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

Abstract

The invention provides an anti-albumin antibody or an antigen-binding fragment thereof and application thereof, and an affinity-enhanced anti-albumin nano antibody or an antigen-binding fragment thereof, the anti-albumin antibody or the antigen-binding fragment thereof can bind to albumin of different species with high affinity, and meanwhile, the affinity-enhanced anti-albumin nano antibody or the antigen-binding fragment thereof can bind to albumin of different species with high affinity. 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

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 (concentration is about 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] The molecular weight of albumin is 66.5kDa, and it can bind to the cell surface receptor FcRn (neonatal Fc receptor) under weakly acidic conditions, but the binding force is weaker 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 to the FcRn of their corresponding species is also different. Therefore, for albumin-bound small molecule drugs or albumin fusion proteins, the pharmacokinetic evaluation and efficacy evaluation conducted in small animals may not necessarily reflect the effect in humans. Therefore, the sequence differences of albumin from different species have brought great 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 affinity, small molecule compounds are coupled to anti-albumin antibodies, or antibodies that bind to specific antigens are fused with anti-albumin antibodies for expression. The pharmacokinetic and efficacy tests of the drug molecules in small animals (such as mice and rats) can provide reliable reference value for the pharmacokinetics and efficacy performance of future drugs in humans.

[0006] Single-domain antibodies or nanobodies are the variable region domains of heavy-chain antibodies that naturally lack light chains in camelids. They have a simple structure and are the smallest antibody unit with complete antigen-binding activity. Nanobodies have a molecular weight of about 13 kDa and are easy to modify, have high thermal stability and good water solubility.

[0007] In order to predict the future effect of active molecules in the human body, it is usually necessary to verify its behavior in animals in relevant animal models, such as analyzing the efficacy, pharmacokinetics, drug distribution and drug metabolism of 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 of multiple species, such as humans, mice and rats, so as 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] In order to overcome the defects of the prior art, the present application provides an anti-albumin antibody or an antigen-binding fragment thereof and its application, as well as an anti-albumin antibody or an antigen-binding fragment thereof with improved affinity, which can bind to albumins of different species with high affinity, and at the same time, the anti-albumin antibody can be connected with a biologically active effector molecule to form a fusion construct without affecting the activity of the biologically active 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 having at least 80% identity to the amino acid sequence shown in 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 having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 7-8, 59.

[0013] Wherein, the SEQ ID NO: 58 (X 1 YYMS), SEQ ID NO: 40 (GISVX 2 X 3 X 4 X 5 LDYADAVX 6 G), SEQ ID NO: 59 (ASGP X 7 X 8 LRX 9 X 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, the SEQ ID NO: 58 (X 1 X in YYMS 1 Represents N or E;

[0015] The SEQ ID NO: 40 (GISVX 2 X 3 X 4 X 5 LDYADAVX 6 X in G) 2 X 3 Represents DS, DA, EG, DM or DG, X 4 X 5 Represents SF or WY, X 6 represents K, A, R or H; preferably,

[0016] The X 2 X 3 Represents DG, X 4 X 5 Represents SF, X 6 represents K (SEQ ID NO: 5);

[0017] The X 2 X 3 Represents DA, X 4 X 5 Represents SF, X 6represents K (SEQ ID NO: 20);

[0018] The X 2 X 3 Represents DS, X 4 X 5 Represents SF, X 6 represents K (SEQ ID NO: 21);

[0019] The X 2 X 3 Represents EG, X 4 X 5 Represents SF, X 6 represents K (SEQ ID NO: 22);

[0020] The X 2 X 3 Represents DG, X 4 X 5 Represents SF, X 6 represents A (SEQ ID NO: 31);

[0021] The X 2 X 3 Represents DG, X 4 X 5 Represents SF, X 6 represents R (SEQ ID NO: 32);

[0022] The X 2 X 3 Represents DG, X 4 X 5 Represents SF, X 6 represents H (SEQ ID NO: 33);

[0023] The X 2 X 3 Represents DG, X 4 X 5 Represents WY, X 6 represents K (SEQ ID NO: 50); or,

[0024] The X 2 X 3 Represents DM, X 4 X 5 Represents SF, X 6 represents K (SEQ ID NO: 51).

[0025] The SEQ ID NO: 59 (ASGPX 7 X 8 LRX 9 X10 X in AP 7 X 8 Represents QG, IW, LW or VG, X 9 X 10 represents LG or WW. Preferably,

[0026] The X 7 X 8 Represents QG, X 9 X 10 represents LG (SEQ ID NO: 9);

[0027] The X 7 X 8 Represents IW, X 9 X 10 represents LG (SEQ ID NO: 52);

[0028] The X 7 X 8 On behalf of LW, X 9 X 10 represents LG (SEQ ID NO: 53);

[0029] The X 7 X 8 Represents QG, X 9 X 10 represents WW (SEQ ID NO: 54); or,

[0030] The X 7 X 8 Represents VG, X 9 X 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 include any 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]

[0049] Preferably, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are arranged in order from N-terminus to C-terminus. The division of the amino acids in 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.

[0050] 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.

[0051] Preferably, the structure of the anti-albumin antibody or its antigen-binding fragment includes nanobody, chimeric antibody, Fab fragment, Fab' fragment, Fd fragment, Fv fragment, dAb fragment, F(ab')2 fragment, single chain antibody (single chain antibody fragment, scFv) or linear antibody.

[0052] The anti-albumin antibody or antigen-binding fragment thereof may be a single domain antibody or a nanobody.

[0053] 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 antibody, Fab, and scFv, at the same mass, the nanobody has a higher molar concentration and can bind to more antigen molecules.

[0054] The anti-albumin antibody or antigen-binding fragment thereof can bind to mammalian albumin.

[0055] Preferably, the mammals include humans or non-human mammals, and the non-human mammals may be wild animals, zoo animals, economic 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.

[0056] Preferably, the amino acid sequence of the anti-albumin antibody or its antigen-binding fragment 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.

[0057] 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, 44-48.

[0058] The anti-albumin antibody or antigen-binding fragment thereof can be constructed by any conventional method in the prior art, such as artificial synthesis or eukaryotic expression or prokaryotic expression.

[0059] The second aspect of the present invention provides an application of the above-mentioned anti-albumin antibody or antigen-binding fragment thereof, the application comprising:

[0060] A use of A in preparing a fusion construct, wherein the fusion construct comprises the 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;

[0061] B. Use in screening bioactive effector molecules, connecting candidate bioactive effector molecules with the anti-albumin antibody or antigen-binding fragment thereof to detect the biological activity of the candidate bioactive effector molecules; or

[0062] Application of C in the detection of albumin.

[0063] 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.

[0064] Preferably, the fusion construct comprises a plurality of anti-albumin antibodies or antigen-binding fragments thereof, and the plurality of anti-albumin antibodies or antigen-binding fragments thereof may be the same or different.

[0065] Preferably, the biologically active effector molecule may be one or more, and the multiple biologically active effector molecules may be the same or different.

[0066] Preferably, the bioactive effector molecules include but are not limited to small molecule compounds or macromolecular compounds.

[0067] 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 disease drugs, anti-Parkinson's disease drugs, anti-inflammatory drugs, anti-allergic drugs, antihypertensive drugs, anti-thromboembolic drugs, anti-epileptic drugs, antidepressant drugs, stroke drugs or autism drugs, etc.

[0068] 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 derutech.

[0069] In a specific embodiment, the small molecule drug is deludecant.

[0070] Preferably, the macromolecular compound includes but is not limited to antibodies, ligands for activating or inhibiting receptors and proteins, biologically active enzymes, nucleic acid drugs, or combinations thereof.

[0071] Preferably, the antibody in the macromolecular compound includes antibodies or antigen-binding fragments thereof to other targets, and the other targets are targets different from albumin.

[0072] 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 (scFv) or a linear antibody.

[0073] 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 (Interleukin Receptor, such as IL1R1, IL2Rα, IL3R, IL4Rα, IL10R, IL12R, IL15Rα, IL17R, IL23R, etc.), Proprotein convertase subtilisin / kexin type 9 (PCSK9), Tumor necrosis factorα (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 (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 155 (CD155), Cluster of Differentiation 171 (CD171), Claudin 18.2 (CD18.2), Cluster of Differentiation 111 (CD111), Cluster of Differentiation 112 (CD112), Cluster of Differentiation 123 (CD123), Cluster of Differentiation 133 (CD133), Cluster of Differentiation 138 (CD138), Cluster of Differentiation 155 (CD155), Cluster of Differentiation 171 (CD171), Claudin 18.2 (CD18.2), Cluster of Differentiation 111 (CD111), Cluster of Differentiation 112 (CD112), Cluster of Differentiation 123 (CD123), Cluster of Differentiation 133 (CD133), Cluster of Differentiation 138 (CD138), Cluster of Differentiation 155 (CD155), Cluster of Differentiation 171 (CD171), Claudin 18.2 (CD18.2), Cluster of Differentiation 111 (CD111), Cluster of Differentiation 112 (CD112), Cluster of Differentiation 123 (CD123), Cluster of Differentiation 133 (CD133), Cluster of Differentiation 138 (CD138), Cluster of Differentiation 155 (CD155), Cluster of Differentiation 171 (CD171), Claudin 18.2 (CD18.2), Cluster of Differentiation 111 (CD111), Cluster of Differentiation 112 (CD112), Cluster of Differentiation 123 (CD123), Cluster of Differentiation 1CLDN18.2), TNF receptor superfamily member 4 (Tumor necrosis factor 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 (Tumor necrosis factor receptor superfamily, member9, TNFRSF9 / 4-1BB / CD137), T cell antigen receptor (T cell receptor, TCR), B / T lymphocyte attenuator (B-and T-Lymphocyte Attenuator, BTLA), T cell immunoglobulin domain and mucin domain-3 (TIM-3), Lymphocyte Activation gene 3 (Lymphocyte Activation Gene3, LAG3), Galectin-9 (GAL9), Programmed cell death 1ligand 1 (PD-L1), Programmed cell death 1ligand 2 (PD-L2), Programmed cell death receptor 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),The antibodies included FAP, epidermal growth factor receptor variant typeⅢ (EGFRvIII), B cell maturation antigen (BCMA), prostate specific membrane antigen (PSMA), carbohydrate antigen 125 (CA125), tyrosine protein kinase receptor A2 (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), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), Mucin 1 (MUC1), Mucin 16 (MUC16), Mesothelin, Cluster of Differentiation 174 (Lewis Y / 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).

