Anti-albumin antibody or its antigen-binding fragment and its applications

Abstract

The present invention provides an anti-albumin antibody or its antigen-binding fragment and its applications, and an anti-albumin nanobody or its antigen-binding fragment with enhanced affinity, wherein the anti-albumin antibody or its antigen-binding fragment can bind to albumin of different species with high affinity, and at the same time the anti-albumin antibody can be linked to a biologically active effector molecule to form a fusion construct, and does not affect the activity of the biologically active effector molecule.

Description

Technology Field

[0001] The present invention relates to the field of biomedical technology, and specifically to an anti-albumin antibody or an antigen-binding fragment thereof and its applications. Background Technology

[0002] Albumin is the most abundant protein in plasma (concentration about 40 mg / mL) and is essential for maintaining plasma osmotic pressure. At the same time, albumin is an important transport carrier for endogenous ligands (e.g., fatty acids, metal ions, hormones, etc.) and exogenous ligands (e.g., drugs).

[0003] Albumin has a molecular weight of 66.5 kDa and can bind to the neonatal Fc receptor (FcRn) on the cell surface under weakly acidic conditions, but its binding affinity is weak under neutral conditions. Therefore, after binding to FcRn, albumin is absorbed into the endosome; as the endosome environment becomes acidic, the binding affinity between albumin and FcRn strengthens, leading to its recirculation back to the cell surface by FcRn or transcellular transport. Consequently, albumin can achieve a long half-life by utilizing the recycling mechanism of FcRn. Since the plasma half-life of human serum albumin is approximately 3 weeks, small molecule compounds that bind to albumin or proteins fused with albumin (e.g., commercially available drugs Tanzeum® and Idelvion®) exhibit significantly improved pharmacokinetic properties and can achieve longer half-lives, potentially extending the dosing interval and providing better administration options for patients.

[0004] However, albumins of different species have sequence differences and exhibit different affinities for the corresponding FcRn. Therefore, for small molecule drugs or albumin fusion proteins that bind to albumin, pharmacokinetic and efficacy evaluations performed in small animals may not necessarily reflect the effects in humans. Consequently, sequence differences in albumins of different species pose significant challenges to conversion studies of albumin-carrier drugs or albumin fusion protein drugs.

[0005] Therefore, developing antibodies capable of cross-binding to various types of albumin could potentially solve this problem. Since anti-albumin antibodies can bind to different types of albumin with similar affinities, pharmacokinetic and pharmacokinetic studies performed on small animals (e.g., mice and rats) of drug molecules when small molecule compounds are conjugated to anti-albumin antibodies, or when antibodies binding to specific antigens are fused with anti-albumin antibodies, can provide reliable reference data for future pharmacokinetic and pharmacokinetic characteristics in humans.

[0006] Single-domain antibodies or nanobodies are variable domains of heavy-chain antibodies that naturally lack light chains in camelid animals; they are the smallest antibody units with a simple structure and full antigen-binding activity. The molecular weight of the nanobodies is approximately 13 kDa, and they possess characteristics such as easy modification, high thermal stability, and excellent water solubility.

[0007] In order to predict the future effects of active molecules in the human body, it is typically necessary to verify the behavior of the active molecule in animals (e.g., pharmacokinetics, drug distribution, and drug metabolism) in relevant animal models. Rodents such as mice and rats are commonly used experimental animal models because they are small in size, have short reproductive cycles, share similar genetic backgrounds among individuals, and have low experimental costs. Therefore, active molecules must possess the ability to bind to targets derived from humans, mice, and rats. In the case of anti-albumin single-domain antibodies or nanobodies, said antibodies must be able to bind to albumin from various species, such as humans, mice, and rats, in order to perform in vivo evaluation experiments of anti-albumin nanobodies and their fusion constructs in rodents (e.g., mice and rats). The problem to be solved

[0008] To overcome the defects of the prior art, the present application provides an anti-albumin antibody or an antigen-binding fragment thereof and its applications, and an anti-albumin antibody or its antigen-binding fragment with enhanced affinity, wherein the anti-albumin antibody or its antigen-binding fragment can bind to albumin of different species with high affinity, and at the same time, the anti-albumin antibody can be linked to a biologically active effector molecule to form a fusion construct without affecting the activity of the biologically active effector molecule. Specifically, as follows. means of solving the problem

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

[0010] The amino acid sequence of CDR-H1 comprises an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO. 1 or 58, or SEQ ID NO. 1 or 58;

[0011] The amino acid sequence of CDR-H2 comprises any one of SEQ ID NOs 3-4, 6, and 40, or an amino acid sequence having at least 80% identity with the amino acid sequence represented by any one of SEQ ID NOs 3-4, 6, and 40;

[0012] The amino acid sequence of CDR-H3 includes an amino acid sequence having at least 80% identity with any one of SEQ ID NOs 7-8 and 59, or any one of SEQ ID NOs 7-8 and 59.

[0013] Herein, the above sequence no. 58 (X1YYMS), sequence no. 40 (GISVX2X3X4X5LDYADAVX6G), and sequence no. 59 (ASGP X7X8LRX9X 10 X among AP) 1-10 can be any natural amino acid residue, e.g., 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), valine (V).

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

[0015] In the above sequence number 40 (GISVX2X3X4X5LDYADAVX6G), X2X3 represents DS, DA, EG, DM, or DG, X4X5 represents SF or WY, and X6 represents K, A, R, or H; preferably,

[0016] The above X2X3 represents DG, X4X5 represents SF, and X6 represents K (sequence number 5);

[0017] The above X2X3 represents DA, X4X5 represents SF, and X6 represents K (sequence number 20);

[0018] The above X2X3 represents DS, X4X5 represents SF, and X6 represents K (sequence number 21);

[0019] The above X2X3 represents EG, X4X5 represents SF, and X6 represents K (sequence number 22);

[0020] The above X2X3 represents DG, X4X5 represents SF, and X6 represents A (sequence number 31);

[0021] The above X2X3 represents DG, X4X5 represents SF, and X6 represents R (sequence number 32);

[0022] The above X2X3 represents DG, X4X5 represents SF, and X6 represents H (sequence number 33);

[0023] The above X2X3 represents DG, X4X5 represents WY, and X6 represents K (sequence number 50); or,

[0024] The above X2X3 represents DM, X4X5 represents SF, and X6 represents K (sequence number 51).

[0025] The above sequence number 59 (ASGPX7X8LRX9X 10 X7X8 in AP represents QG, IW, LW, or VG, and X9X 10 represents LG or WW. Preferably,

[0026] The above X7X8 represents QG, and X9X 10 ... represents LG (sequence number 9);

[0027] The above X7X8 represents IW, and X9X 10 ... represents LG (sequence number 52);

[0028] The above X7X8 represents LW, and X9X 10 ... represents LG (sequence number 53);

[0029] The above X7X8 represents QG, and X9X 10 represents WW (sequence number 54); or,

[0030] The above X7X8 represents VG, and X9X 10 represents LG (sequence number 55).

[0031] In one specific embodiment of the present invention, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are,

[0032] A) Sequence numbers 1, 3, 7;

[0033] B) Sequence numbers 1, 4, 8;

[0034] C) Sequence numbers 2, 5, 9;

[0035] D) Sequence numbers 2, 6, 9;

[0036] E) Sequence numbers 2, 20, 9;

[0037] F) Sequence numbers 2, 21, 9;

[0038] G) Sequence numbers 2, 22, 9;

[0039] H) Sequence numbers 2, 31, 9;

[0040] I) Sequence numbers 2, 32, 9;

[0041] J) Sequence numbers 2, 33, 9;

[0042] K) Sequence numbers 2, 5, 52;

[0043] L) Sequence numbers 2, 50, 53;

[0044] M) Sequence numbers 49, 5, 53;

[0045] N) Sequence numbers 49, 5, 54;

[0046] O) Includes any one of the groups of sequence numbers 2, 51, and 55 (specifically, see Table 1).

[0047] Table 1 Amino acid sequences of candidate antibodies 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 the N-terminus to the C-terminus. In this application, the Kabat numbering system is used for classifying amino acids of the antibody CDR region. The anti-albumin antibody or its antigen-binding fragment includes a heavy chain variable region.

[0050] Preferably, the anti-albumin antibody or its antigen-binding fragment comprises a humanization sequence, and a modified site of the humanization sequence is located in a non-CDR region; more preferably, the modified site of the humanization sequence is located in a framework region and / or an invariant region of the antibody.

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

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

[0053] In one specific embodiment of the present invention, the anti-albumin antibody or its antigen-binding fragment is a nanobody, and compared to full-length IgG antibody, Fab, scFv, the nanobody has a higher molar concentration and can bind to more antigen molecules under the same mass conditions.

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

[0055] Preferably, the mammal includes human or non-human mammals, and the non-human mammal may be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, a pig, a cow, a sheep, a horse, a donkey, a fox, a raccoon, a mink, a camel, a dog, a cat, a rabbit, a rat (e.g., a rat, a mouse, a guinea pig, a hamster, a gerbil, a chinchilla, a squirrel), or a monkey, etc.

[0056] Preferably, the amino acid sequence of the anti-albumin antibody or its antigen-binding fragment comprises any one of the amino acid sequences of SEQ ID NOs 10-13, 16-19, 23-25, 34-36, and 44-48, or has at least 80% identity with any one of the amino acid sequences of SEQ ID NOs 10-13, 16-19, 23-25, 34-36, and 44-48.

[0057] In one specific embodiment of the present invention, the amino acid sequence of the anti-albumin antibody or its antigen-binding fragment is represented by any one of the amino acid sequences of SEQ ID NOs 10-13, 16-19, 23-25, 34-36, and 44-48.

[0058] The above anti-albumin antibody or its antigen-binding fragment can be constructed using any general method of the prior art, such as artificial synthesis or eukaryotic expression or prokaryotic expression.

[0059] In a second aspect of the present invention, an application of the anti-albumin antibody or its antigen-binding fragment is provided, said application being,

[0060] A. Application in the manufacture of fusion constructs - The fusion construct comprises the anti-albumin antibody or its antigen-binding fragment and a biologically active effector molecule, wherein the biologically active effector molecule is connected to the anti-albumin antibody or its antigen-binding fragment - ;

[0061] B. Application in the screening of biologically active effector molecules - linking a candidate biologically active effector molecule to the anti-albumin antibody or its antigen-binding fragment, and detecting the biological activity of the candidate biologically active effector molecule - ; or,

[0062] C. Includes applications in the detection of albumin.

[0063] In a third aspect of the present invention, a fusion construct is provided, said fusion construct comprises one or more of said anti-albumin antibodies or antigen-binding fragments thereof and / or biologically active effector molecules, said biologically active effector molecules are connected to said 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 plurality of biologically active effector molecules may be the same or different.

[0066] Preferably, the biologically active effector molecule comprises, but is 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, e.g., antitumor 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, antithrombotic drugs, antiepileptic drugs, antidepressants, stroke drugs, or autism drugs.

[0068] Preferably, the small molecule drug comprises, but is not limited to, one or more of donepezil, rasagiline, entacapone, rotigotine, camptothecin, paclitaxel, sunitinib, sorafenib, warfarin, curcumin, methotrexate, docetaxel, carbamazepine, seroxat, olanzapine, glibenclamide, nimodipine, idebenone, sulpiride, or deruxtecan.

[0069] In one specific embodiment, the small molecule drug is deruxtecan.

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

[0071] Preferably, the antibody in the macromolecular compound comprises an antibody of another target or an antigen-binding fragment thereof, and the other target is a target different from albumin.

[0072] More preferably, the structure of the antibody of the other target or its antigen-binding fragment comprises one or more combinations of nanobodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, F(ab')2 fragments, single-chain antibody fragments (scFv), or linear antibodies.

