ANTIBODY SPECIFICALLY BINDING TO ICAM-1 AND USE THEREOF

Abstract

The present invention relates to an anti-ICAM-1 antibody or an antigen-binding fragment thereof that specifically binds to ICAM-1, and the use thereof. Specifically, provided are an anti-ICAM-1 antibody or an antigen-binding fragment thereof, a pharmaceutical composition for regulating differentiation and/or function of dendritic cell, and a pharmaceutical composition for preventing and/or treating immune cell-mediated disease, the composition comprising the antibody or the antigen-binding fragment as an active ingredient

Claims

1. An anti-ICAM-1 antibody or an antigen-binding fragment thereof, comprising the following complementarity determining regions (CDRs): CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

2. The anti-ICAM-1 antibody or the antigen-binding fragment thereof according to claim 1, comprising as a framework: a heavy chain framework 1 comprising the amino acid sequence of SEQ ID NO: 97 or SEQ ID NO: 98; a heavy chain framework 2 comprising the amino acid sequence of SEQ ID NO: 99 or SEQ ID NO: 100; a heavy chain framework 3 comprising the amino acid sequence of SEQ ID NO: 101 or SEQ ID NO: 102; a heavy chain framework 4 comprising the amino acid sequence of SEQ ID NO: 81; a light chain framework 1 comprising the amino acid sequence of SEQ ID NO: 103 or SEQ ID NO: 104; a light chain framework 2 comprising the amino acid sequence of SEQ ID NO: 105; a light chain framework 3 comprising the amino acid sequence of SEQ ID NO: 106; and a light chain framework 4 comprising the amino acid sequence of SEQ ID NO: 107.

3. The anti-ICAM-1 antibody or the antigen-binding fragment thereof according to claim 1, comprising as a framework: a heavy chain framework 1 comprising an amino acid sequence selected from SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48; a heavy chain framework 2 comprising an amino acid sequence selected from SEQ ID NOs: 49, 50, 51, 52, 53, 54, and 55; a heavy chain framework 3 comprising an amino acid sequence selected from SEQ ID NOs: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, and 80; a heavy chain framework 4 comprising the amino acid sequence of SEQ ID NO: 81; a light chain framework 1 comprising an amino acid sequence selected from SEQ ID NOs: 82, 83, and 84; a light chain framework 2 comprising an amino acid sequence selected from SEQ ID NOs: 85, 86, 87, 88, and 89; a light chain framework 3 comprising an amino acid sequence selected from SEQ ID NOs: 90, 91, and 92; and a light chain framework 4 comprising the amino acid sequence of SEQ ID NO: 93 or SEQ ID NO: 94.

4. The anti-ICAM-1 antibody or the antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 11 to 34; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 35 to 38.

5. The anti-ICAM-1 antibody or the antigen-binding fragment thereof according to claim 1, wherein the anti-ICAM-1 antibody is an animal antibody, a chimeric antibody, or a humanized antibody.

6. The anti-ICAM-1 antibody or the antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is scFv, (scFv).sub.2, Fab, Fab, or F(ab).sub.2 of the anti-ICAM-1 antibody.

7. A method of prevention or treatment of immune cell mediated disease, comprising administering a pharmaceutically effective amount of the anti-ICAM-1 antibody or the antigen-binding fragment thereof according to claim 1 to a subject in need of prevention or treatment of the immune cell mediated disease.

8. The method of claim 7, wherein the immune cell mediated disease is transplant rejection, graft versus host disease, asthma, obesity, type 2 diabetes, immune cell mediated inflammation, or autoimmune disease.

9. The method of claim 8, wherein the autoimmune disease is encephalomyelitis, rheumatoid arthritis, systemic lupus erythematosus, atopic dermatitis, multiple sclerosis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's disease, Sjogren's syndrome, myasthenia gravis, scleroderma, polyarteritis nodosa, Kikuchi disease, collagen disease, Hashimoko thyroiditis, psoriasis, vitiligo, hyperthyroidism, fibromyalgia, alopecia areata, or allergy.

10. A nucleic acid molecule encoding an amino acid sequence selected from SEQ ID NOs: 1 to 6.

11. A nucleic acid molecule encoding an amino acid sequence selected from SEQ ID NOs: 7 and 11 to 34; an amino acid sequence selected from SEQ ID NOs: 8 and 35 to 38; or both of them.

12. A recombinant vector comprising the nucleic acid molecule of claim 10.

13. A recombinant cell comprising the recombinant vector of claim 12.

14. A method for producing an anti-ICAM-1 antibody or an antigen-binding fragment thereof, comprising a step of culturing the recombinant cell of claim 13.

15. A method for detecting ICAM-1, comprising treating a biological sample with the anti-ICAM-1 antibody or the antigen-binding fragment thereof of claim 1.

16. A pharmaceutical composition comprising the anti-ICAM-1 antibody or the antigen-binding fragment thereof according to claim 1.

17. A recombinant vector comprising the nucleic acid molecule of claim 11.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 is a result of confirming the binding of the chimeric antibody SI9 prepared in one example to Du145 cells, the ICAM-1 expressing cell line, by flow cytometry.

[0082] FIG. 2a to 2d are graphs showing results of quantifying purified antibodies by measuring OD (optical density) at 280 nm in one example.

[0083] FIG. 3a to 3d are graphs showing the results of measuring the peak symmetry factor by performing size exclusion HPLC (hereinafter, SE-HPLC) analysis on the anti-ICAM-1 antibody obtained according to one example.

[0084] FIG. 4a to 4d are graphs showing the results of measuring the fluorescence change by the ANS reagent while leaving the anti-ICAM-1 antibody obtained according to one example at 61? C. for 1 hour.

[0085] FIG. 5 is a graph showing the results of measuring ANS reactivity after leaving the anti-ICAM-1 antibody obtained according to one example at 67? C. for 1 hour.

[0086] FIG. 6 is a graph comparing the binding power of the anti-ICAM-1 antibody to the ICAM-1 antigen obtained according to one example by ELISA test (y-axis: 450 nm OD value; x-axis: antibody concentration (ng/ml)).

[0087] FIG. 7a to 7c are graphs showing the melting point of the anti-ICAM-1 antibody obtained according to one example (7a: chimeric antibody, 7b: H17L1, 7c: H17L4).

[0088] FIG. 8 is a graph showing the affinity (KD) by measuring the binding constant (Kon) and the dissociation constant (Koff) of the anti-ICAM-1 antibody obtained according to one example.

[0089] FIG. 9 is a heat map graph showing changes in gene expression profiles by administration of a humanized antibody obtained according to one example under various peripheral blood conditions.

[0090] FIG. 10 is a graph showing changes in the gene expression profile obtained by administration of the humanized antibody DNP007 obtained according to one example under various peripheral blood conditions by analyzing the KEGG pathway (?10 log (adjusted p-value)).

[0091] FIG. 11 is a graph showing changes in the gene expression profile obtained by administration of the humanized antibody DNP007 obtained according to one example under various peripheral blood conditions by analyzing the GO Biological process (?10 log (adjusted p-value)).

[0092] FIGS. 12a and 12b are graphs showing changes in the gene expression profile obtained by administration of the humanized antibody DNP007 obtained according to one example under various peripheral blood conditions by analyzing the increase and decrease of cytokine genes.

[0093] FIG. 13 is a graph showing changes in cytokines obtained by administration of the humanized antibody DNP007 obtained according to one example under various peripheral blood conditions by analyzing the changed increase and decrease in the protein level.

[0094] FIG. 14 is a graph showing changes in cytokines obtained by administration of the humanized antibody DNP007 obtained according to one example under conditions of co-culture with dendritic cells and their own T cells by analyzing the changed increase and decrease in the protein level.

[0095] FIG. 15 is a graph showing the expression level of maturation cofactors in dendritic cells treated with the humanized antibody DNP007 obtained according to one example.

[0096] FIG. 16 is a graph showing the degree of secretion of inflammatory cytokines in dendritic cells treated with the humanized antibody DNP007 obtained according to one example.

[0097] FIG. 17 is a graph showing the degree of apoptosis of dendritic cells treated with the humanized antibody DNP007 obtained according to one example.

[0098] FIGS. 18a and 18b are graphs showing the expression levels of dendritic cell surface factors treated with the humanized antibody DNP007 obtained according to one example, and show whether or not tolerogenic dendritic cells are induced.

[0099] FIGS. 19a and 19b are graphs showing the expression level of IDO (Indoleamine 2,3-Dioxygenase) in dendritic cells treated with the humanized antibody DNP007 obtained according to one example, FIG. 19a is a Canonical pathway result, FIG. 19b is a non-canonical pathway result. Show each.

[0100] FIG. 20 is a graph showing the level of production of interferon gamma (IFN-r), an inflammatory cytokine and degree of proliferation of T cells cultured with dendritic cells treated with humanized antibody DNP007 obtained according to one example.

[0101] FIG. 21 shows the degree of T cell proliferation according to various dendritic cell maturation pathways (LPS stimulation, TNF-a stimulation, and CD40L stimulation) and the degree of production of interferon gamma (IFN-r), an inflammatory cytokine, of T cells cultured with dendritic cells treated with humanized antibody DNP007 obtained according to one example.

