Binding site for conditional activation of immunoglobulin molecules

Abstract

The present invention provides a binding moiety comprising a non-CDR loop for masking the binding of a binding molecule to its target and a CDR for binding to a bulk serum protein, and a conditionally active target-binding protein containing the binding moiety. Furthermore, it provides a pharmaceutical composition containing the binding protein and a method for using such a formulation. [Solution] A binding portion comprising a non-CDR loop and a cleavable linker, wherein the binding portion can mask the binding of a binding molecule to its target, wherein the binding molecule comprises an immunoglobulin molecule or a non-immunoglobulin molecule.

Description

[Technical Field]

[0001] cross reference This application claims the benefits under U.S. Provisional Patent Applications No. 62 / 671,344, filed May 14, 2018; No. 62 / 671,349, filed May 14, 2018; No. 62 / 756,429, filed November 6, 2018; and No. 62 / 756,453, filed November 6, 2018, each of which is incorporated herein by reference in whole.

[0002] Embedding by citation All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference, and as if they were described in whole. [Background technology]

[0003] T-cell engagers temporarily tether T cells to tumor cells, mediating T-cell-mediated tumor killing. T-cell engagers such as blinatumomab (BLINCYTO®) have shown clinical activity in several hematological malignancies. The uptake of T-cell engagers in solid tumors is limited by a lack of tumor antigens with sufficient differential expression between tumor and normal tissue. T-cell engagers that are preferentially active in the tumor microenvironment could enable safer targeting of a wider range of solid tumor antigens.

[0004] There is a need to extend the half-life of therapeutic, diagnostic, or imaging molecules in circulation and improve their ability to reach their intended targets within the intended site (e.g., tumor cells) without nonspecific binding. [Overview of the project]

[0005] In some embodiments, a binding portion is provided comprising a non-CDR loop and a cleavable linker, wherein the binding portion can mask the binding of a binding molecule to its target, wherein the binding molecule comprises an immunoglobulin molecule or a non-immunoglobulin molecule. In some embodiments, the portion is a native peptide, a synthetic peptide, an engineered scaffold, or an engineered bulk serum protein. In some embodiments, the engineered scaffold comprises sdAb, scFv, Fab, VHH, a fibronectin type III domain, an immunoglobulin-like scaffold, DARPin, a cystine knot peptide, lipocalin, a 3-helix bundle scaffold, a protein G-associated albumin-binding module, or an aptamer scaffold of DNA or RNA. In some embodiments, the portion can bind to a bulk serum protein. In some embodiments, the non-CDR loop is derived from a variable domain, a constant domain, a C1 set domain, a C2 set domain, an I domain, or any combination thereof. In some embodiments, the above portion further comprises a complementation-determining region (CDR). In some embodiments, the above portion can bind to a bulk serum protein. In some embodiments, the bulk serum protein is a half-life extension protein. In some embodiments, the bulk serum protein is albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentameric IgM. In some embodiments, the bulk serum protein is albumin, transferrin, factor XIII, or fibrinogen. In some embodiments, the CDR within the binding portion provides a binding site specific to the bulk serum protein. In some embodiments, the binding portion can mask the binding of its target antigen-binding domain (such as an immunoglobulin molecule) or non-immunoglobulin-binding molecule to its target via specific intermolecular interactions between the binding portion and the target antigen-binding domain or non-immunoglobulin-binding portion.In some embodiments, the non-CDR loop within the binding portion provides a binding site specific to the binding portion to a target antigen-binding domain (such as an immunoglobulin molecule) or a non-immunoglobulin-binding molecule.

[0006] In some embodiments, the binding portion includes a binding site specific to an immunoglobulin light chain. In some embodiments, the immunoglobulin light chain is an Igκ free light chain. In some embodiments, the CDR provides a binding site specific to bulk serum protein or an immunoglobulin light chain. In some embodiments, the immunoglobulin molecule is a target antigen-binding domain. In some embodiments, the binding portion binds to the target antigen-binding domain. In some embodiments, the binding portion covalently binds to the target antigen-binding domain. In some embodiments, the binding portion can mask the binding of the target antigen-binding domain to its target through a specific intermolecular interaction between the binding portion and the target antigen-binding domain. In some embodiments, a non-CDR loop provides a binding site specific to the binding of the binding portion to the target antigen-binding domain. In some embodiments, when a cleavable linker is cleaved, the binding portion separates from the target antigen-binding domain, and the target antigen-binding domain binds to its target. In some embodiments, the target antigen domain binds to a tumor antigen. In some embodiments, tumor antigens include EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, PSMA, CD38, BCMA, and CEA.5T4, AFP, B7-H3, CDH-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, and HPV-16 The domains include E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, CDH-3, CDH-17, EPHB2, ITGAV, ITGB6, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLITRK5, SLITRK6, STEAP1, TIM1, Trop2, or WT1. In some embodiments, the target antigen domain binds to an immune checkpoint protein.In some embodiments, the immune checkpoint protein is CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA. In some embodiments, the target antigen-binding domain binds to T cells. In some embodiments, the target antigen-binding domain binds to CD3. In some embodiments, the cleavable linker includes a cleavage site. In some embodiments, the cleavage site is recognized by a protease. In some embodiments, the protease cleavage site is recognized by serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, metalloproteinase, gelatinase, or asparagine peptide lyase.In some embodiments, the protease cleavage sites include cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, plasmepsin, and nepenthes It is recognized by metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM9, ADAM10, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, and mast cell tryptase.

[0007] One embodiment provides a conditionally active binding protein comprising a binding moiety (M) including a non-CDR loop, a cleavable linker (L), a first target antigen-binding domain (T1), and a second target antigen-binding domain (T2), wherein the first target antigen-binding domain (T1) comprises an immunoglobulin molecule, the non-CDR loop can bind to the first target antigen-binding domain, and the binding moiety can mask the binding of the first target antigen-binding domain to its target. In some embodiments, the binding moiety can bind to a half-life extension protein. In some embodiments, the binding moiety can bind to a native peptide, a synthetic peptide, an engineered scaffold, or an engineered serum bulk protein.In some embodiments, the manipulated scaffold comprises sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffold, DARPin, cystine knot peptide, lipocalin, 3-helix bundle scaffold, protein G-associated albumin-binding module, or a DNA or RNA aptamer scaffold. In some embodiments, the non-CDR loop is derived from a variable domain, a constant domain, a C1 set domain, a C2 set domain, an I domain, or any combination thereof. In some embodiments, the binding portion further comprises a complementarity-determining region (CDR). In some embodiments, the binding portion comprises a binding site specific to a bulk serum protein. In some embodiments, the bulk serum protein is albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentamer IgM. In some embodiments, the binding portion further comprises a binding site specific to an immunoglobulin light chain. In some embodiments, the immunoglobulin light chain is an Igκ free light chain. In some embodiments, the CDR provides a binding site specific to bulk serum protein or immunoglobulin light chain, or any combination thereof. In some embodiments, the binding site can mask the binding of the first target antigen-binding domain to its target via a specific intermolecular interaction between the binding site and the first target antigen-binding domain. In some embodiments, a non-CDR loop provides a binding site specific to the binding of the binding site to the first target antigen-binding domain. In some embodiments, the first or second target antigen-binding domain binds to a tumor antigen. In some embodiments, the tumor antigen includes at least one of the following: EpCAM (exemplary protein sequence includes UniProtkB ID No. P16422), EGFR (exemplary protein sequence includes UniProtkB ID No. P00533), HER-2 (exemplary protein sequence includes UniProtkB ID No. P04626), HER-3 (exemplary protein sequence includes UniProtkB ID No.(including No. P21860), c-Met (exemplary protein sequence includes UniProtkB ID No. P08581), FoIR (exemplary protein sequence includes UniProtkB ID No. P15238), PSMA (exemplary protein sequence includes UniProtkB ID No. Q04609), CD38 (exemplary protein sequence includes UniProtkB ID No. P28907), BCMA (exemplary protein sequence includes UniProtkB ID No. Q02223), and CEA (exemplary protein sequence includes UniProtkB ID No. P06731), 5T4 (exemplary protein sequence includes UniProtkB ID No. Q13641), AFP (exemplary protein sequence includes, includes, UniProtkB ID No. P02771), B7-H3 (exemplary protein sequence includes UniProtkB ID CDH-6 (exemplary protein sequence includes UniProtkB ID No. P97326), CAIX (exemplary protein sequence includes UniProtkB ID No. Q16790), CD117 (exemplary protein sequence includes UniProtkB ID No. P10721), CD123 (exemplary protein sequence includes UniProtkB ID No. P26951), CD138 (exemplary protein sequence includes UniProtkB ID No. P18827), CD166 (exemplary protein sequence includes UniProtkB ID No. Q13740), CD19 (exemplary protein sequence includes UniProtkB ID No. P15931), CD20 (exemplary protein sequence includes UniProtkB ID No. P11836), CD205 (exemplary protein sequence includes UniProtkB ID CD22 (exemplary protein sequence includes UniProtkB ID No. P20273), CD30 (exemplary protein sequence includes UniProtkB ID No. P28908), CD33 (exemplary protein sequence includes UniProtkB ID No. P20138), CD352 (exemplary protein sequence includes UniProtkB IDCD37 (exemplary protein sequence includes UniProtkB ID No. P11049), CD44 (exemplary protein sequence includes UniProtkB ID No. P16070), CD52 (exemplary protein sequence includes UniProtkB ID No. P31358), CD56 (exemplary protein sequence includes UniProtkB ID No. P13591), CD70 (exemplary protein sequence includes UniProtkB ID No. P32970), CD71 (exemplary protein sequence includes UniProtkB ID No. P02786), CD74 (exemplary protein sequence includes UniProtkB ID No. P04233), CD79b (exemplary protein sequence includes UniProtkB ID No. P40259), DLL3 (exemplary protein sequence includes UniProtkB ID (including No.Q9NYJ7), EphA2 (exemplary protein sequence includes UniProtkB ID No.P29317), FAP (exemplary protein sequence includes UniProtkB ID No.Q12884), FGFR2 (exemplary protein sequence includes UniProtkB ID No.P21802), FGFR3 (exemplary protein sequence includes UniProtkB ID No.P22607), GPC3 (exemplary protein sequence includes UniProtkB ID No.P51654), gpA33 (exemplary protein sequence includes UniProtkB ID No.Q99795), FLT-3 (exemplary protein sequence includes UniProtkB ID No.P36888), gpNMB (exemplary protein sequence includes UniProtkB ID No.Q14956), HPV-16 E6 (exemplary protein sequence includes UniProtkB ID (including No. P03126), HPV-16 E7 (exemplary protein sequence includes UniProtkB ID No. P03129), ITGA2 (exemplary protein sequence includes UniProtkB ID No. P17301), ITGA3 (exemplary protein sequence includes UniProtkB ID No. P26006), SLC39A6 (exemplary protein sequence includes UniProtkB ID(including No. Q13433), MAGE (exemplary protein sequence includes UniProtkB ID No. Q9HC15), Mesothelin (exemplary protein sequence includes UniProtkB ID No. Q13421), Muc1 (exemplary protein sequence includes UniProtkB ID No. P15941), Muc16 (exemplary protein sequence includes UniProtkB ID No. Q8WX17), NaPi2b (exemplary protein sequence includes UniProtkB ID No. O95436), Nectin-4 (exemplary protein sequence includes UniProtkB ID No. Q96918), CDH-3 (exemplary protein sequence includes UniProtkB ID No. Q8WX17), CDH-17 (exemplary protein sequence includes UniProtkB ID No. E5RJT3), EPHB2 (exemplary protein sequence includes UniProtkB ID (including No. P29323), ITGAV (exemplary protein sequence includes UniProtkB ID No. P06756), ITGB6 (exemplary protein sequence includes UniProtkB ID No. P18564), NY-ESO-1 (exemplary protein sequence includes UniProtkB ID No. P78358), PRLR (exemplary protein sequence includes UniProtkB ID No. P16471), PSCA (exemplary protein sequence includes UniProtkB ID No. O43653), PTK7 (exemplary protein sequence includes UniProtkB ID No. Q13308), ROR1 (exemplary protein sequence includes UniProtkB ID No. Q01973), SLC44A4 (exemplary protein sequence includes UniProtkB ID No. Q53GD3), SLITRK5 (exemplary protein sequence includes UniProtkB ID (including No. Q8IW52), SLITRK6 (exemplary protein sequence includes UniProtkB ID No. Q9HY7), STEAP1 (exemplary protein sequence includes UniProtkB ID No. Q9UHE8), TIM1 (exemplary protein sequence includes UniProtkB ID No. Q96D42), Trop2 (exemplary protein sequence includes UniProtkB ID(including No. P09758), or WT1 (exemplary protein sequence includes UniProtkB ID No. P19544), or any combination thereof. In some embodiments, the first or second target antigen-binding domain binds to an immune checkpoint protein. In some embodiments, the immune checkpoint protein is at least one of the following: CD27 (exemplary protein sequence includes UniProtkB ID No. P26842), CD137 (exemplary protein sequence includes UniProtkB ID No. Q07011), 2B4 (exemplary protein sequence includes UniProtkB ID No. Q9bZW8), TIGIT (exemplary protein sequence includes UniProtkB ID No. Q495A1), CD155 (exemplary protein sequence includes UniProtkB ID No. P15151), ICOSA (exemplary protein sequence includes UniProtkB ID No. Q9Y6W8), HVEM (exemplary protein sequence includes UniProtkB ID No. O43557), CD40L (exemplary protein sequence includes UniProtkB ID No. P29965), LIGHT (exemplary protein sequence includes UniProtkB ID (including No. O43557), OX40 (exemplary protein sequence includes UniProtkB ID No.), DNAM-1 (exemplary protein sequence includes, including UniProtkB ID No. Q15762), PD-L1 (exemplary protein sequence includes UniProtkB ID No. Q9ZQ7), PD1 (exemplary protein sequence includes UniProtkB ID No. Q15116), PD-L2 (exemplary protein sequence includes UniProtkB ID No. Q9BQ51), CTLA-4 (exemplary protein sequence is U CD8 (exemplary protein sequences include UniProtkB ID No. P10966 and P01732), CD40 (exemplary protein sequences include UniProtkB ID No. P25942), CEACAM1 (exemplary protein sequences include UniProtkB ID No. P13688), CD48 (exemplary protein sequences include UniProtkB ID No. P09326), CD70 (exemplary protein sequences include UniProtkB ID No. P32970), AA2AR (exemplary protein sequences include UniProtkB ID No. P29274), CD39 (exemplary protein sequences include UniProtkB ID No. P49961), CD73 (exemplary protein sequences include UniProtkB ID No. P21589), B7-H3 (exemplary protein sequences include UniProtkB ID (including No. Q5ZPR3), B7-H4 (exemplary protein sequence includes UniProtkB ID No. Q7Z7D3), BTLA (exemplary protein sequence includes UniProtkB ID No. Q76A9), IDO1 (exemplary protein sequence includes UniProtkB ID No. P14902), IDO2 (exemplary protein sequence includes UniProtkB ID No. Q6ZQW0), TDO (exemplary protein sequence includes UniProtkB ID No. P48755), KIR (exemplary protein sequence includes UniProtkB ID No. Q99706), LAG-3 (exemplary protein sequence includes UniProtkB ID No. P18627), TIM-3 (also known as HAVCR2, exemplary protein sequence includes UniProtkB ID No. Q8TDQ0), or VISTA (exemplary protein sequence includes UniProtkB ID No. Q9D659).

[0008] In some embodiments, the first or second target antigen-binding domain binds to immune cells. In some embodiments, the first or second target antigen-binding domain binds to T cells. In some embodiments, the first or second target antigen-binding domain binds to CD3. In some embodiments, the binding moiety (M), the cleavable linker (L), the first target antigen-binding domain (T1), and the second target antigen-binding domain (T2) are one of the following: configuration M:L:T1:T2 and configuration T2:T1:L:M. In some embodiments, the binding moiety includes an albumin-binding domain (anti-Alb), the first target antigen-binding domain (T1) includes a CD3-binding domain (e.g., anti-CD3scFV), and the ProTriTAC molecule has an anti-Alb:anti-CD3:T2 orientation. In some embodiments, the binding portion includes an albumin-binding domain (anti-Alb), the second target antigen-binding domain (T2) includes a CD3-binding domain (e.g., anti-CD3scFV), and the ProTriTAC molecule has an anti-Alb:T1:anti-CD3 orientation. The T1 domain is, in some examples, but not limited to, a tumor antigen-binding domain such as an anti-EGFR domain, anti-MSLN domain, anti-BCMA domain, anti-EpCAM domain, anti-PSMA domain, or anti-DLL3 domain.

[0009] In some embodiments, the cleavable linker includes a cleavage site. In some embodiments, the cleavage site is recognized by a protease. In some embodiments, the protease cleavage site is recognized by serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, metalloproteinase, gelatinase, or asparagine peptide lyase. In some embodiments, the protease cleavage sites include cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidine, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, plasmepsin, nepenthesin, meta It is recognized by roexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM9, ADAM10, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, and mast cell tryptase.In some embodiments, the conditionally active protein comprises a half-life extension domain attached to the binding moiety, where the half-life extension domain provides a safety switch to the binding protein, and where, upon cleavage of the linker, the binding moiety and the half-life extension domain are separated from the first target antigen-binding domain, thereby activating the binding protein, whereby the binding protein is separated from the safety switch. In some embodiments, cleavage of the linker is within the tumor microenvironment.

