Activatable multispecific molecules and methods for using them

Abstract

To provide activatable multispecific molecules and methods for using them. [Solution] An activatable protein comprising: a first target-binding protein (TB1) that specifically binds to a first target; a second target-binding protein (TB2) that specifically binds to a second target, wherein TB2 is directly or indirectly coupled to TB1; a first masking portion (MM1) that inhibits the binding of TB1 to the first target and is directly or indirectly coupled to TB1 via a cleavable portion, for example, via one or more linkers or other components; a second masking portion (MM2) that inhibits the binding of TB2 to the second target and is directly or indirectly coupled to either TB1 or TB2 via a cleavable portion; and a half-life extension portion (EM) that is directly or indirectly coupled to either TB1 or TB2 via a cleavable portion.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. US63 / 326,692, filed 1 April 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence List The sequence listing filed with this application by the EFS, titled "4862-122PCT.xml", was created on 30 March 2023, is 639,463 bytes in size, and is incorporated herein by reference in its entirety.

[0003] This disclosure relates to the field of biotechnology, and more specifically, to activatable multispecific molecules. [Background technology]

[0004] Antibody-based therapies offer proven and effective treatments for a variety of diseases. However, in some cases, their therapeutic efficacy is limited by toxicity resulting from broad target expression. Furthermore, antibody-based therapeutics exhibit other limitations, such as rapid clearance from the circulation after administration.

[0005] Activatable antibodies are driving attempts to expand the therapeutic index of antibody-based therapies. These molecules are administered as activatable prodrugs that are activated in vivo at or near the desired site of action. This mechanism of action may lead to an increase in the therapeutic index of the parent antibody. However, other strategies are continuously needed to increase the therapeutic index of antibody-based therapies. [Overview of the Initiative]

[0006] This disclosure provides activatable proteins, as well as related compositions and methods.

[0007] In one embodiment, the present disclosure provides an activatable protein comprising: a first target-binding domain (TB1) that specifically binds to a first target; a second target-binding domain (TB2) that specifically binds to a second target, wherein TB2 is coupled to TB1; a first masking portion (MM1) coupled to TB1 via a first cleavable portion (CM1), wherein MM1 comprises: a first masking portion (MM1) that inhibits the binding of TB1 to the first target; a second masking portion (MM2) that inhibits the binding of TB2 to the second target; a second cleavable portion (CM2); and a half-life extension portion (EM) directly or indirectly coupled to MM1 or MM2, wherein the components of the activatable protein are configured such that, upon cleavage of CM1 and CM2, the resulting activated protein contains TB1 and TB2 but does not contain MM1, MM2, and EM. As used herein and unless otherwise specified, the components of an activatable molecule to be “coupled” may be coupled either directly or indirectly via covalent bonds, for example, one or more linked peptides (also called “linkers”), cleavable moieties, or other components of an activatable protein.

[0008] In one embodiment, the Disclosure provides a first antigen-binding domain (AB1) that specifically binds to a first target, comprising a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); a second antigen-binding domain (AB2) that specifically binds to a second target, comprising a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), wherein AB2 is directly or indirectly coupled to the C-terminus of HVD1 or the C-terminus of LVD1; and via a first cleavable moiety (CM1) (directly or indirectly, for example, one or more phosphorus molecules) The present invention provides an activatable protein comprising: a first masking moiety (MM1) coupled to AB1 (either via a Kerr), wherein MM1 inhibits the binding of AB1 to a first target; and a half-life extension moiety (EM) directly or indirectly coupled to a second masking moiety (MM2), wherein EM is coupled to AB1 or AB2 via a second cleavable moiety (CM2) (either directly or indirectly, for example, via one or more linkers or other components of the activatable protein), wherein MM2 inhibits the binding of AB2 to a second target.

[0009] In one embodiment, the Disclosure provides a first antigen-binding domain (AB1) that specifically binds to a first target, comprising a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); a second antigen-binding domain (AB2) that specifically binds to a second target, comprising a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), wherein AB2 is directly or indirectly coupled to the C-terminus of HVD1 or LVD1; and via a first cleavable moiety (CM1) (directly or indirectly). The present invention provides an activatable protein comprising: a first masking moiety (MM1) indirectly coupled to AB1 (for example, via one or more linkers), wherein MM1 inhibits the binding of AB1 to a first target; and a half-life extension moiety (EM) directly or indirectly coupled to a second masking moiety (MM2), wherein EM and MM2 are coupled (directly or indirectly) to either AB1 or AB2 via a second cleavable moiety (CM2), wherein MM2 inhibits the binding of AB2 to a second target.

[0010] In one embodiment, the Disclosure comprises: a first antigen-binding domain (AB1) that specifically binds to a first target, comprising a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); a second antigen-binding domain (AB2) that specifically binds to a second target, comprising a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), wherein AB2 is directly or indirectly coupled to the C-terminus of HVD1 or LVD1; and a first cleavable moiety (CM1) (directly or indirectly, for example, via one or more linkers). The present invention provides an activatable protein comprising: a first masking moiety (MM1) coupled to AB1, wherein MM1 inhibits the binding of AB1 to a first target; and a half-life extension moiety (EM) comprising a dimer of a first half-life extension moiety (EM1) and a second half-life extension moiety (EM2), wherein EM1 is coupled to AB1 via a second cleavable moiety (CM2) (directly or indirectly, for example, via one or more linkers), and EM2 is directly or indirectly coupled to a second masking moiety (MM2), wherein MM2 inhibits the binding of AB2 to a second target.

[0011] In one aspect, the present disclosure provides an activatable protein comprising: a first target binding domain (TB1) that specifically binds to a first target; a second target binding domain (TB2) that specifically binds to a second target, wherein TB2 is coupled directly or indirectly to TB1; a first masking portion (MM1) coupled to TB1 via a first cleavable moiety (CM1) (either directly or indirectly, e.g., via one or more linkers), wherein MM1 inhibits binding of TB1 to the first target; a half-life extension moiety (EM) and a second masking portion (MM2) coupled to TB1 or TB2 via a second cleavable moiety (CM2) (either directly or indirectly, e.g., via one or more linkers), wherein MM2 inhibits binding of TB2 to the second target; and components of the activatable molecule are configured such that cleavage of CM1 and CM2 releases MM1, MM2, and EM from TB1 and TB2 (where applicable), optionally, TB1 is an antigen-binding molecule (AB1) comprising HVD1 and LVD1, and optionally, TB2 is an antigen-binding molecule (AB2) comprising HVD2 and LVD2.

[0012] In one embodiment, the Disclosure provides a first antigen-binding domain (AB1) that specifically binds to a first target, comprising a first heavy chain variable domain (HVD1) and a light chain variable domain (LVD1); a second antigen-binding domain (AB2) that specifically binds to a second target, comprising a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), wherein AB2 is coupled either directly or indirectly (e.g., via a linker) to the C-terminus of HVD1 or LVD1; and via a first cleavable moiety (CM1) (directly or indirectly). The present invention provides an activatable protein comprising: a first masking moiety (MM1) coupled to AB1 (for example, via one or more linkers), wherein MM1 inhibits the binding of AB1 to a first target; a second masking moiety (MM2) coupled to AB2 (directly or indirectly, for example, via one or more linkers) via a second cleavable moiety (CM2), wherein MM2 inhibits the binding of AB2 to a second target; and a half-life extension moiety (EM) directly or indirectly coupled to MM1 or MM2.

[0013] In another aspect, the present disclosure provides an activatable protein comprising: a first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy-chain variable domain (HVD1) and a first light-chain variable domain (LVD1); a second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy-chain variable domain (HVD2) and a second light-chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the N-terminus of HVD1 or the N-terminus of LVD1; a first masking moiety (MM1) coupled to AB1 via a first cleavable moiety (CM1) and optionally one or more linkers, wherein MM1 inhibits the binding of AB1 to the first target; a second masking moiety (MM2) coupled to AB2 via a second cleavable moiety (CM2) and optionally one or more linkers, wherein MM2 inhibits the binding of AB2 to the second target; and a half-life extension moiety (EM) coupled to the C-terminus of HVD1 or the C-terminus of LVD1 via a third cleavable moiety (CM3) and optionally one or more linkers.

[0014] In some embodiments, EM is a dimer formed by a first fragment crystallizable (Fc) domain and a second Fc domain. In some embodiments, the protein comprises at least a first polypeptide and a second polypeptide.

[0015] In some embodiments, the first polypeptide comprises MM1, CM1, and VLD1 (including one or more optional linkers between the elements) in the order from the N-terminus to the C-terminus. In some embodiments, the second polypeptide comprises VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain, and the activatable protein further comprises a third polypeptide comprising a second Fc domain. In some embodiments, the second polypeptide comprises VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain in the order from the N-terminus to the C-terminus. In some embodiments, the second polypeptide comprises VHD1, CM2, MM2, and a first Fc domain in the order from the N-terminus to the C-terminus. In some embodiments, the second polypeptide comprises VHD1, CM2, and a first Fc domain in the order from the N-terminus to the C-terminus. In some embodiments, the first polypeptide comprises MM1, CM1, and VLD1, VHD2, and VLD2. In some embodiments, the first polypeptide comprises MM1, CM1, VLD1, VHD2, and VLD2 in order from the N-terminus to the C-terminus. In some embodiments, the first polypeptide comprises MM1, CM1, VLD1, VLD2, and VHD2 in order from the N-terminus to the C-terminus. In some embodiments, the protein comprises a third polypeptide, the third polypeptide comprising a second Fc domain and MM2. In each of the embodiments described above, and unless otherwise specified, the polypeptide may include, for example, one or more optional linkers between each of the enumerated elements.

[0016] In some embodiments, MM2 is linked to the C-terminus of the second Fc domain via a linking peptide. In some embodiments, MM2 is linked to the N-terminus of the second Fc domain via a linking peptide (also called a “linker”). In some embodiments, the second polypeptide further comprises a linker (L1) between MM2 and the first Fc domain. In some embodiments, L1 is a peptide having a length of 5–30, 6–29, 7–28, 8–27, 9–26, 10–25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids. In the structural configurations disclosed in the preceding paragraphs and throughout this disclosure, one or more linkers may optionally be present between the elements. Furthermore, the disclosure also envisions and includes an activatable protein in which one or more of the disclosed elements are in direct contact with each other at any option, and as a result there are no linkers or other amino acid sequences between the elements.

[0017] In some embodiments, the first Fc domain is a hole variant of the Fc domain, and the second Fc domain is a knob variant of the Fc domain. In some embodiments, the hole variant of the Fc domain contains the sequence of SEQ ID NO: 2, and the knob variant of the Fc domain contains the sequence of SEQ ID NO: 1.

[0018] In some embodiments, the first target or epitope is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2). In some embodiments, AB1 is the Fab of trastuzumab. In some embodiments, HVD1 includes the sequence of SEQ ID NO: 27, and LVD1 includes the sequence of SEQ ID NO: 17. In some embodiments, AB2 is an immune effector cell associated with scFv, a leukocyte associated with scFv, a T cell associated with scFv, an NK cell associated with scFv, a macrophage associated with scFv, or a mononuclear cell associated with scFv. In some embodiments, AB2 is anti-CD3 epsilon scFv or anti-CTLA-4 scFv, or derived therefrom. In some embodiments, AB2 is anti-CD3 epsilon scFv, or derived therefrom. In some embodiments, HVD2 includes the sequence of SEQ ID NO: 30, and LVD2 includes the sequence of SEQ ID NO: 31.

[0019] In some embodiments, AB1 is or is derived from an anti-HER2 antibody. In some embodiments, AB1 is scFv, and the activatable protein is an activatable bispecific T cell engager (BiTE) or a biaffinity retargeting antibody (DART). In some embodiments, AB1 is a fragment antigen binder (Fab). In some embodiments, the second target is a costimulatory molecule. In some embodiments, the costimulatory molecule is CD3.

[0020] In some embodiments, CM1 and CM2 each contain the substrate of the same protease. In some embodiments, CM1 and CM2 contain the substrates of different proteases. In some embodiments, CM1 and CM2 each independently contain substrates of ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartate protease, BACE, renin, aspartate cathepsin, cathepsin D, cathepsin E, caspase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cysteine ​​Cathepsin, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Cysteine ​​Proteinase, Crudipain, Regmine, Otsubine-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Metalloproteinase, Meprin, Neprila Icin, PSMA, BMP-1, MMP, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, serine protease, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor protease, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, It contains substrates for proteases selected from the group consisting of granzyme B, guanidinobenzoate, HtrA1, human neutrophil elastase, lactoferrin, malapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane, serine protease, TTSP, DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, and TMPRSS4.

[0021] In some embodiments, MM1 and MM2 are each independently 2 to 40 amino acid lengths. In some embodiments, MM1 and MM2 are each independently 4 to 30 amino acid lengths. In some embodiments, the heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1, the N-terminus of MM2 is coupled to the C-terminus of the light chain variable region of AB2 via CM2 (directly or indirectly, for example, via one or more linkers), EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain of EM. In some embodiments, the heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1, the N-terminus of MM2 is coupled to the C-terminus of the heavy chain fragment of AB1 via CM2 (directly or indirectly, for example, via one or more linkers), EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain. In some embodiments, the heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1, EM comprises a dimer of a first Fc domain and a second Fc domain, the N-terminus of the first Fc domain is coupled to the C-terminus of the heavy chain fragment of AB1 via CM2 (directly or indirectly, for example, via one or more linkers), and the C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the second Fc domain.

[0022] In some embodiments, the activatable protein further comprises a linker between MM2 and a first or second Fc domain directly or indirectly coupled to MM2. In some embodiments, MM1 comprises the sequence of SEQ ID NO: 40, and MM2 comprises one of the sequences of SEQ ID NOs: 34-37 or 66-70. In some embodiments, MM1 has a dissociation constant for binding to AB1 that is greater than the dissociation constant of AB1 for binding to a first target or epitope, and MM2 has a dissociation constant for binding to AB2 that is greater than the dissociation constant of AB2 for binding to a second target or epitope. In some embodiments, the activating molecule has a shorter half-life compared to a corresponding molecule that is the same as the activating molecule but contains EM. In some embodiments, the activating molecule has higher target-binding activity compared to a corresponding molecule that is the same as the activating molecule but contains EM. In some embodiments, the activating molecule has higher target-binding activity compared to the activatable molecule.

[0023] In some embodiments, the second polypeptide further includes a linker (L2) between MM2 and AB2. In some embodiments, L2 has an amino acid length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27. In some embodiments, the second polypeptide further includes a linker (L3) between AB2 and AB1. In some embodiments, L3 has an amino acid length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27. In general, in each embodiment of this specification, unless otherwise specified, the polypeptide may include one or more optional linkers between each of the listed elements, such linkers may be 1-30, 6-29, 7-28, 8-27, 9-26, 10-25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acid lengths.

[0024] In another embodiment, the present disclosure provides compositions comprising activatable proteins and carriers as herein. In some embodiments, the composition is a pharmaceutical composition, and the carrier is a pharmaceutically acceptable carrier.

[0025] In another embodiment, the Disclosure provides a container, vial, syringe, injector pen, or kit containing at least one dose of the composition of this Specified.

[0026] In another aspect, the Disclosure provides a nucleic acid comprising a sequence encoding a second polypeptide of this Specified.

[0027] In another aspect, the Disclosure provides a vector comprising nucleic acids as defined herein.

[0028] In another aspect, the present disclosure provides cells comprising nucleic acids or vectors as herein.

[0029] In another embodiment, the present disclosure provides conjugated activatable proteins, including activatable proteins of this specification conjugated to a drug. In some embodiments, the drug is a therapeutic agent, an antitumor agent, a toxin, a diagnostic agent, a therapeutic polymer, a targeting moiety, or a detectable moiety. In some embodiments, the drug is conjugated to an antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0030] In another embodiment, the present disclosure provides a method for treating a subject in need of treatment, comprising administering a therapeutically effective amount of an activatable protein, composition, or conjugated activatable protein as herein to the subject. In some embodiments, the subject is identified or diagnosed with cancer.

[0031] In another embodiment, the Disclosure provides a method for producing an activatable protein, comprising culturing cells of the Specified in a culture medium under conditions sufficient to produce an activatable protein, and recovering the activatable protein from the cells or culture medium. In some embodiments, the method further comprises isolating the activatable protein recovered from the cells or culture medium. In some embodiments, the isolation of the activatable protein is performed using protein purification tag and / or size exclusion chromatography. In some embodiments, the method further comprises incorporating the activatable protein into a pharmaceutical composition.

[0032] An understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention may be utilized, and to the accompanying drawings. [Brief explanation of the drawing]

[0033] [Figure 1]An exemplary configuration of an activatable molecule is shown. The molecule is designed so that the activating molecule resulting from the activation of the activatable molecule does not contain a half-life extension portion, and therefore has a shorter half-life than the corresponding molecule which is identical to the activating molecule but contains a half-life extension portion. [Figure 2] An exemplary configuration of an activatable molecule is shown. The molecule is designed so that the activating molecule resulting from the activation of the activatable molecule does not contain a half-life extension portion, and therefore has a shorter half-life than the corresponding molecule which is identical to the activating molecule but contains a half-life extension portion. [Figure 3] An exemplary configuration of an activatable molecule is shown. The molecule is designed so that the activating molecule resulting from the activation of the activatable molecule does not contain a half-life extension portion, and therefore has a shorter half-life than the corresponding molecule which is identical to the activating molecule but contains a half-life extension portion. [Figure 4] An exemplary configuration of an activatable molecule is shown. The molecule is designed so that the activating molecule resulting from the activation of the activatable molecule does not contain a half-life extension portion, and therefore has a shorter half-life than the corresponding molecule which is identical to the activating molecule but contains a half-life extension portion. [Figure 5] This is a schematic diagram of exemplary activatable (double-masked) bispecific antibodies according to some embodiments of the present disclosure, before and after activation by protease. On the left, the activatable double-masked protein is schematically illustrated. The dashed lines between elements 501 and 505, and between elements 502 and 503, indicate cleavable regions. On the right, the activated protein is schematically illustrated. Because the activated bispecific antibody does not contain a masking region, it has increased binding affinity to its target compared to the activatable bispecific antibody. The activated bispecific antibody also does not contain a half-life extension region and therefore has a shorter half-life than the activatable bispecific antibody. [Figure 6A]This is a schematic diagram of the components of an exemplary double-masked, bispecific, activatable antibody having a masked Fab fragment that recognizes Her2, a masked scFV component that recognizes CD3, and a half-life extension portion containing a knob-and-hole Fc domain pair. [Figure 6B] Figure 6A is a schematic diagram showing the components of three polypeptides encoding an exemplary double-masked, bispecific, activatable antibody. Dashed lines indicate cleavable regions. [Figure 7A] These are images of SDS-PAGE gels run under reducing conditions. The gels were loaded as follows: (1) a bispecific activatable antibody (ProC1446, SEQ ID NO: 21) double-masked with a 20GG CD3 mask, (2) the product of ProC1446 and uPA (ProC1446+uPA), (3) a bispecific activatable antibody (ProC1447, SEQ ID NO: 22) double-masked with an MN15a CD3 mask, (4) the product of ProC1447 and uPA (ProC1447+uPA), (5) a bispecific activatable antibody (ProC1448, SEQ ID NO: 23) double-masked with an MN15b CD3 mask, and (6) the product of ProC1448 and uPA (ProC1448+uPA). [Figure 7B] This table summarizes the expected molecular weights of the components of each activatable antibody construct before and after protease activation. [Figure 8]This paper provides results from ELISA binding assays to determine the ability of activatable and activating molecules to bind to plate-bound CD3 antigens: an unmasked reference bispecific molecule (ProC531), an activatable bispecific molecule double-masked with a 20GG CD3 mask (ProC1446, SEQ ID NO: 21), a product of ProC1446 and uPA (ProC1446+uPA), a molecule double-masked with an MN15a mask (ProC1447, SEQ ID NO: 22), a product of ProC1447 and uPA (ProC1447+uPA), a molecule double-masked with an MN15b mask (ProC1448, SEQ ID NO: 23), and a product of ProC1448 and uPA (ProC1448+uPA). These results indicate that each CD3 mask between the anti-CD3 scFv and the Fc region attenuated the binding of anti-CD3 scFv to the plate-coated CD3 antigen. Treatment of double-masked, activatable bispecific antibodies with the protease uPA resulted in CD3 antigen binding equivalent to that of the unmasked reference bispecific molecule ProC531. [Figure 9A] This provides the results of a HER2-dependent cytotoxicity assay to determine the in vitro potency of a double-masked, activatable, bispecific antibody (Figure 9A: ProC1446). These results demonstrate that the protease-treated (activated) bispecific molecule of this disclosure was more active than a monovalent, unmasked bispecific antibody control ("ProC306") which has the same HER2 and CD3 binding domains but is arranged in a different configuration. [Figure 9B] This provides the results of a HER2-dependent cytotoxicity assay to determine the in vitro potency of a double-masked, activatable, bispecific antibody (Figure 9B: ProC1447). These results demonstrate that the protease-treated (activated) bispecific molecule of this disclosure was more active than a monovalent, unmasked bispecific antibody control ("ProC306") which has the same HER2 and CD3 binding domains but is arranged in a different configuration. [Figure 9C]This provides the results of a HER2-dependent cytotoxicity assay to determine the in vitro potency of a double-masked, activatable, bispecific antibody (Figure 9C: ProC1448). The results show that the protease-treated (activated) bispecific molecule of this disclosure was more active than a monovalent, unmasked bispecific antibody control ("ProC306") which has the same HER2 and CD3 binding domains but is arranged in a different configuration. [Figure 10] An exemplary configuration of an activatable molecule is shown. This molecule contains a double-masked, activatable, bispecific antibody having an EM coupled to the C-terminus via a third cleavable moiety. The molecule is designed so that the activating molecule resulting from the activation of the activatable molecule does not contain a half-life extension moiety and therefore has a shorter half-life than the corresponding molecule which is identical to the activating molecule but contains a half-life extension moiety. [Figure 11] An exemplary configuration of an activatable molecule is shown. This molecule contains a double-masked, activatable, bispecific antibody having an EM coupled to the C-terminus via a third cleavable moiety. The molecule is designed so that the activating molecule resulting from the activation of the activatable molecule does not contain a half-life extension moiety and therefore has a shorter half-life than the corresponding molecule which is identical to the activating molecule but contains a half-life extension moiety. [Figure 12] This is a schematic diagram of exemplary activatable (double-masked) bispecific antibodies according to some embodiments of the present disclosure, before and after activation by protease. The left side schematically illustrates the activatable double-masked protein. The right side schematically illustrates the activated protein. Because the activated bispecific antibody does not contain a masking moiety, it has increased binding affinity to its target compared to the activatable bispecific antibody. The activated bispecific antibody also does not contain a half-life extension moiety and therefore has a shorter half-life than the activatable bispecific antibody. Dashed lines indicate cleavable moieties. [Figure 13A]This provides the results of the binding (i.e., HER2 binding) of masked, activatable, short-half-life antibodies ProC1446(SHL1), ProC3007(SHL2), ProC3008(SHL2), and masked antibodies ProC1441(1 / 2TCB, non-activatable, short-half-life antibody), and unmasked (ProC1963(SHL1, unmasked, no Fc), ProC1965(SHL2, unmasked, no Fc), and ProC306) anti-CD3, anti-HER2 bispecific antibodies, as well as secondary antibodies ("Sec only", negative control) to NCI-N87 and SKOV3 cells, respectively. [Figure 13B] This provides the results of the binding (i.e., HER2 binding) of masked, activatable, short-half-life antibodies ProC1446(SHL1), ProC3007(SHL2), ProC3008(SHL2), and masked antibodies ProC1441(1 / 2TCB, non-activatable, short-half-life antibody), and unmasked (ProC1963(SHL1, unmasked, no Fc), ProC1965(SHL2, unmasked, no Fc), and ProC306) anti-CD3, anti-HER2 bispecific antibodies, as well as secondary antibodies ("Sec only", negative control) to NCI-N87 and SKOV3 cells, respectively. [Figure 13C] This provides the result of binding the same molecule to Jurkat cells (i.e., CD3 binding). [Figure 14A] Using NCI-N87 cells (Figure 14A), we provide the results of cytotoxicity assays demonstrating the dose-response of ProC1963(SHL1), ProC1965(SHL2), and ProC306 at the indicated concentrations. [Figure 14B] Using SKOV3 cells (Figure 14B), we provide the results of cytotoxicity assays demonstrating the dose-response relationship between ProC1963 (SHL1), ProC1965 (SHL2), and ProC306 at the indicated concentrations. [Figure 15A] Using NCI-N87 cells (Figure 15A), we provide the results of cytotoxicity assays demonstrating the dose-response of ProC1963, ProC1965, ProC1446, ProC3007, and ProC3008 at the indicated concentrations. [Figure 15B] Using SKOV3 cells (Figure 15B), we provide the results of cytotoxicity assays demonstrating the dose-response of ProC1963, ProC1965, ProC1446, ProC3007, and ProC3008 at the indicated concentrations. [Figure 16A] The results of cytotoxicity assays are provided. A shows the dose-response of ProC1963, ProC1965, ProC3007, ProC3008, ProC306, and ProC1441 using NCI-N87 cells (Figure 16A). [Figure 16B] The results of cytotoxicity assays are provided. Figure B shows the dose-response of ProC1963, ProC1965, ProC3007, ProC3008, ProC306, and ProC1441 using SKOV3 cells (Figure 16B). [Figure 16C] The results of cytotoxicity assays are provided. C shows the dose-response of ProC1963, ProC1965, ProC1446, ProC306, and ProC1441 using NCI-N87 cells (Figure 16C). [Figure 16D] The results of cytotoxicity assays are provided. Figure D shows the dose-response of ProC1963, ProC1965, ProC1446, ProC306, and ProC1441 using SKOV3 cells (Figure 16D). [Figure 17] This report provides results from an in vivo tumor growth assay using the NCI-N87 xenograft model. The plots show tumor volume as a function of days after initial treatment with ProC1965, ProC3007, ProC3008, and ProC1441 administered at doses indicated in milligrams / kilogram (mpk). [Modes for carrying out the invention]