[0102] FIGS. 22a to 22c are results of confirming the therapeutic effect of rheumatoid arthritis of the humanized antibody DNP007 obtained according to one example in an animal model of rheumatoid arthritis. FIGS. 22a and 22b show arthritis scores before and after antibody administration, and FIG. 22c shows anti-collagen antibody levels, respectively.

[0103] FIGS. 23a and 23b are the results of confirming the graft-versus-host disease inhibitory effect of the humanized antibody DNP007 obtained according to one example in the graft-versus-host disease animal model. FIG. 22a shows the survival rate, and FIG. 22b shows the degree of T cell re-establishment, respectively.

MODE FOR CARRYING OUT THE INVENTION

[0104] Hereinafter, the present invention will be described in more detail by way of examples, but this is only illustrative and is not intended to limit the scope of the present invention.

<Example 1> Preparation of Mouse Anti-ICAM-1 Monoclonal Antibody

[0105] In order to develop an antibody specific for ICAM-1, the following experiment was performed.

1-1: Mouse Immunity

[0106] Recombinant human ICAM-1 protein (0.5 mg/ml; NCBI accession number NP_000192.2) was mixed with an equal volume of adjuvant (Invivogen, #vac-adx-10) to prepare an immune substance. A 6-week-old Balb/c female mouse intraperitoneal cavity (IP) was injected with 200 uL of the prepared immune substance three times at intervals of 3 weeks.

1-2: Preparation of Mouse Anti ICAM-1 Monoclonal Antibody

[0107] The spleen of the immunized mouse as described above was excised to obtain a single cell suspension. The obtained cells were washed twice with RPMI (GIBCO, #21875034), and then mixed with 0.4% (w/v) trypan blue (sigma) at 1:1 (v/v), and then then the number of cells was counted by staining with trypan blue (Sigma-aldrich, #T8154). The X63 mouse myeloma cell line (ATCC CRL-1580) was washed, counted, and used as a cell fusion partner.

[0108] The myeloma cells and splenocytes were mixed at a ratio of 1:5, and the supernatant was removed after centrifugation. 1 ml of 50% (w/v) PEG (polyethylene glycol) 1500 preheated to 37? C. was slowly added over 1 minute. After holding for about 1 minute, RPMI medium was slowly added and diluted stepwise. After centrifugation, it was suspended in RPMI (20% FBS, hypoxanthine-aminopterin-thymidine; Gibco) containing 1?HAT (hypoxanthine, aminopterin, thymidine), dispensed into a 96-well plate at 150 uL/well, and then cultured in a 37? C. 5% CO.sub.2 incubator. After the fusion, HAT feeding was performed for a certain period of time, and when a well formed colony was observed, 150 uL of HT medium (hypoxanthine, thymidine) was added and cultured in a 37? C. 5% CO.sub.2 incubator for 48 hours.

[0109] 100 uL of the cultured medium was taken from a hybridoma culture 96-well plate to confirm whether or not ICAM-1 was reactive. ICAM-1 (R&D system, #ADP4-050) was diluted in PBS at a concentration of 1.0 ug (microgram)/ml, then dispensed into Nunc-immunoplate (Thermo, #439454) at 100 uL/well, stored in a 37? C. incubator for 1 hour, and coated. After completely removing the coating solution, 1? casein blocking solution (sigma-aldrich, #B6429) was dispensed at 200 uL/well and stored in a 37? C. incubator for 1 hour to perform blocking. After completely removing the blocking solution, the hybridoma cultured solution was dispensed at 100 uL/well and stored in a 37? C. incubator for 1 hour to induce an antigen/antibody reaction. After completely removing the cultured solution, it was washed three times with a washing solution (0.02% Tween 20 in PBS). The secondary antibody Goat anti-mouse IgG-HRP (Jackson, #) was diluted in a blocking solution at a ratio of 1:10,000 (v/v), dispensed at 100 uL/well, and stored in a 37? C. incubator for 30 minutes to induce a secondary antibody reaction. After washing three times, TMB (Thermo, #) was dispensed at 80 uL/well to induce color development and then 1.0 N sulfuric acid (H.sub.2SO.sub.4) was added to terminate the reaction. Flow cytometry was additionally performed to confirm whether the obtained anti-ICAM-1 antibody recognized ICAM-1 expressed on the cell surface. To the prostate cancer cell line Du145 (ATCC, #HTB-81) 1?10.sup.6 cells expressing ICAM-1, 100 uL of the cultured solution was added and allowed to stand at room temperature for 15 minutes to induce antibody binding. After washing by adding PBS, 100 ul of the secondary antibody Goat anti-Mouse IgG-FITC (Jackson, #) diluted in PBS at a ratio of 1:100 (v/v) was added and reacted at room temperature for 10 minutes. After washing was performed to remove the unreacted secondary antibody, reactivity was confirmed by flow cytometry. As in the above-described test method, a positive antibody that binds to the ICAM-1 antigen was first selected, and a monoclonal antibody capable of fluorescent staining in the Du145 cell line was additionally selected. The selected monoclonal antibody (named SI9) exhibited high reactivity to the ICAM-1 protein antigen and the property of effectively binding to the surface of the Du145 cell line.

<Example 2> Preparation of Chimeric Anti-ICAM-1 Monoclonal Antibody

2-1. Anti-ICAM-1 Antibody Gene Sequence Cloning

[0110] The gene of the mouse monoclonal antibody SI9 selected in Example 1 was cloned using Mouse Ig-Primer Set (Millipore, Cat. #: 69831). The monoclonal antibody SI9-producing hybridoma developed in Example 1 was cultured, and total RNA was extracted from the fusion cell line. PCR was performed using the RNA extracted using Mouse Ig-Primer Set as a template, and after inserting it into the pGem-T vector (Promega, Cat. #: A3600), the DNA sequence was confirmed through sequencing, and the mouse antibody gene was identified through the IMGT site (www.imgt.org). The heavy and light chain variable region amino acid sequences and coding nucleic acid sequences of the analyzed SI9 antibody are summarized in Table 1:

TABLE-US-00001 TABLE1 name Sequence SEQIDNO SI9Chimeric GYTFTDYA 1 V.sub.H-CDR1 SI9Chimeric ISTYSGNT 2 V.sub.H-CDR2 SI9Chimeric ARSLYFGSSGFDY 3 V.sub.H-CDR3 SI9Chimeric QTLVYRNGNTY 4 V.sub.L-CDR1 SI9Chimeric KVS 5 V.sub.L-CDR2 SI9Chimeric SQNTHFPYT 6 V.sub.L-CDR3 SI9Chimeric QVQLQQSGAELVRPGVSVKISCKGSGYTFTDYALHW 7 V.sub.H(amino VKQSHAKSLEWIGVISTYSGNTDYNQKFRGKATMTV acidsequence) DKSSTTAYLELARLTSEDSAIHYCARSLYFGSSGFDY WGQGTALTVSS SI9Chimeric DVVLTQTPLSLPVNLGDQASISCRSSQTLVYRNGNTY 8 V.sub.L(amino LHWYLQKAGQSPKLLIYKVSNRFSGVPDRFSGSGSGT acidsequence) DFTLKISRVEAEDLGVYFCSQNTHFPYTFGGGTKIKR SI9Chimeric caggtgcagctgcagcagagcggcgcggaactggtgcgcccgggcgtgagcgtg 9 V.sub.H(coding aaaattagctgcaaaggcagcggctatacctttaccgattatgcgctgcattgggtga nucleicsequence) aacagagccatgcgaaaagcctggaatggattggcgtgattagcacctatagcggc acid aacaccgattataaccagaaatttcgcggcaaagcgaccatgaccgtggataaaag cagcaccaccgcgtatctggaactggcgcgcctgaccagcgaagatagcgcgattc attattgcgcgcgcagcctgtattttggcagcagcggctttgattattggggccaggg caccgcgctgaccgtgagcagctaa SI9Chimeric gatgtggtgctgacccagaccccgctgagcctgccggtgaacctgggcgatcagg 10 V.sub.L(coding cgagcattagctgccgcagcagccagaccctggtgtatcgcaacggcaacacctat nucleicacid ctgcattggtatctgcagaaagcgggccagagcccgaaactgctgatttataaagtg sequence) agcaaccgctttagcggcgtgccggatcgctttagcggcagcggcagcggcaccg attttaccctgaaaattagccgcgtggaagcggaagatctgggcgtgtatttttgcagc cagaacacccattttccgtatacctttggcggcggcaccaaaattaaacgc

2-2. Preparation of Chimeric Antibody

[0111] Based on the amino acid sequence of the prepared anti-ICAM-1 mouse antibody SI9 above, an anti-ICAM-1 chimeric antibody was prepared.

2-2-1. Plasmid Construction

[0112] For expression of the anti-ICAM-1 chimeric antibody, a plasmid for heavy chain expression and a plasmid for light chain expression were prepared, respectively. The plasmid for light chain expression was prepared using the pOptiVEC (Invitrogen) vector, and the plasmid for heavy chain expression was prepared using the pcDNA3.3 (Invitrogen) vector.