[0010] One embodiment provides a conditionally active binding protein comprising a binding moiety that binds to a target antigen-binding domain by a non-CDR loop within the binding moiety, where the binding moiety is further attached to a half-life extension domain and comprises a cleavable linker, where the target antigen-binding domain comprises an immunoglobulin molecule, where the binding protein has an extended half-life prior to its activation by cleavage of the linker, and where, upon activation, the binding moiety and the half-life extension domain are separated from the target antigen-binding domain, and where the binding protein in the activated state does not have an extended half-life. In some embodiments, cleavage of the linker is within the tumor microenvironment.

[0011] In some embodiments, the non-CDR loop comprises at least one CC’ loop of a camelid VHH domain, a human VH domain, a humanized VH domain, or a single domain antibody. In some embodiments, the binding moiety comprises a binding site specific for the CD3e domain, and the binding site specific for the CD3e domain comprises at least one of the following motifs: QDGNE, QDGNEE, DGNE, and DGNEE. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the present invention are, among other things, specified within the scope of the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which describes exemplary embodiments in which the principles of the present invention are used, and to the following appended drawings.

[0013] [Figure 1] An exemplary variable domain of an immunoglobulin domain is illustrated that includes complementarity determining regions (CDR1, CDR2, and CDR3) and non-CDR loops connecting β strands (AB, CC’, C’, D, EF, and DE). [Figure 2] Exemplary configurations of various domains of the conditionally active binding proteins of the present disclosure are provided. A: Version 1. B: Version 2. [Figure 3] An exemplary conditionally active target binding protein of the present disclosure is shown. [Figure 4] Activation and possible modes of action of a trispecific molecule (ProTriTAC) are shown. A shows ProTriTAC molecules in circulation, in the tumor environment, and in circulation. B shows an exemplary sequence for a protease cleavable site in a linker constrained to an anti-albumin binding moiety, and C shows an SDS-PAGE gel showing ProTriTAC in its activatable state (prodrug) as well as its activated state (active drug). [Figure 5] The process of making and purifying the molecules described herein is illustrated. A in FIG. 5 shows a schematic flowchart for the manufacture of ProTriTAC molecules, and C in FIG. 5 shows an SDS-PAGE gel showing three purified ProTriTAC molecules. [Figure 6] Analytical size exclusion chromatograms of ProTriTAC molecules exposed to various stress conditions in the graphical form in A are shown together with the corresponding data in B. B provides the data for FIG. 6A. [Figure 7] Exemplary ProTriTAC molecules exhibit protease-dependent antitumor activity in a colorectal tumor xenograft model of HCT116 in NSG mice. [Figure 8] Various designs of exemplary ProTriTAC and control molecules are shown. A: Control #1; B: Control #2; C: ProTriTAC; and D: Activated ProTriTAC. [Figure 9] Exemplary pharmacokinetic profiles of ProTriTAC and a control molecule are shown. [Figure 10] The transformation and half-life of an exemplary ProTriTAC molecule are shown. [Figure 11] This image illustrates the plasma clearance of an exemplary ProTriTAC molecule and its converted active drug format. [Figure 12] This paper shows an exemplary ProTriTAC molecule, its converted active drug format, and the CD3 binding ability of a control, non-cleavable ProTriTAC molecule. [Figure 13] This paper demonstrates the human primary T cell binding ability of an exemplary ProTriTAC molecule, its converted active drug format, and a control, non-cleavable ProTriTAC molecule. [Figure 14] This paper demonstrates an exemplary ProTriTAC molecule, its converted active drug format, and the T-cell killing potential of a control, non-cleavable ProTriTAC molecule. [Figure 15] The schematic structure of an exemplary triple-specific molecule containing the binding site described herein is shown (hereinafter also referred to herein as ProTriTAC or activatable ProTriTAC). [Figure 16]Exemplary schematic structures for prodrug molecules combining functional masking and half-life extension are shown. A shows a ProDrug molecule comprising an anti-albumin moiety containing a masking moiety and a cleavable linker connecting the anti-albumin moiety to the drug. B shows a ProDrug molecule comprising an anti-albumin moiety containing two peptide motifs (one of which contains the masking moiety) linked by a linker and a cleavable linker connecting the albumin-binding moiety to the drug. C shows a ProDrug molecule containing modified albumin (containing the masking moiety) bound to the drug by a cleavable linker. D shows a ProDrug molecule containing modified albumin (containing the masking moiety and protease cleavage site) bound to the drug. E shows an activated ProDrug. In each schematic structure (AD in Figure 16), the drug molecule is functionally masked by an anti-albumin moiety or modified albumin, either by binding to its target, being activated at an undesirable site, or by forming a drug sink by binding at a non-target site. [Figure 17]The following are exemplary schematic structures for ProTriTAC molecules that combine functional masking and half-life extension. A shows a ProTriTAC molecule containing an anti-albumin moiety with a masking moiety, and a cleavable linker connecting the anti-albumin moiety to a T-cell engager molecule. B shows a ProTriTAC molecule containing an anti-albumin moiety with two peptide motifs (one of which contains the masking moiety) linked by a linker, and a cleavable linker connecting the albumin-binding moiety to a T-cell engager molecule. C shows a ProTriTAC molecule containing modified albumin (containing the masking moiety) bound to a T-cell engager by a cleavable linker. D shows a ProTriTAC molecule containing modified albumin (containing the masking moiety and a protease cleavage site) bound to a T-cell engager. E shows activated ProTriTAC. In each schematic structure (AD in Figure 17), the target binding interface within the ProTriTAC molecule is functionally masked by the anti-albumin moiety or modified albumin, either by binding to the target, being activated at an undesirable site, or binding at a non-target site, thereby creating a sink. [Figure 18] The antitumor activity of exemplary ProTriTAC and TriTAC molecules of this disclosure is shown. [Figure 19] The pharmacokinetic profiles of exemplary ProTriTAC and TriTAC molecules of this disclosure are shown. [Figure 20] The following shows the respective tumor volumes of admix xenografts after administration of the exemplary ProTriTAC molecules or TriTAC molecules of this disclosure. A shows the results for GFP TriTAC. B shows the results for EGFR ProTriTAC (NCLV). C shows the results for EGFR ProTriTAC (L001). D shows the results for EGFR ProTriTAC (L041). E shows the results for EGFR ProTriTAC (L040). F shows the results for EGFR ProTriTAC (L045). [Figure 21]The cytokine levels (IFN-gamma (A in Figure 21), IL-6 (B in Figure 21), and IL-10 (C in Figure 21)) after administration of exemplary ProTriTAC molecules or TriTAC molecules of this disclosure are shown. [Figure 22] The figures show the percentage change in body weight in mice after administration of exemplary ProTriTAC and TriTAC molecules of this disclosure. A shows the result at 30 μg / kg; B shows the result at 100 μg / kg; C shows the result at 300 μg / kg; D shows the result at 1000 μg / kg; and E shows fold protection at various concentrations. [Figure 23] The percentage change in body weight in mice after administration of exemplary ProTriTAC molecules of this disclosure at various concentrations, containing either an uncleavable or cleavable linker. A shows the result at 300 μg / kg; B shows the result at 1000 μg / kg; C shows the multiplier protection at various concentrations. [Figure 24A] This shows the serum concentrations of aspartate aminotransferase (AST) in mice after administration of ProTriTAC molecules containing various concentrations of an uncleavable linker (ProTriTAC(NCLV)) (Figure 24C), TriTAC molecules (Figure 24A), or ProtriTAC molecules containing a cleavable linker (Figure 24B). [Figure 24B] This shows the serum concentrations of aspartate aminotransferase (AST) in mice after administration of ProTriTAC molecules containing various concentrations of an uncleavable linker (ProTriTAC(NCLV)) (Figure 24C), TriTAC molecules (Figure 24A), or ProtriTAC molecules containing a cleavable linker (Figure 24B). [Figure 24C] This shows the serum concentrations of aspartate aminotransferase (AST) in mice after administration of ProTriTAC molecules containing various concentrations of an uncleavable linker (ProTriTAC(NCLV)) (Figure 24C), TriTAC molecules (Figure 24A), or ProtriTAC molecules containing a cleavable linker (Figure 24B). [Figure 25A] The figures show serum alanine aminotransferase (ALT) concentrations in mice after administration of ProTriTAC molecules containing various concentrations of an uncleavable linker (ProTriTAC(NCLV)) (Figure 25C), TriTAC molecules (Figure 25A), or ProtriTAC molecules containing a cleavable linker (Figure 25B). [Figure 25B] The figures show serum alanine aminotransferase (ALT) concentrations in mice after administration of ProTriTAC molecules containing various concentrations of an uncleavable linker (ProTriTAC(NCLV)) (Figure 25C), TriTAC molecules (Figure 25A), or ProtriTAC molecules containing a cleavable linker (Figure 25B). [Figure 25C] The figures show serum alanine aminotransferase (ALT) concentrations in mice after administration of ProTriTAC molecules containing various concentrations of an uncleavable linker (ProTriTAC(NCLV)) (Figure 25C), TriTAC molecules (Figure 25A), or ProtriTAC molecules containing a cleavable linker (Figure 25B). [Figure 26] The serum concentrations of ALT (right panel; Figure 26B) or AST (left panel; Figure 26A) in cynomolgus monkeys after administration of various concentrations of EGFR ProTriTAC molecules or EGFR ProTriTAC (NCLV) molecules are shown. [Figure 27A] This chart shows tumor volume in mice after administration of GFP TriTAC, EGFR TriTAC, or EGFR ProTriTAC molecules at various concentrations. A represents GFP TriTAC (at 300 μg / kg) and EGFR TriTAC (at 10 μg / kg). [Figure 27B] This chart shows tumor volume in mice after administration of GFP TriTAC, EGFR TriTAC, or EGFR ProTriTAC molecules at various concentrations. B represents EGFR TriTAC (at 30 μg / kg and 100 μg / kg). [Figure 27C]This chart shows tumor volume in mice after administration of GFP TriTAC, EGFR TriTAC, or EGFR ProTriTAC molecules at various concentrations. C represents EGFR TriTAC (at 300 μg / kg) and EGFR ProTriTAC (at 30 μg / kg and 100 μg / kg). [Figure 27D] This chart shows tumor volume in mice after administration of GFP TriTAC, EGFR TriTAC, or EGFR ProTriTAC molecules at various concentrations. D represents EGFR ProTriTAC (at 300 μg / kg and 1000 μg / kg). [Figure 28] This shows serum ALT and AST concentrations in mice after administration of various concentrations of GFP TriTAC, EGFR TriTAC, and EGFR ProTriTAC molecules. [Figure 29] This disclosure shows the transplantation of the CD3ε epitope into the CC' loop of the coupling portion: HuCD3e:SEQ ID NO:901; CC10:SEQ ID NO:260; CC12:SEQ ID NO:259; and CC16:SEQ ID NO:261. [Figure 30] This shows the separation of the binding site of this disclosure from a ProTriTAC molecule containing the binding site during tumor-associated protease activation by matriptase. [Figure 31] This disclosure shows the CD3 binding of a ProTriTAC molecule, with or without activation, containing the exemplary binding moieties of this disclosure. [Figure 32] This disclosure demonstrates the cytotoxicity of ProTriTAC molecules, with or without activation, that include the exemplary binding moieties of this disclosure. [Figure 33] This disclosure describes a soft library mutagenesis approach performed to explore non-CDR loops within the exemplary junctions of this disclosure. [Figure 34]The results of a soft library mutagenesis approach performed to explore non-CDR loops within the exemplary binding region of this disclosure, after panning for HSA (human serum albumin, or albumin as referred herein), are illustrated. [Figure 35] This disclosure illustrates that both steric and specific masking can extend the therapeutic range of the binding moiety containing it (e.g., the ProTriTAC molecule). [Figure 36] This specification provides results from a representative T cell-dependent cytotoxicity assay using NCI-H508 cells, employing the exemplary fusion protein of this disclosure containing the anti-EpCAM and anti-CD3 domains described herein. [Figure 37] This specification provides results from a representative T cell-dependent cytotoxicity assay using an exemplary fusion protein of this disclosure containing the anti-EpCAM domain and anti-CD3 domain described herein. [Figure 38] This specification provides results from a representative T cell-dependent cytotoxicity assay using an exemplary fusion protein of this disclosure containing the anti-EpCAM domain and anti-CD3 domain described herein. [Figure 39] This specification provides results from a representative T cell-dependent cytotoxicity assay using an exemplary fusion protein of this disclosure containing the anti-EpCAM domain and anti-CD3 domain described herein. [Figure 40] This specification provides results from a representative T cell-dependent cytotoxicity assay using an exemplary fusion protein of this disclosure containing the humanized anti-EpCAM domain and anti-CD3 domain described herein. [Figure 41A] The percentage change in body weight in mice after administration of the exemplary EpCAM ProTriTAC and EpCAM TriTAC molecules described herein is shown. [Figure 41B] The percentage change in body weight in mice after administration of the exemplary EpCAM ProTriTAC and EpCAM TriTAC molecules described herein is shown. [Figure 41C] The percentage change in body weight in mice after administration of the exemplary EpCAM ProTriTAC and EpCAM TriTAC molecules described herein is shown. [Modes for carrying out the invention]

[0014] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided only as examples. Many modifications, changes, and substitutions will be conceivable without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents are encompassed thereby.

[0015] In one embodiment, a ProTriTAC molecule (also referred to herein as a protrisecific molecule) is provided herein, which is a T cell engager prodrug designed to be conditionally active in the tumor microenvironment. In some cases, this can target a broad selection of tumor antigens (e.g., solid tumor antigens). The ProTriTAC molecule combines, in some examples, desirable attributes of several prodrug approaches, including, but is not limited to: a combination of steric and specific masking, where steric masking is, in some cases, due to albumin recognized by the anti-albumin domain in the ProTriTAC molecule, and specific masking is, in some examples, due to specific intermolecular interactions between the anti-albumin domain (in some examples) and the target antigen-binding domain of the ProTriTAC molecule (in some examples, such as the anti-CD3scFv domain); enhanced safety provided by the prodrug-to-active drug half-life differential induced by the activation of the conditionally activated ProTriTAC molecule; and the ability to plug-and-play with various tumor target binders.

[0016] Specific definition The terms used herein are intended to illustrate only specific cases and are not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context otherwise expressly indicates. Furthermore, the terms “including,” “includes,” “having,” “has,” “with,” or any variation thereof, are intended to be comprehensive in a manner similar to the term “comprising,” to the extent that they are used in any of the detailed descriptions and / or claims.

[0017] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value, as determined by those skilled in the art, and this depends in part on how that value is measured or determined, for example, on the limitations of the measuring system. For example, “about” could mean a standard deviation of 1 or more for any given value. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” should be assumed to mean within an acceptable margin of error for that particular value.

[0018] The terms “individual,” “patient,” or “subject” are used interchangeably. None of the terms require, or are not limited to, a condition characterized by supervision (e.g., continuous or intermittent) of a healthcare worker (e.g., physician, registered nurse, nurse-practitioner, physician’s assistant, janitor, or hospice staff).

[0019] "Single-chain Fv" or "scFv," as used herein, refers to a binding protein in which the variable domains of the heavy chain and light chain of a conventional double-chain antibody bind to form a single chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and generation of the active binding site.

[0020] "Protease cleavage site" or "protease cleavage site" is, as used herein, an amino acid sequence that can be cleaved by a protease, such as matrix metalloproteinase or furin. Examples of such sites include Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln or Ala-Val-Arg-Trp-Leu-Leu-Thr-Ala, which can be cleaved by metalloproteinase, and Arg-Arg-Arg-Arg-Arg-Arg, which can be cleaved by furin. In therapeutic applications, protease cleavage sites can be cleaved by proteases produced by target cells (e.g., cancer cells, infected cells, pathogens).

[0021] As used herein, “elimination half-life” is used in its usual sense, as described in Goodman and Gilman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). In short, the term encompasses a quantitative measure of the time course of drug elimination. Since the drug concentration typically does not reach the concentration required for saturation of the elimination process, the elimination of most drugs is exponential (i.e., follows first-order kinetics). The rate of a rapid process is expressed by its rate constant k, which represents the fractional rate of change per unit of time, or by its half-life t, which is the time required for 50% of the process to be completed. 1 / 2 It can be expressed by the following. The units of these two constants are time, respectively. -1 and time. The first-order reaction rate constant and the reaction half-life are simply related (k × t). 1 / 2 (=0.693), and can be replaced as appropriate. Since the first-order elimination kinetics stipulates that a constant proportion of the drug is lost per unit of time, the logarithmic plot of drug concentration versus time is linear for all time after the initial distribution phase (i.e., after drug absorption and distribution are complete). The half-life of drug elimination can be accurately determined from such a graph.

[0022] "Therapeutic agent" includes "binding molecule" as used herein.

[0023] The term “binding molecule” as used herein is any molecule, or part or fragment thereof, that can bind to a target molecule, cell, complex, and / or tissue, and such binding molecules include proteins, nucleic acids, carbohydrates, lipids, low molecular weight compounds, and fragments thereof, each having the ability to bind to one or more soluble proteins, cell surface proteins, cell surface receptor proteins, intracellular proteins, carbohydrates, nucleic acids, hormones, or low molecular weight compounds (small molecule drugs), or fragments thereof. In some examples, binding molecules are proteins belonging to the immunoglobulin superfamily or non-immunoglobulin binding molecules. “Binding molecules” do not include cytokines.

