Pro-antibodies that reduce off-target toxicity

Abstract

To provide pro-antibodies that reduce normal tissue targeting and that enhance tumor targeting.SOLUTION: The present invention includes proteins, nucleic acids and methods of making and using an activatable antibody (aAb) comprising, in order, the following structure: a first light chain comprising a first variable light region, a cleavable linker, and a first heavy chain comprising a first variable heavy region, wherein the cleavable linker prevents or reduces the first light chain and the first heavy chain from forming a first antigen binding site against a first antigen, and wherein cleavage of the cleavable linker releases the first heavy chain to allow formation of the first antigen binding site to bind a first antigen.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention generally relates to the field of pro - antibodies that reduce the targeting of normal tissues and enhance the targeting of tumors.

[0002] Description of Research Funded by the Federal Government Not applicable.

[0003] Reference to Sequence Listing This application includes a separately filed sequence listing in accordance with the requirements of 37 CFR 1.821 - 1.825.

Background Art

[0004] Without limiting the scope of the present invention, its background art is described in relation to pro - bodies or activatable antibodies.

[0005] One such pro - body is taught in U.S. Patent No. 10,059,762 to Stagliano, et al., entitled "Anti - EGFR activatable antibodies". The inventors of this patent teach a modified antibody or antibody fragment containing an antibody or antibody fragment modified by a masking moiety (MM) that is further coupled to a cleavable moiety (CM), resulting in an activatable antibody (AA). In the AA, the CM has the ability to be cleaved, reduced, photolyzed, or otherwise modified. For example, in the presence of an agent having the ability to cleave, reduce, or photolyze the CM, when the CM is cleaved, reduced, or photolyzed, after the MM is removed, the AA can assume an activatable conformation such that the AB becomes more accessible to the target. However, with respect to the binding to the target binding moiety (TBM), between the MM and the target Competition is a major limitation of this technology. Furthermore, cleavage of MM causes significant off-target effects.

[0006] Another such probody is U.S. Patent Application No. 10, titled "Anti-ITGA3 antibody, activatable anti-ITGA3 antibody, and methods of use thereof" by Sagert, et al. This is taught in Patent Nos. 233 and 244. The invention generally relates to antibodies that bind to ITGa3, activatable antibodies that specifically bind to ITGa3, and methods for producing and using these anti-ITGa3 antibodies and anti-ITGa3 activatable antibodies in various therapeutic, diagnostic, and prophylactic applications.

[0007] Another such probody is taught in U.S. Patent No. 8,541,203, filed by Daugherty, et al., entitled “Activatable binding polypeptides and methods of identification and use thereof.” The inventors of this patent teach an activatable binding polypeptide (ABP) comprising a target binding moiety (TBM), a masking moiety (MM), and a cleavable moiety (CM). The masking portion covers the homologous binding site of the TBM, and when the CM is cleaved, the TBM is exposed. Certain activatable antibody compositions include a TBM containing an antigen-binding domain (ABD), MM, and CM. The ABP includes an "activatable" conformation such that at least one of the TBMs is less accessible to the target when the CM is not cleaved than when it is cleaved in the presence of a cleavage agent capable of cleaving the CM. The application is for a library of ABP candidates. The instructions will provide guidance on screening methods for identifying such ABPs and methods of use. The instructions will describe ABPs specific to VEGF, CTLA4, or VCAM, having a first TBM that binds to VEGF and a second TBM that binds to FGF, as well as their compositions and methods of use. However, a major limitation of this technology is the competition between the MM and the target regarding binding to the TBM. Furthermore, cleavage of the MM causes significant off-target effects.

[0008] Another such probody is taught in U.S. Patent Publication No. 20190359714, filed by Tipton, et al., “Activatable Anti-CTLA-4 Antibodies and Uses Thereof”. The applicants of this patent teach an activatable anti-human CTLA4 antibody comprising a heavy chain containing a VH domain and a light chain containing a masking moiety (MM), a cleavable moiety (CM), and a VL domain. When the cleavable moiety is proteolytically cleaved by a tumor-specific protease, thereby removing the masking moiety, such an activatable anti-human CTLA4 antibody has CTLA4 binding activity in the tumor microenvironment but exhibits significantly reduced binding to CTLA4 outside the tumor.

[0009] Another such probody is Wang's application "Methods and Reagents to Treat Tumor and This is taught in U.S. Patent Publication No. 20180271997, titled "Cancer." The applicant of this patent teaches a reagent for treating tumors and cancer, and a method of using the reagent to treat tumors and cancer with a pro-antibody, which is an antibody that can be activated within the tumor. Another type of reagent is a conjugate of sialidase with an affinity ligand that can bind to the surface of immune cells, or a conjugate of sialidase with an affinity ligand that can bind to another antibody, thus providing a sialidase-based cancer immunotherapy.

[0010] Three approaches to activatable antibodies have been used to date. First, probodies contain masking peptides linked via a proteolytic linker to block antigen-antibody interactions. The development of each probodyceps requires a screening process using phage display for the masking peptides. After cleavage of the linker, the masking peptides are expected to detach and release the antigen-binding site of the antibody. Problems associated with this approach include: (1) the heterogeneity of the masking peptides themselves can trigger an immune response; (2) failure to release the masking peptides reduces effectiveness; (3) some antigen-antibody interactions are too strong to be shielded by short-chain (e.g., 10 amino acids) peptides; and (4) the peptides may degrade before reaching the tumor, potentially exposing normal tissue to the toxicity of the antibody.

[0011] The second approach suggests that dual variable domain immunoglobulin (DVD-Ig) reduces the toxicity of anti-CTLA4 antibodies. Yes. VL and VH of anti-tumor targeting antigen (TTA) antibodies are The anti-CTLA4 antibody is linked via a proteolytic linker to cover the CTLA4 binding site. Unfortunately, this approach requires pairing of tumor-associated antigens (TAAs) with CTLA4, necessitating the addition of a precise set of VL and VH molecules, thus resulting in a larger molecule. Furthermore, precise VL and VH molecules can always interact with off-target antigens and potentially trigger an immune response.

[0012] The third approach physically blocks the binding of the anti-CD3 variable light (VL) and variable heavy (VH) chains by linking "false VL" and "false VH" pairs to active and functional VL and VH. When false VL interacts with VH, and when false VH interacts with VL, the antibody is inactive. Zelinkers are used to link VL, VH, pseudoVL, and pseudoVH together to create very large molecules. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] U.S. Patent No. 10,059,762 [Patent Document 2] U.S. Patent No. 10,233,244 [Patent Document 3] U.S. Patent No. 8,541,203 [Patent Document 4] U.S. Patent Publication No. 20190359714 [Patent Document 5] U.S. Patent Publication No. 20180271997 [Overview of the Initiative] [Problems that the invention aims to solve]

[0014] Novel fusion proteins are needed to overcome the problems of prior art by avoiding targeting normal tissue and enhancing the activity of tumor target sites. [Means for solving the problem]

