Anti-serum albumin single-domain antibody and application thereof

Abstract

The invention provides an anti-serum albumin single-domain antibody and application thereof. Specifically, the single-domain antibody provided by the invention can specifically target serum albumin of human, monkey and mouse, does not influence the combination of HSA and FcRn, and can still maintain the combination activity in an endosome acidification environment. The single-domain antibody can be used for prolonging the half-life period of a medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to single-domain antibodies against serum albumin and their applications. Background Art

[0002] Serum Albumin is an important component of blood, accounting for about half of the serum proteins. The concentration of albumin in human serum is approximately 35 - 50 g / L. Since serum proteins can specifically bind to the neonatal receptor (FcRn) in an acidic environment, and after being endocytosed by endothelial cells, they can return to the cell surface through the protein recycling mechanism mediated by FcRn and avoid being degraded in lysosomes, the half-life of serum proteins is relatively long, and the half-life of human serum albumin can reach 21 days.

[0003] Single-domain antibodies are a novel type of antibody molecule discovered by the research group of Belgian immunologist Hamers-Casterman in camelids. Compared with traditional antibodies, they have the advantages of small molecular weight, simple structure, strong tissue infiltration, and low immunogenicity. Single-domain antibodies do not contain the Fc domain and will not mediate the ADCC effect to cause cytotoxicity during the process of acting as a targeting molecule. In addition, they also have the advantages of good stability, high temperature resistance and extreme pH environment, and low production cost. Therefore, single-domain antibodies are a good targeting molecule and have great value in the development of targeting drugs.

[0004] Therefore, there is a need in the art to develop a single-domain antibody against serum albumin. Summary of the Invention

[0005] The object of the present invention is to provide single-domain antibodies targeting serum albumin and their applications.

[0006] In the first aspect of the present invention, there is provided a single-domain antibody against serum albumin (SA), wherein the VHH chain of the single-domain antibody has three CDRs of the VHH chain shown in any one of SEQ ID NO: 1 - 29, and wherein the CDRs are CDR1, CDR2, and CDR3 determined by any one of the IMGT rule, Kabat rule, Chothia rule, AbM rule, or Contact rule.

[0007] In another preferred embodiment, the VHH chain of the single-domain antibody has the following complementarity-determining regions CDR:

[0008] CDR1 shown in any one of SEQ ID NO: 30 - 34,

[0009] CDR2 shown in any one of SEQ ID NO: 35 - 38, and

[0010] A CDR3 as shown in any one of SEQ ID NO:39 - 47.

[0011] In another preferred embodiment, the CDR region of the single - domain antibody VHH chain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity with any one of the above - mentioned sequences.

[0012] In another preferred embodiment, any one of the above - mentioned amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain the SA - binding affinity.

[0013] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1 - 3, preferably 1 - 2, more preferably 1.

[0014] In another preferred embodiment, the CDR1 sequence of the single - domain antibody VHH chain is as shown in SEQ ID NO.32, the CDR2 sequence is as shown in SEQ ID NO.37, and the CDR3 sequence is as shown in SEQ ID NO.44.

[0015] In another preferred embodiment, the CDR1 sequence of the nanobody VHH chain is as shown in SEQ ID NO.33, the CDR2 sequence is as shown in SEQ ID NO.35, and the CDR3 sequence is as shown in SEQ ID NO.47.

[0016] In another preferred embodiment, the CDR1 sequence of the nanobody VHH chain is as shown in SEQ ID NO.33, the CDR2 sequence is as shown in SEQ ID NO.35, and the CDR3 sequence is as shown in SEQ ID NO.46.

[0017] In another preferred embodiment, the CDR1 sequence of the nanobody VHH chain is as shown in SEQ ID NO.33, the CDR2 sequence is as shown in SEQ ID NO.35, and the CDR3 sequence is as shown in SEQ ID NO.45.

[0018] In another preferred embodiment, the CDR1 sequence of the nanobody VHH chain is as shown in SEQ ID NO.32, the CDR2 sequence is as shown in SEQ ID NO.37, and the CDR3 sequence is as shown in SEQ ID NO.39.

[0019] In another preferred embodiment, the CDR1 sequence of the nanobody VHH chain is as shown in SEQ ID NO.30, the CDR2 sequence is as shown in SEQ ID NO.36, and the CDR3 sequence is as shown in SEQ ID NO.43.

[0020] In another preferred example, the CDR1 sequence of the nanobody VHH chain is as shown in SEQ ID NO.31, the CDR2 sequence is as shown in SEQ ID NO.38, and the CDR3 sequence is as shown in SEQ ID NO.40.

[0021] In another preferred example, the CDR1 sequence of the nanobody VHH chain is as shown in SEQ ID NO.31, the CDR2 sequence is as shown in SEQ ID NO.38, and the CDR3 sequence is as shown in SEQ ID NO.41.

[0022] In another preferred example, the CDR1 sequence of the nanobody VHH chain is as shown in SEQ ID NO.34, the CDR2 sequence is as shown in SEQ ID NO.38, and the CDR3 sequence is as shown in SEQ ID NO.40.

[0023] In another preferred example, the CDR1 sequence of the nanobody VHH chain is as shown in SEQ ID NO.31, the CDR2 sequence is as shown in SEQ ID NO.38, and the CDR3 sequence is as shown in SEQ ID NO.42.

[0024] In another preferred example, the VHH chain of the single-domain antibody further includes a framework region (FR).

[0025] In another preferred example, the CDR1, CDR2, and CDR3 are separated by the framework regions FR1, FR2, FR3, and FR4 of the VHH chain.

[0026] In another preferred example, the framework region FR is of human, murine, rabbit, or camel origin.

[0027] In another preferred example, the single-domain antibody binds to human, murine, or simian SA.

[0028] In another preferred example, the single-domain antibody binds to human serum albumin (HSA).

[0029] In another preferred example, the single-domain antibody includes monomers, bivalent bodies (bivalent antibodies), tetravalent bodies (tetravalent antibodies), and / or multivalent bodies (multivalent antibodies).

[0030] In another preferred example, the amino acid sequence of the VHH chain of the single-domain antibody has a homology of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% with the amino acid sequence shown in any one of SEQ ID NO: 1-29 or 49-66.

[0031] In another preferred embodiment, the VHH chain of the single-domain antibody has an amino acid sequence as shown in any one of SEQ ID NO: 1-29 or 49-66.

[0032] In another preferred embodiment, the amino acid sequence of the VHH chain of the single-domain antibody is selected from any one of SEQ ID NO: 1-29 or 49-66.

[0033] In another preferred embodiment, the single-domain antibody does not affect the binding of HSA to FcRn.

[0034] In another preferred embodiment, the single-domain antibody further comprises an Fc segment of an immunoglobulin, preferably an Fc segment of IgG, such as an Fc segment of human IgG1.

[0035] In a second aspect of the present invention, there is provided a single-domain antibody fusion protein which has a structure as shown in Formula Ia or Ib from the N-terminus to the C-terminus:

[0036] Ab1-F-L-P (Formula Ia);

[0037] P-L-Ab1-F (Formula Ib)

[0038] In the formula,

[0039] Ab1 is the VHH chain of the anti-SA single-domain antibody as described in the first aspect of the present invention;

[0040] F is none or an Fc segment of an immunoglobulin;

[0041] L is a linker sequence;

[0042] P is a therapeutic protein.

[0043] In another preferred embodiment, the L is a flexible linker peptide.

[0044] In another preferred embodiment, the L sequence is as shown in (GGGS)n, where n is an integer selected from 1-6, preferably n is 2, 3 or 4.

[0045] In another preferred embodiment, the P is a therapeutic antibody or a natural ligand.

[0046] In another preferred embodiment, the therapeutic antibody is a monoclonal antibody fragment or a tandem form of monoclonal antibody fragments, preferably scFv, tandem scFv, Fab, tandem Fab, single-domain antibody, tandem single-domain antibody or a combination thereof.

[0047] In another preferred embodiment, the therapeutic antibody targets a tumor-associated antigen.

[0048] In another preferred example, the tumor-associated antigen is selected from the following group: BCMA, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CDlla, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123, CD125, CD132 (IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor α (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TfR1, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or a combination thereof.

[0049] In another preferred embodiment, the therapeutic antibody sequence is as shown in SEQ ID NO:67.

[0050] In another preferred embodiment, the ratio (T1 / T0) of the half-life (T1) of the single-domain antibody fusion protein to the half-life (T0) of the therapeutic protein alone is ≥ 200%, preferably ≥ 250%, more preferably ≥ 300%.

[0051] In a third aspect of the present invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: the anti-SA single-domain antibody of the first aspect of the present invention, the single-domain antibody fusion protein of the second aspect of the present invention, or a combination thereof.

[0052] In another preferred embodiment, the polynucleotide comprises DNA, RNA or cDNA.

[0053] In a fourth aspect of the present invention, there is provided an expression vector containing the polynucleotide of the third aspect of the present invention.

[0054] In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0055] In another preferred embodiment, the expression vector comprises a viral vector, such as a lentivirus, an adenovirus, an AAV virus, a retrovirus.

[0056] In another preferred embodiment, the expression vector is the pCMV3 vector.

[0057] In a fifth aspect of the present invention, there is provided a host cell containing the expression vector of the fourth aspect of the present invention, or the polynucleotide of the third aspect of the present invention is integrated into its genome.

[0058] In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0059] In another preferred embodiment, the host cell is selected from the group consisting of: Escherichia coli, yeast cells, mammalian cells, phages, or combinations thereof.

[0060] In another preferred embodiment, the host cell is selected from HEK293 cells, Pichia pastoris cells, or combinations thereof.

[0061] In a sixth aspect of the present invention, there is provided a method for producing an anti-SA single-domain antibody, comprising the steps of:

[0062] (a) culturing the host cell of the fifth aspect of the present invention under conditions suitable for producing a single-domain antibody, thereby obtaining a culture containing the anti-SA single-domain antibody; and

[0063] (b) isolating or recovering the anti-SA single-domain antibody from the culture; and

[0064] (c) Optionally, purify and / or modify the anti-SA single-domain antibody obtained in step (b).