[0024] The terms “proteins belonging to the immunoglobulin superfamily” or “immunoglobulin molecules” include, as used herein, proteins including immunoglobulin folds, such as antibodies and their target antigen-binding fragments, antigen receptors, antigen-presenting molecules, receptors on natural killer cells, antigen receptor accessory molecules, receptors on leukocytes, IgSF cell adhesion molecules, growth factor receptors, and receptor tyrosine kinases / phosphatases.

[0025] The term “antibody” includes any isotype of antibody or immunoglobulin, or a fragment of an antibody that retains specific binding to an antigen. Examples include, but are not limited to, fragments of Fab, Fv, scFv, and Fd, chimeric antibodies, humanized antibodies, single-chain antibodies (scAb), single-domain antibodies (dAb), single-domain heavy-chain antibodies, single-domain light-chain antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins containing the antigen-binding (also referred herein as antigen-binding) portion of an antibody as well as non-antibody proteins. Antibodies are, in some examples, detectably labeled using, for example, radioisotopes, enzymes that produce detectable products, or fluorescent proteins. Antibodies may, in some cases, further bind to other portions, such as members of a specific binding pair, for example, biotin (a member of the biotin-avidin specific binding pair). Antibodies may, in some cases, bind to a solid support, including, but not limited to, a polystyrene plate or beads. The above terms further encompass Fab', Fv, F(ab')2, and / or other antigen-binding fragments that retain specific binding to an antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a population of identical cells, all of which are produced from a single cell by repetitive cell replication. That is, a clone of a cell produces only a single antibody species. Monoclonal antibodies can be produced using hybridoma production techniques, but other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). Antibodies are monovalent or bivalent in some examples. Antibodies are Ig monomers in some examples and are a "Y-shaped" molecule consisting of four polypeptide chains, i.e., two heavy chains and two light chains linked by disulfide bonds.

[0026] The term "non-immunoglobulin-binding molecule" as used herein includes, but is not limited to, growth factors, hormones, signaling proteins, inflammatory mediators, ligands, receptors or fragments thereof, native hormones or their variants that can bind to their native receptors; nucleic acid or polynucleotide sequences that can bind to complementary sequences or soluble cell surface or intracellular nucleic acid / polynucleotide-binding proteins, other carbohydrate-binding moieties, carbohydrate-binding moieties that can bind to cell surface or intracellular proteins, and low molecular weight compounds (drugs) of soluble or cell surface or intracellular target proteins. Non-immunoglobulin-binding molecules may also include coagulation factors, plasma proteins, fusion proteins, and imaging agents. Non-immunoglobulin-binding molecules do not include cytokines.

[0027] "Cytokines," as used herein, refer to intercellular signaling molecules, and their active fragments and portions, which are involved in the regulation of mammalian somatic cells. Cytokines include many families, such as interleukins, interferons, and transforming growth factors.

[0028] As used herein, the “non-CDR loop” within an immunoglobulin (Ig) molecule is a region of the polypeptide other than the antibody’s complementarity-determining region (CDR). These regions may originate from the antibody or antibody fragment. These regions may also be synthetically or artificially induced, such as by mutagenesis or polypeptide synthesis.

[0029] In Ig, Ig-like, or β-sandwich scaffolds with nine β-strands (e.g., VH, VL, Camelidae VHH, sdAb), non-CDR loops may refer to AB, CC'C''D, EF loops, or loops connecting the proximal β-strand to the C-terminus. In Ig, Ig-like, or β-sandwich scaffolds with seven β-strands (e.g., CH, CL, adnectin, Fn-III), non-CDR loops may refer to AB, CD, and EF loops, or loops connecting the proximal β-strand to the C-terminus. In other Ig-like or β-sandwich scaffolds, non-CDR loops are loops connecting the proximal β-strand to the C-terminus or a topologically equivalent residue, using the framework established in Halaby's 1999 publication (Prot Eng Des Sel 12:563-571).

[0030] In non-β sandwich scaffolds (e.g., DARPin, affimer, affibody), the “non-CDR loop” refers to a region that (1) is susceptible to sequence randomization to enable manipulated specificity for a second antigen, and (2) is distal to the primary specificity-determining region typically used on the scaffold to enable simultaneous engagement of the scaffold to both antigens without steric hindrance. For this purpose, the primary specificity-determining region can be defined using the framework established in Skrlec’s 2015 publication (Trends in Biotechnol, 33:408-418). An excerpt of the above framework is shown below.

[0031] [Table 1]

[0032] The term "target antigen-binding domain" refers to a region that targets a specific antigen, as used herein. A target antigen-binding domain includes, for example, sdAb, scFv, variable heavy chain antibodies (VHH), variable heavy domains (VH) or variable light domains (VL), full-length antibodies, or other peptides having binding affinity to a specific antigen. A target antigen-binding domain does not include cytokines.

[0033] "TriTAC" refers to a conditionally inactive triple-specific binding protein, as used herein.

[0034] The binding site, the cleavable linker, and the conditionally active binding protein. This disclosure provides binding moieties that, in some embodiments, can mask the interaction between binding molecules and their targets. In some embodiments, the binding moieties of this disclosure include a masking moiety and a cleavable linker, such as a protease-cleavable linker. In some embodiments, the binding moieties of this disclosure include a masking moiety (e.g., a modified non-CDR loop sequence) and a non-cleavable linker. As illustrated in Figure 35, binding moieties can synergistically extend the therapeutic range of molecules containing the moiety through both steric and specific masking. In some examples, the binding molecule is a protein belonging to the immunoglobulin superfamily, such as a target antigen-binding domain containing an immunoglobulin fold. In some embodiments, the binding molecule is a non-immunoglobulin protein. In some embodiments, the binding moiety combines both steric masking (e.g., by binding to a large amount of serum albumin) and specific masking (e.g., by a non-CDR loop that binds to the CDR of an anti-CD3scFv domain). In some cases, modification of the non-CDR loop within the binding moiety does not affect albumin binding. Protease-cleavable linkers, in some cases, enable the activation of prodrug molecules containing binding sites (such as ProTriTAC molecules containing the binding sites, CD3-binding domains, and albumin-binding domains described herein) in a single proteolytic event, thereby enabling more efficient conversion of prodrug molecules in the tumor microenvironment. Furthermore, activation of tumor-associated proteolytic events may, in some cases, reveal active T cell engagers (such as ProTriTAC molecules containing the binding sites, CD3-binding domains, and albumin-binding domains described herein) that exhibit minimal off-tumor activity after activation. In some embodiments, this disclosure provides extended half-lives for T cell engager formats containing the binding sites described herein (ProTriTAC), which in some cases represent a novel and improved approach to manipulating conditionally active T cell engagers.

[0035] Figure 4B provides a schematic diagram of a gel showing an exemplary ProTriTAC molecule containing the exemplary binding site (α-albumin sdAb) described herein, and ProTriTAC before and after activation by cleaving a protease-cleavable linker, while Figure 4A shows a possible mode of action of ProTriTAC. Figure 15 shows a schematic structure of an exemplary tripspecific molecule (also referred to herein as ProTriTAC or activatable ProTriTAC) containing the binding site described herein, having an engineered non-CDR loop. The exemplary tripspecific molecule contains an anti-albumin domain, which includes a cleavable linker (also referred to herein as a substrate linker, such as a linker containing a protease-cleavable site) and a masking domain; an anti-CD3 binding domain; and optionally an anti-target domain (specific to a tumor antigen), which is a non-immunoglobulin molecule. Optionally, the non-CDR loop of the anti-albumin domain can bind to and mask the anti-target domain. In some cases, the non-CDR loop of the anti-albumin domain can bind to and mask the anti-CD3 domain. In some embodiments, the binding portion includes a CDR loop specific to the bound albumin.

[0036] In a first embodiment, a binding portion is provided herein that can mask the binding of a target antigen-binding domain and bind to a bulk serum protein such as a half-life extension protein. In some examples, the binding portion of the first embodiment further includes a cleavable linker attached thereto. The cleavable linker includes, for example, a protease cleavage site or a pH-dependent cleavage site. In some examples, the cleavable linker is cleaved only within the tumor microenvironment. Thus, in some examples, the binding portion of the first embodiment, which is bound to a half-life extension protein, linked to a cleavable linker, and further bound to a target antigen-binding domain, maintains the target antigen-binding domain in an inactive state in circulation until the cleavable linker is cleaved within the tumor microenvironment. Thus, the half-life of the target antigen-binding domain, such as an antibody or its antigen-binding fragment, is extended in systemic circulation by the use of the binding portion of the first embodiment acting as a safety switch, the safety switch maintaining the target antigen-binding domain in an inactive state until it reaches the tumor microenvironment where the target antigen-binding domain is conditionally activated by cleavage of the linker and can bind its target antigen.

[0037] In a second embodiment, a binding portion is provided that masks the interaction between a non-immunoglobulin-binding molecule and its target. In some examples, the binding portion of the second embodiment can bind to a bulk serum protein. In some examples, the binding portion of the second embodiment further includes a cleavable linker attached thereto. The cleavable linker includes, for example, a protease cleavage site or a pH-dependent cleavage site. In some examples, the cleavable linker is cleaved only within the tumor microenvironment. The non-immunoglobulin-binding molecule is, in some cases, kept inactive by the binding portion of the second embodiment and activated, for example, by cleavage of the linker in the target environment. In some examples, the cleavable linker is cleaved within the tumor microenvironment, in which case the tumor microenvironment is the target environment. Thus, the half-life of the non-immunoglobulin-binding molecule is extended in systemic circulation by the use of the binding portion of the second embodiment acting as a safety switch, which keeps the non-immunoglobulin-binding molecule inactive until it reaches a target environment where it is conditionally activated by cleavage of the linker. In some examples of the second embodiment, where the non-immunoglobulin-binding molecule is the contrast agent, the contrast agent is activated in the target environment when a cleavable linker is cleaved. The target environment is, in such cases, a tissue or cell, or any biological environment to be imaged using the contrast agent.

[0038] The safety switch described above offers several advantages, which in some cases include: (i) extending the therapeutic range of immunoglobulin molecules, such as target antigen-binding domains, and non-immunoglobulin-binding molecules; (ii) reducing target-mediated pharmacokinetics by maintaining immunoglobulin molecules, such as target antigen-binding domains, and non-immunoglobulin-binding molecules in an inactive state when conditionally active proteins containing binding portions according to the first or second embodiment are in systemic circulation; (iii) reducing the concentration of undesirable activated proteins in systemic circulation, thereby minimizing the diffusion of chemical, product-related, and control-related impurities, such as pre-activated drug products, endogenous viruses, host cell proteins, DNA, leachables, anti-foam, antibiotics, toxins, solvents, and heavy metals; and (iv) reducing the concentration of undesirable activated proteins in systemic circulation, thereby minimizing product-related impurities, aggregates, degradation products, oxidation, deamide, denaturation, and C-term in MAb. To minimize the spread of product variants due to the loss of Lys: (v) to prevent abnormal activation of immunoglobulin molecules or non-immunoglobulin-binding molecules, such as target antigen-binding domains, in circulation; (vi) to reduce toxicity associated with the leakage of reactive species from diseased tissue or other pathophysiological conditions, such as tumors, autoimmune diseases, inflammation, viral infections, tissue remodeling events (such as myocardial infarction and skin wound healing), or external injuries (X-ray, CT scan, ultraviolet exposure); and (vii) to reduce nonspecific binding of immunoglobulin molecules or non-immunoglobulin-binding molecules, such as target antigen-binding domains. Furthermore, after activation, or in other words, after the disruption of the safety switch, immunoglobulin molecules and non-immunoglobulin-binding molecules, such as target antigen-binding domains, are separated from the safety switch, which has provided an extended half-life, and are therefore removed from circulation.

[0039] In addition, the binding portions of the first, second, and third embodiments are sometimes used to generate a "biobetter" version of an organism. Typically, preparing a biobetter form of a molecule (e.g., an antibody, or its antigen-binding fragment) involves taking an originator molecule and making specific modifications to it to improve its parameters, thereby making the molecule a more effective, less frequently administered, better targeted, and / or better tolerated drug. Thus, a target antigen-binding domain masked by the binding portion of the first embodiment, which is bound to a half-life-extending protein and conditionally activated in the tumor microenvironment by cleavage of a cleavable linker, gives the target antigen-binding domain a significantly longer serum half-life and reduces the likelihood of its undesirable activation in circulation, thereby producing a "biobetter" version of the target antigen-binding domain. Similarly, the binding portion of the second embodiment is sometimes used to generate a biobetter version of a non-immunoglobulin-binding molecule. Therefore, in various embodiments, a biobetter version of the immunoglobulin molecule and the non-immunoglobulin-binding molecule is provided, where the biobetter function is due to the binding portion of the first or second embodiment, respectively.

[0040] The binding portions described herein include at least one non-CDR loop. In some embodiments, the non-CDR loop provides a binding site for binding the binding portion of the first embodiment to a target antigen-binding domain. In some examples of the first embodiment, the non-CDR loop provides a binding site for binding the binding portion of the first embodiment to an immunoglobulin molecule, such as a target antigen-binding domain. In some examples of the second embodiment, the non-CDR loop provides a binding site for binding the binding portion of the second embodiment to a non-immunoglobulin-binding molecule. In some cases, the binding portion of the first embodiment masks the binding of the target binding domain to the target antigen, for example, via steric occlusion, via specific intermolecular interactions, or a combination of both. The binding portion of the second embodiment also, in some cases, masks the binding of non-immunoglobulin-binding molecules to their targets, via steric occlusion, via specific intermolecular interactions, or a combination of both.

[0041] In some embodiments, the binding region described herein further comprises a complementarity-determining region (CDR). In some examples, the binding region is a domain derived from an immunoglobulin molecule (Ig molecule). Ig may be any class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM). The polypeptide chain of the Ig molecule folds into a series of parallel β-strands linked by loops. In the variable region, three of the loops constitute a "complementarity-determining region" (CDR) that determines the antigen-binding specificity of the molecule. The IgG molecule comprises at least two heavy (H) chains and two light (L) chains, or their antigen-binding fragments, interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are hypervariable in sequence and / or involved in antigen recognition and / or typically form a structurally defined loop interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of the following three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of this disclosure, at least some, or all, of the amino acid sequences of FR1, FR2, FR3, and FR4 are the “non-CDR loop” portion of the binding region described herein. As shown in Figure 1, the variable domain of an immunoglobulin molecule has several β-strands arranged on two sheets. Both the light-chain and heavy-chain immunoglobulins have variable domains containing three hypervariable loops, or complementarity-determining regions (CDRs). The three CDRs of the V domain (CDR1, CDR2, CDR3) cluster at one end of the β-barrel.The CDR is a loop connecting the β-strands BC, C'-C'', and FG of the immunoglobulin fold, while the bottom loop connecting the β-strands AB, CC', C''-D, and EF of the immunoglobulin fold, and the top loop connecting the DE strand of the immunoglobulin fold, are non-CDR loops. In some embodiments of this disclosure, at least several amino acid residues of the constant domain CH1, CH2, or CH3 are part of the “non-CDR loops” of the binding moieties described herein. In some embodiments, the non-CDR loops include the AB, CD, EF, and DE loops of the C1 set domain of Ig or an Ig-like molecule; the AB, CC', EF, FG, BC, and EC' loops of the C2 set domain of Ig or an Ig-like molecule; and one or more of the DE, BD, GF, A(A1A2)B, and EF loops of the I(intermediate) set domain of Ig or an Ig-like molecule.

[0042] Within the variable domain, CDRs are thought to be responsible for antigen recognition and binding, while FR residues are considered to be scaffolds for CDRs. However, in some cases, some FR residues play a crucial role in antigen recognition and binding. Framework region residues that influence Ag binding can be divided into two categories. The first category consists of FR residues that connect the antigen and are therefore part of the binding site, and some of these residues are close to the CDR in the sequence. The other residues are far from the CDR in the sequence but are close to the CDR in the 3D structure of the molecule (e.g., loops in the heavy chain).

[0043] In some embodiments, non-CDR loops are modified to generate antigen-binding sites specific to bulk serum proteins such as albumin. Various techniques, e.g., site-directed mutagenesis, random mutagenesis, insertion of at least one exogenous amino acid into the amino acid sequence of the non-CDR loop, and amino acid substitution, are intended to be used to modify the non-CDR loops. In some examples, antigen peptides are inserted into the non-CDR loops. In some examples, antigen peptides are used instead of the non-CDR loops. Modifications may be located in as few as one non-CDR loop to generate an antigen-binding site. In other examples, more than one non-CDR loop is modified. For example, a modification may be located in any one of the non-CDR loops shown in Figure 1 (i.e., AB, CC', C''-D, EF, and DE). In some cases, the modification may be located in the DE loop. In other cases, the modification may be located in all four loops: AB, CC', C''-D, and EF. In some cases, the binding sites described herein bind to immunoglobulin molecules (e.g., target antigen-binding domains) and non-immunoglobulin-binding molecules via their AB, CC', C''D, or EF loops, and to bulk serum proteins such as albumin via their BC, C'-C'', or FG loops.

[0044] In one example, the binding portion of the first embodiment binds to the target antigen-binding domain via its AB, CC', C''D, and EF loops, and to bulk serum proteins such as albumin via its BC, C'C'', and FG loops. In one example, the binding portion of the first embodiment binds to the target antigen-binding domain via one or more of the AB, CC', C''D, and EF loops, and to bulk serum proteins such as albumin via one or more of the BC, C'C'', and FG loops. In one example, the binding portion of the first embodiment binds to bulk serum proteins such as albumin via its AB, CC', C''D, or EF loops, and to the target antigen-binding domain via its BC, C'C'', or FG loops. In one example, the binding portion of the first embodiment binds to bulk serum proteins such as albumin via its AB, CC', C''D, and EF loops, and to the target antigen-binding domain via its BC, C'C'', and FG loops. In one example, the binding portion of the first embodiment binds to a bulk serum protein such as albumin via one or more of the AB, CC', C''D, and EF loops, and to a target antigen-binding protein via one or more of the BC, C'C'', and FG loops.