[0113] In order to express each variable region coding cDNA and constant region coding cDNA of the antibody as a continuous amino acid sequence without additional amino acid insertion, each of the genes obtained by linking the cloned variable region coding nucleic acid sequence (see Table 1) with the known human IgG4 constant region (heavy chain; GenBank_AIC59040.1) coding nucleic acid sequence or the kappa constant region (light chain; GenBank_AAA58989.1) coding nucleic acid sequence was synthesized (Bioneer). The CPSCP sequence of the middle hinge region of the light chain constant region was further modified to CPPCP like the IgG1 isotype to prevent antibody shuffling. The heavy and light chain expression genes synthesized as described above were cut with restriction enzymes Xho I and Sal I, and then the light chain expression gene was ligated to the pOptiVec vector, and the heavy chain expression gene was ligated to the pcDNA3.3 vector, respectively, thereby constructing a plasmid for complete antibody expression. (pcDNA3.3-anti-ICAM-1 heavy chain expression plasmid and pOptiVEC-anti-ICAM-1 light chain expression plasmid).

2-2-2. Transformation

[0114] The prepared pcDNA3.3-anti-ICAM-1 heavy chain expression plasmid and pOptiVEC-anti-ICAM-1 light chain expression plasmid were transfected into DG44 cells (Invitrogen) derived from CHO cells to perform a transformation process.

[0115] First, 3 days before transfection, suspended DG44 cells were cultured in MEMa medium containing 5% FBS to induce adherent cells. Transformation was performed in a 6 well plate using ViaFect transfection regent (Promega, Cat. #: E4981). One day before transformation, DG44 cells adapted to adherent state were prepared by subculture at a concentration of 1?10.sup.5 cells/well. And the amount of DNA used for transformation was used in a combination of 1:2 ratio of pcDNA3.3-anti-ICAM-1 heavy chain expression plasmid and pOptiVEC-anti-ICAM-1 light chain expression plasmid, respectively, 1.0 ?g and 2.0 ?g. Transformation was carried out for 48 hours. The transformed cell population was analyzed using a flow cytometer, and the results are shown in FIG. 1. As shown in FIG. 1, it is possible to confirm the binding phenomenon of the chimeric antibody obtained by inserting the variable region coding gene of the mouse SI9 antibody into the constant region coding gene of the human antibody, to the ICAM-1 expressing cell line.

<Example 3> Preparation of Humanized Anti-ICAM-1 Antibody

3.1 Recombinant Antibody Sequence Selection by in Silico Humanization

[0116] The humanized antibody sequence obtained by recombining the germline framework region encoding the human antibody gene while maintaining the amino acid sequence of each of the heavy and light chain CDRs (CDRH1: GYTFTDYA (SEQ ID NO: 1), CDRH2: ISTYSGNT (SEQ ID NO: 2), CDRH3: ARSLYFGSSGFDY (SEQ ID NO: 3), CDRL1: QTLVYRNGNTY (SEQ ID NO: 4), CDRL2: KVS (SEQ ID NO: 5), CDRL3: SQNTHFPYT (SEQ ID NO: 6) of the chimeric ICAM-1 antibody (SI9) was selected in silico method.

[0117] As a result, as humanized DNP007 antibody sequences, 24 kinds of heavy chain variable regions and 4 kinds of light chain variable regions were selected, respectively. The amino acid sequences of the heavy chain variable region, light chain variable region, CDR, and framework of the selected humanized antibody are shown in Tables 2, 3, 4, and 5 below. In Tables 2 and 3 below, the regions in bold underlined are the amino acid sequences of the CDRs (CDR1, CDR2, and CDR3 in order).

TABLE-US-00002 TABLE2 Heavychainvariableregionsequenceof humanizedDNP007antibodyclassification classification name Aminoaicdsequence SEQIDNO VH1 QVQLVQSGAEVKKPGASVKISCKGSGYTFTDYAL 11 HWVRQAPGQRLEWIGVISTYSGNTDYNQKFRGRA TITRDTSASTAYMELSSLRSEDTAVYYCARSLYFG SSGFDYWGQGTALTVSS VH2 QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAL 12 HWVRQAPGQRLEWMGVISTYSGNTDYNQKFRGR VTMTVDTSASTAYMELSSLRSEDTAVHYCARSLY FGSSGFDYWGQGTALTVSS VH3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAL 13 HWVRQAPGQRLEWMGVISTYSGNTDYNQKFRGR ATITRDKSASTAYLELSSLRSEDTAVHYCARSLYF GSSGFDYWGQGTALTVSS VH4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYAL 14 HWVRQAPGQRLEWIGVISTYSGNTDYNQKFRGRV TMTRDTSATTAYLELSSLRSEDTAVYYCARSLYFG SSGFDYWGQGTALTVSS VH5 QVQLVQSGAEVKKPGASVKISCKASGYTFTDYAL 15 HWVRQAPGQRLEWMGVISTYSGNTDYNQKFRGR VTITVDKSATTAYMELSSLRSEDTAVYYCARSLYF GSSGFDYWGQGTALTVSS VH6 QVQLVQSGAEVVKPGASVKVSCKGSGYTFTDYAL 16 HWVRQAPGQRLEWIGVISTYSGNTKYSQKFQGKA TITRDKSASTAYLELSSLRSEDTAVYYCARSLYFGS SGFDYWGQGTALTVSS VH7 QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAL 17 HWVKQAPGQRLEWMGVISTYSGNTKYSQKFQGR ATMTRDTSATTAYMELSSLRSEDTAIHYCARSLYF GSSGFDYWGQGTALTVSS VH8 QVQLQQSGAEVKKPGASVKISCKGSGYTFTDYAL 18 HWVRQAPGQSLEWMGVISTYSGNTKYSQKFQGR ATMTRDKSASTAYMELSSLRSEDTAVYYCARSLY FGSSGFDYWGQGTALTVSS VH9 QVQLQQSGAEVVKPGASVKVSCKASGYTFTDYAL 19 HWVRQAPGQSLEWMGVISTYSGNTKYSQKFQGK VTITVDTSATTAYMELSSLRSEDTAVHYCARSLYF GSSGFDYWGQGTALTVSS VH10 QVQLVQSGAEVVKPGASVKISCKASGYTFTDYAL 20 HWVKQAPGQSLEWMGVISTYSGNTKYSQKFQGR VTITVDTSASTAYLELSSLRSEDTAIYYCARSLYFG SSGFDYWGQGTALTVSS VH11 QVQLQQSGAEVKKPGASVKISCKASGYTFTDYAL 21 HWVRQAPGQRLEWIGVISTYSGNTKYSQKFQGKV TITRDTSASTAYLELSSLRSEDTAIHYCARSLYFGSS GFDYWGQGTALTVSS VH12 QVQLQQSGAEVKKPGASVKVSCKASGYTFTDYAL 22 HWVKQAPGQRLEWIGVISTYSGNTKYSQKFQGRV TMTVDKSATTAYMELSSLRSEDTAVYYCARSLYF GSSGFDYWGQGTALTVSS VH13 QVQLVQSGAEVKKPGASVKISCKGSGYTFTDYAL 23 HWVRQAPGQRLEWMGVISTYSGNTDYNQKFRGR VTITRDKSASTAYLELSSLRSEDTAVYYCARSLYF GSSGFDYWGQGTALTVSS VH14 QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAL 24 HWVRQAPGQRLEWMGVISTYSGNTDYNQKFRGR VTITVDTSATTAYMELSSLRSEDTAVHYCARSLYF GSSGFDYWGQGTALTVSS VH15 QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAL 25 HWVRQAPGQRLEWIGVISTYSGNTDYNQKFRGRV TMTRDTSASTAYMELSSLRSEDTAVHYCARSLYF GSSGFDYWGQGTALTVSS VH16 QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAL 26 HWVRQAPGQRLEWMGVISTYSGNTDYNQKFRGR ATMTRDTSASTAYLELSSLRSEDTAVYYCARSLYF GSSGFDYWGQGTALTVSS VH17 QVQLVQSGAEVKKPGASVKISCKGSGYTFTDYAL 27 HWVRQAPGQRLEWMGVISTYSGNTDYNQKFRGR ATITVDTSASTAYMELSSLRSEDTAVYYCARSLYF GSSGFDYWGQGTALTVSS VH18 QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAL 28 HWVRQAPGQRLEWIGVISTYSGNTDYNQKFRGRV TITRDKSATTAYMELSSLRSEDTAVYYCARSLYFG SSGFDYWGQGTALTVSS VH19 QVQLVQSGAEVKKPGASVKISCKGSGYTFTDYAL 29 HWVRQAPGQRLEWIGVISTYSGNTKYSQKFQGRA TITVDTSATTAYLELSSLRSEDTAVYYCARSLYFGS SGFDYWGQGTALTVSS VH20 QVQLVQSGAEVKKPGASVKISCKGSGYTFTDYAL 30 HWVRQAPGQRLEWIGVISTYSGNTKYSQKFQGRA TMTRDTSASTAYLELSSLRSEDTAVHYCARSLYFG SSGFDYWGQGTALTVSS VH21 QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAL 31 HWVRQAPGQRLEWIGVISTYSGNTKYSQKFQGRV TMTVDKSATTAYLELSSLRSEDTAVYYCARSLYFG SSGFDYWGQGTALTVSS VH22 QVQLVQSGAEVKKPGASVKISCKGSGYTFTDYAL 32 HWVRQAPGQRLEWMGVISTYSGNTKYSQKFQGR ATMTRDKSATTAYMELSSLRSEDTAVHYCARSLY FGSSGFDYWGQGTALTVSS VH23 QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAL 33 HWVRQAPGQRLEWMGVISTYSGNTKYSQKFQGR ATMTVDKSASTAYLELSSLRSEDTAVHYCARSLYF GSSGFDYWGQGTALTVSS VH24 QVQLVQSGAEVKKPGASVKISCKGSGYTFTDYAL 34 HWVRQAPGQRLEWIGVISTYSGNTKYSQKFQGRV TITVDKSATTAYMELSSLRSEDTAVHYCARSLYFG SSGFDYWGQGTALTVSS

