Antibody-drug conjugates

Abstract

This provides an antibody-drug conjugate containing an antibody or its antigen-binding fragment that binds to ITGB4.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and merit of International Application PCT / CN2023 / 086988 filed on 7 April 2023 and International Application PCT / CN2024 / 084464 filed on 28 March 2024, which are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention relates to an antibody-drug conjugate comprising an antibody that binds to integrin β4 (ITGB4) or an antigen-binding fragment thereof.

[0003] Sequence List This application includes a sequence listing, titled C24W8581-Sequence Listing.xml, which is an XML file with a size of 103,642 bytes and was created on March 29, 2024. The information contained in the sequence listing is incorporated herein by reference. [Background technology]

[0004] Integrin β4 (ITGB4) is a transmembrane protein that is part of the integrin family. Integrins are heterodimers composed of an alpha subunit and a beta subunit that bind non-covalently to transmembrane glycoprotein receptors. Only integrin β4 binds to integrin α6, forming a heterodimer (called α6β4) with its extracellular ligand, laminin. Unlike other beta subunits, the cytoplasmic tail of ITGB4 is long, contains approximately 1000 amino acids, and includes four fibronectin type III repeats.

[0005] ITGB4 is primarily found on epithelial cells, which are the cells that line the surface and cavities of the body. The α6β4 integrin protein plays a particularly important role in skin strengthening and stabilization. ITGB4 is involved in cell adhesion, migration, and signaling (J Cell Biol (1996) 134 (2): 559-572). It has been widely reported as a prognostic and predictive molecule in patients with various cancers (Biomolecules. 2021 Aug; 11(8): 1197). High expression of ITGB4 is associated with invasive tumor dynamics and poor prognosis. In the TCGA project, high ITGB4 expression levels were positively associated with poor overall survival in adrenocortical carcinoma (P=0.0044), chromophobic renal cell carcinoma (P=0.041), low-grade glioma (P<0.001), and lung adenocarcinoma (P=0.00073) (Int J Gen Med. 2021 Dec 11;14:9629-9645). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Therefore, the object of this disclosure is to provide, in particular, highly specific and effective anti-ITGB4 antibodies and antibody-drug conjugates thereof for the treatment of cancer. [Means for solving the problem]

[0007] In the first embodiment, equation (I): Ab-(LD) p (I) An antibody-drug conjugate (i.e., ADC) in which, Ab is an antibody or antigen-binding fragment that binds to ITGB4. L is the linker, D is the drug part, We provide an ADC where p is in the range of 1 to approximately 20.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) including LCDR1, LCDR2, and LCDR3, wherein HCDR1-3 and LCDR1-3 belong to the following groups: 1) Sequence numbers 11-16, 2) Sequence numbers 31, 32, 3, and 46-48 respectively, 3) Sequence numbers 52-55, 15, and 56 respectively, 4) Sequence numbers 61-63 and 66-68 respectively, or sequence numbers 64, 65, 63, and 66-68 respectively, 5) Sequence numbers 1-6, 6) Sequence numbers 21-26, 7) Sequence numbers 31, 32, 3, and 33-35 respectively, and 8) Sequence numbers 1-3 and 40-42 respectively It contains the amino acid sequence described in any of the following.

[0009] In some embodiments, VH and VL belong to the following groups: 1) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 17 and 18, respectively. 2) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 36 and 49, respectively. 3) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 57 and 58, respectively. 4) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 69 and 70, respectively, or SEQ ID NO: 69 and 70, 5) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 7 and 8, respectively, or SEQ ID NO: 7 and 8, 6) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27 and 28, respectively, or SEQ ID NO: 27 and 28, 7) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 36 and 37, respectively, or SEQ ID NO: 36 and 37, as well as 8) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 7 and 43, respectively, or SEQ ID NO: 7 and 43 comprises the amino acid sequence according to any one of the above.

[0010] In some embodiments, VH and VL are in the following group: 1) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 73 and 74, respectively, or SEQ ID NO: 73 and 74, 2) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 75 and 74, respectively. 3) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 76 and 74, respectively. 4) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 77 and 74, respectively. 5) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 78 and 74, respectively. 6) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 79 and 74, respectively. 7) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 80 and 74, respectively. 8) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 81 and 74, respectively. 9) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 82 and 74, respectively. 10) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 83 and 74, respectively. 11) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 84 and 74, respectively. 12) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 85 and 74, respectively. 13) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 86 and 74, respectively. 14) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 73 and 87, respectively. 15) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 75 and 87, respectively. 16) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 76 and 87, respectively. 17) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 77 and 87, respectively. 18) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 78 and 87, respectively. 19) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 79 and 87, respectively. 20) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 80 and 87, respectively. 21) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 81 and 87, respectively. 22) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 82 and 87, respectively. 23) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 83 and 87, respectively. 24) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 84 and 87, respectively. 25) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 85 and 87, respectively, and 26) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 86 and 87, respectively. It contains the amino acid sequence described in any of the following.

[0011] In some embodiments, the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the antibody is a humanized antibody.

[0012] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0013] In some embodiments, the antibody is a bispecific antibody comprising an additional antigen-binding domain that binds to a second antigen, preferably the second antigen being CD3, CD138, CD20, CD40, CD19, CD33, CD16, CD38, CD22, CD40, CD8, IL-6, CSPG, RGM A, CTLA-4, BTN02, IGF1, IGF2, Erb2B, IGF-1R, EGFR, CD13, ErbB3, EGFR-2, IGFR, VEGFR-2, Met, VEGF-A, angiopoietin 2 (Ang-2), IL-12, TWEAK, IL-13, IL-1 beta, RGM The group is selected from A, NgR, NogoA, OMGp, PDL-1, PD-1, PD-1, TIM-3, VEGF, Lag-3, OX40, DLL-4, PDGFR, EpCAM, CD16a, PSMA, CEA, TROP-2, HSG, HGF, PDGF, ICOS, CD47, RGM B, Te38, TNFa, Blys, GP130, IL-12, RANK ligand, ITGB6, Nectin 4, and folate receptors.

[0014] In some embodiments, the antibody is an isotype antibody selected from the group consisting of IgG, IgA, IgM, IgE, and IgD, and preferably, the antibody is a subtype antibody selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0015] In some embodiments of any one of the antibody or antigen-binding fragments described herein, the antibody or antigen-binding fragment is a full-length antibody or a single-chain variable fragment (scFv). In some embodiments, the antibody or antigen-binding fragment is a VHH antibody, also called a nanobody, which is an antigen-binding fragment of an antibody consisting only of a heavy chain. In some embodiments, the antibody or antigen-binding fragment is a diabody, which is a non-covalent dimer of a single-chain Fv(scFv) fragment. In some embodiments, the antibody or antigen-binding fragment is a Fab fragment containing VH and CH1 on the heavy chain and VL and CL on the light chain. In some embodiments, the antibody or antigen-binding fragment is a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain. In some embodiments, the antibody or antigen-binding fragment is an F(ab')2 fragment containing two Fab' fragments linked by disulfide crosslinking in the hinge region.

[0016] In some embodiments, the antibody comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), where HC and LC belong to the following groups: 1) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 19 and 20, respectively. 2) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 50 and 51, respectively. 3) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 59 and 60, respectively. 4) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 71 and 72, respectively. 5) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 9 and 10, respectively. 6) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 29 and 30, respectively. 7) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 38 and 39, respectively, and 8) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 44 and 45, respectively. It contains the amino acid sequence described in any of the following.

[0017] In some embodiments, the antibody comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), where HC and LC belong to the following groups: 1) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 88 and 89, respectively. 2) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 90 and 89, respectively. 3) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 91 and 89, respectively. 4) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 92 and 89, respectively. 5) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 93 and 89, respectively. 6) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 94 and 89, respectively. 7) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 95 and 89, respectively. 8) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 96 and 89, respectively. 9) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 97 and 89, respectively. 10) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 98 and 89, respectively. 11) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 99 and 89, respectively. 12) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 100 and 89, respectively. 13) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 101 and 89, respectively. 14) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 88 and 102, respectively. 15) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 90 and 102, respectively. 16) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 91 and 102, respectively. 17) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 92 and 102, 18) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 93 and 102, 19) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 94 and 102, 20) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 95 and 102, respectively. 21) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 96 and 102, respectively. 22) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 97 and 102, 23) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 98 and 102, respectively. 24) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 99 and 102, respectively. 25) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 100 and 102, respectively, and 26) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 101 and 102, respectively. It contains the amino acid sequence described in any of the following.

[0018] In some embodiments, the linker includes a cut-type linker or a non-cut-type linker.

[0019] In some embodiments, the cleavage linker includes an acid-unstable linker, a hydrophilic linker, a protease-sensitive linker, a photosensitive linker, a hydrazone linker, a dimethyl linker, or a disulfide-containing linker.

[0020] In some embodiments, the linker is sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate), MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), para-aminobenzyloxycarbonyl (PABC), dimethylethylamine (DMEA), Val-Cit-PABC, MC-Val-Cit-PABC, CL2A, Mal-PEG8-Val-Ala-PABC, Selected from the group consisting of MC-VC-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), MC-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), dimethylhydrazide (3-methyl-3-mercaptobutanehydrazide), AcBut-dimethylhydrazide, or SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-carboxylate).

[0021] In some embodiments, -LD is,

[0022] [ka]

[0023] [ka]

[0024] [ka]

[0025] [ka]

[0026] [ka]

[0027] [ka]

[0028] [ka]

[0029] [ka] or

[0030] [ka] Selected from the group consisting of, in the formula,

[0031] [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0032] In some embodiments, the drug portion is selected from the group consisting of tubulin inhibitors and microtubule polymerization inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA polymerase II inhibitors, RNA spliceosome inhibitors, and immunostimulants.

[0033] In some embodiments, the drug portion is a tubulin inhibitor and a microtubule polymerization inhibitor selected from the group consisting of auristatin, mytansin, tubulosin, cryptophycin, and rhizoxin.

[0034] In some embodiments, the drug portion is an antibiotic selected from the group consisting of calicheamycin, doxorubicin, and anthracyclines.

[0035] In some embodiments, the drug portion is a DNA synthesis inhibitor selected from the group consisting of duocalmycin, PBD (benzodiazepine), and IGN (indolinobenzodiazepine).

[0036] In some embodiments, the drug portion is a topoisomerase II inhibitor selected from the group consisting of anthracycline analogs (e.g., doxorubicin).

[0037] In some embodiments, the drug portion is a topoisomerase I inhibitor selected from the group consisting of camptothecin analogs.

[0038] In some embodiments, the drug portion is an RNA polymerase II inhibitor selected from the group consisting of amanitin.

[0039] In some embodiments, the drug portion is an RNA spliceosome inhibitor selected from the group consisting of spliceostatins and thailanstatins.

[0040] In some embodiments, the drug portion is an immunostimulant selected from the group consisting of Toll-like receptor agonists and STING agonists.

[0041] In some embodiments, the drug portion is [Table 1-1] [Table 1-2] Selected from, in the formula,

[0042] [ka] This indicates the bonding site to the linker.

[0043] In some embodiments, the antibody-drug conjugate is

[0044] [ka]

[0045] [ka]

[0046] [ka]

[0047] [ka]

[0048] [ka]

[0049] [ka]

[0050] [ka]

[0051] [ka]

[0052] [ka] Selected from the formula, where Ab is an antibody or its antigen-binding fragment that binds to ITGB4, p is in the range of 1 to approximately 20.

[0053] In some preferred embodiments, the antibody-drug conjugate is [ka] In this formula, Ab is an antibody comprising an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), wherein the HC and LC contain amino acid sequences described in any of the following groups: SEQ ID NOs: 9 and 10, SEQ ID NOs: 19 and 20, SEQ ID NOs: 38 and 39, SEQ ID NOs: 44 and 45, SEQ ID NOs: 50 and 51, SEQ ID NOs: 71 and 72, SEQ ID NOs: 94 and 102, and SEQ ID NOs: 92 and 102.

[0054] In some preferred embodiments, the antibody-drug conjugate is [ka] In the formula, Ab is an antibody comprising HC and LC, wherein HC and LC comprise the amino acid sequences described in any of the following groups: SEQ ID NOs: 9 and 10, SEQ ID NOs: 19 and 20, SEQ ID NOs: 38 and 39, SEQ ID NOs: 44 and 45, SEQ ID NOs: 50 and 51, SEQ ID NOs: 71 and 72, SEQ ID NOs: 94 and 102, and SEQ ID NOs: 92 and 102. In a more preferred embodiment, HC and LC comprise the amino acid sequences described in any of the following groups: SEQ ID NOs: 19 and 20, SEQ ID NOs: 71 and 72, SEQ ID NOs: 94 and 102, and SEQ ID NOs: 92 and 102.

[0055] In some preferred embodiments, the antibody-drug conjugate is [ka] In the formula, Ab is an antibody comprising HC and LC, wherein HC and LC comprise any of the following groups: SEQ ID NOs: 9 and 10, SEQ ID NOs: 19 and 20, SEQ ID NOs: 38 and 39, SEQ ID NOs: 44 and 45, SEQ ID NOs: 50 and 51, SEQ ID NOs: 71 and 72, SEQ ID NOs: 94 and 102, and SEQ ID NOs: 92 and 102. In a more preferred embodiment, HC and LC comprise the following groups: SEQ ID NOs: 19 and 20 or SEQ ID NOs: 94 and 102.

[0056] In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0057] In a second embodiment, a humanized antibody or its antigen-binding fragment comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the VH and VL belong to the following groups: 1) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 73 and 74, respectively. 2) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 75 and 74, respectively. 3) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 76 and 74, respectively. 4) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 77 and 74, respectively. 5) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 78 and 74, respectively. 6) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 79 and 74, respectively. 7) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 80 and 74, respectively. 8) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 81 and 74, respectively. 9) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 82 and 74, respectively. 10) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 83 and 74, respectively. 11) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 84 and 74, respectively. 12) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 85 and 74, respectively. 13) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 86 and 74, respectively. 14) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 73 and 87, respectively. 15) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 75 and 87, respectively. 16) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 76 and 87, respectively. 17) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 77 and 87, respectively. 18) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 78 and 87, respectively. 19) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 79 and 87, respectively. 20) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 80 and 87, respectively. 21) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 81 and 87, respectively. 22) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 82 and 87, respectively. 23) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 83 and 87, respectively. 24) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 84 and 87, respectively. 25) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 85 and 87, respectively, and 26) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 86 and 87, respectively. The present invention provides a humanized antibody or antigen-binding fragment containing the amino acid sequence described in any of the following.