[0045] In one example, the binding portion of the second embodiment binds to a non-immunoglobulin molecule via one or more AB, CC', C''D, and EF loops, and to a bulk serum protein such as albumin via one or more BC, C'C'', and FG loops. In one example, the binding portion of the second embodiment binds to a bulk serum protein such as albumin via its AB, CC'C''D, or EF loop, and to a non-immunoglobulin molecule via its BC, C'C'', or FG loop. In one example, the binding portion of the second embodiment binds to a bulk serum protein such as albumin via its AB, CC', C''D, and EF loop, and to a non-immunoglobulin molecule via its BC, C'C'', and FG loop. In one example, the binding portion of the second embodiment binds to a bulk serum protein such as albumin via one or more AB, CC', C'', D, and EF loops, and to a non-immunoglobulin molecule via one or more BC, C'C'', and FG loops. The binding portion is any type of polypeptide.

[0046] For example, in some cases, the binding site is a native peptide, a synthetic peptide, or a fibronectin scaffold, or an engineered bulk serum protein. Bulk serum proteins include, for example, albumin, fibrinogen, or globulin. In some embodiments, the binding site is an engineered scaffold. Engineered scaffolds include, for example, sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffolds (as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine knot peptide, lipocalin, a 3-helix bundle scaffold, a protein G-associated albumin-binding module, or an aptamer scaffold of DNA or RNA.

[0047] In some cases, the binding portion of the first embodiment binds to at least one target antigen-binding domain. In further embodiments, a non-CDR loop within the binding portion of the first embodiment provides a binding site to at least one target antigen-binding domain. The target antigen-binding domain may optionally bind to a target antigen expressed on the surface of a diseased cell or abnormal tissue (e.g., tumor or cancer cells). Target antigens include, but are not limited to, EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, PSMA, CD38, BCMA, and CEA.5T4, AFP, B7-H3, CDH-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, and HPV-16. Includes E7, ITGA2, ITGA3, SLC39A6, MAGE, Mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, CDH-3, CDH-17, EPHB2, ITGAV, ITGB6, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLITRK5, SLITRK6, STEAP1, TIM1, Trop2, or WT1.

[0048] In some cases, the binding portion of the first embodiment binds to a first target antigen-binding domain via its non-CDR loop, and the first target antigen-binding domain is further connected to a second target antigen-binding domain. Examples of the first and second target antigen-binding domains include, but are not limited to, T cell engagers, bispecific T cell engagers, biaffinity retargeting antibodies, variable heavy domains (VH), variable light domains (VL), scFv containing VH and VL domains, soluble TCR fragments containing Valpha and Vbeta domains, single-domain antibodies (sdAb), or variable domains (VHH), non-Ig binding domains of camelid nanobodies, namely antikalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPin, fynomers, Knitz domain peptides, and monobodies, ligands, or peptides. In some examples, the first or second target antigen-binding domain is a VHH domain. In some examples, the first or second target antigen-binding domain is sdAb. In some examples, the first target antigen-binding domain is specific to a tumor antigen such as EGFR, and the second target antigen-binding domain is specific to CD3. The binding of the first target antigen-binding domain to its target, e.g., a tumor antigen such as EGFR, is masked by the binding moiety of the first embodiment via its non-CDR loop. One exemplary conditional active protein containing the binding moiety of the first embodiment is shown in Figure 3.

[0049] In some cases, the non-CDR loop within the binding portion of the second embodiment provides a binding site for non-immunoglobulin-binding molecules. In some cases, the binding portion includes a binding site for bulk serum proteins. In some embodiments, the CDR within the binding portion provides a binding site for bulk serum proteins. Bulk serum proteins are, in some examples, globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentamer IgM.

[0050] In some embodiments, the binding portion includes a binding site for an immunoglobulin light chain. In some embodiments, the CDR provides a binding site for the immunoglobulin light chain. The immunoglobulin light chain is, in some examples, an Igκ free light chain or an Igλ free light chain.

[0051] In some examples, the binding site includes any type of binding domain, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding site is a single-chain variable fragment (scFv), a soluble TCR fragment, a single-domain antibody, e.g., a heavy-chain variable domain (VH), a light-chain variable domain (VL), and a variable domain (VHH) from a camelid nanobody. In other embodiments, the binding site is a non-Ig binding domain, i.e., antikalin, affilin, affibody molecule, affimer, afitin, alpha-body, avimer, DARPin, finomer, Knitz domain peptide, and mono-body.

[0052] [Table 2-1]

[0053] [Table 2-2]

[0054] In some embodiments of this disclosure, the binding moieties described herein are intended to include at least one cleavable linker. In one embodiment, the cleavable linker comprises a polypeptide having a sequence that is recognized in a sequence-specific manner and cleaved. The cleavage is, in some examples, enzymatic or chemical, based on the pH sensitivity of the cleavable linker. The conditionally active binding proteins intended herein may optionally include a protease-cleavable linker recognized in a sequence-specific manner by a matrix metalloproteinase (MMP), e.g., MMP9. In some cases, the protease-cleavable linker comprises a polypeptide recognized by MMP9 and having the amino acid sequence PR(S / T)(L / I)(S / T). In some cases, the protease-cleavable linker recognized by MMP9 comprises a polypeptide having the amino acid sequence LEATA. In some cases, the protease-cleavable linker is recognized in a sequence-specific manner by MMP11. In some cases, the protease-cleavable linker recognized by MMP11 contains a polypeptide having the amino acid sequence GGAANLVRGG (SEQ ID NO: 3). In some cases, the protease-cleavable linker is recognized by the proteases disclosed in Table 3. In some cases, the protease-cleavable linker is recognized by the proteases disclosed in Table 3 and contains a polypeptide having an amino acid sequence selected from the sequences disclosed in Table 3 (SEQ ID NO: 1-42, 53, and 58-62). In some examples, the cleavable linker has the amino acid sequence described in SEQ ID NO: 19. In some cases, the cleavable linker is recognized by MMP9, matryptase, and urokinase-type plasminogen activator (uPA) and has the amino acid sequence described in SEQ ID No. 59.

[0055] In some embodiments of this disclosure, the linking portion described herein includes at least one non-cuttable linker. In some examples, the non-cuttable linker includes the sequence described in SEQ ID No. 51, SEQ ID No. 302, SEQ ID No. 303, SEQ ID No. 304, or SEQ ID No. 305.

[0056] [Table 3]

[0057] Proteases are proteins that, in some cases, cleave other proteins in a sequence-specific manner. Proteases include, but are not limited to, serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, metalloprotease, asparagine peptide lyase, serum protease, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, and stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidine, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, and HSV protease. Examples include mast cell chymase, CMV protease, chymosin, renin, pepsin, matryptase, regmine, plasmmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, type II transmembrane serine protease (TTSP), neutrophil serine protease, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, and mast cell tryptase.

[0058] [Table 4-1]

[0059] [Table 4-2]

[0060] Proteases are known to be secreted by several abnormal cells and tissues (e.g., tumors, cancer cells) and create a protease-rich or protease-enriched microenvironment. In some cases, the subject's blood is protease-rich. In some cases, cells surrounding a tumor secrete proteases into the tumor microenvironment. Cells surrounding a tumor that secrete proteases include, but are not limited to, tumor stromal cells, myofibroblasts, blood cells, mast cells, B cells, NK cells, regulatory T cells, macrophages, cytotoxic T lymphocytes, dendritic cells, mesenchymal stem cells, polymorphonuclear leukocytes, and other cells. In some cases, proteases are present in the subject's blood (e.g., proteases that target amino acid sequences found in microbial peptides). Since T cells are not bound by antigen-binding proteins except in the protease-rich microenvironment of the target cell or tissue, this characteristic allows targeted therapeutic agents, such as antigen-binding proteins, to have greater specificity.

[0061] Therefore, the binding moiety containing a cleavable linker masks binding to the respective targets of the first or second target antigen-binding domain. In some embodiments, the binding moiety binds to the first target antigen-binding domain in the order of binding moiety (M): cleavable linker (L): first target antigen-binding domain (T1): second antigen-binding domain (T2), which is then further bound to the second target antigen-binding domain. In other examples, the domain is constructed in one of the following orders: M:L:T2:T1;T2:T1:L:M, T1:T2:L:M. The binding moiety binds to half-life extension proteins, such as albumin or any other target of its kind described below. In some examples, the binding moiety is albumin or contains a binding site for albumin. In some examples, the binding moiety contains a binding site for IgE. In some embodiments, the binding moiety contains a binding site for Igκ free light chain.

[0062] [Table 5]

[0063] Targets of conditionally active binding proteins The conditionally active binding proteins described herein are activated by the cleavage of at least one cleavable linker attached to the binding site within the conditionally active protein. In some cases, the activated binding proteins are intended to bind to target antigens involved in and / or associated with a disease, disorder, or condition. In particular, target antigens associated with proliferative disorders, neoplastic diseases, inflammatory diseases, immunodeficiencies, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases are intended to be targets for the activated binding proteins described herein.

[0064] In some embodiments, the target antigen is a tumor antigen expressed on tumor cells. Tumor antigens are well known in the art and include, for example, EpCAM, EGFRHER-2, HER-3, c-Met, FoIR, PSMA, CD38, BCMA, and CEA.5T4, AFP, B7-H3, CDH-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 Includes E7, ITGA2, ITGA3, SLC39A6, MAGE, Mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, CDH-3, CDH-17, EPHB2, ITGAV, ITGB6, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLITRK5, SLITRK6, STEAP1, TIM1, Trop2, or WT1.

[0065] In some embodiments, the target antigen is an immune checkpoint protein. Examples of immune checkpoint proteins include, but are not limited to, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, KIR, LAG-3, TIM-3, or VISTA.

[0066] In some embodiments, the target antigen is a cell surface molecule such as a protein, lipid, or polysaccharide. In some embodiments, the target antigen is tumor cells, virus-infected cells, bacterial-infected cells, damaged red blood cells, arterial plaque cells, inflammatory tissue cells, or fibrotic tissue cells. In some embodiments, the target antigen includes an immune response modulator that is not a cytokine. Examples of immune response modulators, but not limited to, include B7-1 (CD80), B7-2 (CD86), CD3, or GITR.

[0067] In some embodiments, the first or second target antigen-binding domain includes an anti-EGFR domain, an anti-EpCAM domain, an anti-DLL3 domain, an anti-MSLN domain, an anti-PSMA domain, an anti-BDMA domain, or any combination thereof.

[0068] In some embodiments, the anti-EGFR domain of the Disclosure comprises amino acids selected from the group consisting of SEQ ID No. 55 and 737-785. In some embodiments, the anti-PSMA domain of the Disclosure comprises amino acids selected from the group consisting of SEQ ID No. 57-73. In some embodiments, the anti-BCMA domain of the Disclosure comprises amino acids selected from the group consisting of SEQ ID No. 91-214. In some embodiments, the anti-MSLN domain of the Disclosure comprises amino acids selected from the group consisting of SEQ ID No. 215-258. In some embodiments, the anti-DLL3 domain of the Disclosure comprises amino acids selected from the group consisting of SEQ ID No. 306-736. In some embodiments, the anti-EpCAM domain of the Disclosure comprises amino acids selected from the group consisting of SEQ ID No. 804-841.

[0069] In some embodiments, the first or second target antigen-binding domain includes an anti-CD3 domain. In some embodiments, the anti-CD3 domain includes an anti-CD3 scFV. In some embodiments, the anti-CD3 scFV includes an amino acid sequence selected from the group consisting of SEQ ID Nos. 74-90 and 794.

[0070] Binding protein variants As used herein, the term “binding protein variant” refers to variants and derivatives of the conditionally active target-binding proteins described herein, which contain the binding moieties described herein, including a non-CDR loop that binds to an immunoglobulin-binding molecule, such as a first or second target antigen-binding domain, or to a non-immunoglobulin-binding molecule. In some embodiments, amino acid sequence variants of the conditionally active target-binding proteins described herein are intended. For example, in some embodiments, amino acid sequence variants of the conditionally active target-binding proteins described herein are intended to improve the binding affinity and / or other bioproperties of the binding protein. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications to the nucleotide sequence encoding the antibody or peptide synthesis. Such modifications include, for example, deletions from and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of such residues.

[0071] Any combination of deletions, insertions, and substitutions can be performed on various domains to arrive at the final construct, provided that the final construct has the desired characteristics (e.g., antigen-binding). In one embodiment, binding protein variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDRs and framework regions. The amino acid substitutions may be introduced into the variable domain of the conditionally active protein of interest, and the product is screened for desired activity, e.g., maintained / improved antigen-binding, reduced immunogenicity, or improved antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Conservative and non-conservative amino acid substitutions are intended for the preparation of antibody variants.

[0072] Another example of substitution to produce conditionally active mutant antibodies involves the substitution of one or more hypervariable region residues of the parent antibody. Generally, mutants are selected based on improvements in desired properties compared to the parent antibody, such as increased affinity, decreased affinity, decreased immunogenicity, or increased pH dependence of binding. For example, affinity-matured mutant antibodies can be produced using phage display-based affinity maturation techniques, such as those described herein and known in the art.

[0073] In another embodiment, substitutions can be made in the hypervariable region (HVR) of a parentally conditionally active antibody to generate mutants, which are selected based on binding affinity, i.e., by affinity maturation. In some embodiments of affinity maturation, the diversity is introduced into a variable gene selected for maturation by one of several methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A second library is then created. The library is then screened to identify any antibody mutant with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling. The substitutions can be at one, two, three or more sites within the parental antibody sequence.

[0074] In some embodiments, the conditionally active binding proteins described herein include a VL domain or a VH domain, or both, and their amino acid sequences correspond to the amino acid sequences of a naturally occurring VL domain or VH domain, respectively; however, the conditionally active binding proteins are “humanized,” that is, they are “humanized” by replacing one or more amino acids in the amino acid sequence of the naturally occurring VL domain or VH domain (and, in particular, within the framework sequence) with one or more amino acid residues that would occur at the corresponding positions in the VL domain or VH domain of a conventional human-derived 4-chain antibody (e.g., as shown above). This can be carried out, for example, in a manner known to those skilled in the art, based on further descriptions herein. Furthermore, it should be noted that such humanized conditionally active target-binding antibodies of this disclosure can be obtained in any suitable manner known on their own and are therefore not strictly limited to polypeptides obtained using polypeptides containing naturally occurring VL domains and / or VH domains as starting materials. In some further embodiments, the conditionally active target-binding antibodies described herein comprise a VL domain and a VH domain, the amino acid sequence of which corresponds to the amino acid sequence of a naturally occurring VL domain or VH domain, but the conditionally active target-binding antibodies are “camelized,” that is, “camelized” by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VL domain or VH domain of a conventional 4-chain antibody with one or more amino acid residues that occur at the corresponding positions in the VL domain or VH domain of a heavy-chain antibody. Such “camelized” substitutions preferably form residues prominent at the VH-VL interface and / or so-called camelid, and / or are inserted at amino acid positions present therein (see, e.g., WO94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as a starting material or starting point for generating or designing a camelized single domain is preferably a VH sequence from a mammal, more preferably a human VH sequence such as a VH3 sequence.However, it should be noted that such camelized conditionally active antibodies in this disclosure can, in some embodiments, be obtained in any suitable manner known in the art, and are therefore not strictly limited to polypeptides obtained using polypeptides containing naturally occurring VL and / or VH domains as starting materials. For example, both "humanization" and "camelization" are performed by providing a nucleotide sequence encoding naturally occurring VL and / or VH domains, respectively, and then altering one or more codons in the nucleotide sequence such that the new nucleotide sequence encodes a "humanized" or "camelized" conditionally active antibody, respectively. This nucleic acid can then be expressed to yield a desired target antigen-binding ability. Alternatively, in other embodiments, a "humanized" or "camelized" conditionally active antibody is novelly synthesized from the amino acid sequence of a naturally occurring antibody containing VL and / or VH domains using known peptide synthesis techniques. In some embodiments, “humanized” or “camelized” conditionally active antibodies are novelly synthesized using known peptide synthesis techniques from amino acid or nucleotide sequences of naturally occurring antibodies containing VL and / or VH domains, respectively, to design nucleotide sequences encoding the desired humanized or camelized conditionally active domain antibody of the Disclosure, respectively, which are then novelly synthesized using known techniques for nucleic acid synthesis, and the nucleic acids thus obtained are then expressed using known expression techniques to yield the desired conditionally active antibody of the Disclosure.

[0075] For example, other suitable methods and techniques for obtaining a conditionally active binding protein and / or nucleic acid encoding the same of the present disclosure, starting from a spontaneously occurring sequence for a VL domain or VH domain, include, in a suitable manner, combining one or more portions of one or more spontaneously occurring VL or VH sequences (one or more framework (FR) sequences and / or complementarity-determining region (CDR) sequences), and / or one or more synthetic or semi-synthetic sequences, and / or a spontaneously occurring sequence for a CH2 domain, and a spontaneously occurring sequence for a CH3 domain, including amino acid substitutions that support the formation of a heterodimer rather than a homodimer, to yield a conditionally active binding protein or nucleotide sequence or nucleic acid encoding the present disclosure.