TABLE-US-00003 TABLE3 LightchainvariableregionsequenceofhumanizedDNP007antibody classification name Aminoaicdsequence SEQIDNO VL VL1 DVVLTQSPLSLPVTLGQPASISCRSSQTLV 35 YRNGNTYLHWYQQRPGQSPRLLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDV GVYFCSQNTHFPYTFGGGTKLEIK VL2 DVVLTQSPLSLPVTLGQPASISCRSSQTLV 36 YRNGNTYLHWYQQRAGQSPRLLIYKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAED VGVYFCSQNTHFPYTFGGGTKLEIK VL3 DVVLTQTPLSSPVTLGQPASISCRSSQTLV 37 YRNGNTYLHWYLQRAGQPPRLLIYKVSN RFSGVPDRFSGSGAGTDFTLKISRVEAED VGVYFCSQNTHFPYTFGGGTKLEIK VL4 DVVLTQTPLSSPVTLGQPASISCRSSQTLV 38 YRNGNTYLHWYQQRPGQPPRLLIYKVSN RFSGVPDRESGSGAGTDFTLKISRVEAED VGVYFCSQNTHFPYTFGGGTKLEIK

TABLE-US-00004 TABLE4 FrameworksequenceofhumanizedDNP007antibodyheavychainvariableregion classification Sequence SEQIDNO VHnumberapplied VH-FR1 QVQLQQSGAELVRPGVSVKISCKGS 39 Chimeric QVQLVQSGAEVKKPGASVKISCKGS 40 VH1,VH13,VH17, VH19,VH20, VH22,VH24 QVQLVQSGAEVKKPGASVKVSCKAS 41 VH3,VH4 QVQLVQSGAEVKKPGASVKISCKAS 42 VH5 QVQLVQSGAEVVKPGASVKVSCKGS 43 VH6 QVQLQQSGAEVKKPGASVKISCKGS 44 VH8 QVQLQQSGAEVVKPGASVKVSCKAS 45 VH9 QVQLVQSGAEVVKPGASVKISCKAS 46 VH10 QVQLQQSGAEVKKPGASVKISCKAS 47 VH11 QVQLQQSGAEVKKPGASVKVSCKAS 48 VH12 VH-FR2 LHWVKQSHAKSLEWIGV 49 Chimeric LHWVRQAPGQRLEWIGV 50 VH1,VH4,VH6, VH11,VH15, VH18,VH19, VH20,VH21,VH24 LHWVRQAPGQRLEWMGV 51 VH2,VH3,VH5, VH13,VH14, VH16,VH17, VH22,VH23 LHWVKQAPGQRLEWMGV 52 VH7 LHWVRQAPGQSLEWMGV 53 VH8,VH9 LHWVKQAPGQSLEWMGV 54 VH10 LHWVKQAPGQRLEWIGV 55 VH12 VH-FR3 DYNQKFRGKATMTVDKSSTTA 56 Chimeric YLELARLTSEDSAIHYC DYNQKFRGRATITRDTSASTAY 57 VH1 MELSSLRSEDTAVYYC DYNQKFRGRVTMTVDTSASTAY 58 VH2 MELSSLRSEDTAVHYC DYNQKFRGRATITRDKSASTAY 59 VH3 LELSSLRSEDTAVHYC DYNQKFRGRVTMTRDTSATTAY 60 VH4 LELSSLRSEDTAVYYC DYNQKFRGRVTITVDKSATTAY 61 VH5 MELSSLRSEDTAVYYC KYSQKFQGKATITRDKSASTAY 62 VH6 LELSSLRSEDTAVYYC KYSQKFQGRATMTRDTSATTAY 63 VH7 MELSSLRSEDTAIHYC KYSQKFQGRATMTRDKSASTAY 64 VH8 MELSSLRSEDTAVYYC KYSQKFQGKVTITVDTSATTAY 65 VH9 MELSSLRSEDTAVHYC KYSQKFQGRVTITVDTSASTAYL 66 VH10 ELSSLRSEDTAIYYC KYSQKFQGKVTITRDTSASTAYL 67 VH11 ELSSLRSEDTAIHYC KYSQKFQGKVTITRDTSASTAYL 68 VH12 ELSSLRSEDTAIHYC DYNQKFRGRVTITRDKSASTAY 69 VH13 LELSSLRSEDTAVYYC DYNQKFRGRVTITVDTSATTAY 70 VH14 MELSSLRSEDTAVHYC DYNQKFRGRVTMTRDTSASTAY 71 VH15 MELSSLRSEDTAVHYC DYNQKFRGRATMTRDTSASTAY 72 VH16 LELSSLRSEDTAVYYC DYNQKFRGRATITVDTSASTAY 73 VH17 MELSSLRSEDTAVYYC DYNQKFRGRVTITRDKSATTAY 74 VH18 MELSSLRSEDTAVYYC KYSQKFQGRATITVDTSATTAYL 75 VH19 ELSSLRSEDTAVYYC KYSQKFQGRATMTRDTSASTAY 76 VH20 LELSSLRSEDTAVHYC KYSQKFQGRVTMTVDKSATTA 77 VH21 YLELSSLRSEDTAVYYC KYSQKFQGRATMTRDKSATTAY 78 VH22 MELSSLRSEDTAVHYC KYSQKFQGRATMTVDKSASTAY 79 VH23 LELSSLRSEDTAVHYC KYSQKFQGRVTITVDKSATTAY 80 VH24 MELSSLRSEDTAVHYC VH-FR4 WGQGTALTVSS 81 Chimeric,VH1, VH2,VH3,VH4, VH5,VH6,VH7, VH8,VH9,VH10, VH11,VH12, VH13,VH14, VH15,VH16, VH17,VH18, VH19,VH20, VH21,VH22, VH23,VH24

TABLE-US-00005 TABLE5 FrameworksequenceofhumanizedDNP007antibody lightchainvariableregion classification Sequence SEQIDNO VLnumberapplied VL-FR1 DVVLTQTPLSLPVNLGDQASISCRSS 82 Chimeric DVVLTQSPLSLPVTLGQPASISCRSS 83 VL1,VL2 DVVLTQTPLSSPVTLGQPASISCRSS 84 VL3,VL4 VL-FR2 LHWYLQKAGQSPKLLIY 85 Chimeric LHWYLQKAGQSPKLLI 86 VL1 LHWYQQRAGQSPRLLIY 87 VL2 LHWYLQRAGQPPRLLIY 88 VL3 LHWYQQRPGQPPRLLIY 89 VL4 VL-FR3 NRFSGVPDRFSGSGSGTDFTLKISR 90 Chimeric,VL1 VEAEDLGVYFC NRFSGVPDRFSGSGSGTDFTLKISR 91 VL2 VEAEDVGVYFC NRFSGVPDRFSGSGAGTDFTLKISR 92 VL3,VL4 VEAEDVGVYFC VL-FR4 FGGGTKIKRQ 93 Chimeric FGGGTKLEIKR 94 VL1,VL2,VL3, VL4

3.2 Expression and Analysis of Humanized Recombinant Antibody

[0118] The selected antibody sequences were ligated to human IgG4 heavy chain constant region and kappa light chain constant region, respectively, and expressed in HEK293 cells (ATCC CRL-1573) in the form of human IgG4.

TABLE-US-00006 TABLE6 HumanizedDNP007antibodyconstantregionsequence classification Aminoancidsequence SEQIDNO CH ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV 95 SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK CL TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ 96 WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0119] After 7 days of transfection, the humanized recombinant antibody was purified from the cultured solution using KanCap A resin (Kaneca).

[0120] The purified antibody was quantified by measuring OD (optical density) at 280 nm, and the results are shown in FIGS. 2a to 2d. As shown in FIGS. 2a to 2d, most of the antibodies produced regardless of the type of heavy and light chains to be combined showed relatively high productivity.

