Binding moieties for conditionally activating immunoglobulin molecules

Abstract

The present application relates to binding moieties for conditionally activating immunoglobulin molecules. Disclosed herein are binding moieties comprising a non-CDR ring for masking binding of a binding molecule to its target and a CDR for binding large volume serum proteins. Also provided are conditionally active target binding proteins containing the binding moieties. Further provided are pharmaceutical compositions comprising the binding proteins disclosed herein and methods of using such formulations.

Description

[0001] This application is a divisional application of an application filed on May 14, 2019, with application number 201980047378.8 and invention name “Binding portion for conditionally activating immunoglobulin molecules”.

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Application Nos. 62 / 671,344, filed May 14, 2018, 62 / 671,349, filed May 14, 2018, 62 / 756,429, filed November 6, 2018, and 62 / 756,453, filed November 6, 2018, each of which is incorporated herein by reference in its entirety.

[0004] Incorporation by reference

[0005] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference and was set forth in its entirety. Background Art

[0006] T cell engagers temporarily tether T cells to tumor cells and mediate T cell-directed tumor killing. T cell engagers, such as blinatumomab Clinical activity has been shown in several hematological malignancies. The use of T cell engagers in solid tumors is limited by the lack of tumor antigens that are sufficiently differentially expressed between tumor and normal tissues. T cell engagers that are preferentially active in the tumor microenvironment could safely target more solid tumor antigens.

[0007] There is a need to prolong the half-life of therapeutic, diagnostic or imaging molecules in the circulation and also improve their ability to reach their target within the intended location (eg, tumor cells) without non-specific binding. Summary of the invention

[0008] One embodiment provides a binding portion comprising a non-CDR loop and a cleavable connector, wherein the portion is capable of masking 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 natural peptide, a synthetic peptide, an engineered scaffold, or an engineered bulk serum protein. In some embodiments, the engineered scaffold comprises an sdAb, scFv, Fab, VHH, a type III fibronectin domain, an immunoglobulin-like scaffold, a DARPin, a cystine knot peptide, a lipocalin, a three-helix bundle scaffold, an albumin binding module associated with protein G, or a DNA or RNA aptamer scaffold. In some embodiments, the portion is capable of binding to bulk serum proteins. In some embodiments, the non-CDR loop is 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 portion further comprises a complementary determining region (CDR). In some embodiments, the portion is capable of binding to bulk serum proteins. In some embodiments, the bulk serum protein is a protein that prolongs half-life. 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 bulk serum protein is albumin, transferrin, factor XIII or fibrinogen. In some embodiments, the CDR in the binding portion provides a binding site specific for the bulk serum protein. In some embodiments, the binding portion is capable of masking the binding of the target antigen binding domain (such as an immunoglobulin molecule) or a non-immunoglobulin binding molecule to its target through specific intermolecular interactions between the binding portion and the target antigen binding domain or non-immunoglobulin binding portion. In some embodiments, the non-CDR loops in the binding portion provide a binding site specific for the binding of the binding portion to the target antigen binding domain (such as an immunoglobulin molecule) or a non-immunoglobulin binding molecule.

[0009] In some embodiments, the binding portion comprises a binding site specific for an immunoglobulin light chain. In some embodiments, the immunoglobulin light chain is a light chain that does not contain Igκ. In some embodiments, the CDR provides a binding site specific for the bulk serum protein or the immunoglobulin light chain. In some embodiments, the immunoglobulin molecule is a target antigen binding domain. In some embodiments, the portion binds to the target antigen binding domain. In some embodiments, the portion is covalently linked to the target antigen binding domain. In some embodiments, the portion is capable of masking the binding of the target antigen binding domain to its target through specific intermolecular interactions between the binding portion and the target antigen binding domain. In some embodiments, the non-CDR loop provides a binding site specific for the binding of the portion to the target antigen binding domain. In some embodiments, when the cleavable linker is cut, the binding portion is separated 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, the 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-16E6, HPV-16E7, 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 comprises a cleavage site. In some embodiments, the cleavage site is recognized by a protease. In some embodiments, the protease cleavage site is recognized by a serine protease, a cysteine ​​protease, an aspartic acid protease, a threonine protease, a glutamic acid protease, a metalloprotease, a gelatinase, or an asparagine peptide lyase. In some embodiments, the protease cleavage site is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, collagenase type IV, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidin, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, protease, legumin, Plasmodium aspartic protease, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP 12, MMP13, MMP14, ADAM9, ADAM10, ADAM12, urokinase plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine proteinase 4, mast cell chymotrypsin and mast cell tryptase recognition.

[0010] In some embodiments, the non-CDR loops include the CC' loop of at least one of: a camelid VHH domain, a human VH domain, a humanized VH domain, or a single domain antibody.

[0011] In some embodiments, the binding moiety comprises a binding site specific for the CD3 epsilon domain, and wherein the binding site specific for the CD3 epsilon domain comprises at least one of the following motifs: QDGNE, QDGNEE, DGNE, and DGNEE.

[0012] One embodiment provides a conditionally active binding protein comprising a binding portion (M), the binding portion comprising 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, wherein the non-CDR loop is capable of binding to the first target antigen binding domain, and wherein the binding portion is capable of masking the binding of the first target antigen binding domain to its target. In some embodiments, the binding portion is capable of binding to a protein that extends half-life. In some embodiments, the binding portion is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered serum bulk protein. In some embodiments, the engineered scaffold includes sdAb, scFv, Fab, VHH, a type III fibronectin domain, an immunoglobulin-like scaffold, a DARPin, a cystine knot peptide, a lipocalin, a three-helix bundle scaffold, a protein G-related albumin binding module, or a DNA or RNA aptamer scaffold. In some embodiments, the non-CDR loop is 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 complementary determining region (CDR). In some embodiments, the binding portion comprises a binding site specific for a large volume serum protein. In some embodiments, the large volume 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 for an immunoglobulin light chain. In some embodiments, the immunoglobulin light chain is a light chain that does not contain Igκ. In some embodiments, the CDR provides a binding site specific for the large volume serum protein or the immunoglobulin light chain, or any combination thereof. In some embodiments, the binding portion is capable of masking the binding of the first target antigen binding domain to its target by a specific intermolecular interaction between the binding portion and the first target antigen binding domain. In some embodiments, the non-CDR loops provide a binding site that is specific for binding of the binding moiety 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 comprises at least one of: EpCAM (exemplary protein sequence comprises UniProtkB ID No. P16422), EGFR (exemplary protein sequence comprises UniProtkB ID No. P00533), HER-2 (exemplary protein sequence comprises UniProtkB ID No. P04626), HER-3 (exemplary protein sequence comprises UniProtkB ID No.P21860), c-Met (exemplary protein sequence comprises UniProtkB ID No. P08581), FoIR (exemplary protein sequence comprises UniProtkB ID No. P15238), PSMA (exemplary protein sequence comprises UniProtkB ID No. Q04609), CD38 (exemplary protein sequence comprises UniProtkB ID No. P28907), BCMA (exemplary protein sequence comprises UniProtkB ID No. Q02223) and CEA (exemplary protein sequence comprises UniProtkB ID No. P06731), 5T4 (exemplary protein sequence comprises UniProtkB ID No. Q13641), AFP (exemplary protein sequence comprises UniProtkB ID No. P02771), B7-H3 (exemplary protein sequence comprises UniProtkB ID No. Q5ZPR3), CDH-6 (exemplary protein sequence comprises UniProtkB ID No. P97326), CAIX (exemplary protein sequence comprises UniProtkB ID No. Q16790), CD117 (exemplary protein sequence comprises UniProtkB ID No. P10721), CD123 (exemplary protein sequence comprises UniProtkB ID No. P26951), CD138 (exemplary protein sequence comprises UniProtkB ID No. P18827), CD166 (exemplary protein sequence comprises UniProtkB ID No. Q13740), CD19 (exemplary protein sequence comprises UniProtkB ID No. P15931), CD20 (exemplary protein sequence comprises UniProtkB ID No. P11836), CD205 (exemplary protein sequence comprises UniProtkB ID No. O60449), CD22 (exemplary protein sequence comprises UniProtkB ID No. P20273), CD30 (exemplary protein sequence comprises UniProtkB ID No. P28908), CD33 (exemplary protein sequence comprises UniProtkB ID No. P20138), CD352 (exemplary protein sequence comprises UniProtkB ID No. Q96DU3), CD37 (exemplary protein sequence comprises UniProtkB ID No. P11049), CD44 (exemplary protein sequence comprises UniProtkB ID No. P16070), CD52 (exemplary protein sequence comprises UniProtkB ID No.P31358), CD56 (exemplary protein sequence comprises UniProtkB ID No. P13591), CD70 (exemplary protein sequence comprises UniProtkB ID No. P32970), CD71 (exemplary protein sequence comprises UniProtkB ID No. P02786), CD74 (exemplary protein sequence comprises UniProtkB ID No. P04233), CD79b (exemplary protein sequence comprises UniProtkB ID No. P40259), DLL3 (exemplary protein sequence comprises UniProtkB ID No. Q9NYJ7), EphA2 (exemplary protein sequence comprises UniProtkB ID No. P29317), FAP (exemplary protein sequence comprises UniProtkB ID No. Q12884), FGFR2 (exemplary protein sequence comprises UniProtkB ID No. P21802), FGFR3 (exemplary protein sequence comprises UniProtkB ID No. No. P22607), GPC3 (exemplary protein sequence comprises UniProtkB ID No. P51654), gpA33 (exemplary protein sequence comprises UniProtkB ID No. Q99795), FLT-3 (exemplary protein sequence comprises UniProtkB ID No. P36888), gpNMB (exemplary protein sequence comprises UniProtkB ID No. Q14956), HPV-16E6 (exemplary protein sequence comprises UniProtkB ID No. P03126), HPV-16E7 (exemplary protein sequence comprises UniProtkB ID No. P03129), ITGA2 (exemplary protein sequence comprises UniProtkB ID No. P17301), ITGA3 (exemplary protein sequence comprises UniProtkB ID No. P26006), SLC39A6 (exemplary protein sequence comprises UniProtkB ID No.Q13433), MAGE (exemplary protein sequence comprises UniProtkB ID No.Q9HC15), mesothelin (exemplary protein sequence comprises UniProtkB ID No.Q13421), Muc1 (exemplary protein sequence comprises UniProtkB ID No.P15941), Muc16 (exemplary protein sequence comprises UniProtkB ID No.Q8WX17), NaPi2b (exemplary protein sequence comprises UniProtkB ID No.O95436), adhesion protein-4 (exemplary protein sequence comprises UniProtkB ID No.Q96918), CDH-3 (exemplary protein sequence comprises UniProtkB ID No. Q8WX17), CDH-17 (exemplary protein sequence comprises UniProtkB ID No. E5RJT3), EPHB2 (exemplary protein sequence comprises UniProtkB ID No. P29323), ITGAV (exemplary protein sequence comprises UniProtkB ID No. P06756), ITGB6 (exemplary protein sequence comprises UniProtkB ID No. P18564), NY-ESO-1 (exemplary protein sequence comprises UniProtkB ID No. P78358), PRLR (exemplary protein sequence comprises UniProtkB ID No. P16471), PSCA (exemplary protein sequence comprises UniProtkB ID No. O43653), PTK7 (exemplary protein sequence comprises UniProtkB ID No. Q13308), ROR1 (exemplary protein sequence comprises UniProtkB ID No.Q01973), SLC44A4 (exemplary protein sequence comprises UniProtkB ID No.Q53GD3), SLITRK5 (exemplary protein sequence comprises UniProtkB ID No.Q8IW52), SLITRK6 (exemplary protein sequence comprises UniProtkB ID No.Q9HY7), STEAP1 (exemplary protein sequence comprises UniProtkB ID No.Q9UHE8), TIM1 (exemplary protein sequence comprises UniProtkB ID No.Q96D42), Trop2 (exemplary protein sequence comprises UniProtkB ID No.P09758) or WT1 (exemplary protein sequence comprises 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 comprises UniProtkB ID No. P26842), CD137 (exemplary protein sequence comprises UniProtkB ID No. Q07011), 2B4 (exemplary protein sequence comprises UniProtkB ID No. Q9bZW8), TIGIT (exemplary protein sequence comprises UniProtkB ID No. Q495A1), CD155 (exemplary protein sequence comprises UniProtkB ID No. P15151), ICOS (exemplary protein sequence comprises UniProtkB ID No.Q9Y6W8), HVEM (exemplary protein sequence comprises UniProtkB ID No.043557), CD40L (exemplary protein sequence comprises UniProtkB ID No.P29965), LIGHT (exemplary protein sequence comprises UniProtkB ID No.043557), OX40 (exemplary protein sequence comprises UniProtkB ID No.), DNAM-1 (exemplary protein sequence comprises UniProtkB ID No.Q15762), PD-L1 (exemplary protein sequence comprises UniProtkB ID No.Q9ZQ7), PD1 (exemplary protein sequence comprises UniProtkB ID No.Q15116), PD-L2 (exemplary protein sequence comprises UniProtkB ID No.Q9BQ51), CTLA-4 (exemplary protein sequence comprises UniProtkB ID No.P16410), CD8 (exemplary protein sequence comprises UniProtkB ID No. P10966, P01732), CD40 (exemplary protein sequence comprises UniProtkB ID No. P25942), CEACAM1 (exemplary protein sequence comprises UniProtkB ID No. P13688), CD48 (exemplary protein sequence comprises UniProtkB ID No. P09326), CD70 (exemplary protein sequence comprises UniProtkB ID No. P32970), AA2AR (exemplary protein sequence comprises UniProtkB ID No. P29274), CD39 (exemplary protein sequence comprises UniProtkB ID No. P49961), CD73 (exemplary protein sequence comprises UniProtkB ID No. P21589), B7-H3 (exemplary protein sequence comprises UniProtkB ID No. Q5ZPR3), B7-H4 (exemplary protein sequence comprises UniProtkB ID No.Q7Z7D3), BTLA (exemplary protein sequence comprises UniProtkB ID No.Q76A9), IDO1 (exemplary protein sequence comprises UniProtkB ID No.P14902), IDO2 (exemplary protein sequence comprises UniProtkB ID No.Q6ZQW0), TDO (exemplary protein sequence comprises UniProtkB ID No.P48755), KIR (exemplary protein sequence comprises UniProtkB ID No.Q99706), LAG-3 (exemplary protein sequence comprises UniProtkB ID No.P18627), TIM-3 (also known as HAVCR2, exemplary protein sequence comprises UniProtkB ID No. Q8TDQ0) or VISTA (exemplary protein sequence comprises UniProtkB ID No. Q9D659). In some embodiments, the first or second target antigen binding domain binds to an immune cell. .

[0013] 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 configurations: M:L:T1:T2 and T2:T1:L:M. In some embodiments, the binding moiety comprises an albumin binding domain (anti-Alb), the first target antigen binding domain (T1) comprises a CD3 binding domain (e.g., anti-CD3 scFV), and the ProTriTAC molecule has the following orientation: anti-Alb: anti-CD3:T2. In some embodiments, the binding moiety comprises an albumin binding domain (anti-Alb), the second target antigen binding domain (T2) comprises a CD3 binding domain (e.g., anti-CD3 scFV), and the ProTriTAC molecule has the following orientation: anti-Alb:T1:anti-CD3. In certain examples, the T1 domain is a tumor antigen binding domain, such as, but not limited to, an anti-EGFR domain, an anti-MSLN domain, an anti-BCMA domain, an anti-EpCAM domain, an anti-PSMA domain, or an anti-DLL3 domain.

