Anti-albumin antibody or antigen-binding fragment thereof and use thereof

Abstract

Provided are an anti-albumin antibody or an antigen-binding fragment thereof and the use thereof, and an anti-albumin nanobody with improved affinity or an antigen-binding fragment thereof. The anti-albumin antibodies or antigen-binding fragments thereof can bind to albumins from different species with high affinity. In addition, the anti-albumin antibody can be linked to a bioactive effector molecule to form a fusion construct without affecting the activity of the bioactive effector molecule.

Description

Anti-albumin antibody or antigen-binding fragment thereof and application thereof Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-albumin antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] Albumin is the most abundant protein in plasma (with a concentration of approximately 40 mg / mL). It is essential for maintaining plasma osmotic pressure. Albumin is also an important transport carrier for endogenous ligands (such as fatty acids, metal ions, hormones, etc.) and exogenous ligands (such as drugs).

[0003] Albumin has a molecular weight of 66.5kDa and can bind to the cell surface receptor FcRn (neonatal Fc receptor) under weakly acidic conditions, but has a weaker binding force under neutral conditions. Therefore, after albumin binds to FcRn, it is internalized into the endosome. As the endosomal environment becomes acidic, the binding force between albumin and FcRn increases, and it is brought back to the cell surface by FcRn, or transported across cells. Therefore, albumin can achieve a longer half-life with the help of the recycling mechanism of FcRn. The half-life of human albumin in plasma is about 3 weeks. Therefore, small molecule compounds that bind to albumin, or proteins expressed by fusion with albumin (such as marketed drugs) and ), has significantly improved pharmacokinetic properties, obtains a longer half-life, can potentially extend the dosing interval, and provide patients with a better medication regimen.

[0004] However, due to differences in albumin sequences between species, the affinity of albumin from different species for the FcRn of their corresponding species also varies. Therefore, for albumin-binding small molecule drugs or albumin fusion proteins, pharmacokinetic and efficacy evaluations conducted in small animals may not necessarily reflect their effects in humans. Consequently, the sequence differences between albumins of different species pose significant challenges to the translational research of drugs using albumin as a carrier or albumin fusion protein drugs.

[0005] Therefore, developing antibodies that can cross-bind to albumins from multiple species can potentially solve this problem. Since anti-albumin antibodies can bind to albumins from different species with similar affinities, conjugating small molecule compounds to anti-albumin antibodies, or fusing antibodies that bind to specific antigens with anti-albumin antibodies, and conducting pharmacokinetic and efficacy tests of these drug molecules in small animals (such as mice and rats) can provide reliable reference value for the pharmacokinetic and efficacy performance of future drugs in humans.

[0006] Single-domain antibodies, or nanobodies, are the variable domains of heavy-chain antibodies naturally found in camelids, lacking light chains. They have a simple structure and are the smallest antibody unit with complete antigen-binding activity. With a molecular weight of approximately 13 kDa, nanobodies are easy to engineer, highly thermally stable, and water-soluble.

[0007] In order to predict the future effects of active molecules in humans, it is usually necessary to verify their behavior in animals in relevant animal models, such as analyzing the efficacy, pharmacokinetics, drug distribution, and drug metabolism of the active molecules. Rodents, such as mice and rats, are commonly used experimental animal models because of their small size, short breeding cycle, similar genetic background between individuals, and low experimental costs. This requires active molecules to have the ability to bind to human, mouse, and rat targets. For single-domain antibodies or nanobodies against albumin, the antibody needs to be able to bind to albumins from multiple species, such as human, mouse, and rat, to facilitate in vivo evaluation experiments of anti-albumin nanobodies and their fusion constructs in rodents, such as mice and rats. Summary of the Invention

[0008] To overcome the deficiencies of the prior art, the present application provides an anti-albumin antibody or antigen-binding fragment thereof and its application, as well as an anti-albumin antibody or antigen-binding fragment thereof with enhanced affinity, which can bind to albumins of different species with high affinity. At the same time, the anti-albumin antibody can be linked to a bioactive effector molecule to form a fusion construct without affecting the activity of the bioactive effector molecule. Specifically,

[0009] In a first aspect, the present invention provides an anti-albumin antibody or an antigen-binding fragment thereof, wherein the anti-albumin antibody or the antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and / or CDR-H3 of the heavy chain variable region.

[0010] The amino acid sequence of CDR-H1 comprises SEQ ID NO: 1 or 58, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 or 58;

[0011] The amino acid sequence of CDR-H2 comprises any one of SEQ ID NOs: 3-4, 6, 40, or an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 3-4, 6, 40;

[0012] The amino acid sequence of CDR-H3 comprises any one of SEQ ID NOs: 7-8, 59, or an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NOs: 7-8, 59.

[0013] Among them, the SEQ ID NO: 58 (X1YYMS), SEQ ID NO: 40 (GISVX2X3X4X5LDYADAVX6G), SEQ ID NO: 59 (ASGP X7X8LRX9X 10 X in AP 1-10 It can be any natural amino acid residue, such as alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), proline (P), serine (S), tryptophan (W), tyrosine (Y), and valine (V).

[0014] In a specific embodiment of the present invention, X1 in SEQ ID NO: 58 (X1YYMS) represents N or E;

[0015] In the SEQ ID NO: 40 (GISVX2X3X4X5LDYADAVX6G), X2X3 represents DS, DA, EG, DM or DG, X4X5 represents SF or WY, and X6 represents K, A, R or H; preferably,

[0016] X2X3 represents DG, X4X5 represents SF, and X6 represents K (SEQ ID NO: 5);

[0017] X2X3 represents DA, X4X5 represents SF, and X6 represents K (SEQ ID NO: 20);

[0018] X2X3 represents DS, X4X5 represents SF, and X6 represents K (SEQ ID NO: 21);

[0019] X2X3 represents EG, X4X5 represents SF, and X6 represents K (SEQ ID NO: 22);

[0020] X2X3 represents DG, X4X5 represents SF, and X6 represents A (SEQ ID NO: 31);

[0021] X2X3 represents DG, X4X5 represents SF, and X6 represents R (SEQ ID NO: 32);

[0022] X2X3 represents DG, X4X5 represents SF, and X6 represents H (SEQ ID NO: 33);

[0023] Said X2X3 represents DG, X4X5 represents WY, and X6 represents K (SEQ ID NO: 50); or,

[0024] X2X3 represents DM, X4X5 represents SF, and X6 represents K (SEQ ID NO: 51).

[0025] The SEQ ID NO: 59 (ASGPX7X8LRX9X 10 AP) in X7X8 represents QG, IW, LW or VG, X9X 10 Represents LG or WW. Preferably,

[0026] The X7X8 represents QG, X9X 10 represents LG (SEQ ID NO: 9);

[0027] The X7X8 represents IW, X9X 10 represents LG (SEQ ID NO: 52);

[0028] The X7X8 represents LW, X9X 10 represents LG (SEQ ID NO: 53);

[0029] The X7X8 represents QG, X9X 10 represents WW (SEQ ID NO: 54); or,

[0030] The X7X8 represents VG, X9X 10 Represents LG (SEQ ID NO: 55).

[0031] In a specific embodiment of the present invention, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 comprise any one of the following groups (see Table 1 for details):

[0032] A) SEQ ID NO: 1, 3, 7;

[0033] B) SEQ ID NO: 1, 4, 8;

[0034] C) SEQ ID NO: 2, 5, 9;

[0035] D) SEQ ID NO: 2, 6, 9;

[0036] E) SEQ ID NO: 2, 20, 9;

[0037] F) SEQ ID NO: 2, 21, 9;

[0038] G) SEQ ID NO: 2, 22, 9;

[0039] H) SEQ ID NO: 2, 31, 9;

[0040] I) SEQ ID NO: 2, 32, 9;

[0041] J) SEQ ID NO: 2, 33, 9;

[0042] K) SEQ ID NO: 2, 5, 52;

[0043] L) SEQ ID NO: 2, 50, 53;

[0044] M) SEQ ID NO: 49, 5, 53;

[0045] N) SEQ ID NO: 49, 5, 54;

[0046] O) SEQ ID NO: 2, 51, 55.

[0047] Table 1 Amino acid sequences of candidate antibody CDR-H1, CDR-H2 and CDR-H3

[0048] Preferably, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are arranged in order from N-terminus to C-terminus. The amino acid division of the antibody CDR region in this application adopts the Kabat numbering system. The anti-albumin antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

[0049] Preferably, the anti-albumin antibody or antigen-binding fragment thereof comprises a humanized sequence, and the modification site of the humanized sequence is located in a non-CDR region. Further preferably, the modification site of the humanized sequence is located in the framework region and / or constant region of the antibody.

[0050] Preferably, the structure of the anti-albumin antibody or its antigen-binding fragment includes a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single chain antibody (scFv) or a linear antibody.

[0051] The anti-albumin antibody or antigen-binding fragment thereof can be a single domain antibody or a nanobody.

[0052] In a specific embodiment of the present invention, the anti-albumin antibody or antigen-binding fragment thereof is a nanobody. Compared with full-length IgG antibodies, Fab, and scFv, nanobodies have a higher molar concentration at the same mass and can bind to more antigen molecules.

[0053] The anti-albumin antibody or antigen-binding fragment thereof can bind to mammalian albumin.

[0054] Preferably, the mammals include humans or non-human mammals, and the non-human mammals may be wild animals, zoo animals, commercial animals, pets, experimental animals, etc. Preferably, the non-human mammals include, but are not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

[0055] Preferably, the amino acid sequence of the anti-albumin antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 10-13, 16-19, 23-25, 34-36, 44-48, or has at least 80% identity with any one of SEQ ID NOs: 10-13, 16-19, 23-25, 34-36, 44-48.

[0056] In a specific embodiment of the present invention, the amino acid sequence of the anti-albumin antibody or antigen-binding fragment thereof is as shown in any one of SEQ ID NOs: 10-13, 16-19, 23-25, 34-36, and 44-48.

[0057] The anti-albumin antibody or antigen-binding fragment thereof can be constructed using any conventional method in the prior art, such as artificial synthesis or eukaryotic or prokaryotic expression.

[0058] The second aspect of the present invention provides a use of the above-mentioned anti-albumin antibody or antigen-binding fragment thereof, the use comprising:

[0059] A use of A in preparing a fusion construct, wherein the fusion construct comprises the above-mentioned anti-albumin antibody or antigen-binding fragment thereof and a biologically active effector molecule, wherein the biologically active effector molecule is linked to the anti-albumin antibody or antigen-binding fragment thereof;

[0060] B. The use of method B in screening bioactive effector molecules, wherein the candidate bioactive effector molecule is linked to the anti-albumin antibody or the antigen-binding fragment thereof, and the bioactivity of the candidate bioactive effector molecule is detected; or

[0061] Application of C in the detection of albumin.

[0062] The third aspect of the present invention provides a fusion construct, which comprises one or more of the above-mentioned anti-albumin antibodies or antigen-binding fragments thereof and / or biologically active effector molecules, and the biologically active effector molecules are connected to the anti-albumin antibodies or antigen-binding fragments thereof.

[0063] Preferably, the fusion construct comprises a plurality of anti-albumin antibodies or antigen-binding fragments thereof, which may be the same or different anti-albumin antibodies or antigen-binding fragments thereof.

[0064] Preferably, the biologically active effector molecule may be one or more, and the multiple biologically active effector molecules may be the same or different.

[0065] Preferably, the bioactive effector molecules include but are not limited to small molecule compounds or macromolecular compounds.

[0066] Preferably, the small molecule compound includes but is not limited to any small molecule drug, such as anti-tumor drugs, protein kinase inhibitors, antiviral drugs, antibiotics, anti-Alzheimer's drugs, anti-Parkinson's drugs, anti-inflammatory drugs, anti-allergic drugs, antihypertensive drugs, anti-thromboembolic drugs, anti-epileptic drugs, antidepressants, stroke drugs or autism drugs, etc.

[0067] Preferably, the small molecule drugs include but are not limited to one or more of donepezil, rasagiline, entacapone, rotigotine, camptothecin, paclitaxel, sunitinib, sorafenib, warfarin, curcumin, methotrexate, docetaxel, carbamazepine, seroxate, olanzapine, glibenclamide, nimodipine, idebenone, sulpiride or delutec.

[0068] In one embodiment, the small molecule drug is deludec.

[0069] Preferably, the macromolecular compound includes but is not limited to antibodies, activating or inhibiting receptors, protein ligands, biologically active enzymes, nucleic acid drugs, or combinations thereof.

[0070] Preferably, the antibody in the macromolecular compound includes an antibody or an antigen-binding fragment thereof against other targets, and the other targets are targets other than albumin.