[0034] The figures provided herein are for illustrative purposes only and are not necessarily drawn to a specific scale.

[0035] This specification provides activatable molecules having a structure with relatively low binding activity and a half-life extension moiety (EM) (e.g., activatable antibodies and activatable proteins such as other activatable therapeutic or diagnostic proteins). When activated by exposure to specific activation conditions (e.g., when the activatable molecule is delivered to a tumor), the resulting activatable molecule has higher binding activity and a shorter half-life compared to the activatable molecule. In one embodiment, the activatable molecule may be an activatable therapeutic polymer. In some embodiments, the activatable therapeutic polymer may be an activatable antibody or any other desired protein, e.g., a therapeutic protein.

[0036] Generally, the activatable molecules described herein may comprise one or more target-binding domains (TBs), one or more masking moieties (MMs) that reduce, inhibit, or interfere with the binding of the TBs to their targets, one or more cleavable moieties (CMs) that couple one or more MMs to one or more TBs, and one or more half-life-extending moieties (EMs) coupled to the TBs via one or more CMs. The coupling of two components within a polypeptide may be direct or indirect. When two components are directly coupled, the C-terminal amino acid residue of one component forms a peptide bond with the N-terminal amino acid residue of the other component. When two components are indirectly coupled, a sequence of amino acids exists between the two components. In some examples, two components of a polypeptide may be indirectly coupled via one or more other components within the polypeptide; that is, one or more other components lie between the two coupled components. When indirect coupling or linking occurs via another component, one or more other components may be a linker, TBs (plural) (e.g., ABs (plural)), CMs (plural), MMs (plural), or any combination thereof.

[0037] CM is a polypeptide containing a substrate for a sequence-specific protease, such as a protease that is present in higher amounts (or in a higher amount in an active state) in the environment of diseased tissue, such as a tumor, than in healthy tissue. The activatable molecules MM and EM described herein can be released from TB by cleaving CM to create an activated molecule. The activated molecule exhibits a higher binding affinity to its target compared to the corresponding activatable molecule containing MM(or more). In some embodiments, the activated molecule has a shorter half-life compared to the corresponding molecule which is the same as the activated molecule but contains EM. The activated molecule may have reduced toxicity and reduced off-target effects compared to the corresponding molecule which is the same as the activated molecule but contains EM.

[0038] In some embodiments, the activatable molecule may be a double-masked, bispecific target-binding molecule. In some embodiments, such a molecule may comprise at least two target-binding proteins and at least two masking moieties, each of which inhibits the binding of the target-binding protein to its target. For example, the activatable molecule may comprise a first target-binding protein (TB1) that specifically binds to a first target, a first masking moiety (MM1) that inhibits the binding of TB1 to the first target, a cleavable moiety (CM1) positioned between MM1 and TB1, a second target-binding protein (TB2) that specifically binds to a second target, a second masking moiety (MM2) that inhibits the binding of TB2 to the second target, a cleavable moiety (CM2) positioned between MM2 and TB2, and an EM coupled to TB1 or TB2 via the cleavable moieties (CM). In some embodiments, the EM may be coupled to TB via a CM that also binds MM to TB. In some embodiments, the EM may be coupled to TB via a CM different from CM1 and CM2 (e.g., a third CM or "CM3"). In the activated state, EM can be released from the activating molecule. Therefore, the activating molecule (containing TB1 and TB2 but not MM1, MM2, or EM) has a shorter half-life compared to a reference molecule containing TB1, TB2, and EM but not MM1 or MM2. The activating molecule (containing TB1 and TB2 but not MM1, MM2, and EM) has higher target binding activity compared to a reference molecule containing TB1, TB2, and EM but not MM1 or MM2.

[0039] This specification also provides related methods, including methods for using and preparing any of the related compositions, kits, nucleic acids and recombinant cells, as well as any of the activatable molecules described herein (e.g., activatable antibodies and other proteins).

[0040] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. This specification describes methods and materials for use in this disclosure. Other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to limit. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification shall prevail, including definitions.

[0041] The terms "a" and "an" refer to one or more (i.e., at least one) grammatical objects of the article. For example, "a cell" includes one or more cells.

[0042] As used herein, the terms “about” and “approximately” are used to modify quantities expressed numerically or within a range, and indicate not only the numerical value but also a reasonable deviation from that value, as known to those skilled in the art. For example, where appropriate, ±20%, ±10%, or ±5% are within the range of meaning intended for the detailed value.

[0043] Concentration, quantity, and other numerical data may be expressed or indicated in range form as specified herein. Such range form is used solely for convenience and simplicity, and should therefore be interpreted flexibly to include not only the numerical values ​​explicitly listed as limits to the range, but also all individual numerical values ​​or partial ranges contained within that range, as if each numerical value and partial range were explicitly listed. For example, the numerical range "approximately 0.01 to 2.0" should be interpreted to include not only the explicitly listed values ​​of approximately 0.01 to approximately 2.0, but also the individual numerical values ​​and partial ranges within the indicated range. Therefore, this numerical range includes individual values, e.g., 0.5, 0.7, and 1.5, as well as partial ranges, e.g., 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5. Furthermore, such interpretation should apply regardless of the width or characteristics of the range described. In addition, note that all percentages are calculated on a weight basis unless otherwise specified.

[0044] For understanding the scope of this disclosure, the terms “including” or “comprising” and their derivatives are intended, as used herein, to be open-ended terms that specify the existence of described features, elements, components, groups, integers and / or processes, but do not exclude the existence of other undescribed features, elements, components, groups, integers and / or processes. The foregoing also applies to words with similar meanings, such as the terms “including” and “having” and their derivatives. The terms “comprising” and their derivatives are intended, as used herein, to be closed-ended terms that specify the existence of described features, elements, components, groups, integers and / or processes, but exclude the existence of other undescribed features, elements, components, groups, integers and / or processes. As used herein, the term “consisting essentially of” is intended to define the presence of any described feature, element, component, group, integer and / or process, and the presence of any feature, element, component, group, integer and / or process that does not substantially affect the basic and novel features(s) of the feature, element, component, group, integer and / or process. Any reference to any one of these transitional clauses (i.e., “comprising,” “consisting,” or “consisting essentially”) is understood to provide direct support for any substitution of the other transitional clauses, which are not specifically used. For example, a modification of the term “comprising” to “consisting essentially of” or “consisting of” would find direct support for any element disclosed through this disclosure to be defined in this way. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0045] For the purposes of this specification, multiple compounds, elements, or processes may be presented in a general list for convenience. However, these lists should be interpreted as if each member of the list were individually identified as a distinct and unique member. Therefore, individual members of such lists should not be interpreted as being substantially equivalent to any other member of the same list based solely on their presentation in a general group, unless otherwise indicated.

[0046] In this specification, the term “exemplary” is used to mean an example, illustration, or explanatory element. Any aspect or design described herein as “exemplary” should not necessarily be construed as being preferable or advantageous to other aspects or designs. Rather, the use of the word “exemplary” is intended to present the concept in a more concrete way.

[0047] Furthermore, specific molecules, constructs, compositions, elements, parts, excipients, diseases, conditions, properties, steps, etc., may be discussed in the context of a particular embodiment or aspect of this disclosure, or in a separate paragraph or section. This is merely for convenience and brevity, and any such disclosure is equally applicable to and intended to be combined with any other embodiment or aspect found anywhere in this disclosure and the claims, and it is understood that all of these together constitute the invention claimed in this application and the claims as of the filing date. For example, any list of constructs, molecules, method steps, kits, or compositions described in relation to a construct, composition, or method is intended to find direct support for embodiments relating to constructs, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, effective agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.

[0048] Activatable molecules In one embodiment, the activatable molecule provided herein may be an activatable target-binding protein (TB), e.g., an activatable antibody, or another protein that specifically binds to a target. In some embodiments, the activatable molecule includes: a TB that specifically binds to a target (e.g., an antigen-binding protein (AB)); a cleavable moiety (CM) that is directly covalently bonded to the TB (e.g., AB) (also called "directly coupled") or indirectly covalently bonded to the TB (also called "indirectly coupled"), wherein the CM is located between the TB and a masking moiety (MM) that reduces, inhibits, or interferes with the binding of the TB (e.g., AB) to its target(s); and one or more half-life extension moieties (EMs) that are coupled to the TB (e.g., AB) via one or more CMs. The MMs and EMs may be released from the TB by cleaving the CMs to generate an activating molecule. In some embodiments, the activatable molecule may include: a first antigen-binding protein (AB1) that specifically binds to a first target; a first masking moiety (MM1) that inhibits the binding of AB1 to the first target and is coupled to AB1 via a first cleavable moiety (CM1); a second antigen-binding protein (AB2) that specifically binds to a second target (AB2); a second masking moiety (MM2) that inhibits the binding of AB2 to the second target and is coupled to either AB1 or either AB2 via a second cleavable moiety (CM2); and an EM coupled to either AB1 or either AB2 via a cleavable moiety. In some embodiments, the EM may be coupled to AB1 via the same cleavable moiety (CM1) that couples MM1 to AB1. In some embodiments, the EM may be coupled to AB2 via the same cleavable moiety (CM2) that couples MM2 to AB2. In some embodiments, the EM may be coupled to AB1 via the same cleavable moiety (CM2) that couples MM2 to AB1. In some embodiments, EM can be coupled to AB1 or AB2 via a third severable portion (CM3).In each of the embodiments described above, the elements of the activatable molecule may be directly coupled or indirectly coupled via one or more arbitrary linkers between the elements. In the activated state, EM may be released from the activatable protein, resulting in an activated protein containing AB1 and AB2 but not MM1, MM2, or EM, the activated protein having a shorter half-life compared to a reference antibody containing AB1, AB2, and EM but not MM1 or MM2.

[0049] In some embodiments, the activatable protein provides a reduction in toxicity and / or off-target side effects that may result from the binding of TB (e.g., AB) at non-therapeutic sites, if TB does not mask or otherwise inhibit its binding to its target. In its activatable state, MM can interfere with the binding of TB to its target molecule.

[0050] In some embodiments, the activatable protein includes: a first antigen-binding protein (AB1) that specifically binds to a first target, wherein AB1 comprises an antibody or fragment thereof comprising a heavy chain fragment and a light chain fragment; a second antigen-binding protein (AB2) that specifically binds to a second target, wherein AB2 comprises a single-chain fragment variable (scFv) comprising a heavy chain variable region and a light chain variable region, and AB2 is coupled to the C-terminus of the heavy chain fragment or light chain fragment of AB1; a first masking moiety (MM1) coupled to AB1 via a first cleavable moiety (CM1) that inhibits the binding of AB1 to the first target when the activatable protein is in an uncleaved state; a second masking moiety (MM2) coupled to AB2 that inhibits the binding of AB2 to the second target when the activatable protein is in an uncleaved state; and a half-life extension moiety (EM) coupled to a component of AB1 or AB2 via a second cleavable moiety (CM2). In some examples, AB1 may be Fab. In some examples, AB1 may be scFv. In some embodiments, EM is optionally coupled to AB1 or AB2 via one or more linkers between one or more components by a masking moiety, e.g., an EM-MM-CM-AB or AB-CM-MM-EM structure. As used herein, the symbol "-" in a structural formula indicates direct or indirect coupling between two components (e.g., any linker may be present between the components). The structural arrangements of the molecules of this disclosure are described in detail below and shown, for example, in Figures 1 to 6.

[0051] As used herein, the terms “activatable protein” and “activatable target-binding protein” (e.g., “activatable antibody”), as well as any of the foregoing terms, including “intact,” “uncleaved,” and / or “inactive,” are used interchangeably to refer to a protein comprising at least one set of MM, CM, and TB, which exhibits reduced binding to a biological target compared to the binding of a corresponding “activated” protein (e.g., an activated antibody) containing the same TB to the same biological target. It will be apparent to those skilled in the art that exposure of an activatable protein to a CM-specific protease may produce an “activated” protein in which the MM does not reduce, inhibit, or interfere with the binding between the TB (e.g., AB) and its target. In some embodiments, cleavage of CM by a suitable protease may result in the release of MM. In some embodiments, cleavage of CM by a suitable protease may result in the release of EM. The terms “activated protein,” “activated target-binding protein” (e.g., “activated antibody”), “cleavable activatable protein,” and “cleavable activatable target-binding protein” (e.g., “cleavable activatable antibody”) are interchangeable in this specification to refer to TB-containing cleavage products generated after exposure of an activatable protein to a CM-specific protease. Throughout this disclosure, descriptions relating to activatable antibodies shall be interpreted as also applying to activatable target-binding proteins.

[0052] As used herein, the terms “masking moiety” and “MM” are used interchangeably to refer to a peptide or protein that, when positioned proximal to TB (e.g., AB), interferes with the binding of TB to a biological target. The terms “cleavable moiety” and “CM” are used interchangeably herein and mean a peptide containing a substrate for a sequence-specific protease. In an activatable protein, the CM is positioned relative to the MM and TB, resulting in cleavage yielding a molecule that can bind to the biological target of TB. Thus, an activatable protein exhibits reduced binding to a biological target compared to an activated protein. In some embodiments, an activatable protein may be designed so that the MM provides masking of TB or reduced binding of TB to its target by selecting a TB of interest and constructing the rest of the activatable protein. Structural design criteria may be considered to provide this functional feature.

[0053] An activatable protein can be a multispecific (e.g., bispecific, triplicate, quadruplicate, and other multispecific activatable proteins) that can bind to multiple distinct antigens when activated. In some embodiments, a multispecific activatable protein can be polyvalent, for example, containing multiple target binding sites, regardless of whether the binding sites recognize the same or different antigens or epitopes. In some embodiments, an activatable protein can be monospecific, for example, that can bind to only one antigen when activated.

[0054] In some embodiments, the activatable protein is bispecific. The term "bispecific" means that, when activated, the activatable protein can specifically bind to two different targets. Typically, a bispecific activatable protein comprises two TBs, namely a first TB and a second TB, each of which can specifically bind to different targets (i.e., a first target and a second target, respectively) after activation. In some embodiments, after activation, the resulting bispecific target-binding molecule may be capable of simultaneously binding to two targets, for example, two target proteins expressed on two separate cells.

[0055] In some embodiments, the activatable protein may include AB1, which can bind to molecules on the surface of disease-associated cells (e.g., tumor cells), and AB2, which can bind to molecules on the surface of immune cells. When activated, such a bispecific activatable protein can simultaneously bind to immune cells and disease-associated cells (e.g., tumor cells), thereby activating the immune cells and cross-linking the activated immune cells to the disease-associated cells. In some embodiments, the activatable protein may be incorporated as part of a pro-bispecific T cell engager (pro-BiTE) molecule, a prochimeric antigen receptor (pro-CAR) modified T cell, or other engineered receptor or other immune effector cells, such as CAR modified NK cells.

[0056] In some cases, the activatable protein may be an activatable T cell-associated bispecific antibody (TCB) or a fragment thereof. For example, the activatable protein may include AB1, which targets disease-associated cells, and AB2, which targets T cell receptors.

[0057] This disclosure includes activatable proteins in various structural configurations described herein. Exemplary configurations of activatable proteins are provided below. The N-terminus-C-terminus order of TB, MM, CM, and EM can be reversed within the activatable protein. CM and MM may have overlapping amino acid sequences; for example, the CM sequence recognized by a sequence-specific protease may be at least partially contained within MM. Various structural configurations of activatable antigen-binding proteins are envisioned, for example, in which AB1 is the antigen-binding fragment (Fab) and AB2 is a single-chain fragment variable, and can be represented by the following formula (in order from the amino (N)-terminal region to the carboxyl (C)-terminal region). In the following formulas, ":" separates two different polypeptides, where "Fab_L" and "Fab_H" are the light chain and heavy chain fragments of Fab, respectively ("Fab_L" includes the variable light chain region (VL) and the light chain constant region, and "Fab_H" includes the variable heavy chain region (VH) and the CH1 region), and "VL*" and "VH*" are the light chain and heavy chain variable regions of scFv. Furthermore, as used herein and unless otherwise specified, each dash (-) between components of an activatable molecule represents either a direct bond or an indirect bond via one or more linkers. (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-MM2-EM) (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM-MM2) Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-MM2-EM)Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM-MM2)(MM1-CM1-Fab_L-VH*-VL *-CM2-MM2-EM):Fab_H(MM1-CM1-Fab_L-VH*-VL*-CM2-EM-MM2):Fab_H(Fab_L-VH*-VL*-CM2-MM2-EM):(MM1-CM1-Fab_H) (Fab_L-VH*-VL*-CM2-EM-MM2):(MM1-CM1-Fab_H) (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-MM2-EM) (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM-MM2) (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-MM2-EM) (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM-MM2) (MM1-CM1-Fab_L-CM2-MM2-EM):(Fab_H-VH*-VL*) (MM1-CM1-Fab_L-CM2-EM-MM2):(Fab_H-VH*-VL*) (Fab_L-CM2-MM2-EM):(MM1-CM1-Fab_H-VH*-VL*) (Fab_L-CM2-EM-MM2):(MM1-CM1-Fab_H-VH*-VL*) (MM1-CM1-Fab_L):(MM2-CM2-VH*-VL*-Fab_H-CM3-EM) (MM1-CM1-Fab_H):(MM2-CM2-VH*-VL*-Fab_L-CM3-EM) (MM2-CM2-VH*-VL*-Fab_L):(MM1-CM1-Fab_H-CM3-EM) (MM2-CM2-VH*-VL*-Fab_H):(MM1-CM1-Fab_L-CM3-EM)

[0058] In any configuration, an activatable protein may contain one or more linkers between any two of its components. For example, an activatable protein may contain a linker between MM1 and CM1, a linker between CM1 and Fab_L, a linker between Fab_H and VH*, a linker between VH* and VL*, a linker between VL* and CM2, a linker between CM2 and MM2, a linker between MM2 and EM, a linker between Fab_L and VH*, a linker between CM1 and Fab_H, or any combination thereof.