[0073] Preferably, the other targets are vascular endothelial growth factor A (VEGFFA), vascular endothelial growth factor B (VEGFFB), vascular endothelial growth factor receptor (VEGFFR), fibroblast growth factor (FGF), fibroblast growth factor receptor (FGFR), placental growth factor (PIGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), integrin, integrin receptor, interleukin (e.g., IL-1β, IL-2, IL-3, IL-4, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin Receptors (Interleukin Receptors, e.g., IL1R1, IL2Rα, IL3R, IL4Rα, IL10R, IL12R, IL15Rα, IL17R, IL23R, etc.), Proprotein convertase subtilisin / kexin type 9 (PCSK9), Tumor necrosis factor α (TNFα), Tumor necrosis factor receptor (TNFR), Receptor Activator of Nuclear Factor-κB Ligand (RANKL), G Protein-Coupled Receptor (GPCR), Glucagon-like peptide-1 receptor,GLP1R), Differentiation Cluster 3 (Cluster of Differentiation 3, CD3), Differentiation Cluster 105 (Cluster of Differentiation 105, CD105), Differentiation Cluster 19 (Cluster of Differentiation 19, CD19), Differentiation Cluster 20 (Cluster of Differentiation 20, CD20), Differentiation Cluster 22 (Cluster of Differentiation 22, CD22), Differentiation Cluster 25 (Cluster of Differentiation 25, CD25), Differentiation Cluster 27 (Cluster of Differentiation 27, CD27), Differentiation Cluster 28 (Cluster of Differentiation 28, CD28), Differentiation Cluster 30 (Cluster of Differentiation 30, CD30), Differentiation Cluster 33 (Cluster of Differentiation 33, CD33), Differentiation Cluster 38 (Cluster of Differentiation 38, CD38), Differentiation Cluster 40 (Cluster of Differentiation 40, CD40), Differentiation Cluster 47 (Cluster of Differentiation 47, CD47), Differentiation Cluster 80 (Cluster of Differentiation 80, CD80 / B7-1), Differentiation Cluster 86 (Cluster of Differentiation 86, CD86 / B7-2), Differentiation Cluster 96 (Cluster of Differentiation 96, CD96), Differentiation Cluster 99 (Cluster of Differentiation 99, CD99), Differentiation Cluster 111 (Cluster of Differentiation 111, CD111), Differentiation Cluster 112 (Cluster of Differentiation 112, CD112), Differentiation Cluster 123 (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 (CLDN18.2), Tumor necrosis factor receptor superfamily, member 4 (TNFRSF4 / OX40 / CD134), Inducible T-Cell Co-Stimulator (ICOS), Cytotoxic T-lymphocyte-associated protein 4 (CTLA4), Tumor necrosis factor receptor superfamily, member 9, TNFRSF9 / 4-1BB / CD137), T cell antigen receptor (TCR), B- and T-Lymphocyte Attenuator (BTLA), T cell immunoglobulin domain and mucin domain-3 (TIM-3), Lymphocyte Activation Gene 3 (LAG3), Galectin-9 (GAL9), Programmed cell death 1 ligand 1 (PD-L1), Programmed cell death 1 ligand 2 (PD-L2), Programmed cell death protein 1 (PD-1),T cell immune receptor with Ig and ITIM domains (TIGIT), Epidermal growth factor receptor (EGFR), Human Epidermal Growth Factor Receptor 2 (HER2), Prostate Stem Cell Antigen (PSCA), Carcinoembryonic Antigen (CEA), Familial Adenomatous Polyposis (FAP), Epidermal growth factor receptor variant type III (EGFRvIII), B cell maturation antigen (BCMA), Prostate Specific Membrane Antigen (PSMA), Cancer antigen 125 (CA125), Ephrin A Receptor 2, EphA2), Cellular-mesenchymal epithelial transition factor (c-Met), L1-Cell Adhesion Molecule (L1CAM), Signaling Lymphocytic Activation Molecule Family, Member 7 (SLAMF7 / CS1), Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Recombinant Mycobacterium Tuberculosis Fusion Protein,It is selected from New York esophageal squamous cell carcinoma-1 (NY-ESO-1), Mucin 1 (MUC1), Mucin 16 (MUC16), Mesothelin, Cluster of Differentiation 174 (LewisY / CD174), Glypican 3 (GPC3), Disialoganglioside-GD2 (GD2), Eukaryotic division factor-like and proliferation-associated protein (EPG), Delta-Like Ligand 3 (DLL3), or Trophoblast Glycoprotein (TPBG / 5T4).

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

[0075] Preferably, the anti-albumin antibody or its antigen-binding fragment and the biologically active effector molecule are directly or indirectly connected.

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

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

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

[0079] The above-mentioned bonding linker includes a functional group linker.

[0080] The above functional group linker may include mercapto, amino, hydroxyl and / or carboxyl reaction active groups, thereby enabling a covalent bond between an anti-albumin antibody or its antigen-binding fragment and a biologically active effector molecule.

[0081] Preferably, the biologically active effector molecule is directly or indirectly connected to the N-terminus, C-terminus, and / or internal residue of the anti-albumin antibody or its antigen-binding fragment.

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

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

[0084] Anti-albumin antibody or its antigen-binding fragment, linking peptide, biologically active effector molecule;

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

[0086] Anti-albumin antibody or its antigen-binding fragment, linking peptide, first biologically active effector molecule, linking peptide, second biologically active effector molecule; or,

[0087] A first biologically active effector molecule, a linking peptide, an anti-albumin antibody or its antigen-binding fragment, a linking peptide, a second biologically active effector molecule, etc.; wherein the link may not include a linking peptide.

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

[0089] In one specific embodiment, the fusion construct comprises any one of the amino acid sequences of SEQ ID NOs 28-30 and 61, or has at least 80% identity with any one of the amino acid sequences of SEQ ID NOs 28-30 and 61.

[0090] In one specific embodiment, the fusion structure includes an Fc fragment.

[0091] More preferably, the Fc fragment comprises SEQ ID NO. 38 or has at least 80% identity with the amino acid sequence represented by SEQ ID NO. 38.

[0092] In one specific embodiment, the fusion construct comprises any one of the amino acid sequences of SEQ ID NOs 14-15, or has at least 80% identity with any one of the amino acid sequences of SEQ ID NOs 14-15.

[0093] Preferably, the fusion construct further comprises a secretion peptide. The secretion peptide is connected to the N-terminus of the fusion construct. More preferably, the amino acid sequence of the secretion peptide may be SEQ ID NO. 37 or 42.

[0094] Preferably, the fusion structure further includes a tag.

[0095] Preferably, the tag is connected to any one of the antibodies or its antigen-binding fragments, or to the C-terminus of the fusion construct.

[0096] Preferably, the fusion construct comprises a conjugate of an anti-albumin antibody and a drug, which comprises any anti-albumin antibody or an antigen-binding fragment thereof, and the biologically active effector molecule is a drug, and said drug is covalently bonded to any anti-albumin antibody or an antigen-binding fragment thereof.

[0097] In a fourth aspect of the present invention, a nucleic acid is provided, said nucleic acid encoding the anti-albumin antibody or its antigen-binding fragment or said fusion construct. For example, said nucleic acid comprises DNA and / or mRNA.

[0098] In some embodiments, the nucleic acid is an antibody or antigen-binding fragment according to the present application, or DNA encoding the fusion construct.

[0099] Preferably, the nucleotide sequence encoding the anti-albumin antibody or its antigen-binding fragment comprises any one of the nucleotide sequences of SEQ ID NOs 63-75, 79-81, and 83-87 or their degenerate sequences, or has at least 80% identity with any one of the nucleotide sequences of SEQ ID NOs 63-75, 79-81, and 83-87 and has a nucleotide sequence encoding the function of the anti-albumin antibody or its antigen-binding fragment.

[0100] Preferably, the nucleotide sequence encoding the fusion construct comprises any one of the nucleotide sequences of SEQ ID NOs 76-78 and 82 or the degenerate sequence thereof, or has at least 80% identity with any one of the nucleotide sequences of SEQ ID NOs 76-78 and 82 and has a nucleotide sequence encoding the function of the fusion construct.

[0101] In a fifth aspect of the present invention, a vector is provided, said vector comprises said nucleic acid.

[0102] The above vector may 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. Examples include E. coli-based vectors and phages.

[0103] In a sixth aspect of the present invention, a host cell is provided, said host cell comprises said nucleic acid or said vector.

[0104] The above 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.

[0106] Prokaryotic cells include, for example, E. coli.

[0107] In a seventh aspect of the present invention, a method for preparing a host cell is provided, said method comprising the step of introducing the nucleic acid or vector into the host cell.

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

[0109] In a ninth aspect of the present invention, a product for treating and / or diagnosing a disease is provided, said product for treating and / or diagnosing the disease, said product is

[0110] A) The above anti-albumin antibody or its antigen-binding fragment;

[0111] B) The above-mentioned fusion structure;

[0112] C) The above nucleic acid;

[0113] D) The above vector; or,

[0114] F) Includes any one of the above host cells.

[0115] Preferably, the anti-albumin antibody or its antigen-binding fragment and / or the biological effector molecule have therapeutic and / or diagnostic activity for a disease, and more preferably, the anti-albumin antibody or its antigen-binding fragment is used as a vector in a product for the treatment and / or diagnosis of a disease.

[0116] The above 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 the signaling pathways of the other targets.

[0117] Preferably, the fusion construct is a conjugate of an anti-albumin antibody and a drug, comprising any anti-albumin antibody or an antigen-binding fragment thereof, and the biologically active effector molecule is a drug, said drug being covalently bonded to any anti-albumin antibody or an antigen-binding fragment thereof.

[0118] In the tenth aspect of the present invention, a method for detecting albumin is provided, the method comprising the step of contacting a sample to be detected with the anti-albumin antibody or its antigen-binding fragment, and then detecting the content of a complex formed of albumin and the anti-albumin antibody or its antigen-binding fragment.

[0119] The above detection method is to detect the presence or content of albumin. Here, the presence indicates the absence or absence, and the content may be an expression amount or protein concentration, etc. Preferably, the albumin is derived from a mammal, and more preferably, the mammal is derived from a human, mouse, or monkey.

[0120] In the first aspect of the present invention, a method for treating and / or preventing a disease is provided, the method comprises the step of administering to an individual the anti-albumin antibody or its antigen-binding fragment, the fusion construct, the nucleic acid, the vector, the host cell, or a product for treating and / or diagnosing the disease.

[0121] The above 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 the signaling pathways of the other targets.

[0122] In one specific embodiment, the disease includes a disease related to the VEGFA signaling pathway. More preferably, it may be an ophthalmic disease related to tumors, abnormal angiogenesis, or neovascularization (e.g., retinal vascular disease).

[0123] Preferably, the target cells are selected from cells expressing VEGFA, such as cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granulosa cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells of the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, periareolar cells, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells, or tumor cells.

[0124] In a 12th aspect of the present invention, the application of the anti-albumin antibody or its antigen-binding fragment, the fusion construct, the nucleic acid, the vector, or the host cell in the manufacture of a product for the treatment and / or prevention of a disease is provided, wherein the anti-albumin antibody or its antigen-binding fragment and / or the biologically active effector molecule have therapeutic and / or diagnostic activity for the disease.

[0125] Preferably, the anti-albumin antibody or its antigen-binding fragment is used as a vector in a product for the treatment and / or diagnosis of a disease.

[0126] The above 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 the signaling pathways of the other targets.

[0127] In one specific embodiment, the disease includes a disease related to the VEGFA signaling pathway. More preferably, it may be an ophthalmic disease related to tumors, abnormal angiogenesis, or neovascularization (e.g., retinal vascular disease).

[0128] The term “fusion construct” as used in the text defines the fusion of the anti-albumin antibody of the present invention or its antigen-binding fragment with another compound. The fusion construct may comprise one or more anti-albumin antibodies or their antigen-binding fragments, and said multiple anti-albumin antibodies or their antigen-binding fragments may be identical or different. The fusion construct may comprise one or more other compounds, and said multiple other compounds may also be identical or different. The compounds may be protein compounds or non-protein compounds. If a compound is a protein compound or if the fusion construct comprises only multiple anti-albumin antibodies or their antigen-binding fragments, the fusion construct may also be referred to as a fusion protein. If a compound is fused with the anti-albumin antibody or its antigen-binding fragment in a conjugated form, the fusion construct may also be referred to as a conjugate.

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

[0130] The “drug” according to the present invention may be used for the treatment of human or non-human animals, e.g., non-human mammals. The drug may comprise pharmaceutically acceptable carriers, excipients, or salts commonly found in the prior art. The drug may be administered via any suitable route of administration, e.g., gastrointestinal administration (e.g., oral) or non-gastrointestinal administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organic, intranasal, intraocular, intracerebral, intrathecal, transdermal, rectal, etc.). The above drug may be any suitable formulation, for example, a formulation for gastrointestinal administration or a formulation for nasogastric administration, and preferably includes, but is not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder aerosols, detergents, ointments, warning agents, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, etc. Various formulations of the above drug may be manufactured according to general production methods in the pharmaceutical field. The above drug may contain the above anti-albumin antibody or its antigen-binding fragment, the above fusion construct, the above nucleic acid, the above vector, the above host cell, the above immune cell, etc., with a weight ratio of 0.01 to 99.5% (specifically, for example, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%).The above drug can be prepared using a reagent with a protein concentration of 1 to 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). A single dose of the above drug may be 0.1 to 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] "Pharmaceuticalally acceptable" according to the present invention means not inhibiting the biological activity and properties of the active substance of the administered product without significantly stimulating the organism.

[0132] The “…method” according to the present invention may be for the purpose of diagnosing and treating a disease, or for the purpose of diagnosing and treating a non-disease.