[0015] In one embodiment, the present invention comprises an activatable antibody (aAb) having the following structure: a first light chain including a first variable light chain region, a cleavable linker, and a first heavy chain including a first variable heavy chain region, wherein the cleavable linker prevents or reduces the formation of a first antigen-binding site for a first antigen by the first light chain and the first heavy chain, and when the cleavable linker is cleaved, the first heavy chain is released, enabling the formation of the first antigen-binding site that binds to the first antigen. In one embodiment, aAb comprises a second antibody-binding site that binds to a second antigen, formed by a second variable light chain and a second steady light chain connected to the first heavy chain, and may further include a flexible non-cleavable linker between the second variable light chain and the second steady light chain. In another embodiment, aAb further includes at least one of the first steady heavy chain region, the first steady heavy chain region, or both. In another embodiment, the first light chain region, the first heavy chain region, or both, further comprises an Fc region, a wild-type Fc region, a mutated Fc region, a monomeric wild-type Fc region, a monomeric mutant Fc region, a dimeric wild-type Fc region, a dimeric mutant Fc region, a second variable heavy chain region and a second Fc region, or a second variable heavy chain region and a second Fc region and an uncleavable flexible linker and a second variable light chain region and a second heavy chain variable region, or a second Fc region and an uncleavable flexible linker and a cytokine. In another embodiment, the first and second antigens are the same antigen; the first and second antigens are different; or the first and second antigens are the same antigen, but the first antigen-binding site and the second antigen-binding site bind to different epitopes of the same antigen; at least one of these. In another embodiment, the first antigen-binding site or the second antigen-binding site binds to a tumor target.In another aspect, the first antigen is ICAM1; VCAM1; EpCAM; the extracellular domain B of fibronectin; melanoma-associated chondroitin sulfate proteoglycan (MCSP); melanoma-associated proteoglycan (MAPG); high molecular weight melanoma associated antigen (HMV-MAA); prostate specific membrane antigen (PSMA); epidermal growth factor receptor (EGFR); hepatocyte growth factor receptor (HGFR); fibroblast activation protein (FAP); carcinoembryonic antigen (CEA). Cell-adhesion molecule (CAM); B-cell maturation target (BCMA); placental growth factor (PLGF); folate receptor, insulin-like growth factor receptor ( ILGFR); CD133; CD40; CD3 7; CD33; CD30; CD28; CD24; CD23; CD22; CD21; CD2 0; CD19; CD13; CD10; HER3; HER2; nonmuscle myosin heavy chain type A (nmMHCA); transferrin; epithelial cell adhesion molecule (EpCAM); annexin A1; nuc The first antigen is a tissue-specific surface antigen selected from nucleotin, tenascin, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR-2), aminopeptidase N, tie-1, tie-2, or c-Met. In another embodiment, the first antigen is ABCF1;ACVR1;ACVR1B;ACVR2;ACVR2B;ACVRL1;ADORA2A;agrecan;AGR2;AICDA;AIF1;AIG1;AKAP1;AKAP2;AMH;AMHR2;ANGPT1;ANGPT2;ANGPTL3;ANGPTL4;ANPEP;APC;APOC1;AR;AZGP1;B7.1;B7.2;BAD;BAFF;BAG1;BAI1;BCL2;BCL6;B DNF;BLNK;BLR1(MDR15);BlyS;BMP1;BMP2;BMP3B(GDF10);BMP4;BMP6;BMP8;BMPR1A;BMPR1B;BMPR2;BPAG1(plectin);BRCA1; C19orf10(IL27w);C3;C4A;C5;C5R1;CANT1;CASP1;CASP4;CAV1;CCBP2(D6 / JAB61);CCL1(1-309);CCL11(eotaxin);CCL13(M CP-4);CCL15(MIP-1d);CCL16(HCC-4);CCL17(TARC);CCL18(PARC);CCL19(MIP-3b);CCL2(MCP-1);MCAF;CCL20(MIP-3a); CCL21(MIP-2);SLC;exodus-2;CCL22(MDC / STC-1);CCL23(MPIF-1);CCL24(MPIF-2 / Eotaxin-2);CCL25(TECK);CCL2 6(eotaxin-3);CCL27(CTACK / ILC);CCL28;CCL3(MIP-1a);CCL4(MIP-1b);CCL5(RANTES);CCL7(MCP-3);CCL8(mcp-2);CCNA1 ;CCNA2;CCND1;CCNE1;CCNE2;CCR1(CKR1 / HM145);CCR2(mcp-1RB / RA);CCR3(CKR3 / CMKBR3);CCR4;CCR5(CMKBR5 / ChemR13);CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6);CCR7(CKR7 / EBI1);CCR8(CMKBR8 / TER1 / CKR-L1);CCR9(GPR-9-6);CCRL1(VSHK1);CCRL2(L-CCR);CD164;CD19;CD1C;CD20;CD200;CD-22;CD24;CD28;CD3;CD37;CD38;CD3E;CD3G;CD3Z;CD4;CD40;CD40L;CD44;CD45RB;CD52;CD69;CD72;CD74;CD79A;CD79B;CD8;CD80;CD81;CD83;CD86;CDH1(E-カドヘリン);CDH10;CDH12;CDH13;CDH18;CDH19;CDH20;CDH5;CDH7;CDH8;CDH9;CDK2;CDK3;CDK4;CDK5;CDK6;CDK7;CDK9;CDKN1A(p21Wap1 / Cip1);CDKNIB(p27Kip1);CDKNIC;CDKN2A(pl6INK4a);CDKN2B;CDKN2C;CDKN3;CEBPB;CER1;CHGA;CHGB;キチナーゼ;CHST10;CKLFSF2;CKLFSF3;CKLFSF4;CKLFSF5;CKLFSF6;CKLFSF7;CKLFSF8;CLDN3;CLDN7(クローディン-7);CLN3;CXCL10(IP-10);CXCL11(I-TAC / IP-9);CXCL12(SDF1);CXCL13;CXCL14;CXCL16;CXCL2(GR02);CXCL3(GR03);CXCL5(ENA-78 / LIX);CXCL6(GCP-2);CXCL9(MIG);CXCR3(GPR9 / CKR-L2);CXCR4;CXCR6(TYMSTR / STRL33 / Bonzo);CYB5;CYC1;CYSLTR1;CGRP;Clq;Clr;CI;C4a;C4b;C2a;C2b;C3a;C3b;DAB2IP;DES;DKFZp451J0118;DNCL1;DPP4;E-セレクチン;E2F1;ECGF1;EDG1;EFNA1;EFNA3;EFNB2;EGF;EGFR;ELAC2;ENG;EN01;EN02;EN03; EPHB4;EPO;ERBB2(Her-2);EREG;ERK8;ESR1;ESR2;F3(TF);Factor VII;Factor IX;Factor V;Factor VIIa;Factor X;Factor XII Factor; Factor XIII; FADD; FasL; FASN; FCER1A; FCER2; Fc gamma receptor; FCGR3A; FGF; FGF1(aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2(bFGF); FGF20; FGF21; FGF22; FGF23; FGF3(int-2); FGF4(HST); FGF5; FGF6(HST-2); FGF7(KGF); FGF8; FGF9; FGFR3; FIGF(VEGFD); FIL1(epsilon); FIL1(zeta); FLJ12584;FLJ25530;FLRT1 (fibronectin);FLT1;FOS;FOSL1 (FRA-1);FY (DARC);GABRP (GABAa);GAGEB1;GAGEC1;GALNAC4S-6ST;GATA3;GDF5;GFI1;GGT1;GMCSF;GNAS1;GNRH1;GPR2 (CCR10);GPR31;GPR44;GPR81 (FKSG80);GRCC10 (CIO);GRP;GSN (gelsolin);GSTP1;glycoprotein (GP) IIb / IIIa;HAVCR2;HDAC4;HDAC5 ;HDAC7A;HDAC9;Her2;HGF;ITGB4 (b4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); L-selectin; LAMA⑤; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III) (metallothionectin-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2;Neurocan;NKG2D;NFKB1;NFKB2;NGF;NGFB(NGF);NGFR;NgR-Lingo;NgR-Nogo66(Nogo);NgR-p75;NgR-Troy;NM E1(NM23A);NOX5;NPPB;NR0B1;NR0B2;NR1D1;NR1D2;NR1H2;NR1H3;NR1H4;NRII2;NRII3;NR2C1;NR2C2;NR2E1;NR2E 3;NR2F1;NR2F2;NR2F6;NR3C1;NR3C2;NR4A1;NR4A2;NR4A3;NR5A1;NR5A2;NR6A1;NRP1;NRP2;NT5E;NTN4;ODZ1;OP RD1;P2RX7;PAP;PARTI;PATE;PAWR;PCA3;PCNA;PDGFA;PDGFB;PECAM1;PF4(CXCL4);PGE2;PGF;PGR;phosphacan;PIAS2; PIK3CG; Plasminogen Activator; PLAU(uPA); PLG; PLXDC1; PPBP(CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; Protein C; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2(COX-2); PTN; RAC2(p21Rac2); RAGE; RARB; RGS1; RGS13; RGS3; RNF110(ZNF144) ;ROB02;SI00A2;SCGB1D2(Lipophyllin B);SCGB2A1(Mammaglobin 2);SCGB2A2(Mammaglobin 1);SCYE1(Endothelial Monocyte Activating Cytokine);SDF2;SERPINA1;SERPINA3;SERPINB5(Maspin);SERPINE1(PAI-1);SERPINF1;SHBG;SLA2;SLC2A2;SLC33A1;SLC43A1;SLIT2;S PP1;SPRR1B(Spr1);ST6GAL1;STAB1;STAT6;STEAP;STEAP2;Substance P;TB4R2;TBX21;TCP10;TDGF1;TEK;TGFA;TGFB1;TGFB111;TGFB2;TGFB3;TGFBI;TGFBR1;TGFBR2;TGFBR3;TH1L;THBS1(Thrombospongin-1);THBS2;THBS4;THP O;TIE(Tie-1);TIMP3;Tissue factor;TLR10;TLR2;TLR3;TLR4;TLR5;TLR6;TLR7;TLR8;TLR9;TNF;TNF-a;TNFAIP2(B94) ;TNFAIP3;TNFRSF11A;TNFRSF1A;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF6(Fas);TNFRSF7;TNFRSF8;TNFRSF9;T NFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEML); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptor; TOP2A (topoisomerase IIa); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; thrombomodulin; thrombin; VEGF; VEGFB; VEGFC; versican; VHL The protein, protein segment, or peptide is selected from at least one gene selected from C5;VLA-4;XCL1 (lymphotactin);XCL2 (SCM-1b);XCR1 (GPR5 / CCXCR1);YY1; and ZFPM2.In another embodiment, tumor targets include tumor target antigens such as HER1, HER2, HER3, GD2, carcinoembryonic antigen (CEA), epidermal growth factor receptor active mutant (EGFRVIII), CD133, and fibroblast activation protein (FAP). a, epithelial cell adhesion molecule (Epcam), glypican 3 (GPC3), EPH receptor A4 (EphA), tyrosine protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor-1 (WT-1), or The linker is selected from the claudin family of proteins. In another embodiment, the first or second antigen-binding site binds to a T cell marker. In another embodiment, the T cell marker is selected from CTLA4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, or TIM-3. In another embodiment, the first or second antigen-binding site binds to a T cell activator. In another embodiment, the T cell activator is selected from CD3, 41BB, or OX40. In another embodiment, the cleavable linker is a protease-cleavable linker. In another embodiment, the cleavable linker is cleaved by tumor-related proteases: MMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, uPA, FAPa, or cathepsin B. In another embodiment, the cleavable linker is cleaved by proteases that are upregulated during apoptosis or inflammation-related response periods. In another embodiment, the cleavable linker is cleaved by caspases. In another embodiment, the caspases are caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, and caspase The value is 12. In another embodiment, the cleavable linker does not shield the antigen-binding site. In another embodiment, aAb further comprises a drug conjugated to aAb. In another embodiment, aAb further comprises a cytokine that is bound to aAb or an Fc region, or that is a fusion protein containing aAb or an Fc region. In another aspect, cytokines include growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormone; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; TNF-α; Müllerian inhibitory factor; gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor; platelet growth factor; placental growth factor, transforming growth factor (TGF); insulin-like growth factor-1 and -11; erythropoietin (EPO); bone induction factor; interferon; colony-stimulating factor (CSF); lymphotoxin-alpha; lymphotoxin -beta;CD27L;CD30L;FASL;4-1BBL;OX40L;TRAIL;IL-1;IL-2;IL-3;IL-4;IL-5;IL-6;IL-7;IL-8;IL-9;IL-10; IL-11;IL-12;IL-13;IL-15;IL-18;IL-21;IL-22;IL-23;IL-33;IFN-a;IFN-b;IFN-g bone morphogenetic protein The agent is selected from at least one of the following: (BMP, bone morphogenetic protein); leukemia inhibitory factor (LIF); or kit ligand (KL, kitligand). In another embodiment, aAb is selected from SEQ ID NOs: 1, 2, or 3. In another embodiment, the agent is at least one of the following: toxin or toxic fragment thereof; microtubule inhibitor; nucleic acid damaging agent; detectable portion; or diagnostic agent.

[0016] In another embodiment, the present invention includes a pharmaceutical composition comprising an activatable Ab. In another embodiment, the present invention includes a method for reducing the binding activity of an activatable Ab to normal tissue and targeting cancer cells, comprising the step of administering an effective amount of the activatable Ab to a subject in need. In another embodiment, the present invention includes a method for treating, alleviating, or slowing the progression of cancer, comprising the step of administering an effective amount of the activatable Ab to a subject in need. In one embodiment, cancer is a cancer that expresses an enzyme that cleaves a cleavable linker. In another embodiment, cancer is selected from bladder cancer, bone cancer, breast cancer, oncogenic body, cervical cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, genitourinary cancer, or urothelial cancer. In another aspect, cancer includes acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, urothelial carcinoma of the bladder, ductal carcinoma of the mammary gland, lobular carcinoma of the mammary gland, esophageal cancer, castration-resistant prostate cancer (CRPC), cervical cancer, bile duct cancer, chronic myeloid leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal cancer, gastric adenocarcinoma, and pleomorphic cancer. Glioblastoma, head and neck squamous cell carcinoma, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), renal pigmentophobic carcinoma, clear cell carcinoma, papillary cell carcinoma, low-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and chrome affinity cell tumor, prostate adenocarcinoma, renal cell carcinoma (RCC), sarcoma, skincutaneous melanoma, head and neck squamous cell carcinoma, T-cell lymphoma, thymoma, papillary thyroid carcinoma, The selection is made from the group consisting of uterine carcinosarcoma, endometrioid carcinoma, and uveal melanoma.