[0059] In some embodiments, EM may comprise two or more moieties (e.g., a pair of Fc domains). For example, EM may be a protein complex comprising two moieties, EM1 and EM2. In such cases, an example of such an activatable protein may be represented by the following formula (from the amino (N)-terminal region to the carboxyl (C)-terminal region): (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-MM2-EM1):EM2 (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM1-MM2):EM2 Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-MM2-EM1):EM2 Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM1-MM2):EM2 (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM1):(MM2-EM2) (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM1):(EM2-MM2) Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM1):(MM2-EM2) Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM1):(EM2-MM2) (MM1-CM1-Fab_L-VH*-VL*-CM2-MM2-EM1):Fab_H:EM2 (MM1-CM1-Fab_L-VH*-VL*-CM2-EM1-MM2):Fab_H:EM2 (Fab_L-VH*-VL*-CM2-MM2-EM1):(MM1-CM1-Fab_H):EM2 (Fab_L-VH*-VL*-CM2-EM1-MM2):(MM1-CM1-Fab_H):EM2 (MM1-CM1-Fab_L-VH*-VL*-CM2-EM1):Fab_H:MM2-EM2 (MM1-CM1-Fab_L-VH*-VL*-CM2-EM1):Fab_H:EM2-MM2 (Fab_L-VH*-VL*-CM2-EM1):(MM1-CM1-Fab_H):MM2-EM2 (Fab_L-VH*-VL*-CM2-EM1):(MM1-CM1-Fab_H):EM2-MM2 (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-MM2-EM1):EM2 (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM1-MM2):EM2 (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-MM2-EM1):EM2 (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM1-MM2):EM2 (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM1):MM2-EM2 (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM1):EM2-MM2 (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM1):MM2-EM2 (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM1):EM2-MM2 (MM1-CM1-Fab_L-CM2-MM2-EM1):(Fab_H-VH*-VL*):EM2 (MM1-CM1-Fab_L-CM2-EM1-MM2):(Fab_H-VH*-VL*):EM2 (Fab_L-CM2-MM2-EM1):(MM1-CM1-Fab_H-VH*-VL*):EM2 (Fab_L-CM2-EM1-MM2):(MM1-CM1-Fab_H-VH*-VL*):EM2 (MM1-CM1-Fab_L-CM2-EM1):(Fab_H-VH*-VL*):MM2-EM2 (MM1-CM1-Fab_L-CM2-EM1):(Fab_H-VH*-VL*):EM2-MM2 (Fab_L-CM2-EM1):(MM1-CM1-Fab_H-VH*-VL*):MM2-EM2 (Fab_L-CM2-EM1):(MM1-CM1-Fab_H-VH*-VL*):EM2-MM2 (MM1-CM1-Fab_L):(MM2-CM2-VH*-VL*-Fab_H-CM3-EM1):EM2 (MM1-CM1-Fab_H):(MM2-CM2-VH*-VL*-Fab_L-CM3-EM1):EM2 (MM2-CM2-VH*-VL*-Fab_L):(MM1-CM1-Fab_H-CM3-EM1):EM2 (MM2-CM2-VH*-VL*-Fab_H):(MM1-CM1-Fab_L-CM3-EM):EM2

[0060] In some embodiments, EM1 and EM2 may be two fragment crystallizable (Fc) domains. The two Fc domains may form a dimer as a half-life extension portion. In some examples, EM1 and EM2 may be two identical Fc domains and thus may form a homodimer. In some constructs, EM1 and EM2 contain Fc domains having two different amino acid sequences that together form a heterodimer. In some examples, the two Fc domains may be a whole variant of the Fc domain and a knob variant of the Fc domain and may form a heterodimer. In any of these configurations, the activatable protein may contain one or more linkers between any two of the components. For example, activatable proteins may include linkers between MM1 and CM1, between CM1 and Fab_L, between Fab_H and VH*, between VH* and VL*, between VL* and CM2, between CM2 and MM2, between MM2 and EM, between Fab_L and VH*, between CM1 and Fab_H, between CM2 and EM1, between MM2 and EM1, between MM2 and EM2, or any combination thereof.

[0061] Figures 1-4 show exemplary configurations of activatable molecules disclosed herein. In these non-limiting examples, the activatable molecule includes AB1, which may be Fab; AB2, which may be scFv; EM, a dimer formed by two Fc domains; MM1, which can be coupled to AB1 via CM1 and can interfere with the binding of AB1 and its target; MM2, which can interfere with the binding of AB2 and its target; and CM2, between the Fc domain of EM and AB1 or AB2. It will be understood that the activatable molecular structures exemplified in Figures 1-4 are similarly applicable to molecules where AB1 and AB2 are antigen-binding proteins other than Fab and scFv. Similarly, the activatable molecular structures exemplified in Figures 1-4 can be similarly applied to molecules where AB1 and AB2 are replaced by TB1 and TB2, which may be target-binding proteins that do not necessarily contain antigen-binding domains, respectively.

[0062] Figure 1 shows an exemplary activatable protein 100 containing three polypeptides. The first polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, MM1 101, an optional linker 102, CM1 103, an optional linker 104, and a light chain fragment 105 of AB1. The second polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, a heavy chain fragment 121 of AB1, a linker 122, a heavy chain variable region 123 of AB2, a linker 124, a light chain variable region 125 of AB2, a linker 126, CM2 127, an optional linker 128, MM2 129, a linker 130, and the first Fc domain 131 of EM. The third polypeptide contains the second Fc domain 141 of EM. In the alternative exemplary configuration, in Figure 1, 105 is the heavy chain fragment of AB1 and 121 is the light chain fragment of AB1. In the alternative exemplary configuration, in Figure 1, 123 is the light chain variable region of AB2 and 125 is the heavy chain variable region of AB2. In the alternative exemplary configuration, in Figure 1, the first Fc domain 131 of EM is a hole variant of the Fc domain and the second Fc domain 141 of EM is a knob variant of the Fc domain. In the alternative exemplary configuration, in Figure 1, the first Fc domain 131 of EM is a knob variant of the Fc domain and the second Fc domain 141 of EM is a hole variant of the Fc domain.

[0063] Figure 2 shows another exemplary activatable protein 200 containing three polypeptides. The first polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, MM1 201, an optional linker 202, CM1 203, an optional linker 204, the light chain fragment of AB1 205, linker 206, the heavy chain variable region of AB2 207, linker 208, and the light chain variable region of AB2 209. The second polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, the heavy chain fragment of AB1 221, an optional linker 222, CM2 223, an optional linker 224, MM2 225, linker 226, and the first Fc domain of EM 227. The third polypeptide contains the second Fc domain of EM 241. In the alternative exemplary configuration, in Figure 2, 205 is the heavy chain fragment of AB1 and 221 is the light chain fragment of AB1. In the alternative exemplary configuration, in Figure 2, 207 is the light chain variable region of AB2 and 209 is the heavy chain variable region of AB2. In the alternative exemplary configuration, in Figure 2, the first Fc domain 227 of EM is a hole variant of the Fc domain and the second Fc domain 241 of EM is a knob variant of the Fc domain. In the alternative exemplary configuration, in Figure 2, the first Fc domain 227 of EM is a knob variant of the Fc domain and the second Fc domain 241 of EM is a hole variant of the Fc domain.

[0064] Figure 3 shows another exemplary activatable protein 300 containing three polypeptides. The first polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, MM1 301, an optional linker 302, CM1 303, an optional linker 304, a light chain fragment of AB1 305, a linker 306, a heavy chain variable region of AB2 307, a linker 308, and a light chain variable region of AB2 309. The second polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, a heavy chain fragment of AB1 321, an optional linker 322, CM2 323, an optional linker 324, and the first Fc domain of EM 325. The third polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, a second Fc domain of EM 341, a linker 342, and MM2 343. In the alternative exemplary configuration, in Figure 3, 305 is the heavy chain fragment of AB1 and 321 is the light chain fragment of AB1. In the alternative exemplary configuration, in Figure 3, 307 is the light chain variable region of AB2 and 309 is the heavy chain variable region of AB2. In the alternative exemplary configuration, in Figure 3, the first Fc domain 325 of EM is a hole variant of the Fc domain and the second Fc domain 341 of EM is a knob variant of the Fc domain. In the alternative exemplary configuration, in Figure 3, the first Fc domain 325 of EM is a knob variant of the Fc domain and the second Fc domain 341 of EM is a hole variant of the Fc domain.

[0065] Figure 4 shows another exemplary activatable protein 400 containing three polypeptides. The first polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, MM1 401, an optional linker 402, CM1 403, an optional linker 404, a light chain fragment of AB1 405, a linker 406, a heavy chain variable region of AB2 407, a linker 408, and a light chain variable region of AB2 409. The second polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, a heavy chain fragment of AB1 421, an optional linker 422, CM2 423, an optional linker 424, and the first Fc domain of EM 425. The third polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, MM2 441, a linker 442, and the second Fc domain of EM 443. In the alternative exemplary configuration, in Figure 4, 405 is the heavy chain fragment of AB1 and 421 is the light chain fragment of AB1. In the alternative exemplary configuration, in Figure 4, 407 is the light chain variable region of AB2 and 409 is the heavy chain variable region of AB2. In the alternative exemplary configuration, in Figure 4, the first Fc domain 425 of EM is a hole variant of the Fc domain and the second Fc domain 443 of EM is a knob variant of the Fc domain. In the alternative exemplary configuration, in Figure 4, the first Fc domain 425 of EM is a knob variant of the Fc domain and the second Fc domain 443 of EM is a hole variant of the Fc domain.

[0066] Figure 5 shows an exemplary activatable bispecific antibody comprising a Fab component (501) that binds to a first target; a first prodomain (505) containing CM1 and MM1 that masks the Fab component; an scFv component (502) that binds to a second target; a second prodomain (503) containing CM2 and MM2 that masks the scFv component; and an EM (504) containing a knob-and-hole Fc domain pair. Upon activation, CM1 is cleaved from the activated bispecific antibody to release MM1, and CM2 is cleaved to release both MM2 and the Fc domain (504). Activated bispecific antibodies lacking the Fc domain have a relatively shorter half-life compared to their parent activatable bispecific antibodies.

[0067] Figure 6A shows an exemplary activatable bispecific antibody targeting Her2 and CD3. In this example, the activatable bispecific antibody comprises three polypeptides. The first polypeptide comprises, from N-terminus to C-terminus, the heavy chain fragment of trastuzumab Fab (anti-HER2 antibody); a linker with 25 amino acids; anti-CD3 scFv; a GSAT linker with 27 amino acids; CM1; MM1 masking anti-CD3 scFv; a GS linker with 24 amino acids; and a hole variant of the Fc domain. The second polypeptide comprises MM2, CM2, and the light chain fragment of Fab for constructing Fab. The third polypeptide comprises a knob variant of the Fc domain. An example of an activatable bispecific antibody having the configuration of Figure 6 may include the first polypeptide containing one of the sequences of SEQ ID NOs. 21-24, the second polypeptide containing the sequence of SEQ ID NO. 18, and the third polypeptide containing the sequence of SEQ ID NO. 1. Figure 6B is a schematic diagram of the three polypeptides that form the activatable bispecific antibody shown in Figure 6A.

[0068] Figure 10 shows another exemplary activatable protein 1000 containing three polypeptides. The first polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, MM1 1001, an optional linker 1002, CM1 1003, an optional linker 1004, and a light chain fragment of AB1 1005. The second polypeptide contains, from the amino (N) terminal to the carboxyl (C) terminal, MM2 1021, an optional linker 1022, CM2 1023, an optional linker 1024, a heavy chain variable region of AB2 1025, a linker 1026, a light chain variable region of AB2 1027, a linker 1028, a heavy chain fragment of AB1 1029, an optional linker 1030, a third cleavable portion (CM3) 1031, an optional linker 1032, and a first domain of EM (EM1) 1033. The third polypeptide contains the second domain of EM (EM2) 1041. In an alternative exemplary configuration, as shown in Figure 10, 1005 is the heavy chain fragment of AB1 and 1029 is the light chain fragment of AB1. In an alternative exemplary configuration, as shown in Figure 10, 1025 is the light chain variable region of AB2 and 1027 is the heavy chain variable region of AB2. In an alternative exemplary configuration, as shown in Figure 10, the first domain of EM (EM1) 1033 is a hole variant of the Fc domain and the second domain of EM (EM2) 1041 is a knob variant of the Fc domain. In an alternative exemplary configuration, as shown in Figure 10, EM1 1033 is a knob variant of the Fc domain and EM2 1041 is a hole variant of the Fc domain.

[0069] Figure 11 shows another exemplary activatable protein 1100 containing three polypeptides. The first polypeptide, in order from the amino (N)-terminal region to the carboxyl (C)-terminal region, contains MM2 1101, an optional linker 1102, CM2 1103, an optional linker 1104, the heavy chain variable region of AB2 1105, linker 1106, the light chain variable region of AB2 1107, linker 1108, and the light chain fragment of AB1 1109. The second polypeptide, in order from the amino (N)-terminal region to the carboxyl (C)-terminal region, contains MM1 1121, an optional linker 1122, CM1 1123, an optional linker 1124, the heavy chain fragment of AB1 1125, an optional linker 1126, a third cleavable portion (CM3) 1127, an optional 1128, and the first domain of EM (EM1) 1129. The third polypeptide contains the second domain of EM (EM2) 1141. In an alternative exemplary configuration, as shown in Figure 11, 1109 is the heavy chain fragment of AB1 and 1125 is the light chain fragment of AB1. In an alternative exemplary configuration, as shown in Figure 11, 1105 is the light chain variable region of AB2 and 1107 is the heavy chain variable region of AB2. In an alternative exemplary configuration, as shown in Figure 11, EM1 1129 is a hole variant of the Fc domain and EM2 1141 is a knob variant of the Fc domain. In an alternative exemplary configuration, as shown in Figure 11, EM1 1129 is a knob variant of the Fc domain and EM2 1141 is a hole variant of the Fc domain.

[0070] Figure 12 shows an exemplary activatable bispecific antibody comprising: a Fab component (1204) that binds to a first target; a first prodomain (1203) containing CM1 (dashed line) and MM1 (triangle) that masks the Fab component; an scFv component (1202) that binds to a second target; a second prodomain (1201) containing CM2 (dashed line) and MM2 (triangle) that masks the scFv component; an EM (1206) containing a pair of knob and hole Fc domains; and a third cleavable portion (CM3) (1205) between the EM and Fab. Upon activation, the activated bispecific antibody cleaves CM1 to release MM1, CM2 to release MM2, and CM3 to release EM (1206). Activated bispecific antibodies lacking EM have a relatively shorter half-life compared to their parent activatable bispecific antibodies.

[0071] In some embodiments, the activated protein resulting from the activation of the activatable protein of this disclosure is not attached to EM. Such an activated protein may have a shorter half-life compared to the activatable protein. Such an activated protein may have a shorter half-life compared to the corresponding protein which is the same as the activated protein but contains EM. As used herein, the term “half-life” is the time it takes for the concentration of a molecule or molecular complex to reach 50% of its original concentration in the environment. In some examples, the environment may be serum, and the half-life is the serum half-life, which is the time it takes for the concentration of a molecule or molecular complex to reach 50% of its original concentration in the serum (e.g., in circulation of the subject). In some examples, an activated protein which contains AB1 and AB2 but does not contain MM1, MM2, or EM (i.e., a protein resulting from the activation of the activatable protein) may have a shorter half-life compared to the corresponding protein which is the same as the activated protein but contains EM. That is, the half-life of the activated molecule (AB1-AB2) is shorter than the half-life of the corresponding protein (AB1-AB2-EM).

[0072] For example, the activated protein resulting from the activation of the activatable protein of this specification may have a half-life of 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, or less than 3 hours (e.g., serum half-life). In one example, the activated protein resulting from the activation of the activatable protein of this specification may have a half-life of 5, 4, 3, or 2 days or less (e.g., serum half-life). In some examples, the activated protein resulting from the activation of the activatable protein herein may have a half-life (e.g., serum half-life) of up to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the corresponding protein containing EM, which is the same as the activated protein.

[0073] In some embodiments, the activated protein resulting from the activation of the activatable proteins herein (i.e., an activated protein not attached to EM or MM) may have higher target binding activity compared to a corresponding protein that is the same as the activated protein but contains attached EM. In some examples, an activated protein containing TB1 and TB2 but not MM1, MM2, or EM has a greater level of target binding activity than a corresponding protein that is the same as the activated protein but contains EM (i.e., TB1-TB2-EM). For example, an activated protein resulting from the activation of an activatable protein disclosed herein may have a target binding activity that is the same as the activated protein but at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 3 times, 4 times, 6 times, 8 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, or 500 times greater than the target binding activity of the corresponding protein containing EM.

[0074] In some embodiments, the activatable protein (before activation) may be characterized, either directly or indirectly, by a target binding activity lower than a control level of target binding activity of TB without MM coupling to it. For example, in some embodiments, the activatable protein may be characterized by a target binding activity that is at least 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or 10000 times lower than a control level of target binding activity of TB without MM coupling to it.

[0075] Target-binding proteins The activatable proteins according to this disclosure may comprise one or more target-binding proteins (TBs). In some examples, the activatable protein may be multispecific. For example, the activatable protein may comprise multiple TBs, each having specificity for different epitopes on the same target. In some examples, the TBs of the activatable protein herein may bind to different targets, e.g., targets on different cell types. Thus, in the activated protein resulting from the activation of the activatable protein disclosed herein, the TBs may colocalize different cell types. In some examples of the multispecific activatable proteins of this disclosure, one TB binds to a target on immune cells, and the other TB binds to disease-associated cells. By targeting immune cells and disease-associated cells and colocalizing them, the activated protein may provide targeted therapy for disease.

[0076] In some embodiments, the target-binding protein (TB) may be an antigen-binding protein (AB). In some embodiments, AB may be an antibody or a fragment thereof, e.g., a monoclonal antibody, a single-chain antibody, a Fab fragment, an F(ab')2 fragment, a single-chain variable fragment (scFv), a diabody (a non-covalent dimer of scFv), a single-chain antibody (scab), a VHH, a domain antibody (dAb), or a single-domain antibody (nanobody, e.g., a single-domain heavy-chain antibody, a single-domain light-chain antibody). A single-domain antibody may be an antibody fragment that is a single monomeric variable antibody domain. A single-domain antibody may have similar affinity to the corresponding full-length antibody. In some embodiments, AB may be a full-length antibody. In some embodiments, AB may be an immunologically active fragment. In some embodiments, AB may be an antigen-binding fragment ("Fab"). In one example, the activatable protein includes Fab as the first AB and scFv as the second AB. In some embodiments, AB may be scFv. In some embodiments, AB may be a mouse, other rodent, chimeric, humanized, or fully human monoclonal antibody. The disclosure includes structures having a combination of one or more polypeptides containing any of the above domains, e.g., one or more of SDA, Fv, ScFv, Fab, scFab, VHH, and dAb, and one or more selected from SDA, Fv, scFv, Fab, VHH, scFab, and dAb.

[0077] The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Specifically, the term “antibody” includes, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific and multispecific antibodies. An example of an antibody is V H -V L This is an antigen-binding domain formed by a dimer. Further examples of antibodies are described herein. Further examples of antibodies are known in the art.

[0078] A "light chain" consists of one variable domain (VL) and one constant domain (CL). There are two different light chain types or classes, called kappa or lambda.

[0079] A "heavy chain" consists of one variable domain (VH) and three constant domains (CH1, CH2, CH3). There are five major heavy chain classes or isotypes, some of which have several subtypes, which determine the functional activity of the antibody molecule. The five major classes of immunoglobulins are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). IgG is by far the most abundant immunoglobulin and has several subclasses (IgG1, 2, 3, and 4 in humans).

[0080] The "fragment antigen binding" (Fab) contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The AF(ab')2 fragment is formed when the antibody is cleaved by pepsin below the hinge region, in which case the two fragment antigen binding domains (Fab) of the antibody molecule remain linked. The AF(ab')2 fragment contains two complete light chains paired with the two VH and CH1 domains of the heavy chain, which are joined to each other by the hinge region. The "fragment crystallizable" (Fc) fragment (F) is used herein. C The Fab domain (also called the Fab fragment) corresponds to the paired CH2 and CH3 domains and is part of the antibody molecule that interacts with effector molecules and cells. The functional differences between heavy chain isotypes are mainly in the Fc fragment. "Single-chain Fv" (scFv) contains only the variable domain (VL) of the light chain, which is linked to the variable domain (VH) of the heavy chain by a series of synthetic peptides. The name single-chain Fv derives from fragment variability. The "hinge region" or "interdomain" is a flexible amino acid extension that joins or links the Fab fragment to the Fc domain. The "synthetic hinge region" is an amino acid sequence that joins or links the Fab fragment to the Fc domain.

[0081] The "prodomain" has a portion that inhibits antigen binding, called the masking moiety (MM), and a portion that includes a protease-cleavable substrate, called the cleavable peptide (CM). When linked to a target-binding protein (TB) (e.g., an antigen-binding protein (AB) such as an antibody or an antigen-binding fragment thereof), it functions to inhibit antigen binding by the TB or AB. The prodomain may include a linker peptide (L1) between the MM and the CM. The prodomain may also include a linker peptide (L2) at the carboxyl terminus of the prodomain to facilitate binding of the prodomain to an antibody. In certain embodiments, the prodomain includes one of the following formulas (the following formulas represent the amino acid sequence in the N-terminal to C-terminal direction), namely, (MM)-(CM), (MM)-L1-(CM), (MM)-(CM)-L2, or (MM)-L1-(CM)-L2.

[0082] TB (e.g., AB) binds specifically to a target. As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to that type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (K d ) of the interaction, with a smaller K d representing a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both the "on-rate constant" (K on ) and the "off-rate constant" (K off ) can be determined by calculating the concentrations as well as the actual rates of association and dissociation (see Nature 361:186-87 (1993)). The ratio of K off / K on allows the cancellation of all parameters not related to affinity and gives the dissociation constant Kd This is equal to (see Davies et al. (1990) Annual Rev Biochem 59:439-473 in general). The TB or antibody-binding domain (AB) of this disclosure is measured by an assay such as a radioligand-binding assay or a similar assay known to those skilled in the art, with a dissociation constant (K d When the concentration is 100 μM or less, 1 μM or less in some embodiments, 100 nM or less in some embodiments, 10 nM or less in some embodiments, and 100 pM or less to about 1 pM in some embodiments, it is said to "specifically bind" to the target or "immunospecifically bind".

[0083] The targets of TB (e.g., AB) can be proteins or other types of molecules. Exemplary targets of TB include cell surface receptors and secretory binding proteins (e.g., growth factors), soluble enzymes, and structural proteins (e.g., collagen, fibronectin). In some cases, the targets of TB may be proteins associated with a disease in the subject (e.g., cancer).

[0084] In some embodiments, TB in an activatable protein may bind to a target which is a disease-related molecule on or inside a cell. For example, TB in an activatable protein may bind to tumor cells. In such cases, TB may bind to tumor-associated antigens. As used herein, the term “tumor-associated antigen” means any antigen, including cancer-related proteins, glycoproteins, gangliosides, carbohydrates, and lipids. Such antigens may be expressed on tumor cells (e.g., malignant cells) or in the tumor microenvironment, for example, on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immunoinfiltrates. In some embodiments, the tumor-associated antigen targeted by AB may be human epidermal growth factor receptor 2 (HER2). For example, AB may be trastuzumab or a fragment thereof, for example, Fab of trastuzumab.