[0058] In some embodiments, the humanized antibody comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), where the HC and LC belong to the following groups: 1) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 88 and 89, respectively. 2) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 90 and 89, respectively. 3) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 91 and 89, respectively. 4) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 92 and 89, respectively. 5) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 93 and 89, respectively. 6) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 94 and 89, respectively. 7) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 95 and 89, respectively. 8) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 96 and 89, respectively. 9) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 97 and 89, respectively. 10) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 98 and 89, respectively. 11) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 99 and 89, respectively. 12) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 100 and 89, respectively. 13) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 101 and 89, respectively. 14) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 88 and 102, respectively. 15) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 90 and 102, respectively. 16) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 91 and 102, respectively. 17) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 92 and 102, 18) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 93 and 102, 19) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 94 and 102, 20) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 95 and 102, respectively. 21) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 96 and 102, respectively. 22) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 97 and 102, 23) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 98 and 102, respectively. 24) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to sequence numbers 99 and 102, respectively. 25) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 100 and 102, respectively, and 26) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 101 and 102, respectively. It contains the amino acid sequence described in any of the following.

[0059] In a third aspect, a composition is provided comprising an antibody-drug conjugate or humanized antibody of the present disclosure and, optionally, a pharmaceutically acceptable carrier or excipient.

[0060] A fourth aspect provides a method for preventing and / or treating a disease in a subject requiring such treatment, comprising administering an antibody-drug conjugate or humanized antibody or composition thereof to the subject.

[0061] In some embodiments, the disease is cancer.

[0062] In some embodiments, the cancer is ITGB4 positive, and preferably, the cancer is selected from the group consisting of head and neck cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, stomach cancer, pancreatic cancer, ovarian cancer, prostate cancer, breast cancer, leukemia, myeloma, squamous cell carcinoma, melanoma, brain cancer, cervical cancer, liver cancer, bladder cancer, breast cancer, kidney cancer, testicular cancer, and thyroid cancer.

[0063] In some embodiments, the method further comprises administering a second therapeutic substance, preferably selected from antibodies, chemotherapeutic agents, and small molecule drugs.

[0064] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the Disclosure. These and other embodiments of the Disclosure will be apparent to those skilled in the art. These and other embodiments of the Disclosure will be further described in the following detailed description. [Brief explanation of the drawing]

[0065] [Figure 1] Figure 1 shows ITGB4, which is highly expressed in many tumor types as listed in bioinformatics databases.

[0066] [Figure 2-1] Figure 2 shows the high expression of ITGB4 in IHC-stained CRC, NSCLC, ESCC, and SCCHN samples. [Figure 2-2] Same as above [Figure 2-3] Same as above

[0067] [Figure 3] Figure 3 shows the high expression of ITGB4 in many tumor cell lines, as determined by flow cytometry.

[0068] [Figure 4] Figure 4 shows that the test antibody can bind to ITGB4-expressing tumor cells.

[0069] [Figure 5] Figure 5 shows some antibodies that can bind to cynomolgus monkey or mouse ITGB4 but not to human ITGB1.

[0070] [Figure 6] Figure 6 shows that the test antibody induces internal targeting in HCT-116 and H292 cells.

[0071] [Figure 7-1] Figure 7 shows that the conjugated ADC maintained its tumor cell binding ability. [Figure 7-2] Same as above

[0072] [Figure 8] Figure 8 shows that the conjugated ADC maintained its ability to induce internal migration into tumor cells.

[0073] [Figure 9-1] Figure 9 shows that the tested ADC exhibits in vitro cytotoxicity against tumor cell line expressing ITGB4. [Figure 9-2] Same as above

[0074] [Figure 10-1] Figure 10 shows that the ADC tested inhibited the growth of xenograft tumors in BALB / c nude mice or SCID Beige mice. [Figure 10-2] Same as above

[0075] [Figure 11] Figure 11 shows, by epitope binning evaluation, that the humanized clones examined recognize the same epitopes as those bound to the parent clones.

[0076] [Figure 12-1] Figure 12 shows that the humanized antibody tested can bind to ITGB4-expressing tumor cells. [Figure 12-2] Same as above

[0077] [Figure 13] Figure 13 shows the results of internal migration of humanized antibodies in SW620 cells.

[0078] [Figure 14] Figure 14 shows that ADCs containing humanized antibodies maintained their tumor cell binding ability.

[0079] [Figure 15] Figure 15 shows the results of internal migration of ADCs containing humanized antibodies into tumor cells.

[0080] [Figure 16-1] Figure 16 shows the cytotoxicity of ADCs containing humanized antibodies in tumor cells. [Figure 16-2] Same as above

[0081] [Figure 17] Figure 17 shows that ADCs containing humanized antibodies inhibited the growth of xenograft tumors in BALB / c nude mice.

[0082] [Figure 18-1] Figure 18 shows the PK profile of ADC in rats. [Figure 18-2] Same as above [Modes for carrying out the invention]

[0083] Sequence List The present invention includes parental anti-ITGB4 antibodies (001, 002, 003, 004, 005, 006, 007, and 008) and humanized clones (H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H The CDR sequences (following the Kabat numbering system), VH sequences, and VL sequences, as well as the full heavy-chain and full light-chain sequences, for 81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, and H813.2) are shown in the table below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] [Table 2-18] [Table 2-19] [Table 2-20]

[0084] definition Before describing embodiments in detail, it should be understood that this disclosure is not limited to any particular composition or biological system, which may naturally vary. Furthermore, it should be understood that the terminology used herein is solely for the purpose of describing specific embodiments and is not intended to be limiting.

[0085] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. Therefore, for example, when referring to "a molecule," it may include, for example, a combination of two or more such molecules.

[0086] The term "approximately" generally refers to plus or minus 10% of the number it indicates. For example, "approximately 10%" could mean a range of 9% to 11%, and "approximately 1" could mean 0.9 to 1.1. Other meanings of "approximately" can become clear from the context; for example, in rounding, "approximately 1" could also mean 0.5 to 1.4.

[0087] Many ranges of values ​​are presented as disclosed herein. Furthermore, unless the context clearly indicates otherwise, each value located between the upper and lower limits of a range is understood to be specifically disclosed to a unit of one-tenth of the lower limit. Each subrange between any displayed or located value within the displayed range and any other displayed or located value within that displayed range is included in this disclosure. The upper and lower limits of such subranges are independently included in or excluded from this range. Each range that includes one or both of the limits, or does not include either, within a subrange is also included in this disclosure and may be subject to the limits excluded in detail within the displayed range. If a displayed range includes one or both limits, the range that excludes either or both of the limits thus included is also included in this disclosure.

[0088] As used herein, the terms “percent identity” and “% identity” applied to nucleic acid or polynucleotide sequences refer to the percentage of matching residues between at least two nucleic acid or polynucleotide sequences aligned using a standardized algorithm. Such algorithms optimize the alignment between two sequences by inserting gaps into the sequences being compared in a standardized and reproducible manner, thereby enabling a more meaningful comparison of the two sequences.

[0089] Percent identity between nucleic acid or polynucleotide sequences may also be determined using a set of commonly used, freely available sequence comparison algorithms provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, SF et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including NCBI, Bethesda, Md., and on the internet at http: / / www.ncbi.nlm.nih.gov / BLAST / .

[0090] Nevertheless, nucleic acids or polynucleotide sequences that do not exhibit a high degree of identity may encode similar amino acid sequences through degeneracy of gene coding. It is understood that this degeneracy can be used to alter nucleic acid sequences to generate multiple nucleic acid sequences that all encode substantially identical proteins. In detail, degenerate codon substitution can be performed by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed group and / or a deoxyinosine residue (Batzer et al., (1991) Nucleic Acid Res 19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol Cell Probes 8:91-98). The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded forms. Unless otherwise specified, this term encompasses nucleic acids, including known analogues of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized similarly to naturally occurring nucleotides. The term nucleic acid is used interchangeably (in appropriate contexts) with polynucleotides, as well as genes, cDNA, and mRNA encoded by genes.

[0091] As used herein, “percent (%) amino acid sequence identity” with respect to a peptide, polypeptide, or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in another peptide or polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after achieving maximum percentage sequence identity by aligning the sequences and introducing gaps as necessary. Percent amino acid sequence identity in this disclosure is measured using BLAST software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared. The term “pharmaceutical composition” refers to a combination of an active substance and an inactive or active carrier that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0092] As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic agent, and absorption retarder. A “pharmaceutically acceptable carrier” does not produce undesirable physiological effects after or during administration to a subject. A carrier in a pharmaceutical composition must also be “acceptable” in the sense that it is compatible with and capable of stabilizing the active ingredient. One or more solubilizers may be used as a pharmaceutical carrier for the delivery of the active ingredient. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible media, adjuvants, additives, and diluents for making a composition usable as a dosage form. Other examples of carriers include colloidal silicon dioxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Further suitable pharmaceutical carriers and diluents, as well as substances pharmaceutically necessary for their use, are described in Remington's Pharmaceutical Sciences. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, non-enteral, spinal, or epidermal administration (e.g., by injection or infusion). The therapeutic compound may contain one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not confer any undesirable toxicological effects (see, for example, Berge, SM, et al. J. Pharm. Sci. 1997, 66, 1-19).

[0093] As used herein, “treating” or “treatment” means the administration of a compound or substance to a person who has a disorder or is at risk of developing a disorder, for the purpose of curing, reducing, alleviating, improving, delaying the onset of, preventing or restoring a disorder, its signs, a condition secondary to a disorder, or a predisposition to a disorder.

[0094] The "effective dose" refers to the amount of active compound / substance required to impart a therapeutic effect to the target of treatment. The effective dose varies depending on the type of symptom being treated, the route of administration, the use of excipients, and the possibility of concomitant use of other therapeutic measures, as recognized by those skilled in the art. The therapeutically effective dose of concomitant therapy for neoplasms is, for example, the amount that produces a reduction in tumor size, a reduction in the number of tumor lesions, or a slowing of tumor growth compared to an untreated animal.

[0095] The aspects and embodiments of this disclosure are understood to include the terms “including,” “consisting of,” and “essentially consisting of.”

[0096] As used herein, the term “antibody” is used in a broad sense and, insofar as it exhibits the desired biological activity (e.g., epitope binding), more specifically, includes, but is not limited to, intact antibodies (e.g., full-length antibodies), antibody fragments (Fab, F(ab')2, scFv, scFv-Fc, single-domain antibodies (sdAb, also known as nanobodies), single heavy-chain antibodies, and single light-chain antibodies), monoclonal antibodies, and polyclonal antibodies.

[0097] The term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Such molecules often contain two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The variable regions of the heavy and light chains of an antibody contain binding domains that interact with the antigen. The constant regions of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component of the classical pathway of complement activation.

[0098] The heavy chain of immunoglobulins can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment). The Fd region contains the VH domain and the CH1 domain and, together with the light chain, forms the Fab (antigen-binding fragment). The Fc fragment is responsible for immunoglobulin effector functions, such as complement binding and binding to homologous Fc receptors on effector cells. The hinge region is found in the immunoglobulin classes IgG, IgA, and IgD, and acts as a mobile spacer, allowing the Fab portion to move freely in space compared to the Fc region. The hinge domain is structurally diverse, with both its sequence and length differing between immunoglobulin classes and subclasses.

[0099] The “light chain variable region” (VL) or “heavy chain variable region” (VH) consists of a “framework” region interrupted by three “complementarity-determining regions” or “CDRs.” The framework region acts to align the CDRs for specific binding to the antigen epitope. The CDRs contain the antibody amino acid residues that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions, from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, CDR2, and CDR3 of the VL domain are also referred herein as LCDR1, LCDR2, and LCDR3, respectively, and CDR1, CDR2, and CDR3 of the VH domain are also referred herein as HCDR1, HCDR2, and HCDR3, respectively.

[0100] The amino acid assignments for each VL and VH domain follow the conventional arbitrary definitions of CDR. Conventional definitions include Kabat's definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), Chothia's definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989), the composite Chothia-Kabat CDR where CDR-H1 is a composite of Chothia CDR and Kabat CDR, the AbM definition used by Oxford Molecular's antibody modeling software, and Martin et al.'s CONTACT definition (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering scheme (Kabat numbering scheme) in which corresponding residues between different heavy chains or different light chains are assigned the same number. Unless otherwise specified, when referring to the numbering of the positions of specific amino acid residues within the antibody variable region, the Kabat numbering scheme is followed. [Table 3]

[0101] Based on the amino acid sequence of the constant region of the antibody's heavy chain, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and further divided into various subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. The light chain of an antibody can be classified as either a lambda (λ) chain or a kappa (κ) chain based on the amino acid sequence of the light chain.

[0102] As used herein, the term “chimeric antibody” refers to an antibody molecule, as well as fragments of such an antibody, insofar as it exhibits the desired biological activity, in which portions of the heavy chain and / or light chain are identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and at the same time, the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass. A chimeric antibody molecule may, for example, include a human constant region along with one or more VH antigen-binding domains and / or VL antigen-binding domains derived from an antibody of a mouse, rat, or other species.

[0103] As used herein, the term “humanized antibody” refers to an antibody molecule derived from a non-human species that binds to a desired antigen and has a framework region and a constant region derived from one or more CDRs of a non-human species as well as a human immunoglobulin molecule. Framework residues within the human framework region are often substituted with corresponding residues derived from the CDR donor antibody to alter, preferably enhance, antigen binding. Such framework substitutions are identified by methods well known in the art, such as modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding, and sequence comparison to identify abnormal framework residues at specific locations.

[0104] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, each antibody constituting the population is identical except for the possibility of small amounts of naturally occurring mutations. Monoclonal antibodies are highly specific and directed to a single antigen. The term “monoclonal antibody” is not limited herein to antibodies produced by hybridoma technology and should not be interpreted as requiring antibody production by any particular method.

[0105] As used herein, the term “bispecific antibody” should be understood in the context of the present invention as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes in a single target. As used herein, the term “bispecific antibody” should be understood in a broad sense and include full-length bispecific antibodies and their antigen-binding fragments. Bispecific antibodies may include further modifications, such as amino acids not naturally occurring, mutations in the Fc region, and mutations in the glycosylation site. Bispecific antibodies also include post-translation modified antibodies, fusion proteins containing antigenic determinants of the antibody, and immunoglobulin molecules containing any other modifications to the antigen recognition site, insofar as such antibodies exhibit the desired biological activity.

