KR20190108308A - Single-domain antibody targeting alpha-v beta-3 integrin - Google Patents

Abstract

The present invention relates to a single-domain antibody targeting αvβ3 integrin and various applications thereof. The single-domain antibody targeting αvβ3 integrin of the present invention shows high binding ability to αvβ3 integrin associated with neovascularization, good tissue penetration and biostability compared to conventional antibodies. In addition, the single-domain antibody of the present invention can be easily measured in vitro, in vivo or ex vivo by binding to fluorescent particles, which is effective for detecting angiogenesis and diagnosing related diseases, and thus can be usefully used in related industries.

Description

Single-domain antibody targeting alpha-v beta-3 integrin

The present invention relates to α _v β ₃ integrin target single domain antibodies and various applications thereof.

Integrins are cell surface receptors that regulate important physiological functions of cells, such as cell adhesion and migration, differentiation, and proliferation. Integrins function as heterodimers in which α and β subunits are non-covalent bonds, and a pair of α and β subunits form 22 integrin families. Integrins mainly bind to extracellular matrix proteins such as bibronectin, fibronectin, collagen, laminin, vWF, and fibrinogen, but differ in ligand specificity by type of integrin, and one type of integrin can bind to several ligands simultaneously. have. Among these, integrin α _v β ₃ is expressed in most aggressive tumor cells of various cancers, including skin cancer, prostate cancer, breast cancer, cervical cancer, colon cancer, lung cancer, gallbladder cancer, pancreatic cancer, gastric cancer, and tumors dependent on adhesion. It is known to improve the malignancy of various human tumors by regulating the growth, survival and penetration of cells. Recently, it has been shown that β integrins regulate intracellular signal transduction to increase tumor growth and metastasis as mediators independent of adhesion (David A Cheresh et al., Nature Medicine 2009, 15 (10): 1163). In addition, the α _v β ₃ integrin is known to be expressed a lot in neovascular microvascularity.

Angiogenesis means that new capillaries are made from existing blood vessels. Strictly controlled phenomena rarely occur under normal physiological conditions, embryo development during fertilization, fertilization of wounds in adults, and changes in reproductive system in the female reproductive cycle. In adults, capillary endothelial cells do not divide relatively well and the rate of division usually ranges from months to years. Angiogenesis occurs as a complex process by the interaction of various types of cells with soluble factors and extracellular matrix components, and its mechanism of action is not yet fully understood. Angiogenesis is a cause of various diseases.

Although more than 300,000 antibodies are reported to be commercially available, they can only be observed in immobilized cells, and because of this, protein folding and interaction between proteins cannot be observed in real time. In addition, existing antibodies are too large or chemically unstable and could not be usefully used in living cells. However, in 1993, Hamers-Casterman derived the camel from the camel, but the antibody consists of only the heavy chain, a structure that is different from the existing antibodies (two heavy chains and two light chains), but functionally complete. Single domain antibodies that act as antibodies have been reported (Hamers-Casterman, C. et al. 1993. Nature 363: 446-448). Conventional antibodies are 150kDa, recombinant antibodies are 25-50kDa, but single-domain antibodies derived from camels, llamas, and sharks are the smallest antibodies, 12-13kDa, and are easily transported into cells (Cortez-Retamozo, V. et. al. 2004. Cancer Res. 64: 2853-2857,) It has the advantage of being easily expressed in bacteria and yeast due to its easy genetic manipulation (Arbabi-Ghahroudii, M. et al. 1997. FEBS Lett. 414: 521- 526). In addition, single domain antibodies are highly water soluble and stable in extreme pH conditions and temperatures up to 90 ° C. (Dumoulin, M. et al. 2002. Protein Sciii. 11: 500-515, Dumoulin, M. et. al. 2003. Nature 424: 783-788).

It is an object of the present invention to provide an α _v β ₃ Integrin target single domain antibody.

In addition, an object of the present invention is the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 It can provide a recombinant vector comprising one or more selected from the group consisting of.

It is also an object of the present invention can provide a recombinant microorganism transformed with the preparation vector.

It is also an object of the present invention can provide a composition for detecting neovascularization.

It is also an object of the present invention can provide a kit for neovascularization detection.

It is also an object of the present invention can provide a composition for diagnosing neovascularization-related diseases comprising the single domain antibody.

It is another object of the present invention can provide a method of producing α _v β ₃ integrin target single domain antibodies.

It is also an object of the present invention can provide a method for screening angiogenesis inhibitors or promoters.

It is also an object of the present invention can provide an information providing method for the diagnosis of neovascularization-related diseases.

In order to achieve the above object, the present invention is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 Provided is a α _v β ₃ Integrin target single domain antibody (encoded with one or more selected from the group consisting of the nucleotide sequences indicated).

In addition, the present invention consists of a nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 Provided is a recombinant vector comprising one or more selected from the group.

The present invention also provides a recombinant microorganism transformed with the recombinant vector.

In another aspect, the present invention provides a composition for detecting neovascularization comprising the single domain antibody.

In another aspect, the present invention provides a kit for neovascularization detection comprising the single domain antibody.

The present invention also provides a composition for diagnosing neovascularization-related diseases comprising the single domain antibody.

The present invention also provides a base represented by (1) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 Culturing the recombinant microorganism transformed with the recombinant vector comprising at least one selected from the group consisting of sequences; And (2) expressing a single domain antibody against α _v β ₃ integrins in the microorganism, the method of producing αvβ3 integrin target single domain antibody.

In another aspect, the present invention comprises the steps of (1) processing the candidate drug to be analyzed in a biological sample or animal model isolated from the sample; (2) binding the α _v β ₃ integrin target single domain antibody to the sample and measuring the level of α _v β ₃ integrin protein; And (3) selecting candidate drugs whose levels of the protein of (2) are inhibited or enhanced as compared to the control group.

In another aspect, the present invention (A) binding the α _v β ₃ integrin target single domain antibody to a biological sample isolated from the sample and measuring the level of α _v β ₃ integrin protein; And (b) comparing the level of the α _v β ₃ integrin protein with a reference value obtained from a control sample; provides an information providing method for the diagnosis of neovascularization-related diseases comprising a.

The α _v β ₃ integrin target single domain antibody of the present invention exhibits high binding ability, good tissue permeability and biostability to α _v β ₃ integrins associated with neovascularization compared to conventional antibodies. In addition, the single domain antibody of the present invention can be easily measured in vitro, in vivo or ex vivo by binding to fluorescent particles, and thus effective in detecting angiogenesis and diagnosing related diseases, and thus can be usefully used in related industries. .

1 is a diagram showing the results of performing the first PCR amplification for amplification of the single domain antibody expression cassette of the present invention.
Figure 2 is a diagram showing the results of the second PCR amplification for amplification of the single domain antibody expression cassette of the present invention.
3 shows fragments of truncated single domain genes.
4 shows a fragment of the cleaved phagemid.
5 is a diagram showing the results of confirming the gene insertion of the single domain antibody clone of the present invention.
6a to 6i show the results of DNA sequencing of 20 single-domain antibody clones and their alignment.
Figure 7 is a diagram showing the SDS-PAGE results of the α _v β ₃ integrin protein used to confirm the α _v β ₃ integrin target efficacy of the present invention.
8a to 6c are diagrams showing the amino acid sequence of clones showing a strong positive after ELISA.
9 is a diagram showing the results of the surface plasma analysis (SPR) analysis of VHH-13, VHH-22 and VHH-25 in a single domain antibody clone.
Figure 10a is a diagram showing a vector domain (pALT-Avb3 VHH25) expression of a single domain antibody VHH-25 containing His6 tag.
10B is a diagram showing a vector map (pALT-Avb3 VHH25K1) of a single domain antibody VHH-25 including Lysin and His6 tag.
Figure 11a is a diagram showing the reduced SDS-PAGE results of the single domain antibody VHH-13 containing His6 tag.
Figure 11b is a diagram showing a reduced SDS-PAGE results of the single domain antibody VHH-22 containing His6 tag.
Figure 11c is a diagram showing a reduced SDS-PAGE results of a single domain antibody VHH-25 containing His6 tag.
Figure 11d is a diagram showing the reduced SDS-PAGE results of a single domain antibody VHH-25 containing Lysin and His6 tag.
Figure 12 shows the results confirming the α _v β ₃ integrin target effect of the single domain antibody VHH-25 of the present invention in cancer cells.
Figure 13 shows the results of confirming the α _v β ₃ integrin target effect of the single domain antibody VHH-25 of the present invention at the tumor site.
14 is a diagram showing the results of confirming the α _v β ₃ integrin target effect of the single domain antibody VHH-25 of the present invention in the atherosclerotic site.