[0085] In some embodiments, AB in an activatable protein may bind to a target that is a molecule on an immune cell and / or can activate the immune cell. In some examples, the target of AB may also be a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand required for a highly efficient immune response. Examples of co-stimulatory molecules that may be targets of AB include components of the T cell receptor (TCR), CD3 zeta, CD3 gamma, CD3 delta, and CD3 epsilon.

[0086] In some cases, AB can bind to costimulatory molecules expressed on the surface of T lymphocytes, such as cytotoxic T lymphocytes, which can interact with antigen-binding molecules to induce T cell activation. The interaction between the antigen-binding molecule and the activated T cell antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. Once activated, AB can bind to such costimulatory molecules to activate T cells. As used herein, “T cell activation” refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. The T cell activation bispecific antigen-binding molecules of the present invention can induce T cell activation.

[0087] In some cases, AB may bind to CD3. For example, CD3 may be the epsilon subunit of CD3, e.g., NCBI RefSeq number NP_000724.1. In some cases, AB may be anti-CD3 scFv. Anti-CD3 scFv may contain one or more sequences from sequence numbers 1-9, 143-145, 149, and 150 of U.S. Patent No. 20190135943 (which is incorporated herein by reference in its entirety). Examples of such sequences include: [Table 6-1] [Table 6-2] [Table 6-3]

[0088] Examples of CD3-binding antibody CDR sequences include: [Table 7]

[0089] Examples of additional anti-CD3 AB agents include: [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6]

[0090] In some embodiments, the activatable proteins described herein may include AB1, which binds to tumor-associated antigens, and AB2, which binds to costimulatory molecules. In one example, the activatable proteins may include AB1, which binds to HER2, and AB2, which binds to CD3. In a specific example, the activatable proteins may include AB1, which is an anti-HER2 Fab (e.g., the Fab of trastuzumab), and AB2, which is an anti-CD3 scFv.

[0091] In some embodiments, AB2 can bind to a target that is an antigen on any immune effector cell. Examples of immune effector cells include leukocytes, T cells, natural killer (NK) cells, macrophages, mononuclear cells, and myeloid mononuclear cells. In some examples, the activatable protein may include immune effector cells that associate with a bispecific activatable antibody that crosslinks the immune effector cell with another cell (e.g., a disease-associated cell such as cancer or an infection).

[0092] Activatable proteins may include bispecific activatable antibodies that associate with leukocytes, bispecific activatable antibodies that associate with T cells, bispecific activatable antibodies that associate with NK cells, bispecific activatable antibodies that associate with macrophage cells, bispecific activatable antibodies that associate with mononuclear cells, or bispecific activatable antibodies that associate with myeloid mononuclear cells. For example, the activatable antibody may include a bispecific antibody that associates with T cells.

[0093] Half-life extension portion Activatable proteins may contain an extended half-life (EM). In activatable proteins, the EM may be coupled to the TB or its components in the activatable protein via a CM. During activation of the activatable protein, the EM may be cleaved from the TB. In some embodiments, for example, the CM is located between the C-terminus of the TB and the N-terminus of the EM. In the specifics of these embodiments, the CM is located between the C-terminus of the TB and the N-terminus of the EM, and the MM is located at the C-terminus of the CM and either the N-terminus or C-terminus of the EM (e.g., N-terminus to C-terminus, TB-CM-EM, TB-CM-EM-MM, TB-CM-MM-EM, etc., where each "-" independently indicates direct or indirect (e.g., via a linker) coupling) (see, for example, Figures 1 and 2). In some embodiments, the EM may be a dimer, for example, a pair of Fc domains of an immunoglobulin. In such embodiments, the first polypeptide may comprise TB, CM, and a first Fc domain, and the second polypeptide may comprise MM and a second Fc domain, with the two polypeptides covalently bonded between the first and second Fc domains via one or more disulfide bonds. In such embodiments, MM may be located at either the N-terminus or C-terminus of the second Fc domain (see, for example, Figures 3 and 4), and when CM on the first polypeptide is cleaved, MM and EM (e.g., both Fc domains) are released from the activated protein. Thus, in some embodiments, the activated protein resulting from the activation of the activatable protein does not contain EM. In some examples, the half-life extension portion may be a serum half-life extension portion, i.e., the half-life of a molecule bound to EM in serum can be extended.

[0094] In some examples, the EM may include a fragment crystallizable region (Fc domain) of an antibody. For example, the EM may be an Fc domain of IgG (e.g., IgG1, IgG2, or IgG4). In some examples, the EM may include a dimer formed by two Fc domains. The Fc domains may be wild-type Fc domains or their variants. For example, the EM may include a dimer formed by two Fc domain variants. In such cases, the two Fc domain variants may include a hole variant of the Fc domain and a knob variant of the Fc domain. The knob and hole variants may interact with each other to promote the dimerization of the two Fc domains. In some embodiments, the knob and hole variants may include one or more amino acid modifications within the interface between the two Fc domains (e.g., in the CH3 domain). For example, modifications include the amino acid substitution T366W and optionally S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other of the antibody heavy chains (numbered according to the EU numbering system). An example of a knob variant of the Fc domain includes the sequence of SEQ ID NO: 1. An example of a hole variant of the Fc domain includes the sequence of SEQ ID NO: 2.

[0095] Examples of Fc domain variants include those described in U.S. Patent No. 7,695,936 (which is incorporated herein by reference in its entirety). One example shows a modification comprising the amino acid substitution T366Y in one IgG Fc domain and the amino acid substitution Y407T in the other IgG Fc domain. Another example shows a modification comprising the amino acid substitution T366W in one IgG Fc domain and the amino acid substitution Y407A in the other IgG Fc domain. Another example shows a modification comprising the amino acid substitution F405A in one IgG Fc domain and the amino acid substitution T394W in the other IgG Fc domain. Another example shows a modification comprising the amino acid substitutions T366Y and F405A in one IgG Fc domain and the amino acid substitutions T394W and Y407T in the other IgG Fc domain. In one example, the modification includes amino acid substitutions T366W and F405W in one IgG Fc domain, and amino acid substitutions T394S and Y407A in the other IgG Fc domain. In another example, the modification includes amino acid substitutions F405W and Y407A in one IgG Fc domain, and amino acid substitutions T366W and T394S in the other IgG Fc domain. In another example, the modification includes amino acid substitution F405W in one IgG Fc domain, and amino acid substitution T394S in the other IgG Fc domain. The mutation sites within the Fc domains are numbered according to the EU numbering system. The IgG Fc domains may contain sequences of SEQ ID NOs. 3-6 (IgG1, IgG2, IgG3, or IgG4). In these sequences, amino acids 1-107 correspond to EU numbering 341-447.

[0096] In some cases, Fc domain variants may exhibit reduced effector function. An example of such an Fc domain is disclosed in U.S. Patent No. 20190135943, which is incorporated herein by reference in its entirety.

[0097] Further examples of EMs include immunoglobulins (e.g., IgG), serum albumin (e.g., human serum albumin (HSA)), hexa-hat GST (glutathione S-transferase) glutathione affinity, calmodulin-binding peptide (CBP), strep tags, cellulose-binding domains, maltose-binding proteins, S-peptide tags, chitin-binding tags, immunoreactive epitopes, epitope tags, E2Tag, HA epitope tags, Myc epitopes, FLAG epitopes, AU1 and AU5 epitopes, Glu-Glu epitopes, KT3 epitopes, IRS epitopes, Btag epitopes, protein kinase-C epitopes, and VSV epitopes.

[0098] In some embodiments, the serum half-life of the activatable protein may be longer than that of the corresponding protein, which is the same as the activatable protein but does not have a half-life extension portion. In some embodiments, the serum half-life of the activatable protein may be longer than the serum half-life of the activated protein. In some embodiments, the serum half-life of the activatable protein, when administered to an organism, is at least 15 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour.

[0099] Masking area (MM) The activatable proteins described herein may contain one or more masking moieties (MMs) that can interfere with the binding of TB to a target. Masking moieties within an activatable molecule “mask” or reduce, or otherwise inhibit, the binding of the activatable polymer to its target and / or epitope. In some embodiments, coupling or modification of a target-binding protein (TB) (e.g., AB or other therapeutic or diagnostic protein) with an MM may inhibit the ability of TB to specifically bind to its target and / or epitope by inhibition known in the art (e.g., structural changes, competition of antigen-binding domains, etc.). In some embodiments, coupling or modification of TB with an MM may result in a structural change that reduces or inhibits the ability of TB to specifically bind to its target and / or epitope. In some embodiments, coupling or modification of a protein containing an antigen-binding domain with an MM may sterically block, reduce or inhibit the ability of the antigen-binding domain to specifically bind to its target and / or epitope. MM can be coupled to TB (e.g., AB) by CM either directly or indirectly (e.g., via one or more linkers as described herein).

[0100] Alternatively, MMs that interfere with the target binding of TB may be coupled to components of an activatable protein other than TB. For example, as illustrated in Figure 2, the activatable protein may include TB1 and TB2, and an MM that interferes with TB2 may be coupled to TB1. In another example, as illustrated in Figures 3 and 4, the activatable protein may include TB1, TB2, and EM, and an MM that interferes with TB2 may be coupled to EM. In either case, in the tertiary or quaternary structure of the activatable structure, the MM may be located in a position that allows the MM to mask TB (e.g., proximal to the TB to be masked).

[0101] In some embodiments, MM may interact with TB to reduce or inhibit the interaction between TB and its binding partner. In some embodiments, MM may comprise at least a partial or complete amino acid sequence of a naturally occurring binding partner of TB. MM may be a fragment of a naturally occurring binding partner. The fragment may retain nucleic acid or amino acid sequence homology with the naturally occurring binding partner of 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, or 20% or less. In some embodiments, MM may be a homologous polypeptide of TB (e.g., AB). For example, MM may comprise a sequence of an epitope of TB or a sequence of a fragment thereof. As used herein, the term “naturally occurring” means, when applied to a subject, the fact that the subject can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by humans in a laboratory, or otherwise naturally occurring.

[0102] In some embodiments, MM may include amino acid sequences that do not exist naturally or that do not contain amino acid sequences of naturally occurring binding partners or target proteins. In certain embodiments, MM is not a natural binding partner of TB. In some embodiments, MM does not include a partial sequence of 4, 5, 6, 7, 8, 9, or more than 10 consecutive amino acid residues of a natural binding partner of TB. MM may be a modified binding partner of TB that contains amino acid changes that reduce binding affinity and / or binding activity to TB. In some embodiments, MM may not contain, or substantially not contain, nucleic acid or amino acid homology with a natural binding partner of TB. In other embodiments, MM is similar to a natural binding partner of TB by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% or less.

[0103] In some embodiments, MM may not specifically bind to TB (or other activatable proteins), but may prevent the binding of target-binding proteins (e.g., AB) to their binding partners via nonspecific interactions, such as steric hindrance. For example, the MM may be positioned within the activatable protein such that, due to the tertiary or quaternary structure of the activatable protein, the MM masks AB via charge-based interactions, thereby holding the MM in the appropriate position and preventing its binding partner from approaching TB.

[0104] In some embodiments, MM may have a dissociation constant for binding to a target-binding protein (e.g., AB) that is less than or equal to the dissociation constant of TB for the target. In some embodiments, MM may not interfere with TB or compete with TB for binding to a cleaved target.

[0105] The structural properties of MMs can be selected according to factors such as the minimum amino acid sequence required to prevent the protein from binding to the target, the target protein-protein binding pair, the size of the TB, and the presence or absence of a linker.

[0106] In some embodiments, the MM may be specific to the coupled TB. Examples of MMs include those specifically screened to bind to the binding domain of a TB, e.g., AB, or a fragment thereof (e.g., an affinity mask). Methods for screening MMs to obtain MMs that are specific to a TB and that bind specifically and / or selectively to the binding domain of a binding partner / target are provided herein and may include protein display methods.

[0107] As used herein, the term “masking efficiency” or “ME” refers to the activity of an activatable protein divided by the activity of a control target-binding protein (e.g., antibody) (e.g., EC50), where the control target-binding protein (e.g., antibody) may be either a cleavage product of the activatable protein (i.e., the activated protein) or a target-binding protein (e.g., an antibody or a fragment thereof) used as a TB of the activatable protein. An activatable protein with a reduced level of target-binding activity may have a masking efficiency greater than 10. In some embodiments, the activatable proteins described herein may have masking efficiencies greater than 10, 100, 1000, or 5000.

[0108] In some embodiments, MM may be a peptide with a length of approximately 2 to 50 amino acids. For example, MM may be a peptide with a length of 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, or 40 to 50 amino acids. For example, MM may be a peptide with a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some cases, MM may be polypeptides longer than 50 amino acids, such as polypeptides with 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids.

[0109] In some embodiments, when the activatable protein has TB and interfering MM in the presence of a TB target, for example, when measured in vivo as described in U.S. Patent No. 20200308243A1, or when measured by a masking efficiency assay, or in When measured by a vitro immunoadsorption assay, there is no or substantially no binding of TB to the target, or the binding of TB to that target is 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 30%, 45%, 60%, 90%, 120%, 150%, 180 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, compared to the binding of the corresponding antibody without interfering MM. For example, the ability of MM to inhibit the binding of activatable proteins to their binding partners at therapeutically relevant concentrations and times can be measured. For this measurement, an immunoabsorption assay (MEA, mask efficiency assay) has been developed to measure the time-dependent binding of activatable proteins to their binding partners, as described in U.S. Patent No. 20200308243A1, which is incorporated herein by reference in its entirety.

[0110] The binding affinity of TB to a target or binding partner with interfering MM is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, or 50,000,000 times lower than the binding affinity of TB to that binding partner without interfering MM, or 5-10, 10-100, 10-1,000, or 10-1 0,000x, 10~100,000x, 10~1,000,000x, 10~10,000,000x, 100~1,000x, 100~10,000 times, 100~100,000 times, 100~1,000,000 times, 100~10,000,000 times, 1,000~10,000 times, 1,000~100, It can be 000 times, 1,000 to 1,000,000 times, 1,000 to 10,000,000 times, 10,000 to 100,000 times, 10,000 to 1,000,000 times, 10,000 to 10,000,000 times, or 100,000 to 10,000,000 times lower.

[0111] The dissociation constant (K) of MM with respect to TB (e.g., AB) that is masked by this is the MM. d ) is the K of TB (e.g., AB) relative to the target. d It may be larger than the K of MM relative to the masked TB. d , TB's K against the target dIt may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 times, or even 10,000,000 times greater than the target. Conversely, the binding affinity of MM to masked TB may be lower than the binding affinity of TB to the target. The binding affinity of MM to TB may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 times, or even 10,000,000 times lower than the binding affinity of TB to the target.

[0112] In some embodiments, MM may contain genetically encoded or unencoded amino acids. Examples of unencoded amino acids include, but are not limited to, D-amino acids, β-amino acids, and γ-amino acids. In certain embodiments, MM may contain 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% or less of unencoded amino acids.

[0113] In some embodiments, when released from an activatable protein and released into a free state, MM may possess biological activity or therapeutic effects, such as binding ability. For example, free MM may bind to the same or different binding partners. In certain embodiments, free MM may exert therapeutic effects to provide secondary functions to the compositions disclosed herein. In some embodiments, when cleaved from an activatable protein and released into a free state, MM may not exhibit advantageous biological activity. For example, in some embodiments, free MM does not induce an immune response in a subject.

[0114] Suitable MMs can be identified and / or further optimized from a library of candidate activatable proteins having various MMs through a screening procedure. For example, TBs and CMs can be selected to provide a desired enzyme / target combination, and the amino acid sequences of MMs can be identified by the screening procedures described below to identify MMs that provide an activatable phenotype. For example, a random peptide library (e.g., a peptide library containing 2 to 40 or more amino acids) may be used in the screening methods disclosed herein to identify suitable MMs.

[0115] In some embodiments, MMs having a specific binding affinity to TB (e.g., AB) can be identified through a screening procedure that includes providing a library of peptide scaffolds containing candidate MMs, each scaffold consisting of a transmembrane protein and a candidate MM. The library may then be contacted with whole or partial proteins, e.g., full-length proteins, naturally occurring protein fragments, or unnaturally occurring protein-containing fragments (capable of binding to a target binding partner) to identify one or more candidate MMs having detectably bound proteins. The screening may be performed by one or more magnetically activated sorting (MACS) or fluorescence-activated sorting (FACS), as well as by determining the binding affinity of the MMs to AB and subsequently determining the masking efficiency, as described, for example, in WO2009025846 and U.S. Patent 20200308243A1 (both incorporated herein by reference in their entirety).

[0116] In some embodiments, MM may be selected for use with a specific protein, antibody, or antibody fragment. For example, a suitable MM for use with an epitope-binding AB may include the epitope sequence. In one example, if the activatables include AB1, which is anti-HER2 Fab, and AB2, which is anti-CD3 scFv, then MM1 (to mask AB1) may include the HER2 sequence to which AB1 binds, and MM2 (to mask AB2) may include the CD3 sequence to which AB2 binds. In other embodiments, MM may not include the sequence of the TB's natural binding partner. In some examples, anti-HER2 A suitable MM1 for masking Fab includes the sequence ALICCSDVSGLCRWC (SEQ ID NO: 40). In some examples, suitable MM2s for masking anti-CD3 scFv include MMs containing the sequences GYLWGCEWNCGGITT (SEQ ID NO: 34), NAFRCWWDPPCQPMT (SEQ ID NO: 35), ARGLCWWDPPCTHDL (SEQ ID NO: 36), or NHSLCYWDPPCEPST (SEQ ID NO: 37). In some examples, suitable MM2s for masking anti-CD3 scFv include MMs containing the sequences MMYCGGNEVLCGPRV (SEQ ID NO: 66), GYRWGCEWNCGGITT (SEQ ID NO: 67), MMYCGGNEIFCEPRG (SEQ ID NO: 68), GYGWGCEWNCGGSSP (SEQ ID NO: 69), or MMYCGGNEIFCGPRG (SEQ ID NO: 70).

[0117] Further preferred MMs are WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, WO201907 Disclosed in 5405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974, which are incorporated herein by reference in their entirety.

[0118] Cuttable portion (CM) Activatable proteins may contain one or more cleavable regions (CMs) as defined above.

[0119] In some embodiments, the activatable protein may contain CM between TB (e.g., AB) and MM. The activatable protein may further contain CM between TB and EM. In some examples, the CM between TB and MM is also located between TB and EM (see, for example, Figure 1, where CM127 is located between TB123 / 125 and MM129, and CM127 is also located between TB123 / 125 and MM131 / 141). In such cases, cleavage of CM can cause both MM and EM to be released from TB. In some examples, the CM is located between the first TB (TB1) and the MM (MM2) that binds to the second TB (TB2) (see, for example, Figure 2, where CM223 is located between the first TB (TB221) and MM225, and MM225 is a masking moiety that inhibits the binding of the second TB (TB207 / 209). See also Figure 3, where CM323 is located between the first TB (TB321) and MM343, and MM343 is a masking moiety that inhibits the binding of the second TB (TB307 / 309) to its target). In certain examples, the CM between TB and MM is not located between TB and EM. In such cases, the activatable protein may include the first CM between TB and MM, and the second CM between TB and EM. In some examples, an activatable protein may have three CMs: a first CM between the first TB and the first MM, a second CM between the second TB and the second MM, and a third CM between the EM and the first or second TB (see, for example, Figures 10 and 11). Activation of the activatable protein can cleave both CMs, so both MM and EM are released from the EM.

[0120] The activatable proteins CM and TB may be selected such that the TB contains a binding site to a given target, the CM contains a substrate for one or more proteases, and the one or more proteases colocalize with the target in the tissue (e.g., at the treatment or diagnostic site of the subject). In some embodiments, the activatable proteins may find particular use when, for example, one or more proteases capable of cleaving the CM site are present at relatively high levels (or are more active) in the target-containing tissue at the treatment or diagnostic site than in the tissue at the non-treatment site (e.g., healthy tissue).

[0121] In some embodiments, the CMs of this specification may include substrates of proteases reported in cancer or certain cancers. See, for example, La Roca et al., British J. Cancer 90(7):1414-1421, 2004. Suitable substrates for use in the CM components used herein include those more widely found in cancer cells and tissues. Therefore, in certain embodiments, the CMs may include substrates of proteases more widely found in cancer-related disease tissues. Examples of cancers include gastric cancer, breast cancer, osteosarcoma, esophageal cancer, HER2-positive cancer, Kaposi's sarcoma, pilocytic cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, malignant cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, ovarian cancer, bladder cancer, BCG-resistant nonmuscle-invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer, or testicular cancer. In some embodiments, the CM component includes a substrate of protease(s) that is more widespread in the tumor tissue. For example, protease(s) may be produced by the tumor in the subject. In some embodiments, the activatable protein may include a first CM between MM and TB (e.g., AB), and a second CM between EM and the same or a different TB. In the activated state, both CMs may be cleaved, resulting in the release of MM and EM from the TB(s). In some examples, the first and second CMs may contain substrates of the same protease. In some examples, the first and second CMs may contain substrates of different proteases. In some examples, the first and second CMs may contain or consist of the same sequence. In some examples, the first and second CMs may contain or consist of different sequences.

[0122] The second CM may be located in an activatable protein where its cleavage facilitates the dissociation of EM from TB. In some examples, the second CM may be between the C-terminus of TB (or, if TB contains multiple polypeptides, its components) and the N-terminus of MM, with the C-terminus of MM being coupled to the N-terminus of EM (or, if EM contains multiple polypeptides, its components). In some examples, the second CM may be between the N-terminus of TB (or, if TB contains multiple polypeptides, its components) and the C-terminus of MM, with the N-terminus of MM being coupled to the C-terminus of EM (or, if EM contains multiple polypeptides, its components). In some examples, the second CM may be between the C-terminus of TB (or, if TB contains multiple polypeptides, its components) and the N-terminus of EM (or, if EM contains multiple polypeptides, its components) being coupled to the N-terminus of MM. In some examples, the second CM may be between the N-terminus of the TB (or, if the TB contains multiple polypeptides, one of its components) and the C-terminus of the EM (or, if the EM contains multiple polypeptides, one of its components), with the N-terminus of the EM (or, if the EM contains multiple polypeptides, one of its components) being coupled to the C-terminus of the MM. In these examples, the MM may be a masking region of the TB or a different TB (e.g., on the same or a different polypeptide) in the activatable protein.