[0071] Further preferably, the structure of the antibody or antigen-binding fragment thereof for other targets includes one or a combination of two or more of a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single-chain antibody fragment (scFv) or a linear antibody.

[0072] Preferably, the other targets are selected from vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor (FGF), fibroblast growth factor receptor (FGFR), placental growth factor (PLacental growth factor, PIGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), integrin, integrin receptor, interleukin (such as IL-1β, IL-2, IL-3, IL-4, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptor ( ... Receptors, such as IL1R1, IL2Rα, IL3R, IL4Rα, IL10R, IL12R, IL15Rα, IL17R, IL23R, etc.), Proprotein convertase subtilisin / kexin type 9 (PCSK9), Tumor necrosis factor α (TNFα), Tumor necrosis factor receptor (TNFR), Receptor Activator of Nuclear Factor-κB Ligand (RANKL), G Protein-Coupled Receptor (GPCR), glucagon-like peptide-1 receptor (GLP1R), Cluster of Differentiation 3 (CD3), Cluster of Differentiation 105 (Cluster of Differentiation 105),Cluster of Differentiation 20 (CD105), Cluster of Differentiation 19 (CD19), Cluster of Differentiation 20 (CD20), Cluster of Differentiation 22 (CD22), Cluster of Differentiation 25 (CD25), Cluster of Differentiation 27 (CD27), Cluster of Differentiation 28 (CD28), Cluster of Differentiation 30 (CD30), Cluster of Differentiation 33 (CD33), Cluster of Differentiation 38 (CD38), Cluster of Differentiation 40 (CD40), Cluster of Differentiation 47 (CD47), Cluster of Differentiation 80 (CD80 / B7-1), Cluster of Differentiation 86 (CD80 / B7-1), Cluster of Differentiation 87 (CD80 / B7-1), Cluster of Differentiation 88 (CD80 / B7-1), Cluster of Differentiation 89 (CD80 / B7-1), Cluster of Differentiation 90 (CD80 / B7-1), Cluster of Differentiation 91 (CD80 / B7-1), Cluster of Differentiation 92 (CD80 / B7-1), Cluster of Differentiation 93 (CD80 / B7-1), Cluster of Differentiation 94 (CD80 / B7-1), Cluster of Differentiation 95 (CD80 / B7-1), Cluster of Differentiation 96 (CD80 / B7-1), Cluster of Differentiation 97 (CD80 / B7-1), Cluster of Differentiation 98 (CD80 / B7-1), Cluster of Differentiation 99 (CD80 / B7-1), Cluster of Differentiation 100 (CD80 / B7-1), Cluster of Differentiation 101 (CD80 / B7-1), Cluster of Differentiation 102 (CD80 / B7-1), Cluster of Differentiation 103 (CD80 Cluster of Differentiation 155 (CD155), Cluster of Differentiation 171 (CD171), Claudin 18.2 (CD18.2), and Cluster of Differentiation 111 (CD111).CLDN18.2), TNF receptor superfamily, member 4 (TNFRSF4 / OX40 / CD134), Inducible T-Cell Co-Stimulator (ICOS), Cytotoxic T-lymphocyte-associated protein 4 (CTLA4), TNF receptor superfamily, member 9 (TNFRSF9 / 4-1BB / CD137), T cell antigen receptor (TCR), B- and T-Lymphocyte Attenuator (BTLA), T cell immunoglobulin domain and mucin domain-3 (TIM-3), Lymphocyte Activation Gene 3 (LYA337), TNF receptor superfamily, member 9 (TNFRSF9 / 4-1BB / CD137), T cell antigen receptor (TCR), B- and T-Lymphocyte Attenuator (BTLA), T cell immunoglobulin domain and mucin domain-3 (TIM-3), Lymphocyte Activation Gene 3 (LYA337), TNF receptor superfamily, member 4 (TNFRSF4 / OX40 / CD134), T cell antigen receptor (TCR), B- and T-Lymphocyte Attenuator (BTLA), T cell immunoglobulin domain and mucin domain-3 (TIM-3), Lymphocyte Activation Gene 3 (LYA337), TNF receptor superfamily, member 9 (TNFRSF9 / 4-1BB / CD137), T cell antigen receptor (TCR), B- and T-Lymphocyte Attenuator (BTLA), T cell immunoglobulin domain and mucin domain-3 (TIM-3), Lymphocyte Activation Gene 3, LAG3), Galectin-9 (GAL9), Programmed cell death 1 ligand 1 (PD-L1), Programmed cell death 1 ligand 2 (PD-L2), Programmed cell death protein 1 (PD-1), T cell immune receptor with Ig and ITIM domains (TIGIT), Epidermal growth factor receptor (EGFR), Human epidermal growth factor receptor 2 (HER2), Prostate stem cell antigen (PSCA), Carcinoembryonic antigen (CEA), Familial adenomatous polyposis (FANPs),FAP), epidermal growth factor receptor variant type Ⅲ (EGFRvIII), B cell maturation antigen (BCMA), prostate specific membrane antigen (PSMA), carbohydrate antigen 125 (CA125), ephrin A receptor 2 (EphA2), cellular-mesenchymal epithelial transition factor (c-Met), L1-cell adhesion molecule (L1CAM), signaling lymphocytic activation molecule family, member 7 (SLAMF7 / CS1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), recombinant tuberculosis fusion protein (Recombinant Mycobacterium tuberculosis fusion protein, Re ... Mycobacterium Tuberculosis Fusion Protein), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), Mucin 1 (MUC1), Mucin 16 (MUC16), Mesothelin, Cluster of Differentiation 174 (LewisY / CD174), Glypican 3 (GPC3), Disialoganglioside-GD2 (GD2), Eukaryotic division factor-like and proliferation-associated protein (EPG), Delta-Like Ligand 3 (DLL3), or Trophoblast Glycoprotein (TPBG / 5T4).

[0073] In one embodiment, the other target is VEGFA.

[0074] Preferably, the anti-albumin antibody or antigen-binding fragment thereof is directly or indirectly linked to the biologically active effector molecule.

[0075] Preferably, the indirect connection may be through a linker, a functional domain and / or a linker for coupling.

[0076] Wherein, the linker is selected from connecting peptides, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, PEG, nucleic acids, polysaccharides, fatty chains, biotin, streptavidin or avidin.

[0077] The functional domain is one or a combination of two or more of Fc fragment, serum albumin, cytokine, transferrin, and scaffold protein.

[0078] The linkers used for coupling include functional group linkers.

[0079] The functional group linker includes sulfhydryl, amino, hydroxyl and / or carboxyl reactive groups, which can covalently couple the anti-albumin antibody or its antigen-binding fragment with the bioactive effector molecule.

[0080] Preferably, the bioactive effector molecule is directly or indirectly linked to the N-terminus, C-terminus and / or internal residues of the anti-albumin antibody or antigen-binding fragment thereof.

[0081] Preferably, the fusion construct comprises one or more biologically active effector molecules.

[0082] For example, in the fusion construct, the sequence of connection of the antibody or antigen-binding fragment thereof and the biologically active effector molecule from N-terminus to C-terminus is as follows:

[0083] anti-albumin antibodies or antigen-binding fragments thereof, connecting peptides, and biologically active effector molecules;

[0084] biologically active effector molecules, connecting peptides, anti-albumin antibodies or antigen-binding fragments thereof;

[0085] anti-albumin antibody or antigen-binding fragment thereof, connecting peptide, first biologically active effector molecule, connecting peptide, second biologically active effector molecule; or,

[0086] A first biologically active effector molecule, a connecting peptide, an anti-albumin antibody or an antigen-binding fragment thereof, a connecting peptide, a second biologically active effector molecule, etc.; wherein the connection may not include a connecting peptide.

[0087] In one embodiment, the biologically active effector molecule comprises an amino acid sequence of any one of SEQ ID NOs: 26-27, or has at least 80% identity with an amino acid sequence of any one of SEQ ID NOs: 26-27.

[0088] In a specific embodiment, the fusion construct comprises any one of the amino acid sequences in SEQ ID NOs: 28-30, 61, or has at least 80% identity with any one of the amino acid sequences in SEQ ID NOs: 28-30, 61.

[0089] In one embodiment, the fusion construct comprises an Fc fragment.

[0090] Further preferably, the Fc fragment comprises SEQ ID NO: 38, or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 38.

[0091] In a specific embodiment, the fusion construct comprises any one of the amino acid sequences in SEQ ID NOs: 14-15, or has at least 80% identity to any one of the amino acid sequences in SEQ ID NOs: 14-15.

[0092] Preferably, the fusion construct further comprises a secretory peptide. The secretory peptide is linked to the N-terminus of the fusion construct. Further preferably, the amino acid sequence of the secretory peptide may be SEQ ID NO: 37 or 42.

[0093] Preferably, the fusion construct further comprises a tag.

[0094] Preferably, the tag is connected to the C-terminus of any antibody or antigen-binding fragment thereof, or a fusion construct.

[0095] Preferably, the fusion construct comprises a conjugate of an anti-albumin antibody and a drug, which comprises any of the above-mentioned anti-albumin antibodies or antigen-binding fragments thereof, and the biologically active effector molecule is a drug, and the drug is covalently bound to any of the above-mentioned anti-albumin antibodies or antigen-binding fragments thereof.

[0096] A fourth aspect of the present invention provides a nucleic acid encoding the anti-albumin antibody or antigen-binding fragment thereof or the fusion construct, for example, comprising DNA and / or mRNA.

[0097] In some embodiments, the nucleic acid is DNA, which encodes the antibody or antigen-binding fragment described herein, or the fusion construct described above.

[0098] Preferably, the nucleotide sequence encoding the anti-albumin antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 63-75, 79-81, 83-87 or a degenerate sequence thereof, or has at least 80% identity with any one of SEQ ID NOs: 63-75, 79-81, 83-87 and has a nucleotide sequence that encodes the anti-albumin antibody or antigen-binding fragment thereof.

[0099] Preferably, the nucleotide sequence encoding the above-mentioned fusion construct comprises any one of SEQ ID NOs: 76-78, 82 or a degenerate sequence thereof, or has at least 80% identity with any one of SEQ ID NOs: 76-78, 82, and has a nucleotide sequence encoding the function of the above-mentioned fusion construct.

[0100] The fifth aspect of the present invention provides a vector comprising the above-mentioned nucleic acid.

[0101] The vector can be expressed in vivo, in vitro, or in vitro. Preferably, the vector is a prokaryotic expression vector, a viral expression vector, or a eukaryotic expression vector, such as an E. coli vector, a bacteriophage, or the like.

[0102] In a sixth aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or the aforementioned vector.

[0103] The host cell can be a eukaryotic cell or a prokaryotic cell.

[0104] Eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells, 293F cells, or CHO cells.

[0105] Prokaryotic cells such as Escherichia coli.

[0106] The seventh aspect of the present invention provides a method for preparing a host cell, which comprises introducing the above-mentioned nucleic acid or vector into the host cell.

[0107] In an eighth aspect, the present invention provides a method for preparing the above-mentioned anti-albumin antibody or its antigen-binding fragment or the above-mentioned fusion construct, which comprises culturing the above-mentioned host cell to express the anti-albumin antibody or its antigen-binding fragment or the above-mentioned fusion construct.

[0108] In a ninth aspect, the present invention provides a product for treating and / or diagnosing a disease, wherein the product for treating and / or diagnosing a disease comprises any one of the following:

[0109] A) the above-mentioned anti-albumin antibody or antigen-binding fragment thereof;

[0110] B) the fusion construct described above;

[0111] C) the nucleic acid described above;

[0112] D) the vector described above; or

[0113] F) The host cell described above.

[0114] Preferably, the anti-albumin antibody or antigen-binding fragment thereof and / or the biological effector molecule has activity in treating and / or diagnosing a disease. Further preferably, the anti-albumin antibody or antigen-binding fragment thereof is used as a carrier in a product for treating and / or diagnosing a disease.

[0115] The diseases include but are not limited to tumors, infectious diseases, Alzheimer's disease, Parkinson's disease, inflammatory diseases, epilepsy, depression, stroke, autism, allergic diseases, hypertension, thrombosis or diseases related to other target signaling pathways.

[0116] Preferably, the fusion construct is a conjugate of an anti-albumin antibody and a drug, which comprises any of the above-mentioned anti-albumin antibodies or antigen-binding fragments thereof, and the biologically active effector molecule is a drug, and the drug is covalently bound to any of the above-mentioned anti-albumin antibodies or antigen-binding fragments thereof.