[0076] affinity maturation When designing conditionally active binding proteins for therapeutic application, it is desirable to create, for example, proteins that modulate the functional activity of the target, and / or improved binding proteins, such as binding proteins with higher specificity and / or affinity, and / or binding proteins that are more bioavailable, stable, or soluble in specific cell or tissue environments.

[0077] The conditionally active binding proteins described herein exhibit improved binding affinity to targets, such as tumor antigens expressed on the cell surface. In some embodiments, the conditionally active binding proteins of this disclosure are affinity-matured to increase their binding affinity to targets using any known technique for affinity maturation (e.g., mutagenesis, chain shuffling, CDR amino acid substitution). Amino acid substitutions may be conservative or semi-conservative. For example, the amino acids glycine, alanine, valine, leucine, and isoleucine can often be substituted for each other (amino acids with aliphatic side chains). Of these possible substitutions, typically glycine and alanine are used for substitution with each other because they have relatively short side chains, and valine, leucine, and isoleucine are used for substitution with each other because they have larger, hydrophobic aliphatic side chains. Other amino acids that are often substituted for each other include, but are not limited to, phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains). In some embodiments, conditionally active target-binding proteins are isolated by screening combinatorial libraries, for example, by generating a phage display library and screening such a library for antibodies that possess the desired binding properties to target antigens such as tumor antigens expressed on the cell surface.

[0078] Conditionally active binding protein modification The conditionally active binding proteins described herein include derivatives or analogs such as leader sequences or secretion sequences, or sequences for blocking immunogenic domains and / or purifying proteins, in which (i) an amino acid is substituted with an amino acid residue not encoded by the gene code, (ii) a mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein.

[0079] Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristylation, oxidation, protein processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginylation and ubiquitination.

[0080] Modifications can be performed anywhere on the conditionally active binding proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Specific common peptide modifications useful for modifying conditionally active binding proteins include glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamate residues, hydroxylation, covalent modification of amino or carboxyl groups in polypeptides, or both, and ADP-ribosylation.

[0081] In some embodiments, the conditionally active binding proteins of this disclosure bind to a drug to form an antibody-drug conjugate (ADC). Generally, ADCs are used in oncological applications, where the use of antibody-drug conjugates for local delivery of cytotoxic or cell division inhibitors enables targeted delivery of the drug portion to the tumor, thereby enabling higher efficacy, lower toxicity, and so on.

[0082] Polynucleotides encoding a binding site or a conditionally active binding protein In some embodiments, polynucleotide molecules encoding the binding sites described herein are also provided. In some embodiments, the polynucleotide molecules are provided as DNA constructs. In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0083] In some embodiments, polynucleotide molecules encoding conditionally active binding proteins described herein are also provided. In some embodiments, the polynucleotide molecules are provided as DNA constructs. In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0084] Polynucleotide molecules are constructed by combining genes encoding various domains (e.g., binding sites, target antigen-binding domains, etc.) that are separated by a peptide linker or, in other embodiments, directly linked by peptide bonds, into a single gene construct operably linked to a suitable promoter and optionally a suitable transcriptional terminator, and then expressed in bacteria or other suitable expression systems such as CHO cells, by known methods. Depending on the vector system and host used, any number of suitable transcriptional and translational elements, including constitutively and conditionally active promoters, may be used. The promoters are selected to promote polynucleotide expression in each host cell.

[0085] In some embodiments, the polynucleotides described herein are inserted into vectors such as expression vectors, which represent further embodiments. These recombinant vectors can be constructed according to known methods. Among other things, the vectors of interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, lentiviruses, etc.), and cosmids.

[0086] Various expression vectors / host systems can be used to express polynucleotides encoding the polypeptide of the conditionally active binding protein described. Examples of expression vectors for expression in E. coli include pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0087] Therefore, in some embodiments, a binding moiety or a conditionally active binding protein containing the binding moiety described herein is produced by introducing a vector encoding the binding moiety or the binding protein into a host cell and culturing the host cell under certain conditions, thereby expressing the binding moiety, the binding protein, or its domain.

[0088] Pharmaceutical composition In some embodiments, pharmaceutical compositions are further provided comprising a therapeutically effective amount of the conditionally active binding protein of this disclosure and at least one pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” includes, but is not limited to, carriers that do not interfere with the efficacy of the biological activity of the component and are not toxic to the patient to whom they are administered. Examples of suitable pharmaceutically acceptable carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and administered to a subject in an appropriate dose. Preferably, the compositions are sterilized. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersants. Prevention of microbial action can, in some examples, be ensured by including various antimicrobial and antifungal agents.

[0089] The conditionally active binding proteins described herein are intended to be used as pharmaceuticals. Administration is achieved by different methods, e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition. The administration regimen is determined by the attending physician and other clinical factors. The dosage for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound being administered, the time and route of administration, the type of treatment, health status, and other drugs administered concurrently. “Effective dose” refers to a sufficient amount of the active ingredient to affect the course and severity of the disease and to result in a reduction or remission of such condition, and can be determined using known methods.

[0090] Treatment method In some embodiments, methods and uses for stimulating an individual's immune system, including the administration of conditionally active binding proteins described herein, are further provided herein. In some examples, the administration induces and / or maintains cytotoxicity against cells expressing a target antigen. In some examples, cells expressing a target antigen are cancer cells or tumor cells, virus-infected cells, bacterial-infected cells, autoreactive T cells or B cells, damaged erythrocytes, arterial plaque, or fibrotic tissue. In some embodiments, the target antigen is an immune checkpoint protein.

[0091] Furthermore, methods and uses for treating diseases, disorders, or conditions associated with a target antigen are also provided herein, comprising the step of administering a conditionally active binding protein described herein to an individual. Diseases, disorders, or conditions associated with a target antigen include, but are not limited to, viral infections, bacterial infections, autoimmune diseases, transplant rejection, atherosclerosis, or fibrosis. In other embodiments, diseases, disorders, or conditions associated with a target antigen include proliferative disorders, neoplastic diseases, inflammatory diseases, immunodeficiencies, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases. In one embodiment, the disease, disorder, or condition associated with a target antigen is cancer. In one example, the cancer is a hematological cancer. In another example, the cancer is melanoma. In yet another example, the cancer is non-small cell lung cancer. In yet another example, the cancer is breast cancer.

[0092] As used herein, in some embodiments, “treatment,” “to treat,” or “treated” means a therapeutic treatment aimed at delaying (alleviating) an undesirable physiological illness, disorder, or disease, or obtaining a beneficial or desirable clinical outcome. Beneficial or desirable clinical outcomes for the purposes described herein include, but are not limited to, symptom relief; reduction of the severity of the illness, disorder, or disease; stabilization (i.e., no worsening) of the state of the illness, disorder, or disease; delaying the onset or progression of the illness, disorder, or disease; improvement of the state of the illness, disorder, or disease; and remission (whether partial or whole) (whether detectable or undetectable), or improvement or enhancement of the illness, disorder, or disease. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment further includes extending survival time compared to the survival time expected without treatment. In other embodiments, “treatment,” “treating,” or “treated” refers to a preventative treatment whose purpose is to delay the onset or reduce the severity of an undesirable physiological illness, disorder, or disease, such as in a person predisposed to a disease (for example, an individual carrying genetic markers for a disease such as breast cancer).

[0093] In some embodiments of the methods described herein, the conditionally active binding proteins described herein are administered in combination with agents for the treatment of specific diseases, disorders, or illnesses. These agents include, but are not limited to, therapeutic agents including antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiotherapeutic agents (gamma rays, X-rays, and / or directional delivery such as radioisotopes, microwaves, UV radiation, etc.), gene therapies (e.g., antisense, retroviral treatments, etc.), and other immunotherapeutic agents. In some embodiments, the conditionally active binding proteins described herein are administered in combination with antidiarrheal agents, antiemetic agents, analgesics, opioids, and / or nonsteroidal anti-inflammatory drugs. In some embodiments, the conditionally active binding proteins described herein are administered before, during, or after surgery. [Examples]

[0094] The following embodiments further illustrate the embodiments described herein without limiting the scope of this disclosure.

[0095] Example 1: Construction of an exemplary binding moiety that binds to albumin and its target antigen-binding domain whose target is EGFR. A modified protein scaffold sequence containing albumin-binding CDR loops and non-CDR loops is obtained. Overlapping PCR is used to introduce random mutations into the non-CDR loop region, thereby generating a library. The resulting sequence is cloned into a phage display vector, thereby generating a phage display library. E. coli cells are transformed with the library and used to construct the phage display library. ELISA is performed using immobilized target antigen-binding domains specific to EGFR. Clones with high specificity to EGFR are selected. Affinity maturation is performed by re-randomizing residues in the non-CDR loop region as before.

[0096] Sequence alignment of the non-CDR loop region of the resulting protein is performed to determine sequence conservedness between proteins with high affinity for the EGFR-binding target antigen-binding domain. Site-directed mutagenesis is performed on one or more amino acids within these sequence-conserved regions to generate further proteins. The binding of the resulting protein to the immobilized target antigen-binding domain, whose target is EGFR, is measured by ELISA. The protein with the highest affinity for the target antigen-binding domain is selected.

[0097] The sequence of this binding region is cloned into a vector containing a sequence for a cleavable linker and a sequence for a second target antigen-binding domain that binds to a second target antigen (e.g., CD3). The resulting vector is expressed in a heterologous expression system to obtain a conditionally active target-binding protein containing a binding region that includes a cleavable linker, a non-CDR loop providing a binding site specific to the target antigen-binding domain whose target is EGFR, and a CDR loop specific to albumin.

[0098] Example 2: Construction of an exemplary binding moiety that binds to albumin and a target antigen-binding domain whose target is CD3. A modified protein scaffold sequence containing a CDR loop and a non-CDR loop capable of binding albumin is obtained. Overlapping PCR is used to introduce random mutations into the non-CDR loop region, thereby generating a library. The resulting sequence is cloned into a phage display vector, thereby generating a phage display library. E. coli cells are transformed with the library and used to construct the phage display library. ELISA is performed using an immobilized target antigen-binding domain with specificity for CD3. Clones with high specificity for CD3 are selected. Affinity maturation is performed by re-randomizing residues in the non-CDR loop region as before.

[0099] Sequence alignment of the non-CDR loop region of the resulting protein is performed to determine sequence conservedness between proteins with high affinity for the EGFR-binding target antigen-binding domain. Site-directed mutagenesis is performed on one or more amino acids within these sequence-conserved regions to generate further proteins. Binding of the resulting protein to the immobilized target antigen-binding domain, which targets CD3, is measured by ELISA. The protein with the highest affinity for the target antigen-binding domain is selected.

[0100] The sequence of this binding region is cloned into a vector containing a sequence for a cleavable linker and a sequence for a second target antigen-binding domain that binds to a second target antigen (e.g., EGFR). The resulting vector is expressed in a heterologous expression system to obtain a conditionally active target-binding protein containing a binding region that includes a cleavable linker, a non-CDR loop providing a binding site specific to the target antigen-binding domain whose target is CD3, and a CDR loop specific to albumin.

[0101] Example 3: The conditionally active binding protein of this disclosure exhibits reduced specificity to cell lines that overexpress EGFR but lack the protease. Cells overexpressing EGFR and showing low expression of matrix metalloproteinases are incubated separately with a typical conditionally active binding protein and an unconditionally active control binding protein of this disclosure. Cells expressing normal levels of EGFR and protease are further incubated with a conditionally active binding protein and an unconditionally active control binding protein of this disclosure. Both proteins contain a target antigen-binding domain specific to PSMA.

[0102] The results show that, in the absence of protease secretion, the conditionally active binding protein of this disclosure interacts with protease-expressing cells but does not interact with EGFR expressed on the surface of protease-deficient cells. In contrast, the non-strained active control binding protein lacks the ability to selectively bind to protease-expressing cells compared to protease-deficient cells. Therefore, the exemplary conditionally active binding protein receptors of this disclosure are advantageous, for example, in reducing off-tumor toxicity.

[0103] Example 4: Treatment with the exemplary conditionally active binding protein of this disclosure inhibits tumor growth in vivo. The mouse tumor cell CT26 was transplanted subcutaneously into Balb / c mice, and the mean tumor size was measured 7 days post-transplant. Test mice were treated with an exemplary conditionally active binding protein having a CTLA4-specific target antigen-binding domain and another CD3-specific target antigen-binding domain, where either the CTLA4-specific or CD3-specific domain binds to the binding moiety via its non-CDR loop, the binding moiety containing a cleavable linker, and is bound to albumin. Control mice were treated with a binding protein containing a CD3 / CTLA4-specific domain but lacking a binding moiety or cleavable linker, and which is not conditionally active. The results show that treatment with the exemplary conditionally active binding protein of this disclosure inhibits tumors more efficiently than the control drug-binding protein that does not contain a non-CDR loop portion.

[0104] Example 5: Exemplary conditionally active binding proteins exhibit reduced specificity to cell lines that overexpress the antigen but lack the protease. Cells overexpressing TLA-4 and showing low expression of matrix metalloproteinases are incubated separately with a typical CTLA4-specific conditionally active binding protein of this disclosure, which contains a binding moiety that binds to the CTLA4-binding domain via a non-CDR loop and binds to albumin via a CDR, or with a control CTLA-4 binding antibody that does not contain a binding moiety that binds to the CTLA4-binding domain via a non-CDR loop and binds to albumin via a CDR. Cells expressing normal levels of antigen and protease are also incubated with an exemplary CTLA4-specific conditionally active binding protein or a control CTLA4 binding antibody.

[0105] The results show that, in the absence of protease secretion, the conditionally active binding proteins of this disclosure bind to protease-expressing cells but not to protease-deficient antigen-expressing cells. In contrast, the control antibody lacks the ability to selectively bind to protease-expressing cells more than to protease-deficient cells. Therefore, the exemplary conditionally active binding proteins of this disclosure are advantageous, for example, in reducing off-tumor toxicity.

[0106] Example 6: Purification, pharmacokinetic analysis, and efficacy testing of exemplary ProTriTAC molecules. A) Expression, purification, and stability of exemplary ProTriTAC molecules

[0107] Protein production

[0108] The sequence of an exemplary ProTriTAC (also known as Pro-triply specific) molecule was cloned into the mammalian expression vector pcDNA 3.4 (Invitrogen), with the leader sequence preceding a 6x histidine tag. Expi293F cells (Life Technologies A14527) were maintained in Optimum Growth Flasks (Thomson) suspension at concentrations of 0.2–8x1e6 cells / mL in Expi 293 medium. Purified plasmid DNA was transfected into Expi293 cells according to the Expi293 Expression System Kit (Life Technologies A14635) protocol and maintained for 4–6 days post-transfection. Alternatively, the sequence of the triple-specific molecule was cloned into CHO-DG44 dhfr- cells, transfected into the resulting stable pool of mammalian expression vector pDEF38 (CMC ICOS), and cultured in the production medium for up to 12 days prior to purification. The amount of exemplary trispecific protein in the conditional medium was quantified using an Octet RED 96 instrument equipped with Protein A tips (ForteBio / Pall) using a control trispecific protein for calibration. Conditional medium from any of the host cells was filtered and partially purified by affinity and desalting chromatography. The trispecific protein was then polished by ion exchange and formulated in a neutral buffer containing excipients during fraction pooling. Final purity was assessed by SDS-PAGE and analytical SEC using a 4.6 × 150 mm column (Waters Corporation) of Acquity BEH SEC 200 1.7u separated in an aqueous / organic mobile phase with neutral pH excipients on a 1290 LC system, and peaks were evaluated using Chemstation CDS software (Agilent). As shown in Figure 5, the trispecific protein purified from CHO host cells was analyzed by performing SDS-PAGE.

[0109] Stability evaluation

[0110] Purified Pro-TriTriTAC trispecific proteins from the two formulations were sub-alicoated into sterile tubes and stressed by five freeze-thaw cycles (each consisting of -80°C and room temperature for longer than 1 hour) or by incubation at 37°C for 1 week. Stressed samples were evaluated for concentration and turbidity by UV spectroscopy using SpectraMax M2 and SoftMaxPro software (Molecular Devices), SDS-PAGE, and a clear 96-well plate (Corning 3635) with analytical SEC, and compared to the same analysis of unstressed control samples. Overlays of chromatograms from analytical SEC of control and stressed samples for a single exemplary trispecific ProTriTAC molecule purified from 293 host cells are shown in Figure 6.

[0111] B) ProTriTAC exhibits potent protease-dependent antitumor activity in rodent tumor xenograft models.

[0112] An exemplary ProTriTAC molecule (SEQ ID NO: 46) containing an EGFR-binding domain, a CD3-binding domain, and an albumin-binding domain with a masking moiety (SEQ ID NO: 50) and a cleavable linker (SEQ ID NO: 53) as target-binding domains was evaluated for in vivo antitumor activity in HCT116 subcutaneous xenograft tumors mixed with human T cells proliferated in immunosuppressed NCG mice. Non-cleavable EGFR-targeted ProTriTAC molecule (SEQ ID NO: 47) and GFP-targeted ProTriTAC molecule (SEQ ID NO: 49) were also used in the study. In particular, 5x10 6 HCT116 cells were administered to each mouse at a rate of 2.5 x 10⁶ cells on day 0. 6The proliferated T cells were mixed with the test molecules. The test molecules (EGFR-targeted ProTriTAC, non-cleavable EGFR-targeted Pro-TriTAC, and GFP-targeted ProTriTAC) were administered by intraperitoneal injection at a dose of 0.03 mg / kg once daily for 10 days (a single daily dose for 10 days), starting the following day. Tumor volume was determined using caliper measurements and calculated at the indicated time using the formula V = (length x width x width) / 2. The results shown in Figure 7 show that tumor growth was suppressed in mice administered with the activatable EGFR-targeted ProTriTAC molecule 10 days after the final administration of the test molecules. However, administration of the GFP-targeted ProTriTAC molecule was not effective in inhibiting tumor growth, and administration of the EGFR-targeted non-cleavable ProTriTAC molecule was not as potent in inhibiting tumor growth as activatable ProTriTAC.