[0123] Suitable CMs for use with the activatable proteins of this specification include any of the protease substrates known in the art. In some examples, CMs may include substrates of serine proteases (e.g., u-plasminogen activator (uPA, also called urokinase)) and matryptases (also referred herein as MT-SP1 or MTSP1). In some examples, CMs may include substrates of matrix metalloproteinases (MMPs). In some examples, CMs may include substrates of cysteine ​​proteases (CPs) (e.g., regmine).

[0124] In some embodiments, CM is a disintegrin and metalloproteinase (ADAM) or a disintegrin and metalloproteinase having a thrombospongin motif (ADAMTS) (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5), aspartate protease (e.g., BACE, renin), aspartate cathepsin (e.g., cathepsin D Cathepsin E), caspases (e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14), cysteine ​​cathepsins (e.g., cathepsin A, cathepsin B, cathepsin C, cathepsin G, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P), cysteine ​​proteases (e.g., cruzipain, regmine, otubain) -2) Chymase, DESC1, DPP-4, FAP, Elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoate, Hepsin, HtrA1, Human neutrophil elastase, KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14), Metalloproteinase (e.g., Meprin, Neprilysin, PSMA, BMP-1), Lactoferrin, Marapsin, Mat It may also contain substrates for liptase-2, MT-SP1 / matryptase, NS3 / 4A, PACE4, plasmin, PSA, MMP (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27), TMPRSS2, TMPRSS3, TMPRSS4, tPA, thrombin, tryptase, and uPA.

[0125] In some embodiments, the protease substrate of CM may include a polypeptide sequence that is not substantially identical (e.g., 90%, 80%, 70%, 60%, or 50% or less identical) to any polypeptide sequence that is spontaneously cleaved by the same protease.

[0126] In some embodiments, CM is or may include the sequence LSGRSDDH (sequence number 33) or ISSGLLSGRSDNH (sequence number 41). In some embodiments, CM may be or may include any one of the sequences in the following table, or may be incorporated into a consensus of sequences: [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9]

[0127] Examples of CMs are also WO2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, W This includes those listed in O2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974, which are incorporated herein by reference in their entirety for all purposes.

[0128] In some embodiments, the CM may be, or include, a combination of the exemplary sequences described above, a C-terminal truncated variant, or an N-terminal truncated variant. Suitable truncated variants of the above-described amino acid sequences for use in the CM may be any truncated variant that retains the recognition site of the corresponding protease. These include C-terminal and / or N-terminal truncated variants containing at least three consecutive amino acids of the above-described amino acid sequence, or at least four, five, six, seven, eight, nine, or ten amino acids of the aforementioned amino acid sequence that retain the protease recognition site. In certain embodiments, the truncated variants of the above-described amino acid sequences may correspond to any of the above but be amino acid sequences in which 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, or 1 to 3 amino acids are truncated at the C-terminus and / or N-terminus, and (1) have at least 3 amino acid residues; and (2) retain the protease recognition site. In some of the embodiments described above, the truncated CM is a CM truncated at the N-terminus. In some embodiments, the truncated CM is a CM truncated at the C-terminus. In some embodiments, the truncated CM is a CM truncated at both the C-terminus and the N-terminus.

[0129] In some embodiments, CM may contain 3 to 25 amino acids. In some embodiments, CM may contain a total of 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25 amino acids. In some embodiments, CM is approximately 0.001 to 1500 × 10 4 M -1 S -1, or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 × 10 4 M -1 S -1 It can be specifically cleaved by at least a protease at a rate of . The rate is the reaction rate of substrate cleavage (k) as disclosed in WO2016118629. cat / K m It can be measured as follows:

[0130] Linker An activatable protein may contain one or more linkers. A linker may contain a sequence of amino acids that links two components in the activatable protein. The linker cannot be cleaved by any protease. In some embodiments, one or more linkers (e.g., flexible linkers) can be introduced into an activatable protein to provide flexibility at one or more junctions between domains, between subdomains, between subdomains and domains, or at any other junctions where a linker would be beneficial. In some embodiments, if the activatable protein is provided as a structurally constrained construct, a flexible linker can be inserted to facilitate the formation and maintenance of the structure in the uncleaved activatable protein. Any linker described herein can provide desired flexibility to facilitate inhibition of target binding or facilitate cleavage of CM by proteases. In some embodiments, the linkers contained in the activatable protein may be fully or partially flexible so that the linker contains one or more subdomains in addition to the flexible linker that confer a less flexible structure, thereby providing the desired activatable protein. Some linkers may contain cysteine ​​residues, which may form disulfide bonds and reduce the flexibility of the constituent.

[0131] In some embodiments, the linker coupled to the MM may have a length that allows the MM to be in a tertiary or quaternary position that effectively masks the TB, for example, in a position proximal to the TB being masked.

[0132] In most cases, the length of a linker can be determined by counting the number of amino acids from the N-terminus to the C-terminus, from the N-terminus of the linker adjacent to the C-terminal amino acid of the previous component to the C-terminus of the linker adjacent to the N-terminal amino acid of the next component (i.e., the length of the linker does not contain either the C-terminal amino acid of the previous component or the N-terminal amino acid of the next component).

[0133] In some embodiments, the linkers total 1-50, 1-40, 1-30, 1-25 (for example, 1-24, 1-22, 1-20, 1-18, 1-16, 1-15, 1-14, 1-12, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 1-2, 2-25, 2-24, 2-22, 2-20, 2-18, 2-16, 2-15, 2-14, 2-12, 2-10, 2-8, 2-6, 2 ~5, 2~4, 2~3, 4~25, 4~24, 4~22, 4~20, 4~18, 4~16, 4~15, 4~14, 4~12, 4~10, 4~8, 4~6, 4~5, 5~25, 5~24, 5~22, 5~20, 5~18, 5~16, 5~15, 5~14, 5~12, 5~10, 5~8, 5~6, 6~25, 6~24, 6~22, 6~20, 6~18, 6~16, 6~15, 6~14, 6~ 12, 6~10, 6~8, 8~25, 8~24, 8~22, 8~20, 8~18, 8~16, 8~15, 8~14, 8~12, 8~10, 10~25, 10~24, 10~22, 10~20, 10~18, 10~16, 10~15, 10~14, 10~12, 12~25, 12~24, 12~22, 12~20, 12~18, 12~16, 12~15, 12~14, 14~25, 14~ The linker may contain 24, 14-22, 14-20, 14-18, 14-16, 14-15, 15-25, 15-24, 15-22, 15-20, 15-18, 15-16, 16-25, 16-24, 16-22, 16-20, 16-18, 18-25, 18-24, 18-22, 18-20, 20-25, 20-24, 20-22, 22-25, 22-24, or 24-25 amino acids. In some embodiments, the linker may contain a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0134] In some embodiments, the linker may be rich in glycine (Gly or G) residues. In some embodiments, the linker may be rich in serine (Ser or S) residues. In some embodiments, the linker may be rich in both glycine and serine residues. In some embodiments, the linker may have one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs).

[0135] In some embodiments, the linker may have one or more Gly-Gly-Gly-Ser (GGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences). In some embodiments, the linker may have one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences). In some embodiments, the linker may have one or more Gly-Gly-Ser-Gly (GGSG) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences). Examples of flexible linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GGS)n, (GSGGS)n, and (GGGS)n, where n is at least an integer of 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Because glycine and glycine-serine polymers may be relatively unstructured, they can function as neutral links between components. Glycine has significantly more access to the phi-psey space than alanine and is less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary flexible linkers include GGSG (SEQ ID NO: 71), GGSGG (SEQ ID NO: 72), GGSSG (SEQ ID NO: 73), GGGGG (SEQ ID NO: 74), GGGSG (SEQ ID NO: 75), GSSSG (SEQ ID NO: 76), GSSGGSGGSGG (SEQ ID NO: 77), GGGS (SEQ ID NO: 78), GGGSGGGS (SEQ ID NO: 79), GGGSGGGSGGGS (SEQ ID NO: 80), GGGGSGGGGSGGGGS (SEQ ID NO: 81), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 82), GGGGSGGGGS (SEQ ID NO: 83), GGGGS (SEQ ID NO: 84), GS,GGGGGSGS (SEQ ID NO: 85), GGGGSGGGGSGGGGGSGS (SEQ ID NO: 86), GGS This includes one or more combinations of LDPKGGGGS (sequence number 87), PKSCDKTHTCPPCPAPELLG (sequence number 88), SKYGPPCPPCPAPEFLG (sequence number 89), GKSSGSGSESKS (sequence number 90), GTSGSSGKSSEGKG (sequence number 91), GTSGSSGKSSEGSGSTKG (sequence number 92), GTSGSSGKPGSGEGSTKG (sequence number 93), GTSGSSGKPGSSEGST (sequence number 94), GGGGSGGS (sequence number 95), GGGGSGGGGSS (sequence number 96), GGGGSSGGSGGSSGGS (sequence number 97), and GTSGSSGKPGSEGST (sequence number 98).

[0136] Examples of linkers may further include sequences that are at least 70% identical to the exemplary linkers described herein (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 96%, 97%, at least 98%, at least 99%, or 100% identical). Those skilled in the art will recognize that the design of an activatable protein may include linkers that are all or partially flexible so that the linker may include not only a flexible linker but also one or more parts that confer a less flexible structure.

[0137] In some embodiments, the activatable protein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (for example, linker sequences identical or different from any of the exemplary linker sequences described herein or known in the art). In some embodiments, the linker may include sulfoSIAB, SMPB, and sulfoSMPB, and the linker reacts with a primary amine sulfhydryl.

[0138] Conjugate In some embodiments, the activatable molecule (e.g., an activatable protein such as an activatable antibody) may further include one or more additional agents, such as a targeting portion to facilitate delivery to a cell or tissue of interest, a therapeutic agent (e.g., an anti-cancer drug such as a chemotherapeutic agent or an antitumor agent), a toxin, or a fragment thereof. The additional agents may be conjugated to the activatable antibody. The term “agent” is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract obtained from a biological material.

[0139] In some embodiments, the activatable protein can be conjugated with a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope.

[0140] Examples of cytotoxic agents that can be conjugated to activatable proteins include drastatin and its derivatives (e.g., auristatin E, AFP, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), desmethyl auristatin E (DMAE), auristatin F, desmethyl auristatin F (DMAF), drastatin 16 (DmJ), drastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone), metansinoids (e.g., DM-1, DM-4), metansinoid derivatives, duocalmycin, α-amanitin, turvostatin, fenstatin, hydroxy Examples include civenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocompstatin, pyrrolobenzimidazole (PBI), cibrostatin 6, doxaliform, semadin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracyclines, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecin, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaprysiatoxin, KahaMide-F, discodermold, and ecteinacidin.

[0141] Examples of enzymatically active toxins that can be conjugated into activatable proteins include diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, lysine A chain, abrin A chain, modesin A chain, α-sarcin, and Aleuriies. Examples include fordii protein, dianfhin protein, Phytoiaca Americana protein (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitors, crucin, crotirs, sapaonaria officinalis inhibitors, geionin, mitogeliin, restrictosin, phenomycin, neomycin, and trichothecenes.

[0142] Examples of anti-cancer drugs that can be conjugated to activatable proteins include Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Methotrexate, Thiotepa, Bisanthren, Novantrone, Thioguanine, Procarbazine, and Cytarabine.

[0143] Examples of antiviral drugs that can be conjugated to activatable proteins include acyclovir, virus A, and simmerel. An example of an antifungal drug that can be conjugated to activatable proteins is nistatin. Examples of detection reagents that can be conjugated to activatable proteins include fluorescein and its derivatives, and fluorescein isothiocyanate (FITC). Examples of antibacterial drugs that can be conjugated to activatable proteins include aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin. Examples of 3β,16β,17α-trihydroxycholesta-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-α-L-arabinopyranoside)(OSW-1) that can be conjugated to activatable proteins include s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, toposysomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepines, calcicheamicins, podophyritoxins, taxanes, and vinca alkaloids. Examples of radiopharmaceuticals that can be conjugated to activatable proteins include: 123 I, 89 Zr, 125 I, 131 I, 99 mTC, 201 T1, 62 Cu, 18 F, 68 Ga, 13 N, 15 O, 38 K, 82 Rb, 111 In, 133 Xe, 11 C, and 99Examples of mTc (technetium) include heavy metals that can be conjugated to activatable proteins, such as barium, gold, and platinum. Examples of anti-mycoplasma agents that can be conjugated to activatable proteins include tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0144] In some embodiments, the activatable protein may include a signal peptide. If it includes multiple polypeptides, the activatable protein may include multiple signal peptides, for example, one signal peptide for each of the multiple polypeptides. The signal peptide may be a peptide (e.g., 10-30 amino acids long) present at the end (e.g., N-terminus or C-terminus) of a newly synthesized protein directed toward the secretory pathway. In some embodiments, the signal peptide may be conjugated to the activatable protein via a spacer. In some embodiments, the spacer may be conjugated to the activatable protein if the signal peptide is not present.

[0145] Those skilled in the art will understand that a wide variety of possible agents can be conjugated to any of the activatable proteins described herein. The agent may be conjugated to another component of the activatable protein by a conjugation moiety. The conjugate may involve any chemical reaction that joins the two molecules, insofar as the activatable protein and the other moiety retain their respective activities. The conjugate may involve many chemical reaction mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complexation. In some embodiments, the bond may be covalent. Covalent bonding can be achieved either by direct condensation of existing side chains or by the incorporation of an external crosslinking molecule. Many divalent or polyvalent linkers may be useful for conjugating any of the activatable proteins described herein. For example, the conjugate may contain organic compounds, such as thioesters, carbodiimides, succinimides, glutaraldehyde, diazobenzene, and hexamethylenediamine. In some embodiments, the activatable protein may contain, or otherwise introduce, one or more non-natural amino acid residues to provide a site suitable for conjugation.

[0146] In some embodiments, the drug and / or conjugate may attach to the antigen-binding domain via disulfide bonds (e.g., disulfide bonds on a cysteine ​​molecule). Since many cancers spontaneously release high levels of glutathione, i.e., a reducing agent, glutathione present in the cancer tissue microenvironment can reduce disulfide bonds, subsequently releasing the drug and / or conjugate at the delivery site.

[0147] In some embodiments, when a conjugate binds to a target within a target site (e.g., diseased tissue (e.g., cancerous tissue)) in the presence of complement, the amide or ester bond attaching the conjugate and / or drug to the linker is cleaved, resulting in the release of the conjugate and / or drug in an activated state. When administered to a subject, these conjugates and / or drugs can achieve delivery and release at the target site (e.g., diseased tissue (e.g., cancerous tissue)). These conjugates and / or drugs may be effective for in vivo delivery of any of the conjugates and / or drugs described herein.

[0148] In some embodiments, the conjugate portion is not cleavable by complement system enzymes. For example, the conjugate and / or drug are released without complement activation, as the target cells are ultimately lysed by complement activation. In such embodiments, the conjugate and / or drug are delivered to the target cells (e.g., hormones, enzymes, corticosteroids, neurotransmitters, or genes). Furthermore, the conjugate portion becomes more readily cleavable by serum proteases, and the conjugate and / or drug are slowly released at the target site.

[0149] In some embodiments, the conjugate and / or drug may be designed so that the conjugate and / or drug is delivered to a target site (e.g., diseased tissue (e.g., cancerous tissue)) but the conjugate and / or drug is not released.

[0150] In some embodiments, the conjugate and / or drug may be attached directly to the antigen-binding domain, or via an amino acid (e.g., a D-amino acid), a peptide, a thiol-containing moiety, or other organic compounds that may be modified to include functional groups that can subsequently be used for attachment to the antigen-binding domain by the methods described herein.

[0151] In some embodiments, the activatable protein may contain at least one conjugation site for a drug. In some embodiments, all possible conjugation sites are available for conjugation to a drug. In some embodiments, one or more conjugation sites may include sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain sulfide bonds but not in intrachain disulfide bonds, and / or sulfur atoms of cysteine ​​or other amino acid residues containing sulfur atoms. In such cases, the residues may be naturally present in the protein structure or may be incorporated into the protein structure by means of site-directed mutagenesis, chemical transformation, or accidental incorporation of unnatural amino acids.

[0152] This disclosure also provides methods and materials for preparing proteins that can be activated by one or more conjugated agents. In some embodiments, the activatable proteins may be modified to include one or more interchain disulfide bonds. For example, the disulfide bonds can be reduced after exposure to a reducing agent, for example, but not limited to TCEP, DTT, or β-mercaptoethanol. In some cases, the reduction of the disulfide bonds may be only partial. As used herein, the term partial reduction refers to a situation in which the activatable protein comes into contact with a reducing agent and some of all possible conjugation sites are reduced (e.g., not all disulfide bonds are reduced). In some embodiments, an activatable protein may be partially reduced after contact with a reducing agent if less than 99% of all available conjugate sites (e.g., less than 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) are reduced. In some embodiments, an activatable protein having reduction at one or more interchain disulfide bonds may be conjugated with a drug that is reactive with free thiols.

[0153] This disclosure also provides methods and materials for conjugating therapeutic agents to specific locations on activatable proteins. In some embodiments, activatable proteins may be modified so that therapeutic agents can be conjugated to them at specific locations on the activatable protein. For example, an activatable protein may be partially reduced to facilitate conjugation to the activatable protein. In such cases, the partial reduction of the activatable protein may occur so that the conjugation site of the activatable protein is not reduced. In some embodiments, the conjugation site(s) on the activatable protein may be selected to facilitate conjugation of the drug at specific locations on the protein construct. In treatment with a reducing agent, various factors may affect the “reduction level” of the activatable protein. For example, to achieve partial reduction of an activatable protein using the methods and materials described herein, optimization of the ratio of the reducing agent to the activatable protein, incubation length, incubation temperature, and / or pH of the reduction reaction solution may be required, but are not limited to these. Partial reduction of the activatable protein can be achieved using any suitable combination of factors (e.g., the ratio of reducing agent to the activatable protein, the incubation period and temperature with the reducing agent, and / or the pH of the reducing agent) (e.g., overall reduction of available conjugate sites, or reduction of specific conjugate sites).

[0154] The effective ratio of a reducing agent to an activatable protein can be any ratio that reduces the activatable protein at least partially (e.g., overall reduction of available conjugate sites, or reduction of specific conjugate sites) in a manner that allows for conjugation to the drug. In some embodiments, the ratio of reducing agent to activatable protein may range from approximately 20:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 20:1 to 1:1.5, 10:1 to 1:1.5, 9:1 to 1:1.5, 8:1 to 1:1.5, 7:1 to 1:1.5, 6:1 to 1:1.5, 5:1 to 1:1.5, 4:1 to 1:1.5, 3:1 to 1:1.5, 2:1 to 1:1.5, 1.5:1 to 1:1.5, or 1:1 to 1:1.5.

[0155] The effective incubation time and temperature for treating a reducing agent-activatable protein can be any time and temperature that at least partially reduces the activatable protein (e.g., overall reduction of available conjugate sites, or reduction of specific conjugate sites) in a manner that allows the drug to conjugate to the activatable protein. In some embodiments, the incubation time and temperature for treating the activatable protein may range from about 1 hour at 37°C to about 12 hours at 37°C (or any partial range thereof).

[0156] The effective pH for a reduction reaction to treat a protein activatable with a reducing agent can be any pH that reduces the activatable protein at least partially (e.g., overall reduction of the available conjugate sites, or reduction of specific conjugate sites) in a manner that allows the drug to conjugate to the activatable protein.

[0157] When a partially reduced activatable protein comes into contact with a thiol-containing agent, the agent can conjugate to interchain thiols within the activatable protein. The agent can be modified to contain thiols using a thiol-containing reagent (e.g., cysteine ​​or N-acetylcysteine). For example, an activatable protein can be partially reduced after incubation with a reducing agent (e.g., TEPC) at a desired ratio of reducing agent to the activatable protein for about 1 hour at about 37°C. The effective ratio of reducing agent to the activatable protein can be any ratio that partially reduces at least two interchain disulfide bonds in the activatable protein (e.g., overall reduction of available conjugate sites or reduction of specific conjugate sites) in a manner that allows conjugate to a thiol-containing agent.

[0158] In some embodiments, the activatable protein may be reduced by a reducing agent in a manner that avoids the reduction of any intrachain disulfide bonds. In some embodiments, the activatable protein may be reduced by a reducing agent in a manner that avoids the reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0159] In some embodiments, the drug (e.g., a drug conjugated to an activatable protein) may be a detectable moiety, such as a label or other marker. For example, the drug may be, or include, a radiolabeled amino acid, one or more biotinyl moieties detectable by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity detectable by optical or calorimetry), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety may be attached by spacer molecules. In some embodiments, the detectable label may include a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the contrast agent may include a radioisotope. In some embodiments, the radioisotope may be indium or technetium. In some embodiments, the contrast agent may include iodine, gadolinium, or iron oxide. In some embodiments, the enzyme may include horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label may include yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or europium derivatives. In some embodiments, the luminescence label may include N-methylacridium derivatives. In some embodiments, the label may include Alexa Fluor® labels such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label may include biotin, avidin, streptavidin, or one or more haptens. Further examples of detectable labels include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances.Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. An example of a luminescent substance is luminol. Examples of bioluminescent substances include luciferase, luciferin, and aequorin. Examples of suitable radioactive substances include... 125 I, 131 I, 35 S, or 3 H can be mentioned.

[0160] In some embodiments, the drug may be conjugated to an activatable protein using a carbohydrate moiety, a sulfhydryl group, an amino group, or a carboxylate group. In some embodiments, the drug may be conjugated to an activatable protein via a linker and / or CM described herein. In some embodiments, the drug may be conjugated to cysteine ​​or lysine in the activatable protein. In some embodiments, the drug may be conjugated to a residue of the activatable protein, for example, a residue disclosed herein.

[0161] In some embodiments, drugs can be conjugated to activatable proteins using various bifunctional protein coupling agents, including N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyl HCl adipiimidoate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, radioactive nucleotides can be conjugated to activatable proteins using a carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agent (see, for example, WO94 / 11026).

[0162] Appropriate conjugation components are those described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester)). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to activatable proteins via oligopeptides. In some embodiments, preferred conjugation moieties include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridinedithio)-toluene (Pierce Chem. Co., catalog (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamide]hexanoate (Pierce Chem. Co., catalog number 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamide]hexanoate (Pierce Chem. Co. catalog #2165-G), and (v) sulfo-NHS (N-hydroxysulfosuccinimidide: Pierce Chem. Co.) conjugated to EDC. Examples include Chem.Co., catalog number 24510. Additional exemplary conjugation parts include SMCC, sulfo-SMCC, SPDB, and sulfo-SPDB.