[0117] In the tenth aspect of the present invention, a method for detecting albumin is provided, which comprises contacting a sample to be tested with the above-mentioned anti-albumin antibody or its antigen-binding fragment, and then detecting the content of a complex formed by albumin and the anti-albumin antibody or its antigen-binding fragment.

[0118] The detection method is to detect the presence or content of albumin. The presence refers to the presence or absence of the protein, and the content may be the expression level or protein concentration. Preferably, the albumin is derived from a mammal, more preferably, the mammal is human, mouse, or monkey.

[0119] In the eleventh aspect of the present invention, a method for treating and / or preventing a disease is provided, which comprises administering the above-mentioned anti-albumin antibody or antigen-binding fragment thereof, the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell or the above-mentioned product for treating and / or diagnosing the disease to an individual.

[0120] The diseases include but are not limited to tumors, infectious diseases, Alzheimer's disease, Parkinson's disease, inflammatory diseases, epilepsy, depression, stroke, autism, allergic diseases, hypertension, thrombosis or diseases related to other target signaling pathways.

[0121] In one embodiment, the disease includes diseases related to the VEGFA signaling pathway, and more preferably, the disease may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.

[0122] Preferably, the target cells are selected from cells expressing VEGFA, such as cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granular cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.

[0123] The twelfth aspect of the present invention provides a use of the above-mentioned anti-albumin antibody or antigen-binding fragment thereof, the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector or the above-mentioned host cell in the preparation of a product for treating and / or preventing a disease, wherein the anti-albumin antibody or antigen-binding fragment thereof and / or the biologically active effector molecule has activity in treating and / or diagnosing a disease.

[0124] Preferably, the anti-albumin antibody or antigen-binding fragment thereof is used as a carrier in a product for treating and / or diagnosing a disease.

[0125] The diseases include but are not limited to tumors, infectious diseases, Alzheimer's disease, Parkinson's disease, inflammatory diseases, epilepsy, depression, stroke, autism, allergic diseases, hypertension, thrombosis or diseases related to other target signaling pathways.

[0126] In one embodiment, the disease includes diseases related to the VEGFA signaling pathway, and more preferably, the disease may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.

[0127] As used herein, a "fusion construct" defines a fusion of an anti-albumin antibody or antigen-binding fragment thereof of the present invention with another compound. The fusion construct may comprise one or more anti-albumin antibodies or antigen-binding fragments thereof, and the multiple anti-albumin antibodies or antigen-binding fragments thereof may be the same or different. The fusion construct may comprise one or more additional compounds, and the multiple additional compounds may be the same or different. The compounds may be proteinaceous compounds or non-proteinaceous compounds. Where the compounds are proteinaceous compounds or the fusion construct comprises only multiple anti-albumin antibodies or antigen-binding fragments thereof, the fusion construct may also be referred to as a fusion protein. Where the compound is fused to the anti-albumin antibody or antigen-binding fragment thereof in a conjugated form, the fusion construct may also be referred to as a conjugate.

[0128] The product described in this application may be a kit, a drug, a chip, an antibody-drug conjugate, etc.

[0129] The "medicine" of the present invention can be used to treat humans or non-human animals, such as non-human mammals. The medicine can include pharmaceutically acceptable carriers, excipients or salts common in the prior art. The medicine can be administered by any suitable route, such as enteral administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.). The medicine can be in any suitable dosage form, such as enteral administration or parenteral administration, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, etc. The various dosage forms of the medicine can be prepared according to the conventional production methods of pharmaceutical field. The drug may contain 0.01-99.5% (e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of the anti-albumin antibody or antigen-binding fragment thereof, the fusion construct, the nucleic acid, the vector, the host cell, the immune cell, etc. by weight. The drug can be prepared as a reagent with a protein concentration of 1-300 mg / mL (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg / mL). The single dose of the drug can be 0.1-1000 mg, for example, 0.1, 0.2, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 5, 10, 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg.

[0130] The term "pharmaceutically acceptable" as used herein means that the pharmaceutical composition neither significantly stimulates the organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0131] The "method" described in the present invention can be used for the diagnosis and treatment of diseases or for non-disease diagnosis and treatment purposes.

[0132] The "antigen-binding fragment" of the present invention is a part of an antibody that retains the specific binding activity of the antibody, that is, any part of the antibody that can specifically bind to the epitope on the target molecule of the antibody. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd and variants of these fragments. For example, the heavy chain and / or light chain of an antibody, the heavy chain variable region and / or light chain variable region of an antibody, or a single or two or more CDRs from the heavy chain or light chain of an antibody. Among them,

[0133] Nanobodies or single-domain antibodies refer to the variable domain of heavy chain (VHH) of an antibody, which has independent antigen-binding activity.

[0134] Chimeric antibodies are antibodies in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0135] Single-chain antibodies are antibodies composed of the variable regions of the heavy chain and the light chain connected by a linker peptide.

[0136] Fab, a monovalent fragment consisting of the VL, VH, CL and CH1 domains.

[0137] Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0138] F(ab')2 is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

[0139] Fd, Fd fragment consisting of VH and CH1 domains.

[0140] Fv, an Fv fragment consisting of the VL and VH domains of a single antibody arm.

[0141] dAb fragments are antibody fragments composed of the VH domain.

[0142] The “linear antibody” described in the present invention comprises one or more pairs of antibody fragments connected in series, wherein the antibody fragment can be an Fd segment (VH-CH1), a single-chain antibody (scFv), an antibody fragment (Fab), or a single-domain antibody (VHH), and these fragments are connected in series through connecting peptides to form a continuous antibody structure.

[0143] Among them, VH represents the heavy chain variable region, VL represents the light chain variable region, CL represents the light chain constant region, and CH represents the heavy chain constant region.

[0144] The "Fc" region of the present invention contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments form a dimer by two or more disulfide bonds in the hinge region and are held together by the hydrophobic interaction of the CH3 domain.

[0145] The terms "comprising" or "including" as used in the present invention are open-ended. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0146] The "homology" or "identity" mentioned in the present invention refers to the fact that when using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 11 %, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology.

[0147] The "humanized antibody" of the present invention refers to an antibody whose framework region and / or constant region portion (e.g., CH region) or all or part of an antibody is encoded by a human antibody gene. In one embodiment of the present invention, the CDR region of the antibody has not been humanized.

[0148] The "individual" described in the present invention can be a human or a non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, a commercial animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

[0149] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0150] The term "prevention" used in the present invention refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in the body.

[0151] The term "diagnosis" as used herein refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to determining the progression or possible future progression of a disease.

[0152] As used herein, a "tumor" can be any undesirable cell proliferation (or any disease manifesting as undesirable cell proliferation), neoplasm, or a predisposition or increased risk of undesirable cell proliferation, neoplasm, or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). A neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, leukocytes. Further preferably, the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (e.g., glioma), intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, rhabdomyosarcoma, tongue squamous cell carcinoma, nasopharyngeal cancer, ovarian cancer, placental choriocarcinoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, neurilemoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.

[0153] The term "retinal vascular disease" as used herein refers to a general term for diseases that occur in the retinal arteries or veins. These include, but are not limited to, age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, and neovascular glaucoma.

[0154] The present invention obtains an antibody or antigen-binding fragment thereof that binds to albumin with high affinity. The anti-albumin nanobody has the following advantages:

[0155] 1. The anti-albumin nanobodies obtained can bind with high affinity to albumins from different species, including human, monkey, mouse, and rat. Furthermore, the humanized anti-albumin antibodies have been humanized, binding to human and mouse albumin, and also to rat albumin. Mice and rats are commonly used experimental animals, which makes the anti-albumin nanobodies of the present invention have a wider range of applications.

[0156] 2. By performing affinity enhancement modification on the selected humanized anti-albumin antibodies, antibodies with affinity to human albumin increased by 3.7 times or 35.7 times were obtained.

[0157] 3. Albumin has the characteristic of being enriched in inflammation and tumor sites. Humanized anti-albumin nanoantibodies can be used to prepare drugs for treating immune diseases, tumors and other diseases. By fusing anti-albumin nanoantibodies with other antibodies, the resulting fusion construct can bind to albumin in vivo and has the characteristic of being enriched in inflammation and tumor sites similar to albumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0159] Figure 1: Cootisol-stained gel images of His-tagged antibodies Alb Nb, 2049, 3004, 3007, and 3005 after Ni column purification. (A) is the cootisol-stained gel image of antibodies Alb Nb, 2049, 3004, and 3007, and (B) is the cootisol-stained gel image of antibody 3005.

[0160] Figure 2: ELISA tests of the binding of fusion proteins 3005-Fc, 3007-Fc, and Alb Nb-Fc to albumins from different species. (A) HSA binding ELISA, (B) MSA binding ELISA, and (C) rat albumin binding ELISA.

[0161] Figure 3: stained results of 3005 humanized antibody containing His tag after Ni column purification.

[0162] Figure 4: ELISA activity test of antibody 3005 and its humanized antibody binding to MSA.

[0163] Figure 5: ELISA activity test of antibody 3005, its humanized antibody 3005Hz6, and control antibody Alb Nb binding to HSA and rat albumin, (A) is HSA binding test (HSA binding ELISA), (B) is rat albumin binding test (Rat albumin binding ELISA).

[0164] Figure 6: Binding and dissociation curves of humanized antibody 3005Hz6 with HSA and MSA (SPR method), (A) is the binding and dissociation curves of antibody 3005Hz6 with HSA, (B) is the binding and dissociation curves of antibody 3005Hz6 with MSA, the ordinate is the response value, and the abscissa is the time.

[0165] Figure 7: SDS-PAGE gel images of the purified humanized antibody 3005Hz6 mutant proteins 3005Hz6(EG), 3005Hz6(DS) and 3005Hz6(DA), (A) is the SDS-PAGE gel image of 3005Hz6(EG) and 3005Hz6(DS), (B) is the SDS-PAGE gel image of 3005Hz6(DA).

[0166] Figure 8: ELISA activity detection of HSA binding of humanized antibody 3005Hz6 and its mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG) (HSA binding ELISA).

[0167] Figure 9: SDS-PAGE gel images of the fusion proteins V1SA-3005Hz6, 3005Hz6-V1SA and V1DP-3005Hz6 after purification by Ni column, (A) is the SDS-PAGE gel image of V1SA-3005Hz6 and 3005Hz6-V1SA, (B) is the SDS-PAGE gel image of V1DP-3005Hz6.

[0168] Figure 10: ELISA activity detection of the fusion protein of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody, and the binding of humanized antibody 3005Hz6 to HSA.

[0169] Figure 11: VEGFR2 competition ELISA activity test of fusion protein and monovalent nanobody V1-SA1 without pre-incubation or with pre-incubation of HSA, (A) is the result without pre-incubation of HSA, (B) is the result with pre-incubation of HSA.

[0170] Figure 12: Flowchart of the conjugation and purification of Nanobody-Drutecan Conjugate (NDC).

[0171] Figure 13: (A) is a schematic structural diagram of 3005K65A-drutecan conjugate (NDC), (B) is an SDS-PAGE gel image of 3005K65A and three different 3005K65A-drutecan conjugates (NDC-1, NDC-2, NDC-3).

[0172] Figure 14: (A) is the ELISA activity test (HSA binding ELISA) of 3005K65A and 3005K65A-drutecan conjugate (NDC-3) binding to HSA, (B) is the results of the proliferation inhibition experiment of Mia Paca-2 cells by drutecan and 3005K65A-drutecan conjugate (NDC-3).

[0173] Figure 15: Co-stained results of purified affinity-enhanced candidate antibodies.

[0174] Figure 16: ELISA results of HSA binding of candidate antibodies with improved affinity (coat antibody method, coat antibody), (A) is the ELISA result at pH = 7.4, (B) is the ELISA result at pH = 6.0.

[0175] Figure 17: ELISA results of affinity-enhanced candidate antibodies binding to rat albumin (Rat albumin binding ELISA) (antigen coating method), (A) is the ELISA result at pH = 7.4, (B) is the ELISA result at pH = 6.0.

[0176] Figure 18: Binding and dissociation curves of affinity-enhanced antibodies and control antibodies with HSA (SPR method), (A) is the binding and dissociation curve of antibody 3005Hz6 with HSA, (B) is the binding and dissociation curve of antibody 1068 with HSA, (C) is the binding and dissociation curve of antibody 2028 with HSA, and (D) is the binding and dissociation curve of control antibody BI-V2 with HSA. The ordinate is the response value and the abscissa is time. DETAILED DESCRIPTION

[0177] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0178] Example 1: Alpaca immunization and antibody library construction

[0179] 1. Alpaca immunity and potency testing

[0180] Alpacas were immunized with emulsified human serum albumin (HSA) (purchased from Sigma-Aldrich, Catalog No. A3782). Each immunization was administered with 1 mg of HSA, once every two weeks, for a total of three immunizations. Serum was collected one week after the second immunization for titer determination.