The present invention comprises a base sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 It provides α _v β ₃ Integrin target single domain antibody, which is encoded by one or more selected from.

The term "α _v β ₃ Integrin" of the present invention refers to an integrin that is present on the cell surface and is a receptor molecule that acts when cells adhere to an extracellular matrix such as fibronectin, collagen, and the like. It is a transmembrane glycoprotein consisting of heterodimers of two subunits of α and β, and the existence of 21 types of integrins has been revealed. Among them, α _v β ₃ integrin has been reported to play an important role in maintaining the structure of the cardiovascular system and bone tissue.

As used herein, the term "single-domain antibody" refers to an antibody in which CDRs are part of a single domain polypeptide, heavy chain antibodies, antibodies naturally free of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered Antibodies and single domain scaffolds other than those derived from antibodies. It is called a variable region of a heavy chain antibody (VHH), nanobody or sdAb to distinguish it from the VH of 4-chain immunoglobulins.

Single domain antibodies of the invention are naturally occurring single domain antibodies derived from heavy chains that are naturally free of light chains, and are specific antibodies against α _v β ₃ integrins and have a molecular weight of about 14-15 KDa. Single domain antibodies of the invention are VHH antibodies derived from Camelidae and may be derived from camels, dromedaries, llamas, alpacas and wild llamas, and are aimed at targeting α _v β ₃ integrins associated with neovascularization. In order to achieve this, other species other than Camellia can be naturally produced with light chain-free heavy chain antibodies, but are not limited thereto.

The single domain antibodies of the present invention are about 10 times smaller than IgG molecules, which are very stable as single polypeptides and are stable even at extreme pH and temperature conditions. In addition, they are resistant to the action of proteases, unlike conventional antibodies, and can be mass-produced in high yield when expressed in vitro.

The single-domain antibody is a nucleotide sequence represented by SEQ ID NO: 1 encodes an amino acid sequence represented by SEQ ID NO: 11, a nucleotide sequence represented by SEQ ID NO: 2 codes an amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: The base sequence represented by 3 may encode an amino acid sequence represented by SEQ ID NO: 13. In addition, the amino acid sequence containing a His6 tag in SEQ ID NO: 1, 2 or 3 may be represented by the amino acids of SEQ ID NO: 14, 15 and 16, respectively.

In one embodiment of the present invention, a camel was immunized by injecting a target protein with recombinant ανβ3 integrin having cross reactivity in mice and humans. Then, mRNA was isolated and purified from peripheral lymphocytes (pheripheral lymphocytes) isolated from camel blood to synthesize cDNA. Antibodies After amplifying the variable domains (VHH) of heavy chain-only antibodies by PCR. Cloned to M13 bacteriophage gene. Phage display using immobiized immunogen (phosphatidylserine) was performed to select phage clones that specifically react with immobilized antigens.

As a result of analyzing 40 clone sequences among the phage clones, DNA sequences of 10 candidate groups were identified. Of these, 20 DNA sequences are shown in Figs. 6A to 6I. Among them, clones 14, 11, 29, 17, 15, 28, 12, 19, 13, 21, 27, 16, 18, 20, 30, 23, 24, 25, 22, 26 were selected and their amino acid sequences are shown in FIGS. 8A-8C. The nucleotide sequences of the clones VHH-25, VHH-13, VHH-22, VHH-11, VHH-17, VHH-15, VHH-12, VHH-16, VHH-20, and VHH-23 show strong positive sequences, respectively. Numbers 1 to 10 are shown. Among them, amino acid sequences of VHH-25, VHH-13, and VHH-22 are shown in SEQ ID NOs: 11, 12, or 13, respectively.

Variants of the above nucleotide sequences are included within the scope of the present invention. Nucleic acid molecules which can be used as genes encoding the proteins constituting the single domain antibody of the present invention are functional equivalents of nucleic acid molecules constituting the same, for example, some nucleotide sequences of the nucleic acid molecules are deleted, substituted (substitution). Or by insertion, but includes variants that can function functionally as nucleic acid molecules. Specifically, the gene comprises a base sequence each having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the base sequence of the present invention. can do. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. May include additions or deletions (ie, gaps) as compared to

The single domain antibody of the present invention may include variants of amino acid sequences described in the attached sequence listing within the scope of specifically recognizing α _v β ₃ integrins. For example, changes can be made to the amino acid sequence of the antibody to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody.

The amino acid variation is made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

In introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index depending on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids which exhibit a hydrophobicity index difference of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids which exhibit a hydrophilicity value difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5.

Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

The single domain antibody of the present invention may be additionally bonded to a functional molecule or element, the functional molecule is one or more selected from the group consisting of inorganic particles, chemicals, peptides, polypeptides, nucleic acids, carbohydrates, radioisotopes and lipids Can be.

The inorganic particles are fluorescent markers or staining materials, Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), Blue fluorescent protein (BFP), Cyan fluorescent protein (CFP), Acridine dyes, Cyanine It may be selected from the group consisting of dyes (Cyanine dyes), fluorine dyes (Fluorone dyes), oxazine dyes (Oxazine dyes), phenanthridine dyes and Rhodamine dyes, but is not limited thereto.

The radioisotope is ¹⁸ F (fluorine), ¹¹ C (carbon), ¹⁵ O (oxygen), ¹³ N (nitrogen), ⁸⁹ Zr (zirconium), C ¹⁵ O, ¹³ N (ammonia), H ₂ ¹⁵ O And ¹⁸ FDG (F-Deoxy Glucose) is characterized in that one or more selected from the group consisting of, but not limited to.

The chemical is a substance that inhibits a disease associated with neovascularization, and a chemical that inhibits a disease related thereto may be combined with the single domain antibody of the present invention without limitation. For example, it may be a chemical such as an angiogenesis inhibitor, an anticancer agent, an atherosclerosis inhibitor, and the like.

As the angiogenesis inhibitor, for example, avastin, itraconazole, carboxyamidotriazole, suramin, SU5416, thrombospondin, sorafenib, sunitinib, Pazopanib, everolimus, and mixtures thereof, but are not limited to these.

As the anticancer agent, for example, acibaicin, aclarubicin, acodazole, acromycin, adozelesin, alanosine, aldesleukin, allopurinol sodium, altretamine, aminoglutetimide, amona Fide, Ampligen, Amsacrine, Androgens, Anguidine, Apidicholine Glycinate, Asarei, Asparaginase, 5-Azacytidine, Azathioprine, Bacillus Calmette-Guerin (BCG), Baker's antipol, beta-2-dioxythioguanosine, bisanthrene HCl, bleomycin sulfate, lysine, butionine sulfoximine, BWA 773U82, BW 502U83 / HCl, BW 7U85 mesylate, ceracemide, Carbetimers, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladribine, corticosteroids, corinbacterium parboom, CPT -11, crisnatol, cyclocytidine, cyclopo Pamide, cytarabine, cytembena, davis maleate, decarbazine, dactinomycin, daunorubicin HCl, diazaziridine, dexlaoxane, dihydrohydrogalactitol, diazikuone, dibromodulitol , Didemin B, diethyldithiocarbamate, diclicoaldehyde, dihydro-5-azacytin, doxorubicin, echinomycin, deda trexate, edelfosine, epronitine, eliot solution, elsamite Leucine, Epirubicin, Esorubicin, Estramustine Phosphate, Estrogen, Etanidazole, Ethiophos, Etoposide, Padrazole, Pazarabine, Penretinide, Filgrastim, Finasteride, Flavone Acetic Acid, Phloxyuri Dean, Fludarabine Phosphate, 5'-Fluorouracil, Fluosol, Flutamide, Gallium Nitrate, Gemcitabine, Goserelin Acetate, Hepsulpam, Hexamethylene Bisacetamide, Homo Ringtonin, Hydrazine Sulfate, 4-hydroxyandrostenedione, Hydroziurea, Idarubicin HCl, Iphosphamide, 4-Ipomeanol, Iproplatin, Isotretinoin, Leucovorin Calcium, Lyuprolide Acetate, Leva Misole, liposome daunorubicin, liposome capture doxorubicin, romastin, rotamine, maytansine, mechloretamine hydrochloride, melphalan, menogaryl, merbarone, 6-mercaptopurine, mesna, bacillus calete-gu Methanol extract of erin, methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin-C, mitotan, mitoxantrone hydrochloride, monosite / macrophage colony-stimulating factor, nabilone, napoxidine Neocardinostatin, octreotide acetate, ormaplatin, oxaliplatin, paclitaxel, pala, pentostatin, piperazindione, pipeobroman, Pyrurubicin, pyretrexime, pyroxanthrone hydrochloride, PIXY-321, plicamycin, porpimer sodium, prednismustine, procarbazine, progestins, pyrazopurin, lazoic acid, sargramostim, tax Styrene, Spigermanium, Spyromustine, Streptonigreen, Streptozosin, Sulofenner, Suramin Sodium, Tamoxifen, Taxorere, Tegapur, Teniposide, Terephthalamidine, Theroxylone, Thioguanine, Thiotepa, Timi Dean injection, thiazopurin, topotecan, toremifene, tretinoin, trifluoroperazine hydrochloride, trifluridine, trimetrexate, tumor necrosis factor, uracil mustard, vinblastine sulfate, vincristine sulfate, Bindesin, vinorelbine, vinzolidine, Yoshi 864, zorubicin, cytosine arabinoside, etoposide, melfaran, taxol and mixtures thereof, but not limited to .