[0163] The conjugation portion described above may contain components with different attributes, thus resulting in conjugates with different physiological and chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Linkers containing NHS esters are less soluble than sulfo-NHS esters. Furthermore, SMPT contains sterically hindered disulfide bonds, which can form highly stable conjugates. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, making it difficult to obtain usable conjugates. Sulfo-NHS can particularly enhance the stability of carbodiimide coupling. When carboimidide coupling (e.g., EDC) is conjugated with sulfo-NHS, it forms esters that are more resistant to hydrolysis than the carboimidide coupling reaction alone.

[0164] Those skilled in the art will understand that a wide variety of possible parts can be coupled to the activatable proteins of this disclosure (see, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, JMCruse and RELewis, Jr(eds), Carger Press, New York, (1989) (the entire contents of which are incorporated herein by reference)). In general, effective conjugation of a drug (e.g., a cytotoxic agent) to an activatable protein can be achieved by any chemical reaction that binds the drug to the activatable protein while allowing both the drug and the activatable protein to retain their functionality.

[0165] Nucleic acids and vectors In some embodiments, the Disclosure further provides nucleic acids comprising sequences encoding activatable molecules as defined herein (e.g., activatable antibodies) or components or fragments thereof. Nucleic acids may comprise coding sequences for TB, CM, MM, EM, and linker(s) in an activatable protein. If the activatable protein comprises multiple polypeptides (e.g., multiple TBs on different polypeptides, or one TB comprising multiple polypeptides), the nucleic acid may comprise coding sequences for multiple polypeptides. In some examples, one coding sequence of polypeptides is contained in a nucleic acid, and another coding sequence of polypeptides is contained in another nucleic acid. In some examples, two or more coding sequences of multiple polypeptides are contained in the same nucleic acid. The Disclosure includes polynucleotides encoding proteins or parts thereof as defined herein, and the use of such polynucleotides for generating proteins and / or for therapeutic purposes. Such polynucleotides may comprise DNA and RNA molecules encoding proteins as defined herein (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.). The Disclosure includes compositions comprising such polynucleotides. In some embodiments, such compositions may be used therapeutically or prophylactically.

[0166] Unless otherwise specified, “protein-coding nucleic acid sequences” include all nucleotide sequences that are degenerate versions of each other and therefore code for the same amino acid sequence. The term “nucleic acid” means deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in single-stranded or double-stranded form. Unless otherwise specified, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties to the reference nucleotide. Unless otherwise specified, a particular nucleic acid sequence also implicitly includes complementary sequences in addition to the explicitly indicated sequence. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0167] The term "N-terminal" refers to the position of a first domain or sequence relative to a second domain or sequence in the primary amino acid sequence of a polypeptide, meaning that the first domain is located closer to the N-terminus of the polypeptide's primary amino acid sequence. In some embodiments, there may be further sequences and / or domains between the first domain or sequence and the second domain or sequence. The term "C-terminal" refers to the position of a first domain or sequence relative to a second domain or sequence in the primary amino acid sequence of a polypeptide, meaning that the first domain is located closer to the C-terminus of the polypeptide's primary amino acid sequence. In some embodiments, there may be further sequences and / or domains between the first domain or sequence and the second domain or sequence.

[0168] Modifications to nucleotide sequences can be introduced by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with acidic side chains (e.g., aspartate and glutamate), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine), and hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include aliphatic hydroxyamino acids (e.g., serine and threonine), amide families (e.g., asparagine and glutamine), aliphatic families (e.g., alanine, valine, leucine and isoleucine), and aromatic families (e.g., phenylalanine, tryptophan and tyrosine).

[0169] This disclosure further provides vectors and sets of vectors containing any of the nucleic acids described herein. Those skilled in the art will be able to select a suitable vector or set of vectors (e.g., an expression vector) to produce any of the activatable proteins described herein, and to express any of the activatable proteins described herein using the vector or set of vectors. For example, in the selection of a vector or set of vectors, the cell type may be selected such that the vector(s) may need to be able to be incorporated into and / or replicate therein in the chromosomes of a cell. Exemplary vectors that can be used to produce activatable proteins are also described herein. As used herein, the term “vector” means a polynucleotide capable of inducing the expression of a recombinant protein (e.g., a first or second monomer) within a cell (e.g., any of the cells described herein). A “vector” can deliver nucleic acids and fragments thereof into a host cell and includes regulatory sequences (e.g., promoters, enhancers, poly(A) signals). Exogenous polynucleotides may be inserted into an expression vector for expression. The term “vector” also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0170] Methods for creating vectors containing any one of the nucleic acids described herein and suitable for transforming cells (e.g., mammalian cells) are well known in the art. For example, Sambrook et al., Eds. "Molecular Cloning: A See Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, 1989 and Ausubel et., Eds., Current Protocols in Molecular Biology, Current Protocols, 1993.

[0171] Examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenovirus vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector), as well as any Gateway® vector. A vector may contain, for example, sufficient cis-active elements for expression. Other elements for expression may be supplied by host mammalian cells or in an in vitro expression system. Those skilled in the art will be able to select suitable vectors and mammalian cells for producing any of the activatable proteins described herein.

[0172] In some embodiments, the activatable protein may be biosynthetically produced using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0173] cell In some embodiments, this disclosure provides recombinant host cells comprising either a vector or nucleic acid as described herein. The cells may be used to generate activatable molecules (e.g., activatable antibodies) as described herein. In some embodiments, the cells may be animal cells, mammalian cells (e.g., human cells), rodent cells (e.g., mouse cells, rat cells, hamster cells, or guinea pig cells), non-human primate cells, insect cells, bacterial cells, fungal cells, or plant cells. In some embodiments, the cells may be eukaryotic cells. As used herein, the term “eukaryotic cell” means a cell having a different membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, e.g., Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, e.g., a mammalian, bird, plant, or insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human fetal kidney cells (e.g., HEK293 cells). In some embodiments, the cells may be prokaryotic cells.

[0174] Methods for introducing nucleic acids and vectors (e.g., any of the vectors or sets of vectors described herein) into cells are known in the art. Examples of methods available for introducing nucleic acids into cells include lipofection, transfusion, calcium phosphate transfusion, cationic polymer transfusion, viral transfusion (e.g., adenovirus transfusion, lentivirus transfusion), nanoparticle transfusion, and electroporation.

[0175] In some embodiments, the introduction step includes introducing into a cell a vector (e.g., any of the vectors or sets of vectors described herein) containing nucleic acids encoding monomers that constitute any activatable protein described herein.

[0176] Compositions and kits This disclosure also provides compositions and kits comprising the activatable molecules described herein (e.g., activatable antibodies). The compositions and kits may further include one or more excipients, carriers, reagents, and instructions necessary for the use of the activatable protein.

[0177] In some embodiments, the composition may be a pharmaceutical composition comprising an activatable protein, its derivatives, fragments, analogs, and homologs. The pharmaceutical composition may comprise an activatable protein and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent, and absorption retarder that is compatible with the administration of the pharmaceutical. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, the standard reference text in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous media such as liposomes and non-volatile oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is intended for use in the composition unless it is incompatible with the active compound. Complementary active ingredients may also be incorporated into the composition.

[0178] Pharmaceutical compositions can be formulated to suit their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain one or more of the following components: sterile diluents such as water for injection, physiological saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and isotonic modifiers such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In some cases, any of the activatable proteins described herein are prepared with a carrier that protects against rapid elimination from the body, such as implants and microencapsulated delivery systems, for both sustained-release and controlled-release formulations. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, polylactic acid-glycolic acid copolymers, and polylactic acid can be used. Methods for preparing such pharmaceutical compositions and formulations will be apparent to those skilled in the art. For example, the activatable protein may be incorporated into a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsifies, nanoparticles, and nanocapsules) or a macroemulsify, for example, in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively.

[0179] Sustained-release preparations may be prepared. Preferred examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which may be in the form of molded articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release over 100 days, while certain hydrogels release proteins over shorter periods.

[0180] In some embodiments, pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders, for the immediate preparation of injectable sterile solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The composition may be sterile, fluid, and must have a viscosity that allows for easy injection. It may also be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be, for example, water, ethanol, a solvent or dispersion medium containing polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In the case of dispersed particle compositions, suitable fluidity can be maintained, for example, by the use of a coating on the particles such as lecithin, and in the case of dispersions, by maintaining the required particle size, and by the use of surfactants. In some embodiments, the pharmaceutical composition may further contain one or more antimicrobial and / or antimicrobial antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In some embodiments, the composition may contain isotonic agents, such as sugars, polyhydric alcohols such as mannitol and sorbitol, and salts such as sodium chloride. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0181] In some embodiments, the pharmaceutical composition may include a sterile injection solution. A sterile injection solution can be prepared by incorporating the required amount of the active compound into a suitable solvent having, if necessary, one or a combination of the components listed above, and then sterilizing by filtration. Generally, dispersions can be prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injection solutions, the preparation method is vacuum drying and freeze-drying, where a powder of the active component plus any additional desired components is obtained from a pre-sterilized filtered solution.

[0182] In some embodiments, the pharmaceutical composition may include an oral composition. The oral composition may include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and may be used in the form of tablets, lozenges, or capsules. The oral composition may also be prepared using a fluid carrier for use as a mouthwash, in which the compound in the fluid carrier is used orally, rinsed and spat out, or swallowed. A pharmaceutically suitable binder and / or adjuvant may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar properties: binders (such as microcrystalline cellulose, tragacanth gum, or gelatin), excipients (such as starch or lactose), disintegrants (such as alginic acid, Primogel, or corn starch), lubricants (such as magnesium stearate or Sterotes), fluidizers (such as colloidal silicon dioxide), sweeteners (such as sucrose or saccharin), or flavorings (such as peppermint, methyl salicylate, or orange flavor).

[0183] In some embodiments, pharmaceutical compositions may be formulated for administration by inhalation. For example, the compound may be delivered in the form of an aerosol spray from a suitable spray, such as a pressurized container or dispenser containing a gas, such as carbon dioxide, or from a nebulizer.

[0184] In some embodiments, pharmaceutical compositions may be formulated for systemic administration. For example, systemic administration may be intravenous, and by mucosal or transdermal means. For mucosal or transdermal administration, a suitable penetrating agent may be used in the formulation for the barrier to which penetration is to occur. Such penetrating agents are generally known in the art and include, for example, surfactants, bile salts, and fusidic acid derivatives for mucosal administration. Mucosal administration may be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound may be formulated in the form of an ointment, salve, gel, or cream, as is generally known in the art.

[0185] In some embodiments, the pharmaceutical composition may be prepared for rectal delivery in the form of a suppository (for example, with a conventional suppository base such as cocoa butter and other glycerides) or in the form of a retained enema.

[0186] In one embodiment, the pharmaceutical composition may be prepared with a controlled-release formulation that includes a carrier, such as an implant and a microencapsulation delivery system, to protect the composition from rapid elimination from the body. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, polylactic acid-coglycolic acid, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0187] For ease of administration and uniformity of dosage, it may be particularly advantageous to formulate oral or parenteral compositions into unit dosage forms. As used herein, unit dosage forms refer to physically separate units suitable as unitary dosages for treating a subject, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit dosage forms in this disclosure may be determined, and may depend directly, on the inherent characteristics of the active compound, the specific therapeutic effect to be achieved, and the essential limitations of the art for formulating such active compounds for the treatment of an individual.

[0188] In some embodiments, the composition (e.g., a pharmaceutical composition) may be contained in a container, vial, syringe, injection pen, pack, or dispenser, optionally together with instructions for administration.

[0189] This specification also provides a kit containing any of the activatable proteins described herein, any of the compositions containing any of the activatable proteins described herein, or any of the pharmaceutical compositions containing any of the activatable proteins described herein. Furthermore, a kit is also provided containing, in addition to the activatable proteins described herein, one or more second therapeutic agents. The second therapeutic agent may be provided in a separate dosage form from the activatable protein. Alternatively, the second therapeutic agent may be formulated together with the activatable protein.

[0190] Any of the kits described herein may include instructions for using any of the compositions (e.g., pharmaceutical compositions) and / or any of the activatable proteins described herein. In some embodiments, the kit may include instructions for carrying out any of the methods described herein. In some embodiments, the kit may include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kit may provide a syringe for administering any of the pharmaceutical compositions described herein.

[0191] This specification also provides activatable proteins produced by any of the methods described herein. Compositions (e.g., pharmaceutical compositions) comprising any of the activatable proteins produced by any of the methods described herein are also provided. This specification also provides kits containing at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0192] Method for generating activatable molecules This specification provides a method for producing any of the activatable molecules (e.g., activatable proteins) described herein, comprising: (a) culturing any of the recombinant host cells described herein in a liquid medium under conditions sufficient to produce an activatable protein; and (b) recovering the activatable protein from the host cells and / or the liquid medium.

[0193] Methods for culturing cells are well known in the art. In some embodiments, cells can be maintained in vitro under conditions favorable for cell proliferation, cell differentiation, and cell growth. For example, recombinant cells can be cultured by contacting cells (e.g., any of the cells described herein) with a cell medium containing the necessary growth factors and sufficient adjuvants to support cell viability and growth.

[0194] In some embodiments, the method may further include isolating the recovered activatable protein. Isolation of the activatable protein can be performed using any separation or purification technique for separating protein species, such as affinity tag-based protein purification (e.g., polyhistidine (His) tag, glutathione-S-transferase tag, etc.), ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand affinity chromatography (e.g., protein A chromatography), ion exchange chromatography (e.g., anionic or cationic), hydrophobic interaction chromatography, etc.

[0195] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that enable the formation of disulfide bonds between the MM and TB of the activatable protein.

[0196] In some embodiments, the method further comprises incorporating an isolated activatable protein into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any isolated activatable protein described herein can be prepared for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0197] Methods using activatable molecules In some embodiments, the Disclosure further provides methods for using the activatable molecules (e.g., activatable antibodies) described herein. In some embodiments, the Disclosure provides methods for treating a disease of interest (e.g., cancer (e.g., any of the cancers described herein)) which comprises administering a therapeutically effective amount of one of the activatable proteins described herein to a subject. In some embodiments, the Disclosure provides methods for preventing, slowing the progression of, treating, alleviating, or improving a disease in a subject by administering a therapeutically effective amount of one of the activatable proteins described herein to a subject in need thereof. The term “treatment” means improving at least one symptom of the disease. In some embodiments, the disease to be treated may be cancer or an autoimmune disease, and may be for alleviating at least one symptom of cancer or an autoimmune disease. As used herein, the term “subject” means any mammal. In some embodiments, the subject is a feline (e.g., cat), a canid (e.g., dog), an equid (e.g., horse), a rabbit, a pig, a rodent (e.g., mouse, rat, hamster, or guinea pig), a non-human primate (e.g., Simian (e.g., monkey (e.g., baboon, marmoset), or ape (e.g., chimpanzee, gorilla, orangutan, or gibbon)), or a human. In some embodiments, the subject is a human. The terms subject and patient are used interchangeably. In some embodiments, the subject is pre-identified or diagnosed with a disease (e.g., cancer (e.g., any of the cancers described herein)).

[0198] In some embodiments, subjects may be identified as having mutations in the HER2 gene that increase the expression and / or activity of HER2 in mammalian cells (e.g., any of the mammalian cells described herein). For example, mutations in the HER2 gene that increase the expression and / or activity of HER2 in mammalian cells may be mutations resulting in the expression of HER2 with gene duplication, one or more amino acid substitutions (e.g., one or more amino acid substitutions selected from the group consisting of G309A, G309E, S310F, R678Q, L755S, L755W, I767M, D769H, D769Y, V777L, Y835F, V842I, R896C, and G1201V) (compared to wild-type protein). See, for example, Weigelt and Reis-Filho, Cancer Discov. 2013, 3(2):145-147.

[0199] Non-exclusive examples of methods for detecting HER2-related disease in a subject include immunohistochemistry, fluorescence in situ hybridization (FISH), and chromogenic in situ hybridization (CISH). See, for example, Yan et al., Cancer Metastasis Rev. 2015, 34:157-164.

[0200] The therapeutically effective dose of the activatable protein of this disclosure generally relates to the amount required to achieve a therapeutic objective. As described above, this can be the binding interaction between the antibody and its target antigen, which in certain cases interferes with the function of the target. The amount required to be administered further depends on the binding affinity of the activatable protein to its particular target and also on the rate at which the administered activatable protein is depleted from other subjects in the free deposit to which it is administered. A general range of therapeutically effective doses of the activatable protein of this disclosure may, as non-limiting examples, be about 0.001, 0.01, 0.1, 0.3, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg / kg body weight or more. The structure of the activatable protein of this disclosure allows for a reduction in the dose of the activatable protein administered to a subject compared to conventional activatable antibodies and conventional antibodies. For example, the dose based on a unit dosage form, or the total dose over the duration of the administration regimen, may be reduced by 10, 20, 30, 40, or 50% compared to the corresponding dose of the corresponding conventional activatable protein or the corresponding conventional antibody.

[0201] Typical administration frequencies may range from once or twice a day, once or twice a week, once or twice every two weeks, or once or twice a month.

[0202] The effectiveness of the treatment is determined in relation to any known method for diagnosing or treating a specific disorder. Methods for screening activatable proteins with desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISA) and other immunological intervention techniques known within the art.

[0203] In another embodiment, an activatable protein directed to two or more targets is used in methods known in the art for target localization and / or quantification (e.g., use in measuring the level of one or more targets in a suitable physiological sample, use in diagnostic methods, use in protein imaging, etc.). In a given embodiment, an activatable protein directed to two or more targets, or its derivatives, fragments, analogs, or homologs containing an antigen-binding domain derived from an antibody, is used as a pharmacologically active compound (hereinafter referred to as "therapeutic agent").

[0204] The activatable proteins used in any of these methods and embodiments of use can be administered at any stage of the disease. For example, such activatable proteins may be administered to patients with cancer at any stage, from early to metastatic. In some embodiments, the activatable proteins and their formulations may be administered to subjects suffering from or susceptible to diseases or disorders associated with abnormal target expression and / or activity.

[0205] Subjects suffering from or susceptible to diseases or disorders associated with abnormal target expression and / or activity may be identified using any of the various methods known in the art. For example, subjects suffering from cancer or other neoplastic conditions may be identified using a physical examination to assess their health status and any of the various clinical and / or laboratory tests, such as blood, urine and / or stool analysis. For example, subjects suffering from inflammation and / or inflammatory disorders may be identified using a physical examination to assess their health status and any of the various clinical and / or laboratory tests, such as blood, urine and / or stool analysis.

[0206] In some embodiments, administration of an activatable protein to a patient suffering from a disease or disorder associated with abnormal target expression and / or activity may be considered successful if any of the various experimental or clinical objectives are achieved. For example, administration of an activatable protein to a patient suffering from a disease or disorder associated with abnormal target expression and / or activity may be considered successful if one or more symptoms associated with the disease or disorder are alleviated, reduced, suppressed, or worsened, i.e., do not progress to a worse state. Administration of an activatable protein to a patient suffering from a disease or disorder associated with abnormal target expression and / or activity may be considered successful if the disease or disorder enters remission or worsened, i.e., does not progress to a worse state.

[0207] As used herein, the term “treat” includes reducing the severity, frequency, or number of one or more symptoms or signs of a disease (e.g., cancer (e.g., any of the cancers described herein)) in a subject (e.g., any of the subjects described herein). In some embodiments where the disease is cancer, treatment results in a reduction of cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduction in the risk of cancer recurrence in a subject with cancer.