[0181] Serum titer detection method is:

[0182] HSA antigen was diluted to 1 μg / mL with CBS buffer, and 100 μL / well was added to the ELISA plate and incubated at 4°C overnight;

[0183] Wash the plate three times with PBST (PBS containing 0.1% Tween 20, pH = 7.4). Add 200 μL of skim milk powder to each well and let it stand at room temperature for 1 hour. Wash the plate three times with PBST.

[0184] Sample addition: Dilute the immunized serum and negative serum by 1000-fold, 3000-fold, 9000-fold, 27000-fold, 81000-fold, and 243000-fold, respectively, and add 100 μL of each to the sealed wells. Incubate at room temperature for 1 hour, and wash the plate three times with PBST.

[0185] Secondary antibody: Add 100 μL of HRP-labeled Goat Anti-Alpaca IgG to each well, incubate at room temperature for 1 h, and wash the plate three times with PBST.

[0186] TMB color development: Add 100 μL / well of TMB color development solution to the ELISA plate and develop for 15 minutes; add 50 μL / well of stop solution, place the ELISA plate in a microplate reader, and read the absorbance at 450 nm (OD450).

[0187] The serum titer test after the second and third alpaca immunization is shown in Table 2. It can be seen that after the serum was diluted 243,000 times, the signal value of binding to human serum albumin was above 0.59, which met the library construction standard. The alpaca serum after the third immunization was used to construct a phage library.

[0188] Table 2 Serum titer detection after the second and third albumin alpaca immunization

[0189] 2. Phage library construction

[0190] Peripheral blood was drawn from alpacas, and total RNA was extracted from the isolated alpaca PBMC using Trizol. Reverse transcription of cDNA was performed, and the variable region VH fragment (approximately 700 bp in length) was amplified twice using nanobody-specific primers. The resulting fragment and the pcomb3X vector were digested with SfiI enzyme, mixed in appropriate proportions, and ligated with T4 ligase. After ligation, the fragments were used for electroporation into XL1-Blue competent cells. Based on the colony growth of the dilution of the competent cells on the resistance-containing plate, the transformation library capacity of the alpaca was calculated to be 6.24×10 8 .

[0191] Example 2: Screening of antibody libraries to obtain antibodies that cross-bind to human, mouse, and cynomolgus monkey albumin

[0192] 1. Phage library screening

[0193] The screening process is:

[0194] Antigens: human serum albumin (HSA) and mouse albumin (MSA) (Sigma-Aldrich, Cat. No. A3559). A negative control (3% skim milk powder coated ELISA plate) was also set up. The plates were incubated at room temperature for 1 hour. The blocking buffer was discarded and the plates were washed with PBST.

[0195] Add about 5×10 12 The pfu library was incubated with antigen on antigen plates at 37°C for 2 hours, and the plates were washed with 0.1% PBST.

[0196] Elution was performed with low pH glycine-HCl, followed by neutralization with Tris-HCl until the pH was 7.4.

[0197] The eluate was mixed with E. coli XL1-Blue and incubated at 37°C with shaking.

[0198] After adding phage, the cells were cultured at 30°C overnight and the supernatant was collected.

[0199] Repeat 1)-5) to obtain the second round library.

[0200] The second-round amplified library obtained under HSA coating conditions was used for ELISA assays. The antigen HSA was coated using either PBS or CBS buffer. ELISA binding assays were then performed. Phage detection was performed using an HRP-labeled Anti-M13 Antibody (purchased from Sino Biological, Cat. No. 11973-MM05T-H) diluted 2500-fold. After incubation with the detection antibody, the plates were washed, developed with TMB, and the OD450 reading was performed. The results of the second-round amplified library are shown in Table 3. As can be seen, strong HSA-binding phage signals were generated after the second round of enrichment.

[0201] Table 3 Detection results of the second round of phage library enriched under HSA coating conditions

[0202] Antigen binding ELISAs were performed on the second-round amplified library obtained under MSA-coating conditions. CBS buffer was used to coat HSA and MSA, respectively. The ELISA results are shown in Table 4. As can be seen, phages with strong binding to HSA and MSA were also obtained under this enrichment condition.

[0203] Table 4 Detection results of the second round of phage library obtained by enrichment under MSA coating conditions combined with HSA and MSA

[0204] Summary: The antigen binding ELISA test of the second-round phage library obtained by coating HSA and MSA enrichment was positive, indicating that phages binding to HSA and MSA respectively were enriched, and binding screening at the phage monoclonal level can be carried out.

[0205] 2. Phage monoclonal screening and sequencing

[0206] The experimental process is:

[0207] 96 monoclonal colonies were selected from the first round phage library enriched by HSA coating for phage expression and coated with HSA protein for ELISA binding activity detection.

[0208] 96 monoclonal colonies were selected from the second-round phage library enriched by coating with MSA for phage expression and coated with HSA, MSA and monkey albumin for ELISA binding activity detection.

[0209] The coating antigens were HSA, MSA, and monkey albumin. The protein concentration during coating was 0.5 μg / mL and the coating was carried out at 4°C overnight.

[0210] Blocking: 3% milk powder, 200 μL / well, room temperature for 1 hour.

[0211] Add 10-fold diluted phage expression supernatant, 100 μL / well, and incubate at room temperature for 1 hour.

[0212] Add detection antibody Anti-M13 Antibody (HRP labeled) (concentration: 0.2 μg / mL) at 100 μL / well and incubate at room temperature for 1 hour.

[0213] Add 200 μL / well of TMB colorimetric solution, develop for 20 minutes, then add 50 μL / well of stop solution. Detect OD450 using a microplate reader.

[0214] From the 96 phage clones in the first round of phage library enriched by coating HSA, one clone was selected and numbered 2049. From the 96 phage clones in the second round of phage library enriched by coating MSA, three clones were selected and numbered 3004, 3005, and 3007, respectively. The ELISA test results are shown in Tables 5 and 6.

[0215] Table 5 Detection of HSA binding activity of phage monoclone 2049

[0216] Table 6 Activity detection of phage monoclonal 3004, 3005, 3007 binding to HSA, MSA and monkey albumin

[0217] The sequences of the four phage clones were obtained by DNA sequencing. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the four antibodies are shown in Table 7 , and the amino acid and nucleotide sequences of the four nanobodies are shown in Table 8 .

[0218] Table 7 Amino acid sequences of the CDR regions of four anti-albumin nanobodies

[0219] Table 8 Amino acid sequences and nucleotide sequences of four anti-albumin nanobodies

[0220] Example 3: Activity detection of anti-albumin candidate antibodies binding to albumins from different species

[0221] 1. Expression of anti-albumin candidate antibodies

[0222] These four candidate antibodies were expressed in mammalian cells, and after protein purification, antigen binding activity was tested to verify the activity of the candidate antibodies in binding to albumin at the protein level.

[0223] By gene synthesis, expression plasmids for His-tagged antibodies 2049, 3004, 3005, 3007 and positive control antibody Alb Nb were constructed. The positive control antibody Alb Nb is a nanobody against human serum albumin. The amino acid sequence of the antibody is derived from SEQ ID NO: 62 in patent US2007 / 0269422 A1. The amino acid sequence is:

[0224] The tPA secretion signal peptide (MDAMKRGLCCVLLLCGAVFVSPS) (SEQ ID NO: 37) was added to the N-terminus of the above-mentioned antibody. The linker GGGGS (SEQ ID NO: 41) and 6xHis were added to the C-terminus of the antibody. The gene sequence was ligated into the expression vector pCDNA3.1(+) via the restriction enzyme cleavage sites NheI and XbaI. After correct plasmid sequencing, the plasmid was extracted in an endotoxin-free manner and then transiently expressed in suspension 293F cells using the transfection reagent polyethylenimine (PEI). Ni column affinity purification was then performed.

[0225] The SDS-PAGE staining of the cell culture supernatant (Input), the flow-through (FT) that could not bind to the Ni column, and the eluted purification product (Eluate) of transiently expressed His-tagged antibodies Alb Nb, 2049, 3004, and 3007 is shown in Figure 1 (A). The SDS-PAGE staining results of the product of the His-tagged antibody 3005 after Ni column purification are shown in Figure 1 (B). Their molecular weights are consistent with expectations (~15 kDa).

[0226] At the same time, fusion proteins of antibodies 3005, 3007 and the positive control antibody Alb Nb with Fc were constructed. The tPA secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 37) was added to the N-terminus of the antibody coding sequence through primer design. The amino acid sequence of human IgG1 Fc was added to the C-terminus of the antibody sequence through homologous recombination:

[0227] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38), and the gene fragment was inserted into the mammalian cell expression vector pCDNA3.1(+).

[0228] The amino acid and nucleotide sequences of the anti-albumin nanobody-Fc fusion protein are shown in Table 9.

[0229] Table 9 Amino acid sequence and nucleotide sequence of anti-albumin nanobody-Fc fusion protein

[0230] After the expression plasmid of the Fc fusion protein is sequenced correctly, a large amount of endotoxin-free extraction is performed, and transient expression in suspended 293F cells and Protein A affinity purification are carried out to obtain the target protein.

[0231] 2. Detection of albumin binding activity of candidate anti-albumin antibodies

[0232] Bio-layer interferometry (BLI) was used to test the affinity of monovalent nanobodies for binding to albumins from different species. Eight NTA sensors were used to bind to the same his-tag monovalent nanobody (at a concentration of 1 μg / mL). The antigens were human serum albumin (HSA) (Baxter AG), mouse albumin (MSA) (purchased from Equitech-bio, Catalog No. MSA62-1000), and rat albumin (purchased from abcam, Catalog No. ab198656). The BLI assay was performed at eight antigen concentration gradients of 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM. The binding time was 180 seconds, and the dissociation time was 360 seconds.

[0233] Table 10 shows the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of the monovalent His-tagged nanobodies 2049, 3004, 3005, and 3007 binding to albumin from different species after fitting the "1:1" model. D ).

[0234] Table 10 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of nanobodies 2049, 3004, 3005 and 3007 for binding to albumin from different species D )

[0235] The data showed that 2049, 3004, 3005 and 3007 all bind to HSA and MSA with high affinity, and 3005 and 3007 can also bind to rat albumin.

[0236] The ELISA method was used to compare the binding activity of 3005 and 3007 to albumin from different species. The experimental method was:

[0237] Dilute the antigen in ELISA coating buffer to a final concentration of 1 μg / mL for human serum albumin (HSA) (Baxter AG), 2 μg / mL for mouse albumin (MSA) (Equitech-bio, Catalog No. MSA62-1000), and 2 μg / mL for rat albumin (abcam, Catalog No. ab198656). Add 100 μL of the antigen dilution to the ELISA plate and coat overnight at 4°C. After blocking with 5% skim milk powder, serial dilutions of candidate antibodies 3005-Fc and 3007-Fc, as well as the positive control antibody Alb Nb-Fc, in PBST (0.1% Tween 20 in PBS, pH 7.4) were added (0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 nM). The plates were incubated at 37°C for 1 hour. After washing, HRP-conjugated goat anti-human IgG Fc antibody (Abbkine, Catalog No. A21050) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (Tiangen Biochemical, Catalog No. PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader.

[0238] The ELISA test results of 3005-Fc, 3007-Fc, and Alb Nb-Fc binding to albumins from different species are shown in Figure 2. The EC50 of 3005-Fc and 3007-Fc binding ELISA experiments are summarized in Table 11.

[0239] Table 11 Summary of EC50 of ELISA experiments of 3005-Fc and 3007-Fc binding to albumin from different species

[0240] Referring to Table 11, the results show that both antibodies 3005-Fc and 3007-Fc efficiently bind to HSA and MSA. Referring to Figure 2, the positive control antibody Alb Nb-Fc showed very weak binding activity to rat albumin. However, both antibodies 3005-Fc and 3007-Fc exhibited strong binding activity to rat serum albumin, with 3005-Fc showing superior binding activity to rat albumin compared to 3007-Fc. Therefore, antibody 3005 was preferred for subsequent studies.

[0241] Example 4: Humanization of anti-albumin candidate antibodies

[0242] 1. Humanization transformation, expression and purification of candidate antibodies

[0243] Humanization was performed by transplanting the CDR region of the antibody. Using candidate antibody 3005 (SEQ ID NO: 12) as the starting antibody, humanization of the antibody was performed. The homology between the candidate antibody 3005 sequence and the human antibody sequence was first compared using the antibody databases IGBLAST and IMGT. The humanized antibody was then constructed by combining the human antibody framework sequence with the three CDR region sequences of candidate antibody 3005 (SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 9).