[0065] In a seventh aspect of the present invention, there is provided an immunoconjugate comprising:

[0066] (a) an anti-SA single-domain antibody as described in the first aspect of the present invention; and

[0067] (b) a conjugate moiety conjugated to the single-domain antibody moiety, the conjugate moiety being selected from the group consisting of: a detectable label, a drug, or a combination thereof.

[0068] In another preferred embodiment, the immunoconjugate is an antibody-drug conjugate.

[0069] In another preferred embodiment, the antibody moiety is conjugated to the conjugate moiety via a chemical bond or a linker.

[0070] In another preferred embodiment, the detectable label is a chemical label, a biological label, or a combination thereof.

[0071] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0072] In another preferred embodiment, the biological label is biotin, avidin, or an enzyme label.

[0073] In another preferred embodiment, the drug is a small molecule drug, a biological factor, an oligonucleotide, or a combination thereof.

[0074] In another preferred embodiment, the oligonucleotide is an antisense oligonucleotide, a small interfering ribonucleic acid, a micro ribonucleic acid, a nucleic acid aptamer, or a combination thereof.

[0075] In another preferred embodiment, the drug is a cytotoxic drug (toxin).

[0076] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding reagent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

[0077] In another preferred embodiment, the conjugate moiety is a detectable label.

[0078] In another preferred embodiment, the detectable label includes a radionuclide, and the radionuclide includes:

[0079] (i) Isotopes for detection, said isotopes for detection are selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0080] (ii) Isotopes for treatment, said isotopes for treatment 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 a combination thereof.

[0081] 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, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, virus particles, virus-like particles (VLPs), liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, etc.

[0082] In another preferred embodiment, the immunoconjugate has the following molecular formula: [[ID=ID=10]]

[0083]

[0084] Wherein:

[0085] nAb is a single-domain antibody against SA as described in the first aspect of the present invention;

[0086] LU is a chemical bond or linker;

[0087] D is a conjugate moiety;

[0088] p is the average number of conjugate moieties in the immunoconjugate, and p is a value selected from 1 to 10.

[0089] In the eighth aspect of the present invention, a recombinant protein is provided, said recombinant protein having:

[0090] (i) The sequences of the single-domain antibody as described in the first aspect of the present invention and the single-domain antibody fusion protein as described in the second aspect of the present invention; and

[0091] (ii) A tag sequence for assisting expression and / or purification.

[0092] In another preferred embodiment, the tag sequence includes a His tag and an HA tag

[0093] In another preferred embodiment, the recombinant protein specifically binds to the SA protein.

[0094] In the ninth aspect of the present invention, there is provided a multimeric protein complex based on a complementary nucleic acid backbone, wherein the complex is a multimer formed by the complexation of n monomers having complementary nucleic acid backbones, and wherein the n monomers consist of:

[0095] (i) At least one half-life extension module, which consists of an anti-SA single-domain antibody as described in the first aspect of the present invention and a single-stranded nucleic acid linked thereto;

[0096] (ii) Optionally none or one or more targeting modules, which consist of a targeting molecule and a single-stranded nucleic acid linked thereto;

[0097] (iii) Optionally none or one or more pharmacodynamic modules, which consist of a pharmacodynamic molecule and a single-stranded nucleic acid linked thereto;

[0098] (iv) Optionally none or one or more backbone monomers, which consist of single-stranded nucleic acids;

[0099] wherein n is a positive integer from 2 to 8, and the single-stranded nucleic acids of each monomer form complementary double-strands with the single-stranded nucleic acids of 1, 2 or 3 other monomers, thereby forming a complementary nucleic acid backbone structure.

[0100] In another preferred embodiment, n = 3, 4, 5 or 6.

[0101] In another preferred embodiment, the multimeric protein complex is a tetra-specific antibody, which consists of one half-life extension module and three targeting modules targeting different targets.

[0102] In another preferred embodiment, the multimeric protein complex is a trivalent protein, which consists of one half-life extension module and three pharmacodynamic modules, and the three pharmacodynamic modules are respectively composed of the same or different pharmacodynamic molecules and single-stranded nucleic acids linked thereto.

[0103] In another preferred example, the multimeric protein complex is a tetrameric ADC, which consists of a half-life extension module, a targeting module, and two pharmacodynamic modules. The two pharmacodynamic modules are respectively composed of the same or different toxin molecules and single-stranded nucleic acids linked thereto.

[0104] In another preferred example, the multimeric protein complex is a tetrameric AOC, which consists of a half-life extension module, a targeting module, and two pharmacodynamic modules. The two pharmacodynamic modules are respectively composed of the same or different oligonucleotide molecules and single-stranded nucleic acids linked thereto.

[0105] In another preferred example, the half-life extension module has the structure of formula IIa or IIb:

[0106] A-W (IIa); W-A (IIb)

[0107] The pharmacodynamic module has the structure of formula IIIa or IIIb:

[0108] D-W (IIIa); W-D (IIIb)

[0109] The targeting module has the structure of formula IVa or IVb:

[0110] T-W(IVa); W-T(IVb)

[0111] In the formula,

[0112] A is an anti-SA single-domain antibody as described in the first aspect of the present invention;

[0113] W is a single-stranded nucleic acid;

[0114] D is a pharmacodynamic molecule;

[0115] T is a targeting molecule;

[0116] Each "-" is independently a linker or a bond.

[0117] In another preferred example, "-" is a covalent bond, a linker, or a combination of both.

[0118] In another preferred example, in the multimer, the element D in each pharmacodynamic module is independently selected from the group consisting of: small cell-killing molecules, polypeptide toxins, oligonucleotide molecules that regulate gene expression, or combinations thereof.

[0119] In another preferred example, the single-stranded nucleic acid is degradation-resistant.

[0120] In another preferred embodiment, the single-stranded nucleic acid is selected from the group consisting of: L-nucleic acid, peptide nucleic acid, locked nucleic acid, morpholino phosphorodiamidate nucleic acid, L-morpholino phosphorodiamidate nucleic acid, sulfur-modified nucleic acid, 2'-fluoro-modified nucleic acid, 5-hydroxymethylcytosine nucleic acid, or a combination thereof. Preferably, the single-stranded nucleic acid is morpholino phosphorodiamidate nucleic acid.

[0121] In another preferred embodiment, in the polymer, the element D in each pharmacodynamic module is the same or different.

[0122] In another preferred embodiment, in the polymer, each element T in each targeting module is independently selected from the group consisting of: an antibody, a natural ligand, a targeting small molecule, or a combination thereof.

[0123] In another preferred embodiment, the T is a single-domain antibody.

[0124] In another preferred embodiment, in the polymer, the W of each monomer is different.

[0125] In another preferred embodiment, the nucleotides at both ends and / or in the middle of W are chemically modified to connect the pharmacodynamic molecule D, the targeting molecule T or the anti-SA single-domain antibody.

[0126] In another preferred embodiment, the nucleic acid single-strand sequence W in the half-life extension module and the drug module has the structure shown in Formula V:

[0127] X1-R1-X2-R2-X3 (V)

[0128] Wherein,

[0129] R1 is base complementary pairing region 1;

[0130] R2 is base complementary pairing region 2;

[0131] X1, X2 and X3 are each independently none or redundant nucleic acid;

[0132] "-" is a bond.

[0133] In another preferred embodiment, the lengths of R1 and R2 are each independently 10-20 bases, preferably 14-16 bases.

[0134] In another preferred embodiment, the length of X1 is 0-5 bases.

[0135] In another preferred embodiment, the length of X3 is 0-5 bases.

[0136] In another preferred embodiment, the length of X2 is 0-3 bases.

[0137] In another preferred embodiment, the sequence of X2 is selected from the group consisting of: A, AA, AGA or AAA.

[0138] In another preferred example, R1 of each monomer forms a base complementary pairing structure with R2 of the left adjacent (or left side) monomer; while R2 forms a base complementary pairing structure with R1 of the right adjacent (or right side) monomer.

[0139] In the eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0140] (i) an anti-SA single-domain antibody as described in the first aspect of the present invention, or a fusion protein as described in the second aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention, or a recombinant protein as described in the eighth aspect of the present invention, or a multimeric protein complex as described in the ninth aspect of the present invention, or a combination thereof; and

[0141] (ii) a pharmaceutically acceptable carrier.

[0142] In another preferred example, the pharmaceutical composition is in an injectable dosage form.

[0143] In another preferred example, the pharmaceutical composition is used for preparing a drug for treating tumors, and the tumors are selected from the group consisting of: colon cancer, renal chromophobe cell carcinoma, renal papillary cell carcinoma, mesothelioma, pancreatic cancer, prostate cancer, ovarian germ cell carcinoma, thyroid cancer, gastric cancer, esophageal cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), malignant glioma, liver cancer, bladder cancer, endometrial cancer, cervical cancer, leukemia, myeloma, osteosarcoma, angiosarcoma, or a combination thereof.

[0144] In another preferred example, the pharmaceutical composition further contains other drugs for treating immune system diseases or tumor diseases.

[0145] In another preferred example, the other drugs for treating immune system diseases or tumor diseases are selected from the group consisting of: budesonide, fluticasone, beclomethasone, mometasone furoate, salbutamol, theophylline, formoterol, tiotropium bromide, sulfasalazine, methotrexate, cyclophosphamide, fluorouracil, bleomycin, anastrozole, or a combination thereof.

[0146] In the twelfth aspect of the present invention, there is provided a use of an active ingredient, and the active ingredient is selected from the group consisting of: an anti-SA single-domain antibody as described in the first aspect of the present invention, or a fusion protein as described in the second aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention, or a recombinant protein as described in the eighth aspect of the present invention, or a multimeric protein complex as described in the ninth aspect of the present invention, or a pharmaceutical composition as described in the eleventh aspect of the present invention, or a combination thereof, and the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; and / or (b) preparing a drug for preventing and / or treating diseases.

[0147] In another preferred example, the detection reagent, the detection plate or the kit is used for:

[0148] (1) Detect the SA protein in the sample; and / or

[0149] (2) Detect the cells expressing the SA protein.