[0208] In some embodiments, the disease may be cancer. In some embodiments, the subject may be identified or diagnosed with cancer. Examples of cancers include solid tumors, hematological malignancies, sarcomas, osteosarcomas, gliablastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, lymphomas (e.g., B-cell lymphoma, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, malignant cutaneous T-cell lymphoma), leukemias (e.g., pilocytic cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic bone cancer). Examples of cancers that can be treated include myelin leukemia (CML), acute lymphoblastic leukemia (ALL), spinal cord dysplasia syndrome (MDS), Kaposi's sarcoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colorectal cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), squamous cell head and neck cancer, endometrial cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is Burkitt lymphoma. In some embodiments, the subjects are identified or diagnosed with familial cancer syndromes, such as Lifraumeni syndrome or familial breast and ovarian cancer (BRCA1 or BRAC2 mutation) syndrome. The disclosed methods are also useful for treating non-solid tumors. Exemplary solid tumors include malignant tumors (e.g., sarcomas, adenocarcinomas, and carcinomas) of various organ systems, such as the lungs, breasts, lymphatic system, digestive tract (e.g., colon), genitourinary tract (e.g., tumors of the kidneys, urothelium, or testes), pharynx, prostate, and ovaries. Exemplary adenocarcinomas include colorectal cancer, renal cell tumors, liver cancer, non-small cell lung cancer, and small intestine cancer. Further examples of cancers that can be treated by the compositions and methods herein include: acute lymphoblastic leukemia (adult); acute lymphoblastic leukemia (child); acute myeloid leukemia (adult); adrenocortical carcinoma; adrenocortical carcinoma (child); AIDS-associated lymphoma; AIDS-associated malignant tumor; anal cancer; pediatric cerebellar astrocytoma; pediatric cerebral astrocytoma; extrahepatic cholangiocarcinoma; bladder cancer; bladder cancer (child); bone cancer, osteosarcoma / malignant fibrous histiocytoma; brainstem glioma (child); brain tumor (adult); brain tumor, brainstem glioma (child); brain tumor, cerebellar astrocytoma (child); brain tumor, cerebral astrocytoma / malignant glioma (child); brain tumor, ependymoma (child);Brain tumors, medulloblastoma (childhood); brain tumors, supratentorial primitive neuroectoderm tumor (childhood); brain tumors, visual tract and hypothalamic glioma (childhood); other brain tumors (childhood); breast cancer; breast cancer and pregnancy; breast cancer (childhood); breast cancer (male); bronchial adenoma / carcinoid (childhood); carcinoid tumor (childhood); gastrointestinal carcinoid tumor; adrenal cortical carcinoma; islet cell carcinoma; cancer of unknown primary origin; primary central nervous system lymphoma; cerebellar astrocytoma (childhood); cerebral astrocytoma / malignant glioma (childhood); cervical cancer; childhood cancer; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorder; tenosynovial clear cell sarcoma; colorectal cancer; colorectal cancer (childhood) Childhood); Cutaneous T-cell lymphoma; Endometrial cancer; Ependymoma (child); Ovarian epithelial carcinoma; Esophageal cancer; Esophageal cancer (child); Ewing's sarcoma family tumor; Extracranial germ cell tumor (child); Extragonadal germ cell tumor; Extrahepatic bile duct cancer; Ocular cancer (intraocular melanoma); Ocular cancer (retinoblastoma); Gallbladder cancer; Gastric (stomach) cancer; Gastric (stomach) cancer (child); Gastrointestinal carcinoid tumor; Extracranial germ cell tumor (child); Extracranial germ cell tumor; Ovarian germ cell tumor; Gestational choriocarcinoma; Pediatric brainstem glioma; Pediatric visual pathway and hypothalamic glioma; Pilocytic cell leukemia; Head and neck Laryngeal cancer; Primary hepatocellular carcinoma (adult); Primary hepatocellular carcinoma (child); Hodgkin's lymphoma (adult); Hodgkin's lymphoma (child); Hodgkin's lymphoma during pregnancy; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma (child); Intraocular melanoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Kidney cancer; Laryngeal cancer; Laryngeal cancer (child); Acute lymphoblastic leukemia (adult); Acute lymphoblastic leukemia (child); Acute myeloid leukemia (adult); Acute myeloid leukemia (child); Chronic lymphocytic leukemia; Chronic myeloid leukemia; Pilocytic cell leukemia; Lip and oral cancer; Primary liver cancer (adult); Primary liver cancer (child) Non-small cell lung cancer; Small cell lung cancer; Acute lymphoblastic leukemia (adult); Acute lymphoblastic leukemia (child); Chronic lymphocytic leukemia; AIDS-associated lymphoma; Primary central nervous system lymphoma; Cutaneous T-cell lymphoma; Hodgkin lymphoma (adult); Hodgkin lymphoma (child); Hodgkin lymphoma during pregnancy; Non-Hodgkin lymphoma (adult); Non-Hodgkin lymphoma (child); Non-Hodgkin lymphoma during pregnancy; Primary central nervous system lymphoma; Waldenström macroglobulinemia; Male breast cancer; Malignant mesothelioma (adult); Malignant mesothelioma (child); Malignant thymoma; Medulloblastoma (child); Melanoma;Intraocular melanoma; Merkel cell carcinoma; Malignant mesothelioma; Metastatic squamous cell carcinoma of unknown primary origin; Multiple endocrine neoplasia syndrome (childhood); Multiple myeloma / plasmacytic neoplasm; Mycosis fungoides; Myelodysplastic syndrome; Myeloid leukemia (chronic); Acute myeloid leukemia (childhood); Multiple myeloma; Chronic myeloproliferative disorder; Nasal cavity and paranasal sinus cancer; Nasopharyngeal cancer; Nasopharyngeal cancer (childhood); Neuroblastoma; Non-Hodgkin lymphoma (adult); Non-Hodgkin lymphoma (childhood); Non-Hodgkin lymphoma during pregnancy; Non-small cell lung cancer; Oral cancer (childhood); Oral and lip cancer; Oral and pharyngeal cancer; Osteosarcoma / Malignant fibrous bone Histiocytoma; ovarian cancer (childhood); ovarian epithelial carcinoma; ovarian germ cell tumor; low-grade ovarian tumor; pancreatic cancer; pancreatic cancer (childhood); islet cell pancreatic cancer; sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pheochromocytoma; primary neuroectodermal tumors of the pineal gland and upper ventricles (childhood); pituitary tumor; plasma cell tumor / multiple myeloma; pleuroblastoma; pregnancy and breast cancer; pregnancy and Hodgkin lymphoma; pregnancy and non-Hodgkin lymphoma; primary central nervous system lymphoma; primary liver cancer (adult); primary liver cancer (childhood); prostate cancer; rectal cancer; renal cell carcinoma; renal cell carcinoma (childhood); renal-pelvic and ureteral junction Skin cancer; retinoblastoma; rhabdomyosarcoma (childhood); salivary gland cancer; salivary gland cancer (childhood); Ewing's sarcoma family tumors; Kaposi's sarcoma; sarcoma (osteosarcoma) / malignant fibrous histiocytoma; rhabdomyosarcoma (childhood); soft tissue sarcoma (adult); soft tissue sarcoma (childhood); Sézary syndrome; skin cancer; skin cancer (childhood); skin cancer (melanoma); Merkel cell carcinoma; small cell lung cancer; small intestine cancer; soft tissue sarcoma (adult); soft tissue sarcoma (childhood); primary and metastatic squamous cell carcinoma; stomach cancer; stomach Examples include cancer (childhood); primary supraventricular neuroectodermal tumor (childhood); cutaneous T-cell lymphoma; testicular cancer; thymoma (childhood); malignant thymoma; thyroid cancer; thyroid cancer (childhood); transitional cell carcinoma of the renal pelvis and ureter; choriocarcinoma during pregnancy; cancer of unknown primary site (childhood); unusual cancers in childhood; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; glioma of the visual pathway and hypothalamic nerve (childhood); vulvar cancer; Waldenström giant cell globulinemia; Wilms' tumor; diffuse large B-cell lymphoma (DLBCL); and mantle cell lymphoma (MCL). Metastasis of the above cancers can also be treated or prevented according to the methods described herein.

[0209] In some embodiments, the disease can be an autoimmune disease or condition. In some embodiments, the subject may be identified or diagnosed as having an autoimmune disease or condition, or may be at high risk of developing an autoimmune disease or condition. Examples of autoimmune diseases include type 1 diabetes, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, celiac disease), infectious diseases (e.g., chickenpox, cold, diphtheria, Escherichia coli, diarrhea, HIV / AIDS, infectious mononucleosis, influenza, Lyme disease, malaria, measles, meningitis, mumps, polio, pneumonia, Rocky Mountain spotted fever, rubella (German measles), salmonella infection, severe acute respiratory syndrome (SARS), sexually transmitted infections, shingles, tetanus, toxic shock syndrome, tuberculosis, viral hepatitis, West Nile virus, pertussis), chronic inflammation, transplant rejection (e.g., in kidney, liver, or heart transplantation), autoimmune diseases, infectious diseases, chronic inflammation, or transplant rejection.

[0210] In some embodiments, the methods herein can result in a reduction in the number, severity, or frequency of one or more symptoms of cancer in a subject (e.g., as compared to the number, severity or frequency of one or more symptoms of cancer in the subject prior to treatment).

[0211] The method can further include administering to the subject one or more additional agents.

[0212] In some embodiments, the activatable protein may be administered in combination with one or more additional agents during and / or after treatment. In some embodiments, the activatable protein may be formulated in a single therapeutic composition, and the activatable protein and additional agents may be administered simultaneously. Alternatively, the activatable protein and additional agents may be separate, for example, each incorporated into a different therapeutic composition, and the activatable protein and additional agents may be administered simultaneously or at different points in time during the treatment regimen. For example, the activatable protein may be administered before, following, or alternately with the additional agents. The activatable protein and additional agents may be administered in single or multiple doses.

[0213] One or more of the activatable proteins described herein may be co-formulated with and / or co-administered with one or more anti-inflammatory agents, immunosuppressants, or metabolic or enzyme inhibitors. In some embodiments, one or more of the activatable proteins described herein may be combined with one or more other types of activatable proteins (e.g., activatable proteins that do not have EM, or activatable proteins whose activated form includes EM).

[0214] The present disclosure also provides a method for detecting the presence or absence of a cleaving agent and / or a target in a subject or sample. Such a method can include (i) contacting the subject or biological sample with an activatable protein, wherein the activatable protein comprises a detectable label disposed on a portion of the activatable protein that is released after cleavage of the CM, and (ii) measuring the level of the activated protein in the subject or biological sample. A detectable level of the activated protein in the subject or biological sample indicates that the cleaving agent, the target, or both the cleaving agent and the target are not present and / or not present sufficiently in the subject or biological sample. Thus, target binding and / or protease cleavage of the activatable protein cannot be detected in the subject or biological sample, and a decrease in the detectable level of the activated protein in the subject or biological sample indicates the presence of the cleaving agent and the target in the subject or biological sample.

[0215] A decrease in the level of the detectable label can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or a substantially 100% decrease. In some embodiments, the detectable label can be conjugated to a component of the activatable protein, such as TB. In some embodiments, measuring the level of the activatable protein in the subject or sample can be accomplished using a secondary reagent that specifically binds to the activated protein, the reagent comprising a detectable label. The secondary reagent can be an antibody comprising a detectable label.

[0216] In some embodiments, activatable proteins may also be useful for detecting targets in patient samples and are therefore useful as diagnostic agents. For example, activatable proteins may be used in in vitro assays, such as ELISA, to detect target levels in patient samples. For example, activatable proteins may be immobilized on a solid support (e.g., the wells of a microtiter plate). The immobilized activatable protein can function as a capture protein for any target that may be present in the test sample. Before contacting the immobilized activatable protein with the patient sample, the solid support may be rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.

[0217] In some embodiments, the stage of a disease in a subject may be determined based on the expression level of a target protein (e.g., antigen) based on results obtained using an activatable protein in an in vitro diagnostic assay. For a given disease, blood samples may be collected from subjects diagnosed as being at various stages of disease progression and / or at various points in time during therapeutic intervention for the disease. A population of samples that provides statistically significant results for each stage of progression or treatment is used to specify a concentration range of a target protein (e.g., antigen) that may be considered specific to each stage.

[0218] The activatable proteins described herein may also be used in diagnostic and / or imaging methods. In some embodiments, such methods may be in vitro methods. In some embodiments, such methods may be in vivo methods. In some embodiments, such methods may be in situ methods. In some embodiments, such methods may be ex vivo methods. For example, an activatable protein having CM can be used to detect the presence or absence of an enzyme capable of cleaving CM. Such an activatable protein can be used for diagnostic purposes, which may include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment with an elevated reduction potential that can provide reduction of disulfide bonds) by the measured accumulation of an activating antibody (i.e., an antibody resulting from the cleavage of the activatable protein) in a given cell or tissue of a given host organism. Such accumulation of the activating protein indicates not only that the tissue expresses enzyme activity (or an increase in reduction potential depending on the nature of the CM), but also that the tissue expresses a target to which the activating protein binds.

[0219] For example, CMs may be selected to be substrates for proteases found in tumor sites, viral or bacterial infection sites, or biologically restricted sites (e.g., abscesses, intra-organs, etc.). TBs may bind to target proteins (e.g., antigens). Detectable labels (e.g., fluorescent or radioactive labels or radioactive tracers) can be conjugated to TBs or other regions of the activatable protein using methods well known to those skilled in the art. Suitable detectable labels can be discussed in relation to the screening methods described above, and additional specific examples are provided below. When TBs specific to a disease-state protein or peptide are used with a protease whose activity is elevated in the disease tissue of interest, the activatable protein may show increased binding to the disease tissue compared to tissues where the CM-specific enzyme is not present at detectable levels, is present at lower levels than in the disease tissue, or is inactive (e.g., in complex with a zymogen or inhibitor). Because small proteins and peptides are rapidly removed from the blood by the renal filtration system, and CM-specific enzymes are not present at detectable levels (or are present at lower levels or in an inactive structure in non-disease tissues), the accumulation of activated proteins may be increased in diseased tissues compared to non-disease tissues.

[0220] In some embodiments, the activatable protein may be useful for in vivo imaging, where detection of a fluorescence signal in a subject, e.g., a mammal including humans, indicates that the disease site contains the target and a protease specific to the CM of the activatable protein. In vivo imaging may be used to identify or select a patient population suitable for treatment with the activatable protein of this disclosure. For example, a patient who tests positive for both the target being tested and the protease that cleaves the substrate in the CM of the activatable protein (e.g., accumulating the activatable protein at the disease site) is identified as a suitable candidate for treatment with such an activatable protein containing such a CM. Similarly, a patient who tests negative may be identified as a suitable candidate for another form of treatment (i.e., not suitable for treatment with the activatable protein being tested). In some embodiments, a patient who tests negative for a first activatable protein may be tested with other activatable proteins containing different CMs until a therapeutically suitable activatable protein (e.g., an activatable protein containing a CM cleaved by the patient at the disease site) is identified.

[0221] In some embodiments, in situ imaging may be useful in identifying which patients to treat. For example, in situ imaging can be used to screen patient samples using an activatable protein to identify patients with appropriate locations, e.g., proteases and targets at the tumor site. In some embodiments, in situ imaging can be used to identify or select a patient population suitable for treatment with the activatable protein of this disclosure. For example, patients who test positive for both the target being tested and the protease that cleaves the substrate in the CM of the activatable protein (e.g., accumulating activated antibodies at the disease site) are identified as suitable candidates for treatment with such an activatable protein containing such a CM. Similarly, patients who test negative for either or both the target and the protease that cleave the CM used in the activatable protein being tested using these methods are identified as suitable candidates for another form of treatment (i.e., not suitable for treatment with the activatable protein being tested). In some embodiments, patients who test negative for a first activatable protein may be tested with other activatable proteins containing different CMs until a therapeutically suitable activatable protein is identified (e.g., an activatable protein containing a CM that is cleaved by the patient at the disease site). [Examples]

[0222] The present invention will be further illustrated in the following examples, which are not intended to limit the scope of the present invention as described in the claims, but rather to provide a proof of concept for the advantageous structures of the activatable polymers described herein.

[0223] Example 1: Generation of activatable bispecific molecules This example demonstrates the generation of exemplary activatable bispecific proteins in which the activated protein does not contain a half-life extension region (e.g., an Fc domain). Double-masked activatable bispecific molecules were prepared by a recombinant method. The proteins were prepared by transforming host cells with three polynucleotides: one having the sequence of SEQ ID NO: 21 (for ProC1446), 22 (for ProC1447), or 23 (for ProC1448); one having the sequence of SEQ ID NO: 1; and one having the sequence of SEQ ID NO: 18; and then culturing the resulting recombinant host cells. These proteins contain a masked Fab (AB1) that specifically binds to HER2 in the activated state, a masked scFv (AB2) that specifically binds to CD3 in the activated state, and a pair of knob and whole mutant Fc domains (EM). The structures of these activatable proteins are shown in Figure 6A.

[0224] Reference molecules ProC306 and ProC531 (non-mask bispecific molecules containing Fab that specifically binds to HER2; scFv that specifically binds to CD3; and pairs of knob and hole Fc domains, in a configuration different from the exemplary activatable bispecific molecules described above) were also prepared by recombinant methods.

[0225] Example 2. Protease treatment of activatable bispecific molecules To release the masking peptide, the double-masked, activatable, bispecific binding molecules prepared in Example 1 were treated overnight at 37°C with a recombinant human protease such as matrix metalloproteinase (MMP) or uPA. Complete protease treatment was tested by reducing SDS-PAGE. Protein aliquots (2 μg) were denatured in sample buffer (with reducing agent added if necessary) at 75°C for 10 minutes, separated at 175V for 1 hour on a 4-12% NuPAGE® Bis-Tris gel (Thermo Fisher Scientific, Waltham, MA catalog no. NP0321) in MOPS buffer, stained with InstantBlue® for 1 hour, followed by destaining in water for at least 4 hours before visualization.

[0226] The untreated protein was confirmed to possess all three chains in the reducing gel (Figures 7A and 7B). After overnight protease treatment, activation was incomplete, but the majority of the protease products had the expected molecular weight.

[0227] Example 3: CD3 antigen-binding ELISA The ability of the double-masked, activatable, bispecific molecule prepared in Example 1 to bind to the CD3 antigen was tested using CD3-binding ELISA. 100 μg of CD3e-his antigen (ACRO Biosystems), dissolved in 0.05 M carbonate-bicarbonate buffer, was adsorbed overnight at 4°C onto the wells of a 96-well microtiter plate. The plate was washed and blocked with blocking buffer (1X PBS, pH 7.4, 0.05% Tween®-20, 1% BSA). The double-masked, activatable, bispecific molecule was serially diluted four-fold with or without protease treatment along with an unmasked reference protein (ProC531) and applied to antigen-coated plates. The degree of protein binding to the peptide was measured by anti-human IgG (Fab-specific) immunoassay. The absorbance of A450 was measured using a plate reader. Dose-response curves were constructed using S-shaped fit nonlinear regression with GraphPad Prism software, and EC50 values ​​were obtained. The results are shown in Figure 8. These results demonstrate that each of the three double-masked, activatable bispecific molecules (ProC1446, ProC1447, and ProC1448) exhibits reduced binding to CD3 compared to the unmasked reference bispecific molecule (ProC531) and compared to the corresponding protease-treated activated molecule. The activity of the double-masked, activatable bispecific molecules recovered to the same or nearly the same level as the corresponding reference bispecific molecule (ProC531) upon treatment with uPA.

[0228] Example 4: HER2-dependent cytotoxicity of a double-masked, activatable, bispecific molecule The in vitro efficacy of the double-masked, activatable, bispecific molecules prepared in Example 1 was determined by a cytotoxicity assay. SKOV3-luc2 target cells and human PBMC effector cells (Stemcell Technologies) were plated together in a co-culture in RPMI medium (Gibco catalog number 22400071) supplemented with 5% human serum (MP Bio catalog number 2930949) at a target-to-effector cell ratio of 1:10. To this co-culture, titrations of intact ProC1446, ProC1447, and ProC1448, as well as their protease-activated forms (uPA-treated ProC1446, uPA-treated ProC1447, and uPA-treated ProC1448), and an unmasked reference ProC306 were added. The plates were incubated at 37°C and 5% CO2 for approximately 48 hours. After incubation, cytotoxicity was evaluated using the ONE-Glo® luciferase assay system (Promega catalog number E6130), and luminescence was measured using a plate reader (TECAN). The cytotoxicity percentage was calculated as follows: (1 - (experimental RLU / untreated mean RLU)) * 100. Using GraphPad PRISM, the cytotoxicity percentage data were plotted and the EC50 values ​​were calculated. The results are shown in Figures 9A-9C. These results demonstrate that each of the three double-masked, activatable bispecific molecules (ProC1446, ProC1447, and ProC1448) exhibits reduced cytotoxicity compared to the unmasked reference bispecific molecule (ProC306) and compared to the corresponding protease-treated activated molecule. The cytotoxic activity of the activated molecules after treatment with uPA was more potent than that of the unmasked reference bispecific molecule (ProC306).

[0229] Example 5: Binding of double-masked bispecific antibodies to Her2+NCI-N87, SKOV3, and CD3ε+Jurkat cells To determine whether the described Her2 and CD3ε masking peptides can inhibit binding in association with double-masked bispecific antibodies, a flow cytometry-based binding assay was performed.

[0230] NCI-N87 (ATCC), SKOV3 (ATCC), and Jurkat (clone E6-1, ATCC, TIB-152) cells were cultured in RPMI-1640 + glutamax (Life Technologies, catalog 72400-047), 10% heat-inactivated fetal bovine serum (HI-FBS, Life Technologies, catalog 10438-026), and, in the case of NCI-N87 cells, in puromycin (Gibco, catalog A11138-03, @2ug / ml). The following bispecific antibodies were tested: recombinantly generated activated SHL1-ProC1963, SHL2-ProC1965, 1 / 2TCB ProC306, and their respective double-masked, activatable bispecific antibodies, ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2), and ProC1441 (1 / 2TCB). We utilized two versions of the cleavable portion (CM) located between the EM (extended half-life portion) and the C-terminus: CM1 for ProC3007 and CM2 for ProC3008.

[0231] NCI-N87 and SKOV3 cells were separated using Versene® (Life Technologies, catalog 15040-066), washed, plated in 96-well plates at 150,000 cells / well, and resuspended in 50 μL of primary antibody (bispecific antibody). Jurkat cells were counted and plated as described for NCI-N87 and SKOV3. Primary antibody titration was started at the concentrations shown in Figures 13A-13B, followed by titration in 3-fold serial dilutions in FACS staining buffer + 2% FBS (BD Pharmingen, catalog 554656), which were then added to the cells. Cells were incubated at 4°C for approximately 1 hour with shaking, harvested, and washed with 2 x 200 μL of FACS staining buffer. Cells were resuspended in 50 μL of Alexa Fluor® 647 conjugate anti-human IgG, F(ab')2 fragment-specific antibody (1.5 μg / ml, Jackson ImmunoResearch, catalog 109-605-097) and incubated at 4°C with shaking for approximately 1 hour. Cells were harvested, washed, and resuspended in 200 μL of FACS staining buffer containing 2.5 μg / ml of 7-AAD (BD Biosciences, catalog 559925). Cells stained with the secondary antibody alone were used as a negative control. Data were acquired using an Attun® NxT flow cytometer, and the median fluorescence intensity (MFI) of live cells was calculated using FlowJo® V10.8.1. Raw MFI data were graphed using curve fitting analysis in GraphPad Prism.

[0232] Figures 13A to 13B show the binding (i.e., HER2 binding) of masked activatable short half-life antibodies, ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2), and masked antibody, ProC1441 (1 / 2TCB, non-activatable, short half-life antibody), and unmasked (ProC1963 (SHL1, unmasked, without Fc), ProC1965, and ProC306) anti-CD3, anti-HER2 bispecific antibodies, as well as a secondary antibody ("Sec only", negative control) to NCI-N87 and SKOV3 cells, respectively. Figure 13C shows the binding (i.e., CD3 binding) of the same molecules to Jurkat cells. The results show that all masked molecules exhibited a reduction in binding to both HER2 and CD3 compared to their corresponding unmasked forms, as represented by a right shift in the binding curves of the masked molecules (very low / no binding even at the highest concentration). The EC50 values were determined from replicate experiments. The mean EC50 values are shown in Table 1 below.

Table 1

[0233] The results show that the unmasked anti-HER2, anti-CD3 TCBs in the activatable short half-life forms (SHL1 and SHL2) exhibited binding to CD3 and HER2 to the same extent as the corresponding unmasked 1 / 2TCB forms. A moderate tendency for increased HER2 binding was observed for the activatable short half-life forms compared to the 1 / 2TCB forms. The masked activatable short half-life molecules exhibited highly attenuated HER2 and CD3 binding to the same extent as that observed for the masked 1 / 2TCB molecules. The masked 1 / 2TCB molecule (ProC1441), which is the molecule shown on the left side of Figure 12, lacks the third cleavable portion (CM3) (1205) between the EM and Fab, meaning that this molecule is not cleavable to release the EM.