Peptides or polypeptides as functional molecules are not particularly limited and include hormones, hormone analogs, enzymes, enzyme inhibitors, signaling proteins or parts thereof, antibodies or parts thereof, short chain antibodies, binding proteins or binding domains, antigens, adhesion proteins thereof , Structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulators, blood clotting factors and vaccines, and the like. More specifically, the peptide or polypeptide additionally bound to the single domain antibody of the present invention is insulin, insulin-like growth factor 1 (IGF-1), growth hormone, erythropoietin, G-CSFs (granulocyte-colony) stimulating factors), GM-CSFs (granulocyte / macrophage-colony stimulating factors), interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin-3, interleukin-4, interleukin-6, interleukin-2, EGFs (epidermal growth factors), calcitonin (calcitonin), adrenocorticotropic hormone (ACTH), tumor necrosis factor (TNF), atobisban, buserelin, cetrorelix, deslorerine deslorelin, desmopressin, dynorphin A (1-13), elcatonin, eleidosin, eptifibatide, growth hormone releasing hormone-II), gonadorelin, goserelin, he Trerin, leuprorelin, lypressin, octreotide, oxytocin, pitressin, secretin, sincalide, ter Terlipressin, thymopentin, thymosine α1, triptorelin, bivalirudin, carbetocin, cyclosporin, exedine, lan Leotard, luteinizing hormone-releasing hormone (LHRH), nafarelin, parathyroid hormone, pramlintide, T-20 (enfuvirtide), thymalfasin, ziconotide, Lysine, Lysine A Chain, Pseudomonas Exotoxin, Diphtheria Toxin, Pokeweed Antiviral Protein, Abrin, Abrin A Chain, Cobra Venom Factor, Gelonin, Saporin, Modesin (modeccin), volkensin, viscumin, clostridium ferpring 'S phospholipase C and can include but is the Bordj pancreatic ribonuclease, but is not limited thereto.

On the other hand, the sequence of a suitable peptide linker used when the functional molecule bound to the single domain antibody is a peptide or polypeptide can be selected in consideration of the following factors: (a) which can be applied to a flexible extended structure ability; (b) the ability not to create secondary structures that interact with epitopes; And (c) absence of hydrophobic residues or charge-bearing residues capable of reacting with epitopes, preferred peptide linkers include Gly, Asn and Ser residues. Other neutral amino acids such as Thr and Ala can also be included in the linker sequence.

In addition, the present invention consists of a nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 Provided is a recombinant vector comprising one or more selected from the group.

As used herein, the term “vector” refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Phagemid vector; Cosmid vector; And viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors, and the like, and are preferably phageimide vectors or plasmid vectors.

The recombinant vector of Figure 10a or 10b 10A shows a vector map (pALT-Avb3 VHH25) expressing a single domain antibody VHH-25 including His6 tag, and FIG. 10B includes Lysin and His6 tag. To express a single domain antibody VHH-25 expression vector map (pALT-Avb3 VHH25K1), to achieve the single domain antibody expression object of the present invention, it is not limited thereto.

According to a preferred embodiment of the invention, the nucleic acid molecule encoding a single domain antibody in the vector of the invention is operatively linked with a promoter.

In the present invention, the term “operably linked” means functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, thereby The regulatory sequence will control transcription and / or translation of the other nucleic acid sequence.

The vector system of the present invention may be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, This document is incorporated herein by reference.

Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. When the vector of the present invention is an expression vector and the prokaryotic cell is a host, powerful promoters capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoters, recA promoters, SP6 promoters, trp promoters and T7 promoters, etc.), ribosome binding sites for initiation of translation, and transcription / detox termination sequences. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator sites of the E. coli tryptophan biosynthesis pathway (Yanofsky, C. (1984), J. Bacteriol., 158: 1018-). 1024) and a phage λ left promoter (pLλ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14: 399-445) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14). , pGEX series, pET series and pUC19, etc.), phagemids (eg pComb3X), phages (eg λgt4 · B, λ-Charon and M13, etc.) or viruses (eg SV40, etc.) may be produced.

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the mammalian cell genome (eg, metallothionine promoter) or a promoter derived from a mammalian virus (eg adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and four promoters of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences as needed to facilitate purification of the protein, and the sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA). ), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA) and the like can be used, but are not limited thereto. In addition, the expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selectable label, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin Resistance genes to neomycin and tetracycline.

The present invention also provides a recombinant microorganism transformed with the recombinant vector.

Host cells capable of stably and continuously cloning and expressing the vectors of the present invention can use any host cell known in the art, such as Escherichia coli, Bacillus subtilis and Bacillus thuringin Bacillus strains such as cis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g. For example, prokaryotic host cells, such as, but not limited to, Staphylocus carnosus. The host cell is preferably E. coli and more preferably E. coli ER2537, E. coli ER2738, E. coli XL-1 Blue, E. coli BL21 (DE3), E. coli JM109, E. coli DH Series, E. coli TOP10, E. coli TG1 and E. coli HB101. In the present invention, E. coli can be used for mass production at low cost by overexpressing a single domain antibody.

The method of carrying the vector of the present invention into a host cell may be performed by CaCl2 method (Cohen, SN et al. (1973), Proc. Natl. Acac. Sci. USA, 9: 2110-2114), one method (Cohen, SN et al. (1973), Proc. Natl. Acac. Sci. USA, 9: 2110-2114; and Hanahan, D. (1983), J. Mol. Biol., 166: 557-580) and electroporation methods ( Dower, WJ et al. (1988), Nucleic. Acids Res., 16: 6127-6145).

Vectors injected into a host cell can be expressed in the host cell, in which case a large amount of the single domain antibody of the invention can be obtained.

In another aspect, the present invention provides a composition for detecting neovascularization comprising the single domain antibody.

As used herein, the term "detection" is for detecting an antigen-antibody complex and can be carried out using various labels. Specific examples of the label include enzymes, fluorescent, ligands, luminescent, microparticles or radioisotopes. Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase and β-latamase and the like. Phosphorus, Eu ³⁺ , Eu ³⁺ chelate or cryptate, and the like, and ligands include biotin derivatives and the like, and luminescent materials include acridinium esters and isoluminol derivatives, and microparticles as colloids. Gold and colored latex, and the like, and radioisotopes include ⁵⁷ Co, ³ H, ¹²⁵ I and ¹²⁵ I-Bolton Hunter reagents, and the like.

In another aspect, the present invention provides a kit for neovascularization detection comprising the single domain antibody.

The kit of the present invention may include not only the single domain antibody of the present invention, but also a color substrate solution to react with a label, a washing solution to be used for each reaction step, and an enzyme stopping solution.

The present invention also provides a composition for diagnosing neovascularization-related diseases comprising the single domain antibody.

The neovascularization-related diseases include atherosclerosis, cancer, diabetic retinopathy, neovascular glaucoma, posterior capsular fibrosis, proliferative vitreoretinopathy, immature retinopathy, ocular inflammation, corneal ulcer, cone thin film, macular degeneration, Sjogren's syndrome, Myopic and tumor, corneal rejection, abnormal wound union, trachoma, bone disease, rheumatoid arthritis, osteoarthritis, sepsis, hemangiomas, angiofibroma, psoriasis ), Pyogenic granuloma, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint insufficiency, nervous system demyelination disease, cirrhosis, renal glomerular disease, immature rupture of the embryonic membrane, inflammatory bowel disease, periodontal disease, ash In the group consisting of stenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging, hyperthyroidism and Grave's disease At least one selected, preferably atherosclerosis or cancer, but not limited thereto.