[0074] In a specific embodiment, the other target is VEGFA.

[0075] Preferably, the anti-albumin antibody or antigen-binding fragment thereof is directly or indirectly linked to the biologically active effector molecule.

[0076] Preferably, the indirect connection may be through a linker, a functional domain and / or a linker for coupling.

[0077] Wherein, the linker is selected from connecting peptides, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, PEG, nucleic acids, polysaccharides, fatty chains, biotin, streptavidin or avidin.

[0078] The functional domain is one or a combination of two or more of Fc fragment, serum albumin, cytokine, transferrin and scaffold protein.

[0079] The linkers used for coupling include functional group linkers.

[0080] The functional group linker includes thiol, amino, hydroxyl and / or carboxyl reactive groups, which can covalently couple the anti-albumin antibody or its antigen-binding fragment with the biologically active effector molecule.

[0081] 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.

[0082] Preferably, the fusion construct comprises one or more biologically active effector molecules.

[0083] For example, in the fusion construct, the connection order of the antibody or antigen-binding fragment thereof and the biologically active effector molecule from the N-terminus to the C-terminus is as follows:

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

[0085] biologically active effector molecules, connecting peptides, anti-albumin antibodies or antigen-binding fragments thereof;

[0086] anti-albumin antibody or antigen-binding fragment thereof, a connecting peptide, a first biologically active effector molecule, a connecting peptide, a second biologically active effector molecule; or,

[0087] 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, and the like; wherein the connection may not include a connecting peptide.

[0088] In one embodiment, the biologically active effector molecule comprises any one of the amino acid sequences in SEQ ID NOs: 26-27, or has at least 80% identity with any one of the amino acid sequences in SEQ ID NOs: 26-27.

[0089] 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.

[0090] In one embodiment, the fusion construct comprises an Fc fragment.

[0091] 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.

[0092] 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 with any one of the amino acid sequences in SEQ ID NOs: 14-15.

[0093] Preferably, the fusion construct further comprises a secretory peptide. The secretory peptide is connected 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.

[0094] Preferably, the fusion construct further comprises a tag.

[0095] Preferably, the tag is connected to the C-terminus of any antibody or antigen-binding fragment thereof, or a fusion construct.

[0096] 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.

[0097] The fourth aspect of the present invention provides a nucleic acid encoding the above-mentioned anti-albumin antibody or antigen-binding fragment thereof or the above-mentioned fusion construct. For example, the nucleic acid comprises DNA and / or mRNA.

[0098] In some embodiments, the nucleic acid is DNA, which encodes the antibody or antigen-binding fragment described herein, or the fusion construct described above.

[0099] Preferably, the nucleotide sequence encoding the anti-albumin antibody or its antigen-binding fragment comprises any one of SEQ ID NOs: 63-75, 79-81, 83-87 or its degenerate sequence, 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 function of the anti-albumin antibody or its antigen-binding fragment.

[0100] Preferably, the nucleotide sequence encoding the above-mentioned fusion construct comprises any one of nucleotide sequences in SEQ ID NO: 76-78, 82 or a degenerate sequence thereof, or has at least 80% identity with any one of nucleotide sequences in SEQ ID NO: 76-78, 82, and has a nucleotide sequence encoding the function of the above-mentioned fusion construct.

[0101] The fifth aspect of the present invention provides a vector, wherein the vector comprises the above-mentioned nucleic acid.

[0102] 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 series vector, a bacteriophage, etc.

[0103] The sixth aspect of the present invention provides a host cell, wherein the host cell comprises the above-mentioned nucleic acid or the above-mentioned vector.

[0104] The host cell may be a eukaryotic cell or a prokaryotic cell.

[0105] Eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells, 293F cells or CHO cells, etc.

[0106] Prokaryotic cells such as Escherichia coli.

[0107] 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.

[0108] In an eighth aspect of the present invention, a method for preparing the above-mentioned anti-albumin antibody or its antigen-binding fragment or the above-mentioned fusion construct is provided, the preparation method comprising culturing the above-mentioned host cell to express the anti-albumin antibody or its antigen-binding fragment or the above-mentioned fusion construct.

[0109] In a ninth aspect of the present invention, there is provided 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:

[0110] A) the above-mentioned anti-albumin antibody or antigen-binding fragment thereof;

[0111] B) the above fusion construct;

[0112] C) the above nucleic acid;

[0113] D) the above-mentioned vector; or,

[0114] F) The host cell described above.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] The detection method is to detect the presence or content of albumin. The presence refers to the presence or absence, and the content can be the expression level or protein concentration, etc. Preferably, the albumin is from a mammal, and more preferably, the mammal is from a human, a mouse or a monkey.

[0120] In the eleventh aspect of the present invention, a method for treating and / or preventing a disease is provided, the method comprising administering the above-mentioned anti-albumin antibody or its antigen-binding fragment, 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.

[0121] 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.

[0122] In a specific 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 (eg, fundus vascular disease), and the like.

[0123] 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.

[0124] The twelfth aspect of the present invention provides a use of the above-mentioned anti-albumin antibody or its antigen-binding fragment, 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 its antigen-binding fragment and / or the above-mentioned biologically active effector molecule has activity in treating and / or diagnosing a disease.

[0125] 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.

[0126] 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.

[0127] In a specific 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 (eg, fundus vascular disease), and the like.

[0128] As used herein, "fusion construct" defines the fusion of an anti-albumin antibody or antigen-binding fragment thereof of the present invention with another compound. The fusion construct may contain 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 contain one or more additional compounds, and the multiple additional compounds may also be the same or different. The compound may be a protein compound or a non-protein compound. In the case where the compound is a protein compound or the fusion construct only contains multiple anti-albumin antibodies or antigen-binding fragments thereof, the fusion construct may also be referred to as a fusion protein. In the case where the compound is fused to the anti-albumin antibody or antigen-binding fragment thereof in a coupled form, the fusion construct may also be referred to as a conjugate.

[0129] The product described in the present application may be a kit, a drug, a chip, an antibody-drug conjugate, etc.

[0130] The "drug" of the present invention can be used to treat humans or non-human animals, such as non-human mammals. The drug can contain pharmaceutically acceptable carriers, excipients or salts common in the prior art. The drug can be administered by any suitable route, such as gastrointestinal administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, rectal, etc.). The drug can be in any suitable dosage form, such as a gastrointestinal dosage form or a parenteral dosage form, 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, drop pills, gels, etc. The various dosage forms of the drug can be prepared according to conventional production methods in the pharmaceutical field. The drug may contain 0.01-99.5% (specifically, 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 its antigen-binding fragment, 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.

[0131] The term "pharmaceutically acceptable" as used herein refers to a substance that neither significantly irritates an organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0132] The "method" described in the present invention can be used for the purpose of diagnosis and treatment of diseases, or for the purpose of diagnosis and treatment of non-diseases.

[0133] 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 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 more than two CDRs from the heavy chain or light chain of an antibody. Among them,

[0134] Nanobodies or single-domain antibodies refer to the variable domain of heavy chain (VHH) of an antibody, which has independent antigen-binding activity.

[0135] 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.

[0136] Single-chain antibody is an antibody composed of the heavy chain variable region and the light chain variable region connected by a linker.

[0137] Fab, a monovalent fragment consisting of the VL, VH, CL and CH1 domains.

[0138] Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain.

[0139] F(ab')2 is a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region.

[0140] Fd, Fd fragment consisting of VH and CH1 domains.

[0141] Fv, Fv fragment consists of the VL and VH domains of a single antibody arm.

[0142] The dAb fragment is an antibody fragment consisting of the VH domain.

[0143] The "linear antibody" described in the present invention includes 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.

[0144] 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.

[0145] The "Fc" region of the present invention contains two heavy chain fragments including the CH2 and CH3 domains of an antibody. The two heavy chain fragments form dimers by two or more disulfide bonds in the hinge region and are held together by the hydrophobic interaction of the CH3 domain.

[0146] The words “comprising” or “including” described in the present invention are open-ended expressions. 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.

[0147] The "homology" or "identity" mentioned in the present invention refers to that in terms of 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%, %, 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.

[0148] The "humanized antibody" of the present invention refers to the framework region and / or constant region portion (such as CH region) of the antibody or all or part of the antibody encoded by human antibody genes. In a specific embodiment of the present invention, the CDR region of the antibody has not been humanized.

[0149] 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, an economic 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 (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0150] 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-related signs, symptoms, conditions, or disorders.

[0151] The term "prevention" as used herein refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in an organism.

[0152] The term "diagnosis" as used herein refers to finding out whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to finding out the progression of a disease or its possible future progression.

[0153] The "tumor" of the present invention can be any undesirable cell proliferation (or any disease that manifests itself as undesirable cell proliferation), neoplasm, or increased tendency or risk of undesirable cell proliferation, neoplasm or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). 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), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblastic cells, 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, 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 carcinoma, 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.

[0154] The "retinal vascular disease" mentioned in the present invention refers to the general term for diseases occurring in the retinal arteries or veins, including but not limited to age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, neovascular glaucoma, etc.