[0106] As used herein, the term “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It is known that fragments of a full-length antibody can perform the antigen-binding function of an antibody.

[0107] Examples of antigen-binding fragments encompassed by the term “antigen-binding portion” of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinking in a hinge region; (iii) Fab' fragments, which are essentially Fab fragments with a portion of the hinge region; (iv) Fd fragments, which consist of a VH domain and a CH1 domain; (v) Fd' fragments, which have a VH domain and a CH1 domain with one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) Fv fragments, which consist of a single arm of the antibody with a VL domain and a VH domain; (vii) dAb fragments, which consist of a VH domain; (viii) isolation complementarity-determining regions (CDRs); and (ix) nanobodies, which are heavy chain variable regions containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by a synthetic linker using recombination methods, allowing them to form a single protein chain, and this VL and VH region can be paired to form a monovalent molecule (known as single-chain Fv (scFv)). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of the antibody. Moreover, this term also includes "linear antibodies" containing a pair of tandem Fd segments (VH-CH1-VH-CH1) that form an antigen-binding region together with a complementary light chain polypeptide, and any modified form of the aforementioned fragment that retains antigen-binding activity.

[0108] Such antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the usefulness of the fragments is screened in the same way as that of intact antibodies.

[0109] As used herein, the terms “binding” or “specific binding” refer to a non-random binding reaction between two molecules, for example, between an antibody and a target antigen. The binding specificity of an antibody can be determined based on affinity and / or binding strength. The equilibrium constant (K) of dissociation between antigen and antibody. D) The affinity represented by is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antibody, and the smaller the value of K D , the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as an affinity constant (K A ), which is 1 / K D .

[0110] Binding force is a measure of the binding strength between an antibody and a suitable antigen. Binding force is related to both the affinity between an antigenic determinant and an antigen-binding site on an antibody and the number of suitable binding sites present on the antibody. Typically, an antibody has a dissociation constant (K -5 ~10 -12 M or less, preferably 10 -7 ~10 -12 M or less, more preferably 10 -8 ~10 -12 M and / or at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , for example, at least 10 12 M -1 and binds to an antigen with a binding affinity of at least 10 -1 M. Any K -4 value exceeding 10 D is generally judged to indicate non-specific binding. Specific binding between an antibody and an antigen or antigenic determinant can be determined by, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, as well as any known suitable method including various modifications of these known in the art.

[0111] The term "epitope" refers to the site on an antigen to which an antibody binds. Epitopes can be formed from continuous amino acids or from discontinuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed from continuous amino acids (also known as linear epitopes) are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding (also known as structural epitopes) are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly, at least five or eight to ten amino acids in a unique spatial structure. Because epitopes define the minimal binding site of an antibody, they are specific targets for the antibody or its antigen-binding fragment.

[0112] As used herein, the term “sequence identity” refers to the degree to which two sequences (amino acids) have identical residues at the same positions in an alignment. For example, “the amino acid sequence is X% identical to sequence number Y” refers to the % identity of the amino acid sequence to sequence number Y, meaning that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in sequence number Y. Generally, computer programs are used for such calculations. Examples of programs that compare and align pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gap-introduced BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0113] Furthermore, in determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conservative" amino acid substitutions, which are commonly described as replacing one amino acid residue with another amino acid residue having a similar chemical structure, and which have little to no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0114] Such conservation substitutions are preferably those in which one amino acid from the following groups (a) to (e): (a) small, aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar charged residues and their amides (uncharged): Asp, Asn, Glu, and Gln; (c) polar positive charged residues: His, Arg, and Lys; (d) large, aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp is replaced by another amino acid residue from the same group.

[0115] Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0116] I. Antibody-drug conjugates In one aspect of this disclosure, formula (I): Ab-(LD) p (I) An antibody-drug conjugate, in which, Ab is an antibody or antigen-binding fragment that binds to ITGB4. L is the linker, D is the drug part, The present invention provides an antibody-drug conjugate in which p is in the range of 1 to approximately 20.

[0117] In the antibody-drug conjugate of formula (I), the drug portion D can be bound to the antibody via a linker L. L is any chemical moiety to which the antibody Ab can be bound to the drug portion D. Linker L binds the antibody Ab to the drug D via covalent bonds (or multiple bonds). The linker reagent is a bifunctional or polyfunctional moiety that can be used to conjugate the drug portion D and the antibody Ab to form an antibody-drug conjugate. The antibody-drug conjugate can be prepared using a linker having reactive functionality for binding to the drug portion D and the antibody Ab. The cysteine, thiol, or amine of the antibody, e.g., the N-terminus, or the amino acid side chain, e.g., lysine, can form a bond with the functional group of the linker reagent.

[0118] In some embodiments, the antibody-drug conjugate is

[0119] [ka] In the formula, Ab is an antibody or its antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20. In some embodiments, Ab is antibody 001, 002, 003, 004, 005, 006, 007 or 008 or its antigen-binding fragment. In some embodiments, Ab is a humanized antibody H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, or H813.2 or its antigen-binding fragment. In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0120] In some embodiments, the antibody-drug conjugate is

[0121] [ka] In the formula, Ab is an antibody or its antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20. In some embodiments, Ab is antibody 001, 002, 003, 004, 005, 006, 007 or 008 or its antigen-binding fragment. In some embodiments, Ab is a humanized antibody H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, or H813.2 or its antigen-binding fragment. In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0122] In some embodiments, the antibody-drug conjugate is

[0123] [ka] In the formula, Ab is an antibody or its antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20. In some embodiments, Ab is antibody 001, 002, 003, 004, 005, 006, 007 or 008 or its antigen-binding fragment. In some embodiments, Ab is a humanized antibody H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, or H813.2 or its antigen-binding fragment. In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0124] In some embodiments, the antibody-drug conjugate is

[0125] [ka] In the formula, Ab is an antibody or its antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20. In some embodiments, Ab is antibody 001, 002, 003, 004, 005, 006, 007 or 008 or its antigen-binding fragment. In some embodiments, Ab is a humanized antibody H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, or H813.2 or its antigen-binding fragment. In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0126] In some embodiments, the antibody-drug conjugate is

[0127] [ka] In the formula, Ab is an antibody or its antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20. In some embodiments, Ab is antibody 001, 002, 003, 004, 005, 006, 007 or 008 or its antigen-binding fragment. In some embodiments, Ab is a humanized antibody H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, or H813.2 or its antigen-binding fragment. In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0128] In some embodiments, the antibody-drug conjugate is

[0129] [ka] In the formula, Ab is an antibody or its antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20. In some embodiments, Ab is antibody 001, 002, 003, 004, 005, 006, 007 or 008 or its antigen-binding fragment. In some embodiments, Ab is a humanized antibody H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, or H813.2 or its antigen-binding fragment. In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0130] In some embodiments, the antibody-drug conjugate is

[0131] [ka] In the formula, Ab is an antibody or its antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20. In some embodiments, Ab is antibody 001, 002, 003, 004, 005, 006, 007 or 008 or its antigen-binding fragment. In some embodiments, Ab is a humanized antibody H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, or H813.2 or its antigen-binding fragment. In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0132] In some embodiments, the antibody-drug conjugate is

[0133] [ka] In the formula, Ab is an antibody or its antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20. In some embodiments, Ab is antibody 001, 002, 003, 004, 005, 006, 007 or 008 or its antigen-binding fragment. In some embodiments, Ab is a humanized antibody H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, or H813.2 or its antigen-binding fragment. In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0134] In some embodiments, the antibody-drug conjugate is

[0135] [ka] In the formula, Ab is an antibody or its antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20. In some embodiments, Ab is antibody 001, 002, 003, 004, 005, 006, 007 or 008 or its antigen-binding fragment. In some embodiments, Ab is a humanized antibody H81.1, H82.1, H83.1, H84.1, H85.1, H86.1, H87.1, H88.1, H89.1, H810.1, H811.1, H812.1, H813.1, H81.2, H82.2, H83.2, H84.2, H85.2, H86.2, H87.2, H88.2, H89.2, H810.2, H811.2, H812.2, or H813.2 or its antigen-binding fragment. In some embodiments, p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0136] In some embodiments, the antibody-drug conjugate is [ka] In this formula, Ab is an antibody comprising an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), wherein the HC and LC contain amino acid sequences described in any of the following groups: SEQ ID NOs: 9 and 10, SEQ ID NOs: 19 and 20, SEQ ID NOs: 38 and 39, SEQ ID NOs: 44 and 45, SEQ ID NOs: 50 and 51, SEQ ID NOs: 71 and 72, SEQ ID NOs: 94 and 102, and SEQ ID NOs: 92 and 102.

[0137] In some preferred embodiments, the antibody-drug conjugate is [ka] where Ab is an antibody comprising HC and LC, and HC and LC comprise an amino acid sequence set forth in any of the following groups: SEQ ID NO: 9 and 10, SEQ ID NO: 19 and 20, SEQ ID NO: 38 and 39, SEQ ID NO: 44 and 45, SEQ ID NO: 50 and 51, SEQ ID NO: 71 and 72, SEQ ID NO: 94 and 102, and SEQ ID NO: 92 and 102. In a more preferred embodiment, HC and LC comprise an amino acid sequence set forth in any of the following groups: SEQ ID NO: 19 and 20, SEQ ID NO: 71 and 72, SEQ ID NO: 94 and 102, and SEQ ID NO: 92 and 102.

[0138] In some preferred embodiments, the antibody-drug conjugate is

Chemical formula

[0139] II. Antibody An antibody or an antigen-binding fragment thereof that binds to ITGB4 of the present disclosure comprises an immunoglobulin heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 and an immunoglobulin light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3.

[0140] In some embodiments, the CDR is determined by any numbering scheme known in the art, such as the Kabat numbering scheme, the Chothia numbering scheme, the AbM numbering scheme, the CONTACT numbering scheme, etc. In some preferred embodiments, the CDR is determined by the Kabat numbering scheme.

[0141] In some embodiments, HCDR1-3 and LCDR1-3 each contain the amino acid sequences described in SEQ ID NOs: 11-16.

[0142] In some embodiments, HCDR1-3 and LCDR1-3 include the amino acid sequences described in SEQ ID NOs. 31, 32, 3, and 46-48, respectively.

[0143] In some embodiments, HCDR1-3 and LCDR1-3 include the amino acid sequences described in SEQ ID NOs. 52-55, 15, and 56, respectively.

[0144] In some embodiments, HCDR1-3 and LCDR1-3 include the amino acid sequences described in SEQ ID NOs. 61-63 and 66-68, respectively.

[0145] In some embodiments, HCDR1-3 and LCDR1-3 each contain the amino acid sequences described in SEQ ID NOs: 1-6.

[0146] In some embodiments, HCDR1-3 and LCDR1-3 each contain the amino acid sequences described in SEQ ID NOs. 21-26.

[0147] In some embodiments, HCDR1-3 and LCDR1-3 include the amino acid sequences described in SEQ ID NOs. 31, 32, 3, and 33-35, respectively.

[0148] In some embodiments, HCDR1-3 and LCDR1-3 include the amino acid sequences described in SEQ ID NOs: 1-3 and 40-42, respectively.

[0149] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs: 17 and 18, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 17 and 18.

[0150] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 36 and 49, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 36 and 49.

[0151] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 57 and 58, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 57 and 58.

[0152] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 69 and 70, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 69 and 70.

[0153] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs: 7 and 8, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 7 and 8.

[0154] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 27 and 28, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 27 and 28.

[0155] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 36 and 37, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 36 and 37.

[0156] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 7 and 43, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 7 and 43.

[0157] In some embodiments, VH comprises a functional variant of the amino acid sequence set forth in any one of SEQ ID NOs: 17, 36, 57, 67, 7, and 27 formed by insertion, deletion, and / or substitution of one or more amino acids (s), as long as the functional variant retains the ability to bind to ITGB4. In some embodiments, VL comprises a functional variant of the amino acid sequence set forth in any one of SEQ ID NOs: 18, 49, 58, 68, 8, 28, 37, and 43 formed by insertion, deletion, and / or substitution of one or more amino acids (s), as long as the functional variant retains the ability to bind to ITGB4. The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0158] In some embodiments, VH and VL each comprise the amino acid sequence set forth in SEQ ID NOs: 73 and 74, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NOs: 73 and 74.

[0159] In some embodiments, VH and VL each comprise the amino acid sequence set forth in SEQ ID NOs: 75 and 74, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NOs: 75 and 74.

[0160] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 76 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 76 and 74.

[0161] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 77 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 77 and 74.

[0162] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 78 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 78 and 74.

[0163] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 79 and 74, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 79 and 74.

[0164] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 80 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 80 and 74.

[0165] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 81 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 81 and 74.

[0166] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 82 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 82 and 74.

[0167] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 83 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 83 and 74.

[0168] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 84 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 84 and 74.

[0169] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 85 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 85 and 74.

[0170] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 86 and 74, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 86 and 74.

[0171] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 73 and 87, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 73 and 87.

[0172] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 75 and 87, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 75 and 87.

[0173] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 76 and 87, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 76 and 87.

[0174] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 77 and 87, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 77 and 87.

[0175] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 78 and 87, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 78 and 87.

[0176] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 79 and 87, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 79 and 87.

[0177] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 80 and 87, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 80 and 87.

[0178] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 81 and 87, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 81 and 87.

[0179] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 82 and 87, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 82 and 87.

[0180] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 83 and 87, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 83 and 87.

[0181] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 84 and 87, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 84 and 87.

[0182] In some embodiments, VH and VL include the amino acid sequences described in SEQ ID NOs. 85 and 87, respectively, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 85 and 87.

[0183] In some embodiments, VH and VL each include the amino acid sequences described in SEQ ID NOs. 86 and 87, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 86 and 87.

[0184] In the context of functional variants, the number of inserted, deleted, and / or substituted amino acids is preferably 40% or less of the total number of amino acids in the parent amino acid sequence, more preferably 35% or less, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%. For example, the number of inserted, deleted, and / or substituted amino acids may be 1-20, preferably 1-10, more preferably 1-7, even more preferably 1-5, and most preferably 1-2. In preferred embodiments, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0185] In some embodiments, insertions, deletions, and / or replacements can be performed in framework (FR) regions, such as FR1, FR2, FR3, and / or FR4.

[0186] In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably those in which one amino acid from the following groups (a) to (e): (a) small, aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar charged residues and their amides (uncharged): Asp, Asn, Glu, and Gln; (c) polar positive charged residues: His, Arg, and Lys; (d) large, aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp is substituted with another amino acid residue from the same group.

