200 acccctcccg tgctggacag cgacggctcc ttctttctgt actccaagct caccgtggac 1260 aagtccaggt ggcaacaggg aaacgtgttc tcctgctccg tgatgcacga ggccctccac 1320 aaccactaca cccagaagag cctgagcctg tcccct 1356 <210> 56 <211> 678 <212> DNA <213> Artificial sequence <220> <223> Bi-204 nucleotide sequence <400> 56 gaggtgcagc tggtggagag cggaggagga ctggtgcagc ccggcggctc tctgagactg 60 agctgcgccg cctccggaag gacctttagc tccatgacca tcggctggtt cagacaagcc 120 cccggcaagg gactggagtt cgtgagcggc atcggctgga caagcggact gaccgtgtat 180 gccgacagcg tcaagggaag gttcaccatc tctagggaca acgccaagaa cagcatgtat 240 ctgcagatga actctctgag ggccgaggac accgccgtgt actactgcgc cgccgacagc 300 atgaacagag gccagttcga ctactggggc caaggcacac tggtgacagt gagcagccgg 360 accgtggccg ccccttccgt gttcatcttt cccccctccg acgagcagct gaagtccgga 420 accgccagcg tggtgtgcct cctgaacaac ttttaccccc gggaggccaa ggtgcagtgg 480 aaggtggaca acgccctgca aagcggcaac tcccaggaat ccgtcaccga gcaggattcc 540 aaggattcca cctacagcct gtcctccacc ctgacactgt ccaaggccga ctacgagaag 600 cacaaggtgt acgcctgcga ggtgacacac cagggcctga gcagccccgt gaccaagtcc 660 ttcaaccggg gcgagtgt 678 <210> 57 <211> 8 <212> PRT <213> Artificial sequence <220> <223> D‑Na‑96 CDR1 <400> 57 Gly Arg Thr Phe Ser Ser Met Thr 1 5 <210> 58 <211> 9 <212> PRT <213> Artificial sequence <220> <223> D‑Na‑96 CDR2 <400> 58 Gly Ile Gly Trp Thr Ser Gly Leu Thr 1 5 <210> 59 <211> 12​​​​​​​​​​​​​​​​​​​​​​​​​​ <400> 60 Gly Ser Glu Phe Ser Phe Ile Pro 1 5 <210> 61 <211> 8 <212> PRT <213> Artificial sequence <220> <223> D-Ye-29 CDR2 <400> 61 Val Phe Thr Ser Gly Gly Leu Thr 1 5 <210> 62 <211> 17 <212> PRT <213> Artificial sequence <220> <223> D-Ye-29 CDR3 <400> 62 Ser Gly Val Pro Gly Pro His Tyr Asn Gly Pro Thr Ser Gly Ile Asn 1 5 10 15 Tyr <210> 63 <211> 8 <212> PRT <213> Artificial sequence <220> <223> K-Yr-13&14-02 CDR1 <400> 63 Gly His Thr Phe Ile Ile Tyr Ala 1 5 <210> 64 <211> 8 <212> PRT <213> Artificial sequence <220> <223> K-Yr-13&14-02 CDR2 <400> 64 Ile Asn Trp Ser Gly Ser Met Thr 1 5 <210> 65 <211> 17 <212> PRT <213> Artificial sequence <220> <223> K-Yr-13&14-02 CDR3 <400> 65 Ala Ala Tyr Val Gly Ala Thr Ile Ser Thr Ala His Ser Arg Tyr Asp 1 5 10 15 Tyr <210> 66 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 66 gtcctggctg ctcttctaca agg 23 <210> 67 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 67 ggtacgtgct gttgaactgt tcc 23 <210> 68 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 68 ctagtgcggc cgcctggaga cggtgacctg ggt 33 <210> 69 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 69 cgcggatccc aggtgcagct gcaggagtct ggrggagg 38 <210> 70 <211> 89 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 70 atttttactg ctgttttatt cgcagcatcc tccgcattag ctaaaagaga ggctgaagca 60 caggtgcagc tgcaggagtc tggrggagg 89 <210> 71 <211> 81 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 71 agttgtcagt tcctgtgccc cccctcctcc cgcgccacct ccgcccgcac ctccgccacc 60 actggagacg gtgacctggg t 81

Claims

1. An anti-PD-L2 nanobody, characterized in that, The complementarity-determining regions (CDRs) of the VHH chain of the PD-L2 nanobody are composed of the following: The amino acid sequences are as shown in SEQ ID NO:57 for CDR1; as shown in SEQ ID NO:58 for CDR2; and as shown in SEQ ID NO:59 for CDR3.