[0244] A secretion signal peptide was added to the antibody's N-terminus, and the linkers GGGGS (SEQ ID NO: 41) and 6xHis were added to the antibody's C-terminus. The coding DNA sequence was synthesized and ligated into the expression vector pCDNA3.1(+) via the restriction enzyme sites NheI and XbaI. The plasmid was sequenced correctly and extracted in an endotoxin-free, large-scale, and transiently expressed in suspension 293F cells and purified by Ni affinity column.

[0245] The amino acid and nucleotide sequences of the humanized modified protein of antibody 3005 are shown in Table 12.

[0246] Table 12 Amino acid sequence and nucleotide sequence of 3005 humanized antibody

[0247] The humanized protein containing the His tag was purified by Ni column and separated by SDS-PAGE electrophoresis. The molecular weight was consistent with the expectation (~13 kDa). The staining results are shown in Figure 3.

[0248] 2. Activity detection of humanized antibodies

[0249] First, the ELISA method was used to detect the activity of the original candidate antibody 3005 and the humanized antibody in binding to mouse albumin MSA.

[0250] In the experiment, mouse albumin MSA (coating protein concentration of 5 μg / mL) was coated in an ELISA plate at 100 μL / well at 4°C overnight. After blocking with 5% skim milk powder, dilutions of the His-tagged proteins to be tested (0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 nM) were prepared in PBST (PBS containing 0.1% Tween 20, pH = 7.4). The proteins tested were the original antibody 3005 and four humanized antibodies. The protein dilutions were added to the ELISA plate and incubated at 37°C for 1 hour. After washing, HRP-conjugated mouse anti-His tag monoclonal antibody (purchased from Proteintech, Cat. No. HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (purchased from Tiangen Biochemical, catalog number: PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader.

[0251] The ELISA test results of antibody 3005 and its humanized antibody binding to MSA are shown in Figure 4. The EC50 of the binding ELISA experiments is summarized in Table 13.

[0252] Table 13 Summary of EC50 and maximum binding values ​​of antibody 3005 and its humanized antibodies binding to MSA in ELISA experiments

[0253] The results showed that the humanized antibody 3005Hz6 had similar binding activity to the original antibody 3005 in terms of MSA.

[0254] The ELISA method was used to detect the binding activity of the original candidate antibody 3005 and the humanized 3005Hz6 to HSA and rat albumin.

[0255] In the experiment, HSA or rat albumin (coating protein concentration of 2 μg / mL) was coated in an ELISA plate at 100 μL / well overnight at 4°C. After blocking with 5% skim milk powder, dilutions of the His-tagged protein to be tested (0.0001, 0.001, 0.01, 0.1, 0.3, 1, 10, and 100 nM) were prepared in PBST (PBS containing 0.1% Tween 20, pH = 7.4). The tested proteins included the positive control antibody Alb Nb, the original antibody 3005, and the humanized antibody 3005Hz6. The protein dilutions were added to the ELISA plate and incubated at 37°C for 1 hour. After washing, HRP-conjugated mouse anti-His tag monoclonal antibody (purchased from Proteintech, Cat. No. HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (purchased from Tiangen Biochemical, catalog number: PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader.

[0256] The ELISA results are shown in Figure 5 (A) and Figure 5 (B). The EC50 values ​​of the binding ELISA curve fitting of Figure 5 are shown in Table 14.

[0257] Table 14 Summary of EC50 of candidate antibody 3005 and its humanized antibody 3005Hz6 binding to HSA and rat albumin

[0258] As can be seen from Figure 5 and Table 14, the binding activity of humanized antibody 3005Hz6 to HSA and rat albumin is comparable to that of original antibody 3005, and both are stronger than the positive control antibody Alb Nb.

[0259] The surface plasmon resonance (SPR) method was used to detect the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of humanized antibody 3005Hz6 binding to HSA and MSA. D ).

[0260] The experiments were performed using a Biacore 8K instrument (Cytiva) and a Series S Sensor Chip NTA to capture the His-tagged humanized antibody 3005Hz6. Analytes were HSA or MSA at gradient concentrations, diluted in a mobile phase buffer (10 mM HEPES, 150 mM NaCl, 0.05% v / v Tween-20, pH 7.4). The association time was 150 seconds, and the dissociation time was 900 seconds. The binding and dissociation curves of the antibody 3005Hz6 with HSA are shown in Figure 6 (A), and the binding and dissociation curves of the antibody 3005Hz6 with MSA are shown in Figure 6 (B).

[0261] The “Two state reaction” model was used to fit the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of 3005Hz6 binding to HSA and MSA. D ), the results are shown in Table 15.

[0262] Table 15 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of humanized antibody 3005Hz6 with HSA and MSA D )

[0263] As shown in Table 15, the humanized antibody 3005Hz6 binds to HSA and MSA with similar affinities, and the KD determined by the SPR method is approximately 11 nM.

[0264] In combination with Table 10 of Example 3, the KD value of the original antibody 3005 for HSA and MSA determined by the BLI method was approximately 5 nM, which is within 3 times of the KD value in Table 15. Therefore, it is believed that the affinity of the humanized antibody 3005Hz6 for binding to HSA and MSA is similar to that of the original antibody 3005.

[0265] Example 5: Potential chemical modification site modification and activity testing of humanized antibody 3005Hz6

[0266] 1. Potential chemical modification site modification scheme and expression and purification of humanized antibody 3005Hz6

[0267] The 3005Hz6 antibody contains the amino acid motif DG in its CDR-H2 region (SEQ ID NO: 5). Compared to other amino acid motifs (such as DS and DA), DG is more likely to promote the isomerization of aspartic acid D to isoaspartic acid, potentially altering the antibody's structure and antigen-binding activity. Aspartic acid isomerization may also accelerate proteolysis and enhance protein immunogenicity.

[0268] Therefore, in this example, point mutations were performed on the amino acid combination DG in the CDR-H2 region of 3005Hz6 to reduce the risk of structural and activity changes in the protein. Specifically, the point mutation scheme for the amino acid combination DG in 3005Hz6 involved replacing aspartic acid D with glutamic acid E, or mutating glycine G to either A, which is structurally simple, or S, which is more flexible. The numbers of the modified antibodies and the amino acid sequences of their CDR regions are shown in Table 16. The full-length amino acid and nucleotide sequences of mutants of the humanized antibody 3005Hz6, including potential chemical modification sites, are shown in Table 17.

[0269] Table 16 CDR region amino acid sequences of mutants of potential chemical modification sites of humanized antibody 3005Hz6

[0270] Table 17 Full-length amino acid sequence and nucleotide sequence of mutants of potential chemical modification sites of humanized antibody 3005Hz6

[0271] A secretory signal peptide (SEQ ID NO: 37) was added to the N-terminus of the mutant protein of antibody 3005Hz6, and linkers GGGGS (SEQ ID NO: 41) and 6xHis were added to the C-terminus of the antibody. The coding DNA sequence was obtained by gene synthesis and then ligated into the expression vector pCDNA3.1(+) via the restriction enzyme cleavage sites NheI and XhoI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin. Transient expression was performed in suspension 293F cells, and the cell culture supernatant was affinity purified using a Ni column. During the purification process, the supernatant (Input) of the culture medium of cells transiently expressing 3005Hz6 (EG) and 3005Hz6 (DS), the flow-through (FT) that did not bind to the Ni column, and the eluted purified product (E) were analyzed by SDS-PAGE (Figure 7 (A). The supernatant of cells transiently expressing 3005Hz6 (DA) was purified by Ni column and then by cation exchange. The SDS-PAGE of the purified protein is shown in Figure 7 (B). The molecular weight of the three mutant proteins was 13 kDa.

[0272] II. Activity testing of potential chemically modified variants of humanized antibody 3005Hz6

[0273] ELISA assays were performed to assess HSA binding activity of the humanized 3005Hz6 antibody mutants, 3005Hz6(DA), 3005Hz6(DS), and 3005Hz6(EG). In the ELISA assay, 2 μg / mL HSA was coated and a concentration gradient of 3005Hz6 or its mutants was added. HSA binding was measured using an HRP-labeled mouse anti-His tag monoclonal antibody (purchased from Proteintech, Cat. No. HRP-66005). The ELISA results are shown in Figure 8 , and the EC50 values ​​are shown in Table 18 .

[0274] Table 18 Summary of EC50 of humanized antibody 3005Hz6 mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG) binding to HSA

[0275] As can be seen from Table 18, the humanized antibody 3005Hz6 and its mutant proteins have similar HSA binding activities.

[0276] Example 6: Fusion expression and activity detection of humanized anti-albumin antibodies and other antigen-binding proteins

[0277] 1. Fusion Expression and Purification of Humanized Antibody 3005Hz6 and Monovalent Anti-VEGFA Nanobody

[0278] After the humanized antibody 3005Hz6 is fused with antibodies that bind to other antigens, the fusion protein binds to albumin in vivo, increasing its molecular weight. Alternatively, after binding to albumin, it participates in the binding and recycling mechanism of albumin and FcRn, potentially extending the half-life of the fusion protein in vivo.

[0279] Two anti-VEGFA nanobodies: V1-SA1 (SEQ ID NO: 26) and V1-DP (SEQ ID NO: 27) (Table 19) were selected for fusion expression with the humanized antibody 3005Hz6, and the activities of the fusion proteins obtained in different tandem manners were compared.

[0280] Table 19 Anti-VEGFA Nanobody Sequences

[0281] When the humanized antibody 3005Hz6 was concatenated with the monovalent anti-VEGFA nanobody V1-SA1 (SEQ ID NO: 26) and V1-DP (SEQ ID NO: 27), the linker GGS was used. The amino acid sequence and nucleotide sequence of the fusion protein are shown in Table 20.

[0282] Table 20 Amino acid sequence and nucleotide sequence of the fusion protein of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0283] A secretion signal peptide was added to the N-terminus of the fusion protein, and linkers GGGGS and 6xHis were added to the C-terminus of the antibody. The coding DNA sequence was obtained by gene synthesis (see Table 20) and then ligated into the expression vector pCDNA3.1(+) via the restriction sites NheI and XhoI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin. Transient expression was performed in suspended 293F cells, and Ni column affinity purification was performed from the cell culture supernatant. During the purification process, the SDS-PAGE results of the cell culture supernatant (Input) after transient transfection of each protein, the flow-through (FT) that did not bind to the Ni column, and the eluted purified product are shown in Figure 9. The molecular weight of the target protein is approximately 28 kDa.

[0284] II. Activity detection of fusion protein of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0285] 1. Detect whether the two nanoantibody components that constitute the fusion protein maintain their original functions.

[0286] The HSA binding activity of the anti-albumin nanobody 3005Hz6 in the fusion protein was detected using the same method as in "2. Activity detection of humanized antibodies" in Example 4. The ELISA results are shown in Figure 10, and the EC50 values ​​of the ELISA experiments are shown in Table 21.

[0287] Table 21 Summary of EC50 of fusion protein or 3005Hz6 binding to HSA

[0288] As shown in Table 21, the HSA binding activity of the fusion protein is similar to that of 3005Hz6 alone, indicating that the fusion of 3005Hz6 with other antibody fragments does not affect the activity of 3005Hz6. Furthermore, the bioactive effector molecule can be located at the N- or C-terminus of the anti-albumin antibody; different positions do not substantially affect the albumin binding activity of the fusion protein.

[0289] 2. Detection of the activity of anti-VEGFA nanobody in fusion protein

[0290] The VEGFR2 competitive ELISA method was used to detect the competitive activity of the fusion protein VEGFR2 when the fusion protein was bound to or not bound to HSA.

[0291] In the competition ELISA assay, the microplate was coated with goat anti-human IgG Fc protein (purchased from Solarbio, Cat. No. SPA105) at a concentration of 5 μg / mL overnight at 4°C. After washing with PBST and blocking with milk powder, a VEGFR2 extracellular domain-human IgG1 Fc fusion protein (VEGFR2-Fc, prepared in-house) was added. A concentration gradient of the fusion protein was incubated with a constant concentration of 1 μM HSA or PBST for 30 minutes, followed by incubation with a constant final concentration (0.4 nM) of biotinylated VEGFA165-Avi-His for 1 hour. VEGFA165-Avi-His was prepared in-house by adding a linker, an Avi tag, and a 6xHis tag to the C-terminus of human VEGFA165. The VEGFA165-Avi-His expression plasmid was co-transfected with a BirA enzyme expression plasmid into 293-F cells. During protein expression, the Avi tag was biotinylated by BirA enzyme. The amino acid sequence of VEGFA165-Avi-His is:

[0292] The amino acid sequence of VEGFR2-Fc is:

[0293] The mixture of fusion protein and VEGFA165-Avi-His was added to an ELISA plate adsorbed with VEGFR2-Fc and incubated at 37°C for 1 hour. The biotin signal of VEGFA165-Avi-His was detected using HRP-labeled streptavidin-HRP (purchased from Sangon Biotechnology, catalog number: D111054-0001). The plate was incubated at room temperature for 45 minutes. After washing, 100 μL / well of TMB colorimetric solution was added to the plate and color was developed for 15 minutes. 50 μL / well of stop solution was added, and the plate was placed in a microplate reader to read the absorbance at 450 nm (OD450).