[0121] In order to analyze the purity of the purified antibody, a size exclusion HPLC (hereinafter, SE-HPLC) analysis was performed using a Sepax Zenix-C SEC-300 size exclusion column (Sepax technologies) to measure the peak symmetry factor. The obtained results are shown in FIGS. 3a to 3d. When the high-purity antibody is analyzed by SE-HPLC, one symmetric peak is observed, but when heterogeneous substances such as macromolecule/small molecule are included, two or more peaks are observed or the symmetric peak factor is observed to be low. As shown in FIGS. 3a to 3d, in most cases, the anti-ICAM-1 humanized antibody exhibited a relatively high symmetric peak factor.

3-3. Humanized Antibody Selection According to Physicochemical/Biological Properties

3-3-1. High Stability Humanized Antibody Screening

3-3-1-1. Primary Screening of High-Stability Humanized Antibodies

[0122] In order to preferentially select antibodies with high stability, humanized antibodies showing resistance to heat denaturation were selected by leaving the antibodies under severe conditions of high temperature and observing changes in their physical properties.

[0123] The measurement of heat denaturation was confirmed through a binding experiment using 8-anilino-1-naphthalenesulfonic acid (hereinafter ANS; Sigma). ANS is a compound that can confirm the presence or absence of protein denaturation because it binds to the hydrophobic site exposed during protein denaturation and emits light with a wavelength of 470 nm.

[0124] 96 kinds of humanized recombinant antibodies prepared in Example 2-1 were constantly diluted to a concentration of 0.2 mg/ml using PBS (phosphate buffered saline), and left at a high temperature (61? C.) for an appropriate time (1 hour). 20 uL of a 0.2 mg/ml ANS solution was added to 200 uL of the antibody sample, mixed, reacted for 15 minutes, and analyzed under 360 nm excitation and 460 nm emission conditions. The 460 nm wavelength emitted by the ANS reaction was measured with a fluorometer (BioTek, SynergyHT), and relative evaluation based on the H1L1 antibody (100%) was shown in FIGS. 4a to 4d. In the case of heavy chains VH7 and VH22, irrespective of the light chain to be combined, 470 nm fluorescence emission by ANS appeared to increase rapidly, and it was confirmed that it was slightly denatured by heat, but other antibodies showed relatively stability against heat.

3-3-1-2. Secondary Screening of Highly Stable Humanized Antibodies

[0125] A total of 47 kinds of humanized antibodies showing relatively high antibody productivity, relatively high peak symmetry factor, and/or relatively high thermal stability under high temperature (61? C.) conditions in FIGS. 2a-2d, 3a-3d, and 4a-4d, were selected primarily (see FIG. 5).

[0126] For more stringent stability evaluation, the 47 kinds of humanized antibodies were constantly diluted to a concentration of 0.2 mg/ml, left at 67? C. for 1 hour, and the ANS reactivity was measured, and the relative reactivity based on the H1L1 antibody was shown in FIG. 5. Compared with the ANS reactivity at 61? C. (4a to 4d), the ANS reactivity at 67? C. tended to be slightly higher, but 6 types of humanized antibodies of the combination of H17L1, H4L3, H5L3, H11L3, H17L3, and H17L4 (See Table 7) showed relatively low ANS reactivity, and were evaluated as antibodies having high resistance to heat denaturation.

TABLE-US-00007 TABLE 7 Antibody name HC & LC combination H17L1 VH17 + VL1 H4L3 VH4 + VL3 H5L3 VH5 + VL3 H11L3 VH11 + VL3 H17L3 VH17 + VL3 H17L4 VH17 + VL4

3-3-2. Antigen Binding Assay

[0127] As a stability test evaluation, as shown in Table 7, humanized antibodies of 6 leading antibodies were selected, and the binding ability of 5 antibodies excluding H11L3 was compared with the parent antibody (chimeric antibody; see Example 1.3).

[0128] The antigen ICAM-1 (ICAM-1; R&D System) was coated on a 96 well plate at 100 ng per well, followed by blocking. The amount of the primary antibody was serially diluted twice from 80 ng/ml and bound for 1 hour at 37? C. and a secondary antibody diluted 1:10000 with goat anti-Human Ig-HRP conjugate (Jackson ImmunoResearch) was combined at 37? C. for 30 minutes. Each step was washed three times and reacted with TMB (3,3,5,5-Tetramethylbenzidine). After the reaction was stopped by treatment with 1N H.sub.2SO.sub.4 solution in the same amount (100 ul) as the TMB solution, the OD value was measured at 450 nm.

[0129] The binding ability of the parent antibody and the five humanized recombinant antibodies obtained as described above to the ICAM-1 antigen is shown in FIG. 6 and Table 8.

TABLE-US-00008 TABLE 8 Antibody concentration (ng/ml) Chimeric H17L1 H4L3 H5L3 H17L3 H17L4 80 1.138 1.108 1.229 1.246 1.197 1.238 40 0.724 0.802 0.832 0.862 0.848 0.983 20 0.473 0.506 0.520 0.592 0.567 0.637 10 0.298 0.297 0.305 0.35 0.344 0.349 5 0.182 0.186 0.189 0.208 0.201 0.236 2.5 0.118 0.119 0.115 0.128 0.135 0.136

[0130] All 5 types of the evaluated humanized antibodies generally showed higher reactivity than the chimeric parent antibody, and among them, the antibody of the H17L4 combination showed the highest reactivity.

3-3-3. Melting Temperature Analysis

[0131] In particular, the melting temperatures of the humanized antibodies H17L1 and H17L4 exhibiting stable physical properties were compared with the parent antibody (chimeric antibody; see Example 1.3) to evaluate the stability.

[0132] Protein thermal shift dye (Lifetechnologies, #4466038) was added to the antibody sample to prepare a reaction sample, and the temperature was continuously raised from 25? C. to 95? C. to induce denaturation of the antibody sample. Antibody denaturation was confirmed by irradiating the reaction solution with a wavelength of 580 nm and measuring the wavelength of 623 nm emitted from the protein thermal shift dye, and the melting temperature was analyzed with ViiA? 7 software. The obtained results are shown in Table 9 below and in FIGS. 7a (chimeric parent antibody), 7b (H17L1) and 7c (H17L4):

TABLE-US-00009 TABLE 9 Melting temperature 1 Melting temperature 2 Chimeric 67.26? C. 71.47? C. H17L1 67.14? C. 82.33? C. H17L4 66.91? C. 82.57? C.

[0133] As shown in Table 9 and FIGS. 7a to 7c, two melting temperatures were identified in each antibody sample. Melting temperature 1 was similarly measured at about 67? C. for all three antibodies including the chimeric parental antibody, but melting temperature 2 was 10? C. or more in the two humanized antibodies compared to the chimeric parental antibody. This means that 2 types of humanized antibodies have strong resistance to heat denaturation compared to the chimeric parent antibody.

3-3-4. Affinity Measurement

[0134] The Octet system (ForteBio) was used to compare the affinity of the H17L1 and H17L4 humanized antibodies to the antigen with the parent antibody. Each of three antibodies was attached to the amine reactive Bio-sensor AR2G (ForteBio), and 5 different concentrations of ICAM-1 antigen solution were added to induce antigen/antibody reactions to occur. Using the antigen/antibody reaction results, the binding constant (Kon) and the dissociation constant (Koff) were measured, and the affinity (KD) was calculated, and the results are shown in Table 10 and FIG. 8:

TABLE-US-00010 TABLE 10 Kon(1/Ms) Koff(1/s) Rmax(nm) KD(M) Full X.sup.2 Full R.sup.2 Chimeric 1.65 ? 10.sup.5 1.69 ? 10.sup.?3 0.3511 1.03 ? 10.sup.?8 0.0635 0.9869 H17L1 2.49 ? 10.sup.5 8.53 ? 10.sup.?4 0.4283 3.43 ? 10.sup.?9 0.0487 0.9927 H17L4 2.56 ? 10.sup.5 9.29 ? 10.sup.?4 0.4282 3.62 ? 10.sup.?9 0.1555 0.9702

[0135] As shown in Table 10 and FIG. 8, the affinity (KD) of the chimeric parent antibody was measured as 1.03?10.sup.?8 M, and the H17L1 and H17L4 humanized antibodies were 3.43?10.sup.?9 and 3.62?10.sup.?9 M, respectively. It was possible to confirm the improved affinity in the humanized antibody.

<Example 4> Immunomodulatory Efficacy Test in Peripheral Blood (In Vitro)

4-1. Isolation of Peripheral Blood Mononuclear Cell and Measurement of IFNg Changes

[0136] In order to confirm the immunomodulatory efficacy of the antibodies provided herein, peripheral blood mononuclear cells were treated with the DNP007 antibody (H17L4 antibody) prepared in Example 3, and the amount of secretion of IFN-gamma, a representative pro-inflammatory cytokine, was measured. Blood was collected from normal volunteers, and peripheral blood mononuclear cells were isolated with Ficoll-Paque Plus (GE healthcare, #17144002). The isolated peripheral blood mononuclear cells were suspended in RPMI medium supplemented with 10% FBS, and GM-CSF (Creagene) and IL-4 (Creagene) were added at a concentration of 100 ng/ml, respectively, so that dendritic cells could differentiate from monocytes. DNP007 antibody was added to peripheral blood mononuclear cells at a concentration of 10.0 ug/ml.