[0187] Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0188] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 17 and 18, respectively.

[0189] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 36 and 49, respectively.

[0190] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs. 57 and 58, respectively.

[0191] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 69 and 70, respectively.

[0192] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 7 and 8, respectively.

[0193] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 27 and 28, respectively.

[0194] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 36 and 37, respectively.

[0195] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 7 and 43, respectively.

[0196] In some embodiments, the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0197] In some embodiments, the antibody is a humanized antibody.

[0198] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 73 and 74, respectively.

[0199] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 75 and 74, respectively.

[0200] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 76 and 74, respectively.

[0201] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 77 and 74, respectively.

[0202] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 78 and 74, respectively.

[0203] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 79 and 74, respectively.

[0204] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 80 and 74, respectively.

[0205] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 81 and 74, respectively.

[0206] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 82 and 74, respectively.

[0207] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 83 and 74, respectively.

[0208] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 84 and 74, respectively.

[0209] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs. 85 and 74, respectively.

[0210] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 86 and 74, respectively.

[0211] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 73 and 87, respectively.

[0212] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs. 75 and 87, respectively.

[0213] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 76 and 87, respectively.

[0214] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 77 and 87, respectively.

[0215] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 78 and 87, respectively.

[0216] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 79 and 87, respectively.

[0217] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 80 and 87, respectively.

[0218] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 81 and 87, respectively.

[0219] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 82 and 87, respectively.

[0220] In preferred embodiments, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 83 and 87, respectively.

[0221] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 84 and 87, respectively.

[0222] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs. 85 and 87, respectively.

[0223] In a preferred embodiment, VH and VL comprise the amino acid sequences described in SEQ ID NOs: 86 and 87, respectively.

[0224] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0225] In some embodiments, the antibody is a monoclonal antibody.

[0226] In some embodiments, the antibody comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC).

[0227] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 88 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 88 and 89.

[0228] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 90 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 90 and 89.

[0229] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 91 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 91 and 89.

[0230] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 92 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 92 and 89.

[0231] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 93 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 93 and 89.

[0232] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 94 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 94 and 89.

[0233] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 95 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 95 and 89.

[0234] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 96 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 96 and 89.

[0235] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 97 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 97 and 89.

[0236] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 98 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 98 and 89.

[0237] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 99 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 99 and 89.

[0238] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 100 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 100 and 89.

[0239] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 101 and 89, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 101 and 89.

[0240] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 88 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 88 and 102.

[0241] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 90 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 90 and 102.

[0242] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 91 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 91 and 102.

[0243] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 92 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 92 and 102.

[0244] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 93 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 93 and 102.

[0245] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 94 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 94 and 102.

[0246] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 95 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 95 and 102.

[0247] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 96 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 96 and 102.

[0248] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 97 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 97 and 102.

[0249] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 98 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 98 and 102.

[0250] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 99 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 99 and 102.

[0251] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs: 100 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 100 and 102.

[0252] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs: 101 and 102, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 101 and 102.

[0253] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 19 and 20, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 19 and 20.

[0254] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 50 and 51, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 50 and 51.

[0255] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 59 and 60, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 59 and 60.

[0256] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 71 and 72, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 71 and 72.

[0257] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs: 9 and 10, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 9 and 10.

[0258] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 29 and 30, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 29 and 30.

[0259] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 38 and 39, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 38 and 39.

[0260] In some embodiments, HC and LC each include the amino acid sequences described in SEQ ID NOs. 44 and 45, or amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 44 and 45.

[0261] In some embodiments, the heavy chain includes a functional variant of the amino acid sequence described in any of SEQ ID NOs: 19, 50, 59, 69, 9, 29, 38, and 44, formed by the insertion, deletion, and / or substitution of one or more amino acids, insofar as the functional variant retains the ability to bind to ITGB4. In some embodiments, the light chain includes a functional variant of the amino acid sequence described in any of SEQ ID NOs: 20, 51, 60, 70, 10, 30, 39, and 45, formed by the insertion, deletion, and / or substitution of one or more amino acids, insofar as the functional variant retains the ability to bind to ITGB4.

[0262] Functional variants contain or consist of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0263] In some embodiments, the number of inserted, deleted, and / or substituted amino acids is preferably 40% or less of the total number of amino acids in the parent amino acid sequence, more preferably 35% or less, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20%. For example, the number of inserted, deleted, and / or substituted amino acids may be 1 to 50, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. In preferred embodiments, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0264] In some embodiments, insertions, deletions, and / or substitutions may be performed in framework (FR) regions, e.g., FR1, FR2, FR3, and / or FR4, and / or steady-state regions, e.g., CL, CH1, CH2, and / or CH3.

[0265] In some embodiments, the substitution of one or more amino acids may be a conservative substitution of one or more amino acids. Examples of conservative substitutions are as described above.

[0266] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 88 and 89, respectively.

[0267] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 90 and 89, respectively.

[0268] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 91 and 89, respectively.

[0269] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 92 and 89, respectively.

[0270] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 93 and 89, respectively.

[0271] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 94 and 89, respectively.

[0272] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs. 95 and 89, respectively.

[0273] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 96 and 89, respectively.

[0274] In a preferred embodiment, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 97 and 89, respectively.

[0275] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 98 and 89, respectively.

[0276] In a preferred embodiment, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 99 and 89, respectively.

[0277] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 100 and 89, respectively.

[0278] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 101 and 89, respectively.

[0279] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 88 and 102, respectively.

[0280] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 90 and 102, respectively.

[0281] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 91 and 102, respectively.

[0282] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 92 and 102, respectively.

[0283] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 93 and 102, respectively.

[0284] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 94 and 102, respectively.

[0285] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 95 and 102, respectively.

[0286] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 96 and 102, respectively.

[0287] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 97 and 102, respectively.

[0288] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 98 and 102, respectively.

[0289] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 99 and 102, respectively.

[0290] In a preferred embodiment, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 100 and 102, respectively.

[0291] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 101 and 102, respectively.

[0292] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 19 and 20, respectively.

[0293] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs. 50 and 51, respectively.

[0294] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs. 59 and 60, respectively.

[0295] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 71 and 72, respectively.

[0296] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 9 and 10, respectively.

[0297] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs: 29 and 30, respectively.

[0298] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs. 38 and 39, respectively.

[0299] In preferred embodiments, HC and LC comprise the amino acid sequences described in SEQ ID NOs. 44 and 45, respectively.

[0300] In some embodiments, the antibody is a bispecific antibody that includes an additional antigen-binding domain that binds to a second antigen.

[0301] In some embodiments, the second antigen is a tumor-associated antigen or an immune cell antigen.

[0302] "Tumor-associated antigens" refer to antigens that are specifically expressed in cancer cells compared to normal cells, and can therefore be used against target cancer cells. Many tumor-associated antigens associated with specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens that can potentially stimulate an obvious tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. Such antigens can usually be characterized as silent (i.e., not expressed) antigens in normal cells, antigens that are expressed only at certain differentiation stages, and antigens that are expressed over time, such as embryonic and fetal antigens. Other cancer antigens are those encoded by mutant cell genes, e.g., oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), and fusion proteins produced by internal deletions or chromosomal translocations. Other cancer antigens may be encoded by viral genes, e.g., viral genes harbored on RNA and DNA oncoviruses. Many other tumor-associated antigens and antibodies against them are known and / or commercially available and can also be produced by those skilled in the art.

[0303] Examples of tumor-associated antigens include, but are not limited to, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, type C lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate-binding protein, GD2, GD3, HIV-1 coat glycoprotein gp41, HIV-1 coat glycoprotein gpl20, melanoma-associated antigen, MUC-1, mutant p53, mutant ras, ROR1, GPC3, VEGFR2, and combinations thereof.

[0304] In some embodiments of the bispecific antibodies described herein, which include an additional antigen-binding domain that binds to a second antigen, the second antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D or any combination thereof. In some embodiments, the T cell antigen is CD3, and the second antigen-binding domain binds to any of the γ, δ, ε, ζ, and η chains of CD3.

[0305] In some preferred embodiments, the second antigen is CD3, CD138, CD20, CD40, CD19, CD33, CD16, CD38, CD22, CD40, CD8, IL-6, CSPG, RGM A, CTLA-4, BTN02, IGF1, IGF2, Erb2B, IGF-1R, EGFR, CD13, ErbB3, EGFR-2, IGFR, VEGFR-2, Met, VEGF-A, Angiopoietin 2 (Ang-2), IL-12, TWEAK, IL-13, IL-1 beta, RGM The group is selected from A, NgR, NogoA, OMGp, PDL-1, PD-1, PD-1, TIM-3, VEGF, Lag-3, OX40, DLL-4, PDGFR, EpCAM, CD16a, PSMA, CEA, TROP-2, HSG, HGF, PDGF, ICOS, CD47, RGM B, Te38, TNFa, Blys, GP130, IL-12, RANK ligand, ITGB6, Nectin 4, and folate receptors.

[0306] Based on the amino acid sequence of the constant region of the heavy chain of an antibody, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and further divided into various subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. The light chain of an antibody can be classified as either a lambda (λ) chain or a kappa (κ) chain based on the amino acid sequence of the light chain. The antibodies disclosed herein may be antibodies of any of the above classes or subtypes.

[0307] In some embodiments, the antibody is an isotype antibody selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is a subtype antibody selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In preferred embodiments, the antibody is an IgG1 antibody.

[0308] The antibodies disclosed herein may be intact antibodies or their antigen-binding fragments. Antigen-binding fragments may be any fragment of an antibody that retains the ability to specifically bind to ITGB4. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fd' fragments, Fv fragments, scFv fragments, VHH antibodies, dAb fragments, isolation complementarity-determining regions (CDRs), nanobodies, linear antibodies containing a pair of tandem Fd segments (VH-CH1-VH-CH1), and any modified form of the aforementioned fragments that retain antigen-binding activity.

[0309] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, VHH, and ds-scFv. In a preferred embodiment, the antigen-binding fragment is Fab. In another preferred embodiment, the antigen-binding fragment is Fv. In yet another preferred embodiment, the antigen-binding fragment is scFv.

[0310] III. Linker One or more drug moieties (e.g., therapeutic and / or diagnostic substances) can be indirectly conjugated to an anti-ITGB4 antibody (e.g., via a linker through direct covalent or non-covalent interactions). The linker may be a chemically binding substance, such as a homobifunctional crosslinker or a heterobifunctional crosslinker, which are available from many commercial suppliers.

[0311] The linker may be susceptible to cleavage under conditions that allow the compound or antibody to maintain its activity, such as acid-inducible cleavage, photo-inducible cleavage, peptidase-inducible cleavage, esterase-inducible cleavage, and disulfide bond cleavage (cleavage-type linker). Alternatively, the linker may be substantially resistant to cleavage (e.g., stable linker or non-cleavage-type linker).

[0312] In some embodiments, the linker is an acid-unstable linker. In some embodiments, the linker is a photo-unstable linker. In some embodiments, the linker is a protease-sensitive linker. In some embodiments, the linker is a hydrazone linker. In some embodiments, the linker is an esterase-cleaving linker. In some embodiments, the linker is a dimethyl linker. In some embodiments, the linker is a disulfide-containing linker. In some embodiments, the linker is a hydrophilic linker. In some embodiments, the linker is a procharged linker. In some embodiments, the linker is an acid-based linker.

[0313] In some embodiments, the linker includes an acid-unstable linker. In some embodiments, the linker includes a hydrophilic linker. In some embodiments, the linker includes a protease-sensitive linker. In some embodiments, the linker includes a photo-unstable linker. In some embodiments, the linker includes a hydrazone linker. In some embodiments, the linker includes a dimethyl linker. In some embodiments, the linker includes a disulfide-containing linker.

[0314] In some embodiments, the linker may include amino acid units. In such embodiments, the amino acid units may facilitate the release of the drug from the antibody-drug conjugate upon exposure to an intracellular protease, such as a lysosomal enzyme, by enabling the linker to be cleaved by a protease. Examples of amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Examples of dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), or N-methyl-valine-citrulline (Me-val-cit). Examples of tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, linkers containing vc (valine-citrulline) units are preferred. The amino acid units can be designed and optimized for selectivity in enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsin B, cathepsin C, and cathepsin D, or plasmin proteases.

[0315] In one embodiment, the linker used in this disclosure is a crosslinking reagent, for example, mc(6-maleimidocaproyl), PABC (para-amino-benzyloxycarbonyl), DMEA (dimethylethylamine), Val-Cit-PABC, mc-Val-Cit-PABC, CL2A, mal-PEG8-Val-Ala-PABC, MC-VC-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), mc-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), dimethylhydrazide (3-methyl-3-methyl Lucaptobutane hydrazide, AcBut-dimethylhydrazide, SPDP (N-succinimidyl-3-(2-pyridyldithio)propionic acid), SPP (N-succinimidyl-4-(2-pyridyldithio)pentanoate), SPDB (N-succinimidyl-4-(2-pyridyldithio)butanoate), sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate), SIA (N-succinimidyliodoacetate), SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate), maleimide PEG It is derived from NHS, SMCC (N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate), sulfo-SMCC (N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate), or 2,5-dioxopyrrolidine-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecane-1-oate (CX1-1).

[0316] In another embodiment, the linker used in this disclosure is derived from a crosslinking material, such as sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate), mc (6-maleimidocaproyl), PABC (para-aminobenzyloxycarbonyl), DMEA (dimethylethylamine), Val-Cit-PABC, mc-Val-Cit-PABC, CL2A, mal-PEG8-Val-Ala-PABC, MC-VC-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), mc-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), dimethylhydrazide (3-methyl-3-mercaptobutanehydrazide), AcBut-dimethylhydrazide, or SMCC (N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate).