[0017] In another embodiment, the present invention comprises, in order: a first light chain including a first variable light chain region; a cleavable linker; and a first heavy chain including a first variable heavy chain region, wherein the cleavable linker prevents or reduces the formation of an antigen-binding site for a first antigen by the first light chain and the first heavy chain; and when the cleavable linker is cleaved, the first heavy chain is released and, together with the first light chain, forms an antibody-binding site that binds to the first antigen. The present invention comprises an activatable antibody (aAb). In another embodiment, aAb further comprises at least one of a first constant light chain region or a first constant heavy chain region. In another embodiment, aAb further comprises an Fc region (which is a wild-type domain or a mutant domain that modifies Fc receptor binding) bound to the first constant heavy chain region, a second variable heavy chain region and a second Fc region, or a second variable heavy chain region and a second Fc region and an uncleavable flexible linker and a second variable light chain region and a second heavy chain variable region, or a second Fc region and an uncleavable flexible linker and a cytokine. In another embodiment, aAb further comprises a second antibody-binding site for binding to a second antigen, formed by a second variable light chain and a second constant light chain attached to the first heavy chain, and may include a flexible, uncleavable linker between the second variable light chain and the second constant light chain. In another embodiment, aAb further comprises a first constant heavy chain region, at least one of the first constant heavy chain regions, or both. In another embodiment, the Fc region is a wild-type Fc region, a mutated Fc region, a monomeric wild-type Fc region, a monomeric mutant Fc region, a dimeric wild-type Fc region, or a dimeric mutant Fc region. In another embodiment, aAb further comprises a cytokine that is bound to aAb or an Fc region, or that is a fusion protein containing aAb or an Fc region.In another embodiment, cytokines include growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormone; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; TNF-alpha; Müllerian inhibitor; gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor; platelet growth factor; placental growth factor, transforming growth factor (TGF); insulin-like growth factor-1 and -11; erythropoietin (EPO); bone induction factor; interferon; colony stimulation At least one of the following is selected: factor (CSF); lymphotoxin-alpha; lymphotoxin-beta; CD27L; CD30L; FASL; 4-1BBL; OX40L; TRAIL; IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-15; IL-18; IL-21; IL-22; IL-23; IL-33; IFN-a; IFN-beta; IFN-gamma; IFN-gamma inducer (IGIF); bone morphogenetic protein (BMP); leukemia suppressor (LIF); or kit ligand (KL).In another embodiment, the first antigen is ICAM1; VCAM1; EpCAM; fibronectin extradomain B; melanoma-associated chondroitin sulfate proteoglycan (MCSP); melanoma-associated proteoglycan (MAPG); high molecular weight melanoma-associated antigen (HMV-MAA); prostate-specific membrane antigen (PSMA); epidermal growth factor receptor (EGFR); hepatocyte growth factor receptor (HGFR); fibroblast-activating protein (FAP); carcinoembryonic antigen (CEA); cell adhesion molecule (CAM); human B cell maturation target (BCMA); placental growth factor (PLGF); folate receptor, insulin-like growth factor Growth factor receptor (ILGFR); CD133; CD40; CD37; CD33; CD30; CD28; CD24; CD23; CD22; CD21; CD20; CD19; CD13; CD10; HER3; HER2; Non-muscle myosin heavy chain type A (nmMHCA); Transferrin; Epithelial cell adhesion molecule (EpCAM); Annexin A1; Nucleotin, Tenascin, Vascular endothelial growth factor receptor 1 (VEGFR1), Vascular endothelial growth factor receptor 2 (VEGFR-2); Aminopeptidase N, tie-1, tie-2, or c-Met are selected tissue-specific surface antigens. In another embodiment, the first antigen is ABCF1;ACVR1;ACVR1B;ACVR2;ACVR2B;ACVRL1;ADORA2A;Aggrecan;AGR2;AICDA;AIF1;AIG1;AKAP1;AKAP2;AMH;AMHR2;ANGPT1;ANGPT2;ANGPTL3;ANGPTL4;ANPEP;APC;APOC1;AR;AZGP1 (zinc-α-glycoprotein);B7.1 ;B7.2;BAD;BAFF;BAG1;BAI1;BCL2;BCL6;BDNF;BLNK;BLR1(MDR15);BlyS;BMP1;BMP2;BMP3B(GDF10);BMP4;B MP6;BMP8;BMPR1A;BMPR1B;BMPR2;BPAG1 (plectin);BRCA1;C19orf10(IL27w);C3;C4A;C5;C5R1;CANT1;CASP1; CASP4;CAV1;CCBP2(D6 / JAB61);CCL1(1-309);CCL11(エオタキシン);CCL13(MCP-4);CCL15(MIP-1d);CCL16(HCC-4);CCL17(TARC);CCL18(PARC);CCL19(MIP-3b);CCL2(MCP-1);MCAF;CCL20(MIP-3a);CCL21(MIP-2);SLC;エクソダス-2;CCL22(MDC / STC-1);CCL23(MPIF-1);CCL24(MPIF-2 / エオタキシン-2);CCL25(TECK);CCL26(エオタキシン-3);CCL27(CTACK / ILC);CCL28;CCL3(MIP-1a);CCL4(MIP-1b);CCL5(RANTES);CCL7(MCP-3);CCL8(mcp-2);CCNA1;CCNA2;CCND1;CCNE1;CCNE2;CCR1(CKR1 / HM145);CCR2(mcp-1RB / RA);CCR3(CKR3 / CMKBR3);CCR4;CCR5(CMKBR5 / ChemR13);CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6);CCR7(CKR7 / EBI1);CCR8(CMKBR8 / TER1 / CKR-L1);CCR9(GPR-9-6);CCRL1(VSHK1);CCRL2(L-CCR);CD164;CD19;CD1C;CD20;CD200;CD-22;CD24;CD28;CD3;CD37;CD38;CD3E;CD3G;CD3Z;CD4;CD40;CD40L;CD44;CD45RB;CD52;CD69;CD72;CD74;CD79A;CD79B;CD8;CD80;CD81;CD83;CD86;CDH1(E-カドヘリン);CDH10;CDH12;CDH13;CDH18;CDH19;CDH20;CDH5;CDH7;CDH8;CDH9;CDK2;CDK3;CDK4;CDK5;CDK6;CDK7;CDK9;CDKN1A(p21Wap1 / Cip1);CDKNIB(p27Kip1);CDKNIC;CDKN2A(pl6INK4a);CDKN2B;CDKN2C;CDKN3;CEBPB;CER1;CHGA;CHGB;キチナーゼ;CHST10;CKLFSF2;CKLFSF3;CKLFSF4;CKLFSF5;CKLFSF6;CKLFSF7;CKLFSF8; CLDN3; CLDN7 (Claudin-7); CLN3; CXCL10 (IP-10); CXCL11 (I-TAC / IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03); CXCL5 (ENA-78 / LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9 / CKR-L2); CXCR4; CXCR6 (TYMSTR / STRL33 / Bonzo); CYB5; CYC1; CYSLTR1; CGRP; Clq; CIR protein; CI; C4a; C4b; C2a; C2b; C3a; C3b; DAB2IP; DES; DKFZp451J0118; DNCL1; DPP4; E-selectin; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; EN01; EN02; EN03; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); Factor VII; Factor IX; Factor V; Factor VIIa; Factor X; Factor XII; Factor XIII Factor; FADD; FasL; FASN; FCER1A; FCER2; Fc gamma receptor; FCGR3A; FGF; FGF1(aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; Fgf17; Fgf18; FGF19; FGF2(bFGF); FGF20; FGF21; FGF22; FGF23; FGF3(int-2); FGF4(HST); FGF5; FGF6(HST-2); FGF7(KGF); FGF8; FGF9; FGFR3; FIGF(VEGFD); FIL1(epsilon); FIL1(zeta); FLJ1 2584;FLJ25530;FLRT1(fibronectin);FLT1;FOS;FOSL1(FRA-1);FY(DARC);GABRP(GABAa);GAGEB1;GAGEC1;GALNAC4S-6ST;GATA3;GDF5;GFI1;GGT1;GMCSF;GNAS1;GNRH1;GPR2(CCR10);GPR31;GPR44;GPR81(FKSG80);GRCC10(CIO);GRP;GSN(gelsolin);GSTP1;glycoprotein IIb;glycoprotein IIIa;HAVCR2;HDAC4;HDAC5;HDAC7A;HDA C9;Her2;HGF;HIF1A;HIP1;Histamine and histamine receptors;HLA-A;HLA-DRA;HM74;HMGB1;HMOX1;HUMCYT2A;ICEBERG;ICOSL;ID2;IFN-alpha;IFNA1;IFNA2;IFNA4;IFNA5;IFNA6;IFNA7;IFNB1;IFN-gamma;IFNW1;IGBP1;IGF1;IGF1R;IGF2;IGFBP2;IGFBP3;IGFBP6;IL-1; IL1A;IL1B;IL10;IL10RA;IL10RB;IL11;IL11RA;IL-12;IL12A;IL12B;IL12RB1;IL12RB2;IL13;IL13RA1;IL13RA2;IL14;IL15 ;IL15RA;IL16;IL17;IL17B;IL17C;IL17R;IL18;IL18BP;IL18R1;IL18RAP;IL19;IL1A;IL1B;IL1F10;IL1F5;IL1F6;IL1F7;IL1 F8;IL1F9;IL1HY1;IL1R1;IL1R2;IL1RAP;IL1RAPL1;IL1RAPL2;IL1RL1;IL1RL2;IL1RN;IL2;IL20;IL20RA;IL21R;IL22;IL22R ;IL22RA2;IL23;IL24;IL25;IL26;IL27;IL28A;IL28B;IL29;IL2RA;IL2RB;IL2RG;IL3;IL30;IL3RA;IL4;IL4R;IL5;IL5RA;IL 6;IL6R;IL6ST (glycoprotein 130);IL7;IL7R;IL8;IL8RA;IL8RB;IL8RB;IL9;IL9R;ILK;INHA;INHBA;INSL3;INSL4;IRAK1;IRAK2;IT GA1;ITGA2;ITGA3;ITGA6(a6 integrin);ITGAV;ITGB3;ITGB4(b4 integrin);JAG1;JAK1;JAK3;JUN;K6HF;KAI1;KDR;KITLG;KLF5(GC Box BP);KLF6;KLK10;KLK12;KLK13;KLK14;KLK15;KLK3;KLK4;KLK5;KLK6;KLK9;KRT1;KRT19( Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); L-selectin; LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; Midokaine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS 1;MUC1(mucin);MYC;MYD88;NCK2;neurocan;NKG2D;NFKB1;NFKB2;NGF;NGFB(NGF);NGFR;NgR-Lingo;NgR-Nogo66(Nogo);NgR-p75;NgR- Troy;NME1(NM23A);NOX5;NPPB;NR0B1;NR0B2;NR1D1;NR1D2;NR1H2;NR1H3;NR1H4;NRII2;NRII3;NR2C1;NR2C2;NR2E1;NR2E3;NR2F1 ;NR2F2;NR2F6;NR3C1;NR3C2;NR4A1;NR4A2;NR4A3;NR5A1;NR5A2;NR6A1;NRP1;NRP2;NT5E;NTN4;ODZ1;OPRD1;P2RX7;PAP;PARTI;PATE;PAWR;PCA3;PCNA;PDGFA;PDGFB;PECAM1;PF4(CXCL4);PGE2;PGF;PGR;Phosphacan;PIAS2;PIK3CG;Plasminogen Activator;PLAU(uPA);PLG ;PLXDC1;PPBP(CXCL7);PPID;PR1;PRKCQ;PRKD1;PRL;PROC;Protein C;PROK2;PSAP;PSCA;PTAFR;PTEN;PTGS2(COX-2);PTN;RAC2(p21Rac2);RAGE;RARB;RGS1;RGS13;RGS3;RNF110(ZNF144);ROB02;SI00A2;SCGB1D2(Lipophyllin B);SCGB2A1(Mammaglobin 2);SCGB2A2(Mammaglobin 1);SCYE1(Endothelial monocyte-activating cytokine);SDF2; SERPINA1;SERPINA3;SERPINB5(maspin);SERPINE1(PAI-1);SERPINF1;SHBG;SLA2;SLC2A2;SLC33A1;SLC43A1;SLIT2;SPP1;SPRR1B(Spr1);ST6GAL1;ST AB1;STAT6;STEAP;STEAP2;Substance P;TB4R2;TBX21;TCP10;TDGF1;TEK;TGFA;TGFB1;TGFB111;TGFB2;TGFB3;TGFBI;TGFBR1;TGFBR2;TGFBR3;TH1L;THB S1(Thrombospongin-1);THBS2;THBS4;THPO;TIE(Tie-1);TIMP3;Tissue Factor;TLR10;TLR2;TLR3;TLR4;TLR5;TLR6;TLR7;TLR8;TLR9;TNF-Alpha;TNFAIP2(B94);TNFAIP3;TNFRSF11A;TNFRSF1A;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF6(Fas);TNFRSF7;TNFRSF8;TNFRSF9;TNFSF10(TRAIL);TNFSF11(TRANCE);TNFSF 12(AP03L);TNFSF13(April);TNFSF13B;TNFSF14(HVEML);TNFSF15(VEGI);TNFSF18;TNFSF4(OX40 ligand);TNFSF5(CD40 ligand);TNFSF6(FasL);TNFSF7(CD27 ligand);TNFSF8(CD30 ligand);TNFSF9(4-1BB ligand);TOLLIP;Toll-like receptor;TOP2A(topoisomerase IIa);TP53;TPM1;TPM2;TRADD;TRAF1;TRAF2;TRAF3;TRAF4;TRAF5;TRAF6;TREM1;TREM2;TRPC6;TSLP;TWEAK;Trombomodulin;Thrombin;VEGF;VEGFB;VEGFC;Versican;VHL; The first antigen is selected from a protein, protein segment, or peptide encoded by at least one gene selected from C5;VLA-4;XCL1 (lymphotactin);XCL2 (SCM-1b);XCR1 (GPR5 / CCXCR1);YY1; and ZFPM2. In another embodiment, the first and second antigens are the same antigen; the first and second antigens are different; or the first and second antigens are the same antigen, but the first antigen-binding site and the second antigen-binding site bind to different epitopes of the same antigen; in another embodiment, the first antigen-binding site or the second antigen-binding site binds to a tumor target. In another embodiment, the tumor target is selected from tumor target antigens, HER1, HER2, HER3, GD2, carcinoembryonic antigen (CEA), epidermal growth factor receptor activating mutant (EGFRVIII), CD133, fibroblast-activating protein alpha (FAP), epithelial cell adhesion molecule (Epcam), glypican 3 (GPC3), EPH receptor A4 (EphA), tyrosine protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor-1 (WT-1), or a member of the Claudin family. In another embodiment, the first or second antigen-binding site binds to a T cell marker. In another embodiment, the T cell marker is selected from CTLA4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, and TIM-3. In another embodiment, the first or second antigen-binding site binds to a T cell activator. In another embodiment, the T cell activator is selected from CD3, 41BB, or OX40. In another embodiment, the cleavable linker is a protease-cleavable linker. In another embodiment, the cleavable linker is a tumor-associated protease: MMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MM In another embodiment, the cleavable linker is cleaved by P20, MMP21, uPA, FAPa, or cathepsin B. In yet another embodiment, the cleavable linker is cleaved by a protease that is upregulated during an apoptosis or inflammation-related response. In yet another embodiment, the cleavable linker is cleaved by a caspase. In yet another embodiment, the caspase is caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, and caspase 12. In yet another embodiment, the cleavable linker does not shield the antigen-binding site. In yet another embodiment, aAb further comprises an agent conjugated to aAb. In yet another embodiment, the agent is at least one of a toxin or a toxic fragment thereof; a microtubule inhibitor; a nucleic acid damaging agent; a detectable portion; or a diagnostic agent. In yet another embodiment, aAb is selected from SEQ ID NOs: 1, 2, or 3.