[0137] In order to confirm the efficacy of DNP007 antibody on the dendritic progenitor cell monocyte, the isolated peripheral blood mononuclear cells were cultured in a culture dish for 2 hours to induce adhesion to the culture dish, and then the adherent cells and suspended cells were divided, respectively, GM-CSF and IL-4 were added at a concentration of 100 ng/ml, respectively, and DNP007 antibody (10.0 ug/ml) was added together.

[0138] On the 6th day of culture, peripheral blood mononuclear cells, adherent cells, and suspension cells were washed, respectively, and Lipopolysaccharides (LPS, Sigma-Aldrich, #L2630) were added at 5.0 ug/ml and cultured. The cultured solution was taken the next day and the concentration of IFN-gamma secreted by the Human IFN gamma Uncoated ELISA kit (Invitrogen, #88-7316-77) was measured.

[0139] In the sample group treated with the DNP007 antibody under all conditions of peripheral blood mononuclear cells, adherent cells, and suspended cells, the amount of INF-gamma secretion was markedly reduced (Table 11). These results were understood as a phenomenon that appears when the DNP007 antibody suppresses the mechanism of immune cell activation by LPS.

TABLE-US-00011 TABLE 11 IFN- Relative Treatment gamma (pg/ml) change (%) Whole () 559.7 100% PBMC DNP007 (10.0 ug/ml) 166.9 29.8% Adherent () 612.7 100% PBMC DNP007 (10.0 ug/ml) 425.9 69.5% Suspended () 513.9 100% PBMC DNP007 (10.0 ug/ml) 130.0 25.3%

4-2. Analysis of the Effect of DNP007 Antibody on Peripheral Blood Mononuclear CellsGene Expression Profile Analysis (Transcriptome Analysis by RNA Sequencing)

[0140] To determine whether the immune response regulation of DNP007 is only due to dendritic cells or contact with immune cells including dendritic cells and T cells, human peripheral blood mononuclear cells were isolated and divided into 5 kinds of peripheral blood groups, and 100 ng/ml GM-CSF and 100 ng/ml IL-4 were added. And cells were obtained by culturing for 6 days under conditions with or without the addition of 10 ug/ml of DNP007 antibody. For induction into mature dendritic cells, the cells were washed on the 6th day of culture and treated with 5 ug/ml LPS (Sigma-Aldrich) for one day to obtain cells for each group, and total RNA was isolated and gene expression profiles were analyzed. [0141] (1) Whole PBMC: Human peripheral blood mononuclear cells were isolated from healthy volunteer blood by performing concentration gradient centrifugation with Ficoll-Paque (GE Healthcare). [0142] (2) CD14+ monocyte-derived dendritic cells: CD14+ monocytes were isolated from human peripheral blood mononuclear cells separated by concentration gradient centrifugation of healthy volunteer blood with Ficoll-Paque (GE Healthcare), using magnetic separation (using of magnetic beads). [0143] (3) CD14 deplete PBMC: The CD14+ monocytes of (2) were separated by magnetic separation (using magnetic beads) and the remaining peripheral blood population was used. [0144] (4) Attached PBMC: Human peripheral blood mononuclear cells isolated by performing concentration gradient centrifugation with Ficoll-Paque (GE Healthcare) from healthy volunteers are attached to the cells in a 37? C., 5% CO.sub.2 incubator for 2 hours in RPMI medium supplemented with 10% FBS, and then the supernatant (including suspension cells) was removed and only adherent cells were separated. [0145] (5) The upper layer of suspension cells removed from the experiment in (4) was used.

[0146] The results are shown in FIG. 9. As shown in FIG. 9, dendritic cells differentiated from CD14+ monocytes alone (A) and peripheral blood populations without CD14+ monocytes (B) showed a gene expression profile similar to that of the control group not treated with DNP007. On the other hand, in the condition (C,D,E) in which several other immune cells are present together, rather than the condition in which CD14+ derived dendritic cells exist alone, when DNP007 was added, the gene expression profile was significantly different from that of the control group not treated with DNP007.

[0147] In addition, as a result of analyzing the KEGG pathway & GO Biological process by dividing genes up and down twice or more compared to the control group not treated with DNP007 into gene groups with similar functions in these total gene expression profiles, the gene expression profiles changed by DNP007 were mostly genes involved in regulating immune responses including inflammatory responses (FIGS. 10 and 11). A schematic diagram of a part for cytokines among genes having a clear pattern of change is shown in FIGS. 12a and 12b. As a result of gene expression analysis of the upper layer of suspension cells and whole peripheral blood mononuclear cell (whole PBMC) samples excluding adherent cells in peripheral blood, as shown in FIGS. 12a and 12b, it was found that gene expression of cytokines, which are targets of representative autoimmune diseases such as IL6, IL17, IL23, and IL36, was suppressed by treatment with the DNP007 antibody.

[0148] Through these results, the ICAM-1 antibody DNP007 proposed in the present specification does not affect only dendritic cells, but acts on the contact process or cross-reaction between dendritic cells and other immune cells, through this, it can be seen that the expression of various immune response genes is regulated, resulting in an immunosuppressive response.

4-3. Analysis of the Effect of DNP007 Antibody on Peripheral Blood Mononuclear CellsCytokine Analysis

[0149] When preparing the 4-2 experiment set above, in order to confirm whether the gene expression change showed a correlation with the actual protein expression change, in the 4-2 experiment, the supernatant was separated and the actual cytokines were quantitatively analyzed.

[0150] IFNgamma, IL1 beta, IL-6, IL-10, IL-12 (p70), IL-17, IL-27 were analyzed using Human Luminex Screening assay (LXSAH, R&D) kit, and TGF-beta was analyzed using a TGF-beta premixed assay kit (TGTBMAG-64K-03; Merck Millipore).

[0151] All of the cytokines analyzed as shown in FIG. 13 showed a reduction in actual protein expression of 40 to 80% level by administration of the DNP007 antibody. This is a result consistent with the gene expression profile result of Example 4-2, and it can be said that the immunosuppressive response by the DNP007 antibody was reconfirmed.

[0152] In addition, even when the immune activation was induced by LPS treatment under conditions of co-culture with T cells of the same person after separating into peripheral blood of normal humans and differentiating into dendritic cells, the sample administered with the DNP007 antibody showed remarkable inhibition of expression of immune cytokines at the protein level as shown in FIG. 14.

Example 5. Effect Test of Antibody on Dendritic Cells

5-1. Semi-Matured Dendritic Cells Induction Activity Test in Peripheral Blood Mononuclear Cells

[0153] In this example, the effect of the antibody provided herein on the differentiation and maturation of dendritic cells was confirmed. More specifically, after attaching CD14 microbead (Miltenyi Biotec, 130-050-201) to peripheral blood prepared by collecting blood from normal volunteers, human peripheral blood mononuclear cell was separated using a cell collection device (Miltenyi Biotec. Cat. 000403).

[0154] 10% FBS containing RPMI culture solution treated with GM-CSF (Creagene) and IL-4 (Creagene) at a concentration of 100 ng/ml, respectively, was added thereto to induce differentiation into immature dendritic cells (immature DCs) for 5 days. On the 5th day of culture, 5 ug/ml of LPS (Lipopolysaccharides, Sigma-Aldrich, #L2630) was added, and the dendritic cells were stimulated for 24 hours to differentiate into mature dendritic cells (mature DC). Antibody DNP007 (H17L4 antibody) and control antibody (hIgG) provided herein were added at a concentration of 5.0 ug/mL on days 0 and 3 of the induction process of dendritic cell differentiation. On the 6th day of differentiation, the expression level of CD80 (Invitrogen, Catalog #11-0809-42), CD86 (BD, Catalog #555657), CD40 (BD, Catalog #555588), CD54 (BD, Catalog #347977) and HLA-DR (BioLegend, Catalog #307616) (Above, in parentheses are antibodies against the factor), a maturation surface factor of dendritic cells, was confirmed and compared using a flow cytometer.

[0155] The obtained expression level (Mean of Fluorescent Intensity, MFI) of each factor was converted into a relative value for the group treated with only LPS, and is shown in FIG. 15.

[0156] As shown in FIG. 15, in the case of dendritic cells treated with a control antibody (hIgG), the expression of cofactors such as CD80, CD54, CD40, and HLA-DR was all increased by LPS stimulation, it was confirmed that the expression of these cofactors was significantly reduced in the group treated with the test antibody DNP007 and LPS. These results show that the antibodies provided herein above limit the maturation of dendritic cells by antigen stimulation and induce an immune suppression environment.