[0155] The present invention obtains an antibody or an antigen-binding fragment thereof that binds to albumin with high affinity. The anti-albumin nanobody has the following advantages:

[0156] 1. The obtained anti-albumin nanobody can bind to albumins of different species with high affinity, including human, monkey, mouse, rat and other species, and has completed humanization transformation. The humanized anti-albumin antibody binds to human albumin and mouse albumin, and can bind to rat albumin. Mice and rats are commonly used experimental animals, which makes the anti-albumin nanobody of the present invention have a wider application prospect.

[0157] 2. By carrying out affinity enhancement modification on the preferred humanized anti-albumin antibodies, antibodies with affinity to human albumin increased by 3.7 times or 35.7 times were obtained.

[0158] 3. Albumin has the characteristic of being enriched in inflammation and tumor sites. Humanized anti-albumin nanoantibodies can be used to prepare drugs for the treatment of 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

[0159] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0160] Figure 1 : Gel staining of His-tagged antibodies Alb Nb, 2049, 3004, 3007 and 3005 after purification by Ni column, (A) is the gel staining of antibodies Alb Nb, 2049, 3004, 3007, (B) is the gel staining of antibody 3005.

[0161] Figure 2 : ELISA test of fusion proteins 3005-Fc, 3007-Fc and Alb Nb-Fc binding to albumins of different species, (A) is HSA binding test (HSA binding ELISA), (B) is MSA binding test (MSA binding ELISA), and (C) is rat albumin binding test (Rat albumin binding ELISA).

[0162] Figure 3 : The staining results of 3005 humanized antibody containing His tag after Ni column purification.

[0163] Figure 4 : ELISA activity test of antibody 3005 and its humanized antibody binding to MSA.

[0164] Figure 5 : ELISA activity test of antibody 3005 and 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).

[0165] 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.

[0166] 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).

[0167] Figure 8 : ELISA activity detection (HSA binding ELISA) of humanized antibody 3005Hz6 and its mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG) binding to HSA.

[0168] Fig. 9 : SDS-PAGE gel images of 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.

[0169] Fig.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.

[0170] Fig.11 : VEGFR2 competitive ELISA activity test of fusion protein and monovalent nanobody V1-SA1 without preincubation or with preincubation of HSA, (A) is the result without preincubation of HSA, (B) is the result with preincubation of HSA.

[0171] Fig.12 : Flow chart of the conjugation and purification of nanobody-derutecan conjugate (NDC).

[0172] Fig.13: (A) is a schematic diagram of the structure of 3005K65A-derutecan conjugate (NDC), (B) is an SDS-PAGE gel image of 3005K65A and three different 3005K65A-derutecan conjugates (NDC-1, NDC-2, NDC-3).

[0173] Fig.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 drutecan and 3005K65A-drutecan conjugate (NDC-3) on MiaPaca-2 cells.

[0174] Fig.15 : The results of staining after purification of candidate antibodies with improved affinity.

[0175] Fig.16 : ELISA experimental results of candidate antibodies with improved affinity binding to HSA (HSA binding ELISA) (coating antibody method, coat antibody), (A) is the ELISA experimental result at pH = 7.4, (B) is the ELISA experimental result at pH = 6.0.

[0176] Fig.17 : ELISA results of candidate antibodies with improved affinity 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.

[0177] Fig.18 : Binding and dissociation curves of affinity-enhanced antibodies and control antibodies with HSA (SPR method), (A) is the binding and dissociation curves of antibody 3005Hz6 with HSA, (B) is the binding and dissociation curves of antibody 1068 with HSA, (C) is the binding and dissociation curves of antibody 2028 with HSA, (D) is the binding and dissociation curves of control antibody BI-V2 with HSA, the ordinate is the response value, and the abscissa is the time. DETAILED DESCRIPTION

[0178] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0179] Example 1: Alpaca immunization and antibody library construction

[0180] 1. Alpaca immunization and titer testing

[0181] Human serum albumin HSA (purchased from Sigma-Aldrich, catalog number: A3782) was emulsified and used to immunize alpacas. 1 mg of human serum albumin was used for each immunization, and the immunization was performed once every 2 weeks for a total of three times. From the second immunization, serum was collected one week after the immunization to test the titer.

[0182] The serum titer test method is:

[0183] HSA antigen was diluted to 1 μg / mL with CBS buffer, and 100 μL / well was added to the ELISA plate at 4°C overnight;

[0184] Wash the plate 3 times with PBST (PBS containing 0.1% Tween20, pH=7.4). Add 200 μL skim milk powder to each well, leave at room temperature for 1 hour, and wash the plate 3 times with PBST;

[0185] Sample addition: Dilute the immunized serum and negative serum by 1000 times, 3000 times, 9000 times, 27000 times, 81000 times, and 243000 times, respectively, take 100 μL and add it to the sealed wells, place at room temperature for 1 hour, and wash the plate 3 times with PBST;

[0186] Secondary antibody: add 100 μL of HRP-labeled Goat Anti-Alpaca IgG to each well, leave at room temperature for 1 h, and wash the plate three times with PBST;

[0187] TMB color development: Add 100 μL / well of TMB color development solution to the ELISA plate and color development for 15 min; add 50 μL / well of stop solution, place the ELISA plate into the ELISA reader, and read the light absorption value (OD450) at 450 nm.

[0188] The serum titer detection 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 for phage library construction.

[0189] Table 2 Serum titer detection after the second and third albumin alpaca immunization

[0190]

[0191] 2. Phage library construction

[0192] Peripheral blood was drawn from alpacas, and total RNA was extracted from the isolated alpaca PBMC using Trizol, and reverse transcription of cDNA was performed. The variable region VH fragment (about 700 bp in length) was amplified twice using nanoantibody-specific primers. The above-obtained fragments and the pcomb3X vector were digested with SfiI enzyme, and then mixed in appropriate proportions and connected with T4 ligase. After connection, it was used for electroporation of XL1-Blue competent cells. According to the colony growth of the dilution of competent cells on the resistance-containing plate, the transformation library capacity of the alpaca was calculated to be 6.24×10 8 .

[0193] Example 2: Screening of antibody library to obtain antibodies that cross-bind to human, mouse, and cynomolgus monkey albumin

[0194] 1. Phage library screening

[0195] The screening process is:

[0196] The antigens human serum albumin HSA and mouse albumin MSA (purchased from Sigma-Aldrich, catalog number: A3559) were coated, and a negative control (enzyme-labeled plate coated with 3% skim milk powder) was set up at the same time, and incubated at room temperature for 1 hour. The blocking buffer was discarded and washed with PBST.

[0197] Add about 5 × 10 12 pfu library was incubated with antigen at 37°C for 2 hours, and the plate was washed with 0.1% PBST.

[0198] Elution was performed with low pH glycine-HCl, followed by neutralization with Tris-HCl until the pH was 7.4.

[0199] The eluate was mixed with E. coli XL1-Blue and incubated at 37 °C with shaking.

[0200] After adding phage, the cells were cultured overnight at 30°C and the supernatant was collected.

[0201] Repeat 1)-5) to obtain the second round library.

[0202] When the second round amplification library obtained under the condition of coating HSA was used for ELISA detection, the antigen HSA was coated with two coating buffers: PBS buffer or CBS buffer, followed by ELISA binding detection. The phage was detected using HRP-labeled Anti-M13 Antibody (purchased from Sino Biological, Cat. No.: 11973-MM05T-H), which was diluted 2500 times. After incubation with the detection antibody, the plate was washed, TMB color was developed, and OD450 was read. The detection results of the second round amplification library are shown in Table 3. It can be seen that after the second round of enrichment, a phage signal that strongly binds to HSA was generated.

[0203] Table 3 Detection results of the second round of phage library obtained by enrichment under HSA coating conditions and binding to HSA

[0204]

[0205] For the second round amplification library obtained under the condition of coating with MSA, an antigen binding ELISA test was performed. In the ELISA experiment, CBS buffer was used to coat HSA and MSA respectively, and the ELISA test results are shown in Table 4. It can be seen that phages that can strongly bind to HSA and MSA were also obtained under this enrichment condition.

[0206] Table 4 Detection results of the second round of phage library enriched under MSA coating conditions combined with HSA and MSA

[0207]

[0208] 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.

[0209] 2. Phage monoclonal screening and sequencing

[0210] The experimental process is:

[0211] 96 monoclonal colonies were selected from the first round phage library enriched by coating HSA for phage expression and coated with HSA protein for ELISA binding activity detection.

[0212] 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.

[0213] 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.

[0214] Blocking: 3% milk powder, 200 μL / well, room temperature for 1 hour.

[0215] Add 10-fold diluted phage expression supernatant, 100 μL / well, at room temperature for 1 hour.

[0216] Add detection antibody Anti-M13 Antibody (HRP labeled) (concentration: 0.2 μg / mL), 100 μL / well, at room temperature for 1 hour.

[0217] Add 200 μL / well of TMB colorimetric solution, color for 20 minutes, add 50 μL / well of stop solution, and measure OD450 with a microplate reader.

[0218] From the 96 phage clones of the first round phage library enriched by coating HSA, one clone was selected and numbered 2049. From the 96 phage clones of the second round 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.

[0219] Table 5 Detection of phage monoclonal 2049 binding activity to HSA

[0220]

[0221] Table 6 Activity detection of phage monoclonal 3004, 3005, 3007 binding to HSA, MSA and monkey albumin

[0222]

[0223] 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. The amino acid and nucleotide sequences of the four nanobodies are shown in Table 8.

[0224] Table 7 Amino acid sequences of the CDR regions of four anti-albumin nanobodies

[0225]

[0226] Table 8 Amino acid sequences and nucleotide sequences of four anti-albumin nanobodies

[0227]

[0228]

[0229] Example 3: Activity detection of anti-albumin candidate antibodies binding to albumins from different species

[0230] 1. Expression of anti-albumin candidate antibodies

[0231] These four candidate antibodies were expressed in mammalian cells, and the antigen binding activity was tested after protein purification to verify the activity of the candidate antibodies in binding to albumin at the protein level.