[0133] The “antigen-binding fragment” according to the present invention is a part of an antibody that maintains a specific binding activity of the antibody, that is, any part of the antibody can specifically bind to an epitope on the target molecule of the antibody. This includes, for example, Fab, Fab', F(ab')2, Fv, Fd, and variants of these fragments. For example, it is a heavy chain and / or light chain of the antibody, a variable region of the heavy chain and / or light chain of the antibody, or a single or more CDRs derived from the heavy chain or light chain of the antibody. Here,

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

[0135] Chimeric antibodies refer to antibodies in which part of the heavy chain and / or light chain originates from a specific origin or species, and the remainder of the heavy chain and / or light chain originates from a different origin or species.

[0136] A single-chain antibody is an antibody formed by connecting the antibody's heavy chain variable region and light chain variable region via a linker peptide.

[0137] Fab is a monovalent fragment consisting of 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 divalent fragment containing two Fab fragments connected by a disulfide bond in a hinge region.

[0140] Fd is an Fd fragment composed of VH and CH1 domains.

[0141] Fv is an Fv fragment composed of the VL and VH domains of a single antibody arm.

[0142] The dAb fragment is an antibody fragment composed of a VH domain.

[0143] The “linear antibody” according to the present invention comprises one or more pairs of serially connected antibody fragments, wherein the antibody fragments may be an Fd fragment (VH-CH1), a single-chain antibody (scFv), an antibody fragment (Fab), or a single-domain antibody (VHH), and these fragments are serially connected by a linking peptide to form a continuous antibody structure.

[0144] Here, VH represents the heavy chain variable region, VL represents the light chain variable region, CL represents the light chain invariant region, and CH represents the heavy chain invariant region.

[0145] The “Fc” region according to the present invention contains two heavy chain fragments comprising CH2 and CH3 domains of the antibody. The two heavy chain fragments form a dimer by two or more disulfide bonds in the hinge region and are held together through the hydrophobic action of the CH3 domain.

[0146] The “containing” or “containing” according to the present invention is an open description method, and when describing the sequence of a protein or nucleic acid, the protein or nucleic acid may consist 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] "Homology" or "identity" according to the present invention means that in the use of a protein sequence or nucleotide sequence, a person skilled in the art can adjust the sequence according to actual work needs, and the sequence used, when compared with a sequence obtained from the prior art, is 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%, 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%, It means to have homology of 99.7%, 99.8%, and 99.9% (including, but not limited to).

[0148] “Humanized antibody” according to the present invention means that a framework region and / or portion of an invariant region (e.g., CH region) of an antibody or all or part of an antibody is encoded by a human antibody gene. In one specific embodiment of the present invention, the CDR region of the antibody is not humanized.

[0149] The “individual” according to the present invention may be a human or non-human mammal, and the non-human mammal may be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, a pig, a cow, a sheep, a horse, a donkey, a fox, a raccoon, a mink, a camel, a dog, a cat, a rabbit, a rat (e.g., a rat, a mouse, a guinea pig, a hamster, a gerbil, a chinchilla, a squirrel), or a monkey, etc.

[0150] The “treatment” according to the present invention means alleviating, stopping, blocking, controlling, halting, reducing, or reversing the signs, symptoms, disorders, pathologies, or progression or severity of the disease after the onset of the disease, but does not necessarily mean the complete elimination of all disease-related signs, symptoms, pathologies, or disorders.

[0151] “Prevention” according to the present invention means a method performed to prevent or delay the occurrence of a disease, pathology, or symptom within an organism.

[0152] The term “diagnosis” according to the present invention means examining whether a patient has suffered from a disease or pathology in the past, at the time of diagnosis, or in the future, or examining the progression of the disease or possible future progression.

[0153] The “tumor” according to the present invention may be any harmful cell proliferation (or any disease manifesting as harmful cell proliferation itself), a neoplasm, or a predisposition or increased risk of harmful cell proliferation, a neoplasm, or a tumor. It may be benign or malignant, and may be primary or secondary (metastatic). A neoplasm may be any abnormal growth or proliferation of cells and may be located in any tissue. Examples of tissue embodiments include the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, appendix, central nervous system (with or without 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, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, and leukocytes. More preferably, the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (e.g., glioma), colorectal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, stomach cancer, bladder cancer, skin cancer, rhabdomyocarcinoma, lingual squamous cell carcinoma, nasopharyngeal cancer, ovarian cancer, placental choriocarcinoma, lymphoma (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermal carcinoma, colon cancer, thymoma, blood cancer, head and neck cancer, or oropharyngeal cancer.

[0154] “Fundus vascular disease” according to the present invention refers to a general term for diseases occurring in the retinal arteries or veins. This includes, but is not limited to, age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, neovascular glaucoma, etc. Effects of the invention

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

[0156] 1. The obtained anti-albumin nanobodies can bind to albumin of various species, including humans, monkeys, mice, and rats, with high affinity, and have simultaneously undergone humanization modification. The humanized anti-albumin antibodies bind to human albumin and mouse albumin, and can also bind to rat albumin. Since mice and rats are commonly used experimental animals, the anti-albumin nanobodies of the present invention have broader application prospects.

[0157] 2. Affinity enhancement was performed on a desirable humanized anti-albumin antibody to obtain an antibody with a 3.7-fold or 35.7-fold improved affinity for human albumin.

[0158] 3. Albumin has the characteristic of accumulating in inflammatory and tumor sites. Humanized anti-albumin nanobodies can be used in the manufacture of drugs for treating diseases such as immune diseases and tumors, and fusion constructs obtained by fusing anti-albumin nanobodies with other antibodies can bind to albumin in vivo and have concentration characteristics in inflammatory and tumor sites similar to albumin. Brief explanation of the drawing

[0159] Embodiments of the present invention are described in detail below with reference to the drawings. Here, Figure 1 is a Coomassie Brilliant Blue stained gel image of His-tagged antibodies Alb Nb, 2049, 3004, 3007, and 3005 after purification with a Ni column. (A) is a Coomassie Brilliant Blue stained gel image of antibodies Alb Nb, 2049, 3004, and 3007, and (B) is a Coomassie Brilliant Blue stained gel image of antibody 3005. Figure 2 shows ELISA tests for albumin binding of different species of fusion proteins 3005-Fc, 3007-Fc and Alb Nb-Fc. (A) is the HSA binding test (HSA binding ELISA), (B) is the MSA binding test (MSA binding ELISA), and (C) is the rat albumin binding test (Rat albumin binding ELISA). Figure 3 shows the Coomassie Brilliant Blue staining results after purifying the 3005 humanized antibody containing His tag with a Ni column. Figure 4 shows the ELISA activity test for MSA binding of antibody 3005 and its humanized antibody. Figure 5 shows the ELISA activity tests for HSA and rat albumin binding of antibody 3005, its humanized antibody 3005Hz6, and the control antibody Alb Nb. (A) is the HSA binding test (HSA binding ELISA), and (B) is the rat albumin binding test (Rat albumin binding ELISA). Figure 6 shows the binding and dissociation curves (SPR method) between the humanized antibody 3005Hz6 and HSA and MSA. (A) is the binding and dissociation curve of antibody 3005Hz6 and HSA, and (B) is the binding and dissociation curve of antibody 3005Hz6 and MSA, with the vertical axis representing the reaction value and the horizontal axis representing time. Figure 7 shows SDS-PAGE Coomassie Brilliant Blue stained gel images of humanized antibody 3005Hz6 mutant proteins 3005Hz6(EG), 3005Hz6(DS), and 3005Hz6(DA) after purification. (A) is an SDS-PAGE Coomassie Brilliant Blue stained gel image of 3005Hz6(EG) and 3005Hz6(DS), and (B) is an SDS-PAGE Coomassie Brilliant Blue stained gel image of 3005Hz6(DA). Figure 8 shows the detection of ELISA activity for HSA binding of the humanized antibody 3005Hz6 and its mutant proteins 3005Hz6(DA), 3005Hz6(DS), and 3005Hz6(EG) (HSA binding ELISA). Figure 9 is an SDS-PAGE gel image of fusion proteins V1SA-3005Hz6, 3005Hz6-V1SA, and V1DP-3005Hz6 after purification with a Ni column. (A) is an SDS-PAGE Coomassie Brilliant Blue stained gel image of V1SA-3005Hz6 and 3005Hz6-V1SA, and (B) is an SDS-PAGE Coomassie Brilliant Blue stained gel image of V1DP-3005Hz6. Figure 10 shows the ELISA activity detection for the binding of humanized antibody 3005Hz6 to HSA of the fusion protein of the humanized antibody 3005Hz6 and the monovalent anti-VEGFA nanobody. Figure 11 shows the VEGFR2 competitive ELISA activity test of the fusion protein and the monovalent nanobody V1-SA1 in HSA non-pre-culture or pre-culture. (A) is the result of HSA non-pre-culture, and (B) is the result of HSA pre-culture. Figure 12 shows the conjugation and purification flowchart of a nanobody-deruxtecan conjugate (NDC). (A) of Fig. 13 is a schematic diagram of the structure of a 3005 K65A-deruxtecan conjugate (NDC), and (B) is an SDS-PAGE Coomassie Brilliant Blue stained gel image of 3005 K65A and three different 3005 K65A-deruxtecan conjugates (NDC-1, NDC-2, NDC-3). Figure 14 (A) shows the ELISA activity test (HSA binding ELISA) for HSA binding of 3005 K65A and 3005 K65A-deruxtecan conjugate (NDC-3), and (B) shows the experimental results of inhibition of Mia Paca-2 cell proliferation by deruxtecan and 3005 K65A-deruxtecan conjugate (NDC-3). Figure 15 shows the Coomassie Brilliant Blue staining result after purification of the candidate antibody with improved affinity. Figure 16 shows the results of an ELISA experiment on HSA binding of a candidate antibody with enhanced affinity (HSA binding ELISA) (antibody coating method, coat antibody). (A) is the result of an ELISA experiment at pH=7.4, and (B) is the result of an ELISA experiment at pH=6.0. Figure 17 shows the results of an ELISA experiment on rat albumin binding of a candidate antibody with enhanced affinity (Rat albumin binding ELISA) (antigen coating method). (A) is the result of an ELISA experiment at pH=7.4, and (B) is the result of an ELISA experiment at pH=6.0. Figure 18 shows the binding and dissociation curves (SPR method) between the antibody with enhanced affinity, the control antibody, and HSA. (A) is the binding and dissociation curve of antibody 3005Hz6 and HSA, (B) is the binding and dissociation curve of antibody 1068 and HSA, (C) is the binding and dissociation curve of antibody 2028 and HSA, and (D) is the binding and dissociation curve of control antibody BI-V2 and HSA, where the vertical axis represents the reaction value and the horizontal axis represents time. Specific details for implementing the invention

[0160] Below, the technical means of the embodiment of the present invention will be clearly and completely described together with the drawings of the embodiment of the present invention; of course, the described embodiment is only a part of the embodiment of the present invention and not the whole. All other embodiments obtained by a person skilled in the art without inventive effort based on the embodiment of the present invention fall within the scope of protection of the present invention.

[0161] Example 1: Alpaca Immunity and Antibody Library Construction

[0162] 1. Alpaca Immunity and Titer Detection

[0163] 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 immunized at each dose, for a total of 3 doses at 2-week intervals. From the second immunization onwards, serum was collected 1 week after immunization to detect the titer.

[0164] Serum titer detection method:

[0165] After diluting the HSA antigen with CBS buffer to 1 μg / mL, 100 μL / well was added to the ELISA plate and left overnight at 4°C.

[0166] The plates were washed three times with PBST (PBS containing 0.1% Tween20, pH=7.4). 200 μL of skim milk powder was added to each well, left at room temperature for 1 hour, and then the plates were washed three times with PBST.

[0167] Sample addition: Post-immunization serum and negative serum were diluted 1,000, 3,000, 9,000, 27,000, 81,000, and 243,000 times, respectively, and 100 μL was added to each block-treated well. After leaving at room temperature for 1 hour, the plates were washed 3 times with PBST.

[0168] Secondary antibody: 100 μL of HRP-labeled Goat Anti-Alpaca IgG was added to each well, left at room temperature for 1 hour, and then the plate was washed 3 times with PBST.

[0169] TMB color development: 100 μL / well of TMB color development solution was added to the ELISA plate and color developed for 15 minutes; 50 μL / well of stop solution was added, and the ELISA plate was placed in a microplate reader to read the absorbance (OD450) at 450 nm.

[0170] The results of alpaca serum titer detection after the second and third immunization are shown in Table 2. As can be seen, even after diluting the serum 243,000 times, the binding signal value to human serum albumin was 0.59 or higher, satisfying all criteria for library construction, and a phage library was constructed using alpaca serum after the third immunization.