[0018] In another embodiment, the present invention comprises, in order: a first light chain including a first variable light chain region; a cleavable linker; and a first heavy chain including a first variable heavy chain region, wherein the cleavable linker prevents or reduces the formation of a first antigen-binding site for a first antigen by the first light chain and the first heavy chain; and when the cleavable linker is cleaved, the first heavy chain is released, enabling the formation of a first antigen-binding site for binding to the first antigen, and includes a nucleic acid encoding an activatable antibody (aAb).

[0019] In another embodiment, the present invention comprises, in order: a first light chain including a first variable light chain region; a cleavable linker; and a first heavy chain including a first variable heavy chain region, wherein the cleavable linker prevents or reduces the formation of an antigen-binding site for a first antigen by the first light chain and the first heavy chain; and when the cleavable linker is cleaved, the first heavy chain is released and, together with the first light chain, comprises a nucleic acid encoding an activatable antibody (aAb) that forms an antibody-binding site for binding to the first antigen.

[0020] In another embodiment, the present invention includes a cell comprising: a first light chain including a first variable light chain region; a cleavable linker; and a first heavy chain including a first variable heavy chain region, wherein the cleavable linker prevents or reduces the formation of a first antigen-binding site for a first antigen by the first light chain and the first heavy chain; and when the cleavable linker is cleaved, the first heavy chain is released, enabling the formation of a first antigen-binding site for binding to the first antigen, thereby comprising a cell containing a nucleic acid encoding an activatable antibody (aAb).

[0021] In another embodiment, the present invention comprises, in order: a first light chain including a first variable light chain region; a cleavable linker; and a first heavy chain including a first variable heavy chain region, wherein the cleavable linker prevents or reduces the formation of an antigen-binding site for a first antigen by the first light chain and the first heavy chain; and when the cleavable linker is cleaved, the first heavy chain is released and, together with the first light chain, forms an antibody-binding site for the first antigen, comprising a cell containing nucleic acid encoding an activatable antibody (aAb).

[0022] In another embodiment, the present invention includes a pharmaceutical composition comprising an activatable Ab and a carrier. In yet another embodiment, the present invention includes a method for reducing the binding activity of an antibody to normal tissue and targeting cancer cells, comprising the step of administering an effective amount of an activatable Ab to a subject in need thereof.

[0023] In another embodiment, the present invention includes a method for treating, alleviating, or slowing the progression of cancer symptoms, comprising the step of administering an effective amount of an activatable Ab to a subject in need thereof. In one embodiment, cancer is a cancer that expresses an enzyme that cleaves a cleavable linker. In another embodiment, cancer is bladder cancer, bone cancer, breast cancer, oncogenic body, cervical cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, The cancer is selected from thyroid cancer, genitourinary cancer, or urothelial carcinoma. In another embodiment, the cancer is selected from acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, urothelial carcinoma of the bladder, ductal carcinoma of the mammary gland, lobular carcinoma of the mammary gland, cancer of the esophagus, castration-resistant prostate cancer (CRPC), cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, adenocarcinoma of the colon and rectum, colorectal cancer (CRC), esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), renal pigmentophobic carcinoma, kidney The group is selected from clear cell carcinoma, papillary cell carcinoma of the kidney, low-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and chrome affinity cell tumor, prostate adenocarcinoma, renal cell carcinoma (RCC), sarcoma, cutaneous melanoma, squamous cell carcinoma of the head and neck, T-cell lymphoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrioid carcinoma of the uterus, and uveal melanoma.

[0024] In another embodiment, the present invention includes an activatable antibody (aAb) comprising, in order, a first variable light chain region, a cleavable linker, a first variable heavy chain region, and an Fc region, wherein the cleavable linker prevents the first variable light chain region and the first variable heavy chain region from forming a first antigen-binding site with respect to a first antigen; the cleavable linker does not shield the antigen-binding site, and when the cleavable linker is cleaved, the first variable heavy chain region is released, enabling the formation of a first antigen-binding site that binds to the first antigen.

[0025] In another embodiment, the present invention comprises a cell expressing an activatable antibody (aAb) comprising, in order: a first light chain including a first variable light chain region; a cleavable linker; and a first heavy chain including a first variable heavy chain region, wherein the cleavable linker prevents or reduces the formation of a first antigen-binding site for a first antigen by the first light chain and the first heavy chain; and when the cleavable linker is cleaved, the first heavy chain is released, enabling the formation of a first antigen-binding site for binding to the first antigen. In one embodiment, the cell is a T cell or a mesenchymal stem cell. In another embodiment, the aAb further comprises a transmembrane sequence that anchors the aAb to the surface of a T cell to form a chimeric antigen receptor, in which case the cell is a CAR T cell. [Brief explanation of the drawing]

[0026] To better understand the characteristics and advantages of the present invention, a detailed description of the invention is provided herewith, along with the accompanying drawings. [Figure 1A] ~ [Figure 1C] This figure shows a schematic diagram of the protein and a diagram illustrating how the pro-antibody design regains its antigen-binding ability after cleavage. Figure 1A shows a schematic diagram of AAB. Figure 1B shows the first type of antigen-binding activation. CL and VH are linked by a proteolytic linker sensitive to MMP14. Before cleavage, VH cannot pair with VL to form a stable antigen-binding site. After cleavage by MMP14, VH is released and can pair with VL to form an antigen-binding site. Figure 1C shows the second type of antigen-binding activation. The first Fc and VH are linked by a proteolytic linker sensitive to MMP14. Before cleavage, VH cannot pair with VL to form a stable antigen-binding site. After cleavage by MMP14, VH is released and can pair with VL to form an antigen-binding site. [Figure 2]This figure shows that pro-anti-CLDN18.2-Fc regains its antigen-binding ability after the linker is cleaved by MMP14. Cell-based ELISA data show that pro-anti-CLDN18.2-Fc, after cleavage by MMP14, binds to CLDN18.2-expressing KatoIII cells as strongly as the positive control. Binding of the uncleaved protein is approximately 1 / 100th that of the positive control. [Figure 3] This figure shows that after the linker is cleaved by MMP14, the bispecific pro-anti-CLDN18.2-Fc-anti-hCD3 regains its antigen-binding ability. Cell-based ELISA data show that pro-anti-CLDN18.2-Fc-anti-hCD3, after cleavage by MMP14, binds to CLDN18.2-expressing KatoIII cells more than 20 times more strongly than the uncleaved protein. [Figure 4] This figure shows that bispecific pro-anti-CLDN18.2-Fc-anti-hCD3 after MMP14 cleavage activates T cells more than 10 times more strongly than activation by the uncleaved protein. Reporter jarcut cells were mixed with Kato III cells and pro-anti-CLDN18.2-Fc-anti-hCD3, with or without MMP14 cleavage. After 24 hours of incubation, T cell activation levels were measured by luciferase production. [Figure 5] This figure shows that pro-anti-hCTLA4 ScFv-Fc, after cleavage by MMP14, binds to surface-coated hCTLA4 protein more than 10 times more strongly than the uncleaved protein. [Figure 6] This figure shows that pro-anti-CLDN18.2 clone 2 regains its antigen-binding ability after the linker is cleaved by MMP14. Cell-based ELISA data show that pro-anti-CLDN18.2 clone 2, after cleavage by MMP14, binds to CLDN18.2-expressing KatoIII approximately 100 × stronger than the binding of the uncleaved protein. [Figure 7]This figure shows that pro-anti-hCD3 ScFab regains its hCD3e binding ability after the linker is cleaved by MMP14. ELISA data show that pro-anti-hCD3 ScFab, after cleavage by MMP14, binds to surface-coated hCD3e more than 20 × stronger than the binding of the uncleaved protein. [Figure 8] This figure shows that pro-anti-hCD3(AAB7) regains its hCD3e binding ability after the linker is cleaved by MMP14. ELISA data show that pro-anti-hCD3(AAB7) after cleavage by MMP14 binds to coated hCD3e more than 10 × stronger than the binding of the uncleaved protein. [Figure 9] This figure shows that pro-anti-hCTLA4 (AAB7) regains its antigen-binding ability after the linker is cleaved by MMP14. Flow cytometry-based binding assay data show that pro-anti-hCTLA4 (AAB7) after cleavage by MMP14 binds to hCTLA4-expressing cells as strongly as the positive control, while the uncleaved protein shows almost no binding ability. [Figure 10] This figure shows that after the linker is cleaved by MMP14, pro-anti-hCD3 (AAB7) regains its ability to stimulate T cell activation. Reporter T cell activation assay data show that pro-anti-hCD3 (AAB7) after cleavage by MMP14 stimulates T cell activation more than 20 × stronger than activation by the uncleaved protein. The level of T cell activation is measured by the amount of luciferase produced. [Figure 11] This figure shows that after linker cleavage by MMP14, bispecific pro-anti-hCD3(AAB8)-anti-hPD-L1 regains its ability to stimulate T cell activation. Reporter T cell activation assay data show that bispecific pro-anti-hCD3(AAB8)-anti-hPD-L1 after cleavage by MMP14 stimulates T cell activation more than 20 × stronger than activation by the uncleaved protein. The level of T cell activation is measured by the amount of luciferase produced. [Figure 12] This figure shows that after the linker is cleaved by MMP14, the bispecific pro-anti-hCD3(AAB8)-anti-CLDN18.2 ScFv regains its ability to stimulate T cell activation. Reporter T cell activation assay data show that bispecific pro-anti-hCD3(AAB8)-anti-CLDN18.2 ScFv stimulates T cell activation more than 20 × stronger than activation by the uncleaved protein after cleavage by MMP14. The level of T cell activation is measured by the amount of luciferase produced. [Figure 13] This figure shows that after linker cleavage by MMP14, bispecific pro-anti-hCD3(AAB8)-pro-anti-hPD-L1 regains its ability to stimulate T cell activation in PBMCs. Bispecific pro-anti-hCD3(AAB8)-pro-anti-hPD-L1, after cleavage by MMP14, stimulates T cell activation in PBMCs more than 20 × stronger than activation by the uncleaved protein. The level of T cell activation is measured by IFN-gamma production. [Figure 14] This figure shows that pro-anti-hCTLA4(AAB1)-Fc regains its antigen-binding ability after the linker is cleaved by MMP14. Flow cytometry-based binding assay data show that pro-anti-hCTLA4(AAB1)-Fc, after cleavage by MMP14, binds to hCTLA4-expressing cells as strongly as the positive control, while the uncleaved protein shows almost no binding ability. [Modes for carrying out the invention]

[0027] While various embodiments of the present invention will be discussed in detail below, it should be recognized that the present invention provides numerous applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein merely illustrate specific ways of creating and using the present invention and do not limit the scope of the invention.

[0028] To facilitate understanding of the present invention, several terms are defined below. Terms as defined herein have meanings that are generally understood by those skilled in the art relating to the present invention. Terms such as "a," "an," and "the" are not intended to refer only to singular entities, but include general classes for which specific examples may be used for illustrative purposes. Technical terms used herein are used to describe specific embodiments of the present invention, but their use does not limit the present invention except as outlined in the claims.

[0029] Therapeutic monoclonal antibodies are being developed to treat various human diseases, including cancer. In cancer therapy, for example, anti-CTLA4 antibodies or anti-CD3 antibodies have been used to activate T cells by reducing immunosuppressive signals in the tumor microenvironment. However, systemic overactivation of T cells due to off-target Ag-Ab interactions can lead to serious adverse events. Antibodies against tumor-associated antigens (TAAs) often target non-tumor tissues that express the same antigen. In the case of prior art anti-CTLA4 activatable antibodies, the construct includes both a cleavable linker that blocks anti-CTLA4 activity and a synthetic peptide / external peptide (masking portion). These two extra peptides may trigger an immune response against the linker, and furthermore, cleavage of the linker does not guarantee the release of the masking portion from the antigen-binding site.

[0030] This invention reduces off-target toxicity in monoclonal antibody therapy by eliminating the use of masking moieties, thereby increasing the therapeutic index and drug tolerability in patients. The pro-antibodies taught herein have been found to be little to no activity until the drug reaches the tumor, thus achieving a long half-life, protein stability, and manufacturability. The novel pro-antibodies are designed so that the heavy and light chains of the antibody are shortened by a short linker that reduces binding affinity to the target molecule. Since this short linker is sensitive to tumor-associated proteases, cleavage of the linker restores the morphological position of the heavy and light chains in tumor tissue, and therefore their binding affinity to the target.