[0317] In some embodiments, the linker used in this disclosure is sulfo-SPDB, and the antibody-drug conjugate is [ka] It is expressed by, and in the formula, [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0318] In some embodiments, the linker used in this disclosure is mc, and the antibody-drug conjugate is [ka] It is expressed by, and in the formula, [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0319] In some embodiments, the linker used in this disclosure is mc-Val-Cit-PABC, and the antibody-drug conjugate is [ka] It is expressed by, and in the formula, [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0320] In some embodiments, the linker used in this disclosure is CL2A, and the antibody-drug conjugate is [ka] It is expressed by, and in the formula, [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0321] In some embodiments, the linker used in this disclosure is mal-PEG8-Val-Ala-PABC, and the antibody-drug conjugate is [ka] It is expressed by, and in the formula, [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0322] In some embodiments, the linker used in this disclosure is mc-GGFG-aminomethyl, and the antibody-drug conjugate is [ka] It is expressed by, and in the formula, [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0323] In some embodiments, the linker used in this disclosure is SMCC: [ka] And in the formula, [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0324] In some embodiments, the linker used in this disclosure is AcBut-dimethylhydrazide, and the antibody-drug conjugate is [ka] And in the formula, [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0325] In some embodiments, the linker used in this disclosure is MC-VC-PAB-DMEA, and the antibody-drug conjugate is [ka] And in the formula, [ka] This indicates the binding site for the antibody or its antigen-binding fragment.

[0326] IV. Drugs In this specification, the terms “drug portion,” “drug payload,” “therapeutic molecule,” “therapeutic payload,” “therapeutic substance,” and “therapeutic portion” as used interchangeably refer to the chemical or biological portion that conjugates to an antibody or its antigen-binding fragment that binds to ITGB4.

[0327] Examples of drugs that can be used in ADCs, i.e., drugs that can be conjugated to antibodies, are listed below and include antibiotics, DNA synthesis inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors, mitotic inhibitors (e.g., tubulin inhibitors and microtubule polymerization inhibitors), antitumor antibiotics, immunomodulators, gene therapy vectors, alkylating agents, anti-angiogenic agents, anti-metabolites, boron-containing substances, chemoprotective agents, hormonal substances, glucocorticoids, photoactive therapeutic agents, oligonucleotides, radioisotopes, radiosensitizers, topoisomerase inhibitors (e.g., topoisomerase I inhibitors), tyrosine kinase inhibitors, and combinations thereof.

[0328] In some embodiments, tubulin inhibitors, microtubule polymerization inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase I inhibitors, RNA polymerase II inhibitors, and RNA spliceosome inhibitors are preferred.

[0329] In some embodiments, the drug portion is a tubulin inhibitor and a microtubule polymerization inhibitor. In some specific embodiments, the drug portion is auristatin. In some specific embodiments, the drug portion is mytansin. In some specific embodiments, the drug portion is tubulicin. In some specific embodiments, the drug portion is cryptophycin. In some specific embodiments, the drug portion is rhizoxin.

[0330] In some embodiments, the drug portion is an antibiotic. In some specific embodiments, the drug portion is calicheamicin. In some specific embodiments, the drug portion is doxorubicin. In some specific embodiments, the drug portion is an anthracycline.

[0331] In some embodiments, the drug portion is a DNA synthesis inhibitor. In some specific embodiments, the drug portion is duocalmycin. In some specific embodiments, the drug portion is PBD (benzodiazepine). In some specific embodiments, the drug portion is IGN (indolinobenzodiazepine).

[0332] In some embodiments, the drug portion is a topoisomerase I inhibitor. In some specific embodiments, the drug portion is a camptothecin analog.

[0333] In some embodiments, the drug portion is an RNA polymerase II inhibitor. In some specific embodiments, the drug portion is amanitin.

[0334] In some embodiments, the drug portion is an RNA spliceosome inhibitor selected from the group consisting of spliceostatins and tylanstatins.

[0335] In some embodiments, mytansinoids (DM1, DM2, DM3, DM4, mytansin, and anthamitosin) and their analogues are preferred.

[0336] In some specific embodiments, the mytansinoid drug moiety is N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-mytansin (also known as DM1). DM1 is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0337] [ka]

[0338] In some specific embodiments, the mytansinoid drug moiety is N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-mytansin (also known as DM3). DM3 is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0339] [ka]

[0340] In some specific embodiments, the mytansinoid drug moiety is N2'-deacetyl-N2'-(4-methyl-4-mercapto-1-oxopentyl)-mytansin (also known as DM4). DM4 is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0341] [ka]

[0342] In some embodiments, auristatins (MMAE, MMAF, MMAD, and anthamitocin) and their analogues are preferred.

[0343] In some specific embodiments, the auristatin drug portion is monomethyl auristatin E (also known as MMAE). MMAE is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0344] [ka]

[0345] In some specific embodiments, the auristatin drug portion is monomethyl auristatin F (also known as MMAF). MMAF is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0346] [ka]

[0347] In some embodiments, PBD (pyrrolobenzodiazepine or pyrrolo[2,lc][l,4]-benzodiazepine, e.g., SG3199) is preferred. PBD is a sequence-selective DNA alkylating antibiotic with significant antitumor properties. PBD has the ability to recognize and bind to specific sequences in DNA, one such sequence being PuGPu (purine-guanine-purine). Furthermore, PBD may bind to PuGPy (purine-guanine-pyrimidine) or PyGPu sequences rather than PyGPy sequences.

[0348] In some embodiments, the PBD drug portion is SG3199, which is a cytotoxic DNA subgroove interstrand crosslinked pyrrolobenzodiazepine (PBD) dimer. SG3199 is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0349] [ka]

[0350] In some embodiments, camptothecin analogs are preferred. Camptothecin analogs are a type of DNA topoisomerase I inhibitor. Unlike monomethyl auristatin E, peripheral neuropathy does not clinically occur with camptothecin-based therapy, suggesting that SGN-CD30C may have the potential to avoid one of the most common adverse events associated with BV.

[0351] In some specific embodiments, the camptothecin drug moiety is 7-ethyl-10-hydroxycamptothecin (also known as SN38). SN38 is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0352] [ka]

[0353] In some specific embodiments, the camptothecin drug portion is exatecan (also known as DX-8951). Exatecan is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0354] [ka]

[0355] In some specific embodiments, the camptothecin drug portion is an exatecan derivative (also known as DXd). DXd is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0356] [ka]

[0357] In some embodiments, calicheamicin is preferred, which is an antitumor antibiotic and a cytotoxic substance that causes DNA double-strand breaks. N-acetylcalicheamicin is a derivative of calicheamicin and is a potent engine antitumor antibiotic.

[0358] In some specific embodiments, the calicheamycin drug portion is N-acetylcalicheamycin γ1. This is represented by the following structural formula, where, [ka] This indicates the bonding site to the linker.

[0359] [ka]

[0360] V. Pharmaceutical Compositions Furthermore, this disclosure provides compositions comprising the antibody-drug conjugate of this disclosure, formulated with a pharmaceutically acceptable carrier, such as pharmaceutical compositions.

[0361] The therapeutic formulations of the present disclosure can be prepared by appropriately mixing an antibody-drug conjugate having a desired degree of purity with a physiologically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or aqueous solution. The acceptable carrier, excipient, or stabilizer is nontoxic to the recipient at the dosage and concentration used and includes buffers, e.g., phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (about 1 This includes proteins (less than 0 amino acid residues), e.g., serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., Tween, Pluronic, or PEG.

[0362] Furthermore, the formulation may contain two or more active compounds, preferably active compounds having complementary activity that does not adversely affect each other, as required for the specific indication being treated. For example, the formulation may further contain another antibody or bispecific antibody, a cytotoxic substance, a chemotherapeutic substance, or an ADC. Such molecules are suitably used in combination in amounts effective for the intended purpose.

[0363] Furthermore, the active ingredient may be encapsulated in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or a macroemulsion, for example, in microcapsules prepared by coacervation technology or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules, and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0364] The pharmaceutical compositions of this disclosure can be administered in combination therapy, i.e., in combination with other substances. Examples of therapeutic substances that can be used in combination therapy are described in more detail below.

[0365] Preparations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane. Sterile injections can be prepared by mixing the required amount of the active compound in a suitable solvent having, as needed, one or a combination of the components listed above, followed by sterile microfiltration. Generally, dispersants are prepared by mixing the active compound in a sterile medium containing a basic dispersion medium and other components required from those listed above. In the case of sterile powders for sterile injection preparation, preferred preparation methods are vacuum drying and freeze-drying, which yield powders of the active ingredient and any further desired components from a previously sterile-filtered solution.

[0366] VI. Dosage The amount of active ingredient that can be mixed with a carrier substance to produce a single dosage form varies depending on the target being treated and the specific method of administration. Generally, the amount of active ingredient that can be mixed with a carrier substance to produce a single dosage form is the amount of the composition that produces the therapeutic effect. Generally, out of 100 percent, this amount, when combined with a pharmaceutically acceptable carrier, is in the range of 0.01% to about 99%, preferably about 0.1% to about 70%, and most preferably about 1% to about 30%.

[0367] The drug regimen is adjusted to produce the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally increased or decreased depending on the urgency of the treatment situation. Formulating non-enteral compositions into unit dosage forms is particularly advantageous to facilitate uniformity of administration and dosage. When used herein, a unit dosage form refers to a physically separate unit suitable as a unit dose for the target to be treated, each unit containing a predetermined amount of the active compound calculated to work in conjunction with the required pharmaceutical carrier to produce the desired therapeutic effect. The specifications of the unit dosage forms in this disclosure are indicated by and directly depend on (a) the unique properties of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations on the care of individual sensitivity inherent in the compound, for example, in the art of the active compound.

[0368] In the administration of the antibody-drug conjugates of this disclosure, the dosage is approximately 0.0001 to 100 mg / kg, more typically in the range of 0.01 to 50 mg / kg of host body weight. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or in the range of 1 to 10 mg / kg. An example treatment plan may require administration daily, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. A preferred dosing regimen for the antibody-drug conjugate of this disclosure includes intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight, and the antibody-drug conjugate is administered using one of the following dosing schedules: (i) six doses every four weeks, then every three months, (ii) every three weeks, or (iii) one dose of 3 mg / kg body weight followed by 1 mg / kg body weight every three weeks.

[0369] Alternatively, antibody-drug conjugates can be administered as sustained-release formulations, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody-drug conjugate in the patient. Generally, human antibodies have the longest half-lives, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency may vary depending on whether the treatment is prophylactic or therapeutic. Prophylactic applications involve relatively low doses administered over a long period at relatively infrequent intervals. Some patients receive lifelong treatment. Therapeutic applications may require relatively high doses at relatively short intervals until disease progression is reduced or halted, preferably until the patient shows partial or complete recovery of symptoms. Subsequently, the patient can be given a prophylactic plan.

[0370] The actual dosage level of the active ingredient in the pharmaceutical compositions of this disclosure may be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the particular composition of this disclosure used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, symptoms, general condition, and medical history of the patient being treated, as well as similar factors well known in the medical technology.

[0371] The therapeutically effective dose of the ADCs of this disclosure preferably results in a reduction in the severity of symptoms, an improvement in the frequency and duration of symptom-free periods, or prevention of functional impairment or disability due to the distress of the disease. For example, in the treatment of tumors, the therapeutically effective dose preferably inhibits cell proliferation or tumor growth or metastasis by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80% compared to an untreated subject. The ability of a substance or compound to inhibit tumor growth can be evaluated in animal model systems that predict efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the inhibitory compound, and such inhibition in vitro can be evaluated by assays known to those skilled in the art. A therapeutically effective dose of a therapeutic compound can reduce tumor size, metastasis, or otherwise restore symptoms in a subject. Those skilled in the art can determine such a dose based on factors such as the size of the subject, the severity of the symptoms in the subject, and a particular composition or selected route of administration.

[0372] VII. Administration The compositions of this disclosure can be administered by one or more routes of administration using one or more of the diverse methods known in the art. As will be understood by those skilled in the art, the route and / or method of administration will vary depending on the desired outcome. Preferred routes of administration of the antibody drug conjugates of this disclosure include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other non-enteral administration routes by injection or infusion. The phrase “non-enteral administration,” as used herein, usually means methods of administration other than intestinal and local administration by injection, and includes, but is not limited to, injections and infusions into intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal regions. Alternatively, the antibody-drug conjugates of this disclosure may be administered via an enteral route, such as a topical, epidermal or mucosal route, for example, intranasal, oral, transvaginal, rectal, sublingual or topical.

[0373] The active compound can be prepared using controlled-release formulations, including carriers that protect the compound from rapid release, such as implantable tablets, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0374] Therapeutic compositions can be administered by medical devices known in the art. For example, the therapeutic compositions of this disclosure can be administered by needleless subcutaneous injectors, such as those disclosed in U.S. Patents 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556. Examples of well-known implantable tablets and modules useful in this disclosure include those described in U.S. Patents 4,487,603, 4,486,194, 4,447,233, 4,447,224, 4,439,196, and 4,475,196. Such patents are incorporated herein by reference. Many other such implantable tablets, delivery systems, and modules are known to those skilled in the art.

[0375] VIII. Treatment method In one embodiment, the present disclosure relates to an in vivo treatment of a target disease using the antibody-drug conjugate, wherein the disease is manifested by ITGB4. In one embodiment, the present disclosure provides a method for preventing and / or treating a disease in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate or a composition comprising an antibody-drug conjugate to the subject, wherein the disease is manifested by ITGB4. In a particular embodiment, the disease is cancer.

[0376] In another embodiment, the Disclosure relates to the in vivo treatment of a subject using the antibody-drug conjugate described above to inhibit the growth and / or metastasis of a cancerous tumor. In one embodiment, the Disclosure provides a method for inhibiting the growth of tumor cells in a subject and / or limiting the metastatic spread thereof, the method comprising administering a therapeutically effective amount of antibody-drug conjugate to the subject.

[0377] Non-limiting examples of cancers favored for treatment include chronic or acute leukemia, head and neck cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, stomach cancer, pancreatic cancer, ovarian cancer, prostate cancer, breast cancer, leukemia, myeloma, squamous cell carcinoma, melanoma, leukemia, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, kidney cancer, testicular cancer, and thyroid cancer, as well as head and neck cancer.

[0378] As used herein, the term “subject” is intended to include humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-human mammals, e.g., non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, but mammals, e.g., non-human primates, sheep, dogs, cats, cattle, and horses are preferred. Preferred subjects include human patients requiring enhancement of the immune response. The method is particularly suited to the treatment of human patients with disorders that can be treated by increasing the immune response.

[0379] Furthermore, the above treatments can be combined with standard cancer treatments. For example, they can be effectively combined with chemotherapy regimens. In such cases, it may be possible to reduce the dose of chemotherapy reagents administered (Mokyr, M. et al. Cancer Res., 1998, 58, 5301-5304).