[0014] In some embodiments, the cleavable linker comprises a cleavage site. In some embodiments, the cleavage site is recognized by a protease. In some embodiments, the protease cleavage site is recognized by a serine protease, a cysteine ​​protease, an aspartic protease, a threonine protease, a glutamic protease, a metalloprotease, a gelatinase, or an asparagine peptide lyase. In some embodiments, the protease cleavage site is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, collagenase type IV, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidin, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, protease, legumin, Plasmodium aspartic protease, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP 12, MMP13, MMP14, ADAM9, ADAM10, ADAM12, urokinase plasminogen activator (uPA), enterokinase, prostate specific target (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymotrypsin and mast cell trypsin recognition. In some embodiments, the conditionally active binding protein further comprises a half-life extension domain bound to the binding portion, wherein the half-life extension domain provides a safety switch for the binding protein, and wherein when the linker is cut, the binding protein is activated by separating the binding portion and the half-life extension domain from the first target antigen binding domain, thereby separating the binding protein from the safety switch. In some embodiments, the cleavage of the linker occurs in the tumor microenvironment.

[0015] One embodiment provides a conditionally active binding protein comprising a binding portion connected to a target antigen binding domain via a non-CDR loop within the binding portion, wherein the binding portion is further connected to a half-life extension domain, and comprises a cleavable linker, wherein the target antigen binding domain comprises an immunoglobulin molecule, wherein the binding protein has an extended half-life before it is activated by cleavage of the linker, and wherein upon activation, the binding portion and the half-life extension domain are separated from the target antigen binding domain, and wherein the binding protein does not have an extended half-life in its activated state. In some embodiments, cleavage of the linker occurs in a tumor microenvironment.

[0016] In some embodiments, the non-CDR loops include the CC' loop of at least one 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 that is specific for the CD3 epsilon domain, and wherein the binding site that is specific for the CD3 epsilon domain comprises at least one of the following motifs: QDGNE, QDGNEE, DGNE, and DGNEE.

[0017] One embodiment provides a method of treatment comprising administering to a subject a conditionally active binding protein described herein, or a pharmaceutical composition comprising the conditionally active binding protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description of illustrative embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0019] Figure 1 The variable domain of an exemplary immunoglobulin domain is shown, comprising the complementarity determining regions (CDR1, CDR2, and CDR3) and the non-CDR loops (AB, CC', C"D, EF, and DE) connecting the beta strands.

[0020] Figure 2 A-2B provides an exemplary arrangement of the various domains of the conditionally active binding proteins of the present disclosure. Figure 2 A: Version 1. Figure 2 B: Version 2.

[0021] Figure 3 Exemplary conditionally active target binding proteins of the present disclosure are shown.

[0022] Figure 4 A-4C shows the activation and possible mode of action of a trispecific molecule (ProTriTAC). Figure 4A shows ProTriTAC molecules in circulation, in the tumor environment, and in circulation. Figure 4 B shows an exemplary sequence of a protease cleavable site in a linker tethered to an anti-albumin binding moiety, and Figure 4 C shows an SDS-PAGE gel showing ProTriTAC in both activatable (prodrug) and activated (active drug) states.

[0023] Figure 5 AB show the processes used to prepare and purify the molecules described herein. Figure 5 A shows a schematic flow chart for preparing ProTriTAC molecules, and Figure 5 B shows a SDS-PAGE gel showing three purified ProTriTAC molecules.

[0024] Figure 6 AB shows analytical size exclusion chromatograms of ProTriTAC molecules exposed to different stress conditions. Figure 6 A is in graphical form. Figure 6 B shows the corresponding data. Figure 6 B provides Figure 6 A's data.

[0025] Figure 7 Protease-dependent antitumor activity of exemplary ProTriTAC molecules in the HCT116 colorectal tumor xenograft model in NSG mice is shown.

[0026] Figure 8 A-8D show various designs of exemplary ProTriTACs and control molecules. Figure 8 A: Control #1; Figure 8 B: control #2; Figure 8 C: ProTriTAC; Figure 8 D: Activated ProTriTAC.

[0027] Fig. 9 Pharmacokinetic profiles of exemplary ProTriTACs and control molecules are shown.

[0028] Fig.10 The conversion and half-life of exemplary ProTriTAC molecules are shown.

[0029] Fig.11 Plasma clearance of exemplary ProTriTAC molecules and their converted active drug forms are shown.

[0030] Fig.12The CD3 binding potential of exemplary ProTriTAC molecules, their converted active drug forms, and a control uncleavable ProTriTAC molecule are shown.

[0031] Fig.13 Human primary T cell binding potential of exemplary ProTriTAC molecules, their converted active drug forms, and control uncleavable ProTriTAC molecules are shown.

[0032] Fig.14 The T cell killing potential of exemplary ProTriTAC molecules, their converted active drug forms, and control uncleavable ProTriTAC molecules are shown.

[0033] Fig.15 Shown is the schematic structure of an exemplary trispecific molecule comprising a binding moiety as described herein (also referred to herein as a ProTriTAC or an activatable ProTriTAC).

[0034] Fig.16 A-16E shows an exemplary schematic structure of a prodrug molecule combining functionality masking and half-life extension. Fig.16 A shows a prodrug molecule comprising an anti-albumin moiety including a masking moiety and a cleavable linker connecting the anti-albumin moiety and the drug. Fig.16 B shows a prodrug molecule comprising an anti-albumin moiety comprising two peptide motifs connected by a linker, one of which comprises a masking moiety, and a cleavable linker connecting the albumin binding moiety to the drug. Fig.16 C shows a prodrug molecule comprising a modified albumin (containing a masking moiety) attached to the drug via a cleavable linker. Fig.16 D shows a prodrug molecule comprising a modified albumin (containing a masking moiety and a protease cleavage site) linked to the drug. Fig.16 E shows the activated prodrug. In each schematic structure ( Fig.16 A-16D), the drug molecule is functionally masked by the anti-albumin moiety or modified albumin so that it does not bind to its target, or is not activated at an undesired site, or is not bound at a non-target site, thereby creating a drug sink.

[0035] Fig.17 A-17E shows an exemplary schematic structure of a ProTriTAC molecule combining functional masking and half-life extension. Fig.17A shows a ProTriTAC molecule comprising an anti-albumin moiety including a masking moiety and a cleavable linker connecting the anti-albumin moiety and the T cell engager molecule. Fig.17 B shows a ProTriTAC molecule comprising an anti-albumin moiety comprising two peptide motifs connected by a linker, one of which comprises a masking moiety, and a cleavable linker connecting the albumin binding moiety to a T cell engager molecule. Fig.17 C shows a ProTriTAC molecule comprising a modified albumin (containing a masking moiety) linked to a T cell engager via a cleavable linker. Fig.17 D shows a ProTriTAC molecule comprising a modified albumin (containing a masking moiety and a protease cleavage site) linked to a T cell engager. Fig.17 E shows the activated ProTriTAC. In each schematic structure ( Fig.17 A-17D), the target binding interface within the ProTriTAC molecule is functionally masked by an anti-albumin moiety or modified albumin so that it does not bind its target, or is not activated at an undesired site, or is not bound at a non-target site, thereby creating a sink.

[0036] Fig.18 The anti-tumor activities of exemplary ProTriTAC molecules and TriTAC molecules of the present disclosure are shown.

[0037] Fig.19 Pharmacokinetic profiles of exemplary ProTriTAC molecules and TriTAC molecules of the present disclosure are shown.

[0038] Fig. 20 A-20F shows mixed xenograft individual tumor volumes after administration of exemplary ProTriTAC molecules or TriTAC molecules of the present disclosure. Fig. 20 A shows the results of GFP TriTAC. Fig. 20 B shows the results of EGFR ProTriTAC (NCLV). Fig. 20 C shows the results of EGFR ProTriTAC (L001). Fig. 20 D shows the results of EGFR ProTriTAC (L041). Fig. 20 E shows the results of EGFR ProTriTAC (L040). Fig. 20 F shows the results of EGFR ProTriTAC (L045).

[0039] Fig.21A-21C shows cytokine levels (IFN-γ ( Fig.21 A) IL-6 ( Fig.21 B) and IL-10; Fig.21 C).

[0040] Fig. 22 A-22E shows the percent body weight change of mice after administration of exemplary ProTriTAC molecules and TriTAC molecules of the present disclosure. Fig. 22 A shows the results for 30 μg / kg; Fig. 22 B shows the results for 100 μg / kg; Fig. 22 C shows the results for 300 μg / kg; Fig. 22 D shows the results for 1000 μg / kg; and Fig. 22 E provided protection multiples at various concentrations.

[0041] Fig.23 A-23C shows the percent body weight change of mice after administration of different concentrations of exemplary ProTriTAC molecules of the present disclosure (containing non-cleavable or cleavable linkers). Fig.23 A shows the results for 300 μg / kg; Fig.23 B shows the results for 1000 μg / kg; Fig.23 C provided protection folds at various concentrations.

[0042] Figures 24A-24C The results show that the non-cleavable linker-containing ProTriTAC molecule (ProTriTAC (NCLV)) ( Fig.24C )、TriTAC molecules( Fig.24A ) or a ProtriTAC molecule containing a cleavable linker ( Fig. 24B ) after , serum concentration of aspartate aminotransferase (AST) in mice.

[0043] Figures 25A-25C The results show that the non-cleavable linker-containing ProTriTAC molecule (ProTriTAC (NCLV)) ( Fig.25C )、TriTAC molecules( Fig.25A ) or a ProtriTAC molecule containing a cleavable linker ( Fig.25B ) after the serum concentration of alanine aminotransferase (ALT) in mice.

[0044] Fig.26A-26B shows ALT in cynomolgus monkeys after administration of different concentrations of EGFR ProTriTAC molecules or EGFR ProTriTAC(NCLV) molecules (right panel; Fig.26 B) or AST (left; Fig.26 A) serum concentration.

[0045] Figures 27A-27D Shown are tumor volumes in mice after administration of GFP TriTAC molecules, EGFR TriTAC molecules, or EGFRProTriTAC molecules at different concentrations. Fig.27A GFP TriTAC (300 μg / kg) and EGFR TriTAC (10 μg / kg) are shown. Fig.27B EGFR TriTAC (30 μg / kg and 100 μg / kg) is shown. Fig.27C EGFR TriTAC (300 μg / kg) and EGFR ProTriTAC (30 μg / kg and 100 μg / kg) are shown. Fig.27D EGFR ProTriTAC (300 μg / kg and 1000 μg / kg) is shown.

[0046] Fig.28 Shown are serum concentrations of ALT and AST in mice after administration of different concentrations of GFP TriTAC, EGFR TriTAC, and EGFR ProTriTAC molecules.

[0047] Fig.29 Grafting of the CD3 epsilon epitope into the CC' loop of the binding moieties of the present disclosure is shown. HuCD3e: SEQ ID NO:901; CC10: SEQ ID NO:260; CC12: SEQ ID NO:259; and CC16: SEQ ID NO:261.

[0048] Fig.30 The dissociation of a binding moiety of the present disclosure from a ProTriTAC molecule containing the binding moiety following activation of a tumor-associated protease by a matriptase is shown.

[0049] Fig.31 CD3 binding of ProTriTAC molecules, with and without activation, containing exemplary binding moieties of the disclosure are shown.

[0050] Fig.32 The cell killing potential of ProTriTAC molecules, with and without activation, containing exemplary binding moieties of the present disclosure are shown.

[0051] Fig.33Shown is a soft library mutagenesis approach that was performed to explore non-CDR loops within exemplary binding moieties of the disclosure.

[0052] Fig.34 Shown are the results of a soft library mutagenesis approach performed to explore non-CDR loops within exemplary binding moieties of the present disclosure following panning against HSA (human serum albumin, or also referred to herein as albumin).

[0053] Fig.35 It is shown that the binding moiety of the present disclosure is capable of expanding the therapeutic window of the molecule containing it (eg, ProTriTAC molecule) through steric masking and specific masking.

[0054] Fig.36 Provided are the results of a representative T cell-dependent cytotoxicity assay performed with NCI-H508 cells using exemplary fusion proteins of the disclosure containing an anti-EpCAM domain and an anti-CD3 domain as described herein.

[0055] Fig.37 Provided are the results of a representative T cell-dependent cytotoxicity assay performed using exemplary fusion proteins of the present disclosure containing an anti-EpCAM domain and an anti-CD3 domain as described herein.

[0056] Fig.38 Provided are the results of a representative T cell-dependent cytotoxicity assay performed using exemplary fusion proteins of the present disclosure containing an anti-EpCAM domain and an anti-CD3 domain as described herein.

[0057] Fig.39 Provided are the results of a representative T cell-dependent cytotoxicity assay performed using exemplary fusion proteins of the present disclosure containing an anti-EpCAM domain and an anti-CD3 domain as described herein.

[0058] Fig.40 Provided are the results of a representative T cell-dependent cytotoxicity assay performed using exemplary fusion proteins of the present disclosure containing a humanized anti-EpCAM domain and an anti-CD3 domain as described herein.

[0059] Figures 41A-41C Shown are the percent changes in body weight of mice following administration of exemplary EpCAM ProTriTAC molecules and EpCAM TriTAC molecules of the present disclosure. DETAILED DESCRIPTION

[0060] Although 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 by way of example only. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes and substitutions may be employed in the process of implementing the present invention. It is intended that the scope of the present invention be limited to the appended claims, thereby encompassing methods and structures and their equivalents within the scope of these claims.

[0061] In certain embodiments, provided herein are ProTriTAC molecules (also referred to herein as protrispecific molecules) that are T cell engager prodrugs designed to be conditionally active in the tumor microenvironment. In some cases, this makes it possible to target a wider selection of tumor antigens (e.g., solid tumor antigens). In some instances, ProTriTAC molecules combine the desirable attributes of several prodrug approaches, including, but not limited to: a combination of steric masking and specific masking, wherein in some cases, steric masking is achieved by albumin recognized by the anti-albumin domain in the ProTriTAC molecule, and in some cases, specific masking is achieved by specific intermolecular interactions between the anti-albumin domain (in some instances) and the target antigen binding domain of the ProTriTAC molecule (e.g., in some instances, the anti-CD3 scFv domain); additional safety conferred by the difference in half-life between the prodrug and the active drug obtained by activating the conditionally activated ProTriTAC molecule; and the ability to plug and play with different tumor target binders.

[0062] Some definitions

[0063] The terms used herein are for the purpose of describing a particular situation only and are not intended to be limiting. Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" as used herein are intended to include the plural forms as well. In addition, to the extent that the terms "includes", "comprising", "having", or variations thereof are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising".

[0064] The term "about" or "approximately" means within an acceptable error range for a particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., limitations of the measurement system. For example, depending on the practice of a given value, "about" can mean within 1 or more than 1 standard deviation. Where specific values ​​are described in the application and claims, the term "about" should be considered to mean an acceptable error range for that specific value unless otherwise stated.

[0065] The terms "individual," "patient," or "subject" are used interchangeably. None of these terms require or are limited to situations characterized by monitoring (e.g., continuous or intermittent) by a health care worker (e.g., a physician, registered nurse, nurse practitioner, physician assistant, caregiver, or hospice worker).

[0066] As used herein, "single-chain Fv" or "scFv" refers to a binding protein in which the variable domains of the heavy and light chains of a traditional two-chain antibody are linked to form one chain. Typically, a connecting peptide is inserted between the two chains to allow correct folding and generation of an active binding site.

[0067] As used herein, a "cleavage site for a protease" or "protease cleavage site" is an amino acid sequence that can be cleaved by a protease, such as a 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 a metalloproteinase, and Arg-Arg-Arg-Arg-Arg-Arg, which can be cleaved by furin. In therapeutic applications, the protease cleavage site can be cleaved by a protease produced by a target cell, such as a cancer cell or an infected cell or pathogen.