In the present invention, the cancer is bile duct cancer, bladder cancer, brain tumor, breast cancer, cervical cancer, chorionic cancer, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, multiple myeloma, AIDS-related leukemia and adult T-cell lymphoma / leukemia, epithelial cancer, Liver cancer, lung cancer, lymphoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer or renal cell carcinoma, and the cancer is not limited to α _v β ₃ integrin.

The present invention also provides a base represented by (1) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 Culturing the recombinant microorganism transformed with the recombinant vector comprising at least one selected from the group consisting of sequences; And (2) expressing a single domain antibody against α _v β ₃ integrins in the microorganism, the method providing a α _v β ₃ integrin target single domain antibody.

The term "culture" in the present invention means to grow microorganisms under environmental conditions that are appropriately artificially controlled.

The microorganisms can be grown in a conventional medium, and the medium contains nutrients required by the microorganisms to be cultured, that is, the culture medium to cultivate specific microorganisms, and may be a mixture of additional substances for special purposes. have. The medium may also be referred to as an incubator or a culture medium, and is a concept that includes all natural, synthetic, or selective media.

The medium used for the cultivation should meet the requirements of the particular strain in an appropriate manner while controlling the temperature, pH, etc. in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like. Carbon sources that can be used include mixed sugars of glucose and xylose as the main carbon source, and sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, coconut Oils such as oils and fats, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, skim soy cake or its degradation product Can be. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, suitable precursors to the culture medium may be used. The raw materials described above may be added batchwise, fed-batch or continuous in a suitable manner to the culture in the culture process, but is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.

In another aspect, the present invention comprises the steps of (1) processing the candidate drug to be analyzed in a biological sample or animal model isolated from the sample; (2) binding the α _v β ₃ integrin target single domain antibody to the sample and measuring the level of α _v β ₃ integrin protein; And (3) selecting a candidate drug that induces inhibition or enhancement by comparing the level of the protein of (2) with a control, and provides an angiogenesis inhibitor or promoter screening method.

In another aspect, the present invention (A) binding the α _v β ₃ integrin target single domain antibody to a biological sample isolated from the sample and measuring the level of α _v β ₃ integrin protein; And (b) comparing the level of the α _v β ₃ integrin protein with a reference value obtained from a control sample; provides an information providing method for the diagnosis of neovascularization-related diseases comprising a.

The single domain antibody of the present invention may be provided as a pharmaceutical composition for preventing and treating angiogenesis-related diseases when combined with a functional molecule.

The pharmaceutical composition of the present invention may further comprise an adjuvant in addition to the single domain antibody. The adjuvant may be used without any limitation as long as it is known in the art, but may further include the Freund's complete adjuvant or incomplete adjuvant to increase its effect.

The pharmaceutical composition according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients and diluents commonly used in the pharmaceutical art. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. .

When formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations contain at least one excipient in the active ingredient, for example starch, calcium carbonate, sucrose, lactose, gelatin It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the individual. It is apparent that the concentration of a single domain antibody included in the pharmaceutical composition can be variously selected according to a subject, and preferably, the pharmaceutical composition is included in a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, the pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg / ml, the human body may be toxic.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1 Induction of Immune Response and Serum Quantitation in Camel

<1-1> Inducing Immune Responses in Camels

In order to induce an immune response after inoculating a camel using a recombinant α _v β ₃ intergrin protein having cross activity in humans or mice, the following experiment was performed.

Specifically, 2 mg / ml human α _v β ₃ integrin ITGAVB3 was used as an antigen, premixed in 1 mg / ml PBS, and divided into 800 ul per EP tube at 4 ° C. As adjuvant, Complete Freund's Adjuvant (CFA) (Sigma) was used first, followed by Incomplete Freund's Adjuvant (IFA) (Sigma), and the antigen and adjuvant were thoroughly mixed. To prepare a stable emulsion. Camelus bacteria nus was administered subcutaneously around the back. Subsequently, primary immunization was induced by mixing 2 ml of collected blood (from 1 ml of preimmune serum) with 600 μg antigen mix / camel with complete Freund's adjuvant (CFA). Boost immunization was induced by immunization with 600 μg antigen mix / camel with incomplete Freund's adjuvant (IFA), performed four times at two-week intervals, and 50 ml of blood was obtained 15 days after the fourth booster immunization. Was collected.

<1-2> Serum Titer of Immunized Induced Camels

In order to confirm the titer of the serum of the immuno-induced camel of Example 1-1, ELISA was performed as follows.

Serum titers were measured the day after the fourth booster immunization was performed and 100 uL of target protein (coating buffer containing 10 uL antigen) was coated and incubated overnight at 4 degrees. After washing twice with wash buffer (PBST, PBS with 0.05% Tween-20), the wells were blocked using 350 μL blocking buffer (PBSM, PBS with 4% milk) and allowed to stand at room temperature for 2 hours. Put it. Thereafter, the blocking buffer was shaken off and washed five times using the wash buffer, and the plate was placed on the clean side of the paper towel. 100 μL diluted serum was added to incubate for 1 hour at room temperature and washed 5 times with wash buffer. Rabbit anti-camel IgG pAb-HRP was diluted with PBS and 100 μL diluted antibody per well was added and then incubated at room temperature for 1 hour. A horseradish peroxidase (HRP) substrate solution was prepared as follows: A stock solution of o-phenylenediamine (OPD) was prepared by dissolving 22 mg OPD (Sigma) containing 100 mL of 50 mM sodium citrate, pH 4.0. It was. After the filter was disinfected and stored at 4 degrees, 36 uL 30% H ₂ O ₂ was added to the 21 mL OPD stock solution before the detection step. 100 uL substrate solution was added per well and incubated for 30 minutes at room temperature. Each plate was analyzed using a microplate reader set at 490 nm. The results are shown in Table 1 below.

As shown in Table 1, it was confirmed that the serum of the immuno-induced camel of Example 1-1 showed a titer of 1/12800. Therefore, the last booster immunization was performed the day after the serum titer was measured and peripheral blood leukocytes (PBLs) were obtained the next day and used for RNA extraction and cDNA analysis.

Dilution Coating: A5B3 1/400 1.399 1/800 1.052 1/1600 0.714 1/3200 0.416 1/6400 0.262 1/12800 * 0.156 * 1/25600 0.1 1/51200 0.126 PBS 0.078 [*]: the end titer a. Coating: A5B3, 0.5 ug / well b. 1st Ab: antisera c. 2nd Ab: HRP-rabbit anti-camel IgG

Example 2 Gene Amplification and Gene Library Generation of Single Domain Antibodies

<2-1> RNA Isolation and cDNA Synthesis of Isolated Peripheral Blood Leukocytes (PBLs)

For gene amplification of single domain antibodies of the invention, RNA isolation and cDNA synthesis of isolated peripheral blood leukocytes (PBLs) were performed as follows.

Specifically, peripheral blood leukocytes (PBLs) were isolated from the camel blood induced with the immunity of Example 1-1 by density gradient centrifugation using a Ficoll Hypaque technique. Total RNA was isolated according to the manufacturer's protocol by the Trizol method. The isolated RNA pellet was dried and dissolved in 100ul DEPC-treated water and used for RT-PCR.

An RNase-free 0.5 ml reaction mixture was prepared comprising 2ul dNTP mix (Pharmacia, 25 mM each dNTP), 2ul Oligo-dT primer (200 pMol), 5ul Super RT buffer and 26.5ul DEPC-treated water. Thereafter, 10 ul RNA (including mRNA up to 1.5 ug) isolated to the tube was added and mixed and pipetted. In order to drop a drop of mineral oil (Sigma) to the mixed solution and to destroy the secondary structure, a thermocycler (Biometra, Trio Thermoblock) was heated at 67 degrees for 5 minutes. Then, after cooling to 42 degrees, 2ul RNAsin (Promega) and 2.5ul Super RT (HT Biotechnology Ltd, Cambridge, UK) were added and incubated at 42 degrees for 1 hour, and then heated at 100 degrees for 3 minutes (killed group) Puncture with a needle). The product containing single-stranded cDNA was then centrifuged and stored at -20 degrees.