[0113] C) Demonstration of functional masking and in vivo stability of ProTriTAC in a 3-week pharmacokinetic study in cynomolgus monkeys.

[0114] Active drugs mimicking PSMA-targeted ProTriTAC (SEQ ID NO: 43), non-cleavable PSMA-targeted ProTriTAC (SEQ ID NO: 44), unmasked / non-cleavable TriTAC (SEQ ID NO: 52), and protease-activated PSMA-targeted ProTriTAC (SEQ ID NO: 45), containing a PSMA-binding domain as a target-binding domain, a CD3-binding domain, and an albumin-binding domain (SEQ ID NO: 50) with a masking moiety (SEQ ID NO: 50) and a cleavable linker (SEQ ID NO: 53), were administered intravenously to cynomolgus monkeys at a dose of 0.1 mg / kg. Plasma samples were collected at the time points shown in Figure 9. The design of the test molecules described above is shown in Figure 8. As described above, the concentrations of various test molecules were determined using a ligand-binding assay with biotinylated recombinant human PSMA (R&D systems) and using sulfolabeled anti-CD3 idiotype antibody cloned 11D3 in the MSD assay (Meso Scale Diagnostic, LLC). Pharmacokinetic parameters were evaluated using Phoenix WinNonlin pharmacokinetic software with a non-compartmental approach consistent with the rapid intravenous administration route.

[0115] To calculate the in vivo conversion rate of the evaluated test molecules (i.e., PSMA-targeted ProTriTAC, non-cleavable PSMA-targeted ProTriTAC, and unmasked / non-cleavable PSMA-targeted ProTriTAC), the concentration of the active drug in circulation was estimated by solving the following differential equation, where P is the concentration of the prodrug, A is the concentration of the active drug, and k a k is the rate of prodrug activation in circulation, ,cP is the clearance rate of the prodrug, and k c,A This is the clearance rate of the active drug.

[0116]

number

[0117] Prodrugs, active drugs, non-masked non-cleavable prodrug controls, and non-cleavable prodrug controls (k c,NCLV ) clearance rates were empirically determined in cynomolgus monkeys. To estimate the rate of prodrug activation in circulation, it was assumed that the difference between the clearance rate of the cleavable prodrug and the non-cleavable prodrug arose solely from non-specific activation in circulation. Therefore, the rate of prodrug conversion to active drug in circulation was evaluated by subtracting the clearance rate of the non-cleavable prodrug from that of the cleavable prodrug.

[0118]

Number

[0119] The initial concentration of the prodrug in circulation was empirically determined, assuming that the initial concentration of the active drug was 0. Further calculations showed that ProTriTAC containing a protease-cleavable linker was sufficiently stable in circulation, with 50% non-tumor-mediated conversion every 194 hours, and the t 1 / 2 was empirically determined to be approximately 211 hours. This indicated that the ProTriTAC molecule was sufficiently stable and protected from off-tumor effects. In contrast, the t 1 / 2 of the active drug fragment mimicking the activated ProTRITAC molecule was empirically determined to be 0.97 hours. Therefore, the active drug was rapidly eliminated from circulation. The results are shown in Figure 10.

[0120] Figure 11 shows a significant >200-fold difference between circulating exposure to ProTriTAC and activated ProTriTAC, as the active drug mimicking the activated ProTriTAC molecule was rapidly removed from circulation, and both ProTriTAC and the control non-cleavable ProTriTAC had longer half-lives. It was also observed that the masked but non-cleavable ProTriTAC control molecule remained in circulation longer than the non-cleavable ProTriTAC control, thus demonstrating that masking has a longer circulating half-life (t 1 / 2 It has been shown to play a role in increasing ) and limiting peripheral T cell binding. The combination of functional masking that inactivates ProTriTAC outside the tumor environment and half-life differences that ensure rapid removal of abnormally activated ProTriTAC in vivo works together to ensure on-target activity and minimize off-tumor activity of ProTriTAC. Supporting pharmacokinetic parameters are shown in Table 5.

[0121] [Table 6]

[0122] D) Protease activation of the ProTriTAC molecule leads to significantly enhanced activity in vitro.

[0123] The objective of this study was to evaluate the relative potency of a protease-activatable ProTriTAC molecule, a non-cleavable ProTriTAC molecule, and a recombinant active drug fragment mimicking a protease-activatable ProTriTAC molecule in CD3 binding and T cell-mediated cell killing. The active drug fragment mimicking a protease-activatable ProTriTAC molecule contained a CD3-binding domain and a target antigen-binding domain, but lacked an albumin-binding domain. On the other hand, the protease-activatable ProTriTAC molecule contained an albumin-binding domain with a masking domain and a protease-cleavable site, a CD3-binding domain, and a target antigen-binding domain. The non-cleavable ProTriTAC molecule lacked a protease-cleavable site, but was otherwise identical to the protease-activatable ProTriTAC molecule.

[0124] Purified ProTriTAC (labeled as a prodrug in Figures 12-14), incleavable ProTriTAC (labeled as a prodrug (incleavable) in Figures 12-14), and recombinant active drug fragments mimicking protease-activated ProTriTAC (labeled as active drugs in Figures 12-14) were tested for binding to recombinant human CD3 in ELISA assays (Figure 12), binding to purified human primary T cells in flow cytometry assays (Figure 13), and functional capacity in cytotoxicity assays of T cell-dependent cells (Figure 14).

[0125] For ELISA, soluble test molecules at specified concentrations (i.e., active drug, prodrug, and prodrug (non-cleavable)) were incubated for 1 hour at room temperature in a multiwell plate with immobilized recombinant human CD3ε (R&D Systems) in PBS supplemented with 15 mg / mL human serum albumin. The plate was blocked using SuperBlock (Thermo Fisher), washed with PBS containing 0.05% Tween 20, and detected using non-competitive anti-CD3 idiotype monoclonal antibody 11D3, followed by detection using a peroxidase-labeled secondary antibody and TMB-ELISA substrate solution (Thermo Fisher). The results shown in Figure 12 demonstrate that the active drug fragment mimicking the protease-activated ProTriTAC molecule was approximately 250-fold potent on bound CD3 compared to the non-cleavable prodrug. 50 The values ​​are provided in Table 6. The masking rate is the active drug EC. 50 Prodrug EC 50 This is the ratio: the larger the value, the greater the doubling shift between the prodrug and the active drug, and therefore the greater the functional masking.

[0126] [Table 7]

[0127] For binding to human primary T cells as determined by flow cytometry, soluble test molecules (i.e., active drug, prodrug, and prodrug (uncleavable)) at specified concentrations (shown in Figure 13) were incubated for 1 hour at 4°C in multiwell plates containing purified human primary T cells in the presence of PBS with 2% fetal bovine serum and 15 mg / ml human serum albumin. The plates were washed with PBS containing 2% fetal bovine serum and detected using AlexaFluor 647-labeled non-competitive anti-CD3 idiotype monoclonal antibody 11D3, and the data were analyzed using FlowJo 10 (FlowJo, LLC). The results shown in Figure 13 demonstrate that the active drug fragment mimicking the protease-activated ProTriTAC molecule was 1000-fold potent in bound human primary T cells compared to the uncleavable prodrug. 50 The values ​​are provided in Table 7.

[0128] [Table 8]

[0129] To assess the functional capability of soluble test molecules in T cell-dependent cell cytotoxicity assays, specified concentrations of soluble test molecules (i.e., active drug, prodrug, and prodrug (non-cleavable)) were incubated for 48 hours at 37°C in multiwell plates containing purified quiescent human T cells (effector cells) and HCT116 cancer cells (target cells) in a 10:1 effector:target cell ratio, as shown in Figure 14. Stable transfection of the HCT116 target cell line with a luciferase reporter gene enabled the measurement of specific T cell-mediated cell killing by ONE-Glo(Promega). The results shown in Figure 14 demonstrate that the active drug fragment mimicking the protease-activated ProTriTAC molecule was approximately 500-fold more potent in T cell-mediated killing of cancer cells compared to the non-cleavable prodrug. EC 50 The values ​​are provided in Table 8.

[0130] [Table 9]

[0131] Example 7: Antitumor activity of exemplary ProTriTAC molecules containing various exemplary linkers in a mixed mouse tumor model. The goal of this study was to investigate the antitumor activity of ProTriTAC molecules containing various linkers. Seven-week-old NSG female mice were used in this study. On day 0, the start of the study, 2.5 × 10⁶ 6 Proliferated human T cells, and 5 × 10 6 HCT116 (human colorectal cancer) tumor cells were injected into NSG female mice. On day 1, the following day, the mice were divided into groups, and each group was treated with at least one of the ProTriTAC molecules listed in Table 9 (SEQ ID No. 786-790), either with a control GFP TriTAC molecule (SEQ ID No. 792) or with a ProTriTAC molecule containing an uncleavable linker (NCLV) (SEQ ID No. 791).

[0132] The ProTriTAC and ProTriTAC NCLV molecules used in the following examples were targeted to EGFR and had the following orientation of their individual domains: (anti-albumin-binding domain (sdAb): anti-CD3 domain (scFV): anti-EGFR domain (sdAb)). The only difference between the ProTriTAC molecules listed in Table 6 was the linker sequence. The ProTriTAC molecule, ProTriTAC NCLV molecule, or GFP TriTAC molecule (GFP TriTAC molecule had the following orientation of its individual domains: anti-GFP sdAb: anti-Alb sdAb: anti-CD3 scFv) was administered daily for 10 days (i.e., the final dose was administered on day 10 after injection of tumor cells and proliferating cells into the animals), and tumor volume was measured at regular intervals for several days prior to the administration of the final dose on day 10.

[0133] [Table 10]

[0134] As shown in Figure 18, ProTriTAC molecules containing linker sequences L001, L045, L040, and L041 exhibited more potent antitumor activity compared to the GFP TriTAC control of ProTriTAC NCLV molecules. Statistical significance of the data was determined by repeated measures one-way ANOVA-Dunnett post-hoc test. The mean tumor volume of each mouse group was compared to the mean tumor volume of the mouse group that received the NCLV molecule.

[0135] The pharmacokinetics after administration of various molecules were evaluated as described above, and the data are shown in Figure 19. The control GFP TriTAC molecule was rapidly removed from circulation after administration, while the NCLV molecule remained in circulation for the longest time. The pharmacokinetic clearance profile of the experimental ProTriTAC molecules was between that of GFP TriTAC and NCLV, with the exception of ProTriTAC containing linker sequence L001, which was removed almost as rapidly as the control GFP TriTAC.

[0136] Example 8: Individual tumor volumes of mixed xenograft tumors after treatment with exemplary ProTriTAC molecules containing various exemplary linkers. The ProTriTAC molecules, control GFP TriTAC molecules, and ProTriTAC NCLV molecules listed in Table 9 were evaluated in a mixed xenograft model to determine the efficacy of ProTriTAC molecules containing various linkers in vivo. As described in the previous example (Example 7), 2.5 × 10⁶ mice were given to 7-week-old NSG mice. 6 Proliferated human T cells, and 5 × 10 6 A xenograft tumor model was generated by injecting HCT116 (human colorectal cancer) tumor cells. The mice were divided into groups, and each group was treated with at least one of the ProTriTAC molecules listed in Table 9: either the GFP TriTAC molecule or the ProTriTAC NCLV molecule. Tumor volume was measured at regular intervals starting 10 days after injection of tumor cells and proliferated T cells.

[0137] In animals treated with an exemplary ProTriTAC molecule containing linker L040, a statistically significant delay in tumor growth was observed compared to mice treated with the control GFP TriTAC molecule or the ProTriTAC NCLV molecule. Similar observations were made for ProTriTAC molecules containing linker sequences L001, L041, and L045. The data are shown in Figure 20.

[0138] Similar tests can also be performed using xenograft models with other cell lines such as A549 (non-small cell lung cancer) cells, DU-145 (prostate) cells, MCF-7 (breast) cells, Colo 205 (colon) cells, 3T3 / GF-IR (mouse fibroblast) cells, NCI H441 cells, HEP G2 (hepatocellular carcinoma) cells, MDA MB 231 (breast) cells, HT-29 (colon) cells, MDA-MB435s (breast) cells, U266 cells, SH-SYSY cells, Sk-Mel-2 cells, NCI-H929, RPM18226, and A431 cells.

[0139] Example 9: Demonstration of a decrease in cytokine levels in cynomolgus monkeys correlated with masking of TriTAC molecules. In this study, cynomolgus monkeys were treated with either an exemplary EGFR-targeted ProTriTAC molecule (ProTriTAC(NCLV)) containing an uncleavable linker at three different concentrations (30 μg / kg; 300 μg / kg; 1000 μg / kg), or an exemplary EGFR-targeted TriTAC molecule (SEQ ID No. 793) at three different concentrations (10 μg / kg; 30 μg / kg; 100 μg / kg).

[0140] As shown in Figure 21, IFN-gamma IL-6 and IL-10 levels 4 hours after administration of the ProTriTAC(NCLV) molecule were significantly lower compared to administration of the EGFR-targeted TriTAC molecule.

[0141] Example 10: Demonstration of improved tolerability in mice provided by an exemplary EGFR-targeted ProTriTAC molecule. This study evaluated the tolerance of exemplary EGFR-targeted ProTriTAC molecules. 2 × 10¹⁶ molecules were injected into the peritoneal cavity of 7-week-old NSG female tumor-free mice. 7 Proliferated human T cells were injected at the start of the study (i.e., day 0). On day 2, mice were divided into various groups and treatment was initiated by administering exemplary EGFR-targeted ProTriTAC molecules containing linker sequence L001 at varying concentrations, EGFR-targeted TriTAC molecules, and EGFR-targeted ProTriTAC molecules containing an uncleavable linker (ProTriTAC(NCLV)). These molecules were administered once daily for 10 days at the following doses: 30 μg / kg, 100 μg / kg, and 300 μg / kg. Animal body weight was recorded daily from day 2.

[0142] As shown in Figure 22, the EGFR-targeted ProTriTAC molecule containing an uncleavable linker (ProTriTAC(NCLV)) and GFP TriTAC (used as a negative control) were well-tolerated in mice, even at a maximum dose of 1000 μg / kg. The EGFR-targeted ProTriTAC molecule containing the L001 linker sequence was well-tolerated at a dose of 100 μg / kg, while the EGFR-targeted TriTAC was well-tolerated at 30 μg / kg. Therefore, in mice, it was observed that ProTriTAC containing the L001 linker sequence increased tolerability by 3-fold, and ProTriTAC(NCLV) increased tolerability by 30-fold. The maximum tolerable doses of the ProTriTAC(NCLV) and TriTAC molecules in mice were consistent with those observed in cynomolgus monkeys.

[0143] To further explore the role of linkers in the tolerability of EGFR-targeted ProTriTAC molecules in mice, the linker sequence was changed from L001 to L040. In this experiment, at the start of the study (i.e., day 0), 7-week-old NSG female tumor-free mice were given 5x10⁶ doses. 6HCT116 tumor cells were subcutaneously injected. Seven days after tumor cell injection, the tumor volume was approximately 180-200 mm². 3 (For example, 183mm) 3 ) When this was the case, the mouse was 2x10 7 Proliferated human T cells were injected intraperitoneally. Mice were divided into various groups, and treatment was initiated on day 9 by treating each group with an EGFR-targeted TriTAC molecule, an EGFR-targeted ProTriTAC molecule with linker sequence L040 (ProTriTAC(L040)), and a ProTriTAC molecule containing an uncleavable linker (ProTriTAC(NCLV)). The above molecules were administered once daily for 10 days at the following doses: 300 μg / kg and 1000 μg / kg. The body weight of the animals was recorded daily from day 2. The results shown in Figure 23 are for the molecule with linker sequence L040. This study demonstrates that EGFR-targeted ProTriTAC molecules exhibit higher tolerability than those using the L001 linker sequence. ProTriTAC(L040) showed high tolerability at 300 μg / kg and 1000 μg / kg, and the rate of body weight change was comparable to that of the ProTriTAC(NCLV) molecule. Therefore, compared to the TriTAC molecule, ProTriTAC containing the L040 linker sequence was observed to increase tolerability by 30-fold in mice, similar to the 30-fold increase in tolerability observed with the ProTriTAC(NCLV) molecule.

[0144] Example 11: Clinical and pathological studies in mice and cynomolgus monkeys In this study, mice were treated with various concentrations of EFFR-targeted TriTAC molecules, EGFR-targeted ProTriTAC molecules containing linker sequence L001 (ProTriTAC(L001)), and EGFR-targeted ProTriTAC molecules containing an uncleavable linker (ProTriTAC(NCLV)). Tolerability was evaluated by measuring serum concentrations of ALT (alanine aminotransferase) and AST (aspartate aminotransferase). The results are shown in Figure 1. Figures 24A, 24B, and 24C, and Figures 25A, 25B, and 25C, show that serum concentrations of AST and ALT did not increase after administration of ProTriTAC(L001) at the maximum dose of 0.3 mg / kg, and also after administration of ProTriTAC(NCLV) at the maximum dose of 1 mg / kg. In contrast, serum concentrations of AST and ALT did not increase after administration of the TriTAC molecule at a dose of 0.3 mg / kg.