[0234] Example 6: Biological Activity of Doubly Masked Activatable Bispecific Antibodies The biological activity of intact, activatable bispecific antibodies and recombinantly activated bispecific antibodies was assayed using cytotoxicity assays. Human PBMCs were purchased from HemaCare Inc (Van Nuys, CA) and co-cultured with Her2-expressing cancer cell lines NCI-N87 (ATCC) or SKOV3 (ATCC) in RPMI-1640 + glutamax at a ratio of 10:1 with 5% thermally inactivated human serum (Sigma, catalog H3667). The dose-response was tested at the initial concentrations shown in Figures 14A-14B and 15A-15B in co-culture medium of intact ProC1446(SHL1), ProC3007(SHL2), ProC3008(SHL2), and ProC1441(1 / 2TCB) with recombinantly generated activated bispecific antibodies ProC1963(SHL1), ProC1965(SHL2), and ProC306(1 / 2TCB), followed by 3-fold serial dilutions. After 48 hours, cytotoxicity was evaluated using the ONE-Glo® luciferase assay system (Promega, Madison, WI catalog E6130). Luminescence was measured using Infinite® M200 Pro (Tecantting AG, Switzerland). Cytotoxicity percentages were calculated and plotted using curve-fit analysis with GraphPad PRISM.

[0235] Figures 14A and 14B show that the recombinantly generated activated bispecific proteins ProC1963(SHL1) and ProC1965(SHL2) exhibit increased potency compared to ProC306 (EC50 more than 1200 times and 50 times lower, respectively), as indicated by the left shift in the dose-response curve.

[0236] Figures 15A and 15B show that in these assays, intact bispecific ProC1446, ProC3007, ProC3008, and ProC1446 are strongly masked compared to their recombinantly generated activated versions, ProC1963 and ProC1965, as indicated by the right-shifted dose-response curves.

[0237] Figures 16A and 16D show that in these assays, intact bispecific ProC1446, ProC3007, ProC3008, and ProC1441 are strongly masked compared to their recombinantly generated activated versions, ProC1963, ProC1965, and ProC306, respectively, as indicated by the right-shifted dose-response curves.

[0238] The EC50 value and masking efficiency (ME) were determined from repeated experiments. The results are provided in Tables 2A and 2B below. [Table 2A]

[0239] The EC50 of ProC1963 was approximately 1200–3000 times lower than that of ProC306. The EC50 of ProC1965 was 50 times lower than that of ProC306. These results suggest that the unmasked, activatable, short-lived TCB form exhibits higher potency with respect to half-life compared to the control (ProC306), which is not activatable, i.e., not cleavable to release EM. [Table 2B]

[0240] These results indicate that masking attenuated the activity of all three forms: SHL1, SHL2, and 1 / 2TCB.

[0241] Example 7: Double-masked, bispecific, activatable antibodies ProC3007 and its corresponding activated form ProC1965 induced regression of established NCI-N87 tumors in mice.

[0242] In this example, intact, activatable bispecific antibodies ProC3007(SHL2TCB), ProC3008(SHL2TCB), ProC1441, and recombinantly generated activated bispecific ProC1965 targeting Her2 and CD3ε were analyzed for their ability to induce regression or reduce proliferation of established NCI-N87 xenograft tumors in NOD scid gamma (NSG) mice transplanted with human PBMCs.

[0243] Human gastric cancer cell line NCI-N87 was obtained from ATCC and cultured in RPMI + Glutamax + 10% FBS according to established procedures. Purified and frozen human PBMCs were obtained from Hemacare Inc (Van Nuys, CA) (Donor ID number D163477; Lot number 22077550). NSG(trademark) (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) mice were obtained from Theackson Laboratories (Bar Harbor, ME).

[0244] On day 0, 1 × 10¹⁶ cells were placed in 100 μL of RPMI + Glutamax serum-free medium containing Matrigel®. 6 NCI-N87 cells were subcutaneously inoculated into the right flank of each mouse. Pre-frozen PBMCs from a single donor were thawed and administered on day 7 in a 1:1 ratio of CD3+ T cells to tumor cells (ip). Tumor volume was approximately 125 mm². 3 Upon reaching this stage, mice were randomized and assigned to treatment groups, receiving IV administration according to Table 3. Recombinant bispecific ProC1965 (SHL2 form, unmasked and without Fc domain) was administered three times weekly to compensate for the expected increase in clearance rate due to the absence of the half-life-extending (Fc) domain. The dose level of ProC1965 was adjusted to account for differences in molecular weight. Tumor volume was measured twice weekly. One mouse from the 0.5 mpk cohort was euthanized early. Subsequently, at days 21 and 25, the cohort had n=7. [Table 3]

[0245] Figure 17 is a plot of tumor volume against the number of days after the initial treatment dose. The results demonstrate that both intact, activatable bispecific antibody ProC3007 and recombinantly produced activated bispecific antibody ProC1965 induced regression of NCI-N87 xenograft tumors at doses of 1 and 0.5 mpk, respectively. The results showed that ProC1965 and ProC3007 were more effective than ProC1441 in this study. ProC3008 had similar efficacy to ProC1441 at equivalent dose levels.

[0246] A second in vivo experiment was performed as described above, but using PBMCs from a different donor (Hemacare, donor ID number D327579, lot number 21070049). This study included a panel of bispecific activating antibodies, including a recombinantly generated, activatable, short-half-life bispecific antibody (ProC1446) and its corresponding recombinantly generated activated version (ProC1963, i.e., having the structure of ProC1446 but lacking the mask and Fc domain). ProC1446 and ProC1963 were administered as described in Table 4 and evaluated for their ability to induce regression or reduce the proliferation of established NCI-N87 xenograft tumors in human PBMC-grafted NSG mice. Both ProC1963 and ProC1446 appeared to have antitumor activity in this experiment, and therefore ProC1963 retains the ability to induce tumor regression. [Table 4]

[0247] The molecular sequences of the examples and other sequences disclosed herein are listed in Table 5 below. [Table 5-1] [Table 5-2] Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12 Table 5-13 Table 5-14 Table 5-15 Table 5-16 Table 5-17 Table 5-18 Table 5-19 Table 5-20 Table 5-21 Table 5-22 Table 5-23 Table 5-24 Table 5-25 Table 5-26 Table 5-27 Table 5-28 Table 5-29 Table 5-30 Table 5-31 Table 5-32 Table 5-33 Table 5-34 Table 5-35 Table 5-36 Table 5-37 Table 5-38 Table 5-39 Table 5-40 Table 5-41 Table 5-42 Table 5-43 Table 5-44 Table 5-45 Table 5-46 Table 5-47 Table 5-48 Table 5-49 Table 5-50 Table 5-51 Table 5-52 Table 5-53 [Table 5-54] [Table 5-55] [Table 5-56] [Table 5-57] [Table 5-58] [Table 5-59] [Table 5-60] [Table 5-61] [Table 5-62] [Table 5-63] [Table 5-64] [Table 5-65] [Table 5-66] [Table 5-67]

[0248] Although the present invention has been described in relation to embodiments for carrying out the above invention, the foregoing description is intended to be illustrative and not to limit the scope of the invention, and it should be understood that the scope of the invention is defined by the appended claims. Other embodiments, advantages and modifications are included in the following claims.

[0249] All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In the event of any conflict, this specification shall prevail, including definitions. In addition to section headings, materials, methods, and examples are illustrative and not intended to be limiting. The present invention provides, for example, the following items: (Item 1) A protein that can be activated, A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the C-terminus of HVD1 or the C-terminus of LVD1; A first masking portion (MM1) is coupled to the AB1 either directly or indirectly via a first cleavable portion (CM1), wherein the MM1 inhibits the binding of the AB1 to the first target; A second masking portion (MM2) is coupled directly or indirectly to a half-life extension portion (EM); The EM is coupled to AB1 or AB2 either directly or indirectly via a second detachable portion (CM2), The MM2 is the activatable protein that inhibits the binding of AB2 to the second target. (Item 2) The EM is a dimer formed by a first fragment crystallizable (Fc) domain and a second Fc domain, as described in item 1, which is an activatable protein. (Item 3) The activatable protein according to item 1 or item 2, wherein the EM is the C-terminus of AB1 or AB2, and the EM is the C-terminus of AB1 or AB2 that is directly or indirectly coupled via CM2, and the MM1 is the N-terminus of AB1. (Item 4) A protein that can be activated, A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the C-terminus of HVD1 or LVD1; A first cleavable portion (CM1) and a first masking portion (MM1) optionally coupled to AB1 via one or more linkers, wherein the MM1 inhibits the binding of AB1 to the first target; A half-life extension portion (EM) directly or indirectly coupled to a second masking portion (MM2), wherein the EM and the MM2 are coupled to the AB1 or AB2 via a second cleavable portion (CM2) and optionally one or more linkers; The MM2 is the activatable protein that inhibits the binding of AB2 to the second target. (Item 5) The activatable protein according to item 4, wherein CM2 is the N-terminus of MM2, MM2 is the N-terminus of EM, and CM2 is the C-terminus of AB1 or AB2, to which EM and MM2 are coupled with CM2 and optionally one or more linkers. (Item 6) A protein that can be activated, A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the C-terminus of HVD1 or LVD1; A first cleavable portion (CM1) and a first masking portion (MM1) optionally coupled to AB1 via one or more linkers, wherein the MM1 inhibits the binding of AB1 to the first target; A half-life extension portion (EM) comprising a dimer of a first half-life extension portion (EM1) and a second half-life extension portion (EM2); The EM1 is coupled to the AB1 via a second detachable portion (CM2) and optionally one or more linkers. The aforementioned EM2 is directly or indirectly coupled to the second masking portion (MM2), The MM2 is the activatable protein that inhibits the binding of AB2 to the second target. (Item 7) The activatable protein described in item 6, wherein CM2 is the N-terminus of EM1 and CM2 is the C-terminus of AB1. (Item 8) The activatable protein is an activatable protein according to any one of items 1 to 7, comprising at least a first polypeptide and a second polypeptide. (Item 9) The first polypeptide is an activatable protein as described in item 8, comprising MM1, CM1, and VLD1 in order from the N-terminus to the C-terminus. (Item 10) The activatable protein according to any one of items 8 to 9, wherein the second polypeptide comprises the VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain, and the activatable protein further comprises a third polypeptide comprising a second Fc domain. (Item 11) The second polypeptide is an activatable protein as described in item 10, comprising, in order from the N-terminus to the C-terminus, VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain. (Item 12) The second polypeptide is an activatable protein as described in item 9, comprising, in order from the N-terminus to the C-terminus, the VHD1, the CM2, the MM2, and the first Fc domain. (Item 13) The second polypeptide is an activatable protein as described in item 9, comprising, in order from the N-terminus to the C-terminus, the VHD1, the CM2, and the first Fc domain. (Item 14) The first polypeptide is an activatable protein according to any one of items 9 or 12-13, comprising MM1, CM1, VLD1, VHD2, and VLD2. (Item 15) The first polypeptide is an activatable protein as described in item 13, comprising MM1, CM1, VLD1, VHD2, and VLD2 in order from the N-terminus to the C-terminus. (Item 16) The first polypeptide is an activatable protein as described in item 13, comprising MM1, CM1, VLD1, VLD2, and VHD2 in order from the N-terminus to the C-terminus. (Item 17) The protein comprises a third polypeptide, the third polypeptide comprising a second Fc domain and the MM2, the activatable protein according to any one of items 13 to 16. (Item 18) The MM2 is an activatable protein as described in item 17, wherein the MM2 is linked to the C-terminus of the second Fc domain via a linker. (Item 19) The MM2 is an activatable protein as described in item 17, wherein the MM2 is linked to the N-terminus of the second Fc domain via a linker. (Item 20) (i) MM1 and CM1, (ii) CM1 and VLD1, (iii) VHD1 and VLD2, (iv) VHD1 and VHD2, (v) VHD1 and CM2, (vi) VLD2 and VHD2, (vii) CM2 and MM2, (viii) CM2 and EM, (ix) EM and MM2, (x) VLD1 and VHD2, and / or (xi) An activatable protein according to any of items 1 to 19, further comprising a linker that couples VLD1 and VLD2. (Item 21) The linker is an activatable protein as described in any one of items 18-20, which is a peptide having 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids. (Item 22) The activatable protein described in item 2 and any one of items 10-19, wherein the first Fc domain is a whole variant of the Fc domain, and the second Fc domain is a knob variant of the Fc domain. (Item 23) The activatable protein described in item 22, wherein the whole variant of the Fc domain contains the sequence of SEQ ID NO: 2, and the knob variant of the Fc domain contains the sequence of SEQ ID NO: 1. (Item 24) The CM2 is an activatable protein as described in item 1, wherein CM2 is directly or indirectly coupled to the C-terminus of AB1 or the C-terminus of AB2. (Item 25) The activatable protein according to item 24, wherein MM2 is directly or indirectly coupled to the C-terminus of CM2, and MM2 is directly or indirectly coupled to the N-terminus of EM. (Item 26) The EM is an activatable protein as described in item 1 or item 24, comprising a dimer of a first half-life extension portion (EM1) and a second half-life extension portion (EM2). (Item 27) The CM2 is an activatable protein as described in item 26, which is directly or indirectly coupled to the N-terminus of the EM1. (Item 28) The activatable protein according to item 26, wherein CM2 is directly or indirectly coupled to the N-terminus of MM2, and MM2 is directly or indirectly coupled to the N-terminus of EM1. (Item 29) The MM2 is an activatable protein according to any one of item 26, which is directly or indirectly coupled to the N-terminus of the EM2. (Item 30) The first target or epitope is an activatable protein as described in any one of items 1 to 29, which is a tumor-associated antigen. (Item 31) The tumor-associated antigen is the human epidermal growth factor receptor 2 (HER2), an activatable protein as described in item 30. (Item 32) The aforementioned AB1 is the fab of trastuzumab, an activatable protein as described in item 31. (Item 33) The activatable protein described in item 31, wherein HVD1 comprises the sequence of SEQ ID NO: 27, and LVD1 comprises the sequence of SEQ ID NO: 17. (Item 34) The aforementioned AB2 is Immune effector cells that associate with Fv, White blood cells that associate with scFv, T cells that associate with scFv, NK cells that associate with scFv, Macrophages that associate with SCFv, or A mononuclear cell that associates with scFv, and an activatable protein as described in any one of items 1-33. (Item 35) The AB2 is an activatable protein as described in any one of items 1 to 34, which is either anti-CD3 epsilon scFv or anti-CTLA-4 scFv, or derived therefrom. (Item 36) The AB2 is an activatable protein as described in item 35, which is anti-CD3 epsilon scFv or derived therefrom. (Item 37) The activatable protein described in item 36, wherein the HVD2 comprises the sequence of SEQ ID NO: 30, and the LVD2 comprises the sequence of SEQ ID NO: 31. (Item 38) The aforementioned AB1 is an anti-HER2 antibody or an activatable protein derived therefrom, as described in any one of items 1 to 37. (Item 39) The CM1 and CM2 are each independently activatable proteins according to any one of items 1 to 38, comprising a substrate for a protease that is upregulated within the tumor microenvironment. (Item 40) The aforementioned AB1 is an activatable protein as described in any one of items 1 to 39, which is a fragment antigen-binding (Fab). (Item 41) The second target is a co-stimulatory molecule, an activatable protein as described in any one of items 1 to 40. (Item 42) The aforementioned co-stimulatory molecule is CD3, an activatable protein as described in item 41. (Item 43) Each of CM1 and CM2 is an activatable protein according to any one of items 1 to 42, containing the same protease substrate. (Item 44) The CM1 and CM2 are activatable proteins according to any one of items 1 to 42, comprising substrates for different proteases. (Item 45) A molecule that can be activated, A first target-binding domain (TB1) that specifically binds to the first target; A second target-binding domain (TB2) that specifically binds to a second target, wherein the TB2 is directly or indirectly coupled to the TB1; A first masking portion (MM1) directly or indirectly coupled to the TB1 via a first cleavable portion (CM1), wherein the MM1 inhibits the binding of the TB1 to the first target; A half-life extension portion (EM) and a second masking portion (MM2) directly or indirectly coupled to TB1 or TB2 via a second cleavable portion (CM2), wherein the MM2 inhibits the binding of TB2 to the second target; The components of the activatable molecule are configured such that the cleavage of CM1 and CM2 releases MM1, MM2, and EM from TB1, which is directly or indirectly coupled to TB2. The activatable molecule wherein, optionally, TB1 is an antigen-binding molecule (AB1) containing HVD1 and LVD1, and optionally, TB2 is an antigen-binding molecule (AB2) containing HVD2 and LVD2. (Item 46) Each of the aforementioned CM1 and CM2 independently contains ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartate protease, BACE, renin, aspartate cathepsin, cathepsin D, cathepsin E, caspase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, and Spase 8, caspase 9, caspase 10, caspase 14, cysteine ​​cathepsin, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cysteine ​​proteinase, cruzipain, regmine, otubein-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, metalloproteinase, meprin, neprilysin, PSMA, BMP-1, MMP MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, serine protease, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor protease, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, granzyme B, guanidinobenzoate, Ht An activatable protein as described in any one of items 1 to 45, comprising a substrate for a protease selected from the group consisting of rA1, human neutrophil elastase, lactoferrin, malapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane, serine protease, TTSP, DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, and TMPRSS4. (Item 47) The aforementioned MM1 is an activatable protein as described in any one of items 1 to 46, which is a peptide with a length of 2 to 40 amino acids. (Item 48) The aforementioned MM2 is an activatable protein as described in any one of items 1 to 47, which is a peptide with a length of 2 to 40 amino acids. (Item 49) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1. The N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the light chain variable region of AB2 via CM2. The activatable protein described in item 47 or 48, wherein the EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of the MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain of the EM. (Item 50) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1. The N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1 via CM2. The activatable protein described in item 47 or 48, wherein the EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of the MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain of the EM. (Item 51) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1. The EM comprises a dimer of a first Fc domain and a second Fc domain, wherein the N-terminus of the first Fc domain is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1 via the CM2. The activatable protein described in item 47 or 48, wherein the N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the second Fc domain. (Item 52) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1. The EM comprises a dimer of a first Fc domain and a second Fc domain, wherein the N-terminus of the first Fc domain is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1 via the CM2. The activatable protein described in item 47 or 48, wherein the C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the second Fc domain. (Item 53) An activatable protein according to any one of items 49 to 52, further comprising a linker between the MM2 and the first or second Fc domain directly or indirectly coupled to the MM2. (Item 54) The activatable protein according to any one of items 1 to 53, wherein MM1 comprises the sequence of SEQ ID NO: 40, and MM2 comprises one of the sequences of SEQ ID NOs: 34-37 or 66-70. (Item 55) The activatable protein according to any one of items 1 to 54, wherein MM1 has a dissociation constant for binding to AB1 that is greater than the dissociation constant of AB1 for binding to the first target or epitope, and MM2 has a dissociation constant for binding to AB2 that is greater than the dissociation constant of AB2 for binding to the second target or epitope. (Item 56) The activatable protein according to any one of items 1 to 55, wherein the components of the activatable protein are configured such that the cleavage of CM1 and CM2 cleaves MM1, MM2, and EM from the activatable protein, thereby producing an activated protein having a shorter half-life compared to a corresponding molecule comprising TB1, TB2, and EM. (Item 57) The activatable protein according to any one of items 1 to 55, wherein the components of the activatable protein are configured such that the cleavage of CM1 and CM2 cleaves MM1, MM2, and EM from the activatable protein, thereby producing an activated protein having higher target-binding activity compared to a corresponding molecule comprising TB1, TB2, and EM. (Item 58) The second polypeptide further comprises a linker (L2) between MM2 and AB2, and is an activatable protein according to any one of items 1 to 57. (Item 59) The L2 is an activatable protein as described in item 58, which is a peptide having an amino acid length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids. (Item 60) The second polypeptide further comprises a linker (L3) between AB2 and AB1, and is an activatable protein according to any one of items 1 to 59. (Item 61) The L3 is an activatable protein as described in item 60, which is a peptide having an amino acid length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids. (Item 62) An activatable molecule as described in item 45, further comprising one or more of the characteristics described in items 1 through 44. (Item 63) A protein that can be activated, A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the N-terminus of HVD1 or the N-terminus of LVD1; A first cleavable portion (CM1) and a first masking portion (MM1) optionally coupled to AB1 via one or more linkers, wherein the MM1 inhibits the binding of AB1 to the first target; A second cleavable portion (CM2) and a second masking portion (MM2) optionally coupled to AB1 via one or more linkers, wherein the MM2 inhibits the binding of AB2 to the second target; The activatable protein comprising a third cleavable portion (CM3) and a half-life extension portion (EM) that is optionally coupled directly or indirectly to the C-terminus of HVD1 or the C-terminus of LVD1 via one or more linkers. (Item 64) An activatable molecule as described in item 63, further comprising the characteristics described in one or more of items 1 through 66. (Item 65) A composition comprising an activatable protein and a carrier as described in any one of items 1 to 64. (Item 66) The composition according to item 65, wherein the composition is a pharmaceutical composition, and the carrier is a pharmaceutically acceptable carrier. (Item 67) A container, vial, syringe, injection pen, or kit comprising at least one dose of the composition described in item 66. (Item 68) A nucleic acid comprising a sequence encoding a second polypeptide as described in any of items 8, 10-13, 58, or 60. (Item 69) A vector containing the nucleic acid described in item 68. (Item 70) Cells containing nucleic acids as described in item 68, or vectors as described in item 69. (Item 71) A conjugated activatable protein containing an activatable protein described in any one of items 1 to 64, which is conjugated to a drug. (Item 72) The aforementioned drug is a therapeutic agent, an antitumor agent, a toxin, a diagnostic agent, a therapeutic polymer, a targeting moiety, or a detectable moiety, as described in item 71, which is a conjugated, activatable protein. (Item 73) The drug is a conjugated activatable protein according to item 71 or 72, which is conjugated to the antibody via a linker. (Item 74) The linker is a cleavable linker, a conjugated activatable protein as described in item 72. (Item 75) The linker is a non-cleaving linker, a conjugated activatable protein as described in item 72. (Item 76) A method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an activatable protein according to any one of items 1 to 64, a composition according to item 65 or 66, or a conjugated activatable protein according to any one of items 71 to 75. (Item 77) The subject is identified or diagnosed with cancer, according to the method described in item 76. (Item 78) A method for generating an activatable protein, The cells described in item 70 are cultured in a culture medium under conditions sufficient to produce the activatable protein, The method comprising recovering the activatable protein from the cells or the culture medium. (Item 79) The method according to item 78, further comprising isolating the activatable protein recovered from the cells or the culture medium. (Item 80) The isolation of the activatable protein is performed using protein purification tag and / or size exclusion chromatography as described in item 79. (Item 81) The method according to any one of items 78 to 80, further comprising incorporating the activatable protein into the pharmaceutical composition.

Claims

[Claim 1] The invention described in the present specification.

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