[0171] Table 2 Detection of Alpaca Serum Tit after Secondary and Tertiary Albumin Immunization

[0172]

[0173] 2. Building the Fragment Library

[0174] Peripheral alpaca blood was collected, and total RNA was extracted from alpaca PBMCs isolated using Trizol. Subsequently, cDNA reverse transcription was performed, and the variable region VH fragment (approx. 700 bp in length) was amplified using nanobody-specific primers and amplified twice. The fragment obtained above and the pcomb3X vector were cleaved with the SfiI enzyme, mixed in appropriate proportions, and ligated with T4 ligase. After ligation, XL1-Blue competent cells were transformed using electroporation. The capacity of the alpaca transformation library, calculated based on the growth status of colonies formed by culturing competent cell dilutions on antibiotic-containing plates, was 6.24 × 10⁶. 8 It was.

[0175] Example 2: Acquisition of antibodies cross-binding to human, mouse, and cyanomorphic monkey albumin via antibody library screening

[0176] 1. Fragment library screening

[0177] Screening Procedure:

[0178] Antigens human serum albumin HSA and mouse albumin MSA (purchased from Sigma-Aldrich, catalog number: A3559) were coated, and a negative control (ELISA plate coated with 3% skim milk powder) was simultaneously set up and incubated at room temperature for 1 hour. The blocking buffer was discarded and washed with PBST.

[0179] Approximately 5×10 12 The pfu library was added to the antigen plate, reacted with the antigen at 37°C for 2 hours, and then the plate was washed with 0.1% PBST.

[0180] After eluting with low pH glycine-HCl, the pH was neutralized using Tris-HCl until it reached 7.4.

[0181] The eluent was mixed with E. coli XL1-Blue and cultured at 37°C while shaking.

[0182] After adding the phage, the mixture was incubated overnight at 30°C, and the supernatant was collected.

[0183] You can obtain a secondary library by repeating steps 1) to 5).

[0184] When using the secondary amplification library obtained under HSA coating conditions for ELISA detection, the antigen HSA was coated with two coating buffers (PBS buffer or CBS buffer), and then ELISA binding detection was performed. For phage detection, HRP-labeled Anti-M13 Antibody (purchased from Sino Biological Inc., catalog number: 11973-MM05T-H) was used after diluting it 2500-fold. After incubating the detection antibody, the plate was washed, color developed with TMB, and the OD450 was read. The detection results of the secondary amplification library are shown in Table 3. As can be seen, a phage signal strongly binding to HSA was generated after secondary enrichment.

[0185] Table 3 HSA binding detection results of secondary phage libraries concentrated under HSA coating conditions

[0186]

[0187] Antigen binding ELISA tests were performed on the secondary amplification library obtained under MSA coating conditions. In the ELISA experiments, HSA and MSA were coated using CBS buffer, respectively, and the ELISA detection results are shown in Table 4. As is known, phages that strongly bind to HSA and MSA were obtained even under the corresponding concentration conditions.

[0188] Table 4. HSA and MSA binding detection results of secondary phage libraries concentrated under MSA coating conditions

[0189]

[0190] Summary: Antigens of secondary phage libraries enriched via HSA and MSA coatings

[0191] The binding ELISA detection result was positive, indicating that phages binding to HSA and MSA, respectively, were concentrated, and binding screening at the phage monoclonal level can be performed.

[0192] 2. Phage Monoclonal Screening and Sequencing

[0193] Experimental Procedure:

[0194] 96 monoclonal colonies were selected from a primary phage library enriched through HSA coating and used for phage expression, and ELISA binding activity was detected by coating with HSA protein.

[0195] 96 monoclonal colonies were selected from a secondary phage library concentrated via MSA coating and used for phage expression, and ELISA binding activity was detected by coating them with HSA, MSA, and monkey albumin, respectively.

[0196] Antigens HSA, MSA, and monkey albumin were coated, with a protein concentration of 0.5 μg / mL at the time of coating, and coated overnight at 4℃.

[0197] Blocking: 3% milk powder, 200μL / well, room temperature for 1 hour.

[0198] 100 μL / well of phage expression supernatant diluted 10-fold was added, and the mixture was left at room temperature for 1 hour.

[0199] 100 μL / well of detection antibody Anti-M13 Antibody (HRP label) (concentration 0.2 μg / mL) was added, and the mixture was left at room temperature for 1 hour.

[0200] 200 μL / well of TMB colorimetric solution was added and the color was developed for 20 minutes, after which 50 μL / well of stop solution was added. OD450 was detected using a microplate reader.

[0201] One clone (No. 2049) of the 96 phage clones of the primary phage library enriched by HSA coating was selected, and three clones (Nos. 3004, 3005, 3007) of the 96 phage clones of the secondary phage library enriched by MSA coating were selected, and the ELISA detection results are as shown in Tables 5 and 6.

[0202] Table 5 Detection of HSA binding activity of phage monoclonal 2049

[0203]

[0204] Table 6 Detection of HSA, MSA, and monkey albumin binding activity of phage monoclones 3004, 3005, and 3007

[0205]

[0206] The sequences of four phage clones were obtained through DNA sequencing, and the amino acid sequences of the four antibodies CDR-H1, CDR-H2, and CDR-H3 are as shown in Table 7, and the amino acid and nucleotide sequences of the four nanobodies are as shown in Table 8.

[0207] Table 7. Amino acid sequences of the CDR regions of four anti-albumin nanobodies

[0208]

[0209] Table 8 Amino acid sequences and nucleotide sequences of four anti-albumin nanobodies

[0210]

[0211]

[0212] Example 3: Detection of albumin binding activity of different species of anti-albumin candidate antibodies

[0213] 1. Expression of anti-albumin candidate antibodies

[0214] These four candidate antibodies were expressed in mammalian cells, and after protein purification, an antigen binding activity test was performed to verify the albumin binding activity of the candidate antibodies at the protein level.

[0215] Expression plasmids of His-tagged antibodies 2049, 3004, 3005, and 3007 and a positive control antibody Alb Nb, respectively, were constructed via gene synthesis, wherein the positive control antibody Alb Nb is an anti-human serum albumin nanobody, the antibody amino acid sequence is derived from SEQ ID No. 62 of Patent US 2007 / 0269422 A1, and the amino acid sequence is as follows:

[0216] EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 62).

[0217] The tPA secretion signal peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ No. 37) was added to the N-terminus of the above antibody, and the linker GGGGS (SEQ No. 41) and 6xHis were added to the C-terminus of the antibody, and the gene sequence was inserted into the expression vector pCDNA3.1(+) through restriction sites NheI and XbaI. After confirming that the plasmid sequencing was correct, the endotoxin was removed and the sample was extracted in large quantities. Transient expression and Ni column affinity purification were performed on floating 293F cells using the transformation reagent polyethyleneimine (PEI).

[0218] The SDS-PAGE staining results of the transient expression cell culture supernatant (Input), effluent not bound to the Ni column (FT), and eluted purified product (Eluate) of His-tagged antibodies Alb Nb, 2049, 3004, and 3007 are shown in Figure 1 (A), and the SDS-PAGE staining results of the product purified by the Ni column with His-tagged antibody 3005 are shown in Figure 1 (B), and their molecular weights were (~15 kDa) as expected.

[0219] Simultaneously, fusion proteins of antibodies 3005, 3007, and positive control antibodies Alb Nb and Fc were constructed, and the tPA secretion signal peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID No. 37) was added to the N-terminus of the antibody coding sequences via primer design. The amino acid sequence of human IgG1 Fc: PPCPAPELLGGPSVFLFPPKPK was added to the C-terminus of the antibody sequences via a homologous recombination method. DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence No. 38) was added, and the gene fragment was inserted into the mammalian cell expression vector pCDNA3.1(+).

[0220] The amino acid and nucleotide sequences of the anti-albumin nanobody-Fc fusion protein are as shown in Table 9.

[0221] Table 9 Amino acid sequence and nucleotide sequence of anti-albumin nanobody-Fc fusion protein

[0222]

[0223]

[0224] After confirming that the expression plasmid sequencing of the Fc fusion protein was correct, the endotoxin was removed and the target protein was obtained through bulk extraction, transient expression in suspended 293F cells, and Protein A affinity purification.

[0225] 2. Detection of albumin binding activity of anti-albumin candidate antibodies

[0226] The binding affinity of monovalent nanobodies to albumins of different species was tested using Bio-Layer Interferometry (BLI). Eight NTA sensors were used to bind monovalent nanobodies (concentration 1 μg / mL) tagged with the same his tag, and the antigens were human serum albumin HSA (Baxter AG), mouse albumin MSA (purchased from Equitech-bio, catalog number: MSA62-1000), and rat albumin (purchased from abcam, catalog number: ab198656), respectively. In the BLI experiment, the eight antigen concentration gradients were set to 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM, and the antigens and antibodies were bound for 180 seconds and dissociated for 360 seconds.

[0227] Table 10 shows the binding rates (ka), dissociation rates (kd), and equilibrium dissociation constants (K) for albumin binding of different species of his-tagged monovalent nanobodies 2049, 3004, 3005, and 3007 after fitting to a “1:1” model. D Shows ).

[0228] Table 10. Binding rates (ka), dissociation rates (kd), and equilibrium dissociation constants (K) for albumin binding of different species of nanobodies 2049, 3004, 3005, and 3007 D )

[0229]

[0230] According to the data, 2049, 3004, 3005, and 3007 were all found to be able to bind to HSA and MSA with high affinity, and 3005 and 3007 were also found to be able to bind to rat albumin.

[0231] The albumin binding activity of different species 3005 and 3007 was compared again using the ELISA method, and the experimental method is as follows.

[0232] The antigens were diluted with ELISA coating buffer, and 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. 100 μL / well of the antigen dilution was added to the ELISA plates, and the plates were coated overnight at 4°C. After blocking with 5% skim milk powder, gradient dilutions (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 nM) of candidate antibodies 3005-Fc, 3007-Fc and positive control antibody Alb Nb-Fc, composed of PBST (PBS containing 0.1% Tween20, pH=7.4), were added and incubated at 37°C for 1 hour. After washing the plate, HRP-labeled goat anti-human IgG Fc antibody (purchased from Abbkine, catalog number: A21050) was added and incubated at room temperature for 45 minutes. After washing the plate, 100 μL of TMB (purchased from Tiangen Biotech, catalog number: PA107-01) was added to each well, and after developing the color at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured using a microplate reader.

[0233] The ELISA test results for albumin binding of different species of 3005-Fc, 3007-Fc, and Alb Nb-Fc are shown in Figure 2. The EC50 summary of the ELISA experiments for 3005-Fc and 3007-Fc binding is shown in Table 11.

[0234] Table 11. EC50 Summary of ELISA Experiments on Albumin Binding of Different Species of 3005-Fc and 3007-Fc

[0235]

[0236] Referring to Table 11, antibodies 3005-Fc and 3007-Fc were found to be able to efficiently bind to HSA and MSA. Referring to Figure 2, the positive control antibody Alb Nb-Fc showed very weak rat albumin binding activity, while antibodies 3005-Fc and 3007-Fc both exhibited excellent rat serum albumin binding activity, and the rat albumin binding activity of antibody 3005-Fc was superior to that of 3007-Fc. Therefore, subsequent studies were preferably conducted using antibody 3005.

[0237] Example 4: Humanized modification of an anti-albumin candidate antibody

[0238] 1. Humanized modification of candidate antibodies and expression purification

[0239] Humanization modification was performed by transplanting the CDR region of the antibody. Humanization modification of the antibody was performed using candidate antibody 3005 (Sequence No. 12) as the starting antibody. First, homology between the sequence of candidate antibody 3005 and the human antibody sequence was compared using the antibody databases IGBLAST and IMGT. Then, a humanized antibody was constructed by combining the human antibody framework region sequence with high homology and the three CDR region sequences of candidate antibody 3005 (Sequence No. 2, Sequence No. 5, and Sequence No. 9).

[0240] A secretion signal peptide was added to the N-terminus of the antibody, and the linkers GGGGS (SEQ No. 41) and 6xHis were added to the C-terminus of the antibody, and the coding DNA sequence was obtained through gene synthesis. It was then inserted into the expression vector pCDNA3.1(+) via restriction sites NheI and XbaI. After confirming that the plasmid sequencing was correct and removing endotoxins, the sample was subjected to bulk extraction, followed by transient expression and Ni column affinity purification in suspended 293F cells.

[0241] The amino acid and nucleotide sequences of the humanized modified protein of antibody 3005 are as shown in Table 12.

[0242] Table 12 Amino acid and nucleotide sequences of 3005 humanized antibodies

[0243]

[0244] After purifying the humanized protein containing the His tag with a Ni column and separating it by SDS-PAGE electrophoresis, the molecular weight was as expected (~13 kDa), and the Coomassie Brilliant Blue staining results are shown in Fig. 3.

[0245] 2. Detection of humanized antibody activity

[0246] First, the mouse albumin MSA binding activity of the raw candidate antibody 3005 and the humanized modified antibody was detected using the ELISA method.