[0031] As used herein, the terms “activatable antibody,” “aAb,” “pro-antibody,” or “pro-body” refer to a fusion protein containing the antigen-binding domain of an antibody (separated by a cleavable linker). The basic structure of a fusion protein, from amino to carboxyl, includes: variable light chain region - cleavable linker - variable heavy chain region, or variable heavy chain region - cleavable linker - variable light chain region. The protein (which binds to the first antigen) can be co-expressed with a second fusion protein that targets a second antigen. The first and second antigens may be the same antigen, different antigens, or even if they are the same antigen, the fusion protein may bind to different epitopes of the antigen. The fusion protein may also include one or more of the following: a constant light chain region, a constant heavy chain region, an Fc region (wild-type or mutant), and a second linker between the Fc and the second protein (e.g., a cytokine).

[0032] The nucleic acid encoding aAb may be part of a vector used to express aAb in host cells, such as bacterial, fungal, plant, or mammalian cells.

[0033] As used herein, the terms “antibody” or “antibody peptide” refer to an intact antibody or its binding fragment that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain variable fragment (scFv) antibodies. Antibodies other than “bispecific” or “bifunctional” antibodies are understood to have the same binding site. An antibody substantially inhibits the adhesion between the receptor and the counter-receptor when an excess of antibody reduces the amount of receptor bound to the counter-receptor by at least about 20%, 40%, 60%, or 80%, and more commonly about 85% (as measured in in vitro competitive binding assays).

[0034] As used herein, the terms “bispecific” or “bifunctional” antibody are understood to have two distinct antigen-binding sites. For example, the bispecific antibody of the present invention comprises two distinct antigen-binding domains, e.g., a first and a second antigen-binding domain, each binding to a first and a second antigen, respectively. A bispecific antibody may also have two distinct antigen-binding regions that bind to the same antigen, but to two different epitopes. More generally, a bispecific antibody binds to two different antigens. The first or second antigen is typically a tumor-specific antigen, while the other antigen-binding region binds to a T cell-activating molecule on a T cell.

[0035] As used herein, the term “antibody” is used in its broadest sense and specifically covers monoclonal antibodies (full-length antibodies, or other bivalent, Fc-region-containing antibodies, e.g., bivalent scFv-Fc fusion antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, scFv) insofar as they exhibit the desired biological activity. Antibodies (Ab, Antibody) and immunoglobulins (Ig, immunoglobulin) are glycoproteins with identical structural properties. Antibodies exhibit binding specificity to specific antigens, while immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter type of polypeptide is produced, for example, at low levels by the lymphatic system and at increased levels by myeloma. In other words, the present invention comprises a fully recombinant monoclonal antibody (and its conjugated fragment), in which the complementarity determining region (CDR) is genetically spliced ​​into the human antibody backbone, often referred to as antibody veneering. Thus, in certain embodiments, the monoclonal antibody is a fully synthesized antibody. In certain embodiments, the monoclonal antibody (and its conjugated fragment) may be produced in eukaryotic cells, including bacterial or plant cells.

[0036] As used herein, the term “antibody fragment” refers to a portion of a full-length antibody, generally the antigen-binding region or variable region, and includes the Fab, Fab', F(ab')2, Fv, and scFv fragments. When an antibody is digested with papain, two identical antigen-binding fragments called Fab fragments (each possessing a single antigen-binding site) and the remaining “Fc” fragment (so named due to its ability to readily crystallize) are produced. Pepsin treatment further enhances the antigen binding. The result is an F(ab')2 fragment having two antigen-binding fragments capable of crosslinking, and the remaining fragment (referred to as pFc'). As used herein, “functional fragment” refers to the Fv, F(ab), and F(ab')2 fragments with respect to an antibody.

[0037] As used herein, the "Fv" fragment is the smallest antibody fragment possessing a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in a non-covalently tightly associated state (V H -V L Dimer). The three CDRs in each variable domain interact to form V H -V L It is in this configuration that antigen-binding sites are defined on the surface of the dimer. Overall, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the entire binding site.

[0038] The Fab fragment, also known as F(ab), contains the constant domain of the light chain and the first constant domain (CH1, firstconstant domain) of the heavy chain. The Fab' fragment contains the heavy chain CH1 The Fab fragment is distinguished by the addition of several residues containing one or more cysteines derived from the antibody hinge region to the carboxyl terminus of the domain. Fab'-SH is the herein designation for Fab' when the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab') fragment is generated by cleaving the disulfide bond of the hinge cysteine ​​of the F(ab')2 pepsin digestion product. Further chemical coupling of antibody fragments is known to those skilled in the art.

[0039] Natural antibodies and immunoglobulins typically consist of two identical light chains (L,light) and two identical... A heterotetrameric glycotan with approximately 150,000 daltons, composed of heavy chains (H, heavy). It is a protein. Each light chain is linked to a heavy chain by one covalent disulfide bond. However, the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end. H ), followed by several constant domains. Each light chain has a variable domain (V) at one end. L ), and the other end has a constant domain. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the variable domain of the light chain and the variable domain of the heavy chain (Clothia et al., J.Mol. Biol. 186, 651-66, 1985); Novotny and Haber, Proc.Natl. Acad. Sci. USA 82 4592-4596 (1985), relevant parts of which are incorporated herein by reference.

[0040] As used herein, “isolated” antibody is an antibody identified, separated, and / or recovered from components of the environment in which it was produced. Contaminant components of its production environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified to be measurable by at least three different methods: 1) to more than 50% by weight of the antibody, e.g., more than 75% by weight, or more than 85% by weight, or more than 95% by weight, or more than 99% by weight, etc., when determined by the Lowry method; 2) to a degree sufficient to obtain at least 10 residues of the N-terminal or internal amino acid sequence, e.g., at least 15 residues of the sequence, etc., when using a spinning cup sequencer; or 3) by SDS-PAGE under reducing or non-reducing conditions, until homogeneous using Coomassie blue, or preferably silver staining. Because at least one component of the antibody’s natural environment is absent, isolated antibodies include in situ antibodies from recombinant cells. However, isolated antibodies are typically prepared through at least one purification step.

[0041] As used herein, the term “antibody mutant” means an amino acid sequence variant of an antibody in which one or more amino acid residues are modified. Such mutants necessarily have less than 100% sequence identity, or similarity to an amino acid sequence having at least 75% amino acid sequence identity, or similarity to the amino acid sequence of either the heavy chain or light chain variable domain of the antibody, for example, at least 80%, at least 85%, at least 90%, or at least 95%, 96%, 97%, 98%, or 99%.

[0042] As used herein, the term “variable” refers to the fact that, in the context of the variable domain of an antibody, certain portions of the variable domain differ extensively in sequence among antibodies, and that these differences are used in the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the entire variable domain of an antibody. Variability is concentrated in three segments called complementarity-determining regions (CDRs), also known as high-frequency variable regions, in both the light-chain and heavy-chain variable domains. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variation (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)); and (2) an approach based on antigen-antibody complex crystallography (Chothia, C. et al. (1989), Nature 342: 877); or both, i.e., the Chothia method + the Kabat method. The more highly conserved portions of the variable domain are called frameworks (FRs). Variable doping of natural heavy and light chains Each of the four FR regions primarily incorporates a β-sheet configuration, which is linked together by three CDRs that form loops connecting β-sheet structures, and in some cases forming part of them. The CDRs within each chain are held together in close proximity by the FR region and, together with CDRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al.). The constant domain does not directly participate in antibody binding to the antigen, but exhibits various effector functions, such as antibody involvement in antibody-dependent cytotoxicity.

[0043] The light chain of an antibody (immunoglobulin) derived from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0044] Immunoglobulins can be classified into different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0045] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for small amounts of naturally occurring variations. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which generally contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. In addition to its specificity, monoclonal antibodies are advantageous in that they are synthesized by culturing hybridomas and are not contaminated by other immunoglobulins. The modifier “monoclonal” describes the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not interpreted as requiring antibody production by any special method. For example, the monoclonal antibody used in the invention disclosed and claimed herein is described in Kohler and Milstein, Nature 256, 495 (1975) (Relevant parts are incorporated by reference herein) It can be prepared by the hybridoma method described.

[0046] All monoclonal antibodies used in accordance with the inventions disclosed and claimed herein are (1) the result of a planned immunization protocol as described in more detail below herein; or (2) the result of an immune response that spontaneously induces antibody production in the course of disease or cancer.

[0047] To use the monoclonal antibodies of the inventions disclosed and claimed herein, it may be necessary to administer such or similar monoclonal antibodies to a target, such as a human. However, when monoclonal antibodies are produced in non-human animals, such as rodents or chickens, administering such antibodies to a human patient usually induces an immune response, but in that case, the immune response is directed towards the antibody itself. Such a reaction limits the duration and effectiveness of such therapy. To overcome such problems, the monoclonal antibodies of the inventions disclosed and claimed herein can be "humanized," that is, the antibody is modified so that its antigenic portion is removed and thus replaced with a human antibody analog portion, while retaining the antibody's affinity for a specific antigen. This modification may involve only a few amino acids, or it may involve the entire framework region of the antibody, leaving only the complementarity-determining region of the antibody intact. Several methods for humanizing antibodies are known in the art, and relevant parts are incorporated herein by reference, U.S. Patent No. 6,180,370 filed by Queenet et al. on January 30, 2001; Brickell U.S. Patent No. 6,054,927, filed on April 25, 2000; Studnicka, 199 U.S. Patent No. 5,869,619, filed on February 9, 1999; Lin, January 19, 1999 U.S. Patent No. 5,861,155 filed by Rodriquezet et al. on January 27, 1998. U.S. Patent No. 5,712,120 filed on [date]; and Cabilly et al. filed on March 2, 1989. It is disclosed in U.S. Patent No. 4,816,567, filed on the 8th.

[0048] Humanized forms of antibodies are, in principle, constructed from the sequences of human immunoglobulins and containing minimal sequences derived from non-human immunoglobulins, as chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies). Humanization can be carried out by substituting non-human (i.e., rodent, chicken) CDRs or CDR sequences with corresponding sequences of human antibodies, according to the method of Winter and collaborators (Jones et al., 1986; Riechmannet al., 1988; Verhoeyen et al., 1988) (see also U.S. Patent No. 5,225,539). In some cases, the F of human immunoglobulins is used. v Framework residues are replaced with corresponding non-human residues derived from the donor antibody. Humanized antibodies may also contain residues not found in the recipient antibody or in the transferred CDR or framework sequence. Generally, humanized antibodies contain at least one, typically two, variable domains where all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the framework region is the framework region of the human immunoglobulin consensus sequence. Humanized antibodies also optimally contain the immunoglobulin constant region (Fc), and generally at least a portion of the Fc of a human immunoglobulin.

[0049] The aAb of the present invention may also include modified sequences or glycosylation sites that confer a favorable level of activity in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD), as can be measured in animal models by bead-based or cell-based assays or in vivo studies.

[0050] aAb can be a single-chain variable fragment (scFv), which is a fusion of the variable regions of the heavy and light chains of an immunoglobulin. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide between the two antigen-binding domains. In this modification, the specificity is usually invariant even after linker cleavage. Such molecules have been created to facilitate phage display, which is highly convenient for expressing the antigen-binding domain as a single peptide. scFv can be created directly from subcloned heavy and light chains derived from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region found in complete antibody molecules, and therefore a common binding site (e.g., protein A / G) is used to purify the antibody. Since protein L interacts with the variable region of the κ light chain, these fragments can often be purified / immobilized using protein L.