[0232] By gene synthesis, expression plasmids of His-tagged antibodies 2049, 3004, 3005, 3007 and positive control antibody Alb Nb were constructed respectively, wherein the positive control Alb Nb antibody is a nanobody against human serum albumin, and the antibody amino acid sequence is from SEQ ID NO: 62 in patent US2007 / 0269422 A1, and the amino acid sequence is:

[0233] EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRD NAKTTLYLQMNSLRPEDTAVYYCTIGGSSLRSSQGTLVTVSS (SEQ ID NO: 62).

[0234] The tPA secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 37) was added to the N-terminus of the above antibody, and the linker GGGGS (SEQ ID NO: 41) and 6xHis were added to the C-terminus of the antibody. The gene sequence was connected to the expression vector pCDNA3.1 (+) through the restriction sites NheI and XbaI. After the plasmid sequencing was correct, it was extracted in large quantities without endotoxin, and the transfection reagent polyethylenimine (PEI) was used to carry out transient expression in suspended 293F cells and Ni column affinity purification.

[0235] SDS-PAGE staining of the cell culture supernatant (Input) of His-tagged antibodies Alb Nb, 2049, 3004, and 3007 transiently expressed, the flow-through (FT) that cannot bind to the Ni column, and the eluted purified product (Eluate) is shown in Figure 1 (A) The product of His-tagged antibody 3005 purified by Ni column, its SDS-PAGE staining result is shown in Figure 1 (B), their molecular weight is consistent with the expected (~15 kDa).

[0236] At the same time, the fusion proteins of antibodies 3005, 3007 and positive control antibody Alb Nb and Fc were constructed, and the tPA secretion signal peptide was added to the N-terminus of the antibody coding sequence through primer design: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 37). The amino acid sequence of human IgG1 Fc was added to the C-terminus of the antibody sequence through homologous recombination:

[0237] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

[0238] VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38), and the gene fragment was inserted into the mammalian cell expression vector pCDNA3.1(+).

[0239] The amino acid and nucleotide sequences of the anti-albumin nanobody-Fc fusion protein are shown in Table 9.

[0240] Table 9 Amino acid sequence and nucleotide sequence of anti-albumin nanobody-Fc fusion protein

[0241]

[0242]

[0243] After the expression plasmid of 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.

[0244] 2. Detection of albumin binding activity of anti-albumin candidate antibodies

[0245] The bio-layer interferometry (BLI) method was used to test the affinity of monovalent nanoantibodies binding to albumins of different species. Eight NTA sensors were used to bind to the same his tag monovalent nanoantibody (concentration of 1 μg / mL), and the antigens were human serum albumin HSA (Baxter AG), mouse albumin MSA (purchased from: Equitech-bio, catalog number: MSA62-1000) and rat albumin Rat albumin (purchased from: abcam, catalog number: ab198656). In the BLI experiment, the eight concentration gradients of the antigen were set to 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM, and the antigen and antibody bound for 180 seconds and dissociated for 360 seconds.

[0246] Table 10 shows the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of monovalent His-tagged nanobodies 2049, 3004, 3005, and 3007 for albumin from different species after fitting the "1:1" model. D ).

[0247] Table 10 The association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of nanoantibodies 2049, 3004, 3005 and 3007 for binding to albumin from different species D )

[0248]

[0249]

[0250] The data showed that 2049, 3004, 3005 and 3007 all bind HSA and MSA with high affinity, and 3005 and 3007 can also bind rat albumin.

[0251] The ELISA method was used to compare the activity of 3005 and 3007 binding to albumin of different species. The experimental method was:

[0252] The antigen was diluted with ELISA coating buffer, the final concentration of human serum albumin HSA (Baxter AG) was 1 μg / mL, the final concentration of mouse albumin MSA (purchased from: Equitech-bio, catalog number: MSA62-1000) was 2 μg / mL, and the final concentration of rat albumin (purchased from: abcam, catalog number: ab198656) was 2 μg / mL. The antigen dilution was added to the ELISA plate, 100 μL / well, and coated overnight at 4°C. After blocking with 5% skim milk powder, the candidate antibodies 3005-Fc and 3007-Fc and the gradient dilution of the positive control antibody Alb Nb-Fc (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 nM) prepared with PBST (PBS containing 0.1% Tween20, pH = 7.4) were added, and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human IgG Fc antibody (purchased from Abbkine, item number: A21050) was added and incubated at room temperature for 45 minutes. After washing, 100 μL TMB (purchased from Tiangen Biochemical, item number: PA107-01) was added to each well, and after color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured with an enzyme marker.

[0253] The ELISA test results of 3005-Fc, 3007-Fc and Alb Nb-Fc binding to albumin from different species are shown in Figure 2The EC50 of 3005-Fc and 3007-Fc binding ELISA experiments are summarized in Table 11.

[0254] Table 11 Summary of EC50 of ELISA experiments of 3005-Fc and 3007-Fc binding to albumin from different species

[0255] protein HSA binding EC50 (nM) Binding to MSA EC50 (nM) Binding to rat albumin EC50 (nM) 3005-Fc 0.24 0.23 0.40 3007-Fc 0.25 0.26 0.68

[0256] Referring to Table 11, the results show that antibodies 3005-Fc and 3007-Fc can both efficiently bind to HSA and MSA. Figure 2 The positive control antibody Alb Nb-Fc had very weak binding activity to rat albumin, but antibodies 3005-Fc and 3007-Fc both had good binding activity to rat serum albumin, and antibody 3005-Fc had better binding activity to rat albumin than 3007-Fc. Therefore, antibody 3005 was preferred for subsequent studies.

[0257] Example 4: Humanization of anti-albumin candidate antibodies

[0258] 1. Humanization transformation, expression and purification of candidate antibodies

[0259] Humanization was performed by transplanting the CDR region of the antibody. The candidate antibody 3005 (SEQ ID NO: 12) was used as the starting antibody to perform humanization of the antibody. The homology between the candidate antibody 3005 sequence and the human antibody sequence was first compared through the antibody databases IGBLAST and IMGT, and then the human antibody framework region sequence with high homology was combined with the three CDR region sequences of the candidate antibody 3005 (SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 9) to construct a humanized antibody.

[0260] A secretion signal peptide was added to the N-terminus of the antibody, 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 linked to the expression vector pCDNA3.1(+) through the restriction sites NheI and XbaI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin, and transiently expressed in suspended 293F cells and purified by Ni column affinity.

[0261] The amino acid and nucleotide sequences of the humanized modified protein of antibody 3005 are shown in Table 12.

[0262] Table 12 Amino acid sequence and nucleotide sequence of 3005 humanized antibody

[0263]

[0264] After the His-tagged humanized protein was purified by Ni column and separated by SDS-PAGE electrophoresis, the molecular weight was consistent with the expected value (~13 kDa). The results of Coomassie staining are shown in Figure 3 .

[0265] 2. Activity detection of humanized antibody

[0266] First, the activity of the original candidate antibody 3005 and the humanized antibody binding to mouse serum albumin (MSA) was detected by ELISA method.

[0267] In the experiment, mouse serum albumin (MSA) (coating protein concentration: 5 μg / mL) was coated on the ELISA plate, 100 μL / well, and incubated overnight at 4°C. After blocking with 5% skim milk powder, diluents of His-tagged proteins to be tested (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 nM) were prepared with PBST (PBS containing 0.1% Tween 20, pH = 7.4). The proteins to be tested were the original antibody 3005 and four humanized antibodies. After adding the protein diluents to the ELISA plate, they were incubated at 37°C for 1 hour. After washing the plate, HRP-labeled mouse anti-His-tag monoclonal antibody (purchased from: Proteintech, product number: HRP-66005) was added and incubated at room temperature for 45 minutes. After washing the plate, 100 μL of TMB (purchased from: Tiangen Biochemical, product number: PA107-01) was added to each well, and after color development at 37°C for 15 minutes, 50 μL of the termination solution was added. The absorbance (OD450) was measured at 450 nm with an ELISA reader.

[0268] The ELISA test results of antibody 3005 and its humanized antibody binding to MSA are shown in Figure 4 . The EC50 summary of the ELISA experiment is shown in Table 13.

[0269] Table 13 Summary of EC50 and maximum binding values of antibody 3005 and its humanized antibody binding to MSA in ELISA experiment

[0270] Antibody Binding to MSA EC50 (nM) Maximum binding value OD450 3005 2.3 1.38 3005Hz3 22.0 0.89 3005Hz4 49.8 0.79 3005Hz5 31.6 0.80 3005Hz6 2.9 1.34

[0271] The results showed that the activity of the humanized antibody 3005Hz6 binding to MSA was similar to that of the original antibody 3005.

[0272] The activity of the original candidate antibody 3005 and the humanized 3005Hz6 binding to human serum albumin (HSA) and rat albumin was detected by ELISA method.

[0273] In the experiment, HSA or rat albumin (coated protein concentration is 2 μg / mL) was coated in the ELISA plate, 100 μL / well, and coated overnight at 4°C. After blocking with 5% skim milk powder, PBST (PBS containing 0.1% Tween20, pH=7.4) was used to prepare the dilution solution (0.0001, 0.001, 0.01, 0.1, 0.3, 1, 10, 100 nM) of the His-tagged protein to be tested. The proteins to be tested were the positive control antibody Alb Nb, as well as the original antibody 3005 and the humanized antibody 3005Hz6. After the protein dilution solution was added to the ELISA plate, it was incubated at 37°C for 1 hour. After washing the plate, HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, catalog number: HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL TMB (purchased from Tiangen Biochemical, catalog number: PA107-01) was added to each well, and 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 an enzyme reader.