[0247] In the experiment, mouse albumin MSA (coated protein concentration 5 μg / mL) was coated onto ELISA plates at a rate of 100 μL / well and coated overnight at 4°C. After blocking with 5% skim milk powder, dilutions containing the His-tag protein to be tested (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 nM) were prepared using PBST (PBS containing 0.1% Tween20, pH=7.4), and the proteins to be tested were the original antibody 3005 and four humanized modified antibodies. The protein dilutions were added to the ELISA plates and incubated at 37°C for 1 hour; after washing the plates, 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 the plate, 100 μL of TMB (purchased from Tiangen Biotech, catalog number: PA107-01) was added to each well, and after developing the color at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured using a microplate reader.

[0248] The ELISA test results for MSA binding of antibody 3005 and its humanized antibody are shown in Fig. 4. The EC50 summary of the binding ELISA experiment is as shown in Table 13.

[0249] Table 13 Summary of EC50 and maximum binding values ​​from ELISA experiments on MSA binding of antibody 3005 and its humanized antibody

[0250]

[0251] The results showed that the MSA binding activity of the humanized antibody 3005Hz6 was similar to that of the original antibody 3005.

[0252] The binding activity of raw candidate antibody 3005 and humanized 3005Hz6 to HSA and rat albumin was detected using the ELISA method.

[0253] In the experiment, HSA or rat albumin (coated protein concentration 2 μg / mL) was coated at a rate of 100 μL / well onto ELISA plates and coated overnight at 4°C. After blocking with 5% skim milk powder, dilutions containing His tag proteins to be tested (0.0001, 0.001, 0.01, 0.1, 0.3, 1, 10, 100 nM) were prepared using PBST (PBS containing 0.1% Tween20, pH=7.4), and the proteins to be tested were positive control antibody Alb Nb, raw antibody 3005, and humanized antibody 3005Hz6. The protein diluent was added to the ELISA plate and 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 the plate, 100 μL of TMB (purchased from Tiangen Biotech, catalog number: PA107-01) was added to each well and incubated at 37°C for 15 minutes, after which 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured using a microplate reader.

[0254] The ELISA results are shown in Figure 5 (A) and Figure 5 (B), respectively. The EC50 values ​​fitted from the binding ELISA curves of Figure 5 are as shown in Table 14.

[0255] Table 14. EC50 Summary for HSA and Rat Albumin Binding of Candidate Antibody 3005 and Its Humanized Antibody 3005Hz6

[0256]

[0257] As can be seen in Figure 5 and Table 14, the HSA and rat albumin binding activity of the humanized antibody 3005Hz6 was equivalent to that of the original antibody 3005 and was stronger than that of the positive control antibody Alb Nb.

[0258] Binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K) for HSA and MSA binding of humanized antibody 3005Hz6 using the Surface Plasmon Resonance (SPR) method D ) was detected.

[0259] In the experiment, a Biacore 8K instrument (Cytiva) was used, and the His-tagged humanized antibody 3005Hz6 was captured using the Series S Sensor Chip NTA. The analytes were HSA or MSA at gradient concentrations, and the analytes were diluted with mobile phase buffer (components: 10 mM HEPES, 150 mM 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 antibody 3005Hz6 and HSA are shown in Fig. 6 (A), and the binding and dissociation curves of antibody 3005Hz6 and MSA are shown in Fig. 6 (B).

[0260] Using the “Two-state reaction” model, the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K) for the binding of HSA and MSA at 3005 Hz6 D ) was fitted, and the results are as shown in Table 15.

[0261] Table 15. Binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K) of humanized antibody 3005Hz6 against HSA and MSA D )

[0262]

[0263] As shown in Table 15, the binding affinity of the humanized antibody 3005Hz6 for HSA and MSA is similar, and K measured by the SPR method D It was about 11 nM.

[0264] K for HSA and MSA of raw antibody 3005 measured by the BLI method in Table 10 of Example 3 D Compared to the value of approximately 5 nM, K in Table 15 D Since the difference between the values ​​is within three times, the binding affinity of humanized antibody 3005Hz6 for HSA and MSA is considered to be similar to that of raw antibody 3005.

[0265] Example 5: Potential Chemical Modification Site Modification and Activity Test of Humanized Antibody 3005Hz6

[0266] A. Methods for Modifying Potential Chemical Modification Sites of Humanized Antibody 3005Hz6 and Expression Purification

[0267] The CDR-H2 region (SEQ No. 5) of the 3005Hz6 antibody contains the amino acid motif DG. Compared to other amino acid motifs (e.g., DS, DA), DG can further promote the isomerization of aspartate D to isoaspartate, which can alter the structure and antigen-binding activity of the antibody. Aspartate isomerization can accelerate protein hydrolysis and enhance the immunogenicity of the protein.

[0268] Accordingly, in this embodiment, point mutation modifications were performed on the amino acid combination DG of the CDR-H2 region of 3005Hz6 to reduce the risk of changes in the protein's structure and activity. Specifically, the point mutation scheme for the amino acid combination DG of 3005Hz6 is as follows: aspartate D was replaced with glutamate E, or glycine G was mutated to A, which has a simple structure, or S, which is prone to forming a flexible structure. The number 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 site of the humanized antibody 3005Hz6 are shown in Table 17.

[0269] Table 16 Amino acid sequences of the mutant CDR region of the potential chemical modification site of the humanized antibody 3005Hz6

[0270]

[0271] Table 17 Full-length amino acid sequences and nucleotide sequences of mutants at the potential chemical modification site of humanized antibody 3005Hz6

[0272]

[0273] A secretion signal peptide (SEQ No. 37) was added to the N-terminus of the mutant protein of antibody 3005Hz6, and the linkers GGGGS (SEQ No. 41) and 6xHis were added to the C-terminus of the antibody, and the coding DNA sequence was obtained through gene synthesis. Then, it was inserted into the expression vector pCDNA3.1(+) via restriction sites NheI and XhoI. After confirming that the plasmid sequencing was correct and removing endotoxins, the sample was subjected to large-scale extraction, transient expression was performed 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 medium supernatant (Input), the effluent that did not bind to the Ni column (FT), and the eluted purified product (E) of transiently expressed 3005Hz6 (EG) and 3005Hz6 (DS) are shown in Figure 7 (A). The cell supernatant of transiently expressed 3005Hz6(DA) protein was purified using a Ni column and then purified by cation exchange chromatography. The SDS-PAGE results of the protein after purification are shown in Figure 7 (B). The molecular weight of the three mutant proteins was 13 kDa.

[0274] B. Activity testing of potential chemical modification site modifiers of humanized antibody 3005Hz6

[0275] ELISA detection of HSA binding activity was performed on humanized antibody 3005Hz6 mutant proteins 3005Hz6(DA), 3005Hz6(DS), and 3005Hz6(EG). In the ELISA experiment, 2 μg / mL of HSA was coated, a concentration gradient of 3005Hz6 or its mutants was added, and the antibody levels binding to HSA were detected using an 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, and the EC50 of the ELISA experiment is shown in Table 18.

[0276] Table 18. Summary of EC50 for HSA binding of humanized antibody 3005Hz6 mutant proteins 3005Hz6(DA), 3005Hz6(DS), and 3005Hz6(EG).

[0277]

[0278] As can be seen in Table 18, the humanized antibody 3005Hz6 and its mutant proteins have similar HSA binding activity.

[0279] Example 6: Fusion expression and activity detection of humanized anti-albumin antibody and other antigen-binding proteins

[0280] A. Fusion Expression and Purification of Humanized Antibody 3005Hz6 and Monovalent Anti-VEGFA Nanobodies

[0281] After fusion expression of the humanized antibody 3005Hz6 with an antibody that binds to another antigen, the fusion protein may bind to albumin in vivo and increase its molecular weight, or after binding to albumin, participate in the binding and recycling mechanism between albumin and FcRn, thereby potentially extending the half-life of the fusion protein in vivo.

[0282] Two anti-VEGFA nanobodies (V1-SA1 (SEQ No. 26) and V1-DP (SEQ No. 27)) (Table 19) were selected and fused with the humanized antibody 3005Hz6, and the activity of the fusion proteins obtained according to different serial linkage methods was compared.

[0283] Table 19 Item - VEGFA nanobody sequence

[0284]

[0285] When humanizing antibody 3005Hz6 and monovalent anti-VEGFA nanobodies V1-SA1 (SEQ No. 26) and V1-DP (SEQ No. 27) were serially linked using a linker GGS, the amino acid sequence and nucleotide sequence of the fusion protein are as shown in Table 20.

[0286] Table 20 Amino acid and nucleotide sequences of the fusion protein of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0287]

[0288]

[0289] A secretion signal peptide was added to the N-terminus of the fusion protein, and the linkers GGGGS and 6xHis were added to the C-terminus of the antibody; the coding DNA sequence (see Table 20) was obtained through gene synthesis. It was then inserted into the expression vector pCDNA3.1(+) via the restriction sites NheI and XhoI. After confirming the plasmid sequencing was correct and removing endotoxins for large-scale extraction, transient expression was performed using suspended 293F cells, followed by Ni column affinity purification from the cell culture supernatant. During the purification process, the SDS-PAGE results of the cell medium supernatant (Input), the effluent not bound to the Ni column (FT), and the eluted purification product following transient transformation of each protein are shown in Figure 9, and the molecular weight of the target protein was approximately 28 kDa.

[0290] B. Detection of fusion protein activity of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0291] 1. Detection of whether the two nanobody elements constituting the fusion protein maintain their original functions.

[0292] The HSA binding activity of the anti-albumin nanobody 3005Hz6 within the fusion protein was detected. The detection method is the same as that in “2. Detection of activity of humanized antibody” of Example 4. The ELISA results are shown in Fig. 10, and the EC50 of the ELISA experiment is as shown in Table 21.

[0293] Table 21 Summary of EC50 for HSA binding of fusion proteins or 3005Hz6

[0294]

[0295] As can be seen in Table 21, the HSA binding activity of the fusion protein is similar to that of 3005Hz6 alone, indicating that fusing other antibody fragments to 3005Hz6 does not affect the activity of 3005Hz6. Additionally, biologically active effector molecules can be located at the N-terminus or C-terminus of the anti-albumin antibody, and different locations do not fundamentally affect the albumin binding activity of the fusion protein.

[0296] 2. Detection of activity of anti-VEGFA nanobodies within fusion proteins

[0297] VEGFR2 competitive ELISA was used for detection. In addition, the effect of the fusion protein binding to HSA and not binding on the VEGFR2 competitive activity of the fusion protein was compared.

[0298] In competitive ELISA experiments, goat anti-human IgG Fc protein (purchased from Solarbio, catalog number: SPA105) was coated onto ELISA plates at a coating concentration of 5 μg / mL overnight at 4°C. After washing the plates with PBST and blocking with milk powder, VEGFR2 extracellular domain-human IgG1 Fc fusion protein (VEGFR2-Fc, internally prepared) was added. The fusion protein of the concentration gradient was incubated with a constant 1 μM HSA or PBST for 30 minutes, followed by incubation with a constant final concentration (0.4 nM) of biotin-modified VEGFA165-Avi-His for 1 hour. VEGFA165-Avi-His was internally synthesized by adding a linker, an Avi tag, and a 6xHis tag 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, and biotin modification on the Avi tag was performed via the catalytic action of the BirA enzyme during protein expression. The amino acid sequence of VEGFA165-Avi-His is as follows: APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKK D RARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRRGSGSGLNDIFEAQKIEWHEGGGGSHHHHH (SEQ ID NO. 39).

[0299] The amino acid sequence of VEGFR2-Fc is as follows:

[0300] ASVGLPSVSLDLPRLSIQK DILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPQIGEKSLISPVDSYQYGTTQTLTCTVYAIPPPHHIHWYWQLEEECANEPSQAVSVTNPYPCEEWRSVEDFQGGNKIEVNKNQFALIEGKNKTVSTLVIQAANVSALYKCEAVNKVGRGERVISFHVTRGPEITLQPDMQPTEQESVSLWCTADRSTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSNSTNDILIMELKNASLQDQGDYVCLAQDRKTKKRHCVVRQLTVLERVAPTITGNLENQTTSIGESIEVSCTASGNPPPQIMWFK D NETLVEDSGIVLK D GNRNLTIRRVRKEDEGLYTCQACSVLGCAKVEAFFIIEGAQEKTNLEPPCPAPELLGGPSVFLFPPKPK D TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(서열번호 43).

[0301] A mixture of the fusion protein and VEGFA165-Avi-His was added to an ELISA plate adsorbed with VEGFR2-Fc and incubated at 37°C for 1 hour. Biotin signal of VEGFA165-Avi-His was detected using the HRP-labeled streptavidin antibody Streptavidin-HRP (purchased from Sangon Biotech, catalog number: D111054-0001), incubated at room temperature for 45 minutes, and after washing the plate, 100 μL / well of TMB colorimetric solution was added to the ELISA plate and color developed for 15 minutes; 50 μL / well of stop solution was added, and the ELISA plate was placed in a microplate reader to read the absorbance (OD450) at 450 nm.

