(12) STANDARD PATENT APPLICATION (11) Application No. AU 2020205249 Al (19) AUSTRALIAN PATENT OFFICE (54) Title Matriptase and u-plasminogen activator substrates and other cleavable moieties and methods of use thereof (51) International Patent Classification(s) C07K 16/00 (2006.01) A61K 39/00 (2006.01) (21) Application No: 2020205249 (22) Date of Filing: 2020.07.14 (43) Publication Date: 2020.08.06 (43) Publication Journal Date: 2020.08.06 (62) Divisional of: 2015210862 (71) Applicant(s) Cytomx Therapeutics, Inc. (72) Inventor(s) Nguyen, Margaret Thy Luu;Moore,Stephen James;Hostetter, Daniel Robert;Vasiljeva, Olga (74) Agent / Attorney Spruson & Ferguson, GPO Box 3898, Sydney, NSW, 2001, AU Abstract The invention relates generally to polypeptides that include a cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator (uPA), to activatable antibodies and other larger molecules that include the cleavable moiety that is a substrate for at least one protease selected from matriptase and u-plasminogen activator, and to methods of making and using these polypeptides that include a cleavable moiety that is a substrate for at least one protease selected from matriptase and uplasminogen activator in a variety of therapeutic, diagnostic and prophylactic indications. MATRIPTASE AND U-PLASMINOGEN ACTIVATOR SUBSTRATES AND OTHER CLEAVABLE MOIETIES AND METHODS OF USE THEREOF Related Applications [00011 This application is a divisional of No. 2015210862, and claims the benefit of U.S. Provisional Application No. 61/934,619, filed January 31, 2014 and U.S. Provisional Application No. 61/971,009, filed March 27, 2014, the contents of each of which are incorporated herein by reference in their entireties. Field of the Invention 100021 The invention relates generally to polypeptides that include a cleavable moiety that is a substrate for at least one protease selected from matriptase and u plasminogen activator (uPA), to activatable antibodies and other larger molecules that include the cleavable moiety that is a substrate for at least one protease selected from matriptase and uPA, and to methods of making and using these polypeptides that include a cleavable moiety that is a substrate for at least one protease selected from matriptase and uPA in a variety of therapeutic, diagnostic and prophylactic indications. BackgroundoftheInvention 100031 Proteases are enzymes that cleave the peptide bonds between amino acid residues. Some proteases are known to break specific peptide bonds based on the presence of a particular amino acid sequence within a protein. Proteases occur naturally in all organisms and are involved in a variety of physiological reactions from simple degradation to highly regulated pathways. However, many pathological conditions are associated with deregulated expression and/or activity of proteases. As such, inappropriate proteolysis can have a major role in development and progression of cancer as well as cardiovascular, inflammatory, neurodegenerative, eukaryotic, bacterial and viral and parasitic diseases. 100041 Accordingly, there exists a need to identify new substrates for proteases and to use these substrates in a variety of therapeutic, diagnostic and prophylactic indications. Summary of the Invention 100051 The disclosure provides amino acid sequences that include a cleavable moiety (CM) that is a substrate for at least one protease selected from matriptase (also I referred to herein as MT-SP1, matriptase-1, and similar terms denoting matriptase) and u plasminogen activator (also referred to herein as uPA, urokinase, urokinase-type plasminogen activator, and similar terms denoting uPA). These CMs are useful in a variety of therapeutic, diagnostic and prophylactic indications. [0006] In some embodiments, the CM is linked or otherwise attached to an antibody. For example, the CM is used to link one or more agents to the antibody or antigen binding fragment thereof that binds a given target, such that the CM is cleaved when exposed to the protease, i.e., matriptase and/or uPA, and the agent is released from the antibody or antigen-binding fragment. Exemplary targets include, but are not limited to the targets shown in Table 1. Exemplary antibodies or antigen-binding fragments thereof include, but are not limited to, the targets shown in Table 2. In some embodiments, the antibody in the uncleaved state has the structural arrangement from N-terminus to C terminus as follows: Agent-CM-(Antibody or Antigen-Binding Fragment) or (Antibody or Antigen-Binding Fragment)-CM-Agent. In some embodiments, the antibody comprises a linking peptide between the antibody or antigen-binding fragment and the CM. In some embodiments, the antibody or antigen-binding fragment comprises a linking peptide between the CM and the conjugated agent. [0007] In some embodiments, the antibody comprises a first linking peptide (LP1) and a second linking peptide (LP2), wherein the antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: Agent-LP1-CM-LP2- (Antibody or Antigen-Binding Fragment) or (Antibody or Antigen-Binding Fragment)-LP2 CM-LP1-Agent. In some embodiments, the two linking peptides need not be identical to each other. [0008] In some embodiments, at least one of LPl or LP2 comprises an amino acid sequence selected from the group consisting of (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 385) and (GGGS)n (SEQ ID NO: 386), where n is an integer of at least one. [0009] In some embodiments, at least one of LPl or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO: 387), GGSGG (SEQ ID NO: 388), GSGSG (SEQ ID NO: 389), GSGGG (SEQ ID NO: 390), GGGSG (SEQ ID NO: 391), and GSSSG (SEQ ID NO: 392). [00010] In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 393), GSSGGSGGSGG (SEQ ID NO: 394), 2 GSSGGSGGSGGS (SEQ ID NO: 395), GSSGGSGGSGGSGGGS (SEQ ID NO: 396), GSSGGSGGSG (SEQ ID NO: 397), or GSSGGSGGSGS (SEQ ID NO: 398). [00011] In some embodiments, LP2 comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 399), GSSGT (SEQ ID NO: 400) or GSSG (SEQ ID NO: 401). [00012] In some embodiments, the antibody or antigen-binding fragment has an equilibrium dissociation constant of about 100 nM or less for binding to the target. [000131 In some embodiments, the antibody or antigen-binding fragment thereof specifically binds a target. In some embodiments, the antibody or immunologically active fragment thereof that binds the target is a monoclonal antibody, domain antibody, single chain, Fab fragment, a F(ab') 2 fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such an antibody or immunologically active fragment thereof that binds the target is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody. [00014] In some embodiments, the protease, i.e., matriptase and/or uPA is co localized with the target in a tissue, and the protease cleaves the CM in the antibody when the antibody is exposed to the protease. [00015] In some embodiments, the CM is a polypeptide of up to 15 amino acids in length. [000161 In some embodiments, the CM is a substrate for at least matriptase. In some embodiments, the CM is a substrate for at least uPA. In some embodiments, the CM is a substrate for at least matriptase and uPA. [00017] In some embodiments, the CM is a substrate for matriptase and/or uPA, and is resistant to cleavage by at least one other protease. In some embodiments, the CM is a substrate for matriptase and/or uPA, and is resistant to cleavage by at least plasmin. In some embodiments, the CM is a substrate for matriptase and/or uPA, and is resistant to cleavage by at least tissue plasminogen activator (tPA). [00018] In some embodiments, the CM is a substrate for matriptase and/or uPA and includes a motif sequence that is recognized by matriptase and/or uPA, provided that for any given motif sequence of the disclosure: (i) the CM does not comprise any of the following amino acid sequences TGRGPSWV (SEQ ID NO: 402), SARGPSRW (SEQ ID NO: 403), or TARGPSFK (SEQ ID NO: 404); and the CM does not comprise a consensus 3 amino acid sequence based on these amino acid sequences, such as for example, TARGPSW (SEQ ID NO: 405); (ii) the CM does not comprise any of the following amino acid sequences LSGRSDNH (SEQ ID NO: 406), GGWHTGRN (SEQ ID NO: 407), HTGRSGAL (SEQ ID NO: 408), or PLTGRSGG (SEQ ID NO: 409); and the CM does not comprise a consensus amino acid sequence based on these amino acid sequences, such as for example, LTGRSGA (SEQ ID NO: 410); and/or (iii) the CM does not comprise any of the following amino acid sequences AARGPAIH (SEQ ID NO: 411), RGPAFNPM (SEQ ID NO: 412), SSRGPAYL (SEQ ID NO: 413), or RGPATPIM (SEQ ID NO: 414); and the CM does not comprise a consensus amino acid sequence based on these amino acid sequences, such as for example, RGPA (SEQ ID NO: 415). [00019] In some embodiments, the motif sequence is a substrate for at least matriptase and includes a core CM consensus sequence shown in Tables 8A-8J below. In some embodiments, the motif sequence includes a subgenus, i.e., a subset, of the core CM consensus sequence shown in Tables 8A-8J below. Table 8A. Matriptase Cleavable Core CM Consensus Sequence 1 Core CM Consensus 1 Subgenus of Core CM Consensus 1 X1X2X 3X4 X 5 (SEQ ID NO: 2), wherein X1 is A, G, P, R, S, or V; X 2 is A, L, M, P, S, or V; X3 is G, L, or P; X 4 is R; and X 5 is A, G, R, S, or V X X X X X (SEQ ID 1 2 3 4 5 NO: 1), wherein: X1X2X 3X4 X 5 (SEQ ID NO: 3), wherein X1 is A, X 1 is A, G, H, K, L, N, P, R, S, or V; P, R, S, or V; X 2 is A, L, M, S, or V; X 3 is P; X4 X 2 is A, H, L, M, P, Q, R, S, or V; is R; and X5 is A, G, S, or V X 3 is A, E, F, G, I, L, P, R, S, T, or V; X1X2X 3 X4 X 5 (SEQ ID NO: 4), wherein: X 1 is A, X 4 is A, I, K, N, R, T, or W; and P, or R; X 2 is A, S, or V; X 3 is P; X4 is R; and X5 X 5 is A, G, I, L, M, Q, R, S, or V is S or V X1X2X 3X4 X 5(SEQ ID NO: 5), wherein: X 1 is A, P, or R; X 2 is A or S; X3 is P; X4 is R; and X5 is S 4 Table 8B.