[0274] The ELISA results were Figure 5 (A) and Figure 5 (B). Figure 5 The EC50 values ​​of the binding ELISA curve fitting are shown in Table 14.

[0275] Table 14 Summary of EC50 of candidate antibody 3005 and its humanized antibody 3005Hz6 binding to HSA and rat albumin

[0276] Antibody Name EC50 of HSA binding (nM) EC50 for binding to rat albumin (nM) 3005 0.31 2.74 3005Hz6 0.26 1.64 Alb 0.66 Weak binding

[0277] from Figure 5 As can be seen from Table 14, the activity of humanized antibody 3005Hz6 in binding to HSA and rat albumin is comparable to that of original antibody 3005, and both are stronger than positive control antibody Alb Nb.

[0278] The surface plasmon resonance (SPR) method was used to detect the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of the humanized antibody 3005Hz6 binding to HSA and MSA. D ).

[0279] In the experiment, the Biacore 8K instrument (Cytiva) was used to capture the humanized antibody 3005Hz6 with the His tag using the Series S Sensor Chip NTA. The analytes were HSA or MSA at gradient concentrations, and the analytes were diluted with a mobile phase buffer (components: 10mM HEPES, 150mM NaCl, 0.05% v / v Tween-20, pH = 7.4). The binding time was 150 seconds and the dissociation time was 900 seconds. The binding and dissociation curves of the antibody 3005Hz6 and HSA are shown in Figure 2. Figure 6 As shown in (A), the binding and dissociation curves of antibody 3005Hz6 and MSA are shown in Figure 6 As shown in (B).

[0280] The “Two state reaction” model was used to fit the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of 3005Hz6 binding to HSA and MSA. D ), the results are shown in Table 15.

[0281] Table 15 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of humanized antibody 3005Hz6 with HSA and MSA D )

[0282]

[0283] As can be seen from 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.

[0284] 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 was within 3 times of the KD 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.

[0285] Example 5: Potential chemical modification site modification and activity testing of humanized antibody 3005Hz6

[0286] 1. Potential chemical modification site modification scheme and expression and purification of humanized antibody 3005Hz6

[0287] For the 3005Hz6 antibody, its CDR-H2 region (SEQ ID NO: 5) contains the amino acid motif DG. Compared with other amino acid motifs (such as DS, DA), DG is more likely to promote the isomerization of aspartic acid D to isoaspartic acid, which may change the structure and antigen binding activity of the antibody. Aspartic acid isomerization may also accelerate proteolysis and enhance the immunogenicity of the protein.

[0288] Therefore, in this embodiment, the amino acid combination DG in the CDR-H2 region of 3005Hz6 is subjected to point mutation modification to reduce the risk of structural and activity changes in the protein. The specific point mutation scheme of the amino acid combination DG of 3005Hz6 is to replace aspartic acid D with glutamic acid E, or to mutate glycine G to A with a simple structure or S that is easy to form a flexible structure. The numbering of the modified antibody and the amino acid sequence of its CDR region are shown in Table 16, and the full-length amino acid and nucleotide sequences of the mutants of the potential chemical modification sites of the humanized antibody 3005Hz6 are shown in Table 17.

[0289] Table 16 CDR region amino acid sequences of mutants of potential chemical modification sites of humanized antibody 3005Hz6

[0290]

[0291] Table 17 Full-length amino acid sequence and nucleotide sequence of mutants of potential chemical modification sites of humanized antibody 3005Hz6

[0292]

[0293]

[0294] A secretion signal peptide (SEQ ID NO: 37) was added to the N-terminus of the mutant protein of antibody 3005Hz6, 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 linked to the expression vector pCDNA3.1 (+) through the restriction sites NheI and XhoI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin. It was transiently expressed using suspended 293F cells and Ni column affinity purification was performed from the cell culture supernatant. During the purification process, the supernatant (Input) of the cell culture medium that transiently expressed 3005Hz6 (EG) and 3005Hz6 (DS), the flow-through (FT) that could not bind to the Ni column, and the eluted purified product (E), their SDS-PAGE results are shown in Figure 7 (A). The supernatant of cells transiently expressing 3005Hz6 (DA) protein was purified by Ni column and then purified by cation exchange. The SDS-PAGE results of the purified protein are shown in Figure 7 (B) The molecular weight of the three mutant proteins is 13 kDa.

[0295] II. Activity test of potential chemical modification sites of humanized antibody 3005Hz6

[0296] ELISA was performed to test the HSA binding activity of the humanized antibody 3005Hz6 mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG). In the ELISA experiment, 2μg / mL HSA was coated, and 3005Hz6 or its mutants were added in a concentration gradient. The antibody level binding to HSA was detected by HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, catalog number: HRP-66005). The results of the ELISA experiment are shown in Figure 8 The EC50 of ELISA experiment is shown in Table 18.

[0297] Table 18 Summary of EC50 of humanized antibody 3005Hz6 mutant proteins 3005Hz6 (DA), 3005Hz6 (DS) and 3005Hz6 (EG) binding to HSA

[0298] Antibody Name EC50 of HSA binding (nM) 3005Hz6 0.35 3005Hz6(DA) 0.44 3005Hz6(DS) 0.28 3005Hz6(EG) 0.40

[0299] As can be seen from Table 18, the humanized antibody 3005Hz6 and its mutant proteins have similar HSA binding activities.

[0300] Example 6: Fusion expression and activity detection of humanized anti-albumin antibodies and other antigen-binding proteins

[0301] 1. Fusion expression and purification of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0302] 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, or after binding to albumin, it participates in the binding and recycling mechanism of albumin and FcRn, which can potentially prolong the half-life of the fusion protein in vivo.

[0303] 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 by different tandem methods were compared.

[0304] Table 19 Anti-VEGFA Nanobody Sequences

[0305]

[0306] When the humanized antibody 3005Hz6 was connected in series with the monovalent anti-VEGFA nanoantibodies 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.

[0307] Table 20 Amino acid sequence and nucleotide sequence of the fusion protein of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0308]

[0309]

[0310] 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 linked to the expression vector pCDNA3.1(+) through the restriction sites NheI and XhoI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin. It was transiently expressed using 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 could not bind to the Ni column, and the eluted purified product were shown in Fig. 9 , the molecular weight of the target protein is approximately 28kDa.

[0311] II. Activity detection of fusion protein of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0312] 1. Detect whether the two nanoantibody elements that constitute the fusion protein maintain their original functions.

[0313] The activity of anti-albumin nanobody 3005Hz6 in the fusion protein in binding to HSA was detected. The detection method was the same as "2. Activity detection of humanized antibodies" in Example 4. The ELISA results are as follows: Fig.10 The EC50 of the ELISA experiment is shown in Table 21.

[0314] Table 21 Summary of EC50 of fusion protein or 3005Hz6 binding to HSA

[0315] Protein name EC50 of HSA binding (nM) V1SA-3005Hz6 0.32 3005Hz6-V1SA 0.33 V1DP-3005Hz6 0.39 3005Hz6 0.25

[0316] As can be seen from Table 21, the activity of the fusion protein in binding to HSA is similar to that of 3005Hz6 alone, indicating that the fusion of 3005Hz6 with other antibody fragments does not affect the activity of 3005Hz6. Moreover, the bioactive effector molecule can be at the N-terminus or C-terminus of the anti-albumin antibody, and different positions do not substantially affect the activity of the fusion protein in binding to albumin.

[0317] 2. Detection of the activity of anti-VEGFA nanoantibodies in fusion proteins

[0318] The VEGFR2 competitive ELISA method was used to detect and compare the effects of the fusion protein on the competitive activity of VEGFR2 when it was bound or not bound to HSA.

[0319] In the competition ELISA experiment, the ELISA plate was coated with goat anti-human IgG Fc protein (purchased from Solarbio, catalog number: SPA105) at a coating concentration of 5 μg / mL at 4°C overnight. After washing the plate with PBST and blocking with milk powder, VEGFR2 extracellular region-human IgG1 Fc fusion protein (VEGFR2-Fc, prepared in-house) was added. The fusion protein with a concentration gradient was incubated with a constant 1 μM HSA or PBST for 30 minutes, and then incubated with a constant final concentration (0.4 nM) of biotin-modified VEGFA165-Avi-His for 1 hour. VEGFA165-Avi-His was prepared in-house, and a linker, Avi tag and 6xHis tag were added to the C-terminus of human VEGFA165. The VEGFA165-Avi-His expression plasmid and the BirA enzyme expression plasmid were co-transfected into 293-F cells. During the expression process, the protein was biotin-modified on the Avi tag by catalysis of BirA enzyme. The amino acid sequence of VEGFA165-Avi-His is: APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNI TMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQL ELNERTCRCDKPRRGSGSGLNDIFEAQKIEWHEGGGGSHHHHHH (SEQ ID NO: 39).

[0320] The amino acid sequence of VEGFR2-Fc is:

[0321] ASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPQIGEKSLISPVDSYQYGTTQTLTCTVYAIPPPHHIHWYWQLEEECANEPSQAVSVTNPYPCEEWRSVEDFQGGNKIEVNKNQFALIEGKNKTVSTLVIQAANVSALYKCEAVNKVGRGERVISFHVTRGPEITLQPDMQPTEQESVSLWCTADRSTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSNSTNDILIMELKNASLQDQGDYVCLAQDRKTKKRHCVVRQLTVLERVAPTITGNLENQTTSIGESIEVSCTASGNPPPQIMWFKDNETLVEDSGIVLKDGNRNLTIRRVRKEDEGLYTCQACSVLGCAKVEAFFIIEGAQEKTNLEPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:43)

[0322] The mixture of fusion protein and VEGFA165-Avi-His was added to the ELISA plate adsorbed with VEGFR2-Fc and incubated at 37°C for 1 hour. The biotin signal of VEGFA165-Avi-His was detected with HRP-labeled streptavidin antibody Streptavidin-HRP (purchased from: Sangon Biotechnology, catalog number: D111054-0001), incubated at room temperature for 45 minutes, and after washing the plate, 100 μL / well of TMB color development solution was added to the ELISA plate for 15 minutes; 50 μL / well of stop solution was added, and the ELISA plate was placed in an ELISA reader to read the light absorption value (OD450) at 450 nm.