[0150] In another preferred example, the detection reagent, detection plate or kit is used for diagnosing SA-related diseases.

[0151] In another preferred example, the detection includes flow cytometry and cellular immunofluorescence detection.

[0152] In the twelfth aspect of the present invention, a method for detecting the SA protein in a sample is provided. The method includes the steps:

[0153] (1) Contact the sample with the anti-SA single-domain antibody as described in the first aspect of the present invention;

[0154] (2) Detect whether an antigen-antibody complex is formed. The formation of the complex indicates the presence of the SA protein in the sample.

[0155] In the thirteenth aspect of the present invention, a detection reagent for the SA protein is provided. The detection reagent comprises:

[0156] (i) The anti-SA single-domain antibody as described in the first aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention, or the recombinant protein as described in the eighth aspect of the present invention; and

[0157] (ii) A pharmaceutically acceptable carrier.

[0158] In another preferred example, the conjugate part of the immunoconjugate is a detection isotope.

[0159] In another preferred example, the pharmaceutically acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0160] In another preferred example, the detection reagent is one or more reagents selected from the group consisting of: isotope tracers, contrast agents, flow cytometry reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agents.

[0161] In another preferred example, the detection reagent is used for in vivo detection.

[0162] In another preferred example, the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, buccal tablet, aerosol).

[0163] In the fourteenth aspect of the present invention, a detection kit for the SA protein is provided. The kit contains the immunoconjugate as described in the seventh aspect of the present invention or the detection reagent as described in the thirteenth aspect of the present invention, and an instruction manual

[0164] In another preferred example, the specification states that the kit is used for non-invasively detecting the expression of SA in a subject to be tested.

[0165] In the fifteenth aspect of the present invention, a method for treating a disease is provided, the method comprising administering to a subject in need thereof the anti-SA single-domain antibody as described in the first aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention, or the recombinant protein as described in the eighth aspect of the present invention, or the multimeric protein complex as described in the ninth aspect of the present invention, or the pharmaceutical composition as described in the tenth aspect of the present invention.

[0166] In another preferred example, the subject includes a human or a non-human mammal.

[0167] In another preferred example, the non-human mammal includes a rodent (such as a mouse, a rabbit), a non-human primate (such as a monkey).

[0168] In another preferred example, the disease is a tumor.

[0169] In the sixteenth aspect of the present invention, a method for prolonging the half-life of a drug is provided, comprising the steps of:

[0170] Linking the drug to the anti-SA single-domain antibody as described in the first aspect of the present invention.

[0171] In another preferred example, the drug is linked to the anti-SA single-domain antibody to form a single-domain antibody fusion protein as described in the second aspect of the present invention, an immunoconjugate as described in the seventh aspect of the present invention, or a multimeric protein complex as described in the ninth aspect of the present invention.

[0172] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] The following drawings are used to illustrate the specific embodiments of the present invention, and are not used to limit the scope of the present invention defined by the claims.

[0174] Figure 1 Shows the protein sequence alignment results of SA antigens of three species: human, cynomolgus monkey, and mouse.

[0175] Figures 2A-2B Shows the phylogenetic tree analysis of the diverse amino acid sequences of the anti-SA single-domain antibody yeast library.

[0176] Figure 3 Shows the binding activity of the anti-SA single-domain antibody to human SA antigen detected by ELISA.

[0177] Figure 4 It shows the binding activity of the anti-SA single-domain antibody detected by ELISA with monkey SA antigen.

[0178] Figure 5 It shows the binding activity of the anti-SA single-domain antibody detected by ELISA with mouse SA antigen.

[0179] Figure 6 It shows the binding activity of the anti-SA single-domain antibody detected by ELISA with SA antigen in human serum.

[0180] Figure 7 It shows the binding activity of the anti-SA single-domain antibody detected by ELISA with SA antigen in mouse serum.

[0181] Figure 8 It shows the binding activity of the anti-SA single-domain antibody detected by ELISA with SA antigen in rat serum.

[0182] Figure 9 It shows the SDS-PAGE gel images of the anti-TfR1 single-domain antibody and the tandem form of the anti-TfR1-HSA single-domain antibody.

[0183] Figure 10 It shows the curve graph of the relative body weight change after drug administration in BALB / c Nude mice.

[0184] Figure 11 It shows the linear fitting of the standard curves of each drug by double logarithmic analysis and the schematic diagram of the LISA detection method.

[0185] Figure 12 It shows the curve graph of the drug concentration-time in mouse serum.

[0186] Figure 13 It shows the binding activity of the humanized anti-SA VHH-Fc detected by ELISA with human SA antigen.

[0187] Figure 14 It shows the binding activity of the humanized anti-SA VHH-Fc detected by ELISA with monkey SA antigen.

[0188] Figure 15 It shows the binding activity of the humanized anti-SA VHH-Fc detected by ELISA with mouse SA antigen.

[0189] Figure 16 It shows the SDS-PAGE identification of the coupling of the single-domain antibody with SM(PEG)2-PMO1.

[0190] Figure 17 It shows the purification and separation results of the single-domain antibody and the single-domain antibody-PMO1 mixture.

[0191] Figure 18 Shows the PMO-NAPPA4-HSA(1)-TfR1(4) structure and the SDS-PAGE identification results of the tetramer assembly.

[0192] Figure 19 Shows the ELISA test results of the binding of the Anti-TfR1 single-domain antibody and the PMO-NAPPA4-HSA(1)-TfR1(4) tetramer to the TfR1 protein.

[0193] Figure 20 Shows the body weight changes of mice treated with the Anti-TfR1 single-domain antibody and PMO-NAPPA4-HSA(1)-TfR1(4).

[0194] Figure 21 Shows the drug concentration-time curve of the Anti-TfR1 single-domain antibody and PMO-NAPPA4-HSA(1)-TfR1(4) in mouse serum. Detailed implementation mode

[0195] After extensive and in-depth research, the present inventors have developed for the first time a single-domain antibody targeting serum albumin. The single-domain antibody of the present invention can specifically target serum albumin of three species, namely human, monkey and mouse, has universality, does not affect the binding of HSA to FcRn, and can still maintain the binding activity in the endosomal acidification environment. Therefore, the single-domain antibody of the present invention can be used to extend the half-life of drugs. On this basis, the present invention has been completed.

[0196] Terms

[0197] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0198] As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0199] As used herein, the terms "comprising", "including", and "containing" are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of" and "consisting essentially of".

[0200] As used herein, the components of the term "pharmaceutically acceptable carrier" refer to substances that are applicable to humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio.

[0201] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art should understand that the "therapeutically effective amount" may vary depending on factors such as the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs.

[0202] Anti-SA nanobody

[0203] As used herein, the terms "the anti-SA single-domain antibody of the present invention", "the single-domain antibody of the present invention", and "the SA single-domain antibody of the present invention" are used interchangeably and all refer to nanobodies that specifically recognize and bind to SA (such as HSA). Particularly preferred are nanobodies in which the amino acid sequence of the VHH chain is as shown in any one of SEQ ID NO.: 1-29 or 49-66.

[0204] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of approximately 150,000 daltons with the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0205] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning and refer to cloning the variable region of the antibody heavy chain to construct a single-domain antibody (VHH) consisting only of one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody that is naturally lacking the light chain and the first constant region (CH1) of the heavy chain, the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting only of one heavy chain variable region.

[0206] As used herein, the term "variable" indicates that certain portions of the variable regions in an antibody differ in sequence, and it forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions in the variable regions are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration and are connected by three CDRs forming connecting loops, and in some cases can form a partial β-sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0207] As is known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding a drug, toxin, cytokine, radionuclide, enzyme, and other diagnostic or therapeutic molecules to the antibody or fragment thereof of the present invention. The present invention also includes cell surface markers or antigens that bind to the anti-SA protein single-domain antibody or fragment thereof described above.

[0208] As used herein, the term "heavy chain variable region" is interchangeable with "V H ".

[0209] As used herein, the term "variable region" is interchangeable with "complementarity determining region (CDR)".

[0210] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

[0211] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the above heavy chain variable region and a heavy chain constant region.

[0212] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are interchangeable and all refer to polypeptides that specifically bind to the SA protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0213] 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 (such as immunoconjugates and fusion proteins) having a heavy chain containing a variable region, provided that the variable region is the same as or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibody of the present invention.

[0214] The present invention includes not only intact antibodies, but also fragments, derivatives and analogs of the antibodies.

[0215] As used herein, the terms "fragment", "derivative" and "analog" 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 conservative or non-conservative amino acid residues (preferably conservative 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 a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing a mature polypeptide with another compound (such as a compound that prolongs 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 (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0216] The antibodies of the present invention refer to polypeptides having SA protein binding activity and including the above CDR regions. The term also includes variant forms of polypeptides containing the above CDR regions that have the same function as the antibodies of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar or similar properties, the function of the protein usually does not change. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0217] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the coding DNA of the antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using the antiserum against the antibodies of the present invention.

[0218] The present invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of the nanobodies of the present invention. Generally, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 80 consecutive amino acids, more preferably at least about 100 consecutive amino acids.

[0219] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or close properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0220] Table A

[0221] Initial residue Representative substitution Preferred substitution 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

[0222] The single-domain antibodies of the present invention specifically bind to HSA. Through the specific binding of HSA to FcRn and the FcRn-mediated protein recycling mechanism, the single-domain antibodies of the present invention can be transported back to the cell surface together with HSA, thereby obtaining a longer half-life. In one embodiment, the single-domain antibodies of the present invention have cross-reactive activity in human, murine, and simian HSA. In one embodiment, the KD value of the affinity of the single-domain antibodies of the present invention for human, murine, or simian HSA at pH 5.0 is ≤10 - 7 M, preferably ≤10 -8 M, more preferably ≤10 -9 M. In one embodiment, the KD value of the affinity of the single-domain antibodies of the present invention for human HSA at pH 7.4 is ≤10 -7 M, preferably ≤10 -8 M, more preferably ≤0.5×10 -9 M.