[0145] In another study, cynomolgus monkeys were treated with EGFR-targeted TriTAC molecules at various concentrations, as well as EGFR-targeted ProTriTAC molecules containing an uncleavable linker (ProTriTAC(NCLV)). Tolerability was assessed by measuring serum concentrations of ALT (alanine aminotransferase) and AST (aspartate aminotransferase). The results are shown in Figure 26. After administration of ProTriTAC(NCLV) at the maximum dose of 1000 μg / kg, serum concentrations of AST and ALT did not increase. In contrast, after administration of TriTAC molecules at a dose of 10 μg / kg, serum concentrations of AST and ALT did not increase.

[0146] Example 12: Demonstration of expanded therapeutic range using exemplary ProTriTAC molecules of this disclosure in a mouse model with cancer. The goal of this study was to evaluate the expansion of the therapeutic range by measuring antitumor activity and observable on-target toxicity in mice with the same cancer. Seven-week-old NSG female mice were used in this study.

[0147] On day 0, which is the start of the exam, 2.5 × 10 6 Proliferated human T cells, and 5 × 10 6 HCT116 (human colorectal cancer) tumor cells were injected into NSG female mice. On day 1, the mice were divided into groups, and each group was treated with either GFP TriTAC molecule (SEQ ID No. 792), EGFR TriTAC molecule (SEQ ID No. 793), or EGFR-targeted ProTriTAC molecule (SEQ ID No. 787) containing linker L040 at the dose levels specified in Figures 27A-297D (for GFP TriTAC, the dose was 300 μg / kg; for EGFR TriTAC, the doses were 10 μg / kg, 30 μg / kg, 100 μg / kg, and 300 μg / kg; EGFR In the case of ProTriTAC, the doses were 30 μg / kg, 100 μg / kg, 300 μg / kg, and 1000 μg / kg), and administered daily for 10 days (i.e., the final dose was administered on the 10th day after injection of tumor cells and proliferated cells into the animals). Tumor volume was measured at regular intervals for several days prior to the administration of the final dose on the 10th day. The results are shown in Figures 27A-27D.

[0148] Using calipers, the radius of the red scarring skin lesion above the original tumor transplant site was measured, and on day 14, the equation was: region = π * (Radius of the lesion) 2On-target EGFR-related toxicity was determined by applying [specific method / method]. The results provided in Figure 28 show that exemplary EGFR ProTriTAC is 30-fold more tolerable than EGFR TriTAC in mice with the same HCT116 cancer, as measured by the development of red scarring skin lesions on the original tumor transplant site. The therapeutic range is defined as the difference between the minimum dose level required for antitumor activity and the maximum dose level without skin lesions.

[0149] The results (from Figures 27 and 28), summarized in the table below, show that when measured in mice with the same cancer, exemplary protease-cleavable EGFR ProTriTAC is 3 times less potent but 30 times more tolerable (i.e., has a 10-fold improved therapeutic range) than EGFR TriTAC. This makes it possible to administer ProTriTAC at approximately 3 times higher doses than TriTAC to achieve at least the same potency and higher tolerability.

[0150] [Table 11]

[0151] Example 13: Exemplary ProTriTAC molecule containing a binding moiety with an extended non-CDR loop to which a human CD3ε epitope is implanted. The sequences of the binding region, including non-CDR loops (AB, EF, C''D, and CC'), were obtained. A portion of the human CD3ε sequence, along with glycine residues, was transplanted into the CC' loop of the non-CDR loop within the binding region, further extending the CC' loop. Figure 29 illustrates three different mutants, each containing 10, 12, or 16 amino acid extensions to the CC' loop. In mutant CC10, the portion of the human CD3ε sequence transplanted into the CC' loop to replace the wild-type sequence of APGKG was QDGNEE (SEQ ID NO. 801), with four additional glycine residues inserted to extend the CC' loop. In mutant CC12, the portion of the human CD3ε sequence transplanted into the CC' loop to replace the wild-type sequence of APGKG was QDGNEEMGG (SEQ ID No. 802), with three additional glycine residues inserted to extend the CC' loop. In the case of mutant CC12, the portion of the human CD3ε sequence transplanted into the CC' loop to replace the wild-type sequence 21APGKG was QDGNEEMGG (SEQ ID No. 803), and in addition, seven glycine residues were inserted to extend the CC' loop. As described above, the binding moiety containing the extended non-CDR loop with the CD3ε sequence was cloned into a vector further containing a coding sequence for a protease-cleavable linker, an scFv containing the CD3-binding domain, and an EGFR-binding domain to express the ProTriTAC molecule. The ProTriTAC molecule contained the exemplary binding moiety, CD3-binding scFv, and EGFR-binding domain of this disclosure. Subsequently, the ProTriTAC molecule was exposed to the tumor-associated protease matryptase to analyze the activation of the molecule upon cleavage of the protease-cleavable linker. This cleavage separated the binding portion containing the cleavable linker (shown as aALB in Figures 29 and 30) from the rest of the molecule, namely the scFv containing the CD3-binding domain (shown as aCD3 in Figures 29 and 30) and the EGFR-binding domain (aEGFR).Figure 30 shows the activation of ProTriTAC molecules containing variants of CC10, CC12, or CC16, or CD3-binding scFv, in the non-CDR loop of CC' in VH-VL (left panel) or VL-VH (right panel) format, upon treatment with matryptase. For the protease activation assay, ProTriTAC molecules containing the wild-type CC' loop at the binding site were used as a control. In addition, TriTAC molecules in a non-pro form, i.e., molecules containing the same domain as ProTriTAC molecules except that they have a half-life extension domain such as albumin instead of a binding site, were also treated with matryptase and used as controls. The results showed that ProTriTAC molecules were activated upon cleavage and generated a free albumin-binding domain (shown as free aALB in Figure 30), but the albumin domain did not separate from the TriTAC molecule. Therefore, unlike the TriTAC version, the ProTriTAC molecule containing the binding portion of this disclosure could be readily separated from the half-life extension domain upon cleavage in the tumor microenvironment, thereby making it more susceptible to rapid clearance from the systemic circulation upon activation.

[0152] Further tests were conducted to analyze the binding of the human CD3ε-containing binding site to CD3. In the presence of human serum albumin, the activated form of the ProTriTAC molecule containing the human CD3ε-containing binding site was observed to be approximately 20 times stronger in binding to CD3 than the activated form without the binding site. The results are shown in Figure 31.

[0153] The potential for cell killing by ProTriTAC molecules containing the binding domain described herein was also analyzed in tests, in which CaOV4 cell lines were treated with ProTriTAC molecules or their activated form in the presence of human serum albumin. As shown in Figure 32, ProTriTAC molecules containing the CC16 variant of the CC' non-CDR loop were approximately 50 times more potent in killing cancer cells compared to their activated form isolated from the albumin-binding domain. The observed total masking was a combination of steric masking due to binding to human serum albumin and specific masking from the CC16 mask of the CC' non-CDR loop.

[0154] Example 14: The CC' loop was identified as the most easily modified by soft library mutagenesis. To create masking capabilities and identify the most easily modified non-CDR loops (AB, CC', C''D, and EF), libraries were constructed and generated using the following four groups of overlapping DNA oligos with different loop lengths containing randomized degenerate "NNK" codons, as outlined below.

[0155] AB Loop Oligo: WT:LVQPGN(20%)(SEQ ID NO:903) AB0:XXXXXX(20%) AB1:XXXXXXX(20%) AB2:XXXXXXXX(20%) AB3:XXXXXXXXX(20%)

[0156] CC' Loop Oligo: WT:APGKG (20%) CC0:XXXXX(20%) CC1:XXXXXX(20%) CC2:XXXXXXX(20%) CC3:XXXXXXXX(20%)

[0157] C''D loop oligo: WT:DSVKGR(20%)(SEQ ID NO:904) CD0:XXXXXX(20%) CD1:XXXXXXX(20%) CD2:XXXXXXXX(20%) CD3:XXXXXXXXX(20%)

[0158] EF Loop Oligo: WT:SLRPED(20%)(SEQ ID NO:905) EF0:XXXXXX(20%) EF1:XXXXXXX(20%) EF2:XXXXXXXX(20%) EF3:XXXXXXXXX(20%)

[0159] Note: "X" represents a randomized residue ("NNK" codon) which can be any of the 20 native amino acids and stop codons. The objective was to make approximately 20% of each non-CDR loop wild-type. These wild-type oligos served as an internal benchmark to measure each loop's resistance to modification (changes in sequence composition and / or length). Loops that were less resistant to change were likely to revert to wild-type easily; in contrast, loops that were highly susceptible to change maintained a diverse sequence repertoire. For this purpose, 24 clones were sequenced from a naive library to validate the randomization of non-CDR loops before panning with HAS, as shown in Figure 33. After two rounds of phage panning with HSA, 30 clones were sequenced to validate the non-CDR loop composition. The results showed that three of the four non-CDR loops (AB, C''D, and EF) reverted to wild-type primarily using 20% ​​wild-type oligos, suggesting that they are less preferred compared to the wild-type sequence. However, the CC' loop maintains a diverse sequence repertoire (both sequence and length), which suggests that the CC' loop may be the most randomization-resistant loop that can be used for specific masking of adjacent domains, as shown in Figure 34.

[0160] Example 15: Screening of a phage display library for identification of EpCAM-binding domains Llamas were immunized with purified EpCAM protein expressed in Expi293 cells. A phage display library for the expression of the heavy variable antibody domain was constructed from circulating B cells. See van der Linden, de Geus, Stok, Bos, van Wassenaar, Verrips, and Frenken. 2000. J Immunol Methods 240:185-195. Phage clones were screened for EpCAM binding by expressing anti-EpCAM protein in E. coli and preparing periplasm extracts, and proteins were screened for human and cynomolgus monkey EpCAM binding activity using colorimetric ELISA. Compared to controls with human and / or cynomolgus monkey EpCAM protein, 38 unique heavy-chain-only sequences that generated a signal in ELISA screening were identified (SEQ ID No. 804-841) (shown in Table 10).

[0161] [Table 12-1]

[0162] [Table 12-2]

[0163] Example 16: Incorporation of a single-domain antibody containing only the EpCAM-conjugated heavy chain into a cytotoxicity assay of fusion proteins and T cell-dependent cells. The single-domain antibodies of the selected anti-EpCAM heavy chain from Example 15 were cloned into DNA constructs for the expression of recombinant proteins. All of these expression constructs encoded signal peptides. One set of anti-EpCAM constructs (SEQ ID No. 842-868) was designed to express a fusion protein having a humanized anti-CD3 scFv domain on the N-terminus of a mature secreted fusion protein, and then a fusion protein having a llama anti-EpCAM domain, with the two domains linked by the sequence GGGGSGGGS and having HHHHHH at the C-terminus. Another set of anti-EpCAM constructs (SEQ ID No. 869-895) was designed to express a fusion protein having a llama anti-EpCAM domain on the N-terminus of a mature secreted fusion protein, and then a fusion protein having a humanized anti-CD3-scFv domain, with the two domains linked by the sequence GGGGSGGGS and having HHHHHH at the C-terminus.

[0164] These anti-EpCAM / anti-CD3 (N-terminus to C-terminus) or anti-CD3 / anti-EpCAM (N-terminus to C-terminus) fusion protein constructs were transfected into Expi293 cells. The amount of anti-EpCAM / anti-CD3 fusion protein in the condition medium from the transfected Expi293 cells was quantified using an Octet instrument with streptavidin, and biotinylated CD3-Fc fusion proteins were loaded using anti-CD3 fusion proteins of the same molecular weight as the reference anti-EpCAM / anti-CD3 protein.

[0165] Conditional media were tested in a cytotoxicity assay of T cell-dependent cells. See Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27. In this assay, luciferase-labeled NCI-H508 cells expressing EpCAM were combined with purified human T cells, and titrated with anti-EpCAM / anti-CD3 fusion proteins or anti-CD3 / anti-EpCAM. It was hypothesized that if the fusion protein instructed T cells to kill NCI-H508 cells, the signal in the luciferase assay performed 48 hours after the start of the experiment should decrease. Figures 36-39 provide TDCC data in graphical format. EC from TDCC assay 50 The values ​​are from Table 11 (EC for SEQ ID No. 842-868). 50 (Data is shown) and Table 12 (EC for SEQ ID No. 869-895) 50 The data is shown in (indicated). The most potent molecule (EPL13) has an EC of approximately 1.6 pM. 50 The values ​​were present. Some anti-EpCAM binding proteins were active only when present in an anti-CD3 / anti-EpCAM configuration. One anti-EpCAM sequence (EPL34) was active only in an anti-EpCAM / anti-CD3 configuration. The negative control for the TDCC assay was an anti-GFP / anti-CD3 protein, which did not instruct T cells to kill NCI-H508 cells (not shown).

[0166] [Table 13]

[0167] [Table 14]

[0168] The binding affinity of fusion proteins to human and cynomolgus monkey EpCAM proteins was measured using conditional media containing known concentrations of anti-EpCAM / anti-CD3 or anti-CD3 / anti-EpCAM fusion proteins. Biotinylated human or cynomolgus monkey EpCAM proteins were loaded into Octet instruments equipped with streptavidin tips, and the on-rate and off-rate of binding of anti-EpCAM / anti-CD3 fusion proteins or anti-CD3 / anti-EpCAM fusion proteins to biotinylated EpCAM proteins was measured. D The value was calculated using a single 50 nM concentration of anti-EPCAM / anti-CD3 or anti-CD3 / anti-EpCAM fusion protein. D By performing the measurements, it became possible to rank the potency. The measured relative affinity is shown in Table 13. All fusion proteins bound to cynomolgus monkey EpCAM and K D The values ​​were in the range of 1.6 to 56 nM. K was in the range of 0.8 to 74 nM. D We measured most, though not all, of the fusion proteins that bind to human EpCAM and have a value.

[0169] [Table 15]

[0170] Example 17: Humanization of a single-domain antibody containing only the EpCAM-conjugated heavy chain, and cytotoxicity assay of T cell-dependent cells. Three of the llama anti-EpCAM antibody sequences identified in Example 15 were humanized into human germline sequences (SEQ ID No. 896-898) by transplanting their CDR sequences while ensuring that the antibodies did not lose activity by retaining several llama framework sequences.

[0171] As described in Example 16, these sequences were cloned into expression constructs for the expression of anti-EpCAM / anti-CD3 fusion proteins (SEQ ID No. 899-901) in Expi293 cells.

[0172] As described in Example 16, the amount of anti-EpCAM / anti-CD3 fusion protein present in the culture medium was quantified. As described in Example 16, the affinity of these humanized proteins for human, cynomolgus monkey, and mouse EpCAM was measured. The relative K values ​​calculated from these measurements were also determined. D The values ​​are shown in Table 14. All three sequences bind to human and cynomolgus monkey EpCAM, and relative K D The values ​​ranged from approximately 0.3 to approximately 18 nM. Two of the above sequences further bound to mouse EpCAM, K D The values ​​ranged from approximately 1.4 to 1.8 nM.

[0173] [Table 16]

[0174] The T-cell killing potential of anti-EpCAM / anti-CD3 fusion proteins present in the culture medium was evaluated as described in Example 16. The results are provided in Table 15 and Figure 40.

[0175] [Table 17]

[0176] Example 18: Demonstration of improved tolerability in mice given by an exemplary EpCAM-targeted ProTriTAC molecule This study evaluated the tolerability of exemplary EpCAM-targeted ProTriTAC molecules. 2 × 10¹⁶ molecules were injected into the peritoneal cavity of 7-week-old NSG female tumor-free mice. 7Proliferated human T cells were injected at the start of the study (i.e., on day 0). On day 2, mice were divided into various groups, and treatment was initiated by administering exemplary EpCAM-targeted ProTriTAC molecules at various concentrations containing linker sequence L040, EpCAMR-targeted TriTAC molecule, EpCAM-targeted ProTriTAC molecule containing an uncleavable linker (EpCAM ProTriTAC (NCLV)), and GFP TriTAC molecule (SEQ ID No. 792) as a control to the mice. The above molecules were administered once daily for 10 days at the following doses: 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg. The body weight of the animals was recorded daily from day 2.

[0177] As shown in Figures 41A-41C, the EpCAM-targeted ProTriTAC molecule containing an uncleavable linker (ProTriTAC(NCLV)) (SEQ ID No. 908) and GFP TriTAC (used as a negative control) were well-tolerated in mice, even at the highest dose of 1 mg / kg. The EpCAM-targeted ProTriTAC molecule containing the L040 linker sequence (SEQ ID No. 907) was well-tolerated at the highest tested dose of 1 mg / kg, while EpCAM-targeted TriTAC (SEQ ID No. 906) was well-tolerated at 0.1 mg / kg. Therefore, it was observed that EpCAM-targeted ProTriTAC containing the L040 linker sequence provided at least approximately 10-fold improved tolerability in mice compared to EpCAM-targeted TriTAC.