[0294] The competition activity of the fusion protein and the monovalent nanobody V1-SA1 for VEGFR2 without pre-incubation with HSA is shown in Figure 11 (A), and the competition activity for VEGFR2 after pre-incubation with HSA is shown in Figure 11 (B). The IC50 values ​​of the VEGFR2 competition experiments are summarized in Table 22.

[0295] Table 22 Summary of IC50 of fusion protein and monovalent nanobody V1-SA1 competing for VEGFR2

[0296] As can be seen from Table 22, the VEGFR2 competitive activity of the three fusion proteins composed of 3005Hz6 and V1-SA1 or V1-DP in series is similar to that of the monovalent nanoantibody V1-SA1 when HSA is not incubated (IC50 ratio is 0.81-1.08), indicating that the fusion of anti-VEGFA antibody with 3005Hz6 does not affect the anti-VEGFA activity. Moreover, the bioactive effector molecule can be at the N-terminus or C-terminus of the anti-albumin antibody, and different positions basically do not affect the activity of the bioactive effector molecule in the fusion protein.

[0297] To simulate the anti-VEGFA activity of antibodies or fusion proteins in the presence of albumin in vivo, HSA was pre-incubated with the fusion protein or monovalent Nanobody V1-SA1 in a competitive ELISA experimental system to detect the activity of competing with VEGFR2 for binding to VEGFA. Table 22 shows that under the condition of pre-incubation with HSA, the IC50 value of the monovalent Nanobody V1-SA1 was approximately twice that when the experimental system did not contain HSA, indicating that HSA would have a systemic effect on the IC50 value of the competition ELISA experiment. The ratio of the IC50 value of the fusion protein to the IC50 of V1-SA1 was 0.88-1.40, indicating that the VEGFR2 competitive activity of the fusion protein was similar to that of the monovalent Nanobody V1-SA1.

[0298] The data in Tables 21 and 22 show that the fusion protein of the monovalent anti-VEGFA Nanobody (V1-SA1 or V1-DP) and the anti-albumin Nanobody (3005Hz6) has strong competitive activity for VEGFR2 and binding to HSA. This indicates that in the fusion protein, the antibody components that bind to different antigens can function independently, and there is little mutual interference between the antibody components. Therefore, antibodies that bind to other antigens can be fused with the humanized anti-albumin antibody 3005Hz6 to construct multispecific and multifunctional fusion proteins.

[0299] Example 7: Chemical Conjugation of Anti-Albumin Nanobody 3005 Mutant with Small Molecule Toxin Derutocan and Activity Determination of the Conjugate

[0300] 1. Construction, expression and purification of anti-albumin nanobody 3005 mutant suitable for coupling reaction

[0301] For the anti-albumin nanobody 3005, the 65th amino acid in its amino acid sequence is a highly reactive lysine residue K, which is located in the CDR-H2 region. Covalent coupling of K65 in CDR-H2 with a small molecule may significantly reduce the affinity of the conjugate for the antigen albumin. Therefore, in this example, K65 in the nanobody 3005 sequence was subjected to point mutation modification, mutating the lysine residue with a primary amino group to an unreactive amino acid, such as alanine A, arginine R, or histidine H. The newly constructed mutants were named 3005K65A, 3005K65R, 3005K65H, etc.

[0302] Table 23 CDR region amino acid sequences of antibody 3005 mutants

[0303] Table 24 Amino acid sequence and nucleotide sequence of antibody 3005 mutants

[0304] The amino acid sequence of the CDR region of the anti-albumin nanobody 3005 mutant is shown in Table 23, and the amino acid sequence and nucleotide sequence of the mutant are shown in Table 24. The expression and purification methods of the mutant are consistent with "1. Expression of anti-albumin candidate antibodies" in Example 3 of this patent. In short, a secretion signal peptide (SEQ ID NO: 37) was added to the N-terminus of the mutant protein of antibody 3005, and a linker GGGGS (SEQ ID NO: 41) and 6xHis were added to the C-terminus of the antibody. The coding DNA sequence was obtained by gene synthesis and then connected to the expression vector pCDNA3.1 (+) through the enzyme cutting sites NheI and XhoI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin. Transient expression was performed using suspended 293F cells. Ni column affinity purification was performed from the cell culture supernatant. After purification, the Q-tof detection of the three mutant proteins showed that the molecular weight of the mutant was 13.8 kDa.

[0305] Bio-layer interferometry (BLI) was used to test the affinity of antibody 3005 and its mutants 3005K65A, 3005K65R, and 3005K65H for albumins from different species. The antibody was bound to a his-tag at a concentration of 5 μg / mL using an NTA sensor. The antigens used were human serum albumin (HSA) (Baxter AG), mouse albumin (MSA) (Equitech-bio, Catalog No. MSA62-1000), and rat albumin (abcam, Catalog No. ab198656). BLI experiments were performed at eight antigen concentrations: 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM. The binding time was 180 seconds, and the dissociation time was 360 seconds.

[0306] The BLI detection results are shown in Table 25, which show that the mutant proteins 3005K65A, 3005K65R, and 3005K65H bind to albumins from different species with affinities similar to that of antibody 3005.

[0307] Table 25 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of antibody 3005 and its mutant proteins 3005K65A, 3005K65R, 3005K65H binding to albumin from different species D )

[0308] 2. Preparation Method of 3005K65A and Delutec Conjugate (Nanobody Drug Conjugate, NDC)

[0309] In this example, mutant 3005K65A is used as an example to illustrate the preparation, purification and activity determination of the conjugate. 3005K65A can be prepared into a nanobody intermediate (3005K65A-SH) having a thiol group by using methods known to those skilled in the art, for example, by reacting the primary amino group of a lysine residue with 2-iminothiolane hydrochloride (Traut's Reagent) to produce a thiol intermediate; by reacting the primary amino group of a lysine residue with N-succinimidyl S-acetylthioalkanoate (N-Succinimidyl The covalent coupling of S-Acetylthioglycolate (SATA) type linkers releases hydroxylamine to produce highly active thiol intermediates; after the lysine residue is reacted with N-succinimidyl 3-(pyridyldithio) propionate (N-Succinimidyl3-(2-pyridyldithio) propionate, SPDP) is used to reduce the disulfide bonds with reducing agents such as dithiothreitol (DTT), 2-mercaptoethanol, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to obtain thiol intermediates; or using a certain concentration of reducing agents such as dithiothreitol (DTT), 2-mercaptoethanol, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to reduce the disulfide bonds in the nanobody molecule to produce thiol groups; etc., but are not limited to these methods.

[0310] The thiolated active nanobody intermediate is covalently coupled to drutican via a thioether bond to obtain a nanobody drutican conjugate. In this example, the primary amino group and 2-iminothiolane hydrochloride are used as an example to describe the reaction of the primary amino group of the lysine residue in the 3005K65A framework with the amidine bond of 2-iminothiolane hydrochloride at a pH of 7-10 to generate a free thiol group. The free thiol group on the thiolated nanobody intermediate 3005K65A-SH framework reacts with the maleimide group of drutican via a Michael addition reaction to generate a structurally stable 3005K65A-drutican conjugate (NDC).

[0311] The conjugation and purification flow chart of the nanobody-derutex conjugate is shown in Figure 12.

[0312] 3. Purification and Structural Characterization of 3005K65A Derutec Conjugate

[0313] The preparation process for the 3005K65A-drutecan conjugate is as follows: a solution of drutecan is added dropwise to a buffer containing the thiol-modified nanobody intermediate 3005K65A-SH, and the reaction is allowed to proceed with continuous shaking. The reaction buffer for 3005K65A-SH and drutecan can be sodium phosphate, sodium acetate, or sodium borate. The pH during the reaction is generally between 5 and 9, preferably around pH 7. The organic cosolvent for dissolving drutecan can be selected from organic solvents such as N,N-dimethylacetamide (DMAC), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and N-methyl-2-pyridone (NMP). In this example, a DMAC solution containing drutecan was added to a buffer containing the thiol-modified 3005K65A-SH at 8-15% v / v and the reaction was carried out. The reaction temperature was 10-25°C, and the reaction time was 1 hour. After the reaction is completed, 1 to 2 molar equivalents of a thiol-containing reagent, cysteine ​​or N-acetyl-L-cysteine ​​(NAC), is added to the reaction system and incubated at room temperature for 20 minutes to inactivate the unreacted delutec and terminate the reaction.

[0314] The conjugate purification strategy is as follows: purification is performed using a commercially available HiTrap Desalting column (purchased from GE, catalog number 17140801). The desalting column is equilibrated with 5-10 column volumes of phosphate buffer (PBS, pH 7.4). The NDC reaction aqueous solution (approximately 1.5 mL) is loaded onto the HiTrap Desalting column and eluted using a fast protein purifier (AKTA Purifier 100, GE) according to the desalting column instructions. Unreacted drutecan, N-acetyl-L-cysteine ​​(NAC), and small organic cosolvents are removed based on molecular weight to obtain a 3005K65A-drutecan conjugate solution. The purified 3005K65A-drutecan conjugate is analyzed by SDS-PAGE and other methods to confirm successful conjugation. The content of free small molecules, such as drutecan, in the purified 3005K65A-drutecan conjugate is then determined by RP-HPLC. The free small molecule content of the purified conjugate should not exceed 1% before subsequent cell activity assays can be performed.

[0315] The structure of the 3005K65A-derutexan conjugate (NDC) described in this example is shown in Figure 13 (A). Figure 13 (B) shows SDS-PAGE gel images of three different 3005K65A-derutexan conjugates (NDC-1, NDC-2, and NDC-3). The conjugate labeled NDC-3 was used as an example for subsequent ELISA experiments binding to human serum albumin and evaluation of its proliferation inhibitory activity against pancreatic cancer cells Mia Paca-2.

[0316] IV. Affinity and in vitro activity determination of 3005K65A-Drutecan conjugate

[0317] The 3005 mutant protein 3005K65A and the 3005K65A-drutecan conjugate (NDC-3) were tested for human serum albumin binding activity using an ELISA. In the ELISA, 2 μg / mL human serum albumin was coated, and a concentration gradient of the 3005 mutant protein 3005K65A and the 3005K65A-drutecan conjugate (NDC-3) was added. Albumin binding was detected using an HRP-labeled mouse anti-His tag monoclonal antibody (purchased from Proteintech, Cat. No. HRP-66005). The ELISA results for HSA binding of the 3005 mutant protein 3005K65A and the 3005K65A-drutecan conjugate (NDC-3) are shown in Figure 14 (A). As shown in Figure 14 (A), after coupling with the small molecule drutecan, the 3005K65A-drutecan conjugate (NDC-3) still has a high binding activity to human serum albumin. The EC50 of the ELISA experiment for binding to HSA is summarized in Table 26.

[0318] Table 26 Summary of EC50 values ​​of 3005 mutant protein 3005K65A and 3005K65A-derutec conjugate (NDC-3) binding to HSA

[0319] The cell proliferation inhibition effect of the conjugate was evaluated using pancreatic cancer cells Mia Paca-2. The experimental process is briefly described as follows: Mia Paca-2 cells were prepared with DMEM medium (Gibco) containing 10% fetal bovine serum to 2×10 4 Cells were seeded into 384-well cell culture microplates at a concentration of 25 μL per well. Furthermore, 25 μL of different concentrations of druteccan or 3005K65A-druteccan conjugate (NDC-3) diluted with DMEM medium was added to each well, so that the final concentrations of druteccan or 3005K65A-druteccan conjugate (NDC-3) were: 0.96 nM, 9.6 nM, 96 nM, 960 nM and 19340 nM. After adding the test substance, the cells were cultured at 37°C and 5% CO2 for 5 days. After culture, the microplate was removed from the incubator, 25 μL was removed and an equal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added. The mixture was shaken on a mixer at room temperature for 10 minutes, and the luminescence of each sample in the microplate was measured with a microplate reader. The survival rate of tumor cells after 5 consecutive days of culture was calculated using the formula:

[0320] Cell survival rate (%) = luminescence of the drug group ÷ luminescence of the control group × 100%

[0321] After Mia Paca-2 cells were treated with the test substances at concentrations of 96 nM, 960 nM, and 19340 nM, the viability of Mia Paca-2 cells is shown in FIG14(B) and Table 27.