[0323] The fusion protein and monovalent nanobody V1-SA1 compete for the activity of VEGFR2 without pre-incubation with HSA. Fig.11 (A), VEGFR2 competitive activity after pre-incubation with HSA Fig.11 (B) The IC50 of VEGFR2 competition experiment is summarized in Table 22.

[0324] Table 22 Summary of IC50 of fusion protein and monovalent nanobody V1-SA1 competing for VEGFR2

[0325]

[0326]

[0327] As can be seen from Table 22, the VEGFR2 competitive activity of the three fusion proteins formed by 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, and the biologically active effector molecule can be at the N or C terminus of the anti-albumin antibody, and different positions basically do not affect the activity of the biologically active effector molecule in the fusion protein.

[0328] In order 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 the 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 is about twice that when the experimental system does not contain HSA, indicating that HSA will have a systematic effect on the IC50 value of the competitive ELISA experiment, and the ratio of the IC50 value of the fusion protein to the IC50 of V1-SA1 is 0.88-1.40, indicating that the VEGFR2 competitive activity of the fusion protein is similar to that of the monovalent nanobody V1-SA1.

[0329] The data of Table 21 and Table 22 show that the fusion protein of monovalent anti-VEGFA nanobody (V1-SA1 or V1-DP) and anti-albumin nanobody (3005Hz6) has strong competition for VEGFR2 and binding activity to HSA. This shows that in the fusion protein, the antibody elements binding to different antigens can function independently, and the mutual interference between the antibody elements is small. Therefore, antibodies binding to other antigens can be fused with the anti-albumin humanized antibody 3005Hz6 to construct a multi-specific and multifunctional fusion protein.

[0330] Example 7: Chemical conjugation of anti-albumin nanobody 3005 mutant with small molecule toxin derutocan and activity determination of the conjugate

[0331] 1. Construction, expression and purification of anti-albumin nanobody 3005 mutant suitable for coupling reaction

[0332] For anti-albumin nanobody 3005, the 65th amino acid in its amino acid sequence is a lysine residue K with high reactivity, and the reaction site is located in the CDR-H2 region. The K65 located in CDR-H2 may significantly reduce the affinity of the conjugate to the antigen albumin after covalent coupling with a small molecule. Therefore, in this embodiment, the K65 in the nanobody 3005 sequence is subjected to point mutation transformation, and the lysine residue with a primary amino group is mutated into an amino acid without reactivity such as alanine A, arginine R, histidine H, etc. The newly constructed mutants are named 3005K65A, 3005K65R, 3005K65H, etc.

[0333] Table 23 CDR region amino acid sequences of antibody 3005 mutants

[0334]

[0335] Table 24 Amino acid sequence and nucleotide sequence of antibody 3005 mutant

[0336]

[0337]

[0338] 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 brief, a secretion signal peptide (SEQ ID NO: 37) is added to the N-terminus of the mutant protein of antibody 3005, and a linker GGGGS (SEQ ID NO: 41) and 6xHis are added to the C-terminus of the antibody. The coding DNA sequence is obtained by gene synthesis, and then connected to the expression vector pCDNA3.1 (+) through the restriction sites NheI and XhoI. The plasmid is sequenced correctly and extracted in large quantities without endotoxin, and transiently expressed using suspended 293F cells, and Ni column affinity purification is carried out from the cell culture supernatant. After purification, the molecular weight of the mutant detected by Q-tof of the three mutant proteins is 13.8kDa.

[0339] Bio-Layer Interferometry (BLI) was used to test the affinity of antibody 3005 and its mutant proteins 3005K65A, 3005K65R and 3005K65H to albumins from different species. The antibody (concentration of 5 μg / mL) combined with his tag was used with NTAvsensor, and the antigens were human serum albumin HSA (Baxter AG), mouse albumin MSA (purchased from: Equitech-bio, catalog number: MSA62-1000) and rat albumin Rat albumin (purchased from: abcam, catalog number: ab198656). In the BLI experiment, the eight concentration gradients of the antigen were set to 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM, and the antigen and antibody were bound for 180 seconds and dissociated for 360 seconds.

[0340] The BLI detection results are shown in Table 25, which show that the affinities of mutant proteins 3005K65A, 3005K65R, and 3005K65H for binding to albumins from different species are similar to that of antibody 3005.

[0341] Table 25 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of antibody 3005 and its mutant proteins 3005K65A, 3005K65R, 3005K65H binding to albumin from different species D )

[0342]

[0343]

[0344] 2. Preparation method of 3005K65A and delutec conjugate (Nanobody Drug Conjugate, NDC)

[0345] In this example, mutant 3005K65A is used as an example to illustrate the preparation, purification and activity determination of the conjugate. 3005K65A can be obtained by using methods known to those skilled in the art to obtain a nano antibody intermediate (3005K65A-SH) with a thiol group, for example: reacting the primary amino group of a lysine residue with 2-iminothiolane hydrochloride (2-Iminothiolanehydrochloride, Traut's Reagent) to produce a thiol intermediate; reacting the primary amino group of a lysine residue with N-succinimidyl S-acetylthioalkanoate (N-Succinimidyl The invention relates to the covalent coupling of a 3-(S-Acetylthioglycolate, SATA) type linker to release hydroxylamine to produce a highly active thiolated intermediate; after the lysine residue is reacted with N-succinimidyl 3-(pyridyldithio) propionate (N-Succinimidyl3-(2-pyridyldithio) propionate, SPDP), the disulfide bond is reduced by a reducing agent such as dithiothreitol (DTT), 2-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to obtain a thiolated intermediate; or a certain concentration of reducing agent dithiothreitol (DTT), 2-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is used to reduce the disulfide bond in the nano antibody molecule to produce a thiol group; etc., but are not limited to these methods.

[0346] The thiolated nanobody active intermediate is covalently coupled to derutec via a thioether bond to obtain a nanobody derutec conjugate. In this embodiment, 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, and the free thiol group on the thiolated nanobody intermediate 3005K65A-SH framework reacts with the maleimide group of derutec via a Michael addition reaction to generate a structurally stable 3005K65A-derutec conjugate (NDC).

[0347] Flow chart of the conjugation and purification of the nanobody-drutecan conjugate, e.g. Fig.12 shown.

[0348] 3. Purification and structural characterization of 3005K65A delutec conjugate

[0349] The preparation process of the 3005K65A-Drutican conjugate is as follows: in a buffer containing a thiol-modified nano antibody intermediate 3005K65A-SH, a solution of Drutican is added dropwise and continuously shaken to react. The reaction buffer of 3005K65A-SH and Drutican can use sodium phosphate, sodium acetate solution or sodium borate, etc. The pH during the reaction is usually between 5 and 9, preferably around pH=7. The organic co-solvent for dissolving Drutican 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 embodiment, the DMAC solution containing Drutican is added to the buffer containing thiol-modified 3005K65A-SH at 8-15% v / v and reacted. The reaction temperature is 10-25°C and the reaction time is 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.

[0350] The conjugate purification strategy is as follows: a commercially available HiTrap Desalting column is used for purification (purchased from GE, item number: 17140801). The desalting column is balanced with 5-10 column volumes of phosphate buffer PBS pH=7.4. The NDC reaction aqueous solution (about 1.5 mL) is loaded on the HiTrap Desalting column, and eluted according to the instructions for use of the desalting column on a fast protein purifier (AKTA purifier 100, GE), and unreacted drutecan, N-acetyl-L-cysteine ​​(NAC), small molecule organic co-solvents, etc. are removed according to the molecular weight to obtain a 3005K65A-drutecan conjugate solution. The purified 3005K65A drutecan conjugate is subjected to SDS-PAGE and the like to determine whether the conjugate is successfully coupled, and the content of free small molecules such as drutecan in the purified 3005K65A drutecan conjugate is detected by RP-HPLC. The free small molecule content of the purified conjugate should not exceed 1% before subsequent cell activity assays can be performed.

[0351] The structure of the 3005K65A-delutec conjugate (NDC) described in this example is as follows Fig.13 As shown in (A), Fig.13 (B) shows the SDS-PAGE gel image of three different 3005K65A-derutecan conjugates (NDC-1, NDC-2, NDC-3), among which the conjugate labeled NDC-3 is used as an example for subsequent ELISA experiments binding to human serum albumin and evaluation of the proliferation inhibitory activity on pancreatic cancer cells Mia Paca-2.

[0352] IV. Affinity and in vitro activity determination of 3005K65A-Drutecan conjugate

[0353] The ELISA method was used to detect the human serum albumin binding activity of the 3005 mutant protein 3005K65A and 3005K65A-drutecan conjugate (NDC-3). In the ELISA experiment, 2 μg / mL human serum albumin was coated, and concentration gradients of the 3005 mutant protein 3005K65A and 3005K65A-drutecan conjugate (NDC-3) were added. The antibody level bound to albumin was detected by HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, catalog number: HRP-66005). The ELISA experimental results of the 3005 mutant protein 3005K65A and 3005K65A-drutecan conjugate (NDC-3) binding to HSA are shown in Fig.14 (A). Fig.14 As shown in (A), after coupling with the small molecule derutecan, the 3005K65A-derutecan conjugate (NDC-3) still has a high binding activity with human serum albumin. The EC50 of the ELISA experiment binding to HSA is summarized in Table 26.