[0178] Example 19: Xenograft tumor model The EpCAM-targeted fusion proteins described herein (e.g., fusion proteins that are triply specific proteins containing a single-domain antibody against the anti-EpCAM heavy chain only, an anti-CD3scFv, and an anti-albumin domain) were evaluated in xenograft models. Multiple xenograft tumor models were used to determine the efficacy of exemplary EpCAM-targeted fusion proteins in vivo. Examples of common tumor cell lines used in xenograft tumor studies include A549 (non-small cell lung cancer) cells, DU-145 (prostate) cells, MCF-7 (mammary gland) cells, Colo 205 (colon) cells, 3T3 (mouse fibroblast) cells, NCI H441 cells, HEP G2 (hepatocellular carcinoma) cells, MDA MB 231 (mammary gland) cells, HT-29 (colon) cells, MDA-MB435s (mammary gland) cells, U266 cells, SH-SYSY cells, Sk-Mel-2 cells, NCI H929, RPM18226, and A431 cells. Immunodeficient NOD / SCID mice were exposed to lethal radiation (2 Gy) and transplanted 1 x 10⁻¹⁰ 6 Tumor cells (e.g., NCI H441 cells) were subcutaneously inoculated. The tumor was 100-200 mm in size. 3 Upon reaching this point, the animals are assigned to three treatment groups. Groups 2 and 3 receive 1.5 × 10⁻¹⁰ treatment. 7 Activated human T cells were injected intraperitoneally. Three days later, animals in group 3 were treated with exemplary EPCAM-targeted trispecific antigen-binding protein. Groups 1 and 2 were treated with vehicle alone. Body weight and tumor volume were measured for 30 days, starting at least 5 days after treatment with exemplary EPCAM-targeted trispecific protein.

[0179] Animals treated with exemplary EpCAM-targeted trispecific proteins are predicted to exhibit statistically significant delays in tumor growth compared to control groups treated with each respective vehicle.

[0180] Example 20: Protocol of a proof-of-concept clinical trial for administration of the EpCAM-targeted trispecific antigen-binding protein of Example 19 to ovarian cancer patients. This is a Phase I / II clinical trial to test an exemplary EpCAM-targeted trispecific antigen-binding protein as a treatment for ovarian epithelial cancer. [[ID= 1]] [[ID= 2]]1. Evaluation items: [[ID= 3]] [[ID= 4]]2. Primary: The maximum tolerated dose of the exemplary EpCAM-targeted trispecific protein. [[ID= 5]] [[ID= 6]]3. Secondary: Determine whether the in vitro response of the exemplary EpCAM-targeted trispecific protein is related to the clinical response. [[ID= 7]] [[ID= 8]]

[0181] [[ID= 9]] [[ID= 10]]4. Determine the Phase I maximum tolerated dose (MTD) in the Phase I section of the trial. [[ID= 11]] [[ID= 12]]1.1 Determine the maximum tolerated dose (MTD) in the Phase I section of the trial. [[ID= 13]] [[ID= 14]]1.2 Enroll patients who meet the eligibility criteria in the previous trial of the EPCAM-targeted trispecific protein. [[ID= 15]] [[ID= 16]]1.3 The aim is to identify the maximum dose of the previous example's EpCAM-targeted trispecific protein that can be safely administered without severe or intractable side effects in participants. The dose given depends on how tolerant the number of participants and doses registered in the previous trial are. Not all participants will receive the same dose. [[ID= 17]] [[ID= 18]]

[0182] [[ID= 19]] [[ID= 20]]Phase II [[ID= 21]] [[ID= 22]]2.1 In the subsequent Phase II section, treatment is carried out at the MTD with the aim of determining whether treatment with the exemplary EpCAM-targeted trispecific protein results in a response rate of at least 20%. [[ID= 23]] [[ID= 24]]Primary evaluation item for Phase II --- Determine whether at least 20% of patients achieve a clinical response (explosive response, slight response, partial response, or complete response) as a result of treatment with the EPCAM-targeted trispecific protein [[ID= 25]] [[ID= 26]]

[0183] [[ID= 27]] [[ID= 28]]Eligibility: [[ID= 29]] [[ID= 30]]· Ovarian cancer confirmed histologically or cytologically. Recurrent ovarian epithelial cancer or potentially progressive disease following failure of primary platinum-based chemotherapy using no more than 1 prior platinum-based regimen. [[ID= 31]] ·Appropriate clinical test values for bone marrow function, renal function, liver function, and echocardiogram

[0184] Phase III 3.1 Using an exemplary EpCAM-targeted trispecific protein, perform the following Phase III section, which evaluates secondary assessment items such as response rate (RR), patient-reported outcome (PRO), progression-free survival (PFS), progression-free survival time, time to progression (TIP), overall survival period, evaluation of health-related quality of life, number of participants with overall survival, duration of response, time to response, number of participants with response, and time to tumor growth.

[0185] [Table 18-1]

[0186] [Table 18-2]

[0187] [Table 18-3]

[0188] [Table 18-4]

[0189] [Table 18-5]

[0190] [Table 18-6]

[0191] [Table 18-7]

[0192] Table 18-8

[0193] Table 18-9

[0194] Table 18-10

[0195] Table 18-11

[0196] Table 18-12

[0197] Table 18-13

[0198] Table 18-14

[0199] Table 18-15

[0200] Table 18-16

[0201] Table 18-17

[0202] Table 18-18

[0203] Table 18-19

[0204] Table 18-20

[0205] Table 18-21

[0206] Table 18-22

[0207] Table 18-23

[0208] Table 18-24

[0209] Table 18-25

[0210] Table 18-26

[0211] Table 18-27

[0212] Table 18-28

[0213] Table 18-29

[0214] Table 18-30

[0215] Table 18-31

[0216] Table 18-32

[0217] Table 18-33

[0218] Table 18-34

[0219] Table 18-35

[0220] Table 18-36

[0221] Table 18-37

[0222] Table 18-38

[0223] Table 18-39

[0224] Table 18-40

[0225] Table 18-41

[0226] Table 18-42

[0227] Table 18-43

[0228] Table 18-44

[0229] Table 18-45

[0230] Table 18-46

[0231] Table 18-47

[0232] Table 18-48

[0233] Table 18-49

[0234] Table 18-50

[0235] Table 18-51

[0236] Table 18-52

[0237] Table 18-53

[0238] Table 18-54

[0239] Table 18-55

[0240] Table 18-56

[0241] Table 18-57

[0242] Table 18-58

[0243] Table 18-59

[0244] Table 18-60

[0245] Table 18-61

[0246] Table 18-62

[0247] Table 18-63

[0248] Table 18-64

[0249] Table 18-65

[0250] Table 18-66

[0251] Table 18-67

[0252] Table 18-68

[0253] Table 18-69

[0254] Table 18-70

[0255] Table 18-71

[0256] Table 18-72

[0257] Table 18-73

[0258] Table 18-74

[0259] Table 18-75

[0260] Table 18-76

[0261] Table 18-77

[0262] Table 18-78

[0263] Table 18-79

[0264] Table 18-80

[0265] Table 18-81

[0266] Table 18-82

[0267] Table 18-83

[0268] Table 18-84

[0269] Table 18-85

[0270] Table 18-86

[0271] Table 18-87

[0272] Table 18-88

[0273] Table 18-89

[0274] Table 18-90

[0275] Table 18-91

[0276] Table 18-92

[0277] Table 18-93

[0278] Table 18-94

[0279] Table 18-95

[0280] Table 18-96

[0281] Table 18-97

[0282] Table 18-98

[0283] Table 18-99

[0284] Table 18-100

[0285] Table 18-101

[0286] Table 18-102

Claims

1. A binding portion comprising a non-CDR loop and a cleavable linker, wherein the binding portion can mask the binding of the binding molecule to its target, wherein the binding molecule comprises an immunoglobulin molecule or a non-immunoglobulin molecule. joining part.

2. The binding portion according to claim 1, wherein the binding portion is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered bulk serum protein.

3. The binding portion according to claim 1 or 2, wherein the manipulated scaffold comprises sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffold, DARPin, cystine knot peptide, lipocalin, 3-helix bundle scaffold, protein G-associated albumin-binding module, or DNA or RNA aptamer scaffold.

4. The binding portion according to any one of claims 1 to 3, wherein the binding portion can bind to bulk serum protein.

5. The coupling portion according to any one of claims 1-4, wherein the non-CDR loop is a variable domain, a steady domain, a C1 set domain, a C2 set domain, an I domain, or any combination thereof.

6. The coupling portion according to any one of claims 1 to 5, further comprising a complementarity determination region (CDR).

7. The binding portion according to claim 6, wherein the binding portion can bind to bulk serum protein.

8. The binding portion according to claim 7, wherein the bulk serum protein is a half-life prolonging protein.

9. The binding portion according to claim 7 or 8, wherein the bulk serum protein is albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentamer IgM.

10. The binding portion according to any one of claims 1 to 9, wherein the binding portion includes a binding site specific to an immunoglobulin light chain.

11. The binding portion according to claim 10, wherein the immunoglobulin light chain is an Igκ free light chain.

12. The binding portion according to any one of claims 6-11, wherein the CDR provides a binding site specific to the bulk serum protein or the immunoglobulin light chain.

13. The binding portion according to any one of claims 1-12, wherein the immunoglobulin molecule is a target antigen-binding domain.

14. The binding portion according to claim 13, wherein the binding portion is bound to the target antigen-binding domain.

15. The binding portion according to claim 13 or 14, wherein the binding portion is covalently bound to the target antigen-binding domain.

16. The binding portion according to claim 13, 14, or 15, wherein the binding portion can mask the binding of the target antigen-binding domain to its target through a specific intermolecular interaction between the binding portion and the target antigen-binding domain.

17. The binding portion according to any one of claims 13-16, wherein the non-CDR loop provides a binding site specific to the binding of the binding portion to the target antigen-binding domain.

18. The binding portion according to any one of claims 13-17, wherein when the cleavable linker is cleaved, the binding portion is separated from the target antigen-binding domain, and the target antigen-binding domain binds to its target.

19. The binding portion according to any one of claims 13-18, wherein the target antigen domain binds to a tumor antigen.

20. The tumor antigens are EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, PSMA, CD38, BCMA, and CEA, 5T4, AFP, B7-H3, CDH-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, and HPV-16 The binding portion according to claim 19, comprising E7, ITGA2, ITGA3, SLC39A6, MAGE, Mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, CDH-3, CDH-17, EPHB2, ITGAV, ITGB6, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLITRK5, SLITRK6, STEAP1, TIM1, Trop2, or WT1.

21. The binding portion according to any one of claims 13-18, wherein the target antigen domain binds to an immune checkpoint protein.

22. The binding portion according to claim 21, wherein the immune checkpoint protein is CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA.

23. The binding portion according to any one of claims 13-18, wherein the target antigen-binding domain binds to T cells.

24. The binding portion according to any one of claims 13-18, wherein the target antigen-binding domain binds to CD3.

25. The connecting portion according to any one of claims 1-24, wherein the severable linker includes a cutting portion.

26. The binding portion according to claim 25, wherein the cleavage site is recognized by a protease.

27. The binding portion according to claim 26, wherein the protease cleavage site is recognized by serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, metalloproteinase, gelatinase, or asparagine peptide lyase.

28. The protease cleavage sites include: cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, and actinin. Dyne, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, prasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloprotease (MMP), MMP1, M MP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, or dipeptidyl The binding portion according to claim 26, which is recognized by peptidase-IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

29. A conditionally active binding protein comprising a non-CDR loop, a cleavable linker (L), a binding moiety (M) including a first target antigen-binding domain (T1), and a second target antigen-binding domain (T2), wherein the first target antigen-binding domain (T1) comprises an immunoglobulin molecule, the non-CDR loop can bind to the first target antigen-binding domain, and the binding moiety can mask the binding of the first target antigen-binding domain to its target. A conditionally active binding protein.

30. The binding portion is capable of binding to the half-life extension protein, as described in claim 29, which is a conditionally active binding protein.

31. The conditionally active binding protein according to claim 29 or 30, wherein the binding portion is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered serum bulk protein.

32. The conditionally active binding protein according to claim 31, wherein the manipulated scaffold comprises sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffold, DARPin, cystine knot peptide, lipocalin, 3-helix bundle scaffold, protein G-associated albumin-binding module, or DNA or RNA aptamer scaffold.

33. The non-CDR loop is derived from a variable domain, a constant domain, a C1 set domain, a C2 set domain, an I domain, or any combination thereof, according to any one of claims 29-32, which is a conditionally active binding protein.

34. The conditionally active binding protein according to any one of claims 29-33, wherein the binding portion further comprises a complementarity-determining region (CDR).

35. The conditionally active binding protein according to any one of claims 29-34, wherein the binding portion includes a binding site specific to bulk serum protein.

36. The conditionally active binding protein according to claim 35, wherein the bulk serum protein is albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentamer IgM.

37. The binding portion further comprises a binding site specific to immunoglobulin light chains, according to any one of claims 29-36, wherein the conditionally active binding protein is described above.

38. The immunoglobulin light chain is an Igκ free light chain, according to claim 37, which is a conditionally active binding protein.

39. The CDR provides a binding site specific to bulk serum proteins or immunoglobulin light chains, or any combination thereof, according to any one of claims 34-38, a conditionally active binding protein.

40. The conditionally active binding protein according to any one of claims 34-39, wherein the binding portion can mask the binding of the first target antigen-binding domain to its target through a specific intermolecular interaction between the binding portion and the first target antigen-binding domain.

41. The conditionally active binding protein according to any one of claims 29-40, wherein the non-CDR loop provides a binding site specific to the binding of the binding portion to the first target antigen-binding domain.

42. The conditionally active binding protein according to any one of claims 29-41, wherein the first target antigen-binding domain or the second target antigen-binding domain binds to a tumor antigen.

43. The tumor antigens are EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, PSMA, CD38, BCMA, and CEA, 5T4, AFP, B7-H3, CDH-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, and HPV-16 A conditionally active binding protein according to claim 42, comprising E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, CDH-3, CDH-17, EPHB2, ITGAV, ITGB6, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLITRK5, SLITRK6, STEAP1, TIM1, Trop2, or WT1.

44. The conditionally active binding protein according to any one of claims 29-41, wherein the first target antigen-binding domain or the second target antigen-binding domain binds to an immune checkpoint protein.

45. The conditionally active binding protein according to claim 44, wherein the immune checkpoint protein is CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA.

46. The first target antigen-binding domain or the second target antigen-binding domain binds to immune cells, the conditionally active binding protein according to any one of claims 29-41.

47. The first target antigen-binding domain or the second target antigen-binding domain binds to T cells, the conditionally active binding protein according to any one of claims 29-46.

48. The conditionally active binding protein according to any one of claims 29-47, wherein the first target antigen-binding domain or the second target antigen-binding domain binds to CD3.

49. The conditionally active binding protein according to any one of claims 29-48, wherein the binding portion (M), the cleavable linker (L), the first target antigen-binding domain (T1), and the second target antigen-binding domain (T2) are one of the following: configuration M:L:T1:T2 and configuration T2:T1:L:M.

50. The conditionally active binding protein according to any one of claims 29-49, wherein the cleavable linker includes a cleavage site.

51. The cleavage site is recognized by a protease, the conditionally active binding protein according to claim 50.

52. The conditionally active binding protein according to claim 51, wherein the protease cleavage site is recognized by serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, metalloproteinase, gelatinase, or asparagine peptide lyase.

53. The protease cleavage sites include: cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, and actinida. In, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, prasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloprotease (MMP), MMP1, MMP2 MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, or dipeptidyl peptidase The conditionally active binding protein according to claim 51, which is recognized by dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

54. The conditionally active binding protein according to claim 29, further comprising a half-life extension domain bound to the binding portion, wherein the half-life extension domain provides a safety switch to the binding protein, and when the linker is cleaved, the binding protein is activated by separating the binding portion and the half-life extension domain from the first target antigen binding domain, and thus the binding protein is separated from the safety switch.

55. The linker cleavage occurs within the tumor microenvironment, the conditionally active binding protein according to claim 54.

56. A conditionally active binding protein comprising a binding portion bound to a target antigen-binding domain via a non-CDR loop within the binding portion, wherein the binding portion comprises a linker further bound to a half-life extension domain and cleavable, the target antigen-binding domain comprises an immunoglobulin molecule, the binding protein has an extended half-life prior to its activation by cleavage of the linker, the binding portion and the half-life extension domain are separated from the target antigen-binding domain upon activation, and the activated binding protein does not have an extended half-life. A conditionally active binding protein.

57. The linker cleavage occurs within the tumor microenvironment, the conditionally active binding protein according to claim 56.

58. A treatment method comprising the step of administering to a subject a conditionally active binding protein according to any one of claims 1 to 56, or a pharmaceutical composition containing the conditionally active binding protein.

59. The conditionally active binding protein according to any one of claims 29-56, wherein the non-CDR loop comprises at least one CC' loop of a camelid VHH domain, a human VH domain, a humanized VH domain, or a single-domain antibody.

60. The binding portion comprises a binding site specific to the CD3e domain, and the binding site specific to the CD3e domain comprises at least one of the following motifs: QDGNE, QDGNEE, DGNE, and DGNEE, the conditionally active binding protein according to any one of claims 29-56.

61. The conditionally active binding protein according to any one of claims 29-56, wherein the non-CDR loop comprises at least one CC' loop of a camelid VHH domain, a human VH domain, a humanized VH domain, or a single-domain antibody.

62. The binding portion comprises a binding site specific to the CD3e domain, and the binding site specific to the CD3e domain comprises at least one of the following motifs: QDGNE, QDGNEE, DGNE, and DGNEE, the conditionally active binding protein according to any one of claims 29-56.

63. The binding portion according to any one of claims 1 to 28, wherein the non-CDR loop comprises at least one CC' loop of a camelid VHH domain, a human VH domain, a humanized VH domain, or a single-domain antibody.

64. The binding portion according to any one of claims 1 to 28, wherein the binding portion includes a binding site specific to the CD3e domain, and the binding site specific to the CD3e domain includes at least one of the following motifs: QDGNE, QDGNEE, DGNE, and DGNEE.

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