[0302] The competitive activity of the fusion protein and the monovalent nanobody V1-SA1 against VEGFR2 without HSA pre-culture is shown in Fig. 11 (A), and the competitive activity against VEGFR2 after HSA pre-culture is shown in Fig. 11 (B). A summary of the IC50 of the VEGFR2 competitive experiment is shown in Table 22.

[0303] Table 22 Summary of VEGFR2 competitive IC50 of fusion proteins and monovalent nanobody V1-SA1

[0304]

[0305] As shown in Table 22, the VEGFR2 competitive activity of the three fusion proteins formed by serially linking 3005Hz6 with V1-SA1 or V1-DP was similar to that of the monovalent nanobody V1-SA1 under conditions without HSA incubation (IC50 ratio 0.81–1.08). This indicates that the anti-VEGFA antibody does not affect anti-VEGFA activity even when fused with 3005Hz6. Furthermore, biologically active effector molecules can be located at the N-terminus or C-terminus of the anti-albumin antibody, and different locations do not fundamentally affect the activity of the biologically active effector molecules within the fusion protein.

[0306] To simulate the anti-VEGFA activity of antibodies or fusion proteins under conditions containing albumin in vivo, HSA was pre-incubated with the fusion protein or the monovalent nanobody V1-SA1 in a competitive ELISA experimental system, and then the competitive activity of VEGFA binding with VEGFR2 was detected. Table 22 shows that the IC50 value of the monovalent nanobody V1-SA1 under HSA pre-incubation conditions is approximately twice that of the system without HSA. This indicates that HSA can have an overall effect on the IC50 values ​​of the competitive ELISA experiment. On the other hand, the ratio of the IC50 value of the fusion protein to that of V1-SA1 is 0.88–1.40, which indicates that the competitive activity of the fusion protein against VEGFR2 is similar to that of the monovalent nanobody V1-SA1.

[0307] Synthesizing the data from Tables 21 and 22, the fusion protein of the monovalent anti-VEGFA nanobody (V1-SA1 or V1-DP) and the anti-albumin nanobody (3005Hz6) exhibits very strong VEGFR2 competitive activity and HSA binding activity. This demonstrates that antibody elements binding to different antigens within the fusion protein can function independently, and that there is minimal mutual interference between the antibody elements. Therefore, it is possible to construct a multispecific and multifunctional fusion protein by fusion-expressing antibodies binding to different antigens with the anti-albumin humanized antibody 3005Hz6.

[0308] Example 7: Chemical conjugation of anti-albumin nanobody 3005 mutant and small molecule toxin deruxtecan and measurement of conjugate activity

[0309] A. Construction and Expression Purification of Anti-Albumin Nanobody 3005 Mutant for Conjugation Reaction

[0310] The 65th amino acid in the amino acid sequence of the anti-albumin nanobody 3005 is lysine residue K, which has high reactive activity, and this reactive site is located in the CDR-H2 region. K65 located in CDR-H2 can significantly reduce the affinity of the conjugate for antigen albumin after covalently bonding with a small molecule. Therefore, in this example, point mutation modification was performed on K65 within the nanobody 3005 sequence, and the lysine residue having a primary amino acid was mutated into an amino acid with no reactive activity (e.g., alanine A, arginine R, histidine H, etc.). The newly constructed mutants were named 3005 K65A, 3005 K65R, 3005 K65H, etc.

[0311] Table 23 Amino acid sequence of the CDR region of antibody 3005 mutant

[0312]

[0313] Table 24 Amino acid and nucleotide sequences of antibody 3005 mutant

[0314]

[0315] The amino acid sequence of the CDR region of the anti-albumin nanobody 3005 mutant is as shown in Table 23, and the amino acid and nucleotide sequences of the mutant are as shown in Table 24. The method of expression and purification of the mutant is identical to “1. Expression of anti-albumin candidate antibody” in Example 3 of this patent. Briefly, a secretion signal peptide (SEQ No. 37) was added to the N-terminus of the antibody 3005 mutant protein, and the linker GGGGS (SEQ No. 41) and 6xHis were added to the C-terminus of the antibody, and the coding DNA sequence was obtained through gene synthesis. Then, it was inserted into the expression vector pCDNA3.1(+) via the restriction sites NheI and XhoI. After confirming that the plasmid sequencing was correct and removing endotoxins, the sample was subjected to large-scale extraction. Transient expression was then performed using suspended 293F cells, and Ni column affinity purification was performed on the cell culture supernatant. After purification, the Q-tof detection molecular weight of the three mutant proteins was 13.8 kDa.

[0316] The affinity of antibody 3005 and its mutant proteins 3005 K65A, 3005 K65R, and 3005 K65H for albumin of different species was tested using Bio-Layer Interferometry (BLI) technology. An NTA sensor was used to bind the his-tagged antibody (concentration 5 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 (purchased from abcam, catalog number: ab198656), respectively. In the BLI experiment, eight antigen concentration gradients 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.

[0317] The BLI detection results are as shown in Table 25, and the results showed that the binding affinity of the different species of mutant proteins 3005 K65A, 3005 K65R, and 3005 K65H to albumin was similar to that of antibody 3005.

[0318] Table 25. Binding rates (ka), dissociation rates (kd), and equilibrium dissociation constants (K) for albumin binding of different species of antibody 3005 and its mutant proteins 3005 K65A, 3005 K65R, and 3005 K65H D )

[0319]

[0320] B. Method for manufacturing 3005 K65A and deruxtecan conjugate (Nanobody Drug Conjugate, NDC)

[0321] In this embodiment, the preparation, purification, and activity measurement of a conjugate are described using mutant 3005 K65A as an example. For 3005 K65A, a nanobody intermediate (3005 K65A-SH) having a mercapto can be obtained using methods known to those skilled in the art. For example, a thiolization intermediate may be produced by reacting a primary amino of a lysine residue with 2-iminothiolane hydrochloride (Traut's Reagent); or a highly active thiolization intermediate may be released by treating with hydroxylamine after covalent bonding of a primary amino of a lysine residue with an N-succinimidyl S-acetylthioglycolate (SATA) series linker; There are methods, but are not limited to: reacting a lysine residue with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and then reducing the disulfide bonds with a reducing agent such as dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to obtain a thiolization intermediate; or using a certain concentration of the reducing agent dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce the disulfide bonds within the nanobody molecule to produce mercapto.

[0322] A thiolized nanobody active intermediate and deruxtecan are covalently bonded via a thioether bond to obtain a nanobody-deruxtecan conjugate. In this example, a primary amino and 2-iminothiolane hydrochloride are used as examples. A primary amino of a lysine residue in the 3005 K65A framework and 2-iminothiolane hydrochloride form an amidine bond at pH 7-10 to produce a free mercapto, and the free mercapto on the thiolized nanobody intermediate 3005 K65A-SH framework and the deruxtecan maleimide group undergo a Michael addition reaction to produce a structurally stable 3005 K65A-deruxtecan conjugate (NDC).

[0323] The conjugation and purification flowchart of the nanobody-deruxtecan conjugate is as shown in Fig. 12.

[0324] C. Purification and Structural Characterization of 3005 K65A-Deruxtecan Conjugate

[0325] The preparation process for the 3005 K65A-deruxtecan conjugate is as follows: A deruxtecan solution was added dropwise to a buffer containing the thiolized nanobody intermediate 3005 K65A-SH while continuously shaking to allow the reaction to proceed. The reaction buffer for 3005 K65A-SH and deruxtecan may be a sodium phosphate salt, a sodium acetate solution, or sodium borate. During the reaction, the pH is generally between 5 and 9, and a pH of approximately 7 is preferred. The organic cosolvent dissolving the deruxtecan may be selected from organic solvents such as N,N-dimethylacetamide (DMAC), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and N-methyl-2-pyridone (NMP). In this example, a DMAC solution containing dissolved deruxtecan was added to a buffer of 3005 K65A-SH containing mercapto at a concentration of 8–15% v / v to allow the reaction to proceed. The reaction temperature was 10–25°C, and the reaction time was 1 hour. After the reaction was finished, 1–2 molar equivalents of a thiol-containing reagent (cysteine ​​or N-acetyl-L-cysteine ​​(NAC)) were added to the reaction system, and the reaction was terminated by incubating at room temperature for 20 minutes to inactivate the unreacted deruxtecan reactivity.

[0326] The purification strategy for the conjugate is as follows: Purification was performed using a commercially available HiTrap Desalting column (purchased from GE, catalog number: 17140801). The desalting column was equilibrated with 5 to 10 column volumes of phosphate buffer PBS pH=7.5 mL. The above HiTrap Desalting column was packed with an aqueous NDC reaction solution (approx. 1.5 mL), and eluted in a high-speed protein purification system (AKTA purifier 100, GE) according to the desalting column instructions. Unreacted deruxtecan, N-acetyl-L-cysteine ​​(NAC), small molecule organic cosolvents, etc., were removed according to molecular weight to obtain a 3005 K65A-deruxtecan conjugate solution. The success of conjugation of the purified 3005 K65A-deruxtecan conjugate was determined using SDS-PAGE, and the content of free small molecules (e.g., deruxtecan) within the purified 3005 K65A-deruxtecan conjugate was detected using RP-HPLC. Subsequent cell activity measurements can be performed only if the content of free small molecules in the purified conjugate does not exceed 1%.

[0327] The structure of the 3005 K65A-deruxtecan conjugate (NDC) described in this embodiment is illustrated in FIG. 13 (A), and FIG. 13 (B) shows SDS-PAGE gel images of three different 3005 K65A-deruxtecan conjugates (NDC-1, NDC-2, NDC-3), with the conjugate labeled NDC-3 being used as an example and subsequently used for human serum albumin binding ELISA experiments and evaluation of proliferation inhibitory activity against pancreatic cancer cells Mia Paca-2.

[0328] D. Measurement of Affinity and In Vitro Activity of 3005 K65A-Deruxtecan Conjugate

[0329] Human serum albumin binding activity was detected for 3005 mutant proteins 3005 K65A and 3005 K65A-deruxtecan conjugate (NDC-3) using the ELISA method. In the ELISA experiment, 2 μg / mL of human serum albumin was coated, and after adding a concentration gradient of 3005 mutant proteins 3005 K65A and 3005 K65A-deruxtecan conjugate (NDC-3), the antibody levels binding to albumin were detected using an HRP-labeled mouse anti-His tag monoclonal antibody (purchased from Proteintech, catalog number: HRP-66005). The results of the ELISA experiment for HSA binding of 3005 mutant proteins 3005 K65A and 3005 K65A-deruxtecan conjugate (NDC-3) are shown in Figure 14 (A). As shown in Figure 14 (A), the 3005 K65A-deruxtecan conjugate (NDC-3) maintained very high binding activity with human serum albumin even after conjugation with small molecule deruxtecan. The EC50 summary of the ELISA experiment for HSA binding is shown in Table 26.

[0330] Table 26 Summary of EC50 values ​​for HSA binding of 3005 mutant proteins 3005 K65A and 3005 K65A-deruxtecan conjugate (NDC-3)

[0331]

[0332] The inhibitory effect of the zygote on cell proliferation was evaluated using Mia Paca-2 pancreatic cancer cells. The experimental procedure is briefly as follows: 2 × 10⁶ Mia Paca-2 cells were prepared in DMEM medium (Gibco) containing 10% fetal bovine serum. 4Cells were prepared at a rate of cells / mL, and 25 μL of cells were inoculated into 384-well cell culture microplates. Subsequently, 25 μL of different concentrations of deruxtecan or 3005 K65A-deruxtecan conjugate (NDC-3), diluted with DMEM medium, were added per well to achieve final concentrations of deruxtecan or 3005 K65A-deruxtecan conjugate (NDC-3) of 0.96 nM, 9.6 nM, 96 nM, 960 nM, and 19340 nM. After adding the test substances, the cells were cultured for 5 days at 37°C under 5% CO2 conditions. After incubation, the microplate was removed from the incubator, 25 μL was removed, and an equal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega) solution was added. The mixture was then shaken with a mixer at room temperature for 10 minutes, and the luminescence of each sample within the microplate was measured using a microplate reader. The viability of tumor cells after 5 consecutive days of incubation was calculated using the following formula.

[0333] Cell viability (%) = Luminous amount of administration group ÷ Luminous amount of control group × 100%

[0334] After treating Mia Paca-2 cells with test substances at concentrations of 96 nM, 960 nM, and 19340 nM, the viability of Mia Paca-2 cells is shown in Figure 14 (B) and Table 27.