5-2. Proinflammatory Cytokine Secretion Test in Dendritic Cells

[0157] In order to confirm whether the antibody provided herein affects the differentiation and maturation stages of dendritic cells, human peripheral blood mononuclear cells isolated from peripheral blood were cultured with GM-CSF and IL-4 for 5 days to differentiate into dendritic cells, and then stimulating factors such as LPS were added and cultured for 24 hours to induce maturation, the amount of proinflammatory cytokine in the culture solution was analyzed to measure the maturity of dendritic cells.

[0158] More specifically, after attaching a CD14 microbead (Miltenyi Biotec, 130-050-201) to human peripheral blood, human peripheral blood mononuclear cells were isolated using a cell collection device (Miltenyi Biotec. Cat. 000403). 10% FBS containing RPMI culture solution treated with GM-CSF and IL-4 at a concentration of 100 ng/ml was added thereto to induce differentiation into immature DCs for 5 days. On the 5th day of culture, 5 ug/ml of LPS was added and the dendritic cells were stimulated for 24 hours to induce differentiation into mature dendritic cells (mature DC). Antibody DNP007 (H17L4 antibody) and control antibody (hIgG) provided herein were added at concentrations of 0.1 to 10.0 ug/mL, respectively, on days 0 and 3 of the induction process of dendritic cell differentiation. On day 6 of differentiation, the amount of proinflammatory cytokine (IL-6 and TNF-a) in the cultured solution was analyzed using an ELISA technique.

[0159] FIG. 16 shows the results obtained above. As shown in FIG. 16, in mature dendritic cells treated with the control antibody, the secretion of IL-6 and TNF-?, which are representative proinflammatory cytokines, is significantly increased due to LPS stimulation, whereas in dendritic cells treated with the test antibody DNP007 the ability to secrete proinflammatory cytokines was significantly reduced. The maturation of dendritic cells refers to the process by which dendritic cells acquire the ability as antigen-presenting cells, and mature dendritic cells function to induce the activity of antigen-specific T cells. Due to the treatment of the antibodies provided herein, the secretion of the proinflammatory cytokines IL-6 and TNF-?, which is an important measure of dendritic cell maturity, was significantly inhibited, and through this, it was confirmed that the antibody provided herein has a function of inhibiting the maturation of dendritic cells.

5-3. Dendritic Cell Apoptosis Test

[0160] In order to determine whether the antibody provided herein affects apoptosis in the differentiation step of dendritic cells, the degree of death of dendritic cells differentiated and matured for 6 days according to the antibody treatment was measured. The degree of apoptosis was confirmed through flow cytometry after 7AAD staining.

[0161] More specifically, after attaching a CD14 microbead (Miltenyi Biotec, 130-050-201) to human peripheral blood, human peripheral blood mononuclear cells were isolated using a cell collection device (Miltenyi Biotec. Cat. 000403). 10% FBS containing RPMI culture solution treated with GM-CSF and IL-4 at a concentration of 100 ng/ml, respectively, was added to induce differentiation into immature DCs for 5 days. On the 5th day of culture, 5 ug/ml of LPS was added and the dendritic cells were stimulated for 24 hours to differentiate mature dendritic cells. Antibody DNP007 (H17L4 antibody) provided herein and control antibody (hIgG) were added twice at a concentration of 5.0 ug/mL on days 0 and 3 of the induction process of dendritic cell differentiation. On the 6th day after induction of differentiation, mature dendritic cells (mature DCs) were stained with 7AAD (7-Amino-Actinomycin D, BD, 51-68981E 5 ul/test) and analyzed using a flow cytometer.

[0162] The results obtained above are shown in FIG. 17. As shown in FIG. 17, as a result of confirming the degree of apoptosis of dendritic cells that have undergone differentiation and maturation, the control antibody-treated group showed apoptosis of 2.33%, whereas the apoptosis rate of dendritic cells treated with the test antibody DNP007 was 36.3%, it showed a higher apoptosis effect than the control group.

[0163] The cytotoxic effect of dendritic cells by the test antibody can be evaluated as due to apoptosis.

5-4. Induction Test of Immune Tolerogenic Dendritic Cells (Tolerogenic DC)

[0164] In order to confirm whether the antibody provided herein induces tolerogenic dendritic cells, expression of tolerogenic dendritic cell markers was confirmed by flow cytometry in semi-mature dendritic cells that had undergone differentiation and maturation for 6 days.

[0165] More specifically, antibody DNP007 (H17L4 antibody) provided herein and control antibody (hIgG) were added to semi-mature dendritic cells that have undergone differentiation and maturation from human peripheral blood mononuclear cells for 6 days in the same manner as in Examples 5-2 or 5-3 twice at a concentration of 5.0 ug/mL on days 0 and 3 of the induction process of dendritic cell differentiation, and the expression of tolerogenic dendritic cell markers was confirmed by flow cytometry on the 6th day after the differentiation induction.

[0166] Cell surface expression factors PDL1 (Invitrogen, Catalog #17-5983-42), PDL2 (Miltenyi biotec, Catalog #130-098-528), and Adenosin Receptor A2b (Novus, Catalog #NBP2-41312PE) (Above, parentheses are antibodies against the factor) was stained by treating the antibody at an appropriate concentration and reacting for 20 minutes in refrigerator. 2 ml of flow cytometry buffer (0.5% BSA, 0.01% NaN3 in 1?PBS) was added, centrifuged at 2200 rpm for 3 minutes, and the supernatant was removed, washed and analyzed.

[0167] Cell fluid substances, IDO (Indoleamine 2,3-Dioxygenase; Invitrogen, Catalog #12-9477-42), TGF-? (R&D systems, Catalog #IC240P), IL-10 (Miltenyi biotec, Catalog #130-112-729) (Above, parentheses are antibodies against the factor) were stained according to the intracellular antigen staining method kit manufacturer's manual. 100 ul of IC Fixation buffer (Invitrogen, Catalog #00-8222-49) was added to the cell pellet, the light was blocked at room temperature, and reacted for 30 minutes to fix the cells. 2 mL of 1? Permeabilization buffer (Invitrogen, Catalog #00-8333-56) was added, centrifuged at 600 g for 5 minutes, and the supernatant was removed. An appropriate concentration of each antibody was added to 100 ul of 1? Permeabilization Buffer, treated on the remaining cell pellet, and allowed to react for 30 minutes after blocking light at room temperature. 1? Permeabilization buffer 2 mL was added, centrifuged at 600 g for 5 minutes, washed, and flow cytometry buffer 2 mL was added each, followed by centrifugation to perform additional washing. Cells were fixed by adding 1% paraformaldehyde, and the expression levels of each antigen were analyzed using a flow cytometer.

[0168] The obtained results are shown in FIGS. 18a and 18b. As shown in FIGS. 18a and 18b, the dendritic cells treated with the test antibody DNP007 showed a significant increase in the expression of IDO, a representative factor of immune tolerance, compared to the control antibody-treated dendritic cells (control), and increase in the expression levels of PDL1, PDL2 and adenosine R A2b, known as other immunosuppressive factors compared to the control group too.

[0169] As such, it was confirmed that the expression of some factors related to IDO and immune tolerance was increased through analysis of the dendritic cells treated with the antibodies provided herein. In particular, IDO expressed on dendritic cells is known to inhibit the proliferation of T cells and play an important role in immune tolerance. Thus, an increase in IDO expression by the antibodies provided herein suggests a high possibility of inducing immune tolerance.

5-5. IDO Expression Test in Dendritic Cells

[0170] In order to determine whether the inhibition of dendritic cell maturation by the antibodies provided herein is by which of the two known maturation pathways, LPS and CD40L were used to induce maturation of dendritic cells. Expression of IDO, a tolerogenic dendritic cell marker, was confirmed by flow cytometry in semi-mature dendritic cells that had undergone differentiation and maturation for 6 days.

[0171] More specifically, stimulation by LPS known as the Canonical pathway was tested under the same conditions as in Examples 5-2 or 5-3 by preparing semi-mature dendritic cells that had undergone differentiation and maturation for 6 days.

[0172] In the stimulation test by CD40L, known as the non-canonical pathway, immature dendritic cells differentiated for 5 days by adding 10% FBS containing RPMI culture solution treated with GM-CSF and IL-4 at a concentration of 100 ng/ml, respectively, were used. On the 5th day of culture, 1 ug/ml of CD40L (Enzo, ALX-522-110) was added to 10% FBS containing RPMI treated with GM-CSF and IL-4 at a concentration of 100 ng/ml and stimulated for 48 hours, to induce mature dendritic cells (mature DCs). Antibody DNP007 (H17L4 antibody) provided herein and control antibody (hIgG) were treated twice at a concentration of 5.0 ug/mL on days 0 and 3 during the induction process of dendritic cell differentiation, and CD40L was treated in an amount of 5.0 ug/mL on day 5.

[0173] On the 7th day of induction of differentiation, mature dendritic cells (Mature DC) were stained with the above-mentioned staining intracellular antigens and analyzed using a flow cytometer.