<2-2> Amplification of the Single Domain Antibody Expression Cassette of the Invention

The cDNA synthesized in 2-1 was quantified by UV spectroscopy. For amplification of the VH-CH1-FC / VHH-FC gene, FC downstream primer, VH / VHH-FR1 upstream primer and cDNA synthesized in 2-1 diluted 200-fold were used as a template. The first PCR amplification of the VH-CH1-FC / VHH-FC gene was performed. The first PCR amplified DNA band of about 600bp was collected and gel purification was performed using a Qiagen Kit. Then, for amplification of the VHH gene, secondary PCR amplification was performed using the VHH-FR4 downstream primer, the VHH-FR1 upstream primer, and the purified DNA. The results are shown in FIGS. 1 and 2.

As shown in FIG. 1, as a result of performing the first PCR amplification, it was confirmed that bands of about 600 bp and 900 bp appeared.

In addition, as shown in FIG. 2, as a result of performing the second PCR amplification, the DNA band of the single domain antibody of the present invention amplified at about 600 bp was confirmed.

Example 3 Construction of a Single Domain Antibody Library of the Present Invention

The selection and retrieval of antibodies recognizing α _v β ₃ integrins from the single domain antibody gene library prepared in Example 2 was carried out using the phage display method, and a single domain antibody clone library was prepared.

<3-1> Insertion and transformation of phagemid of the single domain antibody gene of the present invention

The single domain antibody gene and phagemid of the present invention were cleaved, the truncated gene was inserted into phagemid, and then transformed into E. coli TG1. The secondary PCR product and pDisplay-3M ^TM obtained in 2-2 were digested with BssHII and NheI and simultaneously purified by Qiagen kit. Thereafter, the DNA fragment was ligated using NEB's T4 DNA ligase, and the product was transformed by electro-modification into E. coli TG1 cells. The results are shown in FIGS. 3 and 4.

As shown in FIGS. 3 and 4, gel-electrophoresis of the cleaved single domain gene for phagemid insertion and the cleaved phagemid for cloning the single domain antibody gene was confirmed, and each band was clearly seen.

<3-2> Phage Packaging and Library Preparation

In order to infect the E. coli TG1 transformed in 3-1 to M13K07 helper phage and to increase the phage that specifically binds to the antigen, it was performed as follows.

To prepare M13K07 helper phage, log-phase TG1 cells and M13K07 phage were infected with different dilutions at 37 ° C. for 30 minutes and inoculated in agar of 2TY plates. Small plaques were inoculated into the 3 mL liquid 2TY medium, followed by incubation, and 30 μL of overnight cultured TG1 cells were added and incubated at 37 ° C. for 2 hours. After diluting the culture medium of 1L 2TY medium and incubating for 1 hour, 50 μg / mL kanamycin was added and incubated at 37 ° C. for 16 hours. Cells were removed by centrifugation (10 min, 5000 g) and phage precipitated from the supernatant by adding 0.25 volume of phage precipitate. After culturing on ice for 30 minutes, centrifugation (10 minutes, 5000 g) was performed to obtain phage particles. The pellet was resuspended in 5 mL PBS and then passed through a 0.22-μm filter. The helper phage was confirmed by counting the number of plaque-forming units (pfu) on the 2TY plate with the top-again layer containing 100 μL TG1 (saturated culture). Phage stock solution was diluted to 1Х10 ¹³ pfu / mL and stored at -20 degrees.

In addition, to prepare library phage, 500 mL 2TY-G with library glycerol stock was incubated and incubated at 37 ° C with shaking at 250 rpm until the optical concentration reached 0.8-0.9 at 600 nm. M13KO7 helper phage was added to a final concentration of 5 × 10 ⁹ pfu / mL and incubated at 37 degrees without shaking for 30 minutes, followed by appropriate shaking (200 rpm) for phage infection for 30 minutes. Cells were then recovered by centrifugation at 2200 g for 15 minutes and the pellet was resuspended in the same amount of 2TY-AK. Rapid shaking (300 rpm) was performed to incubate overnight at 30 degrees. Cells were pelleted by centrifugation at 4 degrees, 15 minutes, and 7000 g, and the supernatant containing phage was obtained in a pre-cooled 1 L bottle. Thereafter, 0.3 volume of phage precipitant was added and mixed to allow the phage to precipitate on ice for 1 hour. Thereafter, the cells were centrifuged twice at 4 ° C. for 15 minutes at 7000 g to pellet the phages. The supernatant was removed and the pellet resuspended in 8 mL PBS. Thereafter, the phages were centrifuged at 12,000 g for 10 minutes and phages were collected through the supernatant. Finally, phage stock dilutions were infected with TG1 cells, plated on 2TY-AG and cultured, and the number of ampicillin resistant colonies appearing was determined. Through the above procedure, a single domain library of the present invention was constructed and its titer was measured. The results are shown in Table 2.

As shown in Table 2, it was confirmed that the single domain antibody library of the present invention showed a titer of 5.16 × 10 ¹³ pfu / mL.

<3-3> Confirmation of gene insertion and alignment of DNA in the single domain antibody library of the present invention

Ten representative clones were selected from the single domain library of the present invention selected in 3-2 above, and subjected to QC colony PCR . The results are shown in FIG.

As shown in FIG. 5, 9 clones out of 10 clones were confirmed to be about 600bp, confirming that the single domain of the present invention was correctly inserted.

Through the above procedure, DNA sequencing was performed for 20 clones in which a single domain of the present invention was correctly inserted in a library. S6 downstream primers were used and aligned. The results are shown in FIGS. 6A and 6I.

As shown in FIG. 6A and FIG. 6I, 20 clones were confirmed to exhibit diversity.

Example 4 Specific Antibody Screening by Panning

In order to perform panning in the single domain antibody clone library of the present invention constructed in Example 3, 5-30 μg / mL of the target was coated and incubated for 2 hours at 4 degrees. The wells were then washed three times with wash buffer, then the blocking buffer was filled and incubated overnight at 4 degrees and washed once with wash buffer. PBS-M (2% milk) was added to pre-blocked wells (non-coated) and the original library phages were mixed and incubated for 30 minutes at room temperature. The phages are then placed in pre-blocked (coated) wells and incubated for 1 hour at room temperature. Wash 10 times with wash buffer and elute bound phage by trypsin digestion. The eluate was titrated and amplified. The results are shown in Figure 7 and Table 3.

As shown in FIG. 7, the α _v β ₃ integrin ITGAVB3 antigen to be used for specific antibody selection by panning was verified using SDS-PAGE.

In addition, as shown in Table 3, Phage library candidates with strong binding to the target protein were selected through a three-step selection process.

Round Conditions Input Output Enriching factor 1st Target protein: ITGAVB3 30 ug / ml
Washing: 0.1% PBS-Tween 20, 9 times
Elution: Trypsin digestion 1.12 × 10 ¹¹ 1.61 × 10 ⁵ 6.96 × 10 ⁵ Pre counter select: 2% M-PBS 2nd -P Target protein: ITGAVB3 30 ug / ml
Washing: 0.2% PBS-Tween 20, 9 times
Elution: Trypsin digestion 9.04 × 10 ¹⁰ 2.93 × 10 ⁴ 3.09 × 10 ⁶ Pre counter select: 2% M-PBS 2nd -N Target protein: no coating
Washing: 0.2% PBS-Tween 20, 9 times
Elution: Trypsin digestion 2.26 × ¹⁰ 10 2.6 × 10 ³ 8.69 × 10 ⁶ Pre counter select: 2% M-PBS 3rd -P Target protein: ITGAVB3 30 ug / ml
Washing: 0.2% PBS-Tween 20, 9 times
Elution: Trypsin digestion 1.1 × 10 ¹¹ 2.0 × 10 ⁶ 5.5 × 10 ⁴ Pre counter select: 2% M-PBS 3rd -N Target protein: no coating
Washing: 0.2% PBS-Tween 20, 9 times
Elution: Trypsin digestion 2.76 × 10 ¹⁰ 6.4 × 10 ² 4.31 × 10 ⁷ Pre counter select: 2% M-PBS

Example 5 α Through ELISA _v β ₃  Integrin Specific Single Domain Antibody Assays

After the panning process of Example 4, 40 single domain antibody phage clones were extracted, and binding to α _v β ₃ integrin specific single domain antibodies was analyzed using ELISA.