[0380] Other antibodies that can be used to activate host immune responses may be used in or in conjunction with the bispecific molecular drug conjugates of this disclosure. These include molecules that target the surface of dendritic cells to activate DC function and antigen presentation. For example, anti-CD40 antibodies can effectively replace T cell helper activity (Ridge, J. et al. Nature, 1998, 393, 474-478) and can be used in combination with the bispecific molecular drug conjugates of this disclosure (Ito, N. et al. Immunobiology, 2000, 201, 527-540). Similarly, T cell costimulatory molecules, such as CTLA-4 (US Patent No. 5811097), CD28 (Haan, J. et al. Immunol. Lett., 2014, 162, 103-112), OX-40 (Weinberg, A. et al. J. Immunol., 2000, 164, 2160-2169), 4-1BB (Melero, I. et al. Nature Med., 1997, 3, 682-685), and ICOS (Hutloff, A. et al. Nature, 1999, 397) Antibodies targeting 262-266), or antibodies targeting PD-1 (US Patent No. 8008449) and PD-L1 (US Patent No. 7943743, US Patent No. 8168179), may also result in increased T cell activation levels. In another example, the bispecific molecular drug conjugates of this disclosure can be used in combination with anti-neoplasmic antibodies such as Rituxan (rituximab), Herceptin (trastuzumab), Bexal (tocitumomab), Zevalin (ibritumomab), Campus (aremtuzumab), Lymphocide (eprtuzumab), Avastin (bevacizumab), and Tarceva (erlotinib). [Examples]

[0381] The following examples are provided to those skilled in the art to provide a complete disclosure and an explanation of how to utilize this disclosure, and are not intended to limit the scope of what the inventors consider to be their disclosure, nor to represent that the following experiments are all and only those conducted. While efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), some experimental errors and deviations should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is degrees Celsius, and pressure is atmospheric pressure or approximately atmospheric pressure.

[0382] [Example 1] ITGB4 is highly expressed in multiple tumor types. The expression levels of ITGB4 were compared with adjacent normal tissue in multiple tumor types and analyzed using a bioinformatics database (PCT / CN2022 / 074991). It was demonstrated that ITGB4 is highly expressed in many tumor types, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), esophageal squamous cell carcinoma (ESCC), and head and neck squamous cell carcinoma (SCCHN) (Figure 1).

[0383] [Example 2] ITGB4 is highly expressed in many tumor samples stained with IHC. The anti-ITGB4 monoclonal antibody ab182120 (Abcam, cat.AB182120) was found to bind to tumor cells expressing ITGB4 with higher sensitivity and specificity compared to isotype controls.

[0384] In the IHC protocol, immunohistochemical staining was performed on 4 μm sections using ab182120 (1:400 dilution) rabbit monoclonal primary antibody (Abcam) on a Ventana Benchmark Ultra Autostainer according to the manufacturer's protocol. Briefly, antigen recovery was performed by placing unstained slides in Ventana Ultra CC1 buffer (Tris-EDTA / EGTA, pH 9) at 64°C for 95 minutes, followed by incubation of the tissue with the primary antibody (ab182120) at 36°C for 16 minutes. The antigen-antibody reaction was visualized using the Ultraview® Universal DAB Detection Kit. After hematoxylin staining for 8 minutes, blue staining reagent (Ventana Medical Systems, Tucson, AZ) was used as a counterstain for 4 minutes. Isotype control was performed as described above, except that the primary antibody was replaced with rabbit IgG to ensure antibody specificity. Immunohistochemical staining was semi-quantitatively evaluated for both percentage positivity and intensity, and an H score ranging from 0 to 300 was obtained based on the percentage of stained tumor cell membranes multiplied by the staining intensity (0 to 3).

[0385] ITGB4 was found to be highly expressed in tumor samples (Bioaitech) from a subset of patients with CRC and NSCLC, as well as from all patients with SCCHN and ESCC (Table 1, Figure 2). In this study, 68% of CRC (Figure 2b), 42% of NSCLC (Figure 2c), 100% of ESCC (Figure 2d), and 100% of SCCHN (Figure 2e) were observed to have a tumor cell membrane ITGB4 expression H score ≥ 100 (Table 1). [Table 4]

[0386] [Example 3] ITGB4 is highly expressed in many tumor cell lines evaluated by FACS. The ITGB4 antigen density is measured for PE rat anti-human CD104 (BD), PE mouse anti-human CD104 (BD), and the clone 002 of this application in COLO 201 (Meisen CTCC-007-0101), DLD-1 (SIBS TCHu134), H441 (Meisen CTCC-001-0355), HCC827 (SIBS TCHu153), HCT116 (SIBS TCHu99), HT-29 (ATCC HTB-38), LoVo (ATCC CCL-229), LS1034 (Meisen CTCC-007-0089), RKO (SIBS TCHu116), SW480 (SIBS SCSP-5033), SW620 (Meisen CTCC-003-0021), and T84 (SIBS Cells from TCHu211) were evaluated by FACS using BD Quantibrite® Beads according to the BD recommended protocol (BD, data summarized in Table 2). All such cells were cultured according to ATCC guidance. [Table 5]

[0387] Similar results were achieved with various anti-ITGB4 clones. LS1034, HT-29, HCT-116, DLD-1, SW480, and SW620 showed that at least one anti-ITGB4 antibody could reduce the amount of 10 cells per cell. 5 It was detected beyond the molecule (Figure 3). In LoVo, T84, Colo201, H441, and HCC827, ITGB4 was present on the surface at a rate of 10 per cell. 3 ~10 5 It was expressed at the molecular level. In RKO, 10 per cell 3 The presence of molecules smaller than 100 was detected, which allowed us to determine that it was a negative control for ITGB4 expression (Figure 3).

[0388] [Example 4] Evaluation of antibodies binding to human ITGB4 using OCTET, ELISA, and FACS. Parental clones 001, 002, 003, 004, 005, 006, 007, and 008, as well as their isotypes, were tested by BLI using ForteBio Octet RED96e with Anti-hIgG Fc Capture (AHC) Biosensors (Sartorius #18-5060). The assay was performed at 30°C and mixed at 1000 rpm. All samples were diluted with 10×KB (Sartorius #18-1105) and 1×Kinetics Buffer (KB) prepared with PBS. 10 μg / mL of the test antibody was added to the sensor for 180 seconds, followed by the addition of 1×KB for 120 seconds. Recombinant human integrin alpha-6 beta-4 protein (ITGα6β4.Sino #CT069-H2508H) was associated at 100 nM, 10 nM, 1 nM, and 0 nM concentrations for 180 seconds, after which 1 × KB was added and dissociation was evaluated after 300 seconds. The data were analyzed using ForteBio Data Analysis software HT12.0. The OCTET binding results are shown in Table 3.

[0389] Parental clones 001, 002, 003, 004, 005, 006, 007, and 008, as well as their isotypes, were tested by ELISA. 96-well ELISA plates were coated overnight at 2-8°C with 100 μL / well of recombinant human antibody solution (Sino #CT069-H2508H) at 2 μg / mL in ELISA coating buffer (Solarbio #C1055). The plates were washed three times with 250 μL / well of 1×PBST, and excess liquid was removed by gently tapping on absorbent paper. The plates were blocked at room temperature with SuperBlock® blocking buffer (Thermo Fisher #37516) according to the manufacturer's protocol. Then, 100 μL / well of diluted test antibody was added to the plates and incubated at 37°C for 1 hour. The test antibody concentrate was then subjected to eight serial dilutions, starting at 10 μg / mL and increasing by a 5-fold dilution ratio. After five washes with 250 μL / well 1×PBST, 100 μL / well of goat anti-human IgG HRP (Sigma #A0170) diluted 1:8000 in blocking buffer was added to the plate and incubated at 37°C for 1 hour. After five further washes with 250 μL / well 1×PBST, 100 μL / well of TMB solution (SeraCare #5120-0077) was added and incubated at room temperature for 10 minutes. Subsequently, 100 μL / well of Stop solution (Beyotime #P0215) was added to stop the disease response. Absorbance at 450 nm was measured using a BioTek Synergy 2 Microplate Reader. Nonlinear regression analysis with four adjustable parameters was performed using GraphPad Prism 9.2 software. The ELISA results are shown in Table 3.

[0390] Parental clones 001, 002, 003, 004, 005, 006, 007, and 008, as well as isotype controls, were tested for binding to tumor cell lines H292 (ATCC CRL-1848) and HCT116 (SIBS TCHu99), and modified cells CHO-S-human ITGA6B4-#5 (Celetrix). All tested cells were cultured according to ATCC guidance. 10 cells per well 5 Cells were seeded in a 96-well V-bottom plate. The cells were washed twice with staining buffer (BD #554656) and centrifuged at 300×g at 2–8°C for 3 minutes. The cells were resuspended and incubated with refrigerated diluted antibody in 100 μL / well of staining buffer as the primary antibody solution at 2–8°C, away from light, for 40 minutes. The concentrate of the test antibody was subjected to eight serial dilutions, starting at 50 μg / mL and increasing by a 5-fold dilution factor. After two washes, the cells were resuspended with 100 μL / well of 1 μg / mL refrigerated diluted Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG (H+L) (Jackson #109-605-088) as the secondary antibody and incubated at 2–8°C, away from light, for 30 minutes. After two washes with staining buffer, cells were resuspended in 100 μL / well of staining buffer and analyzed using a Beckman CytoFlex flow cytometer. 10,000 events were collected in the APC channel for each sample. Nonlinear regression analysis with four adjustable parameters was performed using GraphPad Prism 9.2 software. The relevant results are shown in Figure 4.

[0391] All test antibodies bound to human ITGB4 with high affinity, as detected by Octet and ELISA (Table 3). When the antibodies were applied to tumor cells containing HCT116 and H292 which endogenously express ITGB4, or ITGB4-transduced CHO-S (CHO-S human ITGA6B4#5) which highly expresses ITGB4, all test antibodies showed high binding affinity, with a difference of less than 10-fold, except for 001 and 003 which had slightly lower binding affinity (Table 4 and Figure 4). [Table 6] [Table 7]

[0392] [Example 5] Evaluation of cross-reactivity of test antibodies by FACS binding. The FACS evaluation protocol was similar to the protocol used in Example 4. To evaluate cross-reactivity, the test cell lines included CHO cells stably transfected to highly express either cynomolgus monkey or mouse integrin α6β4 (CHO-S-mouse ITGA6B4-#12, CHO-S-cynomolgus monkey ITGA6B4-#8, Celetrix), 4T1 (ATCC) expressing mouse integrin α6β4, and CHO-S-human integrin α6β1 cells stably transfected to highly express human integrin α6β1 (CHO-S-human ITGA6B1-#24, Celetrix).

[0393] The results showed that all tested antibodies recognized cynomolgus monkey ITGB4. Clones 008 and 002 showed excellent binding affinity to cynomolgus monkey ITGB4 (Figure 5a). In addition, all tested antibodies except clones 002 and 008 bound to 4T1 (Figure 5d) and CHO-S-mouse ITGB4 (Figure 5c). None of the tested antibodies recognized human ITGB1 (Figure 5b).

[0394] [Example 6] Evaluation of test antibodies for internal migration To evaluate internal migration into tumor cell lines, parental clones 001, 002, 004, 005, 006, 007, and 008, as well as isotype controls, were examined using H292 (ATCC CRL-1848), HCT116 (SIBS TCHu99), and DLD-1 (SIBS TCHu134) cells. All such cells were cultured according to ATCC guidance.

[0395] In the indirect method of internal migration (FACS surface binding), H292 cells, HCT116 cells, and DLD-1 cells were examined at 0 and 24 hours using a 2 μg / mL parental clone antibody solution. For the initial 0-hour group, the FACS procedure was the same as that described in Example 4. For the 24-hour group, after incubation with the primary antibody solution, the cells were washed twice with 200 μL / well culture medium, resuspended in 500 μL / well culture medium, transferred to a 24-well plate, and cultured at 37°C for 24 hours. Secondary antibody staining was performed on cells collected after washing twice with staining buffer. The following FACS procedure was the same as in Example 4. The 24-hour internal migration rate was calculated using the formula: 100% - (MFI at 24 hours ÷ MFI at 0 hours) * 100%. The relevant results for the indirect method are shown in Figures 6-a, b, and c.

[0396] For the direct internal transfer method (pHAb), H292 and HCT116 cells were tested for 24 hours using a 2 μg / mL pHAb-labeled parental clone solution. The pH of 100 μg of antibody in PBS was adjusted to approximately 8.5. The antibody solution was diluted to 2 mg / mL with 10 mM, pH 8.5 NaHCO3, then mixed with 1.2 μL of 10 mg / mL pHAb Amine Reactive Dye (Promega, #G9841) and incubated at room temperature in the dark for 1 hour. Unconjugated pHAb was removed using Zeba Spin Desalting Columns (Thermo Fisher, #89882), and the buffer was replaced with PBS. pHAb-labeled antibodies A280 and A532 were tested using NanoDrop® OneC (Thermo Fisher) to calculate the concentration and percentage of pHAb labeling. 5 × 10 in 2 mL of culture medium per well 5Cells were seeded in a 6-well plate and incubated for 24 hours. The culture medium was then replaced with 2 mL of fresh culture medium containing 4 μg of pHAb-labeled antibody. After a further 24 hours of incubation at 37°C in the dark, cells were collected, washed with staining buffer (BD #554656), and then centrifuged. The cells were then resuspended in 100 μL / well of staining buffer and analyzed using a Beckman CytoFlex flow cytometer. Each sample showed 10 PE channel values. 4 Events were collected. The magnification change between the MFI of each sample and the MFI of the blank control was analyzed using GraphPad Prism 9.2 software. The relevant results are shown in Figure 6d.

[0397] The results demonstrated that all test antibodies could induce internal migration to these two tumor cell lines. Clones 002, 007, and 008 exhibited superior efficacy against both cell lines (Tables 5 and 6). [Table 8]

[0398] [Example 7] Synthesis and chemical profiling of anti-ITGB4 antibody drug conjugates Antibody clones 001, 002, 004, 005, 006, and 008 were transiently generated using the CHO-K1 cell line. The antibodies were purified by protein A chromatography according to standard procedures. The antibodies were then conjugated to MC-VC-PABC-MMAE. Approximately 50 mg of 001, 002, 004, 005, or 006 was conjugated to MC-VC-PABC-MMAE. The drug linker MC-VC-PABC-MMAE was alkylated against the cysteine ​​of the reduced antibody according to the procedures described in the literature (Sun et al. (2005) Bioconjugate Chem. 16: 1282-1290; McDonagh et al., (2006) Protein Eng. Design Sel. 19: 299-307). In short, the interchain disulfide of the anti-integrin α6β4 antibody was reduced with 2.2–2.5 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (10 mM) for 1 hour at 37°C. Afterward, a solution of the linker payload (MC-VC-PABC-MMAE) was added to the reaction mixture. The linker payload was added in a stoichiometric excess (5–6 equivalents) sufficient to conjugate all the interchain cysteine ​​generated in the TCEP reduction step relative to the mAb. If the linker payload was insoluble in the buffer solution, 10%–30% (v / v) DMSO was added to the conjugate mixture before the linker payload. The reaction was quenched by adding an excess of N-acetylcysteine. Residual non-conjugated drug was removed by purification, and the final anti-integrin α6β4 antibody drug conjugate was formulated in 20 mM histidine pH 6.0.