[0354] Table 26 Summary of EC50 values ​​of 3005 mutant protein 3005K65A and 3005K65A-derutec conjugate (NDC-3) binding to HSA

[0355] name EC50 of HSA binding (nM) 3005K65A 0.43 3005K65A-Drutecan conjugate (NDC-3) 1.85

[0356] 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 in 384-well cell culture microplates at 25 μL per well. Then, 25 μL of different concentrations of derutecan or 3005K65A-derutecan conjugate (NDC-3) diluted with DMEM medium was added to each well, so that the final concentrations of derutecan or 3005K65A-derutecan 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 incubated at 37°C and 5% CO 2After culturing for 5 days, remove the microplate from the incubator, remove 25 μL and add an equal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega), shake the mixer at room temperature for 10 minutes, and measure the luminescence of each sample in the microplate with an ELISA reader. Calculate the survival rate of tumor cells after 5 days of continuous culture using the formula:

[0357] Cell survival rate (%) = luminescence of the drug group ÷ luminescence of the control group × 100%

[0358] After the Mia Paca-2 cells were treated with the test substance at concentrations of 96nM, 960nM and 19340nM, the viability of Mia Paca-2 cells was Fig.14 (B) and shown in Table 27.

[0359] Table 27 Cell viability of Mia Paca-2 cells treated with derutec and 3005K65A-derutec conjugate (NDC-3)

[0360]

[0361] from Fig.14 As shown in (B) and Table 27, at the same concentration, the activity of derutecan in inhibiting tumor cell proliferation is significantly lower than that of 3005K65A-derutecan conjugate (NDC-3). Therefore, anti-albumin antibody 3005 can be used as a carrier to conjugate with active molecules such as small molecule toxins. The biological activity of the conjugate, such as inhibiting cell proliferation, is not inferior to or even better than the active molecule itself. The conjugate can potentially improve the cell killing ability or target cell selectivity of the active molecule, thereby improving the activity of the small molecule in inhibiting cancer cell growth.

[0362] Example 8: Binding activity of humanized anti-albumin antibodies to albumin under different pH conditions

[0363] Albumin can bind to the cell surface receptor FcRn under weakly acidic conditions, but the binding force is weak 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. Albumin-binding antibodies and their fusion proteins will 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 to achieving long circulation and half-life extension.

[0364] 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 ).

[0365] In the experiment, a Biacore 8K instrument (Cytiva) was used, and the antigen HSA was immobilized by amino coupling using Series S Sensor Chip CM5, and the analyte was 3005Hz6 in a concentration gradient. When the binding and dissociation of HSA and 3005Hz6 were detected under neutral conditions, the components of the mobile phase buffer used were: 10mM HEPES pH=7.4, 150mM NaCl, 3mM EDTA, 0.05% v / v Tween-20, and the analyte 3005Hz6 was diluted with this mobile phase. When the binding and dissociation of HSA and 3005Hz6 were detected under acidic conditions, the components of the mobile phase buffer used were: 10mM sodium acetate pH=5.5, 150mM NaCl, 3mM EDTA, 0.05% v / v Tween 20, and the analyte 3005Hz6 was diluted with this mobile phase. The binding time in the experiment was 150 seconds, and the dissociation time was 300 seconds.

[0366] The “1:1 binding” model was used to fit the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of 3005Hz6 binding to HSA at pH = 7.4 and pH = 5.5. D ), the results are shown in Table 28.

[0367] Table 28 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of humanized antibody 3005Hz6 with HSA at pH = 7.4 and pH = 5.5 D )

[0368] pH conditions for SPR experiments Ligand Analyte Solution ka(1 / Ms) kd(1 / s) <![CDATA[K D (M)]]> pH=7.4 HSA 3005Hz6 5.33E+05 1.52E-03 2.84E-09 pH=5.5 HSA 3005Hz6 1.37E+06 4.53E-04 3.29E-10

[0369] As shown in Table 28, using the SPR method for amino-coupled HSA, at pH = 7.4, the equilibrium dissociation constant K of 3005Hz6 and HSA is D About 2.84nM; at pH = 5.5, the equilibrium dissociation constant K of 3005Hz6 and HSA DIt is about 0.33nM, indicating that the humanized antibody 3005Hz6 can strongly bind to HSA under both neutral and acidic conditions, and the affinity of 3005Hz6 to HSA under acidic conditions is 8.6 times stronger than that under neutral conditions. Therefore, it can be inferred that the humanized antibody 3005Hz6 can strongly bind to HSA at physiological pH, and when the complex of 3005Hz6 and HSA enters the acidic environment of the endosome together, the affinity of 3005Hz6 to HSA is enhanced, so that with the help of the binding of HSA to FcRn, 3005Hz6 can achieve intracellular circulation or transcellular transport.

[0370] Example 9: Fusion expression of 3005Hz6 and Aflibercept and activity detection

[0371] 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 and IgG1 Fc) for expression. 3005Hz6 was connected to the N-terminus of the Aflibercept protein, with a linker of GGSGGS (SEQ ID NO: 60), and the fusion protein was named: 3005Hz6-Aflibercept, and the amino acid sequence of the fusion protein 3005Hz6-Aflibercept was: (SEQ ID NO: 61).

[0372] The secretion signal peptide IgK (METDTLLLWVLLLWVPGSTG, SEQ ID NO: 42) was added to the N-terminus of the fusion protein 3005Hz6-Aflibercept, and the protein coding DNA sequence was linked to the expression vector through the restriction sites HindIII and NotI by gene synthesis and molecular cloning technology. The plasmid was sequenced correctly and extracted in large quantities without endotoxin, and transiently expressed using suspended 293F cells, and Protein A affinity purification was performed from the cell culture supernatant to obtain the fusion protein 3005Hz6-Aflibercept.

[0373] The nucleotide sequence of the fusion protein 3005Hz6-Aflibercept is:

[0374]

[0375] The activity of anti-albumin nanoantibody 3005Hz6 in the fusion protein 3005Hz6-Aflibercept in binding to HSA was detected by ELISA. In the experiment, the final concentration of coated human serum albumin HSA (Baxter AG) was 2μg / mL, 100μL / well, and coated overnight at 4°C. After blocking with 5% skim milk powder, gradient dilutions of the fusion protein were added, incubated at 37°C for 1 hour, and HRP-labeled goat anti-human IgG Fc antibody (purchased from: Abbkine, item number: A21050) was added after washing the plate, and incubated at room temperature for 45 minutes. After washing the plate, 100μL TMB (purchased from: Tiangen Biochemical, item number: PA107-01) was added to each well, and after color development at 37°C for 15 minutes, 50μL of stop solution was added. The absorbance at 450nm (OD450) was measured using an enzyme marker. The EC50 of the fusion protein ELISA binding experiment is shown in Table 29.

[0376] The activity of the anti-VEGF protein Aflibercept in the fusion protein 3005Hz6-Aflibercept was detected by VEGFR2 competitive ELISA method, and the control protein was the marketed drug Aflibercept. The competitive ELISA experimental method was the same as "2. Detection of the activity of anti-VEGFA nanoantibodies in the fusion protein" in Example 6. The IC50 of the fusion protein competitive ELISA experiment is shown in Table 29.

[0377] Table 29 Summary of the results of the activity detection experiment of protein 3005Hz6-Aflibercept

[0378] Protein name Binding to HSA ELISA EC50 (nM) VEGFR2 Competition ELISA IC50(nM) 3005Hz6-Aflibercept 0.17 3.99 Aflibercept No binding 4.52

[0379] 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. The results show 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.

[0380] Example 10: Affinity enhancement of humanized antibody 3005Hz6

[0381] The three CDR regions of the humanized antibody 3005Hz6 were subjected to random mutations at a single amino acid site and two adjacent amino acid sites, and the mutated sequences were displayed on the phage surface using phage display technology. By gradually reducing the amount of antigen input and species cross-screening during the screening process, anti-albumin nanoantibody candidate sequences that can simultaneously bind to HSA, cynomolgus monkey serum albumin, MSA, and rat albumin with higher affinity than 3005Hz6 were obtained. Finally, candidate antibodies 1004, 1012, 1036, 1068, and 2028 were obtained. The amino acid sequences and nucleotide sequences of the candidate antibodies are shown in Table 30, and their CDR region amino acid sequences are shown in Table 31.

[0382] Table 30 Amino acid sequences and nucleotide sequences of candidate antibodies with improved affinity

[0383]

[0384]

[0385] Table 31 Amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of candidate antibodies with improved affinity

[0386]

[0387] Example 11: ELISA Binding Activity Test of Affinity-Enhanced Candidate Antibodies to Albumin

[0388] 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.

[0389] The results of purified candidate antibodies with improved affinity are shown in Fig.15 , the molecular weight of the candidate antibody is approximately 14.7 kDa.

[0390] The ELISA method was used to detect the binding activity of candidate antibodies with improved affinity to albumins from different species.

[0391] 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:

[0392] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCT

[0393] VATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYA

[0394] PELLFFAKRYKAAFTECCQAADKAACLLPCLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA

[0395] EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLP

[0396] SLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEF

[0397] KPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDY

[0398] LSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETTFHADICTLSEKERQIKKQTAL

[0399] VELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGSGLNDIFEAQKIEWHEGGGGSHHHHHH, SEQ ID NO: 56), added to the ELISA plate, incubated at 37°C for 1 hour, and washed with PBST solutions of different pH (pH = 7.4 or 6.0). HRP-labeled streptavidin antibody Streptavidin-HRP (purchased from: Sangon Biotechnology, catalog number: D111054-0001) was added, incubated at room temperature for 45 minutes, and 100 μL TMB (purchased from: Tiangen Biochemical, catalog number: PA107-01) was added to each well after washing. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured with an ELISA reader.