Specifically, in order to prepare a single clone of antibody-displaying phages, eluate of the single clone was inoculated in 5 mL 2YT-AG medium and incubated overnight at 37 degrees. Glycerol stock for each clone was prepared and incubated overnight, and 100 μL culture was placed in 20 mL of 2YT-AG medium and incubated. Incubate for several hours at 37 degrees until the optical density of OD600 reached 0.4-0.5. M13KO7 helper phage was added in 20 multiple infections (ie, the number of phage particles / host cells). The cells were incubated for 30 minutes at 37 ° C and shaken for 30 minutes. Infected cells were collected by centrifugation (10 min at 5000 g), resuspended in 2YT-AK and incubated for 16 hours at 30 degrees. Subsequently, phage particles were precipitated in the supernatant, and the cell debris was removed by resuspending the phage pellet in 1 mL PBS and centrifuging (10 min at 5000 g). Ab fragments unrelated to phage particles were removed, phage pellets were removed in 250 μL PBS and centrifugation was performed again.

In addition, to perform phage ELISA, 2.5-5 μg / mL of the target protein and the control protein were coated at 4 degrees using a coating buffer, and the wells were washed three times with 200 μL wash buffer per well, followed by 2 at room temperature. The wells were blocked with 200 μL PBS-M for hours. The blocking buffer was shaken off and the plate was washed six times with the wash solution. Thereafter, 100 μL of phage solution was added to the wash buffer per well and incubated at room temperature for 1-2 hours. Then wash 6 times with wash buffer. HRP binding anti-M13 antibody (GE healthcare) is diluted with 1: 5,000 blocking buffer. 100 μL of diluted conjugate per well was added and incubated for 1 hour at room temperature and washed 6 times with wash buffer. The HRP substrate solution was prepared as follows: A stock solution of OPD was prepared by dissolving 22 mg OPD (Sigma) in 100 mL of 50 mg sodium citrate (pH 4.0), and 36 uL 30% H ₂ in 21 mL of OPL stock solution just prior to the detection step. O ² was added. 100 uL of substrate solution per well was added and incubated for 30 minutes at room temperature. Plates were analyzed using a micro plate reader set at 490 nm. Table 4 shows the results of analyzing each clone.

As shown in Table 4, all 40 clones were confirmed to bind strongly to the α _v β ₃ integrin.

Example 6 DNA Sequencing and Information Analysis of Single Domain Antibody Clones of the Present Invention

To perform DNA sequencing of the single domain antibody clones of the invention, each positive clone (measured by phage ELISA and / or soluble ELISA) plasmid in 5 mL LB-A medium (LB medium with 100 μg / mL ampicillin added) 2 μL glycerol stock TG1 E.coli cells were seeded and incubated overnight at 37 degrees. Plasmid Isolation Kit (Qiagen Miniprep kit) was used to isolate plasmids for each positive clone from the cells, and DNA sequencing was performed using L1 and S6 primers.

In addition, in order to analyze the information of each single domain antibody clone, the returned sequences were transformed using Vector NTI®, Version 10, and the protein sequences were aligned. The clones encoding the same protein sequence were then grouped and analyzed. The results are shown in Table 5, FIGS. 8A to 8C, and the single domain antibody clone of the present invention is described as VHH for convenience.

As shown in Table 5, Analysis of 40 clone sequences confirmed 10 candidate VHH DNA sequences.

In addition, as shown in Figures 8a to 8c, clones 14, 11, 29, 17, 15, 28, 12, 19, 13, 21, 27, 16, 18, 20, 30, 23, 24, 25, 22, 26 were selected and their amino acid sequences were shown.

Among them, the nucleotide sequences of the clones VHH-25, VHH-13, VHH-22, VHH-11, VHH-17, VHH-15, VHH-12, VHH-16, VHH-20 and VHH-23 showed strong positive results. Are represented by SEQ ID NOs: 1 to 10, respectively. Among them, amino acid sequences of VHH-25, VHH-13, and VHH-22 are shown in SEQ ID NOs: 11, 12, or 13, respectively.

In clones VHH-11, VHH-17, VHH-15, VHH-12, VHH-13, VHH-16, VHH-20, VHH-23, VHH-25, VHH-22, showing the strong positives, Example 5 The method of ELISA was performed to select single domain antibody clones. The results are shown in Table 6 and Table 7.

As shown in Tables 6 and 7, among 10 VHH clone candidates, VHH-12, VHH-13, VHH-16, VHH-22, and VHH-25 showed relatively high binding strength.

Example 7 Surface Plasmon Resonance (SPR) Analysis of the Single Domain Antibody Clones of the Present Invention

Among the single domain antibodies targeting α _v β ₃ integrin in Example 6, VHH-13, VHH-22 and VHH-25 were selected, and SPR analysis was performed in comparison with the conventional α _v β ₃ integrin antibody.

Specifically, a dextran-coated CMDH chip (Carboxymethyl Dextran Hydrogel Surface Sensor Chip, Reichert Technologies) was mounted on an SPR device (Reichert SR7500DC system), and immobilization of α _v β ₃ integrin antigen was performed. Thereafter, affinity analysis for each concentration of the selected antibody was performed. The results are shown in FIG. 9 and Table 8.

9 and Table 8, the excellent α _v β ₃ integrin binding force appears in the VHH-13, 22-VHH and VHH-25 value of _{8.8-15.5 nM K D (Equilibrium dissociation constant} ) of the antibody was confirmed as shown in.

Example 7 Expression of the Single Domain Antibodies VHH-13, VHH-22 and VHH-25 of the Invention

In Example 6, it was finally confirmed that VHH-13, VHH-22, and VHH-25 were the best among the single domain antibody clones of the present invention. A vector was produced to produce the same, and introduced into E. coli, the single domain antibody VHH. -25 was expressed.

Specifically, a vector containing a His6 tag was prepared for the expression of the selected single domain antibody clone of the present invention, VHH-13, VHH-22 or VHH-25, and a vector map for VHH-25 (pALT-Avb3 VHH25). ) Is shown in Figure 10a. In addition, in order to increase the binding force with other functional molecules or elements, a vector comprising a lysine (Lysin) and His6 tag was prepared, a vector map (pALT-Avb3 VHH25K1) for this is shown in Figure 10b. The amino acid sequences containing His6 tag in each of the clones VHH-25, VHH-13 or VHH-22 are shown in SEQ ID NOs: 14, 15 and 16, respectively.

The VHH-13, VHH-22 or VHH-25 expression results including the His6 tag were confirmed by performing reduced SDS-PAGE, and are shown in FIGS. 11A to 11C and VHH including Lysine and His6 tag. -25 expression results are shown in Figure 11d.

As a result, it was confirmed that VHH-13 was about 15.9 kDa (FIG. 11A), and VHH-22 was about 14.1 kDa (FIG. 11B). In addition, it was confirmed that VHH-25 is about 14.5 kDa (FIG. 11C), and lysine-containing VHH-25 is about 14.2 kDa (FIG. 11D).

Example 8 [alpha] of single domain antibody VHH-25 of the present invention in cancer cells _v β ₃ Identify Integrin Targeted Effects

To confirm the α _v β ₃ integrin target effect of the single domain antibody VHH-25 of the present invention on cancer cells in vitro, the following experiment was performed.

Specifically, 0.5 mg of VHH-25-K1 containing lysine and 0.25 mg of Cy5.5 (sulfo-Cy5.5-NHS, Lumiprobe) were dissolved in 2 ml of borate buffer (pH 8.4), and the reaction was performed at room temperature for 30 minutes. It was. After the reaction was completed, purification of antibody VHH-25-K into which Cy5.5 was introduced was performed using a PD-10 column (GE healthcare). The results are shown in FIG.

As shown in FIG. 12, Cy5.5-introduced antibody VHH-25-K synthesis and purification was confirmed by SDS-PAGE, and selectively in α _v β ₃ integrin-activated U87-MG cells (human glioblastoma) Cellular uptake was confirmed.

Example 9 α of Single Domain Antibody VHH-25 of the Present Invention at Tumor Sites _v β ₃ Identify Integrin Targeted Effects

In order to confirm the α _v β ₃ integrin target effect of the single domain antibody VHH-25 of the present invention in a tumor mouse animal model, the following experiment was performed.

Specifically, animal models were prepared by xenografting U87-MG cells in Athymic nude mice (female, 6-8 weeks old). When tumors grew to 0.8-1 cm in size, VHH-25-K was bound to Cy5.5 and injected into the tail vein (1 mg / kg). The contrast effect was analyzed using IVIS (PerkinElmer). The results are shown in FIG.

As shown in FIG. 13, 30 minutes after the injection, the contrast effect could be confirmed at the tumor site, and the contrast signal was strongly observed for 4 hours. Then, it was confirmed that the continuous contrast signal appeared at the tumor site for 24 hours.

Example 10 α of the Single Domain Antibody VHH-25 of the Invention at the Atherosclerotic Site _v β ₃ Identify Integrin Targeted Effects

In order to confirm the α _v β ₃ integrin target effect of the single domain antibody VHH-25 of the present invention in an atherosclerotic mouse animal model, the following experiment was performed.