[0223] The present invention provides fusion protein drugs or immunoconjugate drugs containing the single-domain antibodies of the present invention. The single-domain antibodies of the present invention can significantly extend the half-life of drugs in vivo through protein recycling.

[0224] Polynucleotides, vectors, and host cells

[0225] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0226] The polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; the coding sequences of the mature polypeptides and various additional coding sequences; the coding sequences of the mature polypeptides (and optional additional coding sequences) and non-coding sequences.

[0227] The term "polynucleotide encoding a polypeptide" may be a polynucleotide comprising the polynucleotide encoding this polypeptide, or may also be a polynucleotide further comprising additional coding and / or non-coding sequences.

[0228] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides described in the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0229] The full-length nucleotide sequences or fragments of the antibodies of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis methods. A feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Usually, very long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0230] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually done by cloning them into a vector, then transferring them into cells, and then separating the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0231] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or vectors such as) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0232] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0233] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0234] Transformation of the host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase, treated with the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0235] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell. After the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0236] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0237] The antibody of the present invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination of any of the above substances.

[0238] Detectable labels for diagnostic purposes include but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0239] Therapeutic agents that can bind to or be conjugated with the antibodies of the present invention include, but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles / nanorods; 5. Virus particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, etc.

[0240] Immunoconjugate

[0241] The present invention also provides immunoconjugates based on the antibodies of the present invention, preferably single-domain antibody-drug conjugates (nanobody-drug conjugate, NDC).

[0242] Typically, the antibody-drug conjugate includes the antibody and an effector molecule, and the antibody is conjugated with the effector molecule, preferably by chemical conjugation. Among them, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule can be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, an agonist small molecule (STING, TLR7, TLR8, etc.), an oligonucleotide, or a radionuclide.

[0243] The antibody of the present invention and the effector molecule can be conjugated through a coupling agent. Examples of the coupling agent can be any one or several of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound that forms a covalent bond connection between the effector molecule and the antibody, such as glutaraldehyde, etc. The coupling agent using a carboxyl group can be any one or several of cis-aconitic anhydride-based coupling agents (such as cis-aconitic anhydride) and acylhydrazone-based coupling agents (the coupling site is acylhydrazone).

[0244] Certain residues on the antibody (such as Cys or Lys, etc.) are used to connect with various functional groups, including imaging reagents (e.g., chromophores and fluorescent groups), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (e.g., a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be directly or indirectly connected to the antibody through a linker.

[0245] Single-domain antibodies can be conjugated with drugs to form single-domain antibody-drug conjugates (NDCs). Typically, an NDC comprises a linker positioned between the drug and the antibody. The linker can be a degradable or a non-degradable linker. Degradable linkers typically degrade readily in the intracellular environment, e.g., at the target site, such that the drug is released from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl linkers that can be degraded by intracellular proteases (e.g., lysosomal or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is proteolytically cleaved.

[0246] Prior to attachment to the antibody, the linker has reactive moieties capable of reacting with certain amino acid residues, and the attachment is effected through the reactive moieties. Thiol-specific reactive moieties are preferred and include, for example, maleimide compounds, haloamides (e.g., iodo-, bromo-, or chloro-substituted), haloesters (e.g., iodo-, bromo-, or chloro-substituted), halomethyl ketones (e.g., iodo-, bromo-, or chloro-substituted), benzyl halides (e.g., iodo-, bromo-, or chloro-substituted), vinyl sulfones, pyridyl disulfides, mercury derivatives such as 3,6-di-(mercurimethyl) dioxane with counterions acetate, chloride, or nitrate, and polymethylene dimethyl thiosulfonate. The linker can include, for example, a polyethylene glycolylated SMCC crosslinker (SM(PEG)2), a maleimide attached to the antibody through a succinimide.

[0247] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In embodiments, the linker attaches the antibody and the drug, and the drug has a functional group capable of bonding to the linker. For example, the drug can have an amino, carboxyl, thiol, hydroxyl, or keto group capable of bonding to the linker. In cases where the drug is directly attached to the linker, the drug has a reactive moiety prior to attachment to the antibody.

[0248] Useful drug classes include, for example, antimitotic agents, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. Examples of particularly useful classes of cytotoxic drugs include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Representative cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines, maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines, or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0249] The immunoconjugate drugs of the present invention can also be radionuclide conjugate drugs (RDCs), which are composed of the antibodies of the present invention conjugated with radionuclides.

[0250] The immunoconjugate drugs of the present invention can also be antibody-oligonucleotide conjugates (AOCs), which are composed of the single-domain antibodies of the present invention conjugated with therapeutic oligonucleotides. Oligonucleotides that can be used in the AOCs of the present invention include, but are not limited to, antisense oligonucleotides, small interfering ribonucleic acids, micro ribonucleic acids, nucleic acid aptamers, etc.

[0251] In the present invention, a drug-linker can be used to form an NDC in a single step. In other embodiments, a bifunctional linker compound can be used to form an NDC in a two-step or multi-step method. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an NDC.

[0252] Typically, functional groups on the linker are selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker through a 1,3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other conjugation strategies, such as those described in Bioconjugate Techniques, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for the selective reaction of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used either for the linker or for the drug.

[0253] The present invention also provides a method for preparing an NDC, which may further comprise: binding an antibody to a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).

[0254] In certain embodiments, the method of the present invention comprises: binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: binding the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.

[0255] In some embodiments, the structure of the immunoconjugate, preferably the single-domain antibody conjugate NDC, is shown by the following molecular formula:

[0256]

[0257] Wherein:

[0258] nAb is the above-mentioned single-domain antibody targeting SA, a heavy-chain antibody targeting SA, or a multispecific antibody,

[0259] LU is a linker / spacer;

[0260] D is a conjugate moiety;

[0261] And the subscript p is a value selected from 1 to 10.

[0262] Multimeric protein complexes based on complementary nucleic acid backbones

[0263] The single-domain antibody of the present invention can also be linked to a drug molecule through a mutually complementary nucleic acid backbone, thereby prolonging the half-life of the drug in vivo. Therefore, the present invention also provides a multimeric protein complex based on a mutually complementary nucleic acid backbone. The multimeric protein complex of the present invention is composed of at least one half-life extension module, at least one pharmacodynamic module, an optional targeting module, and an optional backbone monomer. The half-life extension module is composed of the anti-SA single-domain antibody of the present invention and a single-stranded nucleic acid linked thereto. The pharmacodynamic module is composed of a pharmacodynamic molecule (such as a protein drug, a toxin, an oligonucleotide) and a single-stranded nucleic acid linked thereto. The targeting module is composed of a targeting molecule and a single-stranded nucleic acid linked thereto.

[0264] The nucleic acid strand in the multimeric protein complex of the present invention is a nucleic acid strand that is resistant to degradation in vivo and does not cause a strong innate immune response. Typically, the single-stranded nucleic acid includes, but is not limited to, L-nucleic acid, peptide nucleic acid, locked nucleic acid, morpholino phosphate nucleic acid, L-morpholino phosphate nucleic acid, sulfur-modified nucleic acid, 2'-fluoro-modified nucleic acid, 5-hydroxymethylcytosine nucleic acid.

[0265] The pharmacodynamic molecule can be any drug with cytotoxicity, cell growth inhibition, or immunosuppression. For example, small molecule toxins, protein drugs, oligonucleotide drugs.

[0266] The targeting molecule can be any targeting antibody, ligand, or targeting small molecule that specifically binds to a target of interest. The targeting antibody can be a monoclonal antibody or an antigen-binding fragment thereof. In one embodiment, the targeting antibody can be a single-domain antibody targeting TfR1, for example, it can have the sequence shown in SEQ ID NO: 68. The ligand can be a natural ligand or a targeting variant thereof.

[0267] In the multimeric protein complex of the present invention, a bifunctional linker molecule (such as SMCC) can be used to bridge the single-stranded nucleic acid and the protein. The single-stranded nucleic acid can contain modifications of active groups at the 5'-end or 3'-end to facilitate subsequent linker coupling. The linker generally has bifunctional groups, that is, one end can be coupled to the active group of the nucleic acid, and the other end can be linked to a specific site (such as NH2, SH) on the protein. In one embodiment, all single-stranded nucleic acids constituting the mutually complementary nucleic acid backbone are modified with NH2 at the 5'-end. Amino acid mutations can be introduced into the anti-SA single-domain antibody and / or the protein drug to facilitate coupling with the linker. In one embodiment, cysteine mutations can be introduced at the N-terminus or C-terminus of the anti-SA single-domain antibody and / or the protein drug.

[0268] In a preferred embodiment of the present invention, the multimeric protein complex contains four different sequences of morpholino phosphate oligonucleotides, which can accurately self-assemble into a stable tetrameric nucleic acid backbone.

[0269] Drug composition

[0270] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the substances being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0271] The pharmaceutical composition of the present invention may contain any anti-tumor drug (such as an anti-tumor antibody) linked to the anti-SA single-domain antibody of the present invention, and thus can be used for treating tumors. In addition, other therapeutic agents can also be used simultaneously.

[0272] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned nanobody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should match the administration method. The pharmaceutical composition of the present invention can be made into an injectable form, for example, prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectables and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 10 micrograms per kilogram of body weight per day - about 50 milligrams per kilogram of body weight. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0273] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually 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 about 10 micrograms per kilogram of body weight - about 10 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the health condition of the patient, which are within the scope of the skills of a skilled physician.

[0274] Labeled nanobody

[0275] In a preferred embodiment of the present invention, the nanobody is labeled with a detectable label. More preferably, the label is selected from the group consisting of: isotopes, colloidal gold labels, colored labels, or fluorescent labels.

[0276] Colloidal gold labeling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present invention, the nanobody against SA is labeled with colloidal gold to obtain a colloidal gold-labeled nanobody.

[0277] Detection method

[0278] The present invention also relates to a method for detecting SA protein. The steps of this method are generally as follows: obtaining a cell and / or tissue sample; dissolving the sample in a medium; detecting the level of SA protein in the dissolved sample.

[0279] In the detection method of the present invention, the sample used is not particularly limited. Representative examples are cell-containing samples present in cell preservation solutions.