[0068] As used herein, "elimination half-life" is used in its ordinary sense, as described in Goodman and Gillman, The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman and Alfred Gilman, eds., 6th edition 1980). In short, the term is intended to include a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics) because the drug concentration is usually not close to the concentration required for saturation of the elimination process. The rate of an exponential process can be determined by its rate constant, k, or by its half-life, t 1 / 2 The rate constant k represents the fractional change per unit time, and the half-life t 1 / 2 It represents the time required for the process to complete 50%. The units of these two constants are time -1 The first-order rate constant and half-life of this reaction are simply related (k×t 1 / 2 =0.693) and can be interchanged accordingly. Since first-order elimination kinetics indicate that a constant fraction of drug is lost per unit time, a plot of the logarithm of drug concentration versus time is linear 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 plot.

[0069] As used herein, "therapeutic agent" includes "binding molecules".

[0070] As used herein, the term "binding molecule" is any molecule or part or fragment thereof that can be bound to a target molecule, cell, complex and / or tissue, and includes proteins, nucleic acids, carbohydrates, lipids, low molecular weight compounds and fragments thereof, each having the ability to bind to one or more of soluble proteins, cell surface proteins, cell surface receptor proteins, intracellular proteins, carbohydrates, nucleic acids, hormones or low molecular weight compounds (small molecule drugs) or their fragments. In some cases, the binding molecule is a protein or non-immunoglobulin binding molecule belonging to the immunoglobulin superfamily. The "binding molecule" does not include cytokines.

[0071] As used herein, the term "protein belonging to the immunoglobulin superfamily" or "immunoglobulin molecule" includes proteins that comprise the immunoglobulin fold, 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.

[0072] The term "antibody" includes antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv and Fd fragments, 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 comprising antigen binding (also referred to herein as antigen binding) portions of antibodies and non-antibody proteins. In some instances, antibodies are detectably labeled, such as with radioisotopes, enzymes that generate detectable products, fluorescent proteins, and the like. In some cases, antibodies are further conjugated to other moieties, such as members of specific binding pairs, such as biotin (members of biotin-avidin specific binding pairs), etc. In some cases, antibodies are bound to solid supports, including but not limited to polystyrene plates or beads, etc. The term also includes Fab', Fv, F(ab')2 and / or other antigen binding fragments that retain specific binding to an antigen, as well as monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which are produced from a single cell by repeated cell replication. That is, the clone of the cell produces only a single antibody species. Although hybridoma production technology can be used to produce monoclonal antibodies, other production methods known to those skilled in the art (e.g., antibodies obtained by antibody phage display libraries) can also be used. In some cases, the antibody is monovalent or divalent. In some cases, the antibody is an Ig monomer, which is a "Y-shaped" molecule consisting of four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.

[0073] The term "non-immunoglobulin binding molecule" as used herein includes, but is not limited to, for example, growth factors, hormones, signaling proteins, inflammatory mediators, ligands, receptors or fragments thereof, natural hormones or variants thereof that can bind to their natural receptors; nucleic acids or polynucleotide sequences that can bind to complementary sequences or soluble cell surface or intracellular nucleic acid / polynucleotide binding proteins, carbohydrate binding moieties that can bind to other carbohydrate binding moieties, cell surface or intracellular proteins, and low molecular weight compounds (drugs) that bind to soluble or cell surface or intracellular target proteins. In some cases, non-immunoglobulin binding molecules include coagulation factors, plasma proteins, fusion proteins and imaging agents. Non-immunoglobulin binding molecules do not include cytokines.

[0074] As used herein, "cytokines" refer to intercellular signaling molecules and their active fragments and portions, which are involved in the regulation of mammalian somatic cells. Many cytokine families are included, such as interleukins, interferons, and transforming growth factors.

[0075] As used herein, "non-CDR loops" within an immunoglobulin (Ig) molecule are regions of a polypeptide other than the complementarity determining regions (CDRs) of an antibody. These regions may be derived from an antibody or antibody fragment. These regions may also be synthetic or artificially derived, for example by mutagenesis or polypeptide synthesis.

[0076] In Ig, Ig-like or β-sandwich scaffolds with 9 β-strands (e.g., VH, VL, Camelid VHH, sdAb), the non-CDR loops may refer to the AB, CC', C"D, EF loops or the loops connecting the β-strands adjacent to the C-terminus. In Ig, Ig-like or β-sandwich scaffolds with 7 β-strands (e.g., CH, CL, adnectin, Fn-III), the non-CDR loops may refer to the AB, CD and EF loops or the loops connecting the β-strands adjacent to the C-terminus. In other Ig-like or β-sandwich scaffolds, the non-CDR loops are loops that connect the β-strands adjacent to the C-terminus or topologically equivalent residues using the framework established in the Halaby 1999 publication (Prot Eng Des Sel 12:563-571).

[0077] In non-β-sandwich scaffolds (e.g., DARPins, affimers, affibodies), "non-CDR loops" refer to regions that are (1) suitable for sequence randomization to allow engineered specificity for a second antigen, and (2) distal to the primary specificity determining regions typically used on the scaffold to allow the scaffold to engage both antigens simultaneously without steric interference. To this end, the framework established in the Skrlec 2015 publication (Trends in Biotechnol, 33:408-418) can be used to define the primary specificity determining regions. An excerpt of the framework is listed below.

[0078]

[0079] As used herein, "target antigen binding domain" refers to a region that targets a specific antigen. The target antigen binding domain includes, for example, sdAb, scFv, variable heavy chain antibody (VHH), heavy chain variable domain (VH) or light chain variable domain (VL), full-length antibody or any other peptide with binding affinity to a specific antigen. The target antigen binding domain does not include cytokines.

[0080] As used herein, "TriTAC" refers to a trispecific binding protein that is not conditionally activated. Binding Moieties, Cleaved Linkers, and Conditionally Active Binding Proteins

[0081] In some embodiments, the present disclosure provides binding moieties that can mask the interaction of a binding molecule with its target. In some embodiments, the binding moieties of the present disclosure comprise a masking moiety and a cleavable linker, such as a protease cleavable linker. In some embodiments, the binding moieties of the present disclosure comprise a masking moiety (e.g., a modified non-CDR loop sequence) and a non-cleavable linker. Fig.35 As shown, the binding portion can synergistically expand the therapeutic window of the molecule comprising the portion by both spatial masking and specific masking. In some instances, the binding molecule is a protein belonging to the immunoglobulin superfamily, such as a target antigen binding domain comprising an immunoglobulin fold. In some embodiments, the binding molecule is a non-immunoglobulin protein. In some embodiments, the binding portion combines spatial masking (e.g., by binding to bulky serum albumin) and specific masking (e.g., by non-CDR loops that bind to the CDRs of the anti-CD3 scFv domain). In some cases, modifying the non-CDR loops within the binding portion does not affect albumin binding. In some cases, a protease-cleavable linker enables activation of a prodrug molecule comprising a binding portion (e.g., a ProTriTAC molecule comprising a binding portion, a CD3 binding domain, and an albumin binding domain as described herein) in a single proteolytic event, thereby allowing more efficient conversion of the prodrug molecule in the tumor microenvironment. Furthermore, in some cases, tumor-associated proteolytic activation reveals active T cell engagers (e.g., ProTriTAC molecules comprising a binding moiety, a CD3 binding domain, and an albumin binding domain as described herein) with minimal off-tumor activity following activation. In some embodiments, the disclosure provides half-life extended T cell engager formats (ProTriTACs) comprising a binding moiety as described herein, which in some cases represent a new and improved approach to engineering conditionally active T cell engagers.

[0082] Figure 4 B provides a schematic diagram of an exemplary ProTriTAC molecule comprising an exemplary binding moiety (anti-albumin sdAb) as described herein, and a gel showing the ProTriTAC before and after activation by cleavage of the protease cleavable linker, and Figure 4 A shows its possible mode of action. Fig.15Schematic structures of exemplary trispecific molecules (also referred to herein as ProTriTAC or activatable ProTriTAC) comprising binding moieties as described herein are shown, which have engineered non-CDR loops. Exemplary trispecific molecules contain anti-albumin domains and masking domains comprising cleavable linkers (such linkers comprising protease-cleavable sites, also referred to herein as substrate linkers); anti-CD3 binding domains; and anti-target domains that are non-immunoglobulin molecules (specific for tumor antigens) in some cases. In some cases, the non-CDR loops in the anti-albumin domain are capable of binding to and masking the anti-target domain. In some cases, the non-CDR loops in the anti-albumin domain are capable of binding to and masking the anti-CD3 domain. In some embodiments, the binding moiety comprises CDR loops that are specific for binding to albumin.

[0083] In the first embodiment, the binding portion of the masked target antigen binding domain and capable of binding to bulky serum proteins such as half-life extension proteins is provided herein. In some cases, the binding portion of the first embodiment further comprises a cleavable linker attached thereto. The cleavable linker, for example, comprises a protease cleavage site or a pH-dependent cleavage site. In some cases, the cleavable linker is only cleaved in the tumor microenvironment. Therefore, in some instances, the binding portion of the first embodiment that is bound to a protein that extends the half-life, connected to a cleavable linker and further bound to the target antigen binding domain maintains the target antigen binding domain in an inert state in the circulation until the cleavable linker is cut in the tumor microenvironment. Therefore, by using the binding portion of the first embodiment (which acts as a safety switch, the safety switch keeps the target antigen binding portion in an inert state until it reaches the tumor microenvironment, where it is conditionally activated by the cutting of the linker and can bind to its target antigen), the half-life of the target antigen binding domain such as an antibody or its antigen-binding fragment in the systemic circulation is extended.

[0084] In the second embodiment, a binding portion for masking the interaction between a non-immunoglobulin binding molecule and its target is provided. In some cases, the binding portion of the second embodiment can be combined with a large volume of serum protein. In some cases, the binding portion of the second embodiment further comprises a cleavable linker attached thereto. The cleavable linker, for example, comprises a protease cleavage site or a pH-dependent cleavage site. In some cases, the cleavable linker is only cut in the tumor microenvironment. In some cases, the non-immunoglobulin binding molecule is maintained in an inert state by the binding portion of the second embodiment and activated by the cutting of the linker, for example in the target environment. In some cases, the cleavable linker is cut in the tumor microenvironment, and in such cases, the tumor microenvironment is the target environment. Therefore, by using the binding portion of the second embodiment (it acts as a safety switch, which keeps the non-immunoglobulin binding molecule in an inert state until it reaches the target environment, where it is conditionally activated by the cutting of the linker), the half-life of the non-immunoglobulin binding molecule in the systemic circulation is extended. In some examples of the second embodiment where the non-immunoglobulin binding molecule is an imaging agent, the imaging agent is activated in the target environment when the cleavable linker is cleaved. In such cases, the target environment is a tissue or cell or any biological environment to be imaged using the imaging agent.

[0085] The safety switch described above has several advantages; some examples include (i) expanding the therapeutic window of immunoglobulin molecules, such as target antigen binding domains, non-immunoglobulin binding molecules; (ii) reducing target-mediated drug disposal by maintaining immunoglobulin molecules, such as target antigen binding domains, non-immunoglobulin binding molecules in an inert state when the conditionally active protein comprising the binding moiety according to the first or second embodiment is in the systemic circulation; (iii) reducing the concentration of undesired activated proteins in the systemic circulation, thereby minimizing the spread of chemistry, preparation and control related impurities, such as pre-activated drug products, endogenous viruses, host cell proteins, DNA, leachates, antifoaming agents, antibiotics, toxins, solvents, heavy metals; (iv) reducing undesired (v) prevent aberrant activation of immunoglobulin molecules such as target antigen binding domains or non-immunoglobulin binding molecules in the circulation; (vi) reduce toxicity associated with leakage of activated species from diseased tissues or other pathophysiological conditions (e.g., tumors, autoimmune diseases, inflammation, viral infections, tissue remodeling events (e.g., myocardial infarction, skin wound healing) or external insults (e.g., X-rays, CT scans, UV exposure)); and (vii) reduce nonspecific binding of immunoglobulin molecules such as target antigen binding domains or non-immunoglobulin binding molecules. In addition, after activation of the safety switch, or in other words, after its cleavage, immunoglobulin molecules such as target antigen binding domains, non-immunoglobulin binding molecules are separated from the safety switch, thereby providing an extended half-life and thus being cleared from the circulation.

[0086] In addition, in some cases, the binding moieties of the first, second and third embodiments are used to generate a "biologically better" form of a biologic. Typically, the preparation of a biologically better form of a molecule, such as an antibody or an antigen-binding fragment thereof, involves obtaining a starting molecule and making specific changes thereto to improve its parameters, thereby making it a more effective, less frequently administered, better targeted and / or better tolerated drug. Therefore, the target antigen binding domain masked by the binding moiety of the first embodiment, which is conditionally activated in the tumor microenvironment by binding to a protein that extends the half-life and by cutting a cleavable linker, gives the target antigen binding domain a significantly longer serum half-life and reduces the likelihood of its undesirable activation in the circulation, thereby producing a "biologically better" form of the target antigen binding domain. Similarly, in some cases, the binding moiety of the second embodiment is used to generate a biologically better form of a non-immunoglobulin binding molecule. Therefore, in a plurality of embodiments, a biologically better form of an immunoglobulin molecule, a non-immunoglobulin binding molecule, is provided, wherein the biologically better function is attributed to the binding moiety according to the first or second embodiment, respectively.

[0087] The binding moiety described herein comprises at least one non-CDR loop. In some embodiments, the non-CDR loop provides a binding site for the binding of the binding moiety of the first embodiment to the target antigen binding domain. In some examples of the first embodiment, the non-CDR loop provides a binding site for the binding of the binding moiety 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 the binding of the binding moiety of the second embodiment to a non-immunoglobulin binding molecule. In some cases, the binding moiety of the first embodiment, for example, masks the binding of the target binding domain to the target antigen by spatial blocking, by specific intermolecular interactions, or a combination of the two. In some cases, the binding moiety of the second embodiment also masks the binding of a non-immunoglobulin binding molecule to its target by spatial blocking, by specific intermolecular interactions, or a combination of the two.

[0088] In some embodiments, the binding moiety described herein further comprises a complementary determining region (CDR). In some cases, the binding moiety is a domain derived from an immunoglobulin molecule (Ig molecule). Ig can be any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chain of the Ig molecule folds into a series of parallel beta chains connected by loops. In the variable region, three loops constitute the "complementary 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 as VH in this article) 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 as VL in this article) and a light chain constant region. The light chain constant region consists of a domain CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), whose sequences are highly variable and / or are involved in antigen recognition and / or generally form structurally defined loops, interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the present disclosure, at least some or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the "non-CDR loops" of the binding moieties described herein. As Figure 1As shown, the variable domain of the immunoglobulin molecule has several β chains arranged in two folds. The variable domains of the immunoglobulin light chain and heavy chain each contain three hypervariable loops or complementary determining regions (CDRs). The three CDRs (CDR1, CDR2, CDR3) of the V domain are clustered at one end of the β barrel. CDR is a loop connecting the β chains BC, C'-C" and FG of the immunoglobulin fold, while the bottom loop connecting the β chains AB, CC', C"-D and EF of the immunoglobulin fold and the top loop connecting the DE chain of the immunoglobulin fold are non-CDR loops. In some embodiments of the present disclosure, at least some of the amino acid residues of the constant domain CH1, CH2 or CH3 are part of the "non-CDR loop" of the binding portion described herein. In some embodiments, the non-CDR loops include one or more of the AB, CD, EF, and DE loops of the C1-set domain of an Ig or Ig-like molecule; the AB, CC', EF, FG, BC, and EC' loops of the C2-set domain of an Ig or Ig-like molecule; and the DE, BD, GF, A(A1A2)B, and EF loops of the I(middle)-set domain of an Ig or Ig-like molecule.