Specifically, a 36-week high-fat diet was performed on ApoE-/-mice (Female, 10 weeks old) to prepare an animal model in which intravascular plaques were formed. Thereafter, after binding Cy5.5 to VHH-25-K prepared in the same manner as in Example 9 and injecting the tail vein (1 mg / kg), the mice were sacrificed and the aorta was separated after 4 hours. The contrast effect of the isolated aorta was analyzed using IVIS. The results are shown in FIG.

As shown in Figure 14, 4 hours after the injection, it was confirmed that the contrast effect appeared selectively at the site where the plaque was formed.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION          KOREA BASIC SCIENCE INSTITUTE <120> Single-domain antibody targeting alpha-v beta-3 integrin <130> PN1711-423 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> VHH-25 <400> 1 gaggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtgtag tctctggata cacctgtagc gtctacgaca tgatctggta ccgccagcct 120 ccagggaagg agcgcgagtt cgtctcactc attaatagta atggtagaac aacctacgca 180 gactccgtga agggccgatt caccatctcc caaaacaacg ccaagaacac ggtgtatctg 240 cagatgaaca gcctgaaacc tgaggacacg gcaatgtact actgtcatgc ggcctgctat 300 tcaccctctc ggctgaatta ttggggccag gggacccagg tcactgtctc ctca 354 <210> 2 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> VHH-13 <400> 2 catgtgcagc tggtggagtc tgggggaggc tcggtgcagc ctggagagtc tctgagactc 60 tcctgtgtag cctctggata caccgtcagt agctcctgca tggcctggtt ccgccaggct 120 ccaggaaagg agcgcgaggt ggtcgcaact attgttgtta ctagtgatac gaccagcact 180 ttctatgccg actccgtaaa gggccgattc accatctccc cagacaacgc caagaataca 240 ctaaatctgc aaatgaatag cctggaaccg tccgacactg ccatgtacta ctgtgcggca 300 gatcgcaagt ggaacgtttg tagtcgtggt tatcgctaca cccctaattg ggccaaccaa 360 tttacgttct ggggccaggg gacccaggtc accgtctcct ca 402 <210> 3 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> VHH-22 <400> 3 caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtgtag tctctggata cacctgtagc gtctacgaca tgatctggta ccgccagcct 120 ccagggaagg agcgcgagtt cgtctcactc attaatagta atggtagaac aacctacgca 180 gactccgtga agggccgatt caccatctcc caaaacaacg ccaagaacac ggtgtatctg 240 cagatgaaca gcctgaaacc tgaggacacg gcaatgtact actgtcatgc ggcctgctat 300 tcaccctctc ggctgaatta ttggggccag gggaccctgg tcaccgtctc ctca 354 <210> 4 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> VHH-11 <400> 4 gatgtgcagc tggtggagtc tgggggaggc tcggtgcagc ctggagagtc tctgagactc 60 tcctgtgtag cctctggata caccgtcagt agctcctgca tggcctggtt ccgccaggct 120 ccaggaaagg agcgcgaggt ggtcgcaact attgttgtta ctagtgatac gaccagcact 180 ttctatgccg actccgtaaa gggccgattc accatctccc cagacaacgc caagaatacg 240 ctaaatctgc aaatgaatag cctggaaccg tccgacactg ccatgtacta ctgtgcggca 300 gatcgcaagt ggaacgtttg tagtcgtggt tatcgctaca cccctaattg ggccaaccaa 360 tttacgttct ggggccaggg gaccttggtc accgtctcct ca 402 <210> 5 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> VHH-17 <400> 5 gatgtgcagc tggtggagtc tgggggaggc tcggtgcagc ctggagagtc tctgagactc 60 tcctgtgtag cctctggata caccgtcagt agctcctgca tggcctggtt ccgccaggct 120 ccaggaaagg agcgcgaggt ggtcgcaact attgttgtta ctagtgatac gaccagcact 180 ttctatgccg actccgtaaa gggccgattc accatctccc cagacaacgc caagaatacg 240 ctaaatctgc aaatgaatag cctggaaccg tccgacactg ccatgtacta ctgtgcggca 300 gatcgcaagt ggaacgtttg tagtcgtggt tatcgctaca cccctaattg ggccaaccaa 360 tttacgttct ggggccaggg gaccttggtc accgtctcct ca 402 <210> 6 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> VHH-15 <400> 6 gaggtgcagc tggtggagtc tgggggaggc tcggtgcagc ctggagagtc tctgagactc 60 tcctgtgtag cctctggata caccgtcagt agctcctgca tggcctggtt ccgccaggct 120 ccaggaaagg agcgcgaggt ggtcgcaact attgttgtta ctagtgatac gaccagcact 180 ttctatgccg actccgtaaa gggccgattc accatctccc cagacaacgc caagaatacg 240 ctaaatctgc aaatgaatag cctggaaccg cccgacactg ccatgtacta ctgtgcggca 300 gatcgcaagt ggaacgtttg tagtcgtggt tatcgctaca cccctaattg ggccaaccaa 360 tttacgttct ggggccaggg gacccaggtc accgtctcct ca 402 <210> 7 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> VHH-12 <400> 7 gaggtgcagc tggtggagtc tgggggaggc tcggtgcagc ctggagagtc tctgagactc 60 tcctgtgtag cctctggata caccgtcagt agctcctgca tggcctggtt ccgccaggct 120 ccaggaaagg agcgcgaggt ggtcgcaact attgttgtta ctagtgatac gaccagcact 180 ttctatgccg actccgtaaa gggccgattc accatctccc cagacaacgc caagaatacg 240 ctaaatctgc aaatgaatag cctggaaccg tccgacactg ccatgtacta ctgtgcggca 300 gatcgcaagt ggaacgtttg tagtcgtggt tatcgctaca cccctaattg ggccaaccaa 360 tttacgttct ggggccaggg gacccaggtc accgtctcct ca 402 <210> 8 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> VHH-16 <400> 8 gatgtgcagc tggtggagtc tgggggaggc tcggtgcagc ctggagggtc tctgagactc 60 tcctgtgcag cctctggata caccgtcgag aactcctgca tggcctggtt ccgccaggct 120 ccagggaagg agcgcgaggt ggtcgcaact attgttacta ataatgcgac cggtactttc 180 tatgccgact ccgtgaaggg ccgattcacc gtctcccaag acaacgccaa gaatacgcta 240 aatctgcaaa tgaatagcct ggaacctgag gacacagcca tgtactactg tgcggcagat 300 accaagtgga tagtttgtag tcgtggttat cgctacaccc ctaattgggc caaccaattt 360 aagtactggg gccaggggac ccaggtcacc gtctcctca 399 <210> 9 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> VHH-20 <400> 9 gatgtgcagc tggtggagtc tgggggaggc tcggtgcagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggata caccgtcgat aactcctgca tggcctggtt ccgccaggct 120 ccagggaagg agcgcgaggt ggtcgcaact attgttacta ataatgcgac cagcactttc 180 tatgccgact ccgtgaaggg ccgattcacc gtctcccacg acaacgccaa gaatacgcta 240 aatctgcaaa tgaataccct ggaacctgag gacactgcca tgtactactg tgcggcagat 300 accaagtgga tagtttgtag tcgtggttat cgctacaccc ctaattgggc caaccatttt 360 aattactggg gccaggggac cctggtcacc gtctcctca 399 <210> 10 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> VHH-23 <400> 10 catgtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60 tcctgtgcag cctctggata cacctcaagt accgtctaca tggcttggtt ccgccagact 120 ccagggaagc agcgcgaggg ggtcgcagca atttatactg gtggtggtcc tacatactat 180 gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa tacggtgtat 240 ctccaaatga acaccctgaa acctgaagac actgccatgt actactgtgc ggccgatcgc 300 tatgtgtacc ggttagttac taactggtac agaccgtctt tttatacata ctggggccag 360 gggacccagg tcaccgtctc ctca 384 <210> 11 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VHH-25 of amino acid <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Tyr Thr Cys Ser Val Tyr              20 25 30 Asp Met Ile Trp Tyr Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val          35 40 45 Ser Leu Ile Asn Ser Asn Gly Arg Thr Thr Tyr Ala Asp Ser Val Lys      50 55 60 Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Asn Thr Val Tyr Leu  65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys His                  85 90 95 Ala Ala Cys Tyr Ser Pro Ser Arg Leu Asn Tyr Trp Gly Gln Gly Thr             100 105 110 Gln Val Thr Val Ser Ser         115 <210> 12 <211> 134 <212> PRT <213> Artificial Sequence <220> <223> VHH-13 of amino acid <400> 12 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Glu   1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Thr Val Ser Ser Ser              20 25 30 Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val          35 40 45 Ala Thr Ile Val Val Thr Ser Asp Thr Thr Ser Thr Phe Tyr Ala Asp      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Pro Asp Asn Ala Lys Asn Thr  65 70 75 80 Leu Asn Leu Gln Met Asn Ser Leu Glu Pro Ser Asp Thr Ala Met Tyr                  85 90 95 Tyr Cys Ala Ala Asp Arg Lys Trp Asn Val Cys Ser Arg Gly Tyr Arg             100 105 110 Tyr Thr Pro Asn Trp Ala Asn Gln Phe Thr Phe Trp Gly Gln Gly Thr         115 120 125 Gln Val Thr Val Ser Ser     130 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VHH-22 of amino acid <400> 13 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Tyr Thr Cys Ser Val Tyr              20 25 30 Asp Met Ile Trp Tyr Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val          35 40 45 Ser Leu Ile Asn Ser Asn Gly Arg Thr Thr Tyr Ala Asp Ser Val Lys      50 55 60 Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Asn Thr Val Tyr Leu  65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys His                  85 90 95 Ala Ala Cys Tyr Ser Pro Ser Arg Leu Asn Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 14 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> VHH-25 with His6 tag for purification <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Tyr Thr Cys Ser Val Tyr              20 25 30 Asp Met Ile Trp Tyr Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val          35 40 45 Ser Leu Ile Asn Ser Asn Gly Arg Thr Thr Tyr Ala Asp Ser Val Lys      50 55 60 Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Asn Thr Val Tyr Leu  65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys His                  85 90 95 Ala Ala Cys Tyr Ser Pro Ser Arg Leu Asn Tyr Trp Gly Gln Gly Thr             100 105 110 Gln Val Thr Val Ser Ser Leu Glu His His His His His His         115 120 125 <210> 15 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> VHH-13 with His6 tag for purification <400> 15 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Glu   1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Tyr Thr Val Ser Ser Ser              20 25 30 Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val          35 40 45 Ala Thr Ile Val Val Thr Ser Asp Thr Thr Ser Thr Phe Tyr Ala Asp      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Pro Asp Asn Ala Lys Asn Thr  65 70 75 80 Leu Asn Leu Gln Met Asn Ser Leu Glu Pro Ser Asp Thr Ala Met Tyr                  85 90 95 Tyr Cys Ala Ala Asp Arg Lys Trp Asn Val Cys Ser Arg Gly Tyr Arg             100 105 110 Tyr Thr Pro Asn Trp Ala Asn Gln Phe Thr Phe Trp Gly Gln Gly Thr         115 120 125 Gln Val Thr Val Ser Ser Leu Glu His His His His His His     130 135 140 <210> 16 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> VHH-16 with His6 tag for purification <400> 16 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Tyr Thr Cys Ser Val Tyr              20 25 30 Asp Met Ile Trp Tyr Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val          35 40 45 Ser Leu Ile Asn Ser Asn Gly Arg Thr Thr Tyr Ala Asp Ser Val Lys      50 55 60 Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Asn Thr Val Tyr Leu  65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys His                  85 90 95 Ala Ala Cys Tyr Ser Pro Ser Arg Leu Asn Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser Leu Glu His His His His His His         115 120 125