[0280] Kit

[0281] The present invention also provides a kit containing the antibody (or its fragment) of the present invention or a detection plate. In a preferred embodiment of the present invention, the kit further includes a container, an instruction manual, a buffer, etc.

[0282] The present invention also provides a detection kit for detecting the level of SA. The kit includes an antibody that recognizes SA protein, a lysis medium for dissolving the sample, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. This detection kit can be an in vitro diagnostic device.

[0283] Application

[0284] As described above, the nanobody of the present invention has broad biological and clinical application values.

[0285] One of the main uses of the present invention is for extending the drug half-life. The present invention provides a method for extending the drug half-life by connecting the drug to the anti-SA single-domain antibody of the present invention, enabling it to return to the cell surface after endocytosis. The applications of the drug composition and detection reagent of the present invention involve multiple fields such as disease diagnosis and treatment, basic medical research, and biological research. A preferred application is for targeted therapy against tumors.

[0286] The main advantages of the present invention include:

[0287] 1) The antibody of the present invention has a strong binding force to SA and specifically binds to human, murine, and simian SA, showing broad applicability.

[0288] 2) The single-domain antibody of the present invention does not affect the binding of SA to FcRn and still maintains a high binding activity to SA in an acidic environment (such as pH 5.0), thus being able to cycle to the cell surface with FcRn for sorting and extending the drug half-life.

[0289] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out 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 according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0290] Example 1: Generation of anti-SA single-domain antibody by immunizing alpacas and detection of serum titer

[0291] For the first immunization, 0.5 mg of human SA antigen (ACRO, Cat#HSA-H522a) was mixed with an equal volume of complete Freund's adjuvant (CFA) and then subcutaneously injected into 2 healthy alpacas to stimulate the alpaca immune system to produce corresponding anti-SA antibodies. For the second booster immunization, 0.25 mg of human SA antigen was mixed with an equal volume of incomplete Freund's adjuvant (IFA) and then subcutaneously injected into the alpacas 20 days after the first immunization. Subsequently, the third and fourth booster immunizations were carried out at intervals of 20 days, and the dosage of the immunizing antigen and the adjuvant were the same as those in the second immunization. Blood was collected one week after the pre-immunization, the second immunization, the third immunization, and the fourth immunization to detect the titer of the target antibody in the serum.

[0292] The serum titer was detected by coating the enzyme-linked immunosorbent assay (ELISA) plates with SA antigen proteins of three species: human, cynomolgus monkey, and mouse, and using HRP-labeled goat anti-alpaca secondary antibody to detect the titers of the corresponding antibodies against antigens of different species. The detection results of the serum antibody titers after the second, third, and fourth immunizations are shown in Tables 1 to 7. The detection results of the serum titer showed that after immunization with human SA antigen, high-titer anti-human and anti-monkey SA antibodies and medium-titer anti-mouse SA antibodies were produced in the blood of 2 alpacas. The protein sequence alignment results of SA antigens of three species: human, cynomolgus monkey, and mouse showed that the sequence similarity between human and monkey was 93.59%, and the sequence similarity between human and mouse was 72.37%. The sequence alignment results are shown in Figure 1 . The immune antibody titer of the alpaca serum met the expectations, and the antibody library was constructed using the peripheral blood after the third and fourth immunizations.

[0293] Table 1 - Detection of serum titer after the second immunization

[0294]

[0295] Note: The detection antigen was Biotinylated Human Serum Albumin (ACRO, Cat#HSA-H82E3).

[0296] Table 2 - Detection of serum titer after the second immunization (MSA binding)

[0297]

[0298] Note: The antigen for detection is Biotinylated Mouse Serum Albumin (ACRO, Cat# MSA-M82E4).

[0299] Table 3 - Detection of the titer of the secondary immune serum (CSA binding)

[0300]

[0301] Note: The antigen for detection is Biotinylated Cynomolgus Serum Albumin (ACRO, Cat# CSA-C82E5).

[0302] Table 4 - Detection of the titer of the serum of alpaca A after the third immunization

[0303]

[0304] Table 5 - Detection of the titer of the serum of alpaca B after the third immunization

[0305]

[0306] Table 6 - Detection of the titer of the serum of alpaca A after the fourth immunization

[0307]

[0308] Table 7 - Detection of the titer of the serum of alpaca B after the fourth immunization

[0309]

[0310] Example 2: Construction and screening of a yeast library of anti-SA single-domain antibodies

[0311] Take 50 ml of peripheral blood from alpacas after the third and fourth immunizations respectively to isolate PBMC (Peripheral Blood Mononuclear Cell), and extract total RNA using RNAiso Plus reagent. Use PrimeScript TMThe II 1st Strand cDNA Synthesis Kit (Takara, Cat#6210A) was used to reverse transcribe 5 μg of total RNA into cDNA, and the operation steps refer to the kit instruction manual. Using the cDNA as a template, the first round of nested PCR was performed with 5 μl of 5-fold diluted cDNA. The PCR product fragment of about 750 bp was gel-extracted and used as the template for the second round of nested PCR. After two rounds of nested PCR amplification, single-domain antibody fragments were obtained. After the PCR products were purified using the Cycle-Puer Kit, they were co-transformed into yeast competent cells with linearized vector fragments by electroporation, and spread on a 200 mm SD-CAA plate with a total spreading volume of 5 mL, and cultured at 30 °C for 4 days. The number of library transformants was determined by taking 10 μL of the yeast cell suspension after electroporation and diluting it serially and spreading it on a 90 mm plate. 48 monoclonal colonies were randomly picked from the library plate for PCR identification, and the results showed that the insertion rates of the libraries constructed from the peripheral blood of alpacas A and B were both 100%. 48 positive PCR products were randomly selected for sequencing. After the sequencing sequences were translated into proteins, amino acid sequence alignment was performed and phylogenetic trees were drawn ( Figures 2A-2B ). Based on the number of library transformants, library insertion rate, and diversity sequencing analysis results, the library capacity of alpaca A yeast library was 1.05x10 8 , and the library capacity of alpaca B yeast library was 1.15x10 8 .

[0312] Through a method combining magnetic bead sorting and flow sorting, 36 unique sequences were obtained from the yeast libraries of A and B. Subsequently, flow analysis was used to further verify the binding activities of the monoclonal cells corresponding to the unique sequences to human, mouse, and monkey SA antigens. Based on sequence alignment and monoclonal flow binding activity verification analysis, 29 unique sequences positive for binding to human, mouse, and monkey SA antigens were obtained (Table 8), which involved 5 CDR1 sequences, 4 CDR2 sequences, and 9 CDR3 sequences (using the IMGT numbering method) (Tables 9 - 10).

[0313] Table 8 - Single-domain antibody sequences positive for binding to human, mouse, and monkey SA antigens

[0314]

[0315]

[0316]

[0317] Table 9 - CDR sequences positive for binding to human, mouse, and monkey SA antigens

[0318]

[0319]

[0320] Table 10 - CDR sequences corresponding to SA antigen - binding positive single - domain antibodies of human, mouse, and monkey

[0321]

[0322] Example 3: Construction of yeast expression strain of anti - SA single - domain antibody and protein preparation

[0323] After codon - optimizing the gene sequence of the anti - SA single - domain antibody with a his - tag added to the N - terminus, it was constructed into the pPICZ alphaA plasmid, and then the plasmid was linearized with PemⅠ enzyme. 5 μL of the linearized plasmid was added to 100 μL of competent cells, and the plasmid carrying the target gene was transformed into X33 Pichia pastoris cells using an electroporator (Biorad, MicroPulser). The electroporated Pichia pastoris cells were restored with a mixed medium (YPD:Sorbitol = 1:1). Then, 50 μL of Pichia pastoris cells were respectively spread on YPD solid media containing 200, 400, 600, 800 μg / mL zeocin (Invitrogen, Cat#R25001), and high - copy number strains of the target gene were obtained by screening with a Zeocin concentration gradient. Monoclonal screening was carried out using GMGY medium (Sangon, Cat#B540130) to culture monoclonal strains at 30 °C and 250 rpm. After obtaining sufficient bacteria, the target single - domain antibody was induced to secrete and express using GMMY medium (Sangon, Cat#B540131) at 20 °C and 250 rpm. After 24 hours, 20 μL of the supernatant was taken, and the expression levels of each colony were analyzed by gel electrophoresis. The strain with the highest expression level was selected for bacteria preservation and protein expression production. The methods for large - scale protein expression and purification are as follows: 400 μL of bacterial solution was inoculated into 200 mL of BMGY medium and cultured at 30 °C and 250 rpm for 3 days for enrichment. Subsequently, 80 mL of BMMY medium was replaced for induction of expression, and 1% methanol was supplemented every 24 hours during this period. The target single - domain antibody was induced to secrete and express at 20 °C and 250 rpm for 3 days. After the induction ended, the supernatant was collected by centrifugation at 12000 rpm for 15 min in a high - speed refrigerated centrifuge, and then the protein was purified using a nickel - ion affinity chromatography packing material (Cytiva, Cat#17092108). Before use, the nickel - ion affinity chromatography column was equilibrated with a binding buffer, and then the centrifuged supernatant containing the target protein was passed through the column. The protein with a his - tag was retained on the column by binding to nickel ions. Then, the non - specifically bound miscellaneous proteins on the column were washed away with a buffer containing 20 mM imidazole, and finally, the target protein was eluted with an elution buffer containing 250 mM imidazole. The summary of the expression levels of 9 CDR - unique nanobody sequences in a 100 - ml culture system is as follows (Table 11):

[0324] Table 11 - Summary of Yeast Expression Levels of Anti - SA Single - Domain Antibodies

[0325] Name Expression level (mg / L) A-SA-1 55.6 A-SA-117 35.2 A-SA-118 56 A-SA-156 27.2 A-SA-157 55.8 A-SA-56 34 B-SA-100 185 B-SA-101 38 B-SA-160 17.8

[0326] Example 4: Analysis of the Binding Activity of Anti - SA Single - Domain Antibodies to Recombinant Human, Mouse, and Monkey SA Proteins

[0327] Prepare human SA - His (ACRO, Cat#HSA - H5220) antigen solution, monkey SA - His (ACRO, Cat#CSA - C52H4) antigen solution, and mouse SA - His (ACRO, Cat#MSA - M52H8) antigen solution respectively with PBS to a final concentration of 1 μg / mL. Add 100 μL per well to a 96 - well ELISA plate and coat overnight at 4°C. Wash 3 times with PBST (PBS + 0.05% Tween 20). Add 200 μL per well of blocking solution (PBST + 3% BSA) and incubate at room temperature for 1 hour. Wash 3 times with PBST. Add 100 μL per well of the serially diluted single - domain antibody solution and incubate at room temperature for 1 hour. Wash 3 times with PBST. Add 100 μL per well of a 1:5000 - diluted solution of horseradish peroxidase - labeled rabbit anti - camelid VHH antibody (Genscript, Cat#A02016) and incubate at room temperature for 1 hour. Wash 3 times with PBST, pat dry, add 100 μL per well of the chromogenic substrate TMB solution (Beyotime, Cat#P0209), develop color at room temperature for 5 to 30 minutes, and then add 100 μL per well of the color - developing stop solution (Beyotime, Cat#P0215). Measure the absorbance at 450 nm for each well using a multi - functional microplate reader (Molecular Devices, SpectraMax i3x). Fit the data with the 4 - parameter equation of the sigmoidal curve in GraphPad Prism 9 software and calculate the binding EC50 value.