[0051] The present invention comprises an activatable antibody (also called a pro-antibody or pro-body) that targets a specific antigen. Examples of antigens include the first antigen, which is ICAM1; VCAM1; EpCAM; extradomain B of fibronectin; melanoma-associated chondroitin sulfate proteoglycan (MCSP); melanoma-associated proteoglycan (MAPG); high molecular weight melanoma-associated antigen (HMV-MAA); prostate-specific membrane antigen (PSMA); epidermal growth factor receptor (EGFR); hepatocyte growth factor receptor (HGFR); fibroblast-activating protein (FAP); carcinoembryonic antigen (CEA); cell adhesion molecule (CAM); human B cell maturation target (BCMA); placental growth factor (PLGF); folate receptor, etc. The following are tissue-specific surface antigens selected from: thrin-like growth factor receptor (ILGFR); CD133; CD40; CD37; CD33; CD30; CD28; CD24; CD23; CD22; CD21; CD20; CD19; CD13; CD10; HER3; HER2; non-muscle myosin heavy chain type A (nmMHCA); transferrin; epithelial cell adhesion molecule (EpCAM); annexin A1; nucleotin, tenascin, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR-2); aminopeptidase N, tie-1, tie-2, or c-Met.そのhis antigen として、ABCF1;ACVR1;ACVR1B;ACVR2;ACVR2B;ACVRL1;ADORA2A;アグリカン;AGR2;AICDA;AIF1;AIG1;AKAP1;AKAP2;AMH;AMHR2;ANGPT1;ANGPT2 ;ANGPTL3;ANGPTL4;ANPEP;APC;APOC1;AR;AZGP1;B7.1;B7.2;BAD;BAFF;BAG1;BAI1;BCL2;BCL6;BDNF;BLNK;BLR1(MDR15);BlyS;BMP1;BMP2;B MP3B(GDF10);BMP4;BMP6;BMP8;BMPR1A;BMPR1B;BMPR2;BPAG1(プレクチン);BRCA1;C19orf10(IL27w);C3;C4A;C5;C5R1;CANT1;CASP1;CASP4;CAV1 ;CCBP2(D6 / JAB61);CCL1(1-309);CCL11(エオタキシン);CCL13(MCP-4);CCL15(MIP-1d);CCL16(HCC-4);CCL17(TARC);CCL18(PARC);CCL19(MIP-3b) );CCL2(MCP-1);MCAF;CCL20(MIP-3a);CCL21(MIP-2);SLC;エクソダス-2;C CL22(MDC / STC-1);CCL23(MPIF-1);CCL24(MPIF-2 / エオタキシン-2);CCL25(T ECK);CCL26(エオタキシン-3);CCL27(CTACK / ILC);CCL28;CCL3(MIP-1a);CCL4(MIP-1b);CCL5(RANTES);CCL7(MCP-3);CCL8(mcp-2);CCNA1;CCNA2; CCND1;CCNE1;CCNE2;CCR1(CKR1 / HM145);CCR2(mcp-1RB / RA);CCR3(CKR3 / CMKBR3);CCR4;CCR5(CMKBR5 / ChemR13);CCR6(CMKBR6 / CKR-L3 / STRL 22 / DRY6);CCR7(CKR7 / EBI1);CCR8(CMKBR8 / TER1 / CKR-L1);CCR9(GPR-9-6);CCRL1(VSHK1);CCRL2(L-CCR);CD164;CD19;CD1C;CD20;CD200;C. D-22;CD24;CD28;CD3;CD37;CD38;CD3E;CD3G;CD3Z;CD4;CD40;CD40L;CD44;CD45RB;CD52;CD69;CD72;CD74;CD79A;CD79B;CD 8;CD80;CD81;CD83;CD86;CDH1(E-カドヘリン);CDH10;CDH12;CDH13;CDH18;CDH19;CDH20;CDH5;CDH7;CDH8;CDH9;CDK2;CDK3;CDK 4;CDK5;CDK6;CDK7;CDK9;CDKN1A(p21Wap1 / Cip1);CDKNIB(p27Kip1);CDKNIC;CDKN2A(pl6INK4a);CDKN2B;CDKN2C;CDKN3;CE BPB;CER1;CHGA;CHGB;キチナーゼ;CHST10;CKLFSF2;CKLFSF3;CKLFSF4;CKLFSF5;CKLFSF6;CKLFSF7;CKLFSF8;CLDN3;CLDN7(クローディン-7);CLN3;CXCL10(IP-10);CXCL11(I-TAC / IP-9);CXCL12(SDF1);CXCL13;CXCL14;CXCL16;CXCL2(GR02);CXCL3(GR03);CXCL 5(ENA-78 / LIX);CXCL6(GCP-2);CXCL9(MIG);CXCR3(GPR9 / CKR-L2);CXCR4;CXCR6(TYMSTR / STRL33 / Bonzo);CYB5;CYC1;CYSLT R1;CGRP;Clq;Clr;CI;C4a;C4b;C2a;C2b;C3a;C3b;DAB2IP;DES;DKFZp451J0118;DNCL1;DPP4;E-セレクチン;E2F1;ECGF1;EDG1;EF NA1;EFNA3;EFNB2;EGF;EGFR;ELAC2;ENG;EN01;EN02;EN03;EPHB4;EPO;ERBB2(Her-2);EREG;ERK8;ESR1;ESR2;F3(TF);Factor VII Factor IX; Factor V; Factor VIIa; Factor X; Factor XII; Factor XIII; FADD; FasL; FASN; FCER1A; FCER2; Fc gamma receptor; FCGR3A; FGF; FGF1(aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2(bFGF); FGF20; FGF21; FGF22; FGF23; FGF3(int-2); FGF4(HST); FGF5; FGF6(HST-2); FGF7(KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (Epsilon); FIL1 (Zeta); FLJ12584; FLJ25530; FLRT1 (Fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GMCSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (CIO); GRP; GSN (Gelsolin); GSTP1; Glycoprotein (GP) IIb / IIIa; HAVCR2;HDAC4;HDAC5;HDAC7A;HDAC9;Her2;HGF;ITGB4(b4 integrin);JAG1;JAK1;JAK3;JUN;K6HF;KAI1;KDR;KITLG;KLF5(GC Box BP);KLF6;KLK10;KLK12;KLK13;KLK14;KLK15;KLK3;KLK4;KLK5;KLK6;KLK9;KRT1;KRT19(keratin 19);KRT2A;KRTHB6(hair-specific type II keratin);L-selectin;LAMA5;LEP(leptin);Lingo-p75;Lingo-Troy;LPS;LTA(TN F-b);LTB;LTB4R(GPR16);LTB4R2;LTBR;MACMARCKS;MAG or Omgp;MAP2K7(c-Jun);MDK;MIB1;Midocaine;MIF;MIP-2;MKI67(Ki-67);MMP2;MMP9;MS4A1;MSMB;MT3(Metallothionectin-III);MTSS1;MUC1(Mucin);MYC;M YD88;NCK2;neurocan;NKG2D;NFKB1;NFKB2;NGF;NGFB(NGF);NGFR;NgR-Lingo;NgR-Nogo66(Nogo);NgR-p75;NgR-Troy;NME1(NM23A);NOX5;NPPB;NR0B1;NR0B2;NR1D1;NR1D2;NR1H2;NR1H3;NR1H4;NRII2;NRII3;NR2C1;NR2C2;NR2E1;NR2E3;NR 2F1;NR2F2;NR2F6;NR3C1;NR3C2;NR4A1;NR4A2;NR4A3;NR5A1;NR5A2;NR6A1;NRP1;NRP2;NT5E;NTN4;ODZ1;OPRD1;P2RX7;PAP;PARTI;PATE;PAWR;PCA3;PCNA;PDGFA;PDGFB;PECAM1;PF4(CXCL4);PGE2;PGF;PGR;Phosphacane;PIAS2;PIK3CG;Plasminogen Activator;PLAU(uPA);PLG;PLXDC1;PPBP(CXCL7);PPID;P R1;PRKCQ;PRKD1;PRL;PROC;Protein C;PROK2;PSAP;PSCA;PTAFR;PTEN;PTGS2(COX-2);PTN;RAC2(p21Rac2);RAGE;RARB;RGS1;RGS13;RGS3;RNF110(ZNF144);ROB02;SI00A2;SCGB1D2(Lipophyllin B);SCGB2A1(Mammaglobin 2);SCGB2A2(Mammaglobin 1);SCYE1(Endothelial Monocyte Activating Cytokine);SDF2;SERPINA1;SERPINA3;SERPINB5 (Maspine); SERPINE1(PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B(Spr1); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; Substance P; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB111; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1(Thrombospongin-1); THBS2; THB S4;THPO;TIE(Tie-1);TIMP3;Tissue factor;TLR10;TLR2;TLR3;TLR4;TLR5;TLR6;TLR7;TLR8;TLR9;TNF;TNF-a;TNFAIP2(B94);TNFAIP3;TNFRSF11A;TNFRSF1A ;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF6(Fas);TNFRSF7;TNFRSF8;TNFRSF9;TNFSF10(TRAIL);TNFSF11(TRANCE);TNFSF12(AP03L);TNFSF13(April);TNFSF13B; TNFSF14 (HVEML); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptor; TOP2A (topoisomerase IIa); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; thrombomodulin; thrombin; VEGF; VEGFB; VEGFC; versican; VHL Examples include proteins, protein segments, or peptides encoded by at least one gene selected from C5;VLA-4;XCL1 (lymphotactin);XCL2 (SCM-1b);XCR1 (GPR5 / CCXCR1);YY1; and / or ZFPM2.

[0052] The present invention also includes tumor target antigens selected from HER1, HER2, HER3, GD2, carcinoembryonic antigen (CEA), epidermal growth factor receptor activating mutant (EGFRVIII), CD133, fibroblast activating protein alpha (FAP), epithelial cell adhesion molecule (Epcam), glypican 3 (GPC3), EPH receptor A4 (EphA), tyrosine protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor-1 (WT-1), or claudin family proteins.

[0053] The present invention also includes antigen-binding domains that target T cell markers. Examples of T cell markers include CTLA4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, and / or TIM-3. Antibodies may also bind to activated T cell markers, CD3, 41BB, or OX40.

[0054] The present invention also includes cleavable linkers, such as protease-cleavable linkers. Examples of cleavable linkers are tumor-associated proteases: MMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, uPA, FAPa, or peptides containing sequences cleaved by cathepsin B. Other examples include cleavable linkers cleaved by proteases upregulated during apoptosis or inflammation-associated responses, such as caspases. Examples of caspases are caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, and / or caspase 12. Unlike the activatable antibodies of prior art, the cleavable linker of the present invention does not directly shield the antigen-binding site.

[0055] The present invention may also include cytokines bound to aAb, for example, as part of an aAb fusion protein, or individually. Cytokines include growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormone; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; TNF-α; Müllerian inhibitor; gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor; platelet growth factor; placental growth factor, transforming growth factor (TGF); insulin-like growth factor-1 and -11; erythropoietin (EPO); bone induction factor; interferon; colony-stimulating factor ( At least one of the following may be selected: CSF; lymphotoxin-alpha; lymphotoxin-beta; CD27L; CD30L; FASL; 4-1BBL; OX40L; TRAIL; IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-15; IL-18; IL-21; IL-22; IL-23; IL-33; IFN-a; IFN-b; IFN-g; IFN-g inducer (IGIF); bone morphogenetic protein (BMP); leukemia suppressor (LIF); or kit ligand (KL).

[0056] The present invention relates to a pro-antibody design. To avoid off-target Ag-Ab interactions, the strategy of the present invention is to introduce a proteolytic linker to reduce or even block antibody antigen binding by twisting the protein structure of the antigen-binding site. After the linker is cleaved at the target site, the two parts of the antibody (which together form the antibody's antigen-binding region) are released from their twisted structure, and the antibody regains its antigen-binding ability.

[0057] The fusion protein designs and methods disclosed herein can be applied to all types of antibodies without adding extra elements to the antibody structure. Furthermore, it has been found that short-chain linkers reduce immunogenicity and increase antibody productivity. Moreover, the present invention does not include G4S repeat linkers, thereby reducing the problem of fusion protein aggregation before linker cleavage. [Examples]

[0058] Materials and methods. Cloning and protein synthesis. DNA fragments for protein expression are synthesized by Genewiz, or used in PCR. It is then generated and sent via isothermal assembly (Quantabio) to pEE6.4 It was cloned into the vector.

[0059] For protein expression, the plasmid was mixed with PEI (Sigma) in 293 Freestyle Medium (Gibco) and then transfected into 293F cells. The cells were then sterilized at 120°F. The samples were incubated at 37°C with shaking in pm. Five to six days after incubation, The supernatant was collected and filtered. The protein was purified using Protein A resin (Repligen) and stored in neutralized elution buffer (40 mM Tris, pH 7.0 / 100 mM glycine / 100 mM NaCl).

[0060] ELISA and cell-based ELISA. When testing the binding strength of anti-CTLA4 antibody, dilute the CTLA4 protein (SinoBiological) to 2 ug / ml and 9 A 6-well plate was coated. Test antibodies were diluted to different concentrations and added to the wells. After incubation at 37°C for 1 hour, unbound proteins were washed off with washing buffer (PBS / 0.05% Tween 20). Detection antibody (AP-goat-anti-human IgG obtained from Jackson ImmunoResearch) was added. Incubation at 37°C for 1 hour. After rinsing, the unbound protein was washed away with washing buffer. Next, PNPP substrate (Pierce) solution was added. Incubate at room temperature until it turned yellow. Biotek Epoch2 OD405 was measured using and analyzed with Gen5 software. Except when testing the binding strength of anti-CLDN18.2 protein, CLDN18.2-expressing KatoIII cells (ATCC) were coated into 96-well plates instead of the protein. The method is the same as described above. Each well contains 10 cells. 5 It included one.