Claims (20)

1 selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10; Α _v β ₃ Integrin target Single domain antibody, encoded as a species or more. The method of claim 1,
The base sequence represented by SEQ ID NO: 1 encodes an amino acid sequence represented by SEQ ID NO: 11, the base sequence represented by SEQ ID NO: 2 encodes an amino acid sequence represented by SEQ ID NO: 12, and a base represented by SEQ ID NO: 3 The α _v β ₃ integrin target single domain antibody, characterized in that the sequence encodes an amino acid sequence represented by SEQ ID NO: 13. The method of claim 1,
The antibody is a variable region of a heavy chain antibody (VHH) derived from Camelidae, α _v β ₃ integrin target single domain antibody. The method of claim 1,
The antibody is characterized in that the coupling additionally has functional molecules or elements, α _v β ₃ integrin target single domain antibodies. The method of claim 1,
The functional molecule or element is α _v β ₃ integrin target single domain antibody, characterized in that at least one selected from the group consisting of inorganic particles, radioisotopes, chemicals, peptides, polypeptides, nucleic acids, carbohydrates and lipids. The method of claim 5,
Α _v β ₃ integrin target single domain antibody, characterized in that the inorganic particles are fluorescent markers or staining material. The method of claim 6,
The fluorescent marker or staining material may be GFP (Green Fluorescent Protein), YFP (Yellow Fluorescent Protein), BFP (Blue fluorescent protein), CFP (Cyan fluorescent protein), Acridine dyes, Cyanine dyes ), Fluorine dyes, oxazine dyes, phenanthridine dyes and rhodamine dyes, characterized in that at least one selected from the group consisting of α _v β ₃ Integrin target single domain antibody. The method of claim 5,
The radioisotope is ¹⁸ F (fluorine), ¹¹ C (carbon), ¹⁵ O (oxygen), ¹³ N (nitrogen), ⁸⁹ Zr (zirconium), C ¹⁵ O, ¹³ N (ammonia), H ₂ ¹⁵ O And ¹⁸ FDG (F-Deoxy Glucose), characterized in that at least one selected from the group consisting of α _v β ₃ integrin target single domain antibody. 1 selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10; Recombinant vectors comprising more than one species. A recombinant microorganism transformed with the recombinant vector of claim 9. Composition for detecting neovascularization comprising the single domain antibody of claim 1. The kit for detecting neovascularization, comprising the single domain antibody of claim 1. A diagnostic composition for angiogenesis-related diseases comprising a single domain antibody of claim 1. The method of claim 13,
The neovascularization-related diseases include atherosclerosis, cancer, diabetic retinopathy, neovascular glaucoma, posterior capsular fibrosis, proliferative vitreoretinopathy, immature retinopathy, ocular inflammation, corneal ulcer, cone thin film, macular degeneration, Sjogren's syndrome, Myopia and tumors, corneal rejection, aberrant wound union, trachoma, bone disease, rheumatoid arthritis, osteoarthritis, sepsis arthritis, hemangiomas, angiofibroma, psoriasis ), Pyogenic granuloma, proteinuria, abdominal aortic aneurysm, degenerative cartilage loss due to traumatic joint insufficiency, nervous system demyelination disease, cirrhosis, renal glomerular disease, immature rupture of the embryonic membrane, inflammatory bowel disease, periodontal disease, ash In the group consisting of stenosis, inflammatory diseases of the central nervous system, Alzheimer's disease, skin aging, hyperthyroidism and Grave's disease A diagnostic composition for angiogenesis-related diseases, characterized in that at least one selected. (1) a group consisting of the nucleotide sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 Culturing the recombinant microorganism transformed with the recombinant vector comprising at least one selected from the group; And
(2) expressing a single domain antibody against α _v β ₃ integrin in the microorganism; comprising the α _v β ₃ integrin target single domain antibody. (1) processing the candidate drug to be analyzed in a biological sample or animal model isolated from the sample;
(2) combining the first term α _v β ₃ integrin target single domain antibodies in the sample and measuring the levels of α _v β ₃ integrin protein; And
(3) A method for screening angiogenesis inhibitors or promoters comprising selecting a candidate drug whose level of protein (2) is inhibited or enhanced compared to a control. The method of claim 16,
The biological sample of (1) is a blood, plasma, serum, cell or tissue sample, characterized in that the angiogenesis inhibitor or promoter screening method. The method of claim 16,
The animal of (1) is rat, mouse, monkey, dog, cat, cow, horse, pig, sheep or goat screening method of angiogenesis inhibitor or promoter. The method of claim 16,
The measurement of (2) is enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, fluid cytometry (flow cytometry), fluorescence activated cell sorting (FACS), enzymatic substrate coloring and antigen-antibody aggregation method is carried out using any one or more methods selected from the group consisting of angiogenesis inhibitor or promoter screening method. (a) combining a first term α _v β ₃ integrin target single domain antibodies in a biological sample isolated from the subject and measuring the level of α _v β ₃ integrin protein; And
(b) comparing the level of the α _v β ₃ integrin protein with a reference value obtained from a control sample; and an information providing method for diagnosing neovascularization-related diseases.

Images