[0399] 002-DXd, 002-SN38, and 008-DXd were synthesized using the following protocol. Following the procedures described in the literature (Nakada T et al. (2016) Bioorg Med Chem Lett. 26(6):1542-1545, Kang MS et al. (2021). Chem Sci. 12(41):13613-13647), the drug linker MC-GGFG-DXd or MC-VC-PAB-DMEA-SN38 was alkylated against the cysteine ​​of the reduced antibody (002 or 008). Briefly, after buffer exchange to 50mM PB containing 5mM EDTA (pH 7.2) using an Amicon ultracel filter (MWCO 25kDa), the antibody sample was reduced with 8 equivalents of TCEP (10mM) at room temperature for 4 hours. Twelve equivalents of drug linker (5 mM in DMSO) were slowly added (over 3-5 minutes) at 20°C for 30 minutes. The reaction was quenched with 15 × NAC (20 mM) at 20°C for 30 minutes. Residual non-conjugate drug was removed by purification, and the antibody-drug conjugate was formulated in 20 mM histidine pH 6.0. Subsequently, the concentration of all formed ADC samples was analyzed by absorbance at 280 nm. The chemical quality of the synthesized ADCs was confirmed by HIC / RP-HPLC to identify the drug antibody rate (DAR) and SEC to identify purity. The results demonstrated that all ADCs were synthesized of good quality (Table 6). [Table 9]

[0400] [Example 8] ADC protein binding evaluation and cell binding affinity The experimental protocols for Octet (for protein binding evaluation) and FACS (for cell binding affinity) were performed as described in Example 4.

[0401] The results demonstrated that the conjugated ADC maintained its binding ability to ITGB4 compared to the corresponding unconjugated antibody (Table 7, Figure 7). [Table 10]

[0402] [Example 9] Evaluation of conjugated ADCs for internal migrations The pHAb assay was applied to examine the internal migration of ADCs to HT-29 and SW620 cells, and their functionality for internal migration (Figure 8a, b). An indirect internal migration assay was performed to evaluate the internal migration rates to SW620 and COLO205 (Figure 8c). The protocol was as described in Example 6.

[0403] The results demonstrated that all ADCs tested induced high levels of internal migration within 24 hours in both pHAb assays and surface-bound FACS (Figure 8).

[0404] [Example 10] Cytotoxic effects of anti-ITGB4 ADCs on tumor cells The cytotoxicity of MMAE conjugated ADCs using antibodies 001, 002, 004, 005, and 006, as well as non-cytotoxic MMAE (Selleck #S7721), was examined using cells from H292, HCT116, HCC827, HT-29, LoVo, SW480, SW620, COLO 201, RKO (negative control), A549 (SIBS SCSP-503), and H1975 (ATCC CRL-5908).

[0405] The cytotoxicity of SN38 and DXd conjugated with parent clone 002, as well as their isotypes, was examined using cells from COLO201, HT-29, SW620, DLD-1, H292, LS1034, and RKO (negative control).

[0406] In short, 1000 cells per well were seeded in 50 μL of culture medium in a 96-well flat-bottom plate and incubated at 37°C for 24 hours. Test samples were diluted with the corresponding culture medium. For MMAE ADC and associated control samples, these 2× concentrates were subjected to nine serial dilutions, starting at 2 μM and using 5-fold dilutions. For SN38 or DXd ADC and associated control samples, these 2× concentrates were subjected to nine serial dilutions, starting at 16 μM and using 5-fold dilutions. 50 μL of the 2× test sample solution was added to the 96-well plate containing the cells and incubated for 5-6 days. After incubation, 100 μL / well of CellTier-Glo (Promega #G7571) was added. The plate was then placed on a swirling shaker for 2 minutes and left on a workbench for 10 minutes to stabilize the signal. Luminescence intensity was detected using BioTek Synergy 2. The inhibition rate was calculated as 100% - (LUM of the sample ÷ LUM of the blank) * 100%. Nonlinear regression analysis with four adjustable parameters was performed using GraphPad Prism 9.2 software, and absolute IC50 was interpolated. The relevant results are shown in Figure 9. [Table 11]

[0407] The results demonstrated that 002-MMAE achieved the highest efficacy (and lowest IC50) and TI for tumor cell killing among all DAR4-MMAE-based ADCs (Table 8, Figures 9a-k). When the 002 clone was applied to the SN38 conjugate, the 002-SN38 conjugate showed toxic activity against test tumor cells including SW620, HT-29, and Colo201, with TIs of 1.34, 4.19, and 1.73, respectively. This demonstrates the target-dependent nature of 002-SN38 (Table 8, Figures 9l-n). Additionally, 002-DXd showed toxic activity against ITGB4-positive tumor cells, with TIs ranging from 2.55 (LS1034) to 62.96 (Colo201) (Table 8, Figures 9n-q).

[0408] [Example 11] Inhibition of xenograft tumor growth by ADCs In vivo efficacy in cell line-derived xenotransplantation was 5 × 10 6 HCT116, H292, SW620, HT29, or colo201 cells were subcutaneously injected into female nude (BALB / c-nude) mice (GemPharmatech) or SCID / Beige mice (Charles River). Mice were randomly assigned to test groups, and those with tumors approximately 300 mm² were treated. 3 The ADC was administered intravenously when the target was reached. Tumor volume was calculated using the formula (volume = 1 / 2 × length × width × width). No weight loss or treatment-related toxicity was observed during the study. All treatment procedures for the animals were carried out according to protocols approved by the animal facility's animal experimentation committee. Efficacy study grouping information is listed in Table 9, and the results are shown in Figure 10. [Table 12-1] [Table 12-2]

[0409] All ADCs demonstrated efficacy in controlling tumor growth. 002-MMAE inhibited H292 tumor growth in SCID / Beige at 3mpk (Figure 10a) and HCT116 tumor growth in BALB / c nude mice at 10mpk (Figure 10b). 002-DXd showed superior efficacy in SW620 (Figure 10c). Tumor inhibition for SW620 persisted for more than 55 days (Figure 10c). Furthermore, 002-DXd demonstrated target-dependent tumor inhibition of COLO201 tumors at a single dose of 10mpk (Figure 10d). 008-DXd more effectively eradicated SW620 tumors (Figure 10g). Both 008-MMAE and 008-DXd effectively controlled COLO205 tumor growth (Figure 10f). 008-DXd showed significant efficacy against SW620 tumors (Figure 10e).

[0410] [Example 12] Humanization of 008 Various humanized 008 clones with slight differences in framework sequences and complementarity-determining region (CDR) sequences were generated using CDR transplantation, and the CDRs of the 008 clones were transplanted into the human framework. The 008 clones were used as mouse antibodies containing a light chain variable (VL) domain with an amino acid sequence (SEQ ID NO: 70) and a heavy chain variable (VH) domain with an amino acid sequence (SEQ ID NO: 69).

[0411] Several humanized light chain variable domains and heavy chain variable domains were generated. Then, the amino acid sequences of candidate humanized light chains and candidate humanized heavy chains were combined in various ways to generate a panel of 26 humanized complete antibodies. The humanization rates of all candidates are shown in Table 10. [Table 13]

[0412] [Example 13] Evaluation of humanized antibodies that bind to human ITGB4 using OCTET, ELISA, and FACS. The epitope binding of humanized antibodies containing parent clone 008 to ITGα6β4 was examined using BLI (ForteBio Octet RED96e). The assay was performed on anti-hIgG Fc Capture (AHC) Biosensors (Sartorius #18-5060) at 30°C with mixing at 1000 rpm. All samples were diluted in 1×KB or PBS. First, 10 μg / mL of 008 antibody was added to the sensor and allowed to stand for 180 seconds, followed by the addition of 1×KB and the addition of 120 seconds. Then, the added sensor was blocked with 200 μg / mL of isotype control antibody for 600 seconds, followed by the addition of 1×KB and the addition of 120 seconds. Next, association of recombinant human ITGα6β4 (Sino #CT069-H2508H) was performed on the blocked sensor at 200 nM for 180 seconds, followed by the addition of 1×KB and the addition of 120 seconds. Finally, 002, 008, H84.2, and H86.2 were added at 10 μg / mL and allowed to stand for 180 seconds, followed by a dissociation step using 1 × KB for 180 seconds. The response at each time point was analyzed using ForteBio Data Analysis software HT12.0 and GraphPad Prism9.2 software. The results are summarized in Figure 11.

[0413] Humanized antibody clones H83.1, H83.2, H84.1, H84.2, H85.1, H85.2, H86.1, H86.2, H87.1, H87.2, H88.1, H88.2, H89.1, H89.2, H810.1, H810.2, H811.1, H811.2, H812.1, H812.2, H813.1, and H813.2, parental clone 008, and isotype controls were tested for binding to tumor cell lines SW620, CHO-S-cynomolgus monkey ITGA6B4-#8, and CHO-S-human ITGA6B4-#5. The FACS procedure was as described in Example 4. Octet data demonstrated that all humanized clones maintained high binding affinity to ITGB4. H811.1, H811.2, and H812.2 have affinity less than 1 nM KD. All humanized antibodies tested bound to the same epitope as the parental clone (Table 11).

[0414] Tumor cells containing SW620, which endogenously expresses ITGB4 (Figure 12a, b, c), and human (Figure 12d) / cynomolgus monkey (Figure 12e, f) ITGB4-transduced CHOS cells that highly express ITGB4 were identified by FACS assay. Clones H86.1 and H86.2 maintained binding to cynomolgus monkey ITGB4 and exhibited affinity comparable to human ITGB4. [Table 14]

[0415] [Example 14] Evaluation of the internal distribution of humanized antibodies into tumor cells Humanized antibody clones H83.1, H83.2, H84.1, H84.2, H85.1, H85.2, H86.1, H86.2, H87.1, H87.2, H88.1, H88.2, H89.1, and H89.2, parental clones 002 and 008, and isotype controls were tested for internal transfer to SW620. Indirect internal transfer assays were applied using the protocol referred to in Example 6. All humanized antibodies tested exhibited superior internal transfer function compared to the parental clones (Figure 13).

[0416] [Example 15] Feasibility assessment of humanized antibodies To evaluate the developmental potential of humanized antibodies H86.2 and H84.2, the inventors assessed transient expression levels, purity, uniformity, and thermal stability, and analyzed PTM hotspots.

[0417] Differential scanning fluorescence (DSF) was used to determine the time lapse rate (Tm) by recording the change in fluorescence intensity of a hydrophobic fluorescent dye added to a solution. Tm was determined using the ABI7500 Fast Real-Time PCR system. Tm was assigned using the first minimum value of the first derivative of the raw data.

[0418] Furthermore, thermal stability can be demonstrated by aggregating the set temperature (Tagg) measured by dynamic light scattering (DLS). The sample was added in the minimum amount appropriate for the plate to a CORNING assay plate (96 or 384 wells) with sealing tape. The plate was centrifuged using a plate centrifuge to remove air bubbles. The plate was scanned at 25°C to 85°C using an HT-DLS DynaPro Plate Reader III (Wyatt Technology). The results were analyzed using Wyatt Technology's DYNAMICS software.

[0419] For hydrophobic interaction chromatography (HIC) analysis, approximately 20 μg (1 mg / mL) of the sample was added at 25°C to a High Performance Liquid Chromatography (HPLC) system (Thermo Scientific) equipped with a TSKgel butyl-NPR 4.6 mm × 35 mm column (Tosoh Bioscience). The column was equilibrated with 90% buffer A with 10% buffer B added, and elution was performed at 0.7 mL / min over 15 minutes using a linear gradient from 90% buffer A to 90% buffer B, with UV absorbance at 280 nm monitored. Buffer A consisted of 20 mM sodium phosphate and 1.5 M ammonium sulfate, pH 7.0, while buffer B consisted of 20 mM sodium phosphate and 25% isopropanol, pH 7.0.

[0420] Thermal stability tests for H86.2 and H84.2 were performed at 40°C for 28 days using 25 mM His and 2 mM EDTA, pH 6.5. Candidates were then examined on days 0, 7, 14, and 28 by reduced and unreduced SDS-PAGE, SEC-HPLC, and iCIEF. The freeze-thaw stability of the antibodies was also investigated using 25 mM His and 2 mM EDTA, pH 6.5. Candidates were then examined after 3 and 5 cycles from F / T by reduced and unreduced SDS-PAGE, SEC-HPLC, and iCIEF. The results are shown in Table 12. [Table 15]

[0421] [Example 16] Synthesis and chemical profiling of antibody-drug conjugates using humanized antibodies For antibody-drug conjugates using humanized antibodies (referred to as humanized ADCs in later text), the same protocol as that described in Example 7 was applied. The results of the chemical properties are shown in Table 13. [Table 16]

[0422] [Example 17] Evaluation of humanized ADCs that bind to human ITGB4 using OCTET and FACS. The ability of humanized ADCs to bind to the ITGB4 protein in humans was evaluated by Octet and FACS (cell binding) according to the protocol described in Example 4.

[0423] The results demonstrated that all humanized ADCs exhibited similar binding affinity to their corresponding non-conjugate antibodies (Figure 14). Further bladder urothelial carcinoma tumor cell line KU-19-19 (Cobioer CBP60593) was also evaluated in this assay.

[0424] [Example 18] Evaluation of the internal migration of humanized ADCs that bind to tumor cells. To identify the internal distribution ability of humanized ADCs in SW620 tumor cells, an indirect method (described in Example 4) was applied. The results demonstrated that the conjugated ADCs had similar internal distribution ability to the corresponding unconjugated antibody H86.2 (Figure 15).