[0089] In the variable domain, it is believed that CDR is responsible for antigen recognition and binding, while FR residues are believed to be the scaffold of CDR. However, in some cases, some FR residues play an important role in antigen recognition and binding. The framework region residues that affect Ag binding are divided into two categories. The first is the FR residues that are in contact with the antigen, and are therefore part of the binding site, and some of these residues are close to CDR in sequence. Other residues are residues that are far away from CDR in sequence but close to it in the 3-D structure of the molecule (e.g., loops in the heavy chain).

[0090] In some embodiments, the non-CDR loops are modified to generate an antigen binding site that is specific for a bulky serum protein such as albumin. It is contemplated that various techniques may be used to modify the non-CDR loops, such as site-directed mutagenesis, random mutagenesis, insertion of at least one amino acid that is foreign to the non-CDR loop amino acid sequence, amino acid substitution. In some instances, an antigenic peptide is inserted into a non-CDR loop. In some instances, a non-CDR loop is replaced with an antigenic peptide. In some cases, the modifications used to generate an antigen binding site are in only one non-CDR loop. In other cases, more than one non-CDR loop is modified. For example, the modification is in Figure 1In any of the non-CDR loops shown, i.e., AB, CC', C"D, EF and DE. In some cases, the modification is in the DE loop. In other cases, the modification is in all four loops, AB, CC', C"-D, EF. In certain instances, the binding moiety described herein binds to an immunoglobulin molecule, such as a target antigen binding domain, a non-immunoglobulin binding molecule, via its AB, CC', C"D or EF loop, and binds to a bulky serum protein, such as albumin, via its BC, C'-C" or FG loop. In certain instances, the binding moiety of the first embodiment binds to a target antigen binding domain via its AB, CC', C"D and EF loops, and binds to a bulky serum protein, such as albumin, via its BC, C'C" and FG loops.

[0091] In certain instances, the binding moiety 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 binds to bulky serum proteins such as albumin via one or more of the BC, C'C", and FG loops. In certain instances, the binding moiety of the first embodiment binds to bulky serum proteins such as albumin via its AB, CC', C"D, or EF loops, and binds to the target antigen binding domain via its BC, C'C", or FG loops. In certain instances, the binding moiety of the first embodiment binds to bulky serum proteins such as albumin via its AB, CC', C"D, and EF loops, and binds to the target antigen binding domain via its BC, C'C", and FG loops. In certain instances, the binding moiety of the first embodiment binds to bulky serum proteins such as albumin via one or more of the AB, CC', C"D, and EF loops, and binds to the target antigen binding protein via one or more of the BC, C'C", and FG loops.

[0092] In certain instances, the binding portion of the second embodiment binds to non-immunoglobulin molecules via one or more of the AB, CC', C"D, and EF loops, and binds to bulky serum proteins such as albumin via one or more of the BC, C'C", and FG loops. In certain instances, the binding portion of the second embodiment binds to bulky serum proteins such as albumin via its AB, CC', C"D, or EF loops, and binds to non-immunoglobulin molecules via its BC, C'C", or FG loops. In certain instances, the binding portion of the second embodiment binds to bulky serum proteins such as albumin via its AB, CC', C"D, and EF loops, and binds to non-immunoglobulin molecules via its BC, C'C", and FG loops. In certain instances, the binding portion of the second embodiment binds to bulky serum proteins such as albumin via one or more of the AB, CC', C"D, and EF loops, and binds to non-immunoglobulin molecules via one or more of the BC, C'C", and FG loops.

[0093] The binding moiety is any kind of polypeptide. For example, in some cases, the binding moiety is a natural peptide, a synthetic peptide or a fibronectin scaffold or an engineered bulky serum protein. Bulk serum proteins include, for example, albumin, fibrinogen or globulin. In some embodiments, the binding moiety is an engineered scaffold. Engineered scaffolds include, for example, sdAb, scFv, Fab, VHH, type III fibronectin domains, immunoglobulin-like scaffolds (as proposed by Halaby et al., 1999. Prot Eng 12(7): 563-571), DARPin, cystine knot peptides, lipocalin, three-helix bundle scaffolds, albumin binding modules associated with protein G, or DNA or RNA aptamer scaffolds.

[0094] In some cases, the binding portion of the first embodiment binds to at least one target antigen binding domain. In further embodiments, the non-CDR loops within the binding portion of the first embodiment provide binding sites for at least one target antigen binding domain. In some cases, the target antigen binding domain binds to a target antigen expressed on the surface of a diseased cell or tissue, such as a tumor or cancer cell. 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, FGFR 2. FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16E6, HPV-16E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, adhesion protein-4, CDH-3, CDH-17, EPHB2, ITGAV, ITGB6, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLITRK5, SLITRK6, STEAP1, TIM1, Trop2, or WT1.

[0095] In some cases, the binding portion of the first embodiment is bound to the first target antigen binding domain via its non-CDR loop, and the first target antigen binding domain is further connected to the 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, dual affinity retargeting antibodies, heavy chain variable domains (VH), light chain variable domains (VL), scFvs comprising VH and VL domains, soluble TCR fragments comprising Valpha and Vbeta domains, single domain antibodies (sdAb) or variable domains (VHH) of nanoantibodies from camelid sources, non-Ig binding domains, i.e., antibody mimics, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and single antibodies (monobodies), ligands or peptides. In some embodiments, the first or second target antigen binding domain is a VHH domain. In some embodiments, the first or second target antigen binding domain is an sdAb. In some cases, the first target antigen binding domain is specific for a tumor antigen such as EGFR, and the second target antigen binding domain is specific for CD3. The binding of the first target antigen binding domain to its target, e.g., a tumor antigen such as EGFR, is shielded by the binding portion of the first embodiment via its non-CDR loops. Figure 3 An exemplary conditionally active protein comprising a binding moiety according to the first embodiment is shown.

[0096] In some cases, the non-CDR loops within the binding portion of the second embodiment provide a binding site for a non-immunoglobulin binding molecule.

[0097] In some cases, the binding portion comprises a binding site for a large volume serum protein. In some embodiments, the CDRs within the binding portion provide a binding site for a large volume serum protein. In some instances, the large volume serum protein is globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE or pentamer IgM. In some embodiments, the binding portion comprises a binding site for an immunoglobulin light chain. In some embodiments, the CDRs provide a binding site for an immunoglobulin light chain. In some instances, the immunoglobulin light chain is an Igκ-free light chain or an Igλ-free light chain.

[0098] In some examples, the binding moiety comprises 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 moiety is a single-chain variable fragment (scFv), a soluble TCR fragment, a single domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of a nanobody from camelid sources. In other embodiments, the binding moiety is a non-Ig binding domain, i.e., an antibody mimetic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and monobodies.

[0099] Table 1: Exemplary sequences of masking sequences within the binding moieties of the present disclosure are in SEQ ID Nos. 50, 259-301 and Provided in 795.

[0100]

[0101]

[0102] It is contemplated herein that in some embodiments of the present disclosure, the binding moiety described herein comprises at least one cleavable linker. On the one hand, the cleavable linker comprises a polypeptide having a sequence that is recognized and cleaved in a sequence-specific manner. In certain instances, the cleavage is enzymatic, based on the pH sensitivity of the cleavable linker, or by chemical degradation. In some cases, the conditionally active binding protein contemplated herein comprises a protease cleavable linker that is recognized in a sequence-specific manner by a matrix metalloproteinase (MMP), such as MMP9. In some cases, the protease cleavable linker is recognized by MMP9 and comprises a polypeptide having an amino acid sequence PR (S / T) (L / I) (S / T). In some cases, the protease cleavable linker recognized by MMP9 comprises a polypeptide having an amino acid sequence LEATA. In some cases, the protease cleavable linker is recognized by MMP11 in a sequence-specific manner. In some cases, the protease cleavable linker recognized by MMP11 comprises a polypeptide having an amino acid sequence GGAANLVRGG (SEQ IN 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 a protease disclosed in Table 3, comprising a polypeptide having an amino acid sequence selected from the sequences disclosed in Table 3 (SEQ ID NOs: 1-42, 53, and 58-62). In some cases, the cleavable linker has an amino acid sequence as shown in SEQ ID No. 59. In some cases, the cleavable linker is recognized by MMP9, protease, urokinase plasminogen activator (uPA), and has an amino acid sequence as shown in SEQ ID No. 59.

[0103] In some embodiments of the present disclosure, the binding moiety described herein comprises at least one non-cleavable linker. In some examples, the non-cleavable linker comprises a sequence as shown in SEQ ID No.51, SEQ ID No.302, SEQ ID No.303, SEQ ID No.304 or SEQ ID No.305.

[0104] Table 2: Exemplary non-cleavable linker sequences

[0105] Non-cleavable linker ID sequence SEQ ID No. L002 SGGGGSGGVV SEQ ID No.302 L016 SGGGGSGGGGSGGGGS SEQ ID No.303 L017 SGGGGSGGGGSGGGGGS SEQ ID No.304 L046 SGGGGSGGGS SEQ ID No.305

[0106] Proteases are proteins that cleave proteins in a sequence-specific manner in some cases. Proteases include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamic proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, cathepsins B, cathepsins C, cathepsins D, cathepsins E, cathepsins K, cathepsins L, kallikreins, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidin proteases, bromelain, calpains, caspases, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV Proteases, chymosin, renin, pepsin, mavericks, legumin, plasmodium aspartic proteinase, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteinases (MMPs), MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase 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 serine protease, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymotrypsin, and mast cell tryptase.

[0107] Table 3: Exemplary proteases and protease recognition sequences

[0108]

[0109]

[0110] Known proteases are secreted by certain pathological cells and tissues, such as tumors or cancer cells, so as to produce a microenvironment rich in proteases or a microenvironment rich in proteases. In some cases, the blood of the experimenter is rich in proteases. In some cases, the cells around the tumor secrete proteases into the tumor microenvironment. The cells around the tumor that secrete proteases include but are not limited to tumor stroma cells, myofibroblasts, hemocytes, mast cells, B cells, NK cells, regulatory T cells, macrophages, cytotoxic T lymphocytes, dendritic cells, mesenchymal stem cells, polymorphonuclear cells and other cells. In some cases, proteases are present in the blood of the experimenter, such as proteases of the amino acid sequence found in the microbial peptides targeted. This feature allows targeted therapeutic agents such as antigen binding proteins to have additional specificity, because except in the microenvironment rich in proteases in the target cell or tissue, T cells will not be combined by antigen binding proteins.

[0111] Thus, the binding portion comprising a cleavable linker masks the binding of the first or second target antigen binding domain to its respective target. In some embodiments, the binding portion binds to the first target antigen binding domain, which further binds to the second target antigen binding domain in the following order: binding portion (M): cleavable linker (L): first target antigen binding domain (T1): second antigen binding domain (T2). In other examples, the domains are organized in any of the following orders: M:L:T2:T1; T2:T1:L:M; T1:T2:L:M. The binding portion further binds to a protein that extends half-life, such as albumin, or any other target thereof as described below. In some cases, the binding portion is albumin or comprises a binding site for albumin. In some cases, the binding portion comprises a binding site for IgE. In some embodiments, the binding portion comprises a binding site for a light chain without Igκ.

[0112] Table 4: Exemplary sequences of binding moieties comprising cleavable linkers are in SEQ ID No.

[0113] Available in 796-800.

[0114]

[0115]

[0116] Targets of conditionally active binding proteins

[0117] The conditionally active binding proteins described herein are activated by cleaving at least one cleavable linker attached to the binding portion within the conditionally active protein. It is expected that in some cases, the activated binding protein binds to a target antigen involved in and / or associated with a disease, disorder or condition. In particular, it is expected that a target antigen associated with a proliferative disease, a tumor disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergy, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease is a target of the activated binding proteins disclosed herein.

[0118] 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, 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-16E6, HPV-16E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, adhesion protein-4, CDH-3, CDH-17, EPHB2, ITGAV, ITGB6, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLITRK5, SLITRK6, STEAP1, TIM1, Trop2, or WT1.

[0119] 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, TDO, KIR, LAG-3, TIM-3, or VISTA.

[0120] 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 on a tumor cell, a virus-infected cell, a bacterially infected cell, a damaged red blood cell, an arterial plaque cell, an inflamed or fibrotic tissue cell.

[0121] In some embodiments, the target antigen comprises an immune response regulator that is not a cytokine.Examples of immune response regulators include, but are not limited to, B7-1 (CD80), B7-2 (CD86), CD3, or GITR.

[0122] In some embodiments, the first target antigen binding domain or the second target antigen binding domain comprises 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. In some embodiments, the first target antigen binding domain or the second target antigen binding domain comprises an anti-EGFR sdAb, an anti-EpCAM sdAb, an anti-DLL3 sdAb, an anti-MSLN sdAb, an anti-PSMA sdAb, an anti-BDMA sdAb, or any combination thereof.

[0123] In some embodiments, the anti-EGFR domain of the present disclosure comprises an amino acid selected from the group consisting of SEQ ID Nos. 55 and 737-785. In some embodiments, the anti-PSMA domain of the present disclosure comprises an amino acid selected from the group consisting of SEQ ID Nos. 57-73. In some embodiments, the anti-BCMA domain of the present disclosure comprises an amino acid selected from the group consisting of SEQ ID Nos. 91-214. In some embodiments, the anti-MSLN domain of the present disclosure comprises an amino acid selected from the group consisting of SEQ ID Nos. 215-258. In some embodiments, the anti-DLL3 domain of the present disclosure comprises an amino acid selected from the group consisting of SEQ ID Nos. 306-736. In some embodiments, the anti-EpCAM domain of the present disclosure comprises an amino acid selected from the group consisting of SEQ ID Nos. 804-841.

[0124] In some embodiments, the first target antigen binding domain or the second target antigen binding domain comprises an anti-CD3 domain. In some embodiments, the anti-CD30 domain comprises an anti-CD3 scFV. In some embodiments, the anti-CD3 scFv comprises an amino acid sequence selected from SEQ ID No. 74-90 and 794.

[0125] Binding protein variants

[0126] As used herein, the term "binding protein variant" refers to variants and derivatives of conditionally active target binding proteins described herein, which contain a binding portion as described above, which comprises a non-CDR loop that binds to an immunoglobulin binding molecule, such as a first or second target antigen binding domain, a non-immunoglobulin binding molecule. In certain embodiments, it is related to amino acid sequence variants of conditionally active target binding proteins described herein. For example, in certain embodiments, it is expected that amino acid sequence variants of conditionally active target binding proteins described herein improve the binding affinity and / or other biological properties of the binding protein. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody.

[0127] Any combination of deletion, insertion and substitution can be carried out to obtain the final construct to each domain, provided that the final construct has the desired characteristic, such as antigen binding. In certain embodiments, a conjugated protein variant with one or more amino acid substitutions is provided. The site of interest for substitution mutagenesis includes CDR and framework region. Amino acid substitution can be introduced into each domain of the conditionally active protein of interest, and for desired activity, for example, the antigen binding of retention / improvement, the immunogenicity of reduction or the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) of improvement are screened for product. Conservative and non-conservative amino acid substitutions are all considered for the preparation of antibody variants.