[0400] The ELISA results of the candidate antibodies with improved affinity binding to HSA at pH = 7.4 or pH = 6.0 are as follows: Fig.16 (A) and Fig.16 As shown in (B), it can be seen that at pH = 7.4, except for 1004, the affinity of the remaining candidate antibodies for binding to HSA is higher than 3005Hz6, among which the affinity of antibody 1068 is the most significantly improved. At pH = 6.0, the affinity of the five candidate antibodies for binding to HSA is higher than 3005Hz6.

[0401] The antigen-coated ELISA method was used to detect the activity of candidate antibodies with improved affinity in binding to rat albumin. In the experiment, unlabeled rat albumin was coated at a concentration of 5 μg / mL and coated overnight at 4°C. Candidate antibodies (3005Hz6, 1004, 1012, 1036, 1068, 2028) were diluted with PBST solutions of different pH (pH = 7.4 or 6.0), added to the ELISA plate, incubated at 37°C for 1 hour, washed with PBST solutions of different pH (pH = 7.4 or 6.0), added anti-His tag monoclonal antibody (purchased from: Proteintech, catalog number: HRP-66005), incubated at room temperature for 45 minutes, and after washing, 100 μL 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. Determined by microplate reader

[0402] OD450. 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).

[0403] The ELISA results of the candidate antibodies with improved affinity binding to rat albumin at pH = 7.4 or pH = 6.0 are as follows: Fig.17 (A) and Fig.17 As shown in (B), it can be seen that at pH = 7.4 or pH = 6.0, the affinity of antibody 2028 to rat albumin is higher than 3005Hz6, while the control protein BI-V2 hardly binds to rat albumin.

[0404] Example 12: Affinity test of candidate antibodies with improved affinity to HSA (SPR)

[0405] Two SPR methods were used to determine the affinity of the affinity-enhanced candidate antibodies to HSA.

[0406] 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 different concentrations of HSA. 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 Values, see Table 32.

[0407] Table 32 Binding 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 )

[0408] Ligand Analyte Solution ka(1 / Ms) kd(1 / s) <![CDATA[K D (M)]]> 1004 HSA 4.41E+05 1.63E-03 3.71E-09 1012 HSA 4.00E+05 1.41E-03 3.52E-09 1036 HSA 4.34E+05 1.88E-03 4.34E-09 1068 HSA 4.48E+05 9.91E-04 2.21E-09 2028 HSA 2.39E+05 1.31E-03 5.49E-09 3005Hz6 HSA 3.94E+05 2.93E-03 7.42E-09

[0409] It can be seen from Table 32 that 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.

[0410] In the second SPR method, a Biacore 8K instrument (Cytiva) was used to immobilize the antigen HSA by amino coupling using a Series S Sensor Chip CM5 (response value was about 1250RU). The analytes were 3005Hz6, 1068, 2028 and the control protein BI-V2 in a concentration gradient. The protein was in a buffer of 10mM HEPES pH=7.4, 150mM NaCl, 3mM EDTA, 0.05% v / v Tween-20. The binding time in the SPR experiment was 120 seconds and the dissociation time was 1000 seconds (for antibody 2028, the dissociation time was 300 seconds). The binding and dissociation curves of antibodies 3005Hz6, 1068, 2028 and the control protein BI-V2 binding to HSA are shown as follows: Fig.18 (A) Fig.18 (B) Fig.18 (C) Fig.18 (D) as shown.

[0411] The “1:1 binding” model was used to fit the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of the antibody binding to HSA. D ), the results are shown in Table 33.

[0412] Table 33 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of antibodies 3005Hz6, 1068, 2028 and control protein BI-V2 to HSA D )

[0413]

[0414]

[0415] 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).

[0416] 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0417] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable 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 that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 or 58; The amino acid sequence of CDR-H2 comprises any one of SEQ ID NOs: 3-4, 6, 40, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs: 3-4, 6, 40; The amino acid sequence of CDR-H3 comprises any one of SEQ ID NOs: 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 NOs: 7-8, 59.

2. The anti-albumin antibody or antigen-binding fragment thereof according to claim 1, characterized in that: In the SEQ ID NO: 58 (X1YYMS), X1 represents N or E, which is SEQ ID NO: 2 or 49, respectively; 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, The X2X3 represents DG, X4X5 represents SF, and X6 represents K (SEQ ID NO: 5); The X2X3 represents DA, X4X5 represents SF, and X6 represents K (SEQ ID NO: 20); The X2X3 represents DS, X4X5 represents SF, and X6 represents K (SEQ ID NO: 21); The X2X3 represents EG, X4X5 represents SF, and X6 represents K (SEQ ID NO: 22); The X2X3 represents DG, X4X5 represents SF, and X6 represents A (SEQ ID NO: 31); The X2X3 represents DG, X4X5 represents SF, and X6 represents R (SEQ ID NO: 32); The 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, The X2X3 represents DM, X4X5 represents SF, and X6 represents K (SEQ ID NO: 51); or, The SEQ ID NO: 59 (ASGPX7X8LRX9X 10 AP) in X7X8 represents QG, IW, LW or VG, X9X 10 represents LG or WW, preferably, The X7X8 represents QG, X9X 10 represents LG (SEQ ID NO: 9); The X7X8 represents IW, X9X 10 represents LG (SEQ ID NO: 52); The X7X8 represents LW, X9X 10 represents LG (SEQ ID NO: 53); The X7X8 represents QG, X9X 10 represents WW (SEQ ID NO: 54); or, The X7X8 represents VG, 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 the CDR-H1, CDR-H2 and CDR-H3 include any 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 to 3, characterized in that: The anti-albumin antibody or antigen-binding fragment thereof comprises a humanized sequence, wherein the modification site of the humanized sequence is located in a non-CDR region, and 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 to 4, characterized in that: The anti-albumin antibody or antigen-binding fragment thereof includes nanobodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, F(ab')2 fragments, single-chain antibodies or linear antibodies.

6. The anti-albumin antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The amino acid sequence of the anti-albumin antibody or its antigen-binding fragment 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.

7. A use of the anti-albumin antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The applications include: A use of A in preparing a fusion construct, wherein the fusion construct comprises the anti-albumin antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a biologically active effector molecule, wherein the biologically active effector molecule is linked to the anti-albumin antibody or antigen-binding fragment thereof; B. Use in screening bioactive effector molecules, connecting candidate bioactive effector molecules with the anti-albumin antibody or antigen-binding fragment thereof to detect the biological activity of the candidate bioactive effector molecules; or Application of C in the detection of albumin.

8. A fusion construct, characterized in that The fusion construct comprises one or more anti-albumin antibodies or antigen-binding fragments thereof and / or biologically active effector molecules according to any one of claims 1 to 6, and the biologically active effector molecules are connected to the anti-albumin antibodies or antigen-binding fragments thereof. Preferably, the fusion construct comprises multiple 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 biologically active effector molecules may be one or more, and the multiple biologically active effector molecules may be the same or different.

9. The fusion construct according to claim 8, characterized in that The bioactive effector molecules include but are not limited to small molecule compounds or macromolecular compounds. Preferably, the small molecule compounds include but are not limited to any small molecule drugs, and the macromolecular compounds include but are not limited to antibodies, ligands that activate or inhibit receptors and proteins, bioactive enzymes, nucleic acid drugs, or combinations thereof. More preferably, the antibody in the macromolecular compound includes antibodies or antigen-binding fragments thereof to other targets, and the other targets are targets other than albumin; More preferably, the structure of the antibody or antigen-binding fragment thereof for other targets is 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 or a 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, interleukin (such as IL-1β, IL-2, IL-3, IL-4, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptor (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, CD3 0. 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, characterized in that The anti-albumin antibody or antigen-binding fragment thereof is directly or indirectly connected to the biologically active effector molecule. Preferably, the indirect connection may be through a linker, a functional domain and / or a linker for coupling; Wherein, the linker is selected from a connecting peptide, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, PEG, a nucleic acid, a polysaccharide, a fatty chain, biotin, streptavidin or avidin; The functional domain is one or a combination of two or more of an Fc fragment, serum albumin, cytokine, transferrin, and a scaffold protein; The linker used for coupling includes a functional group linker. Preferably, the functional group linker includes a thiol, an amino, a hydroxyl and / or a carboxyl reactive group. 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 antigen-binding fragment thereof.

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

13. The nucleic acid according to claim 12, characterized in that The nucleic acid also comprises any nucleotide sequence of SEQ ID NO: 63-75, 79-81, 83-87 or its degenerate sequence, or a nucleotide sequence that has at least 80% identity with any nucleotide sequence of SEQ ID NO: 63-75, 79-81, 83-87 and has the function of encoding an anti-albumin antibody or its antigen-binding fragment.

14. The nucleic acid according to any one of claims 12 to 13, characterized in that The nucleic acid comprises any nucleotide sequence in SEQ ID NO: 76-78, 82 or a degenerate sequence thereof, or has at least 80% identity with any nucleotide sequence in SEQ ID NO: 76-78, 82, and has a nucleotide sequence encoding the function of the fusion construct.

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

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

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

18. Use of the anti-albumin antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the fusion construct according to any one of claims 8 to 11, the nucleic acid according to any one of claims 12 to 14, the vector according to claim 15 or the 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 biologically active effector molecule have activity in treating and / or diagnosing diseases. Preferably, the anti-albumin antibody or antigen-binding fragment thereof is used 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 a disease comprises any of the following: A) the anti-albumin antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; B) the fusion construct of any one of claims 8 to 11; C) the nucleic acid according to any one of claims 12 to 14; D) the vector according to claim 15; or F) The host cell according to claim 16, The anti-albumin antibody or antigen-binding fragment thereof and / or the biologically active effector molecule has activity in treating and / or diagnosing diseases, 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. 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 the sample to be tested with the anti-albumin antibody or its antigen-binding fragment according to any one of claims 1 to 6, and then detecting the content of the complex formed by the albumin and the anti-albumin antibody or its antigen-binding fragment. 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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