[0425] [Example 19] Cytotoxic effects of humanized ADCs on tumor cells The cytotoxic effects of humanized ADCs against tumor cells, including MDA-MB-468, KU-19-19, T84, HT-29, SW620, and RKO, were evaluated using the protocol described in Example 10. The results are summarized below (Table 14) and shown in Figure 16. [Table 17]

[0426] ADCs containing DAR8-DXd were demonstrated to eliminate tumor cells except RKO (ITGB4-negative) and HT-29, exhibiting high efficacy (IC50 in the range of 5.97 nM to 99.54 nM) and high target dependence (TI in the range of 2.29 to 51.17). ADCs containing DAR4-DXd showed slightly lower efficacy (IC50 in the range of 55.34 nM to 575.44 nM) but exhibited a similar TI in the range of 1.33 to 5.63. DAR4-MMAE-based ADCs more effectively killed tumor cells except RKO, with an IC50 in the range of 0.7 nM to 8.79 nM, achieving an even higher TI for all tumor cells except MDA-MB-468 (Table 14, Figure 16).

[0427] [Example 20] Inhibition of xenograft tumor growth by anti-ITGB4 ADCs The protocol was the same as that described in Example 11. Grouping information was listed below (Table 15). The MTDs for each drug in BALB / c nude mice are shown in Table 16. It is demonstrated that all DXd-based ADCs had a maximum tolerated dose (MTD) of >200 mg / kg in nude mice, regardless of DAR4 or DAR8. ADCs with MMAE achieved an MTD of >50 mg / kg.

[0428] Both H86.2-DXd and H84.2-DXd significantly inhibited SW620 tumor growth, demonstrating efficacy comparable to 008-DXd (parental clonal ADC) (Figure 17a). H86.2-DXd showed higher efficacy than H86.2-MMAE in the SW620 model (Figure 17b). Furthermore, H86.2-MMAE more effectively eradicated HT-29 tumors than H86.2-DXd (Figure 17c). [Table 18] [Table 19]

[0429] [Example 21] Rat PK test Male Sprague Dolly (SD) rats were randomly assigned to test groups. A single dose of H84.2-DXd, H86.2-DXd, H86.2-DXd-DAR4, or the corresponding unconjugated mAb of H84.2 or H86.2 was administered intravenously (Table 17). Blood samples were then sequentially collected by venipuncture, treated with serum, and subsequently subjected to quantitative bioanalysis by ELISA for conjugated mAbs (ADCs), whole mAbs (tAbs), and unconjugated mAbs. No apparent abnormalities were observed during this study. All animal handling procedures were performed according to protocols approved by the laboratory's animal experimentation committee. [Table 20]

[0430] The pharmacokinetic (PK) profiles are shown in Figure 18. Systemic exposure to the conjugated mAb resulted in a PK close to that of the tAb. This indicates that such ADCs were relatively stable in systemic circulation. The terminal phase half-lives of these ADC molecules ranged from 3 to 8 days.

Claims

1. Equation (I): Ab-(L-D) ｐ (I) An antibody-drug conjugate, in which, Ab is an antibody or its antigen-binding fragment that binds to ITGB4. L is the linker, D is the drug part, An antibody-drug conjugate in which p is in the range of 1 to approximately 20.

2. The antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3, and an immunoglobulin light chain variable region (VL) including LCDR1, LCDR2, and LCDR3, wherein the HCDR1-3 and LCDR1-3 belong to the following groups: 1) Sequence numbers 11-16, 2) Sequence numbers 31, 32, 3, and 46-48, respectively 3) Sequence numbers 52-55, 15, and 56, respectively 4) Sequence numbers 61-63 and 66-68 respectively, or sequence numbers 64, 65, 63 and 66-68 respectively, 5) Sequence numbers 1 to 6, 6) Sequence numbers 21 to 26, 7) Sequence numbers 31, 32, 3, and 33-35 respectively, and 8) Sequence IDs 1-3 and 40-42, respectively The antibody-drug conjugate according to claim 1, comprising any of the amino acid sequences described in any of the above.

3. The above VH and VL belong to the following groups: 1) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 17 and 18, respectively. 2) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 36 and 49, respectively. 3) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 57 and 58, respectively. 4) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 69 and 70, respectively. 5) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 7 and 8, respectively. 6) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 27 and 28, respectively. 7) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 36 and 37, respectively. 8) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 7 and 43, respectively. 9) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 73 and 74, respectively. 10) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 75 and 74, respectively. 11) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 76 and 74, respectively. 12) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 77 and 74, respectively. 13) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 78 and 74, respectively. 14) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 79 and 74, respectively. 15) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 80 and 74, respectively. 16) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 81 and 74, respectively. 17) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 82 and 74, respectively. 18) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 83 and 74, respectively. 19) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 84 and 74, respectively. 20) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 85 and 74, respectively. 21) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 86 and 74, respectively. 22) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 73 and 87, respectively. 23) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 75 and 87, respectively. 24) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 76 and 87, respectively. 25) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 77 and 87, respectively. 26) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 78 and 87, respectively. 27) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 79 and 87, respectively. 28) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 80 and 87, respectively. 29) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 81 and 87, respectively. 30) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 82 and 87, respectively. 31) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 83 and 87, respectively. 32) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 84 and 87, respectively. 33) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 85 and 87, respectively, and 34) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 86 and 87, respectively. The antibody-drug conjugate according to claim 2, comprising any of the amino acid sequences described in any of the above.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the antibody is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

6. The antibody is a bispecific antibody comprising a further antigen-binding domain that binds to a second antigen, and preferably the second antigen is CD3, CD138, CD20, CD40, CD19, CD33, CD16, CD38, CD22, CD40, CD8, IL-6, CSPG, RGM A, CTLA-4, BTN02, IGF1, IGF2, Erb2B, IGF-1R, EGFR, CD13, ErbB3, EGFR-2, IGFR, VEGFR-2, Met, VEGFR-A, angiopoietin 2 (Ang-2), IL-12, TWEAK, IL-13, IL-I beta, RGM An antibody-drug conjugate according to claim 5, selected from the group consisting of A, NgR, NogoA, OMGp, PDL-1, PD-1, PD-1, TIM-3, VEGF, Lag-3, OX40, DLL-4, PDGFR, EpCAM, CD16a, PSMA, CEA, TROP-2, HSG, HGF, PDGF, ICOS, CD47, RGM B, Te38, TNFa, Blys, GP130, IL-12, RANK ligand, ITGB6, Nectin 4, and folate receptor.

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein the antibody is an isotype antibody selected from the group consisting of IgG, IgA, IgM, IgE, and IgD, and preferably the antibody is a subtype antibody selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

8. The antigen-binding fragments are Fab, Fab', and F(ab'). ２ An antibody-drug conjugate according to any one of claims 1 to 7, selected from the group consisting of Fv, scFv, VHH, and ds-scFv.

9. The antibody comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), wherein the HC and LC belong to the following groups: 1) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 19 and 20, respectively. 2) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 50 and 51, respectively. 3) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 59 and 60, respectively. 4) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 71 and 72, respectively. 5) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 9 and 10, respectively. 6) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 29 and 30, respectively. 7) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 38 and 39, respectively. 8) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 44 and 45, respectively. 9) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 88 and 89, respectively. 10) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 90 and 89, respectively. 11) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 91 and 89, respectively. 12) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 92 and 89, respectively. 13) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 93 and 89, respectively. 14) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 94 and 89, respectively. 15) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 95 and 89, respectively. 16) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 96 and 89, respectively. 17) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 97 and 89, respectively. 18) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 98 and 89, respectively. 19) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 99 and 89, respectively. 20) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 100 and 89, respectively. 21) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 101 and 89, respectively. 22) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 88 and 102, respectively. 23) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 90 and 102, respectively. 24) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 91 and 102, respectively. 25) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 92 and 102, respectively. 26) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 93 and 102, respectively. 27) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 94 and 102, respectively. 28) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 95 and 102, respectively. 29) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 96 and 102, respectively. 30) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 97 and 102, respectively. 31) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 98 and 102, respectively. 32) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 99 and 102, respectively. 33) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 100 and 102, respectively, and 34) Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 101 and 102, respectively. An antibody-drug conjugate according to any one of claims 1 to 8, comprising the amino acid sequence described in any one of the claims.

10. The antibody-drug conjugate according to any one of claims 1 to 9, wherein the linker includes a cleavage-type linker or a non-cleavage-type linker.

11. The antibody-drug conjugate according to claim 10, wherein the cleavage-type linker includes an acid-unstable linker, a hydrophilic linker, a protease-sensitive linker, a photosensitive linker, a hydrazone linker, a dimethyl linker, or a disulfide-containing linker.

12. The linker is sulfo-SPDB (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate), MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), para-aminobenzyloxycarbonyl (PABC), dimethylethylamine (DMEA), Val-Cit-PABC, MC-Val-Cit-PABC, CL2A, Mal-PEG8-Val-Ala-PABC, MC-VC-PABC-DMEA, GGF An antibody-drug conjugate according to any one of claims 1 to 11, selected from the group consisting of G (glycine-glycine-phenylalanine-glycine), MC-GGFG-aminomethyl, AcBut (4-(4-acetylphenoxy)-butanoic acid), dimethylhydrazide (3-methyl-3-mercaptobutanehydrazide), AcBut-dimethylhydrazide, or SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-carboxylate).

13. The aforementioned -L-D is, 【Chemistry 1】 Selected from the group consisting of, in the formula, 【Chemistry 2】 The antibody-drug conjugate according to any one of claims 1 to 12, wherein it indicates a binding site for the antibody or its antigen-binding fragment.

14. The antibody-drug conjugate according to any one of claims 1 to 13, wherein the drug portion is selected from the group consisting of tubulin inhibitors and microtubule polymerization inhibitors, antibiotics, DNA synthesis inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA polymerase II inhibitors, RNA spliceosome inhibitors, and immunostimulants.

15. The drug portion is a tubulin inhibitor and a microtubule polymerization inhibitor selected from the group consisting of auristatin, mytansin, tubulosin, cryptophycin, and rhizoxin. Alternatively, the drug portion is an antibiotic selected from the group consisting of calicheamicin, doxorubicin, and anthracyclines. Alternatively, the drug portion is a DNA synthesis inhibitor selected from the group consisting of duocalmycin, PBD (benzodiazepine), and IGN (indolinobenzodiazepine). Alternatively, the drug portion is a topoisomerase I inhibitor selected from the group consisting of camptothecin analogs. Alternatively, the drug portion is a topoisomerase II inhibitor selected from the group consisting of anthracycline analogs (e.g., doxorubicin), Alternatively, the drug portion is an RNA polymerase II inhibitor selected from the group consisting of amanitin. Alternatively, the drug portion is an RNA spliceosome inhibitor selected from the group consisting of spliceostatin and tylanstatin. Alternatively, the antibody-drug conjugate according to claim 14, wherein the drug portion is an immunostimulant selected from the group consisting of Toll-like receptor agonists and STING agonists.

16. The drug portion is Table 1 Selected from, in the formula, 【Transformation 3】 The antibody-drug conjugate according to any one of claims 1 to 15, wherein the conjugate indicates a binding site to the linker.

17. The antibody-drug conjugate, 【Chemistry 4】 【change】 An antibody-drug conjugate according to any one of claims 1 to 16, wherein Ab is an antibody or antigen-binding fragment that binds to ITGB4, and p is in the range of 1 to about 20.

18. The antibody-drug conjugate, 【Transformation 5】 The antibody-drug conjugate according to any one of claims 1 to 17, wherein Ab is an antibody comprising an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), and the HC and LC comprise an amino acid sequence described in any of the following groups: SEQ ID NOs: 9 and 10, SEQ ID NOs: 19 and 20, SEQ ID NOs: 38 and 39, SEQ ID NOs: 44 and 45, SEQ ID NOs: 50 and 51, SEQ ID NOs: 71 and 72, SEQ ID NOs: 94 and 102, and SEQ ID NOs: 92 and 102.

19. The antibody-drug conjugate, 【Transformation 6】 In the formula, Ab is an antibody comprising HC and LC, wherein HC and LC comprises an amino acid sequence described in any of the following groups: SEQ ID NOs: 9 and 10, SEQ ID NOs: 19 and 20, SEQ ID NOs: 38 and 39, SEQ ID NOs: 44 and 45, SEQ ID NOs: 50 and 51, SEQ ID NOs: 71 and 72, SEQ ID NOs: 94 and 102, and SEQ ID NOs: 92 and 102. Preferably, the antibody-drug conjugate according to any one of claims 1 to 17, wherein the HC and LC comprise an amino acid sequence described in any of the following groups: SEQ ID NOs: 19 and 20, SEQ ID NOs: 71 and 72, SEQ ID NOs: 94 and 102, and SEQ ID NOs: 92 and 102.

20. The antibody-drug conjugate, 【Transformation 7】 In the formula, Ab is an antibody comprising HC and LC, wherein HC and LC comprises an amino acid sequence described in any of the following groups: SEQ ID NOs: 9 and 10, SEQ ID NOs: 19 and 20, SEQ ID NOs: 38 and 39, SEQ ID NOs: 44 and 45, SEQ ID NOs: 50 and 51, SEQ ID NOs: 71 and 72, SEQ ID NOs: 94 and 102, and SEQ ID NOs: 92 and 102. Preferably, the antibody-drug conjugate according to any one of claims 1 to 17, wherein the HC and LC comprise the amino acid sequences described in SEQ ID NOs: 19 and 20 or SEQ ID NOs: 94 and 102.

21. The antibody-drug conjugate according to any one of claims 1 to 20, wherein p is an integer selected from 2 to 10, preferably from 4 to 8, and more preferably p is 4 or 8.

22. A composition comprising an antibody-drug conjugate according to any one of claims 1 to 21 and, optionally, a pharmaceutically acceptable carrier or excipient.

23. A method for preventing and / or treating a disease in a subject requiring such treatment, comprising administering to the subject an antibody-drug conjugate according to any one of claims 1 to 21 or a composition according to claim 22.

24. The method according to claim 23, wherein the disease is cancer.

25. The method according to claim 24, wherein the cancer is ITGB4 positive, and preferably the cancer is selected from the group consisting of head and neck cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, stomach cancer, pancreatic cancer, ovarian cancer, prostate cancer, breast cancer, leukemia, myeloma, squamous cell carcinoma, melanoma, brain cancer, cervical cancer, liver cancer, bladder cancer, breast cancer, kidney cancer, testicular cancer, and thyroid cancer.

26. The method according to any one of claims 23 to 25, further comprising administering a second therapeutic substance to the subject, preferably the second therapeutic substance being selected from antibodies, chemotherapeutic substances, and small molecule drugs.

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