[0128] In another example of substitutions used to generate variant conditionally active antibodies, one or more hypervariable region residues of a parental antibody are substituted. Typically, variants are then selected based on improvements in desired properties compared to the parental antibody, e.g., increased affinity, reduced affinity, reduced immunogenicity, increased pH dependence of binding. For example, affinity maturation techniques such as those based on phage display, such as those described herein and known in the art, can be used to generate affinity-matured variant antibodies.

[0129] In another example, a substitution is performed in the hypervariable region (HVR) of the parent conditionally active antibody to generate a variant, and then based on binding affinity, the variant is selected by affinity maturation. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling or oligonucleotide site-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variant with the desired affinity. Another method of introducing diversity relates to an HVR directed method, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. The substitution can be at one, two, three, four or more sites within the parent antibody sequence.

[0130] In some embodiments, the conditionally active binding proteins described herein comprise a VL domain or a VH domain, or both, having an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VL or VH domain, respectively, but having been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VL or VH domain (particularly in the framework sequence) with one or more amino acid residues present at the corresponding positions in the VL or VH domain of a conventional 4-chain antibody from a human (e.g., as described above). This can be done in a manner known in the art, which will be apparent to those skilled in the art, for example based on the further description herein. In addition, it should be noted that such humanized conditionally active target binding antibodies disclosed herein are obtained in any suitable manner known per se, and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VL and / or VH domains as starting materials. In some further embodiments, the conditionally active target binding antibodies as described herein comprise VL and VH domains having amino acid sequences corresponding to the amino acid sequences of naturally occurring VL or VH domains, respectively, but having been "camelized", i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VL or VH domain from a conventional 4-chain antibody with one or more amino acid residues present at the corresponding positions in the VL or VH domain of a heavy chain antibody. Such "camelization" substitutions are preferably inserted at amino acid positions forming the VH-VL interface and / or present at the VH-VL interface and / or at the so-called Camelidae signature residues (see, e.g., WO 94 / 04678, and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as the 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 in certain embodiments, such camelized conditionally active antibodies of the present disclosure are obtained in any suitable manner known in the art, and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VL and / or VH domains as starting materials. For example, both "humanization" and "camelization" are performed by providing nucleotide sequences encoding naturally occurring VL and / or VH domains, respectively, and then changing one or more codons in the nucleotide sequence in a manner that the new nucleotide sequence encodes a "humanized" or "camelized" conditionally active antibody, respectively. The nucleic acid can then be expressed so as to provide the desired target antigen binding ability. Alternatively, in other embodiments, a "humanized" or "camelized" conditionally active antibody is synthesized de novo from the amino acid sequence of a naturally occurring antibody comprising a VL and / or VH domain using known peptide synthesis techniques.In some embodiments, a "humanized" or "camelized" conditionally active antibody is synthesized de novo from the amino acid sequence or nucleotide sequence of a naturally occurring antibody comprising a VL and / or VH domain, respectively, using known peptide synthesis techniques, a nucleotide sequence encoding the desired humanized or camelized conditionally active domain antibody of the present disclosure is designed, respectively, and then synthesized de novo using known nucleic acid synthesis techniques, and the nucleic acid thus obtained is then expressed using known expression techniques to provide the desired conditionally active antibody of the present disclosure.

[0131] Other suitable methods and techniques for obtaining the conditionally active binding proteins of the present invention and / or nucleic acids encoding the conditionally active binding proteins starting from the naturally occurring sequences of the VL or VH domains include, for example, combining one or more portions of one or more naturally occurring VL or VH sequences (such as 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 the naturally occurring sequences of the CH2 domains, and the naturally occurring sequences of the CH3 domains (which contain amino acid substitutions that favor the formation of heterodimers over homodimers) in a suitable manner to provide the conditionally active binding proteins of the present invention or the nucleotide sequences or nucleic acids encoding the conditionally active binding proteins.

[0132] Affinity Maturation

[0133] When designing conditionally active binding proteins for therapeutic applications, it is desirable to produce, for example, proteins that modulate the functional activity of the target and / or improved binding proteins (e.g., binding proteins with greater specificity and / or affinity) and / or binding proteins that are more bioavailable or more stable or more soluble in a particular cell or tissue environment.

[0134] The conditional active binding proteins described in the present disclosure show improved binding affinity for tumor antigens expressed on the target, such as cell surfaces. In some embodiments, any known affinity maturation techniques (such as mutagenesis, chain shuffling, CDR amino acid replacement) are used to make the conditional active binding proteins of the present disclosure undergo affinity maturation to increase their binding affinity to the target. Amino acid replacement can be conservative or semi-conservative. For example, amino acids such as glycine, alanine, valine, leucine and isoleucine can usually replace each other (amino acid with aliphatic side chains). In these possible replacements, glycine and alanine are usually used to replace each other because they have relatively short side chains, and valine, leucine and isoleucine are used to replace each other because they have hydrophobic larger aliphatic side chains. Other amino acids that can often be 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 phage display libraries and screening such libraries for antibodies with desired binding properties to a target antigen, such as a tumor antigen expressed on the surface of a cell.

[0135] Conditionally active binding protein modification

[0136] The conditionally active binding proteins described herein include derivatives or analogs in which (i) amino acids are replaced with amino acid residues not encoded by the genetic code, (ii) the mature polypeptide is fused to another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or a sequence used to block immunogenic domains and / or to purify the protein.

[0137] Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation and ubiquitination.

[0138] Modifications are made at any position in the conditionally active binding proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Certain common peptide modifications that can be used to modify conditionally active binding proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blocking of amino or carboxyl groups or both in the polypeptide by covalent modification, and ADP-ribosylation.

[0139] In some embodiments, the conditionally active binding proteins of the present disclosure are conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs are used in oncology applications, where local delivery of cytotoxic or cytostatic agents using antibody-drug conjugates allows targeted delivery of the drug moiety to tumors, which can achieve higher efficacy, lower toxicity, etc.

[0140] Polynucleotides encoding binding moieties or conditionally active binding proteins

[0141] In some embodiments, polynucleotide molecules encoding binding moieties described herein are also provided. In some embodiments, the polynucleotide molecules are provided in the form of DNA constructs. In other embodiments, the polynucleotide molecules are provided in the form of messenger RNA transcripts.

[0142] In some embodiments, polynucleotide molecules encoding conditionally active binding proteins described herein are also provided. In some embodiments, the polynucleotide molecules are provided in the form of DNA constructs. In other embodiments, the polynucleotide molecules are provided in the form of messenger RNA transcripts.

[0143] The polynucleotide molecules are constructed by known methods, for example by combining the genes encoding the various binding domains (e.g., binding moieties, target antigen binding domains, etc.) separated by peptide connectors, or directly connected by peptide bonds in other embodiments into a single gene construct, which is operably connected to a suitable promoter and an optional suitable transcription terminator, and expressed in bacteria or other suitable expression systems such as CHO cells. Depending on the vector system and host used, any number of suitable transcription and translation elements can be used, including constitutive and conditionally active promoters. Select promoters so that they drive the expression of the polynucleotides in the corresponding host cells.

[0144] In some embodiments, the polynucleotides described herein are inserted into vectors such as expression vectors, which represent additional embodiments. The recombinant vectors can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, viruses (virii) (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, slow viruses, etc.) and cosmids.

[0145] A variety of expression vector / host systems can be used to contain and express polynucleotides encoding the polypeptides of the conditionally active binding proteins. Examples of expression vectors are pSKK (Le Gall et al., J Immunol Methods. (2004) 285 (1): 111-27) for expression in E. coli, or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0146] Thus, in some embodiments, a binding portion as described herein or a conditionally active binding protein comprising the binding portion is produced by introducing a vector encoding a binding portion or binding protein as described above into a host cell, and culturing the host cell under conditions such that the binding portion or binding protein or its domain is expressed.

[0147] Pharmaceutical composition

[0148] In some embodiments, pharmaceutical compositions are also provided herein, which comprise a therapeutically effective amount of conditionally active binding proteins of the present disclosure and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the component and is nontoxic to the administered patient. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil-in-water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be prepared by conventional methods and can be applied to a subject at a suitable dose. Preferably, the composition is sterile. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersants. In some cases, the action of microorganisms is prevented by including various antibacterial and antifungal agents.

[0149] Conditionally active binding proteins described herein are contemplated for use as drugs. Administration is achieved in different ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician based on other clinical factors. The dosage for any one patient depends on many factors, including patient size, body surface area, age, sex, specific compound to be administered, administration time and route, type of therapy, overall health and other drugs administered simultaneously. "Effective dose" refers to the amount of active ingredient, which is sufficient to affect the course and severity of the disease, thereby causing the alleviation or relief of this pathology, and can be determined using known methods.

[0150] Treatment

[0151] In some embodiments, the present invention also provides methods and uses for stimulating the immune system of an individual in need, which include administering conditionally active binding proteins as described herein. In some cases, administration induces and / or maintains cytotoxicity to cells expressing target antigens. In some cases, cells expressing target antigens are cancer cells or tumor cells, virally infected cells, bacterially infected cells, autoreactive T or B cells, damaged red blood cells, arterial plaques, or fibrotic tissue. In some embodiments, the target antigen is an immune checkpoint protein.

[0152] Also provided herein are methods and uses for treating a disease, disorder or condition associated with a target antigen, comprising administering a conditionally active binding protein as described herein to an individual in need thereof. 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, the disease, disorder or condition associated with a target antigen is a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergy, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease. In one embodiment, the disease, disorder or condition associated with a target antigen is cancer. In one case, the cancer is a hematological cancer. In another case, the cancer is a melanoma. In a further case, the cancer is non-small cell lung cancer. In a further case, the cancer is breast cancer.

[0153] In some embodiments, as used herein, "treatment" or "treatment" refers to therapeutic treatment, wherein the purpose is to slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the condition, disorder or disease state; delayed onset or slowed progression of the condition, disorder or disease; improvement of the condition, disorder or disease state; and detectable or undetectable relief (partial or complete) or enhancement or improvement of the condition, disorder or disease. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment also includes extending survival compared to the expected survival without treatment. In other embodiments, "treatment" or "treatment" refers to preventive measures, wherein the purpose is, for example, in a person susceptible to the disease (e.g., an individual carrying genetic markers for a disease such as breast cancer), delaying the onset of an undesirable physiological condition, disorder or disease or reducing its severity.

[0154] In some embodiments of the methods described herein, conditionally active binding proteins described herein are administered in combination with agents for treating specific diseases, disorders, or conditions. Agents include, but are not limited to, therapy involving antibodies, small molecules (e.g., chemotherapeutic drugs), hormones (steroids, peptides, etc.), radiotherapy (gamma rays, X-rays, and / or directed delivery of radioisotopes, microwaves, UV radiation, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapies. In some embodiments, conditionally active binding proteins described herein are administered in combination with antidiarrheals, antiemetics, analgesics, opioids, and / or nonsteroidal anti-inflammatory agents. In some embodiments, conditionally active binding proteins described herein are administered before, during, or after surgery.

[0155] Example

[0156] The following examples further illustrate the described embodiments but are not intended to limit the scope of the disclosure.

[0157] Example 1: Construction of exemplary binding moieties that bind to albumin and target antigen binding domains targeting EGFR

[0158] The sequence of an engineered protein scaffold comprising CDR loops and non-CDR loops capable of binding to albumin was obtained. Random mutations were introduced in the non-CDR loop regions using overlapping PCR to generate a library. The resulting sequences were cloned into a phage display vector to generate a phage display library. E. coli cells were transformed with the library and used to construct a phage display library. ELISA was performed using an immobilized target antigen binding domain specific for EGFR. Clones with high specificity for EGFR were selected. Affinity maturation was performed by re-randomizing the residues in the non-CDR loop regions as described above.

[0159] The sequence alignment of the non-CDR loop region of the obtained protein is carried out to determine the sequence conservation between proteins with high affinity for EGFR in combination with the target antigen binding domain. One or more amino acids in these sequence conservation regions are subjected to site-directed mutagenesis to generate other proteins. The binding of the obtained protein to the target antigen binding domain whose fixed target is EGFR is measured in ELISA. The protein with the highest affinity for the target antigen binding domain is selected.

[0160] The sequence of the binding moiety is cloned into a vector comprising the sequence of a cleavable linker and a sequence of a second target antigen binding domain that binds to a second target antigen, such as CD3. The resulting vector is expressed in a heterologous expression system to obtain a conditionally active target binding protein comprising a binding moiety comprising a cleavable linker and non-CDR loops and CDR loops specific for albumin, wherein the non-CDR loops provide a binding site specific for a target antigen binding domain that targets EGFR.

[0161] Example 2: Construction of exemplary binding moieties that bind to albumin and target antigen binding domains targeting CD3

[0162] The sequence of an engineered protein scaffold comprising CDR loops and non-CDR loops capable of binding to albumin was obtained. Random mutations were introduced in the non-CDR loop regions using overlapping PCR to generate a library. The resulting sequences were cloned into a phage display vector to generate a phage display library. E. coli cells were transformed with the library and used to construct a phage display library. ELISA was performed using an immobilized target antigen binding domain specific for CD3. Clones with high specificity for CD3 were selected. Affinity maturation was performed by re-randomizing the residues in the non-CDR loop regions as described above.

[0163] Sequence alignment of the non-CDR loop regions of the resulting protein is performed to determine the sequence conservation between proteins with high affinity for EGFR binding to the target antigen binding domain. Site-directed mutagenesis is performed on one or more amino acids within these sequence conservation regions to generate additional proteins. The binding of the resulting protein to the target antigen binding domain whose fixed target is CD3 is measured in an ELISA. The protein with the highest affinity for the target antigen binding domain is selected.

[0164] The sequence of the binding moiety is cloned into a vector comprising the sequence of a cleavable linker and the sequence of a second target antigen binding domain that binds to a second target antigen, such as EGFR. The resulting vector is expressed in a heterologous expression system to obtain a conditionally active target binding protein comprising a binding moiety comprising a cleavable linker and non-CDR loops and CDR loops specific for albumin, wherein the non-CDR loops provide a binding site specific for a target antigen binding domain whose target is CD3.

[0165] Example 3: Conditionally active binding proteins of the present disclosure are expressed in cell lines that overexpress EGFR but lack protease Reduced specificity

[0166] Cells that overexpress EGFR and exhibit low expression of matrix metalloproteinases were incubated with exemplary conditionally active binding proteins according to the present disclosure and non-conditionally active control binding proteins, respectively. Cells expressing normal levels of EGFR and proteases were also incubated with conditionally active binding proteins according to the present disclosure and non-conditionally active control binding proteins. Both proteins comprise a target antigen binding domain specific for PSMA.

[0167] The results show that in the absence of protease secretion, the conditionally active binding proteins of the present disclosure interact with cells expressing the protease, but do not interact with EGFR expressed on the surface of cells lacking the protease. In contrast, the non-conditionally active control binding protein lacks the ability to selectively bind to cells expressing the protease compared to cells lacking the protease. Therefore, the exemplary conditionally active binding protein receptors of the present disclosure are advantageous, for example, in reducing extra-tumor toxicity.

[0168] Example 4: Treatment with exemplary conditionally active binding proteins of the present disclosure inhibits tumor growth in vivo

[0169] The murine tumor line CT26 was implanted subcutaneously in Balb / c mice, and the average size of the tumors was measured on day 7 post-implantation. Test mice were treated with an exemplary conditionally active binding protein having a target antigen binding domain specific for CTLA4 and another target antigen binding domain specific for CD3, wherein the CTLA4 or CD3 specific domain is bound to a binding moiety via its CDR loops, the binding moiety comprising a cleavable linker, and bound to albumin. Control mice were treated with a binding protein containing a CD3 / CTLA4 specific domain but without a binding moiety or a cleavable linker, and there was no conditional activity. The results show that treatment with the exemplary conditionally active binding protein of the present disclosure inhibited tumors more effectively than a comparative binding protein without a moiety having non-CDR loops.