2. The anti-PD-L2 nanobody as described in claim 1, characterized in that, The amino acid sequence of the VHH chain of the anti-PD-L2 nanobody is shown in SEQ ID NO:3 or 16.

3. The anti-PD-L2 nanobody as described in claim 1, characterized in that, The anti-PD-L2 nanobody is humanized, and the amino acid sequence of the VHH chain of the anti-PD-L2 nanobody is as shown in SEQ ID NO:

16.

4. An anti-PD-L1 nanobody, characterized in that, The complementarity-determining regions (CDRs) of the VHH chain of the PD-L1 nanobody consist of the following: The amino acid sequences are as shown in SEQ ID NO:63 for CDR1; as shown in SEQ ID NO:64 for CDR2; and as shown in SEQ ID NO:65 for CDR3.

5. The anti-PD-L1 nanobody as described in claim 4, characterized in that, The amino acid sequence of the VHH chain of the anti-PD-L1 nanobody is shown in SEQ ID NO:19,20,21 or22.

6. The anti-PD-L1 nanobody as described in claim 4, characterized in that, The anti-PD-L1 nanobody is humanized, and the amino acid sequence of the VHH chain of the anti-PD-L1 nanobody is shown in SEQ ID NO:

22.

7. A bispecific antibody, characterized in that, The bispecific antibodies include: the anti-PD-L1 nanobody as described in claim 4 and the anti-PD-L2 nanobody as described in claim 1.

8. The bispecific antibody as described in claim 7, characterized in that, The bispecific antibody contains a polypeptide with the structure shown in Formula I or Formula II. A-L1-Fc1-L2-B (Formula I) A-L3-B-L4-Fc1 (Formula II) in, A and B are each independently the anti-PD-L1 nanobody as described in claim 4 or the anti-PD-L2 nanobody as described in claim 1, and A and B are different antibodies; L1, L2, L3, and L4 are each independently peptide bond or linker element; Fc1 is the Fc segment of the antibody; where Fc1 is the human IgG domain; "-" represents a peptide bond.

9. The bispecific antibody as described in claim 7, characterized in that, The bispecific antibody contains two polypeptide sequences, and the two polypeptides form a heterodimer α through disulfide bonds. The amino acid sequences of the two polypeptides are shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

10. An isolated polynucleotide, characterized in that, The polynucleotide encodes the anti-PD-L2 nanobody as described in claim 1, the anti-PD-L1 nanobody as described in claim 4, or the bispecific antibody as described in claim 7.

11. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 10.

12. A host cell, characterized in that, The host cell contains the vector as described in claim 11, or its genome is integrated with the polynucleotide as described in claim 10; Alternatively, the host cell expresses the anti-PD-L2 nanobody as described in claim 1, the anti-PD-L1 nanobody as described in claim 4, or the bispecific antibody as described in claim 7.

13. A method for generating bispecific antibodies, characterized in that, Including the following steps: (a) Culturing the host cells as described in claim 12 under suitable conditions to obtain a culture containing the bispecific antibody; and (b) The culture obtained in step (a) is purified and / or separated to obtain the bispecific antibody.

14. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) the anti-PD-L2 nanobody as claimed in claim 1, the anti-PD-L1 nanobody as claimed in claim 4, or the bispecific antibody as claimed in claim 7; and (b) The coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, or enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins, or combinations thereof.

15. The use of the anti-PD-L2 nanobody as claimed in claim 1, the anti-PD-L1 nanobody as claimed in claim 4, the bispecific antibody as claimed in claim 7, or the immunoconjugate as claimed in claim 14, characterized in that, For use in the preparation of agents, reagents, detection plates or kits; wherein the agents, detection plates or kits are used to: detect PD-L1 and / or PD-L2 in a sample; wherein the agents are used to treat or prevent tumors expressing PD-L1 or tumors expressing PD-L2.

16. A pharmaceutical composition, characterized in that, Contains: (i) the anti-PD-L2 nanobody as claimed in claim 1, the anti-PD-L1 nanobody as claimed in claim 4, or the bispecific antibody as claimed in claim 7, or the immunoconjugate as claimed in claim 14; and (ii) a pharmaceutically acceptable carrier.

17. A PD-L1 and / or PD-L2 detection reagent, characterized in that, The detection reagent comprises the immunoconjugate as described in claim 14 and a detection-acceptable carrier.

18. A method for in vitro non-diagnostic and non-therapeutic detection of PD-L1 and / or PD-L2 in a sample, the method comprising the steps of: (1) contacting the sample with the anti-PD-L2 nanobody as claimed in claim 1, the anti-PD-L1 nanobody as claimed in claim 4, or the bispecific antibody as claimed in claim 7; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of PD-L1 and / or PD-L2 in the sample.

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