[0328] The experimental results showed that all candidate antibodies had cross - binding activity to human, mouse, and monkey species ( Figures 3-5 ).

[0329] Example 5: Analysis of the Binding Activity of Anti - SA Single - Domain Antibodies to Human, Mouse, and Rat Serum

[0330] Human serum (XinFan Bio-tech, Cat#XFS118) and mouse serum (Abbkine, Cat#BMS0070) were respectively diluted 15,000 times in PBS, and 100 μl / well was added to a 96-well ELISA plate and coated overnight at 4°C. The plate was washed 3 times with PBST (PBS + 0.05% Tween 20), and 200 μl / well of blocking solution (PBST + 3% BSA) was added and blocked at room temperature for 1 hour. The plate was washed once with PBST, and 100 μl / well of serially diluted anti-SA single-domain antibody solution was added. A secondary antibody control group without anti-SA single-domain antibody was set up and incubated at room temperature for 1 hour. The plate was washed 3 times with PBST, and 100 μl / well of 1:5000 diluted horseradish peroxidase-labeled rabbit anti-camelid VHH antibody (GenScript, Cat#A02016) solution was added and incubated at room temperature for 1 hour. The plate was washed 3 times with PBST, patted dry, 100 μl / well of chromogenic substrate TMB solution (Beyotime, Cat#P0209) was added, and the color was developed at room temperature for 5 to 30 minutes, followed by the addition of 100 μl / well of chromogenic stop solution (Beyotime, Cat#P0215). The absorbance values at 450 nm of each well were measured using a multi-functional microplate reader (Molecular Devices, SpectraMax i3x). The EC50 value was fitted and calculated using the 4-parameter equation of the sigmoidal curve of GraphPad Prism9 software.

[0331] The experimental results showed that all candidate antibodies had strong binding activities to human and mouse sera, and weak binding activities to rat serum ( Figures 6-8 ).

[0332] Example 6: Anti-SA single-domain antibody does not affect the binding of HSA to FcRn

[0333] HSA prolongs its half-life through the protein recycling mechanism mediated by FcRn. Therefore, the binding of HSA to FcRn is crucial in this process. The purpose of the following experiment was to verify whether the anti-SA single-domain antibody affects the binding activity of HSA to FcRn by surface plasmon resonance (SPR).

[0334] FcRn (ACRO, Cat# FCN-H52W7) was immobilized on a CM5 chip. A 1 μM HSA (ACRO, Cat# HSA-H5220) solution containing an excess of anti-SA single-domain antibody (2 μM) was flowed over the chip surface at a flow rate of 30 μl / min for a binding time of 90 s. Then, running buffer (1×PBS with 0.05% Tween-20, pH 6.0) was injected to dissociate the complex for a dissociation time of 210 s. A 1 μM HSA solution without anti-SA single-domain antibody was used as a control, and the effect of the anti-SA single-domain antibody on the binding activity was judged by observing the change in the response signal. The instrument used in the above experiment was a Biacore 8K (Cytiva).

[0335] The experimental results showed that all anti-SA single-domain antibodies had no inhibitory effect on the binding of HSA to FcRn (Table 12).

[0336] Table 12 - Effect of anti-SA single-domain antibody on the binding of HSA to FcRn detected by SPR

[0337]

[0338] Example 7: Analysis of the binding activity of anti-SA single-domain antibody to human, mouse, and monkey serum albumin under different pH conditions

[0339] After HSA binds to FcRn on the cell surface, it undergoes the processes of endocytosis, endosome acidification, and sorting. The pH of the acidified endosome is between 6.5 and 5.0. Therefore, only the anti-SA single-domain antibody that still maintains binding activity within this pH range can be sorted to the cell surface through the FcRn recycling and play a role in prolonging the half-life. In this experiment, surface plasmon resonance (SPR) was used to verify the binding activity of the anti-SA single-domain antibody to human serum albumin under different pH conditions.

[0340] HSA (ACRO, Cat# HSA-H5220), MSA (ACRO, Cat# MSA-M52H8), and CSA (ACRO, Cat# CSA-C52H4) were respectively immobilized on the CM5 chip. Antibody solutions of 7 concentrations of anti-SA single-domain antibodies flowed over the chip surface at a flow rate of 30 μl / min for a binding time of 120 s. Then, the running buffer (Buffer A: 1×HEPES (10 mM HEPES, 150 mM NaCl, 3 mM EDTA) with 0.005% Tween-20, pH 7.4; Buffer B: 10 mM sodium acetate, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, pH 5.0) was injected to dissociate the complex for 300 s. The instrument used in the above experiment was Biacore 8K (Cytiva). The affinity data under different pH conditions were calculated and summarized in the following table (Table 13).

[0341] The experimental results showed that all anti-SA single-domain antibodies could bind albumin under the conditions of pH 7.4 and pH 5.0.

[0342] Table 13 SPR detection of albumin-binding activity of anti-SA single-domain antibodies under different pH conditions

[0343]

[0344] Example 8: Verification of the half-life extension effect of anti-SA single-domain antibodies

[0345] The gene sequence of the anti-SA single-domain antibody with a his tag added to the N-terminus was concatenated with the gene sequence of the anti-TfR1 single-domain antibody (H95), and (GGGS)3 linker was added in the middle.

[0346] The sequence of H95 is as follows:

[0347] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSKTMSWLRQAPGKGLEWV SSINSGGSGTDYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAING PNYYRESTRGQGTLVTVSS (SEQ ID NO: 67)

[0348] Linker:

[0349] The GGGSGGGSGGGS (SEQ ID NO:69) tandem gene sequence was codon-optimized and constructed into the pPICZ alpha A plasmid, and the tandem antibody was expressed in yeast (the yeast expression method was the same as in Example 3). The tandem antibodies were named: H95-1 (H95-HSA-1), H95-56, H95-100, H95-101, H95-118, H95-156, and a non-tandem anti-TfR1 single-domain antibody was used as a control. The yeast expression supernatant was purified by nickel ion affinity chromatography to obtain the target single-domain antibody, and the purity was identified by SDS-PAGE to be greater than 95%( Figure 9 ).

[0350] Female BALB / c Nude mice weighing 18 - 22 g were used in the experiment. They were separately injected once with a dose of 50 nmol / kg of the anti-TfR1 single-domain antibody and the anti-TfR1-HSA tandem-form single-domain antibody. The drug content in the serum at each time point was collected, and the pharmacokinetic situation of the drug in vivo was analyzed to verify the effect of the extended half-life of the anti-SA single-domain antibody. Forty-nine mice were grouped according to body weight and randomly divided into 8 groups, with 6 mice in each group, and the 8th group had 1 mouse. The day of grouped administration was the 0th day (D0). The detailed dosing information is shown in Table 14:

[0351] Table 14 - Grouping and dosing

[0352]

[0353] Note: i.v.: tail vein injection; dosing volume 10 μL / g.

[0354] The blood collection times for the first group were 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h after dosing; the blood collection times for the second to seventh groups were 5 min, 2 h, 4 h, 8 h, 1 d, 3 d, 5 d, 7 d after dosing; blood samples of the non-dosing group mice were collected as a control. Serum was quickly separated after blood collection and stored in a -80 °C refrigerator. The body weight of all mice was measured once a day, and the body weight change was calculated using the formula RCBW%=(BW i -BW0) / BW0×100%, where BW i is the current body weight of the mouse, and BW0 is the body weight of the mouse on the day of grouping. The monitoring data showed that the body weights of all mice fluctuated within the normal range, indicating that the mice had good tolerance to the drugs at the tested doses( Figure 10 ).

[0355] The drug concentration in the serum samples was detected by the ELISA method, and the ELISA method is shown in the schematic diagram( Figure 11(lower right), the drug in the serum was captured by coating the TfR1-His (ACRO, Cat#CD1-H5243) antigen method, and then the drug content in the serum was detected with a 1:5000 diluted horseradish peroxidase-labeled rabbit anti-camelid VHH antibody (Genscript, Cat#A02016). The standard curve was prepared by a two-fold serial dilution method starting from 1000 pM, with 12 concentration gradients set. All samples were set with duplicate wells, and a secondary antibody control group and a blank mouse blood sample control group were set. Net OD450 = T OD -C OD ,T OD is the actual OD450 measurement value of each serum sample, and C OD is the average OD450 measurement value of the secondary antibody control group. The standard curve was linearly fitted by double logarithmic analysis. The detection range of the Anti-TfR1 sdAb (H95) sample was 7.8 pM - 500 pM, and R 2 ≥0.99; the detection range of the Anti-TfR1-HSA sdAb sample was 0.5 pM - 1250 pM, and R 2 ≥0.99; ( Figure 11 ).