[0170] Example 5: Exemplary conditionally active binding proteins show activity against cell lines that overexpress antigen but lack proteases Reduced specificity

[0171] Cells that overexpress CTLA-4 and exhibit low expression of matrix metalloproteinases are incubated with an exemplary CTLA4-specific conditionally active binding protein of the present disclosure that contains a binding portion that binds to the CTLA4 binding domain via its non-CDR loops and to albumin via its CDRs, or a control CTLA-4 binding antibody that does not contain a binding portion that binds to the CTLA4 binding domain via its non-CDR loops and to albumin via its CDRs. Cells that express normal levels of antigen and protease are also incubated with an exemplary CTLA4-specific conditionally active binding protein or a control CTLA4 binding antibody.

[0172] The results show that in the absence of protease secretion, the conditionally active binding proteins of the present disclosure bind to cells expressing the protease, but not to antigen-expressing cells lacking the protease. In contrast, the control antibody lacks the ability to selectively bind to cells expressing the protease compared to cells lacking the protease. Therefore, exemplary conditionally active binding proteins of the present disclosure are advantageous, for example, in reducing off-tumor toxicity.

[0173] Example 6: Purification, Pharmacokinetic Analysis and Potency Determination of Exemplary ProTriTAC Molecules

[0174] A) Expression, purification and stability of exemplary ProTriTAC molecules

[0175] Protein production

[0176] The sequence of the exemplary ProTriTAC (also called pre-trispecific) molecule was cloned into the mammalian expression vector pcDNA3.4 (Invitrogen) preceded by the leader sequence and followed by a 6x histidine tag. Expi293F cells (Life Technologies A14527) were maintained in suspension at 0.2-8×1e6 cells / ml in Expi 293 medium in Optimum Growth Flasks (Thomson). Purified plasmid DNA was transfected into Expi293 cells according to the Expi293 Expression System Kit (LifeTechnologies, A14635) protocol and maintained for 4-6 days after transfection. Alternatively, the sequence of the trispecific molecule was cloned into the mammalian expression vector pDEF38 (CMC ICOS), transfected into CHO-DG44 dhfr- cells, a stable pool was generated, and cultured in production medium for up to 12 days before purification. The amount of exemplary trispecific proteins in conditioned medium was quantified using an Octet RED 96 instrument (ForteBio / Pall) with a protein A tip and a standard curve prepared using a control trispecific protein. Conditioned medium from either host cell was filtered and partially purified by affinity chromatography and desalting chromatography. The trispecific protein was then refined by ion exchange and formulated in a neutral buffer containing excipients after merging the fractions. The final purity was estimated by SDS-PAGE and analytical SEC using an AcquityBEH SEC 2001.7u 4.6x 150mm column (Waters Corporation) and resolved on a 1290LC system in an aqueous / organic mobile phase containing excipients at neutral pH, and the peaks were integrated using Chemstation CDS software (Agilent). Figure 5 As shown, trispecific proteins purified from CHO host cells were analyzed by running SDS-PAGE.

[0177] Stability assessment

[0178] The purified pro-tri-specific proteins from both formulations were redistributed into sterile tubes and stressed by five freeze-thaw cycles, each of which included either more than 1 hour at -80°C and room temperature, or incubation at 37°C for 1 week. The concentration and turbidity of the stressed samples were assessed by UV spectroscopy, SDS-PAGE, and analytical SEC using UV-transparent 96-well plates (Corning 3635) and SpectraMax M2 and SoftMaxPro software (Molecular Devices) and compared to the same analysis of control non-stressed samples. Overlays of the chromatograms of analytical SEC for control and stressed samples for a single exemplary tri-specific ProTriTAC molecule purified from 293 host cells are shown in Figure 6 Shown in.

[0179] B) ProTriTAC exhibits potent, protease-dependent antitumor activity in rodent tumor xenograft models

[0180] The in vivo antitumor activity of an exemplary ProTriTAC molecule (SEQ ID NO: 46) containing an EGFR binding domain as a target binding domain, a CD3 binding domain, and an albumin binding domain comprising a masking moiety (SEQ ID NO: 50) and a cleavable linker (SEQ ID NO: 53) was evaluated in immunocompromised NCG mice in HCT116 subcutaneous xenograft tumors mixed with expanded human T cells. Non-cleavable EGFR-targeted ProTriTAC molecules (SEQ ID NO: 47) and GFP-targeted ProTriTAC molecules (SEQ ID NO: 49) were also used in the study. Specifically, 5x10 6 HCT116 cells and 2.5x10 6 The expanded T cells were mixed. The administration of the test molecules (EGFR-targeted ProTriTAC, non-cleavable EGFR-targeted Pro-TriTAC and GFP-targeted ProTriTAC) was carried out by intraperitoneal injection of 0.03 mg / kg dose at qdx 10 (one dose per day for 10 days) from the next day. Tumor volume was determined by measuring with a caliper at the specified time and calculating using the formula V = (length x width x width) / 2. Figure 7The results shown in show that tumor growth was arrested in mice administered the activatable EGFR-targeted ProTriTAC molecule at day 10 after the last dose of the tested molecule. However, administration of the GFP-targeted ProTriTAC molecule was ineffective in inhibiting tumor growth, and administration of the non-cleavable EGFR-targeted ProTriTAC molecule was not as effective as the activatable ProTriTAC in arresting tumor growth.

[0181] C) Functional masking and stability of ProTriTAC in vivo demonstrated in a three-week pharmacokinetic study in cynomolgus monkeys

[0182] A single dose of active drug of PSMA-targeted ProTriTAC (SEQ ID NO: 43), non-cleavable PSMA-targeted ProTriTAC (SEQ ID NO: 44), non-masked / non-cleavable TriTAC (SEQ ID NO: 52), and a protease-mimicking 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: 53) comprising a masking moiety (SEQ ID NO: 50) and a cleavable linker was administered to cynomolgus monkeys by intravenous injection at 0.1 mg / kg. Fig. 9 Plasma samples were collected at the indicated time points. 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 11D3, a sulfo-labeled anti-CD3 idiotypic antibody clone, in an MSD assay (Meso Scale Diagnostic, LLC). Pharmacokinetic parameters were estimated using Phoenix WinNonlin pharmacokinetic software using a non-compartmental approach consistent with the intravenous bolus route of administration.

[0183] To calculate the in vivo conversion rates of the test molecules (i.e., conversion rates of PSMA-targeted ProTriTAC, non-cleavable PSMA-targeted ProTriTAC, and non-masked / non-cleavable PSMA-targeted ProTriTAC), the concentration of active drug in the circulation was estimated by solving the following system of differential equations, where P is the concentration of the prodrug, A is the concentration of the active drug, and k a is the prodrug activation rate in the circulation, k c,P is the clearance rate of the prodrug, k c,A is the clearance rate of the active drug.

[0184]

[0185] The clearance (k) of prodrug, active drug, non-masked non-cleavable prodrug control, and non-cleavable prodrug control in cynomolgus monkeys was determined empirically. c,NCLV ). To estimate the prodrug activation rate in circulation, it is assumed that the difference between the clearance rates of cleavable and non-cleavable prodrugs is due only to non-specific activation in circulation. Therefore, the rate at which the prodrug is converted to active drug in circulation is estimated by subtracting the clearance rate of the cleavable prodrug from the clearance rate of the non-cleavable prodrug.

[0186] k a =k c,NCLV -k c,P

[0187] The initial concentration of the prodrug in the circulation was determined empirically, and the initial concentration of the active drug was assumed to be zero. Further calculations showed that the ProTriTAC containing the protease-cleavable linker was sufficiently stable in the circulation to have 50% non-tumor-mediated turnover every 194 h, and the t of this molecule was determined empirically. 1 / 2 This indicates that the ProTriTAC molecule is sufficiently stable and prevents extra-tumor effects. In contrast, based on experience, the t of the active drug fragment that simulates the activation of the ProTRITAC molecule is 1 / 2 is 0.97 hours. Therefore, the active drug is rapidly cleared from the circulation. Fig.10 Shown in.

[0188] Fig.11 It was shown that there was a significant >200-fold difference in circulating exposure between ProTriTAC and activated ProTriTAC due to the rapid clearance of active drug from the circulation of the simulated activated ProTriTAC molecule and the longer half-life of ProTriTAC and control non-masked non-cleavable ProTriTAC. It was also observed that the masked but non-cleavable ProTriTAC control molecule remained in the circulation longer than the non-masked non-cleavable ProTriTAC control, indicating that masking has an important role in extending the circulating half-life (t 1 / 2 ) and limiting peripheral T cell binding. The functional masking that renders ProTriTAC inert outside the tumor environment is combined with the half-life difference that causes any abnormally activated ProTriTAC to be rapidly cleared from the body in the circulation, ensuring that the on-target, off-tumor activity of ProTriTAC is minimized. Supportive pharmacokinetic parameters are shown in Table 5.

[0189] Table 5: Pharmacokinetics of ProTriTAC, activated ProTriTAC and control molecules

[0190]

[0191] D) Protease activation of ProTriTAC molecules leads to significant enhancement of in vitro activity

[0192] The purpose of this study was to evaluate the relative potency of recombinant active drug fragments of protease-activatable ProTriTAC molecules, non-cleavable ProTriTAC molecules, and mimetic protease-activatable ProTriTAC molecules in CD3 binding and T cell-mediated cell killing. The active drug fragments of mimetic protease-activatable ProTriTAC molecules contain a CD3-binding domain and a target antigen-binding domain, but lack an albumin-binding domain. Whereas, the protease-activatable ProTriTAC molecules contain an albumin-binding domain, a CD3-binding domain, and a target antigen-binding domain that contain a masking domain and a protease-cleavable site. The non-cleavable ProTriTAC molecules lack a protease-cleavable site but are otherwise identical to the protease-activatable ProTriTAC molecules.

[0193] Purified ProTriTAC (in Figure 12-14 in the labeling as prodrugs), non-cleavable ProTriTAC (in Figure 12-14 Recombinant active drug fragments labeled as prodrugs (non-cleavable) and protease-mimicking ProTriTACs (in Figure 12-14 The active drug was tested in an ELISA assay for binding to recombinant human CD3 ( Fig.12 ), tested for binding to purified human primary T cells in flow cytometry analysis ( Fig.13 ) and tested for functional potency in a T cell-dependent cytotoxicity assay ( Fig.14 ).

[0194] For ELISA, the indicated concentrations of soluble test molecules (i.e., active drug, prodrug, and prodrug (non-cleavable)) were incubated with immobilized recombinant human CD3ε (R&D Systems) in PBS supplemented with 15 mg / mL human serum albumin at room temperature for 1 hour in multiwell plates. Plates were blocked with SuperBlock (Thermo Fisher), washed with PBS containing 0.05% Tween-20, and detected with non-competitive anti-CD3 idiotypic monoclonal antibody 11D3, followed by peroxidase-labeled secondary antibodies and TMB-ELISA substrate solution (Thermo Fisher). Fig.12 The results shown in Table 6 indicate that the active drug fragment of the ProTriTAC molecule that mimics protease activation is approximately 250-fold more potent in binding CD3 compared to the non-cleavable prodrug. 50 The masking ratio is the prodrug EC 50 With active drug EC 50Ratio between: The larger the number, the higher the fold change between the prodrug and the active drug, and therefore the higher the functional masking.

[0195] Table 6: CD3 binding potential

[0196] <![CDATA[EC 50 (nM)]]> Masking ratio Active drug 0.16 - Prodrug 11.91 74 Prodrug (non-cleavable) 39.44 247

[0197] For binding to human primary T cells as determined by flow cytometry, the indicated concentrations ( Fig.13 Soluble test molecules (i.e., active drug, prodrug, and prodrug (non-cleavable)) of the type indicated in the table were incubated with purified human primary T cells for 1 hour at 4° C. The plates were washed with PBS containing 2% fetal bovine serum, detected using AlexaFluor 647-labeled non-competitive anti-CD3 idiotypic monoclonal antibody 11D3, and data analyzed using FlowJo 10 (FlowJo, LLC). Fig.13 The results shown in Table 7 indicate that the active drug fragment of the ProTriTAC molecule that mimics protease activation is more than 1000-fold more potent in binding human primary T cells compared to the non-cleavable prodrug. The EC values ​​for cell killing are provided in Table 7. 50 value.

[0198] Table 7: Human primary T cell binding potential

[0199]

[0200]

[0201] For functional efficacy in T cell-dependent cytotoxicity assays, in multiwell plates, Fig.14 Soluble test molecules (i.e., active drug, prodrug, and prodrug (non-cleavable)) at the indicated concentrations shown in the Table 1 were incubated with purified resting human T cells (effector cells) and HCT116 cancer cells (target cells) at an effector:target ratio of 10:1 at 37°C for 48 hours. The HCT116 target cell line has been stably transfected with a luciferase reporter gene to allow specific T cell-mediated cell killing measurements by ONE-Glo (Promega). Fig.14 The results shown in Table 8 indicate that the active drug fragment of the ProTriTAC molecule that mimics protease activation is approximately 500-fold more potent in T cell-mediated cancer cell killing compared to the non-cleavable prodrug. 50 value.

[0202] Table 8: T cell-mediated cell killing potential

[0203] <![CDATA[EC 50 (nM)]]> Masking ratio Active drug 0.004 - Prodrug 0.485 121 Prodrug (non-cleavable) 2.197 549

[0204] Example 7: Exemplary ProTriTACs containing various exemplary linkers in a mixed mouse tumor model Antitumor activity of the molecules

[0205] The aim of this study was to explore the antitumor activity of ProTriTAC molecules containing different linkers. 7-week-old NSG female mice were used for this study. At the beginning of the study, on day 0, NSG female mice were injected with 2.5 x 10 6 Expanded human T cells and 5x 10 6 HCT116 (human colorectal cancer) tumor cells. The next day, on day 1, the mice were divided into several groups, and each group was treated with at least one of the ProTriTAC molecules listed in Table 9 (SEQ ID No. 786-790) or the control GFP TriTAC molecule (SEQ (SEQ ID No. 792) or the ProTriTAC molecule containing a non-cleavable linker (NCLV) (SEQ ID No. 791).

[0206] The ProTriTAC molecules and ProTriTAC NCLV molecules used in the following examples target EGFR and have the following orientation of the various 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 is the linker sequence. ProTriTAC molecules, ProTriTAC NCLV molecules, or GFP TriTAC molecules (GFP TriTAC molecules have the following orientation of the various domains: anti-GFP sdAb: anti-Alb sdAb: anti-CD3 scFv) were administered daily for 10 days (i.e., the final dose was administered on day 10 after the animals were injected with tumor cells and expanded cells), and tumor volume was measured regularly starting several days before the final dose was administered on day 10.

[0207] Table 9: ProTriTAC sequences and linkers

[0208]

[0209] like Fig.18 As shown, ProTriTAC molecules containing linker sequences L001, L045, L040, and L041 exhibited more potent antitumor activity compared to the GFP TriTAC control of the ProTriTAC NCLV molecule. The statistical significance of the data was determined by repeated measures one-way ANOVA-Dunnett post hoc test. The mean tumor volume of each group of mice was compared to the mean tumor volume of the group of mice receiving the NCLV molecule.