[0322] Table 27 Cell viability of Mia Paca-2 cells treated with delutec and 3005K65A-delutec conjugate (NDC-3)

[0323] As can be seen from Figure 14(B) and Table 27, at the same concentration, the activity of drutecan in inhibiting tumor cell proliferation is significantly lower than that of the 3005K65A-drutecan conjugate (NDC-3). Therefore, anti-albumin antibody 3005 can be used as a carrier for conjugation with active molecules such as small molecule toxins. The biological activity of the conjugate, such as inhibition of cell proliferation, is comparable to or even superior to that of the active molecule itself. The conjugate can potentially improve the cytotoxicity or target cell selectivity of the active molecule, thereby enhancing the activity of the small molecule in inhibiting cancer cell growth.

[0324] Example 8: Binding activity of humanized anti-albumin antibodies to albumin under different pH conditions

[0325] Albumin can bind to the cell surface receptor FcRn under weakly acidic conditions, but the binding force is weaker under neutral conditions. Therefore, after binding to FcRn, albumin is internalized into endosomes. As the endosomal environment becomes acidic, the binding force of albumin to FcRn strengthens, and it is brought back to the cell surface by FcRn or transported across cells. Therefore, albumin can achieve a long half-life by leveraging the recycling mechanism of FcRn. Albumin-binding antibodies and their fusion proteins also undergo a similar intracellular transport process after binding to albumin. Therefore, the ability of albumin-binding antibodies to bind to albumin under neutral and acidic conditions is critical for achieving long circulation and extended half-life.

[0326] The SPR method was used to detect the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of humanized antibody 3005Hz6 to HSA under near neutral conditions (pH = 7.4) and acidic conditions (pH = 5.5, simulating the pH of the endosomal microenvironment). D ).

[0327] The experiment was performed using a Biacore 8K instrument (Cytiva) with a Series S Sensor Chip CM5 amino-coupled immobilized antigen, HSA. A concentration gradient of 3005Hz6 was used as the analyte. The binding and dissociation of HSA and 3005Hz6 were measured under neutral conditions using a mobile phase buffer composed of 10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, and 0.05% v / v Tween-20. The analyte was diluted using this mobile phase. The binding and dissociation of HSA and 3005Hz6 were measured under acidic conditions using a mobile phase buffer composed of 10mM sodium acetate, pH 5.5, 150mM NaCl, 3mM EDTA, and 0.05% v / v Tween 20. The association time was 150 seconds, and the dissociation time was 300 seconds.

[0328] The “1:1 binding” model was used to fit the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of 3005Hz6 binding to HSA at pH = 7.4 and pH = 5.5. D ), the results are shown in Table 28.

[0329] Table 28 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of humanized antibody 3005Hz6 with HSA at pH = 7.4 and pH = 5.5 D )

[0330] As shown in Table 28, the equilibrium dissociation constant K of 3005Hz6 and HSA was obtained when the amino-coupled HSA was coupled to the SPR method at pH 7.4. D About 2.84nM; at pH = 5.5, the equilibrium dissociation constant K of 3005Hz6 and HSA D The affinity of the humanized antibody 3005Hz6 for HSA is approximately 0.33 nM, indicating that the humanized antibody 3005Hz6 strongly binds to HSA under both neutral and acidic conditions, and the affinity of 3005Hz6 for HSA under acidic conditions is 8.6 times stronger than under neutral conditions. Therefore, it can be inferred that the humanized antibody 3005Hz6 strongly binds to HSA at physiological pH. When the complex of 3005Hz6 and HSA enters the acidic environment of the endosome, the affinity of 3005Hz6 for HSA is enhanced, thereby enabling intracellular circulation or transcellular transport through the binding of HSA to FcRn.

[0331] Example 9: Fusion Expression of 3005Hz6 and Aflibercept and Activity Detection

[0332] The humanized anti-albumin antibody 3005Hz6 was fused with the marketed drug Aflibercept (a fusion protein of the extracellular domain 2 of VEGFR1 and the extracellular domain 3 of VEGFR2 with IgG1 Fc). 3005Hz6 was linked to the N-terminus of the Aflibercept protein with a linker of GGSGGS (SEQ ID NO: 60). The fusion protein was named 3005Hz6-Aflibercept. The amino acid sequence of the fusion protein 3005Hz6-Aflibercept is as follows:

[0333] The secretion signal peptide IgK (METDTLLLWVLLLWVPGSTG, SEQ ID NO: 42) was added to the N-terminus of the fusion protein 3005Hz6-Aflibercept. Through gene synthesis and molecular cloning, the protein-coding DNA sequence was ligated into an expression vector via the HindIII and NotI restriction sites. The plasmid was sequenced correctly and subjected to endotoxin-free bulk extraction. Transient expression was achieved in suspension 293F cells, and Protein A affinity purification was performed from the cell culture supernatant to obtain the fusion protein 3005Hz6-Aflibercept.

[0334] The nucleotide sequence of the fusion protein 3005Hz6-Aflibercept is:

[0335] The HSA binding activity of the anti-albumin nanobody 3005Hz6 in the fusion protein 3005Hz6-Aflibercept was assessed using an ELISA. Human serum albumin (HSA) (Baxter AG) was coated at a final concentration of 2 μg / mL, with 100 μL / well applied overnight at 4°C. After blocking with 5% skim milk powder, serial dilutions of the fusion protein were added and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human IgG Fc antibody (Abbkine, Catalog No. A21050) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (Tiangen Biochemical, Catalog No. PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop buffer was added. The absorbance at 450 nm (OD450) was measured using a microplate reader. The EC50 values ​​for the fusion protein ELISA binding assay are shown in Table 29.

[0336] The anti-VEGF protein aflibercept activity in the fusion protein 3005Hz6-Aflibercept was detected using a VEGFR2 competition ELISA, with the marketed drug aflibercept as the control protein. The competition ELISA assay was the same as described in "2. Detection of Anti-VEGFA Nanobody Activity in Fusion Protein" in Example 6. The IC50 values ​​for the fusion protein competition ELISA assay are shown in Table 29.

[0337] Table 29 Summary of activity assay results for protein 3005Hz6-Aflibercept

[0338] As shown in Table 29, the fusion protein 3005Hz6-Aflibercept has strong HSA binding activity, and its VEGFR2 competitive activity is similar to that of the marketed drug Aflibercept. These results indicate that the anti-albumin nanobody 3005Hz6 and the anti-VEGF protein Aflibercept in the fusion protein 3005Hz6-Aflibercept do not affect each other's activity and can function independently.

[0339] Example 10: Affinity enhancement of humanized antibody 3005Hz6

[0340] Random mutations were performed on single amino acid sites and two adjacent amino acid sites in the three CDR regions of the humanized antibody 3005Hz6. Phage display technology was used to display the mutated sequences on the phage surface. By gradually reducing the amount of antigen input and conducting species cross-screening during the screening process, candidate anti-albumin nanobody sequences were obtained that could simultaneously bind to HSA, cynomolgus monkey serum albumin, MSA, and rat albumin with higher affinities than 3005Hz6. Finally, candidate antibodies 1004, 1012, 1036, 1068, and 2028 were obtained. The amino acid and nucleotide sequences of the candidate antibodies are shown in Table 30, and the amino acid sequences of their CDR regions are shown in Table 31.

[0341] Table 30 Amino acid sequences and nucleotide sequences of candidate antibodies with improved affinity

[0342] Table 31 Amino acid sequences of affinity-enhanced candidate antibodies CDR-H1, CDR-H2, and CDR-H3

[0343] Example 11: ELISA Binding Activity Test of Affinity-Enhanced Candidate Antibodies to Albumin

[0344] A tPA secretory peptide was added to the N-terminus of the affinity-enhanced candidate antibodies (antibodies 1004, 1012, 1036, 1068, and 2028 shown in Table 30), and a 6xHis tag was added to the C-terminus. Their expression and purification were the same as the experimental steps of "1. Expression of anti-albumin candidate antibodies" in Example 3.

[0345] The results of the purified affinity-enhanced candidate antibody are shown in FIG15 . The molecular weight of the candidate antibody is approximately 14.7 kDa.

[0346] The ELISA method was used to detect the binding activity of candidate antibodies with improved affinity to albumins from different species.

[0347] In the ELISA experiment, the candidate antibody (concentration of 5 μg / mL) was coated at 4°C overnight. The biotin-modified antigen HSA (with Avi and 6xHis tags, amino acid sequence:

[0348] , SEQ ID NO: 56) was added to an ELISA plate and incubated at 37°C for 1 hour. The plate was then washed with PBST solutions of varying pH (pH = 7.4 or 6.0). HRP-labeled streptavidin-HRP antibody (purchased from Sangon Biotechnology, Catalog No. D111054-0001) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (purchased from Tiangen Biochemical, Catalog No. PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader.

[0349] The ELISA results of the candidate antibodies with improved affinity binding to HSA at pH = 7.4 or pH = 6.0 are shown in Figures 16(A) and 16(B), respectively. It can be seen that at pH = 7.4, except for 1004, the binding affinities of the candidate antibodies to HSA were all higher than 3005Hz6, among which the affinity of antibody 1068 was the most significantly improved. At pH = 6.0, the binding affinities of all five candidate antibodies to HSA were higher than 3005Hz6.

[0350] Antigen-coated ELISA was used to test the binding activity of candidate antibodies with enhanced affinity to rat albumin. Untagged rat albumin was coated at a concentration of 5 μg / mL overnight at 4°C. Candidate antibodies (3005Hz6, 1004Hz6, 1012Hz, 1036Hz, 1068Hz, and 2028Hz) were diluted in PBST solutions of varying pH (7.4 or 6.0) and added to an ELISA plate. The plate was incubated at 37°C for 1 hour. The plate was then washed with PBST solutions of varying pH (7.4 or 6.0). Anti-His tag monoclonal antibody (Proteintech, Cat. No. HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (Tiangen Biochemical, Cat. No. PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop buffer was added. OD450 values ​​were measured using a microplate reader. The control group BI-V2 was selected from SEQ ID NO: 15 in patent US2023 / 0050615 A1, and the amino acid sequence was: EVQLVESGGGLVQAGGSLRLSCAASGLTFSSYAMGWFRQAPGKERERVVSISRGGGYTYYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAAARYWATGSEYEFDYWGQGTLVTVSS (SEQ ID NO: 57).

[0351] ELISA results for binding of the affinity-enhanced candidate antibodies to rat albumin at pH 7.4 or pH 6.0 are shown in Figures 17(A) and 17(B), respectively. As can be seen, at both pH 7.4 and pH 6.0, the affinity of antibody 2028 for rat albumin was higher than 3005Hz6, while the control protein BI-V2 barely bound to rat albumin.

[0352] Example 12: Affinity test of candidate antibodies with improved affinity to HSA (SPR)

[0353] Two SPR methods were used to determine the affinity of the affinity-enhanced candidate antibodies to HSA.

[0354] In the first SPR method, the instrument Biacore T200 (Cytiva) was used to amino-couple the anti-His-tagged antibody on the surface of the CM5 chip to capture the candidate antibody with the His tag. The candidate antibody concentration was 20nM, and the buffer was 10mM HEPES pH=7.4, 150mM NaCl, 0.05% v / v Tween-20. The flow rate was 30μL / min and the capture time was 30s. The chip with the captured antibody was then bound and dissociated with HSA at different concentrations. The highest concentration of HSA was 37.5nM or 18.75nM, and the concentration was diluted 2 times. The binding time to the chip was 180s and the dissociation time was 300s. The binding and dissociation curves were fitted to obtain the equilibrium dissociation constant K D For values, see Table 32.

[0355] Table 32 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of candidate antibodies 1004, 1012, 1036, 1068, 2028 and 3005Hz6 with improved affinity to HSA D )

[0356] As can be seen from Table 32, the affinity-enhanced candidate antibodies 1004, 1012, 1036, 1068, and 2028 all have higher affinities with HSA than 3005Hz6, among which antibody 1068 has the highest affinity with HSA.