[0335] Table 27 Cell viability after treatment of Mia Paca-2 cells with deruxtecan and 3005 K65A-deruxtecan conjugate (NDC-3)

[0336]

[0337] As can be seen in Figure 14 (B) and Table 27, the tumor cell proliferation inhibitory activity of deruxtecan at the same concentration was significantly lower than that of the 3005 K65A-deruxtecan conjugate (NDC-3). Therefore, the anti-albumin antibody 3005 can be conjugated to an active molecule, such as a small molecule toxin, as a carrier, and the biological activity of the conjugate (e.g., inhibition of cell proliferation) is not inferior to or is rather superior to that of the active molecule itself. The conjugate can potentially improve the cytotoxicity or selectivity of the active molecule toward target cells, thereby enhancing the activity of the small molecule in inhibiting cancer cell growth.

[0338] Example 8: Albumin binding activity of humanized anti-albumin antibody under different pH conditions

[0339] Albumin can bind to the cell surface receptor FcRn under weakly acidic conditions, whereas its binding affinity is weak under neutral conditions. Therefore, after binding to FcRn, albumin is absorbed into the endosome; as the endosome environment becomes acidic, the binding affinity between albumin and FcRn strengthens, leading to its recirculation back to the cell surface by FcRn or transcellular transport. Consequently, albumin can possess a long half-life by utilizing the recycling mechanism of FcRn. Since albumin-binding antibodies and their fusion proteins also undergo similar intracellular transport processes after binding to albumin, the ability of albumin-binding antibodies to bind to albumin under neutral and acidic conditions is crucial for achieving long-term circulation and extended half-life.

[0340] Using the SPR method, the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K) of humanized antibody 3005Hz6 and HSA under near-neutral (pH=7.4) and acidic conditions (pH=5.5, pH simulation of the endosome microenvironment) D ) was detected.

[0341] In the experiment, a Biacore 8K instrument (Cytiva) was used, and the antigen HSA was immobilized via amino coupling using the Series S Sensor Chip CM5, and the analyte was 3005Hz6 with a concentration gradient. When detecting the binding and dissociation of HSA and 3005Hz6 under neutral conditions, the components of the mobile phase buffer used were 10 mM HEPES pH=7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v Tween-20, and the analyte 3005Hz6 was diluted using the above mobile phase. When detecting the binding and dissociation of HSA and 3005Hz6 under acidic conditions, the components of the mobile phase buffer used were 10 mM sodium acetate pH=5.5, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v Tween-20, and the analyte 3005Hz6 was diluted using the above mobile phase. In the experiment, the binding time was 150 seconds and the dissociation time was 300 seconds.

[0342] Using the “1:1 binding” model, the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K) for HSA binding at 3005 Hz6 under pH=7.4 and pH=5.5 conditions D ) was fitted, and the results are as shown in Table 28.

[0343] Table 28 Binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K) of humanized antibody 3005Hz6 and HSA under pH=7.4 and pH=5.5 conditions D )

[0344]

[0345] As shown in Table 28, the results of detection using the SPR method of HSA with amino coupling showed that at pH=7.4, 3005Hz6 and the equilibrium dissociation constant K of HSA D is approximately 2.84 nM; at pH=5.5, the equilibrium dissociation constant K of HSA and 3005 Hz6. DThe value is approximately 0.33 nM, which indicates that the humanized antibody 3005Hz6 binds strongly to HSA under both neutral and acidic conditions, and that the affinity of 3005Hz6 for HSA under acidic conditions is 8.6 times stronger than under neutral conditions. Therefore, it can be inferred that the humanized antibody 3005Hz6 binds strongly to HSA at physiological pH, and after the complex of 3005Hz6 and HSA enters the acidic environment of the endosome together, the affinity between 3005Hz6 and HSA is enhanced, allowing 3005Hz6 to perform intracellular recycling or transcellular transport through the binding of HSA to FcRn.

[0346] Example 9: Fusion expression and activity detection of 3005Hz6 and Aflibercept

[0347] The humanized anti-albumin antibody 3005Hz6 was fusion-expressed with the commercially available drug Aflibercept (a fusion protein of VEGFR1 extracellular domain 2, VEGFR2 extracellular domain 3, and IgG1 Fc). 3005Hz6 was linked to the N-terminus of the Aflibercept protein, the linker was GGSGGS (SEQ No. 60), the fusion protein was named 3005Hz6-Aflibercept, and the amino acid sequence of the fusion protein 3005Hz6-Aflibercept is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWARQAPGKGLEWVSGISVDGSFLDYADAVKGRFTISRDNAKNSLYLQMNSLRPEDTAVYYCARASGPQGLRLGAPRGQGTLVTVSSGGSGGSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITV TLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK D KTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 61).

[0348] 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's coding DNA sequence was inserted into an expression vector via restriction sites HindIII and NotI using gene synthesis and molecular cloning techniques. After confirming the plasmid sequencing was correct and removing endotoxins for large-scale extraction, transient expression was performed using suspended 293F cells, and Protein A affinity purification was performed on the cell culture supernatant to obtain the fusion protein 3005Hz6-Aflibercept.

[0349] The nucleotide sequence of the fusion protein 3005Hz6-Aflibercept is as follows:

[0350]

[0351] The HSA binding activity of the anti-albumin nanobody 3005Hz6 within the fusion protein 3005Hz6-Aflibercept was detected using the ELISA method. In the experiment, 100 μL / well of human serum albumin HSA (Baxter AG) was coated at a final concentration of 2 μg / mL and coated overnight at 4°C. After blocking with 5% skim milk powder, a gradient dilution of the fusion protein was added and incubated at 37°C for 1 hour. After washing the plates, HRP-labeled goat anti-human IgG Fc antibody (purchased from Abbkine, catalog number: A21050) was added and incubated at room temperature for 45 minutes. After washing the plates, 100 μL of TMB (purchased from Tiangen Biotech, catalog number: PA107-01) was added to each well and color developed at 37°C for 15 minutes, after which 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured using a microplate reader. The EC50 of the fusion protein ELISA binding experiment is shown in Table 29.

[0352] The activity of the anti-VEGF protein Aflibercept within the fusion protein 3005Hz6-Aflibercept was detected using the VEGFR2 competitive ELISA method, and the commercially available drug Aflibercept was used as the control protein. The competitive ELISA experimental method is the same as that in “2. Detection of activity of anti-VEGFA nanobodies within the fusion protein” of Example 6. The IC50 of the fusion protein competitive ELISA experiment is as shown in Table 29.

[0353] Table 29 Summary of Experimental Results on Activity Detection of Protein 3005Hz6-Aflibercept

[0354]

[0355] As shown in Table 29, the fusion protein 3005Hz6-Aflibercept has strong HSA binding activity, and its VEGFR2 competitive activity was similar to that of the commercial drug Aflibercept. The results indicate that the anti-albumin nanobody 3005Hz6 and the anti-VEGF protein Aflibercept within the fusion protein 3005Hz6-Aflibercept do not affect each other's activities and can function independently.

[0356] Example 10: Improvement of the affinity of humanized antibody 3005Hz6

[0357] Random mutations were performed on a single amino acid region and two adjacent amino acid regions of the three CDR regions of the humanized antibody 3005Hz6, and the mutated sequences were expressed on the surface of the phage using phage display technology. During the screening process, the amount of antigen input was gradually reduced and cross-species screening was performed, thereby obtaining candidate anti-albumin nanobody sequences that simultaneously bind to HSA, cyanomus monkey serum albumin, MSA, and rat albumin while having a higher affinity than 3005Hz6. Finally, candidate antibodies 1004, 1012, 1036, 1068, and 2028 were obtained. The amino acid and nucleotide sequences of the candidate antibodies are shown in Table 30, and the amino acid sequences of their CDR regions are shown in Table 31.

[0358] Table 30 Amino acid and nucleotide sequences of candidate antibodies with enhanced affinity

[0359]

[0360]

[0361] Table 31 Amino acid sequences of candidate antibodies CDR-H1, CDR-H2, and CDR-H3 with enhanced affinity

[0362]

[0363] Example 11: ELISA binding activity test of enhanced affinity candidate antibody and albumin

[0364] A tPA secretion peptide was added to the N-terminus of candidate antibodies with enhanced affinity (antibodies 1004, 1012, 1036, 1068, and 2028 shown in Table 30), and a 6xHis tag was added to the C-terminus. The expression and purification of these antibodies were performed in the same manner as the experimental procedure described in “1. Expression of anti-albumin candidate antibodies” of Example 3.

[0365] The Coomassie Brilliant Blue staining results after purifying the candidate antibody with improved affinity are shown in Fig. 15, and the molecular weight of the candidate antibody was approximately 14.7 kDa.

[0366] The albumin binding activity of different species of candidate antibodies with enhanced affinity was detected using the ELISA method.

[0367] In the ELISA experiment, candidate antibodies (concentration 5 μg / mL) were coated and coated overnight at 4°C. Biotin-modified antigen HSA (containing Avi and 6xHis tags, amino acid sequence:) was prepared using PBST solutions of different pH (pH=7.4 or 6.0).

[0368] DAHKSEVAHRFK D LGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHK D DNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESK D VCKNYAEAK DAfter diluting VFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGSGLNDIFEAQKIEWHEGGGGSHHHHHH, SEQ ID NO. 56), the solution was added to an ELISA plate and incubated at 37°C for 1 hour, and the plate was washed with PBST solution of different pH values ​​(pH=7.4 or 6.0). HRP-labeled streptavidin antibody Streptavidin-HRP (purchased from Sangon Biotech, catalog number: D111054-0001) was added and incubated at room temperature for 45 minutes. After washing the plate, 100 μL of TMB (purchased from Tiangen Biotech, catalog number: PA107-01) was added to each well and color developed at 37°C for 15 minutes, after which 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured using a microplate reader.

[0369] The results of ELISA experiments on HSA binding of candidate antibodies with enhanced affinity at pH=7.4 or pH=6.0 are shown in Fig. 16 (A) and Fig. 16 (B), respectively. As can be seen, at pH=7.4, the HSA binding affinity of all candidate antibodies except 1004 was higher than that of 3005Hz6, and the enhancement of affinity of antibody 1068 was the most significant. At pH=6.0, the HSA binding affinity of all five candidate antibodies was higher than that of 3005Hz6.

[0370] The rat albumin binding activity of candidate antibodies with enhanced affinity was detected using the antigen-coated ELISA method. In the experiment, untagged 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 using PBST solutions of different pH (pH=7.4 or 6.0), added to ELISA plates, and incubated at 37°C for 1 hour. After washing the plates with PBST solutions of different pH (pH=7.4 or 6.0), an anti-His tag monoclonal antibody (purchased from Proteintech, catalog number: HRP-66005) was added and incubated at room temperature for 45 minutes. After washing the plates, 100 μL of TMB (purchased from Tiangen Biotech, catalog number: PA107-01) was added to each well and incubated at 37°C for 15 minutes, after which 50 μL of stop solution was added. OD450 was measured using a microplate reader. Here, the control group BI-V2 is SEQ No. 15 selected from patent US 2023 / 0050615 A1, and the amino acid sequence is as follows: EVQLVESGGGLVQAGGSLRLSCAASGLTFSSYAMGWFRQAPGKERERVVSISRGGGYTYYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAAARYWATGSEYEFDYWGQGTLVTVSS (SEQ No. 57).

[0371] The results of ELISA experiments on rat albumin binding of candidate antibodies with enhanced affinity at pH=7.4 or pH=6.0 are shown in Fig. 17 (A) and Fig. 17 (B), respectively. As can be seen, at pH=7.4 or pH=6.0, the affinity of antibody 2028 for rat albumin was higher than that of 3005Hz6, and the control protein BI-V2 hardly bound to rat albumin.

[0372] Example 12: Affinity test (SPR) of candidate antibody with enhanced affinity and HSA

[0373] The affinity of the candidate antibody with enhanced affinity and HSA was measured using two SPR methods.

[0374] In the first SPR method, a Biacore T200 instrument (Cytiva) was used, and an anti-His tagged antibody was immobilized on the surface of a CM5 chip via amino coupling. A His-tagged candidate antibody was captured, with a candidate antibody concentration of 20 nM. The buffer consisted of 10 mM HEPES (pH=7.4), 150 mM NaCl, and 0.05% v / v Tween-20. The flow rate was 30 μL / min, and the capture time was 30 seconds. Different concentrations of HSA were bound to and dissociated from the antibody-captured chip; the peak HSA concentrations were 37.5 nM or 18.75 nM, and the solution was diluted in a twofold concentration gradient. The binding time with the chip was 180 seconds, and the dissociation time was 300 seconds. The equilibrium dissociation constant K was determined by fitting the binding and dissociation curves. D Obtain the value, and the result is as shown in Table 32.

[0375] Table 32. Binding rates (ka), dissociation rates (kd), and equilibrium dissociation constants (K) of HSA and candidate antibodies 1004, 1012, 1036, 1068, 2028, and 3005Hz6 with enhanced affinity. D )

[0376]

[0377] As can be seen in Table 32, the affinities for HSA of the candidate antibodies 1004, 1012, 1036, 1068, and 2028 with enhanced affinity were all higher than those of 3005Hz6, and among them, antibody 1068 had the highest affinity for HSA.