[0061] T-cell activation assay. To test the ability of Pro-anti-CLDN18.2-Fc-CD3 to activate T cells before and after MMP14 cleavage, Jarcutt NFAT cells (InvivoGen), KatoIII cells (ATCC), and diluted proteins before and after MMP14 cleavage were mixed in each well of a 96-well plate. Each well contained 104 Jarcutt cells and 104 KatoIII cells. 30 hours at 37°C. After incubation, 30 µl of the supernatant was transferred from each well to a Black 96-well plate. Next, QUANTI-Luc assay solution (InvivoGen) was injected, and luciferase activity was measured using a luminometer (Biotek Synergy), followed by the use of Gen5 software. It was used for analysis.

[0062] protein sequence Pro-anti-CTLA4-Fc MINEFSSLAGAQRQRLLGVVVVQSLVHHGAAKHVALLPSALVHCQIASESKAAVGIQHGHGGLVVVLGLAVAFPFHGDIARVEALHQTAQRHLILGQLVSAGRLGVHLRLPRLAFGLADGLLNGGGQSLVGDLALVLLAVEPVLVQHGQHGHHSICGVVLLLSGLGFGVVHFDAVHVPVKLHLGVLMADVHHHACHLGCSRDHQCILGFGREQKHGRSAQQFGSRTARNRYHRALTPIVEVAGLTRTVISRGVLGGYRVNLQKELVFRGVTGDRDTRFDRRVVIGRTFIIDETHPFNFLTWELTDPIPTITGGDGGAGNRARQAERTGRINQTRTRFFQFYLSAYVLTPPFDFQLGARTERVRIVVPLLAVVKRLVFVLNRRNGQREVGTRARTTETVRNAGVTGRRVVDQQFRRLTRLLHVPIQIVGFRVRVEQALRAFARDGRFFTRRHAQRRWRLGHHNVSGGTWNPEQQYP(SEQ ID NO: 1)

[0063] Pro-anti-CLDN18.2-Fc METDTLLLWVLLLWVPGSTGDIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE CGSSGRSENIRTAGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2)

[0064] Pro-anti-CLDN18.2-Fc-CD3 (Sequence ID 3)

[0065] Figure 1 shows a schematic diagram of the protein and a diagram illustrating how the pro-antibody design regains its antigen-binding ability after cleavage. Figure 1A shows a schematic diagram of AAB. Figure 1B shows the first type of antigen-binding activation. CL and VH are linked by a proteolytic linker sensitive to MMP14. Before cleavage, VH cannot pair with VL to form a stable antigen-binding site. After cleavage by MMP14, VH is released and can pair with VL to form an antigen-binding site. Figure 1C shows the second type of antigen-binding activation. The first Fc and VH are linked by a proteolytic linker sensitive to MMP14. Before cleavage, VH cannot pair with VL to form a stable antigen-binding site. After cleavage by MMP14, VH is released and can pair with VL to form an antigen-binding site.

[0066] Figure 2 is a graph showing that pro-anti-CLDN18.2-Fc regains its antigen-binding ability after the linker is cleaved by MMP14. Cell-based ELISA data show that pro-anti-CLDN18.2-Fc, after cleavage by MMP14, binds to CLDN18.2-expressing KatoIII cells as strongly as the positive control. The binding rate of uncleaved proteins is approximately 1 / 100 compared to the positive control.

[0067] Figure 3 is a graph showing that bispecific pro-anti-CLDN18.2-Fc-anti-hCD3 regains its antigen-binding ability after the linker is cleaved by MMP14. Cell-based ELISA data show that pro-anti-CLDN18.2-Fc-anti-hCD3, after cleavage by MMP14, binds to CLDN18.2-expressing KatoIII cells and to the uncleaved protein. This demonstrates a bond that is more than 20 times stronger than that of other materials.

[0068] Figure 4 is a graph showing that bispecific pro-anti-CLDN18.2-Fc-anti-hCD3 after MMP14 cleavage activates T cells more than 10 times more strongly than activation by the uncleaved protein. Reporter jarcut cells were mixed with Kato III cells and pro-anti-CLDN18.2-Fc-anti-hCD3, with or without MMP14 cleavage. After 24 hours of incubation, the level of T cell activation was measured by the amount of luciferase produced.

[0069] Figure 5 is a graph showing that pro-anti-hCTLA4 ScFv-Fc, cleaved by MMP14, binds to surface-coated hCTLA4 protein more than 10 times more strongly than the uncleaved protein.

[0070] Figure 6 is a graph showing that pro-anti-CLDN18.2 clone 2 regains its antigen-binding ability after the linker is cleaved by MMP14. Cell-based ELISA data showed that pro-anti-CLDN18.2 clone 2, after cleavage by MMP14, binds to CLDN18.2-expressing KatoIII approximately 100× stronger than the binding of the uncleaved protein. This is a diagram.

[0071] Figure 7 is a graph showing that pro-anti-hCD3 ScFab regains its hCD3e binding ability after the linker is cleaved by MMP14. ELISA data show that pro-anti-hCD3 ScFab, after cleavage by MMP14, binds to surface-coated hCD3e more than 20 × stronger than the binding of the uncleaved protein.

[0072] Figure 8 is a graph showing that pro-anti-hCD3(AAB7) regains its hCD3e binding ability after the linker is cleaved by MMP14. ELISA data show that pro-anti-hCD3(AAB7) after cleavage by MMP14 binds to coated hCD3e more than 10 × stronger than the binding of the uncleaved protein.

[0073] Figure 9 is a graph showing that pro-anti-hCTLA4 (AAB7) regains its antigen-binding ability after the linker is cleaved by MMP14. Flow cytometry-based binding assay data show that pro-anti-hCTLA4 (AAB7) after cleavage by MMP14 binds to hCTLA4-expressing cells as strongly as the positive control, while the uncleaved protein shows almost no binding ability.

[0074] Figure 10 is a graph showing that after linker cleavage by MMP14, pro-anti-hCD3 (AAB7) regains its ability to stimulate T cell activation. Reporter T cell activation assay data showed that pro-anti-hCD3 (AAB7) after MMP14 cleavage stimulates T cell activation more than 20 × stronger than activation by the uncleaved protein. The level of T cell activation is measured by the amount of luciferase produced.

[0075] Figure 11 is a graph showing that after linker cleavage by MMP14, bispecific pro-anti-hCD3(AAB8)-anti-hPD-L1 regains its ability to stimulate T cell activation. Reporter T cell activation assay data show that bispecific pro-anti-hCD3(AAB8)-anti-hPD-L1 after cleavage by MMP14 stimulates T cell activation more than 20 × stronger than activation by the uncleaved protein. The level of T cell activation is measured by the amount of luciferase produced.

[0076] Figure 12 is a graph showing that after linker cleavage by MMP14, the bispecific pro-anti-hCD3(AAB8)-anti-CLDN18.2 ScFv regains its ability to stimulate T cell activation. Reporter T cell activation assay data show that bispecific pro-anti-hCD3(AAB8)-anti-CLDN18.2 ScFv stimulates T cell activation more than 20 × stronger than activation by the uncleaved protein after cleavage by MMP14. The level of T cell activation is measured by luciferase production.

[0077] Figure 13 is a graph showing that after linker cleavage by MMP14, bispecific pro-anti-hCD3(AAB8)-pro-anti-hPD-L1 regains its ability to stimulate T cell activation in PBMCs. Bispecific pro-anti-hCD3(AAB8)-pro-anti-hPD-L1, after cleavage by MMP14, stimulates T cell activation in PBMCs more than 20 × stronger than activation by the uncleaved protein. The level of T cell activation is measured by IFN-gamma production.

[0078] Figure 14 is a graph showing that pro-anti-hCTLA4(AAB1)-Fc regains its antigen-binding ability after the linker is cleaved by MMP14. Flow cytometry-based binding assay data show that pro-anti-hCTLA4(AAB1)-Fc, after cleavage by MMP14, binds to hCTLA4-expressing cells as strongly as the positive control, while the uncleaved protein shows almost no binding ability.

[0079] Any embodiment discussed herein is considered to be implementable with respect to any method, kit, reagent, or composition of the present invention, and vice versa. Furthermore, the compositions of the present invention can be used to implement the methods of the present invention.

[0080] The specific embodiments described herein are to be understood as being presented for illustrative purposes only, and not as limitations of the invention. The key features of the invention can be adopted in various embodiments without departing from the scope of the invention. Those skilled in the art will be able to recognize or confirm, by conventional experimentation, a great many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the invention and are covered by the claims.

[0081] All published materials and patent applications described herein suggest the skill level of a person skilled in the art to which the present invention relates. All published materials and patent applications are incorporated herein by reference to the same extent as if each individual published material or patent application were specifically and individually indicated to be incorporated by reference.

[0082] The use of the words "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, may mean "one," but the words can also mean "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims means "and / or" unless it is explicitly stated that only substitutes are being referred to, or that the substitutes are mutually exclusive. While used for this purpose, this disclosure supports the definitions of substitutes only and “and / or”. Throughout this application, the term “about” is used to indicate that there may be errors in the devices, methods employed to determine the numerical values, and that the numerical values ​​include their inherent variability or variability present among subjects.

[0083] As used herein and in the claims, the words "comprising" (and any form of "comprising," e.g., "comprise" and "comprises"), "having" (and Any form of having, e.g., "have" and "has"), "including" (and any form of including) Forms, such as "includes" and "include", or "~to "containing" (and any form of "containing," such as "contains" and "contain") is inclusive or This is non-restrictive and does not exclude additional unlisted properties, elements, components, groups, integers, and / or steps, but does not exclude the existence of other unmentioned properties, elements, components, groups, integers, and / or steps. In any of the multiple embodiments of compositions and methods presented herein, “comprising” may be replaced by “consisting essentially of” or “consisting of.” As used herein, the term “consisting” is used to indicate the existence of only the listed integers (e.g., properties, elements, features, characteristics, methods / process steps, or limitations) or groups of integers (e.g., properties, elements, features, characteristics, methods / process steps, or limitations). As used herein, the idiomatic phrase “consisting essentially of” refers to the existence of only the identified properties, features, and Elements, components, groups, integers, and / or steps are required, but the presence of other unmentioned characteristics, elements, components, groups, integers, and / or steps, as well as those that do not significantly affect the basic and novel features and / or functions of the claimed invention, is not excluded.

[0084] The term “or any combination thereof” means, as used herein, all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or any combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where the order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing this example, expressly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A person skilled in the art will generally understand that, unless otherwise evident from the context, there is no limit to the number of items or terms in any combination.

[0085] Where used herein, approximate words, such as non-limitingly "about," "substantial," or "substantially," refer to a state that, when modified in this way, is understood not necessarily absolute or complete, but which a person skilled in the art can reasonably be assured of as existing. The range of variation in the description depends on how significant the change may be, and, given such a change, the magnitude of the change that would still lead a person skilled in the art to perceive the modified characteristic as still possessing the required features and capabilities of the unmodified characteristic. Generally, but given the foregoing considerations, numerical values ​​in this specification modified by approximate words, such as "about," may vary by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15% from the stated numerical value.

[0086] Any composition and / or method disclosed and claimed herein is not applicable in light of this disclosure. Furthermore, these can be produced and carried out without excessive experimentation. Although the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the compositions and / or methods described herein, and to the steps of the methods, or to the order of the steps, without departing from the concept, spirit, and scope of the invention. Any such similar substitutes and modifications, which will be apparent to those skilled in the art, are considered to fall within the spirit, scope, and concept of the invention as defined by the appended claims.

[0087] To assist patent offices and any readers in interpreting the attached claims in relation to this specification of any patent issued pursuant to this application, the applicants would like to point out that, unless the words “means for” or “steps for” are expressly used in a particular claim, none of the attached claims are intended to evoke paragraph 6 of 35 U.S. SC § 112, paragraph (f), or equivalents, as they exist as of the filing date of this specification.

[0088] In each claim, each dependent claim may be dependent on both the independent claim and each of the preferred dependent claims to each of the claims, insofar as the preferred claim provides an appropriate antecedent for the terms or elements of the claim.

Claims

1. Structure below: A first light chain comprising a first variable light chain region and a first steady light chain region, Cuttable linker, A first heavy chain including a first variable heavy chain region and a first steady heavy chain region An activatable antibody (aAb) containing the following in order: The cleavable linker prevents or reduces the formation of a first antigen-binding site for the first antigen by the first variable light chain region and the first variable heavy chain region, and the first steady light chain region and the first steady heavy chain region are not disulfide-bonded, When the cleavable linker is cleaved, the first variable heavy chain region is released, enabling the formation of the first antigen-binding site that binds to the first antigen. The above aAb.