[0210] The pharmacokinetics of various molecules after administration as described above were also evaluated and the data are shown in Fig.19 After administration, the control GFP TriTAC molecule was rapidly cleared from the circulation, while the NCLV molecule remained in the circulation the longest. The pharmacokinetic clearance profiles of the tested ProTriTAC molecules were intermediate between those of GFP TriTAC and NCLV, except for the ProTriTAC containing the linker sequence L001, which was cleared almost as fast as the control GFP TriTAC.

[0211] Example 8: Mixed xenografts after treatment with exemplary ProTriTAC molecules containing various exemplary linkers Tumor volumes of transplanted tumors

[0212] The ProTriTAC molecules listed in Table 9, control GFP TriTAC molecules, and ProTriTAC NCLV molecules were evaluated in a mixed xenograft model to determine the in vivo efficacy of ProTriTAC molecules containing different linkers. As described in the previous example (Example 7), 7-week-old NSG mice were injected with 2.5 x 10 6 Expanded human T cells and 5x 10 6 HCT116 (human colorectal cancer) tumor cells were used to generate a xenograft tumor model. The mice were divided into several groups, and each group was treated with at least one of the ProTriTAC molecules, control GFP TriTAC molecules, or ProTriTAC NCLV molecules listed in Table 9. Tumor volume was measured regularly starting from day 10 after injection of tumor cells and expanded T cells.

[0213] A statistically significant delay in tumor growth was observed in animals treated with the exemplary ProTriTAC molecule containing the linker L040 compared to the group of mice treated with the control GFP TriTAC molecule or the group of mice treated with the ProTriTAC NCLV molecule. Similar observations were made for the ProTriTAC molecules containing the linker sequences L001, L041, and L045. The data are shown in Table 20.

[0214] Similar studies can also be performed using xenograft models using other cell lines, such as A549 (non-small cell lung cancer) cells, DU-145 (prostate cancer) cells, MCF-7 (breast cancer) cells, Colo 205 (colon cancer) cells, 3T3 / ]GF-IR (mouse fibroblast) cells, NCIH441 cells, HEP G2 (hepatoma) cells, MDAMB 231 (breast cancer) cells, HT-29 (colon cancer) cells, MDA-MB-435s (breast cancer) cells, U266 cells, SH-SYSY cells, Sk-Mel-2 cells, NCI-H929, RPM18226 and A431 cells.

[0215] Example 9: Reduced cytokine levels in cynomolgus monkeys correlated with masking of the TriTAC molecule

[0216] In this study, cynomolgus monkeys were treated with three different concentrations (30 μg / kg; 300 μg / kg; and 1000 μg / kg) of an exemplary EGFR-targeted ProTriTAC molecule containing a non-cleavable linker (ProTriTAC(NCLV)) or three different concentrations (10 μg / kg; 30 μg / kg; and 100 μg / kg) of an exemplary EGFR-targeted TriTAC molecule (SEQ ID No. 793).

[0217] like Fig.21 As shown, 4 hours after administration of the ProTriTAC (NCLV) molecule, IFN-γ, IL-6, and IL-10 levels were significantly reduced compared to those of the EGFR-targeted TriTAC molecule.

[0218] Example 10: Demonstration of improved resistance in mice conferred by exemplary EGFR-targeted ProTriTAC molecules receptive

[0219] In this study, the tolerability of an exemplary EGFR-targeted ProTriTAC molecule was evaluated. At the beginning of the study, i.e., day 0, 7-week-old NSG female tumor-free mice were injected intraperitoneally with 2 x 10 7 On day 2, treatment was started by dividing mice into different groups and administering different concentrations of exemplary EGFR-targeted ProTriTAC molecules containing linker sequence L001, EGFR-targeted TriTAC molecules, and EGFR-targeted ProTriTAC molecules containing non-cleavable linkers (ProTriTAC (NCLV). These molecules were administered once a day for 10 days at the following doses: 30 μg / kg, 100 μg / kg, 300 μg / kg. Starting from day 2, the body weight of the animals was recorded every day.

[0220] like Fig. 22As shown, EGFR-targeted ProTriTAC molecules containing a non-cleavable linker (ProTriTAC(NCLV)) and GFP TriTAC (used as a negative control) were well tolerated in mice even at the highest dose of 1000 μg / kg. EGFR-targeted ProTriTAC molecules containing the L001 linker sequence were well tolerated at a dose of 100 μg / kg, while EGFR-targeted TriTAC was well tolerated at a dose of 30 μg / kg. It was thus observed that in mice, ProTriTAC containing the L001 linker sequence improved tolerability by approximately 3-fold, while ProTriTAC(NCLV) improved tolerability by approximately 30-fold. The maximum tolerated doses of ProTriTAC(NCLV) and TriTAC molecules in mice were consistent with those observed in cynomolgus monkeys.

[0221] To further explore the role of the linker in the tolerability of the EGFR-targeted ProTriTAC molecule in mice, the linker sequence was changed from L001 to L040. In this experiment, at the beginning of the study, i.e., day 0, 7-week-old NSG female tumor-free mice were injected subcutaneously with 5x10 6 HCT116 tumor cells. On day 7 after tumor cell injection, when the tumor volume was approximately 180-200 mm 3 (For example, 183mm 3 ) were injected intraperitoneally into mice with 2x 10 7 The treatment was started on day 9 by dividing the mice into different groups, each group was administered with EGFR-targeted TriTAC molecules, EGFR-targeted ProTriTAC molecules with linker sequence L040 (ProTriTAC(L040) and ProTriTAC molecules containing a non-cleavable linker (ProTriTAC(NCLV). These molecules were administered once a day for 10 days at the following doses: 300 μg / kg and 1000 μg / kg. Starting from day 2, the body weight of the animals was recorded daily. Fig.23 The results shown in show that the EGFR-targeted ProTriTAC molecule with the linker sequence L040 is better tolerated than when the linker sequence L001 is used. ProTriTAC (L040) was well tolerated at 300 μg / kg and 1000 μg / kg, with a comparable percentage change in body weight as the ProTriTAC (NCLV) molecule. It is thus observed that the ProTriTAC containing the L040 linker sequence confers approximately a 30-fold increase in tolerance in mice compared to the TriTAC molecule, similar to the 30-fold increase in tolerance observed in the ProTriTAC (NCLV) molecule.

[0222] Example 11: Clinical pathology studies in mice and cynomolgus monkeys

[0223] In this study, mice received various concentrations of EFGR-targeted TriTAC molecules, EGFR-targeted ProTriTAC molecules containing the linker sequence L001 (ProTriTAC(L001)), and EGFR-targeted ProTriTAC molecules containing a non-cleavable linker (ProTriTAC(NCLV)). Tolerability was assessed by measuring serum concentrations of ALT (alanine aminotransferase) and AST (aspartate aminotransferase). Results Fig.24A , 24B and 24C and Fig.25A , 25B 25C. It was observed that the serum concentrations of AST and ALT did not increase after administration of ProTriTAC (L001) at a dose of up to 0.3 mg / kg, and the serum concentrations of AST and ALT did not increase after administration of ProTriTAC (NCLV) at a dose of up to 1 mg / kg. In contrast, the serum concentrations of AST and ALT did not increase after administration of the TriTAC molecule at a dose of 0.3 mg / kg.

[0224] In another study, cynomolgus monkeys were treated with various concentrations of EFGR-targeted TriTAC molecules and EGFR-targeted ProTriTAC molecules containing a non-cleavable linker (ProTriTAC (NCLV)). Tolerability was assessed by measuring serum concentrations of ALT (alanine aminotransferase) and AST (aspartate aminotransferase). Results Fig.26 It was observed that the serum concentrations of AST and ALT did not increase after administration of ProTriTAC (NCLV) at a dose of up to 1000 μg / kg. In contrast, the serum concentrations of AST and ALT did not increase after administration of the TriTAC molecule at a dose of 10 μg / kg.

[0225] Example 12: Exemplary ProTriTAC molecules of the present disclosure show expanded therapeutic window in tumor-bearing mouse models

[0226] The aim of this study was to evaluate the expansion of the therapeutic window by measuring antitumor activity and observable on-target toxicity in the same tumor-bearing mice. 7-week-old NSG female mice were used for this study.

[0227] At the beginning of the study, on day 0, NSG female mice were injected with 2.5 x 10 6 Expanded human T cells and 5x 10 6 The next day, on day 1, the mice were divided into several groups, each with Figures 27A-27DAnimals were treated with GFP TriTAC molecules (SEQ ID No. 792), EGFR TriTAC molecules (SEQ ID No. 793), or EGFR-targeted ProTriTAC molecules containing linker L040 (SEQ ID No. 787) at the indicated dose levels (300 μg / kg for GFP TriTAC; 10 μg / kg, 30 μg / kg, 100 μg / kg, and 300 μg / kg for EGFR TriTAC; and 30 μg / kg, 100 μg / kg, 300 μg / kg, and 1000 μg / kg for EGFR ProTriTAC), administered daily for 10 days (i.e., the final dose was administered on day 10 after the animals were injected with tumor cells and expanded cells), and tumor volume was measured regularly starting from a few days before the final dose was administered on day 10. Results are shown in Figures 27A-27D Shown in.

[0228] The area was determined by measuring the radius of the red scarred skin lesion above the original tumor implantation site with a caliper on day 14 and applying the equation area = π*(lesion radius) 2 , determine on-target toxicity related to EGFR. Fig.28 Results presented in show that the exemplary EGFR ProTriTAC was 30-fold better tolerated than the EGFR TriTAC in the same HCT116 tumor-bearing mice, as measured by the appearance of red, scarring skin lesions above the original tumor implantation site. The therapeutic window is defined as the difference between the minimum dose level required for antitumor activity and the highest dose level without skin lesions.

[0229] The results (of Figures 27 and 28) show that the exemplary protease-cleavable EGFR ProTriTAC is 3-fold less potent than EGFR TriTAC but 30-fold more tolerable (i.e., the therapeutic window is improved 10-fold) when measuring efficacy and toxicity on the same tumor-bearing mice, as summarized in the table below. This would allow the dose of ProTriTAC to be about 3 times that of TriTAC to obtain at least the same efficacy and better tolerability.

[0230] Minimum effective dose Maximum scar-free dose Treatment Window TriTAC 10 μg / kg 10 μg / kg 1 ProTriTAC 30 μg / kg 300 μg / kg 10

[0231] Example 13: Exemplary ProTriTAC molecules containing a binding portion with extended non-CDR loops, wherein a human The CD3ε epitope was grafted into this non-CDR loop

[0232] The sequence of the binding portion comprising the non-CDR loops (AB, EF, C"D and CC') was obtained. A portion of the human CD3ε sequence was grafted together with a glycine residue into the CC' loop of the non-CDR loop within the binding portion to further extend the CC loop. Fig.29Three different variants are shown, which contain an extension of 10, 12 or 16 amino acids of the CC' loop. In the case of variant CC10, the portion of the human CD3ε sequence grafted into the CC' loop to replace the wild-type sequence APGKG is QDGNEE (SEQ ID NO.801), and 4 glycine residues are additionally inserted to extend the CC' loop. In the case of variant CC12, the portion of the human CD3ε sequence grafted into the CC' loop to replace the wild-type sequence APGKG is QDGNEEMGG (SEQ ID No.802), and 3 glycine residues are additionally inserted to extend the CC' loop. In the case of variant CC12, the portion of the human CD3ε sequence grafted into the CC' loop to replace the wild-type sequence APGKG is QDGNEEMGG (SEQ ID NO.803), and 7 glycine residues are additionally inserted to extend the CC' loop. As described above, a binding portion comprising an extended non-CDR loop containing a CD3ε sequence is cloned into a vector that further comprises a protease cleavable linker, a scFv containing a CD3 binding domain, and a coding sequence for an EGFR binding domain to express a ProTriTAC molecule. The ProTriTAC molecule contains an exemplary binding portion of the present disclosure, a CD3 binding scFv, and an EGFR binding domain. The ProTriTAC molecule is then exposed to a tumor-associated protease, a protease, to determine the activation of the molecule after cleavage of the protease cleavable linker, which cleaves the binding portion containing the cleavable linker (in Fig.29 and 30 ) is separated from the rest of the molecule, namely from the part containing the CD3 binding domain (depicted as aALB in Fig.29 and 30 Depicted in Figure 3 are the scFvs that separate the aCD3) and EGFR binding domains (aEGFR). Fig.30 Activation of ProTriTAC molecules containing CC10, CC12 or CC16 variants of the CC' non-CDR loop, CD3 binding scFv, in the form of VH-VL (left panel) or VL-VH (right panel) after treatment with a protease is shown. A ProTriTAC molecule containing a wild-type CC' loop in the binding moiety was used as a control for the protease activation assay. Additionally, a TriTAC molecule that is not in the "pro" form, i.e., a molecule that includes the same domains as the ProTriTAC molecule, except that it has a half-life extension domain, such as albumin, instead of the binding moiety, was also treated with a protease and used as a control. The results show that the ProTriTAC molecule is activated after cleavage to generate a free albumin binding domain (in Fig.30The albumin domain is not separated from the TriTAC molecule. Thus, unlike the TriTAC format, the ProTriTAC molecule containing the binding moiety of the present disclosure can be easily dissociated from the half-life extension domain after cleavage in the tumor microenvironment, thereby enabling rapid clearance from the systemic circulation after activation.

[0233] Further studies were performed to determine the binding of a binding moiety containing human CD3ε to CD3. It was observed that in the presence of human serum albumin, the activated form of the ProTriTAC molecule containing a binding moiety having human CD3ε was approximately 20 times more potent in binding to CD3 than the activated form without the binding moiety. Fig.31 Shown in.

[0234] The cell killing potential of the ProTriTAC molecules containing the binding moieties described herein was also determined in a study in which the CaOV4 cell line was treated with the ProTriTAC molecules or their activated forms in the presence of human serum albumin. Fig.32 As shown, the ProTriTAC molecule containing the CC16 variant of the CC' non-CDR loop is about 50 times more potent in killing cancer cells than its activated form isolated from the albumin binding domain. The overall masking observed is due to a combination of steric masking caused by binding to human serum albumin and specific masking caused by CC16 masking in the CC' non-CDR loop.

[0235] Example 14: Soft library mutagenesis determines that the CC' loop is most suitable for modification

[0236] To determine the positions in the non-CDR loops (AB, CC, C"D, and EF) that were most suitable for modification to create masking capacity, a library was assembled and generated using four sets of overlapping DNA oligonucleotides containing randomized degenerate "NNK" codons and with different loop lengths, as shown in the following schematic:

[0237] AB loop oligonucleotide:

[0238] WT: LVQPGN (20%) (SEQ ID NO:903)

[0239] AB0: XXXXXX (20%)

[0240] AB1: XXXXXXX (20%)

[0241] AB2: XXXXXXXX (20%)

[0242] AB3: XXXXXXXXX (20%)

[0243] CC' Cyclic Oligonucleotide:

[0244] WT: APGKG (20%)

[0245] CC0: XXXXX (20%)

[0246] CC1: XXXXXX (20%)

[0247] CC2: XXXXXXX (20%)

[0248] CC3: XXXXXXXX (20%)

[0249] C"D loop oligonucleotide:

[0250] WT: DSVKGR (20%) (SEQ ID NO:904)

[0251] CD0: XXXXXX (20%)

[0252] CD1: XXXXXXX (20%)

[0253] CD2: XXXXXXXX (20%)

[0254] CD3: XXXXXXXXX (20%)

[0255] EF loop oligonucleotide:

[0256] WT: SLRPED (20%) (SEQ ID NO:905)

[0257] EF0: XXXXXX (20%)

[0258] EF1: XXXXXXX (20%)

[0259] EF2: XXXXXXXX (20%)

[0260] EF3: XXXXXXXXX (20%)

[0261] Note: "X" indicates a randomized residue ("NNK" codon), which can be any of the 20 natural amino acids as well as a stop codon. The goal is to have approximately 20% of each non-CDR loop be wild-type. These wild-type oligonucleotides serve as an internal benchmark to assess each loop's tolerance to modification (sequence composition and / or length changes). Loops that are refractory to change will easily revert to wild-type; conversely, loops that are highly tolerant of change will maintain a diverse sequence repertoire. To this end, Fig.33As shown, 24 clones were sequenced from the original library before panning with HSA to verify the randomization of the non-CDR loops. After two rounds of phage panning against HSA, 30 clones were sequenced to check the non-CDR loop composition. The results showed that 3 out of 4 non-CDR loops (AB, C”D, and EF) mainly utilized 20% of the wild-type oligonucleotides and reverted to the wild type, indicating that they may be less favored compared to the wild-type sequence. However, the CC' loop maintained a diverse sequence repertoire (in both sequence and length), suggesting that the CC' loop may be the most tolerant loop to randomization, and we can use it for specific masking of adjacent domains, such as Fig.34 shown.