[0356] Each serum sample was diluted to a concentration within the detection range, and the corresponding OD450 was measured. Then, the corresponding serum drug concentration was calculated according to the standard curve, and a drug concentration-time curve was plotted ( Figure 12 ). The pharmacokinetic parameters of each drug were calculated using the PK solver 2.0 plug-in, and the parameter summary is shown in Table 15 below:

[0357] Table 15 Summary of kinetic parameters of the test substances

[0358]

[0359] According to the calculation, the half-life of the anti-TfR1 single-domain antibody was 11 h, while the half-life of the anti-TfR1-HSA tandem single-domain antibody was between 28 - 38 h. Therefore, among the above anti-SA single-domain antibodies, except for HSA-56, the half-life of the drug was prolonged to varying degrees by the other anti-SA single-domain antibodies.

[0360] Example 9: Humanization of anti-SA single-domain antibody

[0361] Humanization of the anti-SA single-domain antibody was achieved by aligning the parental sequence AS002-N000-118-P with the human Germline database to define the CDR and framework regions of the parental antibody. Based on the different sites in the framework region and the analysis of the antibody 3D model, sequences with different degrees of humanization were designed. The sequence alignment results showed that the parental sequence had the highest homology with the human Germline IGHV3-23 sequence. Therefore, IGHV3-23 with the highest homology was selected as the template for humanization design, and 18 humanized sequences were designed by back mutation (Table 16).

[0362] Table 16 - Summary of humanized sequences of anti-SA single-domain antibody

[0363]

[0364]

[0365] Example 10: Expression and purification of anti-SA humanized VHH-Fc

[0366] The C-terminus of the humanized VHH sequence was fused with the human IgG1 Fc segment. After codon optimization of the fusion sequence, it was constructed into the pCMV3 vector. The fusion expression plasmid was transiently transfected into HEK293 cells for 7 days of expression. The transfection reagent was TF2 (SinoBiological, Cat#STF02), and the expression medium was 293 serum-free CD medium (Sino Biological, Cat#SMM293-TI). The 293 serum-free feeding solution (Sino Biological, Cat#M293-SUPI-100) was added on the 1st, 3rd, and 5th days after transfection. The cells after the end of expression were centrifuged to obtain the supernatant, which was filtered through a 0.22 μM filter membrane and then purified by Protein A affinity packing to obtain the humanized VHH-Fc protein.

[0367] Example 11: Stability analysis of anti-SA humanized VHH-Fc

[0368] The tendency of aggregate formation of the humanized VHH-Fc was analyzed by SEC-HPLC method. The chromatographic column used in the experiment was TSK G2000SWXL, the flow rate was set at 0.5 mL / min, and the detection wavelength was 280 nm. The detection results are shown in Table 17, and the results indicate that the monomer ratio of all humanized antibodies is higher than 98%, the samples are relatively stable and not prone to aggregate formation.

[0369] Table 17 - Summary of SEC-HPLC results of anti-SA humanized VHH-Fc

[0370]

[0371] Note: " / " means none.

[0372] Example 12: Analysis of the binding activity of humanized VHH-Fc to human, monkey, and mouse SA proteins

[0373] The binding activity of humanized VHH-Fc to human SA protein was analyzed by Surface Plasmon Resonance (SPR). HSA (ACRO, Cat#HSA-H5220) was immobilized on a CM5 chip, and the gradient-diluted anti-SA single-domain antibody solution was bound to the chip surface through anti-human IgG Fc for 120 s. Then, the running buffer (pH 5.0) was injected to dissociate the complex for 120 s. The instrument used was biacore T200 (Cytiva), and the detection results are shown in Table 18. The experimental results showed that all humanized antibodies could bind HSA at pH 5.0. Among them, the affinities of VHH1, 2, 7, and 10 were lower than that of the parent, and the affinities of the remaining antibodies were comparable to or better than that of the parent.

[0374] Table 18 SPR detection results of anti-SA humanized VHH-Fc

[0375]

[0376] The binding activity of humanized VHH-Fc to human, monkey, and mouse SA proteins was analyzed by enzyme-linked immunosorbent assay (ELISA). Antigen solutions of HSA (ACRO, Cat#HSA-H5220), MSA (ACRO, Cat#MSA-M52H8), and CSA (ACRO, Cat#CSA-C52H4) were prepared with PBS at a final concentration of 1 μg / mL and added to a 384-well ELISA plate at 25 μL / well. The plate was coated overnight at 4°C. After washing 3 times with PBST (PBS + 0.05% Tween 20), 50 μL / well of blocking solution (PBST + 3% BSA) was added and incubated at room temperature for 1 hour. After washing once with PBST, 25 μL / well of serially diluted VHH-Fc solution (pH 5.0) was added and incubated at room temperature for 1 hour. After washing 3 times with PBST, 25 μL / well of 1:5000 diluted horseradish peroxidase-labeled goat anti-human IgG antibody (Yeasen, Cat#33501ES60) solution was added and incubated at room temperature for 1 hour. After washing 3 times with PBST and blotting dry, 25 μL / well of chromogenic substrate TMB solution (Beyotime, Cat#P0209) was added and allowed to develop color at room temperature for 5 to 30 minutes. Then, 25 μL / well of color development stop solution (Beyotime, Cat#P0215) was added. The absorbance values at 450 nm of each well were measured using a multimode microplate reader (Molecular Devices, SpectraMax i3x). The binding EC50 values were fitted and calculated using the 4-parameter equation of the sigmoidal curve in GraphPad Prism 9 software. The experimental results showed that all humanized VHH-Fc bound to human SA-His, and the binding activities of VHH5, 7, 15, 16, 17, and 18 were stronger than those of the parent; VHH15, 16, 17, and 18 all bound to MSA and CSA, and their binding activities were comparable to those of the parent ( Figures 13-15 ).

[0377] Example 13: Half-life Enhancement Module: Preparation of Anti-HSA Single-Domain Antibody-Morpholino Phosphorodiamidate Oligonucleotide (PMO) Conjugate

[0378] Taking the anti-HSA single-domain antibody A-HSA-118 as an example of the half-life extension factor, a cysteine mutation was introduced at the carboxyl terminus of the anti-HSA single-domain antibody for nucleic acid conjugation. The gene sequence of the anti-HSA single-domain antibody was optimized for yeast-preferred codons and then subcloned into the pPICZ alpha A plasmid. To facilitate purification, a His tag was added to the N-terminus of the single-domain antibody.

[0379] Amino acid sequence of the A-HSA-118 single-domain antibody mutant (SEQ ID NO:48):

[0380] HHHHHHEVQLVESGGNLVQAGDSLTLSCEASGHLFSNYVLGWFRQRTGEEREYVASISRTGKDVRYSDSVKGRFTIYRDNTKNTVYLKMTRLEPEDTAVYLCAIYNGARGEYGDWGQGTQVTVSSGSC

[0381] The plasmid was linearized and electrotransformed into the Pichia pastoris strain X33, and high-copy strains of the target gene were obtained by screening with YPD agar plates with a gradient of Zeocin concentration. Monoclonal strains were cultured in GMGY medium at 30 °C and 250 rpm. After obtaining sufficient bacterial cells, the target single-domain antibody was induced to secrete and express in GMMY medium at 20 °C and 250 rpm, and 1% methanol was supplemented every 24 hours for a total of 72 hours of induced expression. The yeast culture supernatant was collected and the single-domain antibody in the culture supernatant was purified using a His-tag affinity column.

[0382] The single-stranded PMO1 with an NH2 modification at the 5'-end was dissolved in phosphate buffer (50 mM NaH2PO4, 150 mM NaCl, pH 7.4) to prepare a stock solution with a final concentration of 1 mM. The SM(PEG)2 (linker molecule) powder was dissolved in dimethyl sulfoxide (DMSO) to freshly prepare a 250 mM stock solution of SM(PEG)2. 10 - 50-fold molar amount of the SM(PEG)2 stock solution was added to the PMO1 single-stranded stock solution, and after rapid mixing, the reaction was carried out at room temperature for 30 min - 2 h. After the reaction was completed, 10% (v / v) of 1 M Tris-HCl (pH 7.0) was added to the reaction solution, and after mixing, it was incubated at room temperature for 20 minutes to terminate the continued reaction of the excess SM(PEG)2. After the incubation was completed, the SM(PEG)2-PMO1 and the unreacted SM(PEG)2 linker were separated by acetone precipitation method, and the SM(PEG)2-PMO1 conjugate was purified for standby.

[0383] The single-domain antibody sample eluted by His-tag affinity chromatography (Example 2) was dialyzed with a dialysis buffer containing a reducing agent (20 mM Tris, 15 mM NaCl, pH 7.4). During the dialysis process, the thiol group at the C-terminus was reduced, and at the same time, small impurity molecules such as free -SH groups were removed. The reduced single-domain antibody and the SM(PEG)2-PMO1 single-strand were mixed evenly at a molar ratio of 1:1 - 2 and reacted at room temperature for 2 h.

[0384] Identified by SDS-PAGE, the coupling efficiency of the single-domain antibody and the SM(PEG)2-PMO1 single-strand can reach more than 90% ( Figure 16 ).

[0385] Remove the unreacted SM(PEG)2-PMO1 single strand using a His-tag affinity column, and collect the single-domain antibody and the single-domain antibody-PMO1 mixture. Use Superdex TM 75Increase 10 / 300GL to separate the single-domain antibody and the single-domain antibody-PMO1, and the single-domain antibody and the single-domain antibody-PMO were effectively separated ( Figure 17 ).

[0386] Example 14 Self-assembly of the half-life extension module and the target protein molecule

[0387] Taking the single-domain antibody TfR1-134 targeting the transferrin receptor as an example of the target protein molecule, the process of self-assembly of the half-life extension module and the target protein molecule will be described below.

[0388] The amino acid sequence of TfR1-134-GSC is shown in SEQ ID NO:68:

[0389] HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGFTFSGASMTWARQAPGKGLEWVSGIDRSGSSTQYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARGMAWATRGQGTQVTVSSGSC

[0390] Targeting module: The preparation method of the single-domain antibody-PMO4 conjugate against TfR1 is the same as that in Example 13.