[0378] In the second SPR method, a Biacore 8K instrument (Cytiva) was used, and the antigen HSA was immobilized on the Series S Sensor Chip CM5 via amino coupling (reaction value approximately 1250 RU). The analytes used were concentration gradient proteins 3005Hz6, 1068, 2028, and control protein BI-V2. The protein-containing buffer consisted of 10 mM HEPES pH=7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v Tween-20. In the SPR experiment, the binding time was 120 seconds and the dissociation time was 1000 seconds (300 seconds for antibody 2028). The binding and dissociation curves for HSA binding of antibodies 3005Hz6, 1068, 2028 and control protein BI-V2 are shown in Fig. 18 (A), Fig. 18 (B), Fig. 18 (C), and Fig. 18 (D), respectively.

[0379] Using the “1:1 binding” model, the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (K) for the binding of antibodies to HSA D ) was fitted, and the results are as shown in Table 33.

[0380] Table 33. Binding rates (ka), dissociation rates (kd), and equilibrium dissociation constants (K) of antibodies 3005Hz6, 1068, 2028, and control proteins BI-V2 and HSA D )

[0381]

[0382] As a result, the affinity of antibody 1068 for HSA was very high at approximately 56.6 pM, significantly higher than that of 3005Hz6 (approx. 35.7-fold improvement), and higher than that of the control antibody BI-V2 (approx. 2.8-fold improvement), and the affinity of antibody 2028 for HSA was higher than that of 3005Hz6 (approx. 3.7-fold improvement).

[0383] Although preferred embodiments of the present invention have been described in detail above, the present invention is not limited to the specific details of the above embodiments. Various simple modifications to the technical solution means of the present invention are possible within the scope of the technical spirit of the present invention, and all such simple modifications fall within the scope of protection of the present invention.

[0384] In addition, it should be noted that each specific technical feature described in the above specific embodiments may be combined in any appropriate manner, provided there is no contradiction. To avoid unnecessary repetition, the present invention does not separately describe various possible combination methods.

Claims

Claim 1 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 CDR-H3 of a heavy chain variable region; the amino acid sequence of CDR-H1 comprises an amino acid sequence having at least 80% identity with an amino acid sequence represented by SEQ ID NO. 1 or 58, or by SEQ ID NO. 1 or 58; the amino acid sequence of CDR-H2 comprises an amino acid sequence having at least 80% identity with an amino acid sequence represented by any one of SEQ ID NOs 3-4, 6, 40, or by any one of SEQ ID NOs 3-4, 6, 40; and the amino acid sequence of CDR-H3 comprises an amino acid sequence having at least 80% identity with an amino acid sequence represented by any one of SEQ ID NOs 7-8, 59, or by by any one of SEQ ID NOs 7-8, 59. Claim 2 In claim 1, X1 in SEQ ID NO. 58 (X1YYMS) represents N or E, and is SEQ ID NO. 2 or 49, respectively; X2X3 in SEQ ID NO. 40 (GISVX2X3X4X5LDYADAVX6G) represents DS, DA, EG, DM, or DG, X4X5 represents SF or WY, and X6 represents K, A, R, or H; Preferably, X2X3 represents DG, X4X5 represents SF, and X6 represents K (SEQ No. 5); X2X3 represents DA, X4X5 represents SF, and X6 represents K (SEQ No. 20); X2X3 represents DS, X4X5 represents SF, and X6 represents K (SEQ No. 21); X2X3 represents EG, X4X5 represents SF, and X6 represents K (SEQ No. 22); X2X3 represents DG, X4X5 represents SF, and X6 represents A (SEQ No. 31); X2X3 represents DG, X4X5 represents SF, and X6 represents R (SEQ No. 32); X2X3 represents DG, X4X5 represents SF, and X6 represents R (SEQ No. 32); X6 represents H (SEQ No. 33); said X2X3 represents DG, X4X5 represents WY, and X6 represents K (SEQ No. 50); or said X2X3 represents DM, X4X5 represents SF, and X6 represents K (SEQ No. 51); or said SEQ No. 59 (ASGPX7X8LRX9X 10 X7X8 in AP represents QG, IW, LW, or VG, and X9X 10 represents LG or WW, preferably, the above X7X8 represents QG, and X9X 10 ... represents LG (sequence number 9); the above X7X8 represents IW, and X9X 10 ... represents LG (sequence number 52); the above X7X8 represents LW, and X9X 10 ... represents LG (sequence number 53); the above X7X8 represents QG, and X9X 10 represents WW (sequence number 54); or, the above X7X8 represents VG, and X9X 10 An anti-albumin antibody or its antigen-binding fragment characterized by representing LG (Sequence No. 55). Claim 3 In claim 1 or 2, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are: A) Sequence No. 1, 3, 7; B) Sequence No. 1, 4, 8; C) Sequence No. 2, 5, 9; D) Sequence No. 2, 6, 9; E) Sequence No. 2, 20, 9; F) Sequence No. 2, 21, 9; G) Sequence No. 2, 22, 9; H) Sequence No. 2, 31, 9; I) Sequence No. 2, 32, 9; J) Sequence No. 2, 33, 9; K) Sequence No. 2, 5, 52; L) Sequence No. 2, 50, 53; M) Sequence No. 49, 5, 53; N) Sequence No. 49, 5, 54; O) Sequence No. 2, 51, 55; An anti-albumin antibody or an antigen-binding fragment thereof characterized by comprising any one of the following groups. Claim 4 An anti-albumin antibody or an antigen-binding fragment thereof, characterized in that, in any one of claims 1 to 3, the anti-albumin antibody or the antigen-binding fragment thereof comprises a humanization sequence, and a modified site of the humanization sequence is located in a non-CDR region, preferably, the modified site of the humanization sequence is located in a framework region and / or an invariant region of the antibody. Claim 5 An anti-albumin antibody or its antigen-binding fragment according to any one of claims 1 to 4, wherein the anti-albumin antibody or its antigen-binding fragment comprises a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, an Fd fragment, a Fv fragment, a dAb fragment, an F(ab')2 fragment, a single-chain antibody, or a linear antibody. Claim 6 An anti-albumin antibody or an antigen-binding fragment thereof, characterized in that, in any one of claims 1 to 5, the amino acid sequence of the anti-albumin antibody or the antigen-binding fragment thereof comprises any one of the amino acid sequences of SEQ ID NOs 10-13, 16-19, 23-25, 34-36, and 44-48, or has at least 80% identity with any one of the amino acid sequences of SEQ ID NOs 10-13, 16-19, 23-25, 34-36, and 44-48. Claim 7 An application of an anti-albumin antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein the application comprises: A. an application in the preparation of a fusion construct, wherein the fusion construct comprises an anti-albumin antibody or its antigen-binding fragment according to any one of claims 1 to 6 and a biologically active effector molecule, wherein the biologically active effector molecule is connected to the anti-albumin antibody or its antigen-binding fragment; B. an application in the screening of a biologically active effector molecule, wherein a candidate biologically active effector molecule is connected to the anti-albumin antibody or its antigen-binding fragment, and the biological activity of the candidate biologically active effector molecule is detected; or C. an application in the detection of albumin. Claim 8 A fusion construct comprising, wherein the fusion construct comprises one or more anti-albumin antibodies or antigen-binding fragments thereof according to any one of claims 1 to 6 and / or biologically active effector molecules, wherein the biologically active effector molecules are connected to the anti-albumin antibodies or antigen-binding fragments thereof, and preferably, the fusion construct comprises a plurality of anti-albumin antibodies or antigen-binding fragments thereof, wherein the plurality of anti-albumin antibodies or antigen-binding fragments thereof may be identical or different; wherein the biologically active effector molecules may be one or more, and the plurality of biologically active effector molecules may be identical or different. Claim 9 A fusion construct according to claim 8, wherein the biologically active effector molecule comprises, but is not limited to, a small molecule compound or a macromolecular compound, preferably, the small molecule compound comprises, but is not limited to, any small molecule drug, and the macromolecular compound comprises, but is not limited to, an antibody, an activating or inhibiting receptor, a ligand of a protein, a biologically active enzyme, a nucleic acid drug, or a combination thereof, more preferably, the antibody among the macromolecular compounds comprises an antibody of another target or an antigen-binding fragment thereof, and said other target is a target different from albumin; more preferably, the structure of said antibody of another target or its antigen-binding fragment is one or more combinations of a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, an Fd fragment, an Fv fragment, a dAb fragment, an F(ab')2 fragment, a single-chain antibody, or a linear antibody. Claim 10 In claim 9, the other targets are VEGFA, VEGFB, VEGFR, FGF, FGFR, PlGF, PDGF, TGF, Integrin, Integrin receptor, interleukin (e.g., IL-1β, IL-2, IL-3, IL-4, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptor (e.g., IL1R1, IL2Rα, IL3R, IL4Rα, IL10R, IL12R, IL15Rα, IL17R, IL23R, etc.), PCSK9, TNF-α, TNFR, RANKL, GPCR, GLP1R, CD3, CD105, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD47, CD80, CD86, CD96, CD99, A fusion construct characterized by being selected from 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. Claim 11 In claim 10, the anti-albumin antibody or its antigen-binding fragment and the biologically active effector molecule are directly or indirectly connected, preferably, the indirect connection may be a connection through a linker, a functional domain and / or a conjugation linker; said linker is selected from a linking peptide, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, PEG, nucleic acid, a polysaccharide, an aliphatic chain, biotin, streptavidin, or avidin; said functional domain is one or more combinations of an Fc fragment, serum albumin, a cytokine, transferrin, and a scaffold protein; said conjugation linker comprises a functional group linker, preferably, said functional group linker comprises a mercapto, amino, hydroxyl and / or carboxyl reactive group, and more preferably, said biologically active effector molecule is directly or indirectly connected to the N-terminus, C-terminus, and / or internal residue of said anti-albumin antibody or its antigen-binding fragment. Structures. Claim 12 A nucleic acid characterized in that, as a nucleic acid, the nucleic acid codes for an anti-albumin antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, or codes for a fusion construct according to any one of claims 8 to 11. Claim 13 In claim 12, the nucleic acid is characterized by further comprising any one of SEQ ID NOs 63-75, 79-81, 83-87 or a degenerate sequence thereof, or having at least 80% identity with any one of SEQ ID NOs 63-75, 79-81, 83-87 and having a nucleotide sequence encoding an anti-albumin antibody or an antigen-binding fragment function thereof. Claim 14 A nucleic acid according to any one of claims 12 to 13, characterized in that the nucleic acid comprises a nucleotide sequence of any one of SEQ ID NOs 76-78 or 82 or a degenerate sequence thereof, or has at least 80% identity with any one of SEQ ID NOs 76-78 or 82 and has a nucleotide sequence encoding the fusion construct function. Claim 15 A vector characterized in that the vector comprises a nucleic acid according to any one of claims 12 to 14. Claim 16 A host cell, wherein the host cell comprises a nucleic acid according to any one of claims 12 to 14 or a vector according to claim 15. Claim 17 A method for manufacturing an anti-albumin antibody or its antigen-binding fragment according to any one of claims 1 to 6 or a fusion construct according to any one of claims 8 to 11, wherein the method comprises the step of culturing a host cell according to claim 16 to express the anti-albumin antibody or its antigen-binding fragment or the fusion construct. Claim 18 An application in the manufacture of a product for the treatment and / or diagnosis of a disease, wherein the anti-albumin antibody or its antigen-binding fragment 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, wherein the anti-albumin antibody or its antigen-binding fragment and / or the biologically active effector molecule have therapeutic and / or diagnostic activity for the disease, preferably wherein the anti-albumin antibody or its antigen-binding fragment is used as a vector in a product for the treatment and / or diagnosis of the disease. Claim 19 A product for the treatment and / or diagnosis of a disease, wherein the product for the treatment and / or diagnosis of the disease comprises any one of: A) an anti-albumin antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6; B) a fusion construct according to any one of claims 8 to 11; C) a nucleic acid according to any one of claims 12 to 14; D) a vector according to claim 15; or F) a host cell according to claim 16; wherein the anti-albumin antibody or an antigen-binding fragment thereof and / or the biologically active effector molecule have therapeutic and / or diagnostic activity of the disease, preferably, the anti-albumin antibody or an antigen-binding fragment thereof is used as a vector in the product for the treatment and / or diagnosis of the disease, more preferably, the biologically active effector molecule is a drug, and said drug is covalently bonded to said anti-albumin antibody or an antigen-binding fragment thereof. Claim 20 A method for detecting albumin, wherein the detection method comprises the step of contacting a sample to be detected with an anti-albumin antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, and then detecting the content of a complex formed of albumin and the anti-albumin antibody or the antigen-binding fragment thereof, wherein, preferably, the albumin is derived from a mammal, and more preferably, the mammal is derived from a human, mouse, or monkey.

Images