[0262] Example 15: Screening of phage display libraries for identification of EpCAM binding domains

[0263] Llamas were immunized with purified EpCAM protein expressed in Expi293 cells. Phage display libraries for expressing antibody heavy chain variable domains were 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 binding to EpCAM by expressing anti-EpCAM protein in E. coli, preparing periplasmic extracts, and screening the proteins for human and cynomolgus EpCAM binding activity using colorimetric ELISA. 38 unique heavy chain-only sequences (SEQ ID No. 804-841) were identified that produced signals in ELISA screening relative to controls with human and / or cynomolgus EpCAM proteins (as shown in Table 10). Table 10: Signal in ELISA assay (colorimetric) relative to a heavy chain-only single domain antibody control Llama anti-human EpCAM Binding of heavy chain-only single domain antibodies to human and cynomolgus monkey EpCAM

[0264]

[0265]

[0266] Example 16: Incorporation of heavy chain-only single domain antibodies binding to EpCAM into fusion proteins and T cell dependence Cytotoxicity assay

[0267] The anti-EpCAM heavy chain only single domain antibodies selected from Example 15 were cloned into DNA constructs for expressing recombinant proteins. These expression constructs all encode signal peptides. One set of anti-EpCAM constructs (SEQ ID Nos. 842 to 868) were designed to express fusion proteins having a humanized anti-CD3 scFv domain at the N-terminus of the mature secreted fusion protein, followed by a llama anti-EpCAM domain, the two domains being connected by the sequence GGGGSGGGS, and having HHHHHH at the C-terminus. A second set of anti-EpCAM constructs (SEQ ID Nos. 869 to 895) were designed to express fusion proteins having a llama anti-EpCAM domain at the N-terminus of the mature secreted fusion protein, followed by a humanized anti-CD3-scFv domain, the two domains being connected by the sequence GGGGSGGGS, and having HHHHHH at the C-terminus.

[0268] These anti-EpCAM / anti-CD3 (from N-terminus to C-terminus) or anti-CD3 / anti-EpCAM (from N-terminus to C-terminus) fusion protein constructs were transfected into Expi293 cells. The amount of anti-EpCAM / anti-CD3 fusion protein in the conditioned medium from transfected Expi293 cells was quantified by using an Octet instrument with streptavidin and loading of biotinylated CD3-Fc fusion protein, and using an anti-CD3 fusion protein with a similar molecular weight to the anti-EPCAM / anti-CD3 protein as a standard.

[0269] Conditioned medium was tested in a T cell dependent cytotoxicity assay. See Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20: 519-27. In this assay, EpCAM-expressing luciferase-labeled NCI-H508 cells were combined with purified human T cells and anti-EpCAM / anti-CD3 fusion proteins or anti-CD3 / anti-EpCAM were titrated. It was assumed that if the fusion protein directed T cells to kill NCI-H508 cells, the signal in the luciferase assay performed 48 hours after the start of the experiment should be reduced. Figure 36-39 TDCC data are presented in graphical format. EC from TDCC assay 50 The values ​​are listed in Table 11 (listing the EC values ​​of SEQ ID No. 842 to 868) 50 Data) and Table 12 (listing the EC of SEQ ID No.869-895 50 Data). The EC of the most potent molecule (EPL13) 50The value was about 1.6 pM. Some anti-EpCAM binding proteins are only active when present in an anti-CD3 / anti-EpCAM configuration. One anti-EpCAM sequence, EPL34, is only active in an anti-EpCAM / anti-CD3 configuration. The negative control for the TDCC assay was an anti-GFP / anti-CD3 protein that did not direct T cells to kill NCI-H508 cells (data not shown).

[0270] Table 11. Anti-CD3 / anti-EPCAM proteins containing llama anti-EPCAM sequences redirect T cells in NCI-H508 cells EC cell killing 50 value (n / a = insufficient activity to calculate EC using the protein concentration tested 50 )

[0271]

[0272]

[0273] Table 12. Anti-EpCAM / anti-CD3 proteins containing llama anti-EppCAM sequences redirect T cells in NCI-H508 cells EC cell killing 50 value (n / a = insufficient activity to calculate using the protein concentration tested EC 50 )

[0274]

[0275]

[0276] Using conditioned medium with known concentrations of anti-EpCAM / anti-CD3 or anti-CD3 / anti-EpCAM fusion proteins, measure the binding affinity of the fusion proteins to human and cynomolgus EpCAM proteins. Load biotinylated human or cynomolgus EpCAM proteins in an Octet instrument with a streptavidin tip and calculate K by measuring the association rate and dissociation rate of the binding of anti-EPCAM / anti-CD3 fusion proteins or anti-CD3 / anti-EpCAM fusion proteins to biotinylated EpCAM proteins. D K values ​​were calculated using a single 50 nM concentration of anti-EPCAM / anti-CD3 or anti-CD3 / anti-EpCAM fusion protein. D Table 13 lists the relative affinities measured. All fusion proteins bound to cynomolgus monkey EpCAM, with K D The values ​​ranged from 1.6 to 56 nM. Binding of most, but not all, fusion proteins to human EpCAM was measured with K D The values ​​ranged from 0.8 to 74 nM.

[0277] Table 13. Anti-EPCAM / anti-CD3 or anti-CD3 / anti-EpCAM fusion proteins containing llama anti-EpCAM sequences for human Binding affinity to cynomolgus monkey EpCAM

[0278]

[0279]

[0280] Example 17: Humanization of heavy chain-only single domain antibodies binding to EpCAM and T cell-dependent cellular cytotoxicity assay Certainly

[0281] The three llama anti-EpCAM antibody sequences identified in Example 15 were humanized (SEQ ID Nos. 896 to 898) by grafting their CDR sequences onto human germline sequences while retaining some llama framework sequences to ensure that the antibodies did not lose activity.

[0282] These sequences were cloned into expression constructs for expression of anti-EpCAM / anti-CD3 fusion proteins (SEQ ID Nos. 899 to 901) in Expi293 cells as described in Example 16.

[0283] The amount of anti-EpCAM / anti-CD3 fusion protein present in the conditioned medium was quantified as described in Example 16. The affinity of these humanized proteins for human, cynomolgus monkey and mouse EpCAM was measured as described in Example 16. The relative K values ​​calculated from these measurements were D The values ​​are listed in Table 14. All three sequences bound to human and cynomolgus monkey EpCAM with relative K D The values ​​ranged from about 0.3 to about 18 nM. Two of the sequences also bound to mouse EpCAM, with K D The values ​​ranged from about 1.4 to about 1.8 nM.

[0284] Table 14: Anti-EpCAM / anti-CD3 fusion proteins containing llama anti-EpCAM sequences for humans, cynomolgus monkeys and mice Binding affinity of EpCAM (n / q = not quantifiable under the experimental conditions used)

[0285]

[0286] The T cell killing potential of anti-EpCAM / anti-CD3 fusion proteins present in conditioned medium was evaluated as described in Example 16. The results are shown in Tables 15 and Fig.40 Available in.

[0287] Table 15: Recombination of purified anti-CD3 / anti-EpCAM proteins containing humanized anti-EpCAM sequences on NCI-H508 cells EC directed T cell killing 50 value

[0288]

[0289]

[0290] Example 18: Demonstration of improved resistance to leukemia in mice conferred by exemplary EpCAM-targeted ProTriTAC molecules receptive

[0291] In this study, the tolerability of an exemplary EpCAM-targeted ProTriTAC molecule was evaluated. At the beginning of the study, i.e., on day 0, 7-week-old NSG female tumor-free mice were injected intraperitoneally with 2 x 10 7 The treatment was started on day 2 by dividing the mice into different groups and administering different concentrations of an exemplary EpCAM-targeted ProTriTAC molecule containing the linker sequence L040, an EpCAMR-targeted TriTAC molecule, an EpCAM-targeted ProTriTAC molecule containing a non-cleavable linker (EpCAM ProTriTAC (NCLV)), and a GFP TriTAC molecule (SEQ ID No. 792) as a control. These molecules were administered once a day for 10 days at the following doses: 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg. Starting from day 2, the body weight of the animals was recorded daily.

[0292] like Figures 41A-41C As shown, even at the highest dose of 1 mg / kg, the EpCAM-targeted ProTriTAC molecule (ProTriTAC (NCLV)) (SEQ ID No. 908) and GFP TriTAC (used as a negative control) containing a non-cleavable linker were well tolerated in mice. The EpCAM-targeted ProTriTAC molecule (SEQ ID No. 907) containing the L040 linker sequence was well tolerated at the highest tested dose of 1 mg / kg, while the EpCAM-targeted TriTAC (SEQ ID No. 906) was well tolerated at 0.1 mg / kg. It was therefore observed that the EpCAM-targeted ProTriTAC containing the L040 linker sequence was at least about 10 times more tolerable in mice than the EpCAM-targeted TriTAC.

[0293] Example 19: Xenograft Tumor Model

[0294] EpCAM targeting fusion proteins of the present disclosure (e.g., fusion proteins that are trispecific proteins comprising an anti-EpCAM heavy chain-only single domain antibody, an anti-CD3 scFv, and an anti-albumin domain) were evaluated in xenograft models. In order to determine the efficacy of exemplary EpCAM targeting fusion proteins in vivo, a variety of xenograft tumor models were used. Examples of common tumor cell lines used for xenograft tumor studies include A549 (non-small cell lung cancer) cells, DU-145 (prostate cancer) cells, MCF-7 (breast cancer) cells, Colo 205 (colon cancer) cells, 3T3 (mouse fibroblasts) cells, NCI H441 cells, HEP G2 (hepatoma) cells, MDA MB 231 (breast cancer) cells, HT-29 (colon cancer) cells, MDA-MB-435s (breast cancer) cells, U266 cells, SH-SYSY cells, Sk-Mel-2 cells, NCI-H929, RPM18226, and A431 cells. Immunodeficient NOD / scid mice were sublethally irradiated (2 Gy) and 1X 10 6 Tumor cells (e.g., NCI H441 cells) were inoculated subcutaneously into their right dorsal flank. When the tumor reached 100 to 200 mm 3 The animals were divided into three treatment groups. Groups 2 and 3 were injected intraperitoneally with 1.5×10 7 Three days later, animals in Group 3 were then treated with the exemplary EPCAM-targeting trispecific antigen binding protein. Groups 1 and 2 were treated with vehicle only. Body weight and tumor volume were measured for 30 days, starting at least 5 days after treatment with the exemplary EPCAM-targeting trispecific protein.

[0295] Animals treated with the exemplary EpCAM-targeting trispecific proteins are expected to have a statistically significant tumor growth delay compared to corresponding vehicle-treated controls.

[0296] Example 20: Administration of the EpCAM-targeted trispecific antigen binding protein of Example 19 to ovarian cancer patients Proof-of-concept clinical trial plan

[0297] This is a Phase I / II clinical trial for studying the exemplary EpCAM-targeting trispecific antigen binding proteins of the present disclosure as a treatment for epithelial ovarian cancer.

[0298] 1. Research results:

[0299] 2. Primary: Maximum tolerated dose of an exemplary EpCAM-targeting trispecific protein.

[0300] 3. Secondary: Determine whether the in vitro responses of the exemplary EpCAM-targeted trispecific protein correlate with clinical responses

[0301] Phase I

[0302] 4. The maximum tolerated dose (MTD) was determined in the Phase I portion of the trial.

[0303] 1.1 The maximum tolerated dose (MTD) was determined in the Phase I part of this trial.

[0304] 1.2 Patients meeting the eligibility criteria are enrolled in the trial for the EPCAM targeting trispecific protein of the previous example.

[0305] 1.3 The goal is to determine the highest dose at which the EpCAM-targeted trispecific protein of the aforementioned example can be safely administered to participants without severe or difficult-to-handle side effects. The given dose depends on the number of participants who have previously been enrolled in the study and the dose tolerance. Not all participants receive the same dose.

[0306] Phase II

[0307] 2.1 The subsequent Phase II portion was treated at the MTD with the goal of determining whether therapy with the exemplary EpCAM-targeting trispecific protein would result in a response rate of at least 20%.

[0308] The primary outcome of Phase II is to determine whether therapy targeting the trispecific protein with EPCAM will result in a clinical response (blast response, minor response, partial response or complete response) in at least 20% of patients.

[0309] Eligibility:

[0310] Histologically or cytologically confirmed epithelial ovarian cancer. Epithelial ovarian cancer that may have recurred or progressed after failure of first-line platinum-based chemotherapy and has not been treated with more than one prior platinum-based regimen

[0311] Adequate laboratory values ​​for bone marrow function, renal function, liver function, and echocardiography

[0312] Stage III

[0313] 5.3.1 A subsequent Phase III portion was conducted using an exemplary EpCAM-targeted trispecific protein, in which secondary endpoints such as response rate (RR), patient recorded outcomes (PROs), progression-free survival (PFS), progression-free survival, time to progression (TIP), overall survival, health-related quality of life assessment, number of participants with overall survival, duration of response, time to response, number of participants with response, and time to tumor growth were evaluated.

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Claims

1. A binding moiety comprising a non-CDR loop and a cleavable linker, wherein the moiety is capable of masking the binding of a binding molecule to its target, wherein the binding molecule comprises an immunoglobulin molecule or a non-immunoglobulin molecule.

2. The binding moiety of claim 1, wherein the moiety is a natural peptide, a synthetic peptide, an engineered scaffold or an engineered bulky serum protein.

3. The binding moiety of claim 1 or 2, wherein the engineered scaffold comprises a sdAb, a scFv, a Fab, a VHH, a type III fibronectin domain, an immunoglobulin-like scaffold, a DARPin, a cystine knot peptide, a lipocalin, a three-helix bundle scaffold, a protein G-associated albumin binding module, or a DNA or RNA aptamer scaffold.

4. A binding moiety according to any one of claims 1 to 3, wherein the moiety is capable of binding to bulk serum proteins.

5. The binding portion of any one of claims 1-4, wherein the non-CDR loops are from a variable domain, a constant domain, a C1-set domain, a C2-set domain, an I-domain, or any combination thereof.

6. The binding portion of any one of claims 1-5, wherein the portion further comprises a complementarity determining region (CDR).

7. A binding moiety according to claim 6, wherein the moiety is capable of binding to bulk serum proteins.

8. A binding moiety according to claim 7, wherein the bulky serum protein is a half-life extending protein.

9. A binding moiety according to claim 7 or 8, wherein the bulky serum protein is albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, fibrinogen, IgE or pentameric IgM.

10. A binding moiety according to any one of claims 1 to 9, wherein the moiety comprises a binding site specific for an immunoglobulin light chain.

Images