[0174] The obtained results are shown in FIGS. 19a (Canonical pathway) and 19b (Non-canonical pathway). As shown in FIGS. 19a and 19b, test antibody DNP007 induced a decrease in the expression of IDO in cells during the process of maturation of dendritic cells by CD40L, whereas the maturation of dendritic cells by LPS showed a large difference in IDO expression between individuals. As the maturation pathway of dendritic cells, there are canonical pathways induced by LPS and TNF-? stimulation and non-canonical pathways induced by CD40L. The above results show that the antibodies provided herein affect both dendritic cell maturation pathways, specifically limiting maturation by CD40L and inducing differentiation into tolerogenic dendritic cells.

5-6. T Cell Immune Suppression Test by Water-Soluble Substances

[0175] To confirm whether the factor affecting the T cells of dendritic cells whose maturation is inhibited by the treatment of the antibodies provided herein is due to intercellular conjugation or mediators secreted from cells, dendritic cells and T cells were cultured in separate conditions, and the inhibition ability of T cells activity was confirmed.

[0176] More specifically, human T cells isolated using a cell collection device after attaching CD3 microbeads (Miltenyi, 130-050-201) to human peripheral blood, and mature dendritic cells (Mature DC) obtained under the same conditions as in Examples 5-1 to 5-5, were cultured in separate conditions to confirm the inhibition ability of T cells activity. In the lower chamber of the plate made to pass through the culture medium, 1 ug/ml of anti-CD3 antibody (BD, 557052) and 1 ug/ml of anti-CD28 antibody (BD, 555725) were coated, and autologous T cells isolated from peripheral blood were put. At this time, autologous T cells were subjected to CFSE labeling as a condition to confirm proliferation. Dendritic cells differentiated by treatment with antibody DNP007 (H17L4 antibody) provided herein were placed in the upper chamber. And the two cells were cultured for 4 days while sharing the culture medium in each chamber. The number of dendritic cells and autologous T cells was treated at a ratio of 1:10.

[0177] The obtained results are shown in FIG. 20. As shown in FIG. 20, in the absence of direct contact between the two cells, the proliferation of T cells was inhibited by the test antibody-treated dendritic cells, and the production of interferon gamma (IFN-r), a proinflammatory cytokine, was reduced. The above results show that the antibody-treated dendritic cells provided herein can inhibit the activity and proliferation of T cells through material exchange with T cells, and indicate that the substance secreted from dendritic cells acts as a mediator to inhibit the activity of T cells, and induces immune tolerance.

5-7. T Cell Immune Suppression Test by Antibody-Treated Dendritic Cells

[0178] In order to evaluate the ability to inhibit T cell activity of dendritic cells whose maturation was inhibited by treatment with the antibodies provided herein, the activity and proliferation of T cells were confirmed under conditions in which dendritic cells and T cells were cultured together. First, the maturation of dendritic cells was induced using LPS, TNF-a, and CD40L, which are stimulators of two known maturation pathways, and the proliferation of T cells and the expression level of intracellular proinflammatory cytokines were measured. More specifically, the maturation of dendritic cells was induced using two known maturation pathways (Canonical pathway: LPS, and TNF-alpha/Non-canonical pathway: CD40L). Stimulation with LPS and CD40L was carried out in the same manner as in Example 5-5. And stimulation with TNF-alpha was performed by treating 10% FBS containing RPMI treated with GM-CSF and IL-4 at a concentration of 100 ng/ml, respectively, with 13 ng/ml of TNF-a, 10 ng/ml of IL-1?, and 350 ng/ml of PGE2 on day 5 in the state of being induced to immature dendritic cells and giving stimulation for 48 hours to differentiate into mature DCs. Antibody DNP007 (H17L4 antibody) provided herein and control antibody (hIgG) were treated twice at a concentration of 5.0 ug/ml on days 0 and 3 during the induction process of dendritic cell differentiation.

[0179] In order to induce T cell activity, autologous T cells and mature dendritic cells differentiated in the same manner as above were added to a plate coated with 1 ug/ml of anti-CD3 antibody at a ratio of 1:10. After 3 days, the expression level of intracellular inflammatory cytokines and the proliferation of T cells were measured. At this time, similarly, CFSE labeling was performed to confirm the proliferation of T cells.

[0180] The obtained results are shown in FIG. 21. As shown in FIG. 21, T cells cultured with the control antibody-treated dendritic cells showed active cell proliferation and highly expressed IFN-? and IL-10, which are representative of proinflammatory cytokines. In contrast, T cells treated with dendritic cells with restricted maturation by treatment with test antibody DNP007 inhibited proliferation despite stimulation by the anti-CD3 antibody and markedly reduced proinflammatory cytokine secretion.

[0181] These results indicate that the antibodies provided herein affect both the maturation pathways of dendritic cells (the canonical pathway induced by LPS and TNF-? stimulation and the non-canonical pathway induced by CD40L), and T cells sensitized by dendritic cells whose maturation is inhibited limit proliferation and expression of proinflammatory cytokines. That is, the semi-mature dendritic cells induced by the antibodies provided herein inhibit the activity and proliferation of T cells.

Example 6. Effect of Antibody on the Treatment of Immune-Related Diseases (In Vivo)

6-1. Rheumatoid Arthritis Treatment Effect Test

[0182] In order to confirm whether the antibody provided herein is effective for rheumatoid arthritis, a mild and moderate rheumatoid arthritis model was prepared as follows: On the first day, 4 mg of Type II Bovine collagen (Chodrex inc, Cat. #: 20021) was prepared with CFA (Sigma Cat. #: F5881) in an amount of 0.5 ml+0.5 ml in a 1:1 volume ratio, divided into 10 places of 0.1 ml each, and injected into the epidermis of primate animals (Cynomolgus Macaques (Macaca fascicularis), sex: female, age: 2.5-5 years, weight: 2-6 kg, source: PrimGen). On the 21st and 45th days, the injection was carried out in the same way, except that the second and third injection was replaced with IFA (Sigma, Cat. #: F5506) instead of CFA. The control group was injected with PBS in the same manner. The RA score, which measures the incidence of arthritis, was semi-quantitatively scored on the degree of swelling or red color change in each joint of the animal (FIG. 22a). The antibody DNP007 (H17L4 antibody) provided herein was administered at 8 mg/kg twice a week from the 70th day after the first collagen treatment, and the Arthritis score of 64 joints was measured according to the evaluation criteria. In addition, Bovine collagen-specific antibodies were tested from the plasma of the administered animals. The change of the Bovine collagen specific antibody was measured using the ELISA kit (Cat. #: 2052T) of Chondrex.

[0183] The obtained results are shown in FIGS. 22b and 22c. As a result, the Arthritics score decreased in the group (n=3) administered with the test antibody DNP007 compared to the non-administered group (n=2) (FIG. 22b), and the anti-collagen antibody, a direct immunological factor of the disease, was also significantly reduced (FIG. 22c). These results show that the antibodies provided herein are effective in the treatment of rheumatoid arthritis.

6-2. Graft Versus Host Disease Treatment Effect Test

[0184] Graft-versus-host disease (GVHD) is one of the side effects of transplanted patients (recipients) when allogeneic organs (e.g., bone marrow) are transplanted and a phenomenon that occurs when NK cells or T cells of donor attack the recipient's organs. In order to confirm whether the antibody provided herein is effective in inhibiting graft versus host disease during allogeneic bone marrow transplantation, peripheral blood mononuclear cell (PBMC) were added to NOD-SCID mice, and the reconstitution and survival rate of T cells were compared with those of the control group.

[0185] More specifically, the graft-versus-host disease (GVHD) model was established by irradiating NSG (NOD.Cg-Prkdcscid II2rgtm1Wjl/SzJ) female mice with 1.6 Gy and then injecting human PBMCs. Human PBMC were obtained by centrifugation in a Leucosep? tube (Greiner bio-one, 227290) using Ficoll (GE Healthcare, 17544202) and separating the middle white layer. The isolated human PBMC was administered intraperitoneally at 1?10.sup.7 cells/200 ?L per mouse.

[0186] A test group was prepared by administering the antibody DNP007 (H17L4 antibody) provided herein at a dose of 10 mg/kg twice a week for a total of 12 times for 6 weeks, and a positive control was prepared by administering CTLA-4-Ig (Bio X cell, BE0099) at a dose of 10 mg/kg twice a week for a total of 12 times for 6 weeks. G1 (n=3) is a negative control group without PBMC administration, and G2 (n=7) is a negative control group in which GVHD is induced and PBS is administered as a vehicle. In addition, G3 (n=7) is a positive control group administered with CTLA-4-Ig, and G4 (n=7) is a test group administered with test antibody DNP007. Measurement of GVHD induction and treatment effect was recorded by scoring five clinical criteria (weight loss, posture, activity, fur, skin).

[0187] The obtained results are shown in FIGS. 23a and 23b. As a result, it was confirmed that the test group (G4) to which the antibody provided herein was administered had a significantly improved survival rate than that of the negative control group G2 (FIG. 23a), and it was confirmed that re-establishment of human T cells was normally performed well (FIG. 23b). These results show that the antibody provided herein has an effect of preventing graft versus host disease that occurs during allogeneic bone marrow transplantation and enabling re-establishment of normal donor T cells.