2. The aAb according to claim 1, wherein when the cleavable linker is cleaved, a first variable heavy chain region is released, and the first variable heavy chain region, together with the first variable light chain region, forms an antigen-binding site that binds to a first antigen.

3. The aAb according to claim 1 or 2, further comprising an Fc region on the carboxyl terminal side of the first steady heavy chain region.

4. Further comprising a second antigen-binding site for binding to a second antigen, formed by a second variable light chain region and a second constant light chain region connected to the first heavy chain, aAb according to any one of claims 1 to 3, wherein a flexible non-cleavable linker may be included between the second variable light chain region and the second steady light chain region.

5. The aAb according to any one of claims 1 to 3, wherein the first light chain, the first heavy chain, or both thereof further comprises an Fc region, a wild-type Fc region, a mutated Fc region, a monomeric wild-type Fc region, a monomeric mutant Fc region, a dimeric wild-type Fc region, or a dimeric mutant Fc region, a second variable heavy chain region and a second Fc region, or a second variable heavy chain region and a second Fc region and an uncleavable flexible linker and a second variable light chain region and a second heavy chain variable region, or a second Fc region and an uncleavable flexible linker and a cytokine.

6. The first antigen and the second antigen are They are the same antigen; The first antigen and the second antigen are different; The first antigen and the second antigen are the same antigen, but the first antigen-binding site and the second antigen-binding site bind to different epitopes of the same antigen; or The first antigen-binding site or the second antigen-binding site binds to the tumor target; At least one of the following applies: The first antigen mentioned above is ICAM1; VCAM1; EpCAM; extradomain B of fibronectin; melanoma-associated chondroitin sulfate proteoglycan (MCSP); melanoma-associated proteoglycan (MAPG); high molecular weight melanoma associated antigen (HMV-MAA); prostate-specific membrane antigen (PSMA); epidermal growth factor receptor (EGFR); hepatocyte growth factor receptor (HGFR); fibroblast activation protein (FAP); carcinoembryonic antigen (CEA); cell adhesion molecule (CAM); human B cell maturation target (BCMA) target); placental growth factor (PLGF); folate receptor, insulin-like growth factor receptor (ILGFR); CD133; CD40; CD37; CD33; CD30; CD28; CD24; CD23; CD22; CD21; CD20; CD19; CD13; CD10; HER3; HER2; nonmuscle myosin heavy chain type A (nmMHCA); transferrin; epithelial cell adhesion molecule (EpCAM); annexin A1; nucleotin, tenascin, vascular endothelial growth factor receptor 1 (VEGFR1); vascular endothelial growth factor receptor 2 (VEGFR-2); aminopeptidase N; tie-1; tie-2;It is a tissue-specific surface antigen selected from either c-Met or c-Met; The first antigen is mentioned above, ABCF1; ACVR1; ACVR1B; ACV R2;ACVR2B;ACVRL1;ADORA2A;アグリカン; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP 2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPT L3; ANGPTL4; ANPEP; APC; APOC1; AR; A ZGP1; B7.1; B7.2; BAD; BAFF; BAG1; BA I1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR 15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4;BMP6;BMP8;BMPR1A;BMPR1B;BM PR2; BPAG1 (プレクチン); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CA SP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (1-309); CCL11 (エオタキシン); CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC -4); CCL17 (TARC); CCL18 (PARC); CCL 19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL2 0 (MIP-3a); CCL21 (MIP-2); SLC; エクソダス(exodus)-2; CCL22(MDC / STC-1); CC L23 (MPIF-1); CCL24 (MPIF-2 / エオタキシン-2); CCL25 (TECK); CCL26 (エオタキシン-3) ; CCL27 (CTACK / ILC); CCL28; CCL3 (MI P-1a); CCL4 (MIP-1b); CCL5 (RANTES) ; CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR 1 / HM145); CCR2 (mcp-1RB / RA); CCR3 ( CKR3 / CMKBR3); CCR4; CCR5 (CMKBR5 / C hemR13); CCR6 (CMKBR6 / CKR-L3 / STRL 22 / DRY6); CCR7 (CKR7 / EBI1); CCR8 (CM KBR8 / TER1 / CKR-L1); CCR9 (GPR-9-6) ;CCRL1 (VSHK1); CCRL2 (L-CCR); CD16 4; CD19; CD1C; CD20; CD200; CD-22; CD 24; CD28; CD3; CD37; CD38; CD3E; CD3G;CD3Z; CD4; CD40; CD40L; CD44; CD45RB ;CD52; CD69; CD72; CD74; CD79A; CD79 B; CD8; CD80; CD81; CD83; CD86; CDH1 ( E-カドヘリン);CDH10;CDH12;CDH13;CDH1 8; CDH19; CDH20; CDH5; CDH7; CDH8; CD H9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK 7; CDK9; CDKN1A (p21Wap1 / Cip1); CDK NIB (p27Kip1); CDKNIC; CDKN2A (pl6IN K4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; FSF2; CKLFSF3; CKLFSF4; CKLFSF5; CK LFSF6;CKLFF7; N7(クローディン-7);CLN3;CXCL10(IP-10) ;CXCL11(I-TAC / IP-9);CXCL12(SDF1 ); CXCL13; CXCL14; CXCL16; CXCL2 (GR 02); CXCL3 (GR03); CXCL5 (ENA-78 / LEX ); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9 / CKR-L2); CXCR4; CXCR6 (TYMST R / STRL33 / Bonzo); CYB5; CYC1; CYSLT R1; CGRP; Clq; Clr; CI; C4a; C4b; C2a; C2b; C3a; C3b; DAB2IP; DES; DKFZp451 J0118; DNCL1; DPP4; E-セレクチン; E2F1; E CGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF ;EGFR; ELAC2; ENG; EN01; EN02; EN03; E PHB4; EPO; ERBB2 (Her-2); EREG; ERK8 ; ESR1; ESR2; F3 (TF); Factor VII; Factor IX; Factor V; Factor VIIa; Factor X; Factor XII; Factor XIII; FADD; FasL; FASN; FCER1A; FCER2; Fcガンマacceptor; FCGR3A; FGF; FGF1 (aFGF); FG F10; FGF11; FGF12; FGF12B; FGF13; FG F14; FGF16; FGF17; FGF18; FGF19; FGF 2(bFGF);FGF20;FGF21;FGF22;FGF23;FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (Epsilon); FIL1 (Zeta); FLJ12584; FLJ25530; FLRT1 (Fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1 ;GMCSF;GNAS1;GNRH1;GPR2(CCR10);GPR31;GPR44;GPR81(FKSG80);GRCC10(CIO);GRP;GSN(Gelsolin);GSTP1;Glycoprotein (GP) IIb / IIIa;HAVCR2;HDAC4;HDAC5;HDAC7A;HDAC9;Her2;HGF;ITGB4(b4 integrin);JAG1;JAK1;JAK3;JUN;K6HF;KAI1;KDR;KITLG;KLF5(GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (Hair-Specific Type II Keratin); L-Selectin; LAMA5; LEP (Leptin) ;Lingo-p75;Lingo-Troy;LPS;LTA(TNF-b);LTB;LTB4R(GPR16);LTB4R2;LTBR;MA CMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midocaine; MIF; MIP-2; MKI67 (Ki-67 ); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; Neurocan; NKG2D; NFKB1; NFKB2; NGF; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troi; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NRII2; NRII3; NR2C1; NR2C2;NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PARTI; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGE2; PGF; PGR; Phosphacan; PIAS2; PIK3CG; Plasminogen Activator; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; Protein C; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RAGE; RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROB02; SI00A2; SCGB1D2 (Lipophyllin B); SCGB2A1 (Mammaglobulin 2); SCGB2A2 (Mammaglobulin 1); SCYE1 (Endothelial Monocyte Activating Cytokine); SDF2; SERPINA1; SER PINA3; SERPINB5 (Maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; Substance P; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB111; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (Thrombosponge) THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TL R4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; RSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7 ; TNFSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (AP03L);TNFSF13 (April); TNFSF13B; TNFSF14 (HVEML); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); T Selected from proteins, protein segments, or peptides encoded by at least one gene selected from OLLIP; Toll-like receptor; TOP2A (topoisomerase IIa); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; thrombomodulin; thrombin; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5 / CCXCR1); YY1; and ZFPM2; or; The tumor target is selected from tumor target antigens, HER1, HER2, HER3, GD2, carcinoembryonic antigen (CEA), epidermal growth factor receptor active mutant (EGFRVIII), CD133, fibroblast activation protein (FAP) alpha, epithelial cell adhesion molecule (Epcam), glypican 3 (GPC3), EPH receptor A4 (EphA), tyrosine protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor-1 (WT-1), or claudin family proteins; aAb as described in claim 5.

7. At least one of the first antigen-binding site or the second antigen-binding site is bound to a T cell marker, and the T cell marker may be selected from CTLA4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, or TIM-3. Alternatively, at least one of the first antigen-binding site or the second antigen-binding site may bind to a T cell activator, and the T cell activator may be selected from CD3, 41BB, or OX40. aAb as described in claim 5.

8. The cleavable linker is a protease-cleavable linker, and The aforementioned severable linker Tumor-associated proteases: MMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, may be cleaved by uPA, FAPa, or cathepsin B. They may also be cleaved by proteases that are upregulated during apoptosis or inflammation-related responses. It may be cleaved by caspases, which may be caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, and caspase 12, or The cleavable linker does not obstruct the antigen-binding site. aAb according to claim 1, 2, or 3.

9. Further comprising a drug conjugated to aAb, a cytokine bound to an aAb or Fc region, or a fusion protein containing an aAb or Fc region, The cytokines mentioned above include growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormone; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; TNF-α; Müllerian inhibitory factor; gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor; platelet growth factor; placental growth factor, transforming growth factor (TGF); insulin-like growth factor-1 and -11; erythropoietin (EPO); bone induction factor; interferon; colony stimulating factor (CSF). at least one of the following: factor); lymphotoxin-alpha; lymphotoxin-beta; CD27L; CD30L; FASL; 4-1BBL; OX40L; TRAIL; IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-15; IL-18; IL-21; IL-22; IL-23; IL-33; IFN-a; IFN-b; IFN-g; IFN-g inducing factor (IGIF); bone morphogenetic protein (BMP); leukemia inhibitory factor (LIF); or kit ligand (KL); or The agent is at least one of the following: a toxin or a toxic fragment thereof; a microtubule inhibitor; a nucleic acid damaging agent; a detectable portion; a diagnostic agent; or a pharmaceutically acceptable carrier; aAb according to any one of claims 1 to 3.

10. The aAb according to claim 1, having the amino acid sequence of SEQ ID NO: 1, 2, or 3.

11. A nucleic acid encoding aAb according to claim 1, 2, or 3.

12. A cell comprising the nucleic acid encoding aAb according to claim 1, 2, or 3.

13. The cell according to claim 12, which is a T cell or a mesenchymal stem cell.

14. The cell according to claim 12, wherein aAb further comprises a transmembrane sequence that anchors aAb to the surface of a T cell to form a chimeric antigen receptor, and the cell is a CAR T cell.

15. A therapeutic agent for cancer, comprising ab according to any one of claims 1 to 3, wherein the method reduces the binding activity of an antibody to normal tissue and targets cancer cells; or a method for treating, alleviating, or delaying the progression of cancer symptoms, comprising the step of administering an effective amount of the ab to a subject in need thereof; the therapeutic agent, as used in a method for treating, alleviating, or delaying the progression thereof.

16. The therapeutic agent according to claim 15, wherein the cancer is a cancer that expresses an enzyme that cleaves a cleavable linker.

17. Cancer is bladder cancer, bone cancer, breast cancer, cancerous tumor, cervical cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, urogenital cancer, or urothelial carcinoma, acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, bladder urothelial carcinoma, mammary ductal carcinoma, mammary lobular carcinoma, esophageal cancer, castration-resistant prostate cancer (CRPC), cervical cancer, bile duct cancer, chronic myeloid leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC) A therapeutic agent according to claim 15, selected from carcinoma, renal pigmentophobic carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, low-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and chrome affinity cell tumor, prostate adenocarcinoma, renal cell carcinoma (RCC), sarcoma, cutaneous melanoma, squamous cell carcinoma of the head and neck, T-cell lymphoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrioid carcinoma of the uterus, or uveal melanoma.

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