[0391] Measure the concentrations of the single-domain antibody-PMO1, PMO2, PMO3 against HSA and the single-domain antibody-PMO4 against TfR1 respectively. Take appropriate amounts of the above components and preheat them at 37 °C for 5 min, then mix the above 4 components at a molar ratio of 1:1:1:1 under the condition of 37 °C and incubate for 1 min. Thus, the half-life extension module and the single-domain antibody targeting module targeting the transferrin receptor have been assembled, and the assembled molecule is named PMO-NAPPA4-HSA(1)-TfR1(4). Use SDS-PAGE to identify the homogeneity of the assembled tetramer molecule, and the results show that the homogeneity of the transient assembly can reach more than 95% ( Figure 18 ).

[0392] Example 15: Verification of the binding activity of PMO-NAPPA4-HSA(1)-TfR1(4)

[0393] Prepare a human TfR1-His (ACRO, Cat#CD1-H5243) antigen solution with PBS to a final concentration of 1 μg / mL, and add 100 μL / well to a 96-well ELISA plate. Coat overnight at 4°C. Wash 3 times with PBST (PBS + 0.05% Tween 20), add 200 μL / well of blocking solution (PBST + 3% BSA), and incubate at room temperature for 1 hour. Wash once with PBST, add 100 μL / well of serially diluted anti-TfR1 single-domain antibody and PMO-NAPPA4-HSA(1)-TfR1(4) solution, and incubate at room temperature for 1 hour. Wash 3 times with PBST, pat dry, add 100 μL / well of chromogenic substrate TMB solution (Beyotime, Cat#P0209), develop color at room temperature for 5 to 30 minutes, and then add 100 μL / well of color development stop solution (Beyotime, Cat#P0215). Measure the absorbance at 450 nm for each well using a multi-functional microplate reader (Molecular Devices, SpectraMaxi3x). Fit the data with the 4-parameter equation of the sigmoidal curve in GraphPad Prism 9 software and calculate the binding EC50 value ( Figure 19 ). The EC50 values of the anti-TfR1 single-domain antibody and the assembled PMO-NAPPA4-HSA(1)-TfR1(4) binding to the TfR1 protein were 0.388 nM and 0.111 nM, respectively, indicating that PMO-NAPPA for half-life extension does not affect the binding activity of the single-domain antibody to the corresponding antigen.

[0394] Example 16 Verification of the half-life improvement effect of PMO-NAPPA4-HSA(1)-TfR1(4)

[0395] Female BALB / c Nude mice weighing 18 - 22 g were injected once with a dose of 50 nmol / kg of the anti-TfR1 single-domain antibody and PMO-NAPPA4-HSA(1)-TfR1(4) respectively to verify the pharmacokinetics of the two molecular forms in vivo and analyze the half-life extension effect of PMO-NAPPA on the anti-TfR1 single-domain antibody. Thirteen mice were grouped by body weight and randomly divided into 3 groups, with 6 mice in each of the first 2 groups and 1 mouse in the third group. The day of grouped administration was day 0 (D0).

[0396] The blood sampling times for Group 1 were 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h after drug administration; the blood sampling times for Group 2 were 5 min, 2 h, 4 h, 8 h, 1 d, 4 d, 7 d, and 14 d after drug administration; blood samples of mice in the non-drug administration group were collected as controls. Serum was rapidly separated after blood collection and stored in a -80 °C refrigerator. The body weights of all mice were measured once a day, and the body weight change was calculated using the formula RCBW% = (BW i -BW0) / BW0×100%, where BW i is the current body weight of the mouse and BW0 is the body weight of the mouse on the day of grouping. Monitoring data showed that the body weights of all mice fluctuated within the normal range, indicating that all mice had good tolerance to the drugs at the tested doses ( Figure 20 ).

[0397] The drug concentration in the serum samples was detected by ELISA method, and the operation method was the same as that in Example 15. The standard curve was prepared by a two-fold serial dilution method starting from 1000 pM, with 12 concentration gradients set. All samples were set with duplicate wells, and a secondary antibody control group and a blank mouse blood sample control group were set. Net OD450 = T OD -C OD , where T OD is the actual OD450 measurement value of each serum sample, and C OD is the average OD450 measurement value of the secondary antibody control group. The standard curve was linearly fitted by double logarithmic analysis. The detection range of the Anti-TfR1 sdAb sample was 16 pM - 500 pM, and R 2 ≥0.99; the detection range of the PMO-NAPPA4-HSA(1)-TfR1(4) sample was 2 pM - 125 pM, and R 2 ≥0.99.

[0398] Each serum sample was diluted to a concentration within the detection range, and the corresponding OD450 was measured. Then, the corresponding serum drug concentration was calculated according to the standard curve, and a drug concentration-time curve was plotted ( Figure 21 ). The pharmacokinetic parameters of each drug were calculated using the PK solver 2.0 plug-in, and the parameter summary is shown in the following table:

[0399] Table 19 Summary of kinetic parameters of the test substances

[0400]

[0401]

[0402] According to calculations, the half-lives of the anti-TfR1 single-domain antibody and PMO-NAPPA4-HSA(1)-TfR1(4) are 3.4 h and 35 h, respectively. Using the PMO-NAPPA4-HSA(1)-TfR1(4) of the present invention can extend the half-life by about 10 times without affecting the antibody binding performance compared with ordinary single-domain antibodies.

[0403] All documents mentioned in the present invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A single-domain antibody against serum albumin (SA), characterized in that, The VHH chain of the single-domain antibody has three CDRs of the VHH chain with an amino acid sequence as shown in any one of SEQ ID NOs: 1-29, wherein the CDRs are CDR1, CDR2, and CDR3 determined by any one of the IMGT rule, Kabat rule, Chothia rule, AbM rule, or Contact rule.

2. The single-domain antibody according to claim 1, wherein The CDR1 sequence of the VHH chain of the single-domain antibody is as shown in SEQ ID NO.32, the CDR2 sequence is as shown in SEQ ID NO.37, and the CDR3 sequence is as shown in SEQ ID NO.44; or The CDR1 sequence is as shown in SEQ ID NO.33, the CDR2 sequence is as shown in SEQ ID NO.35, and the CDR3 sequence is as shown in SEQ ID NO.47; or The CDR1 sequence is as shown in SEQ ID NO.33, the CDR2 sequence is as shown in SEQ ID NO.35, and the CDR3 sequence is as shown in SEQ ID NO.46; or The CDR1 sequence is as shown in SEQ ID NO.33, the CDR2 sequence is as shown in SEQ ID NO.35, and the CDR3 sequence is as shown in SEQ ID NO.45; or The CDR1 sequence is as shown in SEQ ID NO.32, the CDR2 sequence is as shown in SEQ ID NO.37, and the CDR3 sequence is as shown in SEQ ID NO.39; or The CDR1 sequence is as shown in SEQ ID NO.30, the CDR2 sequence is as shown in SEQ ID NO.36, and the CDR3 sequence is as shown in SEQ ID NO.43; or The CDR1 sequence is as shown in SEQ ID NO.31, the CDR2 sequence is as shown in SEQ ID NO.38, and the CDR3 sequence is as shown in SEQ ID NO.40; or The CDR1 sequence is as shown in SEQ ID NO.31, the CDR2 sequence is as shown in SEQ ID NO.38, and the CDR3 sequence is as shown in SEQ ID NO.41; or The CDR1 sequence is as shown in SEQ ID NO.34, the CDR2 sequence is as shown in SEQ ID NO.38, and the CDR3 sequence is as shown in SEQ ID NO.40; or The CDR1 sequence is as shown in SEQ ID NO.31, the CDR2 sequence is as shown in SEQ ID NO.38, and the CDR3 sequence is as shown in SEQ ID NO.

42.

3. The single-domain antibody according to claim 1, wherein The amino acid sequence of the VHH chain of the single-domain antibody is as shown in any one of SEQ ID NOs: 1-29 or 49-66.

4. A single-domain antibody fusion protein, characterized in that, The single-domain antibody fusion protein has a structure as shown in formula Ia or Ib from the N-terminus to the C-terminus: Ab1-F-L-P (formula Ia); P-L-Ab1-F (formula Ib) In the formula, Ab1 is the VHH chain of the anti-SA single-domain antibody as described in claim 1; F is none or the Fc segment of an immunoglobulin; L is a linker sequence; P is a therapeutic protein.

5. The single-domain antibody fusion protein according to claim 4, characterized in that, The described P is a therapeutic antibody or a natural ligand.

6. The single-domain antibody fusion protein according to claim 5, wherein, The therapeutic antibody is a monoclonal antibody fragment or a tandem form of monoclonal antibody fragments, preferably an scFv, tandem scFv, Fab, tandem Fab, single-domain antibody, tandem single-domain antibody, or a combination thereof.

7. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of: the anti-SA single-domain antibody of claim 1, the single-domain antibody fusion protein of claim 4, or a combination thereof.

8. An expression vector, characterized in that, The expression vector contains the polynucleotide of claim 7.

9. A host cell, characterized in that, The host cell contains the expression vector of claim 8, or the polynucleotide of claim 7 is integrated into its genome.

10. A multimeric protein complex based on intercomplementary nucleic acid backbones, wherein the complex is a multimer formed by the complexation of n monomers having intercomplementary nucleic acid backbones, where The n monomers consist of: (i) at least one half-life extension module, which consists of the anti-SA single-domain antibody of claim 1 and a single-stranded nucleic acid linked thereto; (ii) optionally none or one or more targeting modules, which consist of a targeting molecule and a single-stranded nucleic acid linked thereto; (iii) optionally none or one or more pharmacodynamic modules, which consist of a pharmacodynamic molecule and a single-stranded nucleic acid linked thereto; (iv) optionally none or one or more backbone monomers, which consist of single-stranded nucleic acids; where n is a positive integer from 2 to 8, and the single-stranded nucleic acid of each monomer forms a complementary double strand with the single-stranded nucleic acids of 1, 2, or 3 other monomers through base complementarity, thereby forming a complementary nucleic acid backbone structure.

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