[0357] In the second SPR method, a Biacore 8K instrument (Cytiva) was used with a Series S Sensor Chip CM5 amino-coupled immobilized antigen (response value approximately 1250 RU). Analytes included a concentration gradient of 3005Hz6, 1068, 2028, and the control protein BI-V2 in a buffer of 10mM HEPES pH=7.4, 150mM NaCl, 3mM EDTA, and 0.05% v / v Tween-20. The SPR experiments were performed with an association time of 120 seconds and a dissociation time of 1000 seconds (for antibody 2028, the dissociation time was 300 seconds). The association and dissociation curves for HSA binding of antibodies 3005Hz6, 1068, 2028, and the control protein BI-V2 are shown in Figures 18(A), 18(B), 18(C), and 18(D), respectively.

[0358] The “1:1 binding” model was used to fit the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of the antibody binding to HSA. D ), the results are shown in Table 33.

[0359] Table 33 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of antibodies 3005Hz6, 1068, 2028 and control protein BI-V2 with HSA D )

[0360] The results showed that the affinity of antibody 1068 to HSA was very high, about 56.6 pM, which was significantly higher than 3005Hz6 (about 35.7 times higher), and higher than the control antibody BI-V2 (about 2.8 times higher). The affinity of antibody 2028 to HSA was higher than 3005Hz6 (about 3.7 times higher).

[0361] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0362] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An anti-albumin antibody or an antigen-binding fragment thereof, characterized in that, The anti-albumin antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; wherein, the amino acid sequence of CDR-H1 comprises SEQ ID NO: 1 or 58, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1 or 58; the amino acid sequence of CDR-H2 comprises any one of SEQ ID NO: 3-4, 6, 40 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NO: 3-4, 6, 40; the amino acid sequence of CDR-H3 comprises any one of SEQ ID NO: 7-8, 59 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NO: 7-8, 59.

2. The anti-albumin antibody or antigen-binding fragment thereof according to claim 1, wherein, X1 in the said SEQ ID NO: 58 (X1YYMS) represents N or E, which are SEQ ID NO: 2 or 49 respectively; X2X3 in the said SEQ ID NO: 40 (GISVX2X3X4X5LDYADAVX6G) represents DS, DA, EG, DM or DG, X4X5 represents SF or WY, and X6 represents K, A, R or H; preferably, said X2X3 represents DG, X4X5 represents SF, and X6 represents K (SEQ ID NO: 5); said X2X3 represents DA, X4X5 represents SF, and X6 represents K (SEQ ID NO: 20); said X2X3 represents DS, X4X5 represents SF, and X6 represents K (SEQ ID NO: 21); said X2X3 represents EG, X4X5 represents SF, and X6 represents K (SEQ ID NO: 22); said X2X3 represents DG, X4X5 represents SF, and X6 represents A (SEQ ID NO: 31); said X2X3 represents DG, X4X5 represents SF, and X6 represents R (SEQ ID NO: 32); said X2X3 represents DG, X4X5 represents SF, and X6 represents H (SEQ ID NO: 33); said X2X3 represents DG, X4X5 represents WY, and X6 represents K (SEQ ID NO: 50); or, said X2X3 represents DM, X4X5 represents SF, and X6 represents K (SEQ ID NO: 51); or, The SEQ ID NO: 59 (ASGPX7X8LRX9X 10 AP) where X7X8 represents QG, IW, LW or VG, and X9X 10 represents LG or WW. Preferably, The X7X8 represents QG, and X9X 10 represents LG (SEQ ID NO: 9); The X7X8 represents IW, X9X 10 represents LG (SEQ ID NO: 52); The X7X8 represents LW, and X9X 10 represents LG (SEQ ID NO: 53); wherein X7X8 represents QG, and X9X 10 represents WW (SEQ ID NO: 54); alternatively, The X7X8 represents VG, and X9X 10 represents LG (SEQ ID NO: 55).

3. The anti-albumin antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, the amino acid sequences of said CDR-H1, CDR-H2 and CDR-H3 comprise any one of the following groups: A) SEQ ID NO: 1, 3, 7; B) SEQ ID NO: 1, 4, 8; C) SEQ ID NO: 2, 5, 9; D) SEQ ID NO: 2, 6, 9; E) SEQ ID NO: 2, 20, 9; F) SEQ ID NO: 2, 21, 9; G) SEQ ID NO: 2, 22, 9; H) SEQ ID NO: 2, 31, 9; I) SEQ ID NO: 2, 32, 9; J) SEQ ID NO: 2, 33, 9; K) SEQ ID NO: 2, 5, 52; L) SEQ ID NO: 2, 50, 53; M) SEQ ID NO: 49, 5, 53; N) SEQ ID NO: 49, 5, 54; O) SEQ ID NO: 2, 51, 55.

4. The anti-albumin antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The anti-albumin antibody or its antigen-binding fragment includes a humanized sequence, and the modification site of the humanized sequence is located in the non-CDR region. Preferably, the modification site of the humanized sequence is located in the framework region and / or constant region of the antibody.

5. The anti-albumin antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The anti-albumin antibody or its antigen-binding fragment includes a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, an Fd fragment, an Fv fragment, a dAb fragment, an F(ab')2 fragment, a single-chain antibody or a linear antibody.

6. The anti-albumin antibody or antigen-binding fragment thereof according to any one of claims 1-5, characterized in that, The amino acid sequence of the anti-albumin antibody or its antigen-binding fragment contains any one of the amino acid sequences of SEQ ID NO: 10-13, 16-19, 23-25, 34-36, 44-48, or has at least 80% identity with any one of the amino acid sequences of SEQ ID NO: 10-13, 16-19, 23-25, 34-36, 44-48.

7. Use of the anti-albumin antibody or antigen-binding fragment thereof according to any one of claims 1-6, characterized in that, The applications include: A. Use in the preparation of a fusion construct, the fusion construct includes the anti-albumin antibody or its antigen-binding fragment according to any one of claims 1-6 and a bioactive effector molecule, and the bioactive effector molecule is linked to the anti-albumin antibody or its antigen-binding fragment; B. Use in screening a bioactive effector molecule, linking a candidate bioactive effector molecule to the anti-albumin antibody or its antigen-binding fragment, and detecting the biological activity of the candidate bioactive effector molecule; or, C. Use in detecting albumin.

8. A fusion construct, characterized in that, The fusion construct contains one or more anti-albumin antibodies or their antigen-binding fragments according to any one of claims 1-6 and / or bioactive effector molecules, and the bioactive effector molecule is linked to the anti-albumin antibody or its antigen-binding fragment. Preferably, the fusion construct includes multiple anti-albumin antibodies or their antigen-binding fragments, and the multiple can be the same or different anti-albumin antibodies or their antigen-binding fragments; the bioactive effector molecule can be one or more, and the multiple can be the same or different bioactive effector molecules.

9. The fusion construct according to claim 8, wherein The bioactive effector molecule includes but is not limited to small molecule compounds or macromolecule compounds. Preferably, the small molecule compounds include but are not limited to any small molecule drugs, and the macromolecule compounds include but are not limited to antibodies, activators or inhibitors of receptors, ligands of proteins, bioactive enzymes, nucleic acid drugs, or combinations thereof. More preferably, the antibodies in the macromolecule compounds include antibodies or their antigen-binding fragments against other targets, and the other targets are targets different from albumin. More preferably, the structure of the antibody or its antigen-binding fragment against other targets is one or a combination of two or more of nanobody, chimeric antibody, Fab fragment, Fab' fragment, Fd fragment, Fv fragment, dAb fragment, F(ab')2 fragment, single-chain antibody or linear antibody.

10. The fusion construct according to claim 9, characterized in that, The other targets are selected from VEGFA, VEGFB, VEGFR, FGF, FGFR, PlGF, PDGF, TGF, Integrin, Integrin receptor, interleukins (such as IL-1β, IL-2, IL-3, IL-4, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptors (such as IL1R1, IL2Rα, IL3R, IL4Rα, IL10R, IL12R, IL15Rα, IL17R, IL23R, etc.), PCSK9, TNF-α, TNFR, RANKL, GPCR, GLP1R, CD3, CD105, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD47, CD80, CD86, CD96, CD99, CD111, CD112, CD123, CD133, CD138, CD155, CD171, Claudin 18.2, OX40, ICOS, CTLA4, 4-1BB, TCR, BTLA, TIM-3, LAG3, Galectin-9, PD-L1, PD-L2, PD-1, TIGIT, EGFR, Her2, PSCA, CEA, FAP, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, CS1, ROR1, recombinant mycobacterium tuberculosis fusion protein, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL 3 or 5T4.

11. The fusion construct according to claim 10, wherein The anti-albumin antibody or its antigen-binding fragment is directly or indirectly linked to the bioactive effector molecule. Preferably, the indirect linkage can be through a linker, a functional domain and / or a linker for conjugation; Among them, the linker is selected from linking peptide, oligopeptide, oligopeptide polymer, polypeptide, polypeptide polymer, PEG, nucleic acid, polysaccharide, aliphatic chain, biotin, streptavidin or avidin; The functional domain is one or a combination of two or more of Fc fragment, serum albumin, cytokine, transferrin, scaffold protein; The linker for conjugation includes a functional group linker. Preferably, the functional group linker includes thiol, amino, hydroxyl and / or carboxyl reactive groups, More preferably, the bioactive effector molecule is directly or indirectly linked to the N-terminus, C-terminus and / or internal residues of the anti-albumin antibody or its antigen-binding fragment.

12. A nucleic acid, characterized in that, The nucleic acid encodes the anti-albumin antibody or antigen-binding fragment thereof as claimed in any one of claims 1-6, or encodes the fusion construct as claimed in any one of claims 8-11.

13. The nucleic acid according to claim 12, wherein The nucleic acid further comprises any nucleotide sequence of SEQ ID NOs: 63-75, 79-81, 83-87 or a degenerate sequence thereof, or a nucleotide sequence having at least 80% identity with any nucleotide sequence of SEQ ID NOs: 63-75, 79-81, 83-87 and having the function of encoding an anti-albumin antibody or antigen-binding fragment thereof.

14. The nucleic acid according to any one of claims 12 - 13, characterized in that, The nucleic acid comprises any nucleotide sequence of SEQ ID NOs: 76-78, 82 or a degenerate sequence thereof, or a nucleotide sequence having at least 80% identity with any nucleotide sequence of SEQ ID NOs: 76-78, 82 and having the function of encoding the fusion construct.

15. A carrier, characterized in that, The vector comprises the nucleic acid as claimed in any one of claims 12-14.

16. A host cell, characterized in that, The host cell comprises the nucleic acid as claimed in any one of claims 12-14 or the vector as claimed in claim 15.

17. A method for preparing an anti-albumin antibody or an antigen-binding fragment thereof according to any one of claims 1-6, or a fusion construct according to any one of claims 8-11, characterized in that, The preparation method comprises culturing the host cell as claimed in claim 16 and expressing the anti-albumin antibody or antigen-binding fragment thereof or the fusion construct.

18. Use of an anti-albumin antibody or an antigen-binding fragment thereof according to any one of claims 1-6, a fusion construct according to any one of claims 8-11, a nucleic acid according to any one of claims 12-14, a vector according to claim 15 or a host cell according to claim 16 in the preparation of a product for treating and / or diagnosing a disease, characterized in that, The anti-albumin antibody or antigen-binding fragment thereof and / or the bioactive effector molecule has the activity of treating and / or diagnosing diseases. Preferably, the anti-albumin antibody or antigen-binding fragment thereof serves as a carrier in a product for treating and / or diagnosing diseases.

19. A product for treating and / or diagnosing a disease, characterized in that, The product for treating and / or diagnosing diseases comprises any one of the following: A) The anti-albumin antibody or antigen-binding fragment thereof as claimed in any one of claims 1-6; B) The fusion construct as claimed in any one of claims 8-11; C) The nucleic acid as claimed in any one of claims 12-14; D) The vector as claimed in claim 15; or, F) The host cell as claimed in claim 16, The anti-albumin antibody or antigen-binding fragment thereof and / or the bioactive effector molecule has the activity of treating and / or diagnosing diseases, Preferably, the anti-albumin antibody or antigen-binding fragment thereof serves as a carrier in a product for treating and / or diagnosing diseases, More preferably, the bioactive effector molecule is a drug, and the drug is covalently bound to the anti-albumin antibody or antigen-binding fragment thereof.

20. A method for detecting albumin, characterized in that, The detection method comprises contacting a sample to be detected with the anti-albumin antibody or antigen-binding fragment thereof as claimed in any one of claims 1-6, and then detecting the content of the complex formed by albumin and the anti-albumin antibody or antigen-binding fragment thereof. Preferably, the albumin is from a mammal, and more preferably, the mammal is from a human, a mouse or a monkey.

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